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Sample records for purkinje cell clusters

  1. The Purkinje cell; 2008 style

    PubMed Central

    Dun, Wen; Boyden, Penelope A.

    2010-01-01

    Cardiac Purkinje fibers, due to their unique anatomical location, cell structure and electrophysiologic characteristics, play an important role in cardiac conduction and arrhythmogenesis. Purkinje cell action potentials are longer than their ventricular counterpart, and display two levels of resting potential. Purkinje cells provide for rapid propagation of the cardiac impulse to ventricular cells and have pacemaker and triggered activity, which differs from ventricular cells. Additionally, a unique intracellular Ca2+ release coordination has been revealed recently for the normal Purkinje cell. However, since the isolation of single Purkinje cells is difficult, particularly in small animals, research using Purkinje cells has been restricted. This review concentrates on comparison of Purkinje and ventricular cells in the morphology of the action potential, ionic channel function and molecular determinants by summarizing our present day knowledge of Purkinje cells. PMID:18778712

  2. Large-conductance calcium-activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains.

    PubMed

    Kaufmann, Walter A; Ferraguti, Francesco; Fukazawa, Yugo; Kasugai, Yu; Shigemoto, Ryuichi; Laake, Petter; Sexton, Joseph A; Ruth, Peter; Wietzorrek, Georg; Knaus, Hans-Günther; Storm, Johan F; Ottersen, Ole Petter

    2009-07-10

    Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.

  3. Selective Loss of Presynaptic Potassium Channel Clusters at the Cerebellar Basket Cell Terminal Pinceau in Adam11 Mutants Reveals Their Role in Ephaptic Control of Purkinje Cell Firing

    PubMed Central

    Kole, Matthew J.; Qian, Jing; Waase, Marc P.; Klassen, Tara L.; Chen, Tim T.; Augustine, George J.

    2015-01-01

    A specialized axonal ending, the basket cell “pinceau,” encapsulates the Purkinje cell axon initial segment (AIS), exerting final inhibitory control over the integrated outflow of the cerebellar cortex. This nonconventional axo-axonic contact extends beyond the perisomatic chemical GABAergic synaptic boutons to the distal AIS, lacks both sodium channels and local exocytotic machinery, and yet contains a dense cluster of voltage-gated potassium channels whose functional contribution is unknown. Here, we show that ADAM11, a transmembrane noncatalytic disintegrin, is the first reported Kv1-interacting protein essential for localizing Kv1.1 and Kv1.2 subunit complexes to the distal terminal. Selective absence of these channels at the pinceau due to mutation of ADAM11 spares spontaneous GABA release from basket cells at the perisomatic synapse yet eliminates ultrarapid ephaptic inhibitory synchronization of Purkinje cell firing. Our findings identify a critical role for presynaptic K+ channels at the pinceau in ephaptic control over the speed and stability of spike rate coding at the Purkinje cell AIS in mice. SIGNIFICANCE STATEMENT This study identifies ADAM11 as the first essential molecule for the proper localization of potassium ion channels at presynaptic nerve terminals, where they modulate excitability and the release of neural transmitters. Genetic truncation of the transmembrane disintegrin and metalloproteinase protein ADAM11 resulted in the absence of Kv1 channels that are normally densely clustered at the terminals of basket cell axons in the cerebellar cortex. These specialized terminals are responsible for the release of the neurotransmitter GABA onto Purkinje cells and also display electrical signaling. In the ADAM11 mutant, GABAergic release was not altered, but the ultrarapid electrical signal was absent, demonstrating that the dense presynaptic cluster of Kv1 ion channels at these terminals mediate electrical transmission. Therefore, ADAM11 plays a

  4. Purkinje cell vulnerability to mild traumatic brain injury.

    PubMed

    Fukuda, K; Aihara, N; Sagar, S M; Sharp, F R; Pitts, L H; Honkaniemi, J; Noble, L J

    1996-05-01

    In this study we examined the cerebellar response to mild traumatic brain injury by assessing microglial activation and Purkinje cell loss. Activated microglia were identified using the antibodies OX-42 and ED-1 as well as isolectin B4. The anti-Purkinje cell antibody PEP-19 was used to evaluate Purkinje cell loss after injury. The mechanism of cell injury was examined using a monoclonal antibody to the inducible 72-kDa heat shock protein. A monoclonal antibody to the N-terminal sequence of Fos was used as a marker for neuronal activation. There was progressive activation of microglia in the cerebellar vermis within a few days after forebrain injury. In coronal sections the processes of activated microglia were oriented in "stripes" perpendicular to the cortical surface. In sagittal sections the activated microglia were in irregularly shaped clusters or in a fan-like distribution that radiated from the Purkinje cell layer toward the cortical surface. There was a significant loss of Purkinje cells 7 days postinjury as compared to the control group. There was no evidence of induction of heat shock protein in the cerebellum. In addition, there was no evidence of induction of c-Fos protein in either the cerebellar cortex or inferior olivary nuclei within the first 3 h after injury. These studies demonstrate that a fluid percussive impact to the forebrain results in cerebellar damage. The close anatomical association between activated microglia and Purkinje cells suggests that Purkinje cell injury is the cause of the microglial activation. The mechanism of Purkinje cell death, however, remains unclear.

  5. Inverse Stochastic Resonance in Cerebellar Purkinje Cells

    PubMed Central

    Häusser, Michael; Gutkin, Boris S.; Roth, Arnd

    2016-01-01

    Purkinje neurons play an important role in cerebellar computation since their axons are the only projection from the cerebellar cortex to deeper cerebellar structures. They have complex internal dynamics, which allow them to fire spontaneously, display bistability, and also to be involved in network phenomena such as high frequency oscillations and travelling waves. Purkinje cells exhibit type II excitability, which can be revealed by a discontinuity in their f-I curves. We show that this excitability mechanism allows Purkinje cells to be efficiently inhibited by noise of a particular variance, a phenomenon known as inverse stochastic resonance (ISR). While ISR has been described in theoretical models of single neurons, here we provide the first experimental evidence for this effect. We find that an adaptive exponential integrate-and-fire model fitted to the basic Purkinje cell characteristics using a modified dynamic IV method displays ISR and bistability between the resting state and a repetitive activity limit cycle. ISR allows the Purkinje cell to operate in different functional regimes: the all-or-none toggle or the linear filter mode, depending on the variance of the synaptic input. We propose that synaptic noise allows Purkinje cells to quickly switch between these functional regimes. Using mutual information analysis, we demonstrate that ISR can lead to a locally optimal information transfer between the input and output spike train of the Purkinje cell. These results provide the first experimental evidence for ISR and suggest a functional role for ISR in cerebellar information processing. PMID:27541958

  6. Cerebellar Zonal Patterning Relies on Purkinje Cell Neurotransmission

    PubMed Central

    White, Joshua J.; Arancillo, Marife; Stay, Trace L.; George-Jones, Nicholas A.; Levy, Sabrina L.; Heck, Detlef H.

    2014-01-01

    Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity. PMID:24920627

  7. THE FINE STRUCTURE OF THE PURKINJE CELL

    PubMed Central

    Herndon, Robert M.

    1963-01-01

    This paper describes the fine structure of the Purkinje cell of the rat cerebellum after fixation by perfusion with 1 per cent buffered osmium tetroxide. Structures described include a large Golgi apparatus, abundant Nissl substance, mitochondria, multivesicular bodies, osmiophilic granules, axodendritic and axosomatic synapses, the nucleus, the nucleolus, and the nucleolar body. A new and possibly unique relationship between mitochondria and subsurface cisterns is described. Possible functional correlations are discussed. PMID:13953993

  8. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells.

    PubMed

    Ohashi, Ryo; Sakata, Shin-ichi; Naito, Asami; Hirashima, Naohide; Tanaka, Masahiko

    2014-04-01

    Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.

  9. Purkinje cell stripes and long-term depression at the parallel fiber-Purkinje cell synapse

    PubMed Central

    Hawkes, Richard

    2014-01-01

    The cerebellar cortex comprises a stereotyped array of transverse zones and parasagittal stripes, built around multiple Purkinje cell subtypes, which is highly conserved across birds and mammals. This architecture is revealed in the restricted expression patterns of numerous molecules, in the terminal fields of the afferent projections, in the distribution of interneurons, and in the functional organization. This review provides an overview of cerebellar architecture with an emphasis on attempts to relate molecular architecture to the expression of long-term depression (LTD) at the parallel fiber-Purkinje cell (pf-PC) synapse. PMID:24734006

  10. Nestin immunoreactivity of Purkinje cells in Creutzfeldt-Jakob disease.

    PubMed

    Mizuno, Yuji; Ohama, Eisaku; Hirato, Junko; Nakazato, Yoichi; Takahashi, Hitoshi; Takatama, Masamitsu; Takeuchi, Toshiyuki; Okamoto, Koichi

    2006-07-15

    Nestin, an intermediate filament protein, is mainly expressed in neural progenitor/stem cells in the central nervous system. Recently, we reported that nestin is expressed in Purkinje cells in patients with Creutzfeldt-Jakob disease (CJD). In this study, we examined a total of 19 CJD cerebella to analyze the intensity and pattern of nestin immunoreactivity of Purkinje cells in different pathological stages of degeneration in the cerebellar cortex. The results showed that the Purkinje cells were immunoreactive with nestin regardless of the severity of degenerative cerebellar cortex. Furthermore, we noted several different types of nestin immunoreactivity, indicated by diffuse and fine, coarse, and inclusion-like immunostainings within Purkinje cell bodies as well as dot-like staining outside of the cell bodies. In contrast, on examination of cerebella from non-CJD patients, 6 of 30 cases showed nestin immunoreactivity to a lesser extent. Thus, nestin-positive Purkinje cells are more common in CJD cerebella than in non-CJD cerebella. Although the mechanism of nestin expression in Purkinje cells is not yet understood, we suggest that such nestin-positive Purkinje cells are being reactivated to survive the cell death.

  11. Dendritic planarity of Purkinje cells is independent of Reelin signaling.

    PubMed

    Kim, Jinkyung; Park, Tae-Ju; Kwon, Namseop; Lee, Dongmyeong; Kim, Seunghwan; Kohmura, Yoshiki; Ishikawa, Tetsuya; Kim, Kyong-Tai; Curran, Tom; Je, Jung Ho

    2015-07-01

    The dendritic planarity of Purkinje cells is critical for cerebellar circuit formation. In the absence of Crk and CrkL, the Reelin pathway does not function resulting in partial Purkinje cell migration and defective dendritogenesis. However, the relationships among Purkinje cell migration, dendritic development and Reelin signaling have not been clearly delineated. Here, we use synchrotron X-ray microscopy to obtain 3-D images of Golgi-stained Purkinje cell dendrites. Purkinje cells that failed to migrate completely exhibited conical dendrites with abnormal 3-D arborization and reduced dendritic complexity. Furthermore, their spines were fewer in number with a distorted morphology. In contrast, Purkinje cells that migrated successfully displayed planar dendritic and spine morphologies similar to normal cells, despite reduced dendritic complexity. These results indicate that, during cerebellar formation, Purkinje cells migrate into an environment that supports development of dendritic planarity and spine formation. While Reelin signaling is important for the migration process, it does not make a direct major contribution to dendrite formation.

  12. Purkinje cell apoptosis in arabian horses with cerebellar abiotrophy.

    PubMed

    Blanco, A; Moyano, R; Vivo, J; Flores-Acuña, R; Molina, A; Blanco, C; Monterde, J G

    2006-08-01

    Purkinje cerebellar cells were studied in three Arabian horses aged between 6 and 8 months with clinical disorders in their movements, tremors and ataxia; the occurrence of apoptosis in this cell population was investigated by the (terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) method. Both optical and electron microscopical images showed a scant number of Purkinje cells, most of them with morphological features of apoptosis such as condensation of the nucleus and cytoplasm as well as segregation and fragmentation of the nucleus into apoptotic bodies. The TUNEL technique revealed a substantial number (65%) of positive immunoreactive Purkinje cells.

  13. The role of Cbln1 on Purkinje cell synapse formation.

    PubMed

    Ito-Ishida, Aya; Okabe, Shigeo; Yuzaki, Michisuke

    2014-06-01

    Cbln1 is a glycoprotein which belongs to the C1q family. In the cerebellum, Cbln1 is produced and secreted from granule cells and works as a strong synapse organizer between Purkinje cells and parallel fibers, the axons of the granule cells. In this update article, we will describe the molecular mechanisms by which Cbln1 induces synapse formation and will review our findings on the axonal structural changes which occur specifically during this process. We will also describe our recent finding that Cbln1 has a suppressive role in inhibitory synapse formation between Purkinje cells and molecular layer interneurons. Our results have revealed that Cbln1 plays an essential role to establish parallel fiber-Purkinje cell synapses and to regulate balance between excitatory and inhibitory input on Purkinje cells.

  14. A Signal Processing Analysis of Purkinje Cells in vitro

    PubMed Central

    Abrams, Ze'ev R.; Warrier, Ajithkumar; Trauner, Dirk; Zhang, Xiang

    2010-01-01

    Cerebellar Purkinje cells in vitro fire recurrent sequences of Sodium and Calcium spikes. Here, we analyze the Purkinje cell using harmonic analysis, and our experiments reveal that its output signal is comprised of three distinct frequency bands, which are combined using Amplitude and Frequency Modulation (AM/FM). We find that the three characteristic frequencies – Sodium, Calcium and Switching – occur in various combinations in all waveforms observed using whole-cell current clamp recordings. We found that the Calcium frequency can display a frequency doubling of its frequency mode, and the Switching frequency can act as a possible generator of pauses that are typically seen in Purkinje output recordings. Using a reversibly photo-switchable kainate receptor agonist, we demonstrate the external modulation of the Calcium and Switching frequencies. These experiments and Fourier analysis suggest that the Purkinje cell can be understood as a harmonic signal oscillator, enabling a higher level of interpretation of Purkinje signaling based on modern signal processing techniques. PMID:20508748

  15. Encoding of whisker input by cerebellar Purkinje cells

    PubMed Central

    Bosman, Laurens W J; Koekkoek, Sebastiaan K E; Shapiro, Joël; Rijken, Bianca F M; Zandstra, Froukje; van der Ende, Barry; Owens, Cullen B; Potters, Jan-Willem; de Gruijl, Jornt R; Ruigrok, Tom J H; De Zeeuw, Chris I

    2010-01-01

    The cerebellar cortex is crucial for sensorimotor integration. Sensorimotor inputs converge on cerebellar Purkinje cells via two afferent pathways: the climbing fibre pathway triggering complex spikes, and the mossy fibre–parallel fibre pathway, modulating the simple spike activities of Purkinje cells. We used, for the first time, the mouse whisker system as a model system to study the encoding of somatosensory input by Purkinje cells. We show that most Purkinje cells in ipsilateral crus 1 and crus 2 of awake mice respond to whisker stimulation with complex spike and/or simple spike responses. Single-whisker stimulation in anaesthetised mice revealed that the receptive fields of complex spike and simple spike responses were strikingly different. Complex spike responses, which proved to be sensitive to the amplitude, speed and direction of whisker movement, were evoked by only one or a few whiskers. Simple spike responses, which were not affected by the direction of movement, could be evoked by many individual whiskers. The receptive fields of Purkinje cells were largely intermingled, and we suggest that this facilitates the rapid integration of sensory inputs from different sources. Furthermore, we describe that individual Purkinje cells, at least under anaesthesia, may be bound in two functional ensembles based on the receptive fields and the synchrony of the complex spike and simple spike responses. The ‘complex spike ensembles’ were oriented in the sagittal plane, following the anatomical organization of the climbing fibres, while the ‘simple spike ensembles’ were oriented in the transversal plane, as are the beams of parallel fibres. PMID:20724365

  16. Abnormal climbing fibre-Purkinje cell synaptic connections in the essential tremor cerebellum.

    PubMed

    Lin, Chi-Ying; Louis, Elan D; Faust, Phyllis L; Koeppen, Arnulf H; Vonsattel, Jean-Paul G; Kuo, Sheng-Han

    2014-12-01

    Structural changes in Purkinje cells have been identified in the essential tremor cerebellum, although the mechanisms that underlie these changes remain poorly understood. Climbing fibres provide one of the major excitatory inputs to Purkinje cells, and climbing fibre-Purkinje cell connections are essential for normal cerebellar-mediated motor control. The distribution of climbing fibre-Purkinje cell synapses on Purkinje cell dendrites is dynamically regulated and may be altered in disease states. The aim of the present study was to examine the density and distribution of climbing fibre-Purkinje cell synapses using post-mortem cerebellar tissue of essential tremor cases and controls. Using vesicular glutamate transporter type 2 immunohistochemistry, we labelled climbing fibre-Purkinje cell synapses of 12 essential tremor cases and 13 age-matched controls from the New York Brain Bank. Normally, climbing fibres form synapses mainly on the thick, proximal Purkinje cell dendrites in the inner portion of the molecular layer, whereas parallel fibres form synapses on the thin, distal Purkinje cell spiny branchlets. We observed that, compared with controls, essential tremor cases had decreased climbing fibre-Purkinje cell synaptic density, more climbing fibres extending to the outer portion of the molecular layer, and more climbing fibre-Purkinje cell synapses on the thin Purkinje cell spiny branchlets. Interestingly, in essential tremor, the increased distribution of climbing fibre-Purkinje cell synapses on the thin Purkinje cell branchlets was inversely associated with clinical tremor severity, indicating a close relationship between the altered distribution of climbing fibre-Purkinje cell connections and tremor. These findings suggest that abnormal climbing fibre-Purkinje cell connections could be of importance in the pathogenesis of essential tremor.

  17. Purkinje Cell Loss in Essential Tremor: Random Sampling Quantification and Nearest Neighbor Analysis

    PubMed Central

    Choe, Matthew; Cortés, Etty; Vonsattel, Jean-Paul G.; Kuo, Sheng-Han; Faust, Phyllis L.; Louis, Elan D.

    2015-01-01

    Objective Purkinje cell loss has been documented in some although not all postmortem studies of essential tremor. Hence, there is considerable controversy concerning the presence of Purkinje cell loss in this disease. To date, few studies have been performed. Methods Over the past eight years, we have assembled 50 prospectively-studied cases and 25 age-matched controls; none were reported in our prior large series of 33 essential tremor and 21 controls. In addition to methods used in prior studies, the current study used a random sampling approach to quantify Purkinje cells along the Purkinje cell layer with a mean of 217 sites examined in each specimen, allowing for extensive sampling of the Purkinje cell layer within the section. For the first time, we also quantified the distance between Purkinje cell bodies - a nearest neighbor analysis. Results In the Purkinje cell count data collected from fifteen 100x-fields, cases had lower counts than controls in all three counting criteria (cell bodies, nuclei, nucleoli, all p<0.001). Purkinje cell linear density was also lower in cases than controls (all p<0.001). Purkinje cell linear density obtained by random sampling was similarly lower in cases than controls in all three counting criteria (cell bodies, nuclei, nucleoli, all p≤0.005). In agreement with the quantitative Purkinje cell counts, the mean distance from one Purkinje cell body to another Purkinje cell body along the Purkinje cell layer was greater in cases than controls (p=0.002). Conclusions These data provide support for the neurodegeneration of cerebellar Purkinje cells in essential tremor. PMID:26861543

  18. Sodium action potentials in the dendrites of cerebellar Purkinje cells.

    PubMed

    Regehr, W G; Konnerth, A; Armstrong, C M

    1992-06-15

    We report here that in cerebellar Purkinje cells from which the axon has been removed, positive voltage steps applied to the voltage-clamped soma produce spikes of active current. The spikes are inward, are all-or-none, have a duration of approximately 1 ms, and are reversibly eliminated by tetrodotoxin, a Na channel poison. From cell to cell, the amplitude of the spikes ranges from 4 to 20 nA. Spike latency decreases as the depolarizing step is made larger. These spikes clearly arise at a site where the voltage is not controlled, remote from the soma. From these facts we conclude that Purkinje cell dendrites contain a sufficient density of Na channels to generate action potentials. Activation by either parallel fiber or climbing fiber synapses produces similar spikes, suggesting that normal input elicits Na action potentials in the dendrites. These findings greatly alter current views of how dendrites in these cells respond to synaptic input.

  19. Emergence of endoplasmic reticulum stress and activated microglia in Purkinje cell degeneration mice.

    PubMed

    Kyuhou, Shin-ichi; Kato, Nobuo; Gemba, Hisae

    2006-03-27

    In the current studies, we characterized the molecular and cellular mechanism of cell death in Purkinje cell degeneration (pcd) mice using real-time quantitative PCR, immunohistochemistry, and Western blotting. It appears that endoplasmic reticulum (ER) stress is involved in this degeneration of Purkinje cells because ER stress-related substrates, such as CHOP and caspase 12, were strongly activated in Purkinje cells of pcd mice during the third postnatal (P) week. A significant increase in the expression of the ER-specific chaperone BiP suggested that unfolded protein responses were induced. We also found that Purkinje cells underwent apoptosis via the activation of caspase 3 and subsequent fragmentation of DNA. In addition to the activation of apoptosis in Purkinje cells, many activated microglial cells are found to be present in the molecular layer of the cerebellar cortex. In the later phase of degeneration, there was conspicuous expression of inducible nitric oxide synthase (iNOS), and some Purkinje cells were strongly labeled with an antibody to nitrotyrosine, suggesting that Purkinje cells in pcd mice are damaged by nitric oxide released from microglial cells. Administration of minocycline, which may inhibit iNOS expression, delayed the death of Purkinje cells in pcd mice and mildly improved their motor abilities. These findings suggest that ER stress participates in the degeneration of Purkinje cells and that activation of microglia accelerates Purkinje cell death in pcd mice.

  20. Kinetic model of excitatory synaptic transmission to cerebellar Purkinje cells.

    PubMed

    Marienhagen, J; Keller, B U; Zippelius, A

    1997-09-21

    We present a minimal kinetic model for excitatory synaptic transmission to cerebellar Purkinje cells. The main components are a kinetic model for a single glutamate receptor, which is calibrated with the help of patch clamp data, and a mean field approximation for the dynamics of a population of channels, which generate an EPSC. The resulting minimal model of the parallel fiber-Purkinje cell synapse is used to estimate the dynamics of glutamate in the synaptic cleft and to clarify the role of receptor desensitization in synaptic transmission. We also apply the model to different aspects of synaptic modulation, like long-term depression and potentiation by pharmacological application of ampakines. In the framework of the minimal model these effects can be understood as the result of modified receptor kinetics.

  1. Motor learning of mice lacking cerebellar Purkinje cells

    PubMed Central

    Porras-García, M. Elena; Ruiz, Rocío; Pérez-Villegas, Eva M.; Armengol, José Á.

    2013-01-01

    The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum. PMID:23630472

  2. Motor learning of mice lacking cerebellar Purkinje cells.

    PubMed

    Porras-García, M Elena; Ruiz, Rocío; Pérez-Villegas, Eva M; Armengol, José Á

    2013-01-01

    The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input-output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum.

  3. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

    PubMed

    Tanabe, Koji; Kani, Shuichi; Shimizu, Takashi; Bae, Young-Ki; Abe, Takaya; Hibi, Masahiko

    2010-12-15

    Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

  4. Molecular mechanism of parallel fiber-Purkinje cell synapse formation.

    PubMed

    Mishina, Masayoshi; Uemura, Takeshi; Yasumura, Misato; Yoshida, Tomoyuki

    2012-01-01

    The cerebellum receives two excitatory afferents, the climbing fiber (CF) and the mossy fiber-parallel fiber (PF) pathway, both converging onto Purkinje cells (PCs) that are the sole neurons sending outputs from the cerebellar cortex. Glutamate receptor δ2 (GluRδ2) is expressed selectively in cerebellar PCs and localized exclusively at the PF-PC synapses. We found that a significant number of PC spines lack synaptic contacts with PF terminals and some of residual PF-PC synapses show mismatching between pre- and postsynaptic specializations in conventional and conditional GluRδ2 knockout mice. Studies with mutant mice revealed that in addition to PF-PC synapse formation, GluRδ2 is essential for synaptic plasticity, motor learning, and the restriction of CF territory. GluRδ2 regulates synapse formation through the amino-terminal domain, while the control of synaptic plasticity, motor learning, and CF territory is mediated through the carboxyl-terminal domain. Thus, GluRδ2 is the molecule that bridges synapse formation and motor learning. We found that the trans-synaptic interaction of postsynaptic GluRδ2 and presynaptic neurexins (NRXNs) through cerebellin 1 (Cbln1) mediates PF-PC synapse formation. The synaptogenic triad is composed of one molecule of tetrameric GluRδ2, two molecules of hexameric Cbln1 and four molecules of monomeric NRXN. Thus, GluRδ2 triggers synapse formation by clustering four NRXNs. These findings provide a molecular insight into the mechanism of synapse formation in the brain.

  5. Differential Purkinje cell simple spike activity and pausing behavior related to cerebellar modules

    PubMed Central

    Zhou, Haibo; Voges, Kai; Lin, Zhanmin; Ju, Chiheng

    2015-01-01

    The massive computational capacity of the cerebellar cortex is conveyed by Purkinje cells onto cerebellar and vestibular nuclei neurons through their GABAergic, inhibitory output. This implies that pauses in Purkinje cell simple spike activity are potentially instrumental in cerebellar information processing, but their occurrence and extent are still heavily debated. The cerebellar cortex, although often treated as such, is not homogeneous. Cerebellar modules with distinct anatomical connectivity and gene expression have been described, and Purkinje cells in these modules also differ in firing rate of simple and complex spikes. In this study we systematically correlate, in awake mice, the pausing in simple spike activity of Purkinje cells recorded throughout the entire cerebellum, with their location in terms of lobule, transverse zone, and zebrin-identified cerebellar module. A subset of Purkinje cells displayed long (>500-ms) pauses, but we found that their occurrence correlated with tissue damage and lower temperature. In contrast to long pauses, short pauses (<500 ms) and the shape of the interspike interval (ISI) distributions can differ between Purkinje cells of different lobules and cerebellar modules. In fact, the ISI distributions can differ both between and within populations of Purkinje cells with the same zebrin identity, and these differences are at least in part caused by differential synaptic inputs. Our results suggest that long pauses are rare but that there are differences related to shorter intersimple spike intervals between and within specific subsets of Purkinje cells, indicating a potential further segregation in the activity of cerebellar Purkinje cells. PMID:25717166

  6. Purkinje cell loss and the noradrenergic system in the cerebellum of pcd mutant mice.

    PubMed

    Ghetti, B; Fuller, R W; Sawyer, B D; Hemrick-Luecke, S K; Schmidt, M J

    1981-12-01

    Purkinje cells in the cerebellum receive inhibitory noradrenergic input from the locus coeruleus. In pcd mutant mice all Purkinje cells degenerate by 45 days of age. The purpose of the present studies was to determine if the loss of these cerebellar neurons affects the amounts of norepinephrine in the cerebellum of mice 25-280 days of age. No significant changes in norepinephrine content were detected during or after Purkinje cell degeneration. However, since degeneration led to a reduction in cerebellar weight, the norepinephrine concentration was increased in pcd mutants. These results indicate that despite the loss of a major postsynaptic target (Purkinje cells), the cerebellar noradrenergic input remains stable.

  7. The action of antidromic impulses on the cerebellar Purkinje cells

    PubMed Central

    Eccles, J. C.; Llinás, R.; Sasaki, K.

    1966-01-01

    1. Antidromic impulses have been set up in the axons of Purkinje cells of the cerebellar vermis by stimulation in the juxta-fastigial (J.F.) region. Most experiments were performed on the normal cat cerebellum, but in nine the cerebellum was chronically deafferented by bilateral pedunculotomy 9-23 days previously. 2. Intra- and extracellular recording from Purkinje cells both showed a characteristic inflexion on the rising phase of the spike potential (the characteristic IS—SD inflexion) that presumably signals a delay in invasion between the axon and the large soma-dendritic expansion. 3. Laminar field analysis of the antidromic spike potentials showed that the antidromic impulses invaded at least 200 μ of the main dendrites as well as the soma, there being then a steep decrement to the surface. At superficial levels there was even an inverse antidromic spike potential. There appeared to be a synchronous invasion of the soma-dendritic complex, perhaps due to trigger zones of low threshold on the dendrites. 4. Antidromic soma-dendritic invasion was modified in the expected manner by a volley in the parallel fibres; there was inhibition of transmission into the soma and up the main dendrites (maximum effect at 200-300 μ depth) due to the inhibitory action of the basket and superficial stellate cells that are excited by the parallel fibres; there was facilitation of transmission in the dendrites at levels superficial to 200 μ due to the direct excitatory action of parallel fibres. Both the inhibitory and excitatory actions had a duration in excess of 100 msec. 5. In the chronically deafferented cerebellum a second J.F. stimulation evoked a full size antidromic spike potential at an interval of 3 msec. There was a gradual decline in size down to intervals of about 2 msec, and at briefer intervals, to 1 msec, there was a small residual spike potential that possibly is due to transmission into the Purkinje cell axon collaterals at intervals too brief for soma

  8. Calcitonin gene-related peptide (CGRP) stimulates purkinje cell dendrite growth in culture.

    PubMed

    D'Antoni, Simona; Zambusi, Laura; Codazzi, Franca; Zacchetti, Daniele; Grohovaz, Fabio; Provini, Luciano; Catania, Maria Vincenza; Morara, Stefano

    2010-12-01

    Previous reports described the transient expression during development of Calcitonin Gene-Related Peptide (CGRP) in rodent cerebellar climbing fibers and CGRP receptor in astrocytes. Here, mixed cerebellar cultures were used to analyze the effects of CGRP on Purkinje cells growth. Our results show that CGRP stimulated Purkinje cell dendrite growth under cell culture conditions mimicking Purkinje cell development in vivo. The stimulation was not blocked by CGRP8-37, a specific antagonist, suggesting the activation of other related receptors. CGRP did not affect survival of Purkinje cells, granule cells or astrocytes. The selective expression of Receptor Component Protein (RCP) (a component of CGRP receptor family) in astrocytes points to a role of these cells as mediators of CGRP effect. Finally, in pure cerebellar astrocyte cultures CGRP induced a transient morphological differentiation from flat, polygonal to stellate form. It is concluded that CGRP influences Purkinje cell dendrite growth in vitro, most likely through the involvement of astrocytes.

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

    PubMed Central

    Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R.; Trimmer, James S.

    2015-01-01

    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 Ni2+ elicited a similar effect on APs, indicating the involvement of Ni2+-sensitive Ca2+ 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. PMID:25948259

  10. 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.…

  11. Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains

    PubMed Central

    Weimann, James M.; Charlton, Carol A.; Brazelton, Timothy R.; Hackman, Robert C.; Blau, Helen M.

    2003-01-01

    We show here that cells within human adult bone marrow can contribute to cells in the adult human brain. Cerebellar tissues from female patients with hematologic malignancies, who had received chemotherapy, radiation, and a bone marrow transplant, were analyzed. Brain samples were obtained at autopsy from female patients who received male (sex-mismatched) or female (sex-matched, control) bone marrow transplants. Cerebella were evaluated in 10-μm-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to ≈50% of a human Purkinje nucleus. A total of 5,860 Purkinje cells from sex-mismatched females and 3,202 Purkinje cells from sex-matched females were screened for Y chromosomes by epifluorescence. Confocal laser scanning microscopy allowed definitive identification of the sex chromosomes within the morphologically distinct Purkinje cells. In the brains of females who received male bone marrow, four Purkinje neurons were found that contained an X and a Y chromosome and two other Purkinje neurons contained more than a diploid number of sex chromosomes. No Y chromosomes were detected in the brains of sex-matched controls. The total frequency of male bone marrow contribution to female Purkinje cells approximated 0.1%. This study demonstrates that although during human development Purkinje neurons are no longer generated after birth, cells within the bone marrow can contribute to these CNS neurons even in adulthood. The underlying mechanism may be caused either by generation de novo of Purkinje neurons from bone marrow-derived cells or by fusion of marrow-derived cells with existing recipient Purkinje neurons. PMID:12576546

  12. Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene.

    PubMed

    Sun, X Z; Harada, Y N; Takahashi, S; Shiomi, N; Shiomi, T

    2001-05-15

    Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells.

  13. Identification of feline panleukopenia virus proteins expressed in Purkinje cell nuclei of cats with cerebellar hypoplasia.

    PubMed

    Poncelet, Luc; Héraud, Céline; Springinsfeld, Marie; Ando, Kunie; Kabova, Anna; Beineke, Andreas; Peeters, Dominique; Op De Beeck, Anne; Brion, Jean-Pierre

    2013-06-01

    Parvoviruses depend on initiation of host cell division for their replication. Undefined parvoviral proteins have been detected in Purkinje cells of the cerebellum after experimental feline panleukopenia virus (FPV) infection of neonatal kittens and in naturally occurring cases of feline cerebellar hypoplasia. In this study, a parvoviral protein in the nucleus of Purkinje cells of kittens with cerebellar hypoplasia was shown by immunoprecipitation to be the FPV viral capsid protein VP2. In PCR-confirmed, FPV-associated feline cerebellar hypoplasia, expression of the FPV VP2 protein was demonstrated by immunohistochemistry in Purkinje cell nuclei in 4/10 cases and expression of the FPV non-structural protein NS1 was demonstrated in Purkinje cell nuclei in 5/10 cases. Increased nuclear ERK1 expression was observed in several Purkinje cells in 1/10 kittens. No expression of the G1 and S mitotic phase marker proliferating cell nuclear antigen (PCNA) was evident in Purkinje cell nuclei. These results support the hypothesis that FPV is able to proceed far into its replication cycle in post-mitotic Purkinje cells.

  14. Climbing Fiber Regulation of Spontaneous Purkinje Cell Activity and Cerebellum-Dependent Blink Responses123

    PubMed Central

    Bengtsson, Fredrik

    2016-01-01

    Abstract It has been known for a long time that GABAergic Purkinje cells in the cerebellar cortex, as well as their target neurons in the cerebellar nuclei, are spontaneously active. The cerebellar output will, therefore, depend on how input is integrated into this spontaneous activity. It has been shown that input from climbing fibers originating in the inferior olive controls the spontaneous activity in Purkinje cells. While blocking climbing fiber input to the Purkinje cells causes a dramatic increase in the firing rate, increased climbing fiber activity results in reduced Purkinje cell activity. However, the exact calibration of this regulation has not been examined systematically. Here we examine the relation between climbing fiber stimulation frequency and Purkinje cell activity in unanesthetized decerebrated ferrets. The results revealed a gradual suppression of Purkinje cell activity, starting at climbing fiber stimulation frequencies as low as 0.5 Hz. At 4 Hz, Purkinje cells were completely silenced. This effect lasted an average of 2 min after the stimulation rate was reduced to a lower level. We also examined the effect of sustained climbing fiber stimulation on overt behavior. Specifically, we analyzed conditioned blink responses, which are known to be dependent on the cerebellum, while stimulating the climbing fibers at different frequencies. In accordance with the neurophysiological data, the conditioned blink responses were suppressed at stimulation frequencies of ≥4 Hz. PMID:26839917

  15. Cerebellar globular cells receive monoaminergic excitation and monosynaptic inhibition from Purkinje cells.

    PubMed

    Hirono, Moritoshi; Saitow, Fumihito; Kudo, Moeko; Suzuki, Hidenori; Yanagawa, Yuchio; Yamada, Masahisa; Nagao, Soichi; Konishi, Shiro; Obata, Kunihiko

    2012-01-01

    Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.

  16. The knockout of secretin in cerebellar Purkinje cells impairs mouse motor coordination and motor learning.

    PubMed

    Zhang, Li; Chung, Sookja Kim; Chow, Billy Kwok Chong

    2014-05-01

    Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells.

  17. Selective rather than inductive mechanisms favour specific replacement of Purkinje cells by embryonic cerebellar cells transplanted to the cerebellum of adult Purkinje cell degeneration (pcd) mutant mice.

    PubMed

    Carletti, Barbara; Rossi, Ferdinando

    2005-09-01

    Cell replacement after neuronal degeneration in the adult CNS depends on the availability of specific cues to direct specification, differentiation and integration of newly born neurons into mature circuits. Following recent reports indicating that neurogenic signals may be reactivated in the adult injured CNS, here we asked whether such signals are expressed in the cerebellum after Purkinje cell degeneration. Thus, we compared the fate of embryonic cerebellar cells transplanted to the cerebella of adult wild-type and Purkinje cell degeneration (pcd) mutant mice. Donor cells were dissected from beta-actin-enhanced green fluorescent protein (EGFP) transgenic mice and transplanted as a single cell suspension. In both hosts, grafted cells generated all major cerebellar phenotypes, with a precise localization in the recipient cortex or white matter. Nevertheless, the phenotypic distributions showed striking quantitative differences. Most notably, in the pcd cerebellum there was a higher amount of Purkinje cells, while other phenotypes were less frequent. Analysis of cell proliferation by 5-bromo-2'-deoxyuridine (BrDU) incorporation revealed that in both hosts mitotic activity was strongly reduced shortly after transplantation, and virtually all donor Purkinje cells were actually generated before grafting. Together, these results indicate that some compensatory mechanisms operate in the pcd environment. However, the very low mitotic rate of transplanted cells suggests that the adult cerebellum, either wild-type or mutant, does not provide instructive neurogenic cues to direct the specification of uncommitted progenitors. Rather, specific replacement in mutant hosts is achieved through selective mechanisms that favour the survival and integration of donor Purkinje cells at the expense of other phenotypes.

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

    PubMed

    Armién, A G; McRuer, D L; Ruder, M G; Wünschmann, A

    2013-01-01

    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 foliation. Heterotopic neurons were identified as immature Purkinje cells by their size, location, immunoreactivity for calbindin D-28 K, and ultrastructural features. The authors suggest that this cerebellar abnormality was likely due to a disruption of molecular mechanisms that dictate Purkinje cell migration, placement, and maturation in early embryonic development. The etiology of this condition remains undetermined. Congenital central nervous system disorders have rarely been reported in birds.

  19. 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

  20. Integration of Purkinje cell inhibition by cerebellar nucleo-olivary neurons.

    PubMed

    Najac, Marion; Raman, Indira M

    2015-01-14

    Neurons in the cerebellar cortex, cerebellar nuclei, and inferior olive (IO) form a trisynaptic loop critical for motor learning. IO neurons excite Purkinje cells via climbing fibers and depress their parallel fiber inputs. Purkinje cells inhibit diverse cells in the cerebellar nuclei, including small GABAergic nucleo-olivary neurons that project to the IO. To investigate how these neurons integrate synaptic signals from Purkinje cells, we retrogradely labeled nucleo-olivary cells in the contralateral interpositus and lateral nuclei with cholera toxin subunit B-Alexa Fluor 488 and recorded their electrophysiological properties in cerebellar slices from weanling mice. Nucleo-olivary cells fired action potentials over a relatively narrow dynamic range (maximal rate, ∼ 70 spikes/s), unlike large cells that project to premotor areas (maximal rate, ∼ 400 spikes/s). GABA(A) receptor-mediated IPSCs evoked by electrical or optogenetic stimulation of Purkinje cells were more than 10-fold slower in nucleo-olivary cells (decay time, ∼ 25 ms) than in large cells (∼ 2 ms), and repetitive stimulation at 20-150 Hz evoked greatly summating IPSCs. Nucleo-olivary firing rates varied inversely with IPSP frequency, and the timing of Purkinje IPSPs and nucleo-olivary spikes was uncorrelated. These attributes contrast with large cells, whose brief IPSCs and rapid firing rates can permit well timed postinhibitory spiking. Thus, the intrinsic and synaptic properties of these two projection neurons from the cerebellar nuclei tailor them for differential integration and transmission of their Purkinje cell input.

  1. Purkinje cell death after uptake of anti-Yo antibodies in cerebellar slice cultures.

    PubMed

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

    2010-10-01

    Paraneoplastic cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic cerebellar degeneration.

  2. New structural aspects of the synaptic contacts on Purkinje cells in an elasmobranch cerebellum.

    PubMed Central

    Alvarez-Otero, R; Regueira, S D; Anadon, R

    1993-01-01

    Nerve fibre contacts on Purkinje cell perikarya in the cerebellum of the small-spotted dogfish (Scyliorhinus canicula) were studied using the Cajal reduced silver technique, Golgi methods and electron microscopy. Silver staining revealed axons with thick swellings close to the base of Purkinje cells. Golgi methods demonstrated the presence of 'pincushions' of somatic spines on Purkinje cells. Electron microscopy revealed flattened fibres that formed extensive synaptic contacts with the Purkinje cell 'pincushions'. It is proposed, on the basis of the ultrastructural features, that these fibres are climbing fibres. Their possible significance in terms of the evolution of cerebellar circuitry is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8509296

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. Morphine inhibits Purkinje cell survival and dendritic differentiation in organotypic cultures of the mouse cerebellum

    PubMed Central

    Hauser, Kurt F.; Gurwell, Julie A.; Turbek, Carol S.

    2015-01-01

    The effects of morphine on the morphogenesis and survival of calbindin-D28kimmunoreactive Purkinje cells was studied in organotypic explant cultures isolated from 1- or 7-day-old mouse cerebella. To reduce experimental variability, bilaterally matched pairs of organotypic cultures were used to compare the effects of opiate drug treatment. One explant within each pair was untreated, while the remaining explant was continuously treated for 7 to 10 days with morphine, morphine plus naloxone, or naloxone alone. In explants derived from 1-day-old mice, morphine treatment significantly reduced Purkinje cell dendritic length compared to symmetrically-matched untreated control explants. The concentration of morphine estimated to cause a half-maximal reduction (EC50) in dendritic length was 4.9 × 10−8 M. At higher concentrations (EC50 = 3.6 × 10−6 M), morphine also significantly decreased the number of Purkinje cells in explants from 1-day-old mice compared to untreated explants. Electron microscopy identified increased numbers of degenerating Purkinje cells in explants derived from 1-day-old mice. This showed that high concentrations (10−5 M) of morphine reduced Purkinje cell numbers by decreasing their rate of survival. In explants derived from 7-day-old mice, morphine (10−5 M) neither affected Purkinje cell dendritic length nor cell numbers compared to symmetrically-matched untreated (control) explants. Collectively, these findings suggest that morphine per se, through a direct action on the cerebellum, can affect Purkinje cell differentiation and survival. The results additionally suggest there is a critical period during development when Purkinje cells are especially vulnerable to the effects of morphine. PMID:7821399

  5. Cbln1 downregulates the formation and function of inhibitory synapses in mouse cerebellar Purkinje cells.

    PubMed

    Ito-Ishida, Aya; Kakegawa, Wataru; Kohda, Kazuhisa; Miura, Eriko; Okabe, Shigeo; Yuzaki, Michisuke

    2014-04-01

    The formation of excitatory and inhibitory synapses must be tightly coordinated to establish functional neuronal circuitry during development. In the cerebellum, the formation of excitatory synapses between parallel fibers and Purkinje cells is strongly induced by Cbln1, which is released from parallel fibers and binds to the postsynaptic δ2 glutamate receptor (GluD2). However, Cbln1's role, if any, in inhibitory synapse formation has been unknown. Here, we show that Cbln1 downregulates the formation and function of inhibitory synapses between Purkinje cells and interneurons. Immunohistochemical analyses with an anti-vesicular GABA transporter antibody revealed an increased density of interneuron-Purkinje cell synapses in the cbln1-null cerebellum. Whole-cell patch-clamp recordings from Purkinje cells showed that both the amplitude and frequency of miniature inhibitory postsynaptic currents were increased in cbln1-null cerebellar slices. A 3-h incubation with recombinant Cbln1 reversed the increased amplitude of inhibitory currents in Purkinje cells in acutely prepared cbln1-null slices. Furthermore, an 8-day incubation with recombinant Cbln1 reversed the increased interneuron-Purkinje cell synapse density in cultured cbln1-null slices. In contrast, recombinant Cbln1 did not affect cerebellar slices from mice lacking both Cbln1 and GluD2. Finally, we found that tyrosine phosphorylation was upregulated in the cbln1-null cerebellum, and acute inhibition of Src-family kinases suppressed the increased inhibitory postsynaptic currents in cbln1-null Purkinje cells. These findings indicate that Cbln1-GluD2 signaling inhibits the number and function of inhibitory synapses, and shifts the excitatory-inhibitory balance towards excitation in Purkinje cells. Cbln1's effect on inhibitory synaptic transmission is probably mediated by a tyrosine kinase pathway.

  6. Constitutive activation of neuronal Src causes aberrant dendritic morphogenesis in mouse cerebellar Purkinje cells.

    PubMed

    Kotani, Takenori; Morone, Nobuhiro; Yuasa, Shigeki; Nada, Shigeyuki; Okada, Masato

    2007-02-01

    Src family tyrosine kinases are essential for neural development, but their in vivo functions remain elusive because of functional compensation among family members. To elucidate the roles of individual Src family members in vivo, we generated transgenic mice expressing the neuronal form of c-Src (n-Src), Fyn, and their constitutively active forms in cerebellar Purkinje cells using the L7 promoter. The expression of the constitutively active n-Src retarded the postnatal development of Purkinje cells and disrupted dendritic morphogenesis, whereas the wild-type n-Src had only moderate effects. Neither wild-type nor constitutively active Fyn over-expression significantly affected Purkinje-cell morphology. The aberrant Purkinje cells in n-Src transgenic mice retained multiple dendritic shafts extending in non-polarized directions and were located heterotopically in the molecular layer. Ultrastructural observation of the dendritic shafts revealed that the microtubules of n-Src transgenic mice were more densely and irregularly arranged, and had structural deformities. In primary culture, Purkinje cells from n-Src transgenic mice developed abnormally thick dendritic shafts and large growth-cone-like structures with poorly extended dendrites, which could be rescued by treatment with a selective inhibitor of Src family kinases, PP2. These results suggest that n-Src activity regulates the dendritic morphogenesis of Purkinje cells through affecting microtubule organization.

  7. A computational model of Purkinje fibre single cell electrophysiology: implications for the long QT syndrome

    PubMed Central

    Sampson, K J; Iyer, V; Marks, A R; Kass, R S

    2010-01-01

    Computer modelling has emerged as a particularly useful tool in understanding the physiology and pathophysiology of cardiac tissues. Models of ventricular, atrial and nodal tissue have evolved and include detailed ion channel kinetics and intercellular Ca2+ handling. Purkinje fibre cells play a central role in the electrophysiology of the heart and in the genesis of cardiac arrhythmias. In this study, a new computational model has been constructed that incorporates the major membrane currents that have been isolated in recent experiments using Purkinje fibre cells. The model, which integrates mathematical models of human ion channels based on detailed biophysical studies of their kinetic and voltage-dependent properties, recapitulates distinct electrophysiological characteristics unique to Purkinje fibre cells compared to neighbouring ventricular myocytes. These characteristics include automaticity, hyperpolarized voltage range of the action potential plateau potential, and prolonged action potential duration. Simulations of selective ion channel blockade reproduce responses to pharmacological challenges characteristic of isolated Purkinje fibres in vitro, and importantly, the model predicts that Purkinje fibre cells are prone to severe arrhythmogenic activity in patients harbouring long QT syndrome 3 but much less so for other common forms of long QT. This new Purkinje cellular model can be a useful tool to study tissue-specific drug interactions and the effects of disease-related ion channel dysfunction on the cardiac conduction system. PMID:20498233

  8. Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element

    SciTech Connect

    Arsenio Nunes, M.L.; Sotelo, C.; Wehrle, R.

    1988-07-01

    The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells.

  9. Purkinje cell activity in the cerebellar anterior lobe after rabbit eyeblink conditioning

    PubMed Central

    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. Rabbits were trained in an interstimulus interval discrimination procedure in which one tone signaled a 250-msec conditioned stimulus-unconditioned stimulus (CS-US) interval and a second tone signaled a 750-msec CS-US interval. All rabbits showed conditioned responses to each CS with mean onset and peak latencies that coincided with the CS-US interval. Many anterior lobe Purkinje cells showed significant learning-related activity after eyeblink conditioning to one or both of the CSs. More Purkinje cells responded with inhibition than with excitation to CS presentation. In addition, when the firing patterns of all conditioning-related Purkinje cells were pooled, it appeared that the population showed a pattern of excitation followed by inhibition during the CS-US interval. Using cholera toxin-conjugated horseradish peroxidase, Purkinje cells in recording areas were found to project to the interpositus nucleus. These data support previous studies that have suggested a role for the anterior cerebellar cortex in eyeblink conditioning as well as models of cerebellar-mediated CR timing that postulate that Purkinje cell activity inhibits conditioned response (CR) generation during the early portion of a trial by inhibiting the deep cerebellar nuclei and permits CR generation during the later portion of a trial through disinhibition of the cerebellar nuclei. PMID:15897252

  10. Molecular mechanisms governing competitive synaptic wiring in cerebellar Purkinje cells.

    PubMed

    Watanabe, Masahiko

    2008-03-01

    Cerebellar Purkinje cells (PCs) play a principal role in motor coordination and motor learning. To fulfill these functions, PCs receive and integrate two types of excitatory inputs, climbing fiber (CF) and parallel fiber (PF). CFs are projection axons from the inferior olive, and convey error signals to PCs. On the other hand, PFs are T-shaped axons of cerebellar granule cells, and convey sensory and motor information carried through the pontocerebellar and spinocerebellar mossy fiber pathways. The most remarkable feature of PC circuits is the highly territorial innervation by these two excitatory afferents. A single climbing CF powerfully and exclusively innervates proximal PC dendrites, whereas hundreds of thousands of PFs innervate distal PC dendrites. Recent studies using gene-manipulated mice have been elucidating that the PC circuitry is formed and maintained by molecular mechanisms that fuel homosynaptic competition among CFs and heterosynaptic competition between CFs and PFs. GluRdelta2 (a PC-specific glutamate receptor) and precerebellin or Cbln1 (a granule cell-derived secretory protein) cooperatively work for selective strengthening of PF-PC synapses, and prevent excessive distal extension of CFs that eventually causes multiple innervation at distal dendrites. In contrast, P/Q-type Ca2+ channels, which mediate Ca2+ influx upon CF activity, selectively strengthen the innervation by a single main CF, and expel PFs and other CFs from proximal dendrites that it innervates. Therefore, we now understand that owing to these mechanisms, territorial innervation by CFs and PFs is properly structured and mono-innervation by CFs is established. Several key issues for future study are also discussed.

  11. Purkinje cell dysfunction and alteration of long-term synaptic plasticity in fetal alcohol syndrome.

    PubMed

    Servais, Laurent; Hourez, Raphaël; Bearzatto, Bertrand; Gall, David; Schiffmann, Serge N; Cheron, Guy

    2007-06-05

    In cerebellum and other brain regions, neuronal cell death because of ethanol consumption by the mother is thought to be the leading cause of neurological deficits in the offspring. However, little is known about how surviving cells function. We studied cerebellar Purkinje cells in vivo and in vitro to determine whether function of these cells was altered after prenatal ethanol exposure. We observed that Purkinje cells that were prenatally exposed to ethanol presented decreased voltage-gated calcium currents because of a decreased expression of the gamma-isoform of protein kinase C. Long-term depression at the parallel fiber-Purkinje cell synapse in the cerebellum was converted into long-term potentiation. This likely explains the dramatic increase in Purkinje cell firing and the rapid oscillations of local field potential observed in alert fetal alcohol syndrome mice. Our data strongly suggest that reversal of long-term synaptic plasticity and increased firing rates of Purkinje cells in vivo are major contributors to the ataxia and motor learning deficits observed in fetal alcohol syndrome. Our results show that calcium-related neuronal dysfunction is central to the pathogenesis of the neurological manifestations of fetal alcohol syndrome and suggest new methods for treatment of this disorder.

  12. Early postweaning social isolation but not environmental enrichment modifies vermal Purkinje cell dendritic outgrowth in rats.

    PubMed

    Pascual, Rodrigo; Bustamante, Carlos

    2013-01-01

    In the present study, we analyzed the effects of enriched, social and isolated experiences on vermal Purkinje cell of the rat, together with anxiety-like behavior in the elevated-plus maze. Sprague-Dawley male rats were randomly submitted to either enriched, social, or isolated environments during the early postweaning period (postnatal days 22-32) and were then behaviorally evaluated in the elevated-plus maze and euthanized for histological analysis. Vermal Purkinje cells (sub-lobules VIa and VIb) were sampled, drawn under camera lucida and morphometrically assessed using the Sholl's concentric ring method. Data obtained indicate that environmental enrichment did not significantly modify the Purkinje cell dendritic branching. On the contrary, Purkinje cell of animals reared in social isolation exhibited a significant reduction in dendritic arborization, which was closely associated with anxiety-like behaviors. The data obtained indicate that, although environmental stimulation in normal animals does not produce significant changes in vermal Purkinje cell dendritic arborization, these cells are vulnerable to early stressful experiences, which is in close association with anxiety-like behaviors.

  13. Reduced Purkinje cell dendritic arborization and loss of dendritic spines in essential tremor.

    PubMed

    Louis, Elan D; Lee, Michelle; Babij, Rachel; Ma, Karen; Cortés, Etty; Vonsattel, Jean-Paul G; Faust, Phyllis L

    2014-12-01

    Based on accumulating post-mortem evidence of abnormalities in Purkinje cell biology in essential tremor, we hypothesized that regressive changes in dendritic morphology would be apparent in the Purkinje cell population in essential tremor cases versus age-matched controls. Cerebellar cortical tissue from 27 cases with essential tremor and 27 age-matched control subjects was processed by the Golgi-Kopsch method. Purkinje cell dendritic anatomy was quantified using a Neurolucida microscopic system interfaced with a motorized stage. In all measures, essential tremor cases demonstrated significant reductions in dendritic complexity compared with controls. Median values in essential tremor cases versus controls were: 5712.1 versus 10 403.2 µm (total dendrite length, P=0.01), 465.9 versus 592.5 µm (branch length, P=0.01), 22.5 versus 29.0 (maximum branch order, P=0.001), and 165.3 versus 311.7 (number of terminations, P=0.008). Furthermore, the dendritic spine density was reduced in essential tremor cases (medians=0.82 versus 1.02 µm(-1), P=0.03). Our demonstration of regressive changes in Purkinje cell dendritic architecture and spines in essential tremor relative to control brains provides additional evidence of a pervasive abnormality of Purkinje cell biology in this disease, which affects multiple neuronal cellular compartments including their axon, cell body, dendrites and spines.

