TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.

PubMed

Hof, Thomas; Sallé, Laurent; Coulbault, Laurent; Richer, Romain; Alexandre, Joachim; Rouet, René; Manrique, Alain; Guinamard, Romain

2016-01-15

The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells. Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate

Structural Basis of Cerebellar Microcircuits in the Rat

PubMed Central

Cerminara, Nadia L.; Aoki, Hanako; Loft, Michaela; Apps, Richard

2013-01-01

The topography of the cerebellar cortex is described by at least three different maps, with the basic units of each map termed “microzones,” “patches,” and “bands.” These are defined, respectively, by different patterns of climbing fiber input, mossy fiber input, and Purkinje cell (PC) phenotype. Based on embryological development, the “one-map” hypothesis proposes that the basic units of each map align in the adult animal and the aim of the present study was to test this possibility. In barbiturate anesthetized adult rats, nanoinjections of bidirectional tracer (Retrobeads and biotinylated dextran amine) were made into somatotopically identified regions within the hindlimb C1 zone in copula pyramidis. Injection sites were mapped relative to PC bands defined by the molecular marker zebrin II and were correlated with the pattern of retrograde cell labeling within the inferior olive and in the basilar pontine nuclei to determine connectivity of microzones and patches, respectively, and also with the distributions of biotinylated dextran amine-labeled PC terminals in the cerebellar nuclei. Zebrin bands were found to be related to both climbing fiber and mossy fiber inputs and also to cortical representation of different parts of the ipsilateral hindpaw, indicating a precise spatial organization within cerebellar microcircuitry. This precise connectivity extends to PC terminal fields in the cerebellar nuclei and olivonuclear projections. These findings strongly support the one-map hypothesis and suggest that, at the microcircuit level of resolution, the cerebellar cortex has a common plan of spatial organization for major inputs, outputs, and PC phenotype. PMID:24133249

[Hering, Vintschgau and the problem of Purkinje's succession].

PubMed

Sablik, K

1989-01-01

The problem of Jan Evangelista Purkinje's succession will be presented according to the results of archival research. The Ministery of Cult and Education in Vienna, and especially Karl Rokitansky, who was the adviser for medical education, in 1867 created a new professorship and Institute for Physiology, beside Purkinje and his Institute. Maximilian Vintschgau was to assist the world-famous 80 years old Purkinje but was not permitted to teach the whole field of physiology and to examine students. The fact that the professors of the Prague Medical Faculty in 1868 started to remove the restrictions for Vintschgau with the argument of academic freedom and in 1869 tried to keep the second institute for the future, is not yet mentioned in the literature. Discussions about the problems of the Czech language and its use in physiological lectures were scarcely mentioned by the Ministery: if one day there should be a Czech-speaking lecturer, the problem would be solved. Unfortunately Purkinje had no genuine pupil in Prague, and after his death, Vintschgau was provisional director of the Institute for half a year. In this situation Rokitansky decided that there should only be one institute for physiology in Prague. The Medical Faculty wanted to have Hermann Helmholtz to succeed Purkinje, but Helmholtz refused to come. Ewald Hering, who was nominated in the second place by the Faculty, accepted the call. Vintschgau had only rank four, third was Conrad Eckhard from Giessen. The Ministery in Vienna, however, made a special decision: The Medical Faculty of Innsbruck was founded in 1869, and there was not professor for physiology at the beginning of 1870. The candidates of the Insbruck Medical Faculty were neglected in favour of Vintschgau, who was considered to be a trustworthy Austrian patriot. Hering and Vintschgau became professors on March 6, 1870, and Hering started his work in Prague in a new institute in the "Wenzelsbad".

Ultrasonic vocalization impairment of Foxp2 (R552H) knockin mice related to speech-language disorder and abnormality of Purkinje cells

PubMed Central

Fujita, Eriko; Tanabe, Yuko; Shiota, Akira; Ueda, Masatsugu; Suwa, Kiyotaka; Momoi, Mariko Y.; Momoi, Takashi

2008-01-01

Previous studies have demonstrated that mutation in the forkhead domain of the forkhead box P2 (FOXP2) protein (R553H) causes speech-language disorders. To further analyze FOXP2 function in speech learning, we generated a knockin (KI) mouse for Foxp2 (R552H) [Foxp2 (R552H)-KI], corresponding to the human FOXP2 (R553H) mutation, by homologous recombination. Homozygous Foxp2 (R552H)-KI mice showed reduced weight, immature development of the cerebellum with incompletely folded folia, Purkinje cells with poor dendritic arbors and less synaptophysin immunoreactivity, and achieved crisis stage for survival 3 weeks after birth. At postnatal day 10, these mice also showed severe ultrasonic vocalization (USV) and motor impairment, whereas the heterozygous Foxp2 (R552H)-KI mice exhibited modest impairments. Similar to the wild-type protein, Foxp2 (R552H) localized in the nuclei of the Purkinje cells and the thalamus, striatum, cortex, and hippocampus (CA1) neurons of the homozygous Foxp2 (R552H)-KI mice (postnatal day 10), and some of the neurons showed nuclear aggregates of Foxp2 (R552H). In addition to the immature development of the cerebellum, Foxp2 (R552H) nuclear aggregates may further compromise the function of the Purkinje cells and cerebral neurons of the homozygous mice, resulting in their death. In contrast, heterozygous Foxp2 (R552H)-KI mice, which showed modest impairment of USVs with different USV qualities and which did not exhibit nuclear aggregates, should provide insights into the common molecular mechanisms between the mouse USV and human speech learning and the relationship between the USV and motor neural systems. PMID:18287060

Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

PubMed

Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

2010-08-20

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R

Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and Purkinje fibers.

PubMed

Nayak, Alok Ranjan; Panfilov, A V; Pandit, Rahul

2017-02-01

We present systematic numerical studies of the possible effects of the coupling of human endocardial and Purkinje cells at cellular and two-dimensional tissue levels. We find that the autorhythmic-activity frequency of the Purkinje cell in a composite decreases with an increase in the coupling strength; this can even eliminate the autorhythmicity. We observe a delay between the beginning of the action potentials of endocardial and Purkinje cells in a composite; such a delay increases as we decrease the diffusive coupling, and eventually a failure of transmission occurs. An increase in the diffusive coupling decreases the slope of the action-potential-duration-restitution curve of an endocardial cell in a composite. By using a minimal model for the Purkinje network, in which we have a two-dimensional, bilayer tissue, with a layer of Purkinje cells on top of a layer of endocardial cells, we can stabilize spiral-wave turbulence; however, for a sparse distribution of Purkinje-ventricular junctions, at which these two layers are coupled, we can also obtain additional focal activity and many complex transient regimes. We also present additional effects resulting from the coupling of Purkinje and endocardial layers and discuss the relation of our results to the studies performed in anatomically accurate models of the Purkinje network.

Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and Purkinje fibers

NASA Astrophysics Data System (ADS)

Nayak, Alok Ranjan; Panfilov, A. V.; Pandit, Rahul

2017-02-01

We present systematic numerical studies of the possible effects of the coupling of human endocardial and Purkinje cells at cellular and two-dimensional tissue levels. We find that the autorhythmic-activity frequency of the Purkinje cell in a composite decreases with an increase in the coupling strength; this can even eliminate the autorhythmicity. We observe a delay between the beginning of the action potentials of endocardial and Purkinje cells in a composite; such a delay increases as we decrease the diffusive coupling, and eventually a failure of transmission occurs. An increase in the diffusive coupling decreases the slope of the action-potential-duration-restitution curve of an endocardial cell in a composite. By using a minimal model for the Purkinje network, in which we have a two-dimensional, bilayer tissue, with a layer of Purkinje cells on top of a layer of endocardial cells, we can stabilize spiral-wave turbulence; however, for a sparse distribution of Purkinje-ventricular junctions, at which these two layers are coupled, we can also obtain additional focal activity and many complex transient regimes. We also present additional effects resulting from the coupling of Purkinje and endocardial layers and discuss the relation of our results to the studies performed in anatomically accurate models of the Purkinje network.

Cerebellar Nuclear Neurons Use Time and Rate Coding to Transmit Purkinje Neuron Pauses.

PubMed

Sudhakar, Shyam Kumar; Torben-Nielsen, Benjamin; De Schutter, Erik

2015-12-01

Neurons of the cerebellar nuclei convey the final output of the cerebellum to their targets in various parts of the brain. Within the cerebellum their direct upstream connections originate from inhibitory Purkinje neurons. Purkinje neurons have a complex firing pattern of regular spikes interrupted by intermittent pauses of variable length. How can the cerebellar nucleus process this complex input pattern? In this modeling study, we investigate different forms of Purkinje neuron simple spike pause synchrony and its influence on candidate coding strategies in the cerebellar nuclei. That is, we investigate how different alignments of synchronous pauses in synthetic Purkinje neuron spike trains affect either time-locking or rate-changes in the downstream nuclei. We find that Purkinje neuron synchrony is mainly represented by changes in the firing rate of cerebellar nuclei neurons. Pause beginning synchronization produced a unique effect on nuclei neuron firing, while the effect of pause ending and pause overlapping synchronization could not be distinguished from each other. Pause beginning synchronization produced better time-locking of nuclear neurons for short length pauses. We also characterize the effect of pause length and spike jitter on the nuclear neuron firing. Additionally, we find that the rate of rebound responses in nuclear neurons after a synchronous pause is controlled by the firing rate of Purkinje neurons preceding it.

Tissue Plasminogen Activator Induction in Purkinje Neurons After Cerebellar Motor Learning

NASA Astrophysics Data System (ADS)

Seeds, Nicholas W.; Williams, Brian L.; Bickford, Paula C.

1995-12-01

The cerebellar cortex is implicated in the learning of complex motor skills. This learning may require synaptic remodeling of Purkinje cell inputs. An extracellular serine protease, tissue plasminogen activator (tPA), is involved in remodeling various nonneural tissues and is associated with developing and regenerating neurons. In situ hybridization showed that expression of tPA messenger RNA was increased in the Purkinje neurons of rats within an hour of their being trained for a complex motor task. Antibody to tPA also showed the induction of tPA protein associated with cerebellar Purkinje cells. Thus, the induction of tPA during motor learning may play a role in activity-dependent synaptic plasticity.

Palisade pattern of mormyrid Purkinje cells: a correlated light and electron microscopic study.

PubMed

Meek, J; Nieuwenhuys, R

1991-04-01

The present study is devoted to a detailed analysis of the structural and synaptic organization of mormyrid Purkinje cells in order to evaluate the possible functional significance of their dendritic palisade pattern. For this purpose, the properties of Golgi-impregnated as well as unimpregnated Purkinje cells in lobe C1 and C3 of the cerebellum of Gnathonemus petersii were light and electron microscopically analyzed, quantified, reconstructed, and mutually compared. Special attention was paid to the degree of regularity of their dendritic trees, their relations with Bergmann glia, and the distribution and numerical properties of their synaptic connections with parallel fibers, stellate cells, "climbing" fibers, and Purkinje axonal boutons. The highest degree of palisade specialization was encountered in lobe C1, where Purkinje cells have on average 50 palisade dendrites with a very regular distribution in a sagittal plane. Their spine density decreases from superficial to deep (from 14 to 6 per micron dendritic length), a gradient correlated with a decreasing parallel fiber density but an increasing parallel fiber diameter. Each Purkinje cell makes on average 75,000 synaptic contacts with parallel fibers, some of which are rather coarse (0.45 microns), and provided with numerous short collaterals. Climbing fibers do not climb, since their synaptic contacts are restricted to the ganglionic layer (i.e., the layer of Purkinje and eurydendroid projection cells), where they make about 130 synaptic contacts per cell with 2 or 3 clusters of thorns on the proximal dendrites. These clusters contain also a type of "shunting" elements that make desmosome-like junctions with both the climbing fiber boutons and the necks of the thorns. The axons of Purkinje cells in lobe C1 make small terminal arborizations, with about 20 boutons, that may be substantially (up to 500 microns) displaced rostrally or caudally with respect to the soma. Purkinje axonal boutons were observed to

Comparison of intraocular lens decentration and tilt measurements using 2 Purkinje meter systems.

PubMed

Maedel, Sophie; Hirnschall, Nino; Bayer, Natascha; Markovic, Sabine; Tabernero, Juan; Artal, Pablo; Schaeffel, Frank; Findl, Oliver

2017-05-01

To evaluate the difference in intraocular lens tilt and decentration measurements with 2 Purkinje meters. Vienna Institute for Research in Ocular Surgery, Hanusch Hospital, Vienna, Austria. Prospective evaluation of diagnostic test. This single-center study included pseudophakic patients in 2 substudies in which 3 consecutive measurements were performed with 2 Purkinje meters (Spanish and German). In substudy 1, an inexperienced examiner performed all measurements after a short learning period. In substudy 2, all measurements were taken by experienced examiners under direct supervision of the inventors of the devices. Substudy 1 included 53 pseudophakic eyes in which all 53 scans were successful with the Spanish device; however, only 35 measurements (66%) were successful with the German Purkinje meter. The mean tilt measured with the Spanish Purkinje meter was 4.35 degrees ± 2.50 (SD) and 9.20 ± 6.96 degrees with the German Purkinje meter. The mean decentration was 0.44 ± 0.19 mm and 0.74 ± 0.91 mm, (P = .44), respectively. In substudy 2 (29 pseudophakic eyes), the number of successful scans was 29 (100%) and 18 (62%) for the Spanish meter and German Purkinje meter, respectively. The mean horizontal and vertical tilt difference vector between the 2 systems was 4.89 ± 3.24 degrees and 7.57 ± 3.82 degrees, respectively. Concerning clinical feasibility, the Spanish Purkinje meter had a greater percentage of successful scans than the German device. In addition, this device measured significantly higher tilt values than the Spanish Purkinje meter. Copyright © 2017 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

PubMed Central

Benton, Mark D.; Lewis, Amanda H.; Bant, Jason S.

2013-01-01

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17–21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell

Purkinje Cell Activity in the Cerebellar Anterior Lobe after Rabbit Eyeblink Conditioning

ERIC Educational Resources Information Center

Green, John T.; Steinmetz, Joseph E.

2005-01-01

The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus.…

Developmental and Regional Patterns of GAP-43 Immunoreactivity in a Metamorphosing Brain

PubMed Central

Simmons, Andrea Megela; Tanyu, Leslie H.; Horowitz, Seth S.; Chapman, Judith A.; Brown, Rebecca A.

2012-01-01

Growth-associated protein-43 is typically expressed at high levels in the nervous system during development. In adult animals, its expression is lower, but still observable in brain areas showing structural or functional plasticity. We examined patterns of GAP-43 immunoreactivity in the brain of the bullfrog, an animal whose nervous system undergoes considerable reorganization across metamorphic development and retains a strong capacity for plasticity in adulthood. Immunolabeling was mostly diffuse in hatchling tadpoles, but became progressively more discrete as larval development proceeded. In many brain areas, intensity of immunolabel peaked at metamorphic climax, the time of final transition from aquatic to semi-terrestrial life. Changes in intensity of GAP-43 expression in the medial vestibular nucleus, superior olivary nucleus, and torus semicircularis appeared correlated with stage-dependent functional changes in processing auditory stimuli. Immunolabeling in the Purkinje cell layer of the cerebellum and in the cerebellar nucleus was detectable at most developmental time points. Heavy immunolabel was present from early larval stages through the end of climax in the thalamus (ventromedial, anterior, posterior, central nuclei). Immunolabel in the tadpole telencephalon was observed around the lateral ventricles, and in the medial septum and ventral striatum. In postmetamorphic animals, immunoreactivity was confined mainly to the ventricular zones and immediately adjacent cell layers. GAP-43 expression was present in olfactory, auditory and optic cranial nerves throughout larval and postmetamorphic life. The continued expression of GAP-43 in brain nuclei and in cranial nerves throughout development and into adulthood reflects the high regenerative potential of the bullfrog’s central nervous system. PMID:18431052

An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

PubMed Central

Urushidani, Tetsuro; Tachibana, Shigehiro

2013-01-01

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers. PMID:24260719

Regulation of spinogenesis in mature Purkinje cells via mGluR/PKC-mediated phosphorylation of CaMKIIβ

PubMed Central

Sugawara, Takeyuki; Hisatsune, Chihiro; Miyamoto, Hiroyuki; Ogawa, Naoko; Mikoshiba, Katsuhiko

2017-01-01

Dendritic spines of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites for cerebellar synaptic plasticity. Nevertheless, how density and morphology of these spines are properly maintained in mature Purkinje cells is not well understood. Here we show an activity-dependent mechanism that represses excessive spine development in mature Purkinje cells. We found that CaMKIIβ promotes spine formation and elongation in Purkinje cells through its F-actin bundling activity. Importantly, activation of group I mGluR, but not AMPAR, triggers PKC-mediated phosphorylation of CaMKIIβ, which results in dissociation of the CaMKIIβ/F-actin complex. Defective function of the PKC-mediated CaMKIIβ phosphorylation promotes excess F-actin bundling and leads to abnormally numerous and elongated spines in mature IP3R1-deficient Purkinje cells. Thus, our data suggest that phosphorylation of CaMKIIβ through the mGluR/IP3R1/PKC signaling pathway represses excessive spine formation and elongation in mature Purkinje cells. PMID:28607044

In vivo analysis of Purkinje cell firing properties during postnatal mouse development

PubMed Central

Arancillo, Marife; White, Joshua J.; Lin, Tao; Stay, Trace L.

2014-01-01

Purkinje cell activity is essential for controlling motor behavior. During motor behavior Purkinje cells fire two types of action potentials: simple spikes that are generated intrinsically and complex spikes that are induced by climbing fiber inputs. Although the functions of these spikes are becoming clear, how they are established is still poorly understood. Here, we used in vivo electrophysiology approaches conducted in anesthetized and awake mice to record Purkinje cell activity starting from the second postnatal week of development through to adulthood. We found that the rate of complex spike firing increases sharply at 3 wk of age whereas the rate of simple spike firing gradually increases until 4 wk of age. We also found that compared with adult, the pattern of simple spike firing during development is more irregular as the cells tend to fire in bursts that are interrupted by long pauses. The regularity in simple spike firing only reached maturity at 4 wk of age. In contrast, the adult complex spike pattern was already evident by the second week of life, remaining consistent across all ages. Analyses of Purkinje cells in alert behaving mice suggested that the adult patterns are attained more than a week after the completion of key morphogenetic processes such as migration, lamination, and foliation. Purkinje cell activity is therefore dynamically sculpted throughout postnatal development, traversing several critical events that are required for circuit formation. Overall, we show that simple spike and complex spike firing develop with unique developmental trajectories. PMID:25355961

Numerical approximation of the electromechanical coupling in the left ventricle with inclusion of the Purkinje network.

PubMed

Landajuela, Mikel; Vergara, Christian; Gerbi, Antonello; Dedé, Luca; Formaggia, Luca; Quarteroni, Alfio

2018-03-25

In this work, we consider the numerical approximation of the electromechanical coupling in the left ventricle with inclusion of the Purkinje network. The mathematical model couples the 3D elastodynamics and bidomain equations for the electrophysiology in the myocardium with the 1D monodomain equation in the Purkinje network. For the numerical solution of the coupled problem, we consider a fixed-point iterative algorithm that enables a partitioned solution of the myocardium and Purkinje network problems. Different levels of myocardium-Purkinje network splitting are considered and analyzed. The results are compared with those obtained using standard strategies proposed in the literature to trigger the electrical activation. Finally, we present a numerical study that, although performed in an idealized computational domain, features all the physiological issues that characterize a heartbeat simulation, including the initiation of the signal in the Purkinje network and the systolic and diastolic phases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake

PubMed Central

Wang, Fushun; Xu, Qiwu; Wang, Weishan; Takano, Takahiro; Nedergaard, Maiken

2012-01-01

Recent studies have shown that cerebellar Bergmann glia display coordinated Ca2+ transients in live mice. However, the functional significance of Bergmann glial Ca2+ signaling remains poorly understood. Using transgenic mice that allow selective stimulation of glial cells, we report here that cytosolic Ca2+ regulates uptake of K+ by Bergmann glia, thus providing a powerful mechanism for control of Purkinje cell-membrane potential. The decline in extracellular K+ evoked by agonist-induced Ca2+ in Bergmann glia transiently increased spike activity of Purkinje cells in cerebellar slices as well as in live anesthetized mice. Thus, Bergmann glia play a previously unappreciated role in controlling the membrane potential and thereby the activity of adjacent Purkinje cells. PMID:22547829

A New Approach for Determining Phase Response Curves Reveals that Purkinje Cells Can Act as Perfect Integrators

PubMed Central

Roth, Arnd; Häusser, Michael

2010-01-01

Cerebellar Purkinje cells display complex intrinsic dynamics. They fire spontaneously, exhibit bistability, and via mutual network interactions are involved in the generation of high frequency oscillations and travelling waves of activity. To probe the dynamical properties of Purkinje cells we measured their phase response curves (PRCs). PRCs quantify the change in spike phase caused by a stimulus as a function of its temporal position within the interspike interval, and are widely used to predict neuronal responses to more complex stimulus patterns. Significant variability in the interspike interval during spontaneous firing can lead to PRCs with a low signal-to-noise ratio, requiring averaging over thousands of trials. We show using electrophysiological experiments and simulations that the PRC calculated in the traditional way by sampling the interspike interval with brief current pulses is biased. We introduce a corrected approach for calculating PRCs which eliminates this bias. Using our new approach, we show that Purkinje cell PRCs change qualitatively depending on the firing frequency of the cell. At high firing rates, Purkinje cells exhibit single-peaked, or monophasic PRCs. Surprisingly, at low firing rates, Purkinje cell PRCs are largely independent of phase, resembling PRCs of ideal non-leaky integrate-and-fire neurons. These results indicate that Purkinje cells can act as perfect integrators at low firing rates, and that the integration mode of Purkinje cells depends on their firing rate. PMID:20442875

Decimetre Waves and Cerebellar Cortex Key Element - Purkinje Cells

NASA Astrophysics Data System (ADS)

Maharramov, Akif A.

2007-04-01

Acute experiments have been carried out on both decerebrated and anaesthetized adult cats exposed to Decimetre Range Microwaves (DRM) (λ=65 cm, duration of exposition -10 min., at least) by the help of a 2 cm radius contact applicator located on the temple part of the head with cerebellum in the centre of projection of irradiation conducted from portable physiotherapeutic apparatus ``Romashka''. Extracelullar registration of Purkinje Cells (PC) impulse activities have been led by glass microelectrodes. Statistical and computational analyses of PC activities have been realized by the help of histograms - the characteristic distribution of the number of interimpulse intervals (II) between electrical discharges of a neuron on the II durations, drawn up by the help of a computer. The results obtained revealed the reaction of PC to DRM as a succession of starting to react of PC electrophysiological parameters, beginning from decreasing of known ``Inhibitory Pause'' duration, and further, at first, ``Simple Spikes'' then ``Complex Spikes'' frequencies' increase, and furthermore, durations' increase in ``Big Interimpulse Intervals'', parameter, introduced for the first time by us, in this way, showing the ``evolutionary'' nature of Electromagnetic Fields and Living object interactions.

Climbing fibers mediate vestibular modulation of both "complex" and "simple spikes" in Purkinje cells.

PubMed

Barmack, N H; Yakhnitsa, V

2015-10-01

Climbing and mossy fibers comprise two distinct afferent paths to the cerebellum. Climbing fibers directly evoke a large multispiked action potential in Purkinje cells termed a "complex spike" (CS). By logical exclusion, the other class of Purkinje cell action potential, termed "simple spike" (SS), has often been attributed to activity conveyed by mossy fibers and relayed to Purkinje cells through granule cells. Here, we investigate the relative importance of climbing and mossy fiber pathways in modulating neuronal activity by recording extracellularly from Purkinje cells, as well as from mossy fiber terminals and interneurons in folia 8-10. Sinusoidal roll-tilt vestibular stimulation vigorously modulates the discharge of climbing and mossy fiber afferents, Purkinje cells, and interneurons in folia 9-10 in anesthetized mice. Roll-tilt onto the side ipsilateral to the recording site increases the discharge of both climbing fibers (CSs) and mossy fibers. However, the discharges of SSs decrease during ipsilateral roll-tilt. Unilateral microlesions of the beta nucleus (β-nucleus) of the inferior olive blocks vestibular modulation of both CSs and SSs in contralateral Purkinje cells. The blockage of SSs occurs even though primary and secondary vestibular mossy fibers remain intact. When mossy fiber afferents are damaged by a unilateral labyrinthectomy (UL), vestibular modulation of SSs in Purkinje cells ipsilateral to the UL remains intact. Two inhibitory interneurons, Golgi and stellate cells, could potentially contribute to climbing fiber-induced modulation of SSs. However, during sinusoidal roll-tilt, only stellate cells discharge appropriately out of phase with the discharge of SSs. Golgi cells discharge in phase with SSs. When the vestibularly modulated discharge is blocked by a microlesion of the inferior olive, the modulated discharge of CSs and SSs is also blocked. When the vestibular mossy fiber pathway is destroyed, vestibular modulation of ipsilateral CSs and

Effect of Early Neonatal Exposure to Deltamethrin on the Purkinje Cell Number in Rat Cerebellum

PubMed Central

Asari, Mohd Asnizam; Abdullah, Mohammad Shukri; Abdullah, Suryati

2008-01-01

Deltamethrin is a widely used insecticide belonging to the class of pyrethroid. Although the neurotoxicity of pyrethroids including deltamethrin is well established, it is still unclear whether exposure to deltamethrin during neonatal period has any deleterious effect on the survival of the Purkinje cells in the cerebellum. In the study, we investigated the total number of Purkinje cells in experimental rats exposed to deltamethrin using a stereological method, the fractionator. Deltamethrin in a dose of 1 mg/kg/day (corresponds to 20% of LD50 ) was administered through oral gavage to male pups from 2nd to 5th postnatal day (PND). At PND 21 the animals were sacrificed and their cerebelli were removed. The cerebelli were systematically sampled using the fractionator method and stained with cresyl fast violet. The number of the Purkinje cells was counted for each cerebellum. The results showed that there was no significant difference in the total number of Purkinje cells in the deltamethrin-treated group as compared to the control animals. This suggests that deltamethrin exposure at the current dosage during the neonatal period do not have any significant effect on the survival of the Purkinje cells in the cerebellum. PMID:22570585

Intracellular calcium dynamics permit a Purkinje neuron model to perform toggle and gain computations upon its inputs

PubMed Central

Forrest, Michael D.

2014-01-01

Without synaptic input, Purkinje neurons can spontaneously fire in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence. Climbing fiber input (CF) switches Purkinje neurons out of the trimodal firing pattern and toggles them between a tonic firing and a quiescent state, while setting the gain of their response to Parallel Fiber (PF) input. The basis to this transition is unclear. We investigate it using a biophysical Purkinje cell model under conditions of CF and PF input. The model can replicate these toggle and gain functions, dependent upon a novel account of intracellular calcium dynamics that we hypothesize to be applicable in real Purkinje cells. PMID:25191262

Functional compatibility between Purkinje cell axon branches and their target neurons in the cerebellum.

PubMed

Yang, Zhilai; Chen, Na; Ge, Rongjing; Qian, Hao; Wang, Jin-Hui

2017-09-22

A neuron sprouts an axon, and its branches to innervate many target neurons that are divergent in their functions. In order to efficiently regulate the diversified cells, the axon branches should differentiate functionally to be compatible with their target neurons, i.e., a function compatibility between presynaptic and postsynaptic partners. We have examined this hypothesis by using electrophysiological method in the cerebellum, in which the main axon of Purkinje cell projected to deep nucleus cells and the recurrent axons innervated the adjacent Purkinje cells. The fidelity of spike propagation is superior in the recurrent branches than the main axon. The capabilities of encoding spikes and processing GABAergic inputs are advanced in Purkinje cells versus deep nucleus cells. The functional differences among Purkinje's axonal branches and their postsynaptic neurons are preset by the variable dynamics of their voltage-gated sodium channels. In addition, activity strengths between presynaptic and postsynaptic partners are proportionally correlated, i.e., active axonal branches innervate active target neurons, or vice versa. The physiological impact of the functional compatibility is to make the neurons in their circuits to be activated appropriately. In conclusion, each cerebellar Purkinje cell sprouts the differentiated axon branches to be compatible with the diversified target cells in their functions, in order to construct the homeostatic and efficient units for their coordinated activity in neural circuits.

Functional compatibility between Purkinje cell axon branches and their target neurons in the cerebellum

PubMed Central

Qian, Hao; Wang, Jin-Hui

2017-01-01

A neuron sprouts an axon, and its branches to innervate many target neurons that are divergent in their functions. In order to efficiently regulate the diversified cells, the axon branches should differentiate functionally to be compatible with their target neurons, i.e., a function compatibility between presynaptic and postsynaptic partners. We have examined this hypothesis by using electrophysiological method in the cerebellum, in which the main axon of Purkinje cell projected to deep nucleus cells and the recurrent axons innervated the adjacent Purkinje cells. The fidelity of spike propagation is superior in the recurrent branches than the main axon. The capabilities of encoding spikes and processing GABAergic inputs are advanced in Purkinje cells versus deep nucleus cells. The functional differences among Purkinje's axonal branches and their postsynaptic neurons are preset by the variable dynamics of their voltage-gated sodium channels. In addition, activity strengths between presynaptic and postsynaptic partners are proportionally correlated, i.e., active axonal branches innervate active target neurons, or vice versa. The physiological impact of the functional compatibility is to make the neurons in their circuits to be activated appropriately. In conclusion, each cerebellar Purkinje cell sprouts the differentiated axon branches to be compatible with the diversified target cells in their functions, in order to construct the homeostatic and efficient units for their coordinated activity in neural circuits. PMID:29069799

Tilt and decentration of intraocular lenses in vivo from Purkinje and Scheimpflug imaging. Validation study.

PubMed

de Castro, Alberto; Rosales, Patricia; Marcos, Susana

2007-03-01

To measure tilt and decentration of intraocular lenses (IOLs) with Scheimpflug and Purkinje imaging systems in physical model eyes with known amounts of tilt and decentration and patients. Instituto de Optica Daza de Valdés, Consejo Superior de Investigaciones Científicas, Madrid, Spain. Measurements of IOL tilt and decentration were obtained using a commercial Scheimpflug system (Pentacam, Oculus), custom algorithms, and a custom-built Purkinje imaging apparatus. Twenty-five Scheimpflug images of the anterior segment of the eye were obtained at different meridians. Custom algorithms were used to process the images (correction of geometrical distortion, edge detection, and curve fittings). Intraocular lens tilt and decentration were estimated by fitting sinusoidal functions to the projections of the pupillary axis and IOL axis in each image. The Purkinje imaging system captures pupil images showing reflections of light from the anterior corneal surface and anterior and posterior lens surfaces. Custom algorithms were used to detect the Purkinje image locations and estimate IOL tilt and decentration based on a linear system equation and computer eye models with individual biometry. Both methods were validated with a physical model eye in which IOL tilt and decentration can be set nominally. Twenty-one eyes of 12 patients with IOLs were measured with both systems. Measurements of the physical model eye showed an absolute discrepancy between nominal and measured values of 0.279 degree (Purkinje) and 0.243 degree (Scheimpflug) for tilt and 0.094 mm (Purkinje) and 0.228 mm (Scheimpflug) for decentration. In patients, the mean tilt was less than 2.6 degrees and the mean decentration less than 0.4 mm. Both techniques showed mirror symmetry between right eyes and left eyes for tilt around the vertical axis and for decentration in the horizontal axis. Both systems showed high reproducibility. Validation experiments on physical model eyes showed slightly higher accuracy

Current source density correlates of cerebellar Golgi and Purkinje cell responses to tactile input

PubMed Central

Tahon, Koen; Wijnants, Mike; De Schutter, Erik

2011-01-01

The overall circuitry of the cerebellar cortex has been known for over a century, but the function of many synaptic connections remains poorly characterized in vivo. We used a one-dimensional multielectrode probe to estimate the current source density (CSD) of Crus IIa in response to perioral tactile stimuli in anesthetized rats and to correlate current sinks and sources to changes in the spike rate of corecorded Golgi and Purkinje cells. The punctate stimuli evoked two distinct early waves of excitation (at <10 and ∼20 ms) associated with current sinks in the granular layer. The second wave was putatively of corticopontine origin, and its associated sink was located higher in the granular layer than the first trigeminal sink. The distinctive patterns of granular-layer sinks correlated with the spike responses of corecorded Golgi cells. In general, Golgi cell spike responses could be linearly reconstructed from the CSD profile. A dip in simple-spike activity of coregistered Purkinje cells correlated with a current source deep in the molecular layer, probably generated by basket cell synapses, interspersed between sparse early sinks presumably generated by synapses from granule cells. The late (>30 ms) enhancement of simple-spike activity in Purkinje cells was characterized by the absence of simultaneous sinks in the granular layer and by the suppression of corecorded Golgi cell activity, pointing at inhibition of Golgi cells by Purkinje axon collaterals as a likely mechanism of late Purkinje cell excitation. PMID:21228303

PCPP-260, PURKINJE CELL-SPECIFIC CYCLE AMP-REGULATED MEMBRANE PHOSPHOPROTEIN OF (M SUB R) 260,000

EPA Science Inventory

The present study reports the existence of Purkinje cell-specific phosphoprotein, Mr260,000 (PCPP-260), a neuronal membrance phosphoprotein, in cerebellar Purkinje cells. PCPP-260, which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has an apparaent molecular mass ...

Axonal propagation of simple and complex spikes in cerebellar Purkinje neurons.

PubMed

Khaliq, Zayd M; Raman, Indira M

2005-01-12

In cerebellar Purkinje neurons, the reliability of propagation of high-frequency simple spikes and spikelets of complex spikes is likely to regulate inhibition of Purkinje target neurons. To test the extent to which a one-to-one correspondence exists between somatic and axonal spikes, we made dual somatic and axonal recordings from Purkinje neurons in mouse cerebellar slices. Somatic action potentials were recorded with a whole-cell pipette, and the corresponding axonal signals were recorded extracellularly with a loose-patch pipette. Propagation of spontaneous and evoked simple spikes was highly reliable. At somatic firing rates of approximately 200 spikes/sec, <10% of spikes failed to propagate, with failures becoming more frequent only at maximal somatic firing rates (approximately 260 spikes/sec). Complex spikes were elicited by climbing fiber stimulation, and their somatic waveforms were modulated by tonic current injection, as well as by paired stimulation to depress the underlying EPSCs. Across conditions, the mean number of propagating action potentials remained just above two spikes per climbing fiber stimulation, but the instantaneous frequency of the propagating spikes changed, from approximately 375 Hz during somatic hyperpolarizations that silenced spontaneous firing to approximately 150 Hz during spontaneous activity. The probability of propagation of individual spikelets could be described quantitatively as a saturating function of spikelet amplitude, rate of rise, or preceding interspike interval. The results suggest that ion channels of Purkinje axons are adapted to produce extremely short refractory periods and that brief bursts of forward-propagating action potentials generated by complex spikes may contribute transiently to inhibition of postsynaptic neurons.

Compartmentalization of the chick cerebellar cortex based on the link between the striped expression pattern of aldolase C and the topographic olivocerebellar projection.

PubMed

Vibulyaseck, Suteera; Luo, Yuanjun; Fujita, Hirofumi; Oh-Nishi, Arata; Ohki-Hamazaki, Hiroko; Sugihara, Izumi

2015-09-01

The avian cerebellum is organized into multiple longitudinal stripes defined by expression profiles of aldolase C (zebrin II) in Purkinje cells. The relationship between the aldolase C striped pattern and the olivocerebellar projection pattern is crucial in understanding cerebellar functional compartmentalization. We identified all aldolase C stripes across all lobules with the serial section alignment analysis method and then looked at this relationship by anterograde and retrograde labeling of olivocerebellar axons in the chick cerebellum. Aldolase C stripes were generally consistent and continuous from lobule I through VII and to the medial part of lobules VIII-IXb. The dorsal and ventral lamellas (DL, VL) of the inferior olive projected to the stripes in these areas with a simple mediolateral topographic relation. A few aldolase C stripes appeared at the lateral edge of lobules VI-VIII. Several more stripes were added in the lateral parts of lobules IXa-IXb and IXc-X. The medial column (MC) of the inferior olive projected to the stripes in lobules VIII-X, including the added lateral stripes, with a complex topographic relation. Sharp boundaries between aldolase C-positive and -negative stripes often accompanied a gap in the Purkinje cell layer and bordered topographically distinct groups of axons. Although the compartmental organization of the chick cerebellum is comparable to that of the mammalian cerebellum, several significant differences in the organization suggest partly separate evolutionary lineages of the mammalian and avian cerebella. We propose that rostral lobules may be evolved by rostral extension of medial stripes from caudal lobules in the avian cerebellum. © 2015 Wiley Periodicals, Inc.

The Vps33a gene regulates behavior and cerebellar Purkinje cell number

PubMed Central

Chintala, Sreenivasulu; Novak, Edward K.; Spernyak, Joseph A.; Mazurchuk, Richard; Torres, German; Patel, Suchith; Busch, Kristie; Meeder, Beth A.; Horowitz, Judith M.; Vaughan, Mary M.; Swank, Richard T.

2015-01-01

A mutation in the Vps33a gene causes Hermansky–Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13–14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (~30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may alsobea clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function. PMID:19254700

Morphological study of the atrioventricular conduction system and Purkinje fibers in yak.

PubMed

Duan, Deyong; Yu, Sijiu; Cui, Yan; Li, Chaoxu

2017-07-01

We studied the morphology of the atrioventricular conduction system (AVCS) and Purkinje fibers of the yak. Light and transmission electron microscopy were used to study the histological features of AVCS. The distributional characteristics of the His-bundle, the left bundle branch (LBB), right bundle branch (RBB), and Purkinje fiber network of yak hearts were examined using gross dissection, ink injection, and ABS casting. The results showed that the atrioventricular node (AVN) of yak located in the right side of interatrial septum and had a flattened ovoid shape. The AVN of yak is composed of the slender, interweaving cells formed almost entirely of the transitional cells (T-cells). The His-bundle extended from the AVN, and split into left LBB and RBB at the crest of the interventricular septum. The LBB descended along the left side of interventricular septum. At approximately the upper 1/3 of the interventricular septum, the LBB typically divided into three branches. The RBB ran under the endocardium of the right side of interventricular septum, and extended to the base of septal papillary muscle, passed into the moderator band, crossed the right ventricular cavity to reach the base of anterior papillary muscle, and divided into four fascicles under the subendocardial layer. The Purkinje fibers in the ventricle formed a complex spatial network. The distributional and cellular component characteristics of the AVCS and Purkinje fibers ensured normal cardiac function. © 2017 Wiley Periodicals, Inc.

Posturography of ataxia induced by Coriolis- and Purkinje-effects.

PubMed

Fitger, C; Brandt, T

1982-02-01

Vestibular Coriolis- and Purkinje-effect, which are known to induce vertigo, were investigated with respect to body posture. One aim of this investigation was to provide information concerning clinical vertigo symptoms. Standing on a rotatable stabilometer, 25 healthy subjects had to execute lateral head tilts during (Coriolis), or after (Purkinje), rotation varied with different constant velocities. The conditions were varied with respect to eyes open vs. eyes closed, head upright vs. head tilt to the right and left, direction of rotation clockwise vs. counterclockwise, active vs. passive head tilt, and active vs. passive body rotation. The results supported the expectation that destabilization was less severe with open than with closed eyes and that sway amplitudes were increased after head tilt as well as with a higher velocity of rotation. The direction of the induced body shift was, as expected, opposite to the initial vestibular stimulus. A forward shift after stop without head tilt was frequently found, being independent of the previous direction of rotation. Reported perceptions coincided mostly not with the initial vestibular signal but rather with the actual movement of compensation. Active instead of passive movements did not produce clearly different effects. The Purkinje experiment appeared to be equivalent to the situation when a patient with an acute lesion of a horizontal vestibular canal bends his head. The stabilogram under this condition may allow a prediction of the side of the lesion.

l-Serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons

PubMed Central

Furuya, Shigeki; Tabata, Toshihide; Mitoma, Junya; Yamada, Keiko; Yamasaki, Miwako; Makino, Asami; Yamamoto, Toshifumi; Watanabe, Masahiko; Kano, Masanobu; Hirabayashi, Yoshio

2000-01-01

Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that l-serine (l-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although l-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that l-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons. PMID:11016963

Evidence that GABA ρ subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

PubMed Central

Harvey, Victoria L; Duguid, Ian C; Krasel, Cornelius; Stephens, Gary J

2006-01-01

Ionotropic γ-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for ρ subunit-containing GABAC over other GABA receptors. Exogenous application of the GABAC-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABAC antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABAA receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABAA/GABAC pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone–Purkinje cell (IN–PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that ρ subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABAA α1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that ρ subunits can form complexes

21 CFR 862.1405 - Immunoreactive insulin test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunoreactive insulin test system. 862.1405... Systems § 862.1405 Immunoreactive insulin test system. (a) Identification. An immunoreactive insulin test system is a device intended to measure immunoreactive insulin in serum and plasma. Immunoreactive insulin...

21 CFR 862.1405 - Immunoreactive insulin test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunoreactive insulin test system. 862.1405... Systems § 862.1405 Immunoreactive insulin test system. (a) Identification. An immunoreactive insulin test system is a device intended to measure immunoreactive insulin in serum and plasma. Immunoreactive insulin...

21 CFR 862.1405 - Immunoreactive insulin test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoreactive insulin test system. 862.1405... Systems § 862.1405 Immunoreactive insulin test system. (a) Identification. An immunoreactive insulin test system is a device intended to measure immunoreactive insulin in serum and plasma. Immunoreactive insulin...

21 CFR 862.1405 - Immunoreactive insulin test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunoreactive insulin test system. 862.1405... Systems § 862.1405 Immunoreactive insulin test system. (a) Identification. An immunoreactive insulin test system is a device intended to measure immunoreactive insulin in serum and plasma. Immunoreactive insulin...

Increased protein kinase C gamma activity induces Purkinje cell pathology in a mouse model of spinocerebellar ataxia 14.

PubMed

Ji, Jingmin; Hassler, Melanie L; Shimobayashi, Etsuko; Paka, Nagendher; Streit, Raphael; Kapfhammer, Josef P

2014-10-01

Spinocerebellar ataxias (SCAs) are hereditary diseases leading to Purkinje cell degeneration and cerebellar dysfunction. Most forms of SCA are caused by expansion of CAG repeats similar to other polyglutamine disorders such as Huntington's disease. In contrast, in the autosomal dominant SCA-14 the disease is caused by mutations in the protein kinase C gamma (PKCγ) gene which is a well characterized signaling molecule in cerebellar Purkinje cells. The study of SCA-14, therefore, offers the unique opportunity to reveal the molecular and pathological mechanism eventually leading to Purkinje cell dysfunction and degeneration. We have created a mouse model of SCA-14 in which PKCγ protein with a mutation found in SCA-14 is specifically expressed in cerebellar Purkinje cells. We find that in mice expressing the mutated PKCγ protein the morphology of Purkinje cells in cerebellar slice cultures is drastically altered and mimics closely the morphology seen after pharmacological PKC activation. Similar morphological abnormalities were seen in localized areas of the cerebellum of juvenile transgenic mice in vivo. In adult transgenic mice there is evidence for some localized loss of Purkinje cells but there is no overall cerebellar atrophy. Transgenic mice show a mild cerebellar ataxia revealed by testing on the rotarod and on the walking beam. Our findings provide evidence for both an increased PKCγ activity in Purkinje cells in vivo and for pathological changes typical for cerebellar disease thus linking the increased and dysregulated activity of PKCγ tightly to the development of cerebellar disease in SCA-14 and possibly also in other forms of SCA. Copyright © 2014 Elsevier Inc. All rights reserved.

Immunohistochemical localization of urotensin I/corticotropin-releasing factor, urotensin II, and serotonin immunoreactivities in the caudal spinal cord of nonteleost fishes.

PubMed

Onstott, D; Elde, R

1986-07-08

Neurosecretory cells in the caudal spinal cord of fishes were first discovered in elasmobranches. However, most of the subsequent work on the caudal neurosecretory system has emphasized its morphology and function in teleosts. Two major peptides, urotensins I and II (UI and UII), have been isolated from this system in teleosts and their amino acid sequences have been determined. We have used immunohistochemical techniques to confirm and expand previous morphological and pharmacological findings regarding the phylogenetic occurrence and the anatomical organization of the caudal neurosecretory system and to localize putative neurohormonal products within its structural elements. UII-immunoreactive neuronal structures were found in the holocephalan, Hydrolagus collei; the elasmobranches, Squalus acanthias, Dasyatis sabina, and Raja binoculata; the dipnoan, Protopterus annectens; the brachiopterygian, Erpetoichthys calabaricus; the chondrostean, Polyodon spathula; and the holosteans Lepisosteus platyrhinchus and Amia calva. UI/corticotropin-releasing-factor immunoreactive elements were detected in the caudal spinal cords of Petromyzon marinus, Raja binoculata, Polydon spathula, Lepisosteus platyrhinchus, L. osseus, L. platostomus, and Amia calva. In addition, in several of these species, immunohistochemical techniques have provided evidence for serotoninergic input to the caudal neurosecretory system.

Regional Alterations in Purkinje Cell Density in Patients with Autism

PubMed Central

Skefos, Jerry; Cummings, Christopher; Enzer, Katelyn; Holiday, Jarrod; Weed, Katrina; Levy, Ezra; Yuce, Tarik; Kemper, Thomas; Bauman, Margaret

2014-01-01

Neuropathological studies, using a variety of techniques, have reported a decrease in Purkinje cell (PC) density in the cerebellum in autism. We have used a systematic sampling technique that significantly reduces experimenter bias and variance to estimate PC densities in the postmortem brains of eight clinically well-documented individuals with autism, and eight age- and gender-matched controls. Four cerebellar regions were analyzed: a sensorimotor area comprised of hemispheric lobules IV–VI, crus I & II of the posterior lobe, and lobule X of the flocculonodular lobe. Overall PC density was thus estimated using data from all three cerebellar lobes and was found to be lower in the cases with autism as compared to controls, an effect that was most prominent in crus I and II (p<0.05). Lobule X demonstrated a trend towards lower PC density in only the males with autism (p = 0.05). Brain weight, a correlate of tissue volume, was found to significantly contribute to the lower lobule X PC density observed in males with autism, but not to the finding of lower PC density in crus I & II. Therefore, lower crus I & II PC density in autism is more likely due to a lower number of PCs. The PC density in lobule X was found to correlate with the ADI-R measure of the patient's use of social eye contact (R2 = −0.75, p = 0.012). These findings support the hypothesis that abnormal PC density may contribute to selected clinical features of the autism phenotype. PMID:24586223

Ultrastructural localization of ChAT-like immunoreactivity in the human vestibular periphery.

PubMed

Kong, W J; Hussl, B; Thumfart, W F; Schrott-Fischer, A

1998-05-01

Acetylcholine (ACh) has long been considered a neurotransmitter candidate in the efferent vestibular system of mammals. Recently, choline acetyltransferase (ChAT), the synthesizing enzyme for ACh, was immunocytochemically localized in all five end-organs of the rat vestibule (Kong et al. (1994) Hear. Res. 75, 192-200). However, there is little information in the literature concerning the cholinergic innervation in the vestibular periphery of man. In the present study the ultrastructural localization of the ChAT-like immunoreactivity in the human vestibular periphery was investigated in order to reveal the cholinergic innervation in the human vestibular end-organs. A modified method of pre-embedding immunoelectron microscopy was applied. It was found that the ChAT-like immunoreactivity was located in the bouton-type vesiculated nerve terminals in the vestibular neurosensory epithelia of man. These ChAT-like immunostained nerve terminals make synaptic contacts either with afferent chalices surrounding type I vestibular sensory hair cells, or with type II vestibular sensory hair cells. These results show that the ChAT-like immunoreactivity in the human vestibular periphery is confined to the efferent vestibular system. The ChAT-containing efferents innervate both type I hair cells and type II hair cells, making postsynaptic and presynaptic contacts, respectively. This study presents evidence that ACh is a neurotransmitter candidate in the efferent vestibular system of man.

A novel approach to non-biased systematic random sampling: a stereologic estimate of Purkinje cells in the human cerebellum.

PubMed

Agashiwala, Rajiv M; Louis, Elan D; Hof, Patrick R; Perl, Daniel P

2008-10-21

Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well.

A novel approach to non-biased systematic random sampling: A stereologic estimate of Purkinje cells in the human cerebellum

PubMed Central

Agashiwala, Rajiv M.; Louis, Elan D.; Hof, Patrick R.; Perl, Daniel P.

2010-01-01

Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well. PMID:18725208

Contribution of cerebellar intracortical inhibition to Purkinje cell response during vestibulo-ocular reflex of alert rabbits.

PubMed Central

Miyashita, Y; Nagao, S

1984-01-01

Ionophoretic application of bicuculline, an antagonist of gamma-aminobutyric acid (GABA), was used to examine the contribution of intracortical inhibition to vestibular responses of Purkinje cells in the cerebellar flocculus of alert rabbits. Purkinje cells were sampled extracellularly (with triple-barrelled micropipettes) from the floccular area where electrical stimulation through the micro-electrode evoked abduction of the ipsilateral eye, indicating its close functional relationship to the horizontal vestibulo-ocular reflex. These cells exhibited frequency modulation of simple spike discharges in-phase or out-phase with sinusoidal head rotation (0.5 cycles/s, 5 degrees peak-to-peak) in the horizontal plane. Bicuculline was ejected ionophoretically through one barrel with a 20-60 nA current. The pharmacological effectiveness of the ejected bicuculline was confirmed for each Purkinje cell by its blocking action upon the depressant action of GABA applied ionophoretically through another barrel. Bicuculline usually shifted the simple spike modulation in the in-phase direction: it reduced the amplitude of out-phase modulation in three cells, converted out-phase modulation to the in-phase type in four cells, and increased in-phase modulation in five cells. In three other cells, however, bicuculline shifted the modulation in the out-phase direction. Because bicuculline application usually increased the resting discharge level of a Purkinje cell, ionophoretic application of DL-homocysteate was used in ten Purkinje cells to control for the effect of a generalized increase in excitability. In contrast to bicuculline, DL-homocysteate generally induced a slight increase of the simple spike modulation regardless of the phase relationship. Since frequency modulation of the simple spike discharges of flocculus Purkinje cells is presumed to contribute to the control of vestibulo-ocular reflexes, these results point to an important functional role of intracortical post

Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging.

PubMed

Fattoretti, P; Bertoni-Freddari, C; Caselli, U; Paoloni, R; Meier-Ruge, W

1998-03-16

The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/microm3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 microm3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

Temporal expression and mitochondrial localization of a Foxp2 isoform lacking the forkhead domain in developing Purkinje cells.

PubMed

Tanabe, Yuko; Fujiwara, Yuji; Matsuzaki, Ayumi; Fujita, Eriko; Kasahara, Tadashi; Yuasa, Shigeki; Momoi, Takashi

2012-07-01

FOXP2, a forkhead box-containing transcription factor, forms homo- or hetero-dimers with FOXP family members and localizes to the nucleus, while FOXP2(R553H), which contains a mutation related to speech/language disorders, features reduced DNA binding activity and both cytoplasmic and nuclear localization. In addition to being a loss-of-function mutation, it is possible that FOXP2(R553H) also may act as a gain-of-function mutation to inhibit the functions of FOXP2 isoforms including FOXP2Ex10+ lacking forkhead domain. Foxp2(R552H) knock-in mouse pups exhibit impaired ultrasonic vocalization and poor dendritic development in Purkinje cells. However, expressions of Foxp2 isoforms in the developing Purkinje are unclear. The appearance of 'apical cytoplasmic swelling' (mitochondria-rich regions that are the source of budding processes) correlates with dendritic development of Purkinje cells. In the present study, we focused on Foxp2 isoforms localizing to the apical cytoplasmic swelling and identified two isoforms lacking forkhead domain: Foxp2Ex12+ and Foxp2Ex15. They partly localized to the membrane fraction that includes mitochondria. Foxp2Ex12+ mainly localized to the apical cytoplasmic swelling in early developing Purkinje cells at the stellate stage (P2-P4). Mitochondrial localization of Foxp2Ex12+ in Purkinje cells was confirmed by immune-electron microscopic analysis. Foxp2Ex12+ may play a role in dendritic development in Purkinje cells. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Immunoreactivities of calbindin-D28k, calretinin and parvalbumin in the somatosensory cortex of rodents during normal aging

PubMed Central

Ahn, Ji Hyeon; Hong, Seongkweon; Park, Joon Ha; Kim, In Hye; Cho, Jeong Hwi; Lee, Tae-Kyeong; Lee, Jae-Chul; Chen, Bai Hui; Shin, Bich-Na; Bae, Eun Joo; Jeon, Yong Hwan; Kim, Young-Myeong; Won, Moo-Ho; Choi, Soo Young

2017-01-01

Calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ-aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age-associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB-immunoreactive neurons were distributed in layers II and III, and age-associated alterations in their number were different according to the species. CR-immunoreactive neurons were not abundant in all layers; however, the number of CR-immunoreactive neurons was the highest in all adult species. Many PV-immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB-, CR- and PV-containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease. PMID:28944879

Forms of immunoreactive beta-endorphin in the intermediate pituitary of the holostean fish, Amia calva.

PubMed

Dores, R M; Sei, C A; Morrissey, M A; Crim, J W; Kawauchi, H

1988-01-01

Acid extracts of the intermediate pituitary of the holostean fish, Amia calva, were fractionated by gel filtration chromatography and analyzed with radioimmunoassays specific for N-acetylated beta-endorphin and C-terminally amidated alpha-MSH. In these extracts beta-endorphin-related immunoreactive material and alpha-MSH-related immunoreactive material were present in roughly equimolar amounts. The immunoreactive beta-endorphin-sized material was tested for opiate receptor binding activity using a beta-endorphin radioreceptor assay. The results of these studies were negative. The immunoreactive beta-endorphin-sized material was further analyzed by cation exchange chromatography at pH 2.5. Two major and three minor peaks of immunoreactive material were isolated. Peak 5 exhibited a net charge of +7 at pH 2.5 and represented 53% of the total immunoreactivity recovered. Peak 2 with a net charge of +3 at this pH represented 38% of the total immunoreactivity recovered. The minor forms, Peaks 1, 3 and 4, exhibited net charges of +2, +4 and +6, respectively. The apparent molecular weights of Peaks 2 and 5 were determined on a Sephadex G-50 column. Peak 2 had an apparent molecular weight of 2.7 Kd and Peak 5 had an apparent molecular weight of 3.5 Kd. Reverse phase HPLC analysis of Peak 5 indicates that this form of Amia beta-endorphin had chromatographic properties similar to salmon beta-endorphin II. These results would suggest that N-terminal acetylation and C-terminal proteolytic cleavage are important post-translational modifications of the forms of Amia beta-endorphin.

Administration of a non-NMDA antagonist, GYKI 52466, increases excitotoxic Purkinje cell degeneration caused by ibogaine.

PubMed

O'Hearn, E; Molliver, M E

2004-01-01

Ibogaine is a tremorigenic hallucinogen that has been proposed for clinical use in treating addiction. We previously reported that ibogaine, administered systemically, produces degeneration of a subset of Purkinje cells in the cerebellum, primarily within the vermis. Ablation of the inferior olive affords protection against ibogaine-induced neurotoxicity leading to the interpretation that ibogaine itself is not directly toxic to Purkinje cells. We postulated that ibogaine produces sustained excitation of inferior olivary neurons that leads to excessive glutamate release at climbing fiber terminals, causing subsequent excitotoxic injury to Purkinje cells. The neuronal degeneration induced by ibogaine provides an animal model for studying excitotoxic injury in order to analyze the contribution of glutamate receptors to this injury and to evaluate neuroprotective strategies. Since non-N-methyl-D-aspartate (NMDA) receptors mediate Purkinje cell excitation by climbing fibers, we hypothesized that 1-4-aminophenyl-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466), which antagonizes non-NMDA receptors, may have a neuroprotective effect by blocking glutamatergic excitation at climbing fiber synapses. To test this hypothesis, rats were administered systemic ibogaine plus GYKI-52466 and the degree of neuronal injury was analyzed in cerebellar sections. The results indicate that the AMPA antagonist GYKI-52466 (10 mg/kg i.p. x 3) does not protect against Purkinje cell injury at the doses used. Rather, co-administration of GYKI-52466 with ibogaine produces increased toxicity evidenced by more extensive Purkinje cell degeneration. Several hypotheses that may underlie this result are discussed. Although the reason for the increased toxicity found in this study is not fully explained, the present results show that a non-NMDA antagonist can produce increased excitotoxic injury under some conditions. Therefore, caution should be exercised before employing glutamate

Sensorimotor-correlated discharge recorded from ensembles of cerebellar Purkinje cells varies across the estrous cycle of the rat.

PubMed

Smith, S S

1995-09-01

1. In the present study, locomotor-correlated activity of cerebellar Purkinje cells, recorded using arrays of microwires chronically implanted in adult female rats, was examined across estrous-cycle-associated fluctuations in endogenous sex steroids. Ongoing studies from this laboratory have shown that systemic and local administration of the sex steroid 17 beta-estradiol (E2) augments excitatory responses of cerebellar Purkinje cells to iontophoretically applied glutamate, recorded in vivo from anesthetized female rats. In addition, this steroid potentiated discharge correlated with limb movement. For the present study, extracellular single-unit activity was recorded from as many as 5-11 Purkinje cells simultaneously during treadmill locomotion paradigms. Motor modulation of activity was recorded across three to five consecutive estrous cycles from behaviorally identified cohorts of neurons to test the hypothesis that fluctuations in endogenous sex steroids alter motor modulation of Purkinje cell discharge. 2. Locomotor-associated discharge correlated with treadmill locomotion was increased by a mean of 47% on proestrus, when E2 levels are elevated, relative to diestrus 1. These changes in discharge rate during treadmill locomotion were of significantly greater magnitude than corresponding cyclic alterations in discharge during stationary periods. 3. Correlations with the circadian cycle were also significant, because peak levels of locomotor-associated discharge on the night of behavioral estrus, following elevations in circulating E2, were on average 67% greater than corresponding discharge recorded during the light (proestrus). 4. Alterations in the step cycle were also observed across the estrous cycle: significant decreases in the duration of the flexion phase (by 265 ms, P < 0.05) were noted on estrus compared with diestrus. 5. When recorded on estrus, Purkinje cell discharge correlated with the stance or flexion phase of the step cycle was greater in

Red sorrel (Hibiscus Sabdariffa) prevents the ethanol-induced deficits of Purkinje cells in the cerebellum.

PubMed

Suryanti, S; Partadiredja, G; Atthobari, J

2015-01-01

The present study is aimed at investigating the possible protective effects of H. sabdariffa on ethanol-elicited deficits of motor coordination and estimated total number of the Purkinje cells of the cerebellums of adolescent male Wistar rats. Forty male Wistar rats aged 21 days were divided into five groups. Na/wtr group was given water orally and injected with normal saline intra peritoneally (ip). Eth/wtr group was given water orally and ethanol (ip). Another three experimental groups (Eth/Hsab) were given different dosages of H. sabdariffa and ethanol (ip). All groups were treated intermittently for the total period of treatment of two weeks. The motor coordination of rats was tested prior and subsequent to the treatments. The rats were euthanized, and their cerebellums were examined. The total number of Purkinje cells was estimated using physical fractionator method. Upon revolving drum test, the number of falls of rats increased following ethanol treatment. There was no significant difference between the total number of falls prior and subsequent to treatment in all Eth/Hsab groups. The estimated total number of Purkinje cells in Eth/Hsab groups was higher than in Eth/wtr group. H. sabdariffa may prevent the ethanol-induced deficits of motor coordination and estimated total number of Purkinje cells of the cerebellums in adolescent rats (Tab. 3, Fig. 1, Ref. 42).

Chick cerebellar Purkinje cells express omega-conotoxin GVIA-sensitive rather than funnel-web spider toxin-sensitive calcium channels.

PubMed

Angulo, M C; Parra, P; Dieudonné, S

1998-03-01

Voltage-gated calcium channels form a complex family of distinct molecular entities which participate in multiple neuronal functions. In cerebellar Purkinje cells these channels contribute to the characteristic electrophysiological pattern of complex spikes, first described in birds and later in mammals. A specific calcium channel, the P-type channel, has been shown to mediate the majority of the voltage-gated calcium flux in mammalian Purkinje cells. P-type channels play an essential role in synaptic transmission of mammalian cerebellum. It is unclear whether the P-type calcium channel is present in birds. Studies in chick synaptosomal preparations show that the pharmacological profile of calcium channels is complex and suggest a minimal expression of the P-type channel in avian central nervous system. In the present work, we studied voltage-gated calcium channels in dissociated chick cerebellar Purkinje cells to examine the presence of different calcium channel types. Purkinje cells were used because, in mammals, they express predominantly P-type channels and because the morphology of these cells is thought to be phylogenetically conserved. We found that omega-conotoxin GVIA (omega-CgTx GVIA), a specific antagonist of N-type calcium channel, rather than the synthetic funnel-web spider toxin (sFTX), a P-type channel antagonist, blocks the majority of the barium current flowing through calcium channels in chick Purkinje neurons.

Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

PubMed

Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

2010-06-01

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

Bursting patterns and mixed-mode oscillations in reduced Purkinje model

NASA Astrophysics Data System (ADS)

Zhan, Feibiao; Liu, Shenquan; Wang, Jing; Lu, Bo

2018-02-01

Bursting discharge is a ubiquitous behavior in neurons, and abundant bursting patterns imply many physiological information. There exists a closely potential link between bifurcation phenomenon and the number of spikes per burst as well as mixed-mode oscillations (MMOs). In this paper, we have mainly explored the dynamical behavior of the reduced Purkinje cell and the existence of MMOs. First, we adopted the codimension-one bifurcation to illustrate the generation mechanism of bursting in the reduced Purkinje cell model via slow-fast dynamics analysis and demonstrate the process of spike-adding. Furthermore, we have computed the first Lyapunov coefficient of Hopf bifurcation to determine whether it is subcritical or supercritical and depicted the diagrams of inter-spike intervals (ISIs) to examine the chaos. Moreover, the bifurcation diagram near the cusp point is obtained by making the codimension-two bifurcation analysis for the fast subsystem. Finally, we have a discussion on mixed-mode oscillations and it is further investigated using the characteristic index that is Devil’s staircase.

Effects of subchronic inhalation of vaporized plastic cement on exploratory behavior and Purkinje cell differentiation in the rat.

PubMed

Pascual, R; Salgado, C; Viancos, L; Figueroa, H R

1996-12-06

In the present study, the effects of preweaning cement vapor inhalation on exploratory behavior and cerebellar Purkinje cell differentiation were assessed. Sprague-Dawley albino rats were daily exposed to glue vapors between postnatal d 2 and 21. At postnatal d 22, all animals were submitted to the open-field test in order to evaluate their exploratory behavior. Then they were sacrificed, their brains dissected out, and cerebella stained according to the Golgi-Cox-Sholl procedure. Purkinje cells sampled from parasagittal sections of the cerebellar vermis were drawn under camera lucida and their dendritic domain was determined. The collected data indicate that glue solvent inhalation impairs both Purkinje cell differentiation and locomotor exploratory behavior.

The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes.

PubMed

Burroughs, Amelia; Wise, Andrew K; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y; Lang, Eric J; Apps, Richard; Cerminara, Nadia L

2017-01-01

Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes. Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets. The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear. We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number. This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition

The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes

PubMed Central

Burroughs, Amelia; Wise, Andrew K.; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y.; Lang, Eric J.

2016-01-01

Key points Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes.Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets.The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear.We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number.This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Abstract Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake

Acute adaptation of the vestibuloocular reflex: signal processing by floccular and ventral parafloccular Purkinje cells.

PubMed

Hirata, Y; Highstein, S M

2001-05-01

The gain of the vertical vestibuloocular reflex (VVOR), defined as eye velocity/head velocity was adapted in squirrel monkeys by employing visual-vestibular mismatch stimuli. VVOR gain, measured in the dark, could be trained to values between 0.4 and 1.5. Single-unit activity of vertical zone Purkinje cells was recorded from the flocculus and ventral paraflocculus in alert squirrel monkeys before and during the gain change training. Our goal was to evaluate the site(s) of learning of the gain change. To aid in the evaluation, a model of the vertical optokinetic reflex (VOKR) and VVOR was constructed consisting of floccular and nonfloccular systems divided into subsystems based on the known anatomy and input and output parameters. Three kinds of input to floccular Purkinje cells via mossy fibers were explicitly described, namely vestibular, visual (retinal slip), and efference copy of eye movement. The characteristics of each subsystem (gain and phase) were identified at different VOR gains by reconstructing single-unit activity of Purkinje cells during VOKR and VVOR with multiple linear regression models consisting of sensory input and motor output signals. Model adequacy was checked by evaluating the residual following the regressions and by predicting Purkinje cells' activity during visual-vestibular mismatch paradigms. As a result, parallel changes in identified characteristics with VVOR adaptation were found in the prefloccular/floccular subsystem that conveys vestibular signals and in the nonfloccular subsystem that conveys vestibular signals, while no change was found in other subsystems, namely prefloccular/floccular subsystems conveying efference copy or visual signals, nonfloccular subsystem conveying visual signals, and postfloccular subsystem transforming Purkinje cell activity to eye movements. The result suggests multiple sites for VVOR motor learning including both flocculus and nonflocculus pathways. The gain change in the nonfloccular vestibular

Multiple subclasses of Purkinje cells in the primate floccular complex provide similar signals to guide learning in the vestibulo-ocular reflex

NASA Technical Reports Server (NTRS)

Raymond, J. L.; Lisberger, S. G.

1997-01-01

The neural "learning rules" governing the induction of plasticity in the cerebellum were analyzed by recording the patterns of neural activity in awake, behaving animals during stimuli that induce a form of cerebellum-dependent learning. We recorded the simple- and complex-spike responses of a broad sample of Purkinje cells in the floccular complex during a number of stimulus conditions that induce motor learning in the vestibulo-ocular reflex (VOR). Each subclass of Purkinje cells carried essentially the same information about required changes in the gain of the VOR. The correlation of simple-spike activity in Purkinje cells with activity in vestibular pathways could guide learning during low-frequency but not high-frequency stimuli. Climbing fiber activity could guide learning during all stimuli tested but only if compared with the activity present approximately 100 msec earlier in either vestibular pathways or Purkinje cells.

Miglustat Improves Purkinje Cell Survival and Alters Microglial Phenotype in Feline Niemann-Pick Disease Type C

PubMed Central

Stein, Veronika M.; Crooks, Alexandra; Ding, Wenge; Prociuk, Maria; O’Donnell, Patricia; Bryan, Caroline; Sikora, Tracey; Dingemanse, Jasper; Vanier, Marie T.; Walkley, Steven U.; Vite, Charles H.

2012-01-01

Niemann-Pick disease type C (NPC disease) is an incurable cellular lipid trafficking disorder characterized by neurodegeneration and intralysosomal accumulation of cholesterol and glycosphingolipids. Treatment with miglustat, a small imino sugar that reversibly inhibits glucosylceramide synthase, which is necessary for glycosphingolipid synthesis, has been shown to benefit patients with NPC disease. The mechanism(s) and extent of brain cellular changes underlying this benefit are not understood. To investigate the basis of the efficacy of miglustat, cats with disease homologous to the juvenile-onset form of human NPC disease received daily miglustat orally beginning at 3 weeks of age. The plasma half-life of miglustat was 6.6 ± 1.1 hours, with a tmax, Cmax, and area under the plasma concentration-time curve of 1.7 ± 0.6 hours, 20.3 ± 4.6 μg/ml, and 104.1 ± 16.6 μg hours/ml, respectively. Miglustat delayed the onset of neurological signs and increased the lifespan of treated cats, and was associated with decreased GM2 ganglioside accumulation in the cerebellum and improved Purkinje cell survival. Ex vivo examination of microglia from the brains of treated cats revealed normalization of CD1c and class II major histocompatibility complex expression, as well as generation of reactive oxygen species. Together, these results suggest that prolonged Purkinje cell survival, reduced glycosphingolipid accumulation, and/or the modulation of microglial immunophenotype and function contribute to miglustat-induced neurological improvement in treated cats. PMID:22487861

Resveratrol Protects Purkinje Neurons and Restores Muscle Activity in Rat Model of Cerebellar Ataxia.

PubMed

Ghorbani, Zeynab; Farahani, Reza Mastery; Aliaghaei, Abbas; Khodagholi, Fariba; Meftahi, Gholam Houssein; Danyali, Samira; Abdollahifar, Mohammad Amin; Daftari, Mahtab; Boroujeni, Mahdi Eskandarian; Sadeghi, Yousef

2018-05-01

Cerebellar ataxia (CA) is regarded as a miscellaneous cluster of brain disorders related to the cerebellum. Resveratrol is a naturally occurring polyphenolic compound. Previous reports suggest that resveratrol confers neuroprotection in various animal models of brain damage. Indeed, we considered it invaluable to investigate whether a treatment with resveratrol has a therapeutic role against CA induced by 3-acetylpyridine (3-AP) in rats. In addition, no investigation has examined neuroprotective effect of resveratrol in rat model of CA. Initially, 3-AP administration generated CA rat models followed by intraperitoneal injection with resveratrol. Then, motor performance and muscle electromyography (EMG) activity were assessed. Moreover, the anti-apoptotic role of resveratrol in CA and its relationship to protection of Purkinje cells were explored. According to what we have found, resveratrol administration improved the muscle activity and movement coordination in 3-AP-lesioned rats. Also under resveratrol treatment, the total number of the Purkinje neurons increased whereas a reduction in apoptotic bodies was observed. In conclusion, post-treatment with resveratrol evidently ameliorated motor performance as well as muscle activity accompanied by a protection of Purkinje cells in ataxic rats.

HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition

PubMed Central

Rinaldi, Arianna; Defterali, Cagla; Mialot, Antoine; Garden, Derek L F; Beraneck, Mathieu; Nolan, Matthew F

2013-01-01

Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. PMID:24000178

Calretinin immunoreactivity in the claustrum of the rat

PubMed Central

Druga, Rastislav; Salaj, Martin; Barinka, Filip; Edelstein, Lawrence; Kubová, Hana

2015-01-01

The claustrum is a telencephalic structure which consists of dorsal segment adjoining the insular cortex and a ventral segment termed also endopiriform nucleus (END). The dorsal segment (claustrum) is divided into a dorsal and ventral zone, while the END is parcellated into dorsal, ventral and intermediate END. The claustrum and the END consist of glutamatergic projection neurons and GABAergic local interneurons coexpressing calcium binding proteins. Among neurons expressing calcium binding proteins the calretinin (CR)-immunoreactive interneurons exert specific functions in neuronal circuits, including disinhibition of excitatory neurons. Previous anatomical data indicate extensive and reciprocally organized claustral projections with cerebral cortex. We asked if the distribution of cells immunoreactive for CR delineates anatomical or functional subdivisions in the claustrum and in the END. Both segments of the claustrum and all subdivisions of the END contained CR immunoreactive neurons with varying distribution. The ventral zone of the claustrum exhibited weak labeling with isolated cell bodies and thin fibers and is devoid of immunoreactive puncta. Within the medial margin of the intermediate END we noted a group of strongly positive neurons. Cells immunoreactive for CR in all subdivisions of the claustrum and END were bipolar, multipolar and oval with smooth, beaded aspiny dendrites. Small number of CR-immunoreactive neurons displayed thin dendrites which enter to adjoining structures. Penetration of dendrites was reciprocal. These results show an inhomogenity over the claustrum and the END in distribution and types of CR immunoreactive neurons. The distribution of the CR-immunoreactive neurons respects the anatomical but not functional zones of the claustral complex. PMID:25653596

Chronic treadmill exercise in rats delicately alters the Purkinje cell structure to improve motor performance and toxin resistance in the cerebellum.

PubMed

Huang, Tung-Yi; Lin, Lung-Sheng; Cho, Keng-Chi; Chen, Shean-Jen; Kuo, Yu-Min; Yu, Lung; Wu, Fong-Sen; Chuang, Jih-Ing; Chen, Hsiun-Ing; Jen, Chauying J

2012-09-01

Although exercise usually improves motor performance, the underlying cellular changes in the cerebellum remain to be elucidated. This study aimed to investigate whether and how chronic treadmill exercise in young rats induced Purkinje cell changes to improve motor performance and rendered the cerebellum less vulnerable to toxin insults. After 1-wk familiarization of treadmill running, 6-wk-old male Wistar rats were divided into exercise and sedentary groups. The exercise group was then subjected to 8 wk of exercise training at moderate intensity. The rotarod test was carried out to evaluate motor performance. Purkinje cells in cerebellar slices were visualized by lucifer yellow labeling in single neurons and by calbindin immunostaining in groups of neurons. Compared with sedentary control rats, exercised rats not only performed better in the rotarod task, but also showed finer Purkinje cell structure (higher dendritic volume and spine density with the same dendritic field). The exercise-improved cerebellar functions were further evaluated by monitoring the long-lasting effects of intraventricular application of OX7-saporin. In the sedentary group, OX7-saporin treatment retarded the rotarod performance and induced ∼60% Purkinje cell loss in 3 wk. As a comparison, the exercise group showed much milder injuries in the cerebellum by the same toxin treatment. In conclusion, exercise training in young rats increased the dendritic density of Purkinje cells, which might play an important role in improving the motor performance. Furthermore, as Purkinje cells in the exercise group were relatively toxin resistant, the exercised rats showed good motor performance, even under toxin-treated conditions.

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

PubMed

Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

2017-06-01

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

PubMed Central

Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

2015-01-01

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

3D gaze tracking method using Purkinje images on eye optical model and pupil

NASA Astrophysics Data System (ADS)

Lee, Ji Woo; Cho, Chul Woo; Shin, Kwang Yong; Lee, Eui Chul; Park, Kang Ryoung

2012-05-01

Gaze tracking is to detect the position a user is looking at. Most research on gaze estimation has focused on calculating the X, Y gaze position on a 2D plane. However, as the importance of stereoscopic displays and 3D applications has increased greatly, research into 3D gaze estimation of not only the X, Y gaze position, but also the Z gaze position has gained attention for the development of next-generation interfaces. In this paper, we propose a new method for estimating the 3D gaze position based on the illuminative reflections (Purkinje images) on the surface of the cornea and lens by considering the 3D optical structure of the human eye model. This research is novel in the following four ways compared with previous work. First, we theoretically analyze the generated models of Purkinje images based on the 3D human eye model for 3D gaze estimation. Second, the relative positions of the first and fourth Purkinje images to the pupil center, inter-distance between these two Purkinje images, and pupil size are used as the features for calculating the Z gaze position. The pupil size is used on the basis of the fact that pupil accommodation happens according to the gaze positions in the Z direction. Third, with these features as inputs, the final Z gaze position is calculated using a multi-layered perceptron (MLP). Fourth, the X, Y gaze position on the 2D plane is calculated by the position of the pupil center based on a geometric transform considering the calculated Z gaze position. Experimental results showed that the average errors of the 3D gaze estimation were about 0.96° (0.48 cm) on the X-axis, 1.60° (0.77 cm) on the Y-axis, and 4.59 cm along the Z-axis in 3D space.

Signals and circuits in the purkinje neuron.

PubMed

Abrams, Zéev R; Zhang, Xiang

2011-01-01

Purkinje neurons (PN) in the cerebellum have over 100,000 inputs organized in an orthogonal geometry, and a single output channel. As the sole output of the cerebellar cortex layer, their complex firing pattern has been associated with motor control and learning. As such they have been extensively modeled and measured using tools ranging from electrophysiology and neuroanatomy, to dynamic systems and artificial intelligence methods. However, there is an alternative approach to analyze and describe the neuronal output of these cells using concepts from electrical engineering, particularly signal processing and digital/analog circuits. By viewing the PN as an unknown circuit to be reverse-engineered, we can use the tools that provide the foundations of today's integrated circuits and communication systems to analyze the Purkinje system at the circuit level. We use Fourier transforms to analyze and isolate the inherent frequency modes in the PN and define three unique frequency ranges associated with the cells' output. Comparing the PN to a signal generator that can be externally modulated adds an entire level of complexity to the functional role of these neurons both in terms of data analysis and information processing, relying on Fourier analysis methods in place of statistical ones. We also re-describe some of the recent literature in the field, using the nomenclature of signal processing. Furthermore, by comparing the experimental data of the past decade with basic electronic circuitry, we can resolve the outstanding controversy in the field, by recognizing that the PN can act as a multivibrator circuit.

Calbindin-immunoreactive cells in the fish enteric nervous system.

PubMed

Olsson, Catharina

2011-01-20

Calbindin is present in a large proportion of the intrinsic primary afferent neurons (IPANs) in the mammalian gut. Little is known about either calbindin or IPANs in fish. In the present study, calbindin immunoreactivity was investigated in the enteric nervous system of the teleost shorthorn sculpin (Myoxocephalus scorpius). Calbindin-immunoreactive nerve cell bodies and nerve fibres were present in all the gut regions except the cardiac stomach. The highest proportion was found in the proximal intestine where calbindin-immunoreactive cells constituted 59±6% (N=3) of the total Hu C/D-immunoreactive myenteric nerve cell population. In other regions, calbindin-immunoreactive cells constituted around 30% of the total population. The cells were generally multipolar with one long axon. The size distribution differed significantly between calbindin-positive and calbindin-negative cells in each of the three animals examined. Calbindin-positive neurons in the proximal intestine had a mean cross-sectional soma area of 163±73μm(2) (n=183 cells) while calbindin-negative cells were 348±221μm(2) (n=127 cells). Calbindin immunoreactivity colocalised to a large extent with serotonin immunoreactivity, but not with choline acetyltransferase (ChAT)-immunoreactivity. Thus, the calbindin-immunoreactive nerve cell population in the shorthorn sculpin gut seems to constitute a homogenous subpopulation of the enteric neurons, at least when considering the size and content of some transmitters. Whether markers other than serotonin and ChAT would differentiate the population remains to be tested. In conclusion, the calbindin-immunoreactive cells in the sculpin differ from mammalian IPANs with regard to several parameters and future functional studies could hopefully add information about the role of this large group of cells in the fish enteric nervous system. Copyright © 2010 Elsevier B.V. All rights reserved.

Electrical properties associated with wide intercellular clefts in rabbit Purkinje fibres.

PubMed Central

Colatsky, T J; Tsien, R W

1979-01-01

1. Rabbit Purkinje fibres were studied using micro-electrode recordings of electrical activity or a two-micro-electrode voltage clamp. Previous morphological work had suggested that these preparations offer structural advantages for the analysis of ionic permeability mechanisms. 2. Viable preparations could be obtained consistently by exposure to a K glutamate Tyrode solution during excision and recovery. In NaCl Tyrode solution, the action potential showed a large overshoot and fully developed plateau, but no pacemaker depolarization at negative potentials. 3. The passive electrical properties were consistent with morphological evidence for the accessibility of cleft membranes within the cell bundle. Electrotonic responses to intracellular current steps showed the behaviour expected for a simple leaky capacitative cable. Capacitative current transients under voltage clamp were changed very little by an eightfold reduction in the external solution conductivity. 4. Slow current changes attributable to K depletion were small compared to those found in other cardiac preparations. The amount of depletion was close to that predicted by a cleft model which assumed free K diffusion in 1 micron clefts. 5. Step depolarizations over the plateau range of potentials evoked a slow inward current which was resistant to tetrodotoxin but blocked by D600. 6. Strong depolarizations to potentials near 0 mV elicited a transient outward current and a slowly activating late outward current. Both components resembled currents found in sheep or calf Purkinje fibres. 7. These experiments support previous interpretations of slow plateau currents in terms of genuine permeability changes. The rabbit Purkinje fibre may allow various ionic channels to be studied with relatively little interference from radial non-uniformities in membrane potential or ion concentration. Images Fig. 7 PMID:469754

BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells.

PubMed

Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R; Trimmer, James S; Rasband, Matthew N; Misonou, Hiroaki

2015-05-06

In myelinated axons, K(+) channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na(+) channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K(+) channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K(+) channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni(2+) elicited a similar effect on APs, indicating the involvement of Ni(2+)-sensitive Ca(2+) channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex. Copyright © 2015 the authors 0270-6474/15/357082-13$15.00/0.

Lrp5/6 are required for cerebellar development and for suppressing TH expression in Purkinje cells via β-catenin.

PubMed

Huang, Ying; Zhang, Qiong; Song, Ning-Ning; Zhang, Lei; Sun, Yu-Ling; Hu, Ling; Chen, Jia-Ying; Zhu, Weidong; Li, Jue; Ding, Yu-Qiang

2016-01-15

The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons. We report here that the low-density lipoprotein receptors (Lrp) 5 and 6, Wnt co-receptors, are required for the development of the cerebellum and for suppressing ectopic TH expression in Purkinje cells. Simultaneous inactivation of Lrp 5 and 6 by Nestin-Cre results in defective lamination and foliation of the cerebellum during postnatal development. Surprisingly, TH is ectopically expressed by Purkinje cells, although they still keep its other neurochemical characteristics. These phenotypes are also observed in the cerebellum of GFAP-Cre;β-catenin(flox/flox) mice, and AAV2-Cre-mediated gene deletion leads to ectopic TH expression in Purkinje cells of β-catenin(flox/flox) mice as well. Our results revealed a new role of the canonical Lrp5/6-β-catenin pathway in regulating the morphogenesis of the cerebellum during postnatal development.

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models

PubMed Central

Yokoi, Fumiaki; Dang, Mai T.; Yang, Guang; Li, JinDong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

2011-01-01

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ε-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally-inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ε-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ε-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally-inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ε-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ε-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. PMID:22040906

Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments.

PubMed

Fukatsu, Kazumi; Bannai, Hiroko; Inoue, Takafumi; Mikoshiba, Katsuhiko

2010-09-01

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 μm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

Changes in Purkinje cell simple spike encoding of reach kinematics during adaption to a mechanical perturbation.

PubMed

Hewitt, Angela L; Popa, Laurentiu S; Ebner, Timothy J

2015-01-21

The cerebellum is essential in motor learning. At the cellular level, changes occur in both the simple spike and complex spike firing of Purkinje cells. Because simple spike discharge reflects the main output of the cerebellar cortex, changes in simple spike firing likely reflect the contribution of the cerebellum to the adapted behavior. Therefore, we investigated in Rhesus monkeys how the representation of arm kinematics in Purkinje cell simple spike discharge changed during adaptation to mechanical perturbations of reach movements. Monkeys rapidly adapted to a novel assistive or resistive perturbation along the direction of the reach. Adaptation consisted of matching the amplitude and timing of the perturbation to minimize its effect on the reach. In a majority of Purkinje cells, simple spike firing recorded before and during adaptation demonstrated significant changes in position, velocity, and acceleration sensitivity. The timing of the simple spike representations change within individual cells, including shifts in predictive versus feedback signals. At the population level, feedback-based encoding of position increases early in learning and velocity decreases. Both timing changes reverse later in learning. The complex spike discharge was only weakly modulated by the perturbations, demonstrating that the changes in simple spike firing can be independent of climbing fiber input. In summary, we observed extensive alterations in individual Purkinje cell encoding of reach kinematics, although the movements were nearly identical in the baseline and adapted states. Therefore, adaption to mechanical perturbation of a reaching movement is accompanied by widespread modifications in the simple spike encoding. Copyright © 2015 the authors 0270-6474/15/351106-19$15.00/0.

Predictive and Feedback Performance Errors are Signaled in the Simple Spike Discharge of Individual Purkinje Cells

PubMed Central

Popa, Laurentiu S.; Hewitt, Angela L.; Ebner, Timothy J.

2012-01-01

The cerebellum has been implicated in processing motor errors required for online control of movement and motor learning. The dominant view is that Purkinje cell complex spike discharge signals motor errors. This study investigated whether errors are encoded in the simple spike discharge of Purkinje cells in monkeys trained to manually track a pseudo-randomly moving target. Four task error signals were evaluated based on cursor movement relative to target movement. Linear regression analyses based on firing residuals ensured that the modulation with a specific error parameter was independent of the other error parameters and kinematics. The results demonstrate that simple spike firing in lobules IV–VI is significantly correlated with position, distance and directional errors. Independent of the error signals, the same Purkinje cells encode kinematics. The strongest error modulation occurs at feedback timing. However, in 72% of cells at least one of the R2 temporal profiles resulting from regressing firing with individual errors exhibit two peak R2 values. For these bimodal profiles, the first peak is at a negative τ (lead) and a second peak at a positive τ (lag), implying that Purkinje cells encode both prediction and feedback about an error. For the majority of the bimodal profiles, the signs of the regression coefficients or preferred directions reverse at the times of the peaks. The sign reversal results in opposing simple spike modulation for the predictive and feedback components. Dual error representations may provide the signals needed to generate sensory prediction errors used to update a forward internal model. PMID:23115173

Changes in Purkinje Cell Simple Spike Encoding of Reach Kinematics during Adaption to a Mechanical Perturbation

PubMed Central

Hewitt, Angela L.; Popa, Laurentiu S.

2015-01-01

The cerebellum is essential in motor learning. At the cellular level, changes occur in both the simple spike and complex spike firing of Purkinje cells. Because simple spike discharge reflects the main output of the cerebellar cortex, changes in simple spike firing likely reflect the contribution of the cerebellum to the adapted behavior. Therefore, we investigated in Rhesus monkeys how the representation of arm kinematics in Purkinje cell simple spike discharge changed during adaptation to mechanical perturbations of reach movements. Monkeys rapidly adapted to a novel assistive or resistive perturbation along the direction of the reach. Adaptation consisted of matching the amplitude and timing of the perturbation to minimize its effect on the reach. In a majority of Purkinje cells, simple spike firing recorded before and during adaptation demonstrated significant changes in position, velocity, and acceleration sensitivity. The timing of the simple spike representations change within individual cells, including shifts in predictive versus feedback signals. At the population level, feedback-based encoding of position increases early in learning and velocity decreases. Both timing changes reverse later in learning. The complex spike discharge was only weakly modulated by the perturbations, demonstrating that the changes in simple spike firing can be independent of climbing fiber input. In summary, we observed extensive alterations in individual Purkinje cell encoding of reach kinematics, although the movements were nearly identical in the baseline and adapted states. Therefore, adaption to mechanical perturbation of a reaching movement is accompanied by widespread modifications in the simple spike encoding. PMID:25609626

[Computer-assisted measurement of ocular misalignment in infants and young children using the digital Purkinje reflection pattern procedure].

PubMed

Barry, J C; Effert, R; Kaupp, A; Kleine, M; Reim, M

1994-02-01

A digital image recording and processing system is presented that allows a quick diagnosis of microstrabismus in non-cooperative children. It is thus particularly suited for screening purposes. The Purkinje Reflection Pattern Evaluation (RPE) method is used: three small flashes are used to produce the desired Purkinje images. Two horizontal rows of the three 1st Purkinje images (anterior corneal reflections) and of the three 4th Purkinje images (posterior crystalline lens reflections) stemming from the three light sources form the characteristic Purkinje image reflection pattern. Each eye's position is calculated from the shift between the upper and lower rows of reflections by means of two simple formulae. From the angles obtained in binocular fixation and monocular fixation the manifest angle of strabismus corresponding to the angle measured in the simultaneous prism-and-cover test is computed. The measurement is performed at a fixation distance of 50 cm under natural viewing conditions. To obtain a picture one only has to get the child's attention for a short moment. The primary position is triggered with the fixation light, which is operated by a switch. The digital image recording is done with a hand-held device comprising two miniaturized video cameras, three photo flashes and a fixation light that is operated manually. An IBM-compatible PC equipped with a hard disk and two frame grabbers was adapted for the storage and processing of the pictures. The pictures are evaluated interactively in a few minutes on the workstation's monitor immediately after the measurement. To this end specially designed menu-driven software was implemented. Examples of the measuring procedure and clinical results in infants with microtropic highlight the potential of the system as a screening apparatus and for the exact measurement of small and large squint angles. Usually even 1-year-old children can cooperate well enough to get good-quality pictures in binocular fixation. The new

Discharge properties of Purkinje cells in the oculomotor vermis during visually guided saccades in the macaque monkey.

PubMed

Ohtsuka, K; Noda, H

1995-11-01

1. We previously described discharge properties of cerebellar output cells in the fastigial nucleus during ipsilateral and contralateral saccades. Fastigial cells exhibited unique responses depending on the direction of saccades and were involved in execution of accurate targeting saccades. Purkinje cells in the oculomotor vermis (lobules VIc and VII) are thought to modulate these discharges of fastigial cells. In this study we reexamine discharge properties of Purkinje cells on the basis of this hypothesis. 2. Initially we physiologically identified the right and left sides of the oculomotor vermis. Saccade-related discharges of 79 Purkinje cells were recorded from both sides of the vermis during visually guided saccades toward the sides ipsilateral and contralateral to the recording side in two trained macaque monkeys. To clarify the correlation of Purkinje cell discharge with burst activities in the fastigial nucleus during saccadic eye movements, we analyzed our data by employing methods used in the study of fastigial neurons. 3. Among the 79 cells, 56 (71%) showed burst discharges during saccades (saccadic burst cells). Of the 56 cells, 29 exhibited a peak of burst discharges in both the contralateral and ipsilateral directions (bidirectional cells). The remaining 27 saccadic burst cells showed a peak of burst discharges during either contralateral or ipsilateral saccades (unidirectional cells). Among the 79 cells, 14 (18%) exhibited a pause of discharges during contralateral saccades (pause cells). Among the 79 cells, 9 (11%) showed burst discharge during contralateral saccades followed by tonic discharge that was correlated with eye position (burst tonic cells). 4. The timing of bursts in bidirectional cells with respect to saccade onset was dependent on the direction of saccade. During ipsilateral saccades, Purkinje cells exhibited a long lead burst that built up gradually, peaked near the onset of the saccade, and terminated sharply near midsaccade. The

Apoptosis of Purkinje and granular cells of the cerebellum following chronic ethanol intake.

PubMed

Oliveira, Suelen A; Chuffa, Luiz Gustavo A; Fioruci-Fontanelli, Beatriz Aparecida; Lizarte Neto, Fermino Sanches; Novais, Paulo Cezar; Tirapelli, Luiz Fernando; Oishi, Jorge Camargo; Takase, Luiz Fernando; Stefanini, Maira Aparecida; Martinez, Marcelo; Martinez, Francisco Eduardo

2014-12-01

Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10% (<2 g ethanol/kg body weight/day). At 120 days old, rats were anesthetized and decapitated, and their cerebella were collected and fixed. Cerebellar sections were subjected to immunohistochemistry for terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), caspase-3, X-linked inhibitor of apoptosis protein (XIAP), and insulin-like growth factor 1-receptor (IGF-1R); real-time PCR (RT-PCR) to determine caspase-3, XIAP, and IGF-1R gene expression; and transmission electron microscopy (TEM). We identified fragmentation of DNA and an increase in caspase-3 protein and XIAP in Purkinje cells, whereas granule cells exhibited increased caspase-3 and XIAP. IGF-1R expression was unchanged. There was no significant difference in gene expression of caspase-3, XIAP, and IGF-1R. There were an increase in lipid droplets, a reduction in the cellular cytoplasm in electron-dense nuclei, and changes in the myelin sheath in the cerebellar cortex. In conclusion, our data demonstrated that ethanol induced apoptosis in the Purkinje and granule cells of the cerebellum of adult UChA rats.

Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models.

PubMed

Yokoi, Fumiaki; Dang, Mai T; Yang, Guang; Li, Jindong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

2012-02-01

Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ɛ-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ɛ-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ɛ-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ɛ-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ɛ-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. Copyright © 2011 Elsevier B.V. All rights reserved.

Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

PubMed

Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

2018-04-01

Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

High dosage of monosodium glutamate causes deficits of the motor coordination and the number of cerebellar Purkinje cells of rats.

PubMed

Prastiwi, D; Djunaidi, A; Partadiredja, G

2015-11-01

Monosodium glutamate (MSG) has been widely used throughout the world as a flavoring agent of food. However, MSG at certain dosages is also thought to cause damage to many organs, including cerebellum. This study aimed at investigating the effects of different doses of MSG on the motor coordination and the number of Purkinje cells of the cerebellum of Wistar rats. A total of 24 male rats aged 4 to 5 weeks were divided into four groups, namely, control (C), T2.5, T3, and T3.5 groups, which received intraperitoneal injection of 0.9% sodium chloride solution, 2.5 mg/g body weight (bw) of MSG, 3.0 mg/g bw of MSG, and 3.5 mg/g bw of MSG, respectively, for 10 consecutive days. The motor coordination of the rats was examined prior and subsequent to the treatment. The number of cerebellar Purkinje cells was estimated using physical fractionator method. It has been found that the administration of MSG at a dosage of 3.5 mg/g bw, but not at lower dosages, caused a significant decrease of motor coordination and the estimated total number of Purkinje cells of rats. There was also a significant correlation between motor coordination and the total number of Purkinje cells. © The Author(s) 2015.

Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy

PubMed Central

Kros, Lieke; Lindeman, Sander; Eelkman Rooda, Oscar H. J.; Murugesan, Pavithra; Bina, Lorenzo; Bosman, Laurens W. J.; De Zeeuw, Chris I.; Hoebeek, Freek E.

2017-01-01

Absence epilepsy is characterized by the occurrence of generalized spike and wave discharges (GSWDs) in electrocorticographical (ECoG) recordings representing oscillatory activity in thalamocortical networks. The oscillatory nature of GSWDs has been shown to be reflected in the simple spike activity of cerebellar Purkinje cells and in the activity of their target neurons in the cerebellar nuclei, but it is unclear to what extent complex spike activity is implicated in generalized epilepsy. Purkinje cell complex spike firing is elicited by climbing fiber activation and reflects action potential firing in the inferior olive. Here, we investigated to what extent modulation of complex spike firing is reflected in the temporal patterns of seizures. Extracellular single-unit recordings in awake, head-restrained homozygous tottering mice, which suffer from a mutation in the voltage-gated CaV2.1 calcium channel, revealed that a substantial proportion of Purkinje cells (26%) showed increased complex spike activity and rhythmicity during GSWDs. Moreover, Purkinje cells, recorded either electrophysiologically or by using Ca2+-imaging, showed a significant increase in complex spike synchronicity for both adjacent and remote Purkinje cells during ictal events. These seizure-related changes in firing frequency, rhythmicity and synchronicity were most prominent in the lateral cerebellum, a region known to receive cerebral input via the inferior olive. These data indicate profound and widespread changes in olivary firing that are most likely induced by seizure-related activity changes in the thalamocortical network, thereby highlighting the possibility that olivary neurons can compensate for pathological brain-state changes by dampening oscillations. PMID:29163057

Purkinje cell heterotopy with cerebellar hypoplasia in two free-living American kestrels (Falco sparverius)

USDA-ARS?s Scientific Manuscript database

Two wild fledgling kestrels exhibited lack of motor coordination, postural reaction deficits, and abnormal propioception. At necropsy, the cerebellum and brainstem were markedly underdeveloped. Microscopically, there was Purkinje cells heterotopy, abnormal circuitry, and hypoplasia with defective fo...

Responses of Purkinje cells in the oculomotor vermis of monkeys during smooth pursuit eye movements and saccades: comparison with floccular complex.

PubMed

Raghavan, Ramanujan T; Lisberger, Stephen G

2017-08-01

We recorded the responses of Purkinje cells in the oculomotor vermis during smooth pursuit and saccadic eye movements. Our goal was to characterize the responses in the vermis using approaches that would allow direct comparisons with responses of Purkinje cells in another cerebellar area for pursuit, the floccular complex. Simple-spike firing of vermis Purkinje cells is direction selective during both pursuit and saccades, but the preferred directions are sufficiently independent so that downstream circuits could decode signals to drive pursuit and saccades separately. Complex spikes also were direction selective during pursuit, and almost all Purkinje cells showed a peak in the probability of complex spikes during the initiation of pursuit in at least one direction. Unlike the floccular complex, the preferred directions for simple spikes and complex spikes were not opposite. The kinematics of smooth eye movement described the simple-spike responses of vermis Purkinje cells well. Sensitivities were similar to those in the floccular complex for eye position and considerably lower for eye velocity and acceleration. The kinematic relations were quite different for saccades vs. pursuit, supporting the idea that the contributions from the vermis to each kind of movement could contribute independently in downstream areas. Finally, neither the complex-spike nor the simple-spike responses of vermis Purkinje cells were appropriate to support direction learning in pursuit. Complex spikes were not triggered reliably by an instructive change in target direction; simple-spike responses showed very small amounts of learning. We conclude that the vermis plays a different role in pursuit eye movements compared with the floccular complex. NEW & NOTEWORTHY The midline oculomotor cerebellum plays a different role in smooth pursuit eye movements compared with the lateral, floccular complex and appears to be much less involved in direction learning in pursuit. The output from the

Dual peroxidase and colloidal gold-labeling study of angiotensin converting enzyme and angiotensin-like immunoreactivity in the rat subfornical organ.

PubMed

Pickel, V M; Chan, J; Ganten, D

1986-08-01

The cellular relationships between angiotensin converting enzyme (ACE) (EC 3.4.14.1) and angiotensin-like immunoreactivity (AGLI) were examined in the subfornical organ (SFO). Brains from adult rats were fixed by vascular perfusion with 3.75% acrolein and 2% paraformaldehyde. The region containing the SFO was then sectioned on a vibrating microtome. Partially permeabilized sections were immunocytochemically labeled using the peroxidase-antiperoxidase (PAP) or combined PAP and immunogold methods. Goat antiserum to ACE was localized to both non-neuronal and neuronal cells within the SFO. Intense peroxidase immunoreactivity for ACE was associated with the ventricular and basal surface of ependymal cells, the luminal surface of the vascular endothelium, portions of glial membranes exposed to extracellular spaces, and membranous organelles within neuronal processes. Two antisera raised in rabbits against angiotensin II showed peroxidase immunoreactivity within the extracellular spaces and throughout the cytoplasm of numerous axon terminals and a few perikarya and dendrites in the SFO. Axon terminals and dendrites also showed aggregates of AGLI in smooth membranes and vesicles near the plasmalemma. Gold labeling for AGLI was evident in only 6% of the axon terminals and in a smaller number of dendrites containing peroxidase immunoreactivity for ACE. The low incidence of terminals containing both markers appeared to at least partially reflect limited penetration of the 10 nm gold particles. These results provide the first ultrastructural evidence that ACE is associated with the plasmalemma and membranous organelles strategically located for interaction with precursors of angiotensin II or other peptides within the cerebrospinal fluid, extracellular spaces and neurons of the SFO.

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

Generation-V dual-Purkinje-image eyetracker

NASA Technical Reports Server (NTRS)

Crane, H. D.; Steele, C. M.

1985-01-01

Major advances characterize the Generation-V dual-Purkinje-image eyetracker compared with the Generation-III version previously described. These advances include a large reduction in size, major improvements in frequency response and noise level, automatic alignment to a subject, and automatic adjustment for different separation between the visual and optic axes, which can vary considerably from subject to subject. In a number of applications described in the paper, the eyetracker is coupled with other highly specialized optical devices. These applications include accurately stabilizing an image on a subject's retina; accurately simulating a visually dead retinal region (i.e., a scotoma) of arbitrary shape, size, and position; and, for clinical purposes, stabilizing the position of a laser coagulator beam on a patient's retina so that the point of contact is unaffected by the patient's own eye movements.

[The effect of modulators of SK channels on simple spike firing frequency in the discharge of the cerebellar Purkinje cells in laboratory mice].

PubMed

Egorova, P A; Karelina, T V; Vlasova, O L; Antonov, S M; Besprozvanny, I B

2014-01-01

The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 μM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

PubMed

Takahashi, Natsumi; Nakamuta, Nobuaki; Yamamoto, Yoshio

2016-02-01

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na(+)-K(+)-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Different populations of parvalbumin- and calbindin-D28k-immunoreactive neurons contain GABA and accumulate 3H-D-aspartate in the dorsal horn of the rat spinal cord.

PubMed

Antal, M; Polgár, E; Chalmers, J; Minson, J B; Llewellyn-Smith, I; Heizmann, C W; Somogyi, P

1991-12-01

The colocalization of parvalbumin (PV), calbindin-D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H-D-aspartate selectively were investigated in neurons of laminae I-IV of the dorsal horn of the rat spinal cord. Following injection of 3H-D-aspartate into the basal dorsal horn (laminae IV-VI), perikarya selectively accumulating 3H-D-aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H-D-aspartate were found exclusively in laminae I-III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H-D-aspartate-labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H-D-aspartate. A high proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP-immunoreactive perikarya were GABA-negative. GABA-immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I-IV. A significant proportion of the GABA-negative but PV-immunoreactive neurons also showed CaBP-immunoreactivity in laminae II and IV. These results show that out of the two calcium-binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium-binding proteins in the dorsal horn of the rat

Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pcd) mutant mice.

PubMed

Goodlett, C R; Hamre, K M; West, J R

1992-04-10

Spatial learning in rodents requires normal functioning of hippocampal and cortical structures. Recent data suggest that the cerebellum may also be essential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups of pcd and control mice were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that the pcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. These data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.

Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex.

PubMed

Crook, J; Hendrickson, A; Robinson, F R

2006-09-15

Previous work demonstrates that the cerebellum uses glycine as a fast inhibitory neurotransmitter [Ottersen OP, Davanger S, Storm-Mathisen J (1987) Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon, Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake. Exp Brain Res 66(1):211-221; Ottersen OP, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450(1-2):342-353; Dieudonne S (1995) Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci U S A 92:1441-1445; Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057; Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498; Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123-141]. In the rat cerebellum glycine is not released by itself but is released together with GABA by Lugaro cells onto Golgi cells [Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057] and by Golgi cells onto unipolar brush and granule cells [Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498]. Here we report, from immunolabeling evidence in Macaca cerebellum, that interneurons in the granular cell layer are glycine+ at a density

Sensory processing and corollary discharge effects in posterior caudal lobe Purkinje cells in a weakly electric mormyrid fish.

PubMed

Alviña, Karina; Sawtell, Nathaniel B

2014-07-15

Although it has been suggested that the cerebellum functions to predict the sensory consequences of motor commands, how such predictions are implemented in cerebellar circuitry remains largely unknown. A detailed and relatively complete account of predictive mechanisms has emerged from studies of cerebellum-like sensory structures in fish, suggesting that comparisons of the cerebellum and cerebellum-like structures may be useful. Here we characterize electrophysiological response properties of Purkinje cells in a region of the cerebellum proper of weakly electric mormyrid fish, the posterior caudal lobe (LCp), which receives the same mossy fiber inputs and projects to the same target structures as the electrosensory lobe (ELL), a well-studied cerebellum-like structure. We describe patterns of simple spike and climbing fiber activation in LCp Purkinje cells in response to motor corollary discharge, electrosensory, and proprioceptive inputs and provide evidence for two functionally distinct Purkinje cell subtypes within LCp. Protocols that induce rapid associative plasticity in ELL fail to induce plasticity in LCp, suggesting differences in the adaptive functions of the two structures. Similarities and differences between LCp and ELL are discussed in light of these results. Copyright © 2014 the American Physiological Society.

Regular Patterns in Cerebellar Purkinje Cell Simple Spike Trains

PubMed Central

Shin, Soon-Lim; Hoebeek, Freek E.; Schonewille, Martijn; De Zeeuw, Chris I.; Aertsen, Ad; De Schutter, Erik

2007-01-01

Background Cerebellar Purkinje cells (PC) in vivo are commonly reported to generate irregular spike trains, documented by high coefficients of variation of interspike-intervals (ISI). In strong contrast, they fire very regularly in the in vitro slice preparation. We studied the nature of this difference in firing properties by focusing on short-term variability and its dependence on behavioral state. Methodology/Principal Findings Using an analysis based on CV2 values, we could isolate precise regular spiking patterns, lasting up to hundreds of milliseconds, in PC simple spike trains recorded in both anesthetized and awake rodents. Regular spike patterns, defined by low variability of successive ISIs, comprised over half of the spikes, showed a wide range of mean ISIs, and were affected by behavioral state and tactile stimulation. Interestingly, regular patterns often coincided in nearby Purkinje cells without precise synchronization of individual spikes. Regular patterns exclusively appeared during the up state of the PC membrane potential, while single ISIs occurred both during up and down states. Possible functional consequences of regular spike patterns were investigated by modeling the synaptic conductance in neurons of the deep cerebellar nuclei (DCN). Simulations showed that these regular patterns caused epochs of relatively constant synaptic conductance in DCN neurons. Conclusions/Significance Our findings indicate that the apparent irregularity in cerebellar PC simple spike trains in vivo is most likely caused by mixing of different regular spike patterns, separated by single long intervals, over time. We propose that PCs may signal information, at least in part, in regular spike patterns to downstream DCN neurons. PMID:17534435

Modulated discharge of Purkinje and stellate cells persists after unilateral loss of vestibular primary afferent mossy fibers in mice

PubMed Central

Yakhnitsa, V.

2013-01-01

Cerebellar Purkinje cells are excited by two afferent pathways: climbing and mossy fibers. Climbing fibers evoke large “complex spikes” (CSs) that discharge at low frequencies. Mossy fibers synapse on granule cells whose parallel fibers excite Purkinje cells and may contribute to the genesis of “simple spikes” (SSs). Both afferent systems convey vestibular information to folia 9c–10. After making a unilateral labyrinthectomy (UL) in mice, we tested how the discharge of CSs and SSs was changed by the loss of primary vestibular afferent mossy fibers during sinusoidal roll tilt. We recorded from cells identified by juxtacellular neurobiotin labeling. The UL preferentially reduced vestibular modulation of CSs and SSs in folia 8–10 contralateral to the UL. The effects of a UL on Purkinje cell discharge were similar in folia 9c–10, to which vestibular primary afferents project, and in folia 8–9a, to which they do not project, suggesting that vestibular primary afferent mossy fibers were not responsible for the UL-induced alteration of SS discharge. UL also induced reduced vestibular modulation of stellate cell discharge contralateral to the UL. We attribute the decreased modulation to reduced vestibular modulation of climbing fibers. In summary, climbing fibers modulate CSs directly and SSs indirectly through activation of stellate cells. Whereas vestibular primary afferent mossy fibers cannot account for the modulated discharge of SSs or stellate cells, the nonspecific excitation of Purkinje cells by parallel fibers may set an operating point about which the discharges of SSs are sculpted by climbing fibers. PMID:23966673

Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collin, Ludovic; Doretto, Sandrine; Department of Psychiatry and Human Behavior, University of California Irvine, 3226 Gillespie Neuroscience Research Facility, Irvine CA 92697

2007-08-01

Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiationmore » and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.« less

Choline acetyltransferase immunoreactivity in the human vestibular end-organs.

PubMed

Ishiyama, A; Lopez, I; Wackym, P A

1994-10-01

Acetylcholine (ACh) is believed to play a major role in the efferent vestibular system in several animal models, however no information regarding the role of ACh in the human efferent vestibular system has been published. Post-embedding immunohistochemistry in a hydrophilic resin was used to investigate the choline acetyltransferase immunoreactivity (ChATi) and acetylcholinesterase (AChE) histochemistry in human vestibular end-organs. ChATi and AChE activity was found in numerous bouton-type terminals at the basal area of the vestibular hair cells. These terminals were found to contact type II vestibular hair cells and the afferent chalices surrounding type I hair cells. This study provides the first evidence that the human efferent vestibular axons and terminals are cholinergic.

Sumoylation of FOXP2 Regulates Motor Function and Vocal Communication Through Purkinje Cell Development.

PubMed

Usui, Noriyoshi; Co, Marissa; Harper, Matthew; Rieger, Michael A; Dougherty, Joseph D; Konopka, Genevieve

2017-02-01

Mutations in the gene encoding the transcription factor forkhead box P2 (FOXP2) result in brain developmental abnormalities, including reduced gray matter in both human patients and rodent models and speech and language deficits. However, neither the region-specific function of FOXP2 in the brain, in particular the cerebellum, nor the effects of any posttranslational modifications of FOXP2 in the brain and disorders have been explored. We characterized sumoylation of FOXP2 biochemically and analyzed the region-specific function and sumoylation of FOXP2 in the developing mouse cerebellum. Using in utero electroporation to manipulate the sumoylation state of FOXP2 as well as Foxp2 expression levels in Purkinje cells of the cerebellum in vivo, we reduced Foxp2 expression approximately 40% in the mouse cerebellum. Such a reduction approximates the haploinsufficiency observed in human patients who demonstrate speech and language impairments. We identified sumoylation of FOXP2 at K674 (K673 in mice) in the cerebellum of neonates. In vitro co-immunoprecipitation and in vivo colocalization experiments suggest that PIAS3 acts as the small ubiquitin-like modifier E3 ligase for FOXP2 sumoylation. This sumoylation modifies transcriptional regulation by FOXP2. We demonstrated that FOXP2 sumoylation is required for regulation of cerebellar motor function and vocal communication, likely through dendritic outgrowth and arborization of Purkinje cells in the mouse cerebellum. Sumoylation of FOXP2 in neonatal mouse cerebellum regulates Purkinje cell development and motor functions and vocal communication, demonstrating evidence for sumoylation in regulating mammalian behaviors. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Distribution of enkephalin-like immunoreactivity in the cat digestive tract.

PubMed

Bagnol, D; Henry, M; Cupo, A; Julé, Y

1997-05-12

Immunohistochemical investigations were carried out to determine the pattern of distribution of methionine- and leucine-enkephalin-like materials in the cat pylorus, duodenum, ileum and proximal and distal colon. The present results indicate that leucine-enkephalin-like materials are less densely distributed than methionine-enkephalin-like materials, but that the two patterns of distribution show some similarities. Considerable regional differences exist however in the distribution of these enkephalin-like materials in the muscular layers. In the duodenum, ileum and proximal colon, the immunoreactivity was mainly confined to the myenteric plexus and the circular muscle layer, where it was present in nerve cell bodies and in numerous fibres. In the longitudinal muscle and submucous layers, a few immunoreactive fibres were observed which sometimes surrounded blood vessels. In the pylorus and the distal colon, however, numerous immunoreactive fibres were observed in the longitudinal and circular muscle layers; the immunoreactivity was detected in the cell bodies of numerous myenteric plexus neurons but those of only a few submucous plexus neurons. In addition, the pylorus tissues contained immunoreactive plexi which were localized either within the longitudinal muscle or between the serosa and the longitudinal muscle layer. These plexi were connected to the myenteric plexus by immunoreactive nerve strands. In all the small intestinal segments studied, numerous immunoreactive varicosities were present in the deep muscular plexus, in the inner part of the circular muscle layer. Our results suggest that in cats, the nervous control of external muscular layers mediated by enkephalins shows regional differences. In the pylorus and the distal colon, it involves both the longitudinal and circular muscle layers, whereas in other intestinal segments, only the circular muscle layer is involved.

Purinergic receptor immunoreactivity in the rostral ventromedial medulla.

PubMed

Close, L N; Cetas, J S; Heinricher, M M; Selden, N R

2009-01-23

The rostral ventromedial medulla (RVM) has long been recognized to play a pivotal role in nociceptive modulation. Pro-nociception within the RVM is associated with a distinct functional class of neurons, ON-cells that begin to discharge immediately before nocifensive reflexes. Anti-nociceptive function within the RVM, including the analgesic response to opiates, is associated with another distinct class, OFF-cells, which pause immediately prior to nocifensive reflexes. A third class of RVM neurons, NEUTRAL-cells, does not alter firing in association with nocifensive reflexes. ON-, OFF- and NEUTRAL-cells show differential responsiveness to various behaviorally relevant neuromodulators, including purinergic ligands. Iontophoresis of semi-selective P2X ligands, which are associated with nociceptive transmission in the spinal cord and dorsal root ganglia, preferentially activate ON-cells. By contrast, P2Y ligands activate OFF-cells and P1 ligands suppress the firing of NEUTRAL cells. The current study investigates the distribution of P2X, P2Y and P1 receptor immunoreactivity in RVM neurons of Sprague-Dawley rats. Co-localization with tryptophan hydroxylase (TPH), a well-established marker for serotonergic neurons was also studied. Immunoreactivity for the four purinergic receptor subtypes examined was abundant in all anatomical subdivisions of the RVM. By contrast, TPH-immunoreactivity was restricted to a relatively small subset of RVM neurons concentrated in the nucleus raphe magnus and pallidus, as expected. There was a significant degree of co-localization of each purinergic receptor subtype with TPH-immunoreactivity. This co-localization was most pronounced for P2Y1 receptor immunoreactivity, although this was the least abundant among the different purinergic receptor subtypes examined. Immunoreactivity for multiple purinergic receptor subtypes was often co-localized in single neurons. These results confirm the physiological finding that purinergic receptors are

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

PubMed

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-05-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-01-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119

A two-step enzymatic modification method to reduce immuno-reactivity of milk proteins.

PubMed

Damodaran, Srinivasan; Li, Yan

2017-12-15

A two-step enzymatic approach to reduce immuno-reactivity of whey protein isolate and casein has been studied. The method involves partial hydrolysis of proteins with proteases, followed by repolymerization with microbial transglutaminase. Whey protein isolate partially hydrolyzed with chymotrypsin, trypsin, or thermolysin retained about 80%, 30%, and 20% of the original immuno-reactivity, respectively. Upon repolymerization the immuno-reactivity decreased to 45%, 35%, and 5%, respectively. The immuno-reactivity of hydrolyzed and repolymerized casein was negligible compared to native casein. The repolymerized products were partially resistant to in vitro digestion. Peptides released during digestion of repolymerized thermolysin-whey protein hydrolysate had less than 5% immuno-reactivity, whereas those of whey protein control exhibited a sinusoidal immuno-reactivity ranging from 5 to 20%. Peptides released during digestion of repolymerized thermolysin-casein hydrolysates had no immuno-reactivity. These results indicated that it is possible to produce hypoallergenic milk protein products using the two-step enzymatic modification method involving thermolysin and transglutaminase. Copyright © 2017. Published by Elsevier Ltd.

Absence of Rapid Propagation through the Purkinje Network as a Potential Cause of Line Block in the Human Heart with Left Bundle Branch Block.

PubMed

Okada, Jun-Ichi; Washio, Takumi; Nakagawa, Machiko; Watanabe, Masahiro; Kadooka, Yoshimasa; Kariya, Taro; Yamashita, Hiroshi; Yamada, Yoko; Momomura, Shin-Ichi; Nagai, Ryozo; Hisada, Toshiaki; Sugiura, Seiryo

2018-01-01

Background: Cardiac resynchronization therapy is an effective device therapy for heart failure patients with conduction block. However, a problem with this invasive technique is the nearly 30% of non-responders. A number of studies have reported a functional line of block of cardiac excitation propagation in responders. However, this can only be detected using non-contact endocardial mapping. Further, although the line of block is considered a sign of responders to therapy, the mechanism remains unclear. Methods: Herein, we created two patient-specific heart models with conduction block and simulated the propagation of excitation based on a cellmodel of electrophysiology. In one model with a relatively narrow QRS width (176 ms), we modeled the Purkinje network using a thin endocardial layer with rapid conduction. To reproduce a wider QRS complex (200 ms) in the second model, we eliminated the Purkinje network, and we simulated the endocardial mapping by solving the inverse problem according to the actual mapping system. Results: We successfully observed the line of block using non-contact mapping in the model without the rapid propagation of excitation through the Purkinje network, although the excitation in the wall propagated smoothly. This model of slow conduction also reproduced the characteristic properties of the line of block, including dense isochronal lines and fractionated local electrocardiograms. Further, simulation of ventricular pacing from the lateral wall shifted the location of the line of block. By contrast, in the model with the Purkinje network, propagation of excitation in the endocardial map faithfully followed the actual propagation in the wall, without showing the line of block. Finally, switching the mode of propagation between the two models completely reversed these findings. Conclusions: Our simulation data suggest that the absence of rapid propagation of excitation through the Purkinje network is the major cause of the functional line

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

PubMed

Chung, Chan; Elrick, Matthew J; Dell'Orco, James M; Qin, Zhaohui S; Kalyana-Sundaram, Shanker; Chinnaiyan, Arul M; Shakkottai, Vikram G; Lieberman, Andrew P

2016-05-01

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs.

PubMed

Dosdall, Derek J; Tabereaux, Paul B; Kim, Jong J; Walcott, Gregory P; Rogers, Jack M; Killingsworth, Cheryl R; Huang, Jian; Robertson, Peter G; Smith, William M; Ideker, Raymond E

2008-08-01

Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution (n = 6) or normal Tyrode solution as a control (n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 x 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 +/- 1.5 vs. 9.2 +/- 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.

Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

PubMed

Daikoku, H; Morisaki, I; Ogawa, Y; Maeda, T; Kurisu, K; Wakisaka, S

1999-06-01

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.

Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea.

PubMed

Altschuler, R A; Kachar, B; Rubio, J A; Parakkal, M H; Fex, J

1985-07-08

The immunocytochemical localization of the enzyme choline acetyltransferase (ChAT) was examined in the guinea pig organ of Corti to determine if both lateral and medial systems of efferents would show immunoreactive labeling for this specific enzyme marker of cholinergic neurons. Cochleae were also examined after lesion of efferents to determine if ChAT-like immunoreactivity is confined to efferents. ChAT-like immunoreactivity was seen in the inner spiral bundle, tunnel spiral bundle and by the bases of inner hair cells corresponding to the lateral system of efferents. ChAT-like immunoreactivity was also seen in crossing fibers and puncta at the bases and by the nuclei of outer hair cells corresponding to the medial system of efferents. With the use of video enhanced contrast microscopy more than 9 ChAT-like immunoreactive puncta at the bases of outer hair cells could be resolved. In cochleae examined 6 weeks after ipsilateral lesion of efferents, no ChAT-like immunoreactivity was observed. These results add strong evidence that acetylcholine is a transmitter of both the medial and lateral systems of efferents.

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates.

PubMed

Mizuno, J; Takeda, N

1988-01-01

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

Cross talk between metabotropic and ionotropic glutamate receptor-mediated signaling in parallel fiber-induced inositol 1,4,5-trisphosphate production in cerebellar Purkinje cells.

PubMed

Okubo, Yohei; Kakizawa, Sho; Hirose, Kenzo; Iino, Masamitsu

2004-10-27

In many excitatory glutamatergic synapses, both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are closely distributed on the postsynaptic membrane. However, the functional significance of the close distribution of the two types of glutamate receptors has not been fully clarified. In this study, we examined the functional interaction between iGluR and mGluR at parallel fiber (PF)--> Purkinje cell synapses in the generation of inositol 1,4,5-trisphosphate (IP3), a key second messenger that regulates many important cellular functions. We visualized local IP3 dynamics in Purkinje cells using the green fluorescent protein-tagged pleckstrin homology domain (GFP-PHD) as a fluorescent IP3 probe. Purkinje cells were transduced with Sindbis virus encoding GFP-PHD and imaged with a two-photon laser scanning microscope. Translocation of GFP-PHD from the plasma membrane to the cytoplasm attributable to an increase in IP3 concentration was observed on PF stimulation in fine dendrites of Purkinje cells. Surprisingly, this PF-induced IP3 production was blocked not only by the group I mGluR antagonist but also by the AMPA receptor (AMPAR) antagonist. The PF-induced IP3 production was blocked by either the inhibition of G-protein activation by GDP-betaS or intracellular Ca2+ buffering by BAPTA. These results show that IP3 production is mediated cooperatively by group I mGluR and AMPAR through G-protein activation and Ca2+ influx at PF--> Purkinje cell synapses, identifying the robust cross talk between iGluR and mGluR for the generation of IP3 signals.

Reduced orexin immunoreactivity in Perry syndrome and multiple system atrophy.

PubMed

Mishima, Takayasu; Kasanuki, Koji; Koga, Shunsuke; Castanedes-Casey, Monica; Wszolek, Zbigniew K; Tsuboi, Yoshio; Dickson, Dennis W

2017-09-01

Orexin is a neuropeptide that plays a key role in maintaining a state of arousal, and possibly associates with sleep apnea syndrome (SAS). Reduced orexin immunoreactivity has been reported in various neurologic conditions such as narcolepsy, Alzheimer's disease, Lewy body disease and multiple system atrophy (MSA); however, there has been no report investigating orexin in Perry syndrome, a rare hereditary neurodegenerative disease characterized by four clinical cardinal signs (parkinsonism, depression/apathy, weight loss, and central hypoventilation). Perry syndrome patients frequently have sleep disturbances, including SAS and insomnia. We evaluated orexin immunoreactivity in Perry syndrome. Using imaging analysis, we quantitatively assessed orexin immunoreactivity in the nucleus basalis of Meynert in three Perry syndrome cases, as well as five cases of frontotemporal lobar degeneration with motor neuron disease, five cases of MSA and five age-matched controls. For these cases, antemortem clinical information on sleep disturbances has been reviewed. In Perry syndrome and MSA, there was reduction of orexin immunoreactivity compared with controls (Perry syndrome: p = 0.020, MSA: p < 0.001). In contrast, FTLD-MND did not have significant reduction of orexin immunoreactivity. Two out of three cases of Perry syndrome had SAS confirmed by polysomnography. This is the first report assessing orexin immunoreactivity in Perry syndrome, and it showed significant reduction, similar to select neurodegenerative diseases, such as MSA. Further analysis with more cases will be needed to elucidate the specific mechanism of orexin loss in these disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ontogeny of cholecystokinin-like immunoreactivity in the Brazilian opossum brain.

PubMed

Fox, C A; Jeyapalan, M; Ross, L R; Jacobson, C D

1991-12-17

We have studied the anatomical distribution of cholecystokinin-like immunoreactive (CCK-IR) somata and fibers in the brain of the adult and developing Brazilian short-tailed opossum, Monodelphis domestica. Animals ranged in age from the day of birth (1PN) to young adulthood (180PN). A nickel enhanced, avidin-biotin, indirect immunohistochemical technique was used to identify CCK-IR structures. Somata containing CCK immunoreactivity were observed in the cerebral cortex, hippocampus, hypothalamus, thalamus, midbrain, and brainstem in the adult. Cholecystokinin immunoreactive fibers had a wide distribution in the adult Monodelphis brain. The only major region of the brain that did not contain CCK-IR fibers was the cerebellum. The earliest expression of CCK immunoreactivity was found in fibers in the dorsal brainstem of 5-day-old opossum pups. It is possible that the CCK-IR fibers in the brainstem at 5PN are of vagal origin. Cholecystokinin immunoreactive somata were observed in the brainstem on 10PN. The CCK-IR cell bodies observed in the brainstem at 10PN may mark the first expression of CCK-IR elements intrinsic to the brain. A broad spectrum of patterns of onset of CCK expression was observed in the opossum brain. The early occurrence and varied ontogenesis of CCK-IR structures indicates CCK may be involved in the function of a variety of circuits from the brainstem to the cerebral cortex. The early expression of CCK-IR structures in the dorsal brainstem suggests that CCK may modulate feeding behavior in the Monodelphis neonate. Cholecystokinin immunoreactivity in forebrain structures such as the suprachiasmatic nucleus, medial preoptic area, thalamus and cortical structures indicates that CCK may also be involved in circadian rhythmicity, reproductive functions, as well as the state of arousal of the Brazilian opossum. The ontogenic timing of CCK immunoreactivity in specific circuitry also indicates that CCK expression does not occur simultaneously throughout the

Interleukin (IL)-8 immunoreactivity of injured axons and surrounding oligodendrocytes in traumatic head injury.

PubMed

Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

2016-06-01

Interleukin (IL)-8 has been suggested to be a positive regulator of myelination in the central nervous system, in addition to its principal role as a chemokine for neutrophils. Immunostaining for beta-amyloid precursor protein (AβPP) is an effective tool for detecting traumatic axonal injury, although AβPP immunoreactivity can also indicate axonal injury due to hypoxic causes. In this study, we examined IL-8 and AβPP immunoreactivity in sections of corpus callosum obtained from deceased patients with blunt head injury and from equivalent control tissue. AβPP immunoreactivity was detected in injured axons, such as axonal bulbs and varicose axons, in 24 of 44 head injury cases. These AβPP immunoreactive cases had survived for more than 3h. The AβPP immunostaining pattern can be classified into two types: traumatic (Pattern 1) and non-traumatic (Pattern 2) axonal injuries, which we described previously [Hayashi et al. Int. J. Legal Med. 129 (2015) 1085-1090]. Three of 44 control cases also showed AβPP immunoreactive injured axons as Pattern 2. In contrast, IL-8 immunoreactivity was detected in 7 AβPP immunoreactive and in 2 non-AβPP immunoreactive head injury cases, but was not detected in any of the 44 control cases, including the 3 AβPP immunoreactive control cases. The IL-8 immunoreactive cases had survived from 3 to 24 days, whereas those cases who survived less than 3 days (n=29) and who survived 90 days (n=1) were not IL-8 immunoreactive. Moreover, IL-8 was detected as Pattern 1 axons only. In addition, double immunofluorescence analysis showed that IL-8 is expressed by oligodendrocytes surrounding injured axons. In conclusion, our results suggest that immunohistochemical detection of IL-8 may be useful as a complementary diagnostic marker of traumatic axonal injury. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cell-specific expression of calcineurin immunoreactivity within the rat basolateral amygdala complex and colocalization with the neuropeptide Y Y1 receptor.

PubMed

Leitermann, Randy J; Sajdyk, Tammy J; Urban, Janice H

2012-10-01

Neuropeptide Y (NPY) produces potent anxiolytic effects via activation of NPY Y1 receptors (Y1r) within the basolateral amygdaloid complex (BLA). The role of NPY in the BLA was recently expanded to include the ability to produce stress resilience and long-lasting reductions in anxiety-like behavior. These persistent behavioral effects are dependent upon activity of the protein phosphatase, calcineurin (CaN), which has long been associated with shaping long-term synaptic signaling. Furthermore, NPY-induced reductions in anxiety-like behavior persist months after intra-BLA delivery, which together indicate a form of neuronal plasticity had likely occurred. To define a site of action for NPY-induced CaN signaling within the BLA, we employed multi-label immunohistochemistry to determine which cell types express CaN and if CaN colocalizes with the Y1r. We have previously reported that both major neuronal cell populations in the BLA, pyramidal projection neurons and GABAergic interneurons, express the Y1r. Therefore, this current study evaluated CaN immunoreactivity in these cell types, along with Y1r immunoreactivity. Antibodies against calcium-calmodulin kinase II (CaMKII) and GABA were used to identify pyramidal neurons and GABAergic interneurons, respectively. A large population of CaN immunoreactive cells displayed Y1r immunoreactivity (90%). Nearly all (98%) pyramidal neurons displayed CaN immunoreactivity, while only a small percentage of interneurons (10%) contained CaN immunoreactivity. Overall, these anatomical findings provide a model whereby NPY could directly regulate CaN activity in the BLA via activation of the Y1r on CaN-expressing, pyramidal neurons. Importantly, they support BLA pyramidal neurons as prime targets for neuronal plasticity associated with the long-term reductions in anxiety-like behavior produced by NPY injections into the BLA. Copyright © 2012 Elsevier B.V. All rights reserved.

Chronic pharmacological blockade of the Na+ /Ca2+ exchanger modulates the growth and development of the Purkinje cell dendritic arbor in mouse cerebellar slice cultures.

PubMed

Sherkhane, Pradeep; Kapfhammer, Josef P

2017-09-01

The Na + /Ca 2+ exchanger (NCX) is a bidirectional plasma membrane antiporter involved in Ca 2+ homeostasis in eukaryotes. NCX has three isoforms, NCX1-3, and all of them are expressed in the cerebellum. Immunostaining on cerebellar slice cultures indicates that NCX is widely expressed in the cerebellum, including expression in Purkinje cells. The pharmacological blockade of the forward mode of NCX (Ca 2+ efflux mode) by bepridil moderately inhibited growth and development of Purkinje cell dendritic arbor in cerebellar slice cultures. However, the blockade of the reverse mode (Ca 2+ influx mode) by KB-R7943 severely reduced the dendritic arbor and induced a morphological change with thickened distal dendrites. The effect of KB-R7943 on dendritic growth was unrelated to the activity of voltage-gated calcium channels and was also apparent in the absence of bioelectrical activity indicating that it was mediated by NCX expressed in Purkinje cells. We have used additional NCX inhibitors including CB-DMB, ORM-10103, SEA0400, YM-244769, and SN-6 which have higher specificity for NCX isoforms and target either the forward, reverse, or both modes. These inhibitors caused a strong dendritic reduction similar to that seen with KB-R7943, but did not elicit thickening of distal dendrites. Our findings indicate that disturbance of the NCX-dependent calcium transport in Purkinje cells induces a reduction of dendritic arbor, which is presumably caused by changes in the calcium handling, and underline the importance of the calcium equilibrium for the dendritic development in cerebellar Purkinje cells. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Unravelling how βCaMKII controls the direction of plasticity at parallel fibre-Purkinje cell synapses

NASA Astrophysics Data System (ADS)

Pinto, Thiago M.; Schilstra, Maria J.; Steuber, Volker; Roque, Antonio C.

2015-12-01

Long-term plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses is thought to mediate cerebellar motor learning. It is known that calcium-calmodulin dependent protein kinase II (CaMKII) is essential for plasticity in the cerebellum. Recently, Van Woerden et al. demonstrated that the β isoform of CaMKII regulates the bidirectional inversion of PF-PC plasticity. Because the cellular events that underlie these experimental findings are still poorly understood, our work aims at unravelling how β CaMKII controls the direction of plasticity at PF-PC synapses. We developed a bidirectional plasticity model that replicates the experimental observations by Van Woerden et al. Simulation results obtained from this model indicate the mechanisms that underlie the bidirectional inversion of cerebellar plasticity. As suggested by Van Woerden et al., the filamentous actin binding enables β CaMKII to regulate the bidirectional plasticity at PF-PC synapses. Our model suggests that the reversal of long-term plasticity in PCs is based on a combination of mechanisms that occur at different calcium concentrations.

Effect of glutamate, aspartate and related derivatives on cerebellar Purkinje cell dendrites in the rat: an in vitro study

PubMed Central

Crepel, F.; Dhanjal, S. S.; Sears, T. A.

1982-01-01

1. The responses of Purkinje cells to short duration (pulse) ionophoretic applications of L-aspartate (L-asp), L-glutamate (L-glu), N-methyl DL-aspartate (NMDLA) and quisqualic acid in their dendritic fields were studied in vitro on sagittal slices of lobules IX and X of the adult rat cerebellum. 2. Pulse application of L-asp or L-glu evoked transient and dose-dependent increases in the firing rate of the simple spikes recorded extracellularly as single units. When the ionophoretic electrode was positioned in the dendritic field of the Purkinje cells, the lowest thresholds for L-glu and L-asp mediated excitations of the cells were as low as 25 and 35 pC respectively, with a latency for maximal responses as brief as 7 ms. 3. In intracellular recordings these excitatory responses consisted of depolarizations of up to 18 mV in amplitude and with depolarizing slopes up to 0·52 mV/ms. They were generally unaccompanied by changes in cell input resistance in contrast to the marked decrease which occurred in response to steady applications of large doses of L-asp and L-glu. 4. The spatial distribution of the excitatory sites confirmed that the dendritic sensitivity to L-glu was greater than that of the soma and showed that the same was true for L-asp. In 34% of cells the sensitivity for L-asp declined markedly in the upper region of the molecular layer, whereas it remained high for L-glu; no such differential sensitivity was detected in the remaining 66% of cells. 5. Inhibitory responses, antagonized by 10-5 M-bicuculline in the bath, were also induced in Purkinje cells by L-glu and L-asp when the ionophoretic electrode was withdrawn from the excitatory sites by as little as 8 μm and up to 40 μm upward or downward along the track of parallel fibres or positioned as far as 250 μm laterally. 6. Whenever it was applied in the molecular layer, the pulse application of NMDLA elicited no excitatory response in Purkinje cells recorded extra or intracellularly. However, slow

Catheter ablation of fatal ventricular tachyarrhythmias storm in acute coronary syndrome--role of Purkinje fiber network.

PubMed

Enjoji, Yoshihisa; Mizobuchi, Masahiro; Muranishi, Hiromi; Miyamoto, Chinae; Utsunomiya, Makoto; Funatsu, Atsushi; Kobayashi, Tomoko; Nakamura, Shigeru

2009-12-01

Ventricular fibrillation (VF) or ventricular tachycardia (VT) storm is a life-threatening arrhythmia. Antiarrhythmic drugs (AADs) are not necessarily effective to rescue life from such conditions. Catheter ablation (CA) targeting triggering premature ventricular contractions (PVCs) of VF or VT that originates from Purkinje fiber network (PFN) is reported to be effective, especially in idiopathic patients. However, in condition of acute coronary syndrome (ACS), the efficacy of CA is not well understood. To clarify the usefulness of CA as an alternative way to AADs, we performed CA in four patients with VF or VT storm. The Purkinje potential was seen just before the myocardial ventricular wave during sinus rhythm that became more prominent and double components during the initiating PVC at the targeted area. Following CA, spontaneous episodes of VF or VT were no longer observed. CA is an efficacious way to bail out PFN-related VF or VT storm even in ACS.

Representation of limb kinematics in Purkinje cell simple spike discharge is conserved across multiple tasks

PubMed Central

Hewitt, Angela L.; Popa, Laurentiu S.; Pasalar, Siavash; Hendrix, Claudia M.

2011-01-01

Encoding of movement kinematics in Purkinje cell simple spike discharge has important implications for hypotheses of cerebellar cortical function. Several outstanding questions remain regarding representation of these kinematic signals. It is uncertain whether kinematic encoding occurs in unpredictable, feedback-dependent tasks or kinematic signals are conserved across tasks. Additionally, there is a need to understand the signals encoded in the instantaneous discharge of single cells without averaging across trials or time. To address these questions, this study recorded Purkinje cell firing in monkeys trained to perform a manual random tracking task in addition to circular tracking and center-out reach. Random tracking provides for extensive coverage of kinematic workspaces. Direction and speed errors are significantly greater during random than circular tracking. Cross-correlation analyses comparing hand and target velocity profiles show that hand velocity lags target velocity during random tracking. Correlations between simple spike firing from 120 Purkinje cells and hand position, velocity, and speed were evaluated with linear regression models including a time constant, τ, as a measure of the firing lead/lag relative to the kinematic parameters. Across the population, velocity accounts for the majority of simple spike firing variability (63 ± 30% of Radj2), followed by position (28 ± 24% of Radj2) and speed (11 ± 19% of Radj2). Simple spike firing often leads hand kinematics. Comparison of regression models based on averaged vs. nonaveraged firing and kinematics reveals lower Radj2 values for nonaveraged data; however, regression coefficients and τ values are highly similar. Finally, for most cells, model coefficients generated from random tracking accurately estimate simple spike firing in either circular tracking or center-out reach. These findings imply that the cerebellum controls movement kinematics, consistent with a forward internal model that

Representation of limb kinematics in Purkinje cell simple spike discharge is conserved across multiple tasks.

PubMed

Hewitt, Angela L; Popa, Laurentiu S; Pasalar, Siavash; Hendrix, Claudia M; Ebner, Timothy J

2011-11-01

Encoding of movement kinematics in Purkinje cell simple spike discharge has important implications for hypotheses of cerebellar cortical function. Several outstanding questions remain regarding representation of these kinematic signals. It is uncertain whether kinematic encoding occurs in unpredictable, feedback-dependent tasks or kinematic signals are conserved across tasks. Additionally, there is a need to understand the signals encoded in the instantaneous discharge of single cells without averaging across trials or time. To address these questions, this study recorded Purkinje cell firing in monkeys trained to perform a manual random tracking task in addition to circular tracking and center-out reach. Random tracking provides for extensive coverage of kinematic workspaces. Direction and speed errors are significantly greater during random than circular tracking. Cross-correlation analyses comparing hand and target velocity profiles show that hand velocity lags target velocity during random tracking. Correlations between simple spike firing from 120 Purkinje cells and hand position, velocity, and speed were evaluated with linear regression models including a time constant, τ, as a measure of the firing lead/lag relative to the kinematic parameters. Across the population, velocity accounts for the majority of simple spike firing variability (63 ± 30% of R(adj)(2)), followed by position (28 ± 24% of R(adj)(2)) and speed (11 ± 19% of R(adj)(2)). Simple spike firing often leads hand kinematics. Comparison of regression models based on averaged vs. nonaveraged firing and kinematics reveals lower R(adj)(2) values for nonaveraged data; however, regression coefficients and τ values are highly similar. Finally, for most cells, model coefficients generated from random tracking accurately estimate simple spike firing in either circular tracking or center-out reach. These findings imply that the cerebellum controls movement kinematics, consistent with a forward

AMPA receptor activation controls type I metabotropic glutamate receptor signalling via a tyrosine kinase at parallel fibre-Purkinje cell synapses.

PubMed

Auger, Céline; Ogden, David

2010-08-15

Metabotropic glutamate receptors type 1 (mGluR1s) and ionotropic AMPA receptors (AMPARs) are colocalized at parallel fibre (PF) to Purkinje cell synapses of the cerebellum. Single stimulation of PFs activates fast AMPAR excitatory postsynaptic currents, whereas the activation of mGluR1s requires burst stimulation. mGluR1s signal through several pathways in Purkinje cells and the most prominent is the activation of a slow EPSC (sEPSC). To separate the two synaptic currents, studies of the sEPSC have commonly been performed in the presence of AMPA/KA receptor antagonists. We show here in rat cerebellar slices that inhibition of the fast EPSC by AMPAR antagonists strongly and selectively potentiates the mGluR1 sEPSC, showing a negative regulation of mGluR1 by AMPAR. This effect is observed with low concentrations of NBQX (300 nM to 1 microM), with the selective AMPAR antagonist GYKI 53655 and also with gamma-DGG, a low affinity glutamate receptor antagonist. When photorelease of glutamate from MNI-glutamate was used to study the postsynaptic responses in isolation, AMPAR inhibition produced a similar potentiation of the mGluR1 sEPSC, showing that the interaction is postsynaptic. Finally, perfusion of the postsynaptic cell with PP1, an inhibitor of src-family tyrosine kinase, increased the amplitude of the mGluR1 sEPSC and occluded the effect of AMPAR inhibition. Thus, at PF to Purkinje cell synapses, AMPAR activation inhibits the mGluR1 sEPSC via activation of a src-family tyrosine kinase. Consequently mGluR1 signalling will be more sensitive to spillover of glutamate than to local synaptic release. Furthermore, it will be enhanced at silent PF synapses which are the majority in Purkinje cells.

Colocalization of numerous immunoreactivities in endocrine cells of the chicken proventriculus at hatching.

PubMed

Martínez, A; Buchan, A M; López, J; Sesma, P

2000-05-01

The colocalization of regulatory peptide immunoreactivities in endocrine cells of the chicken proventriculus at hatching has been investigated using the avidin-biotin technique in serial sections and double immunofluorescence in the same section for light microscopy, and double immunogold staining for electron microscopy. In addition to the eight immunoreactivities previously described in this organ, cells immunoreactive for peptide histidine isoleucine (PHI), peptide gene product 9.5 (PGP), and the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) were observed. All the cells immunoreactive to glucagon were also immunostained by the PHI antiserum. In addition, all the glucagon-like peptide 1, avian pancreatic polypeptide, and some of the neurotensin-like cells costored also glucagon- and PHI-immunoreactive substances. PGP- and PAM-immunoreactivities were also found in the glucagon-positive cells. A small proportion of the somatostatin-containing cells were positive for PHI but not for other regulatory peptides. These results could suggest either the existence of a very complex regulatory system or that the endocrine system of the newborn chickens is not yet fully developed.

Parvalbumin and calbindin immunoreactivity in the cerebral cortex of the hedgehog (Erinaceus europaeus).

PubMed Central

Ferrer, I; Zujar, M J; Admella, C; Alcantara, S

1992-01-01

To investigate the morphology and distribution of nonpyramidal neurons in the brain of insectivores, parvalbumin and calbindin 28 kDa immunoreactivity was examined in the cerebral cortex of the hedgehog (Erinaceus europaeus). Parvalbumin-immunoreactive cells were found in all layers of the isocortex, but in contrast to other mammals, a laminar organisation or specific regional distribution was not seen. Characteristic parvalbumin-immunoreactive neurons were multipolar cells with large ascending and descending dendrites extending throughout several layers. Calbindin-immunoreactive neurons were similar to those found in other species, although appearing in smaller numbers than in the cerebral cortex of more advanced mammals. The morphology and distribution of parvalbumin- and calbindin-immunoreactive cells in the piriform and entorhinal cortices were similar in hedgehogs and rodents. Parvalbumin-immunoreactive cells in the hippocampal complex were pyramidal-like and bitufted neurons, which were mainly found in the stratum oriens and stratum pyramidale of the hippocampus, and in the stratum moleculare and hilus of the fascia dentata. Heavily stained cells were found in the deep part of the stratum granulare. Intense calbindin immunoreactivity occurred mainly in the granule cell and molecular layers of the dentate gyrus and in the mossy fibre layer. The most outstanding feature in the hippocampal complex of the hedgehog was the extension of calbindin immunoreactivity to CA1 field of the hippocampus, suggesting, in agreement with other reports, that mossy fibres can establish synaptic contacts throughout the pyramidal cell layer. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:1452472

A Simple Mathematical Model Inspired by the Purkinje Cells: From Delayed Travelling Waves to Fractional Diffusion.

PubMed

Dipierro, Serena; Valdinoci, Enrico

2018-07-01

Recently, several experiments have demonstrated the existence of fractional diffusion in the neuronal transmission occurring in the Purkinje cells, whose malfunctioning is known to be related to the lack of voluntary coordination and the appearance of tremors. Also, a classical mathematical feature is that (fractional) parabolic equations possess smoothing effects, in contrast with the case of hyperbolic equations, which typically exhibit shocks and discontinuities. In this paper, we show how a simple toy-model of a highly ramified structure, somehow inspired by that of the Purkinje cells, may produce a fractional diffusion via the superposition of travelling waves that solve a hyperbolic equation. This could suggest that the high ramification of the Purkinje cells might have provided an evolutionary advantage of "smoothing" the transmission of signals and avoiding shock propagations (at the price of slowing a bit such transmission). Although an experimental confirmation of the possibility of such evolutionary advantage goes well beyond the goals of this paper, we think that it is intriguing, as a mathematical counterpart, to consider the time fractional diffusion as arising from the superposition of delayed travelling waves in highly ramified transmission media. The case of a travelling concave parabola with sufficiently small curvature is explicitly computed. The new link that we propose between time fractional diffusion and hyperbolic equation also provides a novelty with respect to the usual paradigm relating time fractional diffusion with parabolic equations in the limit. This paper is written in such a way as to be of interest to both biologists and mathematician alike. In order to accomplish this aim, both complete explanations of the objects considered and detailed lists of references are provided.

Intractable Electrical Storm After Coronary Artery Bypass Grafting Originating in Abnormal Purkinje Fibers.

PubMed

Tokunaga, Chiho; Tsukada, Toru; Sakamoto, Hiroaki; Naruse, Yoshihisa; Yoshida, Kentaro; Sekiguchi, Yukio; Imai, Akito; Aonuma, Kazutaka; Hiramatsu, Yuji

2016-01-01

Electrical storm is a rare but critical complication following revascularization in patients with ischemic heart disease. We report the case of a 67-year-old man who developed drug refractory intractable electrical storm after emergent coronary artery bypass grafting for ischemic cardiomyopathy. The electrical storm was successfully eliminated by percutaneous endocardial radiofrequency catheter ablation targeting the abnormal Purkinje-related triggering ventricular premature contractions in a low-voltage zone. © 2015 Wiley Periodicals, Inc.

Immunoreactive dynorphin in pituitary and brain.

PubMed Central

Goldstein, A; Ghazarossian, V E

1980-01-01

Distribution of the potent opioid peptide dynorphin has been determined in pituitary gland (pig, beef, rat), in the various regions of rat brain, and in rat spinal cord, by using a highly specific antiserum. By gel permeation chromatography in 4 M guanidine, the porcine pituitary immunoreactivity is found in a major peak of apparent molecular weight about 1700 and a minor peak of about 3400. Similar peaks are found in rat pituitary extracts, whereas rat brain contains, in addition, two peaks of larger apparent molecular weight. In the pituitary, immunoreactive dynorphin is found predominantly in pars nervosa. In the central nervous system, it is distributed widely, with highest concentrations in hypothalamus, medulla-pons, midbrain, and spinal cord. Although dynorphin contains leucine-enkephalin, the regional distribution of dynorphin is different from that of enkephalin or of any other known opioid peptide. PMID:6108564

Long-Term Predictive and Feedback Encoding of Motor Signals in the Simple Spike Discharge of Purkinje Cells

PubMed Central

Popa, Laurentiu S.; Streng, Martha L.

2017-01-01

Abstract Most hypotheses of cerebellar function emphasize a role in real-time control of movements. However, the cerebellum’s use of current information to adjust future movements and its involvement in sequencing, working memory, and attention argues for predicting and maintaining information over extended time windows. The present study examines the time course of Purkinje cell discharge modulation in the monkey (Macaca mulatta) during manual, pseudo-random tracking. Analysis of the simple spike firing from 183 Purkinje cells during tracking reveals modulation up to 2 s before and after kinematics and position error. Modulation significance was assessed against trial shuffled firing, which decoupled simple spike activity from behavior and abolished long-range encoding while preserving data statistics. Position, velocity, and position errors have the most frequent and strongest long-range feedforward and feedback modulations, with less common, weaker long-term correlations for speed and radial error. Position, velocity, and position errors can be decoded from the population simple spike firing with considerable accuracy for even the longest predictive (-2000 to -1500 ms) and feedback (1500 to 2000 ms) epochs. Separate analysis of the simple spike firing in the initial hold period preceding tracking shows similar long-range feedforward encoding of the upcoming movement and in the final hold period feedback encoding of the just completed movement, respectively. Complex spike analysis reveals little long-term modulation with behavior. We conclude that Purkinje cell simple spike discharge includes short- and long-range representations of both upcoming and preceding behavior that could underlie cerebellar involvement in error correction, working memory, and sequencing. PMID:28413823

Distribution of TRPV1- and TRPV2-immunoreactive afferent nerve endings in rat trachea.