  14. 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

  15. Alteration in 5-hydroxymethylcytosine-mediated epigenetic regulation leads to Purkinje cell vulnerability in ATM deficiency

    PubMed Central

    Jiang, Dewei; Zhang, Ying; Hart, Ronald P.; Chen, Jianmin; Herrup, Karl

    2015-01-01

    A long-standing mystery surrounding ataxia-telangiectasia is why it is mainly cerebellar neurons, Purkinje cells in particular, that appear vulnerable to ATM deficiency. Here we present data showing that 5-hydroxymethylcytosine (5hmC), a newly recognized epigenetic marker found at high levels in neurons, is substantially reduced in human ataxia-telangiectasia and Atm−/− mouse cerebellar Purkinje cells. We further show that TET1, an enzyme that converts 5-methylcytosine (5mC) to 5hmC, responds to DNA damage and manipulation of TET1 activity directly affects the DNA damage signalling and ATM-deficient neuronal cell cycle re-entry and death. Quantitative genome-wide analysis of 5hmC-containing sequences shows that in ATM deficiency there is a cerebellum- and Purkinje cell-specific shift in 5hmC enrichment in both regulatory elements and repeated sequences. Finally, we verify that TET1-mediated 5hmC production is linked to the degenerative process of Purkinje cells and behavioural deficits in Atm−/− mice. Taken together, the selective loss of 5hmC plays a critical role in driving Purkinje cell vulnerability in ATM deficiency. PMID:26510954

  16. Ectopic Purkinje cells in the cerebellar white matter of normal adult rodents: a Golgi study.

    PubMed

    Lafarga, M; Berciano, M T; Blanco, M

    1986-01-01

    In Golgi/Río-Hortega preparations of rat and rabbit cerebellar vermis we have occasionally found isolated ectopic Purkinje cells in the white matter. They were located beneath the bases of the folia and their dendritic branches extended within the confines of the white matter without penetrating into the overlying cortical layers. The general morphology of these ectopic cells was variable, particularly in the extension and shape of the dendritic trees, but all of them exhibited a lower density of dendritic branches than normal Purkinje cells. The less-developed ectopic neurons had multipolar dendritic trees with nonplanar branches irregularly studded with spines. The well-developed ones displayed a more extensive arborization of their processes and they usually preserved some morphological features of normal cortical Purkinje cells: distal dendritic branches studded with numerous spines, a pear-shaped soma, clearly defined morphological polarity and a tendency to display planar arrangement of the dendritic arbors. In semithin sections these neurons also showed cytological features of normal Purkinje cells, such as the Nissl substance forming a nuclear cap oriented toward the dendritic pole. We suggest that the abnormal location of the neurons results from a disorder of Purkinje cell migration which occurs naturally during the prenatal development of the cerebellum. The possible morphogenetic mechanisms involved in the migration and differentiation of these ectopic neurons are also discussed.

  17. Alteration in 5-hydroxymethylcytosine-mediated epigenetic regulation leads to Purkinje cell vulnerability in ATM deficiency.

    PubMed

    Jiang, Dewei; Zhang, Ying; Hart, Ronald P; Chen, Jianmin; Herrup, Karl; Li, Jiali

    2015-12-01

    A long-standing mystery surrounding ataxia-telangiectasia is why it is mainly cerebellar neurons, Purkinje cells in particular, that appear vulnerable to ATM deficiency. Here we present data showing that 5-hydroxymethylcytosine (5hmC), a newly recognized epigenetic marker found at high levels in neurons, is substantially reduced in human ataxia-telangiectasia and Atm(-/-) mouse cerebellar Purkinje cells. We further show that TET1, an enzyme that converts 5-methylcytosine (5mC) to 5hmC, responds to DNA damage and manipulation of TET1 activity directly affects the DNA damage signalling and ATM-deficient neuronal cell cycle re-entry and death. Quantitative genome-wide analysis of 5hmC-containing sequences shows that in ATM deficiency there is a cerebellum- and Purkinje cell-specific shift in 5hmC enrichment in both regulatory elements and repeated sequences. Finally, we verify that TET1-mediated 5hmC production is linked to the degenerative process of Purkinje cells and behavioural deficits in Atm(-/-) mice. Taken together, the selective loss of 5hmC plays a critical role in driving Purkinje cell vulnerability in ATM deficiency.

  18. Systematic Regional Variations in Purkinje Cell Spiking Patterns

    PubMed Central

    Xiao, Jianqiang; Cerminara, Nadia L.; Kotsurovskyy, Yuriy; Aoki, Hanako; Burroughs, Amelia; Wise, Andrew K.; Luo, Yuanjun; Marshall, Sarah P.; Sugihara, Izumi; Apps, Richard; Lang, Eric J.

    2014-01-01

    In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs) in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+) and negative (Z−) bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z− PCs, respectively. In both datasets significant differences in simple spike (SS) activity were observed between cortical regions. Specifically, Z− and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution) was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS) activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions. PMID:25144311

  19. Climbing fiber synapse elimination in cerebellar Purkinje cells.

    PubMed

    Watanabe, Masahiko; Kano, Masanobu

    2011-11-01

    Innervation of Purkinje cells (PCs) by multiple climbing fibers (CFs) is refined into mono-innervation during the first three postnatal weeks of rodents' lives. In this review article, we will integrate the current knowledge on developmental process and mechanisms of CF synapse elimination. In the 'creeper' stage of CF innervation (postnatal day 0 (P0)∼), CFs creep among PC somata to form transient synapses on immature dendrites. In the 'pericellular nest' stage (P5∼), CFs densely surround and innervate PC somata. CF innervation is then displaced to the apical portion of PC somata in the 'capuchon' stage (P9∼), and translocate to dendrites in the 'dendritic' (P12∼) stage. Along with the developmental changes in CF wiring, functional and morphological distinctions become larger among CF inputs. PCs are initially innervated by more than five CFs with similar strengths (∼P3). During P3-7 only a single CF is selectively strengthened (functional differentiation), and it undergoes dendritic translocation from P9 on (dendritic translocation). Following the functional differentiation, perisomatic CF synapses are eliminated nonselectively; this proceeds in two distinct phases. The early phase (P7-11) is conducted independently of parallel fiber (PF)-PC synapse formation, while the late phase (P12-17) critically depends on it. The P/Q-type voltage-dependent Ca(2+) channel in PCs triggers selective strengthening of single CF inputs, promotes dendritic translocation of the strengthened CFs, and drives the early phase of CF synapse elimination. In contrast, the late phase is mediated by the mGluR1-Gαq-PLCβ4-PKCγ signaling cascade in PCs driven at PF-PC synapses, whose structural connectivity is stabilized and maintained by the GluRδ2-Cbln1-neurexin system.

  20. MCT8 deficiency in Purkinje cells disrupts embryonic chicken cerebellar development.

    PubMed

    Delbaere, Joke; Vancamp, Pieter; Van Herck, Stijn L J; Bourgeois, Nele M A; Green, Mary J; Wingate, Richard J T; Darras, Veerle M

    2017-02-01

    Inactivating mutations in the human SLC16A2 gene encoding the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in the Allan-Herndon-Dudley syndrome accompanied by severe locomotor deficits. The underlying mechanisms of the associated cerebellar maldevelopment were studied using the chicken as a model. Electroporation of an MCT8-RNAi vector into the cerebellar anlage of a 3-day-old embryo allowed knockdown of MCT8 in Purkinje cell precursors. This resulted in the downregulation of the thyroid hormone-responsive gene RORα and the Purkinje cell-specific differentiation marker LHX1/5 at day 6. MCT8 knockdown also results in a smaller and less complex dendritic tree at day 18 suggesting a pivotal role of MCT8 for cell-autonomous Purkinje cell maturation. Early administration of the thyroid hormone analogue 3,5,3'-triiodothyroacetic acid partially rescued early Purkinje cell differentiation. MCT8-deficient Purkinje cells also induced non-autonomous effects as they led to a reduced granule cell precursor proliferation, a thinner external germinal layer and a loss of PAX6 expression. By contrast, at day 18, the external germinal layer thickness was increased, with an increase in presence of Axonin-1-positive post-mitotic granule cells in the initial stage of radial migration. The concomitant accumulation of presumptive migrating granule cells in the molecular layer, suggests that inward radial migration to the internal granular layer is stalled. In conclusion, early MCT8 deficiency in Purkinje cells results in both cell-autonomous and non-autonomous effects on cerebellar development and indicates that MCT8 expression is essential from very early stages of development, providing a novel insight into the ontogenesis of the Allan-Herndon-Dudley syndrome.

  1. Mesenchymal stem cells rescue Purkinje cells and improve motor functions in a mouse model of cerebellar ataxia.

    PubMed

    Jones, Jonathan; Jaramillo-Merchán, Jesús; Bueno, Carlos; Pastor, Diego; Viso-León, Maricarmen; Martínez, Salvador

    2010-11-01

    Mesenchymal stem cells have been proven to be potentially effective in the treatment of a large variety of diseases, including neurodegenerative disorders. Of these, cerebellar ataxia is a group of disorders characterized by the degeneration of the cerebellum, particularly the Purkinje cells, responsible for motor coordination and control of the motor functions. To analyze the possibility of using bone marrow-derived mesenchymal stem cells in treating ataxia, we transplanted these cells in the cerebellum of newborn Lurcher mutant mice, a very aggressive mouse model characterized by the selective early post-natal death of Purkinje cells in the cerebellum. Two months after the surgical procedure, the treated mice presented significant improvements in the motor behavior tests performed. Histological analysis of the cerebellum indicated that the donor cells had migrated throughout the cerebellum, as well as a significant increase in the number of Purkinje cells. Many grafted stem cells were located adjacent to the Purkinje cell layer, and expressed BDNF, NT-3 or GDNF, neurotrophic factors implicated in Purkinje cell survival. Also, a small percentage of the grafted stem cells had fused with Purkinje cells. Thus, we have shown that mesenchymal stem cells are capable of integrating into the central nervous system, migrate towards the areas where neurodegenerative processes are occurring, and rescue the degenerating cells through cell trophic effects. This is an adequate and feasible model that could be translated into a therapeutic approach for clinical assays in neurodegenerative diseases.

  2. Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells

    PubMed Central

    Bosman, Laurens W. J.; Hartmann, Jana; Barski, Jaroslaw J.; Lepier, Alexandra; Noll-Hussong, Michael; Reichardt, Louis F.; Konnerth, Arthur

    2009-01-01

    The receptor tyrosine kinase TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), are critically important for growth, survival and activity-dependent synaptic strengthening in the central nervous system. These TrkB-mediated actions occur in a highly cell-type specific manner. Here we report that cerebellar Purkinje cells, which are richly endowed with TrkB receptors, develop a normal morphology in trkB-deficient mice. Thus, in contrast to other types of neurons, Purkinje cells do not need TrkB for dendritic growth and spine formation. Instead, we find a moderate delay in the maturation of GABAergic synapses and, more importantly, an abnormal multiple climbing fiber innervation in Purkinje cells in trkB-deficient mice. Thus, our results demonstrate an involvement of TrkB receptors in synapse elimination and reveal a new role for receptor tyrosine kinases in the brain. PMID:17940915

  3. Kv1 channels selectively prevent dendritic hyperexcitability in rat Purkinje cells

    PubMed Central

    Khavandgar, Simin; Walter, Joy T; Sageser, Kristin; Khodakhah, Kamran

    2005-01-01

    Purkinje cells, the sole output of the cerebellar cortex, encode the timing signals required for motor coordination in their firing rate and activity pattern. Dendrites of Purkinje cells express a high density of P/Q-type voltage-gated calcium channels and fire dendritic calcium spikes. Here we show that dendritic subthreshold Kv1.2 subunit-containing Kv1 potassium channels prevent generation of random spontaneous calcium spikes. With Kv1 channels blocked, dendritic calcium spikes drive bursts of somatic sodium spikes and prevent the cell from faithfully encoding motor timing signals. The selective dendritic function of Kv1 channels in Purkinje cells allows them to effectively suppress dendritic hyperexcitability without hindering the generation of somatic action potentials. Further, we show that Kv1 channels also contribute to dendritic integration of parallel fibre synaptic input. Kv1 channels are often targeted to soma and axon and the data presented support a major dendritic function for these channels. PMID:16210348

  4. Differential GABAergic and glycinergic inputs of inhibitory interneurons and Purkinje cells to principal cells of the cerebellar nuclei.

    PubMed

    Husson, Zoé; Rousseau, Charly V; Broll, Ilja; Zeilhofer, Hanns Ulrich; Dieudonné, Stéphane

    2014-07-09

    The principal neurons of the cerebellar nuclei (CN), the sole output of the olivo-cerebellar system, receive a massive inhibitory input from Purkinje cells (PCs) of the cerebellar cortex. Morphological evidence suggests that CN principal cells are also contacted by inhibitory interneurons, but the properties of this connection are unknown. Using transgenic, tracing, and immunohistochemical approaches in mice, we show that CN interneurons form a large heterogeneous population with GABA/glycinergic phenotypes, distinct from GABAergic olive-projecting neurons. CN interneurons are found to contact principal output neurons, via glycine receptor (GlyR)-enriched synapses, virtually devoid of the main GABA receptor (GABAR) subunits α1 and γ2. Those clusters account for 5% of the total number of inhibitory receptor clusters on principal neurons. Brief optogenetic stimulations of CN interneurons, through selective expression of channelrhodopsin 2 after viral-mediated transfection of the flexed gene in GlyT2-Cre transgenic mice, evoked fast IPSCs in principal cells. GlyR activation accounted for 15% of interneuron IPSC amplitude, while the remaining current was mediated by activation of GABAR. Surprisingly, small GlyR clusters were also found at PC synapses onto principal CN neurons in addition to α1 and γ2 GABAR subunits. However, GlyR activation was found to account for <3% of the PC inhibitory synaptic currents evoked by electrical stimulation. This work establishes CN glycinergic neurons as a significant source of inhibition to CN principal cells, forming contacts molecularly distinct from, but functionally similar to, Purkinje cell synapses. Their impact on CN output, motor learning, and motor execution deserves further investigation.

  5. Immuno-histochemistry and three-dimensional architecture of the intermediate filaments in Purkinje cells in mammalian hearts.

    PubMed

    Yoshimura, Akira; Yamaguchi, Takeshi; Kawazato, Hiroaki; Takahashi, Naohiko; Shimada, Tatsuo

    2014-12-01

    In mammalian hearts, Purkinje cells varied greatly in morphological appearance in different species, and were divided into three groups. Bovine Purkinje cells corresponding to group I were a large size, and had a few myofibrils and abundant intermediate filaments throughout the cytoplasm. The aim of the present study was to clarify the more detailed distribution and three-dimensional architecture of intermediate filaments in Purkinje cells. The hearts in various mammals including humans were investigated by both immuno-histochemistry and scanning electron microscopy (SEM).Immuno-histochemical studies demonstrated that sheep Purkinje cells in group I had a great number of intermediate filaments of 10 nm positive for desmin antibody. Purkinje cells in group II (humans, monkeys and dogs) and group III (mice) were somewhat larger or smaller in size than myocardial cells, but also showed a strong positive reaction for desmin antibody. The saponin or NaOH treatment of cardiac tissues in sheep and humans enabled us to view intermediate filaments by SEM three-dimensionally. Intermediate filaments in sheep Purkinje cells formed a considerably delicate network, and were distributed throughout the cytoplasm. In contrast, those in human Purkinje cells were lower in density, and were present around the nucleus and between myofibrils. It was concluded that a delicate network of intermediate filaments in Purkinje cells of mammalian hearts acted as the cytoskeleton to maintain intercellular stability.

  6. Profound morphological and functional changes of rodent Purkinje cells between the first and the second postnatal weeks: a metamorphosis?

    PubMed Central

    Dusart, Isabelle; Flamant, Frederic

    2012-01-01

    Between the first and the second postnatal week, the development of rodent Purkinje cells is characterized by several profound transitions. Purkinje cells acquire their typical dendritic “espalier” tree morphology and form distal spines. During the first postnatal week, they are multi-innervated by climbing fibers and numerous collateral branches sprout from their axons, whereas from the second postnatal week, the regression of climbing fiber multi-innervation begins, and Purkinje cells become innervated by parallel fibers and inhibitory molecular layer interneurons. Furthermore, their periods of developmental cell death and ability to regenerate their axon stop and their axons become myelinated. Thus a Purkinje cell during the first postnatal week looks and functions differently from a Purkinje cell during the second postnatal week. These fundamental changes occur in parallel with a peak of circulating thyroid hormone in the mouse. All these features suggest to some extent an interesting analogy with amphibian metamorphosis. PMID:22514522

  7. Transient Developmental Purkinje Cell Axonal Torpedoes in Healthy and Ataxic Mouse Cerebellum

    PubMed Central

    Ljungberg, Lovisa; Lang-Ouellette, Daneck; Yang, Angela; Jayabal, Sriram; Quilez, Sabrina; Watt, Alanna J.

    2016-01-01

    Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells – known as torpedoes – have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently during development in rat cerebellum. The function of Purkinje cell axonal torpedoes in health as well as in disease is poorly understood. We investigated the properties of developmental torpedoes in the postnatal mouse cerebellum of wild-type and transgenic mice. We found that Purkinje cell axonal torpedoes transiently appeared on axons of Purkinje neurons, with the largest number of torpedoes observed at postnatal day 11 (P11). This was after peak developmental apoptosis had occurred, when Purkinje cell counts in a lobule were static, suggesting that most developmental torpedoes appear on axons of neurons that persist into adulthood. We found that developmental torpedoes were not associated with a presynaptic GABAergic marker, indicating that they are not synapses. They were seldom found at axonal collateral branch points, and lacked microglia enrichment, suggesting that they are unlikely to be involved in axonal refinement. Interestingly, we found several differences between developmental torpedoes and disease-related torpedoes: developmental torpedoes occurred largely on myelinated axons, and were not associated with changes in basket cell innervation on their parent soma. Disease-related torpedoes are typically reported to contain neurofilament; while the majority of developmental torpedoes did as well, a fraction of smaller developmental torpedoes did not. These differences indicate that developmental torpedoes may not be functionally identical to disease-related torpedoes. To study this further, we used a mouse model of spinocerebellar ataxia type 6 (SCA6), and found elevated disease-related torpedo number at 2

  8. Purkinje Cell Axon Collaterals Terminate on Cat-301+ Neurons in Macaca Monkey Cerebellum

    PubMed Central

    Crook, J.D.; Hendrickson, A.; Erickson, A.; Possin, D.; Robinson, F.R.

    2008-01-01

    The monoclonal antibody Cat-301 identifies perineuronal nets around specific neuronal types, including those in the cerebellum. This report finds in adult Macaca monkey that Basket cells in the deep molecular layer; granule cell layer (GCL) interneurons including Lugaro cells; large neurons in the foliar white matter (WM); and deep cerebellar nuclei (DCN) neurons contain subsets of Cat-301+ cells. Most Cat-301+ GCL interneurons are glycine+ and all are densely innervated by a meshwork of calbindin+/GAD+ Purkinje cell collaterals and their synapses. DCN and WM Cat-301+ neurons also receive a similar but less dense innervation. Due to the heavy labeling of adjacent Purkinje cell dendrites, the innervation of Cat-301+ Basket cells was less certain. These findings suggest that several complex feedback circuits from Purkinje cell to cerebellar interneurons exist in primate cerebellum whose function needs to be investigated. Cat-301 labeling begins postnatally in WM and DCN, but remains sparse until at least 3 months of age. Because the appearance of perineuronal nets is associated with maturation of synaptic circuits, this suggests that the Purkinje cell feedback circuits develop for some time after birth. PMID:17936513

  9. Calcium-dependent chloride current in rat cerebellar Purkinje cell membranes.

    PubMed

    Vykhareva, E A; Zamoyski, V L; Grigoriev, V V; Bachurin, S O

    2015-01-01

    The presence of calcium-dependent potential-activated chloride currents in the membranes of freshly isolated rat cerebellar Purkinje cells (12-15 days) was shown by the whole-cell patch clamp technique. Chloride currents appeared in a sodium-free external solution and reversibly disappeared in the absence of external chloride and calcium ions.

  10. Embryonic origins of ZebrinII parasagittal stripes and establishment of topographic Purkinje cell projections.

    PubMed

    Sillitoe, R V; Gopal, N; Joyner, A L

    2009-09-01

    The establishment of neural circuits involves both the precise positioning of cells within brain regions and projection of axons to specific target cells. In the cerebellum (Cb), the medial-lateral (M-L) and anterior-posterior (A-P) position of each Purkinje cell (PC) and the topography of its axon can be defined with respect to two coordinate systems within the Cb; one based on the pattern of lobules and the other on PC gene expression in parasagittal clusters in the embryo (e.g. Pcp2) and stripes in the adult (e.g. ZebrinII). The relationship between the embryonic clusters of molecularly defined PCs and particular adult PC stripes is not clear. Using a mouse genetic inducible fate mapping (GIFM) approach and a Pcp2-CreER-IRES-hAP transgene, we marked three bilateral clusters of PC clusters with myristolated green fluorescent protein (mGfp) on approximately embryonic day (E) 15 and followed their fate into adulthood. We found that these three clusters contributed specifically to ZebrinII-expressing PCs, including nine of the adult stripes. This result suggests that embryonic PCs maintain a particular molecular identity, and that each embryonic cluster can contribute PCs to more than one adult M-L stripe. Each PC projects a primary axon to one of the deep cerebellar nuclei (DCN) or the vestibular nuclei in the brainstem in an organized fashion that relates to the position of the PCs along the M-L axis. We characterized when PC axons from the three M-L clusters acquire topographic projections. Using a combination of GIFM to mark the PC clusters with mGfp and staining for human placental alkaline phosphatase (hAP) in Pcp2-CreER-IRES-hAP transgenic embryos we found that axons from each embryonic PC cluster intermingled with neurons within particular DCN or projected out of the Cb toward the vestibular nuclei by E14.5. These studies show that PC molecular patterning, efferent circuitry, and DCN nucleogenesis occur simultaneously, suggesting a link between these

  11. Properties and expression of Kv3 channels in cerebellar Purkinje cells.

    PubMed

    Sacco, Tiziana; De Luca, Annarita; Tempia, Filippo

    2006-10-01

    In cerebellar Purkinje cells, Kv3 potassium channels are indispensable for firing at high frequencies. In Purkinje cells from young mice (P4-P7), Kv3 currents, recorded in whole-cell in slices, activated at -30 mV, with rapid activation and deactivation kinetics, and they were partially blocked by blood depressing substance-I (BDS-I, 1 microM). At positive potentials, Kv3 currents were slowly but completely inactivating, while the recovery from inactivation was about eightfold slower, suggesting that a previous firing activity or a small change of the resting potential could in principle accumulate inactivated Kv3 channels, thereby finely tuning Kv3 current availability for subsequent action potentials. Single-cell RT-PCR analysis showed the expression by all Purkinje cells (n=10 for each subunit) of Kv3.1, Kv3.3 and Kv3.4 mRNA, while Kv3.2 was not expressed. These results add to the framework for interpreting the physiological function and the molecular determinants of Kv3 currents in cerebellar Purkinje cells.

  12. Impaired motor coordination and Purkinje cell excitability in mice lacking calretinin

    PubMed Central

    Schiffmann, Serge N.; Cheron, Guy; Lohof, Ann; d’Alcantara, Pablo; Meyer, Michael; Parmentier, Marc; Schurmans, Stéphane

    1999-01-01

    In the cerebellum, the parallel fiber-Purkinje cell synapse can undergo long-term synaptic plasticity suggested to underlie motor learning and resulting from variations in intracellular calcium concentration ([Ca2+]i). Ca2+ binding proteins are enriched in the cerebellum, but their role in information processing is not clear. Here, we show that mice deficient in calretinin (Cr−/−) are impaired in tests of motor coordination. An impairment in Ca2+ homeostasis in Cr−/− Purkinje cells was supported by the high Ca2+-saturation of calbindin-D28k in these cells. The firing behavior of Purkinje cells is severely affected in Cr−/− alert mice, with alterations of simple spike firing rate, complex spike duration, and simple spike pause. In contrast, in slices, transmission at parallel fiber- or climbing fiber-Purkinje cell synapses is unaltered, indicating that marked modifications of the firing behavior in vivo can be undetectable in slice. Thus, these results show that calretinin plays a major role at the network level in cerebellar physiology. PMID:10220453

  13. Investigating complex I deficiency in Purkinje cells and synapses in patients with mitochondrial disease

    PubMed Central

    Chrysostomou, Alexia; Grady, John P.; Laude, Alex; Taylor, Robert W.; Turnbull, Doug M.

    2015-01-01

    Aims Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus. Methods We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age‐matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses. Results Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses. Conclusions This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia. PMID:26337858

  14. Highly 4-aminopyridine sensitive delayed rectifier current modulates the excitability of guinea pig cerebellar Purkinje cells.

    PubMed

    Etzion, Y; Grossman, Y

    2001-08-01

    The effects of low concentrations of 4-aminopyridine (4-AP) on the membrane properties of guinea pig cerebellar Purkinje cells were investigated in slice preparation using intracellular recordings. It was found that 1-10 microM 4-AP did not affect the resting potential or the input resistance of the cells, but reduced markedly the duration of the slowly depolarizing potential (SDP), and thus the latency to the firing of Ca2+ spikes in response to intracellular current pulses. Intradendritic recordings in the presence of tetrodotoxin, Cd2+, and low [Ca2+]o, which blocked all the regenerative responses, exhibited prominent membrane outward rectification in response to depolarizing current pulses. Under these conditions, the SDP was abolished and, in contrast, a slowly developing hyperpolarization was consistently observed. Application of 10 microM 4-AP reduced the outward membrane rectification in a reversible manner, but did not affect the transient hyperpolarization, which is usually attributed to the activation of potassium "A" current. These results demonstrate, for the first time, the presence of a highly 4-AP sensitive delayed rectifier in guinea pig cerebellar Purkinje cells, which prominently affects their excitability. The results also indicate that the slowly depolarizing potential of guinea pig Purkinje cells does not involve inactivation of transient potassium currents, which has been suggested previously as an underlying mechanism for this phenomenon in turtle Purkinje cells.

  15. Vulnerability of Purkinje Cells Generated from Spinocerebellar Ataxia Type 6 Patient-Derived iPSCs.

    PubMed

    Ishida, Yoshihito; Kawakami, Hideshi; Kitajima, Hiroyuki; Nishiyama, Ayaka; Sasai, Yoshiki; Inoue, Haruhisa; Muguruma, Keiko

    2016-11-01

    Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by loss of Purkinje cells in the cerebellum. SCA6 is caused by CAG trinucleotide repeat expansion in CACNA1A, which encodes Cav2.1, α1A subunit of P/Q-type calcium channel. However, the pathogenic mechanism and effective therapeutic treatments are still unknown. Here, we have succeeded in generating differentiated Purkinje cells that carry patient genes by combining disease-specific iPSCs and self-organizing culture technologies. Patient-derived Purkinje cells exhibit increased levels of full-length Cav2.1 protein but decreased levels of its C-terminal fragment and downregulation of the transcriptional targets TAF1 and BTG1. We further demonstrate that SCA6 Purkinje cells exhibit thyroid hormone depletion-dependent degeneration, which can be suppressed by two compounds, thyroid releasing hormone and Riluzole. Thus, we have constructed an in vitro disease model recapitulating both ontogenesis and pathogenesis. This model may be useful for pathogenic investigation and drug screening.

  16. Mild cerebellar neurodegeneration of aged heterozygous PCD mice increases cell fusion of Purkinje and bone marrow-derived cells.

    PubMed

    Díaz, David; Recio, Javier S; Weruaga, Eduardo; Alonso, José R

    2012-01-01

    Bone marrow-derived cells have different plastic properties, especially regarding cell fusion, which increases with time and is prompted by tissue injury. Several recessive mutations, including Purkinje Cell Degeneration, affect the number of Purkinje cells in homozygosis; heterozygous young animals have an apparently normal phenotype but they undergo Purkinje cell loss as they age. Our findings demonstrate that heterozygous pcd mice undergo Purkinje cell loss at postnatal day 300, this slow but steadily progressing cell death starting sooner than has been reported previously and without massive reactive gliosis or inflammation. Here, transplantation of bone marrow stem cells was performed to assess the arrival of bone marrow-derived cells in the cerebellum in these heterozygous mice. Our results reveal that a higher number of cell fusion events occurs in heterozygous animals than in the controls, on days 150 and 300 postnatally. In sum, this study indicates that mild cell death promotes the fusion of bone marrow-derived cells with surviving Purkinje neurons. This phenomenon suggests new therapies for long-lasting neurodegenerative disorders.

  17. Engineered expression of polysialic acid enhances Purkinje cell axonal regeneration in L1/GAP-43 double transgenic mice.

    PubMed

    Zhang, Yi; Zhang, Xinyu; Yeh, John; Richardson, Peter; Bo, Xuenong

    2007-01-01

    Purkinje axons in adult mammals are generally unable to regenerate after axotomy. Our recent work has shown that over-expression of growth related genes, GAP-43 and L1, in Purkinje cells increased their axonal outgrowth into a predegenerated peripheral nerve graft, but not into a fresh graft [Zhang et al., (2005) Proc. Natl Acad. Sci. USA, 102, 14883-14888]. In the current study we investigated whether engineered expression of growth permissive molecule polysialic acid (PSA) in the glial scar or on transplanted Schwann cells could overcome the inhibitory environment and promote Purkinje axonal regeneration. A stab wound was introduced in the cerebellum of the L1/GAP-43 transgenic mice and a lentiviral vector (LV) carrying the polysialyltransferase (PST) cDNA (LV/PST) was injected into the lesion site to transduce the cells in the glial scar. Regenerating Purkinje axons were examined by calbindin immunostaining. There was increased Purkinje axonal sprouting in the area expressing high-level PSA. However, Purkinje axons were unable to grow into the lesion cavity. In the second set of experiments when LV/PST transduced Schwann cells were transplanted into the lesion site, the number of Purkinje axons growing into the transplant was nine times more than that growing into Schwann cell transplant expressing GFP two months post operation. Our result suggests that transplanted Schwann cells engineered to express PSA support axonal regeneration better than naïve Schwann cells.

  18. Potassium accumulation around individual purkinje cells in cerebellar slices from the guinea-pig.

    PubMed Central

    Hounsgaard, J; Nicholson, C

    1983-01-01

    K+-selective micropipettes were used to measure external K+ concentration [( K+]o) in the immediate vicinity of Purkinje cells in slices from guinea-pig cerebellum. The cells were either spontaneously active or were polarized via a separate intracellular micro-electrode. The level of [K+]o rose by 1-3 mM around the soma and dendrites of Purkinje cells during spike activity. The increases in [K+]o were usually greater during Ca2+-mediated spikes than during Na+-mediated spikes. This was even true at the soma where the Ca2+ spike only invaded electrotonically from the dendrites, in contrast to the Na+ spikes which were generated at the soma. No [K+]o changes were seen in the vicinity of Purkinje cells when the cells were hyperpolarized with current passage nor was any [K+]o change seen during subthreshold depolarizations. In glial cells, however, a hyperpolarizing current reduced [K+]o while a depolarizing current increased [K+]o in a symmetrical manner. When Ba2+ was substituted for Ca2+ in the bathing Ringer solution, prolonged plateau-potential spikes could be evoked from Purkinje cells. These spikes were accompanied by large [K+]o elevations but the plateau potentials outlasted the [K+]o elevations. These experiments suggest that large [K+]o increases can occur in the absence of Ca2+-mediated K+ conductances. Substitution of Mn2+ for Ca2+ in the Ringer solution removed some of the [K+]o increases at the Purkinje cell soma. Addition of tetrodotoxin to normal Ringer solution also reduced, but did not abolish the [K+]o increases at the soma. These experiments confirmed that both Na+ and Ca2+ spikes were involved in the [K+]o change. The diffusion characteristics of the slices were determined by an ionophoretic method using tetramethylammonium and ion-selective micropipettes. The extracellular volume fraction of the slice averaged 0.28 while the tortuosity averaged 1.84. These values were close to those found previously in the intact rat cerebellum. These data were

  19. GAP-43 overexpression in adult mouse Purkinje cells overrides myelin-derived inhibition of neurite growth.

    PubMed

    Gianola, Sara; Rossi, Ferdinando

    2004-02-01

    Up-regulation of growth-associated proteins in adult neurons promotes axon regeneration and neuritic elongation onto nonpermissive substrates. To investigate the interaction between these molecules and myelin-related inhibitory factors, we examined transgenic mice in which overexpression of the growth-associated protein GAP-43 is driven by the Purkinje cell-specific promoter L7. Contrary to their wild-type counterparts, which have extremely poor regenerative capabilities, axotomized transgenic Purkinje cells exhibit profuse sprouting along the intracortical neurite and at the severed stump [Buffo et al. (1997) J. Neurosci., 17, 8778-8791]. Here, we investigated the relationship between such sprouting axons and oligodendroglia to ask whether GAP-43 overexpression enables Purkinje neurites to overcome myelin-derived inhibition. Intact transgenic Purkinje axons display normal morphology and myelination. Following injury, however, many GAP-43-overexpressing neurite stumps are devoid of myelin cover and sprout into white matter regions containing densely packed myelin and Nogo-A- or MAG-immunopositive oligodendrocytes. The intracortical segments of these neurites show focal accumulations of GAP-43, which are associated with disrupted or retracted myelin sheaths. Numerous sprouts originate from such demyelinated segments and spread into the granular layer. Some myelin loss, though not axon sprouting, is also evident in wild-type mice, but this phenomenon is definitely more rapid and extensive in transgenic cerebella. Thus, GAP-43-overexpressing Purkinje axons are endowed with enhanced capabilities for growing into nonpermissive territories and show a pronounced tendency to lose myelin. Our observations suggest that accumulation of GAP-43 along precise axon segments disrupts the normal axon-glia interaction and enhances the retraction of oligodendrocytic processes to facilitate the outgrowth of neuritic sprouts.

  20. The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum

    PubMed Central

    Eccles, J. C.; Llinás, R.; Sasaki, K.

    1966-01-01

    1. A single climbing fibre makes an extraordinarily extensive synaptic contact with the dendrites of a Purkinje cell. Investigation of this synaptic mechanism in the cerebellum of the cat has been based on the discovery by Szentagothai & Rajkovits (1959) that the climbing fibres have their cells of origin in the contralateral inferior olive. 2. Stimulation in the accessory olive selectively excites fibres that have a powerful synaptic excitatory action on Purkinje cells in the contralateral vermis, evoking a repetitive spike discharge of 5-7 msec duration. Almost invariably this response had an all-or-nothing character. In every respect it corresponds with the synaptic action that is to be expected from climbing fibres. 3. Intracellular recording from Purkinje cells reveals that this climbing fibre stimulation evokes a large unitary depolarization with an initial spike and later partial spike responses superimposed on a sustained depolarization. 4. Typical climbing fibre responses can be excited, but in a much less selective manner, by stimulation of the olive-cerebellar pathway in the region of the fastigial nucleus, there being often a preceding antidromic spike potential of the Purkinje cell under observation. 5. Impaled Purkinje cells rapidly deteriorate with loss of all spike discharge, the climbing fibre response being then reduced to an excitatory post-synaptic potential. This potential shows that stimulation of the inferior olive may evoke two or more discharges at about 2 msec intervals in the same climbing fibre. The complexity of neuronal connexions in the inferior olive is also indicated by the considerable latency range in responses. 6. A further complication is that, with stimulation in the region of the fastigial nucleus, the initial direct climbing fibre response is often followed by a reflex discharge, presumably from the inferior olive, which resembles the responses produced by inferior olive stimulation in being often repetitive. 7. Typical

  1. 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

  2. The spontaneous ataxic mouse mutant tippy is characterized by a novel Purkinje cell morphogenesis and degeneration phenotype

    PubMed Central

    Shih, Evelyn K.; Sekerková, Gabriella; Ohtsuki, Gen; Aldinger, Kimberly A.; Chizhikov, Victor V.; Hansel, Christian; Mugnaini, Enrico; Millen, Kathleen J.

    2015-01-01

    This study represents the first detailed analysis of the spontaneous neurological mouse mutant, tippy, uncovering its unique cerebellar phenotype. Homozygous tippy mutant mice are small, ataxic and die around weaning. Although the cerebellum shows grossly normal foliation, tippy mutants display a complex cerebellar Purkinje cell phenotype consisting of abnormal dendritic branching with immature spine features and patchy, non-apoptotic cell death that is associated with widespread dystrophy and degeneration of the Purkinje cell axons throughout the white matter, the cerebellar nuclei and the vestibular nuclei. Moderate anatomical abnormalities of climbing fiber innervation of tippy mutant Purkinje cells were not associated with changes in climbing fiber-EPSC amplitudes. However, decreased ESPC amplitudes were observed in response to parallel fiber stimulation and correlated well with anatomical evidence for patchy dark cell degeneration of Purkinje cell dendrites in the molecular layer. The data suggest that the Purkinje neurons are a primary target of the tippy mutation. Furthermore, we hypothesize that the Purkinje cell axonal pathology together with disruptions in the balance of climbing fiber and parallel fiber Purkinje cell input in the cerebellar cortex underlie the ataxic phenotype in these mice. The constellation of Purkinje cell dendritic malformation and degeneration phenotypes in tippy mutants is unique and has not been reported in any other neurologic mutant. Fine mapping of the tippy mutation to a 2.1MB region of distal chromosome 9, which does not encompass any gene previously implicated in cerebellar development or neuronal degeneration, confirms that the tippy mutation identifies novel biology and gene function. PMID:25626522

  3. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    PubMed Central

    Silvestri, Ludovico; Paciscopi, Marco; Soda, Paolo; Biamonte, Filippo; Iannello, Giulio; Frasconi, Paolo; Pavone, Francesco S.

    2015-01-01

    Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells (PCs) across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all PCs are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent PCs. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of PCs, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of PCs with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments. PMID:26074783

  4. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis.

    PubMed

    Silvestri, Ludovico; Paciscopi, Marco; Soda, Paolo; Biamonte, Filippo; Iannello, Giulio; Frasconi, Paolo; Pavone, Francesco S

    2015-01-01

    Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells (PCs) across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all PCs are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent PCs. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of PCs, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of PCs with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  5. Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss.

    PubMed

    Ramadoss, Jayanth; Lunde, Emilie R; Ouyang, Nengtai; Chen, Wei-Jung A; Cudd, Timothy A

    2008-08-01

    Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

  6. Diacylglycerol kinase ε localizes to subsurface cisterns of cerebellar Purkinje cells.

    PubMed

    Hozumi, Yasukazu; Fujiwara, Hiroki; Kaneko, Kenya; Fujii, Satoshi; Topham, Matthew K; Watanabe, Masahiko; Goto, Kaoru

    2017-02-13

    Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGKε is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGKε play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGKε antibody. DGKε was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGKε was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGKε was also distributed in close apposition to DG lipase-α, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGKε-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKε plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.

  7. Alcohol Impairs Long-Term Depression at the Cerebellar Parallel Fiber–Purkinje Cell Synapse

    PubMed Central

    Belmeguenai, Amor; Botta, Paolo; Weber, John T.; Carta, Mario; De Ruiter, Martijn; De Zeeuw, Chris I.; Valenzuela, C. Fernando; Hansel, Christian

    2008-01-01

    Acute alcohol consumption causes deficits in motor coordination and gait, suggesting an involvement of cerebellar circuits, which play a role in the fine adjustment of movements and in motor learning. It has previously been shown that ethanol modulates inhibitory transmission in the cerebellum and affects synaptic transmission and plasticity at excitatory climbing fiber (CF) to Purkinje cell synapses. However, it has not been examined thus far how acute ethanol application affects long-term depression (LTD) and long-term potentiation (LTP) at excitatory parallel fiber (PF) to Purkinje cell synapses, which are assumed to mediate forms of cerebellar motor learning. To examine ethanol effects on PF synaptic transmission and plasticity, we performed whole cell patch-clamp recordings from Purkinje cells in rat cerebellar slices. We found that ethanol (50 mM) selectively blocked PF–LTD induction, whereas it did not change the amplitude of excitatory postsynaptic currents at PF synapses. In contrast, ethanol application reduced voltage-gated calcium currents and type 1 metabotropic glutamate receptor (mGluR1)–dependent responses in Purkinje cells, both of which are involved in PF–LTD induction. The selectivity of these effects is emphasized by the observation that ethanol did not impair PF–LTP and that PF–LTP could readily be induced in the presence of the group I mGluR antagonist AIDA or the mGluR1a antagonist LY367385. Taken together, these findings identify calcium currents and mGluR1-dependent signaling pathways as potential ethanol targets and suggest that an ethanol-induced blockade of PF–LTD could contribute to the motor coordination deficits resulting from alcohol consumption. PMID:18922952

  8. Ischemia deteriorates the spike encoding of rat cerebellar Purkinje cells by raising intracellular Ca{sup 2+}

    SciTech Connect

    Zhao Shidi; Chen Na; Yang Zhilai; Huang Li; Zhu Yan; Guan Sudong; Chen Qianfen; Wang Jinhui

    2008-02-08

    Ischemia-induced excitotoxicity at cerebellar Purkinje cells is presumably due to a persistent glutamate action. To the fact that they are more vulnerable to ischemia than other glutamate-innervated neurons, we studied whether additional mechanisms are present and whether cytoplasm Ca{sup 2+} plays a key role in their ischemic excitotoxicity. Ischemic changes in the excitability of Purkinje cells were measured by whole-cell recording in cerebellar slices of rats with less glutamate action. The role of cytoplasm Ca{sup 2+} was examined by two-photon cellular imaging and BAPTA infusion in Purkinje cells. Lowering perfusion rate to cerebellar slices deteriorated spike timing and raised spike capacity of Purkinje cells. These changes were associated with the reduction of spike refractory periods and threshold potentials, as well as the loss of their control to spike encoding. Ischemia-induced functional deterioration at Purkinje neurons was accompanied by cytoplasm Ca{sup 2+} rise and prevented by BAPTA infusion. Therefore, the ischemia destabilizes the spike encoding of Purkinje cells via raising cytoplasm Ca{sup 2+} without a need for glutamate, which subsequently causes their excitotoxic death.

  9. Activity-dependent plasticity of spike pauses in cerebellar Purkinje cells

    PubMed Central

    Grasselli, Giorgio; He, Qionger; Wan, Vivian; Adelman, John P.; Ohtsuki, Gen; Hansel, Christian

    2016-01-01

    Summary Plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst–pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2−/− mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity—by altering the Purkinje cell output—may be crucial to cerebellar information storage and learning. PMID:26972012

  10. 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.

  11. Spatiotemporal response properties of cerebellar Purkinje cells to animal displacement: a population analysis.

    PubMed

    Pompeiano, O; Andre, P; Manzoni, D

    1997-12-01

    The hypothesis that corticocerebellar units projecting to vestibulospinal neurons contribute to the spatiotemporal response characteristics of forelimb extensors to animal displacement was tested in decerebrate cats in which the activity of Purkinje cells and unidentified cells located in the cerebellar anterior vermis was recorded during wobble of the whole animal. This stimulus imposed to the animal a tilt of fixed amplitude (5 degrees) with a direction moving at a constant angular velocity (56.2 degrees/s), both in the clockwise and counterclockwise directions over the horizontal plane. Eighty-three percent (143/173) of Purkinje cells and 81% (42/52) of unidentified cells responded to clockwise and/or counterclockwise rotations. In particular, 116/143 Purkinje cells (81%) and 32/42 unidentified cells (76%) responded to both clockwise and counterclockwise rotations (bidirectional units), while 27/143 Purkinje cells (19%) and 10/42 unidentified cells (24%) responded to wobble in one direction only (unidirectional units). For the bidirectional units, the direction of maximum sensitivity to tilt (Smax) was identified. Among these units, 24% of the Purkinje cells and 26% of the unidentified cells displayed an equal amplitude of modulation during clockwise and counterclockwise rotations, indicating a cosine-tuned behavior. For this unit type, the temporal phase of the response to a given direction of tilt should remain constant, while the sensitivity would be maximal along the Smax direction, declining with the cosine of the angle between Smax and the tilt direction. The remaining bidirectional units, i.e. 57% of the Purkinje cells and 50% of the unidentified cells displayed unequal amplitudes of modulation during clockwise and counterclockwise rotations. For these neurons, a non-zero sensitivity along the null direction is expected, with a response phase varying as a function of stimulus direction. As to the unidirectional units, their responses to wobble in one

  12. The ins and outs of GluD2--why and how Purkinje cells use the special glutamate receptor.

    PubMed

    Yuzaki, Michisuke

    2012-06-01

    The δ2 glutamate receptor (GluD2) is predominantly expressed in cerebellar Purkinje cells and plays crucial roles in cerebellar functions. Indeed, the number of synapses between parallel fibers (PFs) and Purkinje cells is specifically and severely reduced in GluD2-null cerebellum. In addition, long-term depression (LTD) at PF-Purkinje cell synapses is impaired in these mice. Nevertheless, the mechanism by which GluD2 regulate these two functions-morphological and functional synaptic plasticity at PF synapses-has remained unclear. Recently, we found that Cbln1, a glycoprotein released from granule cells, was bound to the N-terminal domain of GluD2 and regulated formation and maintenance of PF-Purkinje cell synapses. Furthermore, we found that D: -Ser released from Bergmann glia bound the ligand-binding domain of GluD2 and mediated LTD in a manner dependent on the C-terminus. These findings indicate how GluD2 is activated and regulates functions at PF-Purkinje cell synapses. A hypothesis about why GluD2 is employed by PF synapses is also discussed.

  13. Calbindin positive Purkinje cells in the pathology of human cerebellum occurring at the time of its development.

    PubMed

    Laure-Kamionowska, Milena; Maślińska, Danuta

    2009-01-01

    Development of cerebellum continues over an extremely long period of time extending from the early embryonic phase until the first postnatal years. During an extended time of maturation the cerebellum is vulnerable to harmful agents. A group of cytoplasmic proteins that may protect cells against injury are the calcium binding proteins, among others calbindin. The distribution of this protein is not well known in cerebellar pathology, thus in the present study the localisation and appearance of calbindin expressing Purkinje cells in different pathological conditions occurring at the time of cerebellar development was examined. The investigations were carried out on human maturing cerebellar cortex (age range 30 gestational weeks - 2 years) of cases with paraneoplastic cerebellar degeneration and cerebellar neuronal migration disturbances. The Purkinje cells located in cerebellar heterotopias and dysgenesias were morphologically well developed and strongly immunostained with calbindin antibody. In paraneoplastic cerebellar degeneration the progressive decrease of calbindin content and disintegration of Purkinje cells were observed. Our results show that intrauterine harmful agents that disturb migration of the cerebellar neurons do not affect the content of calbindin in misoriented neurons and that this protein may play a role in development of Purkinje cells located in heterotopias and cerebellar dysgenesias. The progressive decrease of calbindin content in the Purkinje cells undergoing degeneration and death during paraneoplastic changes in the cerebellum also supports the hypothesis that this protein is very important component of intracellular homeostasis in cerebellar neurons.

  14. Kv3 K+ channels enable burst output in rat cerebellar Purkinje cells.

    PubMed

    McKay, B E; Turner, R W

    2004-08-01

    The ability of cells to generate an appropriate spike output depends on a balance between membrane depolarizations and the repolarizing actions of K(+) currents. The high-voltage-activated Kv3 class of K(+) channels repolarizes Na(+) spikes to maintain high frequencies of discharge. However, little is known of the ability for these K(+) channels to shape Ca(2+) spike discharge or their ability to regulate Ca(2+) spike-dependent burst output. Here we identify the role of Kv3 K(+) channels in the regulation of Na(+) and Ca(2+) spike discharge, as well as burst output, using somatic and dendritic recordings in rat cerebellar Purkinje cells. Kv3 currents pharmacologically isolated in outside-out somatic membrane patches accounted for approximately 40% of the total K(+) current, were very fast and high voltage activating, and required more than 1 s to fully inactivate. Kv3 currents were differentiated from other tetraethylammonium-sensitive currents to establish their role in Purkinje cells under physiological conditions with current-clamp recordings. Dual somatic-dendritic recordings indicated that Kv3 channels repolarize Na(+) and Ca(2+) spikes, enabling high-frequency discharge for both types of cell output. We further show that during burst output Kv3 channels act together with large-conductance Ca(2+)-activated K(+) channels to ensure an effective coupling between Ca(2+) and Na(+) spike discharge by preventing Na(+) spike inactivation. By contributing significantly to the repolarization of Na(+) and especially Ca(2+) spikes, our data reveal a novel function for Kv3 K(+) channels in the maintenance of high-frequency burst output for cerebellar Purkinje cells.

  15. Temporal effects of thyroid hormone (TH) and decabrominated diphenyl ether (BDE209) on Purkinje cell dendrite arborization.

    PubMed

    Ibhazehiebo, K; Koibuchi, N

    2012-06-07

    Thyroid hormones (TH) 3,3',4-tri-iodothyronine (T3) and 3,3',4,4'-tetra-iodothyronine (T4) plays crucial role in cerebellar development. Deficiency of TH consistently results in aberrant growth and development of the cerebellum including reduced growth and branching of the Purkinje cells. In rodents, the critical period of thyroid hormone action on cerebellum development is within the first two to three weeks, after which thyroid hormone replacement cannot fully reverse abnormal cerebellar development induced by thyroid hormone insult. Decabrominated diphenyl ether (BDE209) is an industrial reagent used as an additive flame retardant to reduce flammability of various commercial and household produce. BDE209 has bio-accumulative potential and is neurotoxic. Previously, we have shown that T4 (10-8 M) induced extensive dendrite arborization of Purkinje cells and low dose BDE209 (10-10 M) remarkably suppressed TH-induced Purkinje cell dendrite arborization. In the present study, we show that the critical period for TH-induced Purkinje cell growth and dendrite arborization in culture is much earlier than reported in animal models. Also, we show for the first time that low dose BDE209 suppressed TH-induced dendrite arborization in a time-dependent manner. Taken together, our study indicates that hypothyroidism and exposure to BDE209 during critical stage of cerebellar development can lead to impaired Purkinje cell growth and dendrite arborization and may consequently disrupt normal cerebellar functions.

  16. Case Study: Somatic Sprouts and Halo-Like Amorphous Materials of the Purkinje Cells in Huntington's Disease.

    PubMed

    Sakai, Kenji; Ishida, Chiho; Morinaga, Akiyoshi; Takahashi, Kazuya; Yamada, Masahito

    2015-12-01

    We described a 63-year-old Japanese female with genetically confirmed Huntington's disease who showed unusual pathological findings in the cerebellum. This case exhibited typical neuropathological features as Huntington's disease, including severe degeneration of the neostriatum and widespread occurrence of ubiquitin and expanded polyglutamine-positive neuronal intranuclear and intracytoplasmic inclusions. The cerebellum was macroscopically unremarkable; however, somatic sprouts and halo-like amorphous materials of Purkinje cell with a large amount of torpedoes were noteworthy. Furthermore, the Purkinje cells were found to have granular cytoplasmic inclusions. Somatic sprouting is a form of degenerated Purkinje cell exhibited in several specific conditions. Although this finding usually appeared in developmental brains, several neurodegenerative disorders, including Menkes kinky hair disease, familial spinocerebellar ataxia, acute encephalopathy linked to familial hemiplegic migraine, and several other conditions, have been reported showing sprouting from the soma of Purkinje cell. We propose that Huntington's disease is another degenerative condition associated with these distinct neuropathological findings of Purkinje cell. Abnormally accumulated huntingtin protein in the cytoplasm could be related to the development of these structures.

  17. Ischemic insult to cerebellar Purkinje cells causes diminished GABAA receptor function and Allopregnanolone neuroprotection is associated with GABAA receptor stabilization

    PubMed Central

    Kelley, MH; Taguchi, N; Ardeshiri, A; Kuroiwa, M; Hurn, PD; Traystman, RJ; Herson, PS

    2009-01-01

    Cerebellar Purkinje cells are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down-regulation of GABAA receptors in cerebellar Purkinje cells, thereby increasing susceptibility to excitotoxicity. Oxygen-glucose deprivation caused a decline in functional GABAA receptors, within the first hour of re-oxygenation. Decreased amplitude of miniature inhibitory post-synaptic potentials confirmed that oxygen-glucose deprivation caused a significant decrease in functional synaptic GABAA receptors and quantitative Western blot analysis demonstrated the loss of GABAA receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone prevented the decline in GABAA receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABAA receptor protein and Purkinje cell specific immunoreactivity. Moreover, allopregnanolone provided strong protection of Purkinje cells and prevented ischemia-induced decline in GABAA receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABAA receptors in Purkinje cells, whereas allopregnanolone prevents the decline in GABAA receptors and protects against ischemia-induced damage. Thus, interventions which prevent ischemia-induced decline in GABAA receptors may represent a novel neuroprotective strategy. PMID:18699862

  18. Cbln1 binds to specific postsynaptic sites at parallel fiber-Purkinje cell synapses in the cerebellum.

    PubMed

    Matsuda, Keiko; Kondo, Tetsuro; Iijima, Takatoshi; Matsuda, Shinji; Watanabe, Masahiko; Yuzaki, Michisuke

    2009-02-01

    Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis.

  19. CHP1-mediated NHE1 biosynthetic maturation is required for Purkinje cell axon homeostasis.

    PubMed

    Liu, Ye; Zaun, Hans C; Orlowski, John; Ackerman, Susan L

    2013-07-31

    Axon degeneration is a critical pathological feature of many neurodegenerative diseases. Misregulation of local axonal ion homeostasis has been recognized as an important yet understudied mechanism for axon degeneration. Here we report a chemically induced, recessive mouse mutation, vacillator (vac), which causes ataxia and concomitant axon degeneration of cerebellar Purkinje cells. By positional cloning, we identified vac as a point mutation in the calcineurin-like EF hand protein 1 (Chp1) gene that resulted in the production of mutant CHP1 isoforms with an amino acid substitution in a functional EF-hand domain or a truncation of this motif by aberrant splicing and significantly reduced protein levels. CHP1 has been previously shown to interact with the sodium hydrogen exchanger 1 (NHE1), a major regulator of intracellular pH. We demonstrated that CHP1 assists in the full glycosylation of NHE1 that is necessary for the membrane localization of this transporter and that truncated isoforms of CHP1 were defective in stimulating NHE1 biosynthetic maturation. Consistent with this, membrane localization of NHE1 at axon terminals was greatly reduced in Chp1-deficient Purkinje cells before axon degeneration. Furthermore, genetic ablation of Nhe1 also resulted in Purkinje cell axon degeneration, pinpointing the functional convergence of the two proteins. Our findings clearly demonstrate that the polarized presynaptic localization of NHE/CHP1 is an important feature of neuronal axons and that selective disruption of NHE1-mediated proton homeostasis in axons can lead to degeneration, suggesting that local regulation of pH is pivotal for axon survival.

  20. Immature chemodifferentiation of Purkinje cell synapses revealed by 5'-nucleotidase ecto-enzyme activity in the cerebellum of the reeler mouse.

    PubMed

    Bailly, Y J; Schoen, S W; Mariani, J; Kreutzberg, G W; Delhaye-Bouchaud, N

    1998-07-01

    During postnatal development of the rodent cerebellum, a transient enzyme activity of ecto-5'-nucleotidase has been shown in the asymmetrical synapses of Purkinje cells. The alterations of the afferent circuitry and microenvironment of the ectopic Purkinje cells present in the cerebellum of the reeler mutant mouse could enlighten parameters that influence the synaptic 5'-nucleotidase activity of these cells. Ecto-enzyme cytochemistry reveals intense 5'-nucleotidase activity in 43% of synapses of the Purkinje cells throughout the cortex and the core of the reeler cerebellar vermis, although the molecular layer displays large areas with less than 1% of labelled synapses. However, enzymatic labelling is found in considerably more Purkinje cells synapses (73%) throughout the granular layer and the subcortical mass. Climbing fiber synapses of monoinnervated Purkinje cells are labelled by 5'-nucleotidase activity in the molecular layer, as well as asymmetrical synapses made on the subjacent ectopic Purkinje cells by the multiple climbing fibers and by the heterologous afferences. The non-innervated dendritic spines of these cells are also labelled, suggesting that 5'-nucleotidase activity at postsynaptic sites of reeler Purkinje cells does not depend on the presynaptic innervation. Rather, 5'-nucleotidase enzyme activity is enhanced at theses sites when the Purkinje cells have not achieved chemodifferentiation but have conserved immature wiring, i.e., low parallel fiber and multiple climbing fiber inputs.

  1. Neuroligins Sculpt Cerebellar Purkinje-Cell Circuits by Differential Control of Distinct Classes of Synapses.

    PubMed

    Zhang, Bo; Chen, Lulu Y; Liu, Xinran; Maxeiner, Stephan; Lee, Sung-Jin; Gokce, Ozgun; Südhof, Thomas C

    2015-08-19

    Neuroligins are postsynaptic cell-adhesion molecules that bind presynaptic neurexins and are genetically linked to autism. Neuroligins are proposed to organize synaptogenesis and/or synaptic transmission, but no systematic analysis of neuroligins in a defined circuit is available. Here, we show that conditional deletion of all neuroligins in cerebellar Purkinje cells caused loss of distal climbing-fiber synapses and weakened climbing-fiber but not parallel-fiber synapses, consistent with alternative use of neuroligins and cerebellins as neurexin ligands for the excitatory climbing-fiber versus parallel-fiber synapses. Moreover, deletion of neuroligins increased the size of inhibitory basket/stellate-cell synapses but simultaneously severely impaired their function. Multiple neuroligin isoforms differentially contributed to climbing-fiber and basket/stellate-cell synapse functions, such that inhibitory synapse-specific neuroligin-2 was unexpectedly essential for maintaining normal climbing-fiber synapse numbers. Using systematic analyses of all neuroligins in a defined neural circuit, our data thus show that neuroligins differentially contribute to various Purkinje-cell synapses in the cerebellum in vivo.

  2. Neuroligins Sculpt Cerebellar Purkinje-Cell Circuits by Differential Control of Distinct Classes of Synapses

    PubMed Central

    Zhang, Bo; Chen, Lulu Y.; Liu, Xinran; Maxeiner, Stephan; Lee, Sung-Jin; Gokce, Ozgun; Südhof, Thomas C.

    2015-01-01

    Neuroligins are postsynaptic cell-adhesion molecules that bind presynaptic neurexins and are genetically linked to autism. Neuroligins are proposed to organize synaptogenesis and/or synaptic transmission, but no systematic analysis of neuroligins in a defined circuit is available. Here, we show that conditional deletion of all neuroligins in cerebellar Purkinje cells caused loss of distal climbing-fiber synapses and weakened climbing-fiber but not parallel-fiber synapses, consistent with alternative use of neuroligins and cerebellins as neurexin ligands for the excitatory climbing-fiber vs. parallel-fiber synapses. Moreover, deletion of neuroligins increased the size of inhibitory basket/stellate-cell synapses but simultaneously severely impaired their function. Multiple neuroligin isoforms differentially contributed to climbing-fiber and basket/stellate-cell synapse functions, such that inhibitory synapse-specific neuroligin-2 was unexpectedly essential for maintaining normal climbing-fiber synapse numbers. Using systematic analyses of all neuroligins in a defined neural circuit, our data thus show that neuroligins differentially contribute to various Purkinje-cell synapses in the cerebellum in vivo. PMID:26291161

  3. Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices.

    PubMed Central

    Llinás, R; Sugimori, M

    1980-01-01

    1. The electrical activity of Purkinje cells was studied in guinea-pig cerebellar slices in vitro. Intracellular recordings from Purkinje cell somata were obtained under direct vision, and antidromic, synaptic and direct electroresponsiveness was demonstrated. Synaptic potentials produced by the activation of the climbing fibre afferent could be reversed by direct membrane depolarization. 2. Input resistance of impaled neurones ranged from 10 to 19 M omega and demonstrated non-linearities in both hyperpolarizing and depolarizing directions. 3. Direct activation of a Purkinje cell indicated that repetitive firing of fast somatic spikes (s.s.) occurs, after a threshold, with a minimum spike frequency of about 30 spikes/sec, resembling the '2-class' response of crab nerve (Hodgkin, 1948). 4. As the amplitude of the stimulus was increased, a second form of electroresponsiveness characterized by depolarizing spike bursts (d.s.b.) was observed and was often accomppanied by momentary inactivation of the s.s. potentials. Upon application of tetrodotoxin (TTX) or removal of Na+ ions from the superfusion fluid, the s.s. potentials were abolished while the burst responses remained intact. However, Ca conductance blockers such as Co, Cd, Mn and D600, or the replacement of Ca by Mg, completely abolish d.s.b.s. 5. If Ca conductance was blocked, or Ca removed from the superfusion fluid without blockage of Na conductance, two types of Na-dependent electroresponsiveness were seen: (a) the s.s. potentials and (b) slow rising all-or-none responses which reached plateau at approximately -15 mV and could last for several seconds. These all-or-none Na-dependent plateau depolarizations outlasted the stimulus and were accompanied by a large increase in membrane conductance. Within certain limits the rate of rise and amplitude of the plateau were independent of stimulus strength. The latency, however, was shortened as stimulus amplitude was increased. These potentials were blocked by TTX

  4. Modulation, Plasticity and Pathophysiology of the Parallel Fiber-Purkinje Cell Synapse

    PubMed Central

    Hoxha, Eriola; Tempia, Filippo; Lippiello, Pellegrino; Miniaci, Maria Concetta

    2016-01-01

    The parallel fiber-Purkinje cell (PF-PC) synapse represents the point of maximal signal divergence in the cerebellar cortex with an estimated number of about 60 billion synaptic contacts in the rat and 100,000 billions in humans. At the same time, the Purkinje cell dendritic tree is a site of remarkable convergence of more than 100,000 parallel fiber synapses. Parallel fiber activity generates fast postsynaptic currents via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and slower signals, mediated by mGlu1 receptors, resulting in Purkinje cell depolarization accompanied by sharp calcium elevation within dendritic regions. Long-term depression (LTD) and long-term potentiation (LTP) have been widely described for the PF-PC synapse and have been proposed as mechanisms for motor learning. The mechanisms of induction for LTP and LTD involve different signaling mechanisms within the presynaptic terminal and/or at the postsynaptic site, promoting enduring modification in the neurotransmitter release and change in responsiveness to the neurotransmitter. The PF-PC synapse is finely modulated by several neurotransmitters, including serotonin, noradrenaline and acetylcholine. The ability of these neuromodulators to gate LTP and LTD at the PF-PC synapse could, at least in part, explain their effect on cerebellar-dependent learning and memory paradigms. Overall, these findings have important implications for understanding the cerebellar involvement in a series of pathological conditions, ranging from ataxia to autism. For example, PF-PC synapse dysfunctions have been identified in several murine models of spino-cerebellar ataxia (SCA) types 1, 3, 5 and 27. In some cases, the defect is specific for the AMPA receptor signaling (SCA27), while in others the mGlu1 pathway is affected (SCA1, 3, 5). Interestingly, the PF-PC synapse has been shown to be hyper-functional in a mutant mouse model of autism spectrum disorder, with a selective deletion of Pten in

  5. 3-D confocal laser scanning microscopy used in morphometric analysis of rat Purkinje cell dendritic spines after chronic ethanol consumption.

    PubMed

    Wenisch, S; Fortmann, B; Steinmetz, T; Kriete, A; Leiser, R; Bitsch, I

    1998-12-01

    A confocal laser scanning microscope (with a 543 nm laser) was used for imaging rat Purkinje cell dendritic spines at high 3-D resolution. In a nutritionally controlled study of the rat, 5 months of ethanol consumption was demonstrated to alter the spines of Purkinje cell dendrites in rat cerebellum. Intact spines showed significant elongation after ethanol exposure, whereas this neuromorphological alteration could not be detected in controls. Spine elongation could be regarded as compensative growth of spines in search of new synaptic contacts due to alcohol induced cell loss.

  6. Rapid development of Purkinje cell excitability, functional cerebellar circuit, and afferent sensory input to cerebellum in zebrafish.

    PubMed

    Hsieh, Jui-Yi; Ulrich, Brittany; Issa, Fadi A; Wan, Jijun; Papazian, Diane M

    2014-01-01

    The zebrafish has significant advantages for studying the morphological development of the brain. However, little is known about the functional development of the zebrafish brain. We used patch clamp electrophysiology in live animals to investigate the emergence of excitability in cerebellar Purkinje cells, functional maturation of the cerebellar circuit, and establishment of sensory input to the cerebellum. Purkinje cells are born at 3 days post-fertilization (dpf). By 4 dpf, Purkinje cells spontaneously fired action potentials in an irregular pattern. By 5 dpf, the frequency and regularity of tonic firing had increased significantly and most cells fired complex spikes in response to climbing fiber activation. Our data suggest that, as in mammals, Purkinje cells are initially innervated by multiple climbing fibers that are winnowed to a single input. To probe the development of functional sensory input to the cerebellum, we investigated the response of Purkinje cells to a visual stimulus consisting of a rapid change in light intensity. At 4 dpf, sudden darkness increased the rate of tonic firing, suggesting that afferent pathways carrying visual information are already active by this stage. By 5 dpf, visual stimuli also activated climbing fibers, increasing the frequency of complex spiking. Our results indicate that the electrical properties of zebrafish and mammalian Purkinje cells are highly conserved and suggest that the same ion channels, Nav1.6 and Kv3.3, underlie spontaneous pacemaking activity. Interestingly, functional development of the cerebellum is temporally correlated with the emergence of complex, visually-guided behaviors such as prey capture. Because of the rapid formation of an electrically-active cerebellum, optical transparency, and ease of genetic manipulation, the zebrafish has great potential for functionally mapping cerebellar afferent and efferent pathways and for investigating cerebellar control of motor behavior.

  7. Inflammatory infiltrates and complete absence of Purkinje cells in anti-Yo-associated paraneoplastic cerebellar degeneration.

    PubMed

    Verschuuren, J; Chuang, L; Rosenblum, M K; Lieberman, F; Pryor, A; Posner, J B; Dalmau, J

    1996-01-01

    We studied the nervous systems and tumors of two patients with anti-Yo-associated paraneoplastic cerebellar degeneration (PCD). In both patients the underlying tumor was an ovarian adenocarcinoma that expressed Yo antigens and contained extensive infiltrates of lymphocytes and plasma cells. The major central nervous system findings were a complete loss of cerebellar Purkinje cells with Bergmann astrogliosis. One patient had inflammatory infiltrates in the medulla and pons, and moderate axonal loss and demyelination involving the spinal cord. No inflammatory infiltrates were identified in the cerebrum, cerebellum or brain-stem of the other patient. Using quantitative Western blot analysis, deposits of anti-Yo IgG could not be demonstrated in the nervous system, possibly as a result of the loss of cells expressing Yo antigens. The detection of the anti-Yo antibody as a common marker of PCD in one patient with inflammatory infiltrates and another without infiltrates suggests that some PCD pathologically classified as "non-inflammatory" may represent a final burn-out stage of a cellular immune-mediated disorder. Our findings indicate that Purkinje cells are the main, but not necessarily the exclusive, targets of this disorder.

  8. Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development.

    PubMed

    Florio, Marta; Leto, Ketty; Muzio, Luca; Tinterri, Andrea; Badaloni, Aurora; Croci, Laura; Zordan, Paola; Barili, Valeria; Albieri, Ilaria; Guillemot, François; Rossi, Ferdinando; Consalez, G Giacomo

    2012-07-01

    By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.

  9. Cellular localization of cerebellar muscarinic receptors: an autoradiographic analysis of weaver, reeler, Purkinje cell degeneration and staggerer mice

    SciTech Connect

    Neustadt, A.; Frostholm, A.; Rotter, A.

    1988-02-01

    Light microscopic autoradiography of (/sup 3/H)quinuclidinyl benzilate binding sites was used to study the distribution of muscarinic cholinergic receptors in mouse mutants which have abnormalities affecting specific cerebellar cell types. In the normal C57BL/6J mouse, binding sites were distributed throughout the cerebellar cortex, with the highest levels in the granule cell layer and deep cerebellar nuclei. Normal binding site density was observed in the cerebellum of the weaver mutant in which the majority of granule cells had degenerated. The density of (/sup 3/H)quinuclidinyl benzilate binding sites was elevated in the cortex of the reeler, despite a reduction in the number of granule cells. The concentration of binding sites was also high over the Purkinje cell masses where granule cells were largely absent. No significant reduction in cortical (/sup 3/H)quinuclidinyl benzilate binding site density was detected in the Purkinje cell degeneration mutant, in which essentially all Purkinje cells had degenerated. In contrast, receptor binding in the deep cerebellar nuclei of this mutant was significantly increased. A substantial increase in labeling was observed in the cortex and deep nuclei of the staggerer cerebellum in which a large fraction of Golgi II cells, Purkinje cells, granule cells and mossy fibers have degenerated. We discuss the possibility that the persistence of (/sup 3/H)quinuclidinyl benzilate binding sites in all four mutants may imply a non-neuronal localization for a large proportion of muscarinic receptors in the mouse cerebellar cortex.

  10. 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.

  11. Parallel fiber to Purkinje cell synaptic impairment in a mouse model of spinocerebellar ataxia type 27

    PubMed Central

    Tempia, Filippo; Hoxha, Eriola; Negro, Giulia; Alshammari, Musaad A.; Alshammari, Tahani K.; Panova-Elektronova, Neli; Laezza, Fernanda

    2015-01-01

    Genetically inherited mutations in the fibroblast growth factor 14 (FGF14) gene lead to spinocerebellar ataxia type 27 (SCA27), an autosomal dominant disorder characterized by heterogeneous motor and cognitive impairments. Consistently, genetic deletion of Fgf14 in Fgf14−/− mice recapitulates salient features of the SCA27 human disease. In vitro molecular studies in cultured neurons indicate that the FGF14F145S SCA27 allele acts as a dominant negative mutant suppressing the FGF14 wild type function and resulting in inhibition of voltage-gated Na+ and Ca2+ channels. To gain insights in the cerebellar deficits in the animal model of the human disease, we applied whole-cell voltage-clamp in the acute cerebellar slice preparation to examine the properties of parallel fibers (PF) to Purkinje neuron synapses in Fgf14−/− mice and wild type littermates. We found that the AMPA receptor-mediated excitatory postsynaptic currents evoked by PF stimulation (PF-EPSCs) were significantly reduced in Fgf14−/− animals, while short-term plasticity, measured as paired-pulse facilitation (PPF), was enhanced. Measuring Sr2+-induced release of quanta from stimulated synapses, we found that the size of the PF-EPSCs was unchanged, ruling out a postsynaptic deficit. This phenotype was corroborated by decreased expression of VGLUT1, a specific presynaptic marker at PF-Purkinje neuron synapses. We next examined the mGluR1 receptor-induced response (mGluR1-EPSC) that under normal conditions requires a gradual build-up of glutamate concentration in the synaptic cleft, and found no changes in these responses in Fgf14−/− mice. These results provide evidence of a critical role of FGF14 in maintaining presynaptic function at PF-Purkinje neuron synapses highlighting critical target mechanisms to recapitulate the complexity of the SCA27 disease. PMID:26089778

  12. Fluoro-jade identification of cerebellar granule cell and purkinje cell death in the alpha1A calcium ion channel mutant mouse, leaner.

    PubMed

    Frank, T C; Nunley, M C; Sons, H D; Ramon, R; Abbott, L C

    2003-01-01

    Cell death is a critical component of normal nervous system development; too little or too much results in abnormal development and function of the nervous system. The leaner mouse exhibits excessive, abnormal cerebellar granule cell and Purkinje cell death during postnatal development, which is a consequence of a mutated calcium ion channel subunit, alpha(1A). Previous studies have shown that leaner cerebellar Purkinje cells die in a specific pattern that appears to be influenced by functional and anatomical boundaries of the cerebellum. However, the mechanism of Purkinje cell death and the specific timing of the spatial pattern of cell death remain unclear. By double labeling both leaner and wild-type cerebella with Fluoro-Jade and terminal deoxynucleotide transferase-mediated, deoxyuridine triphosphate nick-end labeling or Fluoro-Jade and tyrosine hydroxylase immunohistochemistry we demonstrated that the relatively new stain, Fluoro-Jade, will label neurons that are dying secondary to a genetic mutation. Then, by staining leaner and wild-type cerebella between postnatal days 20 and 80 with Fluoro-Jade, we were able to show that Purkinje cell death begins at approximately postnatal day 25, peaks in the vermis about postnatal day 40 and in the hemispheres at postnatal day 50 and persists at a low level at postnatal day 80. In addition, we showed that there is a significant difference in the amount of cerebellar Purkinje cell death between rostral and caudal divisions of the leaner cerebellum, and that there is little to no Purkinje cell death in the wild type cerebellum at the ages we examined. This is the first report of the use of Fluoro-Jade to identify dying neurons in a genetic model for neuronal cell death. By using Fluoro-Jade, we have specifically defined the temporospatial pattern of postnatal Purkinje cell death in the leaner mouse. This information can be used to gain insight into the dynamic mechanisms controlling Purkinje cell death in the leaner

  13. Simulated Responses of Cerebellar Purkinje Cells are Independent of the Dendritic Location of Granule Cell Synaptic Inputs

    NASA Astrophysics Data System (ADS)

    de Schutter, Erik; Bower, James M.

    1994-05-01

    Cerebellar Purkinje cell responses to granule cell synaptic inputs were examined with a computer model including active dendritic conductances. Dendritic P-type Ca2+ channels amplified postsynaptic responses when the model was firing at a physiological rate. Small synchronous excitatory inputs applied distally on the large dendritic tree resulted in somatic responses of similar size to those generated by more proximal inputs. In contrast, in a passive model the somatic postsynaptic potentials to distal inputs were 76% smaller. The model predicts that the somatic firing response of Purkinje cells is relatively insensitive to the exact dendritic location of synaptic inputs. We describe a mechanism of Ca2+-mediated synaptic amplification, based on the subspiking threshold recruitment of P-type Ca2+ channels in the dendritic branches surrounding the input site.

  14. Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice

    PubMed Central

    Borghesani, Paul R.; Alt, Frederick W.; Bottaro, Andrea; Davidson, Laurie; Aksoy, Saime; Rathbun, Gary A.; Roberts, Thomas M.; Swat, Wojciech; Segal, Rosalind A.; Gu, Yansong

    2000-01-01

    Motor incoordination, immune deficiencies, and an increased risk of cancer are the characteristic features of the hereditary disease ataxia–telangiectasia (A-T), which is caused by mutations in the ATM gene. Through gene targeting, we have generated a line of Atm mutant mice, Atmy/y mice. In contrast to other Atm mutant mice, Atmy/y mice show a lower incidence of thymic lymphoma and survive beyond a few months of age. Atmy/y mice exhibit deficits in motor learning indicative of cerebellar dysfunction. Even though we found no gross cerebellar degeneration in older Atmy/y animals, ectopic and abnormally differentiated Purkinje cells were apparent in mutant mice of all ages. These findings establish that some neuropathological abnormalities seen in A-T patients also are present in Atm mutant mice. In addition, we report a previously unrecognized effect of Atm deficiency on development or maintenance of CD4+8+ thymocytes. We discuss these findings in the context of the hypothesis that abnormal development of Purkinje cells and lymphocytes contributes to the pathogenesis of A-T. PMID:10716718

  15. [Dynamics of the activity of cerebellar Purkinje cells induced by changes in the duration of complex spikes].

    PubMed

    Podladchikova, L N; Bondar', G G; Ivlev, S A; Tikidzhi-Khambur'ian, R A; Dunin-Barkovskiĭ, V L

    2008-01-01

    The relationship between complex and simple spikes of Purkinje cells from vermis cerebelli of guinea pigs has been investigated. The ratio of complex spikes innervated by the processes of one and the same liana-like fiber ("twins cells") has also been studied. Three types of complex spikes in each Purkinje cell from vermis cerebelli of guinea pigs (n = 44) have been differentiated, which differ in duration. It was found that long (10.28 +/- 0.27 ms) complex spikes in all cells lead to a more pronounced inhibition of simple spikes than complex spikes of short duration (6.08 +/- 0.25 ms). It was shown that the dynamics of duration of complex spikes coordinates with changes in the activity of some Purkinje cells and their local groups: (a) complex spikes generated before the onset of pauses of simple spikes are longer than complex spikes generated before the termination of pauses; (b) in "twins cells" innervated by one liana-like fiber, the properties of complex spikes change simultaneously; (c) The degree of synchronism of complex spikes in closely-spaced (to 150 microm) Purkinje cells receiving the inputs from different liana-like fibers increases with their duration. A possible functional role and the mechanisms of generation of complex spikes are discussed.

  16. Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction.

    PubMed

    Yip, Jane; Soghomonian, Jean-Jacques; Blatt, Gene J

    2008-02-15

    It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.

  17. Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions.

    PubMed

    Baader, S L; Sanlioglu, S; Berrebi, A S; Parker-Thornburg, J; Oberdick, J

    1998-03-01

    Members of the En and Wnt gene families seem to play a key role in the early specification of the brain territory that gives rise to the cerebellum, the midhindbrain junction. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, we expressed En-2 ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity to the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures that is highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions.

  18. Lack of neurogenesis in the adult rat cerebellum after Purkinje cell degeneration and growth factor infusion.

    PubMed

    Grimaldi, Piercesare; Rossi, Ferdinando

    2006-05-01

    Although constitutive neurogenesis exclusively occurs in restricted regions of the adult mammalian brain, resident progenitors can be isolated from many different CNS sites, and neuronal neogeneration can be stimulated in vivo by injury or infusion of growth factors. To ask whether latent compensatory mechanisms, which may be exploited to promote repair processes, are present throughout the CNS, we examined the neurogenic potentialities of the adult rat cerebellum in normal conditions, following injury, and after infusion of growth factors. Degeneration of Purkinje cells was induced by intracerebroventricular administration of the toxin saporin, conjugated to anti-p75 antibodies. In addition, epidermal growth factor and basic fibroblast growth factor, or FGF8, were infused for 2 weeks to either intact or injured animals. In all conditions, proliferating cells were identified from bromodeoxyuridine (BrdU) incorporation. In the unmanipulated cerebellum there were rare dividing cells, mainly represented by NG2-positive presumptive oligodendrocyte precursors. Mitotic activity was strongly enhanced in cortical areas with Purkinje cell degeneration, being mostly sustained by microglia, plus minor fractions of NG2-expressing cells, astrocytes and oligodendrocytes. In contrast, growth factor infusion had a weak effect on both intact and injured cerebella. In all experimental conditions, we never found any BrdU-positive cells coexpressing distinctive markers for immature or differentiated cerebellar neurons. Therefore, although some progenitor cells reside in the adult cerebellum, the local environment, either intact or injured, does not provide efficient cues to direct their differentiation towards neuronal phenotypes. In addition, neurogenic potentialities cannot be induced or boosted by the application of growth factors which are effective in other CNS regions.

  19. RORα Regulates Multiple Aspects of Dendrite Development in Cerebellar Purkinje Cells In Vivo.

    PubMed

    Takeo, Yukari H; Kakegawa, Wataru; Miura, Eriko; Yuzaki, Michisuke

    2015-09-09

    The establishment of cell-type-specific dendritic arbors is fundamental for proper neural circuit formation. Here, using temporal- and cell-specific knock-down, knock-out, and overexpression approaches, we show that multiple aspects of the dendritic organization of cerebellar Purkinje cells (PCs) are controlled by a single transcriptional factor, retinoic acid-related orphan receptor-alpha (RORα), a gene defective in staggerer mutant mice. As reported earlier, RORα was required for regression of primitive dendrites before postnatal day 4 (P4). RORα was also necessary for PCs to form a single Purkinje layer from P0 to P4. The knock-down of RORα from P4 impaired the elimination of perisomatic dendrites and maturation of single stem dendrites in PCs at P8. Filopodia and spines were also absent in these PCs. The knock-down of RORα from P8 impaired the formation and maintenance of terminal dendritic branches of PCs at P14. Finally, even after dendrite formation was completed at P21, RORα was required for PCs to maintain dendritic complexity and functional synapses, but their mature innervation pattern by single climbing fibers was unaffected. Interestingly, overexpression of RORα in PCs at various developmental stages did not facilitate dendrite development, but had specific detrimental effects on PCs. Because RORα deficiency during development is closely related to the severity of spinocerebellar ataxia type 1, delineating the specific roles of RORα in PCs in vivo at different time windows during development and throughout adulthood would facilitate our understanding of the pathogenesis of cerebellar disorders. Significance statement: The genetic programs by which each neuron subtype develops and maintains dendritic arbors have remained largely unclear. This is partly because dendrite development is modulated dynamically by neuronal activities and interactions with local environmental cues in vivo. In addition, dendrites are formed and maintained by the

  20. TrkB is necessary for pruning at the climbing fibre–Purkinje cell synapse in the developing murine cerebellum

    PubMed Central

    Johnson, Erin M; Craig, Ethan T; Yeh, Hermes H

    2007-01-01

    TrkB, the cognate receptor for brain-derived neurotrophic factor and neurotrophin-4, has been implicated in regulating synapse formation in the central nervous system. Here we asked whether TrkB plays a role in the maturation of the climbing fibre–Purkinje cell (CF–PC) synapse. In rodent cerebellum, Purkinje cells are initially innervated by multiple climbing fibres that are subsequently culled to assume the mature mono-innervated state, and whose contacts translocate from the soma to the dendrites. By employing transgenic mice hypomorphic or null for TrkB expression, our results indicated that perturbation of TrkB in the immature cerebellum resulted in ataxia, that Purkinje cells remained multiply innervated by climbing fibres beyond the normal developmental time frame, and that synaptic transmission at the parallel fibre–Purkinje cell synapse remained functionally unaltered. Mechanistically, we present evidence that attributes the persistence of multiple climbing fibre innervation to an obscured discrimination of relative strengths among competing climbing fibres. Soma-to-dendrite translocation of climbing fibre terminals was unaffected. Thus, TrkB regulates pruning but not translocation of nascent CF–PC synaptic contacts. PMID:17463037

  1. Kv3.3 channels at the Purkinje cell soma are necessary for generation of the classical complex spike waveform.

    PubMed

    Zagha, Edward; Lang, Eric J; Rudy, Bernardo

    2008-02-06

    Voltage-gated potassium channel subunit Kv3.3 is prominently expressed in cerebellar Purkinje cells and is known to be important for cerebellar function, as human and mouse movement disorders result from mutations in Kv3.3. To understand these behavioral deficits, it is necessary to know the role of Kv3.3 channels on the physiological responses of Purkinje cells. We studied the function of Kv3.3 channels in regulating the synaptically evoked Purkinje cell complex spike, the massive postsynaptic response to the activation of climbing fiber afferents, believed to be fundamental to cerebellar physiology. Acute slice recordings revealed that Kv3.3 channels are required for generation of the repetitive spikelets of the complex spike. We found that spikelet expression is regulated by somatic, and not by dendritic, Kv3 activity, which is consistent with dual somatic-dendritic recordings that demonstrate spikelet generation at axosomatic membranes. Simulations of Purkinje cell Na+ currents show that the unique electrical properties of Kv3 and resurgent Na+ channels are coordinated to limit accumulation of Na+ channel inactivation and enable rapid, repetitive firing. We additionally show that Kv3.3 knock-out mice produce altered complex spikes in vitro and in vivo, which is likely a cellular substrate of the cerebellar phenotypes observed in these mice. This characterization presents new tools to study complex spike function, cerebellar signaling, and Kv3.3-dependent human and mouse phenotypes.

  2. 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.

  3. Tetraethylammonium and 4-aminopyridine block calcium-dependent chloride current in rat cerebellum Purkinje cells.

    PubMed

    Zamoyski, V L; Vikhareva, E A; Grigoriev, V V; Bachurin, S O

    2016-09-01

    Using patch-clamp method (whole cell configuration), it was shown that tetraethylammonium (TEA) and 4-aminopyridine (4-AP) block calcium-dependent chloride currents in the membrane of freshly isolated cerebellar Purkinje cells of rats (12-15 days). In the concentration range studied (50 μM-10 mM TEA and 100 μM-1 mM 4-AP), both compounds blocked the chloride current at IC50 130 μM for TEA and 110 μM for 4-AP. TEA blockade was reversible after washing. The effect of 4-AP at concentrations greater than 100 μM was irreversible: both outward and inward chloride currents were blocked even after the removal of 4-AP from the incubation medium.

  4. Altered Purkinje cell responses and calmodulin expression in the spontaneously ataxic mouse, Pogo.

    PubMed

    Lee, Kwan Young; Kim, Jin Seong; Kim, Se Hoon; Park, Hyung Seo; Jeong, Young-Gil; Lee, Nam-Seob; Kim, Dong Kwan

    2011-04-01

    Ataxia is often associated with altered cerebellar motor control, a process in which Purkinje cells (PCs) play a principal role. Pogo mice display severe motor deficits characterized by an ataxic gait accompanying hindlimb hyperextension. Here, using whole-cell patch-clamp recordings, we show that parallel fiber (PF)-excitatory post-synaptic currents (PF-EPSCs) are reduced, paired-pulse facilitation (PPF) is increased and PF-PC long-term depression (LTD) is impaired in Pogo mice; in contrast, climbing-fiber EPSCs are preserved. In control mice, treatment with the calmodulin antagonist calmidazolium (5 μm) impaired PPF and LTD. Notably, cerebellar calmodulin expression was significantly reduced in Pogo mice compared with control mice. Control PCs predominantly exhibited a tonic firing pattern, whereas the firing pattern in Pogo PCs was mainly a complex burst type. These results implicate alterations in PC responses and calmodulin content in the abnormal cerebellar function of Pogo mice.

  5. Model of very fast (>75 Hz) network oscillations generated by electrical coupling between the proximal axons of cerebellar Purkinje cells

    PubMed Central

    Traub, Roger D; Middleton, Steven J; Knöpfel, Thomas; Whittington, Miles A

    2009-01-01

    Very fast oscillations (VFO, >75 Hz) occur transiently in vivo, in the cerebellum of mice genetically modified to model Angelman syndrome, and in a mouse model of fetal alcohol syndrome. We recently reported VFO in slices of mouse cerebellar cortex (Crus I and II of ansiform and paramedian lobules), either in association with gamma oscillations (~40 Hz, evoked by nicotine), or in isolation (evoked by nicotine in combination with GABAA receptor blockade). The experimental data suggest a role for electrical coupling between Purkinje cells (blockade of VFO by drugs reducing gap junction conductance, and spikelets in some Purkinje cells); and the data suggest the specific involvement of Purkinje cell axons (because of field oscillation maxima in the granular layer). We show here that a detailed network model (1,000 multicompartment Purkinje cells) replicates the experimental data, when gap junctions are located on the proximal axons of Purkinje cells, provided sufficient spontaneous firing is present. Unlike other VFO models, most somatic spikelets do not correspond to axonal spikes in the parent axon, but reflect spikes in electrically coupled axons. The model predicts gating of VFO frequency by gNa inactivation, and experiments prolonging this inactivation time constant, with β-pompilidotoxin, are consistent with this prediction. The model also predicts that cerebellar VFO can be explained as an electrically coupled system of axons which are not intrinsic oscillators: the electrically uncoupled cells do not individually oscillate (in the model), and axonal firing rates are much lower in the uncoupled state than in the coupled state. PMID:18973579

  6. 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

  7. Purkinje cells express Angiotensin II AT(2) receptors at different developmental stages.

    PubMed

    Arce, María E; Sánchez, Susana I; Aguilera, Francisco López; Seguin, Leonardo R; Seltzer, Alicia M; Ciuffo, Gladys M

    2011-02-01

    Angiotensin II (Ang II) binds and activates two major receptors subtypes, namely AT(1) and AT(2). In the fetus, AT(2) receptors predominate in all tissues and decline shortly after birth, being restricted to a few organs including brain. Interpretation of the function of Ang II in the cerebellum requires a thorough understanding of the localization of Ang II receptors. The aim of the present paper is to evaluate the localization of Ang II AT(2) receptors in the Purkinje cell (PC) layer during development. By binding autoradiography, a clear complementary pattern of AT(1) and AT(2) binding labeled by [(125)I] Ang II was observed in young rats within the cerebellar cortex. This pattern was present at the stages P8 and P15, but not at P30 and P60, where AT(2) binding appears low and superimposed with AT(1) binding. We demonstrate that AT(2) antibodies recognized postmitotic Purkinje cells, labeling the somata of these cells at all the stages studied, from P8 to P60, suggesting that PCs express these receptors from early stages of development until adulthood. In P8 and P15 animals, we observed a clear correspondence between immunolabeling and the well-defined layer observed by binding autoradiography. Confocal analysis allowed us to discard the co-localization of AT(2) receptors with glial fibrillary acidic protein (GFAP), a glial marker. Double immunolabeling allowed us to demonstrate the co-localization of Ang II AT(2) receptors with zebrin II, a specific PC marker. Since PCs are the sole output signal from the cerebellar cortex and considering the role of cerebellum in movement control, the specific receptor localization suggests a potential role for Ang II AT(2) receptors in the cerebellar function.

  8. AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease.

    PubMed

    Xie, Chang; Gong, Xue-Min; Luo, Jie; Li, Bo-Liang; Song, Bao-Liang

    2017-03-01

    Niemann-Pick type C (NPC) disease is a fatal inherited neurodegenerative disorder caused by loss-of-function mutations in the NPC1 or NPC2 gene. There is no effective way to treat NPC disease. In this study, we used adeno-associated virus (AAV) serotype 9 (AAV9) to deliver a functional NPC1 gene systemically into NPC1(-/-) mice at postnatal day 4. One single AAV9-NPC1 injection resulted in robust NPC1 expression in various tissues, including brain, heart, and lung. Strikingly, AAV9-mediated NPC1 delivery significantly promoted Purkinje cell survival, restored locomotor activity and coordination, and increased the lifespan of NPC1(-/-) mice. Our work suggests that AAV-based gene therapy is a promising means to treat NPC disease.

  9. PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

    PubMed

    Kim, Jun-Dal; Park, Kyung-Eui; Ishida, Junji; Kako, Koichiro; Hamada, Juri; Kani, Shuichi; Takeuchi, Miki; Namiki, Kana; Fukui, Hajime; Fukuhara, Shigetomo; Hibi, Masahiko; Kobayashi, Makoto; Kanaho, Yasunori; Kasuya, Yoshitoshi; Mochizuki, Naoki; Fukamizu, Akiyoshi

    2015-12-01

    The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

  10. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    PubMed Central

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  11. 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.

  12. [Electron microscopy analysis of the structural elements of the vestibular input to nodulus Purkinje's cells in rats exposed to a 9-day space flight].

    PubMed

    Krasnov, I B

    2008-01-01

    Electron microscopy was used to study structural elements of the vestibular afferent input to the cerebellar nodulus Purkinje's cells--terminals of mossy fibers and granular cells in the granular layer and parallel fibers and Purkinje's cell dendrites in the molecular layer in rats decapitated in 2-3 hours and 9 days after the 9-day space flight aboard NASA shuttle Columbia (STS 40, SLS-1 mission). Analysis of the revealed ultrastructural changes on the base of morphofunctional correlations leads to the following conclusions: 1) space flight induced a prolonged reduction in vestibular input to most of the mossy fiber terminals and nodulus Purkinje's cells; 2) within the initial hours of recovery the vestibular input to a part of mossy fiber terminals and granular cells was increasing due to elevation of the sensitivity of vestibular receptors in microgravity; 3) regain of the vestibular input to Purkinje's cells after space flight is hampered by structural, as a result of microgravity effects, and also functional, developing shortly after space flight, impediments, and 4) in 9 d after landing the vestibular input to Purkinje's cells was almost normal. The observed reduction in the vestibular input to the nodulus Purkinje's cells during and after the spaceflight microgravity is presumably the key to the mechanism altering the velocity storage in mammals in microgravity and on return from space flight.

  13. LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum

    PubMed Central

    Zhao, Yangu; Kwan, Kin-Ming; Mailloux, Christina M.; Lee, Woon-Kyu; Grinberg, Alexander; Wurst, Wolfgang; Behringer, Richard R.; Westphal, Heiner

    2007-01-01

    Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum. PMID:17664423

  14. Growth-associated protein GAP-43 and L1 act synergistically to promote regenerative growth of Purkinje cell axons in vivo.

    PubMed

    Zhang, Yi; Bo, Xuenong; Schoepfer, Ralf; Holtmaat, Anthony J D G; Verhaagen, Joost; Emson, Piers C; Lieberman, A Robert; Anderson, Patrick N

    2005-10-11

    Neuronal expression of growth-associated protein 43 (GAP-43) and the cell adhesion molecule L1 has been correlated with CNS axonal growth and regeneration, but it is not known whether expression of these molecules is necessary for axonal regeneration to occur. We have taken advantage of the fact that Purkinje cells do not express GAP-43 or L1 in adult mammals or regenerate axons into peripheral nerve grafts to test the importance of these molecules for axonal regeneration in vivo. Transgenic mice were generated in which Purkinje cells constitutively express L1 or both L1 and GAP-43 under the Purkinje cell-specific L7 promoter, and regeneration of Purkinje cell axons into peripheral nerve grafts implanted into the cerebellum was examined. Purkinje cells expressing GAP-43 or L1 showed minor enhancement of axonal sprouting. Purkinje cells expressing both GAP-43 and L1 showed more extensive axonal sprouting and axonal growth into the proximal portion of the graft. When a predegenerated nerve graft was implanted into double-transgenic mice, penetration of the graft by Purkinje cell axonal sprouts was strongly enhanced, and some axons grew along the entire intracerebral length of the graft (2.5-3.0 mm) and persisted for several months. The results demonstrate that GAP-43 and L1 coexpressed in Purkinje cells can act synergistically to switch these regeneration-incompetent CNS neurons into a regeneration-competent phenotype and show that coexpression of these molecules is a key regulator of the regenerative ability of intrinsic CNS neurons in vivo.

  15. 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.

  16. Do the Purkinje cells have a special type of oligodendrocyte as satellites?

    PubMed Central

    Monteiro, R A

    1983-01-01

    Two types of oligodendrocytes considered to be a constant feature in the cerebellar cortex of the rat are described. One cell type (I) exhibits rounded or elliptical nuclei, whereas the other type (II) presents more irregular nuclear and cellular contours and wider perinuclear cisternae. The latter cell type shows a more electron-dense cytoplasm with more heavily clumped heterochromatin, contrasting strongly with the euchromatin; also long and parallel cisternae of rough endoplasmic reticulum are more frequent. The percentages of both types of oligodendrocytes in relation to the total population of common glial cell types were calculated in the cortical layers and at several levels in these layers. The distribution of oligodendrocytes in the associated white matter was also carried out for purposes of comparison. The results provide evidence the the Purkinje cells may have a special kind of oligodendrocyte (Type II) as satellites. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:6630036

  17. Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar Purkinje cells in vitro and in vivo.

    PubMed

    Ito-Ishida, Aya; Miura, Eriko; Emi, Kyoichi; Matsuda, Keiko; Iijima, Takatoshi; Kondo, Tetsuro; Kohda, Kazuhisa; Watanabe, Masahiko; Yuzaki, Michisuke

    2008-06-04

    Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders.

  18. Ethanol affects NMDA receptor signaling at climbing fiber-Purkinje cell synapses in mice and impairs cerebellar LTD.

    PubMed

    He, Qionger; Titley, Heather; Grasselli, Giorgio; Piochon, Claire; Hansel, Christian

    2013-03-01

    Ethanol profoundly influences cerebellar circuit function and motor control. It has recently been demonstrated that functional N-methyl-(D)-aspartate (NMDA) receptors are postsynaptically expressed at climbing fiber (CF) to Purkinje cell synapses in the adult cerebellum. Using whole cell patch-clamp recordings from mouse cerebellar slices, we examined whether ethanol can affect NMDA receptor signaling in mature Purkinje cells. NMDA receptor-mediated currents were isolated by bath application of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzol[f]quinoxaline (NBQX). The remaining (D)-2-amino-5-phosphonovaleric acid ((D)-APV)-sensitive current was reduced by ethanol at concentrations as low as 10 mM. At a concentration of 50 mM ethanol, the blockade of (D)-APV-sensitive CF-excitatory postsynaptic currents was significantly stronger. Ethanol also altered the waveform of CF-evoked complex spikes by reducing the afterdepolarization. This effect was not seen when NMDA receptors were blocked by (D)-APV before ethanol wash-in. In contrast to CF synaptic transmission, parallel fiber (PF) synaptic inputs were not affected by ethanol. Finally, ethanol (10 mM) impaired long-term depression (LTD) at PF to Purkinje cell synapses as induced under control conditions by paired PF and CF activity. However, LTD induced by pairing PF stimulation with depolarizing voltage steps (substituting for CF activation) was not blocked by ethanol. These observations suggest that the sensitivity of cerebellar circuit function and plasticity to low concentrations of ethanol may be caused by an ethanol-mediated impairment of NMDA receptor signaling at CF synapses onto cerebellar Purkinje cells.

  19. 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.

  20. Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum

    PubMed Central

    Ju, Jun; Liu, Qian; Zhang, Yang; Liu, Yuanxiu; Jiang, Mei; Zhang, Liguo; He, Xuelian; Peng, Chenchen; Zheng, Tao; Lu, Q. Richard; Li, Hedong

    2016-01-01

    The oligodendrocyte transcription factor Olig2 plays a crucial role in the neurogenesis of both spinal cord and brain. In the cerebellum, deletion of both Olig2 and Olig1 results in impaired genesis of Purkinje cells (PCs) and Pax2+ interneurons. Here, we perform an independent study to show that Olig2 protein is transiently expressed in the cerebellar ventricular zone (VZ) during a period when PCs are specified. Further analyses demonstrate that Olig2 is expressed in both cerebellar VZ progenitors and early-born neurons. In addition, unlike in the ganglionic eminence of the embryonic forebrain where Olig2 is mostly expressed in proliferating progenitors, Olig2+ cells in the cerebellar VZ are in the process of leaving the cell cycle and differentiating into postmitotic neurons. Functionally, deletion of Olig2 alone results in a preferential reduction of PCs in the cerebellum, which is likely mediated by decreased neuronal generation from their cerebellar VZ progenitors. Furthermore, our long-term lineage tracing experiments show that cerebellar Olig gene-expressing progenitors produce PCs but rarely Pax2+ interneurons in the developing cerebellum, which opposes the “temporal identity transition” model of the cerebellar VZ progenitors stating that majority of Pax2+ interneuron progenitors are transitioned from Olig2+ PC progenitors. PMID:27469598

  1. Morphological Plasticity of Emerging Purkinje Cells in Response to Exogenous VEGF

    PubMed Central

    Herrfurth, Leonard; Theis, Verena; Matschke, Veronika; May, Caroline; Marcus, Katrin; Theiss, Carsten

    2017-01-01

    Vascular endothelial growth factor (VEGF) is well known as the growth factor with wide-ranging functions even in the central nervous system (CNS). Presently, most attention is given to the investigation of its role in neuronal protection, growth and maturation processes, whereby most effects are mediated through VEGF receptor 2 (VEGFR-2). The purpose of our current study is to provide new insights into the impact of VEGF on immature and mature Purkinje cells (PCs) in accordance with maturity and related receptor expression. Therefore, to expand our knowledge of VEGF effects in PCs development and associated VEGFR-2 expression, we used cultivated organotypic cerebellar slice cultures in immunohistochemical or microinjection studies, followed by confocal laser scanning microscopy (CLSM) and morphometric analysis. Additionally, we incorporated in our study the method of laser microdissection, followed by quantitative polymerase chain reaction (qPCR). For the first time we could show the age-dependent VEGF sensitivity of PCs with the largest promoting effects being on dendritic length and cell soma size in neonatal and juvenile stages. Once mature, PCs were no longer susceptible to VEGF stimulation. Analysis of VEGFR-2 expression revealed its presence in PCs throughout development, which underlined its mediating functions in neuronal cells. PMID:28194096

  2. Localization of Neurensin1 in cerebellar Purkinje cells of the developing chick and its possible function in dendrite formation.

    PubMed

    Kiyonaga-Endou, Keiko; Oshima, Manabu; Sugimoto, Kazuya; Thomas, Mervyn; Taketani, Shigeru; Araki, Masasuke

    2016-03-15

    Neurensin1 (Nrsn1) gene, highly specific to neurons, has been considered to play a role in neurite growth during neuronal development and regeneration in mice. Intense expression of Nrsn1 was found particularly in projecting neurons like retinal ganglion cells and spinal motor neurons, suggesting that Neurensin1 is needed for active neurite growth. In the present study we cloned chick Nrsn1 gene and produced an antibody against cNrsn1 to examine Nrsn1 localization in the chick brain, since the chick is a suitable animal model for the study of developmental neurobiology. We found that there are neurons intensely stained for Nrsn1 antibody localized in the optic tectum, the cerebellum and the brain stem. These neurons are large in size and considered to be projecting neurons. In the cerebellum, Purkinje cells are the only one type of neurons stained for Nrsn1. During Purkinje cell development the arborized dendrites and axons become intensely stained at stages E17-18. A siRNA gene knock down was applied to the cultured embryonic cerebellar tissues and the result showed that Nrsn1 has an important role in dendrite formation of Purkinje cells. These findings suggest that Neurensin1 is also involved in neural development in the chick brain and that the embryonic chick brain is a good model to disclose the molecular and physiological functions of Nrsn1.

  3. Purkinje cell number decreases in the adult female rat cerebellum following exposure to 900 MHz electromagnetic field.

    PubMed

    Sonmez, Osman Fikret; Odaci, Ersan; Bas, Orhan; Kaplan, Süleyman

    2010-10-14

    The biological effects of electromagnetic field (EMF) exposure from mobile phones have growing concern among scientists since there are some reports showing increased risk for human health, especially in the use of mobile phones for a long duration. In the presented study, the effects on the number of Purkinje cells in the cerebellum of 16-week (16 weeks) old female rats were investigated following exposure to 900 MHz EMF. Three groups of rats, a control group (CG), sham exposed group (SG) and an electromagnetic field exposed group (EMFG) were used in this study. While EMFG group rats were exposed to 900 MHz EMF (1h/day for 28 days) in an exposure tube, SG was placed in the exposure tube but not exposed to EMF (1h/day for 28 days). The specific energy absorption rate (SAR) varied between 0.016 (whole body) and 2 W/kg (locally in the head). The CG was not placed into the exposure tube nor was it exposed to EMF during the study period. At the end of the experiment, all of the female rats were sacrificed and the number of Purkinje cells was estimated using a stereological counting technique. Histopathological evaluations were also done on sections of the cerebellum. Results showed that the total number of Purkinje cells in the cerebellum of the EMFG was significantly lower than those of CG (p<0.004) and SG (p<0.002). In addition, there was no significant difference at the 0.05 level between the rats' body and brain weights in the EMFG and CG or SG. Therefore, it is suggested that long duration exposure to 900 MHz EMF leads to decreases of Purkinje cell numbers in the female rat cerebellum.

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

    PubMed Central

    Alviña, Karina

    2014-01-01

    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. PMID:24790163

  5. Subcellular compartment-specific molecular diversity of pre- and postsynaptic GABAB-activated GIRK channels in Purkinje cells

    PubMed Central

    Fernández-Alacid, Laura; Aguado, Carolina; Ciruela, Francisco; Martín, Ricardo; Colón, José; Cabañero, María José; Gassmann, Martin; Watanabe, Masahiko; Shigemoto, Ryuichi; Wickman, Kevin; Bettler, Bernhard; Sánchez-Prieto, José; Luján, Rafael

    2009-01-01

    Activation of G protein-gated inwardly-rectifying K+ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABAB) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABAB receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABAB receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, postsynaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The postsynaptic association of GIRK subunits with GABAB receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At presynaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABAB receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABAB receptors. The association of GIRK channels and GABAB receptors with excitatory synapses at both post- and presynaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum. PMID:19558451

  6. 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

  7. Slow delayed rectifier current and repolarization in canine cardiac Purkinje cells.

    PubMed

    Han, W; Wang, Z; Nattel, S

    2001-03-01

    Although cardiac Purkinje cells (PCs) are believed to be the source of early afterdepolarizations generating ventricular tachyarrhythmias in long Q-T syndromes (LQTS), the ionic determinants of PC repolarization are incompletely known. To evaluate the role of the slow delayed rectifier current (I(Ks)) in PC repolarization, we studied PCs from canine ventricular false tendons with whole cell patch clamp (37 degrees C). Typical I(Ks) voltage- and time-dependent properties were noted. Isoproterenol enhanced I(Ks) in a concentration-dependent fashion (EC(50) approximately 30 nM), negatively shifted I(Ks) activation voltage dependence, and accelerated I(Ks) activation. Block of I(Ks) with 293B did not alter PC action potential duration (APD) in the absence of isoproterenol; however, in the presence of isoproterenol, 293B significantly prolonged APD. We conclude that, without beta-adrenergic stimulation, I(Ks) contributes little to PC repolarization; however, beta-adrenergic stimulation increases the contribution of I(Ks) by increasing current amplitude, accelerating I(Ks) activation, and shifting activation voltage toward the PC plateau voltage range. I(Ks) may therefore provide an important "braking" function to limit PC APD prolongation in the presence of beta-adrenergic stimulation.

  8. Autistic-Like Traits and Cerebellar Dysfunction in Purkinje Cell PTEN Knock-Out Mice.

    PubMed

    Cupolillo, Dario; Hoxha, Eriola; Faralli, Alessio; De Luca, Annarita; Rossi, Ferdinando; Tempia, Filippo; Carulli, Daniela

    2016-05-01

    Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits.

  9. The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

    PubMed Central

    Valkova, Christina; Liebmann, Lutz; Krämer, Andreas; Hübner, Christian A.; Kaether, Christoph

    2017-01-01

    Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs. PMID:28117367

  10. Treadmill exercise ameliorates motor dysfunction through inhibition of Purkinje cell loss in cerebellum of valproic acid-induced autistic rats

    PubMed Central

    Cho, Han-Sam; Kim, Tae-Woon; Ji, Eun-Sang; Park, Hye-Sang; Shin, Mal-Soon; Baek, Seung-Soo

    2016-01-01

    Autism is a complex developmental disorder with impairments in social interaction, communication, repetitive behavior and motor skills. Exercise enhances cognitive function, ameliorates motor dysfunction, and provides protective profits against neurodegeneration. In the present study, we evaluated the effect of treadmill exercise on the motor coordination and Purkinje cell loss in relation with reactive astrocytes and microglial activation in the cerebellum using valproic acid (VPA)-induced autism rat model. On the 12th day of pregnancy, the pregnant rats in the VPA-exposed group received intraperitoneal injections of 600-mg/kg VPA. After birth, the rat pups were divided into four groups: the control group, the exercise group, the VPA-treated group, the VPA-treated and exercise group. The rat pups in the exercise groups were forced to run on a treadmill for 30 min once a day, 5 times a week for 4 weeks. In the present results, motor balance and coordination was disturbed by induction of autism, in contrast, treadmill exercise alleviated motor dysfunction in the autistic rats. Purkinje cell loss, reactive astrocytes, and microglial activation were occurred by induction of autism, in contrast, treadmill exercise enhanced survival rate of Purkinje neurons through inhibition of reactive astrocytes and microglia in the autistic rats. The present study showed that exercise may provide a potential therapeutic strategy for the alleviation of motor dysfunction in autistic patients. PMID:27656625

  11. Paraneoplastic Optic Neuropathy Associated With Purkinje Cell Antibody-2 in a Patient With Small Cell Lung Cancer.

    PubMed

    Micieli, Jonathan A; Margolin, Edward A

    2017-03-01

    Paraneoplastic optic neuropathy (PON) is a rare cause of vision loss usually associated with small cell lung cancer. Patients with this condition usually test positive for anti-collapsin response mediating protein-5 (CRMP-5). We describe a case of a 57-year-old woman with bilateral vision loss with the characteristic features of CRMP-5 PON including bilateral optic disc edema and vitreous cells. However, she was negative for anti-CRMP-5 including a negative Western blot on two occasions, but positive for Purkinje Cell Antibody (PCA)-2. Although paraneoplastic antibodies are more predictive of an underlying cancer than a specific syndrome, previously PON has not been associated with PCA-2. Based on this observation, we recommend that the workup should include PCA-2 antibodies in patients who present with bilateral optic neuropathy and vitreous cells.

  12. Duration of Purkinje cell complex spikes increases with their firing frequency

    PubMed Central

    Warnaar, Pascal; Couto, Joao; Negrello, Mario; Junker, Marc; Smilgin, Aleksandra; Ignashchenkova, Alla; Giugliano, Michele; Thier, Peter; De Schutter, Erik

    2015-01-01

    Climbing fiber (CF) triggered complex spikes (CS) are massive depolarization bursts in the cerebellar Purkinje cell (PC), showing several high frequency spikelet components (±600 Hz). Since its early observations, the CS is known to vary in shape. In this study we describe CS waveforms, extracellularly recorded in awake primates (Macaca mulatta) performing saccades. Every PC analyzed showed a range of CS shapes with profoundly different duration and number of spikelets. The initial part of the CS was rather constant but the later part differed greatly, with a pronounced jitter of the last spikelets causing a large variation in total CS duration. Waveforms did not effect the following pause duration in the simple spike (SS) train, nor were SS firing rates predictive of the waveform shapes or vice versa. The waveforms did not differ between experimental conditions nor was there a preferred sequential order of CS shapes throughout the recordings. Instead, part of their variability, the timing jitter of the CS’s last spikelets, strongly correlated with interval length to the preceding CS: shorter CS intervals resulted in later appearance of the last spikelets in the CS burst, and vice versa. A similar phenomenon was observed in rat PCs recorded in vitro upon repeated extracellular stimulation of CFs at different frequencies in slice experiments. All together these results strongly suggest that the variability in the timing of the last spikelet is due to CS frequency dependent changes in PC excitability. PMID:25918500

  13. Localization of SK2 channels relative to excitatory synaptic sites in the mouse developing Purkinje cells

    PubMed Central

    Ballesteros-Merino, Carmen; Martínez-Hernández, José; Aguado, Carolina; Watanabe, Masahiko; Adelman, John P.; Luján, Rafael

    2014-01-01

    Small-conductance, Ca2+-activated K+ (SK) channels regulate neuronal excitability in a variety of ways. To understand their roles in different neuronal subtypes it is important to determine their precise subcellular distribution. Here, we used biochemical, light microscopy immunohistochemical and immunoelectron microscopy techniques, combined with quantitative approaches, to reveal the expression and subcellular localization patterns of SK2 in the developing cerebellum. Using western blots, the SK2 protein showed a progressive increase during postnatal development. At the light microscopic level, SK2 immunoreactivity was very prominent in the developing Purkinje cells (PC), particularly in the molecular layer (ML). Electron microscopy revealed that throughout development SK2 was mostly detected at the extrasynaptic and perisynaptic plasma membrane of dendritic shafts and dendritic spines of PCs. However, there was some localization at axon terminals as well. Quantitative analyses and 3D reconstructions further revealed a progressive developmental change of SK2 on the surface of PCs from dendritic shafts to dendritic spines. Together, these results indicate that SK2 channels undergo dynamic spatial regulation during cerebellar development, and this process is associated with the formation and maturation of excitatory synaptic contacts to PCs. PMID:25565979

  14. Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice

    PubMed Central

    Peter, Saša; ten Brinke, Michiel M.; Stedehouder, Jeffrey; Reinelt, Claudia M.; Wu, Bin; Zhou, Haibo; Zhou, Kuikui; Boele, Henk-Jan; Kushner, Steven A.; Lee, Min Goo; Schmeisser, Michael J.; Boeckers, Tobias M.; Schonewille, Martijn; Hoebeek, Freek E.; De Zeeuw, Chris I.