PubMed

Yamamoto, Yoshio; Sato, Yoshikazu; Taniguchi, Kazuyuki

2007-12-01

Nociception in the trachea is important for respiratory modulation. We investigated the distribution, neurochemical characteristics, and origin of nerve endings with immunoreactivity for candidate sensor channels, TRPV1 and TRPV2, in rat trachea. In the epithelial layer, the intraepithelial nerve endings and dense subepithelial network of nerve fibers were immunoreactive for TRPV1. In contrast, TRPV2 immunoreactivity was observed mainly in nerve fibers of the tracheal submucosal layer and in several intrinsic ganglion cells in the peritracheal plexus. Double immunostaining revealed that some TRPV1-immunoreactive nerve fibers were also immunoreactive for substance P or calcitonin gene-related peptide, but neither neuropeptide colocalized with TRPV2. Injection of the retrograde tracer, fast blue, into the tracheal wall near the thoracic inlet demonstrated labeled neurons in the jugular, nodose, and dorsal root ganglia at segmental levels of C2-C8. In the jugular and nodose ganglia, 59.3% (70/118) and 10.7% (17/159), respectively, of fast blue-labeled neurons were immunoreactive for TRPV1, compared to 8.8% (8/91) and 2.6% (5/191) for TRPV2-immunoreactive. Our results indicate that TRPV1-immunoreactive nerve endings are important for tracheal nociception, and the different expression patterns of TRPV1 and TRPV2 with neuropeptides may reflect different subpopulations of sensory neurons.

[Measuring the effect of eyeglasses on determination of squint angle with Purkinje reflexes and the prism cover test].

PubMed

Barry, J C; Backes, A

1998-04-01

The alternating prism and cover test is the conventional test for the measurement of the angle of strabismus. The error induced by the prismatic effect of glasses is typically about 27-30%/10 D. Alternatively, the angle of strabismus can be measured with methods based on Purkinje reflex positions. This study examines the differences between three such options, taking into account the influence of glasses. The studied system comprised the eyes with or without glasses, a fixation object and a device for recording the eye position: in the case of the alternate prism and cover test, a prism bar was required; in the case of a Purkinje reflex based device, light sources for generation of reflexes and a camera for the documentation of the reflex positions were used. Measurements performed on model eyes and computer ray traces were used to analyze and compare the options. When a single corneal reflex is used, the misalignment of the corneal axis can be measured; the error in this measurement due to the prismatic effect of glasses was 7.6%/10 D, the smallest found in this study. The individual Hirschberg ratio can be determined by monocular measurements in three gaze directions. The angle of strabismus can be measured with Purkinje reflex based methods if the fundamental differences between these methods and the alternate prism and cover test, and if the influence of glasses and other sources of error are accounted for.

Dendritic excitability modulates dendritic information processing in a purkinje cell model.

PubMed

Coop, Allan D; Cornelis, Hugo; Santamaria, Fidel

2010-01-01

Using an electrophysiological compartmental model of a Purkinje cell we quantified the contribution of individual active dendritic currents to processing of synaptic activity from granule cells. We used mutual information as a measure to quantify the information from the total excitatory input current (I(Glu)) encoded in each dendritic current. In this context, each active current was considered an information channel. Our analyses showed that most of the information was encoded by the calcium (I(CaP)) and calcium activated potassium (I(Kc)) currents. Mutual information between I(Glu) and I(CaP) and I(Kc) was sensitive to different levels of excitatory and inhibitory synaptic activity that, at the same time, resulted in the same firing rate at the soma. Since dendritic excitability could be a mechanism to regulate information processing in neurons we quantified the changes in mutual information between I(Glu) and all Purkinje cell currents as a function of the density of dendritic Ca (g(CaP)) and Kca (g(Kc)) conductances. We extended our analysis to determine the window of temporal integration of I(Glu) by I(CaP) and I(Kc) as a function of channel density and synaptic activity. The window of information integration has a stronger dependence on increasing values of g(Kc) than on g(CaP), but at high levels of synaptic stimulation information integration is reduced to a few milliseconds. Overall, our results show that different dendritic conductances differentially encode synaptic activity and that dendritic excitability and the level of synaptic activity regulate the flow of information in dendrites.

Axonal transports of tripeptidyl peptidase II in rat sciatic nerves.

PubMed

Chikuma, Toshiyuki; Shimizu, Maki; Tsuchiya, Yukihiro; Kato, Takeshi; Hojo, Hiroshi

2007-01-01

Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12-72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.

Canine pancreatic lipase immunoreactivity concentrations associated with intervertebral disc disease in 84 dogs.

PubMed

Schueler, R O; White, G; Schueler, R L; Steiner, J M; Wassef, A

2018-05-01

To determine the differences in serum canine pancreatic lipase immunoreactivity between dogs with intervertebral disc herniation and healthy control dogs. Eighty-four client-owned dogs with intervertebral disc herniation, diagnosed by neurologic examination and imaging, and 18 healthy control dogs. Samples of whole blood were collected within 90 minutes of admission. Serum canine pancreatic lipase immunoreactivity concentrations were measured by a commercial immunoassay and evaluated for association with intervertebral disc herniation, signalment, neurolocalisation and the preadmission administration of glucocorticosteriods or non-steroidal anti-inflammatory drugs. Serum canine pancreatic lipase immunoreactivity concentrations were statistically increased in dogs with intervertebral disc herniation (P<0·01, n=38). A subgroup of dogs (19/38) with elevated canine pancreatic lipase immunoreactivity concentrations was re-evaluated between 2 and 4 weeks later, and 15 had resolution of clinical signs and values less than 200 μg/L. Serum canine pancreatic lipase immunoreactivity concentrations were not significantly correlated with clinical gastrointestinal disease, neurolocalisation or the preadmission administration of corticosteroids or non-steroidal anti-inflammatory drugs. These results suggest that serum canine pancreatic lipase immunoreactivity concentrations are significantly elevated in dogs with intervertebral disc herniation. © 2018 British Small Animal Veterinary Association.

Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis.

PubMed

Shan, X; Hu, J H; Cayabyab, F S; Krieger, C

2005-01-01

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated

GFAP immunoreactivity within the rat nucleus ambiguus after laryngeal nerve injury

PubMed Central

Berdugo-Vega, G; Arias-Gil, G; Rodriguez-Niedenführ, M; Davies, D C; Vázquez, T; Pascual-Font, A

2014-01-01

Changes that occur in astroglial populations of the nucleus ambiguus after recurrent (RLN) or superior (SLN) laryngeal nerve injury have hitherto not been fully characterised. In the present study, rat RLN and SLN were lesioned. After 3, 7, 14, 28 or 56 days of survival, the nucleus ambiguus was investigated by means of glial fibrillary acidic protein (GFAP) immunofluorescence or a combination of GFAP immunofluorescence and the application of retrograde tracers. GFAP immunoreactivity was significantly increased 3 days after RLN resection and it remained significantly elevated until after 28 days post injury (dpi). By 56 dpi it had returned to basal levels. In contrast, following RLN transection with repair, GFAP immunoreactivity was significantly elevated at 7 dpi and remained significantly elevated until 14 dpi. It had returned to basal levels by 28 dpi. Topographical analysis of the distribution of GFAP immunoreactivity revealed that after RLN injury, GFAP immunoreactivity was increased beyond the area of the nucleus ambiguus within which RLN motor neuron somata were located. GFAP immunoreactivity was also observed in the vicinity of neuronal somata that project into the uninjured SLN. Similarly, lesion of the SLN resulted in increased GFAP immunoreactivity around the neuronal somata projecting into it and also in the vicinity of the motor neuron somata projecting into the RLN. The increase in GFAP immunoreactivity outside of the region containing the motor neurons projecting into the injured nerve, may reflect the onset of a regenerative process attempting to compensate for impairment of one of the laryngeal nerves and may occur because of the dual innervation of the posterior cricoarytenoid muscle. This dual innervation of a very specialised muscle could provide a useful model system for studying the molecular mechanisms underlying axonal regeneration process and the results of the current study could provide the basis for studies into functional regeneration

Train stimulation of parallel fibre to Purkinje cell inputs reveals two populations of synaptic responses with different receptor signatures

PubMed Central

Devi, Suma Priya Sudarsana; Howe, James R.

2016-01-01

Key points Purkinje cells of the cerebellum receive ∼180,000 parallel fibre synapses, which have often been viewed as a homogeneous synaptic population and studied using single action potentials.Many parallel fibre synapses might be silent, however, and granule cells in vivo fire in bursts. Here, we used trains of stimuli to study parallel fibre inputs to Purkinje cells in rat cerebellar slices.Analysis of train EPSCs revealed two synaptic components, phase 1 and 2. Phase 1 is initially large and saturates rapidly, whereas phase 2 is initially small and facilitates throughout the train. The two components have a heterogeneous distribution at dendritic sites and different pharmacological profiles.The differential sensitivity of phase 1 and phase 2 to inhibition by pentobarbital and NBQX mirrors the differential sensitivity of AMPA receptors associated with the transmembrane AMPA receptor regulatory protein, γ‐2, gating in the low‐ and high‐open probability modes, respectively. Abstract Cerebellar granule cells fire in bursts, and their parallel fibre axons (PFs) form ∼180,000 excitatory synapses onto the dendritic tree of a Purkinje cell. As many as 85% of these synapses have been proposed to be silent, but most are labelled for AMPA receptors. Here, we studied PF to Purkinje cell synapses using trains of 100 Hz stimulation in rat cerebellar slices. The PF train EPSC consisted of two components that were present in variable proportions at different dendritic sites: one, with large initial EPSC amplitude, saturated after three stimuli and dominated the early phase of the train EPSC; and the other, with small initial amplitude, increased steadily throughout the train of 10 stimuli and dominated the late phase of the train EPSC. The two phases also displayed different pharmacological profiles. Phase 2 was less sensitive to inhibition by NBQX but more sensitive to block by pentobarbital than phase 1. Comparison of synaptic results with fast glutamate

Distinct responses of Purkinje neurons and roles of simple spikes during associative motor learning in larval zebrafish

PubMed Central

Harmon, Thomas C; Magaram, Uri; McLean, David L; Raman, Indira M

2017-01-01

To study cerebellar activity during learning, we made whole-cell recordings from larval zebrafish Purkinje cells while monitoring fictive swimming during associative conditioning. Fish learned to swim in response to visual stimulation preceding tactile stimulation of the tail. Learning was abolished by cerebellar ablation. All Purkinje cells showed task-related activity. Based on how many complex spikes emerged during learned swimming, they were classified as multiple, single, or zero complex spike (MCS, SCS, ZCS) cells. With learning, MCS and ZCS cells developed increased climbing fiber (MCS) or parallel fiber (ZCS) input during visual stimulation; SCS cells fired complex spikes associated with learned swimming episodes. The categories correlated with location. Optogenetically suppressing simple spikes only during visual stimulation demonstrated that simple spikes are required for acquisition and early stages of expression of learned responses, but not their maintenance, consistent with a transient, instructive role for simple spikes during cerebellar learning in larval zebrafish. DOI: http://dx.doi.org/10.7554/eLife.22537.001 PMID:28541889

Step-related discharges of Purkinje cells in the paravermal cortex of the cerebellar anterior lobe in the cat.

PubMed Central

Edgley, S A; Lidierth, M

1988-01-01

1. Extracellular recordings were made of the simple spike discharges of Purkinje cells in the lateral part of the paravermal cortex of lobule V in the cerebellum of awake cats. The cells were located within the c2 and c3 zones of Oscarsson (1979). 2. The peripheral receptive fields in which light mechanical stimuli could evoke simple spikes were examined in 252 Purkinje cells. Ninety-two per cent were activated by stimulation of the ipsilateral forelimb and 52% of 113 tested cells also discharged simple spikes in response to stimulation of the contralateral forelimb. The receptive fields were concentrated on the distal parts of the limbs: 67% of the 139 cells which were examined in most detail responded to stimulation of the paw or wrist of the ipsilateral forelimb. 3. In 135 of the Purkinje cells, the discharges were recorded during locomotion. Simple spikes were discharged at a mean rate of 54.3 +/- 27.8 impulses/s (S.D., n = 135) during steady walking on a belt moving at 0.5-0.7 m/s. The discharges of each cell were rhythmically modulated in time with the movements of stepping and although the timings of the discharges were highly variable between cells, activity in the population was greatest at the times of transition between the stance and swing phases in the ipsilateral forelimb and least during mid-stance. 4. As a population Purkinje cells with simple spike receptive fields on the distal parts of the forelimb(s) exhibited two activity maxima. These occurred during early stance and during the transition from stance to swing in the ipsilateral forelimb. Cells with receptive fields on the proximal parts of the limb achieved an activity maximum during late swing, and their average discharge rate fell at the time of onset of the swing phase in the ipsilateral forelimb instead of rising as was the case for the distal group. 5. The present results are compared with those from cells located more medially in the paravermal cortex. It is shown that medially located

Innervation of the Uvea by Galanin and Somatostatin Immunoreactive Axons in Macaques and Baboons

PubMed Central

Firth, Sally I.; Kaufman, Paul L.; De Jean, Baptiste J.; Byers, John M.; Marshak, David W.

2014-01-01

The neuropeptide galanin has not been localized previously in the primate uvea, and the neuropeptide somatostatin has not been localized in the uvea of any mammal. Here, the distribution of galanin-like and somatostatin-like immunoreactive axons in the iris, ciliary body and choroid of macaques and baboons using double and triple immunofluorescence labeling techniques and confocal microscopy was reported. In the ciliary body, galanin-like immunoreactive axons innervated blood vessels and the ciliary processes, particularly at their bases. In the iris, the majority of these axons was associated with the loose connective tissue in the stroma. Somatostatin-like immunoreactive axons were found in many of the same areas of the uvea supplied by cholinergic nerves. In the ciliary body, there were labelled axons within the ciliary processes and ciliary muscle. They were also found alongside blood vessels in the ciliary stroma. In the iris, somatostatin-like immunoreactive axons were abundant in the sphincter muscle and less so in the dilator muscle. A unilateral sympathectomy had no effect on the distribution of somatostatin-like or galanin-like immunoreactive axons, and these axons did not contain the sympathetic marker tyrosine hydroxylase. They did not contain the parasympathetic marker choline acetyltransferase, either. The galanin-like immunoreactive axons contained other neuropeptides found in sensory nerves, including calcitonin gene-related peptide, substance P and cholecystokinin. Somatostatin-like immunoreactive axons did not contain any of these sensory neuropeptides or galanin-like immunoreactivity, and they were neither labelled with an antibody to 200 kDa neurofilament protein, nor did they bind isolectin-IB4. Nevertheless, they are likely to be of sensory origin because somatostatin-like immunoreactive perikarya have previously been localized in the trigeminal ganglion of primates. Taken together, these findings indicate galanin and somatostatin are present

Ataxia with loss of Purkinje cells in a mouse model for Refsum disease

PubMed Central

Ferdinandusse, Sacha; Zomer, Anna W. M.; Komen, Jasper C.; van den Brink, Christina E.; Thanos, Melissa; Hamers, Frank P. T.; Wanders, Ronald J. A.; van der Saag, Paul T.; Poll-The, Bwee Tien; Brites, Pedro

2008-01-01

Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal α-oxidation system, resulting in the accumulation of the branched-chain fatty acid phytanic acid. The main clinical symptoms are polyneuropathy, cerebellar ataxia, and retinitis pigmentosa. To study the pathogenesis of Refsum disease, we generated and characterized a Phyh knockout mouse. We studied the pathological effects of phytanic acid accumulation in Phyh−/− mice fed a diet supplemented with phytol, the precursor of phytanic acid. Phytanic acid accumulation caused a reduction in body weight, hepatic steatosis, and testicular atrophy with loss of spermatogonia. Phenotype assessment using the SHIRPA protocol and subsequent automated gait analysis using the CatWalk system revealed unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws. Histochemical analyses in the CNS showed astrocytosis and up-regulation of calcium-binding proteins. In addition, a loss of Purkinje cells in the cerebellum was observed. No demyelination was present in the CNS. Motor nerve conduction velocity measurements revealed a peripheral neuropathy. Our results show that, in the mouse, high phytanic acid levels cause a peripheral neuropathy and ataxia with loss of Purkinje cells. These findings provide important insights in the pathophysiology of Refsum disease. PMID:19004801

MICROLESIONS OF THE INFERIOR OLIVE REDUCE VESTIBULAR MODULATION OF PURKINJE CELL COMPLEX AND SIMPLE SPIKES IN MOUSE CEREBELLUM

PubMed Central

Barmack, N.H.; Yakhnitsa, V.

2011-01-01

Cerebellar Purkinje cells have two distinct action potentials: Complex spikes (CSs) are evoked by single climbing fibers that originate from the contralateral inferior olive. Simple spikes (SSs) are often ascribed to mossy fiber---granule cell---parallel fiber inputs to Purkinje cells. Although generally accepted, this view lacks experimental support. Vestibular stimulation independently activates primary afferent mossy fibers and tertiary afferent climbing fibers that project to theuvula-nodulus (folia 8-10). CSs and SSs normally discharge antiphasically during sinusoidal roll-tilt. When CSs increase, SSs decrease. We tested the relative independence of these pathways in mice by making electrolytic microlesions of the two inferior olivary nuclei from which vestibular climbing fibers originate; the β-nucleus and dorsomedial cell column (DMCC). This reduced vestibular climbing fiber signaling to the contralateral folia 8-10, while leaving intact vestibular primary and secondary afferent mossy fibers. We recorded from Purkinje cells and interneurons in folia 8-10, identified by juxtacellular labeling with neurobiotin. Microlesions of the inferior olive increased the spontaneous discharge of SSs in contralateral folia 8-10, but blocked their modulation during vestibular stimulation. The vestibularly-evoked discharge of excitatory cerebellar interneurons (granule cells and unipolar brush cells) was not modified by olivary microlesions. The modulated discharge of stellate cells, but not Golgi cells was reduced by olivary microlesions. We conclude that vestibular modulation of CSs and SSs depends on intact climbing fibers. The absence of vestibularly-modulated SSs following olivary microlesions reflects the loss of climbing fiber-evoked stellate cell discharge. PMID:21734274

Electrophysiological effects of haloperidol on isolated rabbit Purkinje fibers and guinea pigs papillary muscles under normal and simulated ischemia.

PubMed

Yan, Dong; Cheng, Lu-feng; Song, Hong-Yan; Turdi, Subat; Kerram, Parhat

2007-08-01

Overdoses of haloperidol are associated with major ventricular arrhythmias, cardiac conduction block, and sudden death. The aim of this experiment was to study the effect of haloperidol on the action potentials in cardiac Purkinje fibers and papillary muscles under normal and simulated ischemia conditions in rabbits and guinea pigs. Using the standard intracellular microelectrode technique, we examined the effects of haloperidol on the action potential parameters [action potential amplitude (APA), phase 0 maximum upstroke velocity (V(max)), action potential amplitude at 90% of repolarization (APD(90)), and effective refractory period (ERP)] in rabbit cardiac Purkinje fibers and guinea pig cardiac papillary cells, in which both tissues were under simulated ischemic conditions. Under ischemic conditions, different concentrations of haloperidol depressed APA and prolonged APD(90) in a concentration-dependent manner in rabbit Purkinje fibers. Haloperidol (3 micromol/L) significantly depressed APA and prolonged APD(90), and from 1 micromol/L, haloperidol showed significant depression on V(max); ERP was not significantly affected. In guinea pig cardiac papillary muscles, the thresholds of significant reduction in APA, V(max), EPR, and APD(90) were 10, 0.3, 1, and 1 mumol/L, respectively, for haloperidol. Compared with cardiac conductive tissues, papillary muscles were more sensitive to ischemic conditions. Under ischemia, haloperidol prolonged ERP and APD(90) in a concentration-dependent manner and precipitated the decrease in V(max) induced by ischemia. The shortening of ERP and APD(90) in papillary muscle action potentials may be inhibited by haloperidol.

Optoelectrofluidic enhanced immunoreaction based on optically-induced dynamic AC electroosmosis.

PubMed

Han, Dongsik; Park, Je-Kyun

2016-04-07

We report a novel optoelectrofluidic immunoreaction system based on electroosmotic flow for enhancing antibody-analyte binding efficiency on a surface-based sensing system. Two conventional indium tin oxide glass slides are assembled to provide a reaction chamber for a tiny volume of sample droplet (∼5 μL), in which the top layer is employed as an antibody-immobilized substrate and the bottom layer acts as a photoconductive layer of an optoelectrofluidic device. Under the application of an AC voltage, an illuminated light pattern on the photoconductive layer causes strong counter-rotating vortices to transport analytes from the bulk solution to the vicinity of the assay spot on the glass substrate. This configuration overcomes the slow immunoreaction problem of a diffusion-based sensing system, resulting in the enhancement of binding efficiency via an optoelectrofluidic method. Furthermore, we investigate the effect of optically-induced dynamic AC electroosmotic flow on optoelectrofluidic enhancement for surface-based immunoreaction with a mathematical simulation study and real experiments using immunoglobulin G (IgG) and anti-IgG. As a result, dynamic light patterns provided better immunoreaction efficiency than static light patterns due to effective mass transport of the target analyte, resulting in an achievement of 2.18-fold enhancement under a growing circular light pattern compared to the passive mode.

Enkephalin-like immunoreactive principal ganglion cells and nerve fibres in the inferior mesenteric ganglion of the cat.

PubMed

Balayadi, M; Jule, Y; Cupo, A

1988-10-05

The occurrence and distribution of methionine-enkephalin (ME), leucine-enkephalin (LE) and methionine-enkephalin-Arg6-Gly7-Leu8 (MERGL)-like (LI) immunoreactive material in the inferior mesenteric ganglion (IMG) of the cat were studied by immunohistochemical techniques using the peroxidase-antiperoxidase method. Numerous ME-Li, LE-Li and MERGL-Li immunoreactive fibres with the same distribution pattern were observed. They were varicose and often surrounded closely neighbouring unlabelled ganglion cell bodies. Sometimes they ran in strands between ganglion cells. ME-Li immunoreactive material was detected in a number of cell bodies, the diameter of which was similar to that of unlabelled principal ganglion cell bodies, and which were probably Enk-Li-containing principal ganglion cells. These immunoreactive cells were often surrounded by ME-Li immunoreactive fibres. No LE-Li or MERGL-Li immunoreactive ganglion cell bodies were observed. The presence of ME-Li immunoreactive principal ganglion cells raises the possibility that the Enk-Li immunoreactive fibres present in the IMG may have a prevertebral ganglionic source. The possibility that the Enk-Li material present in nerve fibres might be derived from preproenkephalin-A was suggested by the occurrence of MERGL-Li immunoreactivity.

Human and porcine immunoreactive gastric inhibitory polypeptides (IR-GIP) are not identical.

PubMed

Bacarese-Hamilton, A J; Adrian, T E; Bloom, S R

1984-03-12

Immunoreactive gastric inhibitory polypeptide (IR-GIP) from human and porcine intestine was quantified by radioimmunoassay and the molecular forms characterised by gel permeation and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration revealed two major immunoreactive peaks corresponding to the previously described 5-kDa and 8-kDa molecular forms, which appeared similar in both species. Isocratic reverse-phase HPLC revealed that the major immunoreactive GIP peak (5-kDa) in the human tissue eluted earlier than the corresponding porcine molecular form, indicating the latter to be less hydrophobic. These findings suggest significant species differences between human and porcine GIP.

FoxP3 and indoleamine 2,3-dioxygenase immunoreactivity in sentinel nodes from melanoma patients.

PubMed

Ryan, Marisa; Crow, Jennifer; Kahmke, Russel; Fisher, Samuel R; Su, Zuowei; Lee, Walter T

2014-01-01

1) Assess FoxP3/indoleamine 2,3-dioxygenase immunoreactivity in head and neck melanoma sentinel lymph nodes and 2) correlate FoxP3/indoleamine 2,3-dioxygenase with sentinel lymph node metastasis and clinical recurrence. Retrospective cohort study. Patients with sentinel lymph node biopsy for head and neck melanoma between 2004 and 2011 were identified. FoxP3/indoleamine 2,3-dioxygenase prevalence and intensity were determined from the nodes. Poor outcome was defined as local, regional or distant recurrence. The overall immunoreactivity score was correlated with clinical recurrence and sentinel lymph node metastasis using the chi-square test for trend. Fifty-six sentinel lymph nodes were reviewed, with 47 negative and 9 positive for melanoma. Patients with poor outcomes had a statistically significant trend for higher immunoreactivity scores (p=0.03). Positive nodes compared to negative nodes also had a statistically significant trend for higher immunoreactivity scores (p=0.03). Among the negative nodes, there was a statistically significant trend for a poor outcome with higher immunoreactivity scores (p=0.02). FoxP3/indoleamine 2,3-dioxygenase immunoreactivity correlates with sentinel lymph node positivity and poor outcome. Even in negative nodes, higher immunoreactivity correlated with poor outcome. Therefore higher immunoreactivity may portend a worse prognosis even without metastasis in the sentinel lymph node. This could identify a subset of patients that may benefit from future trials and treatment for melanoma through Treg and IDO suppression. Published by Elsevier Inc.

Frequency-dependent reliability of spike propagation is function of axonal voltage-gated sodium channels in cerebellar Purkinje cells.

PubMed

Yang, Zhilai; Wang, Jin-Hui

2013-12-01

The spike propagation on nerve axons, like synaptic transmission, is essential to ensure neuronal communication. The secure propagation of sequential spikes toward axonal terminals has been challenged in the neurons with a high firing rate, such as cerebellar Purkinje cells. The shortfall of spike propagation makes some digital spikes disappearing at axonal terminals, such that the elucidation of the mechanisms underlying spike propagation reliability is crucial to find the strategy of preventing loss of neuronal codes. As the spike propagation failure is influenced by the membrane potentials, this process is likely caused by altering the functional status of voltage-gated sodium channels (VGSC). We examined this hypothesis in Purkinje cells by using pair-recordings at their somata and axonal blebs in cerebellar slices. The reliability of spike propagation was deteriorated by elevating spike frequency. The frequency-dependent reliability of spike propagation was attenuated by inactivating VGSCs and improved by removing their inactivation. Thus, the functional status of axonal VGSCs influences the reliability of spike propagation.

Serial measurement of pancreatic lipase immunoreactivity concentration in dogs with immune-mediated disease treated with prednisolone.

PubMed

Ohta, H; Morita, T; Yokoyama, N; Osuga, T; Sasaki, N; Morishita, K; Nakamura, K; Takiguchi, M

2017-06-01

In this pilot study, serum canine pancreatic lipase immunoreactivity was measured repeatedly in dogs with various immune-mediated diseases that were treated with immunosuppressive doses of prednisolone. Ten client-owned dogs with newly diagnosed immune-mediated disease that had normal canine pancreatic lipase immunoreactivity concentrations (≤200 µg/l) were treated with 2 to 2.2 mg/kg prednisolone orally once daily as the initial treatment. Serum samples were obtained from each of the dogs prior to treatment and at 1- to 4-week intervals during immunosuppressive treatment. The highest canine pancreatic lipase immunoreactivity concentration detected during immunosuppressive treatment was defined as the peak canine pancreatic lipase immunoreactivity. Peak canine pancreatic lipase immunoreactivity concentrations were classified as normal in two dogs, questionable (201 to 399 µg/l) in three dogs, and abnormal (≥400 µg/l) in five dogs. Peak canine pancreatic lipase immunoreactivity concentrations were significantly higher than baseline canine pancreatic lipase immunoreactivity concentrations but there was no evidence of clinical pancreatitis. It remains unclear whether the five of 10 dogs with elevated canine pancreatic lipase immunoreactivity during prednisone treatment had subclinical pancreatitis or whether the abnormal results were a consequence of prednisolone administration. © 2017 British Small Animal Veterinary Association.

Angiotensin II effects on ischemic focal ventricular tachycardia are predominantly mediated through myocardial AT(2) receptor.

PubMed

Gopinathannair, Rakesh; Chaudhary, Ashok K; Xing, Dezhi; Ely, Debra; Zheng, Wei; Martins, James B

2009-11-01

Ischemic focal ventricular tachycardia (VT) occurs in animals and humans. Angiotensin-converting enzyme inhibitors and receptor blockers reduce sudden death in patients with ischemic heart disease. In our dog model of coronary artery occlusion (CAO), we tested the hypothesis that angiotensin II (AGII) will selectively promote focal VT and that the specific AT(2) blocker PD-123319 (PD), or AT(1) blocker losartan, will affect this VT. Anesthetized dogs (n = 90) underwent CAO, followed by three-dimensional activation mapping of inducible VT. Dogs without VT in 1-3 h after CAO received AGII, and those with VT received either PD or losartan. Focal endocardium excised from ischemic sites was studied in vitro with standard microelectrode. Of 33 dogs with no inducible VT, AGII infusion resulted in sustained VT of only focal Purkinje origin in 13 (39%) compared with 0 of 20 dogs with saline. Of 26 dogs with inducible VT at baseline, given PD, reinduction was blocked in 8 of 10 (P < 0.05) focal VT, but only 1 of 15 with reentry. In contrast, of 11 dogs given losartan, reinduction of either mechanism was not blocked. In vitro triggered activity in Purkinje was blocked by PD in 13 of 19 (P < 0.05), but not by losartan in 8. Also, triggered activity was promoted by AGII, losartan, or the combination in 9 of 12 tissues. AGII promotes only focal, mainly Purkinje ischemic VT. PD, but not losartan, preferentially blocked focal VT, which is likely due to triggered activity due to delayed afterdepolarizations in Purkinje.

Immunoreactive transforming growth factor alpha is commonly present in colorectal neoplasia.

PubMed Central

Tanaka, S.; Imanishi, K.; Yoshihara, M.; Haruma, K.; Sumii, K.; Kajiyama, G.; Akamatsu, S.

1991-01-01

Surgical specimens from 19 patients with invasive colorectal cancers and 12 specimens of normal mucosa from the same patients were examined immunohistochemically for the production of the immunoreactive (IR-) transforming growth factor (TGF)-alpha and IR-epidermal growth factor (EGF) with an anti-TGF-alpha monoclonal antibody (MAb) OAL-MTG01 and anti-EGF MAb KEM-10. Immunoreactive TGF-alpha was detected in 16 (84.2%) of 19 colorectal cancers. In contrast, there was no IR-TGF-alpha in the gland cells of normal mucosa. Immunoreactive EGF was detected in 7 (36.8%) of 19 colorectal cancers and 1 (8.3%) of 12 cases of normal mucosa. The production of both IR-TGF-alpha and IR-EGF in colorectal cancer did not differ by histologic type and Dukes' stage. Immunoreactive TGF-alpha was detected at significantly higher incidence than IR-EGF in colorectal cancer. These results indicate that IR-TGF-alpha should prove valuable as a possible tumor marker in colorectal cancers, and it may be very useful in understanding the biology of colorectal cancer. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1853928

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells.

PubMed

Engbers, Jordan D T; Anderson, Dustin; Asmara, Hadhimulya; Rehak, Renata; Mehaffey, W Hamish; Hameed, Shahid; McKay, Bruce E; Kruskic, Mirna; Zamponi, Gerald W; Turner, Ray W

2012-02-14

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

Voltage signals of individual Purkinje cell dendrites in rat cerebellar slices.

PubMed

Borst, A; Heck, D; Thomann, M

1997-11-28

For investigating neuronal information processing at the cellular level, a technique which visualizes the voltage distribution within single neurons in situ would be extremely useful. Voltage-sensitive dyes are, in principle, capable of reporting membrane potential [Cohen, L.B. and Salzberg, B.M., Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-88; Grinvald, A., Lieke, E.E., Frostig, R.D. and Hildesheim, R., J. Neurosci., 14 (1994) 2545-2568; Kleinfeld, D., Delaney, K.R., Fee, M.S., Flores, J.A., Tank, D.W. and Gelperin, A., J. Neurophysiol., 72 (1994) 1402-1419]. However, their application to single cells internally is technically difficult [Antic, S. and Zecevic, D., J. Neurosci., 15 (1995) 1392-1405; Grinvald, A., Salzberg, B.M., Lev-Ram, V. and Hildesheim, R., Biophys. J., 51 (1987) 643-651; Kogan, A., Ross, W.N., Zecevic, D. and Lasser-Ross, N., Brain Res., 700 (1995) 235-239; Zecevic, D., Nature, 381 (1996) 322-325]. An alternative strategy consists in applying the dye from the outside to all cells in the tissue, while manipulating a single cell by current injection [Krauthamer, V. and Ross, W.N., J. Neurosci., 4 (1984) 673-682; Ross, W.N. and Krauthamer, V., J. Neurosci., 4 (1984) 659-672]. Here, we modify this technique to further enhance spatial at the cost of temporal resolution [Borst, A., Z. Naturforsch., 50 (1995) 435-438]. Applied to rat cerebellar slices we demonstrate that the potential spread in individual Purkinje cells can be imaged up to even fine dendritic branches. The acquired optical signals suggest that steadily hyperpolarized Purkinje cells are electrically compact. When permanently depolarized, the somatic input resistance is significantly diminished, yet the spatial voltage drop along the dendrites remains unchanged. As demonstrated by compartmental modeling, this hints to a concentration of outward rectifying currents at the soma of the cells.

Effect of Chitosan Properties on Immunoreactivity

PubMed Central

Ravindranathan, Sruthi; Koppolu, Bhanu prasanth; Smith, Sean G.; Zaharoff, David A.

2016-01-01

Chitosan is a widely investigated biopolymer in drug and gene delivery, tissue engineering and vaccine development. However, the immune response to chitosan is not clearly understood due to contradicting results in literature regarding its immunoreactivity. Thus, in this study, we analyzed effects of various biochemical properties, namely degree of deacetylation (DDA), viscosity/polymer length and endotoxin levels, on immune responses by antigen presenting cells (APCs). Chitosan solutions from various sources were treated with mouse and human APCs (macrophages and/or dendritic cells) and the amount of tumor necrosis factor-α (TNF-α) released by the cells was used as an indicator of immunoreactivity. Our results indicate that only endotoxin content and not DDA or viscosity influenced chitosan-induced immune responses. Our data also indicate that low endotoxin chitosan (<0.01 EU/mg) ranging from 20 to 600 cP and 80% to 97% DDA is essentially inert. This study emphasizes the need for more complete characterization and purification of chitosan in preclinical studies in order for this valuable biomaterial to achieve widespread clinical application. PMID:27187416

Correlation Between Extravasation and Alterations of Cerebrovascular Laminin and β-Dystroglycan Immunoreactivity Following Cryogenic Lesions in Rats.

PubMed

Kálmán, Mihály; Tóth, László; Szöllosi, Dávid; Oszwald, Erzsébet; Mahalek, Judit; Sadeghian, Sam

2017-11-01

The blood-brain barrier becomes "leaky" following lesions. Former studies revealed that following lesions the immunoreactivity of cerebrovascular laminin becomes detectable whereas that of β-dystroglycan disappears. These alterations may be indicators of glio-vascular decoupling that may result in the impairment of the blood-brain-barrier. This study investigates correlation between the post-lesion extravasation and the above-mentioned immunohistochemical alterations. Following cryogenic lesions, the survival periods lasted 5, 10, 30 minutes, 1 or 12 hours, or 1 day. Some brains were fixed immediately post-lesion. Immunofluorescent reactions were performed in floating sections. The extravasation was detected with immunostaining for plasma fibronectin and rat immunoglobulins. When the survival period was 30 minutes or longer, the area of extravasation corresponded to the area of altered laminin and β-dystroglycan immunoreactivities. Following immediate fixation some laminin immunoreactivity was already detected. The extravasation seemed to precede this early appearance of laminin immunoreactivity. The β-dystroglycan immunoreactivity disappeared later. When the extravasation spread into the corpus callosum, vascular laminin immunoreactivity appeared but the β-dystroglycan immunoreactivity persisted. It seems that extravasation separates the glial and vascular basal laminae, which results in the appearance of laminin immunoreactivity. The disappearance of β-dystroglycan immunoreactivity is neither a condition nor an inevitable consequence of the 2 other phenomena. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques.

PubMed

Bacarese-Hamilton, A J; Adrian, T E; Chohan, P; Bloom, S R

1985-06-01

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.

Reproduction-associated immunoreactive peptides in the nervous systems of prosobranch gastropods.

PubMed

Ram, J L; Gallardo, C S; Ram, M L; Croll, R P

1998-12-01

Antibodies against reproductive peptides of Aplysia and Lymnaea were used to localize homologous immunoreactive peptides in the nervous systems of three prosobranch species: Busycon canaliculatum, Concholepas concholepas, and Tegula atra. Positive control experiments in L. stagnalis demonstrated the broad species range of the anti-egg-laying hormone (anti-ELH) antibody used in this study, and showed binding of anti-alpha-caudodorsal-cell peptide (anti-alpha-CDCP) to the same cells in cerebral and buccal ganglia. Dot immunoassays with synthetic ELH confirmed the reactivity and sensitivity (< 0.1 microgram) of the anti-ELH antibody. Experiments with preadsorbed antibody or no primary antibody confirmed its specificity. In B. canaliculatum, clusters of more than 300 neuronal cell bodies immunoreactive to both anti-ELH and anti-alpha-CDCP were observed along the medial margins of left and right cerebral ganglia. Anti-alpha-CDCP reacted with additional small populations of cerebral ganglion neurons not stained by anti-ELH. Anti-ELH and anti-alpha-CDCP also reacted with overlapping but different small populations of neurons in buccal ganglia. In C. concholepas and T atra, ELH-like immunoreactivity was found in cerebral ganglia, and in T. atra in fibers in the cerebral ganglia and cerebral-pedal connectives. Thus, cerebral ganglia are the major locus of the ELH-like immunoreactivity in prosobranchs.

Modeling our understanding of the His-Purkinje system.

PubMed

Vigmond, Edward J; Stuyvers, Bruno D

2016-01-01

The His-Purkinje System (HPS) is responsible for the rapid electric conduction in the ventricles. It relays electrical impulses from the atrioventricular node to the muscle cells and, thus, coordinates the contraction of ventricles in order to ensure proper cardiac pump function. The HPS has been implicated in the genesis of ventricular tachycardia and fibrillation as a source of ectopic beats, as well as forming distinct portions of reentry circuitry. Despite its importance, it remains much less well characterized, structurally and functionally, than the myocardium. Notably, important differences exist with regard to cell structure and electrophysiology, including ion channels, intracellular calcium handling, and gap junctions. Very few computational models address the HPS, and the majority of organ level modeling studies omit it. This review will provide an overview of our current knowledge of structure and function (including electrophysiology) of the HPS. We will review the most recent advances in modeling of the system from the single cell to the organ level, with considerations for relevant interspecies distinctions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immunoreactivity of Biochemically Purified Amandin from Thermally Processed Almonds (Prunus dulcis L.).

PubMed

Zaffran, Valerie D; Sathe, Shridhar K

2018-06-15

Almond seeds were subjected to select thermal processing and amandin was purified from processed and unprocessed (control) seeds using cryoprecipitation. Amandin immunoreactivity was assessed using two murine monoclonal antibodies (mAbs)-4C10 and 4F10 detecting human IgE-relevant conformational and linear epitopes, respectively. Overall amandin immunoreactivity following thermal treatment ranged from 64.9% to 277.8% (4C10) and 81.3% to 270.3% (4F10). Except for autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min), the tested processing conditions resulted in increased immunoreactivity as determined by mAbs 4C10 and 4F10-based enzyme-linked immunosorbent assays (ELISAs). A significant, yet not complete, reduction in immunoreactivity was caused by autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min). Western- and dot-blot immunoassays corroborated the ELISA results, confirming amandin thermal stability. The tested immunoassays indicated amandin to be stable, regardless of the targeted epitope and the processing method that whole almond seeds were subjected to. © 2018 Institute of Food Technologists®.

Development of A-type allatostatin immunoreactivity in antennal lobe neurons of the sphinx moth Manduca sexta.

PubMed

Utz, Sandra; Schachtner, Joachim

2005-04-01

The antennal lobe (AL) of the sphinx moth Manduca sexta is a well-established model system for studying mechanisms of neuronal development. To understand whether neuropeptides are suited to playing a role during AL development, we have studied the cellular localization and temporal expression pattern of neuropeptides of the A-type allatostatin family. Based on morphology and developmental appearance, we distinguished four types of AST-A-immunoreactive cell types. The majority of the cells were local interneurons of the AL (type Ia) which acquired AST-A immunostaining in a complex pattern consisting of three rising (RI-RIII) and two declining phases (DI, DII). Type Ib neurons consisted of two local neurons with large cell bodies not appearing before 7/8 days after pupal ecdysis (P7/P8). Types II and III neurons accounted for single centrifugal neurons, with type II neurons present in the larva and disappearing in the early pupa. The type III neuron did not appear before P7/P8. RI and RII coincided with the rises of the ecdysteroid hemolymph titer. Artificially shifting the pupal 20-hydroxyecdysone (20E) peak to an earlier developmental time point resulted in the precocious appearance of AST-A immunostaining in types Ia, Ib, and III neurons. This result supports the hypothesis that the pupal rise in 20E plays a role in AST-A expression during AL development. Because of their early appearance in newly forming glomeruli, AST-A-immunoreactive fibers could be involved in glomerulus formation. Diffuse AST-A labeling during early AL development is discussed as a possible signal providing information for ingrowing olfactory receptor neurons.

Phase resetting in a model of cardiac Purkinje fiber.

PubMed Central

Guevara, M R; Shrier, A

1987-01-01

The phase-resetting response of a model of spontaneously active cardiac Purkinje fiber is investigated. The effect on the interbeat interval of injecting a 20-ms duration depolarizing current pulse is studied as a function of the phase in the cycle at which the pulse is delivered. At low current amplitudes, a triphasic response is recorded as the pulse is advanced through the cycle. At intermediate current amplitudes, the response becomes quinquephasic, due to the presence of supernormal excitability. At high current amplitudes, a triphasic response is seen once more. At low stimulus amplitudes, type 1 phase resetting occurs; at medium amplitudes, a type could not be ascribed to the phase resetting because of the presence of effectively all-or-none depolarization; at high amplitudes, type 0 phase resetting occurs. The modeling results closely correspond with published experimental data; in particular type 1 and type 0 phase resetting are seen. Implications for the induction of ventricular arrhythmias are considered. PMID:3663827

Development of "Pinceaux" formations and dendritic translocation of climbing fibers during the acquisition of the balance between glutamatergic and gamma-aminobutyric acidergic inputs in developing Purkinje cells.

PubMed

Sotelo, Constantino

2008-01-10

The acquisition of the dynamic balance between excitation and inhibition in developing Purkinje cells, necessary for their proper function, is analyzed. Newborn (P0) mouse cerebellum contains glutamatergic (VGLUT2-IR) and gamma-aminobutyric acid (GABA)-ergic (VIAAT-IR) axons. The former prevail and belong to climbing fibers, whereas the latter neither colabel with calbindin-expressing fibers nor belong to axons of the cortical GABAergic interneurons. During the first postnatal week, VIAAT-IR axons in the Purkinje cell neighborhood remains very low, and the first synapses with basket fibers are formed at P7, when climbing fibers have already established dense pericellular nets. The descending basket fibers reach the Purkinje cell axon initial segment by P9, immediately establishing axoaxonic synapses. The pinceaux appear as primitive vortex-like arrangements by P12, and by P20 interbasket fiber septate-like junctions, typical of fully mature pinceaux, are still missing. The climbing fiber's somatodendritic translocation occurs later than expected, after the regression of the multiple innervation, and follows the ascending collaterals of the basket axons, which are apparently the optimal substrate for the proper subcellular targeting of the climbing fibers. These results emphasize that chemical transmission in the axon initial segment precedes the electrical inhibition generated by field effects. In addition, GABAergic Purkinje cells, as opposed to glutamatergic projection neurons in other cortical structures, do not begin to receive their excitation to inhibition balance until the end of the first postnatal week, despite the early presence of potentially functional GABAergic axons that possess the required vesicular transport system. (c) 2007 Wiley-Liss, Inc.

Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain

PubMed Central

Preston, Chet; Wang, Louis; Yi, Jae Kyo; Lin, Chih-Li; Sun, Wei; Spyropoulos, Demetri D.; Rhee, Soyoung; Li, Mingsong; Zhou, Jie; Ge, Shaoyu; Zhang, Guofeng; Snider, Ashley J.; Hannun, Yusuf A.; Obeid, Lina M.; Mao, Cungui

2015-01-01

Dyshomeostasis of both ceramides and sphingosine-1-phosphate (S1P) in the brain has been implicated in aging-associated neurodegenerative disorders in humans. However, mechanisms that maintain the homeostasis of these bioactive sphingolipids in the brain remain unclear. Mouse alkaline ceramidase 3 (Acer3), which preferentially catalyzes the hydrolysis of C18:1-ceramide, a major unsaturated long-chain ceramide species in the brain, is upregulated with age in the mouse brain. Acer3 knockout causes an age-dependent accumulation of various ceramides and C18:1-monohexosylceramide and abolishes the age-related increase in the levels of sphingosine and S1P in the brain; thereby resulting in Purkinje cell degeneration in the cerebellum and deficits in motor coordination and balance. Our results indicate that Acer3 plays critically protective roles in controlling the homeostasis of various sphingolipids, including ceramides, sphingosine, S1P, and certain complex sphingolipids in the brain and protects Purkinje cells from premature degeneration. PMID:26474409

Ontogenetic organization of the FMRFamide immunoreactivity in the nervus terminalis of the lungfish, Neoceratodus forsteri.

PubMed

Fiorentino, Maria; D'Aniello, Biagio; Joss, Jean; Polese, Gianluca; Rastogi, Rakesh K

2002-08-19

The development of the nervus terminalis system in the lungfish, Neoceratodus forsteri, was investigated by using FMRFamide as a marker. FMRFamide immunoreactivity appears first within the brain, in the dorsal hypothalamus at a stage around hatching. At a slightly later stage, immunoreactivity appears in the olfactory mucosa. These immunoreactive cells move outside the olfactory organ to form the ganglion of the nervus terminalis. Immunoreactive processes emerge from the ganglion of the nervus terminalis in two directions, one which joins the olfactory nerve to travel to the brain and the other which courses below the brain to enter at the level of the preoptic nucleus. Neither the ganglion of the nervus terminalis nor the two branches of the nervus terminalis form after surgical removal of the olfactory placode at a stage before the development of FMRFamide immunoreactivity external to the brain. Because this study has confirmed that the nervus terminalis in lungfish comprises both an anterior and a posterior branch, it forms the basis for discussion of homology between these branches and the nervus terminalis of other anamniote vertebrates. Copyright 2002 Wiley-Liss, Inc.

Effects of the Maillard Reaction on the Immunoreactivity of Amandin in Food Matrices.

PubMed

Chhabra, Guneet S; Liu, Changqi; Su, Mengna; Venkatachalam, Mahesh; Roux, Kenneth H; Sathe, Shridhar K

2017-10-01

Amandin is the major storage protein and allergen in almond seeds. Foods, containing almonds, subjected to thermal processing typically experience Maillard browning reaction. The resulting destruction of amino groups, protein glycation, and/or denaturation may alter amandin immunoreactivity. Amandin immunoreactivity of variously processed almond containing foods was therefore the focus of the current investigation. Commercial and laboratory prepared foods, including those likely to have been subjected to Maillard browning, were objectively assessed by determining Hunter L * , a * , b * values. The L * values for the tested samples were in the range of 31.75 to 85.28 consistent with Maillard browning. Three murine monoclonal antibodies, 4C10, 4F10, and 2A3, were used to determine the immunoreactivity of the targeted samples using immunoassays (ELISA, Western blot, dot blot). The tested foods did not exhibit cross-reactivity indicating that the immunoassays were amandin specific. For sandwich ELISAs, ratio (R) of sample immunoreactivity to reference immunoreactivity was calculated. The ranges of R values were 0.67 to 15.19 (4C10), 1.00 to 11.83 (4F10), and 0.77 to 23.30 (2A3). The results of dot blot and Western blot were consistent with those of ELISAs. Results of these investigations demonstrate that amandin is a stable marker protein for almond detection regardless of the degree of amandin denaturation and/or destruction as a consequence of Maillard reaction encountered under the tested processing conditions. Foods containing almond are often subjected to processing prior to consumption. Amandin, the major allergen in almond, may experience Maillard reaction. Understanding the change in amandin immunoreactivity as a result of Maillard reaction is important for amandin detection and production of hypoallergenic food products. © 2017 Institute of Food Technologists®.

Evaluation of immunoreactivity of normal tissues from dogs, using monoclonal antibody B72.3.

PubMed

Clemo, F A; DeNicola, D B; Zimmermann, J L

1994-08-01

Monoclonal antibody (MAB) B72.3, which recognizes human tumor-associated glycoprotein-72, has immunoreactivity for malignant epithelial neoplasms in human beings and dogs. To further characterize the range of immunoreactivity of MAB B72.3 in canine tissues, MAB B72.3 and 2 other tumor-associated glycoprotein-72 antibodies (MAB CC49 and CC83) were tested against a wide spectrum of normal tissues from dogs. Immunoreactivity was detected, using an avidin-biotin-complex immunoperoxidase method. Monoclonal antibody B72.3 did not stain most types of normal canine tissues, but various types of epithelial cells within the gastrointestinal and respiratory tract mucosae, salivary gland, esophagus, epididymis, uterus, thymus, hair follicle, and apocrine glands of the anal sac had variable staining with MAB B72.3. A similar range of immunoreactivity in comparable types of normal tissues was seen for MAB CC49 and CC83; however, MAB CC49, but not MAB B72.3 and CC83, stained the endothelium of capillaries and small vessels in most normal tissues. Staining of frozen and paraffin-embedded tissues was similar. In conclusion, we found that MAB B72.3, CC49, and CC83 had selected immunoreactivity for specific types of normal canine epithelial cells, especially those involved with mucin production.

Comparison of alpha-synuclein immunoreactivity in the spinal cord between the adult and aged beagle dog

PubMed Central

Ahn, Ji-Hyeon; Choi, Jung-Hoon; Park, Joon-Ha; Yan, Bing-Chun; Kim, In-Hye; Lee, Jae-Chul; Lee, Dae-Hwan; Kim, Jin-Sang

2012-01-01

Alpha-synuclein (α-syn) is a presynaptic protein that is richly expressed in the central and peripheral nervous systems of mammals, and it is related to the pathogenesis of Parkinson's disease and other neurodegenerative disorders. In the present study, we compared the distribution of the immunoreactivity of α-syn and its related gliosis in the spinal cord of young adult (2-3 years) and aged (10-12 years) beagle dogs. We discovered that α-syn immunoreactivity was present in many neurons in the thoracic level of the aged spinal cord, however, its protein level was not distinct inform that of the adult spinal cord. In addition, ionized calcium-binding adapter molecule-1 (a marker for microglia) immunoreactivity, and not glial fibrillary acidic protein (a marker for astrocytes) immunoreactivity, was somewhat increased in the aged group compared to the adult group. These results indicate that α-syn immunoreactivity was not dramatically changed in the dog spinal cord during aging. PMID:23091516

Essential role of axonal VGSC inactivation in time-dependent deceleration and unreliability of spike propagation at cerebellar Purkinje cells

PubMed Central

2014-01-01

Background The output of the neuronal digital spikes is fulfilled by axonal propagation and synaptic transmission to influence postsynaptic cells. Similar to synaptic transmission, spike propagation on the axon is not secure, especially in cerebellar Purkinje cells whose spiking rate is high. The characteristics, mechanisms and physiological impacts of propagation deceleration and infidelity remain elusive. The spike propagation is presumably initiated by local currents that raise membrane potential to the threshold of activating voltage-gated sodium channels (VGSC). Results We have investigated the natures of spike propagation and the role of VGSCs in this process by recording spikes simultaneously on the somata and axonal terminals of Purkinje cells in cerebellar slices. The velocity and fidelity of spike propagation decreased during long-lasting spikes, to which the velocity change was more sensitive than fidelity change. These time-dependent deceleration and infidelity of spike propagation were improved by facilitating axonal VGSC reactivation, and worsen by intensifying VGSC inactivation. Conclusion Our studies indicate that the functional status of axonal VGSCs is essential to influencing the velocity and fidelity of spike propagation. PMID:24382121

The significance of variations in immunoreactive and clottable fibrinogen in health and following thrombosis

PubMed Central

Wolf, P.; Farrell, G. W.; Walton, K. W.

1972-01-01

In individuals in a steady state of fibrinogen metabolism, immunoreactive and clottable fibrinogen estimates of plasma fibrinogen show close agreement. These estimates also detect any increase of plasma fibrinogen (due to increased synthesis) following metabolic stresses but in certain circumstances discrepancies between immunoreactive and clottable fibrinogen values occur which are of diagnostic assistance. During extensive thrombosis, circulating `cryoprofibrin' (fibrin intermediates) may be formed. These fail to give full quantitative immunodiffusion reactions with antifibrinogen. Values for immunoreactive are therefore lower than for clottable fibrinogen. When intravascular catabolism (due to plasmin action) accompanies increased synthesis, since some of the molecular breakdown products of fibrin or fibrinogen react with antifibrinogen but are not clottable, immunoreactive values exceed those for clottable fibrinogen. This discrepancy is therefore an indicator of thrombolysis. Each discrepancy in turn may be encountered during the alternating predominance of thrombosis or thrombolysis in vivo: (a) physiologically, in association with the menstrual cycle; (b) pathologically, following surgical operations or extensive intravascular thrombosis. Images PMID:4259368

Action Potentials Initiate in the Axon Initial Segment and Propagate Through Axon Collaterals Reliably in Cerebellar Purkinje Neurons

PubMed Central

Foust, Amanda; Popovic, Marko; Zecevic, Dejan; McCormick, David A.

2010-01-01

Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By utilizing a recent improvement in voltage sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than approximately 250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission. PMID:20484631

Distribution of FMRFamide-like immunoreactivity in the brain, retina and nervus terminalis of the sockeye salmon parr, Oncorhynchus nerka.

PubMed

Ostholm, T; Ekström, P; Ebbesson, S O

1990-09-01

Neurons displaying FMRFamide(Phe - Met - Arg - Phe - NH2)-like immunoreactivity have recently been implicated in neural plasticity in salmon. We now extend these findings by describing the extent of the FMRF-like immunoreactive (FMRF-IR) system in the brain, retina and olfactory system of sockeye salmon parr using the indirect peroxidase anti-peroxidase technique. FMRF-IR perikarya were found in the periventricular hypothalamus, mesencephalic laminar nucleus, nucleus nervi terminalis and retina (presumed amacrine cells), and along the olfactory nerves. FMRF-IR fibers were distributed throughout the brain with highest densities in the ventral area of the telencephalon, in the medial forebrain bundle, and at the borders between layers III/IV and IV/V in the optic tectum. High densities of immunoreactive fibers were also observed in the area around the torus semicircularis, in the medial hypothalamus, median raphe, ventromedial tegmentum, and central gray. In the retina, immunopositive fibers were localized to the inner plexiform layer, but several fiber elements were also found in the outer plexiform layer. The olfactory system displayed FMRF-IR fibers in the epithelium and along the olfactory nerves. These findings differ from those reported in other species as follows: (i) FMRF-IR cells in the retina have not previously been reported in teleosts; (ii) the presence of FMRF-IR fibers in the outer plexiform layer of the retina is a new finding for any species; (iii) the occurrence of immunopositive cells in the mesencephalic laminar nucleus has to our knowledge not been demonstrated previously.

The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover

PubMed Central

Bay-Jensen, Anne-Christine; Tabassi, Nadine CB; Sondergaard, Lene V; Andersen, Thomas L; Dagnaes-Hansen, Frederik; Garnero, Patrick; Kassem, Moustapha; Delaissé, Jean-Marie

2009-01-01

Introduction The urinary level of the type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomised rats, suggesting that oestrogen deprivation induces cartilage breakdown. Here we investigate whether this response to oestrogen is also true for other type II collagen turnover markers known to be affected in osteoarthritis, and whether it relates to its presence in specific areas of cartilage tissue. Methods The type II collagen degradation markers CTX-II and Helix-II were measured in the body fluids of premenopausal and postmenopausal women and in those of ovariectomised rats receiving oestrogen or not. Levels of PIIANP, a marker of type II collagen synthesis, were also measured in rats. Rat knee cartilage was analysed for immunoreactivity of CTX-II and PIIANP and for type II collagen expression. Results As expected, urinary levels of CTX-II are significantly increased in postmenopausal women and also in oestrogen-deprived rats, although only transiently. However, in neither case were these elevations paralleled by a significant increase of Helix-II levels and PIIANP levels did not change at any time. CTX-II immunoreactivity and collagen expression were detected in different cartilage areas. The upper zone is the area where CTX-II immunoreactivity and collagen expression best reflected the differences in urinary levels of CTX-II measured in response to oestrogen. However, correlations between urinary levels of CTX-II and tissue immunostainings in individual rats were not statistically significant. Conclusions We found only a small effect of oestrogen deprivation on cartilage. It was detected by CTX-II, but not by other type II collagen turnover markers typically affected in osteoarthritis. PMID:20527083

Immunoreactive serum opsonic alpha 2 sb glycoprotein as a noninvasive index of RES systemic defense after trauma.

PubMed

Kaplan, J E; Saba, T M

1979-01-01

Reticuloendothelial system (RES) depression has been correlated with diminished resistance to trauma, shock, and sepsis in man and animals. Previous studies have related the depression of RES hepatic Kupffer cell phagocytic function after trauma to diminished bioassayable opsonic activity. The present study determined if the loss of biological activity and RES alteration correlated with immunoreactive serum opsonic alpha 2 SB glycoprotein levels after trauma. Serum opsonic activity was measured by liver slice bioassay, and immunoreactive opsonic protein was measured by rocket electroimmunoassay. RE function was determined by colloid clearance over a 24-hour post-trauma period. Anesthetized rats (250-300 gm) subjected to sublethal or severe (greater than LD50) whole-body NCD trauma were the shock models investigated. Immunoreactive levels in 63 rats prior to injury were 518 +/- 24 microgram/ml. Neither biological nor immunoreactive levels were altered over 24 hours in anesthetized sham-traumatized controls. Temporal alteration in the initial decrease and recovery pattern of biologically active and immunoreactive opsonic protein levels significantly correlated following both sublethal and severe injury. Moreover, the patterns of immunoreactive levels of the opsonic protein correlated with the functional phagocytic activity of the RES as determined by vascular clearance of a test dose of blood-borne radiolabeled particulates. This glycoprotein falls after trauma, and the magnitude and duration of the decline increases with severity of injury. Immunoreactive opsonic alpha 2 SB glycoprotein appears to be an accurate measurement of circulating opsonic activity and RE Kupffer cell function after trauma, especially with respect to clearance. Thus, immunoreactive opsonic protein warrants clinical consideration as a noninvasive measure of reticuloendothelial systemic defense in patients after trauma and burn.

Quantitative distribution of GABA-immunoreactive neurons in cetacean visual cortex is similar to that in land mammals.

PubMed

Garey, L J; Takács, J; Revishchin, A V; Hámori, J

1989-04-24

Sections of the anterior portion of the visual cortex in the lateral gyrus of the Black Sea porpoise were studied to determine the neuronal architecture and numerical density, and the distribution of neurons immunoreactive to gamma-aminobutyric acid (GABA). Cytoarchitecture and neuronal density are similar to those described in another cetacean, the bottlenose dolphin. GABA-positive neurons are distributed through all layers of the visual cortex but are especially dense in layers II and III, and comprise some 20% of the total neuronal population in this part of the cortex. The distribution of GABA-positive neurons is similar to that found in land mammals.

21 CFR 862.1405 - Immunoreactive insulin test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunoreactive insulin test system. 862.1405 Section 862.1405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test...

Immunoreactivity reduction of soybean meal by fermentation, effect on amino acid composition and antigenicity of commercial soy products.

PubMed

Song, Y-S; Frias, J; Martinez-Villaluenga, C; Vidal-Valdeverde, C; de Mejia, E Gonzalez

2008-05-15

Food allergy has become a public health problem that continues to challenge both the consumer and the food industry. The objectives of this study were to evaluate the reduction of immunoreactivity by natural and induced fermentation of soybean meal (SBM) with Lactobacillus plantarum, Bifidobacterium lactis, Saccharomyces cereviseae, and to assess the effect on amino acid concentration. Immunoreactivity of commercially available fermented soybean products and ingredients was also evaluated. ELISA and western blot were used to measure IgE immunoreactivity using plasma from soy sensitive individuals. Commercial soy products included tempeh, miso and yogurt. Fermented SBM showed reduced immunoreactivity to human plasma, particularly if proteins were <20kDa. S. cereviseae and naturally fermented SBM showed the highest reduction in IgE immunoreactivity, up to 89% and 88%, respectively, against human pooled plasma. When SBM was subjected to fermentation with different microorganisms, most of the total amino acids increased significantly (p<0.05) and only few of them suffered a decrease depending on the type of fermentation. All commercial soy containing products tested showed very low immunoreactivity. Thus, fermentation can decrease soy immunoreactivity and can be optimized to develop nutritious hypoallergenic soy products. However, the clinical relevance of these findings needs to be determined by human challenge studies. Copyright © 2007 Elsevier Ltd. All rights reserved.

Antibody-dendrimer conjugates: the number, not the size of the dendrimers, determines the immunoreactivity.

PubMed

Wängler, C; Moldenhauer, G; Eisenhut, M; Haberkorn, U; Mier, W

2008-04-01

Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect.

Effect of bombesin on serum immunoreactive trypsin in healthy subjects and in patients with chronic pancreatitis.

PubMed

Labò, G; Vezzadini, P; Gullo, L; Sternini, C; Bonora, G

1983-08-01

We studied the effect of bombesin (9 ng/kg X min for 30 min by intravenous infusion) on serum immunoreactive trypsin in healthy subjects and in chronic pancreatitis patients. Bombesin administration caused a marked and significant increase of serum immunoreactive trypsin concentration in healthy subjects. The increase occurred in the first 15 min after the beginning of bombesin infusion and persisted for the duration of the study (2 h). In patients with chronic pancreatitis, the increase was much less pronounced. In these patients, the integrated immunoreactive trypsin response to bombesin was significantly correlated with bicarbonate, lipase, and chymotrypsin outputs into the duodenum. The response of serum immunoreactive trypsin to bombesin stimulation seems to vary according to the degree of pancreatic exocrine dysfunction and to reflect the functional capacity of acinar cell mass.

Calcium, Synaptic Plasticity and Intrinsic Homeostasis in Purkinje Neuron Models

PubMed Central

Achard, Pablo; De Schutter, Erik

2008-01-01

We recently reproduced the complex electrical activity of a Purkinje cell (PC) with very different combinations of ionic channel maximum conductances, suggesting that a large parameter space is available to homeostatic mechanisms. It has been hypothesized that cytoplasmic calcium concentrations control the homeostatic activity sensors. This raises many questions for PCs since in these neurons calcium plays an important role in the induction of synaptic plasticity. To address this question, we generated 148 new PC models. In these models the somatic membrane voltages are stable, but the somatic calcium dynamics are very variable, in agreement with experimental results. Conversely, the calcium signal in spiny dendrites shows only small variability. We demonstrate that this localized control of calcium conductances preserves the induction of long-term depression for all models. We conclude that calcium is unlikely to be the sole activity-sensor in this cell but that there is a strong relationship between activity homeostasis and synaptic plasticity. PMID:19129937

Plasma marinobufagenin-like and ouabain-like immunoreactivity during saline volume expansion in anesthetized dogs.

PubMed

Bagrov, A Y; Fedorova, O V; Dmitrieva, R I; French, A W; Anderson, D E

1996-02-01

This study investigated effects of acute plasma volume expansion on plasma levels and urinary output of two endogenous Na,K-ATPase inhibitors, marinobufagenin-like and ouabain-like immunoreactive substances. Plasma volume was expanded for 3 h via intravenous saline infusion in three groups of anesthetized dogs--nontreated (n = 5); pretreated with rabbit antidigoxin (n = 5); and pretreated with rabbit antimouse (control) antibody (n = 4). Plasma marinobufagenin-like immunoreactivity increased to 11.87 +/- 3.16 nmol.l-1 (vs. 0.30 +/- 0.16 nmol.l-1) within 10 min of volume expansion, in parallel with a 15% increase in LVdP/dt, then decreased to 2.21 +/- 0.59 nmol.l-1, and in 90 min increased to 11.8 +/- 2.8 nmol.l-1, in parallel with the maximal natriuretic response. Plasma concentrations of ouabain-like immunoreactive material were increased after 90 min of saline infusion (0.019 +/- 0.004 nmol.l-1 vs. 0.139 +/- 0.056 nmol.l-1). Pretreatment of the animals with antidigoxin antibody blocked the positive inotropic and reduced natriuretic response to volume expansion, and decreased the urinary release of marinobufagenin-like, but not ouabain-like, material. These results show the presence of marinobufagenin-like immunoreactive substance in dog plasma and suggest that mammalian EDLF may have a bufodienolide nature. Endogenous marinobufagenin-like immunoreactive substance, which is likely to cross-react with antidigoxin antibody, is involved in the natriuretic and positive inotropic responses to plasma volume expansion.

Synaptology of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cells in the nervus terminalis of the gray short-tailed opossum (Monodelphis domestica).

PubMed

Zheng, L M; Pfaff, D W; Schwanzel-Fukuda, M

1990-05-08

Light and electron microscopic immunocytochemistry were used to examine the structure of LHRH neurons and fibers in the nervus terminalis of the gray short-tailed opossum (Monodelphis domestica). LHRH-immunoreactive neurons and fibers form a loose plexus within the fascicular network of the ganglion terminale on the median surface of the olfactory bulb. There are at least two populations of LHRH-immunoreactive neurons within the network of the ganglion terminale: fusiform and round neurons similar to those described in the forebrain. At the ultrastructural level, axosomatic and axodendritic contacts were seen between LHRH-immunoreactive and nonimmunoreactive elements in the ganglion terminale. These contacts were classified as 1) synaptic input, with asymmetric synapses seen between a nonimmunoreactive axon terminal and a LHRH-immunoreactive cell body or a nonimmunoreactive axon terminal and a LHRH-immunoreactive dendritic process. 2) synaptic output, with symmetric synapses seen between LHRH-immunoreactive and nonimmunoreactive processes. This study is the first systematic examination of the ultrastructure of the LHRH-immunoreactive neurons and their synaptic contacts in the nervus terminalis. The possible integrative roles for this LHRH-immunoreactive system are discussed.

Purkinje cells signal hand shape and grasp force during reach-to-grasp in the monkey.

PubMed

Mason, Carolyn R; Hendrix, Claudia M; Ebner, Timothy J

2006-01-01

The cerebellar cortex and nuclei play important roles in the learning, planning, and execution of reach-to-grasp and prehensile movements. However, few studies have investigated the signals carried by cerebellar neurons during reach-to-grasp, particularly signals relating to target object properties, hand shape, and grasp force. In this study, the simple spike discharge of 77 Purkinje cells was recorded as two rhesus monkeys reached and grasped 16 objects. The objects varied systematically in volume, shape, and orientation and each was grasped at five different force levels. Linear multiple regression analyses showed the simple spike discharge was significantly modulated in relation to objects and force levels. Object related modulation occurred preferentially during reach or early in the grasp and was linearly related to grasp aperture. The simple spike discharge was positively correlated with grasp force during both the reach and the grasp. There was no significant interaction between object and grasp force modulation, supporting previous kinematic findings that grasp kinematics and force are signaled independently. Singular value decomposition (SVD) was used to quantify the temporal patterns in the simple spike discharge. Most cells had a predominant discharge pattern that remained relatively constant across object grasp dimensions and force levels. A single predominant simple spike discharge pattern that spans reach and grasp and accounts for most of the variation (>60%) is consistent with the concept that the cerebellum is involved with synergies underlying prehension. Therefore Purkinje cells are involved with the signaling of prehension, providing independent signals for hand shaping and grasp force.

Optimization of cerebellar purkinje neuron cultures and development of a plasmid-based method for purkinje neuron-specific, miRNA-mediated protein knockdown.

PubMed

Alexander, C J; Hammer, J A

2016-01-01

We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture. Copyright © 2016 Elsevier Inc. All rights reserved.

Glial S100B Positive Vacuoles In Purkinje Cells: Earliest Morphological Abnormality In SCA1 Transgenic Mice

PubMed Central

VIG, Parminder J.S.; LOPEZ, Maripar E.; WEI, Jinrong; D’SOUZA, David R.; SUBRAMONY, SH; HENEGAR, Jeffrey; FRATKIN, Jonathan D.

2007-01-01

Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-1. The overexpression of mutant ataxin-1 in SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in Purkinje neurons (PKN) of the cerebellum. PKN are closely associated with neighboring Bergmann glia. To elucidate the role of Bergmann glia in SCA1 pathogenesis, cerebellar tissue from 7 days to 6 wks old SCA1 transgenic and wildtype mice were used. We observed that Bergmann glial S100B protein is localized to the cytoplasmic vacuoles in SCA1 PKN. These S100B positive cytoplasmic vacuoles began appearing much before the onset of behavioral abnormalities, and were negative for other glial and PKN marker proteins. Electron micrographs revealed that vacuoles have a double membrane. In the vacuoles, S100B colocalized with receptors of advanced glycation end-products (RAGE), and S100B co-immunoprecipated with cerebellar RAGE. In SCA1 PKN cultures, exogenous S100B protein interacted with the PKN membranes and was internalized. These data suggest that glial S100B though extrinsic to PKN is sequestered into cytoplasmic vacuoles in SCA1 mice at early postnatal ages. Further, S100B may be binding to RAGE on Purkinje cell membranes before these membranes are internalized. PMID:18176630

Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates.

PubMed

Mizuno, J; Takeda, N

1988-01-01

1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

ChAT-like immunoreactivity of olivocochlear fibres on rat outer hair cells during the postnatal development.

PubMed

Roth, B; Dannhof, B; Bruns, V

1991-01-01

Several studies present a great deal of information about putative efferent neurotransmitters and their distribution in the adult and developing cochlea. Anatomical mapping of outer hair cell efferent fibres during ontogeny is still not available. Using quantitative electron microscopy in combination with immunocytochemistry, the distribution of ChAT-like immunoreactivity in the developing rat was investigated. Adult-like immunoreactivity in the whole cochlea is first observed in 30-day-old rats. We localized the adult-like immunoreactivity in all efferent fibres and synapses of the outer hair cells along the entire cochlear duct. An adult-like reaction in the whole cochlea could be observed on the 25th day after birth in two out of three cases. On the 20th postnatal day, no adult-like ChAT immunoreactivity was found, with the exception of one case where labelling was seen in the basal region only. The adult-like ChAT immunoreactivity on the 30th day, 2-3 weeks after the onset of hearing, is the latest maturation of all features of the organ of Corti so far investigated. Synaptogenesis of the outer hair cell efferents reaches an adult-like appearance already on the 16th day after birth.

The origin of downbeat nystagmus: an asymmetry in the distribution of on-directions of vertical gaze-velocity Purkinje cells.

PubMed

Marti, Sarah; Straumann, Dominik; Glasauer, Stefan

2005-04-01

Various hypotheses on the origin of cerebellar downbeat nystagmus (DBN) have been presented; the exact pathomechanism, however, is still not known. Based on previous anatomical and electrophysiological studies, we propose that an asymmetry in the distribution of on-directions of vertical gaze-velocity Purkinje cells leads to spontaneous upward ocular drift in cerebellar disease, and therefore, to DBN. Our hypothesis is supported by a computational model for vertical eye movements.

Fifteen novel immunoreactive proteins of Chinese virulent Haemophilus parasuis serotype 5 verified by an immunoproteomic assay.

PubMed

Yu, Yanfei; Wu, Guangyan; Zhai, Zhipeng; Yao, Huochun; Lu, Chengping; Zhang, Wei

2015-01-01

Haemophilus parasuis (H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum.

Gonadectomy reduces the density of androgen receptor-immunoreactive neurons in male rat's hippocampus: testosterone replacement compensates it.

PubMed

Moghadami, Sajjad; Jahanshahi, Mehrdad; Sepehri, Hamid; Amini, Hossein

2016-01-28

In the present study, the role of gonadectomy on memory impairment and the density of androgen receptor-immunoreactive neurons in rats' hippocampus as well as the ability of testosterone to compensate of memory and the density of androgen receptors in the hippocampus was evaluated. Adult male rats (except intact-no testosterone group) were bilaterally castrated, and behavioral tests performed 2 weeks later. Animals bilaterally cannulated into lateral ventricles and then received testosterone (10, 40 and 120 µg/0.5 µl DMSO) or vehicle (DMSO; 0.5 µl) for gonadectomized-vehicle group, 30 min before training in water maze test. The androgen receptor-immunoreactive neurons were detected by immunohistochemical technique in the hippocampal areas. In the gonadectomized male rats, a memory deficit was found in Morris water maze test on test day (5th day) after DMSO administration. Gonadectomy decreased density of androgen receptor-immunoreactive neurons in the rats' hippocampus. The treatment with testosterone daily for 5 days attenuated memory deficits induced by gonadectomy. Testosterone also significantly increased the density of androgen receptor-immunoreactive neurons in the hippocampal areas. The intermediate dose of this hormone (40 µg) appeared to have a significant effect on spatial memory and the density of androgen receptor-immunoreactive neurons in gonadectomized rats' hippocampus. The present study suggests that testosterone can compensate memory failure in gonadectomized rats. Also testosterone replacement can compensate the reduction of androgen receptor-immunoreactive neurons density in the rats' hippocampus after gonadectomy.

Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

PubMed

Weber, Thomas; Namikawa, Kazuhiko; Winter, Barbara; Müller-Brown, Karina; Kühn, Ralf; Wurst, Wolfgang; Köster, Reinhard W

2016-11-15

The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTAC TM ) further expands the repertoire of genetic tools for conditional interrogation of cellular functions. © 2016. Published by The Company of Biologists Ltd.

Density of GFAP-immunoreactive astrocytes is decreased in left hippocampi in major depressive disorder.

PubMed

Cobb, J A; O'Neill, K; Milner, J; Mahajan, G J; Lawrence, T J; May, W L; Miguel-Hidalgo, J; Rajkowska, G; Stockmeier, C A

2016-03-01

Neuroimaging and postmortem studies of subjects with major depressive disorder (MDD) reveal smaller hippocampal volume with lengthening duration of illness. Pathology in astrocytes may contribute significantly to this reduced volume and to the involvement of the hippocampus in MDD. Postmortem hippocampal tissues were collected from 17 subjects with MDD and 17 psychiatrically-normal control subjects. Sections from the body of the hippocampus were immunostained for glial fibrillary acidic protein (GFAP), a marker of intermediate filament protein expressed in astrocytes. The density of GFAP-immunoreactive astrocytes was measured in the hippocampus using 3-dimensional cell counting. Hippocampal subfields were also assessed for GFAP-immunoreactive area fraction. In CA1, there was a significant positive correlation between age and either density or area fraction in MDD. The density of astrocytes in the hilus, but not CA1 or CA2/3, was significantly decreased only in depressed subjects not taking an antidepressant drug, but not for depressed subjects taking an antidepressant drug. The area fraction of GFAP-immunoreactivity was significantly decreased in the dentate gyrus in women but not men with depression. In CA2/3, the area fraction of GFAP-immunoreactivity was inversely correlated with the duration of depression in suicide victims. Astrocyte contributions to neuronal function in the hilus may be compromised in depressed subjects not taking antidepressant medication. Due to the cross-sectional nature of the present study of postmortem brain tissue, it remains to be determined whether antidepressant drug treatment prevented a decrease in GFAP-immunoreactive astrocyte density or restored cell density to normal levels. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Cannabinoid CB1 receptor immunoreactivity in the prefrontal cortex: Comparison of schizophrenia and major depressive disorder.

PubMed

Eggan, Stephen M; Stoyak, Samuel R; Verrico, Christopher D; Lewis, David A

2010-09-01

We recently showed that measures of cannabinoid 1 receptor (CB1R) mRNA and protein were significantly reduced in dorsolateral prefrontal cortex (DLPFC) area 9 in schizophrenia subjects relative to matched normal comparison subjects. However, other studies have reported unaltered or higher measures of CB1R levels in schizophrenia. To determine whether these discrepancies reflect differences across brain regions or across subject groups (eg, presence of depression, cannabis exposure, etc), we used immunocytochemical techniques to determine whether lower levels of CB1R immunoreactivity are (1) present in another DLPFC region, area 46, in the same subjects with schizophrenia, (2) present in area 46 in a new cohort of schizophrenia subjects, (3) present in major depressive disorder (MDD) subjects, or (4) attributable to factors other than a diagnosis of schizophrenia, including prior cannabis use. CB1R immunoreactivity levels in area 46 were significantly 19% lower in schizophrenia subjects relative to matched normal comparison subjects, a deficit similar to that observed in area 9 in the same subjects. In a new cohort of subjects, CB1R immunoreactivity levels were significantly 20 and 23% lower in schizophrenia subjects relative to matched comparison and MDD subjects, respectively. The lower levels of CB1R immunoreactivity in schizophrenia subjects were not explained by other factors such as cannabis use, suicide, or pharmacological treatment. In addition, CB1R immunoreactivity levels were not altered in monkeys chronically exposed to haloperidol. Thus, the lower levels of CB1R immunoreactivity may be common in schizophrenia, conserved across DLPFC regions, not present in MDD, and not attributable to other factors, and thus a reflection of the underlying disease process.

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

PubMed

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth Manduca sexta.

PubMed

Lohr, Christian; Bergstein, Sandra; Hirnet, Daniela

2007-01-01

The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6-10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.

Effects of processing and storage on almond (Prunus dulcis L.) amandin immunoreactivity.

PubMed

Su, Mengna; Liu, Changqi; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

2017-10-01

A murine monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) was used to assess amandin immunoreactivity in processed and long-term stored almonds. The results demonstrated that amandin immunoreactivity is stable in variously processed almond seeds. Using the ELISA, amandin immunoreactivity could be detected in commercial whole raw and processed (blanched, sliced, dry roasted, and indicated combinations thereof) almond seeds stored for eleven years and eight months, defatted almond seed flours from several almond varieties/hybrids and their borate saline buffer-solubilized protein extracts stored for ten years and seven months, and several almond varieties grown in different California counties (full fat flours and their defatted flour counterparts). Roasting Nonpareil whole full fat almond seeds, full fat flour, and defatted flour at 170°C for 20min each with 2, 5, 10, and 20% w/w corn syrup or sucrose did not prevent amandin detection by ELISA. Similarly, amandin detection in select food matrices spiked with Nonpareil almond protein extract was not inhibited. In conclusion, amandin is a stable target protein for almond detection under the tested processing and storage conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glutamine synthetase immunoreactivity is present in oligodendroglia of various regions of the central nervous system

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Eng, L. F.; Gibbs, M. A.

1990-01-01

Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

Impact of food processing and simulated gastrointestinal digestion on gliadin immunoreactivity in rolls.

PubMed

Brzozowski, Bartosz

2018-07-01

The enzymatic modification of wheat proteins during dough fermentation and its digestion as supported by peptidases of microbiological origin can result in the degradation of important peptides in the pathogenesis of coeliac disease. However, baking bread and the high temperature associated with this could change the physicochemical and immunological properties of proteins. Thermal changes in the spatial structure of proteins and their hydrolysis can lead to a masking or degrading of immunoreactive peptides. The addition of prolyl endopeptidase (PEP), comprising peptidases isolated from Lactobacillus acidophilus 5e2 (LA) or transglutaminase (TG) in the course of fermentation, decreases its immunoreactivity by 83.9%, 51.9% and 18.5%, respectively. An analysis of the fractional composition of gliadins revealed that γ- and ω-gliadins are the proteins most susceptible to enzymatic modification. Hydrolysis of wheat storage proteins with PEP and LA reduces the content of αβ-, γ- and ω-gliadins by 13.7%, 60.2% and 41.9% for PEP and by 22.1%, 43.5% and 36.9% for LA, respectively. Cross-linking of proteins with TG or their hydrolysis by PEP and LA peptidases during the process of forming wheat dough, followed by digesting bread samples with PEP and LA peptidases, decreases the immunoreactivity of bread hydrolysates from 2.4% to 0.02%. The content of peptide detected in polypeptide sequences is 263.4 ± 3.3, 30.9 ± 1.5 and 7.9 ± 0.4 mg kg -1 in samples of hydrolysates of bread digested with PEP, as produced from dough modified by TG, PEP and LA, respectively. Enzymatic pre-modification of proteins during the process of dough fermentation decreases their immunoreactive potential, such that fewer peptides recognised by R5 antibodies are released during the digestion process from the bread matrix. Immunoreactive peptides are degraded more effectively when digestive enzymes are supported by the addition of PEP. © 2017 Society of Chemical Industry. © 2017

Direct reticular projections of trigeminal sensory fibers immunoreactive to CGRP: potential monosynaptic somatoautonomic projections

PubMed Central

Panneton, W. Michael; Gan, Qi

2014-01-01

Few trigeminal sensory fibers project centrally beyond the trigeminal sensory complex, with only projections of fibers carried in its sensory anterior ethmoidal (AEN) and intraoral nerves described. Fibers of the AEN project into the brainstem reticular formation where immunoreactivity against substance P and CGRP are found. We investigated whether the source of these peptides could be from trigeminal ganglion neurons by performing unilateral rhizotomies of the trigeminal root and looking for absence of label. After an 8–14 days survival, substance P immunoreactivity in the trigeminal sensory complex was diminished, but we could not conclude that the sole source of this peptide in the lateral parabrachial area and lateral reticular formation arises from primary afferent fibers. Immunoreactivity to CGRP after rhizotomy however was greatly diminished in the trigeminal sensory complex, confirming the observations of others. Moreover, CGRP immunoreactivity was nearly eliminated in fibers in the lateral parabrachial area, the caudal ventrolateral medulla, both the peri-ambiguus and ventral parts of the rostral ventrolateral medulla, in the external formation of the nucleus ambiguus, and diminished in the caudal pressor area. The nearly complete elimination of CGRP in the lateral reticular formation after rhizotomy suggests this peptide is carried in primary afferent fibers. Moreover, the arborization of CGRP immunoreactive fibers in these areas mimics that of direct projections from the AEN. Since electrical stimulation of the AEN induces cardiorespiratory adjustments including an apnea, peripheral vasoconstriction, and bradycardia similar to those seen in the mammalian diving response, we suggest these perturbations of autonomic behavior are enhanced by direct somatic primary afferent projections to these reticular neurons. We believe this to be first description of potential direct somatoautonomic projections to brainstem neurons regulating autonomic activity. PMID

Direct reticular projections of trigeminal sensory fibers immunoreactive to CGRP: potential monosynaptic somatoautonomic projections.

PubMed

Panneton, W Michael; Gan, Qi

2014-01-01

Few trigeminal sensory fibers project centrally beyond the trigeminal sensory complex, with only projections of fibers carried in its sensory anterior ethmoidal (AEN) and intraoral nerves described. Fibers of the AEN project into the brainstem reticular formation where immunoreactivity against substance P and CGRP are found. We investigated whether the source of these peptides could be from trigeminal ganglion neurons by performing unilateral rhizotomies of the trigeminal root and looking for absence of label. After an 8-14 days survival, substance P immunoreactivity in the trigeminal sensory complex was diminished, but we could not conclude that the sole source of this peptide in the lateral parabrachial area and lateral reticular formation arises from primary afferent fibers. Immunoreactivity to CGRP after rhizotomy however was greatly diminished in the trigeminal sensory complex, confirming the observations of others. Moreover, CGRP immunoreactivity was nearly eliminated in fibers in the lateral parabrachial area, the caudal ventrolateral medulla, both the peri-ambiguus and ventral parts of the rostral ventrolateral medulla, in the external formation of the nucleus ambiguus, and diminished in the caudal pressor area. The nearly complete elimination of CGRP in the lateral reticular formation after rhizotomy suggests this peptide is carried in primary afferent fibers. Moreover, the arborization of CGRP immunoreactive fibers in these areas mimics that of direct projections from the AEN. Since electrical stimulation of the AEN induces cardiorespiratory adjustments including an apnea, peripheral vasoconstriction, and bradycardia similar to those seen in the mammalian diving response, we suggest these perturbations of autonomic behavior are enhanced by direct somatic primary afferent projections to these reticular neurons. We believe this to be first description of potential direct somatoautonomic projections to brainstem neurons regulating autonomic activity.

Development of neuropeptide Y (NPY) immunoreactive neurons in the rat occipital cortex: A combined immunohistochemical-autoradiographic study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cavanagh, M.E.; Parnavelas, J.G.

1990-07-22

The postnatal development of neuropeptide Y (NPY)-immunoreactive neurons, previously labeled with (3H)thymidine on embryonic days E14-E21, has been studied in the rat occipital cortex. Immunohistochemistry combined with autoradiography showed evidence of a modified inside-out pattern of maturation. NPY-neurons are generated between E14 and E20 and are found in layers II-VI of the cortex and the subcortical white matter. NPY neurons from all these birthdates are overproduced at first, although cells generated at E16 produce the greatest excess, followed by E15 and E17. Some of these transient neurons are found in the wrong layer for their birthdates, and their elimination producesmore » a more correct alignment at maturity. However, most of the NPY neurons that survive are generated at E17, and these cells are found throughout layers II-VI with a preponderance in layer VI. This evidence is strongly suggestive of cell death rather than merely cessation of production of NPY.« less

Calbindin-D28k immunoreactivity in the mice thoracic spinal cord after space flight

NASA Astrophysics Data System (ADS)

Porseva, Valentina V.; Shilkin, Valentin V.; Krasnov, Igor B.; Masliukov, Petr M.

2015-10-01

The aim of the work was to analyse changes in the location and morphological characteristics of calbindin (CB)-immunoreactive (IR) neurons of the thoracic spinal cord of C57BL/6N male mice after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). Space flight induced multidirectional changes of the number and morphological parameters of CB-positive neurons. The number of IR neurons increased in laminae I (from 10 to 17 neurons per section), II (from 42 to 67 cells per section) and IX (from two neurons per segment to two neurons per section), but CB disappeared in neurons of lamina VIII. Weightlessness did not affect the number of CB-IR neurons in laminae III-V and VII, including preganglionic sympathetic neurons. The cross-sectional area of CB-IR neurons decreased in lamina II and VII (group of partition cells) and increased in laminae III-V and IX. After a space flight, few very large neurons with long dendrites appeared in lamina IV. The results obtained give evidence about substantial changes in the calcium buffer system and imbalance of different groups of CB-IR neurons due to reduction of afferent information under microgravity.

GABA-like immunoreactivity in Biomphalaria: Colocalization with tyrosine hydroxylase-like immunoreactivity in the feeding motor systems of panpulmonate snails.

PubMed

Vaasjo, Lee O; Quintana, Alexandra M; Habib, Mohamed R; Mendez de Jesus, Paola A; Croll, Roger P; Miller, Mark W

2018-08-01

The simpler nervous systems of certain invertebrates provide opportunities to examine colocalized classical neurotransmitters in the context of identified neurons and well defined neural circuits. This study examined the distribution of γ-aminobutyric acid-like immunoreactivity (GABAli) in the nervous system of the panpulmonates Biomphalaria glabrata and Biomphalaria alexandrina, major intermediate hosts for intestinal schistosomiasis. GABAli neurons were localized in the cerebral, pedal, and buccal ganglia of each species. With the exception of a projection to the base of the tentacle, GABAli fibers were confined to the CNS. As GABAli was previously reported to be colocalized with markers for dopamine (DA) in five neurons in the feeding network of the euopisthobranch gastropod Aplysia californica (Díaz-Ríos, Oyola, & Miller, 2002), double-labeling protocols were used to compare the distribution of GABAli with tyrosine hydroxylase immunoreactivity (THli). As in Aplysia, GABAli-THli colocalization was limited to five neurons, all of which were located in the buccal ganglion. Five GABAli-THli cells were also observed in the buccal ganglia of two other intensively studied panpulmonate species, Lymnaea stagnalis and Helisoma trivolvis. These findings indicate that colocalization of the classical neurotransmitters GABA and DA in feeding central pattern generator (CPG) interneurons preceded the divergence of euopisthobranch and panpulmonate taxa. These observations also support the hypothesis that heterogastropod feeding CPG networks exhibit a common universal design. © 2018 Wiley Periodicals, Inc.

[Met]- and [Leu]enkephalin-like immunoreactive cell bodies and nerve fibres in the coeliac ganglion of the cat.

PubMed

Julé, Y; Clerc, N; Niel, J P; Condamin, M

1986-06-01

The occurrence and distribution of methionine- and leucine-enkephalin-like immunoreactivity were investigated in the cat coeliac ganglion using either the indirect immunoperoxidase method or the peroxidase-antiperoxidase technique. Several antisera raised to methionine- and leucine-enkephalin were used. Their specificity was assessed by incubating sections of the coeliac ganglion with increasing dilutions of antisera and with antisera saturated with their respective antigen. The present study was performed both in untreated and in colchicine-treated cats. Immunoreactive methionine- and leucine-enkephalin-like cell bodies were only visualized in colchicine-treated cats. Two types of labeled cells were observed. The first type had a size similar to that of unlabeled principal ganglion cells. These labeled cells were numerous and scattered throughout the ganglion; they probably represented enkephalin-containing ganglion cells. The second type of immunoreactive cells were of a much smaller size. They were always gathered in small clusters of about 5-15 cells and were not numerous; they presumably represented enkephalin-containing small intensely fluorescent cells. Immunoreactive nerve fibres were mainly observed in untreated cats and accessorily in colchicine-treated cats. In untreated animals dense networks of methionine- and leucine-enkephalin-like immunoreactive fibres were found in the coeliac ganglion. These fibres had numerous varicosities which often closely surrounded unlabeled principal ganglion cells. In colchicine-treated cats some immunoreactive fibres surrounded labeled principal ganglion cell bodies. The present results establish for the first time the presence of enkephalin-like immunoreactive principal ganglion cells in a mammalian sympathetic prevertebral ganglion. The presence of enkephalin-containing principal ganglion cells, small intensely fluorescent cells and nerve terminals, supports an important role of enkephalins in the integrative synaptic

Striatal infarction in the rat causes a transient reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra.

PubMed

Soriano, M A; Justicia, C; Ferrer, I; Rodríguez-Farré, E; Planas, A M

1997-01-01

Dopaminergic neurons of the substantia nigra pars compacta were examined in the rat brain following striatal infarction subsequent to transient focal cerebral ischemia. Rats had the middle cerebral artery occluded for 2 h or were sham-operated, and tyrosine hydroxylase immunoreactivity was evaluated by Western blot and immunohistochemistry at different times ranging from 1 to 60 days after ischemia. The number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta was counted under the light microscope and compared to that in the contralateral side and controls. No changes of tyrosine hydroxylase immunoreactivity were detected in the ipsilateral versus the contralateral substantia nigra of sham-operated rats or 1 day after ischemia. However, a statistically significant reduction of tyrosine hydroxylase-immunoreactive cells became apparent in the ipsilateral compared with the contralateral substantia nigra at 7 and 14 days after ischemia. This reduction showed a clear recovery at 30 days after ischemia, and no signs of difference between the ipsilateral and the contralateral side were apparent by 60 days. Therefore, the reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra was only transiently seen from 1 to 2 weeks following ischemia. The observed loss of tyrosine hydroxylase was not accompanied by signs of cell death or gliosis in the ipsilateral pars compacta. The present results show a transitory reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra pars compacta after focal ischemia and suggest that striatal infarction causes a transient deficit of dopaminergic function.

Increased neuronal beta-amyloid precursor protein expression in human temporal lobe epilepsy: association with interleukin-1 alpha immunoreactivity.

PubMed

Sheng, J G; Boop, F A; Mrak, R E; Griffin, W S

1994-11-01

Levels of immunoreactive beta-amyloid precursor protein and interleukin-1 alpha were found to be elevated in surgically resected human temporal lobe tissue from patients with intractable epilepsy compared with postmortem tissue from neurologically unaffected patients (controls). In tissue from epileptics, the levels of the 135-kDa beta-amyloid precursor protein isoform were elevated to fourfold (p < 0.05) those of controls and those of the 130-kDa isoform to threefold (p < 0.05), whereas those of the 120-kDa isoform (p > 0.05) were not different from control values. beta-Amyloid precursor protein-immunoreactive neurons were 16 times more numerous, and their cytoplasm and proximal processes were more intensely immunoreactive in tissue sections from epileptics than controls (133 +/- 12 vs. 8 +/- 3/mm2; p < 0.001). However, neither beta-amyloid precursor protein-immunoreactive dystrophic neurites nor beta-amyloid deposits were found in this tissue. Interleukin-1 alpha-immunoreactive cells (microglia) were three times more numerous in epileptics than in controls (80 +/- 8 vs. 25 +/- 5/mm2; p < 0.001), and these cells were often found adjacent to beta-amyloid precursor protein-immunoreactive neuronal cell bodies. Our findings, together with functions established in vitro for interleukin-1, suggest that increased expression of this protein contributes to the increased levels of beta-amyloid precursor protein in epileptics, thus indicating a potential role for both of these proteins in the neuronal dysfunctions, e.g., hyperexcitability, characteristic of epilepsy.

Effects of halothane and methoxyflurane on regional brain and spinal cord substance P-like and beta-endorphin-like immunoreactivities in the rat.

PubMed

Karuri, A R; Agarwal, R K; Engelking, L R; Kumar, M S

1998-03-15

Effects of acute exposure (2 hr) to either 1.5% halothane or 0.5% methoxyflurane were investigated in the Sprague Dawley rat. Pituitary (PIT) and central nervous system (CNS) substance P (SP)-like and beta-endorphin (beta-end)-like immunoreactivities were evaluated immediately after anesthetic exposure (2 h), after righting reflex (4 h) or 24 hr postexposure (24 h). Only halothane significantly reduced SP-like immunoreactivity in olfactory bulbs in both the 2-h and 4-h groups. Halothane elevated SP-like immunoreactivity of hippocampus at all three time periods, and in the hypothalamus at 2 h. Both anesthetics significantly depleted thalamic concentrations of SP-like immunoreactivity. Methoxyflurane anesthesia resulted in a drastic decrease in SP-like immunoreactivity in PIT at all three time periods periods, while halothane elevated PIT concentrations of this peptide at 4 h. Both anesthetics significantly decreased beta-end-like immunoreactivity in the olfactory bulbs and thalami at 2, 4, and 24 h. However, halothane alone significantly elevated beta-end-like immunoreactivity in the spinal cord at 24 h. Halothane significantly elevated PIT beta-end-like immunoreactivity at 2 and 24 h, while methoxyflurane significantly lowered it in the 4-h group, but elevated the levels of the same in the 24-h group. Brain stem beta-end immunoreactivity were significantly reduced at 2 h by both anesthetics, and at 4 h by methoxyflurane. Results indicate that halothane and methoxyflurane may differ significantly in their actions on SP and beta-end secreting neurons in the CNS.

Utilization of Exocellular Mannan from Rhodotorula glutinis as an Immunoreactive Antigen in Diagnosis of Leptospirosis

PubMed Central

Matsuo, Kouki; Isogai, Emiko; Araki, Yoshio

2000-01-01

Previously, Rhodotorula glutinis was reported to produce a large amount of exocellular mannan, having a repeating unit of →3)-d-Manp-(1→4)-d-Manp-(1→. Recently, we found that antigenic polysaccharides of Leptospira biflexa serovar patoc strain Patoc I have the same repeating unit and cross-react with antisera raised against extended strains of other leptospires (K. Matsuo, E. Isogai, and Y. Araki, Carbohydr. Res., in press). This structural identity and the difficulty of producing and isolating antigens led us to confirm the usefulness of Rhodotorula mannan as an immunoreactive antigen in a serological diagnosis of leptospirosis. In the present investigation, we confirmed the structural identity of an exocellular mannan isolated from R. glutinis AHU 3479 and tried to use it as an immunoreactive antigen in a serological diagnosis of leptospirosis. From its chemical analysis and 1H- and 13C-labeled nuclear magnetic resonance spectrometry, the Rhodotorula mannan was confirmed to consist of the same disaccharide units. Furthermore, such a preparation was shown to immunoreact to various sera from patients suffering with leptospirosis as well as to most rabbit antiserum preparations obtained from immunization with various strains of pathogenic leptospires. Therefore, the Rhodotorula mannan preparation is useful as an immunoreactive antigen in the serological diagnosis for leptospirosis. PMID:11015396

Role of cytosolic calcium diffusion in cardiac purkinje cells.

PubMed

Limbu, Bijay; Shah, Kushal; Deo, Makarand

2016-08-01

The Cardiac Purkinje cells (PCs) exhibit distinct calcium (Ca2+) homeostasis than that in ventricular myocytes (VMs). Due to lack of t-tubules in PCs, the Ca2+ ions entering the cell have to diffuse through the cytoplasm to reach the sarcoplasmic reticulum (SR) before triggering Ca2+-induced-Ca2+-release (CICR). In recent experimental studies PCs have been shown to be more susceptible to action potential (AP) abnormalities than the VMs, however the exact mechanisms are poorly understood. In this study, we utilize morphologically realistic detailed biophysical mathematical model of a murine PC to systematically examine the role intracellular Ca2+ diffusion in the APs of PCs. A biphasic spatiotemporal Ca2+ diffusion process, as observed experimentally, was implemented in the model which includes radial Ca2+ wavelets and cell wide longitudinal Ca2+ diffusion wave (CWW). The AP morphology, specifically plateau, is affected due to changes in intracellular Ca2+ dynamics. When Ca2+ concentration in sarcolemmal region is elevated, it activated inward sodium Ca2+ exchanger (NCX) current resulting into prolongation of the plateau at faster diffusion rates. Our results demonstrate that the cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights into the increased arrhythmogeneity of PCs.

Behavior related pauses in simple spike activity of mouse Purkinje cells are linked to spike rate modulation

PubMed Central

Cao, Ying; Maran, Selva K.; Dhamala, Mukesh; Jaeger, Dieter; Heck, Detlef H.

2012-01-01

Purkinje cells (PCs) in the mammalian cerebellum express high frequency spontaneous activity with average spike rates between 30 and 200 Hz. Cerebellar nuclear (CN) neurons receive converging input from many PCs resulting in a continuous barrage of inhibitory inputs. It has been hypothesized that pauses in PC activity trigger increases in CN spiking activity. A prediction derived from this hypothesis is that pauses in PC simple spike activity represent relevant behavioral or sensory events. Here we asked whether pauses in the simple spike activity of PCs related to either fluid licking or respiration, play a special role in representing information about behavior. Both behaviors are widely represented in cerebellar PC simple spike activity. We recorded PC activity in the vermis and lobus simplex of head fixed mice while monitoring licking and respiratory behavior. Using cross correlation and Granger causality analysis we examined whether short ISIs had a different temporal relation to behavior than long ISIs or pauses. Behavior related simple spike pauses occurred during low-rate simple spike activity in both licking and breathing related PCs. Granger causality analysis revealed causal relationships between simple spike pauses and behavior. However, the same results were obtained from an analysis of surrogate spike trains with gamma ISI distributions constructed to match rate modulations of behavior related Purkinje cells. Our results therefore suggest that the occurrence of pauses in simple spike activity does not represent additional information about behavioral or sensory events that goes beyond the simple spike rate modulations. PMID:22723707

Ingestion of dried-bonito broth (dashi) facilitates PV-parvalbumin-immunoreactive neurons in the brain, and affects emotional behaviors in mice.

PubMed

Jargalsaikhan, Undarmaa; Nishimaru, Hiroshi; Matsumoto, Jumpei; Takamura, Yusaku; Nakamura, Tomoya; Hori, Etsuro; Kondoh, Takashi; Ono, Taketoshi; Nishijo, Hisao

2017-12-01

Emerging evidence suggests that traditional diets and nutrition have a significant impact on brain development, and could contribute to the promotion of mental health and prevention of psychiatric disorders in children and adolescents. Moreover, deficits in parvalbumin (PV)-immunoreactive and/or GABAergic neurons are closely associated with various psychiatric disorders in children and adolescents. To investigate the possible neural mechanisms of diet involvement in mental health, we analyzed the effects of dried-bonito dashi (Japanese fish broth) (DBD) on PV-immunoreactive neurons and emotional behaviors in young mice. Male mice after weaning were fed DBD for 60 days, and tested with a resident-intruder test for aggressiveness and a forced swimming test for depression-like symptoms. After the behavioral testing, PV-immunoreactive neurons in the brain were immunohistochemically analyzed. The results indicated that DBD intake decreased aggressiveness and depression-like symptoms, and increased the densities of PV-immunoreactive neurons in the medial prefrontal cortex (mPFC), amygdala, hippocampus, and superior colliculus. These behavioral changes were correlated with the densities of PV-immunoreactive neurons in the mPFC, amygdala, and hippocampus. However, subdiaphragmatic vagotomy did not affect the effects of DBD on emotional behaviors, although it nonspecifically decreased the densities of PV-immunoreactive neurons. The results suggest that DBD might modulate emotional behaviors by promoting PV-immunoreactive and/or GABAergic neuronal activity through parallel routes. The present results highlight a new mechanism for diet involvement in brain functions, and suggest that DBD might have therapeutic potential for the promotion of mental health.

Substance P immunoreactivity in the lumbar spinal cord of the turtle Trachemys dorbigni following peripheral nerve injury.

PubMed

Partata, W A; Krepsky, A M R; Xavier, L L; Marques, M; Achaval, M

2003-04-01

Immunoreactive substance P was investigated in turtle lumbar spinal cord after sciatic nerve transection. In control animals immunoreactive fibers were densest in synaptic field Ia, where the longest axons invaded synaptic field III. Positive neuronal bodies were identified in the lateral column of the dorsal horn and substance P immunoreactive varicosities were observed in the ventral horn, in close relationship with presumed motoneurons. Other varicosities appeared in the lateral and anterior funiculi. After axotomy, substance P immunoreactive fibers were reduced slightly on the side of the lesion, which was located in long fibers that invaded synaptic field III and in the varicosities of the lateral and anterior funiculus. The changes were observed at 7 days after axonal injury and persisted at 15, 30, 60 and 90 days after the lesion. These findings show that turtles should be considered as a model to study the role of substance P in peripheral axonal injury, since the distribution and temporal changes of substance P were similar to those found in mammals.

Abdominal surgery activates nesfatin-1 immunoreactive brain nuclei in rats

PubMed Central

Stengel, Andreas; Goebel, Miriam; Wang, Lixin; Taché, Yvette

2011-01-01

Abdominal surgery-induced postoperative gastric ileus is well established to induce Fos expression in specific brain nuclei in rats within 2-h after surgery. However, the phenotype of activated neurons has not been thoroughly characterized. Nesfatin-1 was recently discovered in the rat hypothalamus as a new anorexigenic peptide that also inhibits gastric emptying and is widely distributed in rat brain autonomic nuclei suggesting an involvement in stress responses. Therefore, we investigated whether abdominal surgery activates nesfatin-1-immunoreactive (ir) neurons in the rat brain. Two hours after abdominal surgery with cecal palpation under short isoflurane anesthesia or anesthesia alone, rats were transcardially perfused and brains processed for double immunohistochemical labeling of Fos and nesfatin-1. Abdominal surgery, compared to anesthesia alone, induced Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), Edinger-Westphal nucleus (EW), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM). Double Fos/nesfatin-1 labeling showed that of the activated cells, 99% were nesfatin-1-immunoreactive in the SON, 91% in the LC, 82% in the rRPa, 74% in the EW and VLM, 71% in the anterior parvicellular PVN, 47% in the lateral magnocellular PVN, 41% in the medial magnocellular PVN, 14 % in the NTS and 9% in the medial parvicellular PVN. These data established nesfatin-1 immunoreactive neurons in specific hypothalamic and pontine nuclei as part of the neuronal response to abdominal surgery and suggest a possible implication of nesfatin-1 in the alterations of food intake and gastric transit associated with such a stressor. PMID:19944727

Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

NASA Astrophysics Data System (ADS)

He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

2015-07-01

Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

Muscarinic Acetylcholine Receptors in Macaque V1 Are Most Frequently Expressed by Parvalbumin-Immunoreactive Neurons

PubMed Central

Disney, Anita A.; Aoki, Chiye

2010-01-01

Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit. PMID:18265004

Effects of heat and high-pressure treatments on the solubility and immunoreactivity of almond proteins.

PubMed

Zhang, Yan; Zhang, Jieqiong; Sheng, Wei; Wang, Shuo; Fu, Tong-Jen

2016-05-15

The effects of dry and moist heat, autoclave sterilization and high-pressure treatment on the biochemical characteristics and immunological properties of almond proteins were investigated. Changes in the solubility and immunoreactivity of almond proteins extracted from treated almond flour were evaluated using a total protein assay, indirect competitive inhibition enzyme-linked immunosorbent assay (IC-ELISA), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Almond proteins were stable during dry-heat treatment at temperatures below 250°C. Dry heat at 400°C, boiling, autoclave sterilization and high-pressure treatment in the presence of water at ⩾ 500 MPa greatly reduced the solubility and immunoreactivity of almond proteins. SDS-PAGE revealed that the protein profiles of almond flour samples treated under these conditions also changed significantly. The synergistic effects of heat, pressure and the presence of water contributed to significant changes in solubility and immunoreactivity of almond proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Midluteal immunoreactive alpha-inhibin serum concentrations as markers of luteal phase deficiency.

PubMed

Balasch, J; Creus, M; Fábregues, F; Casamitjana, R; Ordi, J; Vanrell, J A

1996-12-01

The present prospective clinical study was undertaken to determine the usefulness of midluteal phase serum immunoreactive alpha-inhibin concentrations as markers of luteal phase deficiency and whether they are better indicators of biopsy confirmed luteal phase defect than serum progesterone. Consecutive patients (n = 138) with regular menstrual cycles attending our Infertility Clinic (experimental group) and 15 fertile women who were requesting contraception and had regular menstrual patterns (control group) were included. In all women (patients and controls), basal body temperature, midluteal serum concentrations of oestradiol, prolactin, progesterone and immunoreactive alpha-inhibin, and premenstrual endometrial biopsy were used in the same cycle to assess luteal function. Out-of-phase secretory endometria were detected in 15 of the 138 patients. Thus, hormonal concentrations were compared between the following three groups of women: group 1 (n = 15), infertile patients with defective secretory endometria; group 2 (n = 123), infertile patients with normal secretory endometria; and controls (n = 15), fertile women with normal secretory endometria. Midluteal serum concentrations of progesterone, immunoreactive alpha-inhibin, oestradiol, and prolactin of the two groups studied were similar to those of the control group of fertile women. Our results indicate that midluteal serum inhibin determination does not accurately reflect histological maturation of the endometrium and it is not a better indicator of endometrial luteal phase deficiency than midluteal serum progesterone concentration.

Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat.

PubMed

Roda, Elisa; Coccini, Teresa; Acerbi, Davide; Castoldi, Anna; Bernocchi, Graziella; Manzo, Luigi

2008-05-01

The developing central nervous system (CNS) is a target of the environmental toxicant methylmercury (MeHg), and the cerebellum seems the most susceptible tissue in response to this neurotoxicant. The cholinergic system is essential for brain development, acting as a modulator of neuronal proliferation, migration and differentiation processes; its muscarinic receptors (MRs) play pivotal roles in regulating important basic physiologic functions. By immunohistochemistry, we investigated the effects of perinatal (GD7-PD21) MeHg (0.5 mg/kg bw/day in drinking water) administration on cerebellum of mature (PD36) and immature (PD21) rats, evaluating the: (i) M2- and M3-MR expression; (ii) presence of gliosis; (iii) cytoarchitecture alterations. Regarding to M2-MRs, we showed that: at PD21, MeHg-treated animals did not display any differences compared to controls, while, at PD36 there was a significant increase of M2-immunopositive Bergmann cells in the molecular layer (ML), suggesting a MeHg-related cytotoxic effect. Similarly to M2-MRs, at PD21 the M3-MRs were not affected by MeHg, while, at PD36 a lacking immunoreactivity of the granular layer (IGL) was observed after MeHg treatment. In MeHg-treated rats, at both developmental points, we showed reactive gliosis, e.g. a significant increase in Bergmann glia of the ML and astrocytes of the IGL, identified by their expression of glial fibrillar acidic protein. No MeHg-related effects on Purkinje cells were detected neither at weaning nor at puberty. These findings suggest: (i) a delayed MeHg exposure-related effect on M2- and M3-MRs, (ii) an overt MeHg-related cytotoxic effect on cerebellar oligodendroglia, e.g. reactive gliosis, (iii) a selective vulnerability of granule cells and Purkinje neurons to MeHg, with the latter that remain unharmed.

Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes

PubMed Central

HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN HELMUT; VILA, ALEJANDRO; BRECHA, NICHOLAS C.

2009-01-01

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell (RBC) marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP2/RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin (PNA) established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in

Neuroanatomy of pars intercerebralis neurons with special reference to their connections with neurons immunoreactive for pigment-dispersing factor in the blow fly Protophormia terraenovae.

PubMed

Yasuyama, Kouji; Hase, Hiroaki; Shiga, Sakiko

2015-10-01

Input regions of pars intercerebralis (PI) neurons are examined by confocal and electron microscopies with special reference to their connections with neurons immunoreactive for pigment-dispersing factor (PDF) in the blow fly, Protophormia terraenovae. PI neurons are a prerequisite for ovarian development under long-day conditions. Backfills from the cardiac recurrent nerve after severance of the posterior lateral tracts labeled thin fibers derived from the PI neurons in the superior medial protocerebrum. These PI fibers were mainly synapsin-negative and postsynaptic to unknown varicose profiles containing dense-core vesicles. Backfilled fibers in the periesophageal neuropils, derived from the PI neurons or neurons with somata in the subesophageal zone, were varicose and some were synapsin-positive. Electron microscopy revealed the presence of both presynaptic and postsynaptic sites in backfilled fibers in the periesophageal neuropils. Many PDF-immunoreactive varicosities were found in the superior medial and lateral protocerebrum and double-labeling showed that 60-88 % of PDF-immunoreactive varicosities were also synapsin-immunoreactive. Double-labeling with the backfills and PDF immunocytochemistry showed that the PI fibers and PDF-immunoreactive varicosities were located close to each other in the superior medial protocerebrum. Results of triple-labeling of PI neurons, PDF-immunoreactive neurons and synapsin-immunoreactive terminals demonstrated that the synapsin-positive PDF-immunoreactive varicosities contacted the PI fibers. These data suggest that PI neurons receive synaptic contacts from PDF-immunoreactive fibers, which are derived from circadian clock neurons, of small ventral lateral neurons (previously called OL2) or posterior dorsal (PD) neurons with somata in the pars lateralis.

The effects of ethanol on insulin-like growth factor-I immunoreactive neurons in the central nervous system.

PubMed

Dalcik, Cannur; Yildirim, Guler K; Dalcik, Hakki

2009-08-01

To evaluate the effect of chronically ethanol treatment on insulin-like growth factor-I (IGF-I) synthesis in various adult brain regions using immunocytochemistry. We performed this study at the Faculty of Medicine, Kocaeli University, Kocaeli, Turkey from March 2006 to October 2007. The vascular perfusion was utilized to fix the adult rat brains (10 for each group). After applying the routine histological techniques, the tissues were embedded in the paraffin. The immunohistochemical protocol was applied to the 10 um thick sections and the expression of IGF-I positive cells were observed in the neuro-anatomic areas. The distribution of IGF-I immunoreactive cells differed between the layers of the normal cerebral cortex and in the thalamic areas. In the alcoholic brain, the amount of IGF-I immunoreactive cells were decreased compared to the similar neuro-anatomical areas examined in the normal brains. The presence of IGF-I immunoreactivity in the neurons of the various neuro-anatomic areas demonstrates clearly that, these particular neurons are active in IGF-I synthesis. The decrease in the immunoreactivity of IGF-I in the chronically ethanol treated adult rat brain areas, show clearly that, ethanol effects negatively on the IGF-I synthesis.

Changes in enkephalin immunoreactivity of sympathetic ganglia and digestive tract of the cat after splanchnic nerve ligation.

PubMed

Bagnol, D; Herbrecht, F; Julé, Y; Jarry, T; Cupo, A

1993-09-22

The aim of the present study was to analyze changes in the enkephalin immunoreactivity of sympathetic prevertebral ganglia coeliac plexus and inferior mesenteric ganglion) and intestinal tract (myenteric plexus and external muscle layers) in cats 2 days after left thoracic splanchnic nerve ligation, using radioimmunoassay and immunohistochemical techniques. Specific polyclonal antibodies directed against methionine- and leucine-enkephalin were used. The nerve ligation led to a considerable increase in the enkephalin immunoreactivity in the cranial part of the ligated nerves. This finding confirms the presence, in the cat, of an enkephalin output originating from thoracic spinal structures which are probably enkephalin-containing preganglionic neurons. In prevertebral ganglia the nerve ligation induced a marked decrease in the enkephalin immunoreactivity, which was probably due to the interruption of thoracic enkephalin efferents projecting towards both the coeliac plexus and the inferior mesenteric ganglion. In the digestive tract, the nerve ligation depressed the methionine-enkephalin immunoreactivity only in the gastro-duodenal region, and had no effect on the ileo-colonic region. The results of the present study add to the growing evidence that the sympathetic nervous system is involved in regulating the enteric enkephalinergic innervation, which is probably involved in controlling the intestinal motility.

Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

PubMed

Campeau, Jody L; Wu, Gengshu; Bell, John R; Rasmussen, Jay; Sim, Valerie L

2013-01-01

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Effect of Melanotan-II on Brain Fos Immunoreactivity and Oxytocin Neuronal Activity and Secretion in Rats.

PubMed

Paiva, L; Sabatier, N; Leng, G; Ludwig, M

2017-02-01

Melanocortins stimulate the central oxytocin systems that are involved in regulating social behaviours. Alterations in central oxytocin have been linked to neurological disorders such as autism, and melanocortins have been proposed for therapeutic treatment. In the present study, we investigated how systemic administration of melanotan-II (MT-II), a melanocortin agonist, affects oxytocin neuronal activity and secretion in rats. The results obtained show that i.v., but not intranasal, administration of MT-II markedly induced Fos expression in magnocellular neurones of the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and this response was attenuated by prior i.c.v. administration of the melanocortin antagonist, SHU-9119. Electrophysiological recordings from identified magnocellular neurones of the SON showed that i.v. administration of MT-II increased the firing rate in oxytocin neurones but did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis. Our data suggest that, after i.v., but not intranasal, administration of MT-II, the activity of magnocellular neurones of the SON is increased. Because previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT-II are likely to be mediated at least partly indirectly, possibly by activation of inputs from the caudal brainstem, where MT-II also increased Fos expression. © 2016 British Society for Neuroendocrinology.

ENOS, ET-1 and ETB-R immunoreactivities in the porcine mesometrial lymphatics during the estrous cycle.

PubMed

Doboszyńska, Teresa; Andronowska, Aneta

2002-01-01

Abstract: Immunohistochemical localization and distribution of nitric oxide synthase (eNOS), endothelin (ET-1) and endothelin beta receptor (ETB-R) were investigated in precollector and collector lymph vessels in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and ETB-R and monoclonal antibody for eNOS isoform were used to perform observations on the light microscopic level. Immunoreactivities to ET-1, ETB-R and eNOS were observed in the endothelium of precollector and collector lymphangions but not in smooth muscle cells of the lymphatics examined. The staining for eNOS in the endothelial cells of all studied lymphatic vessels was stronger comparing to ET-1 and ETB-R. During the estrous cycle, only eNOS showed the correlation with the particular phases of the estrous cycle. The differences between ET-1 and ETB-R immunoreactivities were very slight and rather independent of the size or type of the lymphatic lymphangions and estrous cycle. The highest immunoreactivity level for eNOS was displayed by collector lymphangions with widened lumen in the follicular phase comparing to the precollector ones. During the luteal phase, a slight decrease in the reaction intensity was observed. The immunoreactivities for ET-1 in the endothelium of the studied vessels was not comparable with the presence or with the reactivity level of ETB-R. Optically stronger immunoreaction for ETB-R was observed in the cytoplasm of collector lymphangions in the follicular phase. eNOS, ET-1 and ETB-R were also present in the cytoplasm of the lymphatic valves. These results suggest that ET-1 and eNOS can play a role in the mechanisms regulating the vascular contractile activity, promoting lymph flow during the estrous cycle in the porcine broad ligament.

Exploration of the immunoreactivity of the Traditional Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide

PubMed Central

Gu, Yu-Chun; Chen, De-Zhen

1997-01-01

AIM: To study the immunoreactivity of the Chinese medicine Shenrouyangzhentang to vasoactive intestinal polypeptide (VIP) and its therapeutic mechanism. METHODS: The immunoreactivity of the Chinese medicine Shenrouyangzhentang to VIP was detected in the plasma of 20 normal people and 20 patients with Piyinxu (Spleen Yin deficiency) using the radioimmunoassay (RIA) method. RESULTS: The maximum binding rate B0/T was 53.29%, the non-specific binding rate N0/T was 1.170%, and the VIP standard curve was Y = 0.81983 + 0.44319X - 0.28927X2, R2 = 0.990. The VIP content in Shenrouyangzhentang was 106.6 ng/L ± 20 ng/L), while it was 90.16 ng/L ± 15 ng/L in normal human plasma and 63.25 ng/L ± 11 ng/L in the plasma of Pixinxu patients. The difference between normal plasma and Pixinxu patient plasma was statistically significant (P < 0.05). CONCLUSION: The Chinese medicine Shenrouyangzhentang demonstrated VIP immunoreactivity similar to that of normal plasma. The (vasoactive intestinal polypeptide) VIP content in Pixinxu patient plasma was lower than that in healthy subjects (P < 0.05). PMID:27041949

Noradrenergic innervation of the hypothalamus of rhesus monkeys: distribution of dopamine-beta-hydroxylase immunoreactive fibers and quantitative analysis of varicosities in the paraventricular nucleus.

PubMed

Ginsberg, S D; Hof, P R; Young, W G; Morrison, J H

1993-01-22

The distribution of noradrenergic processes within the hypothalamus of rhesus monkeys (Macaca mulatta) was examined by immunohistochemistry with an antibody against dopamine-beta-hydroxylase. The results revealed that the pattern of dopamine-beta-hydroxylase immunoreactivity varied systematically throughout the rhesus monkey hypothalamus. Extremely high densities of dopamine-beta-hydroxylase-immunoreactive processes were observed in the paraventricular and supraoptic nuclei, while relatively lower levels were found in the arcuate and dorsomedial nuclei and in the medial preoptic, perifornical, and suprachiasmatic areas. Moderate levels of dopamine-beta-hydroxylase immunoreactivity were found throughout the lateral hypothalamic area and in the internal lamina of the median eminence. Very few immunoreactive processes were found in the ventromedial nucleus or in the mammillary complex. Other midline diencephalic structures were found to have high densities of dopamine-beta-hydroxylase immunoreactivity, including the paraventricular nucleus of the thalamus and a discrete subregion of nucleus reuniens, the magnocellular subfascicular nucleus. A moderate density of dopamine-beta-hydroxylase immunoreactive processes were found in the rhomboid nucleus and zona incerta whereas little dopamine-beta-hydroxylase immunoreactivity was found in the fields of Forel, nucleus reuniens, or subthalamic nucleus. The differential distribution of dopamine-beta-hydroxylase-immunoreactive processes may reflect a potential role of norepinephrine as a regulator of a variety of functions associated with the nuclei that are most heavily innervated, e.g., neuroendocrine release from the paraventricular and supraoptic nuclei, and gonadotropin release from the medial preoptic area and mediobasal hypothalamus. Additionally, quantitative analysis of dopamine-beta-hydroxylase-immunoreactive varicosities was performed on a laser scanning microscope in both magnocellular and parvicellular regions of

Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

PubMed Central

Geraci, Nicholas S.; Mukbel, Rami M.; Kemp, Michael T.; Wadsworth, Mariha N.; Lesho, Emil; Stayback, Gwen M.; Champion, Matthew M.; Bernard, Megan A.; Abo-Shehada, Mahmoud; Coutinho-Abreu, Iliano V.; Ramalho-Ortigão, Marcelo; Hanafi, Hanafi A.; Fawaz, Emadeldin Y.; El-Hossary, Shabaan S.; Wortmann, Glenn; Hoel, David F.; McDowell, Mary Ann

2014-01-01

Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents. PMID:24615125

Treadmill performance of mice with cerebellar lesions: 1. Purkinje cell degeneration mutant mice.

PubMed

Le Marec, N; Lalonde, R

1998-02-01

The purpose of this study was to evaluate the sensorimotor skills of a spontaneous mouse mutant, Purkinje cell degeneration (PCD), marked by selective cerebellar cortical atrophy on a treadmill activated at 1 of 2 speeds and at 1 of 3 slopes, requiring forward movements to avoid footshocks. There was no difference in latencies before falling from the belt between PCD mutants and controls during acquisition. However, PCD mutants were impaired on the fast treadmill during retention, implicating the cerebellum in the memory of a motor skill. During acquisition of the slow treadmill task at the 2 lowest slopes of inclination, PCD mutants spent more time walking than controls, an indication of a decreased ability of coordinating whole body movements. The same pattern of higher walking time on the slow treadmill in PCD mutants was evident during retention. These results indicate that the cerebellar cortex is involved in the acquisition and the retention of a task requiring equilibrium.

A novel two-chain IGF-II-derived peptide from purified β-cell granules.

PubMed

Buchanan, Christina M; Phillips, Anthony R J; Cooper, Garth J S

2010-10-01

Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic β-cells. Here we isolated secretory granules from a β-cell line, βTC6-F7, and characterized the nature of the IGF-II-LI located therein. Secretory granules were isolated from cultured mouse βTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH(2)-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. βTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 10⁶ cells compared to 340 ± 44 pmol insulin-LI per 10⁶ cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37-40)IGF-II, which we termed 'vesiculin'. This molecule was also detectable in βTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC(50) value of 131 nM ± 1.35. Vesiculin, des(37-40)IGF-II, is a novel two-chain insulin-like hormone and the major "IGF-II-like" peptide found in purified mouse βTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species. Copyright © 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Immunohistochemichal Assessment of the CrkII Proto-oncogene Expression in Common Malignant Salivary Gland Tumors and Pleomorphic Adenoma.

PubMed

Askari, Mitra; Darabi, Masoud; Jahanzad, Esa; Mostakhdemian Hosseini, Zahra; Musavi Chavoshi, Marjan; Darabi, Maryam

2015-01-01

Background and aims. Various morphologies are seen in different salivary gland tumorsor within an individual tumor, and the lesions show divers biological behaviors. Experimental results support the hypothesis that increased CrkII proto-oncogene is associated with cytokine-induced tumor initiation and progression by altering cell motility signaling pathway. The aim of this study was to assess the CrkII expression in common malignant salivary gland tumors and pleomorphic ade-noma. Materials and methods. Immunohistochemical analysis of CrkII expression was performed on paraffin blocks of 64 car-cinomas of salivary glands, 10 pleomorphic adenomas, and 10 normal salivary glands. Biopsies were subjected to immu-nostaining with EnVision detection system using monoclonal anti-CrkII. Evaluation of immunoreactivity of CrkII was based on the immunoreaction intensity and percentage of stained tumor cells which were scored semi-quantitatively on a scale with four grades 0 to 3. Kruskal-wallis test and additional Mann-Whitney statistical test were used for analysis of CrkII expression levels. Results. Increased expression of CrkII was seen (P=0.005) in malignant tumors including: mucoepidermoid carcinoma, adenoid cystic carcinoma, and carcinoma ex pleomorphic adenoma, but CrkII expression in acinic cell carcinoma was weak. CrkII expression in pleomorphic adenoma was weak or negative. A weak staining was sparsely seen in normal acinar serous cell. Conclusion. Increased expression of CrkII and its higher intensity of staining in tumors with more aggressive biologic behavior in carcinomas of salivary gland is consistent with a role for this proto-oncogene in salivary gland tumorigenesis and cancer progression.

Chronic prenatal exposure to carbon monoxide results in a reduction in tyrosine hydroxylase-immunoreactivity and an increase in choline acetyltransferase-immunoreactivity in the fetal medulla: implications for Sudden Infant Death Syndrome.

PubMed

Tolcos, M; McGregor, H; Walker, D; Rees, S

2000-03-01

Maternal cigarette smoking during pregnancy is associated with a significantly increased risk of Sudden Infant Death Syndrome (SIDS). This study investigated the effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, on the neuroglial and neurochemical development of the medulla in the fetal guinea pig. Pregnant guinea pigs were exposed to 200 p.p.m CO for 10 h per day from day 23-25 of gestation (term = 68 days) until day 61-63, at which time fetuses were removed and brains collected for analysis. Using immunohistochemistry and quantitative image analysis, examination of the medulla of CO-exposed fetuses revealed a significant decrease in tyrosine hydroxylase-immunoreactivity (TH-IR) in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), area postrema, intermediate reticular nucleus, and the ventrolateral medulla (VLM), and a significant increase in choline acetyltransferase-immunoreactivity (ChAT-IR) in the DMV and hypoglossal nucleus compared with controls. There was no difference between groups in immunoreactivity for the m2 muscarinic acetylcholine receptor, substance P- or met-enkephalin in any of the medullary nuclei examined, nor was there evidence of reactive astrogliosis. The results show that prenatal exposure to CO affects cholinergic and catecholaminergic pathways in the medulla of the guinea pig fetus, particularly in cardiorespiratory centers, regions thought to be compromised in SIDS.

Activity-dependent regulation of NMDAR1 immunoreactivity in the developing visual cortex.

PubMed

Catalano, S M; Chang, C K; Shatz, C J

1997-11-01

NMDA receptors have been implicated in activity-dependent synaptic plasticity in the developing visual cortex. We examined the distribution of immunocytochemically detectable NMDAR1 in visual cortex of cats and ferrets from late embryonic ages to adulthood. Cortical neurons are initially highly immunostained. This level declines gradually over development, with the notable exception of cortical layers 2/3, where levels of NMDAR1 immunostaining remain high into adulthood. Within layer 4, the decline in NMDAR1 immunostaining to adult levels coincides with the completion of ocular dominance column formation and the end of the critical period for layer 4. To determine whether NMDAR1 immunoreactivity is regulated by retinal activity, animals were dark-reared or retinal activity was completely blocked in one eye with tetrodotoxin (TTX). Dark-rearing does not cause detectable changes in NMDAR1 immunoreactivity. However, 2 weeks of monocular TTX administration decreases NMDAR1 immunoreactivity in layer 4 of the columns of the blocked eye. Thus, high levels of NMDAR1 immunostaining within the visual cortex are temporally correlated with ocular dominance column formation and developmental plasticity; the persistence of staining in layers 2/3 also correlates with the physiological plasticity present in these layers in the adult. In addition, visual experience is not required for the developmental changes in the laminar pattern of NMDAR1 levels, but the presence of high levels of NMDAR1 in layer 4 during the critical period does require retinal activity. These observations are consistent with a central role for NMDA receptors in promoting and ultimately limiting synaptic rearrangements in the developing neocortex.

Ionic mechanisms of transient inward current in the absence of Na(+)-Ca2+ exchange in rabbit cardiac Purkinje fibres.

PubMed Central

Han, X; Ferrier, G R

1992-01-01

1. Membrane currents were measured with a two-microelectrode technique in voltage clamped rabbit cardiac Purkinje fibres under conditions known to cause intracellular calcium overload and to eliminate or minimize Na(+)-Ca2+ exchange. 2. Increasing [Ca2+]o from 2.5 to 5 mM or above and substituting external sodium with either sucrose, choline or Li+ induced an oscillatory transient inward current (TI) which peaked 200-300 ms after repolarization from a previous depolarizing pulse. The TI quickly disappeared upon return to normal Tyrode solution. Both the rate and configuration of action potentials of Purkinje fibres also returned to control upon return to Tyrode solution after 30 min of high Ca2+ exposure, if the Ca2+ concentration was 30 mM or less. 3. The TI in Na(+)-free solution was Ca2+ dependent. Either zero or low (2.5 mM) [Ca2+]o, or replacement of [Ca2+]o by BaCl prevented induction of the TI current upon repolarization from a previous depolarizing pulse. 4. In the presence of 30 mM-CaCl2 and with choline chloride as the substitute for NaCl, TI had a distinct reversal potential (Erev) of -25 mV. The time-to-peak TI, either inward or outward, did not shift significantly with change in voltage. Both inward and outward TI were simultaneously abolished by exposure to 1 microM-ryanodine, suggesting they were both activated by transient release of Ca2+ from the sarcoplasmic reticulum. The occurrence of TI in the absence of [Na+]o is not compatible with an electrogenic Na(+)-Ca2+ exchange mechanism. The existence of a clear-cut reversal potential suggests that an ionic channel may be responsible for the TI under these conditions. 5. Both the magnitude of peak TI and the Erev were affected by changes of CaCl2 concentration. (i) Under steady-state conditions, peak inward TI was significantly increased when the [Ca2+]o was elevated from 5 to 15 mM. The peak TI in the outward direction was significantly increased when [Ca2+]o was elevated from 15 to 30 mM; however

Substance P immunoreactive nerve terminals in the dorsolateral nucleus of the tractus solitarius: roles in the baroreceptor reflex.

PubMed

Massari, V J; Shirahata, M; Johnson, T A; Lauenstein, J M; Gatti, P J

1998-03-02

Physiological and light microscopic evidence suggest that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; however, ultrastructural support for this hypothesis is lacking. We have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase (HRP). The dorsolateral subnucleus of the nucleus tractus solitarius (dlNTS) was processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical visualization of SP by dual labeling light and electron microscopic methods. Either HRP or SP was readily identified in single-labeled unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS, which were simultaneously identified as CSN primary afferents. However, only 15% of CSN terminals in the dlNTS were immunoreactive for SP. Therefore, while the ultrastructural data support the hypothesis that SP immunoreactive first-order neurons are involved in the origination of the baroreceptor reflex, they suggest that only a modest part of the total sensory input conveyed from the carotid sinus baroreceptors to the dlNTS is mediated by SP immunoreactive CSN terminals. Five types of axo-axonic synapses were observed in the dlNTS. SP immunoreactive CSN afferents were very rarely involved in these synapses. Furthermore, SP terminals were never observed to form the presynaptic element in an axo-axonic synapse with a CSN afferent. Therefore, SP does not appear to be involved in the modulation of the baroreceptor reflex in the dlNTS. Copyright 1998 Elsevier Science B.V.

Monoclonal antibody (AFH1) immunoreactive on morphologically abnormal basal melanocytes within dysplastic nevi, nevocellular nevus nests, and melanoma.

PubMed

Aronson, P J; Ito, K; Fukaya, T; Hashimoto, K; Mehregan, A H

1988-04-01

The mouse monoclonal antibody AFH1 was produced using formalin-fixed, sham paraffin-embedded human melanoma cell culture line A375 as immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against formalin- or acid alcohol-fixed paraffin-embedded tissue as well as formalin- or acid alcohol-fixed unembedded lesions. Ninety-seven nevomelanocytic lesions, neurofibromas, epithelial lesions, and a plasmacellular infiltrate were evaluated. AFH1 was immunoreactive on 54 of 55 nevocytic lesions (98.2%), 15 of 16 primary melanomas (93.7%), a lentigo maligna, and nests in 21 of 21 dysplastic nevi (100%). Of 100 consecutive basal melanocytes of intraepidermal melanoma cells counted in each lesion, mean AFH1 immunoreactivity for nonnested basal melanocytes in nevocellular nevi was 3.8%; for dysplastic nevi, 13.8%; and for intraepidermal melanoma cells, 78.0%. When nonnested basal melanocytes were subdivided into cytologically normal and abnormal cell groups, AFH1 immunoreactivity was 9.4% and 72.6%, respectively. AFH1 recognition of the lentiginous portion of dysplastic nevi corresponds statistically to the appearance of abnormal melanocyte cytology, nest formation, or both. Using 50% immunoreactive nonnested melanocytes as the criterion, AFH1 seems to distinguish primary melanoma from dysplastic nevi with a sensitivity of 93.8% and a specificity of 95.8%.

alpha-Smooth muscle actin immunoreactivity may change in nature in interlobular fibrosis of the pancreas in patients with congenital biliary dilatation.

PubMed

Matsubara, Kenro; Suda, Koichi; Suzuki, Fujihiko; Kumasaka, Toshio; Shiotsu, Hidetoshi; Miyano, Takeshi

2004-07-01

Pancreatic fibrosis in patients with congenital biliary dilatation (CBD) or choledochal cyst was studied to determine why biliary pancreatitis seldom progresses to chronic pancreatitis/more progressive state. Pancreatic collagenization in eight patients (three adults with pancreatoduodenectomy and five children with biopsy of the pancreas performed when excising the cyst) with CBD was evaluated histopathologically and immunohistochemically. Interlobular and periductal fibrosis with both collagen Type I and Type III immunoreactivities was found in six out of eight cases and in all four cases in which the pancreatic duct was included, respectively. The interlobular area was seldom immunoreactive for alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, but was usually positive for CD34, a human progenitor cell antigen. In contrast, the periductal area was usually immunoreactive for alpha-SMA, but usually negative for CD34 and immunopositive for bcl-2, indicating a continuously progressive state of fibrosis, in which 'pre-existing'alpha-SMA immunoreactivity in the interlobular area may change in nature and lead to CD34-positive fibrosis or apoptosis. In conclusion, biliary pancreatitis is not likely to evolve into chronic pancreatitis/more progressive state because 'pre-existing'alpha-SMA immunoreactivity in the interlobular area may change in nature.

Cerebellar Purkinje Cells Generate Highly Correlated Spontaneous Slow-Rate Fluctuations.

PubMed

Cao, Ying; Liu, Yu; Jaeger, Dieter; Heck, Detlef H

2017-01-01

Cerebellar Purkinje cells (PC) fire action potentials at high, sustained rates. Changes in spike rate that last a few tens of milliseconds encode sensory and behavioral events. Here we investigated spontaneous fluctuations of PC simple spike rate at a slow time scale of the order of 1 s. Simultaneous recordings from pairs of PCs that were aligned either along the sagittal or transversal axis of the cerebellar cortex revealed that simple spike rate fluctuations at the 1 s time scale were highly correlated. Each pair of PCs had either a predominantly positive or negative slow-rate correlation, with negative correlations observed only in PC pairs aligned along the transversal axis. Slow-rate correlations were independent of faster rate changes that were correlated with fluid licking behavior. Simultaneous recordings from PCs and cerebellar nuclear (CN) neurons showed that slow-rate fluctuations in PC and CN activity were also highly correlated, but their correlations continually alternated between periods of positive and negative correlation. The functional significance of this new aspect of cerebellar spike activity remains to be determined. Correlated slow-rate fluctuations seem too slow to be involved in the real-time control of ongoing behavior. However, slow-rate fluctuations of PCs converging on the same CN neuron are likely to modulate the excitability of the CN neuron, thus introduce a possible slow modulation of cerebellar output activity.

Diagnosis of ocular and glandular toxoplasmosis using the Indian ink immunoreaction.

PubMed

Safar, E H; Azab, M E; Osman, Z M

1984-01-01

The Indian ink immunoreaction (IIR) as a method for diagnosis of Toxoplasma infection has been evaluated. 68 sera from patients with suspected ocular and glandular toxoplasmosis and 30 control sera from normal individuals were tested using both indirect fluorescent antibody test (IFAT) and IIR. The test proved to be specific, simple and rapid especially for screening purposes.

Immunoreactive GnRH Type I Receptors in the Mouse and Sheep Brain

PubMed Central

Albertson, Asher J.; Navratil, Amy; Mignot, Mallory; Dufourny, Laurence; Cherrington, Brian; Skinner, Donal C.

2008-01-01

GnRH has been implicated in an array of functions outside the neuroendocrine reproductive axis. Previous investigations have reported extensive GnRH binding in numerous sites and this has been supported by in situ hybridization studies reporting GnRH receptor mRNA distribution. The present study on mice and sheep supports and extends these earlier investigations by revealing the distribution of cells immunoreactive for the GnRH receptor. In addition to sites previously shown to express GnRH receptors such as the hippocampus, amygdala and the arcuate nucleus, the improved resolution afforded by immunocytochemistry detected cells in the mitral cell lay of the olfactory bulb as well as the central grey of the mesencephalon. In addition, GnRH receptor immunoreactive neurons in the hippocampus and mesencephalon of the sheep were shown to colocalize with estrogen receptor β. Although GnRH may act at some of these sites to regulate reproductive processes, evidence is accumulating to support an extra-reproductive role for this hypothalamic decapeptide. PMID:18439800

Localisation of atrial natriuretic peptide immunoreactivity in the ventricular myocardium and conduction system of the human fetal and adult heart.

PubMed Central

Wharton, J; Anderson, R H; Springall, D; Power, R F; Rose, M; Smith, A; Espejo, R; Khaghani, A; Wallwork, J; Yacoub, M H

1988-01-01

Atrial natriuretic peptide immunoreactivity was found in ventricular and atrial tissues with specific antisera raised to the amino and carboxy terminal regions of the precursor molecule. In 13 developing human hearts (7-24 weeks' gestation) the immunoreactivity was concentrated in the atrial myocardium and ventricular conduction system but it was also detected in the early fetal ventricular myocardium. Immunoreactivity in five normal adults was largely confined to the atrial myocardium although it was also found in the ventricular conduction tissues of hearts removed from 10 patients who were undergoing cardiac transplantation. The ventricular conduction system is an extra-atrial site for the synthesis of atrial natriuretic peptide. In the failing heart this synthesis may be further supplemented by expression of the gene in the ventricular myocardium. It is possible that ventricular production of the peptide contributes to the raised circulating concentrations of atrial natriuretic peptide immunoreactivity found in severe congestive heart disease, particularly in patients with dilated cardiomyopathy. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 PMID:2973340

Distribution of enkephalin immunoreactivity in sympathetic prevertebral ganglia and digestive tract of guinea-pigs and rats.

PubMed

Herbrecht, F; Bagnol, D; Cucumel, K; Jule, Y; Cupo, A

1995-05-04

The aim of the present study was to determine the distribution of methionine-enkephalin (ME) and leucine-enkephalin (LE) immunoreactivity in the sympathetic prevertebral ganglia (coeliac plexus and inferior mesenteric ganglion) and in the myenteric plexus-muscular layer complex of the digestive tract in guinea-pigs and rats. This study was performed using the same immunological approaches including radioimmunoassays and HPLC characterization as those used previously on cats in order to be able to make inter-region and inter-species comparisons. In rat and guinea-pig prevertebral ganglia, the distributions of the enkephalin immunoreactivities were comparable and were characterized by a low ME/LE concentration ratio, of less than 1. In the digestive tract of rats, the enkephalin immunoreactivities were homogeneously distributed, whereas in guinea-pigs, they were found to be very low in the lower oesophageal sphincter and high in the duodenum. In both species, the ME/LE concentration ratio was around 2. The ME/LE concentration ratio determined in the present study in peripheral nervous structures was much lower than that determined previously in the rat brain. Radioimmunoassay and biochemical data might indicate that different mechanisms are responsible for the processing and/or degradation of enkephalins in the central and peripheral nervous systems. The present study provides further evidences that there are tissue- and species-dependent differences in the distribution of enkephalin immunoreactivities. These differences should be taken into consideration when dealing with the effects and the role of enkephalins in the nervous control of intestinal motility in mammals.

A Critical Period in Purkinje Cell Development Is Mediated by Local Estradiol Synthesis, Disrupted by Inflammation, and Has Enduring Consequences Only for Males.

PubMed

Hoffman, Jessica F; Wright, Christopher L; McCarthy, Margaret M

2016-09-28

Identifying and understanding critical periods in brain development is essential to decoding the long-term impact of widespread, poorly defined, and frequently occurring insults such as inflammation. Using the laboratory rat Rattus norvegicus, we have discovered a narrowly constrained critical period in Purkinje neuron development subject to dysregulation by inflammation. The onset and offset of heightened vulnerability are attributed to a tightly orchestrated gene expression profile present only during the second postnatal week and not the first or third weeks. Genes expressed during this time code for enzymes and receptors which are critical not only for prostaglandin production and activity but also for estradiol production via the aromatase enzyme and estradiol action via the α isoform of the estrogen receptor. The two synthetic pathways are connected by prostaglandin E2 (PGE2) activation of the aromatase enzyme, as we reported previously (Dean et al., 2012b) and confirm here. Dysregulation of the PGE2-estradiol pathway during the second week by treatment with PGE2 or lipopolysaccharides produces enduring consequences as a result of reduced growth of Purkinje dendritic trees and impaired juvenile social play behavior, but only in males. The deleterious consequences of inflammation locally in the cerebellum are prevented by peripheral treatment with the cyclooxygenase inhibitor nimesulide or the aromatase inhibitor formestane. These findings highlight a novel regulatory pathway that creates a critical period in brain development vulnerable to dysregulation by inflammation. The cerebellum is increasingly appreciated for its role in social, emotional, and cognitive behaviors. It is consistently and severely affected in neuropsychiatric disorders originating during development, such as autism spectrum disorder and schizophrenia. We have identified a critical period in rat development during the second week of life that is dysregulated by inflammatory insults. An

Bronchial mucosal immunoreactivity of sensory neuropeptides in severe airway diseases.

PubMed

Chanez, P; Springall, D; Vignola, A M; Moradoghi-Hattvani, A; Polak, J M; Godard, P; Bousquet, J

1998-09-01

Neuropeptides act on most of the components of the bronchial environment. They influence bronchomotor tone and bronchial vascular caliber and permeability. To investigate the nonadrenergic, noncholinergic system within the airways in asthma and chronic bronchitis, we performed endobronchial biopsies in 16 normal human volunteers, 49 patients with asthma of varying severity, including 16 patients treated with oral corticosteroids, and 13 patients with chronic bronchitis. Frozen sections of biopsies stained with specific antibodies against the neural marker PGP 9.5, vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and neuropeptide Y (NPY) were analyzed for the presence of nerves through indirect immunofluorescence. Nerves were present in most of the biopsies and were found within and below the epithelium and adjacent to smooth muscle, glands, and blood vessels. By comparison with those in normal subjects, the numbers of VIP-immunoreactive nerves were not significantly decreased in patients with asthma and chronic bronchitis, but NPY-immunoreactive nerves were significantly decreased in the smooth muscle of these latter two groups of patients (p < 0.005). There was no correlation between disease severity and the number of nerves found in the biopsies. This study does not confirm previous findings in autopsy material of some defects in sensory and VIP-containing nerves in severe asthma.

Muscarinic acetylcholine receptor activation blocks long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses via cannabinoid signaling.

PubMed

Rinaldo, Lorenzo; Hansel, Christian

2013-07-02

Muscarinic acetylcholine receptors (mAChRs) are known to modulate synaptic plasticity in various brain areas. A signaling pathway triggered by mAChR activation is the production and release of endocannabinoids that bind to type 1 cannabinoid receptors (CB1R) located on synaptic terminals. Using whole-cell patch-clamp recordings from rat cerebellar slices, we have demonstrated that the muscarinic agonist oxotremorine-m (oxo-m) blocks the induction of presynaptic long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell synapses in a CB1R-dependent manner. Under control conditions, LTP was induced by delivering 120 PF stimuli at 8 Hz. In contrast, no LTP was observed when oxo-m was present during tetanization. PF-LTP was restored when the CB1R antagonist N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) was coapplied with oxo-m. Furthermore, the suppressive effect of oxo-m on PF-LTP was abrogated by the GDP analog GDP-β-S (applied intracellularly), the phospholipase C inhibitor U-73122, and the diacylglycerol lipase inhibitor tetrahydrolipstatin (THL), suggesting that cannabinoid synthesis results from the activation of Gq-coupled mAChRs present on Purkinje cells. The oxo-m-mediated suppression of LTP was also prevented in the presence of the M3 receptor antagonist DAU 5884, and was absent in M1/M3 receptor double-KO mice, identifying M3 receptors as primary oxo-m targets. Our findings allow for the possibility that cholinergic signaling in the cerebellum--which may result from long-term depression (LTD)-related disinhibition of cholinergic neurons in the vestibular nuclei--suppresses presynaptic LTP to prevent an up-regulation of transmitter release that opposes the reduction of postsynaptic responsiveness. This modulatory capacity of mAChR signaling could promote the functional penetrance of LTD.

Substance P-immunoreactive nerves in endobronchial biopsies in cough-variant asthma and classic asthma.

PubMed

Lee, Sang Yeub; Kim, Min Kyung; Shin, Chol; Shim, Jae Jeong; Kim, Han Kyeom; Kang, Kyung Ho; Yoo, Se Hwa; In, Kwang Ho

2003-01-01

Unlike classic asthma, cough-variant asthma does not show any evidence of airway obstruction. The main symptom is a dry cough with little known pathophysiology. Hypersensitivity of the cough receptors in cough-variant asthma and an increase in the sensory nerve density of the airway epithelium in persistent dry cough patients have been reported. Therefore, it is possible that there is a higher sensory nerve density in cough-variant asthma patients than in classic asthma patients. This study was undertaken to compare the substance P (SP)-immunoreactive nerve density in mucosal biopsies of cough-variant asthma patients, classic asthma patients, and in control subjects. Bronchoscopic biopsies were performed in 6 cough-variant asthma patients, 14 classic asthma patients, and 5 normal controls. The tissues obtained were stained immunohistochemically. The SP-immunoreactive nerve density was measured in the bronchial epithelium using a light microscope at 400 x magnification. SP- immunoreactive nerve density for the cough-variant asthma group was significantly higher than that of the classic asthma group (p = 0.001), and of the normal control group (p = 0.006). It is possible that a sensory nerve abnormality within the airway may be related to hypersensitivity of the cough receptor, and that this may be one of the pathophysiologies of cough-variant asthma. Copyright 2003 S. Karger AG, Basel

Uroplakin II Expression in Breast Carcinomas Showing Apocrine Differentiation: Putting Some Emphasis on Invasive Pleomorphic Lobular Carcinoma as a Potential Mimic of Urothelial Carcinoma at Metastatic Sites.