    2016-01-01

    Loss-of-function mutations in the gene encoding the postsynaptic scaffolding protein SHANK2 are a highly penetrant cause of autism spectrum disorders (ASD) involving cerebellum-related motor problems. Recent studies have implicated cerebellar pathology in the aetiology of ASD. Here we evaluate the possibility that cerebellar Purkinje cells (PCs) represent a critical locus of ASD-like pathophysiology in mice lacking Shank2. Absence of Shank2 impairs both PC intrinsic plasticity and induction of long-term potentiation at the parallel fibre to PC synapse. Moreover, inhibitory input onto PCs is significantly enhanced, most prominently in the posterior lobe where simple spike (SS) regularity is most affected. Using PC-specific Shank2 knockouts, we replicate alterations of SS regularity in vivo and establish cerebellar dependence of ASD-like behavioural phenotypes in motor learning and social interaction. These data highlight the importance of Shank2 for PC function, and support a model by which cerebellar pathology is prominent in certain forms of ASD. PMID:27581745

  15. Storage of Correlated Patterns in Standard and Bistable Purkinje Cell Models

    PubMed Central

    Clopath, Claudia; Nadal, Jean-Pierre; Brunel, Nicolas

    2012-01-01

    The cerebellum has long been considered to undergo supervised learning, with climbing fibers acting as a ‘teaching’ or ‘error’ signal. Purkinje cells (PCs), the sole output of the cerebellar cortex, have been considered as analogs of perceptrons storing input/output associations. In support of this hypothesis, a recent study found that the distribution of synaptic weights of a perceptron at maximal capacity is in striking agreement with experimental data in adult rats. However, the calculation was performed using random uncorrelated inputs and outputs. This is a clearly unrealistic assumption since sensory inputs and motor outputs carry a substantial degree of temporal correlations. In this paper, we consider a binary output neuron with a large number of inputs, which is required to store associations between temporally correlated sequences of binary inputs and outputs, modelled as Markov chains. Storage capacity is found to increase with both input and output correlations, and diverges in the limit where both go to unity. We also investigate the capacity of a bistable output unit, since PCs have been shown to be bistable in some experimental conditions. Bistability is shown to enhance storage capacity whenever the output correlation is stronger than the input correlation. Distribution of synaptic weights at maximal capacity is shown to be independent on correlations, and is also unaffected by the presence of bistability. PMID:22570592

  16. Tactile Stimulation Evokes Long-Lasting Potentiation of Purkinje Cell Discharge In Vivo

    PubMed Central

    Ramakrishnan, K. B.; Voges, Kai; De Propris, Licia; De Zeeuw, Chris I.; D’Angelo, Egidio

    2016-01-01

    In the cerebellar network, a precise relationship between plasticity and neuronal discharge has been predicted. However, the potential generation of persistent changes in Purkinje cell (PC) spike discharge as a consequence of plasticity following natural stimulation patterns has not been clearly determined. Here, we show that facial tactile stimuli organized in theta-patterns can induce stereotyped N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA-A) receptor-dependent changes in PCs and molecular layer interneurons (MLIs) firing: invariably, all PCs showed a long-lasting increase (Spike-Related Potentiation or SR-P) and MLIs a long-lasting decrease (Spike-Related Suppression or SR-S) in baseline activity and spike response probability. These observations suggests that tactile sensory stimulation engages multiple long-term plastic changes that are distributed along the mossy fiber-parallel fiber (MF-PF) pathway and operate synergistically to potentiate spike generation in PCs. In contrast, theta-pattern electrical stimulation (ES) of PFs indistinctly induced SR-P and SR-S both in PCs and MLIs, suggesting that tactile sensory stimulation preordinates plasticity upstream of the PF-PC synapse. All these effects occurred in the absence of complex spike changes, supporting the theoretical prediction that PC activity is potentiated when the MF-PF system is activated in the absence of conjunctive climbing fiber (CF) activity. PMID:26924961

  17. A Majority of FXTAS Cases Present with Intranuclear Inclusions Within Purkinje Cells.

    PubMed

    Ariza, Jeanelle; Rogers, Hailee; Monterrubio, Angela; Reyes-Miranda, Adriana; Hagerman, Paul J; Martínez-Cerdeño, Verónica

    2016-10-01

    Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder that affects carriers of a FMR1 premutation. Symptoms include cerebellar ataxia, tremor, and cognitive deficits. The most characteristic pathology of FXTAS is the presence of eosinophilic ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the nervous system and non-nervous tissues. Inclusions are present in neurons throughout the brain but are widely believed not to be present in the Purkinje cells (PCs) of the cerebellum. However, we analyzed 26 postmortem cases of FXTAS and demonstrated that 65 % of cases presented with inclusions within PCs of the cerebellum. We determined that the presence or absence of inclusions in PCs is correlated with age and that those cases with PC inclusions were overall 11 years older than those with no PC inclusions. Half of the cases with PCs with inclusions presented with twin nuclear inclusions. This novel finding demonstrating the presence of inclusions within PCs provides an insight into the understanding of the FXTAS motor symptoms and provides a novel target for the development of therapeutic strategies.

  18. Altered branching patterns of Purkinje cells in mouse model for cortical development disorder.

    PubMed

    Kim, Jinkyung; Kwon, Namseop; Chang, Soeun; Kim, Kyong-Tai; Lee, Dongmyeong; Kim, Seunghwan; Yun, So Jeong; Hwang, Daehee; Kim, Jee Woong; Hwu, Yeukuang; Margaritondo, Giorgio; Je, Jung Ho; Rhyu, Im Joo

    2011-01-01

    Disrupted cortical cytoarchitecture in cerebellum is a typical pathology in reeler. Particularly interesting are structural problems at the cellular level: dendritic morphology has important functional implication in signal processing. Here we describe a combinatorial imaging method of synchrotron X-ray microtomography with Golgi staining, which can deliver 3-dimensional(3-D) micro-architectures of Purkinje cell(PC) dendrites, and give access to quantitative information in 3-D geometry. In reeler, we visualized in 3-D geometry the shape alterations of planar PC dendrites (i.e., abnormal 3-D arborization). Despite these alterations, the 3-D quantitative analysis of the branching patterns showed no significant changes of the 77 ± 8° branch angle, whereas the branch segment length strongly increased with large fluctuations, comparing to control. The 3-D fractal dimension of the PCs decreased from 1.723 to 1.254, indicating a significant reduction of dendritic complexity. This study provides insights into etiologies and further potential treatment options for lissencephaly and various neurodevelopmental disorders.

  19. Oxygen-glucose deprivation increases firing of unipolar brush cells and enhances spontaneous EPSCs in Purkinje cells in the vestibulo-cerebellum.

    PubMed

    Takayasu, Yukihiro; Shino, Masato; Nikkuni, Osamu; Yoshida, Yukari; Furuya, Nobuhiko; Chikamatsu, Kazuaki

    2016-05-01

    Unipolar brush cells (UBCs) are excitatory interneurons in the granular layer of the cerebellar cortex, which are predominantly distributed in the vestibulo-cerebellar region. The unique firing properties and synaptic connections of UBCs may underlie lobular heterogeneity of excitability in the granular layer and the susceptibility to ischemia-induced excitotoxicity. In this study, we investigated the effects of oxygen-glucose deprivation (OGD) on the firing properties of UBCs and granule cells and spontaneous excitatory postsynaptic currents (sEPSCs) of Purkinje cells using whole-cell recordings. Short-term OGD induced increases in spontaneous firing of UBCs by causing membrane depolarization via the activation of NMDA receptors. UBC firing indirectly affected Purkinje cells by altering parallel fiber inputs of a subset granule cells, resulting in a marked increase in sEPSCs in Purkinje cells in vestibulo-cerebellar lobules IX-X, but not in lobules IV-VI, which have fewer UBCs. Similarly, the frequency and amplitude of sEPSCs in Purkinje cells were significantly greater in lobules IX-X than in IV-VI, even in control conditions. These results reveal that UBCs play key roles in regulating local excitability in the granular layer, resulting in lobular heterogeneity in the susceptibility to ischemic insult in the cerebellum.

  20. Repetitive behavior and increased activity in mice with Purkinje cell loss: a model for understanding the role of cerebellar pathology in autism.

    PubMed

    Martin, Loren A; Goldowitz, Dan; Mittleman, Guy

    2010-02-01

    Repetitive behaviors and hyperactivity are common features of developmental disorders, including autism. Neuropathology of the cerebellum is also a frequent occurrence in autism and other developmental disorders. Recent studies have indicated that cerebellar pathology may play a causal role in the generation of repetitive and hyperactive behaviors. In this study, we examined the relationship between cerebellar pathology and these behaviors in a mouse model of Purkinje cell loss. Specifically, we made aggregation chimeras between Lc/+ mutant embryos and +/+ embryos. Lc/+ mice lose 100% of their Purkinje cells postnatally due to a cell-intrinsic gain-of-function mutation. Through our histological examination, we demonstrated that Lc/+<-->+/+ chimeric mice have Purkinje cells ranging from zero to normal numbers. Our analysis of these chimeric cerebella confirmed previous studies on Purkinje cell lineage. The results of both open-field activity and hole-board exploration testing indicated negative relationships between Purkinje cell number and measures of activity and investigatory nose-poking. Additionally, in a progressive-ratio operant paradigm, we found that Lc/+ mice lever-pressed significantly less than +/+ controls, which led to significantly lower breakpoints in this group. In contrast, chimeric mice lever-pressed significantly more than controls and this repetitive lever-pressing behavior was significantly and negatively correlated with total Purkinje cell numbers. Although the performance of Lc/+ mice is probably related to their motor deficits, the significant relationships between Purkinje cell number and repetitive lever-pressing behavior as well as open-field activity measures provide support for a role of cerebellar pathology in generating repetitive behavior and increased activity in chimeric mice.

  1. Natural apoptosis in developing mice dopamine midbrain neurons and vermal Purkinje cells.

    PubMed

    Martí-Clúa, J

    2016-01-01

    Natural cell death by apoptosis was studied in two neuronal populations of BALB/c, C57BL/6 and B6CBA-Aw-j/A hybrid stock mice: (I) dopaminergic (DA) neurons in choosing coronal levels throughout the anteroposterior extent of the substantia nigra pars compacta (SNc), and (II) Purkinje cells (PCs) in each vermal lobe of the cerebellar cortex. Mice were collected at postnatal day (P) 2 and P14 for the midbrain study, and at P4 and P7 for the analysis of the cerebellum. No DA cells with morphologic criteria for apoptosis were found. Moreover, when the combination of tyrosine hydroxylase and TUNEL or tyrosine hydroxylase and active caspase-3 immunohistochemistry were performed in the same tissue section, no DA cells TUNEL positives or active caspase-3-stained DA neurons were seen. On the other hand, when PCs were considered, data analysis revealed that more dying PCs were observed at P4 than at P7. Values of neuron death were highest in the central lobe; this was followed by the posterior and anterior lobes and then by the inferior lobe. To determine if apoptotic death of PCs is linked to their time-of-origin profiles, pregnant dams were administered with [3H]TdR on embryonic days 11-12, 12-13, 13-14 and 14-15. When TUNEL and [3H]TdR autoradiography or active caspase-3 immunohistochemistry and [3H]TdR autoradiography were combined in the same tissue section, results reveal that the naturally occurring PC death is not related to its time of origin but, rather, is random across age.

  2. Modulation of Purkinje cell complex spike waveform by synchrony levels in the olivocerebellar system.

    PubMed

    Lang, Eric J; Tang, Tianyu; Suh, Colleen Y; Xiao, Jianqiang; Kotsurovskyy, Yuriy; Blenkinsop, Timothy A; Marshall, Sarah P; Sugihara, Izumi

    2014-01-01

    Purkinje cells (PCs) generate complex spikes (CSs) when activated by the olivocerebellar system. Unlike most spikes, the CS waveform is highly variable, with the number, amplitude, and timing of the spikelets that comprise it varying with each occurrence. This variability suggests that CS waveform could be an important control parameter of olivocerebellar activity. The origin of this variation is not well known. Thus, we obtained extracellular recordings of CSs to investigate the possibility that the electrical coupling state of the inferior olive (IO) affects the CS waveform. Using multielectrode recordings from arrays of PCs we showed that the variance in the recording signal during the period when the spikelets occur is correlated with CS synchrony levels in local groups of PCs. The correlation was demonstrated under both ketamine and urethane, indicating that it is robust. Moreover, climbing fiber reflex evoked CSs showed an analogous positive correlation between spikelet-related variance and the number of cells that responded to a stimulus. Intra-IO injections of GABA-A receptor antagonists or the gap junction blocker carbenoxolone produced correlated changes in the variance and synchrony levels, indicating the presence of a causal relationship. Control experiments showed that changes in variance with synchrony were primarily due to changes in the CS waveform, as opposed to changes in the strength of field potentials from surrounding cells. Direct counts of spikelets showed that their number increased with synchronization of CS activity. In sum, these results provide evidence of a causal link between two of the distinguishing characteristics of the olivocerebellar system, its ability to generate synchronous activity and the waveform of the CS.

  3. Chronic imaging of movement-related Purkinje cell calcium activity in awake behaving mice

    PubMed Central

    Gaffield, Michael A.; Amat, Samantha B.; Bito, Haruhiko

    2015-01-01

    Purkinje cells (PCs) are a major site of information integration and plasticity in the cerebellum, a brain region involved in motor task refinement. Thus PCs provide an ideal location for studying the mechanisms necessary for cerebellum-dependent motor learning. Increasingly, sophisticated behavior tasks, used in combination with genetic reporters and effectors of activity, have opened up the possibility of studying cerebellar circuits during voluntary movement at an unprecedented level of quantitation. However, current methods used to monitor PC activity do not take full advantage of these advances. For example, single-unit or multiunit electrode recordings, which provide excellent temporal information regarding electrical activity, only monitor a small population of cells and can be quite invasive. Bolus loading of cell-permeant calcium (Ca2+) indicators is short-lived, requiring same-day imaging immediately after surgery and/or indicator injection. Genetically encoded Ca2+ indicators (GECIs) overcome many of these limits and have garnered considerable use in many neuron types but only limited use in PCs. Here we employed these indicators to monitor Ca2+ activity in PCs over several weeks. We could repeatedly image from the same cerebellar regions across multiple days and observed stable activity. We used chronic imaging to monitor PC activity in crus II, an area previously linked to licking behavior, and identified a region of increased activity at the onset of licking. We then monitored this same region after training tasks to initiate voluntary licking behavior in response to different sensory stimuli. In all cases, PC Ca2+ activity increased at the onset of rhythmic licking. PMID:26561609

  4. A-Type potassium currents active at subthreshold potentials in mouse cerebellar purkinje cells

    PubMed Central

    Sacco, Tiziana; Tempia, Filippo

    2002-01-01

    Voltage-dependent and calcium-independent K+ currents were whole-cell recorded from cerebellar Purkinje cells in slices. Tetraethylammonium (TEA, 4 mm) application isolated an A-type K+ current (Ik(a)) with a peak amplitude, at +20 mV, of about one third of the total voltage-dependent and calcium-independent K+ current. The Ik(a) activated at about −60 mV, had a V0.5 of activation of −24.9 mV and a V0.5 of inactivation of −69.2 mV. The deactivation time constant at −70 mV was 3.4 ± 0.4 ms, while the activation time constant at +20 mV was 0.9 ± 0.2 ms. The inactivation kinetics was weakly voltage dependent, with two time constants; those at +20 mV were 19.3 ± 3.1 and 97.6 ± 9.8 ms. The recovery from inactivation had two time constants of 60.8 ms (78.4%) and 962.3 ms (21.6%). The Ik(a) was blocked by 4-aminopyridine with an IC50 of 67.6 μM. Agitoxin-2 (2 nm) blocked 17.4 ± 2.1% of the Ik(a). Flecainide completely blocked the Ik(a) with a biphasic effect with IC50 values of 4.4 and 183.2 μM. In current-clamp recordings the duration of evoked action potentials was affected neither by agitoxin-2 (2 nm) nor by flecainide (3 μM), but action potentials that were already broadened by TEA were further prolonged by 4-aminopyridine (100 μM). The amplitude of the hyperpolarisation at the end of depolarising steps was reduced by all these blockers. PMID:12205185

  5. Ethanol Modulates the Spontaneous Complex Spike Waveform of Cerebellar Purkinje Cells Recorded in vivo in Mice

    PubMed Central

    Zhang, Guang-Jian; Wu, Mao-Cheng; Shi, Jin-Di; Xu, Yin-Hua; Chu, Chun-Ping; Cui, Song-Biao; Qiu, De-Lai

    2017-01-01

    Cerebellar Purkinje cells (PCs) are sensitive to ethanol, but the effect of ethanol on spontaneous complex spike (CS) activity in these cells in vivo is currently unknown. Here, we investigated the effect of ethanol on spontaneous CS activity in PCs in urethane-anesthetized mice using in vivo patch-clamp recordings and pharmacological manipulation. Ethanol (300 mM) induced a decrease in the CS-evoked pause in simple spike (SS) firing and in the amplitude of the afterhyperpolarization (AHP) under current clamp conditions. Under voltage-clamp conditions, ethanol significantly decreased the area under the curve (AUC) and the number of CS spikelets, without changing the spontaneous frequency of the CSs or the instantaneous frequency of the CS spikelets. Ethanol-induced a decrease in the AUC of spontaneous CSs was concentration dependent. The EC50 of ethanol for decreasing the AUC of spontaneous CSs was 168.5 mM. Blocking N-methyl-D-aspartate receptors (NMDARs) failed to prevent the ethanol-induced decreases in the CS waveform parameters. However, blockade of cannabinoid receptor 1 (CB1) significantly suppressed the ethanol-induced effects on the CS-evoked pause in SS firing, amplitude of the AHP, spikelet number and the AUC of CSs. Moreover, a CB1 receptor agonist not only reduced the number of spikelets and the AUC of CSs, but also prevented the ethanol-induced inhibition of CS activity. Our results indicate that ethanol inhibits CS activity via activation of the CB1 receptor in vivo in mice, suggesting that excessive ethanol intake inhibits climbing fiber (CF)–PC synaptic transmission by modulating CB1 receptors in the cerebellar cortex. PMID:28293172

  6. Dendritic morphogenesis of cerebellar Purkinje cells through extension and retraction revealed by long-term tracking of living cells in vitro.

    PubMed

    Tanaka, M; Yanagawa, Y; Obata, K; Marunouchi, T

    2006-08-25

    Cerebellar Purkinje cells have the most elaborate dendritic trees among the neurons in the CNS. To investigate the dynamic aspects of dendritic morphogenesis of Purkinje cells, we performed a long-term analysis of living cells in cerebellar cell cultures derived from glutamate decarboxylase 67-green fluorescent protein mice. Most Purkinje cells had several primary dendrites during the 25-day culture period. Repeated observation of green fluorescent protein-expressing Purkinje cells over a period of 10-25 days in vitro demonstrated that not only extension, but also retraction of primary dendrites occurred during this culture period. Interestingly, both extension and retraction of primary dendrites were active between 10 and 15 days in vitro, and retraction of a primary dendrite occurred concomitantly with elongation of other primary dendrites in the same cell. Analysis of the morphological characteristics of the retracted primary dendrites demonstrated that shorter and less branched primary dendrites tended to retract. Furthermore, treatment with an inhibitor of calcium/calmodulin-dependent protein kinase II reduced the number of primary dendrites specifically during 5-15 days in vitro, the culture period when the extension and retraction of primary dendrites occurred actively. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-type glutamate receptors also reduced the number of primary dendrites during the same culture period, while inhibition of glutamate transporters increased the number. These findings suggest that the final morphology of Purkinje cells is achieved not only through extension, but also through retraction of their dendrites, and that calcium/calmodulin-dependent protein kinase II and neuronal activity are involved in this dendritic morphogenesis.

  7. Purkinje-like cells in the cochlear nucleus of the Common Tree Shrew (Tupaia glis) identified by calbindin immunohistochemistry.

    PubMed

    Spatz, W B

    2003-09-05

    The dorsal cochlear nucleus (DCN) of Tree Shrews (Tupaia glis; n=2) was examined by calbindin (CB) immunohistochemistry for the presence of Purkinje-like cells (PLCs), detected previously in only four different mammals. We found up to eight CB-immunoreactive PLCs in the left and right DCN, and a few axons, likely of PLC origin, that appeared to leave the DCN. These findings suggest that PLCs may have a wider distribution through mammalian species, and may represent more than just misrouted cells.

  8. Why do Purkinje cells die so easily after global brain ischemia? Aldolase C, EAAT4, and the cerebellar contribution to posthypoxic myoclonus.

    PubMed

    Welsh, John P; Yuen, Genevieve; Placantonakis, Dimitris G; Vu, Toan Q; Haiss, Florent; O'Hearn, Elizabeth; Molliver, Mark E; Aicher, Sue A

    2002-01-01

    The experiments strongly suggested that the reason why Purkinje cells die so easily after global brain ischemia relates to deficiencies in aldolase C and EAAT4 that allow them to survive pathologically intense synaptic input from the inferior olive after the restoration of blood flow. This conclusion is based on: (a) the remarkably tight correspondence between the regional absence of aldolase C and EAAT4 in Purkinje cells and the patterned loss of Purkinje cells after a bout of global brain ischemia; (b) the necessity of the olivocerebellar pathway for the ischemic death of Purkinje cells; and (c) the build-up of pathologically synchronous and high-frequency burst activity within the inferior olive during recovery from ischemia. Indeed, the correspondence between the absence of aldolase C and EAAT4 to sensitivity to ischemia could be demonstrated for zones of Purkinje cells as small as two neurons. A second finding was that Purkinje cells are not uniformly sensitive to transient ischemia, since they die most frequently in zones where aldolase C and EAAT4 are absent. One implication of the experiment is that factors beyond the unique synaptic and membrane properties of Purkinje cells play an important role in determining this neuron's high sensitivity to ischemia. The data strongly imply that two properties of Purkinje cells that make them susceptible to ischemic death are their reduced capability to sequester glutamate and reduced ability to generate energy during anoxia. The patterned death of Purkinje cells is sufficient to induce a form of audiogenic myoclonus, as determined with a neurotoxic dose of ibogaine. Ibogaine-induced myoclonus is recognized behaviorally as a reduced ability to habituate to a startle stimulus and resembles the myoclonic jerk of rats during recovery from a prolonged bout of global brain ischemia. Commonalities of ischemia and ibogaine-induced neurodegeneration are the intricately striped Purkinje cell loss in the posterior lobe and a

  9. [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.

  10. 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

  11. 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.

  12. Plasticity of cerebellar Purkinje cells in behavioral training of body balance control

    PubMed Central

    Lee, Ray X.; Huang, Jian-Jia; Huang, Chiming; Tsai, Meng-Li; Yen, Chen-Tung

    2015-01-01

    Neural responses to sensory inputs caused by self-generated movements (reafference) and external passive stimulation (exafference) differ in various brain regions. The ability to differentiate such sensory information can lead to movement execution with better accuracy. However, how sensory responses are adjusted in regard to this distinguishability during motor learning is still poorly understood. The cerebellum has been hypothesized to analyze the functional significance of sensory information during motor learning, and is thought to be a key region of reafference computation in the vestibular system. In this study, we investigated Purkinje cell (PC) spike trains as cerebellar cortical output when rats learned to balance on a suspended dowel. Rats progressively reduced the amplitude of body swing and made fewer foot slips during a 5-min balancing task. Both PC simple (SSs; 17 of 26) and complex spikes (CSs; 7 of 12) were found to code initially on the angle of the heads with respect to a fixed reference. Using periods with comparable degrees of movement, we found that such SS coding of information in most PCs (10 of 17) decreased rapidly during balance learning. In response to unexpected perturbations and under anesthesia, SS coding capability of these PCs recovered. By plotting SS and CS firing frequencies over 15-s time windows in double-logarithmic plots, a negative correlation between SS and CS was found in awake, but not anesthetized, rats. PCs with prominent SS coding attenuation during motor learning showed weaker SS-CS correlation. Hence, we demonstrate that neural plasticity for filtering out sensory reafference from active motion occurs in the cerebellar cortex in rats during balance learning. SS-CS interaction may contribute to this rapid plasticity as a form of receptive field plasticity in the cerebellar cortex between two receptive maps of sensory inputs from the external world and of efference copies from the will center for volitional movements

  13. Relating Cerebellar Purkinje Cell Activity to the Timing and Amplitude of Conditioned Eyelid Responses

    PubMed Central

    Khilkevich, Andrei; Mauk, Michael D.

    2015-01-01

    How Purkinje cell (PC) activity may be altered by learning is central to theories of the cerebellum. Pavlovian eyelid conditioning, because of how directly it engages the cerebellum, has helped reveal many aspects of cerebellar learning and the underlying mechanisms. Theories of cerebellar learning assert that climbing fiber inputs control plasticity at synapses onto PCs, and thus PCs control the expression of learned responses. We tested this assertion by recording 184 eyelid PCs and 240 non-eyelid PCs during the expression of conditioned eyelid responses (CRs) in well trained rabbits. By contrasting the responses of eyelid and non-eyelid PCs and by contrasting the responses of eyelid PCs under conditions that produce differently timed CRs, we test the hypothesis that learning-related changes in eyelid PCs contribute to the learning and adaptive timing of the CRs. We used a variety of analyses to test the quantitative relationships between eyelid PC responses and the kinematic properties of the eyelid CRs. We find that the timing of eyelid PC responses varies systematically with the timing of the behavioral CRs and that there are differences in the magnitude of eyelid PC responses between larger-CR, smaller-CR, and non-CR trials. However, eyelid PC activity does not encode any single kinematic property of the behavioral CRs at a fixed time lag, nor does it linearly encode CR amplitude. Even so, the results are consistent with the hypothesis that learning-dependent changes in PC activity contribute to the adaptively timed expression of conditioned eyelid responses. PMID:25995469

  14. Microtubule-associated protein 2 (MAP2) in Purkinje cell dendrites: Evidence that factors other than binding to microtubules are involved in determining its cytoplasmic distribution

    SciTech Connect

    Matus, A.; Delhaye-Bouchaud, N.; Mariani, J. )

    1990-07-15

    We have studied the distribution of microtubule-associated protein 2 (MAP2) in the Purkinje cell dendrites of rats whose cerebella were exposed to X-irradiation during the second postnatal week. The Purkinje cells of such animals have abnormally elongated apical primary processes that branch in the other molecular layer rather than close to the cell body as in normal tissue. The results show that in these distorted dendrites the MAP2 distribution is shifted distally relative to the normal pattern, in which MAP2 is distributed evenly throughout the dendritic tree. Tubulin and other microtubule-associated proteins, such as MAP1, are not affected and remain evenly distributed throughout the dendritic tree despite the anatomical distortion. We conclude that the distribution of MAP2 in Purkinje cells is not determined solely by its binding to tubulin. Other factors must be involved and these appear to be related to dendritic morphology and possibly to branching.

  15. Purkinje cell compartmentation in the cerebellum of the lysosomal Acid phosphatase 2 mutant mouse (nax - naked-ataxia mutant mouse).

    PubMed

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.

  16. Loss of the calcium channel β4 subunit impairs parallel fibre volley and Purkinje cell firing in cerebellum of adult ataxic mice.

    PubMed

    Benedetti, Bruno; Benedetti, Ariane; Flucher, Bernhard E

    2016-06-01

    The auxiliary voltage-gated calcium channel subunit β4 supports targeting of calcium channels to the cell membrane, modulates ionic currents and promotes synaptic release in the central nervous system. β4 is abundant in cerebellum and its loss causes ataxia. However, the type of calcium channels and cerebellar functions affected by the loss of β4 are currently unknown. We therefore studied the structure and function of Purkinje cells in acute cerebellar slices of the β4 (-/-) ataxic (lethargic) mouse, finding that loss of β4 affected Purkinje cell input, morphology and pacemaker activity. In adult lethargic cerebellum evoked postsynaptic currents from parallel fibres were depressed, while paired-pulse facilitation and spontaneous synaptic currents were unaffected. Because climbing fibre input was spared, the parallel fibre/climbing fibre input ratio was reduced. The dendritic arbor of adult lethargic Purkinje cells displayed fewer and shorter dendrites, but a normal spine density. Accordingly, the width of the molecular and granular layers was reduced. These defects recapitulate the impaired cerebellar maturation observed upon Cav 2.1 ataxic mutations. However, unlike Cav 2.1 mutations, lethargic Purkinje cells also displayed a striking decrease in pacemaker firing frequency, without loss of firing regularity. All these deficiencies appear in late development, indicating the importance of β4 for the normal differentiation and function of mature Purkinje cells networks. The observed reduction of the parallel fibre input, the altered parallel fibre/climbing fibre ratio and the reduced Purkinje cell output can contribute to the severe motor impairment caused by the loss of the calcium channel β4 subunit in lethargic mice.

  17. Physiology, morphology and detailed passive models of guinea-pig cerebellar Purkinje cells.

    PubMed Central

    Rapp, M; Segev, I; Yarom, Y

    1994-01-01

    1. Purkinje cells (PCs) from guinea-pig cerebellar slices were physiologically characterized using intracellular techniques. Extracellular caesium ions were used to linearize the membrane properties of PCs near the resting potential. Under these conditions the average input resistance, RN, was 29 M omega, the average system time constant, tau 0, was 82 ms and the average cable length, LN, was 0.59. 2. Three PCs were fully reconstructed following physiological measurements and staining with horseradish peroxidase. Assuming that each spine has an area of 1 micron 2 and that the spine density over the spiny dendrites is ten spines per micrometre length, the total membrane area of each PC is approximately 150,000 microns 2, of which approximately 100,000 microns 2 is in the spines. 3. Detailed passive cable and compartmental models were built for each of the three reconstructed PCs. Computational methods were devised to incorporate globally the huge number of spines into these models. In all three cells the models predict that the specific membrane resistivity, Rm, of the soma is much lower than the dendritic Rm (approximately 500 and approximately 100,000 omega cm2 respectively). The specific membrane capacitance, Cm, is estimated to be 1.5-2 muF cm-2 and the specific cytoplasm resistivity, Ri, is 250 omega cm. 4. The average cable length of the dendrites according to the model is 0.13 lambda, suggesting that under caesium conditions PCs are electrically very compact. Brief somatic spikes, however, are expected to attenuate 30-fold when spreading passively into the dendritic terminals. A simulated 200 Hz train of fast, 90 mV somatic spikes produced a smooth 12 mV steady depolarization at the dendritic terminals. 5. A transient synaptic conductance increase, with a 1 nS peak at 0.5 ms and a driving force of 60 mV, is expected to produce approximately 20 mV peak depolarization at the spine head membrane. This EPSP then attenuates between 200- and 900-fold into the soma

  18. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia

    PubMed Central

    Zhang, Qifeng; Smethurst, Elizabeth; Segonds-Pichon, Anne; Schrewe, Heinrich; Wakelam, Michael J. O.

    2016-01-01

    Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA), a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO) mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA) regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction. PMID:27658289

  19. Sensorimotor enhancement in mouse mutants lacking the Purkinje cell-specific Gi/o modulator, Pcp2(L7).

    PubMed

    Iscru, Emilia; Serinagaoglu, Yelda; Schilling, Karl; Tian, Jinbin; Bowers-Kidder, Stephanie L; Zhang, Rui; Morgan, James I; DeVries, A Courtney; Nelson, Randy J; Zhu, Michael X; Oberdick, John

    2009-01-01

    Pcp2(L7) is a GoLoco domain protein specifically and abundantly expressed in cerebellar Purkinje cells. It has been hypothesized to "tune" G(i/o)-coupled receptor modulation of physiological effectors, including the P-type Ca(2+) channel. We have analyzed a mouse mutant in which the Pcp2(L7) gene was inactivated and find significant anatomical, behavioral and electrophysiological changes. Anatomically, we observed mild cerebellar hypoplasia. Behaviorally, the mutants were altered in modalities atypical for a traditional cerebellar mutant, and oddly, all of these changes could be considered functional enhancements. This includes increased asymptotic performance in gross motor learning, increased rate of acquisition in tone-conditioned fear, and enhanced pre-pulse inhibition of the acoustic startle response. Electrophysiological analysis of Purkinje cells in the mutants reveals depression of the complex spike waveform that may underlie the behavioral changes. Based on these observations we suggest that the Pcp2(L7) protein acts as a sensorimotor damper that modulates time- and sense-dependent changes in motor responses.

  20. 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

  1. 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.

  2. A promoter element with enhancer properties, and the orphan nuclear receptor RORalpha, are required for Purkinje cell-specific expression of a Gi/o modulator.

    PubMed

    Serinagaoglu, Yelda; Zhang, Rui; Zhang, Yufang; Zhang, Linda; Hartt, Greg; Young, Anthony P; Oberdick, John

    2007-03-01

    The promoter and structural portion of the gene, Pcp-2(L7), has frequently been used to target expression of proteins to cerebellar Purkinje cells. In our continuing analysis of the transcription of this gene and how it relates to the G-protein and Ca2+ channel modulatory functions of the encoded protein, we have dissociated the promoter and structural gene and identified cooperative functions. A 0.9 kb fragment of the proximal promoter has positional properties of a classical enhancer, yet its function requires the presence of the structural gene. We demonstrate that RORalpha, the gene product of the mutant mouse locus called staggerer (Rora(sg)), binds to and activates expression through this promoter element using functional assays in vitro and in vivo. The structural gene has a repressive effect on gene expression outside Purkinje cells, and likely participates in the suppression of Pcp-2(L7) gene expression in the many other brain and non-neuronal cell types, besides Purkinje cells, known to express RORalpha. Additional studies in vivo show that while Pcp-2(L7) expression is dependent on RORalpha throughout the cerebellum, this dependence is greatest in the intermediate region between the vermis and far lateral hemispheres. Thus, in addition to its recently indicated role in Ca2+-mediated reciprocal cell-cell signaling in Purkinje cells, RORalpha may also contribute to functional differences in cerebellar subregions.

  3. Bidirectional modulation of deep cerebellar nuclear cells revealed by optogenetic manipulation of inhibitory inputs from Purkinje cells.

    PubMed

    Han, V Z; Magnus, G; Zhang, Y; Wei, A D; Turner, E E

    2014-09-26

    In the mammalian cerebellum, deep cerebellar nuclear (DCN) cells convey all information from cortical Purkinje cells (PCs) to premotor nuclei and other brain regions. However, how DCN cells integrate inhibitory input from PCs with excitatory inputs from other sources has been difficult to assess, in part due to the large spatial separation between cortical PCs and their target cells in the nuclei. To circumvent this problem we have used a Cre-mediated genetic approach to generate mice in which channelrhodopsin-2 (ChR2), fused with a fluorescent reporter, is selectively expressed by GABAergic neurons, including PCs. In recordings from brain slice preparations from this model, mammalian PCs can be robustly depolarized and discharged by brief photostimulation. In recordings of postsynaptic DCN cells, photostimulation of PC axons induces a strong inhibition that resembles these cells' responses to focal electrical stimulation, but without a requirement for the glutamate receptor blockers typically applied in such experiments. In this optogenetic model, laser pulses as brief as 1 ms can reliably induce an inhibition that shuts down the spontaneous spiking of a DCN cell for ∼50 ms. If bursts of such brief light pulses are delivered, a fixed pattern of bistable bursting emerges. If these pulses are delivered continuously to a spontaneously bistable cell, the immediate response to such photostimulation is inhibitory in the cell's depolarized state and excitatory when the membrane has repolarized; a less regular burst pattern then persists after stimulation has been terminated. These results indicate that the spiking activity of DCN cells can be bidirectionally modulated by the optically activated synaptic inhibition of cortical PCs.

  4. The 40-year history of modeling active dendrites in cerebellar Purkinje cells: emergence of the first single cell “community model”

    PubMed Central

    Bower, James M.

    2015-01-01

    The subject of the effects of the active properties of the Purkinje cell dendrite on neuronal function has been an active subject of study for more than 40 years. Somewhat unusually, some of these investigations, from the outset have involved an interacting combination of experimental and model-based techniques. This article recounts that 40-year history, and the view of the functional significance of the active properties of the Purkinje cell dendrite that has emerged. It specifically considers the emergence from these efforts of what is arguably the first single cell “community” model in neuroscience. The article also considers the implications of the development of this model for future studies of the complex properties of neuronal dendrites. PMID:26539104

  5. Purkinje-cell-restricted restoration of Kv3.3 function restores complex spikes and rescues motor coordination in Kcnc3 mutants.

    PubMed

    Hurlock, Edward C; McMahon, Anne; Joho, Rolf H

    2008-04-30

    The fast-activating/deactivating voltage-gated potassium channel Kv3.3 (Kcnc3) is expressed in various neuronal cell types involved in motor function, including cerebellar Purkinje cells. Spinocerebellar ataxia type 13 (SCA13) patients carrying dominant-negative mutations in Kcnc3 and Kcnc3-null mutant mice both display motor incoordination, suggested in mice by increased lateral deviation while ambulating and slips on a narrow beam. Motor skill learning, however, is spared. Mice lacking Kcnc3 also exhibit muscle twitches. In addition to broadened spikes, recordings of Kcnc3-null Purkinje cells revealed fewer spikelets in complex spikes and a lower intraburst frequency. Targeted reexpression of Kv3.3 channels exclusively in Purkinje cells in Kcnc3-null mice as well as in mice also heterozygous for Kv3.1 sufficed to restore simple spike brevity along with normal complex spikes and to rescue specifically coordination. Therefore, spike parameters requiring Kv3.3 function in Purkinje cells are involved in the ataxic null phenotype and motor coordination, but not motor learning.

  6. Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings

    PubMed Central

    Roth, Arnd; Häusser, Michael

    2001-01-01

    Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective Ih blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (Cm), specific membrane resistance (Rm) and intracellular resistivity (Ri) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: Cm, 0.77 ± 0.17 μF cm−2 (mean ±s.d.; range, 0.64-1.00 μF cm−2), Rm, 122 ± 18 kΩ cm2 (98-141 kΩ cm2) and Ri, 115 ± 20 Ω cm (93-142 Ω cm). The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. A model of Ih was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients

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

    PubMed Central

    Dell’Orco, James M.; Qin, Zhaohui S.; Kalyana-Sundaram, Shanker; Chinnaiyan, Arul M.; Shakkottai, Vikram G.; Lieberman, Andrew P.

    2016-01-01

    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. PMID:27152617

  8. Rescue of motor coordination by Purkinje cell-targeted restoration of Kv3.3 channels in Kcnc3-null mice requires Kcnc1.

    PubMed

    Hurlock, Edward C; Bose, Mitali; Pierce, Ganon; Joho, Rolf H

    2009-12-16

    The role of cerebellar Kv3.1 and Kv3.3 channels in motor coordination was examined with an emphasis on the deep cerebellar nuclei (DCN). Kv3 channel subunits encoded by Kcnc genes are distinguished by rapid activation and deactivation kinetics that support high-frequency, narrow action potential firing. Previously we reported that increased lateral deviation while ambulating and slips while traversing a narrow beam of ataxic Kcnc3-null mice were corrected by restoration of Kv3.3 channels specifically to Purkinje cells, whereas Kcnc3-mutant mice additionally lacking one Kcnc1 allele were partially rescued. Here, we report mice lacking all Kcnc1 and Kcnc3 alleles exhibit no such rescue. For Purkinje cell output to reach the rest of the brain it must be conveyed by neurons of the DCN or vestibular nuclei. As Kcnc1, but not Kcnc3, alleles are lost, mutant mice exhibit increasing gait ataxia accompanied by spike broadening and deceleration in DCN neurons, suggesting the facet of coordination rescued by Purkinje-cell-restricted Kv3.3 restoration in mice lacking just Kcnc3 is hypermetria, while gait ataxia emerges when additionally Kcnc1 alleles are lost. Thus, fast repolarization in Purkinje cells appears important for normal movement velocity, whereas DCN neurons are a prime candidate locus where fast repolarization is necessary for normal gait patterning.

  9. Extracellular divalent and trivalent cation effects on sodium current kinetics in single canine cardiac Purkinje cells.

    PubMed Central

    Hanck, D A; Sheets, M F

    1992-01-01

    1. The effects of the extracellular divalent cations barium, calcium, cadmium, cobalt, magnesium, manganese, nickel and zinc and the trivalent cation lanthanum on macroscopic sodium current (INa) were characterized in enzymatically isolated single canine cardiac Purkinje cells under voltage clamp at 9-14 degrees C. 2. All di(tri)valent cations produced depolarizing shifts in the conductance-voltage relationship. The order of efficacy, taken as the concentration required to produce a 5 mV shift in the mid-point of peak INa conductance, from least to most effective was (mM): Ca2+ (2.97) approximately Mg2+ (2.67) approximately Ba2+ (1.93) > CO2+ (1.02) approximately Mn2+ (0.88) > Ni2+ (0.54) > La3+ (0.095) approximately Cd2+ (0.083) approximately Zn2+ (0.076). 3. Addition of di(tri)valent cations also produced depolarizing shifts in voltage-dependent availability. The order of efficacy from the least to most effective was (mM): Cd2+ (7.70) approximately Mg2+ (6.86) approximately Ba2+ (4.50) > Ca2+ (2.47) approximately CO2+ (1.87) approximately Mn2+ (1.24) approximately Ni2+ (1.20) > Zn2+ (0.300) > La3+ (0.060). 4. The Gouy-Chapman-Stern equations were used to evaluate di(tri)valent cation efficacy in binding to surface charges. Surface charge density was estimated as 0.72 sites nm-2, and it was assumed that Mg2+, the divalent cation that produced the smallest shift, screened but did not bind to surface charges. Based on voltage-dependent availability, KD from lowest to highest affinity were (mM): Ba2+ (2500) > CO2+ (1670) approximately Mn2+ (1430) approximately Ca2+ = Cd2+ = Ni2+ (1200) > Zn2+ (250) > La3+ (30). 5. All di(tri)valent cations also produced a concentration-dependent acceleration of INa tail current relaxation. The addition of Ca2+ and La3+ produced acceleration of tail current relaxations that could be accounted for by the surface charge effects predicted from the shift in voltage-dependent availability. Cd2+, which produced almost no change in voltage

  10. Posterior cerebellar Purkinje cells in an SCA5/SPARCA1 mouse model are especially vulnerable to the synergistic effect of loss of β-III spectrin and GLAST.

    PubMed

    Perkins, Emma M; Suminaite, Daumante; Clarkson, Yvonne L; Lee, Sin Kwan; Lyndon, Alastair R; Rothstein, Jeffrey D; Wyllie, David J A; Tanaka, Kohichi; Jackson, Mandy

    2016-08-15

    Clinical phenotypes of spinocerebellar ataxia type-5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type-1 (SPARCA1) are mirrored in mice lacking β-III spectrin (β-III(-/-)). One function of β-III spectrin is the stabilization of the Purkinje cell-specific glutamate transporter EAAT4 at the plasma membrane. In β-III(-/-) mice EAAT4 levels are reduced from an early age. In contrast levels of the predominant cerebellar glutamate transporter GLAST, expressed in Bergmann glia, only fall progressively from 3 months onwards. Here we elucidated the roles of these two glutamate transporters in cerebellar pathogenesis mediated through loss of β-III spectrin function by studying EAAT4 and GLAST knockout mice as well as crosses of both with β-III(-/-) mice. Our data demonstrate that EAAT4 loss, but not abnormal AMPA receptor composition, in young β-III(-/-) mice underlies early Purkinje cell hyper-excitability and that subsequent loss of GLAST, superimposed on the earlier deficiency of EAAT4, is responsible for Purkinje cell loss and progression of motor deficits. Yet the loss of GLAST appears to be independent of EAAT4 loss, highlighting that other aspects of Purkinje cell dysfunction underpin the pathogenic loss of GLAST. Finally, our results demonstrate that Purkinje cells in the posterior cerebellum of β-III(-/-) mice are most susceptible to the combined loss of EAAT4 and GLAST, with degeneration of proximal dendrites, the site of climbing fibre innervation, most pronounced. This highlights the necessity for efficient glutamate clearance from these regions and identifies dysregulation of glutamatergic neurotransmission particularly within the posterior cerebellum as a key mechanism in SCA5 and SPARCA1 pathogenesis.

  11. Flocculus Purkinje cell signals in mouse Cacna1a calcium channel mutants of escalating severity: an investigation of the role of firing irregularity in ataxia

    PubMed Central

    Thumser, Zachary C.

    2014-01-01

    Mutation of the Cacna1a gene for the P/Q (CaV2.1) calcium channel invariably leads to cerebellar dysfunction. The dysfunction has been attributed to disrupted rhythmicity of cerebellar Purkinje cells, but the hypothesis remains unproven. If irregular firing rates cause cerebellar dysfunction, then the irregularity and behavioral deficits should covary in a series of mutant strains of escalating severity. We compared firing irregularity in floccular and anterior vermis Purkinje cells in the mildly affected rocker and moderately affected tottering Cacna1a mutants and normal C57BL/6 mice. We also measured the amplitude and timing of modulations of floccular Purkinje cell firing rate during the horizontal vestibuloocular reflex (VOR, 0.25–1 Hz) and the horizontal and vertical optokinetic reflex (OKR, 0.125–1 Hz). We recorded Purkinje cells selective for rotational stimulation about the vertical axis (VAPCs) and a horizontal axis (HAPCs). Irregularity scaled with behavioral deficit severity in the flocculus but failed to do so in the vermis, challenging the irregularity hypothesis. Mutant VAPCs exhibited unusually strong modulation during VOR and OKR, the response augmentation scaling with phenotypic severity. HAPCs exhibited increased OKR modulation but in tottering only. The data contradict prior claims that modulation amplitude is unaffected in tottering but support the idea that attenuated compensatory eye movements in Cacna1a mutants arise from defective transfer of Purkinje cell signals to downstream circuitry, rather than attenuated synaptic transmission within the cerebellar cortex. Shifts in the relative sizes of the VAPC and HAPC populations raise the possibility that Cacna1a mutations influence the development of floccular zone architecture. PMID:25143538

  12. Patch-clamp analysis in canine cardiac Purkinje cells of a novel sodium component in the pacemaker range.

    PubMed

    Rota, Marcello; Vassalle, Mario

    2003-04-01

    A putative Na+ component playing a role in the initiation and maintenance of spontaneous discharge in Purkinje fibres was studied by means of the whole-cell patch-clamp technique in canine cardiac single Purkinje cells. In 4 mM [K+]o, during depolarising clamp steps, a slowly inactivating current appeared at approximately -58 mV, negative to the threshold for the fast Na+ current (INa; approximately -50 mV). During depolarising ramps, the current underwent inward rectification with a negative slope region that began at approximately -60 mV. The current underlying the negative slope increased during faster ramps, decreased as a function of time when the initial depolarising ramp was over, decreased during depolarisations positive to approximately -35 mV and was much larger than the current during the symmetrical repolarising ramp. Increasing biphasic ('oscillatory') voltage ramps required much smaller currents at a holding potential (Vh) of -60 mV than at -80 mV and were associated with a marked decrease in slope conductance. At Vh -50/-40 mV, the oscillatory ramp currents and superimposed pulse currents reversed direction. The negative slope in the I-V relation as well as the change in current direction at -50/-40 mV were markedly reduced by tetrodotoxin (15 microM) and lidocaine (lignocaine, 100 microM) and therefore are due to a slowly inactivating Na+ current, labelled here INa3. Lower [K+]o (2.7 mM) reduced the steady state slope conductance as well as the current in the diastolic range, and increased as well as shifted INa3 in a negative direction. High [K+]o had the opposite effects. Cs+ (2 mM) and Ba2+ (2 mM) reduced the initial current during depolarising ramps but not INa3. In current-clamp mode, current-induced voltage oscillations elicited action potentials through a gradual transition between diastolic depolarisation and upstroke, consistent with the activation of INa3. Thus, the initiation and maintenance of spontaneous discharge in Purkinje strands

  13. Discharges of Purkinje cells in the paravermal part of the cerebellar anterior lobe during locomotion in the cat.

    PubMed Central

    Armstrong, D M; Edgley, S A

    1984-01-01

    Extracellular recordings were made from 124 Purkinje cells in the paravermal part of lobule V of the cerebellum in cats walking steadily at a speed of 0.5 m/s on a moving belt. All cells tested had a tactile receptive field from which simple spikes could be evoked and 96% of these were on the ipsilateral forelimb. Seventy-six of the cells were also studied whilst the animals sat or lay quietly without movement. Complex spikes were discharged at 1-2/s and these were accompanied by simple spikes in fifty-nine cells (78%); in the remaining cells there were no or few simple spikes. The over-all mean discharge rate (including both types of spike) was 37.8 +/- 27 impulses/s (+/- S.D.). During locomotion all cells discharged both types of spike and the over-all mean rate was 57.6 +/- 29 impulses/s (+/- S.D.). In all cells but one, the frequency of the simple spikes was modulated rhythmically in time with the stepping movements but the phasing relative to the step cycle varied widely between cells. Peak rates also varied widely, the average being 91.5 +/- 44 impulses/s (+/- S.D.). Most cells (63%) generated one period of accelerated discharge per step but others generated two (35%) or three (2%) such periods. Despite the individual variations in discharge timing the population as a whole was considerably more active during the swing than the stance phase of the step cycle in the ipsilateral forelimb (68 impulses/s as compared with 49 impulses/s on average). Thirty-four cells were electrophysiologically identified as lying in the c1 zone of the cortex and twenty-five as being in the c2 zone (nomenclature of Oscarsson, 1980). During locomotion, the population activity in the two zones differed slightly: activity in the c1 population was phase advanced by approximately one-tenth of the step cycle. The results are discussed, with particular emphasis on the finding that population activity in the Purkinje cells of the c1 zone fluctuated during the step cycle in parallel with

  14. Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

    PubMed Central

    Zhou, Feng C.; Resendiz, Marisol; Lo, Chiao-Ling; Chen, Yuanyuan

    2016-01-01

    Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology. PMID:27583369

  15. Spatial organization of visual messages of the rabbit's cerebellar flocculus. II. Complex and simple spike responses of Purkinje cells.

    PubMed

    Graf, W; Simpson, J I; Leonard, C S

    1988-12-01

    1. Complex and simple spike responses of Purkinje cells were recorded in the flocculus of anesthetized, paralyzed rabbits during rotating full-field visual stimuli produced by a three-axis planetarium projector. 2. On the basis of the spatial properties of their complex spike responses, floccular Purkinje cells could be placed into three distinct classes called Vertical Axis, Anterior (45 degrees) Axis and Posterior (135 degrees) Axis. The first two classes occurred in both monocular and binocular forms; the third class was encountered only in binocular form. For the binocular response forms, stimulation through one eye, called the dominant eye, elicited a stronger modulation of the complex spike firing rate than did stimulation of the other eye. The approximate orientation of that axis about which full-field rotation elicited the deepest modulation (the preferred axis) when presented to the dominant eye served as the class label. These classes are the same as those determined qualitatively for inferior olive neurons in the previous paper (47). The present study provides a quantitative description of their spatial tuning. 3. For Vertical Axis cells, the dominant eye was ipsilateral with respect to the flocculus recording site. The preferred axis was vertical and null (no-response) axes were in the horizontal plane. For the binocular response form of Vertical Axis cells (less than 10% of this class), the direction preferences for the two eyes were synergistic with respect to rotation about the vertical axis. 4. The dominant eye for the Anterior (45 degrees) Axis cells was contralateral, with the preferred axis oriented in the horizontal plane at approximately 45 degrees contralateral azimuth. The modulation depth showed a close to cosine relation with the angle between the preferred axis and the stimulus rotation axis. The average orientation (n = 10) for the dominant eye preferred axis, determined by the best-fit sinusoid, was 47 degrees contralateral azimuth. The

  16. Mapping calcium transients in the dendrites of Purkinje cells from the guinea-pig cerebellum in vitro.

    PubMed Central

    Ross, W N; Werman, R

    1987-01-01

    1. A 10 X 10 photodiode array was used to detect stimulation-dependent absorbance changes simultaneously from many positions in the dendrite field of guinea-pig Purkinje cells which had been injected with the calcium indicator Arsenazo III in thin cerebellar slices. Signals from each element of the array were matched to positions on the cells by mapping them onto fluorescence photographs of Lucifer Yellow which had been co-injected into the cells with the Arsenazo III. 2. In response to intrasomatic stimulation the rising phase of the absorbance signals corresponded in time with the calcium spikes recorded with an intracellular electrode. There was no increase in absorbance during bursts of fast sodium spikes. Absorbance signals persisted after the sodium spikes were blocked by tetrodotoxin (TTX). In addition, the signals were largest at 660 nm and small signals of opposite polarity were found at 540 nm. These results indicate that the absorbance signals came from calcium entry into the cell resulting from the turning on of voltage-dependent calcium conductances. 3. In these experiments signals were usually seen all over the dendritic field and were weak or totally absent over the soma. In some cases signals were seen over a more restricted area. With a spatial resolution of 25 microns we were not able to see any evidence for highly localized sites of calcium entry. 4. Sometimes the rising phase of the calcium signals was separated by almost 13 ms in different parts of the dendritic field, too long to be explained by active propagation delay. This suggests that calcium spikes causing these signals can be evoked separately in different regions of the Purkinje cell dendritic field by long-lasting potentials which may reach local threshold at different times. 5. Calcium signals resulting from slow plateau after-potentials and the calcium spikes produced by them were also detected in all locations in the dendritic field. The relative distribution of amplitudes from

  17. Quantitative Localization of Cav2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels

    PubMed Central

    Indriati, Dwi Wahyu; Kamasawa, Naomi; Matsui, Ko; Meredith, Andrea L.; Watanabe, Masahiko; Shigemoto, Ryuichi

    2014-01-01

    P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Cav2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Cav2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Cav2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Cav2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ~40 nm. Calcium nanodomain created by the opening of Cav2.1 channels likely activates the two channels that limit the extent of depolarization. PMID:23426693

  18. The composite neuron: a realistic one-compartment Purkinje cell model suitable for large-scale neuronal network simulations.

    PubMed

    Coop, A D; Reeke, G N

    2001-01-01

    We present a simple method for the realistic description of neurons that is well suited to the development of large-scale neuronal network models where the interactions within and between neural circuits are the object of study rather than the details of dendritic signal propagation in individual cells. Referred to as the composite approach, it combines in a one-compartment model elements of both the leaky integrator cell and the conductance-based formalism of Hodgkin and Huxley (1952). Composite models treat the cell membrane as an equivalent circuit that contains ligand-gated synaptic, voltage-gated, and voltage- and concentration-dependent conductances. The time dependences of these various conductances are assumed to correlate with their spatial locations in the real cell. Thus, when viewed from the soma, ligand-gated synaptic and other dendritically located conductances can be modeled as either single alpha or double exponential functions of time, whereas, with the exception of discharge-related conductances, somatic and proximal dendritic conductances can be well approximated by simple current-voltage relationships. As an example of the composite approach to neuronal modeling we describe a composite model of a cerebellar Purkinje neuron.

  19. A change in the pattern of activity affects the developmental regression of the Purkinje cell polyinnervation by climbing fibers in the rat cerebellum.

    PubMed

    Andjus, P R; Zhu, L; Cesa, R; Carulli, D; Strata, P

    2003-01-01

    Pattern of activity during development is important for the refinement of the final architecture of the brain. In the cerebellar cortex, the regression from multiple to single climbing fiber innervation of the Purkinje cell occurs during development between postnatal days (P) 5 and 15. However, the regression is hampered by altering in various ways the morpho-functional integrity of the parallel fiber input. In rats we disrupted the normal activity pattern of the climbing fiber, the terminal arbor of the inferior olive neurons, by administering harmaline for 4 days from P9 to P12. At all studied ages (P15-87) after harmaline treatment multiple (double only) climbing fiber EPSC-steps persist in 28% of cells as compared with none in the control. The ratio between the amplitudes of the larger and the smaller climbing fiber-evoked EPSC increases in parallel with the decline of the polyinnervation factor, indicating a gradual enlargement of the synaptic contribution of the winning climbing fiber synapse at the expense of the losing one. Harmaline treatment had no later effects on the climbing fiber EPSC kinetics and I/V relation in Purkinje cells (P15-36). However, there was a rise in the paired-pulse depression indicating a potentiation of the presynaptic mechanisms. In the same period, after harmaline treatment, parallel fiber-Purkinje cell electrophysiology was unaffected. The distribution of parallel fiber synaptic boutons was also not changed. Thus, a change in the pattern of activity during a narrow developmental period may affect climbing fiber-Purkinje cell synapse competition resulting in occurrence of multiple innervation at least up to 3 months of age. Our results extend the current view on the role of the pattern of activity in the refinement of neuronal connections during development. They suggest that many similar results obtained by different gene or receptor manipulations might be simply the consequence of disrupting the pattern of activity.