PubMed

Tajima, Shogo; Koda, Kenji

2016-01-01

Uroplakin II antibody is exclusively specific for urothelial carcinoma. Nonurothelial carcinoma has not been reported to be immunoreactive for uroplakin II. In the present study, we hypothesized that breast carcinoma showing apocrine differentiation, such as invasive pleomorphic lobular carcinoma (IPLC) and apocrine carcinoma (AC), stains positive for uroplakin II. We identified 6 cases of IPLC between 2000 and 2014 by searching a computerized pathological database. We randomly selected 10 cases of each classic invasive lobular carcinoma (cILC) and AC and five cases of apocrine metaplasia (AM) that coexisted in a surgically resected breast carcinoma specimen. Immunohistochemistry was performed for uroplakin II, GATA3, CK7, CK20, and other representative markers positive for urothelial carcinoma. All cases of IPLC, AC, and AM, except those of cILC, showed immunoreactivity for uroplakin II. Poorly differentiated urothelial carcinoma sometimes shows similar morphology to IPLC with the following immunophenotype: CK7+, CK20-, GATA3+, and uroplakin II+. In the present study, this immunophenotype was observed in all the cases of IPLC and AC. Therefore, when studying metastatic, poorly differentiated carcinoma showing the aforementioned immunophenotype, we should consider the possibility of it being IPLC in addition to metastatic urothelial carcinoma.

Dopamine- and Tyrosine Hydroxylase-Immunoreactive Neurons in the Brain of the American Cockroach, Periplaneta americana

PubMed Central

Hamanaka, Yoshitaka; Minoura, Run; Nishino, Hiroshi; Miura, Toru; Mizunami, Makoto

2016-01-01

The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine’s actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach, Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase (TH, an enzyme required for dopamine synthesis), and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of “dopaminergic” neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory

Mitochondrial proteomic profiling reveals increased carbonic anhydrase II in aging and neurodegeneration.

PubMed

Pollard, Amelia; Shephard, Freya; Freed, James; Liddell, Susan; Chakrabarti, Lisa

2016-10-10

Carbonic anhydrase inhibitors are used to treat glaucoma and cancers. Carbonic anhydrases perform a crucial role in the conversion of carbon dioxide and water into bicarbonate and protons. However, there is little information about carbonic anhydrase isoforms during the process of ageing. Mitochondrial dysfunction is implicit in ageing brain and muscle. We have interrogated isolated mitochondrial fractions from young adult and middle aged mouse brain and skeletal muscle. We find an increase of tissue specific carbonic anhydrases in mitochondria from middle-aged brain and skeletal muscle. Mitochondrial carbonic anhydrase II was measured in the Purkinje cell degeneration ( pcd 5J ) mouse model. In pcd 5J we find mitochondrial carbonic anhydrase II is also elevated in brain from young adults undergoing a process of neurodegeneration. We show C.elegans exposed to carbonic anhydrase II have a dose related shorter lifespan suggesting that high CAII levels are in themselves life limiting. We show for the first time that the mitochondrial content of brain and skeletal tissue are exposed to significantly higher levels of active carbonic anhydrases as early as in middle-age. Carbonic anhydrases associated with mitochondria could be targeted to specifically modulate age related impairments and disease.

Micropatterning of neural stem cells and Purkinje neurons using a polydimethylsiloxane (PDMS) stencil.

PubMed

Choi, Jin Ho; Lee, Hyun; Jin, Hee Kyung; Bae, Jae-sung; Kim, Gyu Man

2012-12-07

A new fabrication method of a polydimethylsiloxane (PDMS) stencil embedded microwell plate is proposed and applied to a localized culture of Purkinje neurons (PNs) and neural stem cells (NSCs). A microwell plate combines a PDMS stencil and well plate. The PDMS stencil was fabricated by spin casting from an SU-8 master mold. Gas blowing using nitrogen was adopted to perforate the stencil membrane. An acrylic well plate compartment mold was fabricated using computer numerical control (CNC) machining. By PDMS casting using a stencil placed on an acrylic mold, microwell plates were fabricated without punching or the use of a plasma bonding process. By using the stencil as a physical mask for the cell culture, PNs and NSCs were successfully cultured into micropatterns. The microwell plate could be applied to the localizing and culturing of a cell. The micropatterned NSCs were differentiated into neurons, astrocytes, and oligodendrocytes. The results showed that cells could be cultured and differentiated into micropatterns in a precisely controlled manner in any shape and in specific sizes for bioscience study and bioengineering applications.

Plasma immunoreactive beta-melanocyte-stimulating hormone and skin pigmentation in chronic renal failure.

PubMed Central

Smith, A G; Shuster, S; Comaish, J S; Plummer, N A; Thody, A J; Alvarez-Ude, F; Kerr, D N

1975-01-01

Plasma immunoreactive beta-melanocyte stimulating hormone (beta-MSH) concentrations were greatly increased in patients with chronic renal failure. There was no correlation between the severity of the renal failure or the degree of pigmentation and the plasma beta-MSH levels. PMID:1125653

High feline trypsin-like immunoreactivity in a cat with pancreatitis and hepatic lipidosis.

PubMed

Bruner, J M; Steiner, J M; Williams, D A; Van Alstine, W G; Blevins, W

1997-06-15

A 1.5-year-old domestic shorthair cat was examined because of vomiting and icterus. Clinicopathologic abnormalities included high alanine transaminase, alkaline phosphatase, and gamma-glutamyltransferase activities and high total bilirubin concentration. During abdominal ultrasonography, the left limb and body of the pancreas appeared hypoechoic, and a small quantity of peritoneal effusion was seen. The liver was diffusely hyperechoic, with echogenicity similar to that of the spleen, indicating hepatic lipidosis. Feline trypsin-like immunoreactivity was high, suggesting that the cat also had pancreatitis. The cat was treated with crystalloid fluids and was fed a protein-restricted diet via a percutaneous endoscopically placed gastrostomy tube. The cat's condition continued to deteriorate despite medical treatment, and it was euthanatized. Necropsy confirmed the clinical suspicion of acute pancreatitis and hepatic lipidosis. This case suggests that measurement of trypsin-like immunoreactivity may be useful in cats suspected of having pancreatitis.

[Morphological and laminar distribution of cholecystokinin-immunoreactive neurons in cortex of human inferior parietal lobe and their clinical significance].

PubMed

Puskas, Laslo; Draganić-Gajić, Saveta; Malobabić, Slobodan; Puskas, Nela; Krivokuća, Dragan; Stanković, Gordana

2008-01-01

Cholecystocinine is a neuropeptide whose function in the cortex has not yet been clarified, although its relation with some psychic disorders has been noticed. Previous studies have not provided detailed data about types, or arrangement of neurons that contain those neuropeptide in the cortex of human inferior parietal lobe. The aim of this study was to examine precisely the morphology and typography of neurons containing cholecytocinine in the human cortex of inferior parietal lobule. There were five human brains on which we did the immunocystochemical research of the shape and laminar distribution of cholecystocinine immunoreactive neurons on serial sections of supramarginal gyrus and angular gyrus. The morphological analysis of cholecystocinine-immunoreactive neurons was done on frozen sections using avidin-biotin technique, by antibody to cholecystocinine diluted in the proportion 1:6000 using diamine-benzedine. Cholecystocinine immunoreactive neurons were found in the first three layers of the cortex of inferior parietal lobule, and their densest concentration was in the 2nd and 3rd layer. The following types of neurons were found: bipolar neurons, then its fusiform subtype, Cajal-Retzius neurons (in the 1st layer), reverse pyramidal (triangular) and unipolar neurons. The diameters of some types of neurons were from 15 to 35 microm, and the diameters of dendritic arborization were from 85-207 microm. A special emphasis is put on the finding of Cajal-Retzius neurons that are immunoreactive to cholecystocinine, which demands further research. Bearing in mind numerous clinical studies pointing out the role of cholecystokinine in the pathogenesis of schizophrenia, the presence of a great number of cholecystokinine immunoreactive neurons in the cortex of inferior parietal lobule suggests their role in the pathogenesis of schizophrenia.

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis.

PubMed

Trigo, Gabriela; Ferreira, Paula; Ribeiro, Niza; Dinis, Márcia; Andrade, Elva Bonifácio; Melo-Cristino, José; Ramirez, Mário; Tavares, Delfina

2008-11-01

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.

Localization of amylin-like immunoreactivity in melanocyte-stimulating hormone-containing cells of the pars intermedia but not those of the pars distalis in the axolotl (Ambystoma mexicanum) pituitary.

PubMed

Suzuki, Hirohumi; Yamamoto, Toshiharu

2016-04-01

Immunohistochemical techniques were employed to investigate the distribution of amylin-like immunoreactivity in the axolotl (Ambystoma mexicanum) pituitary. Amylin-immunoreactive cells were observed in the pars intermedia, and these cells were found to be immunoreactive for α-melanocyte-stimulating hormone (αMSH) as well. In contrast, αMSH-immunoreactive cells in the pars distalis were immuno-negaitive for amylin. These light microscopic findings were confirmed by immunoelectron microscopy. Amylin-immunoreactive signals were located on the haloes of presumable secretory granules in association with αMSH-immunoreactive signals in the amylin-positive cells. However, in the pars distalis, the αMSH-positive cells did not contain amylin-immunoreactive secretory granules. Western blot analysis of axolotl pituitary extracts revealed the labeling of a protein band at approximately 10.5-kDa by the anti-rat amylin serum, which was not labeled by the anti-αMSH antibody. These findings indicate that amylin secreted from MSH-producing cells in the pars intermedia may modulate MSH secretion in an autocrine fashion and may participate in MSH functions such as fatty homeostasis together with MSH. Copyright © 2016 Elsevier GmbH. All rights reserved.

[Localization of substance P- and FMRFamide-like immunoreactivity in the atrium of the snail Achatina fulica].

PubMed

Shabel'nikov, S V; Bystrova, O A; Martynova, M G

2008-01-01

By immunohistochemical and immunocytochemical methods localization of Substanse P (SP) and FMRFamide in the atrium of the snail Achatina fulica was investigated. Nerve fibers innervating the snail atrium contact tightly with the granular cells (GC) situated between muscle and endocardial cells, forming neuroendocrine units. Both neuromediators were found in the cells of the neuroendocrine units. By immunohistochemistry SP- and FMRFamide-immunoreactive material was revealed in the granules of the atrial GC. Elecrtonmicroscopical immunocytochemistry has confirmed the presence of SP- and FMRFamide-immunoreactive material in the granules of the GC and shown their presence in the neurosecretory granules of the nerve endings contacting both the atrial GC and cardiomyocytes.

Difference in transient ischemia-induced neuronal damage and glucose transporter-1 immunoreactivity in the hippocampus between adult and young gerbils

PubMed Central

Park, Seung Min; Lee, Jae-Chul; Chen, Bai Hui; Shin, Bich-Na; Cho, Jeong Hwi; Kim, In Hye; Park, Joon Ha; Won, Moo-Ho; Ahn, Ji Hyeon; Tae, Hyun-Jin; Shin, Myoung Cheol; Park, Chan Woo; Cho, Jun Hwi; Lee, Hui Young

2016-01-01

Objective(s): The alteration of glucose transporters is closely related with the pathogenesis of brain edema. We compared neuronal damage/death in the hippocampus between adult and young gerbils following transient cerebral ischemia/reperfusion and changes of glucose transporter-1(GLUT-1)-immunoreactive microvessels in their ischemic hippocampal CA1 region. Materials and Methods: Transient cerebral ischemia was developed by 5-min occlusion of both common carotid arteries. Neuronal damage was examined by cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining and changes in GLUT-1 expression was carried out by immunohistochemistry. Results: About 90% of pyramidal neurons only in the adult CA1 region were damaged after ischemia/reperfusion; in the young, about 53 % of pyramidal neurons were damaged from 7 days after ischemia/reperfusion. The density of GLUT-1-immunoreactive microvessels was significantly higher in the young sham-group than that in the adult sham-group. In the ischemia-operated-groups, the density of GLUT-1-immunoreactive microvessels was significantly decreased in the adult and young at 1 and 4 days post-ischemia, respectively, thereafter, the density of GLUT-1-immunoreactive microvessels was gradually increased in both groups after ischemia/reperfusion. Conclusion: CA1 pyramidal neurons of the young gerbil were damaged much later than that in the adult and that GLUT-1-immunoreactive microvessels were significantly decreased later in the young. These data indicate that GLUT-1 might differently contribute to neuronal damage according to age after ischemic insults. PMID:27403259

Gonadotropin-releasing hormone immunoreactivity in the adult and fetal human olfactory system.

PubMed

Kim, K H; Patel, L; Tobet, S A; King, J C; Rubin, B S; Stopa, E G

1999-05-01

Studies in fetal brain tissue of rodents, nonhuman primates and birds have demonstrated that cells containing gonadotropin-releasing hormone (GnRH) migrate from the olfactory placode across the nasal septum into the forebrain. The purpose of this study was to examine GnRH neurons in components of the adult and fetal human olfactory system. In the adult human brain (n=4), immunoreactive GnRH was evident within diffusely scattered cell bodies and processes in the olfactory bulb, olfactory nerve, olfactory cortex, and nervus terminalis located on the anterior surface of the gyrus rectus. GnRH-immunoreactive structures showed a similar distribution in 20-week human fetal brains (n=2), indicating that the migration of GnRH neurons is complete at this time. In 10-11-week fetal brains (n=2), more cells were noted in the nasal cavity than in the brain. Our data are consistent with observations made in other species, confirming olfactory derivation and migration of GnRH neurons into the brain from the olfactory placode. Copyright 1999 Elsevier Science B.V.

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

PubMed Central

Romero-Fernandez, W.; Borroto-Escuela, D.O.; Vargas-Barroso, V.; Narváez, M.; Di Palma, M.; Agnati, L.F.; Sahd, J. Larriva

2014-01-01

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially

Dopamine D1 and D2 receptor immunoreactivities in the arcuate-median eminence complex and their link to the tubero-infundibular dopamine neurons.

PubMed

Romero-Fernandez, W; Borroto-Escuela, D O; Vargas-Barroso, V; Narváez, M; Di Palma, M; Agnati, L F; Larriva Sahd, J; Fuxe, K

2014-07-18

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tubero-infundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region.  Dopamine D1 and D2 receptors may therefore directly and differentially

Effect of atropine and somatostatin on bombesin-stimulated plasma immunoreactive trypsin release in man.

PubMed

de Jong, A J; Klamer, M; Lamers, C B

1987-01-01

This study was undertaken to determine the effect of atropine and somatostatin, two inhibitors of intraduodenal pancreatic enzyme secretion, on bombesin-stimulated release of plasma immunoreactive trypsin in 6 healthy volunteers. Infusion of 5 ng/kg.min bombesin during 30 min induced significant increases in plasma trypsin from 206 +/- 20 to 334 +/- 44 ng/ml (p less than 0.01). Atropine (15 ng/kg as i.v. bolus followed by 5 ng/kg.h) had no influence on the bombesin-stimulated increase in plasma immunoreactive trypsin (207 +/- 20 to 326 +/- 54 ng/ml). Somatostatin (125 micrograms as i.v. bolus followed by 125 micrograms/h) also failed to inhibit the plasma trypsin response to bombesin (207 +/- 18 to 663 +/- 166 ng/ml). These results point to major differences in the regulation of plasma and intraduodenal trypsin secretion.

The distribution of neuropeptide Y and dynorphin immunoreactivity in the brain and pituitary gland of the platyfish, Xiphophorus maculatus, from birth to sexual maturity

NASA Technical Reports Server (NTRS)

Cepriano, L. M.; Schreibman, M. P.

1993-01-01

Immunoreactive neuropeptide Y and dynorphin have been localized in the brain and pituitary gland of the platyfish, Xiphophorus maculatus, at different ages and stages of development from birth to sexual maturity. Immunoreactive neuropeptide Y was found in perikarya and tracts of the nucleus olfactoretinalis, telencephalon, ventral tegmentum and in the neurohypophysis and in the three regions of the adenohypophysis. Immunoreactive dynorphin was found in nerve tracts in the olfactory bulb and in cells of the pars intermedia and the rostral pars distalis of the pituitary gland.

Aberrant connections between climbing fibres and Purkinje cells induce alterations in the timing of an instrumental response in the rat.

PubMed

Gaytán-Tocavén, Lorena; López-Vázquez, Miguel Ángel; Guevara, Miguel Ángel; Olvera-Cortés, María Esther

2017-09-01

Cerebellar participation in timing and sensory-motor sequences has been supported by several experimental and clinical studies. A relevant role of the cerebellum in timing of conditioned responses in the range of milliseconds has been demonstrated, but less is known regarding the role of the cerebellum in supra-second timing of operant responses. A dissociated role of the cerebellum and striatum in timing in the millisecond and second range had been reported, respectively. The climbing fibre-Purkinje cell synapse is crucial in timing models; thus, the aberrant connection between these cellular elements is a suitable model for evaluating the contribution of the cerebellum in timing in the supra-second range. The aberrant connection between climbing fibres and Purkinje cells was induced by administration of the antagonist of NMDA receptors MK-801 to Sprague-Dawley rats at postnatal days 7-14. The timing of an operant response with two fixed intervals (5 and 8 s) and egocentric sequential learning was evaluated in 60-day-old adult rats. The aberrant connections caused a reduced accuracy in the timing of the instrumental response that was more evident in the 8-s interval and a reduced number of successive correct responses (responses emitted in the correct second without any other response between them) in the 8-s interval. In addition, an inability to incorporate new information in a sequence previously learned in egocentric-based sequence learning was apparent in rats with aberrant CF-PC synapses. These results support a relevant role for the cerebellum in the fine-tuning of the timing of operant responses in the supra-second range.

Potential and tension changes induced by sodium removal in dog Purkinje fibres: role of an electrogenic sodium-calcium exchange.

PubMed Central

Croaboeuf, E; Gautier, P; Giuraudou, P

1981-01-01

1. Isolated dog Purkinje fibres were bathed in K-free media or in the presence of ouabain 10(-4) M in order to depress the electrogenic sodium pump activity. Membrane potential and mechanical tension were recorded in the presence of normal external sodium concentration and during lowering or removal of external Na. 2. Lowering or removal of external Na (Na being replaced by choline, Tris, sucrose or Li) induced a hyperpolarization and a contracture which reached a maximum after 1 or 2 min and then decreased progressively. Using Tris, Em increased from -40 +/- 3 to -72 +/- 10 mV (n = 39). The Na-free contracture and hyperpolarization did not occur in the absence of Na pump depression. 3. Tetrodotoxin (1.2 x 10(-5)M), Mn (4 mM), verapamil (1-4 x 10(-5) M) tetraethylammonium (5 mM), 4-aminopyridine (5 mM) and Cs (20 mM, in the presence of ouabain) did not alter the Na-free contracture and hyperpolarization. On the other hand Mn (20 mM), acid media (external pH less than 6.0) and low temperatures depressed or suppressed both the hyperpolarization and contracture. Lanthanum (0.4 mM) did not suppress the hyperpolarization and the contracture. On the contrary the Na-free contracture was generally increased in the presence of La. 4. Caffeine (10 mM) induced strong contractures with no changes in Em, thus demonstrating the possibility for the Purkinje fibers of developing contractures without concomitant hyperpolarizations. 5. It can be concluded that the Na-free contracture and hyperpolarization are not due to changes in passive conductances but are related to the functioning of an electrogenic Na-Ca exchange mechanism which carries inwardly 1 Ca and outwardly 3 or more Na. Images Fig. 1 PMID:7264984

[Substance P and/or calcitonin gene-related peptide immunoreactive neurons in dorsal root ganglia possibly involved in the transmission of nociception in rat penile frenulum].

PubMed

Wu, Zhong-Min; Ni, Jing-Jing; Ling, Shu-Cai

2007-12-01

To study the relationship between substance P (SP) and/or calcitonin gene-related peptide (CGRP) immunoreactive neurons in dorsal root ganglia (DRG) and the transmission of nociception in the penile frenulum of rats. The fluoro-gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in the penile frenulum of rats. And SP and/or CGRP immunofluorescence labeling was employed to detect the distribution of SP and/or CGRP immunoreactive neurons in DRG. FG retrograde tracing showed that the FG retrolabeled neurons were localized in L6-DRG and S1-DRG. SP and/or CGRP immunofluorescence labeling indicated that a large number of DRG neurons were SP- and CGRP-immunoreactive, different in size, bright red and bright green respectively in color, and arranged in rows or spots among nerve bundles. All the FG/SP and FG/CGRP double-labeled neurons were medium or small-sized. One third of the FG-labeled neurons were SP-immunoreactive, and a half of them CGRP-immunoreactive in L6-DRG and S1-DRG respectively. The FG/SP/CGRP-labeled neurons accounted for one fifth of the FG retro labeled neurons. SP- and CGRP-immunoreactive neurons in L6-DRG and SI-DRG of rats may be involved in the transmission of nociception in rat penile frenulum.

Development of serotonin-like immunoreactivity in the embryos and larvae of nudibranch mollusks with emphasis on the structure and possible function of the apical sensory organ.

PubMed

Kempf, S C; Page, L R; Pires, A

1997-09-29

This investigation provides a light and electron microscopic examination of the development of serotonin-like immunoreactivity and structure of the apical sensory organ (ASO) in embryos and/or larvae of four nudibranch species: Berghia verrucicornis, Phestilla sibogae, Melibe leonina, and Tritonia diomedea. Serotonin-like immunoreactivity is first expressed in somata, dendrites, and axons of a group of five distinct neurons within the ASO. These neurons extend axons into an apical neuropil, a structure that is situated centrally and immediately dorsal to the cerebral commissure. Three of these neurons possess sensory dendrites that extend through the pretrochal epithelium, each supporting two cilia at their distal ends. Later development of serotonin-like immunoreactivity includes 1) axons from the apical neuropil that extend into each of the velar lobes; 2) neuron perikarya in the cerebral and pedal ganglia; 3) axons that extend through the cerebral commissure, cerebral-pedal connectives, pedal commissure, and possibly the visceral loop connective; and 4) axons extending from each pedal ganglion into the larval foot. Ultrastructurally, the ASO can be seen to be composed of three lobes and an apical neuropil that is separately delineated from the cerebral commissure. Four cell types are present within the ASO: ciliary tuft cells, type I and type II parampullary neurons, and ampullary neurons. Immunofluorescence and 3,3' diaminobenzidine tetrahydrochloride (DAB) labeling verify that the serotonergic neurons of the ASO are type I and type II parampullary neurons. The ampullary and type I parampullary neurons possess dendrites that extend through the pretrochal epithelium. These dendrites are partitioned into three bundles, one on either side of the ciliary tuft cells and a third bundle penetrating the pretrochal epithelium centrally between the ciliary tuft cells. One serotonergic type I parampullary neuron is associated with each of these bundles. Two ampullary

Effect of hyperthermia on calbindin-D 28k immunoreactivity in the hippocampal formation following transient global cerebral ischemia in gerbils

PubMed Central

Lee, Jae-Chul; Cho, Jeong-Hwi; Lee, Tae-Kyeong; Kim, In Hye; Won, Moo-Ho; Cho, Geum-Sil; Shin, Bich-Na; Hwang, In Koo; Park, Joon Ha; Ahn, Ji Hyeon; Kang, Il Jun; Lee, Young Joo; Kim, Yang Hee

2017-01-01

Calbindin D-28K (CB), a Ca2+-binding protein, maintains Ca2+ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We investigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei (NeuN) and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia (39.5 ± 0.2°C) was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neuronal damage/death in the pyramidal layer of CA1–3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreactivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immunoreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults. PMID:29089991

Anatomical mapping of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in outer hair cell efferents in adult rats.

PubMed

Dannhof, B J; Roth, B; Bruns, V

1991-10-01

The distribution of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in the cochleae of 15 adult Wistar white rats was investigated using the peroxidase-antiperoxidase (PAP) technique. A monoclonal antibody to ChAT and a polyclonal antiserum to GAD were used. Immunoreaction was investigated quantitatively, in the electron microscope, on tangential sections of the tunnel of Corti and the rows of outer hair cells. ChAT-like and GAD-like immunoreactivity was found in all efferent nerve fibres in the tunnel of Corti and in all efferent synapses on the outer hair cells. A coexistence of ChAT and GAD in the efferent system to the outer hair cells of the rat is therefore assumed.

FOSB immunoreactivity in endothelia of epithelioid hemangioma (angiolymphoid hyperplasia with eosinophilia).

PubMed

Ortins-Pina, Ana; Llamas-Velasco, Mar; Turpin, Sara; Soares-de-Almeida, Luís; Filipe, Paulo; Kutzner, Heinz

2018-06-01

Accurate distinction of epithelioid hemangioma (EH) from its malignant mimics is paramount but remains challenging due to its wide morphological spectrum and lack of objective molecular markers. FOSB oncogenic activation was recently identified as a key event in endothelial proliferation. We sought to investigate the FOSB staining pattern in EH with angiolymphoid hyperplasia with eosinophilia (EH-AHLE) morphology and to evaluate its value in differential diagnosis of epithelioid vascular tumors. From the authors' files, 15 representative cases of EH-ALHE were selected and evaluated for their FOSB immunostaining pattern. Other vascular proliferations which can be morphological mimics were also tested: epithelioid hemangioendothelioma (EHE) (5 cases) and epithelioid angiosarcoma (EAS) (5 cases). All 15 cases of EH-ALHE showed strong and homogeneous FOSB nuclear expression in endothelial cells with ample cytoplasm and intracytoplasmic vacuoles. All cases of EHE and EAS lacked FOSB immunoreactivity or showed only incidental weak FOSB immunoreactivity in less than 5 nuclei per lesion. FOSB immunohistochemistry is sensitive in the diagnosis of EH-ALHE, and allows differentiation from its histological mimics. An immunohistochemical panel including not only pan-cytokeratin AE1/AE3 and endothelial markers, but also FOSB, helps in the diagnosis of epithelioid vascular tumors. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of bombesin and cholecystokinin on plasma immunoreactive trypsin in humans.

PubMed

de Jong, A J; Klamer, M; Jansen, J B; Hopman, W P; Lamers, C B

1987-01-01

Since bombesin is a potent stimulus of the release of cholecystokinin (CCK), it has been suggested that the stimulatory effect of bombesin on pancreatic enzyme secretion is mediated by CCK. The present study was undertaken to determine the role of CCK in the bombesin-induced stimulation of plasma immunoreactive trypsin. Plasma CCK was measured by radioimmunoassay using the antibody T204, which binds to all biologically active sulfated COOH-terminal CCK-peptides. Plasma trypsin was also measured by radioimmunoassay. Infusion of 5 ng/kg/min bombesin in 6 healthy volunteers increased plasma CCK from 1.2 +/- 0.2-8.9 +/- 0.7 pM (p less than 0.0001). The peak increment in plasma CCK during bombesin (9.3 +/- 0.6 pM) was accompanied by a significant rise in plasma trypsin from 206 +/- 21-334 +/- 44 ng/ml (p less than 0.01). However, when similar increases in plasma CCK were achieved by infusion of 0.018 CU/kg/min CCK-33 (9.9 +/- 0.8 pM) or by intraduodenal instillation of 250 ml 20% Intralipid (9.7 +/- 1.9 pM), no significant changes in plasma trypsin were observed. It is therefore concluded that the stimulatory effect of bombesin on plasma immunoreactive trypsin is not mediated by CCK.

Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network.

PubMed

Good, Jean-Marc; Mahoney, Michael; Miyazaki, Taisuke; Tanaka, Kenji F; Sakimura, Kenji; Watanabe, Masahiko; Kitamura, Kazuo; Kano, Masanobu

2017-11-21

Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF) to Purkinje cell (PC) network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor δ2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Distinct patterns of serum immunoreactivity as evidence for multiple brain-directed autoantibodies in juvenile neuronal ceroid lipofuscinosis.

PubMed

Lim, M J; Beake, J; Bible, E; Curran, T M; Ramirez-Montealegre, D; Pearce, D A; Cooper, J D

2006-10-01

Autoantibodies to glutamic acid decarboxylase (GAD65) have been reported in sera from the Cln3(-/-) mouse model of juvenile neuronal ceroid lipofuscinosis (JNCL), and in individuals with this fatal paediatric neurodegenerative disorder. To investigate the existence of other circulating autoreactive antibodies, we used sera from patients with JNCL and other forms of neuronal ceroid lipofuscinosis (NCL) as primary antisera to stain rat and human central nervous system sections. JNCL sera displayed characteristic patterns of IgG, but not IgA, IgE or IgM immunoreactivity that was distinct from the other forms of NCL. Immunoreactivity of JNCL sera was not confined to GAD65-positive (GABAergic) neurons, but also stained multiple other cell populations. Preadsorption of JNCL sera with recombinant GAD65 reduced the intensity of the immunoreactivity, but did not significantly change its staining pattern. Moreover, sera from Stiff Person Syndrome and Type I Diabetes, disorders in which GAD65 autoantibodies are present, stained with profiles that were markedly different from JNCL sera. Collectively, these studies provide evidence of the presence of autoreactive antibodies within multiple forms of NCL, and are not exclusively directed towards GAD65.

Postnatal development of orexin-A and orexin-B like immunoreactivities in the Eastern grey kangaroo (Macropus giganteus) hypothalamus.

PubMed

Yamamoto, Yukiyo; McKinley, Michael J; Nakazato, Masamitsu; Yamashita, Hiroshi; Shirahata, Akira; Ueta, Yoichi

2006-01-09

The Eastern grey kangaroo (Macropus giganteus) is a marsupial, which is born in an extremely undeveloped state and has a long suckling period in the mother's pouch. In the present study, we examined the immunoreactivities of orexin-A (OXA) and orexin-B (OXB) in the hypothalamus of the Eastern grey kangaroo during the preweaning period, postweaning period and adulthood. In the preweaning period, only a few OXA- and OXB-like immunoreactive (LI) neurons and fibers were present and the intensity of staining was very weak. In the postweaning period, there was a pronounced increase in the numbers of OXA- and OXB-LI neurons and fibers and the intensity of the immunoreactivity was considerably stronger in comparison to the preweaning period. In the adult, the numbers of OXA- and OXB-LI neurons and fibers appeared to be slightly increased and the intensity was slightly stronger in comparison to the postweaning period. At all time periods, the distributions of OXA- and OXB-LI neurons was similar. The postnatal development of hypothalamic orexin neurons may be associated with developmental changes, including feeding behavior.

Calbindin D-28k and parvalbumin immunoreactivity in the frontal cortex in patients with frontal lobe dementia of non-Alzheimer type associated with amyotrophic lateral sclerosis.

PubMed Central

Ferrer, I; Tuñón, T; Serrano, M T; Casas, R; Alcántara, S; Zújar, M J; Rivera, R M

1993-01-01

The morphology and distribution of local-circuit neurons (interneurons) were examined, by calbindin D-28k and parvalbumin immunocytochemistry, in the frontal cortex (area 8) in two patients with frontal lobe dementia of non-Alzheimer type associated with classical amyotrophic lateral sclerosis (ALS), and in seven normal cases. The density of calbindin D-28k immunoreactive cells was dramatically reduced in ALS patients, but the density of parvalbumin-immunoreactive neurons was preserved. Decreased density of calbindin D-28k-immunoreactive neurons, which are mainly located in the upper cortical layers, may interfere with the normal processing of cortico-cortical connections, whereas integrity of parvalbumin-immunoreactive cells may be associated with the preservation of the major inhibitory intracortical circuits in patients with frontal lobe dementia. Images PMID:8459241

Vesicular monoamine transporter-1 (VMAT-1) mRNA and immunoreactive proteins in mouse brain.

PubMed

Ashe, Karen M; Chiu, Wan-Ling; Khalifa, Ahmed M; Nicolas, Antoine N; Brown, Bonnie L; De Martino, Randall R; Alexander, Clayton P; Waggener, Christopher T; Fischer-Stenger, Krista; Stewart, Jennifer K

2011-01-01

Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. VMAT-1 mRNA was detected in mouse brain with RT-PCR. The cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four different antibodies to VMAT-1. Sequencing confirmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine and a proton ionophore, but not by tetrabenazine, which has a high affinity for VMAT-2. Antibodies to either the C- or N- terminus of VMAT-1 detected two proteins (73 and 55 kD) in transfected COS-1 cells. The C-terminal antibodies detected both proteins in extracts of mouse medulla/pons, cortex, hypothalamus, and cerebellum but only the 73 kD protein and higher molecular weight immunoreactive proteins in mouse adrenal and rat PC12 cells, which are positive controls for rodent VMAT-1. These findings demonstrate that a functional VMAT-1 mRNA coding sequence is expressed in mouse brain and suggest processing of VMAT-1 protein differs in mouse adrenal and brain.

Fetal myosin immunoreactivity in human dystrophic muscle.

PubMed

Schiaffino, S; Gorza, L; Dones, I; Cornelio, F; Sartore, S

1986-01-01

We report immunofluorescence observations on normal and dystrophic human muscle using an antibody (anti-bF) raised against bovine fetal myosin and specific for fetal myosin heavy chains. In rat skeletal muscle, anti-bF was previously found to react selectively with myosin isoforms expressed during fetal and early postnatal development and in regenerating muscles. Anti-bF stained most fibers in human fetal and neonatal muscle, whereas only nuclear chain fibers of muscle spindles were labeled in normal adult muscle. In muscle biopsies from patients with Duchenne's muscular dystrophy, numerous extrafusal fibers were stained: some were small regenerating fibers, others were larger fibers presumably resulting from previous regenerative events. Fetal myosin immunoreactivity in Duchenne's dystrophy appears to reflect the reexpression of fetal-specific myosin isoforms and provides a new valuable tool for identifying regenerating fibers and following their destiny in dystrophic muscle.

The ontogenetic development of neurons containing calcium-binding proteins in the septum of the guinea pig: Late onset of parvalbumin immunoreactivity versus calbindin and calretinin.

PubMed

Hermanowicz-Sobieraj, Beata; Robak, Anna

2017-01-01

The study describes the immunoreactivity of calbindin (CB), calretinin (CR) and parvalbumin (PV), their distribution pattern and the co-distribution of CB and CR as well as CB and PV in the septum of the guinea pig during development. Immunohistochemistry was conducted on embryonic (E40, E50, E60), newborn (P0) and postnatal (P5, P10, P20, P40, P100) guinea pig brains. The presence of both CB and CR was detected at E40, while PV began to be observed at E60. Immunoreactivity for CB was constant throughout ontogeny. In contrast to CR immunoreactivity, PV immunoreactivity was higher in the postnatal stages than in the prenatal and newborn stages. Double immunostaining showed that CB co-localized with CR from E40 onwards, while with PV from P5 onwards, suggesting that CB co-operates with these proteins in the guinea pig septum during different periods of ontogeny. Our results also indicate that among the studied CaBPs, CB exhibited the highest immunoreactivity during both embryonic and postnatal development. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

PubMed

Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

1998-01-01

In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the

Differential inhibition of Ca2+ channels in mature rat cerebellar Purkinje cells by sFTX-3.3 and FTX-3.3.

PubMed

Dupere, J R; Moya, E; Blagbrough, I S; Usowicz, M M

1996-01-01

Synthetic funnel web spider toxin (sFTX-3.3) is a polyamine amide analogue of FTX, a toxin fraction isolated from the venom of the funnel web spider, Agelenopsis aperta, that blocks P-type Ca2+ channels. The structures of these polyamine containing compounds are not identical: sFTX-3.3 contains an amide carbonyl oxygen that is absent from the predicted structure of native FTX. Recently, a compound called FTX-3.3 was synthesized with the structure predicted for native FTX. We have compared the effects of polyamine amide sFTX-3.3 and polyamine FTX-3.3, on Ca2+ channel currents in the soma of mature rat cerebellar Purkinje neurons, in which the predominant Ca2+ channels are defined as P-type. Differential inhibition by sFTX-3.3 and FTX-3.3 revealed three populations of Ca2+ channels. One group, mediating approximately 66% of the current, was blocked by sFTX-3.3 with an IC50 (concentration producing half maximal inhibition) of 33 nM or by FTX-3.3 with an IC50 of 55 pM. A second population (5-25% of the total current) was inhibited by sFTX-3.3 with an IC50 of 33 nM, but was insensitive to FTX-3.3, while a third (10-30%) was blocked by FTX-3.3 with an IC50 of 125 nM and was resistant to sFTX-3.3. These channels also showed distinctive current-voltage relationships. Our results suggest that P-type Ca2+ channels in mature rat cerebellar Purkinje cells may be subdivided according to pharmacological and biophysical properties.

Treadmill exercise ameliorates symptoms of attention deficit/hyperactivity disorder through reducing Purkinje cell loss and astrocytic reaction in spontaneous hypertensive rats

PubMed Central

Yun, Hyo-Soon; Park, Mi-Sook; Ji, Eun-Sang; Kim, Tae-Woon; Ko, Il-Gyu; Kim, Hyun-Bae; Kim, Hong

2014-01-01

Attention deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder of cognition. We investigated the effects of treadmill exercise on Purkinje cell and astrocytic reaction in the cerebellum of the ADHD rat. Adult male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKYR) weighing 210± 10 g were used. The animals were randomly divided into four groups (n= 15): control group, ADHD group, ADHD and methylphenidate (MPH)-treated group, ADHD and treadmill exercise group. The rats in the MPH-treated group as a positive control received 1 mg/kg MPH orally once a day for 28 consecutive days. The rats in the treadmill exercise group were made to run on a treadmill for 30 min once a day for 28 days. Motor coordination and balance were determined by vertical pole test. Immunohistochemistry for the expression of calbindinD-28 and glial fibrillary acidic protein (GFAP) in the cerebellar vermis and Western blot for GFAP, Bax, and Bcl-2 were conducted. In the present results, ADHD significantly decreased balance and the number of calbindin-positive cells, while GFAP expression and Bax/Bcl-2 ratio in the cerebellum were significantly increased in the ADHD group compared to the control group (P< 0.05, respectively). In contrast, treadmill exercise and MPH alleviated the ADHD-induced the decrease of balance and the number of calbindine-positive cells, and the increase of GFAP expression and Bax/Bcl-2 ratio in the cerebellum (P< 0.05, respectively). Therefore, the present results suggested that treadmill exercise might exert ameliorating effect on ADHD through reduction of Purkinje cell loss and astrocytic reaction in the cerebellum. PMID:24678501

Loss of calretinin immunoreactive fibers in subcortical visual recipient structures of the RCS dystrophic rat.

PubMed

Vugler, Anthony A; Coffey, Peter J

2003-11-01

The retinae of dystrophic Royal College of Surgeons (RCS) rats exhibit progressive photoreceptor degeneration accompanied by pathology of ganglion cells. To date, little work has examined the consequences of retinal degeneration for central visual structures in dystrophic rats. Here, we use immunohistochemistry for calretinin (CR) to label retinal afferents in the superior colliculus (SC), lateral geniculate nucleus, and olivary pretectal nucleus of RCS rats aged between 2 and 26 months of age. Early indications of fiber loss in the medial dystrophic SC were apparent between 9 and 13 months. Quantitative methods reveal a significant reduction in the level of CR immunoreactivity in visual layers of the medial dystrophic SC at 13 months (P < 0.02). In dystrophic animals aged 19-26 months the loss of CR fibers in SC was dramatic, with well-defined patches of fiber degeneration predominating in medial aspects of the structure. This fiber degeneration in SC was accompanied by increased detection of cells immunoreactive for CR. In several animals, regions of fiber loss were also found to contain strongly parvalbumin-immunoreactive cells. Loss of CR fibers was also observed in the lateral geniculate nucleus and olivary pretectal nucleus. Patterns of fiber loss in the dystrophic SC compliment reports of ganglion cell degeneration in these animals and the response of collicular neurons to degeneration is discussed in terms of plasticity of the dystrophic visual system and properties of calcium binding proteins.

Identification of immunoreactive proteins of Streptococcus agalactiae isolated from cultured tilapia in China.

PubMed

Liu, Guangjin; Zhang, Wei; Lu, Chengping

2013-12-01

Streptococcus agalactiae (Group B streptococcus, GBS) is an important zoonotic pathogen that can cause lethal infections in humans and animals, including aquatic species. Immunoreactive proteins of the S. agalactiae strain, GD201008-001, isolated from cultured tilapia in China, were screened by immunoproteomics using hyperimmune sera, convalescent guinea pig sera and GD201008-001-infected tilapia antisera as primary detection antibodies. A total of 16 different proteins were identified including 13 novel immunoreactive proteins of S. agalactiae. Four proteins, serine-rich repeat glycoprotein 1, branched-chain alpha-keto acid dehydrogenase (BKD) subunit E2, 5'-nucleotidase family protein and ornithine carbamoyltransferase, were shown to react with the three types of sera and thus were considered to represent novel S. agalactiae vaccine candidate antigens. Our findings represent the basis for vaccine development for piscine S. agalactiae and are necessary for understanding virulence factors and immunogenicity of S. agalactiae with different hosts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Impaired specific immunoreactivity in cows with hepatic lipidosis.

PubMed

Wentink, G H; Rutten, V P; van den Ingh, T S; Hoek, A; Müller, K E; Wensing, T

1997-05-01

In this study, hepatic lipidosis in cows was experimentally induced by offering an energy surplus during the dry period. Liver triacylglycerol (TAG) was 16% in the experimental group. In the control group fed the same diet in restricted quantities, liver TAG was about 7%. The animals of both groups were vaccinated with tetanus vaccine at Day 3 after parturition. It was demonstrated that the cows with high liver TAG percentages had lower humoral and cellular (P < 0.05) immunological responses compared with the animals with low liver TAG levels at Day 14 after vaccination. The results obtained in the high TAG group support the notion that the frequent occurrence of aspecific infections in cows with hepatic lipidosis may be due to impaired immunoreactivity.

Histamine Immunoreactive Elements in the Central and Peripheral Nervous Systems of the Snail, Biomphalaria spp., Intermediate Host for Schistosoma mansoni

PubMed Central

Habib, Mohamed R.; Mohamed, Azza H.; Osman, Gamalat Y.; Sharaf El-Din, Ahmed T.; Mossalem, Hanan S.; Delgado, Nadia; Torres, Grace; Rolón-Martínez, Solymar; Miller, Mark W.; Croll, Roger P.

2015-01-01

Histamine appears to be an important transmitter throughout the Animal Kingdom. Gastropods, in particular, have been used in numerous studies establishing potential roles for this biogenic amine in the nervous system and showing its involvement in the generation of diverse behaviours. And yet, the distribution of histamine has only previously been described in a small number of molluscan species. The present study examined the localization of histamine-like immunoreactivity in the central and peripheral nervous systems of pulmonate snails of the genus Biomphalaria. This investigation demonstrates immunoreactive cells throughout the buccal, cerebral, pedal, left parietal and visceral ganglia, indicative of diverse regulatory functions in Biomphalaria. Immunoreactivity was also present in statocyst hair cells, supporting a role for histamine in graviception. In the periphery, dense innervation by immunoreactive fibers was observed in the anterior foot, perioral zone, and other regions of the body wall. This study thus shows that histamine is an abundant transmitter in these snails and its distribution suggest involvement in numerous neural circuits. In addition to providing novel subjects for comparative studies of histaminegic neurons in gastropods, Biomphalaria is also the major intermediate host for the digenetic trematode parasite, which causes human schistosomiasis. The study therefore provides a foundation for understanding potential roles for histamine in interactions between the snail hosts and their trematode parasites. PMID:26086611

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

PubMed Central

Kim, In Hye; Jeon, Yong Hwan; Lee, Tae-Kyeong; Cho, Jeong Hwi; Lee, Jae-Chul; Park, Joon Ha; Ahn, Ji Hyeon; Shin, Bich-Na; Kim, Yang Hee; Hong, Seongkweon; Yan, Bing Chun; Won, Moo-Ho; Lee, Yun Lyul

2017-01-01

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investigated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal transient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining calbindin D28k immunoreactivity. PMID:28761424

Orexin-A/hypocretin-1 Immunoreactivity in the Lateral Hypothalamus is Reduced in Genetically Obese but not in Diet-induced Obese Mice.

PubMed

González, J Antonio; Prehn, Jochen H M

2018-01-15

The mechanisms that link diet and body weight are not fully understood. A diet high in fat often leads to obesity, and this in part is the consequence of diet-induced injury to specific hypothalamic nuclei. It has been suggested that a diet high in fat leads to cell loss in the lateral hypothalamus, which contains specific populations of neurons that are essential for regulating energy homoeostasis; however, we do not know which cell types are affected by the diet. We studied the possibility that high-fat diet leads to a reduction in orexin-A/hypocretin-1 (Hcrt1) and/or melanin-concentrating hormone (MCH) immunoreactivity in the lateral hypothalamus. We quantified immuno-labeled Hcrt1 and MCH cells in brain sections of mice fed a diet high in fat for up to 12 weeks starting at 4 weeks of age and found that this diet did not modify the number of Hcrt1- or MCH-immunoreactive neurons. By contrast, there were fewer Hcrt1- (but not MCH-) immunoreactive cells in genetically obese db/db mice compared to wild-type mice. Non-obese, heterozygous db/+ mice also had fewer Hcrt1-immunoreactive cells. Differences in the number of Hcrt1-immunoreactive cells were only a function of the db genotype but not of diet or body weight. Our findings show that the lateral hypothalamus is affected differently in the db genotype and in diet-induced obesity, and support the idea that not all hypothalamic neurons involved in energy balance regulation are sensitive to the effects of diet. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

PubMed

Suzuki, Hirohumi; Yamamoto, Toshiharu

2014-02-01

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH. Copyright © 2013 Elsevier Ltd. All rights reserved.

Prognostic significance of hepatocyte growth factor activator inhibitor type 1 (HAI-1) immunoreactivity in pancreatic ductal adenocarcinoma.

PubMed

Sakugawa, Chihiro; Haruyama, Yukihiro; Tanaka, Hiroyuki; Fukushima, Tsuyoshi; Kawaguchi, Makiko; Kataoka, Hiroaki

2017-12-04

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine protease inhibitor that is expressed on the surface of epithelial cells. Evidence has suggested that decreased cell surface HAI-1 in carcinoma cells results in enhanced invasiveness. However, little is known regarding the expression of HAI-1 in pancreatic ductal adenocarcinoma (PDAC). This study aimed to analyze HAI-1 expression in PDAC and its impact on patient prognosis. HAI-1 immunohistochemistry was performed on samples from 67 PDAC cases. HAI-1 expression was increased in intraepithelial neoplasia compared to the adjacent non-neoplastic ductal epithelium. Of the 67 samples tested, 58% (39/67) of PDAC cases showed diffuse (> 75%) immunoreactivity in PDAC cells. The remaining cases showed reduced HAI-1 immunoreactivity in a substantial number of cancer cells. Although there was no correlation between HAI-1 status and tumor size, histologic grade or lymph node metastasis, diffuse HAI-1 positive cases showed longer disease-free survival (DFS; p = 0.006, log-rank test). In conclusion, HAI-1 is upregulated in pancreatic intraepithelial neoplasia and broadly expressed in PDAC cells. However, PDAC cases having areas of reduced HAI-1 immunoreactivity may show shorter DFS.

Characterization of PDF-immunoreactive neurons in the optic lobe and cerebral lobe of the cricket, Gryllus bimaculatus.

PubMed

Abdelsalam, Salaheldin; Uemura, Hiroyuki; Umezaki, Yujiro; Saifullah, A S M; Shimohigashi, Miki; Tomioka, Kenji

2008-07-01

Pigment-dispersing factor (PDF) is a neuropeptide playing important roles in insect circadian systems. In this study, we morphologically and physiologically characterized PDF-immunoreactive neurons in the optic lobe and the brain of the cricket Gryllus bimaculatus. PDF-immunoreactivity was detected in cells located in the proximal medulla (PDFMe cells) and those in the dorsal and ventral regions of the outer chiasma (PDFLa cells). The PDFMe cells had varicose processes spread over the frontal surface of the medulla and the PDFLa cells had varicose mesh-like innervations in almost whole lamina, suggesting their modulatory role in the optic lobe. Some of PDFMe cells had a hairpin-shaped axonal process running toward the lamina then turning back to project into the brain where they terminated at various protocerebral areas. The PDFMe cells had a low frequency spontaneous spike activity that was higher during the night and was often slightly increased by light pulses. Six pairs of PDF-immunoreactive neurons were also found in the frontal ganglion. Competitive ELISA with anti-PDF antibodies revealed daily cycling of PDF both in the optic lobe and cerebral lobe with an increase during the night that persisted in constant darkness. The physiological role of PDF is discussed based on these results.

Characterization of Insulin-Immunoreactive Cells and Endocrine Cells Within the Duct System of the Adult Human Pancreas.

PubMed

Li, Rong; Zhang, Xiaoxi; Yu, Lan; Zou, Xia; Zhao, Hailu

2016-01-01

The adult pancreatic duct system accommodates endocrine cells that have the potential to produce insulin. Here we report the characterization and distribution of insulin-immunoreactive cells and endocrine cells within the ductal units of adult human pancreas. Sequential pancreas sections from 12 nondiabetic adults were stained with biomarkers of ductal epithelial cells (cytokeratin 19), acinar cells (amylase), endocrine cells (chromogranin A; neuron-specific enolase), islet hormones (insulin, glucagon, somatostatin, pancreatic polypeptide), cell proliferation (Ki-67), and neogenesis (CD29). The number of islet hormone-immunoreactive cells increased from large ducts to the terminal branches. The insulin-producing cells outnumbered endocrine cells reactive for glucagon, somatostatin, or pancreatic polypeptide. The proportions of insulin-immunoreactive count compared with local islets (100% as a baseline) were 1.5% for the main ducts, 7.2% for interlobular ducts, 24.8% for intralobular ducts, 67.9% for intercalated ducts, and 348.9% for centroacinar cells. Both Ki-67- and CD29-labeled cells were predominantly localized in the terminal branches around the islets. The terminal branches also showed cells coexpressing islet hormones and cytokeratin 19. The adult human pancreatic ducts showed islet hormone-producing cells. The insulin-reactive cells predominantly localized in terminal branches where they may retain potential capability for β-cell neogenesis.

Effect on Membrane Potential and Electrical Activity of Adding Sodium to Sodium-Depleted Cardiac Purkinje Fibers

PubMed Central

Wiggins, Jay R.; Cranefield, Paul F.

1974-01-01

Canine cardiac Purkinje fibers exposed to Na-free solutions containing 128 mM TEA and 16 mM Ca show resting potentials in the range -50 to -90 mV; if the concentration of Na in the perfusate is raised from 0 to 4 to 24 mM, hyperpolarization follows. If the initial resting potential is low, the hyperpolarization tends to be greater; the average increase in the presence of 8 mM Na is 14 mV. Such hyperpolarization is not induced by adding Na to K-free solutions, is not seen in cooled fibers, or in fibers exposed to 10-3 M ouabain, nor is it induced by adding Li and thus may result from electrogenic sodium extrusion. Fibers exposed to Na-free solutions are often spontaneously active; if they are quiescent they often show repetitive activity during depolarizing pulses. Such spontaneous or repetitive activity is suppressed by the addition of Na. This suppression may or may not be related to the hyperpolarization. PMID:4418558

Hypoglycemia in a dog with a leiomyoma of the gastric wall producing an insulin-like growth factor II-like peptide.

PubMed

Boari, A; Barreca, A; Bestetti, G E; Minuto, F; Venturoli, M

1995-06-01

A 12-year-old mixed-breed male dog was referred to the Clinica Medica Veterinaria of Bologna University for recurrent episodes of seizures due to hypoglycemia with abnormally low plasma insulin levels (18 pmol/l). Resection of a large leiomyoma (780 g) of the gastric wall resulted in a permanent resolution of the hypoglycemic episodes. Insulin-like growth factors I and II (IGF-I and -II) were measured by RIA in serum before and after surgery and in tumor tissue. Results were compared to the serum concentration of 54 normal and to the tissue concentration observed in eight non-hypoglycemic dog gastric wall extracts. Before surgery, circulating immunoreactive IGF-I was 0.92 nmol/l, which is significantly lower than the control values (16.92 +/- 8.44 nmol/l, range 3.53-35.03), while IGF-II was 152 nmol/l, which is significantly higher than the control values (42.21 +/- 3.75, range 31.99-50.74). After surgery, IGF-I increased to 6.80 nmol/l while IGF-II decreased to 45.52 nmol/l. Tumor tissue IGF-II concentration was higher than normal (5.66 nmol/kg tissue as compared to a range in normal gastric wall tissue of 1.14-3.72 nmol/kg), while IGF-I was 0.08 nmol/kg tissue, which is close to the lowest normal value (range in controls, 0.08-1.18 nmol/kg). Partial characterization of IGF-II immunoreactivity extracted from tissue evidenced a molecular weight similar to that of mature IGF-II, thus excluding that peptide released by the tumor is a precursor molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin-immunoreactive neurons in the ventral nerve cord of Remipedia (Crustacea): support for a sister group relationship of Remipedia and Hexapoda?

PubMed Central

2013-01-01

Background Remipedia were initially seen as a primitive taxon within Pancrustacea based on characters considered ancestral, such as the homonomously segmented trunk. Meanwhile, several morphological and molecular studies proposed a more derived position of Remipedia within Pancrustacea, including a sister group relationship to Hexapoda. Because of these conflicting hypotheses, fresh data are crucial to contribute new insights into euarthropod phylogeny. The architecture of individually identifiable serotonin-immunoreactive neurons has successfully been used for phylogenetic considerations in Euarthropoda. Here, we identified neurons in three species of Remipedia with an antiserum against serotonin and compared our findings to reconstructed ground patterns in other euarthropod taxa. Additionally, we traced neurite connectivity and neuropil outlines using antisera against acetylated α-tubulin and synapsin. Results The ventral nerve cord of Remipedia displays a typical rope-ladder-like arrangement of separate metameric ganglia linked by paired longitudinally projecting connectives. The peripheral projections comprise an intersegmental nerve, consisting of two branches that fuse shortly after exiting the connectives, and the segmental anterior and posterior nerve. The distribution and morphology of serotonin-immunoreactive interneurons in the trunk segments is highly conserved within the remipede species we analyzed, which allows for the reconstruction of a ground pattern: two posterior and one anterior pair of serotonin-immunoreactive neurons that possess a single contralateral projection. Additionally, three pairs of immunoreactive neurons are found in the medial part of each hemiganglion. In one species (Cryptocorynetes haptodiscus), the anterior pair of immunoreactive neurons is missing. Conclusions The anatomy of the remipede ventral nerve cord with its separate metameric ganglia mirrors the external morphology of the animal’s trunk. The rope

Immunoreactivity of specific epitopes of PrPSc is enhanced by pretreatment in a hydrated autoclave.

PubMed Central

Yokoyama, T; Momotani, E; Kimura, K; Yuasa, N

1996-01-01

An abnormal protein (PrPSc) accumulates in animals affected with scrapie. Immunoblotting procedures have been used widely to detect PrPSc. Blotted membranes were subjected to pretreatment in a hydrated autoclave, and the subsequent immunoreactivity of PrPSc was examined. The immunoreactivity of PrPSc to antisera against the synthetic peptides of the mouse PrP amino acid sequences 199 to 208 and 213 to 226 was enhanced by the pretreatment. However, the reactivity to antisera of peptide sequences 100 to 115 and 165 to 174 was not affected. The antibody-binding ability of the specific epitopes which are located close to the C-terminal end of PrP27-30 the proteinase-resistant portion of PrPSc, was enhanced by pretreatment in a hydrated autoclave. This pretreatment increased the sensitivity of PrPSc, and it would be useful for diagnosis of scrapie. PMID:8807215

Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrain.

PubMed

Nakamura, Kouichi; Hioki, Hiroyuki; Fujiyama, Fumino; Kaneko, Takeshi

2005-11-21

Vesicular glutamate transporter 1 (VGluT1) and VGluT2 accumulate neurotransmitter glutamate into synaptic vesicles at presynaptic terminals, and their antibodies are thus considered to be a good marker for glutamatergic axon terminals. In the present study, we investigated the postnatal development and maturation of glutamatergic neuronal systems by single- and double-immunolabelings for VGluT1 and VGluT2 in mouse forebrain including the telencephalon and diencephalon. VGluT2 immunoreactivity was widely distributed in the forebrain, particularly in the diencephalon, from postnatal day 0 (P0) to adulthood, suggesting relatively early maturation of VGluT2-loaded glutamatergic axons. In contrast, VGluT1 immunoreactivity was intense only in the limbic regions at P0, and drastically increased in the other telencephalic and diencephalic regions during three postnatal weeks. Interestingly, VGluT1 immunoreactivity was frequently colocalized with VGluT2 immunoreactivity at single axon terminal-like profiles in layer IV of the primary somatosensory area from P5 to P10 and in the ventral posteromedial thalamic nucleus from P0 to P14. This was in sharp contrast to the finding that almost no colocalization was found in glomeruli of the olfactory bulb, patchy regions of the caudate-putamen, and the ventral posterolateral thalamic nucleus, where moderate to intense immunoreactivities for VGluT1 and VGluT2 were intermingled with each other in neuropil during postnatal development. The present results indicate that VGluT2-loaded glutamatergic axons maturate earlier than VGluT1-laden axons in the mouse telencephalic and diencephalic regions, and suggest that VGluT1 plays a transient developmental role in some glutamatergic systems that mainly use VGluT2 in the adulthood. (c) 2005 Wiley-Liss, Inc.

Laminar distribution of GABAA- and glycine-receptor mediated tonic inhibition in the dorsal horn of the rat lumbar spinal cord: effects of picrotoxin and strychnine on expression of Fos-like immunoreactivity.

PubMed

Cronin, John N; Bradbury, Elizabeth J; Lidierth, Malcolm

2004-11-01

Inhibitory mechanisms are essential in suppressing the development of allodynia and hyperalgesia in the normal animal and there is evidence that loss of inhibition can lead to the development of neuropathic pain. We used Fos expression to map the distribution of tonically inhibited cells in the healthy rat lumbar spinal cord. In a control group, Fos-like immunoreactive (Fos-LI) cells were rare, averaging 7.5+/-2.2 cells (mean+/-SEM; N=13 sections) per 20 microm thick section of dorsal horn. This rose to 103+/-11 (mean+/-SEM; N=20) in picrotoxin-treated rats and to 88+/-11 (mean+/-SEM; N=18) in strychnine-treated rats. These changes were significant (ANOVA; P<0.001). There were marked regional variations in the distribution of Fos-LI cells between picrotoxin- and strychnine-treated animals. Picrotoxin induced a significant increase in the number of Fos-LI cells throughout the dorsal horn (lamina I-VI) while strychnine significantly elevated Fos-like immunoreactivity only in deep laminae (III-VI). For both picrotoxin and strychnine, the increase in Fos-like immunoreactivity peaked in lamina V (at 3579+/-319 and 3649+/-375% of control, respectively; mean+/-SEM) but for picrotoxin an additional peak was observed in the outer part of lamina II (1959+/-196%). Intrathecal administration of both GABAA and glycine receptor antagonists has been shown elsewhere to induce tactile allodynia. The present data suggest that this allodynia could arise due to blockade of tonic GABAA and glycine-receptor mediated inhibition in the deep dorsal horn. GABAA antagonists also induce hypersensitivity to noxious inputs. The blockade of tonic inhibition in the superficial dorsal horn shown here may underlie this hyperalgesia.

Effects of sample handling methods on substance P concentrations and immunoreactivity in bovine blood samples.

PubMed

Mosher, Ruby A; Coetzee, Johann F; Allen, Portia S; Havel, James A; Griffith, Gary R; Wang, Chong

2014-02-01

To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples. Blood samples obtained from a healthy 6-month-old calf. Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography-tandem mass spectrometry. Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition. Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

Estimation of Local Orientations in Fibrous Structures With Applications to the Purkinje System

PubMed Central

Plank, Gernot; Trayanova, Natalia A.; Vidal, René

2011-01-01

The extraction of the cardiac Purkinje system (PS) from intensity images is a critical step toward the development of realistic structural models of the heart. Such models are important for uncovering the mechanisms of cardiac disease and improving its treatment and prevention. Unfortunately, the manual extraction of the PS is a challenging and error-prone task due to the presence of image noise and numerous fiber junctions. To deal with these challenges, we propose a framework that estimates local fiber orientations with high accuracy and reconstructs the fibers via tracking. Our key contribution is the development of a descriptor for estimating the orientation distribution function (ODF), a spherical function encoding the local geometry of the fibers at a point of interest. The fiber/branch orientations are identified as the modes of the ODFs via spherical clustering and guide the extraction of the fiber centerlines. Experiments on synthetic data evaluate the sensitivity of our approach to image noise, width of the fiber, and choice of the mode detection strategy, and show its superior performance compared to those of the existing descriptors. Experiments on the free-running PS in an MR image also demonstrate the accuracy of our method in reconstructing such sparse fibrous structures. PMID:21335301

Reduction of IgE immunoreactivity of whole peanut (Arachis hypogaea L.) after pulsed light illumination

USDA-ARS?s Scientific Manuscript database

Pulsed light (PL), a novel food processing and preservation technology, has been shown in literature to reduce allergen levels on peanut, soybean, almond, and shrimp protein extracts. This study investigated how PL affected the immunoreactivity of whole peanut kernels at two sample-to-lamp distance...

The B2 Alternatively Spliced Isoform of Nonmuscle Myosin II-B Lacks Actin-activated MgATPase Activity and In Vitro Motility

PubMed Central

Kim, Kye-Young; Kawamoto, Sachiyo; Bao, Jianjun; Sellers, James R.; Adelstein, Robert S.

2008-01-01

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the non-spliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells (Ma et al., Mol. Biol. Cell 15 (2006) 2138-2149). In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acids II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific. PMID:18060863

Intrathecal treatment with MK-801 suppresses thermal nociceptive responses and prevents c-fos immunoreactivity induced in rat lumbar spinal cord neurons.

PubMed

Huang, W; Simpson, R K

1999-09-01

Sensitization of the second order neurons in the spinal dorsal horn after somatic noxious stimuli is partly mediated by the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor. These neurons also express c-Fos immunoreactivity in response to the somatic noxious stimuli. The present study assessed the influence of intrathecal pre-treatment with MK-801, a non-competitive antagonist of NMDA receptor, on thermal sensitization following peripheral noxious heat stimulation. In addition, the influence of MK-801 on c-Fos immunoreactivity in the rat lumbar spinal cord neurons after the peripheral noxious heat was examined. Sprague-Dawley rats were subject to intrathecal catheterization and administration of MK-801 or saline before and after noxious heat (52 degrees C) stimulation of rat hindpaws. Thermal sensitization was tested after MK-801 (0.1 mumol 10 microliters-1). Fos-like immunoreactivity was evaluated 2 h after noxious stimulation in a separate group of animals. MK-801 significantly increased the thermal withdrawal threshold by 60% following noxious heat stimulation and reduced c-Fos immunoreactivity in the second order neurons by 70% in the dorsal horn. The study suggests that glutamate plays a pivotal role in the thermal nociceptive pathway and indicates that the NMDA receptor is necessary to maintain normal thermal sensitization, possibly by regulating c-fos gene expression in second order neurons.

Effects of electroacupuncture on orphanin FQ immunoreactivity and preproorphanin FQ mRNA in nucleus of raphe magnus in the neuropathic pain rats.

PubMed

Ma, Fei; Xie, Hong; Dong, Zhi-Qiang; Wang, Yan-Qing; Wu, Gen-Cheng

2004-07-15

Orphanin FQ (OFQ) is an endogenous ligand for opioid receptor-like-1 (ORL1) receptor. Previous studies have shown that both OFQ immunoreactivity and preproorphanin FQ (ppOFQ) mRNA expression could be observed in the brain regions involved in pain modulation, e.g., nucleus of raphe magnus (NRM), dorsal raphe nucleus (DRN), and ventrolateral periaqueductal gray (vlPAG). It was reported that electroacupuncture (EA) has analgesic effect on neuropathic pain, and the analgesic effect was mediated by the endogenous opioid peptides. In the present study, we investigated the effects of EA on the changes of OFQ in the neuropathic pain rats. In the sciatic nerve chronic constriction injury (CCI) model, we investigated the changes of ppOFQ mRNA and OFQ immunoreactivity in NRM after EA by in situ hybridization (ISH) and immunohistochemistry methods, respectively. Then, the ppOFQ mRNA-positive and OFQ immunoreactive cells were counted under a computerized image analysis system. The results showed that expression of ppOFQ mRNA decreased and OFQ immunoreactivity increased after EA treatment in the neuropathic pain rats. These results indicated that EA modulated OFQ synthesis and OFQ peptide level in NRM of the neuropathic pain rats. Copyright 2004 Elsevier Inc.

Parabrachial origin of calcitonin gene-related peptide-immunoreactive axons innervating Meynert's basal nucleus.

PubMed

Knyihár-Csillik, E; Boncz, I; Sáry, G; Nemcsók, J; Csillik, B

1999-06-01

Meynert's basal nucleus is innervated by calcitonin gene-related peptide (CGRP)-immunoreactive axons synapsing with cholinergic principal cells. Origin of CGRP-immunopositive axons was studied in the albino rat. Since beaded axons containing the nicotinic acetylcholine receptor (nAChR) are also present in the basal nucleus, the microstructural arrangement raises the question whether or not an interaction between CGRP and nAChR exists like in the neuromuscular junction. We found that electrolytic lesion of the parabrachial nucleus results in degeneration of CGRP-immunoreactive axons in the ipsilateral nucleus basalis and induces shrinkage of principal cholinergic neurons while the contralateral nucleus basalis remains intact. Electrolytic lesions in the thalamus, caudate-putamen, and hippocampus did not induce alterations in Meynert's basal nucleus. Disappearance of CGRP after lesions of the parabrachial nucleus does not impair presynaptic nAChR in the basal nucleus, suggesting that, unlike in the neuromuscular junction, CGRP is not involved in the maintenance of nAChR in the basal forebrain. It is concluded that the parabrachial nucleus is involved in the activation of the nucleus basalis-prefrontal cortex system, essential in gnostic and mnemonic functions. Copyright 1999 Academic Press.

Amaranth addition to enzymatically modified wheat flour improves dough functionality, bread immunoreactivity and quality.

PubMed

Heredia-Sandoval, N G; Calderón de la Barca, A M; Carvajal-Millán, E; Islas-Rubio, A R

2018-01-24

Consumers with gluten-related disorders require gluten-free (GF) foods to avoid an immune response. Alternative to the use of non-gluten containing grains to prepare GF bread, the gluten reactivity has been greatly reduced using a proline specific cleavage enzyme, however, the gluten functionality was lost. The aim of this study was to evaluate the effect of adding an amaranth flour blend (AFB) to enzymatically modified wheat-flour proteins on dough functionality and to evaluate the immunoreactivity and acceptability of the prepared bread. First, wheat flour (20% w/v, substrate) was hydrolyzed using 8.4 U mg -1 protein Aspergillus niger prolyl-endopeptidase (AnPEP) for 8 h at 40 °C under constant agitation. Four types of breads were prepared with the same formulation except for the type of flour (14% w.b.): wheat flour (WF), WF-AFB unmodified not incubated, WF-AFB unmodified incubated and WF-AFB modified. The protein composition and free thiols were analyzed before and after amaranth addition, and the flour and bread proteins were run using SDS-PAGE and immune-detected in blots with IgA from celiac disease patients. The immunoreactive gluten content, specific volume and bread acceptability were evaluated. The polymeric proteins and free thiol groups of WF decreased after AnPEP treatment. The electrophoretic patterns of the modified flour and bread proteins were different and the IgA-immunodetection in blots was highly reduced, particularly for the higher molecular weight subunits. The addition of AFB to the modified wheat flour prepared using AnPEP improved the dough functionality by increasing the thiol groups and allowed the preparation of a sensorially acceptable bread with only 60 mg kg -1 immunoreactive gluten.

Distribution and heterogeneity of immunoreactive cholecystokinin (CCK) in the mucosa of the porcine gastrointestinal tract.

PubMed

Bacarese-Hamilton, A J; Adrian, T E; Bloom, S R

1984-11-01

The concentration and molecular nature of cholecystokinin-like immunoreactivity (CCK-LI) in extracts of porcine intestinal mucosa were determined using sequence-specific radioimmunoassays. Highest CCK concentrations were measured in duodenal mucosa (258 +/- 60 pmol/g in the distal duodenum) followed by jejunal mucosa (204 +/- 36 pmol/g in the proximal jejunum) and pylorus (51 +/- 9 pmol/g). All other gastrointestinal regions proximal to the pylorus and distal to the jejunum contained less than 20 pmol/g. Pancreas contained less than 1 pmol/g. Gel chromatography in 6 M urea revealed four immunoreactive forms and this was confirmed by reverse-phase high-pressure liquid chromatography (HPLC). The predominant molecular form in acid extracts of duodenal mucosa resembled CCK-33 although high concentrations of the larger CCK form ('CCK-58') and of the form intermediate in size between CCK-33 and CCK-8 were measured. A molecular form resembling CCK-8 was the principal form in neutral extracts of the duodenum.

Dental Fluorosis and Catalase Immunoreactivity of the Brain Tissues in Rats Exposed to High Fluoride Pre- and Postnatally.

PubMed

Güner, Şirin; Uyar-Bozkurt, Süheyla; Haznedaroğlu, Eda; Menteş, Ali

2016-11-01

This study evaluated dental fluorosis of the incisors and immunoreactivity in the brain tissues of rats given chronic fluoride doses pre- and postnatally. Female rats were given drinking water with 0, 30 or 100 ppm fluoride ad libitum throughout gestation and the nursing period. In addition, 63 male offspring were treated with the same water regimens as the mothers after weaning and were followed for 1, 3 or 5 months. The upper and lower incisors were collected, and all teeth were examined under a stereomicroscope and scored by two blinded examiners using a modified rodent enamel fluorosis index. Cortical, hippocampal and cerebellar brain samples were evaluated morphologically and immunohistochemically. All fluoride-treated pups were born with low body weight (p = 0.001). All animals from the fluoride groups had enamel fluorosis with defects of various degrees. The increase in the dental fluorosis scores in the fluoride treatment groups was significant (p < 0.01). The catalase immunoreactivity in the 30- and 100-ppm fluoride groups was significantly higher than that in the controls after 1, 3 and 5 months (p < 0.001). In conclusion, this study showed that rats with dental fluorosis had catalase immunoreactivity in the brain tissues, which may reflect the neurobehavioral toxicity of fluoride.

Distribution of calcium channel Ca(V)1.3 immunoreactivity in the rat spinal cord and brain stem.

PubMed

Sukiasyan, N; Hultborn, H; Zhang, M

2009-03-03

The function of local networks in the CNS depends upon both the connectivity between neurons and their intrinsic properties. An intrinsic property of spinal motoneurons is the presence of persistent inward currents (PICs), which are mediated by non-inactivating calcium (mainly Ca(V)1.3) and/or sodium channels and serve to amplify neuronal input signals. It is of fundamental importance for the prediction of network function to determine the distribution of neurons possessing the ion channels that produce PICs. Although the distribution pattern of Ca(V)1.3 immunoreactivity (Ca(V)1.3-IR) has been studied in some specific central nervous regions in some species, so far no systematic investigations have been performed in both the rat spinal cord and brain stem. In the present study this issue was investigated by immunohistochemistry. The results indicated that the Ca(V)1.3-IR neurons were widely distributed across different parts of the spinal cord and the brain stem although with variable labeling intensities. In the spinal gray matter large neurons in the ventral horn (presumably motoneurons) tended to display higher levels of immunoreactivity than smaller neurons in the dorsal horn. In the white matter, a subset of glial cells labeled by an oligodendrocyte marker was also Ca(V)1.3-positive. In the brain stem, neurons in the motor nuclei appeared to have higher levels of immunoreactivity than those in the sensory nuclei. Moreover, a number of nuclei containing monoaminergic cells, for example the locus coeruleus, were also strongly immunoreactive. Ca(V)1.3-IR was consistently detected in the neuronal perikarya regardless of the neuronal type. However, in the large neurons in the spinal ventral horn and the cranial motor nuclei the Ca(V)1.3-IR was clearly detectable in first and second order dendrites. These results indicate that in the rat spinal cord and brain stem Ca(V)1.3 is probably a common calcium channel used by many kinds of neurons to facilitate the neuronal

Reduced cholecystokinin-like and somatostatin-like immunoreactivity in limbic lobe is associated with negative symptoms in schizophrenia.

PubMed

Ferrier, I N; Roberts, G W; Crow, T J; Johnstone, E C; Owens, D G; Lee, Y C; O'Shaughnessy, D; Adrian, T E; Polak, J M; Bloom, S R

1983-08-01

Cholecystokinin-like immunoreactivity (CCK) and somatostatin-like immunoreactivity (SRIF) were determined in fourteen brains from patients dying with a diagnosis of schizophrenia and in twelve brains from control cases. The schizophrenics had been rated during life and were divided into two groups on the basis of the presence or absence of negative symptoms (affective flattening and poverty of speech). CCK was reduced in temporal cortex of the schizophrenics and in hippocampus and amygdala of those patients with negative symptoms. SRIF was reduced in the hippocampus in samples from the latter group. The selectivity of these changes to limbic lobe may reflect the presence of a degenerative process in that area. The association of changes in hippocampus and amygdala with negative symptoms of schizophrenia suggests a separate mechanism underlying these symptoms.