  20. Motor dysfunction and altered synaptic transmission at the parallel fiber-Purkinje cell synapse in mice lacking potassium channels Kv3.1 and Kv3.3.

    PubMed

    Matsukawa, Hiroshi; Wolf, Alexander M; Matsushita, Shinichi; Joho, Rolf H; Knöpfel, Thomas

    2003-08-20

    Micelacking both Kv3.1 and both Kv3.3 K+ channel alleles display severe motor deficits such as tremor, myoclonus, and ataxic gait. Micelacking one to three alleles at the Kv3.1 and Kv3.3 loci exhibit in an allele dose-dependent manner a modest degree of ataxia. Cerebellar granule cells coexpress Kv3.1 and Kv3.3 K+ channels and are therefore candidate neurons that might be involved in these behavioral deficits. Hence, we investigated the synaptic mechanisms of transmission in the parallel fiber-Purkinje cell system. Action potentials of parallel fibers were broader in mice lacking both Kv3.1 and both Kv3.3 alleles and in mice lacking both Kv3.1 and a single Kv3.3 allele compared with those of wild-type mice. The transmission of high-frequency trains of action potentials was only impaired at 200 Hz but not at 100 Hz in mice lacking both Kv3.1 and Kv3.3 genes. However, paired-pulse facilitation (PPF) at parallel fiber-Purkinje cell synapses was dramatically reduced in a gene dose-dependent manner in mice lacking Kv3.1 or Kv3.3 alleles. Normal PPF could be restored by reducing the extracellular Ca2+ concentration indicating that increased activity-dependent presynaptic Ca2+ influx, at least in part caused the altered PPF in mutant mice. Induction of metabotropic glutamate receptor-mediated EPSCs was facilitated, whereas longterm depression was not impaired but rather facilitated in Kv3.1/Kv3.3 double-knockout mice. These results demonstrate the importance of Kv3 potassium channels in regulating the dynamics of synaptic transmission at the parallel fiber-Purkinje cell synapse and suggest a correlation between short-term plasticity at the parallel fiber-Purkinje cell synapse and motor performance.

  1. Rice Bran Dietary Supplementation Improves Neurological Symptoms and Loss of Purkinje Cells in Vitamin E-Deficient Mice

    PubMed Central

    Takahashi, Toru; Nakaso, Kazuhiro; Horikoshi, Yosuke; Hanaki, Takehiko; Yamakawa, Miho; Nakasone, Masato; Kitagawa, Yoshinori; Koike, Taisuke; Matsura, Tatsuya

    2016-01-01

    Background Vitamin E (VE, α-tocopherol) is a fat-soluble vitamin and is well known as an antioxidant. A deficiency in VE induces oxidative stress in the brain and causes motor and memory dysfunction. The consumption of a VE-rich diet has been given much attention in recent years, in regards to anti-aging and the prevention of age-related neuronal disorders. Methods A VE-deficient mouse model was prepared by feeding the animals a diet lacking VE. In addition, to evaluate the effect of VE-containing rice bran (RB) on VE deficiency, a diet including RB was also provided. VE levels in the brain tissue, as well as in the RB, were measured using an HPLC system. Behavioral tests, including rotarod, wheel running activity, Y-maze, and elevated plus maze were performed. To clarify the effect of VE deficiency and RB, we investigated the induction of heme oxygenase-1 (HO-1). Histological studies were performed using HE staining and immunohistochemical studies were performed using antibodies against glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1). Results VE in the mouse brain under a VE-deficient diet was decreased, and recovered α-tocopherol levels were observed in the brain of mice fed an RB diet. Motor behavioral scores were decreased in VE-deficient conditions, while the supplementation of RB improved motor function. HO-1, a marker of oxidative stress, was upregulated in the mouse brain under VE deficiency, however, RB supplementation inhibited the increase of HO-1. Histological analyses showed neuronal degeneration of Purkinje cells and decreased GFAP-immunoreactivity of Bergmann glia in the cerebellum. In addition, activated astrocytes and microglia were observed in mice fed the VE-deficient diet. Mice fed the RB diet showed improvement in these histological abnormalities. Conclusion A VE-deficient diet induced motor dysfunction in mice due to the degeneration of Purkinje cells in the cerebellum. Oral supplementation of RB

  2. Evidence that the loss of Purkinje cells and deep cerebellar nuclei neurons in homozygous weaver is not related to neurogenetic patterns.

    PubMed

    Martí, J; Wills, K V; Ghetti, B; Bayer, S A

    2001-10-01

    To determine whether the neurogenetic patterns of Purkinje cells and deep cerebellar nuclei neurons were normal in weaver homozygotes and whether the degeneration of those neuronal types was linked to their time of origin, [3H] thymidine autoradiography was applied on sections of homozygous weaver mice and normal controls on postnatal day 90. The experimental animals were the offspring of pregnant dams injected with [3H] thymidine on embryonic days 11-12, 12-13, 13-14 and 14-15. The results show that the onset of neurogenesis, its pattern of peaks and valleys, and its total span were similar between wild type and homozygous weaver in the cerebellar areas analyzed, indicating that the loss of Purkinje cells and deep cerebellar nuclei neurons is not related to neurogenetic patterns. In weaver homozygotes, the loss of Purkinje cells and deep cerebellar nuclei neurons followed a lateral to medial gradient of increasing severity. Thus, the vermis and the fastigial nucleus, which are medially located, presented the most important neuron loss, whereas in the lateral hemisphere and the dentate nucleus, neuron loss was spared.

  3. Postsynaptic inositol 1,4,5-trisphosphate signaling maintains presynaptic function of parallel fiber–Purkinje cell synapses via BDNF

    PubMed Central

    Furutani, Kazuharu; Okubo, Yohei; Kakizawa, Sho; Iino, Masamitsu

    2006-01-01

    The maintenance of synaptic functions is essential for neuronal information processing, but cellular mechanisms that maintain synapses in the adult brain are not well understood. Here, we report an activity-dependent maintenance mechanism of parallel fiber (PF)–Purkinje cell (PC) synapses in the cerebellum. When postsynaptic metabotropic glutamate receptor (mGluR) or inositol 1,4,5-trisphosphate (IP3) signaling was chronically inhibited in vivo, PF–PC synaptic strength decreased because of a decreased transmitter release probability. The same effects were observed when PF activity was inhibited in vivo by the suppression of NMDA receptor-mediated inputs to granule cells. PF–PC synaptic strength similarly decreased after the in vivo application of an antibody against brain-derived neurotrophic factor (BDNF). Furthermore, the weakening of synaptic connection caused by the blockade of mGluR–IP3 signaling was reversed by the in vivo application of BDNF. These results indicate that a signaling cascade comprising PF activity, postsynaptic mGluR–IP3 signaling and subsequent BDNF signaling maintains presynaptic functions in the mature cerebellum. PMID:16709674

  4. SLC26A11 (KBAT) in Purkinje Cells Is Critical for Inhibitory Transmission and Contributes to Locomotor Coordination123

    PubMed Central

    Xu, Jie; Hoebeek, Freek E.

    2016-01-01

    Abstract Chloride homeostasis determines the impact of inhibitory synaptic transmission and thereby mediates the excitability of neurons. Even though cerebellar Purkinje cells (PCs) receive a pronounced inhibitory GABAergic input from stellate and basket cells, the role of chloride homeostasis in these neurons is largely unknown. Here we studied at both the cellular and systems physiological level the function of a recently discovered chloride channel, SLC26A11 or kidney brain anion transporter (KBAT), which is prominently expressed in PCs. Using perforated patch clamp recordings of PCs, we found that a lack of KBAT channel in PC-specific KBAT KO mice (L7-KBAT KOs) induces a negative shift in the reversal potential of chloride as reflected in the GABAA-receptor-evoked currents, indicating a decrease in intracellular chloride concentration. Surprisingly, both in vitro and in vivo PCs in L7-KBAT KOs showed a significantly increased action potential firing frequency of simple spikes, which correlated with impaired motor performance on the Erasmus Ladder. Our findings support an important role for SLC26A11 in moderating chloride homeostasis and neuronal activity in the cerebellum. PMID:27390771

  5. Kv3.3b expression defines the shape of the complex spike in the Purkinje cell.

    PubMed

    Veys, Ken; Snyders, Dirk; De Schutter, Erik

    2013-01-01

    The complex spike (CS) in cerebellar Purkinje Cells (PC) is not an all-or-nothing phenomena as originally proposed, but shows variability depending on the spiking behavior of the Inferior Olive and intrinsic variability in the number and shape of spikelets. The potassium channel Kv3.3b, which has been proposed to undergo developmental changes during the postnatal PC maturation, has been shown to be crucial for the repolarization of the spikelets in the CS. We address here the regulation of the intrinsic CS variability by the expression of inactivating Kv3.3 channels in PCs by combining patch-clamp recordings and single-cell PCR methods on the same neurons, using a technique that we recently optimized to correlate single cell transcription levels with membrane ion channel electrophysiology. We show that while the inactivating TEA sensitive Kv3.3 current peak intensity increases with postnatal age, the channel density does not, arguing against postnatal developmental changes of Kv3.3b expression. Real time PCR of Kv3.3b showed a high variability from cell to cell, correlated with the Kv3.3 current density, and suggesting that there are no mechanisms regulating these currents beyond the mRNA pool. We show a significant correlation between normalized quantity of Kv3.3b mRNA and both the number of CS spikelets and their rate of voltage fluctuation, linking the intrinsic CS shape directly to the Kv3.3b mRNA pool. Comparing the observed cell-to-cell variance with studies on transcriptional noise suggests that fluctuations of the Kv3.3b mRNA pool are possibly not regulated but represent merely transcriptional noise, resulting in intrinsic variability of the CS.

  6. Nucleolar disruption and cajal body disassembly are nuclear hallmarks of DNA damage-induced neurodegeneration in purkinje cells.

    PubMed

    Baltanás, Fernando C; Casafont, Iñigo; Weruaga, Eduardo; Alonso, José R; Berciano, María T; Lafarga, Miguel

    2011-07-01

    The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.

  7. Downregulation of Purkinje Cell Activity by Modulators of Small Conductance Calcium-Activated Potassium Channels In Rat Cerebellum

    PubMed Central

    Karelina, T. V.; Stepanenko, Yu. D.; Abushik, P. A.; Sibarov, D. A.; Antonov, S. M.

    2016-01-01

    Small-conductance calcium-activated potassium channels (SK channels) are widely expressed in CNS tissues. Their functions, however, have not been well studied. Participation of SK channels in Purkinje cell (PC) pacemaker activity has been studied predominantly in vitro. Here we studied for the first time the effects of SK channel activation by NS309 or CyPPA on the PC simple spike frequency in vivo in adult (3 – 6 months) and aged (22 – 28 months) rats using extracellular microelectrode recordings. Both pharmacological agents caused a statistically significant decrease in the PC simple spike frequency. The maximum value of the decrease in the simple spike frequency did not depend on age, whereas a statistically significant inhibition of the spike frequency was achieved faster in aged animals than in adult ones. In experiments on cultured neurons PCs were identified by the expression of calbindin as the PC-specific marker. Registration of transmembrane currents in cerebellar neurons revealed the direct action of NS309 and CyPPA on the SK channels of PC consisted in the enhancement of outward potassium currents and action potential after-hyperpolarization. Thus, SK channel activators can compensate for age-related changes of the autorhythmic functions of the cerebellum. PMID:28050270

  8. Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice

    PubMed Central

    Blosa, M.; Bursch, C.; Weigel, S.; Holzer, M.; Jäger, C.; Janke, C.; Matthews, R. T.; Arendt, T.; Morawski, M.

    2016-01-01

    The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis. PMID:26819763

  9. Intracisternal Cyclodextrin Prevents Cerebellar Dysfunction and Purkinje Cell Death in Feline Niemann-Pick type C1 disease

    PubMed Central

    Vite, C. H.; Bagel, J. H.; Swain, G. P.; Prociuk, M.; Sikora, T. U.; Stein, V. M.; O’Donnell, P.; Ruane, T.; Ward, S.; Crooks, A.; Li, S.; Mauldin, E.; Stellar, S.; De Meulder, M.; Kao, M. L.; Ory, D. S.; Davidson, C.; Vanier, M. T.; Walkley, S. U.

    2015-01-01

    Niemann-Pick type C1 (NPC) disease is a lysosomal storage disease caused by mutations in the NPC1 gene, leading to an increase in unesterified cholesterol and several sphingolipids, and resulting in hepatic disease and progressive neurological disease. Whereas subcutaneous administration of the pharmaceutical excipient 2-hydroxypropyl-beta-cyclodextrin (HPβCD) ameliorated hepatic disease, doses sufficient to reduce neurological disease resulted in pulmonary toxicity. In contrast, direct administration of HPβCD into the cisterna magna of presymptomatic cats with NPC disease prevented the onset of cerebellar dysfunction for greater than a year and resulted in a reduction in Purkinje cell loss and near normal concentrations of cholesterol and sphingolipids. Moreover, administration of intracisternal HPβCD to NPC cats with ongoing cerebellar dysfunction slowed disease progression, increased survival time, and decreased the accumulation of brain gangliosides. An increase in hearing threshold was identified as a potential adverse effect. Together, these studies in the feline animal model have provided critical data on efficacy and safety of drug administration directly into the CNS that will be important for advancing HPβCD into clinical trials. PMID:25717099

  10. Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice.

    PubMed

    Blosa, M; Bursch, C; Weigel, S; Holzer, M; Jäger, C; Janke, C; Matthews, R T; Arendt, T; Morawski, M

    2016-01-01

    The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis.

  11. Type 1 inositol trisphosphate receptor regulates cerebellar circuits by maintaining the spine morphology of purkinje cells in adult mice.

    PubMed

    Sugawara, Takeyuki; Hisatsune, Chihiro; Le, Tung Dinh; Hashikawa, Tsutomu; Hirono, Moritoshi; Hattori, Mitsuharu; Nagao, Soichi; Mikoshiba, Katsuhiko

    2013-07-24

    The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP3R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)-PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP3R1 specifically in PCs (L7-Cre;Itpr1(flox/flox)) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF-PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itpr1(flox/flox) mice. Our findings reveal a specific role for IP3R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF-PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults.

  12. The same oculomotor vermal Purkinje cells encode the different kinematics of saccades and of smooth pursuit eye movements

    PubMed Central

    Sun, Zongpeng; Smilgin, Aleksandra; Junker, Marc; Dicke, Peter W.; Thier, Peter

    2017-01-01

    Saccades and smooth pursuit eye movements (SPEM) are two types of goal-directed eye movements whose kinematics differ profoundly, a fact that may have contributed to the notion that the underlying cerebellar substrates are separated. However, it is suggested that some Purkinje cells (PCs) in the oculomotor vermis (OMV) of monkey cerebellum may be involved in both saccades and SPEM, a puzzling finding in view of the different kinematic demands of the two types of eye movements. Such ‘dual’ OMV PCs might be oddities with little if any functional relevance. On the other hand, they might be representatives of a generic mechanism serving as common ground for saccades and SPEM. In our present study, we found that both saccade- and SPEM-related responses of individual PCs could be predicted well by linear combinations of eye acceleration, velocity and position. The relative weights of the contributions that these three kinematic parameters made depended on the type of eye movement. Whereas in the case of saccades eye position was the most important independent variable, it was velocity in the case of SPEM. This dissociation is in accordance with standard models of saccades and SPEM control which emphasize eye position and velocity respectively as the relevant controlled state variables. PMID:28091557

  13. Low-threshold Ca2+ currents in dendritic recordings from Purkinje cells in rat cerebellar slice cultures.

    PubMed

    Mouginot, D; Bossu, J L; Gähwiler, B H

    1997-01-01

    Voltage-dependent Ca2+ conductances were investigated in Purkinje cells in rat cerebellar slice cultures using the whole-cell and cell-attached configurations of the patch-clamp technique. In the presence of 0.5 mM Ca2+ in the extracellular solution, the inward current activated with a threshold of -55 +/- 1.5 mV and reached a maximal amplitude of 2.3 +/- 0.4 nA at -31 +/- 2 mV. Decay kinetics revealed three distinct components: a fast (24.6 +/- 2 msec time constant), a slow (304 +/- 46 msec time constant), and a nondecaying component. Rundown of the slow and sustained components of the current, or application of antagonists for the P/Q-type Ca2+ channels, allowed isolation of the fast-inactivating Ca2+ current, which had a threshold for activation of -60 mV and reached a maximal amplitude of 0.7 nA at a membrane potential of -33 mV. Both activation and steady-state inactivation of this fast-inactivating Ca2+ current were described with Boltzmann equations, with half-activation and inactivation at -51 mV and -86 mV, respectively. This Ca2+ current was nifedipine-insensitive, but its amplitude was reduced reversibly by bath-application of NiCl2 and amiloride, thus allowing its identification as a T-type Ca2+ current. Channels with a conductance of 7 pS giving rise to a fast T-type ensemble current (insensitive to omega-Aga-IVA) were localized with a high density on the dendritic membrane. Channel activity responsible for the ensemble current sensitive to omega-Aga-IVA was detected with 10 mM Ba2+ as the charge carrier. These channels were distributed with a high density on dendritic membranes and in rare cases were also seen in somatic membrane patches.

  14. Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

    PubMed

    Martí, Joaquín; Santa-Cruz, María C; Hervás, José P; Bayer, Shirley A; Villegas, Sandra

    2016-01-01

    Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally

  15. Mutational analysis of dendritic Ca2+ kinetics in rodent Purkinje cells: role of parvalbumin and calbindin D28k

    PubMed Central

    Schmidt, Hartmut; Stiefel, Klaus M; Racay, Peter; Schwaller, Beat; Eilers, Jens

    2003-01-01

    The mechanisms governing the kinetics of climbing fibre-mediated Ca2+ transients in spiny dendrites of cerebellar Purkinje cells (PCs) were quantified with high-resolution confocal Ca2+ imaging. Ca2+ dynamics in parvalbumin (PV−/−) and parvalbumin/calbindin D28k null-mutant (PV/CB−/−) mice were compared with responses in wild-type (WT) animals. In the WT, Ca2+ transients in dendritic shafts were characterised by double exponential decay kinetics that were not due to buffered Ca2+ diffusion or saturation of the indicator dye. Ca2+ transients in PV−/− PCs reached the same peak amplitude as in the WT but the biphasic nature of the decay was less pronounced, an effect that could be attributed to PV's slow binding kinetics. In contrast, peak amplitudes in PV/CB−/− PCs were about two times higher than in the WT and the decay became nearly monophasic. Numerical simulations indicate that the residual deviation from a single exponential decay in PV/CB−/− is due to saturation of the Ca2+ indicator dye. Furthermore, the simulations imply that the effect of uncharacterised endogenous Ca2+ binding proteins is negligible, that buffered diffusion and dye saturation significantly affects spineous Ca2+ transients but not those in the dendritic shafts, and that neither CB nor PV undergoes saturation in spines or dendrites during climbing fibre-evoked Ca2+ transients. Calbindin's medium-affinity binding sites are fast enough to reduce the peak amplitude of the Ca2+ signal. However, similar to PV, delayed binding by CB leads to biphasic Ca2+ decay kinetics. Our results suggest that the distinct kinetics of PV and CB underlie the biphasic kinetics of synaptically evoked Ca2+ transients in dendritic shafts of PCs. PMID:12813159

  16. Reassessment of long-term depression in cerebellar Purkinje cells in mice carrying mutated GluA2 C terminus

    PubMed Central

    Yamaguchi, Kazuhiko; Itohara, Shigeyoshi; Ito, Masao

    2016-01-01

    Long-term depression (LTD) of synaptic transmission from parallel fibers (PFs) to a Purkinje cell (PC) in the cerebellum has been considered to be a core mechanism of motor learning. Recently, however, discrepancies between LTD and motor learning have been reported in mice with a mutation that targeted the expression of PF–PC LTD by blocking AMPA-subtype glutamate receptor internalization regulated via the phosphorylation of AMPA receptors. In these mice, motor learning behavior was normal, but no PF–PC LTD was observed. We reexamined slices obtained from these GluA2 K882A and GluA2 Δ7 knockin mutants at 3–6 mo of age. The conventional protocols of stimulation did not induce LTD in these mutant mice, as previously reported, but surprisingly, LTD was induced using certain modified protocols. Such modifications involved increases in the number of PF stimulation (from one to two or five), replacement of climbing fiber stimulation with somatic depolarization (50 ms), filling a patch pipette with a Cs+-based solution, or extension of the duration of conjunction. We also found that intracellular infusion of a selective PKCα inhibitor (Gö6976) blocked LTD induction in the mutants, as in WT, suggesting that functional compensation occurred downstream of PKCα. The possibility that LTD in the mutants was caused by changes in membrane resistance, access resistance, or presynaptic property was excluded. The present results demonstrate that LTD is inducible by intensified conjunctive stimulations even in K882A and Δ7 mutants, indicating no contradiction against the LTD hypothesis of motor learning. PMID:27551099

  17. Interaction between Purkinje Cells and Inhibitory Interneurons May Create Adjustable Output Waveforms to Generate Timed Cerebellar Output

    PubMed Central

    Hong, Simon; Optican, Lance M.

    2008-01-01

    We develop a new model that explains how the cerebellum may generate the timing in classical delay eyeblink conditioning. Recent studies show that both Purkinje cells (PCs) and inhibitory interneurons (INs) have parallel signal processing streams with two time scales: an AMPA receptor-mediated fast process and a metabotropic glutamate receptor (mGluR)-mediated slow process. Moreover, one consistent finding is an increased excitability of PC dendrites (in Larsell's lobule HVI) in animals when they acquire the classical delay eyeblink conditioning naturally, in contrast to in vitro studies, where learning involves long-term depression (LTD). Our model proposes that the delayed response comes from the slow dynamics of mGluR-mediated IP3 activation, and the ensuing calcium concentration change, and not from LTP/LTD. The conditioned stimulus (tone), arriving on the parallel fibers, triggers this slow activation in INs and PC spines. These excitatory (from PC spines) and inhibitory (from INs) signals then interact at the PC dendrites to generate variable waveforms of PC activation. When the unconditioned stimulus (puff), arriving on the climbing fibers, is coupled frequently with this slow activation the waveform is amplified (due to an increased excitability) and leads to a timed pause in the PC population. The disinhibition of deep cerebellar nuclei by this timed pause causes the delayed conditioned response. This suggested PC-IN interaction emphasizes a richer role of the INs in learning and also conforms to the recent evidence that mGluR in the cerebellar cortex may participate in slow motor execution. We show that the suggested mechanism can endow the cerebellar cortex with the versatility to learn almost any temporal pattern, in addition to those that arise in classical conditioning. PMID:18648667

  18. Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia

    PubMed Central

    Quillinan, Nidia; Grewal, Himmat; Deng, Guiying; Shimizu, Kaori; Yonchek, Joan C; Strnad, Frank; Traystman, Richard J; Herson, Paco S

    2014-01-01

    Motor deficits are present in cardiac arrest survivors and injury to cerebellar Purkinje cells (PCs) likely contribute to impairments in motor coordination and post-hypoxic myoclonus. NMDA receptor mediated excitotoxicity is a well-established mechanism of cell death in several brain regions, but the role of NMDA receptors in PC injury remains understudied. Emerging data in cortical and hippocampal neurons indicates that the GluN2A-containing NMDA receptors signal to improve cell survival and GluN2B-containing receptors contribute to neuronal injury. This study compared neuronal injury in the hippocampal CA1 region to that in PCs and investigated the role of NMDA receptors in PC injury in our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Analysis of cell density demonstrated a 24% loss of PCs within 24 hours after 8 min CA/CPR and injury stabilized to 33% by 7 days. The subunit promiscuous NMDA receptor antagonist MK-801 protected both CA1 neurons and PCs from ischemic injury following CA/CPR, demonstrating a role for NMDA receptor activation in injury to both brain regions. In contrast, the GluN2B antagonist, Co 101244, had no effect on Purkinje cell loss while protecting against injury in the CA1 region. These data indicate that ischemic injury to cerebellar PCs progresses via different cell death mechanisms compared to hippocampal CA1 neurons. PMID:25450957

  19. Genetic ablation of homeodomain-interacting protein kinase 2 selectively induces apoptosis of cerebellar Purkinje cells during adulthood and generates an ataxic-like phenotype

    PubMed Central

    Anzilotti, S; Tornincasa, M; Gerlini, R; Conte, A; Brancaccio, P; Cuomo, O; Bianco, G; Fusco, A; Annunziato, L; Pignataro, G; Pierantoni, G M

    2015-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2−/− mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2−/− mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2−/− mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype. PMID:26633710

  20. Change of Na+ pump current reversal potential in sheep cardiac Purkinje cells with varying free energy of ATP hydrolysis.

    PubMed Central

    Glitsch, H G; Tappe, A

    1995-01-01

    1. The Na(+)-K+ pump current, Ip, of cardioballs from isolated sheep cardiac Purkinje cells was measured at 30-34 degrees C by means of whole-cell recording. 2. Under physiological conditions Ip is an outward current. Experimental conditions which cause a less negative free energy of intracellular ATP hydrolysis (delta GATP) and steeper sarcolemmal gradients for the pumped Na+ and Cs+ ions evoked an Ip in the inward direction over a wide range of membrane potentials. The reversal of the Ip direction was reversible. 3. The inwardly directed Ip increased with increasingly negative membrane potentials and amounted to -0.13 +/- 0.03 microA cm-2 (mean +/- S.E.M.; n = 6) at -95 mV. 4. The reversal potential (Erev) of Ip was studied as a function of delta GATP at constant sarcolemmal gradients of the pumped cations. 5. In order to vary delta GATP the cell interior was dialysed with patch pipette solutions containing 10 mM ATP and different concentrations of ADP and inorganic phosphate. The media were composed to produce delta GATP levels of about -58, -49 and -39 kJ mol-1. 6. A less negative delta GATP shifted Erev to more positive membrane potentials. From measurements of Ip as a function of membrane potential Erev was estimated to be -195, -115 and -60 mV at delta GATP levels of approximately -58, -49 and -39 kJ mol-1, respectively. The calculated Erev amounted to -224 mV at delta GATP approximately -58 kJ mol-1, -126 mV at delta GATP approximately 49 kJ mol-1 and -24 mV at delta GATP approximately -39 kJ mol-1. 7. Possible reasons for the discrepancy between estimated and calculated Erev values are discussed. 8. Shifting delta GATP to less negative values not only altered Erev but also diminished Ip at each membrane potential tested. The maximal Ip (Ip,max), which can be activated by external Cs+ (Cs+o), decreased under these conditions, whereas [Cs+]o causing half-maximal Ip activation remained unchanged. Similarly, the voltage dependence of Ip activation by Cs+o was

  1. The chromosome 16q-linked autosomal dominant cerebellar ataxia (16q-ADCA): A newly identified degenerative ataxia in Japan showing peculiar morphological changes of the Purkinje cell: The 50th Anniversary of Japanese Society of Neuropathology.

    PubMed

    Ishikawa, Kinya; Mizusawa, Hidehiro

    2010-10-01

    The chromosome 16q22.1-linked autosomal-dominant cerebellar ataxia (16q-ADCA) is a form of spinocerebellar ataxia (SCA) common in Japan. It is clinically characterized by late-onset purely cerebellar ataxia. The neuropathologic hallmark of 16q-ADCA is degeneration of Purkinje cells accompanied by an eosinophilic structure which we named "halo-like amorphous materials". By immunohistochemistry and electron microscopy, the structure has been so far found to contain two components: the somatic sprouts from the Purkinje cells and presynaptic terminals of unknown origin. As far as we are aware, this peculiar morphological change of Purkinje cells has not been previously described. Further investigations may disclose unique pathological processes in SCA.

  2. Developmental enhancement of alpha2-adrenoceptor-mediated suppression of inhibitory synaptic transmission onto mouse cerebellar Purkinje cells.

    PubMed

    Hirono, M; Matsunaga, W; Chimura, T; Obata, K

    2008-09-22

    Noradrenaline (NA) modulates glutamatergic and GABAergic transmission in various areas of the brain. It is reported that some alpha2-adrenoceptor subtypes are expressed in the cerebellar cortex and alpha2-adrenoceptors may play a role in motor coordination. Our previous study demonstrated that the selective alpha2-adrenoceptor agonist clonidine partially depresses spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse cerebellar Purkinje cells (PCs). Here we found that the inhibitory effect of clonidine on sIPSCs was enhanced during postnatal development. The activation of alpha2-adrenoceptors by clonidine did not affect sIPSCs in PCs at postnatal days (P) 8-10, when PCs showed a few sIPSCs and interneurons in the molecular layer (MLIs) did not cause action potential (AP). In the second postnatal week, the frequency of sIPSCs increased temporarily and reached a plateau at P14. By contrast, MLIs began to fire at P11 with the firing rate gradually increasing thereafter and reaching a plateau at P21. In parallel with this rise in the rate of firing, the magnitude of the clonidine-mediated inhibition of sIPSCs increased during postnatal development. Furthermore, the magnitude of the clonidine-mediated firing suppression in MLIs, which seemed to be mediated by a reduction in amplitude of the hyperpolarization-activated nonselective cation current, I(h), was constant across development. Both alpha2A- and alpha2B-, but not alpha2C-, adrenoceptors were strongly expressed in MLIs at P13, and P31. Therefore, the developmental enhancement of the clonidine-mediated inhibition of sIPSCs is attributed to an age-dependent increase in AP-derived sIPSCs, which can be blocked by clonidine. Thus, presynaptic activation of alpha2-adrenoceptors inhibits cerebellar inhibitory synaptic transmission after the second postnatal week, leading to a restriction of NA signaling, which is mainly mediated by alpha1- and beta2-adrenoceptors in the adult cerebellar neuronal circuit.

  3. Effect of trehalose on the properties of mutant {gamma}PKC, which causes spinocerebellar ataxia type 14, in neuronal cell lines and cultured Purkinje cells.

    PubMed

    Seki, Takahiro; Abe-Seki, Nana; Kikawada, Takahiro; Takahashi, Hideyuki; Yamamoto, Kazuhiro; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2010-10-22

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation, which induces apoptotic cell death. The disaccharide trehalose has been reported to inhibit aggregate formation and to alleviate symptoms in cellular and animal models of Huntington disease, Alzheimer disease, and prion disease. Here, we show that trehalose can be incorporated into SH-SY5Y cells and reduces the aggregation of mutant γPKC-GFP, thereby inhibiting apoptotic cell death in SH-SY5Y cells and primary cultured Purkinje cells (PCs). Trehalose acts by directly stabilizing the conformation of mutant γPKC without affecting protein turnover. Trehalose was also found to alleviate the improper development of dendrites in PCs expressing mutant γPKC-GFP without aggregates but not in PCs with aggregates. In PCs without aggregates, trehalose improves the mobility and translocation of mutant γPKC-GFP, probably by inhibiting oligomerization and thereby alleviating the improper development of dendrites. These results suggest that trehalose counteracts various cellular dysfunctions that are triggered by mutant γPKC in both neuronal cell lines and primary cultured PCs by inhibiting oligomerization and aggregation of mutant γPKC.

  4. The effects of a lengthy period of undernutrition from birth and subsequent nutritional rehabilitation on the granule-to-Purkinje cell ratio in the rat cerebellum.

    PubMed Central

    Warren, M A; Bedi, K S

    1988-01-01

    Male rats were undernourished for various lengths of time between birth and 150 days of age, with some rats being nutritionally rehabilitated between 75 and 150 days of age. Eight control and eight experimental rats were anaesthetised and perfused with 2.5% glutaraldehyde at each of 21, 75 and 150 days of age. Stereological procedures were used to estimate granule-to-Purkinje cell ratios in lobes IV, V and VI, using 0.5 micron thick toluidine blue-stained sections. Undernourished rats had significantly lower body and cerebellar weights than controls at all ages examined. These deficits persisted even after a period of nutritional rehabilitation. The granule-to-Purkinje cells ratio did not differ between control and experimental groups at 21 or 75 days of age. However, at 150 days both undernourished and rehabilitated groups of animals had significant deficits in this ratio compared with age-matched controls. These results suggest that undernutrition can have profound effects on brain development in later life even if the effects are not apparent during the period of undernutrition. PMID:3248962

  5. Single course of antenatal betamethasone produces delayed changes in morphology and calbindin-D28k expression in a rat's cerebellar Purkinje cells.

    PubMed

    Pascual, Rodrigo; Valencia, Martina; Larrea, Sebastián; Bustamante, Carlos

    2014-01-01

    In the current study, we analyzed the impact of antenatal betamethasone on macroscopic cerebellar development, Purkinje cell morphology and the expression of the neuroprotective protein calbindin-D28k. Pregnant rats (Sprague-Dawley) were randomly divided into two experimental groups: control (CONT) and betamethasone-treated (BET). At gestational day 20 (G20), BET dams were subcutaneously injected with a solution of 0.17 mg kg⁻¹ of betamethasone, while CONT animals received a similar volume of saline. At postnatal days 22 (P22) and P52, BET and CONT offspring were behaviorally evaluated, and the cerebella were histologically and immunohistochemically processed. Animals that were prenatally treated with a single course of betamethasone exhibited long-lasting behavioral changes consistent with anxiety-like behavior in the open-field test, together with (1) reduced cerebellar weight and volume, (2) Purkinje cell dendritic atrophy, and (3) an overexpression of calbindin-D28k. The current results indicate that an experimental single course of betamethasone in pregnant rats produces long-lasting anxiety-like behaviors, together with macroscopic and microscopic cerebellar alterations.

  6. Congenital multifocal increase of Purkinje fibres in a calf with cardiac conduction delay.

    PubMed

    Sakurai, M; Kuninaga, N; Takeuchi, T; Tsuka, T; Morita, T

    2014-01-01

    A female 4-month-old Holstein-Friesian calf was presented in heart failure. Microscopical examination of samples of the cardiac wall taken at necropsy examination revealed numerous aggregates of Purkinje fibres, particularly in the perivascular areas. Some Purkinje fibres were stained strongly with phosphotungstic acid haematoxylin and immunohistochemically were shown to express alpha smooth muscle actin, indicating an embryonic-like Purkinje fibre phenotype. A diagnosis of congenital multifocal increase of Purkinje fibres was made. The histological features of this case resemble multifocal cardiac Purkinje cell tumour of the heart in man.

  7. Mechanisms of extracellular divalent and trivalent cation block of the sodium current in canine cardiac Purkinje cells.

    PubMed Central

    Sheets, M F; Hanck, D A

    1992-01-01

    1. Single canine cardiac Purkinje cells were internally perfused and voltage clamped with a large-bore perfusion pipette for measurement of sodium ionic current (INa) in the absence and presence of extracellular group IIA divalent cations (Mg2+, Ba2+ and Ca2+), transition divalent cations (CO2+, Mn2+ and Ni2+), group IIB divalent cations (Cd2+ and Zn2+), and the trivalent cation La3+. 2. Open channel block of cardiac INa by external Ca2+, assessed from instantaneous INa-voltage (I-V) relationships, has been well described by a two-barrier, one-well model with a dissociation constant at 0 mV, KB(0), of 37 mM and an electrical distance, z' = delta, of 0.34. At the most negative test potentials there was less block of INa than predicted by the model, but correction of INa for the contribution of Na+ channel gating current (Ig) to the peak current improved the fit by the model. 3. The divalent cations Ba2+, Mg2+, CO2+ and Mn2+ produced voltage-dependent, open channel block of INa, which by the two-barrier, one-well model predicted a similar z' about one-third into the membrane field. The relative efficacy for voltage-dependent block was CO2+ > Mn2+ > Ca2+ > Mg2+ > Ba2+ with respective KB(0)s of 11, 13, 37, 43 and 61 mM. 4. Cd2+, Zn2+ and La3+ produced block of INa at low concentrations that was nearly voltage independent with z' < or = 0.13. Fits of single-site binding curves to peak INa in response to step depolarizations at positive test potentials gave the following apparent KD values: Zn2+ 0.14 mM, Cd2+ 0.27 mM and La3+ 0.50 mM. 5. In the presence of Cd2+, INa tail current relaxations were much faster than could be accounted for by Cd2+ binding to and/or screening of extracellular surface charges. Fits of the data to a model that assumed voltage-dependent open channel block during the tail current relaxations estimated the KB(0) for Cd2+ to be 0.80 mM. 6. Both z' and KB(0) for Ni2+ from fits of the two-barrier, one-well model to instantaneous I-V relationships

  8. Regional distribution of 5-HT transporters in the brain of wild type and 'Purkinje cell degeneration' mutant mice: a quantitative autoradiographic study with [3H]citalopram.

    PubMed

    Le Marec, N; Hébert, C; Amdiss, F; Botez, M I; Reader, T A

    1998-09-01

    The neurological mutant 'Purkinje cell degeneration' (pcd) is characterized by a primary degeneration of Purkinje cells, as well as by retrograde and secondary partial degeneration of cerebellar granule cells and inferior olivary neurons, and can be considered as an animal model of human degenerative ataxias. The serotonin (5-HT) innervation was examined in wild type and pcd mice, by quantifying 5-HT uptake sites, or transporters, using [3H]citalopram binding autoradiography. In both wild type and pcd mutants, the highest densities of 5-HT transporters were in mesencephalic and rostral pontine regions, in limbic structures, in hypothalamus and in discrete thalamic divisions, while the lowest labelling was found in cerebellum and brainstem reticular formation. In pcd mice, although [3H]citalopram labelling was higher in cerebellar cortex and deep cerebellar nuclei, when binding densities were corrected for surface area, the up-regulation of 5-HT transporters was present only in deep cerebellar nuclei. Also, higher labelling was found in nuclei raphe dorsalis and medialis, in ventral divisions of rostral neostriatum, caudal neostriatum, rostral globus pallidus, posteromedial amygdaloid nucleus, septum, olfactory tubercles, vertical limb of Broca's diagonal band, periventricular, latero-ventral and medio-ventral thalamic nuclei, medial geniculate nucleus, anterior hypothalamus and entorhinal cortex. The results indicate a relative integrity of the 5-HT innervation, but with a reorganization of serotoninergic terminals in the cerebellum, in particular in the deep cerebellar nuclei. This suggests that in progressive cerebellar degeneration, as found in the pcd mutant, the modified 5-HT system may still participate in motor functions by exerting an overall modulation of excitatory amino acid neurotransmission, but the availability of 5-HT may be altered in defined brain targets, as is the case for other spontaneous cerebellar mutants, in particular for the 'Lurcher

  9. Alterations in Purkinje cell GABAA receptor pharmacology following oxygen and glucose deprivation and cerebral ischemia reveal novel contribution of β1-subunit-containing receptors

    PubMed Central

    Kelley, Melissa H.; Ortiz, Justin; Shimizu, Kaori; Grewal, Himmat; Quillinan, Nidia; Herson, Paco S.

    2013-01-01

    Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABAA receptor function following injury is thought to contribute to PC sensitivity to ischemia-induced excitotoxicity. Here we examined the functional properties of the GABAA receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit-specific positive modulators of GABAA receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest-induced cerebral ischemia cause a decrease in sensitivity to the β2/3-subunit-preferring compound, etomidate. However, sensitivity to propofol, a β-subunit-acting compound that modulates β1–3-subunits, was not affected by OGD. The α/γ-subunit-act-ing compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single-cell reverse transcription–polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the β1-subunit, contrary to previous reports examining GABAA receptor subunit expression in PCs. GABAA receptor β1-subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 µm) inhibited PC GABA-mediated currents, as previously demonstrated with β1-subunit-containing GABAA receptors expressed in heterologous systems. From our data we conclude that PCs express the β1-subunit and that there is a greater contribution of β1-subunit-containing GABAA receptors following OGD. PMID:23176253

  10. Gating of long-term depression by Ca2+/calmodulin-dependent protein kinase II through enhanced cGMP signalling in cerebellar Purkinje cells

    PubMed Central

    Kawaguchi, Shin-ya; Hirano, Tomoo

    2013-01-01

    Long-term depression (LTD) at parallel fibre synapses on a cerebellar Purkinje cell has been regarded as a cellular basis for motor learning. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the LTD induction as an important Ca2+-sensing molecule, the underlying signalling mechanism remains unclear. Here, we attempted to explore the potential signalling pathway underlying the CaMKII involvement in LTD using a systems biology approach, combined with validation by electrophysiological and FRET imaging experiments on a rat cultured Purkinje cell. Model simulation predicted the following cascade as a candidate mechanism for the CaMKII contribution to LTD: CaMKII negatively regulates phosphodiesterase 1 (PDE1), subsequently facilitates the cGMP/protein kinase G (PKG) signalling pathway and down-regulates protein phosphatase 2A (PP-2A), thus supporting the LTD-inducing positive feedback loop consisting of mutual activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This model suggestion was corroborated by whole-cell patch clamp recording experiments. In addition, FRET measurement of intracellular cGMP concentration revealed that CaMKII activation causes sustained increase of cGMP, supporting the signalling mechanism of LTD induction by CaMKII. Furthermore, we found that activation of the cGMP/PKG pathway by nitric oxide (NO) can support LTD induction without activation of CaMKII. Thus, this study clarified interaction between NO and Ca2+/CaMKII, two important factors required for LTD. PMID:23297306

  11. Aberrant Purkinje cell activity is the cause of dystonia in a shRNA-based mouse model of Rapid Onset Dystonia-Parkinsonism.

    PubMed

    Fremont, Rachel; Tewari, Ambika; Khodakhah, Kamran

    2015-10-01

    Loss-of-function mutations in the α3 isoform of the sodium pump are responsible for Rapid Onset Dystonia-Parkinsonism (RDP). A pharmacologic model of RDP replicates the most salient features of RDP, and implicates both the cerebellum and basal ganglia in the disorder; dystonia is associated with aberrant cerebellar output, and the parkinsonism-like features are attributable to the basal ganglia. The pharmacologic agent used to generate the model, ouabain, is selective for sodium pumps. However, close to the infusion sites in vivo it likely affects all sodium pump isoforms. Therefore, it remains to be established whether selective loss of α3-containing sodium pumps replicates the pharmacologic model. Moreover, while the pharmacologic model suggested that aberrant firing of Purkinje cells was the main cause of abnormal cerebellar output, it did not allow the scrutiny of this hypothesis. To address these questions RNA interference using small hairpin RNAs (shRNAs) delivered via adeno-associated viruses (AAV) was used to specifically knockdown α3-containing sodium pumps in different regions of the adult mouse brain. Knockdown of the α3-containing sodium pumps mimicked both the behavioral and electrophysiological changes seen in the pharmacologic model of RDP, recapitulating key aspects of the human disorder. Further, we found that knockdown of the α3 isoform altered the intrinsic pacemaking of Purkinje cells, but not the neurons of the deep cerebellar nuclei. Therefore, acute knockdown of proteins associated with inherited dystonias may be a good strategy for developing phenotypic genetic mouse models where traditional transgenic models have failed to produce symptomatic mice.

  12. [Age-related changes in activity of cerebellum Purkinje cells, shape of the complex spike, and locomotion of wistar rats under effect of ethanol].

    PubMed

    Karelina, T V

    2012-01-01

    The work deals with study of peculiarities of effect of ethanol upon the Purkinje cell activity, shape of the complex spike, and locomotion of rats at different stages of ontogenesis, such as the stage of the morphofunstional maturation of the cerebellar cortex, the mature stage, and in the process of aging. The experiments were carried out on three age groups of Wistar rats: rat pups (2 weeks), adult rats (3-6 months), and senile animals (22-26 months). The administration of ethanol has been established to produce an increase in frequency of simple spikes, a decrease in frequency of complex spikes, a shortening of duration of depression of simple spikes, a decrease in the total duration of the complex spike, the number and frequency of its impulses as well as reduction of the motor activity of animals of all age groups. The change of the majority of the studied parameters occurred by the common temporal scheme. The earliest responding were the rat pups, later--the adult rats, and the last--the animals of the senior group. The stronger effect of ethanol was observed in adult rats. Their differences of all studied parameters, as compared with rat pups and senile animals, were characterized on the whole by the longer period of time and by the higher percent of changes relative to the initial values. Analysis of the obtained results has shown that the most pronounced changes in parameters of the cerebellum Purkinje cell activity and of the complex spike shape corresponded to the more significant decrease in the locomotion level, i. e., were recorded in adult rats. Thus, the work has demonstrated different sensitivity to administration of ethanol in the Wistar rats at different stages of ontogenetic development.

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

    PubMed Central

    Hoffman, Jessica F.; McCarthy, Margaret M.

    2016-01-01

    inflammatory insults. An intrinsic program of gene expression determines the critical period. The enduring consequences of inflammation during the second postnatal week are stunted dendrites of the cerebellum's principal neurons, Purkinje cells, and impairments in later social behavior. These changes are not evident if inflammation occurs during the first or third week, highlighting the importance of fine-grained analyses of developmental processes and the factors that influence them. PMID:27683901

  14. Kv3.3 channels harbouring a mutation of spinocerebellar ataxia type 13 alter excitability and induce cell death in cultured cerebellar Purkinje cells.

    PubMed

    Irie, Tomohiko; Matsuzaki, Yasunori; Sekino, Yuko; Hirai, Hirokazu

    2014-01-01

    The cerebellum plays crucial roles in controlling sensorimotor functions. The neural output from the cerebellar cortex is transmitted solely by Purkinje cells (PCs), whose impairment causes cerebellar ataxia. Spinocerebellar ataxia type 13 (SCA13) is an autosomal dominant disease, and SCA13 patients exhibit cerebellar atrophy and cerebellar symptoms. Recent studies have shown that missense mutations in the voltage-gated K(+) channel Kv3.3 are responsible for SCA13. In the rodent brain, Kv3.3 mRNAs are expressed most strongly in PCs, suggesting that the mutations severely affect PCs in SCA13 patients. Nevertheless, how these mutations affect the function of Kv3.3 in PCs and, consequently, the morphology and neuronal excitability of PCs remains unclear. To address these questions, we used lentiviral vectors to express mutant mouse Kv3.3 (mKv3.3) channels harbouring an R424H missense mutation, which corresponds to the R423H mutation in the Kv3.3 channels of SCA13 patients, in mouse cerebellar cultures. The R424H mutant-expressing PCs showed decreased outward current density, broadened action potentials and elevated basal [Ca(2+)]i compared with PCs expressing wild-type mKv3.3 subunits or those expressing green fluorescent protein alone. Moreover, expression of R424H mutant subunits induced impaired dendrite development and cell death selectively in PCs, both of which were rescued by blocking P/Q-type Ca(2+) channels in the culture conditions. We therefore concluded that expression of R424H mutant subunits in PCs markedly affects the function of endogenous Kv3 channels, neuronal excitability and, eventually, basal [Ca(2+)]i, leading to cell death. These results suggest that PCs in SCA13 patients also exhibit similar defects in PC excitability and induced cell death, which may explain the pathology of SCA13.

  15. High Frequency Burst Firing of Granule Cells Ensures Transmission at the Parallel Fiber to Purkinje Cell Synapse at the Cost of Temporal Coding

    PubMed Central

    van Beugen, Boeke J.; Gao, Zhenyu; Boele, Henk-Jan; Hoebeek, Freek; De Zeeuw, Chris I.

    2013-01-01

    Cerebellar granule cells (GrCs) convey information from mossy fibers (MFs) to Purkinje cells (PCs) via their parallel fibers (PFs). MF to GrC signaling allows transmission of frequencies up to 1 kHz and GrCs themselves can also fire bursts of action potentials with instantaneous frequencies up to 1 kHz. So far, in the scientific literature no evidence has been shown that these high-frequency bursts also exist in awake, behaving animals. More so, it remains to be shown whether such high-frequency bursts can transmit temporally coded information from MFs to PCs and/or whether these patterns of activity contribute to the spatiotemporal filtering properties of the GrC layer. Here, we show that, upon sensory stimulation in both un-anesthetized rabbits and mice, GrCs can show bursts that consist of tens of spikes at instantaneous frequencies over 800 Hz. In vitro recordings from individual GrC-PC pairs following high-frequency stimulation revealed an overall low initial release probability of ~0.17. Nevertheless, high-frequency burst activity induced a short-lived facilitation to ensure signaling within the first few spikes, which was rapidly followed by a reduction in transmitter release. The facilitation rate among individual GrC-PC pairs was heterogeneously distributed and could be classified as either “reluctant” or “responsive” according to their release characteristics. Despite the variety of efficacy at individual connections, grouped activity in GrCs resulted in a linear relationship between PC response and PF burst duration at frequencies up to 300 Hz allowing rate coding to persist at the network level. Together, these findings support the hypothesis that the cerebellar granular layer acts as a spatiotemporal filter between MF input and PC output (D’Angelo and De Zeeuw, 2009). PMID:23734102

  16. Effects of Gadolinium-Based Contrast Agents on Thyroid Hormone Receptor Action and Thyroid Hormone-Induced Cerebellar Purkinje Cell Morphogenesis

    PubMed Central

    Ariyani, Winda; Iwasaki, Toshiharu; Miyazaki, Wataru; Khongorzul, Erdene; Nakajima, Takahito; Kameo, Satomi; Koyama, Hiroshi; Tsushima, Yoshito; Koibuchi, Noriyuki

    2016-01-01

    Gadolinium (Gd)-based contrast agents (GBCAs) are used in diagnostic imaging to enhance the quality of magnetic resonance imaging or angiography. After intravenous injection, GBCAs can accumulate in the brain. Thyroid hormones (THs) are critical for the development and functional maintenance of the central nervous system. TH actions in brain are mainly exerted through nuclear TH receptors (TRs). We examined the effects of GBCAs on TR-mediated transcription in CV-1 cells using transient transfection-based reporter assay and TH-mediated cerebellar Purkinje cell morphogenesis in primary culture. We also measured the cellular accumulation and viability of Gd after representative GBCA treatments in cultured CV-1 cells. Both linear (Gd-diethylene triamine pentaacetic acid-bis methyl acid, Gd-DTPA-BMA) and macrocyclic (Gd-tetraazacyclododecane tetraacetic acid, Gd-DOTA) GBCAs were accumulated without inducing cell death in CV-1 cells. By contrast, Gd chloride (GdCl3) treatment induced approximately 100 times higher Gd accumulation and significantly reduced the number of cells. Low doses of Gd-DTPA-BMA (10−8 to 10−6M) augmented TR-mediated transcription, but the transcription was suppressed at higher dose (10−5 to 10−4M), with decreased β-galactosidase activity indicating cellular toxicity. TR-mediated transcription was not altered by Gd-DOTA or GdCl3, but the latter induced a significant reduction in β-galactosidase activity at high doses, indicating cellular toxicity. In cerebellar cultures, the dendrite arborization of Purkinje cells induced by 10−9M T4 was augmented by low-dose Gd-DTPA-BMA (10−7M) but was suppressed by higher dose (10−5M). Such augmentation by low-dose Gd-DTPA-BMA was not observed with 10−9M T3, probably because of the greater dendrite arborization by T3; however, the arborization by T3 was suppressed by a higher dose of Gd-DTPA-BMA (10−5M) as seen in T4 treatment. The effect of Gd-DOTA on dendrite arborization was much weaker

  17. Effect of long-chain triglyceride lipid emulsion on bupivacaine-induced changes in electrophysiological parameters of rabbit Purkinje cells.

    PubMed

    Lemoine, Sandrine; Rouet, René; Manrique, Alain; Hanouz, Jean-Luc

    2014-10-01

    Lipid emulsions are used in the reversal of local anesthetic toxicity. The aim of this study was to investigate the cellular electrophysiological effects of long-chain triglyceride lipid emulsion (LCTE) on cardiac action potential characteristics and conduction disturbances induced by bupivacaine. Purkinje fibers were dissected from the left ventricle of New Zealand white rabbit hearts and superfused with either Tyrode's solution during 30 min (control group), with bupivacaine 10(-6) M, 10(-5) M, and 5.10(-5) M alone, or in the presence of LCTE 0.5%, in addition, LCTE at 0.1%, 0.5%, and 1% was perfused alone. Electrophysiological parameters were recorded using the conventional microelectrode technique (37 °C, 1 Hz frequency). Bupivacaine 5.10(-5) M-induced conduction blocks (8/8 preparations): LCTE 0.5% suppressed the bupivacaine 5.10(-5) M-induced conduction blocks (1/8 preparations). Exposure to bupivacaine 10(-6) M, 10(-5) M, and 5.10(-5) M resulted in a significant decrease in the maximal rate of depolarization (Vmax) (respectively, 25%, 55%, 75%; P < 0.002 vs. control group). In the presence of LCTE 0.5%, bupivacaine 10(-6) M did not significantly decreased Vmax (13%; P = 0.10 vs. control group). The decrease in Vmax resulting from bupivacaine 10(-5) M alone was significantly less in the presence of LCTE 0.5% (P < 0.01 vs. bupivacaine 10(-5) M alone). Exposure to bupivacaine 10(-6) M, 10(-5) M, and 5.10(-5) M alone or in the presence of LCTE 0.5% resulted in a significant decrease in action potential duration measured at 50% and 90% repolarization (APD50 and APD90; P < 0.01 vs. control group). LCTE inhibited the Purkinje fibers conduction blocks induced by bupivacaine. Moreover, LCTE 0.5% attenuates the decrease in Vmax induced by bupivacaine 10(-6) M and 10(-5) M.

  18. Model-Founded Explorations of the Roles of Molecular Layer Inhibition in Regulating Purkinje Cell Responses in Cerebellar Cortex: More Trouble for the Beam Hypothesis

    PubMed Central

    Bower, James M.

    2010-01-01

    For most of the last 50 years, the functional interpretation for inhibition in cerebellar cortical circuitry has been dominated by the relatively simple notion that excitatory and inhibitory dendritic inputs sum, and if that sum crosses threshold at the soma the Purkinje cell generates an action potential. Thus, inhibition has traditionally been relegated to a role of sculpting, restricting, or blocking excitation. At the level of networks, this relatively simply notion is manifest in mechanisms like “surround inhibition” which is purported to “shape” or “tune” excitatory neuronal responses. In the cerebellum, where all cell types except one (the granule cell) are inhibitory, these assumptions regarding the role of inhibition continue to dominate. Based on our recent series of modeling and experimental studies, we now suspect that inhibition may play a much more complex, subtle, and central role in the physiological and functional organization of cerebellar cortex. This paper outlines how model-based studies are changing our thinking about the role of feed-forward molecular layer inhibition in the cerebellar cortex. The results not only have important implications for continuing efforts to understand what the cerebellum computes, but might also reveal important features of the evolution of this large and quintessentially vertebrate brain structure. PMID:20877427

  19. KV10.1 opposes activity-dependent increase in Ca2+ influx into the presynaptic terminal of the parallel fibre–Purkinje cell synapse

    PubMed Central

    Mortensen, Lena Sünke; Schmidt, Hartmut; Farsi, Zohreh; Barrantes-Freer, Alonso; Rubio, María E; Ufartes, Roser; Eilers, Jens; Sakaba, Takeshi; Stühmer, Walter; Pardo, Luis A

    2015-01-01

    The voltage-gated potassium channel KV10.1 (Eag1) is widely expressed in the mammalian brain, but its physiological function is not yet understood. Previous studies revealed highest expression levels in hippocampus and cerebellum and suggested a synaptic localization of the channel. The distinct activation kinetics of KV10.1 indicate a role during repetitive activity of the cell. Here, we confirm the synaptic localization of KV10.1 both biochemically and functionally and that the channel is sufficiently fast at physiological temperature to take part in repolarization of the action potential (AP). We studied the role of the channel in cerebellar physiology using patch clamp and two-photon Ca2+ imaging in KV10.1-deficient and wild-type mice. The excitability and action potential waveform recorded at granule cell somata was unchanged, while Ca2+ influx into axonal boutons was enhanced in mutants in response to stimulation with three APs, but not after a single AP. Furthermore, mutants exhibited a frequency-dependent increase in facilitation at the parallel fibre–Purkinje cell synapse at high firing rates. We propose that KV10.1 acts as a modulator of local AP shape specifically during high-frequency burst firing when other potassium channels suffer cumulative inactivation. PMID:25556795

  20. Cbln1 accumulates and colocalizes with Cbln3 and GluRdelta2 at parallel fiber-Purkinje cell synapses in the mouse cerebellum.

    PubMed

    Miura, Eriko; Matsuda, Keiko; Morgan, James I; Yuzaki, Michisuke; Watanabe, Masahiko

    2009-02-01

    Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluRdelta2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluRdelta2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluRdelta2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.

  1. Maturation of rat cerebellar Purkinje cells reveals an atypical Ca2+ channel current that is inhibited by omega-agatoxin IVA and the dihydropyridine (-)-(S)-Bay K8644.

    PubMed

    Tringham, Elizabeth W; Payne, C Elizabeth; Dupere, Jonathan R B; Usowicz, Maria M

    2007-02-01

    To determine if the properties of Ca2+ channels in cerebellar Purkinje cells change during postnatal development, we recorded Ca2+ channel currents from Purkinje cells in cerebellar slices of mature (postnatal days (P) 40-50) and immature (P13-20) rats. We found that at P40-50, the somatic Ca2+ channel current was inhibited by omega-agatoxin IVA at concentrations selective for P-type Ca2+ channels (approximately 85%; IC50, <1 nM) and by the dihydropyridine (-)-(S)-Bay K8644 (approximately 70%; IC50, approximately 40 nM). (-)-(S)-Bay K8644 is known to activate L-type Ca2+ channels, but the decrease in current was not secondary to the activation of L-type channels because inhibition by (-)-(S)-Bay K8644 persisted in the presence of the L-type channel blocker (R,S)-nimodipine. By contrast, at P13-20, the current was inhibited by omega-agatoxin IVA (approximately 86%; IC50, approximately 1 nM) and a minor component was inhibited by (R,S)-nimodipine (approximately 8%). The dihydropyridine (-)-(S)-Bay K8644 had no clear effect when applied alone, but in the presence of (R,S)-nimodipine it reduced the current (approximately 40%), suggesting that activation of L-type channels by (-)-(S)-Bay K8644 masks its inhibition of non-L-type channels. Our findings indicate that Purkinje neurons express a previously unrecognized type of Ca2+ channel that is inhibited by omega-agatoxin IVA, like prototypical P-type channels, and by (-)-(S)-Bay K8644, unlike classical P-type or L-type channels. During maturation, there is a decrease in the size of the L-type current and an increase in the size of the atypical Ca2+ channel current. These changes may contribute to the maturation of the electrical properties of Purkinje cells.

  2. Distinct physiological and developmental properties of hippocampal CA2 subfield revealed by using anti-Purkinje cell protein 4 (PCP4) immunostaining

    PubMed Central

    San Antonio, Andrew; Liban, Kristopher; Ikrar, Taruna; Tsyganovskiy, Eugene; Xu, Xiangmin

    2014-01-01

    The hippocampal CA2 subfield was initially identified by Lorente de Nó as an anatomically distinct region based on its cytoarchitectural features. Although there is an enormous body of literature on other hippocampal subfields (CA1 and CA3), relatively little is known about the physiological and developmental properties of CA2. Here we report identification of the CA2 region in the mouse by immunostaining with a Purkinje cell protein 4 (PCP4) antibody, which effectively delineates CA3/CA2 and CA2/CA1 borders and agrees well with previous cytoarchitectural definitions of CA2. The PCP4 immunostaining–delineated CA2 neurons have distinguishable differences in cell morphology, physiology, and synaptic circuit connections compared with distal CA3 and proximal CA1 regions. The average somatic sizes of excitatory cells differ across CA1–3, with the smallest to largest somatic size being CA1cells have dense dendritic spines, but do not have thorny excrescences associated with bordering CA3 neurons. Photostimulation functional circuit mapping shows that CA2 excitatory neurons receives extensive synaptic input from CA3, but no detectable input from the dentate gyrus. CA2 excitatory cells also differ significantly from CA3 cells in intrinsic electrophysiological parameters, such as membrane capacitance and spiking rates. Although CA2 neurons differ from CA1 neurons for PCP4 and other marker expressions, these neurons have less distinct neurophysiological and morphological properties. Developmental examination revealed that PCP4 immunostaining first appears at postnatal day 4–5 and becomes successively more refined around CA2 until reaching adult form by postnatal day 21. J. Comp. Neurol. J. Comp. Neurol. 522:1333–1354, 2014. © 2013 Wiley Periodicals, Inc. PMID:24166578

  3. Maternal marginal iodine deficiency limits dendritic growth of cerebellar purkinje cells in rat offspring by NF-κB signaling and MAP1B.

    PubMed

    Yu, Ye; Dong, Jing; Wang, Yuan; Wang, Yi; Min, Hui; Shan, Zhongyan; Teng, Weiping; Chen, Jie

    2017-04-01

    Iodine deficiency (ID) during early pregnancy had an adverse effect on children's psychomotor and motor function. It is worth noting that maternal marginal ID tends to be a common public health problem. Whether marginal ID potentially had adverse effects on the development of cerebellum and the underlying mechanisms remain unclear. Therefore, our aim was to study the effects of marginal ID on the dendritic growth in filial cerebellar Purkinje cells (PCs) and the underlying mechanism. In the present study, we established Wistar rat models by feeding dam rats with a diet deficient in iodine and deionized water supplemented with potassium iodide. We examined the total dendritic length using immunofluorescence, and Western blot analysis was conducted to investigate the activity of nuclear factor-κB (NF-κB) signaling and microtubule-associated protein 1B (MAP1B). Our results showed that marginal ID reduced the total dendritic length of cerebellar PCs, slightly down-regulated the activity of NF-κB signaling and decreased MAP1B in cerebellar PCs on postnatal day (PN) 7, PN14, and PN21. Our study may support the hypothesis that decreased T4 induced by marginal ID limits PCs dendritic growth, which may involve in the disturbance of NF-κB signaling and MAP1B on the cerebellum. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1241-1251, 2017.

  4. Developmental Hypothyroxinemia and Hypothyroidism Reduce Parallel Fiber-Purkinje Cell Synapses in Rat Offspring by Downregulation of Neurexin1/Cbln1/GluD2 Tripartite Complex.

    PubMed

    Wang, Yuan; Dong, Jing; Wang, Yi; Wei, Wei; Song, Binbin; Shan, Zhongyan; Teng, Weiping; Chen, Jie

    2016-10-01

    Iodine is a significant micronutrient. Iodine deficiency (ID)-induced hypothyroxinemia and hypothyroidism during developmental period can cause cerebellar dysfunction. However, mechanisms are still unclear. Therefore, the present research aims to study effects of developmental hypothyroxinemia caused by mild ID and hypothyroidism caused by severe ID or methimazole (MMZ) on parallel fiber-Purkinje cell (PF-PC) synapses in filial cerebellum. Maternal hypothyroxinemia and hypothyroidism models were established in Wistar rats using ID diet and deionized water supplemented with different concentrations of potassium iodide or MMZ water. Birth weight and cerebellum weight were measured. We also examined PF-PC synapses using immunofluorescence, and western blot analysis was conducted to investigate the activity of Neurexin1/cerebellin1 (Cbln1)/glutamate receptor d2 (GluD2) tripartite complex. Our results showed that hypothyroxinemia and hypothyroidism decreased birth weight and cerebellum weight and reduced the PF-PC synapses on postnatal day (PN) 14 and PN21. Accordingly, the mean intensity of vesicular glutamate transporter (VGluT1) and Calbindin immunofluorescence was reduced in mild ID, severe ID, and MMZ groups. Moreover, maternal hypothyroxinemia and hypothyroidism reduced expression of Neurexin1/Cbln1/GluD2 tripartite complex. Our study supports the hypothesis that developmental hypothyroxinemia and hypothyroidism reduce PF-PC synapses, which may be attributed to the downregulation of Neurexin1/Cbln1/GluD2 tripartite complex.

  5. 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.

  6. Differentiated pattern of sodium channel expression in dissociated Purkinje neurons maintained in long-term culture.

    PubMed

    Fry, Mark; Boegle, Aimee K; Maue, Robert A

    2007-05-01

    Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.

  7. Distribution and Structure of Synapses on Medial Vestibular Nuclear Neurons Targeted by Cerebellar Flocculus Purkinje Cells and Vestibular Nerve in Mice: Light and Electron Microscopy Studies

    PubMed Central

    Matsuno, Hitomi; Kudoh, Moeko; Watakabe, Akiya; Yamamori, Tetsuo; Shigemoto, Ryuichi; Nagao, Soichi

    2016-01-01

    Adaptations of vestibulo-ocular and optokinetic response eye movements have been studied as an experimental model of cerebellum-dependent motor learning. Several previous physiological and pharmacological studies have consistently suggested that the cerebellar flocculus (FL) Purkinje cells (P-cells) and the medial vestibular nucleus (MVN) neurons targeted by FL (FL-targeted MVN neurons) may respectively maintain the memory traces of short- and long-term adaptation. To study the basic structures of the FL-MVN synapses by light microscopy (LM) and electron microscopy (EM), we injected green florescence protein (GFP)-expressing lentivirus into FL to anterogradely label the FL P-cell axons in C57BL/6J mice. The FL P-cell axonal boutons were distributed in the magnocellular MVN and in the border region of parvocellular MVN and prepositus hypoglossi (PrH). In the magnocellular MVN, the FL-P cell axons mainly terminated on somata and proximal dendrites. On the other hand, in the parvocellular MVN/PrH, the FL P-cell axonal synaptic boutons mainly terminated on the relatively small-diameter (< 1 μm) distal dendrites of MVN neurons, forming symmetrical synapses. The majority of such parvocellular MVN/PrH neurons were determined to be glutamatergic by immunocytochemistry and in-situ hybridization of GFP expressing transgenic mice. To further examine the spatial relationship between the synapses of FL P-cells and those of vestibular nerve on the neurons of the parvocellular MVN/PrH, we added injections of biotinylated dextran amine into the semicircular canal and anterogradely labeled vestibular nerve axons in some mice. The MVN dendrites receiving the FL P-cell axonal synaptic boutons often closely apposed vestibular nerve synaptic boutons in both LM and EM studies. Such a partial overlap of synaptic boutons of FL P-cell axons with those of vestibular nerve axons in the distal dendrites of MVN neurons suggests that inhibitory synapses of FL P-cells may influence the function

  8. Hydroxyurea Treatment and Development of the Rat Cerebellum: Effects on the Neurogenetic Profiles and Settled Patterns of Purkinje Cells and Deep Cerebellar Nuclei Neurons.

    PubMed

    Martí, Joaquín; Santa-Cruz, M C; Serra, Roger; Hervás, José P

    2016-11-01

    The current paper analyzes the development of the male and female rat cerebellum exposed to hydroxyurea (HU) (300 or 600 mg/kg) as embryo and collected at postnatal day 90. Our study reveals that the administration of this drug compromises neither the cytoarchitecture of the cerebellar cortex nor deep nuclei (DCN). However, in comparison with the saline group, we observed that several cerebellar parameters were lower in the HU injected groups. These parameters included area of the cerebellum, cerebellar cortex length, molecular layer area, Purkinje cell number, granule cell counts, internal granular layer, white matter and cerebellar nuclei areas, and number of deep cerebellar nuclei neurons. These features were larger in the rats injected with saline, smaller in those exposed to 300 mg/kg of HU and smallest in the group receiving 600 mg/kg of this agent. No sex differences in the effect of the HU were observed. In addition, we infer the neurogenetic timetables and the neurogenetic gradients of PCs and DCN neurons in rats exposed to either saline or HU as embryos. For this purpose, 5-bromo-2'-deoxyuridine was injected into pregnant rats previously administered with saline or HU. This thymidine analog was administered following a progressively delayed cumulative labeling method. The data presented here show that systematic differences exist in the pattern of neurogenesis and in the spatial location of cerebellar neurons between rats injected with saline or HU. No sex differences in the effect of the HU were observed. These findings have implications for the administration of this compound to women in gestation as the effects of HU on the development of the cerebellum might persist throughout their offsprings' life.

  9. Territories of heterologous inputs onto Purkinje cell dendrites are segregated by mGluR1-dependent parallel fiber synapse elimination

    PubMed Central

    Ichikawa, Ryoichi; Hashimoto, Kouichi; Miyazaki, Taisuke; Uchigashima, Motokazu; Yamasaki, Miwako; Aiba, Atsu; Kano, Masanobu; Watanabe, Masahiko

    2016-01-01

    In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites. PMID:26858447

  10. Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia

    PubMed Central

    Willis, B. Cicero; Pandit, Sandeep V.; Ponce-Balbuena, Daniela; Zarzoso, Manuel; Guerrero-Serna, Guadalupe; Limbu, Bijay; Deo, Makarand; Camors, Emmanuel; Ramirez, Rafael J.; Mironov, Sergey; Herron, Todd J.; Valdivia, Héctor H.

    2016-01-01

    Background— In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca2+ dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2R4496C+/Cx40eGFP), we tested whether PC intracellular Ca2+ ([Ca2+]i) dysregulation results from a constitutive [Na+]i surplus relative to VMs. Methods and Results— Simultaneous optical mapping of voltage and [Ca2+]i in CPVT hearts showed that spontaneous Ca2+ release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca2+ imaging, early and delayed afterdepolarizations trailed spontaneous Ca2+ release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca2+ load, measured by caffeine-induced Ca2+ transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na+]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na+/Ca2+ exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na+]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca2+ release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na+]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca2+ spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na+]i played a central role. Conclusions— In CPVT mice, the constitutive [Na+]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs. PMID:27169737

  11. Mathematical model of bursting in dissociated purkinje neurons.

    PubMed

    Forrest, Michael D

    2013-01-01

    In vitro, Purkinje cell behaviour is sometimes studied in a dissociated soma preparation in which the dendritic projection has been cleaved. A fraction of these dissociated somas spontaneously burst. The mechanism of this bursting is incompletely understood. We have constructed a biophysical Purkinje soma model, guided and constrained by experimental reports in the literature, that can replicate the somatically driven bursting pattern and which hypothesises Persistent Na(+) current (INaP) to be its burst initiator and SK K(+) current (ISK) to be its burst terminator.

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

    PubMed

    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.

  13. Maturation of rat cerebellar Purkinje cells reveals an atypical Ca2+ channel current that is inhibited by ω-agatoxin IVA and the dihydropyridine (−)-(S)-Bay K8644

    PubMed Central

    Tringham, Elizabeth W; Payne, C Elizabeth; Dupere, Jonathan R B; Usowicz, Maria M

    2007-01-01

    To determine if the properties of Ca2+ channels in cerebellar Purkinje cells change during postnatal development, we recorded Ca2+ channel currents from Purkinje cells in cerebellar slices of mature (postnatal days (P) 40–50) and immature (P13–20) rats. We found that at P40–50, the somatic Ca2+ channel current was inhibited by ω-agatoxin IVA at concentrations selective for P-type Ca2+ channels (∼85%; IC50, <1 nm) and by the dihydropyridine (–)-(S)-Bay K8644 (∼70%; IC50, ∼40 nm). (−)-(S)-Bay K8644 is known to activate L-type Ca2+ channels, but the decrease in current was not secondary to the activation of L-type channels because inhibition by (−)-(S)-Bay K8644 persisted in the presence of the L-type channel blocker (R,S)-nimodipine. By contrast, at P13–20, the current was inhibited by ω-agatoxin IVA (∼86%; IC50, ∼1 nm) and a minor component was inhibited by (R,S)-nimodipine (∼8%). The dihydropyridine (−)-(S)-Bay K8644 had no clear effect when applied alone, but in the presence of (R,S)-nimodipine it reduced the current (∼40%), suggesting that activation of L-type channels by (−)-(S)-Bay K8644 masks its inhibition of non-L-type channels. Our findings indicate that Purkinje neurons express a previously unrecognized type of Ca2+ channel that is inhibited by ω-agatoxin IVA, like prototypical P-type channels, and by (−)-(S)-Bay K8644, unlike classical P-type or L-type channels. During maturation, there is a decrease in the size of the L-type current and an increase in the size of the atypical Ca2+ channel current. These changes may contribute to the maturation of the electrical properties of Purkinje cells. PMID:17124267

  14. IgG from Amyotrophic Lateral Sclerosis Patients Increases Current Through P-Type Calcium Channels in Mammalian Cerebellar Purkinje Cells and in Isolated Channel Protein in Lipid Bilayer

    NASA Astrophysics Data System (ADS)

    Llinas, R.; Sugimori, M.; Cherksey, B. D.; Smith, R. Glenn; Delbono, O.; Stefani, E.; Appel, S.

    1993-12-01

    The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IBa) in mammalian dissociated Purkinje cells and in isolated P-type calcium channels in lipid bilayers. Whole cell clamp of Purkinje cells demonstrates that ALS IgG increases the amplitude of IBa without modifying their voltage kinetics. This increased IBa could be blocked by a purified nonpeptide toxin from Agelenopsis aperta venom (purified funnel-web spider toxin) or by a synthetic polyamine analog (synthetic funnel-web spider toxin) and by a peptide toxin from the same spider venom, ω-Aga-IVA. Similar results were obtained on single-channel recordings from purified P channel protein. The addition of ALS IgG increased single-channel IBa open time without affecting slope conductance. The results described above were not seen with normal human IgG nor with boiled ALS IgG. It is concluded that ALS IgG enhances inward current through P-type calcium channels. Since P-type Ca2+ channels are present in motoneuron axon terminals, we propose that the enhanced calcium current triggered by ALS IgG may contribute to neuronal damage in ALS.

  15. Bergmann glial S100B activates myo-inositol monophosphatase 1 and Co-localizes to purkinje cell vacuoles in SCA1 transgenic mice.

    PubMed

    Vig, Parminder J S; Shao, Qingmei; Subramony, S H; Lopez, Mariper E; Safaya, Eshan

    2009-09-01

    Spinocerebellar ataxia-1 (SCA1) is a late onset neurodegenerative disease caused by the expansion of a polyglutamine repeat within ataxin-1 protein. The toxic effects triggered by mutant ataxin-1 result in degeneration of the neurons in cerebellum, brain stem and spinocerebellar tracts. The targeted overexpression of mutant ataxin-1 in cerebellar Purkinje cells (PCs) of the SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in PCs. These vacuoles appear early on before the onset of behavioral abnormalities. Interestingly, we found that vacoules contain S100B and vimentin proteins, which normally localize to neighboring Bergmann glia (BG). Further, immunohistochemical and specialized silver stain analysis revealed that vacuolar formation is associated with alterations in the morphology of dendritic spines of PCs. To gain insights into the mechanisms of vacuolar formation, we investigated if vacuoles in SCA1 PCs have an autophagic origin or are a consequence of some other event. We examined the expression levels (by Western blotting) of microtubule-associated protein light chain 3 (LC3)-I and LC3-II, and the degradation levels of p62 (a LC3 partner) in the cerebellar fractions prepared from pre-symptomatic SCA1 and age-matched wild-type mice. No p62 degradation was observed; however, LC3-II/(LC3-I + LC3-II) ratios were significantly altered in SCA1 mice indicating changes in the autophagic flux. In addition, LC3 localized to PC vacuoles. Further, we observed a co-localization of myo-inositol monophosphatase 1 (IMPA1) with S100B in PC vacuoles. IMPA1 is present in PC spines and has been implicated in autophagy. In vitro studies using purified IMPA1 and S100B demonstrated that S100B interacted with and activated IMPA1. Both apo and Ca(2+)-bound S100B were found to activate IMPA1, depending on substrate concentration. IMPA1 is regulated by another calcium-binding protein calbindin-D28k (CaB), since we reported earlier that the CaB levels are reduced

  16. The effects of black garlic (Allium sativum L.) ethanol extract on the estimated total number of Purkinje cells and motor coordination of male adolescent Wistar rats treated with monosodium glutamate.

    PubMed

    Aminuddin, M; Partadiredja, G; Sari, D C R

    2015-03-01

    A number of studies have indicated that monosodium glutamate (MSG) might cause negative effects on the nervous system, including in the cerebellum. Garlic (Allium sativum) has long been known as a flavouring agent and a traditional remedy for various illnesses. The present study aimed at investigating the effects of garlic on the motor coordination and the number of Purkinje cells present in rats treated with MSG. A total of 25 male Wistar rats aged 4 to 5 weeks old were used in this study and were divided into five groups, namely a negative control (C-) group, which received 0.9 % NaCl solution, a positive control (C+) group, which received MSG, and three treated groups, which received 2 mg/g bw of MSG and 2.5 mg (T2.5), 5 mg (T5), or 10 mg (T10) of black garlic solution per oral administration (per 200 g bw), respectively. All treatments were carried out for 10 days. Upon the end of the treatment, the motor performance of all rats were tested using the rotarod apparatus. The rats were subsequently sacrificed, and the cerebella of the rats were processed for stereological analyses. It has been found that the number of Purkinje cells of the cerebella of all treated groups were significantly higher than that of the group treated with MSG only. No changes in motor coordination function were observed as a result of MSG treatment.

  17. 1,2,5,6,9,10-αHexabromocyclododecane (HBCD) impairs thyroid hormone-induced dendrite arborization of Purkinje cells and suppresses thyroid hormone receptor-mediated transcription.

    PubMed

    Ibhazehiebo, Kingsley; Iwasaki, Toshiharu; Shimokawa, Noriaki; Koibuchi, Noriyuki

    2011-03-01

    1,2,5,6,9,10-αHexabromocyclododecane (HBCD) is a nonaromatic, brominated cyclic alkane used as an additive flame retardant. It bioaccumulates, persists in the environment, and has been detected in humans and wildlife. Its developmental neurotoxicity is of great concern. We investigated the effect of HBCD on thyroid hormone (TH) receptor (TR)-mediated transcription using transient transfection-based reporter gene assays and found that a low-dose (10(-10) M) HBCD suppressed TR-mediated transcription. We further examined the effect of HBCD on interaction of TR with TH response element (TRE) and found a partial dissociation of TR from TRE. HBCD did not dissociate steroid receptor coactivator-1 from TR in the presence of TH; neither did it recruit corepressors (N-CoR and SMRT) to TR in the absence of TH. Furthermore, low-dose HBCD (10(-10) M) significantly suppressed TH-induced dendrite arborization of Purkinje cells in primary cerebellar culture derived from newborn rat. These results show that low-dose HBCD can potentially disrupt TR-mediated transactivation and impairs Purkinje cell dendritogenesis, suggesting that HBCD can interfere with TH action in target organs, including the developing brain.

  18. 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.

  19. Purkinje image eyetracking: A market survey

    NASA Technical Reports Server (NTRS)

    Christy, L. F.

    1979-01-01

    The Purkinje image eyetracking system was analyzed to determine the marketability of the system. The eyetracking system is a synthesis of two separate instruments, the optometer that measures the refractive power of the eye and the dual Purkinje image eyetracker that measures the direction of the visual axis.

  20. Signals and Circuits in the Purkinje Neuron

    PubMed Central

    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. PMID:21980311

  1. 'Medusa-head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 1: Anti-mGluR1, anti-Homer-3, anti-Sj/ITPR1 and anti-CARP VIII.

    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.

  2. 'Medusa head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 2: Anti-PKC-gamma, anti-GluR-delta2, anti-Ca/ARHGAP26 and anti-VGCC.

    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 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.

  3. Nutrient shielding in clusters of cells

    NASA Astrophysics Data System (ADS)

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2013-06-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells' spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude among different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ.

  4. Nutrient Shielding in Clusters of Cells

    PubMed Central

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2014-01-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells’ spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude between different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ. PMID:23848711

  5. Effects on rabbit nodal, atrial, ventricular and Purkinje cell potentials of a new antiarrhythmic drug, cibenzoline, which protects against action potential shortening in hypoxia.

    PubMed Central

    Millar, J. S.; Vaughan Williams, E. M.

    1982-01-01

    1 The effects of cibenzoline (UP 339-01), a new anti-arrhythmic drug, have been investigated in various cardiac tissues. 2 UP 339-01 produced a bradycardia, due partly to prolongation of the intracellularly recorded sinus node action potential duration (APD) and partly to depression of the maximum rate of depolarization (MRD). The slope of the slow diastolic depolarization was not significantly reduced. 3 UP 339-01 was not a beta-adrenoceptor antagonist. 4 UP 339-01 was negatively inotropic, and shifted the relation between [Ca2+]o and force of contractions to the right, and increased A-H conduction time. It was concluded that UP 339-01 restricted slow inward current. 5 In all cardiac tissues depolarized by fast inward current, UP 339-01 caused a reduction in MRD and conduction velocity. The reduction was similar in atrial muscle, His and terminal Purkinje fibres, but in papillary muscle the effect was about half as great. On desheathed frog nerve UP 339-01 had a local anaesthetic potency slightly greater than that of procaine. 6 APD was significantly prolonged in a dose-related manner in ventricular muscle but to a lesser extent in the bundle of His and atrial tissue. In terminal Purkinje fibres APD50 and APD90 were unaltered, but the transient outward current ("notch') was abolished, resulting in a lengthening of APD20. 7 The effective and functional refractory periods of the A-V node and right bundle branch were both lengthened by UP 339-01 in a dose-related manner, and the difference between them was greatly increased. 8 UP 339-01 (2.63 microM) completely prevented the shortening of APD90 induced by hypoxia, and the shortening of APD50 and APD20 was much attenuated. There was no protection against hypoxic depression of contractions. 9 It was concluded that UP 339-01 is a highly active class 1 anti-arrhythmic agent with additional class 3 and 4 properties. PMID:7066601

  6. The pacemaker current in cardiac Purkinje myocytes

    PubMed Central

    1995-01-01

    It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time- dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time

  7. On the Firing Rate Dependency of the Phase Response Curve of Rat Purkinje Neurons In Vitro

    PubMed Central

    Couto, João; Linaro, Daniele; De Schutter, E; Giugliano, Michele

    2015-01-01

    Synchronous spiking during cerebellar tasks has been observed across Purkinje cells: however, little is known about the intrinsic cellular mechanisms responsible for its initiation, cessation and stability. The Phase Response Curve (PRC), a simple input-output characterization of single cells, can provide insights into individual and collective properties of neurons and networks, by quantifying the impact of an infinitesimal depolarizing current pulse on the time of occurrence of subsequent action potentials, while a neuron is firing tonically. Recently, the PRC theory applied to cerebellar Purkinje cells revealed that these behave as phase-independent integrators at low firing rates, and switch to a phase-dependent mode at high rates. Given the implications for computation and information processing in the cerebellum and the possible role of synchrony in the communication with its post-synaptic targets, we further explored the firing rate dependency of the PRC in Purkinje cells. We isolated key factors for the experimental estimation of the PRC and developed a closed-loop approach to reliably compute the PRC across diverse firing rates in the same cell. Our results show unambiguously that the PRC of individual Purkinje cells is firing rate dependent and that it smoothly transitions from phase independent integrator to a phase dependent mode. Using computational models we show that neither channel noise nor a realistic cell morphology are responsible for the rate dependent shift in the phase response curve. PMID:25775448

  8. Image-Based Structural Modeling of the Cardiac Purkinje Network

    PubMed Central

    Liu, Benjamin R.; Cherry, Elizabeth M.

    2015-01-01

    The Purkinje network is a specialized conduction system within the heart that ensures the proper activation of the ventricles to produce effective contraction. Its role during ventricular arrhythmias is less clear, but some experimental studies have suggested that the Purkinje network may significantly affect the genesis and maintenance of ventricular arrhythmias. Despite its importance, few structural models of the Purkinje network have been developed, primarily because current physical limitations prevent examination of the intact Purkinje network. In previous modeling efforts Purkinje-like structures have been developed through either automated or hand-drawn procedures, but these networks have been created according to general principles rather than based on real networks. To allow for greater realism in Purkinje structural models, we present a method for creating three-dimensional Purkinje networks based directly on imaging data. Our approach uses Purkinje network structures extracted from photographs of dissected ventricles and projects these flat networks onto realistic endocardial surfaces. Using this method, we create models for the combined ventricle-Purkinje system that can fully activate the ventricles through a stimulus delivered to the Purkinje network and can produce simulated activation sequences that match experimental observations. The combined models have the potential to help elucidate Purkinje network contributions during ventricular arrhythmias. PMID:26583120

  9. Electrogenic sodium extrusion in cardiac Purkinje fibers.

    PubMed

    Gadsby, D C; Cranefield, P F

    1979-06-01

    Thin canine cardiac Purkinje fibers in a fast flow chamber were exposed to K-free fluid for 15 s to 6 min to initiate "sodium loading," then returned to K-containing fluid to stimulate the sodium pump. The electrophysiological effects of enhanced pump activity may result from extracellular K depletion caused by enhanced cellular uptake of K or from an increase in the current generated as a result of unequal pumped movements of Na and K, or from both. The effects of pump stimulation were therefore studied under three conditions in which lowering the external K concentration ([K]0) causes changes opposite to those expected from an increase in pump current. First, the resting potential of Purkinje fibers may have either a "high" value of a "low" (less negative) value: at the low level of potential, experimental reduction of [K]0 causes depolarization, whereas an increase in pump current should cause hyperpolarization. Second, in regularly stimulated Purkinje fibers, lowering [K]0 prolongs the action potential, whereas an increase in outward pump current should shorten it. Finally, lowering [K]0 enhances spontaneous "pacemaker" activity in Purkinje fibers, whereas an increase in outward pump current should reduce or abolish spontaneous activity. Under all three conditions, we find that the effects of temporary stimulation of the sodium pump are those expected from a transient increase in outward pump current, not those expected from K depletion.

  10. Postnatal Loss of P/Q-type Channels Confined to Rhombic Lip Derived Neurons Alters Synaptic Transmission at the Parallel Fiber to Purkinje Cell Synapse and Replicates Genomic Cacna1a Mutation Phenotype of Ataxia and Seizures in Mice

    PubMed Central

    Maejima, Takashi; Wollenweber, Patric; Teusner, Lena U. C.; Noebels, Jeffrey L.; Herlitze, Stefan; Mark, Melanie D.

    2013-01-01

    Ataxia, episodic dyskinesia and thalamocortical seizures are associated with an inherited loss of P/Q-type voltage-gated Ca2+ channel function. P/Q-type channels are widely expressed throughout the neuraxis, obscuring identification of the critical networks underlying these complex neurological disorders. We recently showed that the conditional postnatal loss of P/Q-type channels in cerebellar Purkinje cells (PCs) in mice (purky) leads to these aberrant phenotypes, suggesting that intrinsic alteration in PC output is a sufficient pathogenic factor for disease initiation. The question arises whether P/Q-type channel deletion confined to a single upstream cerebellar synapse might induce the pathophysiological abnormality of genomically inherited P/Q-type channel disorders. PCs integrate two excitatory inputs, climbing fibers from inferior olive and parallel fibers (PFs) from granule cells (GCs) that receive mossy fiber (MF) input derived from precerebellar nuclei. In this paper, we introduce a new mouse model with a selective knock-out of P/Q-type channels in rhombic lip derived neurons including PF- and MF-pathways (quirky). We found that in quirky mice, PF-PC synaptic transmission is reduced during low-frequency stimulation. Using focal light stimulation of GCs that express optogenetic light-sensitive channels, channelrhodopsin-2, we found that modulation of PC firing via GC input is reduced in quirky mice. Phenotypic analysis revealed that quirky mice display ataxia, dyskinesia and absence epilepsy. These results suggest that developmental alteration of patterned input confined to only one of the main afferent cerebellar excitatory synaptic pathways has a significant role in generating the neurological phenotype associated with the global genomic loss of P/Q-type channel function. PMID:23516282

  11. Interaction of Kv3 potassium channels and resurgent sodium current influences the rate of spontaneous firing of Purkinje neurons.

    PubMed

    Akemann, Walther; Knöpfel, Thomas

    2006-04-26

    Purkinje neurons spontaneously generate action potentials in the absence of synaptic drive and thereby exert a tonic, yet plastic, input to their target cells in the deep cerebellar nuclei. Purkinje neurons express two ionic currents with biophysical properties that are specialized for high-frequency firing: resurgent sodium currents and potassium currents mediated by Kv3.3. How these ionic currents determine the intrinsic activity of Purkinje neurons has only partially been understood. Purkinje neurons from mutant mice lacking Kv3.3 have a reduced rate of spontaneous firing. Dynamic-clamp recordings demonstrated that normal firing rates are rescued by inserting artificial Kv3 currents into Kv3.3 knock-out Purkinje neurons. Numerical simulations indicated that Kv3.3 increases the spontaneous firing rate via cooperation with resurgent sodium currents. We conclude that the rate of spontaneous action potential firing of Purkinje neurons is controlled by the interaction of Kv3.3 potassium currents and resurgent sodium currents.

  12. Red blood cell clustering in Poiseuille microcapillary flow

    NASA Astrophysics Data System (ADS)

    Tomaiuolo, Giovanna; Lanotte, Luca; Ghigliotti, Giovanni; Misbah, Chaouqi; Guido, Stefano

    2012-05-01

    Red blood cells (RBC) flowing in microcapillaries tend to associate into clusters, i.e., small trains of cells separated from each other by a distance comparable to cell size. This process is usually attributed to slower RBCs acting to create a sequence of trailing cells. Here, based on the first systematic investigation of collective RBC flow behavior in microcapillaries in vitro by high-speed video microscopy and numerical simulations, we show that RBC size polydispersity within the physiological range does not affect cluster stability. Lower applied pressure drops and longer residence times favor larger RBC clusters. A limiting cluster length, depending on the number of cells in a cluster, is found by increasing the applied pressure drop. The insight on the mechanism of RBC clustering provided by this work can be applied to further our understanding of RBC aggregability, which is a key parameter implicated in clotting and thrombus formation.

  13. Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons.

    PubMed

    Eitan, Erez; Braverman, Carmel; Tichon, Ailone; Gitler, Daniel; Hutchison, Emmette R; Mattson, Mark P; Priel, Esther

    2016-08-01

    Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

  14. Purkinje fibers after myocardial ischemia-reperfusion.

    PubMed

    García Gómez-Heras, Soledad; Álvarez-Ayuso, Lourdes; Torralba Arranz, Amalia; Fernández-García, Héctor

    2015-07-01

    The purpose of this study was to evaluate the effects of ischemia-reperfusion on Purkinje fibers, comparing them with the adjacent cardiomyocytes. In a model of heterotopic heart transplantation in pigs, the donor heart was subjected to 2 hours of ischemia (n=9), preserved in cold saline, and subjected to 24 hours of ischemia with preservation in Wisconsin solution, alone (n=6), or with an additive consisting of calcium (n=4), Nicorandil (n=6) or Trolox (n=7). After 2 hours of reperfusion, we evaluated the recovery of cardiac electrical activity and took samples of ventricular myocardium for morphological study. The prolonged ischemia significantly affected atrial automaticity and A-V conduction in all the groups subjected to 24 hours of ischemia, as compared to 2 hours. There were no significant differences among the groups that underwent prolonged ischemia. Changes in the electrical activity did not correlate with the morphological changes. In the Purkinje fibers, ischemia-reperfusion produced a marked decrease in the glycogen content in all the groups. In the gap junctions the immunolabeling of connexin-43 decreased significantly, adopting a dispersed distribution, and staining the sarcolemma adjacent to the connective tissue. These changes were less marked in the group preserved exclusively with Wisconsin solution, despite the prolonged ischemia. The addition of other substances did not improve the altered morphology. In all the groups, the injury appeared to be more prominent in the Purkinje fibers than in the neighboring cardiomyocytes, indicating the greater susceptibility of the former to ischemia-reperfusion injury.

  15. Purkinje-related arrhythmias part I: monomorphic ventricular tachycardias.

    PubMed

    Nogami, Akihiko

    2011-05-01

    Purkinje-related monomorphic ventricular tachycardias (VTs) can be classified into four distinct groups: (1) verapamil-sensitive left fascicular VT, (2) Purkinje fiber-mediated VT post infarction, (3) bundle branch reentry (BBR) and interfascicular reentry VTs, and (4) focal Purkinje VT. There are three subtypes of fascicular VTs: (1) left posterior fascicular VT with a right bundle branch block (RBBB) configuration and superior axis; (2) left anterior fascicular VT with an RBBB configuration and right-axis deviation; and (3) upper septal fascicular VT with a narrow QRS configuration. The mechanism of the fascicular VT is macroreentry. While the antegrade limb of the circuit is a midseptal abnormal Purkinje fiber in the anterior and posterior fascicular VTs, the antegrade limb of the upper septal fascicular VT is both the anterior and posterior fascicles, and the retrograde limb is a midseptal abnormal Purkinje fiber. Purkinje fiber-mediated VT post infarction also exhibits verapamil sensitivity, and the surviving muscle bundles within the myocardium and Purkinje system are components of the reentry circuit. BBR-VT and interfascicular reentry VT are amenable to being cured by the creation of bundle or fascicular block. The mechanism of focal Purkinje VT is abnormal automaticity from the distal Purkinje system, and the ablation target is the earliest Purkinje activation during the VT. It is difficult to distinguish verapamil-sensitive fascicular VT from focal Purkinje VT by the 12-lead electrocardiogram; however, focal Purkinje VT is not responsive to verapamil . The recognition of the heterogeneity of these VTs and their unique characteristics should facilitate an appropriate diagnosis and therapy.

  16. Size-controlled insulin-secreting cell clusters.

    PubMed

    Mendelsohn, Adam D; Nyitray, Crystal; Sena, Mark; Desai, Tejal A

    2012-12-01

    The search for an effective cure for type I diabetes from the transplantation of encapsulated pancreatic β-cell clusters has so far produced sub-optimal clinical outcomes. Previous efforts have not controlled the size of transplanted clusters, a parameter implicated in affecting long-term viability and the secretion of therapeutically sufficient insulin. Here we demonstrate a method based on covalent attachment of patterned laminin for fabricating uniformly size-controlled insulin-secreting cell clusters. We show that cluster size within the range 40-120μm in diameter affects a variety of therapeutically relevant cellular responses including insulin expression, content and secretion. Our studies elucidate two size-dependent phenomena: (1) as the cluster size increases from 40μm to 60μm, glucose stimulation results in a greater amount of insulin produced per cell; and (2) as the cluster size increases beyond 60μm, sustained glucose stimulation results in a greater amount of insulin secreted per cell. Our study describes a method for producing uniformly sized insulin-secreting cell clusters, and since larger cluster sizes risk nutrient availability limitations, our data suggest that 100-120μm clusters may provide optimal viability and efficacy for encapsulated β-cell transplants as a treatment for type I diabetes and that further in vivo evaluation is warranted.

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

    SciTech Connect

    Collin, Ludovic; Doretto, Sandrine; Malerba, Monica; Ruat, Martial; Borrelli, Emiliana . E-mail: borrelli@uci.edu

    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, differentiation 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.

  18. Mapping of Purkinje neuron loss and polyglucosan body accumulation in hereditary cerebellar degeneration in Scottish terriers.

    PubMed

    Urkasemsin, G; Linder, K E; Bell, J S; de Lahunta, A; Olby, N J

    2012-09-01

    A hereditary cerebellar degenerative disorder has emerged in Scottish Terriers. The aims of this study were to describe and quantify polyglucosan body accumulation and quantify Purkinje neurons in the cerebellum of affected and control dogs. The brains of 6 affected Scottish Terriers ranging in age from 8 to 15 years and 8 age-matched control dogs were examined histopathologically. Counts of Purkinje neurons and polyglucosan bodies were performed in control and affected dogs on cerebellar sections stained with periodic acid-Schiff. Affected dogs showed a significant loss of Purkinje neurons compared with control dogs (vermis: P < .0001; hemisphere: P = .0104). The degeneration was significantly more pronounced dorsally than ventrally (P < .0001). There were significantly more polyglucosan bodies in the ventral half of the vermis when compared with the dorsal half (P < .0001) in affected dogs. In addition, there were more polyglucosan bodies in the ventral half of the vermis in affected dogs than in control dogs (P = .0005). Polyglucosan bodies in all affected dogs stained positively with toluidine blue and alcian blue. Immunohistochemically, polyglucosan bodies in affected dogs were positive for neurofilament 200 kD and ubiquitin and negative for glial fibrillary acidic protein, synaptophysin, neurospecific enolase, vimentin, and S100; the bodies were negative for all antigens in control dogs. Ultrastructurally, polyglucosan bodies in 1 affected dog were non-membrane-bound, amorphous structures with a dense core. This study demonstrates significant Purkinje cell loss and increased polyglucosan bodies in the cerebellum of affected Scottish Terriers.

  19. 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

  20. Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters

    PubMed Central

    Choong, Meng Ling; Tan, Shan Ho; Tan, Tuan Zea; Manesh, Sravanthy; Ngo, Anna; Yong, Jacklyn W. Y.; Yang, Henry He; Lee, May Ann

    2014-01-01

    Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy. PMID:25343454

  1. Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function

    PubMed Central

    Edgerton, Jeremy R; Reinhart, Peter H

    2003-01-01

    The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca2+ currents and Ca2+-activated K+ channels (KCa channels) is important for Purkinje cell activity, but how many different KCa channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca2+ channel and KCa channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca2+ entering through P-type voltage-gated Ca2+ channels activates both small-conductance (SK) and large-conductance (BK) KCa channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response. PMID:12576503

  2. Clusters of circulating tumor cells traverse capillary-sized vessels

    PubMed Central

    Au, Sam H.; Storey, Brian D.; Moore, John C.; Tang, Qin; Chen, Yeng-Long; Javaid, Sarah; Sarioglu, A. Fatih; Sullivan, Ryan; Madden, Marissa W.; O’Keefe, Ryan; Haber, Daniel A.; Maheswaran, Shyamala; Langenau, David M.; Stott, Shannon L.; Toner, Mehmet

    2016-01-01

    Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis. PMID:27091969

  3. Clusters of circulating tumor cells traverse capillary-sized vessels.

    PubMed

    Au, Sam H; Storey, Brian D; Moore, John C; Tang, Qin; Chen, Yeng-Long; Javaid, Sarah; Sarioglu, A Fatih; Sullivan, Ryan; Madden, Marissa W; O'Keefe, Ryan; Haber, Daniel A; Maheswaran, Shyamala; Langenau, David M; Stott, Shannon L; Toner, Mehmet

    2016-05-03

    Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.

  4. Ionic mechanisms of burst firing in dissociated Purkinje neurons.

    PubMed

    Swensen, Andrew M; Bean, Bruce P

    2003-10-22

    Cerebellar Purkinje neurons have intrinsic membrane properties that favor burst firing, seen not only during complex spikes elicited by climbing fiber input but also with direct electrical stimulation of cell bodies. We examined the ionic conductances that underlie all-or-none burst firing elicited in acutely dissociated mouse Purkinje neurons by short depolarizing current injections. Blocking voltage-dependent calcium entry by cadmium or replacement of external calcium by magnesium enhanced burst firing, but it was blocked by cobalt replacement of calcium, probably reflecting block of sodium channels. In voltage-clamp experiments, we used the burst waveform of each cell as a voltage command and used ionic substitutions and pharmacological manipulations to isolate tetrodotoxin (TTX)-sensitive sodium current, P-type and T-type calcium current, hyperpolarization-activated cation current (Ih), voltage-activated potassium current, large-conductance calcium-activated potassium current, and small-conductance calcium-activated potassium (SK) current. Measured near the middle of the first interspike interval, TTX-sensitive sodium current carried the largest inward current, and T-type calcium current was also substantial. Current through P-type channels was large immediately after a spike but decayed rapidly. These inward currents were opposed by substantial components of voltage-dependent and calcium-dependent potassium current. Termination of the burst is caused partly by decay of sodium current, together with a progressive buildup of SK current after the first interspike interval. Although burst firing depends on the net balance between multiple large currents flowing after a spike, it is surprisingly robust, probably reflecting complex interactions between the exact voltage waveform and voltage and calcium dependence of the various currents.

  5. Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin

    PubMed Central

    Pan, Weichun; Galkin, Oleg; Filobelo, Luis; Nagel, Ronald L.; Vekilov, Peter G.

    2007-01-01

    Sickle cell hemoglobin (HbS) is a mutant, whose polymerization while in deoxy state in the venous circulation underlies the debilitating sickle cell anemia. It has been suggested that the nucleation of the HbS polymers occurs within clusters of dense liquid, existing in HbS solutions. We use dynamic light scattering with solutions of deoxy-HbS, and, for comparison, of oxy-HbS and oxy-normal adult hemoglobin, HbA. We show that solutions of all three Hb variants contain clusters of dense liquid, several hundred nanometers in size, which are metastable with respect to the Hb solutions. The clusters form within a few seconds after solution preparation and their sizes and numbers remain relatively steady for up to 3 h. The lower bound of the cluster lifetime is 15 ms. The clusters exist in broad temperature and Hb concentration ranges, and occupy 10−5–10−2 of the solution volume. The results on the cluster properties can serve as test data for a potential future microscopic theory of cluster stability and kinetics. More importantly, if the clusters are a part of the nucleation mechanism of HbS polymers, the rate of HbS polymerization can be controlled by varying the cluster properties. PMID:17040989

  6. Challenges in imaging cell surface receptor clusters

    NASA Astrophysics Data System (ADS)

    Medda, Rebecca; Giske, Arnold; Cavalcanti-Adam, Elisabetta Ada

    2016-01-01

    Super-resolution microscopy offers unique tools for visualizing and resolving cellular structures at the molecular level. STED microscopy is a purely optical method where neither complex sample preparation nor mathematical post-processing is required. Here we present the use of STED microscopy for imaging receptor cluster composition. We use two-color STED to further determine the distribution of two different receptor subunits of the family of receptor serine/threonine kinases in the presence or absence of their ligands. The implications of receptor clustering on the downstream signaling are discussed, and future challenges are also presented.

  7. Time-variant clustering model for understanding cell fate decisions

    PubMed Central

    Huang, Wei; Cao, Xiaoyi; Biase, Fernando H.; Yu, Pengfei; Zhong, Sheng

    2014-01-01

    Both spatial characteristics and temporal features are often the subjects of concern in physical, social, and biological studies. This work tackles the clustering problems for time course data in which the cluster number and clustering structure change with respect to time, dubbed time-variant clustering. We developed a hierarchical model that simultaneously clusters the objects at every time point and describes the relationships of the clusters between time points. The hidden layer of this model is a generalized form of branching processes. A reversible-jump Markov Chain Monte Carlo method was implemented for model inference, and a feature selection procedure was developed. We applied this method to explore an open question in preimplantation embryonic development. Our analyses using single-cell gene expression data suggested that the earliest cell fate decision could start at the 4-cell stage in mice, earlier than the commonly thought 8- to 16-cell stage. These results together with independent experimental data from single-cell RNA-seq provided support against a prevailing hypothesis in mammalian development. PMID:25339442

  8. Statistical approaches and software for clustering islet cell functional heterogeneity

    PubMed Central

    Wills, Quin F.; Boothe, Tobias; Asadi, Ali; Ao, Ziliang; Warnock, Garth L.; Kieffer, Timothy J.

    2016-01-01

    ABSTRACT Worldwide efforts are underway to replace or repair lost or dysfunctional pancreatic β-cells to cure diabetes. However, it is unclear what the final product of these efforts should be, as β-cells are thought to be heterogeneous. To enable the analysis of β-cell heterogeneity in an unbiased and quantitative way, we developed model-free and model-based statistical clustering approaches, and created new software called TraceCluster. Using an example data set, we illustrate the utility of these approaches by clustering dynamic intracellular Ca2+ responses to high glucose in ∼300 simultaneously imaged single islet cells. Using feature extraction from the Ca2+ traces on this reference data set, we identified 2 distinct populations of cells with β-like responses to glucose. To the best of our knowledge, this report represents the first unbiased cluster-based analysis of human β-cell functional heterogeneity of simultaneous recordings. We hope that the approaches and tools described here will be helpful for those studying heterogeneity in primary islet cells, as well as excitable cells derived from embryonic stem cells or induced pluripotent cells. PMID:26909740

  9. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    PubMed Central

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited. PMID:28074895

  10. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    NASA Astrophysics Data System (ADS)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

  11. Loss of the golgin GM130 causes Golgi disruption, Purkinje neuron loss, and ataxia in mice

    PubMed Central

    Liu, Chunyi; Mei, Mei; Li, Qiuling; Pang, Qianqian; Ying, Zhengzhou; Gao, Fei; Lowe, Martin; Bao, Shilai

    2017-01-01

    The Golgi apparatus lies at the heart of the secretory pathway where it is required for secretory trafficking and cargo modification. Disruption of Golgi architecture and function has been widely observed in neurodegenerative disease, but whether Golgi dysfunction is causal with regard to the neurodegenerative process, or is simply a manifestation of neuronal death, remains unclear. Here we report that targeted loss of the golgin GM130 leads to a profound neurological phenotype in mice. Global KO of mouse GM130 results in developmental delay, severe ataxia, and postnatal death. We further show that selective deletion of GM130 in neurons causes fragmentation and defective positioning of the Golgi apparatus, impaired secretory trafficking, and dendritic atrophy in Purkinje cells. These cellular defects manifest as reduced cerebellar size and Purkinje cell number, leading to ataxia. Purkinje cell loss and ataxia first appear during postnatal development but progressively worsen with age. Our data therefore indicate that targeted disruption of the mammalian Golgi apparatus and secretory traffic results in neuronal degeneration in vivo, supporting the view that Golgi dysfunction can play a causative role in neurodegeneration. PMID:28028212

  12. Continuous network of endoplasmic reticulum in cerebellar Purkinje neurons.

    PubMed Central

    Terasaki, M; Slater, N T; Fein, A; Schmidek, A; Reese, T S

    1994-01-01

    Purkinje neurons in rat cerebellar slices injected with an oil drop saturated with 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3) or DiI] to label the endoplasmic reticulum were observed by confocal microscopy. DiI spread throughout the cell body and dendrites and into the axon. DiI spreading is due to diffusion in a continuous bilayer and is not due to membrane trafficking because it also spreads in fixed neurons. DiI stained such features of the endoplasmic reticulum as densities at branch points, reticular networks in the cell body and dendrites, nuclear envelope, spines, and aggregates formed during anoxia nuclear envelope, spines, and aggregates formed during anoxia in low extracellular Ca2+. In cultured rat hippocampal neurons, where optical conditions provide more detail, DiI labeled a clearly delineated network of endoplasmic reticulum in the cell body. We conclude that there is a continuous compartment of endoplasmic reticulum extending from the cell body throughout the dendrites. This compartment may coordinate and integrate neuronal functions. Images PMID:7519781

  13. Fibroblast morphogenesis on 3D collagen matrices: the balance between cell clustering and cell migration.

    PubMed

    da Rocha-Azevedo, Bruno; Grinnell, Frederick

    2013-10-01

    Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell-cell interactions. In vitro studies have shown that increasing the potential for cell-cell adhesion relative to cell-substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.

  14. FGF14 regulates the intrinsic excitability of cerebellar Purkinje neurons.

    PubMed

    Shakkottai, Vikram G; Xiao, Maolei; Xu, Lin; Wong, Michael; Nerbonne, Jeanne M; Ornitz, David M; Yamada, Kelvin A

    2009-01-01

    A missense mutation in the fibroblast growth factor 14 (FGF14) gene underlies SCA27, an autosomal dominant spinocerebellar ataxia in humans. Mice with a targeted disruption of the Fgf14 locus (Fgf14(-/-)) develop ataxia resembling human SCA27. We tested the hypothesis that loss of FGF14 affects the firing properties of Purkinje neurons, which play an important role in motor control and coordination. Current clamp recordings from Purkinje neurons in cerebellar slices revealed attenuated spontaneous firing in Fgf14(-/-) neurons. Unlike in the wild type animals, more than 80% of Fgf14(-/-) Purkinje neurons were quiescent and failed to fire repetitively in response to depolarizing current injections. Immunohistochemical examination revealed reduced expression of Nav1.6 protein in Fgf14(-/-) Purkinje neurons. Together, these observations suggest that FGF14 is required for normal Nav1.6 expression in Purkinje neurons, and that the loss of FGF14 impairs spontaneous and repetitive firing in Purkinje neurons by altering the expression of Nav1.6 channels.

  15. Resurgent sodium current and action potential formation in dissociated cerebellar Purkinje neurons.

    PubMed

    Raman, I M; Bean, B P

    1997-06-15

    Voltage-dependent sodium channels were studied in dissociated cerebellar Purkinje neurons from rats. In whole-cell recordings, a tetrodotoxin (TTX)-sensitive inward current was elicited when the membrane was repolarized to voltages between -60 and -20 mV after depolarizations to +30 mV long enough to produce maximal inactivation. At -40 mV, this "resurgent" current peaked in 8 msec and decayed with a time constant of 30 msec. With 50 mM sodium as a charge carrier, the resurgent current was on average approximately 120 pA. CA3 pyramidal neurons had no such current. The current may reflect recovery of inactivated channels through open states, because in Purkinje neurons (but not CA3 neurons) there was partial recovery from inactivation at -40 mV, coinciding with the rise of resurgent current. In single-channel recordings, individual channels gave openings corresponding to resurgent and conventional transient current. Action potentials were recorded from dissociated neurons under current clamp to investigate the role of the resurgent current in action potential formation. Purkinje neurons fired spontaneously at approximately 30 Hz. Hyperpolarization to -85 mV prevented spontaneous firing, and brief depolarization then induced all-or-none firing of conglomerate action potentials comprising three to four spikes. When conglomerate action potentials were used as command voltages in voltage-clamp experiments, TTX-sensitive sodium current was elicited between spikes. The falling phase of an action potential is similar to voltage patterns that activate resurgent sodium current, and thus, resurgent sodium current likely contributes to the formation of conglomerate action potentials in Purkinje neurons.

  16. '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.

  17. Ankyrin-based subcellular gradient of neurofascin, an immunoglobulin family protein, directs GABAergic innervation at purkinje axon initial segment.

    PubMed

    Ango, Fabrice; di Cristo, Graziella; Higashiyama, Hiroyuki; Bennett, Vann; Wu, Priscilla; Huang, Z Josh

    2004-10-15

    Distinct classes of GABAergic synapses are segregated into subcellular domains (i.e., dendrite, soma, and axon initial segment-AIS), thereby differentially regulating the input, integration, and output of principal neurons. In cerebellum, for example, basket interneurons make exquisitely precise "pinceau synapses" on AIS of Purkinje neurons, but the underlying mechanism is unknown. Using BAC transgenic reporter mice, we found that basket axons always contacted Purkinje soma before innervating AIS. This synapse targeting process followed the establishment of a subcellular gradient of neurofascin186 (NF186), an L1 family immunoglobulin cell adhesion molecule (L1CAM), along the Purkinje AIS-soma axis. This gradient was dependent on ankyrinG, an AIS-restricted membrane adaptor protein that recruits NF186. In the absence of neurofascin gradient, basket axons lost directional growth along Purkinje neurons and precisely followed NF186 to ectopic locations. Disruption of NF186-ankyrinG interactions at AIS reduced pinceau synapse formation. These results implicate ankyrin-based localization of L1CAMs in subcellular organization of GABAergic synapses.