Pregnancy-associated glycoprotein, chymosin and pepsinogen immunoreactivity of proteins extracted from fetal gastric tissue in bovine species.

PubMed

Bella, A; Sousa, N M; Dehimi, M L; Beckers, J F

2012-06-01

The objective of this work was to investigate the expression of gastric aspartic proteinases in fundic and pyloric mucosa removed from bovine fetuses. For this purpose, fractions issued from classical biochemical protocols were analyzed by proteolytic method, by PAG-RIA and by Western blot with the use of antisera raised against both pepsinogens and PAG. A strong reaction of proteins extracted from the fundic mucosa collected at the beginning of pregnancy was revealed with both anti-bPAG-I and anti-bPAG-II antisera, suggesting the expression of pepsinogen F in bovine species. Concerning pyloric mucosa, the analysis by Western blot highlighted a very strong immunoreaction with the anti-bovine chymosin serum. Amino-terminal sequencing allowed to identify bovine fetuin and albumin in fundic extracts, chymosin in the pyloric mucosa extracts, as well as some unknown proteins in both mucosa. Despite no N-terminal microsequence corresponding to the hypothetical pepsinogen F could be identified, it cannot be excluded that an existing bovine pepsinogen F-like molecule could be degraded during the purification procedure or that co-purified proteins could be responsible for masking its N-terminal microsequence. Copyright © 2011 Elsevier Ltd. All rights reserved.

Robust transmission of rate coding in the inhibitory Purkinje cell to cerebellar nuclei pathway in awake mice

PubMed Central

Abbasi, Samira; Maran, Selva K.; Cao, Ying; Abbasi, Ataollah; Heck, Detlef H.

2017-01-01

Neural coding through inhibitory projection pathways remains poorly understood. We analyze the transmission properties of the Purkinje cell (PC) to cerebellar nucleus (CN) pathway in a modeling study using a data set recorded in awake mice containing respiratory rate modulation. We find that inhibitory transmission from tonically active PCs can transmit a behavioral rate code with high fidelity. We parameterized the required population code in PC activity and determined that 20% of PC inputs to a full compartmental CN neuron model need to be rate-comodulated for transmission of a rate code. Rate covariance in PC inputs also accounts for the high coefficient of variation in CN spike trains, while the balance between excitation and inhibition determines spike rate and local spike train variability. Overall, our modeling study can fully account for observed spike train properties of cerebellar output in awake mice, and strongly supports rate coding in the cerebellum. PMID:28617798

Balance and coordination training, but not endurance training, enhances synaptophysin and neurotrophin-3 immunoreactivity in the lumbar spinal cord after sciatic nerve crush.

PubMed

Bonetti, Leandro Viçosa; Ilha, Jocemar; Schneider, Ana Paula Krauthein; Barbosa, Silvia; Faccioni-Heuser, Maria Cristina

2016-04-01

Numerous rehabilitation treatments have been shown to be useful for peripheral and central restoration after (PNI). After sciatic nerve crush, we investigated 4 weeks of endurance training (ET) and balance and coordination training (BCT) with sciatic function index, hind-paw stride length, and spinal cord dorsal horn synaptophysin and neurotrophin-3 immunoreactivity. Our results demonstrated no significant differences between the non-trained (NT), ET, and BCT groups in sciatic functional index, and in stride-length analysis, but the ET showed higher values compared with the NT group. Synaptophysin immunoreactivity was higher in the BCT group compared with the NT group, and neurotrophin-3 immunoreactivity in the BCT group was greater compared with the other groups. BCT can positively affect spinal cord plasticity after a (PNI), and these modifications are important in the rehabilitation process. © 2015 Wiley Periodicals, Inc.

Soymilk fermentation by Enterococcus faecalis VB43 leads to reduction in the immunoreactivity of allergenic proteins β-conglycinin (7S) and glycinin (11S).

PubMed

Biscola, V; de Olmos, A Rodriguez; Choiset, Y; Rabesona, H; Garro, M S; Mozzi, F; Chobert, J-M; Drouet, M; Haertlé, T; Franco, B D G M

2017-08-24

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the β-subunit from β-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.

Natural Immunoreactivity of Secretory IgA to Indigenous Strains of Streptococcus mutans From Chinese Spousal Pairs

PubMed Central

Nie, Min; Chen, Dong; Gao, Zhenyan; Wu, Xinyu; Li, Tong

2016-01-01

Background Dental caries is a well-known biofilm-mediated disease initiated by Streptococcus mutans, which should infect and colonize in a milieu perfused with components of the mucosal immune system. Little is known, however, regarding the relationship between the natural secretory IgA activity and S. mutans of a variety of diverse genotypes. Objectives The current study aimed to use spousal pairs to investigate the natural immunoreactivity of salivary secretory IgA to different genotype strains of S. mutans. Patients and Methods Indigenous strains were characterized from nine spousal pairs using polymerase reaction chain (PCR) and arbitrarily primed polymerase chain reaction (AP-PCR) by genotype monitoring. Unstimulated submandibular/sublingual secretions were collected and the concentrations of secretory IgA were determined by the enzyme-linked immunosorbent assay (ELISA). Each saliva sample was examined by Western blot to analyze the immunoreactivity of naturally occurring salivary secretory IgA antibodies for his/her own indigenous strain, spouse’s strain and reference strains including S. mutans GS-5 and Ingbritt (C). Results The results showed that naturally induced salivary IgA antibodies against S. mutans were present in all subjects. Almost all subjects had the similar individual immunoblotting profiles to different genotype strains. Conclusions The current study indicated that the immunoreactivity of secretory IgA might have no direct correlation with the colonization of indigenous flora and rejection of exogenous strains in adults. The relationship of microbes, host and dental caries should be in the light of coevolved microecosystem as a whole, but not caused by one factor alone. PMID:27303613

SUBFIELD AND LAYER-SPECIFIC DEPLETION IN CALBINDIN-D28K, CALRETININ AND PARVALBUMIN IMMUNOREACTIVITY IN THE DENTATE GYRUS OF APP/PS1 TRANSGENIC MICE

PubMed Central

Popovi, Miroljub; Caballero-Bleda, María; Kadish, Inga; van Groen, Thomas

2008-01-01

The depletion of neuronal calcium binding proteins deprives neurons of the capacity to buffer high levels of intracellular Ca2+ and this leaves them vulnerable to pathological processes, such as those present in Alzheimer’s disease (AD). The aim of the present study was to investigate the expression of the calcium binding proteins, calbindin-D28K, calretinin and parvalbumin in the dentate gyrus (DG) of APP/PS1 transgenic mice and their non-Tg littermates, as well as the relation with the deposition of human Aβ. We measured the expression of these three proteins at seven different rostro-caudal levels, and in the molecular, granular and polymorphic layers of the DG. We found that, except in the most caudal part of the DG, there is a substantial loss of calbindin-D28K immunoreactivity in all three layers of the DG in APP/PS1 mice compared to the non-Tg mice. Significant loss of calretinin immunoreactivity is present in most of the polymorphic layer of the DG of APP/PS1 mice compared to the non-Tg mice, as well as in the rostral and intermediate part of the inner molecular layer. Compared to the non-Tg mice parvalbumin immunoreactivity is significantly reduced throughout the whole polymorphic layer as well as in the rostral and intermediate part of the granular layer of DG in APP/PS1 mice. The relatively preservation of calbindin immunoreactivity in the caudal part of molecular and granular layers as well as calretinin immunoreactivity in the caudal part of polymorphic layer of the DG is likely related to the lower Aβ expression in those parts of DG. The present data suggest an involvement of calcium-dependent pathways in the pathogenesis of AD and indicate that there exists a subfield and layer-specific decrease in immunoreactivity which is related to the type of calcium-binding protein in APP/PS1 mice. Moreover, it seems that APP expression affects more the calbindin expression then parvalbumin and calretinin expression in the DG of APP/PS1 transgenic mice. PMID

Immunoreactivities for glutathione S-transferases and glutathione peroxidase in the lateral wall of pigmented and albino guinea pig cochlea.

PubMed

Fujimura, Takeyuki; Suzuki, Hideaki; Udaka, Tsuyoshi; Shiomori, Teruo; Mori, Takanori; Inaba, Tsuyoshi; Hiraki, Nobuaki; Kayashima, Kotaro; Doi, Yoshiaki

2008-09-01

Dark-skinned people are known to be more tolerant of ototraumatic noise than are light-skinned people, and pigmented animals are more tolerant of ototraumatic noise and aminoglycoside ototoxicity than are albino animals. Such tolerance may be dependent on the local ability of detoxification and antioxidant enzymes, including glutathione S-transferase (GST) and glutathione peroxidase (GSPx). In the present study, we examined the difference in GST/GSPx expression in the lateral wall of the cochlea between pigmented and albino guinea pigs. Eight-week-old male pigmented and albino guinea pigs were killed by transcardiac perfusion with 2% paraformaldehyde. The cochlear ducts were isolated, further fixed with 4% paraformaldehyde, decalcified, and then embedded in paraffin. Sections prepared at 5-microm thickness were incubated with anti-GST-alpha,-mu,-pi, or anti-GSPx antibody, reacted with Alexa Fluorconjugated secondary antibody, and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The cochlea ducts were also subjected to immunoelectron microscopy for GST-pi by the postembedment method. The stria vascularis of pigmented guinea pigs was strongly immunoreactive for GST-alpha,-mu,-pi, and GSPx, whereas no or only weak immunoreactivities were seen in the stria vascularis of albino guinea pigs. The spiral ligament showed positive but different immunoreactivities for these enzymes between the strains. Double-stained immunofluorescence micrographs for GST-pi and GSPx showed a close resemblance of localization between the two enzymes in both pigmented and albino guinea pigs. At the ultrastructural level, immunoreactivity for GST-pi was localized preferentially in the melanin cells of pigmented guinea pigs. These results suggest that correlation between pigmentation and inner ear susceptibility is, at least partially, attributed to the different distribution of GST/GSPx in the stria vascularis.

Palisade endings in extraocular muscles of the monkey are immunoreactive for choline acetyltransferase and vesicular acetylcholine transporter.

PubMed

Konakci, Kadriye Zeynep; Streicher, Johannes; Hoetzenecker, Wolfram; Haberl, Ines; Blumer, Michael Josef Franz; Wieczorek, Grazyna; Meingassner, Josef Gottfried; Paal, Szabolcs Levente; Holzinger, Daniel; Lukas, Julius-Robert; Blumer, Roland

2005-12-01

To analyze palisade endings in extraocular muscles (EOMs) of a primate species and to examine our previous findings in cat that palisade endings are putative effector organs. Eleven monkeys (Macaca fascicularis) of both sexes, between 4 and 6 years of age were analyzed. Whole EOM myotendons were immunostained with four combinations of triple-fluorescent labeling and examined by confocal laser scanning microscopy. Labeling included antibodies against choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neurofilament, and synaptophysin. Muscle fibers were counterstained with phalloidin. Palisade endings were observed in all monkey EOMs. Nerve fibers extended from the muscle into the tendon and looped back to divide into a terminal arborization (palisade ending) around a single muscle fiber tip. In approximately 30% of the cases, nerve fibers supplying palisade endings often established motor terminals outside the palisade complex. Nerve fibers forming palisade endings were ChAT-neurofilament positive. Axonal branches of palisade endings were ChAT-neurofilament positive as well. All palisade nerve terminals exhibited ChAT-synaptophysin immunoreactivity. Within the palisade complex, palisade nerve terminals exhibited VAChT immunoreactivity. All palisade nerve terminals were VAChT-synaptophysin immunoreactive. The results confirm that in the monkey, palisade endings contain acetylcholine and are therefore most likely effector organs. Palisade endings are also present in human EOMs and because of their location at the myotendinous junction, these organs are of crucial interest for strabismus surgery.

Cholecystokinin immunoreactivity in the digestive tract of bowfin (Amia calva), bluegill (Lepomis macrochirus), and bullfrog (Rana catesbeiana).

PubMed

Rajjo, I M; Vigna, S R; Crim, J W

1988-04-01

The distribution of the intestinal hormone, cholecystokinin (CCK), was studied in the gastrointestinal tract of a holostean fish, the bowfin (Amia calva), and compared to a teleostean fish, the bluegill (Lepomis macrochirus), and an amphibian, the bullfrog (Rana catesbeiana), using an antiserum specific for the carboxyl terminal tetrapeptide of CCK in an unlabeled biotin-avidin immunoperoxidase procedure. In the bowfin CCK immunostained cells were detected only in the anterior and mid-intestine; the stomach and the rest of the gastrointestinal tract were negative. Immunoreactive cells were open in appearance and were scattered along the intestinal mucosal epithelium, with cells in mid-intestine relatively more abundant than in anterior intestine. These relative distributions were confirmed by radioimmunoassay of tissue extracts. Additional immunostained cells of uncertain function were detected in the lamina propria of the intestine. In bluegill gut immunoreactive cells were observed in the anterior and mid-intestine and in the pyloric caeca, where cells were clustered near the intestinal opening. Immunoreactive cells occurred relatively uniformly along the anterior and mid-intestine. Bullfrog CCK-containing cells were detected both in the antral stomach and in the duodenum. CCK in gut tissues likely originated in the intestine. The redistribution of CCK cells toward the anterior part of the intestine during evolution coincides with the development of a compact pancreas in higher classes of vertebrates. Such a redistribution constitutes an adaptive placement of endocrine cells for signaling during the intestinal phase of digestion.

Extrabulbar olfactory system and nervus terminalis FMRFamide immunoreactive components in Xenopus laevis ontogenesis.

PubMed

Pinelli, Claudia; D'Aniello, Biagio; Polese, Gianluca; Rastogi, Rakesh K

2004-09-01

The extrabulbar olfactory system (EBOS) is a collection of nerve fibers which originate from primary olfactory receptor-like neurons and penetrate into the brain bypassing the olfactory bulbs. Our description is based upon the application of two neuronal tracers (biocytin, carbocyanine DiI) in the olfactory sac, at the cut end of the olfactory nerve and in the telencephalon of the developing clawed frog. The extrabulbar olfactory system was observed already at stage 45, which is the first developmental stage compatible with our techniques; at this stage, the extrabulbar olfactory system fibers terminated diffusely in the preoptic area. A little later in development, i.e. at stage 50, the extrabulbar olfactory system was maximally developed, extending as far caudally as the rhombencephalon. In the metamorphosing specimens, the extrabulbar olfactory system appeared reduced in extension; caudally, the fiber terminals did not extend beyond the diencephalon. While a substantial overlapping of biocytin/FMRFamide immunoreactivity was observed along the olfactory pathways as well as in the telencephalon, FMRFamide immunoreactivity was never observed to be colocalized in the same cellular or fiber components visualized by tracer molecules. The question whether the extrabulbar olfactory system and the nervus terminalis (NT) are separate anatomical entities or represent an integrated system is discussed.

A spiking network model of cerebellar Purkinje cells and molecular layer interneurons exhibiting irregular firing

PubMed Central

Lennon, William; Hecht-Nielsen, Robert; Yamazaki, Tadashi

2014-01-01

While the anatomy of the cerebellar microcircuit is well-studied, how it implements cerebellar function is not understood. A number of models have been proposed to describe this mechanism but few emphasize the role of the vast network Purkinje cells (PKJs) form with the molecular layer interneurons (MLIs)—the stellate and basket cells. We propose a model of the MLI-PKJ network composed of simple spiking neurons incorporating the major anatomical and physiological features. In computer simulations, the model reproduces the irregular firing patterns observed in PKJs and MLIs in vitro and a shift toward faster, more regular firing patterns when inhibitory synaptic currents are blocked. In the model, the time between PKJ spikes is shown to be proportional to the amount of feedforward inhibition from an MLI on average. The two key elements of the model are: (1) spontaneously active PKJs and MLIs due to an endogenous depolarizing current, and (2) adherence to known anatomical connectivity along a parasagittal strip of cerebellar cortex. We propose this model to extend previous spiking network models of the cerebellum and for further computational investigation into the role of irregular firing and MLIs in cerebellar learning and function. PMID:25520646

Histamine-immunoreactive local neurons in the antennal lobes of the Hymenoptera

PubMed Central

Dacks, Andrew M.; Reisenman, Carolina E.; Paulk, Angelique C.; Nighorn, Alan J.

2010-01-01

Neural networks receive input which is transformed before being sent as output to higher centers of processing. These transformations are often mediated by local interneurons (LNs) that influence output based on activity across the network. In primary olfactory centers, the LNs that mediate these lateral interactions are extremely diverse. For instance, the antennal lobes (ALs) of bumble bees possess both GABA and histamine-immunoreactive (HA-ir) LNs, and both are neurotransmitters associated with fast forms of inhibition. Although the GABAergic network of the AL has been extensively studied, we sought to examine the anatomical features of the HA-ir LNs in relation to the other cellular elements of the bumble bee AL. As a population, HA-ir LNs densely innervate the glomerular core while sparsely arborizing in the outer glomerular rind, overlapping with the terminals of olfactory receptor neurons. Individual fills of HA-ir LNs revealed heavy arborization of the outer ring of a single “principal” glomerulus and sparse arborization in the core of other glomeruli. In contrast, projection neurons, and GABA-immunoreactive LNs project throughout the glomerular volume. To provide insight as to the selective pressures that resulted in the evolution of HA-ir LNs, we determined the phylogenetic distribution of HA-ir LNs in the AL. HA-ir LNs were present in all but the most basal hymenopteran examined, although there were significant morphological differences between major groups within the Hymenoptera. The ALs of other insect taxa examined lacked HA-ir LNs, suggesting that this population of LNs arose within the Hymenoptera and underwent extensive morphological modification. PMID:20533353

Expression of synaptogyrin-1 in T1R2-expressing type II taste cells and type III taste cells of rat circumvallate taste buds.

PubMed

Kotani, Takeshi; Toyono, Takashi; Seta, Yuji; Kitou, Ayae; Kataoka, Shinji; Toyoshima, Kuniaki

2013-09-01

Synaptogyrins are conserved components of the exocytic apparatus and function as regulators of Ca(2+)-dependent exocytosis. The synaptogyrin family comprises three isoforms: two neuronal (synaptogyrin-1 and -3) and one ubiquitous (synaptogyrin-2) form. Although the expression patterns of the exocytic proteins synaptotagmin-1, SNAP-25, synaptobrevin-2 and synaptophysin have been elucidated in taste buds, the function and expression pattern of synaptogyrin-1 in rat gustatory tissues have not been determined. Therefore, we examined the expression patterns of synaptogyrin-1 and several cell-specific markers of type II and III cells in rat gustatory tissues. Reverse transcription/polymerase chain reaction assays and immunoblot analysis revealed the expression of synaptogyrin-1 mRNA and its protein in circumvallate papillae. In fungiform, foliate and circumvallate papillae, the antibody against synaptogyrin-1 immunolabeled a subset of taste bud cells and intra- and subgemmal nerve processes. Double-labeling experiments revealed the expression of synaptogyrin-1 in most taste cells immunoreactive for aromatic L-amino acid decarboxylase and the neural cell adhesion molecule. A subset of synaptogyrin-1-immunoreactive taste cells also expressed phospholipase Cβ2, gustducin, or sweet taste receptor (T1R2). In addition, most synaptogyrin-1-immunoreactive taste cells expressed synaptobrevin-2. These results suggest that synaptogyrin-1 plays a regulatory role in transmission at the synapses of type III cells and is involved in exocytic function with synaptobrevin-2 in a subset of type II cells in rat taste buds.

Motor learning induces plastic changes in Purkinje cell dendritic spines in the rat cerebellum.

PubMed

González-Tapia, D; González-Ramírez, M M; Vázquez-Hernández, N; González-Burgos, I

2017-12-14

The paramedian lobule of the cerebellum is involved in learning to correctly perform motor skills through practice. Dendritic spines are dynamic structures that regulate excitatory synaptic stimulation. We studied plastic changes occurring in the dendritic spines of Purkinje cells from the paramedian lobule of rats during motor learning. Adult male rats were trained over a 6-day period using an acrobatic motor learning paradigm; the density and type of dendritic spines were determined every day during the study period using a modified version of the Golgi method. The learning curve reflected a considerable decrease in the number of errors made by rats as the training period progressed. We observed more dendritic spines on days 2 and 6, particularly more thin spines on days 1, 3, and 6, fewer mushroom spines on day 3, fewer stubby spines on day 1, and more thick spines on days 4 and 6. The initial stage of motor learning may be associated with fast processing of the underlying synaptic information combined with an apparent "silencing" of memory consolidation processes, based on the regulation of the neuronal excitability. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

Change in platelet endothelial cell adhesion molecule-1 immunoreactivity in the dentate gyrus in gerbils fed a folate-deficient diet.

PubMed

Yoo, Ki-Yeon; Hwang, In Koo; Kim, Young Sup; Kwon, Dae Young; Won, Moo Ho

2008-02-01

Folate deficiency increases stroke risk. We examined whether folate deficiency affects platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-associated cell adhesion molecule and mediates the final common pathway of neutrophil transendothelial migration, in blood vessels in the gerbil dentate gyrus after transient forebrain ischemia. Gerbils were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to common carotid artery occlusion for 5 min. In the control diet (CD)- and FAD-treated sham-operated groups, weak PECAM-1 immunoreactivity was detected in the blood vessels located in the dentate gyrus. PECAM-1 immunoreactivity in both groups was increased by 4 days after ischemic insult. PECAM-1 immunoreactivity in the FAD-treated group was twice as high that in the CD-treated-sham-operated group 4 days after ischemic insult. Western blot analyses showed that the change patterns in PECAM-1 protein levels in the dentate gyrus in both groups after ischemic insult were similar to changes in PECAM-1 immunohistochemistry in the ischemic dentate gyrus. Our results suggest that folate deficiency enhances PECAM-1 in the dentate gyrus induced by transient ischemia.

GABA and glutamate immunoreactivity in tentacles of the sea anemone Phymactis papillosa (LESSON 1830).

PubMed

Delgado, Luz M; Couve, Eduardo; Schmachtenberg, Oliver

2010-07-01

Sea anemones have a structurally simple nervous system that controls behaviors like feeding, locomotion, aggression, and defense. Specific chemical and tactile stimuli are transduced by ectodermal sensory cells and transmitted via a neural network to cnidocytes and epithelio-muscular cells, but the nature of the neurotransmitters operating in these processes is still under discussion. Previous studies demonstrated an important role of peptidergic transmission in cnidarians, but during the last decade the contribution of conventional neurotransmitters became increasingly evident. Here, we used immunohistochemistry on light and electron microscopical preparations to investigate the localization of glutamate and GABA in tentacle cross-sections of the sea anemone Phymactis papillosa. Our results demonstrate strong glutamate immunoreactivity in the nerve plexus, while GABA labeling was most prominent in the underlying epithelio-muscular layer. Immunoreactivity for both molecules was also found in glandular epithelial cells, and putative sensory cells were GABA positive. Under electron microscopy, both glutamate and GABA immunogold labeling was found in putative neural processes within the neural plexus. These data support a function of glutamate and GABA as signaling molecules in the nervous system of sea anemones.

Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its low-affinity receptor in cerebellum.

PubMed

Figueiredo, B C; Otten, U; Strauss, S; Volk, B; Maysinger, D

1993-04-16

Deficits or excesses of thyroid hormones during critical periods of mammalian cerebellar development can lead to profound biochemical and morphological abnormalities in this system. The goal of this study was to investigate the effects of perinatal hypo- and hyperthyroidism on the ontogeny of nerve growth factor (NGF) and its low-affinity receptor (p75NGFR) in the rat cerebellum. The concentration of NGF and of p75NGFR immunoreactivity (IR) were measured, several days after birth, in cerebella of rats which had received propylthiouracil (PTU) or thyroxine. NGF concentration was markedly enhanced only on postnatal day 2 (P2) in hyperthyroid rats, whereas in hypothyroid (PTU-treated) rats NGF values were similar to age-matched controls. These observations suggest that thyroid hormone affects NGF synthesis during early periods of cerebellar development. In Purkinje cells of control animals, p75NGFR IR peaked at P10. In hypothyroid rats, the expression of p75NGFR was retarded, peaking at P15, whereas in hyperthyroid rats it was advanced, peaking at P8. The increased p75NGFR IR found in Purkinje cell bodies and the delayed disappearance of p75NGFR IR from the external granular layer of hypothyroid rats suggest different roles for thyroid hormone in the developing cerebellum. We conclude that p75NGFR and NGF are independently regulated by thyroid hormone during critical periods of cerebellar development. The effect of thyroid hormone deficiency on p75NGFR content in Purkinje cells may involve complex mechanisms such as impaired efficiency of axonal transport.

FMRFamide-like immunoreactive nervus terminalis innervation to the pituitary in the catfish, Clarias batrachus (Linn.): demonstration by lesion and immunocytochemical techniques

NASA Technical Reports Server (NTRS)

Krishna, N. S.; Subhedar, N.; Schreibman, M. P.

1992-01-01

Certain thick FMRFamide-like immunoreactive fibers arising from the ganglion cells of nervus terminalis in the olfactory bulb of Clarias batrachus can be traced centripetally through the medial olfactory tract, telencephalon, lateral preoptic area, tuberal area, and hypothalamohypophysial tract to the pituitary. Following 6 days of bilateral olfactory tract transection, the immunoreactivity in the thick fibers, caudal to the lesion site, was partially eliminated, whereas after 10 and 14 days, it was totally abolished in the processes en route to the pituitary. The results indicate a direct innervation of the pituitary gland by the FMRFamide-like peptide containing fibers of the nervus terminalis.

Immunoreactive "calcitonin-like" material in heroin addicts: varying reactivity with different antibodies.

PubMed

Tagliaro, F; Dorizzi, R; Ghielmi, S; Comberti, E; Alverà, P; Manzato, E; Marigo, M

1992-01-01

High levels of immunoreactive calcitonin (iCT) in the blood of heroin addicts were previously reported. As it is well known that multiple forms of calcitonin exist in the blood and in tissues, the purpose of the present study was to investigate the immunological nature of the CT-like immunoreactive material found in the blood of these subjects. We investigated 25 addicts, who had been using heroin for more than one year and were hospitalized for a 2 week detoxication program. Blood samples were drawn at the start of the program (when the subjects were still on heroin) and after 5 and 12 days of abstinence from heroin. Twenty-five healthy subjects served as controls. We used 2 commercial RIA kits, calibrated against the same reference material (WHO 70-234), but employing different antisera. One antiserum substantially confirmed the previous findings of increased levels of calcitonin during heroin use, but the other seemed to exclude any change in the hormone concentrations. This suggests that the "calcitonin like" material found in heroin addicts contains some epitopes similar to those found in the calcitonin standard which are detected by the first antiserum. However it lacks other epitopes which are also present in calcitonin standard and which are recognized by the second antiserum. Therefore, this substance seems to be different from the standard human calcitonin 1-32. A possible involvement of a calcitonin analogue (precursor or metabolite) in the biochemical changes occurring during chronic opiate use is suggested.

Immunoreactive Proteins of Bifidobacterium longum ssp. longum CCM 7952 and Bifidobacterium longum ssp. longum CCDM 372 Identified by Gnotobiotic Mono-Colonized Mice Sera, Immune Rabbit Sera and Non-immune Human Sera

PubMed Central

Górska, Sabina; Dylus, Ewa; Rudawska, Angelika; Brzozowska, Ewa; Srutkova, Dagmar; Schwarzer, Martin; Razim, Agnieszka; Kozakova, Hana; Gamian, Andrzej

2016-01-01

The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited. Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK (B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase (B. longum ssp. longum CCDM 372). PMID:27746766

Immunoreactive Proteins of Bifidobacterium longum ssp. longum CCM 7952 and Bifidobacterium longum ssp. longum CCDM 372 Identified by Gnotobiotic Mono-Colonized Mice Sera, Immune Rabbit Sera and Non-immune Human Sera.

PubMed

Górska, Sabina; Dylus, Ewa; Rudawska, Angelika; Brzozowska, Ewa; Srutkova, Dagmar; Schwarzer, Martin; Razim, Agnieszka; Kozakova, Hana; Gamian, Andrzej

2016-01-01

The Bifidobacteria show great diversity in the cell surface architecture which may influence the physicochemical properties of the bacterial cell and strain specific properties. The immunomodulatory role of bifidobacteria has been extensively studied, however studies on the immunoreactivity of their protein molecules are very limited. Here, we compared six different methods of protein isolation and purification and we report identification of immunogenic and immunoreactive protein of two human Bifidobacterium longum ssp. longum strains. We evaluated potential immunoreactive properties of proteins employing polyclonal sera obtained from germ free mouse, rabbit and human. The protein yield was isolation method-dependent and the reactivity of proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and have them sequenced. Among the immunoreactive proteins we identified enolase, aspartokinase, pyruvate kinase, DnaK ( B. longum ssp. longum CCM 7952) and sugar ABC transporter ATP-binding protein, phosphoglycerate kinase, peptidoglycan synthethase penicillin-binding protein 3, transaldolase, ribosomal proteins and glyceraldehyde 3-phosphate dehydrogenase ( B. longum ssp. longum CCDM 372).

Quantitative changes of GABA-immunoreactive cells in the hindlimb representation of the rat somatosensory cortex after 14-day hindlimb unloading by tail suspension

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Fox, R. A.; Wu, L. C.; Daunton, N. G.

1996-01-01

The present study was aimed at evaluating quantitatively gamma-aminobutyric acid (GABA) immunoreactivity in the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension. A reduction in the number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-containing terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system of hindlimb-suspended animals, it is suggested that the unloading due to hindlimb suspension alters afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the reduction in immunoreactivity of local circuit GABAergic neurons and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.

Central cardiovascular action of urotensin II in spontaneously hypertensive rats.

PubMed

Lin, Yingzi; Tsuchihashi, Takuya; Matsumura, Kiyoshi; Fukuhara, Masayo; Ohya, Yusuke; Fujii, Koji; Iida, Mitsuo

2003-10-01

We have previously reported that urotensin II acts on the central nervous system to increase blood pressure in normotensive rats. In the present study, we have determined the central cardiovascular action of urotensin II in spontaneously hypertensive rats (SHR). Intracerebroventricular (ICV) injection of urotensin II elicited a dose-dependent increase in blood pressure in both SHR and normotensive Wistar-Kyoto rats (WKY). The changes in mean arterial pressure induced by ICV urotensin II at doses of 1 and 10 nmol in the WKY were 8 +/- 2 and 23 +/- 3 mmHg, respectively. ICV administration of urotensin II caused significantly greater increases in blood pressure in SHR (16 +/- 3 mmHg at 1 nmol and 35 +/- 3 mmHg at 10 nmol, respectively) compared with those in WKY. Urotensin II (10 nmol) elicited significant and comparable increases in heart rate in SHR (107 +/- 10 bpm) and WKY (101 +/- 21 bpm). Plasma epinephrine concentrations after ICV administration of 10 nmol urotensin II were 203 +/- 58 pmol/ml in SHR and 227 +/- 47 pmol/ml in WKY, which tended to be higher than those in artificial cerebrospinal fluid-injected rats (73+/- 7 and 87 +/- 28 pmol/ml, respectively, p < 0.1). The immunoreactivity of urotensin II receptor GPR 14 was expressed extensively in the glial cells within the brainstem, hypothalamus, and thalamus. These results suggest that central urotensin II may play a role in the pathogenesis of hypertension in SHR. Since GPR 14 was expressed in the glial cells of the brain, urotensin II may act as a neuromodulator to regulate blood pressure.

Immunocytochemical localization of glutamic acid decarboxylase (GAD) and substance P in neural areas mediating motion-induced emesis: Effects of vagal stimulation on GAD immunoreactivity

NASA Technical Reports Server (NTRS)

Damelio, F.; Gibbs, M. A.; Mehler, W. R.; Daunton, Nancy G.; Fox, Robert A.

1991-01-01

Immunocytochemical methods were employed to localize the neurotransmitter amino acid gamma-aminobutyric acid (GABA) by means of its biosynthetic enzyme glutamic acid decarboxylase (GAD) and the neuropeptide substance P in the area postrema (AP), area subpostrema (ASP), nucleus of the tractus solitarius (NTS), and gelatinous nucleus (GEL). In addition, electrical stimulation was applied to the night vagus nerve at the cervical level to assess the effects on GAD-immunoreactivity (GAR-IR). GAD-IR terminals and fibers were observed in the AP, ASP, NTS, and GEL. They showed pronounced density at the level of the ASP and gradual decrease towards the solitary complex. Nerve cells were not labelled in our preparations. Ultrastructural studies showed symmetric or asymmetric synaptic contracts between labelled terminals and non-immunoreactive dendrites, axons, or neurons. Some of the labelled terminals contained both clear- and dense-core vesicles. Our preliminary findings, after electrical stimulation of the vagus nerve, revealed a bilateral decrease of GAD-IR that was particularly evident at the level of the ASP. SP-immunoreactive (SP-IR) terminals and fibers showed varying densities in the AP, ASP, NTS, and GEL. In our preparations, the lateral sub-division of the NTS showed the greatest accumulation. The ASP showed medium density of immunoreactive varicosities and terminals and the AP and GEL displayed scattered varicose axon terminals. The electron microscopy revealed that all immunoreactive terminals contained clear-core vesicles which make symmetric or asymmetric synaptic contact with unlabelled dendrites. It is suggested that the GABAergic terminals might correspond to vagal afferent projections and that GAD/GABA and substance P might be co-localized in the same terminal allowing the possibility of a regulated release of the transmitters in relation to demands.

Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

PubMed

Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

2004-11-01

GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

Secretin and autism: a basic morphological study about the distribution of secretin in the nervous system.

PubMed

Köves, Katalin; Kausz, Mária; Reser, Diana; Illyés, György; Takács, József; Heinzlmann, Andrea; Gyenge, Eszter; Horváth, Károly

2004-12-15

For the first time, the relationship between secretin and autism has been demonstrated by one of us. Intravenous administration of secretin in autistic children caused a fivefold higher pancreaticobiliary fluid secretion than in healthy ones and, at least in some of the patients, better mental functions were reported after the secretin test. Because the precise localization of secretin in the brain is still not completely known, the abovementioned observation led us to map secretin immunoreactivity in the nervous system of several mammalian species. In the present work, the distribution of secretin immunoreactivity in cat and human nervous systems was compared with that of rats using an immunohistochemical approach. Secretin immunoreactivity was observed in the following brain structures of both humans and in colchicine-treated rats: (1) Purkinje cells in the cerebellar cortex; (2) central cerebellar nuclei; (3) pyramidal cells in the motor cortex; and (4) primary sensory neurons. Additionally, secretin immnoreactive cells were observed in the human hippocampus and amygdala and in third-order sensory neurons of the rat auditory system. In cats, secretin was only observed in the spinal ganglia. Our findings support the view that secretin is not only a gastrointestinal peptide but that it is also a neuropeptide. Its presence or the lack of its presence may have a role in the development of behavioral disorders.

Suppression of amyloid beta A11 antibody immunoreactivity by vitamin C: possible role of heparan sulfate oligosaccharides derived from glypican-1 by ascorbate-induced, nitric oxide (NO)-catalyzed degradation.

PubMed

Cheng, Fang; Cappai, Roberto; Ciccotosto, Giuseppe D; Svensson, Gabriel; Multhaup, Gerd; Fransson, Lars-Åke; Mani, Katrin

2011-08-05

Amyloid β (Aβ) is generated from the copper- and heparan sulfate (HS)-binding amyloid precursor protein (APP) by proteolytic processing. APP supports S-nitrosylation of the HS proteoglycan glypican-1 (Gpc-1). In the presence of ascorbate, there is NO-catalyzed release of anhydromannose (anMan)-containing oligosaccharides from Gpc-1-nitrosothiol. We investigated whether these oligosaccharides interact with Aβ during APP processing and plaque formation. anMan immunoreactivity was detected in amyloid plaques of Alzheimer (AD) and APP transgenic (Tg2576) mouse brains by immunofluorescence microscopy. APP/APP degradation products detected by antibodies to the C terminus of APP, but not Aβ oligomers detected by the anti-Aβ A11 antibody, colocalized with anMan immunoreactivity in Tg2576 fibroblasts. A 50-55-kDa anionic, sodium dodecyl sulfate-stable, anMan- and Aβ-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C terminus). anMan-containing HS oligo- and disaccharide preparations modulated or suppressed A11 immunoreactivity and oligomerization of Aβ42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by 3-β[2(diethylamino)ethoxy]androst-5-en-17-one (U18666A) and decreased when Gpc-1 autoprocessing was stimulated by ascorbate. Neither overexpression of Gpc-1 in Tg2576 fibroblasts nor addition of copper ion and NO donor to hippocampal slices from 3xTg-AD mice affected A11 immunoreactivity levels. However, A11 immunoreactivity was greatly suppressed by the subsequent addition of ascorbate. We speculate that temporary interaction between the Aβ domain and small, anMan-containing oligosaccharides may preclude formation of toxic Aβ oligomers. A portion of the oligosaccharides are co-secreted with the Aβ peptides and deposited in plaques. These results support the notion that an inadequate supply of vitamin C could contribute to late onset AD

Aquaporin-4 immunoreactivity in Müller and amacrine cells of marine teleost fish retina.

PubMed

Hombrebueno, José R; Lee, Eun-Jin; Martínez-Ruiz, Noemí; García-Alcázar, Alicia; Grzywacz, Norberto M; De Juan, Joaquín

2012-01-13

Aquaporins (AQPs) are membrane proteins that facilitate water transport across biological membranes and are essential for the proper function of neural tissue. Although AQPs have been extensively studied in mammalian retina, their presence in lower vertebrate retina is less frequently characterized. AQP4 expressed in mammalian and chick Müller cells plays a major part in maintaining retinal homeostasis. In this study, we examined the immunoreactivity of AQP4 in the adult retina of gilthead sea bream (Sparus aurata-teleost fish), during light and dark adaptation. The AQP4 expression was detected in Müller cell somas at the inner nuclear layer and in the end-feet processes near the vitreoretinal border. Moreover, AQP4 was also evident in cone photoreceptor cells and in a GABAergic subpopulation of amacrine cells (AQP4-ACs). Four different types of AQP4-ACs were characterized based on their morphology and dendrite stratification. Interestingly, a stronger AQP4 immunoreactivity was observed in the inner nuclear layer during dark adaptation, accompanied by a significant increment in AQP4-ACs cell size. Hence, AQP4 may play an important role in water distribution in the teleost fish retina. Copyright © 2011 Elsevier B.V. All rights reserved.

Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje neurons to Bergmann glia.

PubMed

Sawada, Yusuke; Konno, Ayumu; Nagaoka, Jun; Hirai, Hirokazu

2016-06-13

Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.

Selective activation of group III metabotropic glutamate receptor subtypes produces different patterns of γ-aminobutyric acid immunoreactivity and glutamate release in the retina.

PubMed

Guimarães-Souza, E M; Calaza, K C

2012-12-01

Glutamate, the major excitatory neurotransmitter in the retina, functions by activation of both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. Group III mGluRs, except for mGluR6, are mostly found in the inner plexiform layer (IPL), and their retinal functions are not well known. Therefore, we decided to investigate the effect of mGluRIII on glutamate release and GABAergic amacrine cells in the chick retina. The nonselective mGluRIII agonist L-SOP promoted a decrease in the number of γ-aminobutyric acid (GABA)-positive cells and in the GABA immunoreactivity in all sublayers of the IPL. This effect was prevented by the antagonist MAP-4, by GAT-1 inhibitor, and by antagonists of iGluR. Under the conditions used, L-SOP did not alter endogenous glutamate release. VU0155041, an mGluR4-positive allosteric modulator, reduced GABA immunoreactivity in amacrine cells and in sublayers 2 and 4 of the IPL but evoked an increase in the glutamate released. VU0155041's effect was inhibited by the absence of calcium. AMN082, a selective mGluR7-positive allosteric modulator, also decreased GABA immunoreactivity in amacrine cells and sublayers 1, 2, and 3 and increased glutamate release, and this effect was also inhibited by calcium absence. DCPG, an mGluR8-selective agonist, did not significantly alter GABA immunoreactivity in amacrine cells or glutamate release. However, it did significantly increase GABA immunoreactivity in sublayers 4 and 5. The results suggest that mGluRIIIs are involved in the modulation of glutamate and GABA release in the retina, possibly participating in distinct visual pathways: mGluR4 might be involved with cholinergic circuitry, whereas mGluR7 and mGluR8 might participate, respectively, in the OFF and the ON pathways. Copyright © 2012 Wiley Periodicals, Inc.

The nervus terminalis in the chick: a FMRFamide-immunoreactive and AChE-positive nerve.

PubMed

Wirsig-Wiechmann, C R

1990-07-16

The chick terminal nerve (TN) was examined by immunocytochemical and histochemical methods. Molluscan cardioexcitatory peptide-immunoreactive (FMRFamide-ir) and acetylcholinesterase (AChE)-positive TN perikarya and fibers were distributed along olfactory and trigeminal nerves. FMRFamide-ir TN fibers terminated in the olfactory lamina propria and epithelium and in ganglia along the rostroventral nasal septum. This initial description of several populations of avian TN neurons should provide the foundation for future developmental studies of this system.

Unique Cardiac Purkinje Fiber Transient Outward Current β-Subunit Composition

PubMed Central

Xiao, Ling; Koopmann, Tamara T.; Ördög, Balázs; Postema, Pieter G.; Verkerk, Arie O.; Iyer, Vivek; Sampson, Kevin J.; Boink, Gerard J.J.; Mamarbachi, Maya A.; Varro, Andras; Jordaens, Luc; Res, Jan; Kass, Robert S.; Wilde, Arthur A.; Bezzina, C.R.; Nattel, Stanley

2015-01-01

Rationale A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current (Ito) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF Ito and that its overexpression might specifically alter PF Ito properties and repolarization. Objective To assess the potential role of DPP6 in PF Ito. Methods and Results Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle Ito had similar density, but PF Ito differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, Ito density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K+-channel interacting β-subunit K+-channel interacting protein type-2, essential for normal expression of Ito in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small Ito; Ito amplitude was greatly enhanced by coexpression with K+-channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K+-channel interacting protein type-2 failed to alter Ito compared with Kv4.3/K+-channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF Ito composition) greatly enhanced Ito compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that Ito enhancement can greatly accelerate PF repolarization. Conclusions These results point to a previously unknown central role of DPP6 in PF Ito, with DPP6 gain of function

Nicotinic receptor abnormalities in the cerebellar cortex in autism.

PubMed

Lee, M; Martin-Ruiz, C; Graham, A; Court, J; Jaros, E; Perry, R; Iversen, P; Bauman, M; Perry, E

2002-07-01

Autism is a common developmental disorder associated with structural and inferred neurochemical abnormalities of the brain. Cerebellar abnormalities frequently have been identified, based on neuroimaging or neuropathology. Recently, the cholinergic neurotransmitter system has been implicated on the basis of nicotinic receptor loss in the cerebral cortex. Cerebellar cholinergic activities were therefore investigated in autopsy tissue from a series of autistic individuals. The presynaptic cholinergic enzyme, choline acetyltransferase, together with nicotinic and muscarinic receptor subtypes were compared in the cerebellum from age-matched mentally retarded autistic (eight), normal control (10) and non-autistic mentally retarded individuals (11). The nicotinic receptor binding the agonist epibatidine (the high affinity receptor subtype, consisting primarily of alpha3 and alpha4, together with beta2 receptor subunits) was significantly reduced by 40-50% in the granule cell, Purkinje and molecular layers in the autistic compared with the normal group (P < 0.05). There was an opposite increase (3-fold) in the nicotinic receptor binding alpha-bungarotoxin (to the alpha7 subunit) which reached significance in the granule cell layer (P < 0.05). These receptor changes were paralleled by a significant reduction (P < 0.05) and non-significant increase, respectively, of alpha4 and alpha7 receptor subunit immunoreactivity measured using western blotting. Immunohistochemically loss of alpha(4 )reactivity was apparent from Purkinje and the other cell layers, with increased alpha7 reactivity in the granule cell layer. There were no significant changes in choline acetyltransferase activity, or in muscarinic M1 and M2 receptor subtypes in autism. In the non-autistic mentally retarded group, the only significant abnormality was a reduction in epibatidine binding in the granule cell and Purkinje layers. In two autistic cases examined histologically, Purkinje cell loss was observed in

The recovery of 5-HT transporter and 5-HT immunoreactivity in injured rat spinal cord.

PubMed

Saruhashi, Yasuo; Matsusue, Yoshitaka; Fujimiya, Mineko

2009-09-01

Experimental spinal cord injury. To determine the role of serotonin (5-HT) and 5-HT transporter in recovery from spinal cord injury. We examined 5-HT and 5-HT transporter of spinal cord immunohistologically and assessed locomotor recovery after extradural compression at the thoracic (T8) spinal cord in 21 rats. Eighteen rats had laminectomy and spinal cord injury, while the remaining three rats received laminectomy only. All rats were evaluated every other day for 4 weeks, using a 0-14 point scale open field test. Extradural compression markedly reduced mean hindlimbs scores from 14 to 1.5 +/- 2.0 (mean +/- standard error of mean). The rats recovered apparently normal walking by 4 weeks. The animals were perfused with fixative 1-3 days, 1, 2 and 4 weeks (three rats in each) after a spinal cord injury. The 5-HT transporter immunohistological study revealed a marked reduction of 5-HT transporter-containing terminals by 1 day after injury. By 4 weeks after injury, 5-HT transporter immunoreactive terminals returned to the control level. The 5-HT immunohistological study revealed a reduction of 5-HT-containing terminals by 1 week after injury. By 4 weeks after injury, 5-HT immunoreactive fibers and terminals returned to the control level. We estimated the recovery of 5-HT transporter and 5-HT neural elements in lumbosacral ventral horn by ranking 5-HT transporter and 5-HT staining intensity and counting 5-HT and 5-HT transporter terminals. The return of 5-HT transporter and 5-HT immunoreactivity of the lumbosacral ventral horn correlated with locomotor recovery, while 5-HT transporter showed closer relationship with locomotor recovery than 5-HT. The presence of 5-HT transporter indicates that the 5-HT fibers certainly function. This study shows that return of the function of 5-HT fibers predict the time course and extent of locomotory recovery after thoracic spinal cord injury.

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies

PubMed Central

Sun, Ning; Shibata, Brad; Hess, John F.

2015-01-01

Purpose Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. Methods A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. Results An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ

Reduced expression of the Ca(2+) transporter protein PMCA2 slows Ca(2+) dynamics in mouse cerebellar Purkinje neurones and alters the precision of motor coordination.

PubMed

Empson, Ruth M; Turner, Paul R; Nagaraja, Raghavendra Y; Beesley, Philip W; Knöpfel, Thomas

2010-03-15

Cerebellar Purkinje neurones (PNs) express high levels of the plasma membrane calcium ATPase, PMCA2, a transporter protein critical for the clearance of calcium from excitable cells. Genetic deletion of one PMCA2 encoding gene in heterozygous PMCA2 knock-out (PMCA2(+/-) mice enabled us to determine how PMCA2 influences PN calcium regulation without the complication of the severe morphological changes associated with complete PMCA2 knock-out (PMCA2(-/-) in these cells. The PMCA2(+/-) cerebellum expressed half the normal levels of PMCA2 and this nearly doubled the time taken for PN dendritic calcium transients to recover (mean fast and slow recovery times increased from 70 ms to 110 ms and from 600 ms to 1100 ms). The slower calcium recovery had distinct consequences for PMCA2(+/-) PN physiology. The PNs exhibited weaker climbing fibre responses, prolonged outward Ca(2+)-dependent K(+) current (mean fast and slow recovery times increased from 136 ms to 192 ms and from 595 ms to 1423 ms) and a slower mean frequency of action potential firing (7.4 Hz compared with 15.8 Hz). Our findings were consistent with prolonged calcium accumulation in the cytosol of PMCA2(+/-) Purkinje neurones. Although PMCA2(+/-) mice exhibited outwardly normal behaviour and little change in their gait pattern, when challenged to run on a narrow beam they exhibited clear deficits in hindlimb coordination. Training improved the motor performance of both PMCA2(+/-) and wild-type mice, although PMCA2(+/-) mice were always impaired. We conclude that reduced calcium clearance perturbs calcium dynamics in PN dendrites and that this is sufficient to disrupt the accuracy of cerebellar processing and motor coordination.

Effects of paternal deprivation on cocaine-induced behavioral response and hypothalamic oxytocin immunoreactivity and serum oxytocin level in female mandarin voles.

PubMed

Wang, Jianli; Fang, Qianqian; Yang, Chenxi

2017-09-15

Early paternal behavior plays a critical role in behavioral development in monogamous species. The vast majority of laboratory studies investigating the influence of parental behavior on cocaine vulnerability focus on the effects of early maternal separation. However, comparable studies on whether early paternal deprivation influences cocaine-induced behavioral response are substantially lacking. Mandarin vole (Microtus mandarinus) is a monogamous rodent with high levels of paternal care. After mandarin vole pups were subjected to early paternal deprivation, acute cocaine- induced locomotion, anxiety- like behavior and social behavior were examined in 45day old female pups, while hypothalamic oxytocin immunoreactivity and serum oxytocin level were also assessed. We found that cocaine increased locomotion and decreased social investigation, contact behavior and serum oxytocin level regardless of paternal care. Cocaine increased anxiety levels and decreased oxytocin immunoreactive neurons of the paraventricular nuclei and supraoptic nuclei in the bi-parental care group, whilst there were no specific effects in the paternal deprivation group. These results indicate that paternal deprivation results in different behavioral response to acute cocaine exposure in adolescents, which may be in part associated with the alterations in oxytocin immunoreactivity and peripheral OT level. Copyright © 2017 Elsevier B.V. All rights reserved.

Six commercially available angiotensin II AT1 receptor antibodies are non-specific.

PubMed

Benicky, Julius; Hafko, Roman; Sanchez-Lemus, Enrique; Aguilera, Greti; Saavedra, Juan M

2012-11-01

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization.

Six Commercially Available Angiotensin II AT1 Receptor Antibodies are Non-specific

PubMed Central

Benicky, Julius; Hafko, Roman; Sanchez-Lemus, Enrique; Aguilera, Greti

2012-01-01

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization. PMID:22843099

Brainstem and cervical spinal cord Fos immunoreactivity evoked by nerve growth factor injection into neck muscles in mice.

PubMed

Panfil, C; Makowska, A; Ellrich, J

2006-02-01

Although myofascial tenderness is thought to play a key role in the pathophysiology of tension-type headache, very few studies have addressed neck muscle nociception. The neuronal activation pattern following local nerve growth factor (NGF) administration into semispinal neck muscles in anaesthetized mice was investigated using Fos protein immunohistochemistry. In order to differentiate between the effects of NGF administration on c-fos expression and the effects of surgical preparation, needle insertion and intramuscular injection, the experiments were conducted in three groups. In the sham group (n=7) cannula needles were only inserted without any injection. In the saline (n=7) and NGF groups (n=7) 0.9% physiological saline solution or 0.8 microm NGF solution were injected in both muscles, respectively. In comparison with sham and saline conditions, NGF administration induced significantly stronger Fos immunoreactivity in the mesencephalic periaqueductal grey (PAG), the medullary lateral reticular nucleus (LRN), and superficial layers I and II of cervical spinal dorsal horns C1, C2 and C3. This activation pattern corresponds very well to central nervous system processing of deep noxious input. A knowledge of the central anatomical representation of neck muscle pain is an essential prerequisite for the investigation of neck muscle nociception in order to develop a future model of tension-type headache.

Light microscopic image analysis system to quantify immunoreactive terminal area apposed to nerve cells.

PubMed

Wu, L C; D'Amelio, F; Fox, R A; Polyakov, I; Daunton, N G

1997-06-06

The present report describes a desktop computer-based method for the quantitative assessment of the area occupied by immunoreactive terminals in close apposition to nerve cells in relation to the perimeter of the cell soma. This method is based on Fast Fourier Transform (FFT) routines incorporated in NIH-Image public domain software. Pyramidal cells of layer V of the somatosensory cortex outlined by GABA immunolabeled terminals were chosen for our analysis. A Leitz Diaplan light microscope was employed for the visualization of the sections. A Sierra Scientific Model 4030 CCD camera was used to capture the images into a Macintosh Centris 650 computer. After preprocessing, filtering was performed on the power spectrum in the frequency domain produced by the FFT operation. An inverse FFT with filter procedure was employed to restore the images to the spatial domain. Pasting of the original image to the transformed one using a Boolean logic operation called 'AND'ing produced an image with the terminals enhanced. This procedure allowed the creation of a binary image using a well-defined threshold of 128. Thus, the terminal area appears in black against a white background. This methodology provides an objective means of measurement of area by counting the total number of pixels occupied by immunoreactive terminals in light microscopic sections in which the difficulties of labeling intensity, size, shape and numerical density of terminals are avoided.

Light microscopic image analysis system to quantify immunoreactive terminal area apposed to nerve cells

NASA Technical Reports Server (NTRS)

Wu, L. C.; D'Amelio, F.; Fox, R. A.; Polyakov, I.; Daunton, N. G.

1997-01-01

The present report describes a desktop computer-based method for the quantitative assessment of the area occupied by immunoreactive terminals in close apposition to nerve cells in relation to the perimeter of the cell soma. This method is based on Fast Fourier Transform (FFT) routines incorporated in NIH-Image public domain software. Pyramidal cells of layer V of the somatosensory cortex outlined by GABA immunolabeled terminals were chosen for our analysis. A Leitz Diaplan light microscope was employed for the visualization of the sections. A Sierra Scientific Model 4030 CCD camera was used to capture the images into a Macintosh Centris 650 computer. After preprocessing, filtering was performed on the power spectrum in the frequency domain produced by the FFT operation. An inverse FFT with filter procedure was employed to restore the images to the spatial domain. Pasting of the original image to the transformed one using a Boolean logic operation called 'AND'ing produced an image with the terminals enhanced. This procedure allowed the creation of a binary image using a well-defined threshold of 128. Thus, the terminal area appears in black against a white background. This methodology provides an objective means of measurement of area by counting the total number of pixels occupied by immunoreactive terminals in light microscopic sections in which the difficulties of labeling intensity, size, shape and numerical density of terminals are avoided.

Microbial transglutaminase treatment in pasta-production does not affect the immunoreactivity of gliadin with celiac disease patients' sera.

PubMed

Ruh, Tobias; Ohsam, Jürgen; Pasternack, Ralf; Yokoyama, Keiichi; Kumazawa, Yoshiyuki; Hils, Martin

2014-07-30

The effect of microbial transglutaminase (MTG)-treatment of pasta-dough on the immunoreactivity with celiac disease patient's sera has been investigated. Modification by MTG has been proven by determination of the MTG reaction product ε-(γ-glutamyl)lysine (3.63 μmol/g protein), which was not detectable in non-MTG-treated pasta. Antigenicity has been analyzed by immunoblotting and ELISA using gliadin-extracts from pasta and MTG-treated pasta. Immunoblotting showed that the antibody-population (antigliadin antibodies and antideamidated gliadin antibodies) of the sera is specific for every individual patient. Immunoblotting and ELISA showed that there is no difference in immunoreactivity of gliadin extracted from pasta and MTG-pasta. Recognition pattern and intensity in Western blot as well as antibody titer has also been identical even for sera with a high antideamidated gliadin antibody titer. These results indicate no difference between pasta-gliadin and MTG-pasta-gliadin and especially no increased deamidation in pasta-gliadin by MTG-treatment.

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

PubMed

Tsai, Peter T; Hull, Court; Chu, YunXiang; Greene-Colozzi, Emily; Sadowski, Abbey R; Leech, Jarrett M; Steinberg, Jason; Crawley, Jacqueline N; Regehr, Wade G; Sahin, Mustafa

2012-08-30

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Increased serotonin axons (immunoreactive to 5-HT transporter) in postmortem brains from young autism donors.

PubMed

Azmitia, Efrain C; Singh, Jorawer S; Whitaker-Azmitia, Patricia M

2011-06-01

Imaging studies of serotonin transporter binding or tryptophan retention in autistic patients suggest that the brain serotonin system is decreased. However, treatment with drugs which increase serotonin (5-HT) levels, specific serotonin reuptake inhibitors (SSRIs), commonly produce a worsening of the symptoms. In this study we examined 5-HT axons that were immunoreactive to a serotonin transporter (5-HTT) antibody in a number of postmortem brains from autistic patients and controls with no known diagnosis who ranged in age from 2 to 29 years. Fine, highly branched, and thick straight fibers were found in forebrain pathways (e.g. medial forebrain bundle, stria terminalis and ansa lenticularis). Many immunoreactive varicose fine fibers were seen in target areas (e.g. globus pallidus, amygdala and temporal cortex). Morphometric analysis of the stained axons at all ages studied indicated that the number of serotonin axons was increased in both pathways and terminal regions in cortex from autism donors. Our findings provide morphological evidence to warrant caution when using serotonin enhancing drugs (e.g. SSRIs and receptor agonist) to treat autistic children. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'. Copyright © 2011 Elsevier Ltd. All rights reserved.

HNK-1 immunoreactivity during early morphogenesis of the head region in a nonmodel vertebrate, crocodile embryo

NASA Astrophysics Data System (ADS)

Kundrát, Martin

2008-11-01

The present study examines HNK-1 immunoidentification of a population of the neural crest (NC) during early head morphogenesis in the nonmodel vertebrate, the crocodile ( Crocodylus niloticus) embryos. Although HNK-1 is not an exclusive NC marker among vertebrates, temporospatial immunoreactive patterns found in the crocodile are almost consistent with NC patterns derived from gene expression studies known in birds (the closest living relatives of crocodiles) and mammals. In contrast to birds, the HNK-1 epitope is immunoreactive in NC cells at the neural fold level in crocodile embryos and therefore provides sufficient base to assess early migratory events of the cephalic NC. I found that crocodile NC forms three classic migratory pathways in the head: mandibular, hyoid, and branchial. Further, I demonstrate that, besides this classic phenotype, there is also a forebrain-derived migratory population, which consolidates into a premandibular stream in the crocodile. In contrast to the closely related chick model, crocodilian premandibular and mandibular NC cells arise from the open neural tube suggesting that species-specific heterochronic behavior of NC may be involved in the formation of different vertebrate facial phenotypes.

Immunoreactive opsin and glial fibrillary acidic protein in persistent hyperplastic primary vitreous.

PubMed

Hara, S; Mito, T; Takahashi, M; Ohmura, M; Mizuno, K; Masuda, T

1988-08-01

An 8-month-old boy had an anterior type of persistent hyperplastic primary vitreous in the right eye. Results of needle biopsy, performed because of elevated intraocular pressure, disclosed clusters of blastic cells. The eye was enucleated on the suspicion of retinoblastoma. Histological examination showed retrolental fibrovascular tissue and retinal dysplasia. Immunoreactive opsin was detected in the innermost structures and in photoreceptor-like cells of rosettes. We conclude that photoreceptor cells differentiated to express opsin, even when neighbouring cells were abnormally arranged. An immunocytochemical study of glial fibrillary acidic protein demonstrated glial proliferation in the inner layer of the retina but not in the preretinal space.

No differences in calcium-binding protein immunoreactivity in the posterior cingulate and visual cortex: schizophrenia and controls.

PubMed

Wheeler, David G; Dixon, Gavin; Harper, Clive G

2006-06-01

Schizophrenia-specific alterations in the densities of interneurons immunoreactive (ir) to the calcium binding proteins are reported for several cortical regions. However, no reported studies have searched for such differences within the posterior cingulate cortex using antibodies to a specific calcium binding protein, calbindin (Cb). Compare the (a) relative density of Cb-ir neurons (ratio of labeled neurons to total neurons), (b) relative width of cortical layers II/III and (c) somal areas of Cb-ir neurons in people with schizophrenia and non-psychiatric age-, gender- and postmortem index-matched controls (9 per group). Tissue from Brodmann's area (BA) 30 and 23 and an internal control region, the visual cortex (BA 18) were labeled with polyclonal Cb antibodies then Nissl counter-stained. Cb-ir neurons as well as counter-stained neurons with clearly visible nucleoli were plotted and counted within their area 1 and laminar boundaries. No qualitative or statistical differences in the relative density of Cb-ir neurons were observed. A trend towards a significant effect was detected in BA 30, the relative density of Cb-ir neurons for controls was greater than for schizophrenics (P=0.0518). There were no significant differences in the relative cortical widths or somal areas. The data from this study suggest that the posterior cingulate cortex may not be involved in schizophrenia, at least not as far as Cb-ir neurons are concerned.

Ischemic preconditioning maintains the immunoreactivities of glucokinase and glucokinase regulatory protein in neurons of the gerbil hippocampal CA1 region following transient cerebral ischemia

PubMed Central

CHO, YOUNG SHIN; CHO, JUN HWI; SHIN, BICH-NA; CHO, GEUM-SIL; KIM, IN HYE; PARK, JOON HA; AHN, JI HYEON; OHK, TAEK GEUN; CHO, BYUNG-RYUL; KIM, YOUNG-MYEONG; HONG, SEONGKWEON; WON, MOO-HO; LEE, JAE-CHUL

2015-01-01

Glucokinase (GK) is involved in the control of blood glucose homeostasis. In the present study, the effect of ischemic preconditioning (IPC) on the immunoreactivities of GK and its regulatory protein (GKRP) following 5 min of transient cerebral ischemia was investigated in gerbils. The gerbils were randomly assigned to four groups (sham-operated group, ischemia-operated group, IPC + sham-operated group and IPC + ischemia-operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia, followed by 1 day of recovery. In the ischemia-operated group, a significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) at 5 days post-ischemia; however, in the IPC+ischemia-operated group, the neurons in the SP were well protected. Following immunohistochemical investigation, the immunoreactivities of GK and GKRP in the neurons of the SP were markedly decreased in the CA1, but not the CA2/3, from 2 days post-ischemia, and were almost undetectable in the SP 5 days post-ischemia. In the IPC + ischemia-operated group, the immunoreactivities of GK and GKRP in the SP of the CA1 were similar to those in the sham-group. In brief, the findings of the present study demonstrated that IPC notably maintained the immunoreactivities of GK and GKRP in the neurons of the SP of CA1 following ischemia-reperfusion. This indicated that GK and GKRP may be necessary for neuron survival against transient cerebral ischemia. PMID:26134272

Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

PubMed

Zhou, Liang; Yang, Dong; Wang, De-Juan; Xie, Ya-Jun; Zhou, Jia-Huan; Zhou, Lin; Huang, Hao; Han, Shuo; Shao, Chong-Yu; Li, Hua-Shun; Zhu, J Julius; Qiu, Meng-Sheng; De Zeeuw, Chris I; Shen, Ying

2015-12-15

Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1.

Induction of c-Fos immunoreactivity in the amygdala of mice expressing anxiety-like behavior after local perfusion of veratrine in the prelimbic medial prefrontal cortex.

PubMed

Yamada, Misa; Saitoh, Akiyoshi; Ohashi, Masanori; Suzuki, Satoshi; Oka, Jun-Ichiro; Yamada, Mitsuhiko

2015-08-01

Local perfusion of the sodium channel activator veratrine in mouse prelimbic medial prefrontal cortex (PL) induced c-Fos immunoreactivity in the sub-regions of amygdala. Co-perfusion of the NMDA receptor antagonist MK-801 diminished the c-Fos expression. Significant correlations were observed between c-Fos immunoreactivity and behavioral measures in the open-field test. The PL stimulation activates a neural network projecting to the amygdala via NMDA receptor-mediated glutamatergic neurotransmission. Anxiety-like behavior induced after the PL stimulation may be partly mediated through the activation of amygdala.

Anisakis haemoglobin is a main antigen inducing strong and prolonged immunoreactions in rats.

PubMed

Abe, Niichiro; Teramoto, Isao

2017-07-01

Anisakis simplex larvae are well known to cause gastrointestinal and allergic manifestations after ingestion of parasitized raw or undercooked seafood. The antibody recognition dynamics against the components of Anisakis larval antigen after primary and re-infection with Anisakis live larvae remain unclear. For this study, immunoblot analyses of serum IgG, IgE, and IgM against Anisakis larval somatic extract were performed in rats that had been orally inoculated with A. simplex live larvae. Multiple antigen fractions were recognized after primary infection. Their reaction was enhanced after re-infection. Antibody recognition was observed for 12 weeks after re-infection. The fraction of approximately 35 kDa contained a main antigen that induced strong and prolonged immunoreactions in IgG and IgE. The antibody reaction to this fraction appeared to be enhanced after inoculation of larval homogenates. This fraction was heat tolerant with boiling for 30 min. The fraction was spotted by immunoblotting after two-dimensional electrophoresis and was identified as Anisakis haemoglobin (Ani s 13) using mass spectrometry analysis. The amino acid sequences of haemoglobin mRNAs from two A. simplex sensu stricto and one Anisakis pegreffii were identified by RACE-PCR. They differed from those of two isolates of Pseudoterranova decipiens and A. pegreffii. Results of this study show that Anisakis haemoglobin, which is known to be a major allergen of A. simplex, induces strong and prolonged immunoreaction in rats. This report is the first to show the amino acid sequence variation of Anisakis haemoglobin mRNA between A. simplex sensu stricto and A. pegreffii.

Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity.

PubMed

García-Mesa, Y; García-Piqueras, J; García, B; Feito, J; Cabo, R; Cobo, J; Vega, J A; García-Suárez, O

2017-12-01

The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch-gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell-neurite complexes, Meissner-like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20 + , chromogranin A + and synaptophisin + ) Merkel cells. Most of them were in close contact with Piezo2 - nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2 + , but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non-nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low-threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans. © 2017 Anatomical Society.

Inhibition by enflurane and methoxyflurane of postdrive hyperpolarization in canine Purkinje fibers.

PubMed

Pratila, M; Vogel, S; Sperelakis, N

1984-05-01

When a pacemaker cell is driven with a train of stimuli at a rate faster than its own, the termination of the drive is followed by a transient hyperpolarization, due to the activity of an electrogenic Na+-K+ pump. In this study, the effect of the halogenated ethers, enflurane and methoxyflurane, on postdrive hyperpolarization (PDH) was determined in cardiac Purkinje fibers. The fibers were removed from freshly excised canine hearts and superfused with a Tyrode's solution (containing 2.7 or 3.5 mM K+). The preparation was paced at 0.2 Hz before and after drives, and at 2 Hz during drives. Under control conditions, drives of 2 min produced a PDH of 5.5 +/- 0.2 mV. Enflurane (1.5-5%) significantly reduced the PDH. At 4 to 5%, enflurane reduced the PDH to a mean value of 42% of the control. Methoxyflurane was more potent than enflurane in affecting the PDH. At 0.5 to 0.75%, methoxyflurane reduced the PDH to 5% of the control. At higher (1-1.5%) concentrations of methoxyflurane, the PDH was converted to a depolarization, which varied between 0.5 and 8.0 mV. The PDH was restored to control levels within 10 to 20 min after washout of either anesthetic agent. Methoxyflurane (0.5 or 1%) enhanced the automaticity of spontaneously firing cells (2.35 mM K+ Tyrode's solution used). This positive chronotropic action coincided with a depolarization of 2 to 8 mV. Enflurane, at concentrations of 3 to 5%, gave similar results. On the action potential, methoxyflurane, at 1%, reduced the amplitude and duration (measured at 50% repolarization) of the plateau, and also the maximal upstroke velocity (+Vmax) of the rising phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems.

PubMed

Ma, W; Chabot, J-G; Powell, K J; Jhamandas, K; Dickerson, I M; Quirion, R

2003-01-01

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

PubMed Central

Bielen, F V; Glitsch, H G; Verdonck, F

1991-01-01

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that

Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds.

PubMed

Ohtubo, Yoshitaka; Iwamoto, Masafumi; Yoshii, Kiyonori

2012-06-01

Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ∼60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Neuropeptide Y-immunoreactive neurons in the cerebral cortex of humans and other haplorrhine primates

PubMed Central

Raghanti, Mary Ann; Conley, Tiffini; Sudduth, Jessica; Erwin, Joseph M.; Stimpson, Cheryl D.; Hof, Patrick R.; Sherwood, Chet C.

2012-01-01

We examined the distribution of neurons immunoreactive for neuropeptide Y (NPY) in the posterior part of the superior temporal cortex (Brodmann's area 22 or area Tpt) of humans and nonhuman haplorrhine primates. NPY has been implicated in learning and memory and the density of NPY-expressing cortical neurons and axons is reduced in depression, bipolar disorder, schizophrenia, and Alzheimer's disease. Due to the role that NPY plays in both cognition and neurodegenerative diseases, we tested the hypothesis that the density of cortical and interstitial neurons expressing NPY was increased in humans relative to other primate species. The study sample included great apes (chimpanzee and gorilla), Old World monkeys (pigtailed macaque, moor macaque, and baboon) and New World monkeys (squirrel monkey and capuchin). Stereologic methods were used to estimate the density of NPY-immunoreactive (-ir) neurons in layers I-VI of area Tpt and the subjacent white matter. Adjacent Nissl-stained sections were used to calculate local densities of all neurons. The ratio of NPY-ir neurons to total neurons within area Tpt and the total density of NPY-ir neurons within the white matter were compared among species. Overall, NPY-ir neurons represented only an average of 0.006% of the total neuron population. While there were significant differences among species, phylogenetic trends in NPY-ir neuron distributions were not observed and humans did not differ from other primates. However, variation among species warrants further investigation into the distribution of this neuromodulator system. PMID:23042407

Comparative mapping of serotonin-immunoreactive neurons in the central nervous systems of nudibranch molluscs.

PubMed

Newcomb, James M; Fickbohm, David J; Katz, Paul S

2006-11-20

The serotonergic systems in nudibranch molluscs were compared by mapping the locations of serotonin-immunoreactive (5-HT-ir) neurons in 11 species representing all four suborders of the nudibranch clade: Dendronotoidea (Tritonia diomedea, Tochuina tetraquetra, Dendronotus iris, Dendronotus frondosus, and Melibe leonina), Aeolidoidea (Hermissenda crassicornis and Flabellina trophina), Arminoidea (Dirona albolineata, Janolus fuscus, and Armina californica), and Doridoidea (Triopha catalinae). A nomenclature is proposed to standardize reports of cell location in species with differing brain morphologies. Certain patterns of 5-HT immunoreactivity were found to be consistent for all species, such as the presence of 5-HT-ir neurons in the pedal and cerebral ganglia. Also, particular clusters of 5-HT-ir neurons in the anterior and posterior regions of the dorsal surface of the cerebral ganglion were always present. However, there were interspecies differences in the number of 5-HT-ir neurons in each cluster, and some clusters even exhibited strong intraspecies variability that was only weakly correlated with brain size. Phylogenetic analysis suggests that the presence of particular classes of 5-HT-ir neurons exhibits a great deal of homoplasy. The conserved features of the nudibranch serotonergic system presumably represent the shared ancestral structure, whereas the derived characters suggest substantial independent evolutionary changes in the number and presence of serotonergic neurons. Although a number of studies have demonstrated phylogenetic variability of peptidergic systems, this study suggests that serotonergic systems may also exhibit a high degree of homoplasy in some groups of organisms.

Cell-specific expression of neuropeptide Y Y1 receptor immunoreactivity in the rat basolateral amygdala.

PubMed

Rostkowski, Amanda B; Teppen, Tara L; Peterson, Daniel A; Urban, Janice H

2009-11-10

Activation of neuropeptide Y (NPY) Y1 receptors (Y1r) in the rat basolateral nuclear complex of the amygdala (BLA) produces anxiolysis and interferes with the generation of conditioned fear. NPY is important in regulating the output of the BLA, yet the cell types involved in mediating this response are currently unknown. The current studies employed multiple label immunocytochemistry to determine the distribution of Y1r-immunoreactivity (-ir) in glutamatergic pyramidal and GABAergic cell populations in the BLA using scanning laser confocal stereology. Pyramidal neurons were identified by expression of calcium-calmodulin dependent kinase II (CaMKII-ir) and functionally distinct interneuron subpopulations were distinguished by peptide (cholecystokinin, somatostatin) or calcium-binding protein (parvalbumin, calretinin) content. Throughout the BLA, Y1r-ir was predominately on soma with negligible fiber staining. The high degree of coexpression of Y1r-ir (99.9%) in CaMKII-ir cells suggests that these receptors colocalize on pyramidal cells and that NPY could influence BLA output by directly regulating the activity of these projection neurons. Additionally, Y1r-ir was also colocalized with the interneuronal markers studied. Parvalbumin-ir interneurons, which participate in feedforward inhibition of BLA pyramidal cells, represented the largest number of Y1r expressing interneurons in the BLA ( approximately 4% of the total neuronal population). The anatomical localization of NPY receptors on different cell populations within the BLA provides a testable circuit whereby NPY could modulate the activity of the BLA via actions on both projection cells and interneuronal cell populations.

Activity-Dependent Gating of Calcium Spikes by A-type K+ Channels Controls Climbing Fiber Signaling in Purkinje Cell Dendrites

PubMed Central

Otsu, Yo; Marcaggi, Païkan; Feltz, Anne; Isope, Philippe; Kollo, Mihaly; Nusser, Zoltan; Mathieu, Benjamin; Kano, Masanobu; Tsujita, Mika; Sakimura, Kenji; Dieudonné, Stéphane

2014-01-01

Summary In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules. PMID:25220810

Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in Purkinje cell dendrites.

PubMed

Otsu, Yo; Marcaggi, Païkan; Feltz, Anne; Isope, Philippe; Kollo, Mihaly; Nusser, Zoltan; Mathieu, Benjamin; Kano, Masanobu; Tsujita, Mika; Sakimura, Kenji; Dieudonné, Stéphane

2014-10-01

In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules. Copyright © 2014 Elsevier Inc. All rights reserved.

Ischemic preconditioning protects neurons from damage and maintains the immunoreactivity of kynurenic acid in the gerbil hippocampal CA1 region following transient cerebral ischemia

PubMed Central

LEE, JAE-CHUL; TAE, HYUN-JIN; CHO, GEUM-SIL; KIM, IN HYE; AHN, JI HYEON; PARK, JOON HA; CHEN, BAI HUI; CHO, JEONG-HWI; SHIN, BICH NA; CHO, JUN HWI; BAE, EUN JOO; PARK, JINSEU; KIM, YOUNG-MYEONG; CHOI, SOO YOUNG; WON, MOO-HO

2015-01-01

Pyramidal neurons in region I of hippocampus proper (CA1) are particularly vulnerable to excitotoxic processes following transient forebrain ischemia. Kynurenic acid (KYNA) is a small molecule derived from tryptophan when this amino acid is metabolized through the kynurenine pathway. In the present study, we examined the effects of ischemic preconditioning (IPC) on the immunoreactivity and protein levels of KYNA following 5 min of transient forebrain ischemia in gerbils. The animals were randomly assigned to 4 groups (sham-operated group, ischemia-operated group, IPC + sham-operated group and IPC + ischemia-operated group). IPC was induced by subjecting the gerbils to 2 min of ischemia followed by 1 day of recovery. In the ischemia-operated group, we observed a significant loss of pyramidal neurons in the CA1 stratum pyramidale (SP) at 5 days post-ischemia; however, in the IPC + ischemia-operated group, the pyramidal neurons were well protected. KYNA immunoreactivity in the SP of the ischemia-operated group was significantly altered following ischemia-reperfusion and was very low 5 days following ischemia-reperfusion. In the IPC + ischemia-operated group, however, KYNA immunoreactivity was constitutively detected in the SP of the CA1 region after the ischemic insult. We also found that the alteration pattern of the KYNA protein level in the CA1 region following ischemia was generally similar to the immunohistochemical changes observed. In brief, our findings demonstrated that IPC maintained and even increased KYNA immunoreactivity in the SP of the CA1 region following ischemia-reperfusion. The data from the present study thus indicate that the enhancement of KYNA expression by IPC may be necessary for neuronal survival following transient ischemic injury. PMID:25872573

Intrauterine Growth Restriction Affects Cerebellar Granule Cells in the Developing Guinea Pig Brain.

PubMed

Tolcos, Mary; McDougall, Annie; Shields, Amy; Chung, Yoonyoung; O'Dowd, Rachael; Turnley, Ann; Wallace, Megan; Rees, Sandra

2018-01-01

Intrauterine growth restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. However, the effects of IUGR on the cerebellum are still to be fully elucidated. A major determinant of growth and development of the cerebellum is proliferation and subsequent migration of cerebellar granule cells. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in guinea pigs, results in abnormal cerebellar development due to deficits suggestive of impaired granule cell proliferation and/or migration. CPI was induced by unilateral ligation of the uterine artery at mid-gestation, producing growth-restricted (GR) foetuses at 52 and 60 days of gestation (dg), and neonates at 1 week postnatal age (term approx. 67 dg). Controls were from sham-operated animals. In GR foetuses compared with controls at 52 dg, the external granular layer (EGL) width and internal granular layer (IGL) area were similar. In GR foetuses compared with controls at 60 dg: (a) the EGL width was greater (p < 0.005); (b) the IGL area was smaller (p < 0.005); (c) the density of Ki67-negative (postmitotic) granule cells in the EGL was greater (p < 0.01); (d) the somal area of Purkinje cells was reduced (p < 0.005), and (e) the linear density of Bergmann glia was similar. The EGL width in GR foetuses at 60 dg was comparable to that of 52 dg control and GR foetuses. The pattern of p27-immunoreactivity in the EGL was the inverse of Ki67-immunoreactivity at both foetal ages; there was no difference between control and GR foetuses at either age in the width of p27-immunoreactivity, or in the percentage of the EGL width that it occupied. In the molecular layer of GR neonates compared with controls there was an increase in the areal density of granule cells (p < 0.05) and in the percentage of migrating to total number of granule cells (p < 0.01) at 1 week but not at 60 dg (p > 0.05). Thus, we found no specific evidence that IUGR affects granule cell

Glucose transporter-1 (GLUT-1) immunoreactivity in benign, premalignant and malignant lesions of the gallbladder.

PubMed

Legan, Mateja; Tevžič, Spela; Tolar, Ana; Luzar, Boštjan; Marolt, Vera Ferlan

2011-03-01

GLUT-1 is a transmembrane glucose transport protein that allows the facilitated transport of glucose into cells, normally expressed in tissues which depend mainly on glucose metabolism. Enhanced expression of GLUT-1 can also be found in a large spectrum of carcinomas. This study aimed to investigate GLUT-1 expression in gallbladder tissue: from normal tissue samples, hyperplasias, low-grade and high-grade dysplasias to gallbladder carcinomas. In all, 115 archived samples of gallbladder tissue from 68 patients, presented after cholecystectomy, were immunohistochemically stained for GLUT-1. According to the intensity of GLUT-1 immunoreactivity, samples were divided into negative (stained 0-10% of cells stained), positive with weak to moderate (10-50%) and positive with strong (>50%) GLUT-1 expression. The GLUT-1 immunoreactivity of the samples showed a characteristic increase from premalignant lesions to carcinomas. Normal gallbladder tissue samples did not express GLUT-1 (100%). Weak expression was shown only focally in hyperplasias, but to a greater extent with low-grade dysplasias (20%), high-grade dysplasias (40%) and carcinomas (51.8%). Normal gallbladder tissue is GLUT-1 negative. GLUT-1 expression in carcinoma tissue is significantly higher than in dysplastic lesions. Strong GLUT-1 expression indicates 100% specificity for detecting gallbladder carcinomas. Therefore, GLUT-1 is a candidate as a diagnostic as well as a tissue prognostic marker in gallbladder carcinoma patients.

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon

PubMed Central

2012-01-01

Background Remipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated α-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa. Results The dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes. Conclusions The brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a

Characterisation of recombinant immunoreactive antigens of the scab mite Sarcoptes scabiei.

PubMed

Kuhn, C; Lucius, R; Matthes, H F; Meusel, G; Reich, B; Kalinna, B H

2008-05-31

Sarcoptic mange (or scabies) is an important skin disease which can affect a variety of species including humans, cattle, goats, sheep, horses, pigs, rabbits, and dogs. Approximately 300 million people are affected worldwide and in lifestock animals the infestation may lead to substantial economic losses caused by depression in growth and feed conversion rates. Diagnosis of Sarcoptes infestation is difficult and only a few serological tests have been developed using whole mite antigen for diagnosis of mange in animals. Here we describe the isolation and characterisation of cDNAs of several immunoreactive clones and their recombinant expression in Escherichia coli. Three of the proteins contain repetitive sequences which suggests that they might be involved in immune evasion. The application of these antigens in serodiagnosis and the suitability for diagnosis is discussed.

Early in vivo changes in calcium ions, oxidative stress markers, and ion channel immunoreactivity following partial injury to the optic nerve.

PubMed

Wells, Jonathan; Kilburn, Matthew R; Shaw, Jeremy A; Bartlett, Carole A; Harvey, Alan R; Dunlop, Sarah A; Fitzgerald, Melinda

2012-03-01

CNS injury is often localized but can be followed by more widespread secondary degenerative events that usually result in greater functional loss. Using a partial transection model in rat optic nerve (ON). we recently demonstrated in vivo increases in the oxidative stress-associated enzyme MnSOD 5 min after injury. However, mechanisms by which early oxidative stress spreads remain unclear. In the present study, we assessed ion distributions, additional oxidative stress indicators, and ion channel immunoreactivity in ON in the first 24 hr after partial transection. Using nanoscale secondary ion mass spectroscopy (NanoSIMS), we demonstrate changes in the distribution pattern of Ca ions following partial ON transection. Regions of elevated Ca ions in normal ON in vivo rapidly decrease following partial ON transection, but there is an increasingly punctate distribution at 5 min and 24 hr after injury. We also show rapid decreases in catalase activity and later increases in immunoreactivity of the advanced glycation end product carboxymethyl lysine in astrocytes. Increased oxidative stress in astrocytes is accompanied by significantly increased immunoreactivity of the AMPA receptor subunit GluR1 and aquaporin 4 (AQP4). Taken together, the results indicate that Ca ion changes and oxidative stress are early events following partial ON injury that are associated with changes in GluR1 AMPA receptor subunits and altered ionic balance resulting from increased AQP4. Copyright © 2011 Wiley Periodicals, Inc.

Cytophagic and S-100 protein immunoreactive myeloid leukemia cutis.

PubMed

Thomas, Crystal G; Patel, Rajiv M; Bergfeld, Wilma F

2010-03-01

Myeloid leukemia cutis (LC) is the cutaneous involvement by neoplastic leukocytes of the myeloid series. Myeloid LC may occur de novo or concurrently with acute myeloid leukemias, chronic myeloid leukemias, other myeloproliferative disorders or myelodysplastic syndromes. We describe an unusual case of cytophagic S-100 protein immunoreactive leukemia cutis presenting in an 87-year-old woman without prior history of myeloid leukemia or other hematologic disorders. We outline key histologic and immunohistochemical features that aide in the diagnosis of LC. The presence of cytophagocytosis on histologic examination, a phenomenon more commonly associated with lymphoid rather than myeloid malignancies, provided a clue to the possibility of a malignant process. The atypical myeloid infiltrate showed S-100 protein positivity, an unusual finding that may be seen in LC. Although not commonly reported in LC, the presence of S-100 protein positivity and cytophagocytosis should not lead to the premature exclusion of LC as a possible diagnosis until a thorough clinical, histologic and immunohistochemical evaluation is performed. In addition, the presence of cytophagocytosis has been shown to have prognostic significance for patients with myeloid leukemia.

Sumoylation of FOXP2 regulates motor function and vocal communication through Purkinje cell development

PubMed Central

Usui, Noriyoshi; Co, Marissa; Harper, Matthew; Rieger, Michael A.; Dougherty, Joseph D.; Konopka, Genevieve

2016-01-01

Background Mutations in the gene encoding the transcription factor forkhead box P2, FOXP2, result in brain developmental abnormalities including reduced gray matter in both human patients and rodent models, and speech and language deficits. However, neither the region-specific function of FOXP2 in the brain, in particular the cerebellum, nor the effects of any post-translational modifications of FOXP2 in the brain and disorders have been explored. Methods We characterized sumoylation of FOXP2 biochemically, and analyzed the region-specific function and sumoylation of FOXP2 in the developing mouse cerebellum. Using in utero electroporation to manipulate the sumoylation-state of Foxp2 as well as Foxp2 expression levels in Purkinje cells (PCs) of the cerebellum in vivo, we reduced Foxp2 expression approximately 40% in the mouse cerebellum. Such a reduction approximates the haploinsufficiency observed in human patients who demonstrate speech and language impairments. Results We identified sumoylation of FOXP2 at K674 (K673 in mouse) in the cerebellum of neonates. In vitro co-immunoprecipitation and in vivo colocalization experiments suggest that PIAS3 acts as the SUMO E3 ligase for FOXP2 sumoylation. This sumoylation modifies transcriptional regulation by FOXP2. We demonstrate that Foxp2 sumoylation is required for regulation of cerebellar motor function and vocal communication, likely through dendritic outgrowth and arborization of PCs in the mouse cerebellum. Conclusions Sumoylation of Foxp2 in neonatal mouse cerebellum regulates PC development as well as motor functions and vocal communication, demonstrating evidence for sumoylation in regulating mammalian behaviors. PMID:27009683

Study of the temperature dependent immuno-reaction kinetics for the bio-functionalized magnetic nanoparticle assay of bio-markers of colorectal cancer

NASA Astrophysics Data System (ADS)

Yang, S. Y.; Chang, J. F.; Chen, T. C.; Yang, C. C.; Ho, C. S.

2014-01-01

By conjugating antibodies on magnetic nanoparticles, target antigens can be quantitatively detected by measuring the magnetic signals of the magnetic nanoparticles due to their association with target antigens. This method of detection is called magnetically labeled immunoassay. The assay characteristics of magnetically labeled immunoassay have been reported widely. However, the immuno-reaction kinetics of magnetically labeled immunoassay has not been studied. In this work, the reaction rates between magnetic nanoparticles and target antigens are measured at various temperatures. It is found that the temperature dependent reaction rate obeys Arrhenius's equation, which shows the collision frequency and activation energy for the immuno-reaction between antibody-functionalized magnetic nanoparticles and target antigens. The carcinoembryonic antigen, which is a regular blood bio-marker for in-vitro diagnosis of colorectal cancer, is used as a target antigen for the example.

Changes in CB1 and CB2 receptors in the post-mortem cerebellum of humans affected by spinocerebellar ataxias

PubMed Central

Rodríguez-Cueto, Carmen; Benito, Cristina; Fernández-Ruiz, Javier; Romero, Julián; Hernández-Gálvez, Mariluz; Gómez-Ruiz, María

2014-01-01

Background and PurposeSpinocerebellar ataxias (SCAs) are a family of chronic progressive neurodegenerative diseases, clinically and genetically heterogeneous, characterized by loss of balance and motor coordination due to degeneration of the cerebellum and its afferent and efferent connections. Unlike other motor disorders, the possible role of changes in the endocannabinoid system in the pathogenesis of SCAs has not been investigated. Experimental ApproachThe status of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) receptors in the post-mortem cerebellum of SCA patients and controls was investigated using immunohistochemical procedures. Key ResultsImmunoreactivity for the CB1 receptor, and also for the CB2 receptor, was found in the granular layer, Purkinje cells, neurons of the dentate nucleus and areas of white matter in the cerebellum of SCA patients at levels notably higher than controls. Double-labelling procedures demonstrated co-localization of CB1 and, in particular, CB2 receptors with calbindin, supporting the presence of these receptors in Purkinje neurons. Both receptors also co-localized with Iba-1 and glial fibrillary acidic protein in the granular layer and white matter areas, indicating that they are present in microglia and astrocytes respectively. Conclusions and ImplicationsOur results demonstrate that CB1 and CB2 receptor levels are significantly altered in the cerebellum of SCA patients. Their identification in Purkinje neurons, which are the main cells affected in SCAs, as well as the changes they experienced, suggest that alterations in endocannabinoid receptors may be related to the pathogenesis of SCAs. Therefore, the endocannabinoid system could provide potential therapeutic targets for the treatment of SCAs and its progression. Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6 PMID:23808969

Aldosterone acting through the central nervous system sensitizes angiotensin II-induced hypertension.

PubMed

Xue, Baojian; Zhang, Zhongming; Roncari, Camila F; Guo, Fang; Johnson, Alan Kim

2012-10-01

Previous studies have shown that preconditioning rats with a nonpressor dose of angiotensin II (Ang II) sensitizes the pressor response produced by later treatment with a higher dose of Ang II and that Ang II and aldosterone (Aldo) can modulate each other's pressor effects through actions involving the central nervous system. The current studies tested whether Aldo can cross-sensitize the pressor actions of Ang II to enhance hypertension by employing an induction-delay-expression experimental design. Male rats were implanted for telemetered blood pressure recording. During induction, subpressor doses of either subcutaneous or intracerebroventricular Aldo were delivered for 1 week. Rats were then rested for 1 week (delay) to assure that any exogenous Aldo was metabolized. After this, Ang II was given subcutaneously for 2 weeks (expression). During induction and delay, Aldo had no sustained effect on blood pressure. However, during expression, Ang II-induced hypertension was greater in the groups receiving subcutaneous or intracerebroventricular Aldo during induction in comparison with those groups receiving vehicle. Central administration of mineralocorticoid receptor antagonist blocked sensitization. Brain tissue collected at the end of delay and expression showed increased mRNA expression of several renin-angiotensin-aldosterone system components in cardiovascular-related forebrain regions of cross-sensitized rats. Cultured subfornical organ neurons preincubated with Aldo displayed greater increases in [Ca2+]i after Ang II treatment, and there was a greater Fra-like immunoreactivity present at the end of expression in cardiovascular-related forebrain structures. Taken together, these results indicate that Aldo pretreatment cross-sensitizes the development of Ang II-induced hypertension probably by mechanisms that involve the central nervous system.

Folic acid deficiency increases delayed neuronal death, DNA damage, platelet endothelial cell adhesion molecule-1 immunoreactivity, and gliosis in the hippocampus after transient cerebral ischemia.

PubMed

Hwang, In Koo; Yoo, Ki-Yeon; Suh, Hong-Won; Kim, Young Sup; Kwon, Dae Young; Kwon, Young-Guen; Yoo, Jun-Hyun; Won, Moo-Ho

2008-07-01

Folic acid deficiency increases stroke risk. In the present study, we examined whether folic acid deficiency enhances neuronal damage and gliosis via oxidative stress in the gerbil hippocampus after transient forebrain ischemia. Animals were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to occlusion of both common carotid arteries for 5 min. Exposure to an FAD increased plasma homocysteine levels by five- to eightfold compared with those of animals fed with a control diet (CD). In CD-treated animals, most neurons were dead in the hippocampal CA1 region 4 days after ischemia/reperfusion, whereas, in FAD-treated animals, this occurred 3 days after ischemia/reperfusion. Immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was performed to examine DNA damage in CA1 neurons in both groups after ischemia, and it was found that 8-OHdG immunoreactivity in both FAD and CD groups peaked at 12 hr after reperfusion, although the immunoreactivity in the FAD group was much greater than that in the CD group. Platelet endothelial cell adhesion molecule-1 (PECAM-1; a final mediator of neutrophil transendothelial migration) immunoreactivity in both groups increased with time after ischemia/reperfusion: Its immunoreactivity in the FAD group was much higher than that in the CD group 3 days after ischemia/reperfusion. In addition, reactive gliosis in the ischemic CA1 region increased with time after ischemia in both groups, but astrocytosis and microgliosis in the FAD group were more severe than in the CD group at all times after ischemia. Our results suggest that folic acid deficiency enhances neuronal damage induced by ischemia. 2008 Wiley-Liss, Inc.

Comparative mapping of GABA-immunoreactive neurons in the central nervous systems of nudibranch molluscs.

PubMed

Gunaratne, Charuni A; Sakurai, Akira; Katz, Paul S

2014-03-01

The relative simplicity of certain invertebrate nervous systems, such as those of gastropod molluscs, allows behaviors to be dissected at the level of small neural circuits composed of individually identifiable neurons. Elucidating the neurotransmitter phenotype of neurons in neural circuits is important for understanding how those neural circuits function. In this study, we examined the distribution of γ-aminobutyric-acid;-immunoreactive (GABA-ir) neurons in four species of sea slugs (Mollusca, Gastropoda, Opisthobranchia, Nudibranchia): Tritonia diomedea, Melibe leonina, Dendronotus iris, and Hermissenda crassicornis. We found consistent patterns of GABA immunoreactivity in the pedal and cerebral-pleural ganglia across species. In particular, there were bilateral clusters in the lateral and medial regions of the dorsal surface of the cerebral ganglia as well as a cluster on the ventral surface of the pedal ganglia. There were also individual GABA-ir neurons that were recognizable across species. The invariant presence of these individual neurons and clusters suggests that they are homologous, although there were interspecies differences in the numbers of neurons in the clusters. The GABAergic system was largely restricted to the central nervous system, with the majority of axons confined to ganglionic connectives and commissures, suggesting a central, integrative role for GABA. GABA was a candidate inhibitory neurotransmitter for neurons in central pattern generator (CPG) circuits underlying swimming behaviors in these species, however none of the known swim CPG neurons were GABA-ir. Although the functions of these GABA-ir neurons are not known, it is clear that their presence has been strongly conserved across nudibranchs. Copyright © 2013 Wiley Periodicals, Inc.

Somatostatin-immunoreactive senile plaque-like structures in the frontal cortex and nucleus accumbens of aged tree shrews and Japanese macaques.

PubMed

Yamashita, Akiko; Fuchs, Eberhard; Taira, Masato; Yamamoto, Takamitsu; Hayashi, Motoharu

2012-06-01

Previously, we demonstrated decreased expression of somatostatin mRNA in aged macaque brain, particularly in the prefrontal cortex. To investigate whether or not this age-dependent decrease in mRNA is related to morphological changes, we analyzed somatostatin cells in the cerebra of aged Japanese macaques and compared them with those in rats and tree shrews, the latter of which are closely related to primates. Brains of aged macaques, tree shrews, and rats were investigated by immunohistochemistry with special emphasis on somatostatin. We observed degenerating somatostatin-immunoreactive cells in the cortices of aged macaques and tree shrews. Somatostatin-immunoreactive senile plaque-like structures were found in areas 6 and 8 and in the nucleus accumbens of macaques, as well as in the nucleus accumbens and the cortex of aged tree shrews, where amyloid accumulations were observed. Somatostatin degenerations may be related to amyloid accumulations and may play roles in impairments of cognitive functions during aging. © 2012 John Wiley & Sons A/S.

Regulation of angiotensin II-induced neuromodulation by MARCKS in brain neurons.

PubMed

Lu, D; Yang, H; Lenox, R H; Raizada, M K

1998-07-13

Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. These neuromodulatory actions of Ang II involve Ras-Raf-MAP kinase signal transduction pathways (Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609-1617). In this study, we present evidence to demonstrate participation of another signaling pathway in these neuronal actions of Ang II. It involves activation of protein kinase C (PKC)beta subtype and phosphorylation and redistribution of myristoylated alanine-rich C kinase substrate (MARCKS) in neurites. Ang II caused a dramatic redistribution of MARCKS from neuronal varicosities to neurites. This was accompanied by a time-dependent stimulation of its phosphorylation, that was mediated by the angiotensin type 1 receptor subtype (AT1). Incubation of neurons with PKCbeta subtype specific antisense oligonucleotide (AON) significantly attenuated both redistribution and phosphorylation of MARCKS. Furthermore, depletion of MARCKS by MARCKS-AON treatment of neurons resulted in a significant decrease in Ang II-stimulated accumulation of TH and DbetaH immunoreactivities and [3H]NE uptake activity in synaptosomes. In contrast, mRNA levels of TH, DbetaH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148-165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DbetaH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKCbeta and its redistribution from varicosities to neurites is important in Ang II-induced synaptic accumulation of TH, DbetaH, and NE. They suggest that a coordinated stimulation of transcription of TH, DbetaH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKCbeta-MARCKS pathway are key in persistent

The ionic bases of the action potential in isolated mouse cardiac Purkinje cell.

PubMed

Vaidyanathan, Ravi; O'Connell, Ryan P; Deo, Makarand; Milstein, Michelle L; Furspan, Philip; Herron, Todd J; Pandit, Sandeep V; Musa, Hassan; Berenfeld, Omer; Jalife, José; Anumonwo, Justus M B

2013-01-01

Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse. To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs. Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data. APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs. Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

FMRFamide-like immunoreactive neurons of the nervus terminalis of teleosts innervate both retina and pineal organ.

PubMed

Ekström, P; Honkanen, T; Ebbesson, S O

1988-09-13

The tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) was first isolated from molluscan ganglia. Subsequently, it has become clear that vertebrate brains also contain endogenous FMRFamide-like substances. In teleosts, the neurons of the nervus terminalis contain an FMRFamide-like substance, and provide a direct innervation to the retina (Proc. Natl. Acad. Sci. U.S.A., 81 [1984] 940-944). Here we report the presence of FMRFamide-immunoreactive axonal bundles in the pineal organ of Coho salmon and three-spined sticklebacks. The largest numbers of axons were observed proximal to the brain, in the pineal stalk, while the distal part of the pineal organ contained only few axons. No FMRFamide-like-immunoreactive (IR) cell bodies were observed in the pineal organ. In adult fish it was not possible to determine the origin of these axons, due to the large numbers of FMRFamide-like IR axons in the teleost brain. However, by following the development of FMRFamide-like IR neurons in the embryonic and larval stickleback brain, it was possible to conclude that, at least in newly hatched fish, FMRFamide-like IR axons that originate in the nucleus nervus terminalis reach the pineal organ. Thus, it seems there is a direct connection between a specialized part of the chemosensory system and both the retina and the pineal organ in teleost fish.

Reproduction phase-related expression of GnRH-like immunoreactivity in the olfactory receptor neurons, their projections to the olfactory bulb and in the nervus terminalis in the female Indian major carp Cirrhinus mrigala (Ham.).

PubMed

Biju, K C; Singru, Praful S; Schreibman, Martin P; Subhedar, Nishikant

2003-10-01

The reproductive biology of the Indian major carp Cirrhinus mrigala is tightly synchronized with the seasonal changes in the environment. While the ovaries show growth from February through June, the fish spawn in July-August to coincide with the monsoon; thereafter the fish pass into the postspawning and resting phases. We investigated the pattern of GnRH immunoreactivity in the olfactory system at regular intervals extending over a period of 35 months. Although no signal was detected in the olfactory organ of fish collected from April through February following year, distinct GnRH-like immunoreactivity appeared in the fish collected in March. Intense immunoreactivity was noticed in several olfactory receptor neurons (ORNs) and their axonal fibers as they extend over the olfactory nerve, spread in the periphery of the olfactory bulb (OB), and terminate in the glomerular layer. Strong immunoreactivity was seen in some fascicles of the medial olfactory tracts extending from the OB to the telencephalon. Some neurons of the ganglion cells of nervus terminalis showed GnRH immunostaining during March; no immunoreactivity was detected at other times of the year. Plexus of GnRH immunoreactive fibers extending throughout the bulb represented a different component of the olfactory system; the fiber density showed a seasonal pattern that could be related to the status of gonadal maturity. While it was highest in the prespawning phase, significant reduction in the fiber density was noticed in the fish of spawning and the following regressive phases. Taken together the data suggest that the GnRH in the olfactory system of C. mrigala may play a major role in translation of the environmental cues and influence the downstream signals leading to the stimulation of the brain-pituitary-ovary axis.

An immunohistochemical study of neuropeptides and neuronal cytoskeletal proteins in the neuroepithelial component of a spontaneous murine ovarian teratoma. Primitive neuroepithelium displays immunoreactivity for neuropeptides and neuron-associated beta-tubulin isotype.

PubMed Central

Caccamo, D. V.; Herman, M. M.; Frankfurter, A.; Katsetos, C. D.; Collins, V. P.; Rubinstein, L. J.

1989-01-01

Approximately one third of the female mice of the LTXBO strain develop spontaneous ovarian teratomas. These tumors contain a large neuroepithelial component, which includes primitive neural structures resembling embryonic neural tubes (medulloepithelial rosettes), ependymoblastic and ependymal rosettes, neuroblasts, mature ganglionic neurons, myelinated neurites, and astrocytes. The purpose of this study was to characterize these tumors according to the immunohistochemical location of some well-characterized trophic and regulatory neuropeptides and neurotransmitters, several neuronal-associated cytoskeletal proteins, and other proteins indicative of neuronal and glial differentiation. Medulloepithelial rosettes showed focal serotonin-like, opioid peptide-like and gamma-amino butyric acid-like immunoreactivity, and displayed immunostaining for the neuron-associated class III beta-tubulin isotype. The mature ganglion cells were also immunoreactive for these markers, and, in addition, for somatostatin, cholecystokinin, bombesin, glucagon, vasoactive intestinal peptide, and neuropeptide Y. Mature ganglion cells were also immunoreactive for proteins associated with the neuronal cytoskeleton (including microtubule-associated proteins, MAP2 and tau, and higher molecular weight phosphorylated and non-phosphorylated neurofilament subunits), neuron-specific enolase, and synaptophysin. Undifferentiated stem cells, ependymoblastic and ependymal rosettes, and astroglia all stained with a monoclonal antibody that recognizes all mammalian beta-tubulin isotypes, but did not react with antibodies to neuronal-associated cytoskeletal proteins or neuropeptides. Neuropeptide-like immunoreactivity and demonstration of the class III beta-tubulin isotype indicate early neuronal commitment in neoplastic primitive neuroepithelium. These patterns of immunoreactivity closely follow those encountered in the normal neurocytogenesis of the mammalian and avian forebrain, and increase the

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon.

PubMed

Hibberd, Timothy J; Travis, Lee; Wiklendt, Lukasz; Costa, Marcello; Brookes, Simon J H; Hu, Hongzhen; Keating, Damien J; Spencer, Nick J

2018-01-01

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 μM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways. NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways

FUS/TLS-immunoreactive neuronal and glial cell inclusions increase with disease duration in familial amyotrophic lateral sclerosis with an R521C FUS/TLS mutation.

PubMed

Suzuki, Naoki; Kato, Shinsuke; Kato, Masako; Warita, Hitoshi; Mizuno, Hideki; Kato, Masaaki; Shimakura, Naoko; Akiyama, Haruhiko; Kobayashi, Zen; Konno, Hidehiko; Aoki, Masashi

2012-09-01

Basophilic inclusions (BIs) are pathological features of a subset of frontotemporal lobar degeneration disorders, including sporadic amyotrophic lateral sclerosis (ALS) and familial ALS (FALS). Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have recently been identified as a cause of FALS. The FUS/TLS-immunoreactive inclusions are consistently found in cases of frontotemporal lobar degeneration with BIs; however, the association between ALS cases with BIs and FUS/TLS accumulation is not well understood. We used immunohistochemistry to analyze 3 autopsy cases of FALS with the FUS/TLS mutation and with BIs using anti-FUS/TLS antibodies. The disease durations were 1, 3, and 9 years. As the disease duration becomes longer, there were broader distributions of neuronal and glial FUS/TLS-immunoreactive inclusions. As early as 1 year after the onset, BIs, neuronal cytoplasmic inclusions and glial cytoplasmic inclusions were found in the substantia nigra in addition to the anterior horn of the spinal cord. Glial cytoplasmic inclusions are found earlier and in a wider distribution than neuronal cytoplasmic inclusions. The distribution of FUS/TLS-immunoreactive inclusions in FUS/TLS-mutated FALS with BIs was broader than that of BIs alone, suggesting that the pathogenetic mechanism may have originated from the FUS/TLS proteinopathy.

The spatiotemporal relationships between chondroitin sulfate proteoglycans and terminations of calcitonin gene related peptide and parvalbumin immunoreactive afferents in the spinal cord of mouse embryos.

PubMed

Wang, Liqing; Yu, Chao; Wang, Jun; Zhao, Hui; Chan, Sun-On

2017-08-10

Chondroitin sulfate (CS) proteoglycans (PGs) are a family of complex molecules in the extracellular matrix and cell surface that regulate axon growth and guidance during development of the central nervous system. In this study, the expression of CSPGs was investigated in the mouse spinal cord at late embryonic and neonatal stages using CS-56 antibody. CS immunoreactivity was observed abundantly in ventral regions of spinal cord of embryonic day (E) 15 embryos. At E16 to E18, CS expression spread dorsally, but never reached the superficial layers of the dorsal horn. This pattern was maintained until postnatal day 4, the latest stage examined. Antibodies against calcitonin gene related peptide (CGRP) and parvalbumin (PV) were employed to label primary afferents from nociceptors and proprioceptors, respectively. CGRP-immunoreactive fibers terminated in the superficial regions of the dorsal horn where CSPGs were weakly expressed, whereas PV-immunoreactive fibers were found in CSPG-rich regions in the ventral horn. Therefore, we conclude that CS expression is spatiotemporally regulated in the spinal cord, which correlates to the termination of sensory afferents. This pattern suggests a role of CSPGs on patterning afferents in the spinal cord, probably through a differential response of axons to these growth inhibitory molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of protein kinase C α- and tyrosine hydroxylase-immunoreactive cells in the microbat retina.

PubMed

Park, Eun-Bee; Jeon, Joo-Yeong; Jeon, Chang-Jin

2018-05-09

A growing number of studies have revealed the functional neuroarchitecture of the microbat retina and suggested that microbats can see using their eyes. To better understand the organization of the microbat retina, quantitative analysis of protein kinase C alpha (PKCα)- and tyrosine hydroxylase (TH)-immunoreactive (IR) cells was conducted on the greater horseshoe bat (Rhinolophus ferrumequinum) retina. As a result, PKCα immunoreactivity was observed in rod bipolar cells, consistent with previous studies on other mammalian retinas. PKCα-IR cell distribution in the inner nuclear layer showed regional differences in density, with the highest density found in the nasal retina. The average density of PKCα-IR cells was 10,487±441 cells/mm2 (mean ± S.D.; n=4), with a total of 43,077±1,843 cells/retina. TH-IR cells in the Rhinolophus ferrumequinum retina could be classified into four types based on soma location and ramification in the inner plexiform layer: conventional amacrine, displaced amacrine, interplexiform, and intercalated cells. The majority of TH-IR cells were conventional amacrine cells. TH-IR cells were nonrandomly distributed at low density over the retina. The average density was 29.7±3.1 cells/mm2 (mean ± S.D.; n=3), with a total of 124.0±11.3 cells/retina. TH-IR processes showed varicosities and formed ring-like structures encircling AII amacrine cells. Our study provides the foundation for understanding the neurochemical architecture of the microbat retina and supports the notion that the eyes do play a role in the visual system of microbats.

Choline-acetyltransferase-like immunoreactivity in the organ of Corti of the rat during postnatal development.

PubMed

Merchán Pérez, A; Gil-Loyzaga, P; Eybalin, M; Fernández Mateos, P; Bartolomé, M V

1994-10-14

The mammalian cochlea receives efferent innervation from neurons located in the superior olivary complex. This efferent olivocochlear innervation is divided in two separate systems, lateral and medial, which mainly innervate afferent dendrites connected to inner hair cells and the cell body of outer hair cells, respectively. Besides other substances, lateral and medial efferent terminals of the adult cochlea use acetylcholine (ACh) as a neurotransmitter. In this study, we have used immunocytochemistry to detect the presence of choline acetyltransferase (ChAT), the synthesizing enzyme of ACh, in efferent olivocochlear terminals during the development of the rat. The appearance and distribution of immunoreactivity to ChAT has been studied in developing rat cochleas from birth (postnatal day 1, P1) to adulthood. Attention was paid to the temporal relationships between the expression of ChAT, the presence of other putative neuroactive substances, the onset of hearing and other developmental phenomena. Our results indicate that ChAT-like immunoreactivity is already present at birth (P1) in the region of inner hair cells, that it appears at P3 in the outer hair cell area and that it reaches an adult pattern of distribution by P15. ACh may thus be present early in the developing cochlea, before the onset of hearing, as it also occurs with other putative transmitters/modulators such as enkephalins, CGRP or GABA. It is suggested that ACh could be involved in the modulation of sound-evoked potentials as soon as they appear, and in the regulation of other developmental phenomena such as neurite outgrowth or synaptogenesis.

Serial alterations in digital hemodynamics and endothelin-1 immunoreactivity, platelet-neutrophil aggregation, and concentrations of nitric oxide, insulin, and glucose in blood obtained from horses following carbohydrate overload.

PubMed

Eades, Susan C; Stokes, Ashley M; Johnson, Philip J; LeBlanc, Casey J; Ganjam, Venkataseshu K; Buff, Preston R; Moore, Rustin M

2007-01-01

To quantify changes in endothelium-derived factors and relate those changes to various aspects of digital hemodynamics during the prodromal stages of carbohydrate overload (CHO)-induced laminitis in horses. 20 adult horses without abnormalities of the digit. Digital and jugular venous blood samples were collected at 1-hour intervals (for assessment of endothelin-1 [ET-1] immunoreactivity and measurement of glucose, insulin, and nitric oxide [NO] concentrations) or 4-hour intervals (CBC and platelet-neutrophil aggregate assessment) for 8 hours or 16 hours after induction of CHO-associated laminitis in horses treated with an ET-1 antagonist. Effects of treatment, collection site, and time and the random effects of horse on each variable were analyzed by use of a repeated-measures model. Where treatment and collection site had no significant effect, data were combined. Compared with baseline values, CHO resulted in changes in several variables, including a significant increase from baseline in digital blood ET-like immunoreactivity at 11 hours; digital blood ET-like immunoreactivity was significantly greater than that in jugular venous blood at 8, 9, 11, and 12 hours. Digital and jugular venous blood concentrations of glucose increased from baseline significantly at 3, 4, and 5 hours; insulin concentration increased significantly at 5 hours; and the number of platelet-neutrophil aggregates increased significantly at 12 hours. In horses, concurrent increases in venous blood ET-1 immunoreactivity, insulin and glucose concentrations, and platelet-neutrophil aggregates support a role of endothelial dysfunction in the pathogenesis of CHO-induced laminitis.

Neurochemical characterization of sea lamprey taste buds and afferent gustatory fibers: presence of serotonin, calretinin, and CGRP immunoreactivity in taste bud bi-ciliated cells of the earliest vertebrates.

PubMed

Barreiro-Iglesias, Antón; Villar-Cerviño, Verona; Villar-Cheda, Begoña; Anadón, Ramón; Rodicio, María Celina

2008-12-01

Neuroactive substances such as serotonin and other monoamines have been suggested to be involved in the transmission of gustatory signals from taste bud cells to afferent fibers. Lampreys are the earliest vertebrates that possess taste buds, although these differ in structure from taste buds in jawed vertebrates, and their neurochemistry remains unknown. We used immunofluorescence methods with antibodies raised against serotonin, tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), glutamate, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), calretinin, and acetylated alpha-tubulin to characterize the neurochemistry and innervation of taste buds in the sea lamprey, Petromyzon marinus L. For localization of proliferative cells in taste buds we used bromodeoxyuridine labeling and proliferating cell nuclear antigen immunohistochemistry. Results with both markers indicate that proliferating cells are restricted to a few basal cells and that almost all cells in taste buds are nonproliferating. A large number of serotonin-, calretinin-, and CGRP-immunoreactive bi-ciliated cells were revealed in lamprey taste buds. This suggests that serotonin participates in the transmission of gustatory signals and indicates that this substance appeared early on in vertebrate evolution. The basal surface of the bi-ciliated taste bud cells was contacted by tubulin-immunoreactive fibers. Some of the fibers surrounding the taste bud were calretinin immunoreactive. Lamprey taste bud cells or afferent fibers did not exhibit TH, GABA, glutamate, or NPY immunoreactivity, which suggests that expression of these substances evolved in taste buds of some gnathostomes lines after the separation of gnathostomes and lampreys. (c) 2008 Wiley-Liss, Inc.

Downregulation of missing in metastasis gene (MIM) is associated with the progression of bladder transitional carcinomas.

PubMed

Wang, Ying; Liu, Jiali; Smith, Elizabeth; Zhou, Kang; Liao, Jie; Yang, Guang-Yu; Tan, Ming; Zhan, Xi

2007-03-01

Missing in metastasis (MIM) gene encodes a putative metastasis suppressor. However, the role of MIM in tumorigenesis and metastasis has not yet been established. Western blot analysis using a MIM specific antibody demonstrated that MIM protein is present at varying levels in a variety of normal cells as well as tumor cell lines. Immunohistochemical staining of adult mouse tissues revealed abundant MIM immunoreactivity in uroepithelial cells in the bladder, neuron Purkinje cells in the cerebellum, and megakaryocytes in the bone marrow and spleen in addition. MIM immunoreactivity also was found in human normal bladder transitional epithelial cells. However, the reactivity was not seen in 69 percent of human primary transitional cell carcinoma specimens. Over 51 percent of the tumors at low grade display MIM staining similarly to the normal cells, whereas only 16.7 percent of the tumors at high-grade with poor differentiation show faint or mild staining. Furthermore, full-length MIM protein is highly expressed in SV-HUC-L an immortalized normal transitional epithelial cell line, moderately expressed in T24 and poorly expressed in J82 and TCCSUP transitional cell carcinoma cells. This finding indicates that downegulation of MIM expression may correlate with the transition of tumor cells from distinct epithelium-like morphology to less differentiated carcinomas.

Immunoreactive cortisone in droppings reflect stress levels, diet and growth rate of gull-billed tern chicks.

PubMed

Albano, Noelia; Santiago-Quesada, Francisco; Masero, José A; Sánchez-Guzmán, Juan M; Möstl, Erich

2015-03-01

Blood levels of corticosterone have been traditionally analyzed to assess stress levels in birds; however, measuring steroid hormone metabolites in feces and droppings has gained much interest as a noninvasive technique successfully used for such purposed in vertebrates. Diet may affect these fecal metabolite levels (e.g., due to nutritional stress), however, this variable has not been taken into account in studies with chicks despite the great dietary flexibility of many avian species. In this study, we addressed for the first time this key issue and validated the technique in wild gull-billed tern chicks (Gelochelidon nilotica). Several enzyme immunoassays were used to determine the most appropriate test to measure the stress response. Subsequently, we performed an experiment in captivity to assess adrenocortical activity in gull-billed tern chicks fed with two diets: piscivorous vs. insectivorous. Finally, the relation between the chicks' growth rate and excreted immunoreactive glucocorticoid metabolites (EGMs) was also evaluated. We found the immunoreactive cortisone metabolites to be a good index of stress (as being an index of adrenocortical reactivity) in chicks of this species. Fish-fed chicks had higher levels of cortisone metabolites when comparing both concentration and total daily excreted metabolites. Within each treatment diet, cortisone metabolite levels and growth rates were negatively correlated. These findings suggest that the diet should be considered when using this technique for comparative purposes and highlight the trade-off between stress levels and chicks growth rates. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of different calcium supplies and a single vitamin D injection on vitamin D receptor and calbindin D9k immunoreactivities in the gastrointestinal tract of goat kids.

PubMed

Sidler-Lauff, K; Boos, A; Kraenzlin, M; Liesegang, A

2010-11-01

The purpose of this study was to investigate whether diets differing in Ca concentration would have an influence on vitamin D (VitD) receptor (VDR) and calbindin D9k (Calb9k) immunoreactivities in the gastrointestinal tract of growing goats. In addition, the effect of a single VitD injection was studied, to clarify whether exogenous VitD would further increase the active Ca absorption mechanisms. The hypothesis of the study was that reduced Ca intake would lead to greater active Ca absorption, and with that, to greater amounts of VDR and Calb9k immunoreactivities. The normal Ca kid group (according to age requirements) received 2.5 to 6 g of Ca/d, whereas the lesser Ca kid group (less than requirements) received 1.5 to 4 g of Ca/d from wk 6 (weaning) to 15 (slaughter). In addition, 5 and 6 goat kids, respectively, of each group (normal Ca kid group, lesser Ca kid group), were injected with VitD (0.05 mg of cholecalciferol/kg of BW) in wk 14 of life. Blood samples were taken in wk 14 and 15. Calcium and VitD (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) concentrations were determined in serum. Immediately after slaughter, the duodenum (DD) and rumen (RU) were mounted in conventional Ussing chambers. Unidirectional flux rates of Ca across gastrointestinal tissues were measured. Additionally, tissue specimens of the gastrointestinal tract were collected, and formaldehyde-fixed paraffin sections were used for VDR and Calb9k immunohistochemistry. In all kid groups, a net absorption in the RU and a net secretion of Ca in the DD were observed. Immunoreactions of VDR were greatest in the duodenal mucosa, whereas Calb9k immunoreactions were observed in the forestomach and intestinal tissues. The greatest expression was observed in the duodenal surface epithelium. Additionally, in the VitD-injected groups, an immunoreaction occurred in the jejunal superficial and basal glands and the ileal superficial epithelium. In contrast, the other groups showed no Calb9k

Ultrastructural study of relationships between c-kit immunoreactive interstitial cells and other cellular elements in the human colon.

PubMed

Mazzia, C; Porcher, C; Julé, Y; Christen, M O; Henry, M

2000-05-01

C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".

Intraspinal release of immunoreactive calcitonin gene-related peptide during development of inflammation in the joint in vivo--a study with antibody microprobes in cat and rat.

PubMed

Schaible, H G; Freudenberger, U; Neugebauer, V; Stiller, R U

1994-10-01

This study addressed the intraspinal release of immunoreactive calcitonin gene-related peptide in vivo during mechanical stimulation of the normal joint and during the development of an acute experimental inflammation in the knee joint in the anaesthetized cat (spinalized) and rat (not spinalized). Release was assessed using microprobes coated with antibody to calcitonin gene-related peptide; inhibition of binding of [125I]calcitonin gene-related peptide to these probes following insertion into the spinal cord is equated with intraspinal release of the endogenous (unlabelled) peptide. Probes inserted prior to inflammation showed marked basal release of immunoreactive calcitonin gene-related peptide in the dorsal horn with a maximum in the superficial dorsal horn in the absence of intentional stimulation. The pattern of binding of [125I]calcitonin gene-related peptide was not or only minimally changed by innocuous mechanical stimuli (flexion of and innocuous pressure to the knee in the cat and innocuous pressure to the knee of the rat) but was significantly altered by electrical stimulation of the tibial nerve in the cat (sufficient to excite unmyelinated afferent fibres), indicating release of the peptide by the latter stimulus. During the first hours of the development of an experimental inflammation in the knee joint induced by intra-articular injections of kaolin and carrageenan, the pattern of binding of [125I]calcitonin gene-related peptide changed. In the cat, the level of immunoreactive calcitonin gene-related peptide showed a persistent increase in the gray matter and up to the surface of the cord and release was slightly increased by innocuous stimuli. In the rat, increased levels of immunoreactive calcitonin gene-related peptide were mainly seen in the superficial and deep dorsal horn during innocuous pressure (this stimulus did not evoke release of the peptide prior to inflammation) and noxious pressure applied to the injected knee, whereas increased

An ultrastructural study of calcitonin gene-related peptide-immunoreactive nerve fibers innervating the rat posterior longitudinal ligament. A morphologic basis for their possible efferent actions.

PubMed

Imai, S; Konttinen, Y T; Tokunaga, Y; Maeda, T; Hukuda, S; Santavirta, S

1997-09-01

The present study investigated ultrastructural characteristics of calcitonin gene-related peptide-immunoreactive nerve fibers in the posterior longitudinal ligament of the rat lumbar spine. To provide a morphologic basis for assessment of the afferent and, in particular, efferent functions of calcitonin gene-related peptide immunoreactive nerves in the posterior longitudinal ligament and their eventual role in degenerative spondylarthropathies and low back pain. Previous studies using light-microscopic localization of sensory neuronal markers such as calcitonin gene-related peptide have reported the presence of sensory fibers in the supporting structures of the vertebral column. Meanwhile, accumulating research data have suggested efferent properties for calcitonin gene-related peptide, i.e., a trophic action that alters the intrinsic properties of target cells not through transient action of synaptic transmission, but through long-lasting signal transmission by the secreted neuropeptides. To verify such trophic, paracrine actions of the calcitonin gene-related peptide-containing fibers in the posterior longitudinal ligament, however, ultrastructural details of the terminals and their spatial relationship to their eventual target structures have to be elucidated. Rat posterior longitudinal ligaments were stained immunohistochemically for calcitonin gene-related peptide. Light-microscopic analysis of the semithin sections facilitated subsequent electron microscopy of specific sites of the posterior longitudinal ligament to determine ultrastructural details and nerve fiber-target relationships. The rat lumbar posterior longitudinal ligament was found to be innervated by two distinctive calcitonin gene-related peptide immunoreactive nerve networks. In immunoelectronmicroscopy, the fibers of the deep network had numerous free nerve endings, whereas those of the superficial network showed spatial associations with other non-calcitonin gene-related peptide immunoreactive

Tyrosine hydroxylase-immunoreactivity and its relations with gonadotropin-releasing hormone and neuropeptide Y in the preoptic area of the guinea pig.

PubMed

Bogus-Nowakowska, Krystyna; Równiak, Maciej; Hermanowicz-Sobieraj, Beata; Wasilewska, Barbara; Najdzion, Janusz; Robak, Anna

2016-12-01

The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA. Copyright © 2016 Elsevier B.V. All rights reserved.

IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

PubMed Central

Hisatsune, Chihiro; Miyamoto, Hiroyuki; Hirono, Moritoshi; Yamaguchi, Naohide; Sugawara, Takeyuki; Ogawa, Naoko; Ebisui, Etsuko; Ohshima, Toshio; Yamada, Masahisa; Hensch, Takao K.; Hattori, Mitsuharu; Mikoshiba, Katsuhiko

2013-01-01

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway. PMID:24109434

Purkinje Cell Protein 4 Expression Is Associated With DNA Methylation Status in Aldosterone-Producing Adenoma.

PubMed

Kobuke, Kazuhiro; Oki, Kenji; Gomez-Sanchez, Celso E; Ohno, Haruya; Itcho, Kiyotaka; Yoshii, Yoko; Yoneda, Masayasu; Hattori, Noboru

2018-03-01

Aldosterone production is stimulated by activation of calcium signaling in aldosterone-producing adenomas (APAs), and epigenetic factors such as DNA methylation may be associated with the expression of genes involved in aldosterone regulation. Our aim was to investigate the DNA methylation of genes related to calcium signaling cascades in APAs and the association of mutations in genes linked to APAs with DNA methylation levels. Nonfunctioning adrenocortical adenoma (n = 12) and APA (n = 35) samples were analyzed. The KCNJ5 T158A mutation was introduced into human adrenocortical cell lines (HAC15 cells) using lentiviral delivery. DNA methylation array analysis was conducted using adrenal tumor samples and HAC15 cells. The Purkinje cell protein 4 (PCP4) gene was one of the most hypomethylated in APAs. DNA methylation levels in two sites of PCP4 showed a significant inverse correlation with messenger RNA expression in adrenal tumors. Bioinformatics and multiple regression analysis revealed that CCAAT/enhancer binding protein alpha (CEBPA) may bind to the methylation site of the PCP4 promoter. According to chromatin immunoprecipitation assay, CEBPA was bound to the PCP4 hypomethylated region by chromatin immunoprecipitation assay. There were no significant differences in PCP4 methylation levels among APA genotypes. Moreover, KCNJ5 T158A did not influence PCP4 methylation levels in HAC15 cells. We showed that the PCP4 promoter was one of the most hypomethylated in APAs and that PCP4 transcription may be associated with demethylation as well as with CEBPA in APAs. KCNJ5 mutations known to result in aldosterone overproduction were not related to PCP4 methylation in either clinical or in vitro studies.

Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells

PubMed Central

Miyazaki, Taisuke; Yamasaki, Miwako; Hashimoto, Kouichi; Kohda, Kazuhisa; Yuzaki, Michisuke; Shimamoto, Keiko; Tanaka, Kohichi; Kano, Masanobu; Watanabe, Masahiko

2017-01-01

Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF–PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10–90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs. PMID:28655840

The cellular localization of the neuropeptides substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y in guinea-pig vestibular sensory organs: a high-resolution confocal microscopy study.

PubMed

Scarfone, E; Ulfendahl, M; Lundeberg, T

1996-11-01

Four neuropeptides, substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y, were detected by radioimmunoassay in guinea-pig vestibular end-organs. High-resolution confocal microscopy visualization of immunofluorescence staining was used to determine the cellular localization of these peptides. Substance P- and neurokinin A-like immunoreactivities were found to co-exist in afferent fibers innervating the peripheral regions of both the utricular and ampullar sensory organs. The immunoreactivity was more concentrated in the distal ends of the calyceal-shaped nerve endings that innervate type I sensory cells. While in the guinea-pig, nerve calyces and type I cells are distributed in both the central and peripheral regions of the sensory epithelia, immunoreactive calyces were found only in the peripheral regions. Calcitonin gene-related peptide-like immunoreactivity was localized in small bouton endings situated at the level of the base of the hair cells. These boutons were in a position to make axosomatic contacts with type II sensory cells and axodendritic contacts with afferent nerve endings. Calcitonin gene-related peptide immunoreactivity co-existed with choline acetyltransferase immunoreactivity. The localization and shape of these boutons identified them as the axonal endings of efferent vestibular fibers. Neuropeptide Y-like immunoreactivity was not observed in the actual sensory epithelium but in the underlying connective tissue, where it was located in varicose fibers along blood vessels. The synaptic position of the tachykinins is clearly distinct from that of calcitonin gene-related peptide. This segregation distinguishes the vestibular end-organs from most peripheral tissues where these peptides are co-localized. The tachykinin-immunoreactive afferent fibers are postsynaptic to the hair cells. If, as in somatic sensory endings, these fibers can be triggered to release the neuropeptides by an axon reflex type of activation, then the

Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein.

PubMed

Mao, Shanping; Xiong, Guoxiang; Zhang, Lei; Dong, Huimin; Liu, Baohui; Cohen, Noam A; Cohen, Akiva S

2016-01-01

A60, the mouse monoclonal antibody against the neuronal nuclear protein (NeuN), is the most widely used neuronal marker in neuroscience research and neuropathological assays. Previous studies identified fragments of A60-immunoprecipitated protein as Synapsin I (Syn I), suggesting the antibody will demonstrate cross immunoreactivity. However, the likelihood of cross reactivity has never been verified by immunohistochemical techniques. Using our established tissue processing and immunofluorescent staining protocols, we found that A60 consistently labeled mossy fiber terminals in hippocampal area CA3. These A60-positive mossy fiber terminals could also be labeled by Syn I antibody. After treating brain slices with saponin in order to better preserve various membrane and/or vesicular proteins for immunostaining, we observed that A60 could also label additional synapses in various brain areas. Therefore, we used A60 together with a rabbit monoclonal NeuN antibody to confirm the existence of this cross reactivity. We showed that the putative band positive for A60 and Syn I could not be detected by the rabbit anti-NeuN in Western blotting. As efficient as Millipore A60 to recognize neuronal nuclei, the rabbit NeuN antibody demonstrated no labeling of synaptic structures in immunofluorescent staining. The present study successfully verified the cross reactivity present in immunohistochemistry, cautioning that A60 may not be the ideal biomarker to verify neuronal identity due to its cross immunoreactivity. In contrast, the rabbit monoclonal NeuN antibody used in this study may be a better candidate to substitute for A60.

Genetic basis for loss of immuno-reactive O-chain in Salmonella enterica serovar Enteritidis veterinary isolates.

PubMed

Szabo, Istvan; Grafe, Marianne; Kemper, Nicole; Junker, Ernst; Malorny, Burkhard

2017-05-01

Fifty-two rough Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates from broilers and the environment were characterized for their serological and genotypic properties. Under routine diagnostic serotyping methods such isolates lack the immuno-reactivity of the O-chain of the lipopolysaccharide (LPS), and are referred to as non-typeable. Using a modified slide agglutination method, the isolates could be differentiated into three different serological variants. Twenty-six isolates (50%) were defined as semi-rough, nineteen isolates (37%) as deep-rough, four isolates (8%) as rough and three isolates could not be assigned. Genetically, all semi-rough isolates lacked the wzyB gene encoding the O-antigen polymerase. Two isolates carried a frameshift mutation in wzyB. In 15 of 23 cases deep-rough or rough isolates had a single point mutation, a single - or double-nucleotide insert or deletion in the wbaP gene. The mutational changes lead to expression of truncated (premature) protein, resulting in the loss of the immuno-reactive O-chain. Both rough and smooth S. Enteritidis isolates showed identical or highly similar XbaI-PFGE profiles. Our results indicate that the loss of a functional LPS in S. Enteritidis isolates is caused by a variety of different mutation events within the wzyB (semi-rough) or the wbaP (deep-rough) gene and is not a result of a vertical spread of a specific S. Enteritidis subtype. The defect of the LPS may be a common evolutionary mechanism through which host defence can be escaped. Copyright © 2017. Published by Elsevier B.V.

Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in the brain of the CCK-1 receptor deficient obese OLETF rat

PubMed Central

Abraham, Hajnalka; Covasa, Mihai; Hajnal, Andras

2013-01-01

Cocaine- and amphetamine regulated transcript (CART) peptide is expressed in brain areas involved in homeostatic regulation and reward. CART has been shown to reduce food intake but the underlying mechanisms and the relevance of this effect to obesity yet remain unknown. Therefore, we used immunohistochemistry to investigate expression of CART peptide in various brain regions of the obese Otsuka Long Evans Tokushima Fatty (OLETF) rats lacking the CCK-1 receptor. Analysis revealed that whereas the distribution of CART peptide-immunoreactive neurons and axonal networks was identical in OLETF rats and lean controls, intensity of CART immunoreactivity was significantly reduced in the rostral part of the nucleus accumbens (p<0.01), the basolateral complex of the amygdala (p<0.05), and the rostro-medial nucleus of solitary tract (p<0.001) of the OLETF rats. These areas are involved in reward and integration of taste and viscerosensory information and have been previously associated with altered functions in this strain. The findings suggest that in addition to previously described deficits in peripheral satiety signals and augmented orexigenic regulation, the anorectic effect of CART peptide may also be diminished in OLETF rats. PMID:19533109

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite.

PubMed

Masetti, Massimo; Locci, Teresa; Cecchettini, Antonella; Lucchesi, Paolo; Magi, Marta; Malvaldi, Gino; Bruschi, Fabrizio

2004-05-01

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.

GD3- and O-acetylated GD3-gangliosides in the GM2 synthase-deficient mouse brain and their immunohistochemical localization

PubMed Central

Matsuda, Junko; Vanier, Marie T.; Popa, Iuliana; Portoukalian, Jacques; Suzuki, Kunihiko

2006-01-01

Gangliosides in the brain of the knockout mouse deficient in the activity of β1,4 N-acetylgalactosaminyl transferase (β1,4 GalNAc-T)(GM2 synthase) consisted of nearly exclusively of GM3- and GD3-gangliosides as expected from the known substrate specificity of the enzyme and in confirmation of the initial reports from two laboratories that generated the mutant mouse experimentally. The total molar amount of gangliosides was approximately 30% higher in the mutant mouse brain than that in the wild-type brain. However, contrary to the initial reports, one-fourth of total GD3-ganglioside was O-acetylated. It reacted positively with an anti-O-acetylated GD3 monoclonal antibody and disappeared with a corresponding increase in GD3-ganglioside after mild alkaline treatment. The absence of O-acetylated GD3 in the initial reports can be explained by the saponification step included in their analytical procedures. Although quantitatively much less and identification tentative, we also detected GT3 and O-acetylated GT3. Anti-GD3 and anti-O-acetylated GD3 monoclonal antibodies gave positive reactions in the brain of mutant mouse as expected from the analytical results. Either antibody barely stained wild-type brain except for immunoreactivity of GD3 in the cerebellar Purkinje cells. The distributions of GD3 and O-acetylated GD3 in the brain of mutant mouse were similar but differential localization was noted in the cerebellar Purkinje cells and cerebral cortex. PMID:25792782

Nuclear localization of Klotho in brain: an anti-aging protein

PubMed Central

German, Dwight C.; Khobahy, Ida; Pastor, Johanne; Kuro-o, Makoto; Liu, Xinran

2011-01-01

Klotho is a putative age-suppressing gene whose over-expression in mice results in extension of life span. The klotho gene encodes a single-pass transmembrane protein whose extracellular domain is shed and released into blood, urine, and cerebrospinal fluid, potentially functioning as a humoral factor. The extracellular domain of Klotho has an activity that increases the expression of anti-oxidant enzymes and confers resistance to oxidative stress in cultured cells and in whole animals. The transmembrane form of the Klotho protein directly binds to multiple fibroblast growth factor receptors and modifies their ligand affinity and specificity. The purpose of the present study was to determine the precise cellular localization of Klotho in the mouse brain. Using light microscopic immunohistochemical methods, we found the highest levels of Klotho immunoreactivity in two brain regions: the choroid plexus, and cerebellar Purkinje cells. In the choroid plexus cells, Klotho was found not only on the plasma membrane but also in large amounts near the nuclear membrane. Likewise, in the Purkinje cell Klotho was found throughout the cell including dendrites, axon and soma with large amounts near the nuclear membrane. Using immunoelectron microscopy, we found Klotho in the cell membrane, but the highest concentration was localized in the peripheral portion of the nucleus and the nucleolus in both cell types. This new finding suggests that in addition to Klotho being secreted from cells in brain, it also has a nuclear function. PMID:22245317

Paroxysmal atrioventricular block: Electrophysiological mechanism of phase 4 conduction block in the His-Purkinje system: A comparison with phase 3 block.

PubMed

Shenasa, Mohammad; Josephson, Mark E; Wit, Andrew L

2017-11-01

Paroxysmal atrioventricular (A-V) block is relatively rare, and due to its transient nature, it is often under recognized. It is often triggered by atrial, junctional, or ventricular premature beats, and occurs in the presence of a diseased His-Purkinje system (HPS). Here, we present a 45-year-old white male who was admitted for observation due to recurrent syncope and near-syncope, who had paroxysmal A-V block. The likely cellular electrophysiological mechanisms(s) of paroxysmal A-V block and its differential diagnosis and management are discussed. Continuous electrocardiographic monitoring was done while the patient was in the cardiac unit. Multiple episodes of paroxysmal A-V block were documented in this case. All episodes were initiated and terminated with atrial/junctional premature beats. The patient underwent permanent pacemaker implantation and has remained asymptomatic since then. Paroxysmal A-V block is rare and often causes syncope or near-syncope. Permanent pacemaker implantation is indicated according to the current guidelines. Paroxysmal A-V block occurs in the setting of diseased HPS and is bradycardia-dependent. The detailed electrophysiological mechanisms, which involve phase 4 diastolic depolarization, and differential diagnosis are discussed. © 2017 Wiley Periodicals, Inc.

Chromatographic evidence for high-molecular-mass galanin immunoreactivity in pig and cat adrenal glands.

PubMed

Bauer, F E; Adrian, T E; Yanaihara, N; Polak, J M; Bloom, S R

1986-06-09

Galanin was measured by radioimmunoassay in extracts of pig, cat and rat adrenals using non-C- and mid to C-terminally directed antibodies. The extracts were fractioned by gel chromatography and HPLC. The non-C-terminal galanin immunoreactivity in pig was 92.8 +/- 11.7 pmol/g, in cat 9.1 +/- 0.9 pmol/g and in rat less than 1 pmol/g. Two higher molecular forms of galanin have been identified in both pig and cat adrenal. One major large form behaves as if it was N-terminally extended (Kav pig 0.58, cat 0.48) and the other, a very high-molecular-mass form (Kav pig 0.10, 0.24, cat 0.10), as if it had both N- and C-terminal extensions.

Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses

PubMed Central

Silver, R Angus; Momiyama, Akiko; Cull-Candy, Stuart G

1998-01-01

EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This ‘multiple-probability fluctuation’ (MPF) analysis gave an estimate of 510 ± 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 ± 0.03 nS (n = 6). Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mm Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 ± 0.01 nS and a CV of 0.37 ± 0.01 (n = 4). Similar estimates of q were obtained in 2 mm Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 μm) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (P¯r) was unusually high (0.90 ± 0.03 at 0.033 Hz

Sodium channel Nav1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome

PubMed Central

2010-01-01

Background Voltage gated sodium channels Nav1.7 are involved in nociceptor nerve action potentials and are known to affect pain sensitivity in clinical genetic disorders. Aims and Objectives To study Nav1.7 levels in dental pulpitis pain, an inflammatory condition, and burning mouth syndrome (BMS), considered a neuropathic orofacial pain disorder. Methods Two groups of patients were recruited for this study. One group consisted of patients with dental pulpitis pain (n = 5) and controls (n = 12), and the other patients with BMS (n = 7) and controls (n = 10). BMS patients were diagnosed according to the International Association for the Study of Pain criteria; a pain history was collected, including the visual analogue scale (VAS). Immunohistochemistry with visual intensity and computer image analysis were used to evaluate levels of Nav1.7 in dental pulp tissue samples from the dental pulpitis group, and tongue biopsies from the BMS group. Results There was a significantly increased visual intensity score for Nav1.7 in nerve fibres in the painful dental pulp specimens, compared to controls. Image analysis showed a trend for an increase of the Nav1.7 immunoreactive % area in the painful pulp group, but this was not statistically significant. When expressed as a ratio of the neurofilament % area, there was a strong trend for an increase of Nav1.7 in the painful pulp group. Nav1.7 immunoreactive fibres were seen in abundance in the sub-mucosal layer of tongue biopsies, with no significant difference between BMS and controls. Conclusion Nav1.7 sodium channel may play a significant role in inflammatory dental pain. Clinical trials with selective Nav1.7 channel blockers should prioritise dental pulp pain rather than BMS. PMID:20529324

A pilot study evaluating changes in pancreatic lipase immunoreactivity concentrations in canines treated with L-asparaginase (ASNase), vincristine, or both for lymphoma.

PubMed

Wright, Zachary; Steiner, Joerg; Suchodolski, Jan; Rogers, Kenita; Barton, Claudia; Brown, Marjorie

2009-04-01

L-asparaginase (ASNase) is a common chemotherapy agent for the treatment of lymphoid malignancies. L-asparaginase has been reported to cause clinical pancreatitis in both humans and canines. Canine pancreatic lipase immunoreactivity (cPLI) is now a common diagnostic tool for evaluating pancreatitis in dogs. A total of 52 dogs were enrolled into this study. Canine pancreatic lipase immunoreactivity (cPLI) concentrations were evaluated before and after administration of ASNase, vincristine, or both. All dogs enrolled in the study were evaluated for signs compatible with clinical pancreatitis. No dogs receiving ASNase alone showed evidence of clinical pancreatitis after administration. Also, there was no statistically significant change in cPLI concentrations before or after treatment. Fourteen percent of dogs that received both vincristine and ASNase concurrently had elevated concentrations of cPLI after treatment. Of the 11 dogs with clinical signs compatible with pancreatitis after any chemotherapy treatment, no dog had a cPLI concentration > 400 microg/dL. In conclusion, ASNase did not cause clinical pancreatitis in this cohort of dogs but larger sample sizes are required to further validate this data.

Caspase-6 Activation in Familial Alzheimer Disease Brains Carrying Amyloid Precursor Protein, Presenilin I or Presenilin II Mutations

PubMed Central

Albrecht, Steffen; Bogdanovic, Nenad; Ghetti, Bernardino; Winblad, Bengt; LeBlanc, Andréa C.

2010-01-01

We previously demonstrated the activation of Caspase-6 in the hippocampus and cortex in cases of mild, moderate, severe and very severe Alzheimer disease (AD). To determine whether Caspase-6 is also activated in familial AD, we performed an immunohistochemical analysis of active Caspase-6 and Tau cleaved by Caspase-6 in temporal cortex and hippocampal tissue sections from cases of familial AD. The cases included 5 carrying the amyloid precursor protein K670N, M671L Swedish mutation, 1 carrying the amyloid precursor protein E693G Arctic mutation, 2 each carrying the Presenilin I M146V, F105L, A431E, V261F, Y115C mutations, and 1 with the Presenilin II N141I mutation. Active Caspase-6 immunoreactivity was found in all cases. Caspase-6 immunoreactivity was observed in neuritic plaques or cotton wool plaques in some cases, neuropil threads and neurofibrillary tangles. These results indicate that Caspase-6 is activated in familial forms of AD, as previously observed in sporadic forms. Since sporadic and familial AD cases have similar pathological features, these results support a fundamental role of Caspase-6 in the pathophysiology of both familial and sporadic AD. PMID:19915487

Primary Subcutaneous Synovial Sarcoma: First Reported Subcutaneous Case Showing TLE1 Immunoreactivity.

PubMed

Alegría-Landa, Victoria; Nájera, Laura; Massa, Dolores Suárez; Roustan, Gastón; Río, María Del; Kutzner, Heinz; Requena, Luis

2018-05-08

Synovial sarcoma (SS) accounts for 5%-10% of all soft tissue sarcomas. It is a well-defined soft tissue neoplasm with biphasic and monophasic histologic subtypes and unknown histogenesis. It usually occurs in the extremities, especially the thigh-knee region of young adults. Recurrences are frequent and distant metastasis developed in approximately half of the patients. SSs are characterized by a recurrent nonrandom chromosomal translocation, t(X; 18) (p11; q11), which is considered the primary genetic event in more than 90% of cases. Only 4 cases of cutaneous and subcutaneous SSs have been published in the literature so far. We report a case of primary subcutaneous SS in the forearm of a young woman and discuss the histopathologic differential diagnosis with other similar neoplasms. This is the first reported case of primary cutaneous SS showing immunoreactivity for TLE1 in the nuclei of neoplastic cells, supporting the use of this marker for diagnosis of this rare cutaneous neoplasm.

Calbindin-D28k is a more reliable marker of human Purkinje cells than standard Nissl stains: a stereological experiment.

PubMed

Whitney, Elizabeth R; Kemper, Thomas L; Rosene, Douglas L; Bauman, Margaret L; Blatt, Gene J

2008-02-15

In a study of human Purkinje cell (PC) number, a striking mismatch between the number of PCs observed with the Nissl stain and the number of PCs immunopositive for calbindin-D28k (CB) was identified in 2 of the 10 brains examined. In the remaining eight brains this mismatch was not observed. Further, in these eight brains, analysis of CB immunostained sections counterstained with the Nissl stain revealed that more than 99% Nissl stained PCs were also immunopositive for CB. In contrast, in the two discordant brains, only 10-20% of CB immunopositive PCs were also identified with the Nissl stain. Although this finding was unexpected, a historical survey of the literature revealed that Spielmeyer [Spielmeyer W. Histopathologie des nervensystems. Julius Springer: Berlin; 1922. p. 56-79] described human cases with PCs that lacked the expected Nissl staining intensity, an important historical finding and critical issue when studying postmortem human brains. The reason for this failure in Nissl staining is not entirely clear, but it may result from premortem circumstances since it is not accounted for by postmortem delay or processing variables. Regardless of the exact cause, these observations suggest that Nissl staining may not be a reliable marker for PCs and that CB is an excellent alternative marker.

Separation of the pace-maker and plateau components of delayed rectification in cardiac Purkinje fibres

PubMed Central

Hauswirth, O.; Noble, D.; Tsien, R. W.

1972-01-01

1. Experiments on sheep Purkinje fibres were designed to determine whether the current mechanisms responsible for delayed rectification at the pace-maker (negative to -50 mV) and plateau (positive to -50 mV) ranges of potential are kinetically separable and independent. 2. Hyperpolarizations from the plateau range were shown to produce decay of a single component of outward current within the plateau range, but two components were evident when the hyperpolarizations entered the pace-maker range. 3. The time courses of recovery of the two components were too similar at -25 mV to allow temporal resolution at this potential. Clear temporal resolution was, however, possible at potentials between -55 and -95 mV. An indirect method of resolving the two components at -25 mV was used. 4. The kinetic properties of the two components correspond to those previously described for the pace-maker potassium current, iK2, and the outward plateau current, ix1 (Noble & Tsien, 1968, 1969a). 5. The instantaneous (fully activated) current—voltage relation for iK2 was reconstructed from the analysed current records. It was found that this relation shows a negative slope conductance at all potentials positive to -75 mV and that the current tends towards zero at zero membrane potential. 6. The results are compared with those predicted by two reaction models of the iK2 and ix1 mechanisms. It is concluded that iK2 and ix1 are kinetically separable but that it is not possible with present techniques to decide whether they are controlled by the same or completely independent membrane structures. It is also shown that the instantaneous current—voltage relation calculated for iK2 does not depend on whether the controlling mechanisms are assumed to be independent or linked. PMID:4679715

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

PubMed Central

Taleb, Omar; Kemmel, Véronique; Laux, Alexis; Miehe, Monique; Delalande, François; Roussel, Guy; Van Dorsselaer, Alain; Metz-Boutigue, Marie-Hélène; Aunis, Dominique; Goumon, Yannick

2008-01-01

Background Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system. Methodology and Principal Findings Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10−10 M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies. Conclusions/Significance The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a

Desipramine and citalopram attenuate pretest swim-induced increases in prodynorphin immunoreactivity in the dorsal bed nucleus of the stria terminalis and the lateral division of the central nucleus of the amygdala in the forced swimming test.

PubMed

Chung, Sung; Kim, Hee Jeong; Kim, Hyun Ju; Choi, Sun Hye; Cho, Jin Hee; Cho, Yun Ha; Kim, Dong-Hoon; Shin, Kyung Ho

2014-10-01

Dynorphin in the nucleus accumbens shell plays an important role in antidepressant-like effect in the forced swimming test (FST), but it is unclear whether desipramine and citalopram treatments alter prodynorphin levels in other brain areas. To explore this possibility, we injected mice with desipramine and citalopram 0.5, 19, and 23 h after a 15-min pretest swim and observed changes in prodynorphin expression before the test swim, which was conducted 24 h after the pretest swim. The pretest swim increased prodynorphin immunoreactivity in the dorsal bed nucleus of the stria terminalis (dBNST) and lateral division of the central nucleus of the amygdala (CeL). This increase in prodynorphin immunoreactivity in the dBNST and CeL was blocked by desipramine and citalopram treatments. Similar changes in prodynorphin mRNA levels were observed in the dBNST and CeL, but these changes did not reach significance. To understand the underlying mechanism, we assessed changes in phosphorylated CREB at Ser(133) (pCREB) immunoreactivity in the dBNST and central nucleus of the amygdala (CeA). Treatment with citalopram but not desipramine after the pretest swim significantly increased pCREB immunoreactivity only in the dBNST. These results suggest that regulation of prodynorphin in the dBNST and CeL before the test swim may be involved in the antidepressant-like effect of desipramine and citalopram in the FST and suggest that changes in pCREB immunoreactivity in these areas may not play an important role in the regulation of prodynorphin in the dBNST and CeA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Substance P immunoreactivity exhibits frequent colocalization with kisspeptin and neurokinin B in the human infundibular region.