  18. Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration

    SciTech Connect

    Furneaux, H.M.; Dropcho, E.J.; Barbut, D.; Chen, Yaotseng; Rosenblum, M.K.; Old, L.J.; Posner, J.B. )

    1989-04-01

    Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. The authors have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.

  19. Digital expression profiling of the compartmentalized translatome of Purkinje neurons

    PubMed Central

    Kratz, Anton; Beguin, Pascal; Kaneko, Megumi; Chimura, Takahiko; Suzuki, Ana Maria; Matsunaga, Atsuko; Kato, Sachi; Bertin, Nicolas; Lassmann, Timo; Vigot, Réjan; Carninci, Piero

    2014-01-01

    Underlying the complexity of the mammalian brain is its network of neuronal connections, but also the molecular networks of signaling pathways, protein interactions, and regulated gene expression within each individual neuron. The diversity and complexity of the spatially intermingled neurons pose a serious challenge to the identification and quantification of single neuron components. To address this challenge, we present a novel approach for the study of the ribosome-associated transcriptome—the translatome—from selected subcellular domains of specific neurons, and apply it to the Purkinje cells (PCs) in the rat cerebellum. We combined microdissection, translating ribosome affinity purification (TRAP) in nontransgenic animals, and quantitative nanoCAGE sequencing to obtain a snapshot of RNAs bound to cytoplasmic or rough endoplasmic reticulum (rER)–associated ribosomes in the PC and its dendrites. This allowed us to discover novel markers of PCs, to determine structural aspects of genes, to find hitherto uncharacterized transcripts, and to quantify biophysically relevant genes of membrane proteins controlling ion homeostasis and neuronal electrical activities. PMID:24904046

  20. A microfluidic device for label-free, physical capture of circulating tumor cell clusters.

    PubMed

    Sarioglu, A Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K; Miyamoto, David T; Luo, Xi; Bardia, Aditya; Wittner, Ben S; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David T; Stott, Shannon L; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel A; Toner, Mehmet

    2015-07-01

    Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.

  1. Highly sensitive magnetite nano clusters for MR cell imaging.

    PubMed

    Li, Mingli; Gu, Hongchen; Zhang, Chunfu

    2012-03-31

    High sensitivity and suitable sizes are essential for magnetic iron oxide contrast agents for cell imaging. In this study, we have fabricated highly MR sensitive magnetite nanoclusters (MNCs) with tunable sizes. These clusters demonstrate high MR sensitivity. Especially, water suspensions of the MNCs with average size of 63 nm have transverse relaxivity as high as 630 s-1mM-1, which is among the most sensitive iron oxide contrast agents ever reported. Importantly, such MNCs have no adverse effects on cells (RAW 264.7). When used for cell imaging, they demonstrate much higher efficiency and sensitivity than those of SHU555A (Resovist), a commercially available contrast agent, both in vitro and in vivo, with detection limits of 3,000 and 10,000 labeled cells, respectively. The studied MNCs are sensitive for cell imaging and promising for MR cell tracking in clinics.

  2. Highly sensitive magnetite nano clusters for MR cell imaging

    PubMed Central

    2012-01-01

    High sensitivity and suitable sizes are essential for magnetic iron oxide contrast agents for cell imaging. In this study, we have fabricated highly MR sensitive magnetite nanoclusters (MNCs) with tunable sizes. These clusters demonstrate high MR sensitivity. Especially, water suspensions of the MNCs with average size of 63 nm have transverse relaxivity as high as 630 s-1mM-1, which is among the most sensitive iron oxide contrast agents ever reported. Importantly, such MNCs have no adverse effects on cells (RAW 264.7). When used for cell imaging, they demonstrate much higher efficiency and sensitivity than those of SHU555A (Resovist), a commercially available contrast agent, both in vitro and in vivo, with detection limits of 3,000 and 10,000 labeled cells, respectively. The studied MNCs are sensitive for cell imaging and promising for MR cell tracking in clinics. PMID:22462693

  3. Parallel hyperbolic PDE simulation on clusters: Cell versus GPU

    NASA Astrophysics Data System (ADS)

    Rostrup, Scott; De Sterck, Hans

    2010-12-01

    Increasingly, high-performance computing is looking towards data-parallel computational devices to enhance computational performance. Two technologies that have received significant attention are IBM's Cell Processor and NVIDIA's CUDA programming model for graphics processing unit (GPU) computing. In this paper we investigate the acceleration of parallel hyperbolic partial differential equation simulation on structured grids with explicit time integration on clusters with Cell and GPU backends. The message passing interface (MPI) is used for communication between nodes at the coarsest level of parallelism. Optimizations of the simulation code at the several finer levels of parallelism that the data-parallel devices provide are described in terms of data layout, data flow and data-parallel instructions. Optimized Cell and GPU performance are compared with reference code performance on a single x86 central processing unit (CPU) core in single and double precision. We further compare the CPU, Cell and GPU platforms on a chip-to-chip basis, and compare performance on single cluster nodes with two CPUs, two Cell processors or two GPUs in a shared memory configuration (without MPI). We finally compare performance on clusters with 32 CPUs, 32 Cell processors, and 32 GPUs using MPI. Our GPU cluster results use NVIDIA Tesla GPUs with GT200 architecture, but some preliminary results on recently introduced NVIDIA GPUs with the next-generation Fermi architecture are also included. This paper provides computational scientists and engineers who are considering porting their codes to accelerator environments with insight into how structured grid based explicit algorithms can be optimized for clusters with Cell and GPU accelerators. It also provides insight into the speed-up that may be gained on current and future accelerator architectures for this class of applications. Program summaryProgram title: SWsolver Catalogue identifier: AEGY_v1_0 Program summary URL

  4. Impulse responses of automaticity in the Purkinje fiber.

    PubMed

    Chay, T R; Lee, Y S

    1984-04-01

    We examined the effects of brief current pulses on the pacemaker oscillations of the Purkinje fiber using the model of McAllister , Noble, and Tsien (1975. J. Physiol. [Lond.]. 251:1-57). This model was used to construct phase-response curves for brief electric stimuli to find "black holes," where rhythmic activity of the Purkinje fiber ceases. In our computer simulation, a brief current stimulus of the right magnitude and timing annihilated oscillations in membrane potential. The model also revealed a sequence of alternating periodic and chaotic regimes as the strength of a steady bias current is varied. We compared the results of our computer simulations with experimental work on Purkinje fibers and pointed out the importance of modeling results of this kind for understanding cardiac arrhythmias.

  5. Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters

    PubMed Central

    Bithi, Swastika S.; Vanapalli, Siva A.

    2017-01-01

    Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells. PMID:28150812

  6. Microglia-derived proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta induce Purkinje neuronal apoptosis via their receptors in hypoxic neonatal rat brain.

    PubMed

    Kaur, Charanjit; Sivakumar, Viswanathan; Zou, Zhirong; Ling, Eng-Ang

    2014-01-01

    The developing cerebellum is extremely vulnerable to hypoxia which can damage the Purkinje neurons. We hypothesized that this might be mediated by tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) derived from activated microglia as in other brain areas. One-day-old rats were subjected to hypoxia following, which the expression changes of various proteins in the cerebellum including hypoxia inducible factor-1α, TNF-α, IL-1β, TNF-R1 and IL-1R1 were analyzed. Following hypoxic exposure, TNF-α and IL-1β immunoexpression in microglia was enhanced coupled by that of TNF-R1 and IL-1R1 in the Purkinje neurons. Along with this, hypoxic microglia in vitro showed enhanced release of TNF-α and IL-1β whose receptor expression was concomitantly increased in the Purkinje neurons. In addition, nitric oxide (NO) level was significantly increased in the cerebellum and cultured microglia subjected to hypoxic exposure. Moreover, cultured Purkinje neurons treated with conditioned medium derived from hypoxic microglia underwent apoptosis but the incidence was significantly reduced when the cells were treated with the same medium that was neutralized with TNF-α/IL-1β antibody. We conclude that hypoxic microglia in the neonatal cerebellum produce increased amounts of NO, TNF-α and IL-1β which when acting via their respective receptors could induce Purkinje neuron death.

  7. Analysis of Transcriptional Profiles and Functional Clustering of Global Cerebellar Gene Expression in PCD3J Mice

    PubMed Central

    Ford, Gregory D.; Ford, Byron D.; Steele, Ernest C.; Gates, Alicia; Hood, Darryl; Matthews, Mika A.B.; Mirza, Sophia; MacLeish, Peter R.

    2008-01-01

    The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and Expression Analysis Systematic Explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders. PMID:18930027

  8. How the Purkinje System Determines the Ventricular Activation Sequence

    DTIC Science & Technology

    2007-11-02

    Zipes, Purkinje- muscle coupling and endocardial response to hyperkalemia , hypoxia, and acidosis Am.J.Physiol., vol. 247, pp. H303-H3111984. [12...R.D. Veenstra, R.W. Joyner, R.T. Wiedmann, M.L. Young, and R.C. Tan, Effects of hypoxia, hyperkalemia , and metabolic acidosis on canine subendocardial

  9. Maternal mobile phone exposure adversely affects the electrophysiological properties of Purkinje neurons in rat offspring.

    PubMed

    Haghani, M; Shabani, M; Moazzami, K

    2013-10-10

    Electromagnetic field (EMF) radiations emitted from mobile phones may cause structural damage to neurons. With the increased usage of mobile phones worldwide, concerns about their possible effects on the nervous system are rising. In the present study, we aimed to elucidate the possible effects of prenatal EMF exposure on the cerebellum of offspring Wistar rats. Rats in the EMF group were exposed to 900-MHz pulse-EMF irradiation for 6h per day during all gestation period. Ten offspring per each group were evaluated for behavioral and electrophysiological evaluations. Cerebellum-related behavioral dysfunctions were analyzed using motor learning and cerebellum-dependent functional tasks (Accelerated Rotarod, Hanging and Open field tests). Whole-cell patch clamp recordings were used for electrophysiological evaluations. The results of the present study failed to show any behavioral abnormalities in rats exposed to chronic EMF radiation. However, whole-cell patch clamp recordings revealed decreased neuronal excitability of Purkinje cells in rats exposed to EMF. The most prominent changes included afterhyperpolarization amplitude, spike frequency, half width and first spike latency. In conclusion, the results of the present study show that prenatal EMF exposure results in altered electrophysiological properties of Purkinje neurons. However, these changes may not be severe enough to alter the cerebellum-dependent functional tasks.

  10. Dendritic cell-nerve clusters are sites of T cell proliferation in allergic airway inflammation.

    PubMed

    Veres, Tibor Z; Shevchenko, Marina; Krasteva, Gabriela; Spies, Emma; Prenzler, Frauke; Rochlitzer, Sabine; Tschernig, Thomas; Krug, Norbert; Kummer, Wolfgang; Braun, Armin

    2009-03-01

    Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2'-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semi-automated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.

  11. Depletion induced clustering of red blood cells in microchannels

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Brust, Mathias; Podgorski, Thomas; Coupier, Gwennou

    2012-11-01

    The flow properties of blood are determined by the physical properties of its main constituents, the red blood cells (RBC's). At low shear rates RBC's form aggregates, so called rouleaux. Higher shear rates can break them up and the viscosity of blood shows a shear thinning behavior. The physical origin of the rouleaux formation is not yet fully resolved and there are two competing models available. One predicts that the adhesion is induced by bridging of the plasma (macromolecular) proteins in-between two RBC's. The other is based on the depletion effect and thus predicts the absence of macromolecules in-between the cells of a rouleaux. Recent single cell force measurements by use of an AFM support strongly the depletion model. By varying the concentration of Dextran at different molecular weights we can control the adhesions strength. Measurements at low hematocrit in a microfluidic channel show that the number of size of clusters is determined by the depletion induced adhesion strength.

  12. Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish

    PubMed Central

    Matsui, Takaaki; Ishikawa, Hiroshi; Bessho, Yasumasa

    2015-01-01

    Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called “collective cell migration,” is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as “cell collectivity,” remain largely unknown. During the formation of Kupffer's vesicle (KV, an organ of laterality in zebrafish), KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration. PMID:26000276

  13. Cluster-cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

    NASA Astrophysics Data System (ADS)

    Alves, S. G.; Martins, M. L.

    2010-09-01

    Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster-cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture.

  14. SC3: consensus clustering of single-cell RNA-seq data.

    PubMed

    Kiselev, Vladimir Yu; Kirschner, Kristina; Schaub, Michael T; Andrews, Tallulah; Yiu, Andrew; Chandra, Tamir; Natarajan, Kedar N; Reik, Wolf; Barahona, Mauricio; Green, Anthony R; Hemberg, Martin

    2017-03-27

    Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach (http://bioconductor.org/packages/SC3). We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.

  15. Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

    NASA Technical Reports Server (NTRS)

    Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

    2002-01-01

    Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

  16. Clustering of asbestos fibres in cell damage: A percolational perspective

    NASA Astrophysics Data System (ADS)

    Englman, Robert; Jaurand, Marie-Claude

    1997-03-01

    In vitro researches on rat cells exposed to several types of thin asbestos fibres show a saturation in cytotoxicity as one increases the fibre concentration n on the cell surface. For given average fibre lengths, the saturation occurs at values that are 2-3 times the critical concentration nc for a percolative arrangement of randomly thrown sticks on a surface. Measurements of the threshold for genotoxic damage give concentrations that are about 0.1nc. One expects that, somewhere between these concentrations, large scale "critical fluctuations" will be observed in the data. These fluctuations are indeed seen in chrysotile treated rat pleural mesothelial cells, exhibiting DNA damage and chromosomal-number aberrations. We hypothesize that at such concentrations that fibre-clustering occurs, the fibres lock together and are hindered from traversing the cell membranes and internalizing. Some damage processes are thereby impeded. The kinetics of internalization is worked out with models involving continuum percolation. Pieces of evidence from in vivo results that support the theory are noted.

  17. 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.

  18. Patterning of Mono- and Multi-layered Pancreatic β-cell Clusters

    PubMed Central

    Mendelsohn, Adam D.; Bernards, Daniel A.; Lowe, Rachel D.; Desai, Tejal A.

    2010-01-01

    Cluster-size dependent behavior of pancreatic β-cells has direct implications in islet transplantation therapy for type I diabetes treatment. Control over the cluster-size enables evaluation of cluster-size dependent function, ultimately leading to the production of β-cell clusters with improved transplant efficacy. This work for the first time demonstrates the use of microcontact printing-based cell patterning of discrete two and three-dimensional clusters of pancreatic β-cells. Both single and multiple cell layers are confined to a 2D area by attaching to patterns of covalently linked laminin and not adhering to surrounding polyethylene glycol. Cell clusters were successfully formed within 24 hours for printed patterns in the range 40-120 μm, and simple modulation of the initial cell seeding density leads to the formation of multiple cell layers. Semi-quantitative fluorescence microscopy, x-ray photoelectron spectroscopy, and fourier-transform infrared spectroscopy were used to extensively characterize the surface chemistry. This technique offers exceptional control over cell cluster shape and size and provides an effective tool to study not only the cluster-size dependent behavior of pancreatic β-cells, but also has potential applicability to numerous other cell lines. PMID:20218546

  19. Cluster-cell calculation using the method of generalized homogenization

    SciTech Connect

    Laletin, N.I.; Boyarinov, V.F.

    1988-05-01

    The generalized-homogenization method (GHM), used for solving the neutron transfer equation, was applied to calculating the neutron distribution in the cluster cell with a series of cylindrical cells with cylindrically coaxial zones. Single-group calculations of the technological channel of the cell of an RBMK reactor were performed using GHM. The technological channel was understood to be the reactor channel, comprised of the zirconium rod, the water or steam-water mixture, the uranium dioxide fuel element, and the zirconium tube, together with the adjacent graphite layer. Calculations were performed for channels with no internal sources and with unit incoming current at the external boundary as well as for channels with internal sources and zero current at the external boundary. The PRAKTINETs program was used to calculate the symmetric neutron distributions in the microcell and in channels with homogenized annular zones. The ORAR-TsM program was used to calculate the antisymmetric distribution in the microcell. The accuracy of the calculations were compared for the two channel versions.

  20. Transplanted hematopoietic cells seed in clusters in recipient bone marrow in vivo.

    PubMed

    Askenasy, Nadir; Zorina, Tatiana; Farkas, Daniel L; Shalit, Itamar

    2002-01-01

    The process of hematopoietic stem and progenitor cell (HSPC) seeding in recipient bone marrow (BM) early after transplantation is not fully characterized. In vivo tracking of HSPCs, labeled with PKH dyes, through an optical window surgically implanted on the mouse femur revealed that transplanted cells cluster in the recipient BM. Within the first day after intravenous injection, 86 +/- 6% of the cells seeded in clusters (p < 0.001 versus scattered cells) in the endosteal surfaces of the epiphyses. The primary clusters were formed by concomitant seeding of 6-10 cells over an area of approximately 70 microm, and secondarily injected cells did not join the already existing clusters but formed new clusters. Major antigen-disparate HSPCs participated in formation of the primary clusters, and T lymphocytes were also incorporated. After 4 to 5 days, some cellular clusters were observed in the more central regions of the BM, where the brightness of PKH fluorescence decreased, indicating cellular division. These later clusters were classified as secondary, assuming that the mechanisms of migration in the BM might be different from those of primary seeding. Some clusters remained in the periphery of the BM and retained bright fluorescence, indicating cellular quiescence. The number of brightly fluorescent cells in the clusters decreased exponentially to two to three cells after 24 days (p < 0.001). The data suggest that the hematopoietic niche is a functional unit of the BM stromal microenvironment that hosts seeding of a number of transplanted cells, which form a cluster. This may be the site where auxiliary non-HSPC cells, such as T lymphocytes, act in support of HSPC engraftment.

  1. Circulating tumor cell clusters: What we know and what we expect (Review)

    PubMed Central

    Hong, Yupeng; Fang, Francia; Zhang, Qi

    2016-01-01

    The major cause of cancer-associated mortality is tumor metastasis, a disease that is far from understood. Many studies have observed circulating tumor cells (CTCs) in patients' circulation systems, and a few latest investigations showed that CTC clusters have a potentially high capacity of metastasis. The capture and analysis of CTC clusters offer new insights into tumor metastasis and can facilitate the development of cancer treatments. We reviewed the research history of the CTC clusters, as well as the technologies used for detecting and isolating CTC clusters. In addition, we discuss the characteristics of CTC clusters and their roles in tumor dissemination. Clinical relevance of CTC clusters was also implicated in currently limited data. Moving forward, the next frontier in this field is to develop more efficient capture methods and decipher conundrums of characterization of CTC clusters. This will ultimately identify the clinical value of CTC clusters as a biomarker and therapeutic target. PMID:27779656

  2. A microfluidic device for label-free, physical capture of circulating tumor cell-clusters

    PubMed Central

    Sarioglu, A. Fatih; Aceto, Nicola; Kojic, Nikola; Donaldson, Maria C.; Zeinali, Mahnaz; Hamza, Bashar; Engstrom, Amanda; Zhu, Huili; Sundaresan, Tilak K.; Miyamoto, David T.; Luo, Xi; Bardia, Aditya; Wittner, Ben S.; Ramaswamy, Sridhar; Shioda, Toshi; Ting, David T.; Stott, Shannon L.; Kapur, Ravi; Maheswaran, Shyamala; Haber, Daniel A.; Toner, Mehmet

    2015-01-01

    Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identify CTC-clusters in 30–40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. PMID:25984697

  3. Single-neuron diversity generated by Protocadherin-β cluster in mouse central and peripheral nervous systems.

    PubMed

    Hirano, Keizo; Kaneko, Ryosuke; Izawa, Takeshi; Kawaguchi, Masahumi; Kitsukawa, Takashi; Yagi, Takeshi

    2012-01-01

    The generation of complex neural circuits depends on the correct wiring of neurons with diverse individual characteristics. To understand the complexity of the nervous system, the molecular mechanisms for specifying the identity and diversity of individual neurons must be elucidated. The clustered protocadherins (Pcdh) in mammals consist of approximately 50 Pcdh genes (Pcdh-α, Pcdh-β, and Pcdh-γ) that encode cadherin-family cell surface adhesion proteins. Individual neurons express a random combination of Pcdh-α and Pcdh-γ, whereas the expression patterns for the Pcdh-β genes, 22 one-exon genes in mouse, are not fully understood. Here we show that the Pcdh-β genes are expressed in a 3'-polyadenylated form in mouse brain. In situ hybridization using a pan-Pcdh-β probe against a conserved Pcdh-β sequence showed widespread labeling in the brain, with prominent signals in the olfactory bulb, hippocampus, and cerebellum. In situ hybridization with specific probes for individual Pcdh-β genes showed their expression to be scattered in Purkinje cells from P10 to P150. The scattered expression patterns were confirmed by performing a newly developed single-cell 3'-RACE analysis of Purkinje cells, which clearly demonstrated that the Pcdh-β genes are expressed monoallelically and combinatorially in individual Purkinje cells. Scattered expression patterns of individual Pcdh-β genes were also observed in pyramidal neurons in the hippocampus and cerebral cortex, neurons in the trigeminal and dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous observations of diversity at the single-neuron level generated by Pcdh expression and suggest that the Pcdh-β cluster genes contribute to specifying the identity and diversity of individual neurons.

  4. Three-Dimensional Analysis of the Effect of Epidermal Growth Factor on Cell-Cell Adhesion in Epithelial Cell Clusters

    PubMed Central

    Notbohm, J.; Kim, J.-H.; Asthagiri, A.R.; Ravichandran, G.

    2012-01-01

    The effect that growth factors such as epidermal growth factor (EGF) have on cell-cell adhesion is of interest in the study of cellular processes such as epithelial-mesenchymal transition. Because cell-cell adhesions cannot be measured directly, we use three-dimensional traction force microscopy to measure the tractions applied by clusters of MCF-10A cells to a compliant substrate beneath them before and after stimulating the cells with EGF. To better interpret the results, a finite element model, which simulates a cluster of individual cells adhered to one another and to the substrate with linear springs, is developed to better understand the mechanical interaction between the cells in the experiments. The experiments and simulations show that the cluster of cells acts collectively as a single unit, indicating that cell-cell adhesion remains strong before and after stimulation with EGF. In addition, the experiments and model emphasize the importance of three-dimensional measurements and analysis in these experiments. PMID:22455915

  5. Slowing down light using a dendritic cell cluster metasurface waveguide

    NASA Astrophysics Data System (ADS)

    Fang, Z. H.; Chen, H.; Yang, F. S.; Luo, C. R.; Zhao, X. P.

    2016-11-01

    Slowing down or even stopping light is the first task to realising optical information transmission and storage. Theoretical studies have revealed that metamaterials can slow down or even stop light; however, the difficulty of preparing metamaterials that operate in visible light hinders progress in the research of slowing or stopping light. Metasurfaces provide a new opportunity to make progress in such research. In this paper, we propose a dendritic cell cluster metasurface consisting of dendritic structures. The simulation results show that dendritic structure can realise abnormal reflection and refraction effects. Single- and double-layer dendritic metasurfaces that respond in visible light were prepared by electrochemical deposition. Abnormal Goos-Hänchen (GH) shifts were experimentally obtained. The rainbow trapping effect was observed in a waveguide constructed using the dendritic metasurface sample. The incident white light was separated into seven colours ranging from blue to red light. The measured transmission energy in the waveguide showed that the energy escaping from the waveguide was zero at the resonant frequency of the sample under a certain amount of incident light. The proposed metasurface has a simple preparation process, functions in visible light, and can be readily extended to the infrared band and communication wavelengths.

  6. Slowing down light using a dendritic cell cluster metasurface waveguide.

    PubMed

    Fang, Z H; Chen, H; Yang, F S; Luo, C R; Zhao, X P

    2016-11-25

    Slowing down or even stopping light is the first task to realising optical information transmission and storage. Theoretical studies have revealed that metamaterials can slow down or even stop light; however, the difficulty of preparing metamaterials that operate in visible light hinders progress in the research of slowing or stopping light. Metasurfaces provide a new opportunity to make progress in such research. In this paper, we propose a dendritic cell cluster metasurface consisting of dendritic structures. The simulation results show that dendritic structure can realise abnormal reflection and refraction effects. Single- and double-layer dendritic metasurfaces that respond in visible light were prepared by electrochemical deposition. Abnormal Goos-Hänchen (GH) shifts were experimentally obtained. The rainbow trapping effect was observed in a waveguide constructed using the dendritic metasurface sample. The incident white light was separated into seven colours ranging from blue to red light. The measured transmission energy in the waveguide showed that the energy escaping from the waveguide was zero at the resonant frequency of the sample under a certain amount of incident light. The proposed metasurface has a simple preparation process, functions in visible light, and can be readily extended to the infrared band and communication wavelengths.

  7. Slowing down light using a dendritic cell cluster metasurface waveguide

    PubMed Central

    Fang, Z. H.; Chen, H.; Yang, F. S.; Luo, C. R.; Zhao, X. P.

    2016-01-01

    Slowing down or even stopping light is the first task to realising optical information transmission and storage. Theoretical studies have revealed that metamaterials can slow down or even stop light; however, the difficulty of preparing metamaterials that operate in visible light hinders progress in the research of slowing or stopping light. Metasurfaces provide a new opportunity to make progress in such research. In this paper, we propose a dendritic cell cluster metasurface consisting of dendritic structures. The simulation results show that dendritic structure can realise abnormal reflection and refraction effects. Single- and double-layer dendritic metasurfaces that respond in visible light were prepared by electrochemical deposition. Abnormal Goos-Hänchen (GH) shifts were experimentally obtained. The rainbow trapping effect was observed in a waveguide constructed using the dendritic metasurface sample. The incident white light was separated into seven colours ranging from blue to red light. The measured transmission energy in the waveguide showed that the energy escaping from the waveguide was zero at the resonant frequency of the sample under a certain amount of incident light. The proposed metasurface has a simple preparation process, functions in visible light, and can be readily extended to the infrared band and communication wavelengths. PMID:27886279

  8. Red blood cell cluster separation from digital images for use in sickle cell disease.

    PubMed

    González-Hidalgo, Manuel; Guerrero-Peña, F A; Herold-García, S; Jaume-I-Capó, Antoni; Marrero-Fernández, P D

    2015-07-01

    The study of cell morphology is an important aspect of the diagnosis of some diseases, such as sickle cell disease, because red blood cell deformation is caused by these diseases. Due to the elongated shape of the erythrocyte, ellipse adjustment and concave point detection are applied widely to images of peripheral blood samples, including during the detection of cells that are partially occluded in the clusters generated by the sample preparation process. In the present study, we propose a method for the analysis of the shape of erythrocytes in peripheral blood smear samples of sickle cell disease, which uses ellipse adjustments and a new algorithm for detecting notable points. Furthermore, we apply a set of constraints that allow the elimination of significant image preprocessing steps proposed in previous studies. We used three types of images to validate our method: artificial images, which were automatically generated in a random manner using a computer code; real images from peripheral blood smear sample images that contained normal and elongated erythrocytes; and synthetic images generated from real isolated cells. Using the proposed method, the efficiency of detecting the two types of objects in the three image types exceeded 99.00%, 98.00%, and 99.35%, respectively. These efficiency levels were superior to the results obtained with previously proposed methods using the same database, which is available at http://erythrocytesidb.uib.es/. This method can be extended to clusters of several cells and it requires no user inputs.

  9. Mean-cluster approach indicates cell sorting time scales are determined by collective dynamics

    NASA Astrophysics Data System (ADS)

    Beatrici, Carine P.; de Almeida, Rita M. C.; Brunnet, Leonardo G.

    2017-03-01

    Cell migration is essential to cell segregation, playing a central role in tissue formation, wound healing, and tumor evolution. Considering random mixtures of two cell types, it is still not clear which cell characteristics define clustering time scales. The mass of diffusing clusters merging with one another is expected to grow as td /d +2 when the diffusion constant scales with the inverse of the cluster mass. Cell segregation experiments deviate from that behavior. Explanations for that could arise from specific microscopic mechanisms or from collective effects, typical of active matter. Here we consider a power law connecting diffusion constant and cluster mass to propose an analytic approach to model cell segregation where we explicitly take into account finite-size corrections. The results are compared with active matter model simulations and experiments available in the literature. To investigate the role played by different mechanisms we considered different hypotheses describing cell-cell interaction: differential adhesion hypothesis and different velocities hypothesis. We find that the simulations yield normal diffusion for long time intervals. Analytic and simulation results show that (i) cluster evolution clearly tends to a scaling regime, disrupted only at finite-size limits; (ii) cluster diffusion is greatly enhanced by cell collective behavior, such that for high enough tendency to follow the neighbors, cluster diffusion may become independent of cluster size; (iii) the scaling exponent for cluster growth depends only on the mass-diffusion relation, not on the detailed local segregation mechanism. These results apply for active matter systems in general and, in particular, the mechanisms found underlying the increase in cell sorting speed certainly have deep implications in biological evolution as a selection mechanism.

  10. RNA Polymerase II cluster dynamics predict mRNA output in living cells

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothée; Cisse, Ibrahim I

    2016-01-01

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output. DOI: http://dx.doi.org/10.7554/eLife.13617.001 PMID:27138339

  11. Hydrodynamic and macromolecules induced clusters of red blood cells in microcapillary flow

    NASA Astrophysics Data System (ADS)

    Claveira, Viviana; Aouane, Othmane; Coupier, Gwennou; Misbah, Chaouqi; Abkarian, Manouk; Wagner, Christian

    2015-11-01

    Recent studies have been shown that despite the large shear rates, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of RBC in microcapillaries under flow conditions. The contribution of hydrodynamic interactions and interactions induced by the presence of macromolecules in the cluster formation has not been established. In order to elucidate this mechanism, we compare experimentally in microchannels under flow condition, the pure hydrodynamic cluster formation of RBCs and the cluster formation of RBCs in the presence of macromolecules inducing aggregation. The results reveal strong differences in the cluster morphology. Emphasizing on the case of clusters formed by two cells, the surface to surface interdistances between the cells in the different solutions shows a bimodal distribution. Numerical simulations based on the boundary integral method showed a good agreement with the experimental findings.

  12. Differentiation of human embryonic stem cells into pancreatic endoderm in patterned size-controlled clusters.

    PubMed

    Van Hoof, Dennis; Mendelsohn, Adam D; Seerke, Rina; Desai, Tejal A; German, Michael S

    2011-05-01

    Pancreatic β-cells function optimally when clustered in islet-like structures. However, nutrient and oxygen deprivation limits the viability of cells at the core of excessively large clusters. Hence, production of functional β-cells from human embryonic stem cells (hESCs) for patients with diabetes would benefit from the growth and differentiation of these cells in size-controlled aggregates. In this study, we controlled cluster size by seeding hESCs onto glass cover slips patterned by the covalent microcontact-printing of laminin in circular patches of 120 μm in diameter. These were used as substrates to grow and differentiate hESCs first into SOX17-positive/SOX7-negative definitive endoderm, after which many clusters released and formed uniformly sized three-dimensional clusters. Both released clusters and those that remained attached differentiated into HNF1β-positive primitive gut tube-like cells with high efficiency. Further differentiation yielded pancreatic endoderm-like cells that co-expressed PDX1 and NKX6.1. Controlling aggregate size allows efficient production of uniformly-clustered pancreatic endocrine precursors for in vivo engraftment or further in vitro maturation.

  13. Increased osmolarity and cell clustering preserve canine notochordal cell phenotype in culture.

    PubMed

    Spillekom, Sandra; Smolders, Lucas A; Grinwis, Guy C M; Arkesteijn, Irene T M; Ito, Keita; Meij, Björn P; Tryfonidou, Marianna A

    2014-08-01

    Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive for designing new tissue engineering approaches for IVD regeneration. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture of NCs are not known. The purpose of this study was to investigate the effects of different culture media and osmolarity on the physiology of NCs in vitro. NC clusters isolated from canine IVDs were suspended in alginate beads and cultured at 37°C under normoxic conditions for 28 days. Three different culture conditions were investigated; (1) Dulbecco's modified Eagle's medium (DMEM)/F12 (300 mOsm/L), (2) α-MEM (300 mOsm/L), and (3) α-MEM adjusted to 400 mOsm/L to mimic a hyperosmolar environment. NC morphology, expression of genes related to NC markers, matrix production and remodeling, and DNA- and glycosaminoglycan (GAG) analyses were performed on 1, 7, 14, and 28 days in culture. Large, vesicle-containing cells organized in clusters, characterized as NCs, remained present during 28 days for all culture conditions. However, the proportion of the NC clusters decreased over time, whereas the proportion of spindle-shaped cells increased. Gene expression profiling at 7, 14, and 28 days in culture compared to day 1 indicated a initial loss of NC phenotype followed by some recovery of brachyury and aggrecan gene expression after 28 days of culture supporting a potential recovery of NC phenotype. NCs cultured in α-MEM adjusted to 400 mOsm/L showed the highest gene expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1α1 gene expression. In conclusion, NCs cultured in alginate in native cell clusters, partially retained their

  14. Clustered Simple Cell Mapping: An extension to the Simple Cell Mapping method

    NASA Astrophysics Data System (ADS)

    Gyebrószki, Gergely; Csernák, Gábor

    2017-01-01

    When a dynamical system has a complex structure of fixed points, periodic cycles or even chaotic attractors, Cell Mapping methods are excellent tools to discover and thoroughly analyse all features in the state space. These methods discretize a region of the state space into cells and examine the dynamics in the cell state space. By determining one or more image cells for each cell, the global behaviour within the region can be quickly determined. In the simplest case - Simple Cell Mapping (SCM) method - only one image corresponds to a cell and usually a rectangular grid of cells is used. In typical applications the grid of cells is refined at specific locations. This paper, however, introduces a different approach, which is useful to expand the analysed state space region to include all features which properly characterize the global dynamics of the system. Instead of refining the initial cell state space, we start with a small initial state space region, analyse other interesting regions of the state space and incorporate them into a cluster of cell mapping solutions. By this approach, trajectories escaping the original state space region can be followed automatically and additional objects in the state space can be discovered. To illustrate the benefits of the method, we present the exploration of the phase-space of the micro-chaos map - a simple model of digitally controlled systems.

  15. Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images.

    PubMed

    Wang, Yuliang; Zhang, Zaicheng; Wang, Huimin; Bi, Shusheng

    2015-01-01

    Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.

  16. VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

    PubMed Central

    Zurzolo, C; van't Hof, W; van Meer, G; Rodriguez-Boulan, E

    1994-01-01

    We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins. Images PMID:8306971

  17. Genetically Programmed Clusters of Gold Nanoparticles for Cancer Cell-Targeted Photothermal Therapy.

    PubMed

    Oh, Mi Hwa; Yu, Jeong Heon; Kim, Insu; Nam, Yoon Sung

    2015-10-14

    Interpretations of the interactions of nanocarriers with biological cells are often complicated by complex synthesis of materials, broad size distribution, and heterogeneous surface chemistry. Herein, the major capsid proteins of an icosahedral T7 phage (55 nm in diameter) are genetically engineered to display a gold-binding peptide and a prostate cancer cell-binding peptide in a tandem sequence. The genetically modified phage attracts gold nanoparticles (AuNPs) to form a cluster of gold nanoparticles (about 70 nanoparticles per phage). The cluster of AuNPs maintains cell-targeting functionality and exhibits excellent dispersion stability in serum. Under a very low light irradiation (60 mW cm(-2)), only targeted AuNP clusters kill the prostate cancer cells in minutes (not in other cell types), whereas neither nontargeted AuNP clusters nor citrate-stabilized AuNPs cause any significant cell death. The result suggests that the prostate cancer cell-targeted clusters of AuNPs are targeted to only prostate cancer cells and, when illuminated, generate local heating to more efficiently and selectively kill the targeted cancer cells. Our strategy can be generalized to target other types of cells and assemble other kinds of nanoparticles for a broad range of applications.

  18. AUTOMATED CELL COUNTING AND CLUSTER SEGMENTATION USING CONCAVITY DETECTION AND ELLIPSE FITTING TECHNIQUES

    PubMed Central

    Kothari, Sonal; Chaudry, Qaiser; Wang, May D

    2016-01-01

    This paper presents a novel, fast and semi-automatic method for accurate cell cluster segmentation and cell counting of digital tissue image samples. In pathological conditions, complex cell clusters are a prominent feature in tissue samples. Segmentation of these clusters is a major challenge for development of an accurate cell counting methodology. We address the issue of cluster segmentation by following a three step process. The first step involves pre-processing required to obtain the appropriate nuclei cluster boundary image from the RGB tissue samples. The second step involves concavity detection at the edge of a cluster to find the points of overlap between two nuclei. The third step involves segmentation at these concavities by using an ellipse-fitting technique. Once the clusters are segmented, individual nuclei are counted to give the cell count. The method was tested on four different types of cancerous tissue samples and shows promising results with a low percentage error, high true positive rate and low false discovery rate.

  19. Early degeneration of the cerebellar cortex, particularly the granular cells.

    PubMed

    Bugiani, O; Berio, A; Di Stefano, A; Mangiante, G; Mancardi, G L; Leonardi, A

    1978-12-07

    An 8 month old infant, who died of severe gastroenteritis, presented a degeneration of the cerebellar cortex involving cells arising from the outer granular layer as well as Purkinje and Golgi II cells. Residual Purkinje cells showed vacuolar change of the cell body and dendritic abnormalities. Related lesions were atrophy of the inferior olives and degeneration of the mossy fibers.

  20. Activation of steroid-sensitive TRPM3 channels potentiates glutamatergic transmission at cerebellar Purkinje neurons from developing rats.

    PubMed

    Zamudio-Bulcock, Paula A; Everett, Julie; Harteneck, Christian; Valenzuela, C Fernando

    2011-11-01

    The functional implications of transient receptor potential melastatin 3 (TRPM3) activation, the most recently described member of the melastatin subfamily of cation permeable TRP channels, have begun to be elucidated in recent years. The discovery of TRPM3 activation by the steroid pregnenolone sulfate (PregS) has shed new light on the physiological role of this channel. For example, TRPM3 activation enhances insulin secretion from β pancreatic cells, induces contraction of vascular smooth muscle, and is also involved in the detection of noxious heat. Although TRPM3 expression has been detected in several regions of the developing and mature brain, little is known about the roles of TRPM3 in brain physiology. In this study, we demonstrate the abundant expression of TRPM3 steroid-sensitive channels in the developing cerebellar cortex. We also show that TRPM3-like channels are expressed at glutamatergic synapses in neonatal Purkinje cells. We recently showed that PregS potentiates spontaneous glutamate release onto neonatal Purkinje cells during a period of active glutamatergic synapse formation; we now show that this effect of PregS is mediated by TRPM3-like channels. Mefenamic acid, a recently discovered TRPM3 antagonist, blocked the effect of PregS on glutamate release. The PregS effect on glutamate release was mimicked by other TRPM3 agonists (nifedipine and epipregnanolone sulfate) but not by a TRMP3-inactive steroid (progesterone). Our findings identify TRPM3 channels as novel modulators of glutamatergic transmission in the developing brain.

  1. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    PubMed

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  2. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

    PubMed Central

    Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.; Dustin, Michael L.; Groves, Jay

    2011-01-01

    T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower. PMID:21576490

  3. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

    SciTech Connect

    Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.; Dustin, Michael L.; Groves, Jay

    2011-05-31

    T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.

  4. On the performance limiting behavior of defect clusters in commercial silicon solar cells

    SciTech Connect

    Sopori, B.L.; Chen, W.; Jones, K.; Gee, J.

    1998-09-01

    The authors report the observation of defect clusters in high-quality, commercial silicon solar cell substrates. The nature of the defect clusters, their mechanism of formation, and precipitation of metallic impurities at the defect clusters are discussed. This defect configuration influences the device performance in a unique way--by primarily degrading the voltage-related parameters. Network modeling is used to show that, in an N/P junction device, these regions act as shunts that dissipate power generated within the cell.

  5. Metal etching with reactive gas cluster ion beams using pickup cell

    SciTech Connect

    Toyoda, Noriaki; Yamada, Isao

    2012-11-06

    Mixed gas cluster ion beams were formed using pickup cell for metal etching. O{sub 2} neutral clusters pick up acetic acid and formed mixed cluster beam. By using O{sub 2}-GCIB with acetic acid, enhancement of Cu etching was observed. Because of dense energy deposition by GCIB, etching of Cu proceeds by CuO formation, enhancement of chemical reaction with acetic acid and desorption of etching products. Surface roughening was not observed on poly crystalline Cu because of the small dependence of etching rate on crystal orientation. Halogen free and low-temperature metal etching with GCIB using pickup cell is possible.

  6. ODE, RDE and SDE Models of Cell Cycle Dynamics and Clustering in Yeast

    PubMed Central

    Boczko, Erik M.; Stowers, Chris C.; Gedeon, Tomas; Young, Todd R.

    2009-01-01

    Biologists have long observed periodic-like oxygen consumption oscillations in yeast populations under certain conditions and several unsatisfactory explanations for this phenomenon have been proposed. These “autonomous oscillations” have often appeared with periods that are nearly integer divisors of the calculated doubling time of the culture. We hypothesize that these oscillations could be caused by a form of cell cycle synchronization that we call clustering. We develop some novel ordinary differential equation models of the cell cycle. For these models, and for random and stochastic perturbations, we give both rigorous proofs and simulations showing that both positive and negative growth rate feedback within the cell cycle are possible agents that can cause clustering of populations within the cell cycle. It occurs for a variety of models and for a broad selection of parameter values. These results suggest that the clustering phenomenon is robust and is likely to be observed in nature. Since there are necessarily an integer number of clusters, clustering would lead to periodic-like behavior with periods that are nearly integer divisors of the period of the cell cycle. Related experiments have shown conclusively that cell cycle clustering occurs in some oscillating yeast cultures. PMID:20563236

  7. Numerical Simulation of Bubble Cluster Induced Flow by Three-Dimensional Vortex-in-Cell Method.

    PubMed

    Chen, Bin; Wang, Zhiwei; Uchiyama, Tomomi

    2014-08-01

    The behavior of air bubble clusters rising in water and the induced flow field are numerically studied using a three-dimensional two-way coupling algorithm based on a vortex-in-cell (VIC) method. In this method, vortex elements are convected in the Lagrangian frame and the liquid velocity field is solved from the Poisson equation of potential on the Eulerian grid. Two-way coupling is implemented by introducing a vorticity source term induced by the gradient of void fraction. Present simulation results are favorably compared with the measured results of bubble plume, which verifies the validity of the proposed VIC method. The rising of a single bubble cluster as well as two tandem bubble clusters are simulated. The mechanism of the aggregation effect in the rising process of bubble cluster is revealed and the transient processes of the generation, rising, strengthening, and separation of a vortex ring structure with bubble clusters are illustrated and analyzed in detail. Due to the aggregation, the average rising velocity increases with void fraction and is larger than the terminal rising velocity of single bubble. For the two tandem bubble cluster cases, the aggregation effect is stronger for smaller initial cluster distance, and both the strength of the induced vortex structure and the average bubble rising velocity are larger. For the 20 mm cluster distance case, the peak velocity of the lower cluster is about 2.7 times that of the terminal velocity of the single bubble and the peak average velocity of two clusters is about 2 times larger. While for the 30 mm cluster distance case, both the peak velocity of the lower cluster and two clusters are about 1.7 times that of the terminal velocity of the single bubble.

  8. 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.

  9. Receptor Pre-Clustering and T cell Responses: Insights into Molecular Mechanisms.

    PubMed

    Castro, Mario; van Santen, Hisse M; Férez, María; Alarcón, Balbino; Lythe, Grant; Molina-París, Carmen

    2014-01-01

    T cell activation, initiated by T cell receptor (TCR) mediated recognition of pathogen-derived peptides presented by major histocompatibility complex class I or II molecules (pMHC), shows exquisite specificity and sensitivity, even though the TCR-pMHC binding interaction is of low affinity. Recent experimental work suggests that TCR pre-clustering may be a mechanism via which T cells can achieve such high sensitivity. The unresolved stoichiometry of the TCR makes TCR-pMHC binding and TCR triggering, an open question. We formulate a mathematical model to characterize the pre-clustering of T cell receptors (TCRs) on the surface of T cells, motivated by the experimentally observed distribution of TCR clusters on the surface of naive and memory T cells. We extend a recently introduced stochastic criterion to compute the timescales of T cell responses, assuming that ligand-induced cross-linked TCR is the minimum signaling unit. We derive an approximate formula for the mean time to signal initiation. Our results show that pre-clustering reduces the mean activation time. However, additional mechanisms favoring the existence of clusters are required to explain the difference between naive and memory T cell responses. We discuss the biological implications of our results, and both the compatibility and complementarity of our approach with other existing mathematical models.

  10. Receptor Pre-Clustering and T cell Responses: Insights into Molecular Mechanisms

    PubMed Central

    Castro, Mario; van Santen, Hisse M.; Férez, María; Alarcón, Balbino; Lythe, Grant; Molina-París, Carmen

    2014-01-01

    T cell activation, initiated by T cell receptor (TCR) mediated recognition of pathogen-derived peptides presented by major histocompatibility complex class I or II molecules (pMHC), shows exquisite specificity and sensitivity, even though the TCR–pMHC binding interaction is of low affinity. Recent experimental work suggests that TCR pre-clustering may be a mechanism via which T cells can achieve such high sensitivity. The unresolved stoichiometry of the TCR makes TCR–pMHC binding and TCR triggering, an open question. We formulate a mathematical model to characterize the pre-clustering of T cell receptors (TCRs) on the surface of T cells, motivated by the experimentally observed distribution of TCR clusters on the surface of naive and memory T cells. We extend a recently introduced stochastic criterion to compute the timescales of T cell responses, assuming that ligand-induced cross-linked TCR is the minimum signaling unit. We derive an approximate formula for the mean time to signal initiation. Our results show that pre-clustering reduces the mean activation time. However, additional mechanisms favoring the existence of clusters are required to explain the difference between naive and memory T cell responses. We discuss the biological implications of our results, and both the compatibility and complementarity of our approach with other existing mathematical models. PMID:24817867

  11. 3D Porous Calcium-Alginate Scaffolds Cell Culture System Improved Human Osteoblast Cell Clusters for Cell Therapy

    PubMed Central

    Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

    2015-01-01

    Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects. PMID:25825603

  12. Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters

    PubMed Central

    Ng, Mei Rosa; Besser, Achim; Brugge, Joan S; Danuser, Gaudenz

    2014-01-01

    Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. DOI: http://dx.doi.org/10.7554/eLife.03282.001 PMID:25479385

  13. Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy.

    PubMed

    Chen, Xuanze; Wei, Mian; Zheng, M Mocarlo; Zhao, Jiaxi; Hao, Huiwen; Chang, Lei; Xi, Peng; Sun, Yujie

    2016-02-23

    Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

  14. Influence of red blood cell clustering on phase separation in capillary networks

    NASA Astrophysics Data System (ADS)

    Podgorski, Thomas; Boucly, Celine; Coupier, Gwennou

    2014-11-01

    We investigate the flow of red blood cell suspensions in microfluidic bifurcations and capillary networks. At strong degrees of confinement, such as those encountered in the microcirculation, phase separation takes place at bifurcations of the network, leading to strong heterogeneities and fluctuations of the hematocrit (blood cell concentration). We highlight the influence of the mechanical properties of cells : an increase of membrane or cytoplasm rigidity, as can happen in pathologies such as sickle cell disease tends to reduce the phase separation. The influence of the attractive interaction between cells, that leads to clustering (rouleau formation) was also investigated by varying the concentration of macromolecules in the solution (dextran or fibrinogen). We show that hydrodynamic stresses in bifurcations can lead to rupture of clusters at a critical speed which increases with interaction energy. Overall, the clustering phenomenon tends to increase phase separation and hematocrit heterogeneities.

  15. Heparanase Facilitates Cell Adhesion and Spreading by Clustering of Cell Surface Heparan Sulfate Proteoglycans

    PubMed Central

    Levy-Adam, Flonia; Feld, Sari; Suss-Toby, Edith; Vlodavsky, Israel; Ilan, Neta

    2008-01-01

    Heparanase is a heparan sulfate (HS) degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys158-Asp171, termed KKDC) was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity. PMID:18545691

  16. Growing Bladder-Cancer Cells In Three-Dimensional Clusters

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F.; Prewett, Tacey L.; Goodwin, Thomas J.

    1995-01-01

    Artificial growth process helps fill gaps in cancer research. Cell cultures more accurate as models for in vivo studies and as sources of seed cells for in vivo studies. Effected in horizontal rotating bioreactor described in companion article, "Simplified Bioreactor for Growing Mammalian Cells" (MSC-22060). Provides aggregates of cells needed to fill many of gaps.

  17. Mechanism of alpha-2 adrenergic modulation of canine cardiac Purkinje action potential.

    PubMed

    Lee, H C; Cai, J J; Arnar, D O; Shibata, E F; Martins, J B

    1996-08-01

    We reported recently that stimulation of postjunctional alpha-2 adrenergic receptors prolongs the action potential durations (APD) of isolated canine Purkinje fibers. With standard microelectrode techniques, we examined the ionic mechanism through which alpha-2 adrenergic stimulation prolonged Purkinje APD, by measuring the effects of inhibitors of the various plateau currents on the alpha-2-mediated prolongation of APD. The alpha-2-specific agonist UK 14,304 (0.1 microM) prolonged the Purkinje APD at 50% repolarization and the APD at 90% repolarization, and these effects were inhibited by yohimbine (0.1 microM). The Purkinje APD at 50% repolarization and the APD at 90% repolarization were prolonged significantly with the transient outward potassium current inhibitor 4-aminopyridine (1 mM), the rapid component of delayed rectifier potassium current inhibitor d-sotalol (10 microM), the slow component of delayed rectifier potassium current inhibitor indapamide (0.1 microM) and the chloride current inhibitor mefenamic acid (10 nM) and were shortened significantly with the calcium current inhibitor nifedipine (0.3 microM). Prolongation of Purkinje APD at 50% repolarization and APD at 90% repolarization by UK 14,304 remained intact in the presence of d-sotalol, indapamide, mefenamic acid and nifedipine. All of these UK 14,304 effects were significantly reversed by yohimbine. Only in the presence of 4-aminopyridine did UK 14,304 fail to prolong Purkinje APD. The phase 1 magnitudes of Purkinje action potentials were also significantly inhibited by UK 14,304. This effect was completely abolished only in the presence of 4-aminopyridine. These results suggest that inhibition of the 4-aminopyridine-sensitive transient outward potassium current is the major ionic mechanism by which alpha-2 adrenergic stimulation prolongs Purkinje APD.

  18. Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival

    PubMed Central

    Sage, Evelyne; Harrison, Lynn

    2011-01-01

    A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1–2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990’s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed. PMID:21185841

  19. Clustering in Cell Cycle Dynamics with General Response/Signaling Feedback

    PubMed Central

    Young, Todd R.; Fernandez, Bastien; Buckalew, Richard; Moses, Gregory; Boczko, Erik M.

    2011-01-01

    Motivated by experimental and theoretical work on autonomous oscillations in yeast, we analyze ordinary differential equations models of large populations of cells with cell-cycle dependent feedback. We assume a particular type of feedback that we call Responsive/Signaling (RS), but do not specify a functional form of the feedback. We study the dynamics and emergent behaviour of solutions, particularly temporal clustering and stability of clustered solutions. We establish the existence of certain periodic clustered solutions as well as “uniform” solutions and add to the evidence that cell-cycle dependent feedback robustly leads to cell-cycle clustering. We highlight the fundamental differences in dynamics between systems with negative and positive feedback. For positive feedback systems the most important mechanism seems to be the stability of individual isolated clusters. On the other hand we find that in negative feedback systems, clusters must interact with each other to reinforce coherence. We conclude from various details of the mathematical analysis that negative feedback is most consistent with observations in yeast experiments. PMID:22001733

  20. 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.

  1. Differential migration and proliferation of geometrical ensembles of cell clusters

    SciTech Connect

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi

    2011-06-10

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

  2. Phenotype Clustering of Breast Epithelial Cells in Confocal Imagesbased on Nuclear Protein Distribution Analysis

    SciTech Connect

    Long, Fuhui; Peng, Hanchuan; Sudar, Damir; Levievre, Sophie A.; Knowles, David W.

    2006-09-05

    Background: The distribution of the chromatin-associatedproteins plays a key role in directing nuclear function. Previously, wedeveloped an image-based method to quantify the nuclear distributions ofproteins and showed that these distributions depended on the phenotype ofhuman mammary epithelial cells. Here we describe a method that creates ahierarchical tree of the given cell phenotypes and calculates thestatistical significance between them, based on the clustering analysisof nuclear protein distributions. Results: Nuclear distributions ofnuclear mitotic apparatus protein were previously obtained fornon-neoplastic S1 and malignant T4-2 human mammary epithelial cellscultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 andthe number of days in cultured. A probabilistic ensemble approach wasused to define a set of consensus clusters from the results of multipletraditional cluster analysis techniques applied to the nucleardistribution data. Cluster histograms were constructed to show how cellsin any one phenotype were distributed across the consensus clusters.Grouping various phenotypes allowed us to build phenotype trees andcalculate the statistical difference between each group. The resultsshowed that non-neoplastic S1 cells could be distinguished from malignantT4-2 cells with 94.19 percent accuracy; that proliferating S1 cells couldbe distinguished from differentiated S1 cells with 92.86 percentaccuracy; and showed no significant difference between the variousphenotypes of T4-2 cells corresponding to increasing tumor sizes.Conclusion: This work presents a cluster analysis method that canidentify significant cell phenotypes, based on the nuclear distributionof specific proteins, with high accuracy.

  3. Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons: inhibition by BDS-I toxin.

    PubMed

    Martina, Marco; Metz, Alexia E; Bean, Bruce P

    2007-01-01

    We characterized the kinetics and pharmacological properties of voltage-activated potassium currents in rat cerebellar Purkinje neurons using recordings from nucleated patches, which allowed high resolution of activation and deactivation kinetics. Activation was exceptionally rapid, with 10-90% activation in about 400 mus at +30 mV, near the peak of the spike. Deactivation was also extremely rapid, with a decay time constant of about 300 mus near -80 mV. These rapid activation and deactivation kinetics are consistent with mediation by Kv3-family channels but are even faster than reported for Kv3-family channels in other neurons. The peptide toxin BDS-I had very little blocking effect on potassium currents elicited by 100-ms depolarizing steps, but the potassium current evoked by action potential waveforms was inhibited nearly completely. The mechanism of inhibition by BDS-I involves slowing of activation rather than total channel block, consistent with the effects described in cloned Kv3-family channels and this explains the dramatically different effects on currents evoked by short spikes versus voltage steps. As predicted from this mechanism, the effects of toxin on spike width were relatively modest (broadening by roughly 25%). These results show that BDS-I-sensitive channels with ultrafast activation and deactivation kinetics carry virtually all of the voltage-dependent potassium current underlying repolarization during normal Purkinje cell spikes.