PubMed

Hrabovszky, Erik; Borsay, Beáta Á; Rácz, Kálmán; Herczeg, László; Ciofi, Philippe; Bloom, Stephen R; Ghatei, Mohammad A; Dhillo, Waljit S; Liposits, Zsolt

2013-01-01

Neurons synthesizing neurokinin B (NKB) and kisspeptin (KP) in the hypothalamic arcuate nucleus represent important upstream regulators of pulsatile gonadotropin-releasing hormone (GnRH) neurosecretion. In search of neuropeptides co-expressed in analogous neurons of the human infundibular nucleus (Inf), we have carried out immunohistochemical studies of the tachykinin peptide Substance P (SP) in autopsy samples from men (21-78 years) and postmenopausal (53-83 years) women. Significantly higher numbers of SP-immunoreactive (IR) neurons and darker labeling were observed in the Inf of postmenopausal women than in age-matched men. Triple-immunofluorescent studies localized SP immunoreactivity to considerable subsets of KP-IR and NKB-IR axons and perikarya in the infundibular region. In postmenopausal women, 25.1% of NKB-IR and 30.6% of KP-IR perikarya contained SP and 16.5% of all immunolabeled cell bodies were triple-labeled. Triple-, double- and single-labeled SP-IR axons innervated densely the portal capillaries of the infundibular stalk. In quadruple-labeled sections, these axons formed occasional contacts with GnRH-IR axons. Presence of SP in NKB and KP neurons increases the functional complexity of the putative pulse generator network. First, it is possible that SP modulates the effects of KP and NKB in axo-somatic and axo-dendritic afferents to GnRH neurons. Intrinsic SP may also affect the activity and/or neuropeptide release of NKB and KP neurons via autocrine/paracrine actions. In the infundibular stalk, SP may influence the KP and NKB secretory output via additional autocrine/paracrine mechanisms or regulate GnRH neurosecretion directly. Finally, possible co-release of SP with KP and NKB into the portal circulation could underlie further actions on adenohypophysial gonadotrophs.

The Characterization of AT1 Expression in the Dorsal Root Ganglia After Chronic Constriction Injury.

PubMed

Oroszova, Zuzana; Hricova, Ludmila; Stropkovska, Andrea; Lukacova, Nadezda; Pavel, Jaroslav

2017-04-01

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT 1 expression in neuronal subpopulation of lower lumbar dorsal root ganglia under normal conditions and its alteration in neuropathic pain model. The characterization of AT 1 expression was done under control and after the chronic constriction injury induced by four loose ligatures of the sciatic nerve representing the model of posttraumatic painful peripheral neuropathy. Major Angiotensin II receptor type was expressed in approximately 43 % of small-sized and 62 % of large-sized neurons in control. The AT 1 overexpression after sciatic nerve ligation lasting 7 days was detected predominantly in small-sized AT 1 immunoreactive neurons (about 38 % increase). Chronic constriction injury caused a statistically marked increase in number of the small-sized peptidergic (CGRP immunoreactive) neuronal subpopulation expressing AT 1 (about 64 %). The subpopulations of AT 1 -immunoreactive and nonpeptide-containing primary sensory neurons revealed by IB4 binding, tyrosine hydroxylase- and parvalbumin-immunoreactive neurons were not markedly changed. Our results indicate that: (1) the AT 1 overexpression after the chronic constriction injury is an important factor in Angiotensin II-potentiated pain perception; (2) Angiotensin II is involved in pathological mechanisms of neuropathic pain and this effect can be mediated perhaps in combination with other neuropeptides synthesized in the primary sensory neurons.

Novel experimental results in human cardiac electrophysiology: measurement of the Purkinje fibre action potential from the undiseased human heart.

PubMed

Nagy, Norbert; Szél, Tamás; Jost, Norbert; Tóth, András; Gy Papp, Julius; Varró, András

2015-09-01

Data obtained from canine cardiac electrophysiology studies are often extrapolated to the human heart. However, it has been previously demonstrated that because of the lower density of its K(+) currents, the human ventricular action potential has a less extensive repolarization reserve. Since the relevance of canine data to the human heart has not yet been fully clarified, the aim of the present study was to determine for the first time the action potentials of undiseased human Purkinje fibres (PFs) and to compare them directly with those of dog PFs. All measurements were performed at 37 °C using the conventional microelectrode technique. At a stimulation rate of 1 Hz, the plateau potential of human PFs is more positive (8.0 ± 1.8 vs 8.6 ± 3.4 mV, n = 7), while the amplitude of the spike is less pronounced. The maximal rate of depolarization is significantly lower in human PKs than in canine PFs (406.7 ± 62 vs 643 ± 36 V/s, respectively, n = 7). We assume that the appreciable difference in the protein expression profiles of the 2 species may underlie these important disparities. Therefore, caution is advised when canine PF data are extrapolated to humans, and further experiments are required to investigate the characteristics of human PF repolarization and its possible role in arrhythmogenesis.

A Novel Marker for Purkinje Cells, Ribosomal Protein MPS1/S27: Expression of MPS1 in Human Cerebellum.

PubMed

Fernandez-Pol, J Alberto

2016-01-01

The ribosomal protein metallopanstimulin-1 (MPS1/S27) serves critical survival purposes in cell division, in normal and cancerous cells; for this reason, selective pressures of evolution have conserved the DNA sequences encoding MPS1/S27 in Archaea and Eukariotic cells. The expression of MPS1/S27 protein in human adult cerebellum has not been established. The presence of MPS1/S27, was screened in paraffin-embedded human adult brain specimens processed for tissue inmunohistochemistry. Affinity-purified specific antibodies were directed against the N-terminus of MPS1. The antibodies to MPS1 detected Purkinje cells (PC) and their dendrites. In PC, MPS1 antigen-positive staining was found in: the nucleolus, which was strongly stained; ribosomes attached to the external nuclear membrane; cytoplasm of PC, with strong staining in a punctuate fashion; the soma-attached large dendrite trunks of PC, which were MPS1 antigen-positive; and the granular cell layer, where cellular staining in a few cells that appeared to resemble smaller PC was observed. Since MPS1 is involved in cell division, DNA repair, and ribosomal biogenesis, it may be a useful antigen for studying processes such as protein synthesis, oncogenesis, regeneration, aging, and perhaps diseases of the human cerebellum. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Action potential processing in a detailed Purkinje cell model reveals a critical role for axonal compartmentalization

PubMed Central

Masoli, Stefano; Solinas, Sergio; D'Angelo, Egidio

2015-01-01

The Purkinje cell (PC) is among the most complex neurons in the brain and plays a critical role for cerebellar functioning. PCs operate as fast pacemakers modulated by synaptic inputs but can switch from simple spikes to complex bursts and, in some conditions, show bistability. In contrast to original works emphasizing dendritic Ca-dependent mechanisms, recent experiments have supported a primary role for axonal Na-dependent processing, which could effectively regulate spike generation and transmission to deep cerebellar nuclei (DCN). In order to account for the numerous ionic mechanisms involved (at present including Nav1.6, Cav2.1, Cav3.1, Cav3.2, Cav3.3, Kv1.1, Kv1.5, Kv3.3, Kv3.4, Kv4.3, KCa1.1, KCa2.2, KCa3.1, Kir2.x, HCN1), we have elaborated a multicompartmental model incorporating available knowledge on localization and gating of PC ionic channels. The axon, including initial segment (AIS) and Ranvier nodes (RNs), proved critical to obtain appropriate pacemaking and firing frequency modulation. Simple spikes initiated in the AIS and protracted discharges were stabilized in the soma through Na-dependent mechanisms, while somato-dendritic Ca channels contributed to sustain pacemaking and to generate complex bursting at high discharge regimes. Bistability occurred only following Na and Ca channel down-regulation. In addition, specific properties in RNs K currents were required to limit spike transmission frequency along the axon. The model showed how organized electroresponsive functions could emerge from the molecular complexity of PCs and showed that the axon is fundamental to complement ionic channel compartmentalization enabling action potential processing and transmission of specific spike patterns to DCN. PMID:25759640

Activation patterns of Purkinje fibers during long-duration ventricular fibrillation in an isolated canine heart model.

PubMed

Tabereaux, Paul B; Walcott, Greg P; Rogers, Jack M; Kim, Jong; Dosdall, Derek J; Robertson, Peter G; Killingsworth, Cheryl R; Smith, William M; Ideker, Raymond E

2007-09-04

The roles of Purkinje fibers (PFs) and focal wave fronts, if any, in the maintenance of ventricular fibrillation (VF) are unknown. If PFs are involved in VF maintenance, it should be possible to map wave fronts propagating from PFs into the working ventricular myocardium during VF. If wave fronts ever arise focally during VF, it should be possible to map them appearing de novo. Six canine hearts were isolated, and the left main coronary artery was cannulated and perfused. The left ventricular cavity was exposed, which allowed direct endocardial mapping of the anterior papillary muscle insertion. Nonperfused VF was induced, and 6 segments of data, each 5 seconds long, were analyzed during 10 minutes of VF. During 36 segments of data that were analyzed, 1018 PF or focal wave fronts of activation were identified. In 534 wave fronts, activation was mapped propagating from working ventricular myocardium to PF. In 142 wave fronts, activation was mapped propagating from PF to working ventricular myocardium. In 342 wave fronts, activation was mapped arising focally. More than 1 of these 3 patterns could occur in the same wave front. PFs are highly active throughout the first 10 minutes of VF. In addition to retrograde propagation from the working ventricular myocardium to PFs, antegrade propagation occurs from PFs to working ventricular myocardium, which suggests PFs are important in VF maintenance. Prior plunge needle recordings in dogs indicate activation propagates from the endocardium toward the epicardium after 1 minute of VF, which suggests that focal sites on the endocardium may represent foci and not breakthrough. If so, in addition to reentry, abnormal automaticity or triggered activity may also occur during VF.

Gene expression in the developing cerebellum during perinatal hypo- and hyperthyroidism.

PubMed

Figueiredo, B C; Almazan, G; Ma, Y; Tetzlaff, W; Miller, F D; Cuello, A C

1993-03-01

The intensity of p75NGFR receptor-like immunoreactivity and the mRNAs encoding p75NGFR, T alpha 1 alpha-tubulin, GAP-43 and the myelin proteins MBP and PLP were measured in the developing cerebellum to study the effects of perinatal thyroid hormone imbalance in rats. Results compared to age-matched controls provide in vivo evidence for differential gene regulation by thyroid hormone in the developing cerebellum. We found that p75NGFR immunoreactivity was strikingly elevated in hypothyroid rats, whereas p75NGFR mRNA content remained only twice as high as that of control levels on postnatal day 15 (P15). When p75NGFR immunoreactivity was still elevated in hypothyroid rats, Purkinje cells exhibited proximal axonal varicosities, axonal twisting and differences in axonal caliber. The mRNAs encoding proteins involved with neurite growth-promoting elements, T alpha 1 alpha-tubulin and GAP-43, were also increased in hypothyroidism, possibly reflecting a neuronal response to a deficiency in, or damage to, cerebellar neurons, or a general delay in their down regulation. Similar increases were not observed for the myelin specific genes. MBP and PLP mRNAs were first detected on P2 of hyperthyroid rats, and they increased with age. Hypo- or hyperthyroidism did not affect the initial onset of MBP and PLP expression, however, hyperthyroidism increased levels of PLP and MBP mRNAs between P2 and P10. By contrast, the most consistent decrease in MBP and PLP mRNAs in rats with thyroid hormone deficiency was observed only on P10. At later times (P15 and P30), the two mRNA levels were similar to controls in all groups. These results are consistent with a role for thyroid hormone in the earlier stages of cerebellar myelination. Hypothryoidism led to specific increases in T alpha 1 alpha-tubulin and GAP-43 mRNAs, and in the immunoreactivity and mRNA levels of p75NGFR receptor--all changes that may play a role in the observed abnormal neuronal outgrowth.

Neural stem cells rescue nervous purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets.

PubMed

Li, Jianxue; Imitola, Jaime; Snyder, Evan Y; Sidman, Richard L

2006-07-26

Neural stem cells (NSCs) offer special therapeutic prospects because they can be isolated from the CNS, expanded ex vivo, and re-implanted into diseased CNS where they not only migrate and differentiate according to cues from host tissue but also appear to be capable of affecting host cells. In nervous (nr) mutant mice Purkinje neuron (PN) mitochondria become abnormal by the second postnatal week, and a majority of PNs die in the fourth to fifth weeks. We previously identified in nr cerebellum a 10-fold increase in tissue plasminogen activator (tPA) as a key component of the mechanism causing nr PN death. Here we report that undifferentiated wild-type murine NSCs, when transplanted into the newborn nr cerebellar cortex, do not replace host PNs but contact imperiled PNs and support their mitochondrial function, dendritic growth, and synaptogenesis, subsequently leading to the rescue of host PNs and restoration of motor coordination. This protection of nr PNs also is verified by an in vitro organotypic slice model in which nr cerebellar slices are cocultured with NSCs. Most importantly, the integrated NSCs in young nr cerebellum rectify excessive tPA mRNA and protein to close to normal levels and protect the mitochondrial voltage-dependent anion channel and neurotrophins, downstream targets of the tPA/plasmin proteolytic system. This report demonstrates for the first time that NSCs can rescue imperiled host neurons by rectifying their gene expression, elevating somatic stem cell therapeutic potential beyond solely cell replacement strategy.

Modulation of type I immediate and type IV delayed immunoreactivity using direct suggestion and guided imagery during hypnosis.

PubMed

Zachariae, R; Bjerring, P; Arendt-Nielsen, L

1989-11-01

Cutaneous reactivity against histamine skin prick test (Type I) and purified tuberculin protein derivative (Mantoux reaction, Type IV) was studied in eight volunteers under hypnosis. Types I and IV immunoreactivity were modulated by direct suggestion (Type I) and guided imagery (Type IV). The volunteers were highly susceptible subjects, selected by means of the Harvard Group Scale of Hypnotic Susceptibility, Form A. When the volunteers underwent hypnotic suggestion to decrease the cutaneous reaction to histamine prick test, a significant (P less than 0.02) reduction of the flare reaction (area of erythema) was observed compared with control histamine skin prick tests. The wheal reaction did not respond to hypnotic suggestion. Neither wheal nor flare reaction could be increased in size by hypnotic suggestion compared with control histamine skin prick tests. A hypnotic suggestion of increasing the Type IV reaction on one arm and decreasing the reaction on the other revealed a significant difference in both erythema size (P less than 0.02) and palpable induration (P less than 0.01). In two cases the reactions were monitored by laser doppler blood flowmetry and skin thickness measurement by ultrasound. The difference between the suggested increased and decreased reaction was 19% for the laser doppler bloodflow (in favor of the augmented side), and 44% for the dermal infiltrate thickness. This study objectively supports the numerous uncontrolled case reports of modulation of immunoreactivity in allergic diseases involving both Type I and Type IV skin reactions following hypnotic suggestions.

A previous history of repeated amphetamine exposure modifies brain angiotensin II AT1 receptor functionality.

PubMed

Casarsa, B S; Marinzalda, M Á; Marchese, N A; Paz, M C; Vivas, L; Baiardi, G; Bregonzio, C

2015-10-29

Previous results from our laboratory showed that angiotensin II AT1 receptors (AT1-R) are involved in the neuroadaptative changes induced by amphetamine. The aim of the present work was to study functional and neurochemical responses to angiotensin II (ANG II) mediated by AT1-R activation in animals previously exposed to amphetamine. For this purpose male Wistar rats (250-320 g) were treated with amphetamine (2.5mg/kg/day intraperitoneal) or saline for 5 days and implanted with intracerebroventricular (i.c.v.) cannulae. Seven days after the last amphetamine administration the animals received ANG II (400 pmol) i.c.v. One group was tested in a free choice paradigm for sodium (2% NaCl) and water intake and sacrificed for Fos immunoreactivity (Fos-IR) determinations. In a second group of rats, urine and plasma samples were collected for electrolytes and plasma renin activity determination and then they were sacrificed for Fos-IR determination in Oxytocinergic neurons (Fos-OT-IR). Repeated amphetamine exposure (a) prevented the increase in sodium intake and Fos-IR cells in caudate-putamen and accumbens nucleus induced by ANG II i.c.v. (b) potentiated urinary sodium excretion and Fos-OT-IR in hypothalamus and (c) increased the inhibitory response in plasma renin activity, in response to ANG II i.c.v. Our results indicate a possible functional desensitisation of AT1-R in response to ANG II, induced by repeated amphetamine exposure. This functional AT1-R desensitisation allows to unmask the effects of ANG II i.c.v. mediated by oxytocin. We conclude that the long lasting changes in brain AT1-R functionality should be considered among the psychostimulant-induced neuroadaptations. Published by Elsevier Ltd.

A pilot study evaluating changes in pancreatic lipase immunoreactivity concentrations in canines treated with L-asparaginase (ASNase), vincristine, or both for lymphoma

PubMed Central

Wright, Zachary; Steiner, Joerg; Suchodolski, Jan; Rogers, Kenita; Barton, Claudia; Brown, Marjorie

2009-01-01

L-asparaginase (ASNase) is a common chemotherapy agent for the treatment of lymphoid malignancies. L-asparaginase has been reported to cause clinical pancreatitis in both humans and canines. Canine pancreatic lipase immunoreactivity (cPLI) is now a common diagnostic tool for evaluating pancreatitis in dogs. A total of 52 dogs were enrolled into this study. Canine pancreatic lipase immunoreactivity (cPLI) concentrations were evaluated before and after administration of ASNase, vincristine, or both. All dogs enrolled in the study were evaluated for signs compatible with clinical pancreatitis. No dogs receiving ASNase alone showed evidence of clinical pancreatitis after administration. Also, there was no statistically significant change in cPLI concentrations before or after treatment. Fourteen percent of dogs that received both vincristine and ASNase concurrently had elevated concentrations of cPLI after treatment. Of the 11 dogs with clinical signs compatible with pancreatitis after any chemotherapy treatment, no dog had a cPLI concentration > 400 μg/dL. In conclusion, ASNase did not cause clinical pancreatitis in this cohort of dogs but larger sample sizes are required to further validate this data. PMID:19436578

Comparative analysis of Met-enkephalin, galanin and GABA immunoreactivity in the developing trout preoptic-hypophyseal system.

PubMed

Rodríguez Díaz, M A; Candal, E; Santos-Durán, G N; Adrio, F; Rodríguez-Moldes, I

2011-08-01

We studied the organization of Met-enkephalin-containing cells and fibers in the developing preoptic-hypophyseal system of the brown trout (Salmo trutta fario) by immunohistochemistry and determined the relationship of these cells and fibers to the galaninergic and GABAergic systems. Met-enkephalin immunoreactivity was observed in cells in the preoptic area, the hypothalamus and the pituitary of late larvae. In the hypophysis, a few Met-enkephalin-containing cells were present in all divisions of the adenohypophysis, and some immunoreactive fibers were present in the interdigitations of the neural lobe with the proximal pars distalis. Concurrently, GABAergic fibers innervated the anterior and posterior neural lobe. Galanin cells coexisted with Met-enkephalin cells in neuronal groups of the preoptic-hypophyseal system. Galaninergic and GABAergic fibers innervated the preoptic and hypothalamic areas, but GABAergic fibers containing galanin were not observed. These results indicate that Met-enkephalin, galanin and GABA may modulate neuroendocrine activities in the preoptic area, hypothalamus and pituitary during the transition from larval to juvenile period. To better know how the development of the trout preoptic-hypophyseal system takes place, we studied the patterns of cell proliferation and expression of Pax6, a conserved transcription factor involved in the hypophysis development. Pax6 expressing cells and proliferating cells were present in the Rathke's pouch, the hypothalamus and the hypophysis of early larvae. In late larvae, Pax6 expression was no longer observed in these areas, and the density of proliferating cells largely decreased throughout development, although they remained in the hypophysis of late larvae and juveniles, suggesting that Pax6 might play an important role in the early regionalization of the pituitary in the trout. Copyright © 2011 Elsevier Inc. All rights reserved.

Postischemic alterations of BDNF, NGF, HSP 70 and ubiquitin immunoreactivity in the gerbil hippocampus: pharmacological approach.

PubMed

Himeda, Toshiki; Tounai, Hiroko; Hayakawa, Natsumi; Araki, Tsutomu

2007-03-01

1. We investigated the immunohistochemical alterations of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus 1 h to 14 days after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor pitavastatin against the changes of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus after cerebral ischemia in the hippocampus after ischemia. 2. The transient cerebral ischemia was carried out by clamping the carotid arteries with aneurismal clips for 5 min. 3. In the present study, the alteration of HSP 70 and ubiquitin immunoreactivity in the hippocampal CA1 sector was more pronounced than that of BDNF and NGF immunoreactivity after transient cerebral ischemia. In double-labeled immunostainings, BDNF, NGF and ubiquitin immunostaining was observed both in GFAP-positive astrocytes and MRF-1-positive microglia in the hippocampal CA1 sector after ischemia. Furthermore, prophylactic treatment with pitavastatin prevented the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after ischemia. 4. These findings suggest that the expression of stress protein including HSP 70 and ubiquitin may play a key role in the protection against the hippocampal CA1 neuronal damage after transient cerebral ischemia in comparison with the expression of neurotrophic factor such as BDNF and NGF. The present findings also suggest that the glial BDNF, NGF and ubiquitin may play some role for helping surviving neurons after ischemia. Furthermore, our present study indicates that prophylactic treatment with pitavastatin can prevent the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after transient cerebral ischemia. Thus our study provides further valuable information for the pathogenesis after transient cerebral ischemia.

Lack of ubiquitin immunoreactivities at both ends of neuropil threads. Possible bidirectional growth of neuropil threads.

PubMed

Iwatsubo, T; Hasegawa, M; Esaki, Y; Ihara, Y

1992-02-01

Immunocytochemically, neuropil threads (curly fibers) were investigated in the Alzheimer's disease brain using a confocal laser scanning fluorescence microscope by double labeling with tau/ubiquitin antibodies. Ubiquitin immunoreactivities were found to be lacking at one or both ends in more than 40% of tau-positive threads. Immunoelectron microscopy showed that bundles of paired helical filaments, which constitute neuropil threads, were positive for ubiquitin around their midportions, but often negative at their ends. Since it is reasonable to postulate that tau deposition as paired helical filaments precedes ubiquitination, the aforementioned observation suggests that the ends of the threads are newly formed portions, and thus the threads are often growing bidirectionally in small neuronal processes.

Calretinin and parvalbumin immunoreactive interneurons in the retrosplenial cortex of the rat brain: Qualitative and quantitative analyses.

PubMed

Salaj, Martin; Druga, Rastislav; Cerman, Jiří; Kubová, Hana; Barinka, Filip

2015-11-19

The retrosplenial cortex (RSC) is a mesocortical region broadly involved with memory and navigation. It shares many characteristics with the perirhinal cortex (PRC), both of which appear to be significantly involved in the spreading of epileptic activity. We hypothesized that RSC possesses an interneuronal composition similar to that of PRC. To prove the hypothesis we studied the general pattern of calretinin (CR) and parvalbumin (PV) immunoreactivity in the RSC of the rat brain, its optical density as well as the morphological features and density of CR- and PV-immunoreactive (CR+ and PV+) interneurons. We also analyzed the overall neuronal density on Nissl-stained sections in RSC. Finally, we compared our results with our earlier analysis of PRC (Barinka et al., 2012). Compared to PRC, RSC was observed to have a higher intensity of PV staining and lower intensity of CR staining of neuropil. Vertically-oriented bipolar neurons were the most common morphological type among CR+ neurons. The staining pattern did not allow for a similarly detailed analysis of somatodendritic morphology of PV+ neurons. RSC possessed lower absolute (i.e., neurons/mm(3)) and relative (i.e., percentage of the overall neuronal population) densities of CR+ neurons and similar absolute and lower relative densities of PV+ neurons relative to PRC. CR: PV neuronal ratio in RSC (1:2 in area 29 and 1:2.2 in area 30) differed from PRC (1:1.2 in area 35 and 1:1.7 in area 36). In conclusion, RSC, although similar in many aspects to PRC, differs strikingly in the interneuronal composition relative to PRC. Copyright © 2015 Elsevier B.V. All rights reserved.

FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis

PubMed Central

Deng, Han-Xiang; Zhai, Hong; Bigio, Eileen H.; Yan, Jianhua; Fecto, Faisal; Ajroud, Kaouther; Mishra, Manjari; Ajroud-Driss, Senda; Heller, Scott; Sufit, Robert; Siddique, Nailah; Mugnaini, Enrico; Siddique, Teepu

2014-01-01

Objective Amyotrophic lateral sclerosis (ALS) is a fatal disorder of motor neuron degeneration. Most cases of ALS are sporadic (SALS), but about 5-10% of ALS cases are familial (FALS). Recent studies have shown that mutations in FUS are causal in approximately 4-5% of FALS and some apparent SALS cases. The pathogenic mechanism of the mutant FUS-mediated ALS and potential roles of FUS in non-FUS ALS remain to be investigated. Methods Immunostaining was performed on postmortem spinal cords from 78 ALS cases, including SALS (n=52), ALS with dementia (ALS/dementia, n=10) and FALS (n=16). In addition, postmortem brains or spinal cords from 22 cases with or without frontotemporal lobar degeneration (FTLD) were also studied. In total, 100 cases were studied. Results FUS-immunoreactive inclusions were observed in spinal anterior horn neurons in all sporadic and familial ALS cases, except for those with SOD1 mutations. The FUS-containing inclusions were also immunoreactive with antibodies to TDP43, p62 and ubiquitin. A fraction of tested FUS antibodies recognized FUS inclusions and an unusual antigen retrieval appeared to be important for detection of the skein-like FUS inclusions. Interpretation Although mutations in FUS account for only a small fraction of FALS and SALS, our data suggest that FUS protein may be a common component of the cellular inclusions in non-SOD1 ALS and some other neurodegenerative conditions, implying a shared pathogenic pathway underlying SALS, non-SOD1 FALS, ALS/dementia and related disorders. Our data also indicate that SOD1-linked ALS may have a distinct pathogenic pathway from SALS and other types of FALS. PMID:20517935

Isolation of the most immunoreactive antigenes of echinococcus granulosus from sheep hydatid fluid.

PubMed

Pozzuoli, R; Piantelli, M; Perucci, C; Arru, E; Musiani, P

1975-11-01

This paper describes a simplified procedure for obtaining purified Echinococcus granulosus antigens from sheep hydatid fluid by using affinity chromatography on concanavalin A-Sepharose. The presence of two "major" antigens (4 and 5) was confirmed. Antigen 5 was isolated by preparative polyacrylamide gel electrophoresis. Antigen 4, eluted by diffusion from the gel, was seen to be "contaminated" by antigen 5 and was isolated by using anti-5 Sepharose-linked serum. These two major antigens were then tested separately against the sera of hydatidosis patients by using very simple immunolgic tests. The best results were obtained in passive hemagglutination with antigen 4. Antigen 4 is the most immunoreactive parasitic antigen; antibodies against it were found in the sera of all hydatidosis patients showing positive reaction. Apart from the direct use of this antigen in serologic tests, it appears possible to standarize the most frequently used and commerically available antigenic materials by titrating this component.

Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell

PubMed Central

1985-01-01

A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of a Ca2+ transient. Simultaneously monitoring tension and aequorin bioluminescence provided two methods for estimating the peak myoplasmic [free Ca2+] reached during the spontaneous cyclic Ca2+ release from the SR obtained in the continuous presence of a bulk solution [free Ca2+] sufficiently high to overload the SR. These methods gave results in excellent agreement for the spontaneous Ca2+ release under a variety of conditions of pH and [free Mg2+], and of enhancement of Ca2+ release by calmodulin. Disagreement was observed, however, when the Ca2+ transient was modified during its ascending phase. The experiments also permitted quantification of the aequorin binding within the myofibrils and determination of its operational apparent affinity constant for Ca2+ at various [free Mg2+] levels. An increase of [free Ca2+] at the outer surface of the SR during the ascending phase of the Ca2+ transient induced further release of Ca2+. In contrast, an increase of [free Ca2+] during the descending phase of the Ca2+ transient did not cause further Ca2+ release. Varying [free H+], [free Mg2+], or the [Na+]/[K+] ratio had no significant effect on the Ca2+ transient during which the modification was applied, but it altered the subsequent Ca2+ transient. Therefore, Ca2+ appears to be the major, if not the only, ion controlling Ca2+ release from the SR rapidly enough to alter a Ca2+ transient during its course. PMID:3981128

Functional contributions of glutamate transporters at the parallel fibre to Purkinje neuron synapse-relevance for the progression of cerebellar ataxia.

PubMed

Power, Emmet M; Empson, Ruth M

2014-01-01

Rapid uptake of glutamate by neuronal and glial glutamate transporters (EAATs, a family of excitatory amino acid transporters) is critical for shaping synaptic responses and for preventing excitotoxicity. Two of these transporters, EAAT4 in Purkinje neurons (PN) and EAAT1 in Bergmann glia are both enriched within the cerebellum and altered in a variety of human ataxias. PN excitatory synaptic responses and firing behaviour following high frequency parallel fibre (PF) activity commonly encountered during sensory stimulation in vivo were adversely influenced by acute inhibition of glutamate transporters. In the presence of a non-transportable blocker of glutamate transporters we observed very large amplitude and duration excitatory postsynaptic currents accompanied by excessive firing of the PNs. A combination of AMPA and mGluR1, but not NMDA, type glutamate receptor activation powered the hyper-excitable PN state. The enhanced PN excitability also recruited a presynaptic mGluR4 dependent mechanism that modified short term plasticity at the PF synapse. Our findings indicate that reduced glutamate transporter activity, as occurs in the early stages of some forms of human cerebellar ataxias, excessively excites PNs and disrupts the timing of their output. Our findings raise the possibility that sustaining cerebellar glutamate uptake may provide a therapeutic approach to prevent this disruption and the glutamate excitotoxicity-induced PN death that signals the end point of the disease.

Pattern of calbindin-D28k and calretinin immunoreactivity in the brain of Xenopus laevis during embryonic and larval development.

PubMed

Morona, Ruth; González, Agustín

2013-01-01

The present study represents a detailed spatiotemporal analysis of the localization of calbindin-D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions. Copyright © 2012 Wiley Periodicals, Inc.

Hybridomas using athymic nude mouse injected with Crohn's disease (CD) tissue filtrate. Immunoreactivity of the hybridomas with CD sera.

PubMed Central

Das, K. M.; Vecchi, M.; Novikoff, A.; Mazumdar, S.; Novikoff, P. M.

1990-01-01

Injections of Crohn's disease (CD) tissue filtrates produce lymphoma and hyperplastic lymph nodes from plasma cell hyperplasia (PCH) in athymic nude (nu/nu) mice; these lymphoid tissue contain an antigen(s) recognized by CD serum/gamma G immunoglobulin (IgG). To immortalize the "CD-reactive antigen(s)," the authors fused the lymphoid cells from a CD tissue filtrate primed nu/nu mouse with nonsecretory mouse myeloma cells. Hybrids were screened and selected based on their reactivity with CD serum IgG, but not with control serum IgG in an indirect immunofluorescence assay (IF). Two CD-positive hybridomas were examined by IF with sera from 47 CD, 38 ulcerative colitis (UC), 13 controls with other gastrointestinal diseases, 19 with autoimmune diseases, and 21 normal subjects. Sera from 16 CD patients (34%) reacted with the two hybridomas, but only one of 38 UC sera and none of the 53 other disease or normal control sera reacted. The immunoreactivity of CD sera was significantly higher than UC sera (P less than 0.01) and each of the other groups (P less than 0.007). Using immunoperoxidase techniques at light and electron microscopic levels, the authors localized CD-associated antigen(s) in the plasma membrane of the two hybridomas. Further characterization of these hybridomas and the immunoreactive protein(s) may provide an important probe(s) for the diagnosis and the understanding of the pathogenesis of CD. Images Figure 2 Figure 3 PMID:2192559

Histone Deacetylase 3 Is Necessary for Proper Brain Development*

PubMed Central

Norwood, Jordan; Franklin, Jade M.; Sharma, Dharmendra; D'Mello, Santosh R.

2014-01-01

The functional role of histone deacetylase 3 (HDAC3) in the developing brain has yet to be elucidated. We show that mice lacking HDAC3 in neurons and glia of the central nervous system, Nes-Cre/HDAC3 conditional KO mice, show major abnormalities in the cytoarchitecture of the neocortex and cerebellum and die within 24 h of birth. Later-born neurons do not localize properly in the cortex. A similar mislocalization is observed with cerebellar Purkinje neurons. Although the proportion of astrocytes is higher than normal, the numbers of oligodendrocytes are reduced. In contrast, conditional knockout of HDAC3 in neurons of the forebrain and certain other brain regions, using Thy1-Cre and calcium/calmodulin dependent protein kinase II α-Cre for ablation, produces no overt abnormalities in the organization of cells within the cortex or of cerebellar Purkinje neurons at birth. However, both lines of conditional knockout mice suffer from progressive hind limb paralysis and ataxia and die around 6 weeks after birth. The mice display an increase in overall numbers of cells, higher numbers of astrocytes, and Purkinje neuron degeneration. Taken together, our results demonstrate that HDAC3 plays an essential role in regulating brain development, with effects on both neurons and glia in different brain regions. PMID:25339172

Enhancement of native and phosphorylated TDP-43 immunoreactivity by proteinase K treatment following autoclave heating.

PubMed

Mori, Fumiaki; Tanji, Kunikazu; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

2011-08-01

TDP-43 is a major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 (FTLD-TDP). To evaluate the effectiveness of proteinase K (PK) treatment in antigen retrieval for native and phosphorylated TDP-43 protein, we examined the temporal cortex and spinal cord from patients with sporadic ALS and FTLD-TDP and control subjects. PK treatment following heat retrieval enhanced the immunoreactivity for native TDP-43 in controls as well as for native and phosphorylated TDP-43 in ALS and FTLD-TDP. A significant number of TDP-43-positive neuropil threads were demonstrated in lesions, in which routine immunohistochemistry revealed that the predominant inclusions are cytoplasmic. This retrieval method is the best of immunohistochemical techniques for demonstrating TDP-43 pathology, especially in the neuropil. © 2010 Japanese Society of Neuropathology.

Spatiotemporal expression of MANF in the developing rat brain.

PubMed

Wang, Haiping; Ke, Zunji; Alimov, Alexander; Xu, Mei; Frank, Jacqueline A; Fang, Shengyun; Luo, Jia

2014-01-01

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor which exhibited neuroprotective properties. Recent studies suggested that MANF may play a role in the neural development of Drosophila and zebra fishes. In this study, we investigated the spatiotemporal expression of MANF in the brain of postnatal and adult rats. MANF expression appeared wide spread and mainly localized in neurons. In the cerebral cortex, neurons in layer IV and VI displayed particularly strong MANF immunoreactivity. In the hippocampus, intensive MANF expression was observed throughout the subfields of Cornu Amonis (CA1, CA2, and CA3) and the granular layer of the dentate gyrus (DG). In the substantia nigra, high MANF expression was shown in the substantia nigra pars compacta (SNpc). In the thalamus, the anterodorsal thalamic nucleus (ADTN) exhibited the highest MANF immunoreactivity. In the hypothalamus, intensive MANF immunoreactivity was shown in the supraoptic nucleus (SON) and tuberomammillary nucleus (TMN). In the cerebellum, MANF was localized in the external germinal layer (EGL), Purkinje cell layer (PCL), internal granule layer (IGL) and the deep cerebellar nuclei (DCN). We examined the developmental expression of MANF on postnatal day (PD) 3, 5, 7, 9, 15, 21, 30 and adulthood. In general, the levels of MANF were high in the early PDs (PD3 and PD5), and declined gradually as the brain matured; MANF expression in the adult brain was the lowest among all time points examined. However, in some structures, such as PCL, IGL, SON, TMN and locus coeruleus (LC), high expression of MANF sustained throughout the postnatal period and persisted into adulthood. Our results indicated that MANF was developmentally regulated and may play a role in the maturation of the central nervous system (CNS).

Spatiotemporal Expression of MANF in the Developing Rat Brain

PubMed Central

Wang, Haiping; Ke, Zunji; Alimov, Alexander; Xu, Mei; Frank, Jacqueline A.; Fang, Shengyun; Luo, Jia

2014-01-01

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor which exhibited neuroprotective properties. Recent studies suggested that MANF may play a role in the neural development of Drosophila and zebra fishes. In this study, we investigated the spatiotemporal expression of MANF in the brain of postnatal and adult rats. MANF expression appeared wide spread and mainly localized in neurons. In the cerebral cortex, neurons in layer IV and VI displayed particularly strong MANF immunoreactivity. In the hippocampus, intensive MANF expression was observed throughout the subfields of Cornu Amonis (CA1, CA2, and CA3) and the granular layer of the dentate gyrus (DG). In the substantia nigra, high MANF expression was shown in the substantia nigra pars compacta (SNpc). In the thalamus, the anterodorsal thalamic nucleus (ADTN) exhibited the highest MANF immunoreactivity. In the hypothalamus, intensive MANF immunoreactivity was shown in the supraoptic nucleus (SON) and tuberomammillary nucleus (TMN). In the cerebellum, MANF was localized in the external germinal layer (EGL), Purkinje cell layer (PCL), internal granule layer (IGL) and the deep cerebellar nuclei (DCN). We examined the developmental expression of MANF on postnatal day (PD) 3, 5, 7, 9, 15, 21, 30 and adulthood. In general, the levels of MANF were high in the early PDs (PD3 and PD5), and declined gradually as the brain matured; MANF expression in the adult brain was the lowest among all time points examined. However, in some structures, such as PCL, IGL, SON, TMN and locus coeruleus (LC), high expression of MANF sustained throughout the postnatal period and persisted into adulthood. Our results indicated that MANF was developmentally regulated and may play a role in the maturation of the central nervous system (CNS). PMID:24587361

Immunoreactive LH in long-term frozen human urine samples.

PubMed

Singh, Gurmeet Kaur Surindar; Jimenez, Mark; Newman, Ron; Handelsman, David J

2014-04-01

Urine provides a convenient non-invasive alternative to blood sampling for measurement of certain hormones. Urinary luteinizing hormone (LH) measurements have been used for endocrinology research and anti-doping testing. However, the commercially available LH immunoassays are developed and validated for human blood samples but not urine so that LH assays intended for use with urine samples need thorough validation. Therefore, the present study evaluated the measurement of urinary LH immunoreactivity using previously validated immunofluorometric (IF) and immunochemiluminometric (ICL) LH assays after prolonged frozen storage. LH was measured in serial urine samples following administration of a single injection of one of two doses of recombinant human chorionic hormone (rhCG) with assays run at the end of study (2008) and again after four years of frozen (-20 °C) storage where samples were stored without adding preservatives. The ICL assay showed quantitatively reproducible LH measurements after prolonged -20 °C storage. However, the IF immunoassay gave consistently lower LH levels relative to ICL (2008) with a further proportionate reduction after four years of sample storage (2012). Yet, both the assays displayed similar patterns of the time-course of urine LH measurement both before and after four years of frozen storage. In conclusion, we found that both immunoassays are suitable for urinary LH measurements with ICL assay being more robust for quantitative urinary LH measurement such as for anti-doping purposes, whereas the IF could be applicable for research studies where urine LH levels are compared within-study but not in absolute terms. Copyright © 2013 John Wiley & Sons, Ltd.

Lack of ubiquitin immunoreactivities at both ends of neuropil threads. Possible bidirectional growth of neuropil threads.

PubMed Central

Iwatsubo, T.; Hasegawa, M.; Esaki, Y.; Ihara, Y.

1992-01-01

Immunocytochemically, neuropil threads (curly fibers) were investigated in the Alzheimer's disease brain using a confocal laser scanning fluorescence microscope by double labeling with tau/ubiquitin antibodies. Ubiquitin immunoreactivities were found to be lacking at one or both ends in more than 40% of tau-positive threads. Immunoelectron microscopy showed that bundles of paired helical filaments, which constitute neuropil threads, were positive for ubiquitin around their midportions, but often negative at their ends. Since it is reasonable to postulate that tau deposition as paired helical filaments precedes ubiquitination, the aforementioned observation suggests that the ends of the threads are newly formed portions, and thus the threads are often growing bidirectionally in small neuronal processes. Images Figure 1 Figure 2 PMID:1310831

Second-degree atrioventricular block.

PubMed

Zipes, D P

1979-09-01

1) While it is possible only one type of second-degree AV block exists electrophysiologically, the available data do not justify such a conclusion and it would seem more appropriate to remain a "splitter," and advocate separation and definition of multiple mechanisms, than to be a "lumper," and embrace a unitary concept. 2) The clinical classification of type I and type II AV block, based on present scalar electrocardiographic criteria, for the most part accurately differentiates clinically important categories of patients. Such a classification is descriptive, but serves a useful function and should be preserved, taking into account the caveats mentioned above. The site of block generally determines the clinical course for the patient. For most examples of AV block, the type I and type II classification in present use is based on the site of block. Because block in the His-Purkinje system is preceded by small or nonmeasurable increments, it is called type II AV block; but the very fact that it is preceded by small increments is because it occurs in the His-Purkinje system. Similar logic can be applied to type I AV block in the AV node. Exceptions do occur. If the site of AV block cannot be distinguished with certainity from the scalar ECG, an electrophysiologic study will generally reveal the answer.

Nominal effective immunoreaction volume of magnetic beads at single bead level.

PubMed

Wang, Rui; Chen, Yuan; Fan, Kai; Ji, Feng; Wu, Jian; Yu, Yong-Hua

Immunomagnetic bead (IMB)-based enzyme-linked immunosorbent assay (ELISA) has been the tool frequently used for protein detection in research and clinical laboratories. For most ELISA reactions the recommended dosage of IMBs is usually according to their weight (mg) or mass fraction (w/v) instead of the bead number. Consequently, the processes occurring in the immediate vicinity of the IMBs have always been ignored by researchers and they cannot be revealed in detail during the ELISA reaction. In this paper, we established the relationship between number of IMBs and colorimetric results, and further proposed a new concept of "nominal effective immunoreaction volume (NEIV)" to characterize a single IMB during ELISA reaction. Results showed that the NEIV of a single IMB has a constant value, which is unrelated to the amount of beads and the concentration of antigen. Optimal results of the colorimetric ELISA are achieved when the incubation volume meets each IMB's NEIV and is no longer enhanced by increasing the incubation volume. Thus, the reliable and relatively precise number of IMBs for ELISA detection during practical application could be determined. Most importantly, a study using IMB's NEIV would lay the foundation for a kinetics analysis of IMBs and antigens for future study.

Kinetic and thermodynamic parameters for thermal denaturation of ovine milk lactoferrin determined by its loss of immunoreactivity.

PubMed

Navarro, F; Harouna, S; Calvo, M; Pérez, M D; Sánchez, L

2015-07-01

Lactoferrin is a protein with important biological functions that can be obtained from milk and by-products derived from the dairy industry, such as whey. Although bovine lactoferrin has been extensively studied, ovine lactoferrin is not quite as well known. In the present study, the effect of several heat treatments in 3 different media, over a temperature range from 66 to 75°C, has been studied on lactoferrin isolated from sheep milk. Denaturation of lactoferrin was determined by measuring its immunoreactivity with specific polyclonal antibodies. Kinetic and thermodynamic parameters obtained indicate that lactoferrin denatures by heat more rapidly in whey than in phosphate buffer or milk. The value of activation energy found for the denaturation process of lactoferrin when treated in whey is higher (390kJ/mol) than that obtained in milk (194kJ/mol) or phosphate buffer (179kJ/mol). This indicates that a great amount of energy is necessary to start denaturation of ovine lactoferrin, probably due to the interaction of this protein with other whey proteins. The changes in the hydrophobicity of lactoferrin after heat treatments were determined by fluorescence measurement using acrylamide. The decrease in the hydrophobicity constant was very small for the treatments from 66 to 75°C, up to 20min, which indicates that lactoferrin conformation did not experienced a great change. The results obtained in this study permit the prediction of behavior of ovine lactoferrin under several heat treatments and show that high-temperature, short-time pasteurization (72°C, 15 s) does not cause loss of its immunoreactivity and, consequently, would not affect its conformation and biological activity. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Association of postprandial serum triglyceride concentration and serum canine pancreatic lipase immunoreactivity in overweight and obese dogs.

PubMed

Verkest, K R; Fleeman, L M; Morton, J M; Groen, S J; Suchodolski, J S; Steiner, J M; Rand, J S

2012-01-01

Hypertriglyceridemia has been proposed to contribute to the risk of developing pancreatitis in dogs. To determine associations between postprandial serum triglyceride concentrations and canine pancreatic lipase immunoreactivity (cPLI) concentrations or pancreatic disease. Thirty-five client-owned overweight (n = 25) or obese (n = 10) dogs weighing >10 kg. Healthy dogs were prospectively recruited for a cross-sectional study. Serum triglyceride concentrations were measured before and hourly for 12 hours after a meal. Fasting cPLI and canine trypsin-like immunoreactivity (cTLI) concentrations were assayed. Cut-off values for hypertriglyceridemia were set a priori for fasting (≥ 88, ≥ 177, ≥ 354, ≥ 885 mg/dL) and peak postprandial (≥ 133, ≥ 442, ≥ 885 mg/dL) triglyceride concentrations. The association between hypertriglyceridemia and high cPLI concentrations was assessed by exact logistic regression. Follow-up was performed 4 years later to determine the incidence of pancreatic disease. Eight dogs had peak postprandial triglycerides >442 mg/dL and 3 dogs had fasting serum cPLI concentrations ≥ 400 μg/L. Odds of high cPLI concentrations were 16.7 times higher in dogs with peak postprandial triglyceride concentrations ≥ 442 mg/dL relative to other dogs (P < .001). Fasting triglyceride concentration was not significantly associated with cPLI concentrations. None of the dogs with high triglyceride concentrations and one of the dogs with low fasting and peak postprandial triglyceride concentrations developed clinically important pancreatic disease. Overweight and obese dogs with peak serum postprandial triglyceride concentrations ≥ 442 mg/dL after a standard meal are more likely to have serum cPLI concentrations ≥ 400 μg/L, but did not develop clinically important pancreatic disease. Copyright © 2011 by the American College of Veterinary Internal Medicine.

Cerebellar Expression of Copper Chaperone for Superoxide, Cytosolic Cu/Zn-Superoxide Dismutase, 4-Hydroxy-2-Nonenal, Acrolein and Heat Shock Protein 32 in Patients with Menkes Kinky Hair Disease: Immunohistochemical Study

PubMed Central

Yokoyama, Atsushi; Ohno, Kousaku; Hirano, Asao; Shintaku, Masayuki; Kato, Masako; Hayashi, Kazuhiko; Kato, Shinsuke

2014-01-01

Background To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death. Methods Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32). Results The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively. Conclusion In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism. PMID:25067875

Cerebellar expression of copper chaperone for superoxide, cytosolic cu/zn-superoxide dismutase, 4-hydroxy-2-nonenal, acrolein and heat shock protein 32 in patients with menkes kinky hair disease: immunohistochemical study.

PubMed

Yokoyama, Atsushi; Ohno, Kousaku; Hirano, Asao; Shintaku, Masayuki; Kato, Masako; Hayashi, Kazuhiko; Kato, Shinsuke

2014-03-01

To clarify the pathogenesis of cerebellar Purkinje cell death in patients with Menkes kinky hair disease (MD), a disorder of copper absorption, we investigated the morphological and functional abnormalities of residual Purkinje cells in MD patients and the mechanism of cell death. Seven MD patients and 39 neurologically normal autopsy cases were studied. We performed histopathological and quantitative analyses of the Purkinje cells. In addition, we used immunohistochemistry to detect copper-dependent enzymes [cytosolic Cu/Zn-superoxide dismutase (SOD1) and copper chaperone for superoxide dismutase (CCS)], oxidative stress markers [4-hydroxy-2-nonenal (HNE) and acrolein] and heat shock protein 32 (hsp 32). The surviving MD Purkinje cells showed abnormal development, such as somatic sprouts and heterotopic location. Due to maldevelopment and degeneration, dendrites showed the cactus and weeping willow patterns. Axonal degeneration led to the formation of torpedoes. Quantitative analysis revealed loss of approximately 50% of the Purkinje cells in MD patients. Almost all of the normal Purkinje cells were positive for immunostaining by anti-CCS and anti-SOD1 antibodies, with staining of the cell bodies, dendrites and axons. Normal Purkinje cells were not stained by antibodies for HNE, acrolein or hsp 32. In MD patients, the majority of Purkinje cells were positive for CCS, but the positive rate for SOD1 was only about 23%. Approximately 56%, 42% and 40% of the Purkinje cells of MD patients were positive for HNE, acrolein and hsp 32, respectively. In MD patients, about 50% of the Purkinje cells have been lost due to maldevelopment and degeneration. In the residual Purkinje cells, CCS expression seems to be nearly normal as a protective response to decreased SOD1 activity due to copper deficiency. Because oxidative stress is elevated secondary to decreased SOD1 activity, hsp 32 is induced as another protective mechanism.

Changes in calcitonin gene-related peptide (CGRP) receptor component and nitric oxide receptor (sGC) immunoreactivity in rat trigeminal ganglion following glyceroltrinitrate pretreatment

PubMed Central

2013-01-01

Background Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Infusion of the nitrovasodilator glyceroltrinitrate (nitroglycerin, GTN), which mobilizes NO in the organism, is an approved migraine model in humans. Calcitonin gene-related peptide (CGRP) is regarded as another key mediator in migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. The nociceptive processes and interactions underlying the NO and CGRP mediated headache are poorly known but can be examined in animal experiments. In the present study we examined changes in immunofluorescence of CGRP receptor components (CLR and RAMP1) and soluble guanylyl cyclase (sGC), the intracellular receptor for NO, in rat trigeminal ganglia after pretreatment with GTN. Methods Isoflurane anaesthetised rats were intravenously infused with GTN (1 mg/kg) or saline for four hours and two hours later the trigeminal ganglia were processed for immunohistochemistry. Different primary antibodies recognizing CLR, RAMP1, CGRP and sGC coupled to fluorescent secondary antibodies were used to examine immunoreactive cells in serial sections of trigeminal ganglia with epifluorescence and confocal laser scanning microscopy. Several staining protocols were examined to yield optimized immunolabeling. Results In vehicle-treated animals, 42% of the trigeminal ganglion neurons were immunopositive for RAMP1 and 41% for CLR. After GTN pretreatment CLR-immunopositivity was unchanged, while there was an increase in RAMP1-immunopositive neurons to 46%. RAMP1 and CLR immunoreactivity was also detected in satellite cells. Neurons immunoreactive for sGC were on average smaller than sGC-immunonegative neurons. The percentage of sGC-immunopositive neurons (51% after vehicle) was decreased after GTN infusion (48%). Conclusions Prolonged infusion of GTN caused increased fractions of RAMP1- and decreased fractions of s

Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability

PubMed Central

Dell'Orco, James M.; Wasserman, Aaron H.; Chopra, Ravi; Ingram, Melissa A. C.; Hu, Yuan-Shih; Singh, Vikrant; Wulff, Heike; Opal, Puneet; Orr, Harry T.

2015-01-01

Neuronal atrophy in neurodegenerative diseases is commonly viewed as an early event in a continuum that ultimately results in neuronal loss. In a mouse model of the polyglutamine disorder spinocerebellar ataxia type 1 (SCA1), we tested the hypothesis that cerebellar Purkinje neuron atrophy serves an adaptive role rather than being simply a nonspecific response to injury. In acute cerebellar slices from SCA1 mice, we find that Purkinje neuron pacemaker firing is initially normal but, with the onset of motor dysfunction, becomes disrupted, accompanied by abnormal depolarization. Remarkably, subsequent Purkinje cell atrophy is associated with a restoration of pacemaker firing. The early inability of Purkinje neurons to support repetitive spiking is due to unopposed calcium currents resulting from a reduction in large-conductance calcium-activated potassium (BK) and subthreshold-activated potassium channels. The subsequent restoration of SCA1 Purkinje neuron firing correlates with the recovery of the density of these potassium channels that accompanies cell atrophy. Supporting a critical role for BK channels, viral-mediated increases in BK channel expression in SCA1 Purkinje neurons improves motor dysfunction and partially restores Purkinje neuron morphology. Cerebellar perfusion of flufenamic acid, an agent that restores the depolarized membrane potential of SCA1 Purkinje neurons by activating potassium channels, prevents Purkinje neuron dendritic atrophy. These results suggest that Purkinje neuron dendritic remodeling in ataxia is an adaptive response to increases in intrinsic membrane excitability. Similar adaptive remodeling could apply to other vulnerable neuronal populations in neurodegenerative disease. SIGNIFICANCE STATEMENT In neurodegenerative disease, neuronal atrophy has long been assumed to be an early nonspecific event preceding neuronal loss. However, in a mouse model of spinocerebellar ataxia type 1 (SCA1), we identify a previously unappreciated

Effect of peptidase inhibition on the pattern of intraspinally released immunoreactive substance P detected with antibody microprobes.

PubMed

Duggan, A W; Schaible, H G; Hope, P J; Lang, C W

1992-05-08

Antibody microprobes bearing antibodies to the C-terminus of substance P (SP) were used to measure release of immunoreactive (ir) SP in the dorsal horn of barbiturate anaesthetized spinal cats. Electrical stimulation of unmyelinated primary afferents of the ipsilateral tibial nerve produced a relatively localised release of ir SP in the superficial dorsal horn. Prior microinjection of the peptidase inhibitors kelatorphan and enalaprilat in the dorsal horn resulted in ir SP being detected over the whole of the dorsal horn and the overlying dorsal column. This pattern had previously been observed with evoked release of ir neurokinin A and supports the proposal that a slow degradation results in a neuropeptide accessing many sites remote from sites of release.

Behçet Disease serum is immunoreactive to neurofilament medium which share common epitopes to bacterial HSP-65, a putative trigger.

PubMed

Lule, S; Colpak, A I; Balci-Peynircioglu, B; Gursoy-Ozdemir, Y; Peker, S; Kalyoncu, U; Can, A; Tekin, N; Demiralp, D; Dalkara, T

2017-11-01

Autoimmune and dysimmune inflammatory mechanisms on a genetically susceptible background are implicated in the etiology of Behçet's Disease (BD). Heat-shock protein-65 (HSP-65) derived from Streptococcus sanguinis was proposed as a triggering factor based on its homology with human HSP-60. However, none of the autoantigens identified so far in sera from BD share common epitopes with bacterial HSP-65 or has a high prevalence. Here, we report that sera from BD patients are immunoreactive against filamentous neuronal processes in the mouse brain, retina and scrotal skin in great majority of patients. By using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting, Western blotting and peptide blocking experiments, we have identified neurofilament medium (NF-M) as the probable antigen for the serologic response observed. Clustal Omega analyses detected significant structural homology between the human NF-M and bacterial HSP-65 corresponding to amino acids 111-126, 213-232 and 304-363 of mycobacterial HSP-65, which were previously identified to induce proliferation of lymphocytes obtained from BD patients. We also found that sera immunoreactive against NF-M cross-reacted with bacterial HSP-65. These findings suggest that NF-M may be involved in autoimmunity in BD due to its molecular mimicry with bacterial HSP-65. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig.

PubMed

Wasilewska, Barbara; Najdzion, Janusz; Równiak, Maciej; Bogus-Nowakowska, Krystyna; Hermanowicz, Beata; Kolenkiewicz, Małgorzata; Żakowski, Witold; Robak, Anna

2016-03-01

In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed. Copyright © 2015 Elsevier GmbH. All rights reserved.

Calbindin-D28k immunoreactivity is a marker for a subdivision of the sexually dimorphic nucleus of the preoptic area of the rat: developmental profile and gonadal steroid modulation.

PubMed

Sickel, M J; McCarthy, M M

2000-05-01

Calbindin-D28k (calbindin) is a 28 kilodalton calcium binding protein which potentially plays a role in neuroprotection. We report here the normal development and gonadal steroid modulation of a sexually dimorphic group of calbindin immunoreactive cells within the sexually dimorphic nucleus of the preoptic area (SDN) which we call the calbindin-immunoreactive SDN or CALB-SDN. Beginning on PN2, a faintly immunoreactive CALB-SDN is present, however, the volume is not sexually dimorphic. On PN4, the staining of the CALB-SDN appears more robust but the volume is still not sexually dimorphic. By PN8 and extending through PN12 and PN26, the latest age analysed, the volume of the CALB-SDN is larger in males by two- to fourfold. Cresyl violet counterstain reveals a similar developmental profile of the SDN as well as clusters of darkly staining calbindin immunonegative cells which lie around the CALB-SDN. Castration of males on PN0 decreases the volume of the CALB-SDN by PN12 and administration on the day of birth and PN1 of either testosterone propionate or oestradiol benzoate, but not dihydrotestosterone propionate to females increases the volume of the CALB-SDN by PN12. By demonstrating the sexual dimorphism and gonadal steroid modulation of the CALB-SDN, we hereby establish that calbindin is a specific marker of a subdivision of the SDN and can be used as such in future studies.

The histone deacetylase HDAC3 is essential for Purkinje cell function, potentially complicating the use of HDAC inhibitors in SCA1

PubMed Central

Venkatraman, Anand; Hu, Yuan-Shih; Didonna, Alessandro; Cvetanovic, Marija; Krbanjevic, Aleksandar; Bilesimo, Patrice; Opal, Puneet

2014-01-01

Spinocerebellar ataxia type 1 (SCA1) is an incurable neurodegenerative disease caused by a pathogenic glutamine repeat expansion in the protein ataxin-1 (ATXN1). One likely mechanism mediating pathogenesis is excessive transcriptional repression induced by the expanded ATXN-1. Because ATXN1 binds HDAC3, a Class I histone deacetylase (HDAC) that we have found to be required for ATXN1-induced transcriptional repression, we tested whether genetically depleting HDAC3 improves the phenotype of the SCA1 knock-in mouse (SCA1154Q/2Q), the most physiologically relevant model of SCA1. Given that HDAC3 null mice are embryonic lethal, we used for our analyses a combination of HDAC3 haploinsufficient and Purkinje cell (PC)-specific HDAC3 null mice. Although deleting a single allele of HDAC3 in the context of SCA1 was insufficient to improve cerebellar and cognitive deficits of the disease, a complete loss of PC HDAC3 was highly deleterious both behaviorally, with mice showing early onset ataxia, and pathologically, with progressive histologic evidence of degeneration. Inhibition of HDAC3 may yet have a role in SCA1 therapy, but our study provides cautionary evidence that this approach could produce untoward effects. Indeed, the neurotoxic consequences of HDAC3 depletion could prove relevant, wherever pharmacologic inhibition of HDAC3 is being contemplated, in disorders ranging from cancer to neurodegeneration. PMID:24594842

Immunocytochemical analysis of P2X2 in rat circumvallate taste buds.

PubMed

Yang, Ruibiao; Montoya, Alana; Bond, Amanda; Walton, Jenna; Kinnamon, John C

2012-05-23

Our laboratory has shown that classical synapses and synaptic proteins are associated with Type III cells. Yet it is generally accepted that Type II cells transduce bitter, sweet and umami stimuli. No classical synapses, however, have been found associated with Type II cells. Recent studies indicate that the ionotropic purinergic receptors P2X2/P2X3 are present in rodent taste buds. Taste nerve processes express the ionotropic purinergic receptors (P2X2/P2X3). P2X2/P2X3(Dbl-/-) mice are not responsive to sweet, umami and bitter stimuli, and it has been proposed that ATP acts as a neurotransmitter in taste buds. The goal of the present study is to learn more about the nature of purinergic contacts in rat circumvallate taste buds by examining immunoreactivity to antisera directed against the purinergic receptor P2X2. P2X2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds. Intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina. The P2X2 immunoreactive nerve processes also display syntaxin-1-LIR. The immunoreactive nerves are in close contact with the IP(3)R3-LIR Type II cells and syntaxin-1-LIR and/or 5-HT-LIR Type III cells. Taste cell synapses are observed only from Type III taste cells onto P2X2-LIR nerve processes. Unusually large, "atypical" mitochondria in the Type II taste cells are found only at close appositions with P2X2-LIR nerve processes. P2X2 immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells. Based on our immunofluorescence and immunoelectron microscopical studies we believe that both perigemmal and most all intragemmal nerve processes display P2X2-LIR. Moreover, colloidal gold immunoelectron microscopy indicates that P2X2-LIR in nerve processes is concentrated at sites of close apposition with Type II cells. This supports the hypothesis that ATP may be a key neurotransmitter in taste transduction

Chronic upregulation of activated microglia immunoreactive for galectin-3/Mac-2 and nerve growth factor following diffuse axonal injury

PubMed Central

2010-01-01

Background Diffuse axonal injury in patients with traumatic brain injury (TBI) can be associated with morbidity ranging from cognitive difficulties to coma. Magnetic resonance imaging scans now allow early detection of axonal injury following TBI, and have linked cognitive disability in these patients to white matter signal changes. However, little is known about the pathophysiology of this white matter injury, and the role of microglial activation in this process. It is increasingly recognized that microglia constitute a heterogeneous population with diverse roles following injury. In the present studies, we tested the hypothesis that following diffuse axonal injury involving the corpus callosum, there is upregulation of a subpopulation of microglia that express the lectin galectin-3/Mac-2 and are involved in myelin phagocytosis. Methods Adult mice were subject to midline closed skull injury or sham operation and were sacrificed 1, 8, 14 or 28 days later. Immunohistochemistry and immunofluorescence techniques were used to analyze patterns of labelling within the corpus callosum qualitatively and quantitatively. Results Activated microglia immunoreactive for galectin-3/Mac-2 were most abundant 1 day following injury. Their levels were attenuated at later time points after TBI but still were significantly elevated compared to sham animals. Furthermore, the majority of galectin-3/Mac-2+ microglia were immunoreactive for nerve growth factor in both sham and injured animals. Conclusions Our results suggest that galectin-3/Mac-2+ microglia play an important role in the pathogenesis of diffuse axonal injury both acutely and chronically and that they mediate their effects, at least in part by releasing nerve growth factor. PMID:20507613

Serum immunoreactive erythropoietin in high altitude natives with and without excessive erythrocytosis.

PubMed

León-Velarde, F; Monge, C C; Vidal, A; Carcagno, M; Criscuolo, M; Bozzini, C E

1991-05-01

We report the estimation of blood hemoglobin (Hb), arterial blood oxygen saturation (SaO2), and serum immunoreactive erythropoietin (siEPO) in a group of Peruvian workers residing in Cerro de Pasco at 4300 m showing "excessive erythrocytosis" (EE, Monge's disease, chronic mountain sickness). These estimates were compared with those of humans residing either in Cerro de Pasco and showing "normal erythrocytosis" (NE) or in Lima (sea level, SL) to determine whether Hb and SaO2 are related to siEPO in high altitude (HA) natives with NE or EE. The three parameters showed statistically significant differences between HA and SL groups--the values in SL being lower. Significant differences were also found between NE and EE groups in Hb and SaO2. There was no statistical difference in siEPo between the two groups. The results indicate, therefore, that HA residents who develop EE are not distinguishable from residents who develop NE on the basis of estimates of siEPO. As a result, siEPO and Hb do not show a dose-response relationship in HA residents, and variation in EPO does not explain the striking variation in Hb at high altitudes.

Expression of m1-type muscarinic acetylcholine receptors by parvalbumin-immunoreactive neurons in the primary visual cortex: a comparative study of rat, guinea pig, ferret, macaque, and human.

PubMed

Disney, Anita A; Reynolds, John H

2014-04-01

Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin-immunoreactive (PV) neurons in macaque V1 express the m1-type (pirenzepine-sensitive, Gq-coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine-sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV- and mAChR-immunoreactive somata by dual-immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV-ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only ∼25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq-coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex. Copyright © 2013 Wiley Periodicals, Inc.

Using mass spectrometry to detect, differentiate, and semiquantitate closely related peptide hormones in complex milieu: measurement of IGF-II and vesiculin.

PubMed

Lee, Kate L; Middleditch, Martin J; Williams, Geoffrey M; Brimble, Margaret A; Cooper, Garth J S

2015-03-01

The search for an islet β-cell growth factor has been a key objective in recent diabetes research, because the ability to regenerate and/or protect the functioning β-cell population in patients could result in a great advancement for diabetes treatment. IGF-I and IGF-II are known to play crucial roles in fetal growth and prenatal development, and there is growing evidence that IGF-II increases β-cell proliferation and survival in vitro and in vivo. A search for the source of IGF-II-like immunoreactivity in isolated β-cell secretory granules from the murine cell line βTC6-F7 revealed a novel 2-chain IGF-II-derived peptide, which we named vesiculin and which has been shown to be a full insulin agonist. Here, we present a liquid chromatography-tandem mass spectrometry method that enables selective detection and semiquantitation of the highly related IGF-II and vesiculin molecules. We have used this method to measure these 2 peptides in conditioned media from 2 β-cell lines, produced under increasing glucose concentrations. This technique detected both IGF-II and vesiculin in media conditioned by MIN6 and βTC6-F7 cells at levels in the range of 0 to 6 μM (total insulin, 80-450 μM) and revealed a glucose-stimulated increase in insulin, IGF-II, and vesiculin. IGF-II was detected in adult human and neonatal mouse serum in high levels, but vesiculin was not present. The methodology we present herein has utility for detecting and differentiating active peptides that are highly related and of low abundance.

Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta.

PubMed

Schachtner, Joachim; Trosowski, Björn; D'Hanis, Wolfgang; Stubner, Stephan; Homberg, Uwe

2004-06-01

During metamorphosis, the insect nervous system undergoes considerable remodeling: new neurons are integrated while larval neurons are remodeled or eliminated. To understand further the mechanisms involved in transforming larval to adult tissue we have mapped the metamorphic changes in a particularly well established brain area, the antennal lobe of the sphinx moth Manduca sexta, using an antiserum recognizing RFamide-related neuropeptides. Five types of RFamide-immunoreactive (ir) neurons could be distinguished in the antennal lobe, based on morphology and developmental appearance. Four cell types (types II-V, each consisting of one or two cells) showed RFamide immunostaining in the larva that persisted into metamorphosis. By contrast, the most prominent group (type I), a mixed population of local and projection neurons consisting of about 60 neurons in the adult antennal lobe, acquired immunostaining in a two-step process during metamorphosis. In a first step, from 5 to 7 days after pupal ecdysis, the number of labeled neurons reached about 25. In a second step, starting about 4 days later, the number of RFamide-ir neurons increased within 6 days to about 60. This two-step process parallels the rise and fall of the developmental hormone 20-hydroxyecdysone (20E) in the hemolymph. Artificially shifting the 20E peak to an earlier developmental time point resulted in the precocious appearance of RFamide immunostaining and led to premature formation of glomeruli. Prolonging high 20E concentrations to stages when the hormone titer starts to decline had no effect on the second increase of immunostained cell numbers. These results support the idea that the rise in 20E, which occurs after pupal ecdysis, plays a role in the first phase of RFamide expression and in glomeruli formation in the developing antennal lobes. The role of 20E in the second phase of RFamide expression is less clear, but increased cell numbers showing RFamide-ir do not appear to be a consequence of

Sweat chloride and immunoreactive trypsinogen in infants carrying two CFTR mutations and not affected by cystic fibrosis.

PubMed

Castellani, Carlo; Tridello, Gloria; Tamanini, Anna; Assael, Baroukh M

2017-07-01

Newborns with raised immunotrypsinogen levels who have non-pathological sweat chloride values and carry two cystic fibrosis transmembrane regulator ( CFTR ) mutations of which at least one is not acknowledged to be cystic fibrosis (CF)-causing are at risk of developing clinical manifestations consistent with CFTR-related disorders or even CF. It is not known whether newborns with similar genotypes and normal immunoreactive trypsinogen (IRT) may share the same risk. This study found that newborns with these characteristics and normal IRT have lower sweat chloride values than those with raised IRT (p=0.007). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

'Medusa head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 3: Anti-Yo/CDR2, anti-Nb/AP3B2, PCA-2, anti-Tr/DNER, other antibodies, diagnostic pitfalls, summary and outlook.

PubMed

Jarius, S; Wildemann, B

2015-09-17

Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as 'Medusa head antibodies' due to their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects and provides a summary and outlook.

Cortical orofacial motor representation in Old World monkeys, great apes, and humans. II. Stereologic analysis of chemoarchitecture.

PubMed

Sherwood, Chet C; Holloway, Ralph L; Erwin, Joseph M; Hof, Patrick R

2004-01-01

This study presents a comparative stereologic investigation of neurofilament protein- and calcium-binding protein-immunoreactive neurons within the region of orofacial representation of primary motor cortex (Brodmann's area 4) in several catarrhine primate species (Macaca fascicularis, Papio anubis, Pongo pygmaeus, Gorilla gorilla, Pan troglodytes, and Homo sapiens). Results showed that the density of interneurons involved in vertical interlaminar processing (i.e., calbindin- and calretinin-immunoreactive neurons) as well pyramidal neurons that supply heavily-myelinated projections (i.e., neurofilament protein-immunoreactive neurons) are correlated with overall neuronal density, whereas interneurons making transcolumnar connections (i.e., parvalbumin-immunoreactive neurons) do not exhibit such a relationship. These results suggest that differential scaling rules apply to different neuronal subtypes depending on their functional role in cortical circuitry. For example, cortical columns across catarrhine species appear to involve a similar conserved network of intracolumnar inhibitory interconnections, as represented by the distribution of calbindin- and calretinin-immunoreactive neurons. The subpopulation of horizontally-oriented wide-arbor interneurons, on the other hand, increases in density relative to other interneuron subpopulations in large brains. Due to these scaling trends, the region of orofacial representation of primary motor cortex in great apes and humans is characterized by a greater proportion of neurons enriched in neurofilament protein and parvalbumin compared to the Old World monkeys examined. These modifications might contribute to the voluntary dexterous control of orofacial muscles in great ape and human communication. Copyright 2004 S. Karger AG, Basel

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

NASA Technical Reports Server (NTRS)

Costes, S.; Streuli, C. H.; Barcellos-Hoff, M. H.

2000-01-01

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

Lgr4 Protein Deficiency Induces Ataxia-like Phenotype in Mice and Impairs Long Term Depression at Cerebellar Parallel Fiber-Purkinje Cell Synapses*

PubMed Central

Guan, Xin; Duan, Yanhong; Zeng, Qingwen; Pan, Hongjie; Qian, Yu; Li, Dali; Cao, Xiaohua; Liu, Mingyao

2014-01-01

Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4−/− mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4−/− mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4−/− PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4−/− cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia. PMID:25063812

Patterns of immunoreactivity specific for gustducin and for NCAM differ in developing rat circumvallate papillae and their taste buds.

PubMed

Iwasaki, Shin-Ichi; Aoyagi, Hidekazu; Asami, Tomoichiro; Wanichanon, Chaitip; Jackowiak, Hanna

2012-05-01

α-Gustducin and neural cell adhesion molecule (NCAM) are molecules previously found to be expressed in different cell types of mammalian taste buds. We examined the expression of α-gustducin and NCAM during the morphogenesis of circumvallate papillae and the formation of their taste buds by immunofluorescence staining and laser-scanning microscopy of semi-ultrathin sections of fetal and juvenile rat tongues. Images obtained by confocal laser scanning microscopy in transmission mode were also examined to provide outlines of histology and cell morphology. Morphogenesis of circumvallate papillae had already started on embryonic day 13 (E13) and was evident as the formation of placode. By contrast, taste buds in the circumvallate papillae started to appear between postnatal day 0 (P0) and P7. Although no cells with immunoreactivity specific for α-gustducin were detected in fetuses from E13 to E19, cells with NCAM-specific immunoreactivity were clearly apparent in the entire epithelium of the circumvallate papillary placode, the rudiment of each circumvallate papilla and the developing circumvallate papilla itself from E13 to E19. However, postnatally, both α-gustducin and NCAM became concentrated within taste cells as the formation of taste buds advanced. After P14, neither NCAM nor α-gustducin was detectable in the epithelium around the taste buds. In conclusion, α-gustducin appeared in the cytoplasm of taste cells during their formation after birth, while NCAM appeared in the epithelium of the circumvallate papilla-forming area. However, these two markers of taste cells were similarly distributed within mature taste cells. Copyright © 2011 Elsevier GmbH. All rights reserved.