  4. Monoclonal antibody binding-site diversity assessment with a cell-based clustering assay.

    PubMed

    Liao-Chan, Sindy; Zachwieja, Joseph; Gomez, Steven; Duey, Dana; Lippincott, John; Theunissen, Jan-Willem

    2014-03-01

    The diversity of a panel of antibodies that target a specific antigen can be established in various assay formats. In conventional epitope binning assays purified antibodies are tested in a pairwise manner: two antibodies that compete with each other for binding to an antigen are grouped into the same cluster or bin, while they are assigned to two different clusters when they do not compete. Here we present a high through put assay that enables grouping of crude hybridoma supernatants without a need for antibody purification. In addition, the assay does not require recombinant protein, because it is conducted on cells that express the antigen of interest. Hence, one can use the antibody-clustering assay for cell surface proteins that are not amenable to purification. Heavy chain variable region (VH) sequencing shows that VH composition within clusters is conserved. Finally, the assay is in good agreement with a conventional epitope binning assay with purified antigen.

  5. Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.

    PubMed

    Dillard, Pierre; Pi, Fuwei; Lellouch, Annemarie C; Limozin, Laurent; Sengupta, Kheya

    2016-03-14

    We investigate the adhesion and molecular organization of the plasma membrane of T lymphocytes interacting with a surrogate antigen presenting cell comprising glass supported ordered arrays of antibody (α-CD3) nano-dots dispersed in a non-adhesive matrix of polyethylene glycol (PEG). The local membrane adhesion and topography, as well as the distribution of the T cell receptors (TCRs) and the kinase ZAP-70, are influenced by dot-geometry, whereas the cell spreading area is determined by the overall average density of the ligands rather than specific characteristics of the dots. TCR clusters are recruited preferentially to the nano-dots and the TCR cluster size distribution has a weak dot-size dependence. On the patterns, the clusters are larger, more numerous, and more enriched in TCRs, as compared to the homogeneously distributed ligands at comparable concentrations. These observations support the idea that non-ligated TCRs residing in the non-adhered parts of the proximal membrane are able to diffuse and enrich the existing clusters at the ligand dots. However, long distance transport is impaired and cluster centralization in the form of a central supramolecular cluster (cSMAC) is not observed. Time-lapse imaging of early cell-surface contacts indicates that the ZAP-70 microclusters are directly recruited to the site of the antibody dots and this process is concomitant with membrane adhesion. These results together point to a complex interplay of adhesion, molecular organization and activation in response to spatially modulated stimulation.

  6. [Automatic segmentation of clustered breast cancer cells based on modified watershed algorithm and concavity points searching].

    PubMed

    Tong, Zhen; Pu, Lixin; Dong, Fangjie

    2013-08-01

    As a common malignant tumor, breast cancer has seriously affected women's physical and psychological health even threatened their lives. Breast cancer has even begun to show a gradual trend of high incidence in some places in the world. As a kind of common pathological assist diagnosis technique, immunohistochemical technique plays an important role in the diagnosis of breast cancer. Usually, Pathologists isolate positive cells from the stained specimen which were processed by immunohistochemical technique and calculate the ratio of positive cells which is a core indicator of breast cancer in diagnosis. In this paper, we present a new algorithm which was based on modified watershed algorithm and concavity points searching to identify the positive cells and segment the clustered cells automatically, and then realize automatic counting. By comparison of the results of our experiments with those of other methods, our method can exactly segment the clustered cells without losing any geometrical cell features and give the exact number of separating cells.

  7. Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

    PubMed

    Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

    2005-05-01

    Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

  8. A new algorithm to find earthquake clusters using neighboring cell connection and rate analysis.

    NASA Astrophysics Data System (ADS)

    Peng, W.; Toda, S.

    2015-12-01

    To study earthquake interaction, it is important to objectively find a group of earthquakes occurred closely in space and time. Earthquake clusters are chosen with previous techniques that characterize them as mainshock-aftershock sequences or swarm sequences by empirical laws (e.g., Omori-Utsu; ETAS) or direct assumptions about physical processes such as stress transfer, transient stress loading, and fluid migration. Recent papers instead proposed non-parameterized techniques such as a kernel-based smoothing method. The cumulative rate clustering method (CURATE, Jacobs et al., 2013) is one of the approaches without any direct assumptions. The CURATE method was applied in New Zealand and provided a good result for selecting the swarm sequences comparing with the ETAS model. However, it is still difficult to choose a proper confined area and a time interval for extracting sequences from the catalog. To avoid arbitrariness in space and time parameters, here we propose a new method modifying the CURATE approach. We first identify the spatial clusters by looking into the spatial distribution in a 2-D cell-gridded map. The spatial clusters defined as multiple neighboring cells, each of which contains at least one earthquake in a time period T. From the selected spatial clusters, we then evaluate temporal clustering which is defined as a transient increase of seismicity rate comparing to the rate before the target event. We tested this method focusing on shallow crustal seismicity, northern Honshu, Japan. We chose the parameter range from T = 1 to 100 days and cell size = 0.01°to 0.1°. As a result, the number of the clusters increase with longer T and larger cell size. By choosing the T = 30 days and cell size = 0.05°, we successfully selected the long-lasting aftershock sequences associated with the 2004 M6.8 Chuetsu and 2007 M6.8 Chuetsu-oki earthquakes, while other empirical models and CURATE method failed to decluster.

  9. Germ cell cluster organization and oogenesis in the tardigrade Dactylobiotus parthenogeneticus Bertolani, 1982 (Eutardigrada, Murrayidae).

    PubMed

    Poprawa, Izabela; Hyra, Marta; Rost-Roszkowska, Magdalena Maria

    2015-07-01

    Germ cell cluster organization and the process of oogenesis in Dactylobiotus parthenogeneticus have been described using transmission electron microscopy and light microscopy. The reproductive system of D. parthenogeneticus is composed of a single, sac-like, meroistic ovary and a single oviduct that opens into the cloaca. Two zones can be distinguished in the ovary: a small germarium that is filled with oogonia and a vitellarium that is filled with germ cell clusters. The germ cell cluster, which has the form of a modified rosette, consists of eight cells that are interconnected by stable cytoplasmic bridges. The cell that has the highest number of stable cytoplasmic bridges (four bridges) finally develops into the oocyte, while the remaining cells become trophocytes. Vitellogenesis of a mixed type occurs in D. parthenogeneticus. One part of the yolk material is produced inside the oocyte (autosynthesis), while the second part is synthesized in the trophocytes and transported to the oocyte through the cytoplasmic bridges. The eggs are covered with two envelopes: a thin vitelline envelope and a three-layered chorion. The surface of the chorion forms small conical processes, the shape of which is characteristic for the species that was examined. In our paper, we present the first report on the rosette type of germ cell clusters in Parachela.

  10. A computational investigation of the role of behavioral heterogeneities on cell cluster motion

    NASA Astrophysics Data System (ADS)

    Copenhagen, Katherine; Gov, Nir; Gopinathan, Ajay

    2015-03-01

    Collective motion of cells is a common occurence in many biological systems, including tissue develope- ment and repair, and tumor formation. Recent experiments have shown that malignant B and T lymphocytes form clusters in a chemical gradient of CCL19 which display three different phases: translational, rotational, and random. Could these phases be due to interactions between cells as well as chemotaxis of individuals? If so what types of local interactions can lead to the three phases seen in experiment? We model cell clusters with a continuous two dimensional agent based model. To form a single cell cluster which displays all three of the phases described above, cells interact with a Vicsek alignment interaction, a Lennard-Jones collision- avoidance and cohesiveness interaction, and a long range spring interaction to prevent fracture. By changing the behaviors of individual cells depending on the number of cells they are contacting, we are able to create clusters that occupy these phases with varying likelihood. Our results show that heterogeneous behaviors of individuals based on local environment can lead to novel phases seen in experiments.

  11. Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

    PubMed Central

    Lopez, Ana D.; Kayali, Ayse G.; Hayek, Alberto; King, Charles C.

    2014-01-01

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells. PMID:24895054

  12. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

    PubMed Central

    Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

    2015-01-01

    ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

  13. CIDR: Ultrafast and accurate clustering through imputation for single-cell RNA-seq data.

    PubMed

    Lin, Peijie; Troup, Michael; Ho, Joshua W K

    2017-03-28

    Most existing dimensionality reduction and clustering packages for single-cell RNA-seq (scRNA-seq) data deal with dropouts by heavy modeling and computational machinery. Here, we introduce CIDR (Clustering through Imputation and Dimensionality Reduction), an ultrafast algorithm that uses a novel yet very simple implicit imputation approach to alleviate the impact of dropouts in scRNA-seq data in a principled manner. Using a range of simulated and real data, we show that CIDR improves the standard principal component analysis and outperforms the state-of-the-art methods, namely t-SNE, ZIFA, and RaceID, in terms of clustering accuracy. CIDR typically completes within seconds when processing a data set of hundreds of cells and minutes for a data set of thousands of cells. CIDR can be downloaded at https://github.com/VCCRI/CIDR .

  14. Differential localization of LGR5 and Nanog in clusters of colon cancer stem cells.

    PubMed

    Amsterdam, Abraham; Raanan, Calanit; Schreiber, Letizia; Freyhan, Ora; Fabrikant, Yakov; Melzer, Ehud; Givol, David

    2013-05-01

    One paradigm of cancer development claims that cancer emerges at the niche of tissue stem cells and these cells continue to proliferate in the tumor as cancer stem cells. LGR5, a membrane receptor, was recently found to be a marker of normal colon stem cells in colon polyps and is also expressed in colon cancer stem cells. Nanog, an embryonic stem cell nuclear factor, is expressed in several embryonic tissues, but Nanog expression is not well documented in cancerous stem cells. Our aim was to examine whether both LGR5 and Nanog are expressed in the same clusters of colon stem cells or cancer stem cells, using immunocytochemistry with specific antibodies to each antigen. We analyzed this aspect using paraffin embedded tumor tissue sections obtained from 18 polyps and 36 colon cancer specimens at stages I-IV. Antibodies to LGR5 revealed membrane and cytoplasm immunostaining of scattered labeled cells in normal crypts, with no labeling of Nanog. However, in close proximity to the tumors, staining to LGR5 was much more intensive in the crypts, including that of the epithelial cells. In cancer tissue, positive LGR5 clusters of stem cells were observed mainly in poorly differentiated tumors and in only a few scattered cells in the highly differentiated tumors. In contrast, antibodies to Nanog mainly stained the growing edges of carcinoma cells, leaving the poorly differentiated tumor cells unlabeled, including the clustered stem cells that could be detected even by direct morphological examination. In polyp tissues, scattered labeled cells were immunostained with antibodies to Nanog and to a much lesser extent with antibodies to LGR5. We conclude that expression of LGR5 is probably specific to stem cells of poorly differentiated tumors, whereas Nanog is mainly expressed at the edges of highly differentiated tumors. However, some of the cell layers adjacent to the carcinoma cell layers that still remained undifferentiated, expressed mainly Nanog with only a few cells

  15. Effect of different cell cluster models on the radiobiological output for (211)At-radioimmunotherapy.

    PubMed

    Lin, Hui; Jing, Jia; Xu, Yuanying

    2011-02-01

    The cell cluster modeling is a widely used method to estimate the small-scale dosimetry and provides the implication for a clinic. This work evaluated the effect of different regular cluster models on the radiobiological outputs for (211)At-radioimmunotherapy. The cell activity threshold was estimated using a tumor control probability of 0.90. Basically, regular models show similar features with cluster configuration and cell dimension variation. However, their individual results such as the cumulated activity threshold per cell and the prescription dose per volume should not be substituted reciprocally. The tissue composed of smaller cells or midcell packing will need a little more high prescription dose per volume. The radiation sensitivity parameters in a linear-quadratic model are critical to decide the radiobiological response with dose. The cumulated cell activity threshold increases exponentially with α decreasing, and its influence on the big cell dimension is more than on the small one. The different subsources affect radioresistant organs or tissues more remarkably than radiosensitive ones, especially the cells with large cytoplasm. The heterogeneous activity of Gaussian distribution will decrease the therapeutical effectiveness for the nucleus source, but its influence on the cytoplasm and cell surface sources is a little uncertain, as their real mean value is always higher than its set mean value by assuming the cell activity uptakes from zero. Careful usage of underdose with heterogeneous activity distribution should be practiced in clinics. The deteriorated heterogeneous distribution will salvage the potential subversive and lead to the failure of tumor local control. Some cells with no or little activity that are located on the edge or vertex of cube or corner models will have the ability to survive, as there is a lack of a part of the cross-fire dose effect, and so more attention should be paid in selecting the dosage. Although this work focuses on

  16. Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells.

    PubMed

    Lizunov, Vladimir A; Stenkula, Karin; Troy, Aaron; Cushman, Samuel W; Zimmerberg, Joshua

    2013-01-01

    Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin's effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60-240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.

  17. Regulation of transcription of cell division genes in the Escherichia coli dcw cluster.

    PubMed

    Vicente, M; Gomez, M J; Ayala, J A

    1998-04-01

    The Escherichia coli dcw cluster contains cell division genes, such as the phylogenetically ubiquitous ftsZ, and genes involved in peptidoglycan synthesis. Transcription in the cluster proceeds in the same direction as the progress of the replication fork along the chromosome. Regulation is exerted at the transcriptional and post-transcriptional levels. The absence of transcriptional termination signals may, in principle, allow extension of the transcripts initiated at the up-stream promoter (mraZ1p) even to the furthest down-stream gene (envA). Complementation tests suggest that they extend into ftsW in the central part of the cluster. In addition, the cluster contains other promoters individually regulated by cis- and trans-acting signals. Dissociation of the expression of the ftsZ gene, located after ftsQ and A near the 3' end of the cluster, from its natural regulatory signals leads to an alteration in the physiology of cell division. The complexities observed in the regulation of gene expression in the cluster may then have an important biological role. Among them, LexA-binding SOS boxes have been found at the 5' end of the cluster, preceding promoters which direct the expression of ftsI (coding for PBP3, the penicillin-binding protein involved in septum formation). A gearbox promoter, ftsQ1p, forms part of the signals regulating the transcription of ftsQ, A and Z. It is an inversely growth-dependent mechanism driven by RNA polymerase containing sigma s, the factor involved in the expression of stationary phase-specific genes. Although the dcw cluster is conserved to a different extent in a variety of bacteria, the regulation of gene expression, the presence or absence of individual genes, and even the essentiality of some of them, show variations in the phylogenetic scale which may reflect adaptation to specific life cycles.

  18. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  19. On the coherent behavior of pancreatic beta cell clusters

    NASA Astrophysics Data System (ADS)

    Loppini, Alessandro; Capolupo, Antonio; Cherubini, Christian; Gizzi, Alessio; Bertolaso, Marta; Filippi, Simonetta; Vitiello, Giuseppe

    2014-09-01

    Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with “phase transition” phenomena of field theory.

  20. Intraocular lens alignment from an en face optical coherence tomography image Purkinje-like method

    NASA Astrophysics Data System (ADS)

    Sun, Mengchan; de Castro, Alberto; Ortiz, Sergio; Perez-Merino, Pablo; Birkenfeld, Judith; Marcos, Susana

    2014-06-01

    Measurement of intraocular lens (IOL) alignment implanted in patients in cataract surgery is important to understand their optical performance. We present a method to estimate tilt and decentration of IOLs based on optical coherence tomography (OCT) images. En face OCT images show Purkinje-like images that correspond to the specular reflections from the corneal and IOL surfaces. Unlike in standard Purkinje-imaging, the tomographic nature of OCT allows unequivocal association of the reflection with the corresponding surface. The locations of the Purkinje-like images are linear combinations of IOL tilt, IOL decentration, and eye rotation. The weighting coefficients depend on the individual anterior segment geometry, obtained from the same OCT datasets. The methodology was demonstrated on an artificial model eye with set amounts of lens tilt and decentration and five pseudophakic eyes. Measured tilt and decentration in the artificial eye differed by 3.7% and 0.9%, respectively, from nominal values. In patients, average IOL tilt and decentration from Purkinje were 3.30±4.68 deg and 0.16±0.16 mm, respectively, and differed on average by 0.5 deg and 0.09 mm, respectively, from direct measurements on distortion-corrected OCT images. Purkinje-based methodology from anterior segment en face OCT imaging provided, therefore, reliable measurements of IOL tilt and decentration.

  1. p53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

    PubMed Central

    Yi, Qiyi; Zhao, Xiaoyu; Huang, Yun; Ma, Tieliang; Zhang, Yingyin; Hou, Heli; Cooke, Howard J.; Yang, Da-Qing; Wu, Mian; Shi, Qinghua

    2011-01-01

    Background p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Principal Findings Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Conclusions p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. PMID:22076149

  2. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    NASA Astrophysics Data System (ADS)

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-06-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

  3. Positive feedback can lead to dynamic nanometer-scale clustering on cell membranes

    SciTech Connect

    Wehrens, Martijn; Rein ten Wolde, Pieter; Mugler, Andrew

    2014-11-28

    Clustering of molecules on biological membranes is a widely observed phenomenon. A key example is the clustering of the oncoprotein Ras, which is known to be important for signal transduction in mammalian cells. Yet, the mechanism by which Ras clusters form and are maintained remains unclear. Recently, it has been discovered that activated Ras promotes further Ras activation. Here we show using particle-based simulation that this positive feedback is sufficient to produce persistent clusters of active Ras molecules at the nanometer scale via a dynamic nucleation mechanism. Furthermore, we find that our cluster statistics are consistent with experimental observations of the Ras system. Interestingly, we show that our model does not support a Turing regime of macroscopic reaction-diffusion patterning, and therefore that the clustering we observe is a purely stochastic effect, arising from the coupling of positive feedback with the discrete nature of individual molecules. These results underscore the importance of stochastic and dynamic properties of reaction diffusion systems for biological behavior.

  4. Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae.

    PubMed

    Francis, F; Ramirez-Arcos, S; Salimnia, H; Victor, C; Dillon, J R

    2000-06-27

    A cluster of genes involved in cell division and cell wall (dcw) biosynthesis was identified in Neisseria gonorrhoeae using genomic analysis and through verification of gene order by polymerase chain reaction (PCR) analysis. The gonococcal dcw cluster consists of 17 genes, in the order 5'-mraZ-mraW-ftsI-murE-hyp1-murF- mraY-hyp2-murD-ftsW-murG-murC-ddl -ft sQ-ftsA-ftsZ-hyp3-3'. The gene organization of the dcw cluster of N. gonorrhoeae is more similar to that observed in Gram-negative rods such as Escherichia coli and Haemophilus influenzae than in Gram-positive bacteria. The cluster is characterized by several intergenic spaces. Compared with E. coli, two genes, ftsL and envA, are absent in the gonococcal dcw cluster and three hypothetical genes are novel to the cluster. The cluster is flanked by two transcriptional terminators consisting of paired neisserial uptake sequences and also includes four internal terminators, three of which are paired neisserial uptake sequences. We also found that a repeated sequence on the gonococcal genome, commonly called a Correia element, acts as the fourth transcriptional terminator. All termination sequences were shown to be fully functional by using reverse transcription PCR experiments. Transcriptional start sites upstream of ftsQ, ftsA and ftsZ were determined by primer extension and six promoters were identified; three promoters were located upstream of ftsZ in the intergenic space, two were upstream of ftsA within ftsQ and one was upstream of ftsQ within ddl. Some of these promoters were preferentially used under anaerobic conditions. The location of these promoters differed from those described in E. coli indicating dissimilar transcriptional regulation.

  5. Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

    PubMed Central

    Roy, Nathan H.; Chan, Jany; Lambelé, Marie

    2013-01-01

    HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env's fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag's ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells. PMID:23637402

  6. Human omentum fat-derived mesenchymal stem cells transdifferentiates into pancreatic islet-like cluster.

    PubMed

    Dhanasekaran, M; Indumathi, S; Harikrishnan, R; Mishra, Rashmi; Lissa, R P; Rajkumar, J S; Sudarsanam, D

    2013-10-01

    Current protocols of islet cell transplantation for the treatment of diabetes mellitus have been hampered by islet availability and allograft rejection. Although bone marrow and subcutaneous adipose tissue stem cells feature their tissue repair efficacy, applicability of stem cells from various sources is being researched to develop a promising therapy for diabetes mellitus. Although omentum fat has emerged as an innovative source of stem cells, the dearth of researches confirming its transdifferentiation potential limits its applicability as a regenerative tool in diabetic therapy. Thus, this work is a maiden attempt to explore the colossal potency of omentum fat-derived stem cells on its lucrative differentiation ability. The plasticity of omentum fat stem cells was substantiated by transdifferentiation into pancreatic islet-like clusters, which was confirmed by dithizone staining and immunocytochemistry for insulin. It was also confirmed by the expression of pancreatic endocrine markers nestin and pancreatic duodenal homeobox 1 (Pdx 1) using Fluorescence-activated cell sorting (FACS), neurogenic 3, islet-1 transcription factor, paired box gene 4, Pdx 1 and insulin using quantitative real-time polymerase chain reaction and through insulin secretion assay. This study revealed the in vitro differentiation potency of omentum fat stem cells into pancreatic islet-like clusters. However, further research pursuits exploring its in vivo endocrine efficacy would make omentum fat stem cells a superior source for β-cell replacement therapy.

  7. Effect of amiodarone on retrograde conduction and refractoriness of the His-Purkinje system in man.

    PubMed Central

    Reddy, C P; Kuo, C S

    1984-01-01

    The effects of long term treatment with oral amiodarone on retrograde conduction ( S2H2 interval) and refractoriness of the His-Purkinje system were studied in 11 patients using His bundle electrograms and the ventricular extrastimulus method. Ten patients had ventricular tachycardia and one supraventricular tachycardia. Electrophysiological studies were carried out before and after the patients had been taking their maintenance dose for a mean duration of 84 days. After amiodarone treatment the HV interval was prolonged in seven patients and unchanged in four. At comparable S1S2 intervals, the S2H2 intervals were longer after treatment with amiodarone in all patients than before. Similarly, the longest S2H2 intervals achieved after amiodarone were longer than the control values. Amiodarone significantly increased the relative, effective, and functional refractory periods of the His-Purkinje system. Thus amiodarone exerts important effects on the His-Purkinje system. PMID:6732994

  8. Purkinje fiber dysplasia (histiocytoid cardiomyopathy) with ventricular noncompaction in a savannah kitten.

    PubMed

    Gelberg, H B

    2009-07-01

    In a 2-month-old female savannah kitten that died unexpectedly, the pathologic findings of significance were restricted to the heart and included abnormal Purkinje fibers and biventricular myocardial trabeculation or noncompaction. The Purkinje fibers were large, angular, and tightly packed. They contained few disorganized myofibrils among a rarified cytoplasm. The fibers were distinct from adjacent myocytes and were immunohistochemically positive for desmin, muscle actin, myoglobin, sarcomeric actin, and chromogranin A. These findings are identical to those that occur in children with histiocytoid cardiomyopathy, a fatal genetic mitochondrial disorder of Purkinje fibers. Ventricular noncompaction likely has a multifactoral cause that results from fetal arrest of ventricular organizational development that might occur in conjunction with, or independent of, histiocytoid cardiomyopathy.

  9. Micro RNA-17-92 cluster mediates interleukin-4-suppressed IL-10 expression in B cells.

    PubMed

    Liu, Zhi-Qiang; Yang, Gui; Geng, Xiao-Rui; Liu, Jiang-Qi; Mo, Li-Hua; Liu, Zhi-Gang; Yang, Ping-Chang

    2016-01-01

    The pathogenesis of allergen-related inflammation in the intestine is to be further understood. Micro RNA (miR) can regulate immune responses. This study aims to investigate the role of miR-17-92 cluster in the induction of food allergen-related inflammation in the intestine. In this study, a mouse model of food allergen-related intestinal inflammation was developed. Expression of miR-17-92 cluster in B cells of the intestinal mucosa was analyzed by real time quantitative RT-PCR. The results showed that the levels of miR-19a, one of the members of the miR-17-92 cluster, were detected in the B cells of the intestine of mice sensitized to ovalbumin, which was significantly higher than that in naïve control mice. The expression of IL-10 by B cells was significantly lower in the sensitized mice as compared with naive control mice. Exposure to IL-4 in the culture increased the expression of miR-19a as well as suppression the expression of IL-10 in B cells via remolding DNA structure at the IL-10 promoter locus. We conclude that B cells from sensitized mice show higher levels of miR-19a, which plays an important role in the suppression of IL-10 in the B cells.

  10. Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells.

    PubMed

    Gandía, L; Mayorgas, I; Michelena, P; Cuchillo, I; de Pascual, R; Abad, F; Novalbos, J M; Larrañaga, E; García, A G

    1998-10-01

    Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five

  11. Preparation of multi-coloured different sized fluorescent gold clusters from blue to NIR, structural analysis of the blue emitting Au7 cluster, and cell-imaging by the NIR gold cluster.

    PubMed

    Roy, Subhasish; Baral, Abhishek; Bhattacharjee, Rameswar; Jana, Batakrishna; Datta, Ayan; Ghosh, Surajit; Banerjee, Arindam

    2015-02-07

    Blue, green, orange-red, red and NIR emitting gold quantum clusters have been prepared in aqueous media by using a bioactive peptide glutathione (reduced) at physiological pH. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses show that the core structure sizes of the five different gold clusters are Au7 (blue), Au16 (green), Au19 (orange-red), Au21 (red) and Au22 (NIR). The photo-stability and pH-stability of these quantum clusters have been measured, and these are photo-stable against continuous UV irradiation for a few hours. They also exhibit moderate to good pH-stability within the pH range of 5-12.5. A computational study reveals the organisation of gold atoms in the thiolate-protected blue quantum cluster and its several structural parameters, including the mode of interaction of ligand molecules with Au atoms in the Au7 cluster. Interestingly, it has been found that NIR emitting gold quantum cluster can easily be internalized into the adenocarcinomic human alveolar basal epithelial cell line (A549 cell line). Moreover, a MTT assay indicates that our NIR emitting gold quantum cluster show very low cytotoxicy to A549 cancer cells.

  12. Real-time dynamics of RNA Polymerase II clustering in live human cells

    NASA Astrophysics Data System (ADS)

    Cisse, Ibrahim

    2014-03-01

    Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

  13. Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice.

    PubMed

    Park, In-Su; Chung, Phil-Sang; Ahn, Jin Chul

    2015-07-03

    Human adipose-derived mesenchymal stem cells (hASCs) are attractive cell source for skin tissue engineering. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted cluster hASC in a skin wound animal model. The hASCs were cultured in monolayer or clusters. The LLLT, hASCs, hASC clusters, and hASC clusters transplantation with LLLT (cluster + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining, and elastin van gieson histochemistry. The survival, differentiation, and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the cluster ASC were evaluated by immunohistochemistry and Western blotting. The cluster + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the cluster group. The secretion of growth factors was stimulated in the cluster + LLLT group compared with the ASCs and cluster group. These data suggest that LLLT is an effective biostimulator of cluster hASCs in wound healing that enhances the survival of hASCs and stimulates the secretion of growth factors in the wound bed.

  14. Uruguay Roselli 1938 and Rosellichnus, n. ichnogenus: Two ichnogenera for clusters of fossil bee cells

    USGS Publications Warehouse

    Genise, J.F.; Bown, T.M.

    1996-01-01

    The systematic of the fossil bee nest ichnogenus Uruguay from the Uruguayan Cretaceous or lower Tertiary is reviewed and two new ichnospecies of a new ichnogenus, Rosellichnus, are proposed for fossil bee nests from the Miocene of the United Arab Emirates and Patagonian Argentina. Uruguay contains U. auroranormae (the ichnotype) and U. rivasi, which differs from the type ichnospecies in having clustered cells arranged in three rows, with one central and two marginal rows of subparallel cells. No bee nest architecture known to us unequivocally fits with any ichnospecies described herein. Uruguay was constructed in the subsoil and may be of halictid origin; however, its large cell size, thick cell walls, and the presence of a vestibular cell do not fit the architecture of nests of extant halictids. Rosellichnus arabicus lacks the complete cavity surrounding the cell cluster that is typical in nests of Old World halictids, but it may, nonetheless, still be of halictid origin. R. patagonicus has an architecture more similar to nests of anthophorine bees than to those of Neotropical Halictidae. Therefore, the ichnogenus Rosellichnus may include both halictid and anthophorine constructions. Although the smooth inner cell walls, spiral cell caps, and clustering of cells indicate that both Uruguay and Rosellichnus are trace fossils of bee origin, it is likely that both represent nest architectures that are no longer employed by extant bees, or those that are as yet unknown. The paleoenvironments of both the Argentine and Emirates species of Rosellichnus indicate that the trace-makers of this ichnogenus constructed their nests in open ground sandy soils. ?? 1996 OPA (Overseas Publishers Association) Amsterdam B.V. Published in The Netherlands by Harwood Academic Publishers GmbH.

  15. Plasma protein induced clustering of red blood cells in micro capillaries

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Brust, Mathias; Aouane, Othmane; Flormann, Daniel; Thiebaud, Marine; Verdier, Claude; Coupier, Gwennou; Podgorski, Thomas; Misbah, Chaouqi; Selmi, Hassib

    2013-11-01

    The plasma molecule fibrinogen induces aggregation of RBCs to clusters, the so called rouleaux. Higher shear rates in bulk flow can break them up which results in the pronounced shear thinning of blood. This led to the assumption that rouleaux formation does not take place in the microcapillaries of the vascular network where high shear rates are present. However, the question is of high medical relevance. Cardio vascular disorders are still the main cause of death in the western world and cardiac patients have often higher fibrinogen level. We performed AFM based single cell force spectroscopy to determine the work of separation. Measurements at low hematocrit in a microfluidic channel show that the number of size of clusters is determined by the adhesion strength and we found that cluster formation is strongly enhanced by fibrinogen at physiological concentrations, even at shear rate as high as 1000 1/s. Numerical simulations based on a boundary integral method confirm our findings and the clustering transition takes place both in the experiments and in the simulations at the same interaction energies. In vivo measurements with intravital fluorescence microscopy in a dorsal skin fold chamber in a mouse reveal that RBCs indeed form clusters in the micrcapillary flow. This work was supported by the German Science Foundation research imitative SFB1027.

  16. The role of late I and antiarrhythmic drugs in EAD formation and termination in Purkinje fibers.

    PubMed

    Fedida, David; Orth, Peter M R; Hesketh, J Christian; Ezrin, Alan M

    2006-05-01

    Multiple components of cardiac Na current play a role in determining electrical excitation in the heart. Recently, the role of nonequilibrium components in controlling cardiac action potential plateau duration, and their importance in regulating the occurrence of afterdepolarizations and arrhythmias have garnered more attention. In particular, late Na current (late I(Na)) has been shown to be important in LQT2 and LQT3 arrhythmias. Class III agents like dofetilide, clofilium, and sotalol, which can all cause a drug-induced form of LQT2, significantly lengthen action potential duration at 50% and 90% repolarization in isolated rabbit Purkinje fibers, and can initiate the formation of early afterdepolarizations, and extra beats. These actions can lead to the development of a serious ventricular tachycardia, torsades de pointes, in animal models and patients. However, pretreatment with agents that block late I(Na), like lidocaine, mexiletine, and RSD1235, a novel mixed ion channel blocker for the rapid pharmacologic conversion of atrial fibrillation, significantly attenuates the prolonging effects of Class III agents or those induced by ATX-II, a specific toxin that delays Na channel inactivation and amplifies late I(Na) greatly, mimicking LQT3. The Na channel block caused by lidocaine and RSD1235 can be through the open or inactivated states of the channel, but both equivalently inhibit a late component of Na current (I(Na)), recorded at 22 degrees C using whole-cell patch clamp of Nav 1.5 expressed in HEK cells. These protective actions of lidocaine, mexiletine, and RSD1235 may result, at least in part, from their ability to inhibit late I(Na) during action potential repolarization, and inhibition of the inward currents contributing to EAD and arrhythmia formation.

  17. Endogenous light scattering as an optical signature of circulating tumor cell clusters

    PubMed Central

    Lyons, Joe; Polmear, Michael; Mineva, Nora D.; Romagnoli, Mathilde; Sonenshein, Gail E.; Georgakoudi, Irene

    2016-01-01

    Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC. PMID:27231606

  18. Endogenous light scattering as an optical signature of circulating tumor cell clusters.

    PubMed

    Lyons, Joe; Polmear, Michael; Mineva, Nora D; Romagnoli, Mathilde; Sonenshein, Gail E; Georgakoudi, Irene

    2016-03-01

    Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC.

  19. Hostility is related to clusters of T-cell cytokines and chemokines in healthy men.

    PubMed

    Mommersteeg, Paula M C; Vermetten, Eric; Kavelaars, Annemieke; Geuze, Elbert; Heijnen, Cobi J

    2008-09-01

    Hostility is a risk factor for adverse health outcomes as diverse as cardiovascular disease and post-traumatic stress disorder (PTSD). Cytokines have been suggested to mediate this relationship. We investigated whether in healthy men a relation existed between hostility and T-cell mitogen-induced cytokines and chemokines. Male Dutch military personnel (n=304) were included before deployment. Eleven cytokines and chemokines were measured in supernatants of T-cell mitogen-stimulated whole blood cultures by multiplex immunoassay. Factor analysis was used to identify clusters of cytokines and chemokines. In a regression analysis hostility was related to the cytokine/chemokine clusters, and the potential risk factors age, BMI, smoking, drinking, previous deployment, early life trauma and depression. Explorative factor analysis showed four functional clusters; a pro-inflammatory factor (IL-2, TNFalpha, IFNgamma), an anti-inflammatory factor (IL-4, IL-5, IL-10), IL-6/chemokine factor (IL-6, MCP-1, RANTES, IP-10), and MIF. Hostility was significantly related to decreased IL-6/chemokine secretion and increased pro- and anti-inflammatory cytokines. There was an inverse relation between age and hostility scores. Early life trauma and depression were positively and independently related to hostility as well. This study represents a novel way of investigating the relation between cytokines and psychological characteristics. Cytokines/chemokines clustered into functional factors, which were related to hostility in healthy males. Moreover this relation appeared to be independent of reported depression and early trauma.

  20. The Magea gene cluster regulates male germ cell apoptosis without affecting the fertility in mice

    PubMed Central

    Hou, Siyuan; Xian, Li; Shi, Peiliang; Li, Chaojun; Lin, Zhaoyu; Gao, Xiang

    2016-01-01

    While apoptosis is essential for male germ cell development, improper activation of apoptosis in the testis can affect spermatogenesis and cause reproduction defects. Members of the MAGE-A (melanoma antigen family A) gene family are frequently clustered in mammalian genomes and are exclusively expressed in the testes of normal animals but abnormally activated in a wide variety of cancers. We investigated the potential roles of these genes in spermatogenesis by generating a mouse model with a 210-kb genomic deletion encompassing six members of the Magea gene cluster (Magea1, Magea2, Magea3, Magea5, Magea6 and Magea8). Male mice carrying the deletion displayed smaller testes from 2 months old with a marked increase in apoptotic germ cells in the first wave of spermatogenesis. Furthermore, we found that Magea genes prevented stress-induced spermatogenic apoptosis after N-ethyl-N-nitrosourea (ENU) treatment during the adult stage. Mechanistically, deletion of the Magea gene cluster resulted in a dramatic increase in apoptotic germ cells, predominantly spermatocytes, with activation of p53 and induction of Bax in the testes. These observations demonstrate that the Magea genes are crucial in maintaining normal testicular size and protecting germ cells from excessive apoptosis under genotoxic stress. PMID:27226137

  1. Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer

    PubMed Central

    Servín-González, Luis Steven; Granados-López, Angelica Judith; López, Jesús Adrián

    2015-01-01

    Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s) or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2), which regulated mitogen-activated protein kinases (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design. PMID:26057746

  2. Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer.

    PubMed

    Servín-González, Luis Steven; Granados-López, Angelica Judith; López, Jesús Adrián

    2015-06-05

    Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s) or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2), which regulated mitogen-activated protein kinases (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design.

  3. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data.

    PubMed

    Weber, Lukas M; Robinson, Mark D

    2016-12-01

    Recent technological developments in high-dimensional flow cytometry and mass cytometry (CyTOF) have made it possible to detect expression levels of dozens of protein markers in thousands of cells per second, allowing cell populations to be characterized in unprecedented detail. Traditional data analysis by "manual gating" can be inefficient and unreliable in these high-dimensional settings, which has led to the development of a large number of automated analysis methods. Methods designed for unsupervised analysis use specialized clustering algorithms to detect and define cell populations for further downstream analysis. Here, we have performed an up-to-date, extensible performance comparison of clustering methods for high-dimensional flow and mass cytometry data. We evaluated methods using several publicly available data sets from experiments in immunology, containing both major and rare cell populations, with cell population identities from expert manual gating as the reference standard. Several methods performed well, including FlowSOM, X-shift, PhenoGraph, Rclusterpp, and flowMeans. Among these, FlowSOM had extremely fast runtimes, making this method well-suited for interactive, exploratory analysis of large, high-dimensional data sets on a standard laptop or desktop computer. These results extend previously published comparisons by focusing on high-dimensional data and including new methods developed for CyTOF data. R scripts to reproduce all analyses are available from GitHub (https://github.com/lmweber/cytometry-clustering-comparison), and pre-processed data files are available from FlowRepository (FR-FCM-ZZPH), allowing our comparisons to be extended to include new clustering methods and reference data sets. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

  4. Emergence of organized bursting in clusters of pancreatic beta-cells by channel sharing.

    PubMed Central

    Sherman, A; Rinzel, J; Keizer, J

    1988-01-01

    Pancreatic beta-cells in an intact Islet of Langerhans exhibit bursting electrical behavior. The Chay-Keizer model describes this using a calcium-activated potassium (K-Ca) channel, but cannot account for the irregular spiking of isolated beta-cells. Atwater I., L. Rosario, and E. Rojas, Cell Calcium. 4:451-461, proposed that the K-Ca channels, which are rarely open, are shared by several cells. This suggests that the chaotic behavior of isolated cells is stochastic. We have revised the Chay-Keizer model to incorporate voltage clamp data of Rorsman and Trube and extended it to include stochastic K-Ca channels. This model can describe the behavior of single cells, as well as that of clusters of cells tightly coupled by gap junctions. As the size of the clusters is increased, the electrical activity shows a transition from chaotic spiking to regular bursting. Although the model of coupling is over-simplified, the simulations lend support to the hypothesis that bursting is the result of channel sharing. PMID:2850029

  5. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    SciTech Connect

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  6. Clusters of synaptic inputs on dendrites of layer 5 pyramidal cells in mouse visual cortex

    PubMed Central

    Gökçe, Onur; Bonhoeffer, Tobias; Scheuss, Volker

    2016-01-01

    The spatial organization of synaptic inputs on the dendritic tree of cortical neurons plays a major role for dendritic integration and neural computations, yet, remarkably little is known about it. We mapped the spatial organization of glutamatergic synapses between layer 5 pyramidal cells by combining optogenetics and 2-photon calcium imaging in mouse neocortical slices. To mathematically characterize the organization of inputs we developed an approach based on combinatorial analysis of the likelihoods of specific synapse arrangements. We found that the synapses of intralaminar inputs form clusters on the basal dendrites of layer 5 pyramidal cells. These clusters contain 4 to 14 synapses within ≤30 µm of dendrite. According to the spatiotemporal characteristics of synaptic summation, these numbers suggest that there will be non-linear dendritic integration of synaptic inputs during synchronous activation. DOI: http://dx.doi.org/10.7554/eLife.09222.001 PMID:27431612

  7. Segmentation of White Blood Cells through Nucleus Mark Watershed Operations and Mean Shift Clustering

    PubMed Central

    Liu, Zhi; Liu, Jing; Xiao, Xiaoyan; Yuan, Hui; Li, Xiaomei; Chang, Jun; Zheng, Chengyun

    2015-01-01

    This paper presents a novel method for segmentation of white blood cells (WBCs) in peripheral blood and bone marrow images under different lights through mean shift clustering, color space conversion and nucleus mark watershed operation (NMWO). The proposed method focuses on obtaining seed points. First, color space transformation and image enhancement techniques are used to obtain nucleus groups as inside seeds. Second, mean shift clustering, selection of the C channel component in the CMYK model, and illumination intensity adjustment are employed to acquire WBCs as outside seeds. Third, the seeds and NMWO are employed to precisely determine WBCs and solve the cell adhesion problem. Morphological operations are further used to improve segmentation accuracy. Experimental results demonstrate that the algorithm exhibits higher segmentation accuracy and robustness compared with traditional methods. PMID:26370995

  8. Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis

    PubMed Central

    Taylor, Marcia L.; Sehgal, Devinder; Raffeld, Mark; Obiakor, Harold; Akin, Cem; Mage, Rose G.; Metcalfe, Dean D.

    2004-01-01

    Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering. PMID:15507672

  9. An effective algorithm for the generation of patient-specific Purkinje networks in computational electrocardiology

    NASA Astrophysics Data System (ADS)

    Palamara, Simone; Vergara, Christian; Faggiano, Elena; Nobile, Fabio

    2015-02-01

    The Purkinje network is responsible for the fast and coordinated distribution of the electrical impulse in the ventricle that triggers its contraction. Therefore, it is necessary to model its presence to obtain an accurate patient-specific model of the ventricular electrical activation. In this paper, we present an efficient algorithm for the generation of a patient-specific Purkinje network, driven by measures of the electrical activation acquired on the endocardium. The proposed method provides a correction of an initial network, generated by means of a fractal law, and it is based on the solution of Eikonal problems both in the muscle and in the Purkinje network. We present several numerical results both in an ideal geometry with synthetic data and in a real geometry with patient-specific clinical measures. These results highlight an improvement of the accuracy provided by the patient-specific Purkinje network with respect to the initial one. In particular, a cross-validation test shows an accuracy increase of 19% when only the 3% of the total points are used to generate the network, whereas an increment of 44% is observed when a random noise equal to 20% of the maximum value of the clinical data is added to the measures.

  10. Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

    PubMed Central

    Maio, Nunziata; Rouault, Tracey. A.

    2014-01-01

    Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

  11. Unsupervised unstained cell detection by SIFT keypoint clustering and self-labeling algorithm.

    PubMed

    Muallal, Firas; Schöll, Simon; Sommerfeldt, Björn; Maier, Andreas; Steidl, Stefan; Buchholz, Rainer; Hornegger, Joachim

    2014-01-01

    We propose a novel unstained cell detection algorithm based on unsupervised learning. The algorithm utilizes the scale invariant feature transform (SIFT), a self-labeling algorithm, and two clustering steps in order to achieve high performance in terms of time and detection accuracy. Unstained cell imaging is dominated by phase contrast and bright field microscopy. Therefore, the algorithm was assessed on images acquired using these two modalities. Five cell lines having in total 37 images and 7250 cells were considered for the evaluation: CHO, L929, Sf21, HeLa, and Bovine cells. The obtained F-measures were between 85.1 and 89.5. Compared to the state-of-the-art, the algorithm achieves very close F-measure to the supervised approaches in much less time.

  12. ALA-PDT of glioma cell micro-clusters in BD-IX rat brain

    NASA Astrophysics Data System (ADS)

    Madsen, Steen J.; Angell-Petersen, Even; Spetalen, Signe; Carper, Stephen W.; Ziegler, Sarah A.; Hirschberg, Henry

    2006-02-01

    A significant contributory factor to the poor prognosis of patients with glioblastoma multiforme is the inability of conventional treatments to eradicate infiltrating glioma cells. A syngeneic rat brain tumor model is used to investigate the effects of aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on small clusters of tumor cells sequestered in normal brain. The intrinsic sensitivity of rat glioma cells to PDT was investigated by exposing ALA-incubated cells to a range of radiant exposures and irradiances using 635 nm light. Biodistribution studies were undertaken on tumor-bearing animals in order to determine the tumor selectivity of the photosensitizer following systemic administration (i.p.) of ALA. Effects of ALA-PDT on normal brain and gross tumor were evaluated using histopathology. Effects of PDT on isolated glioma cells in normal brain were investigated by treating animals 48 h after tumor cell implantation: a time when the micro-clusters of cells are protected by an intact blood-brain-barrier (BBB). Rat glioma cells in monolayer are susceptible to ALA-PDT - lower irradiances are more effective than higher ones. Fluorescence microscopy of frozen tissue sections showed that photosensitizer is produced with better than 200:1 tumor-to-normal tissue selectivity following i.p. ALA administration. ALA-PDT resulted in significant damage to both gross tumor and normal brain, however, the treatment failed to prolong survival of animals with newly implanted glioma cells compared to non-treated controls if the drug was delivered either i.p. or directly into the brain. In contrast, animals inoculated with tumor cells pre-incubated in vitro with ALA showed a significant survival advantage in response to PDT.

  13. Prostaglandin E2 stimulates estradiol synthesis in the cerebellum postnatally with associated effects on Purkinje neuron dendritic arbor and electrophysiological properties.

    PubMed

    Dean, Shannon L; Wright, Christopher L; Hoffman, Jessica F; Wang, Meina; Alger, Bradley E; McCarthy, Margaret M

    2012-11-01

    Prostaglandins (PGs) are ubiquitous membrane-derived, lipid-signaling molecules with wide ranging effects throughout the body. In the brain, PGE(2) is the key regulator of fever after inflammation but is also implicated in neural development and synaptic plasticity. The steroid hormone estradiol is also a key regulator of neural development and synaptic plasticity. Recently, we showed that administering cyclooxygenase (COX) inhibitors to block PGE(2) production increased the total length of Purkinje cell dendrites, the number of dendritic spines, and the level of spinophilin protein, which is enriched in dendritic spines. Correspondingly, PGE(2) administration into the cerebellum decreased spinophilin protein content. We now report that PGE(2) stimulates estradiol synthesis in the immature rat cerebellum via enhanced activity of the aromatase enzyme. Treatment with cyclooxygenase inhibitors reduced cerebellar aromatase activity and estradiol content whereas PGE(2) administration increased both. Treatment with either PGE(2) or estradiol stunted Purkinje neuron dendritic length and complexity and produced a corresponding reduction in spinophilin content. Treatment with formestane to inhibit aromatase activity led to excessive sprouting of the dendritic tree, whereas elevated estradiol had the opposite effect. Electrophysiological measurements from Purkinje neurons revealed novel sex differences in input resistance and membrane capacitance that were abolished by estradiol exposure, whereas a sex difference in the amplitude of the afterhyperpolarization after an action potential was not. Correlated changes in action potential threshold suggest that prolonged alterations in neuronal firing activity could be a consequence of increased estradiol content during the second week of life. These findings reveal a previously unappreciated role for PG-stimulated steroidogenesis in the developing brain and a new potential route for inflammation-mediated disruption of neuronal

  14. Biocompatible glutathione capped gold clusters as one- and two-photon excitation fluorescence contrast agents for live cells imaging

    NASA Astrophysics Data System (ADS)

    Polavarapu, Lakshminarayana; Manna, Manoj; Xu, Qing-Hua

    2011-02-01

    The one- and two-photon excitation emission properties of water soluble glutathione monolayer protected gold clusters were investigated. Strong two-photon emission was observed from the gold clusters. The two-photon absorption cross section of these gold clusters in water was deduced from the z-scan measurement to be 189 740 GM, which is much higher compared to organic fluorescent dyes and quantum dots. These gold clusters also showed high photo-stability. The MTT assay showed that these gold clusters have low toxicity even at high concentrations. We have successfully demonstrated their applications for both one and two-photon excitation live cell imaging. The exceptional properties of these gold clusters make them a promising alternative for one- and two-photon bio-imaging and other nonlinear optical applications.The one- and two-photon excitation emission properties of water soluble glutathione monolayer protected gold clusters were investigated. Strong two-photon emission was observed from the gold clusters. The two-photon absorption cross section of these gold clusters in water was deduced from the z-scan measurement to be 189 740 GM, which is much higher compared to organic fluorescent dyes and quantum dots. These gold clusters also showed high photo-stability. The MTT assay showed that these gold clusters have low toxicity even at high concentrations. We have successfully demonstrated their applications for both one and two-photon excitation live cell imaging. The exceptional properties of these gold clusters make them a promising alternative for one- and two-photon bio-imaging and other nonlinear optical applications. Electronic supplementary information (ESI) available: Control experiments; blinking studies of gold clusters. See DOI: 10.1039/c0nr00458h

  15. Quantitative Control of Protein and Cell Interaction with Nanostructured Surfaces by Cluster Assembling.

    PubMed

    Schulte, Carsten; Podestà, Alessandro; Lenardi, Cristina; Tedeschi, Gabriella; Milani, Paolo

    2017-02-21

    of biological entities on surfaces, are strongly influenced and controlled by the nanoscale roughness. By developing a high-throughput method (protein surface interaction microarray, PSIM), we characterized quantitatively the capacity of the nanostructured surfaces to adsorb proteins, showing that with increasing roughness the adsorption rises beyond what could be expected by the increase in specific area, paralleled by an almost linear decrease in protein binding affinity. We also determined that the spatial layout of the surface asperities effectively perceived by the cells mimics at the nanoscale the topographical ECM characteristics. The interaction with these features consequently regulates parameters significant for cell adhesion and mechanotransductive signaling, such as integrin clustering, focal adhesion maturation, and the correlated cellular mechanobiology, eventually impacting the cellular program and differentiation, as we specifically showed for neuronal cells.

  16. Three-Dimensional Characterization of Cell Clusters Using Synchrotron-Radiation-Based Micro-Computed Tomography

    NASA Astrophysics Data System (ADS)

    Müller, Ert; Riedel, Marco; Thurner, Philipp J.

    2006-04-01

    Micro-computed tomography with the highly intense, monochromatic X rays produced by the synchrotron is a superior method to nondestructively measure the local absorption in three-dimensional space. Because biological tissues and cells consist mainly of water as the surrounding medium, higher absorbing agents have to be incorporated into the structures of interest. Even without X-ray optics such as refractive lens, one can uncover the stain distribution with the spatial resolution of about 1 [mu]m. Incorporating the stain at selected cell compartments, for example, binding to the RNA/DNA, their density distribution becomes quantified. In this communication, we demonstrate that tomograms obtained at the beamlines BW2 and W2 (HASYLAB at DESY, Hamburg, Germany) and 4S (SLS, Villigen, Switzerland) clearly show that the RNA/DNA-stained HEK 293 cell clusters have a core of high density and a peripheral part of lower density, which correlate with results of optical microscopy. The inner part of the clusters is associated with nonvital cells as the result of insufficient oxygen and nutrition supply. This necrotic part is surrounded by (6 ± 1) layers of vital cells.

  17. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

    PubMed

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

    2014-07-01

    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

  18. Lesional gene expression profiling in cutaneous T-cell lymphoma reveals natural clusters associated with disease outcome

    PubMed Central

    Shin, Jessica; Monti, Stefano; Aires, Daniel J.; Duvic, Madeleine; Golub, Todd

    2007-01-01

    Cutaneous T-cell lymphoma (CTCL) is defined by infiltration of activated and malignant T cells in the skin. The clinical manifestations and prognosis in CTCL are highly variable. In this study, we hypothesized that gene expression analysis in lesional skin biopsies can improve understanding of the disease and its management. Based on 63 skin samples, we performed consensus clustering, revealing 3 patient clusters. Of these, 2 clusters tended to differentiate limited CTCL (stages IA and IB) from more extensive CTCL (stages IB and III). Stage IB patients appeared in both clusters, but those in the limited CTCL cluster were more responsive to treatment than those in the more extensive CTCL cluster. The third cluster was enriched in lymphocyte activation genes and was associated with a high proportion of tumor (stage IIB) lesions. Survival analysis revealed significant differences in event-free survival between clusters, with poorest survival seen in the activated lymphocyte cluster. Using supervised analysis, we further characterized genes significantly associated with lower-stage/treatment-responsive CTCL versus higher-stage/treatment-resistant CTCL. We conclude that transcriptional profiling of CTCL skin lesions reveals clinically relevant signatures, correlating with differences in survival and response to treatment. Additional prospective long-term studies to validate and refine these findings appear warranted. PMID:17638852

  19. Dysregulation and functional roles of miR-183-96-182 cluster in cancer cell proliferation, invasion and metastasis

    PubMed Central

    Ma, Yi; Liang, A-Juan; Fan, Yu-Ping; Huang, Yi-Ran; Zhao, Xiao-Ming; Sun, Yun; Chen, Xiang-Feng

    2016-01-01

    Previous studies have reported aberrant expression of the miR-183-96-182 cluster in a variety of tumors, which indicates its' diagnostic or prognostic value. However, a key characteristic of the miR-183-96-182 cluster is its varied expression levels, and pleomorphic functional roles in different tumors or under different conditions. In most tumor types, the cluster is highly expressed and promotes tumorigenesis, cancer progression and metastasis; yet tumor suppressive effects have also been reported in some tumors. In the present study, we discuss the upstream regulators and the downstream target genes of miR-183-96-182 cluster, and highlight the dysregulation and functional roles of this cluster in various tumor cells. Newer insights summarized in this review will help readers understand the different facets of the miR-183-96-182 cluster in cancer development and progression. PMID:27081087

  20. Dysregulation and functional roles of miR-183-96-182 cluster in cancer cell proliferation, invasion and metastasis.

    PubMed

    Ma, Yi; Liang, A-Juan; Fan, Yu-Ping; Huang, Yi-Ran; Zhao, Xiao-Ming; Sun, Yun; Chen, Xiang-Feng

    2016-07-05

    Previous studies have reported aberrant expression of the miR-183-96-182 cluster in a variety of tumors, which indicates its' diagnostic or prognostic value. However, a key characteristic of the miR-183-96-182 cluster is its varied expression levels, and pleomorphic functional roles in different tumors or under different conditions. In most tumor types, the cluster is highly expressed and promotes tumorigenesis, cancer progression and metastasis; yet tumor suppressive effects have also been reported in some tumors. In the present study, we discuss the upstream regulators and the downstream target genes of miR-183-96-182 cluster, and highlight the dysregulation and functional roles of this cluster in various tumor cells. Newer insights summarized in this review will help readers understand the different facets of the miR-183-96-182 cluster in cancer development and progression.

  1. Biocompatible glutathione capped gold clusters as one- and two-photon excitation fluorescence contrast agents for live cells imaging.

    PubMed

    Polavarapu, Lakshminarayana; Manna, Manoj; Xu, Qing-Hua

    2011-02-01

    The one- and two-photon excitation emission properties of water soluble glutathione monolayer protected gold clusters were investigated. Strong two-photon emission was observed from the gold clusters. The two-photon absorption cross section of these gold clusters in water was deduced from the z-scan measurement to be 189 740 GM, which is much higher compared to organic fluorescent dyes and quantum dots. These gold clusters also showed high photo-stability. The MTT assay showed that these gold clusters have low toxicity even at high concentrations. We have successfully demonstrated their applications for both one and two-photon excitation live cell imaging. The exceptional properties of these gold clusters make them a promising alternative for one- and two-photon bio-imaging and other nonlinear optical applications.

  2. Biomimetic 3D Clusters Using Human Adipose Derived Mesenchymal Stem Cells and Breast Cancer Cells: A Study on Migration and Invasion of Breast Cancer Cells.

    PubMed

    Park, Min Hee; Song, Boa; Hong, Seungpyo; Kim, Sang Heon; Lee, Kangwon

    2016-07-05

    Invasion and metastasis of cancer directly related to human death have been associated with interactions among many different types of cells and three-dimensional (3D) tissue matrices. Precise mechanisms related to cancer invasion and metastasis still remain unknown due to their complexities. Development of tumor microenvironment (TME)-mimicking system could play a key role in understanding cancer environments and in elucidating the relating phenomena and their driving forces. Here we report a facile and novel platform of 3D cancer cell-clusters using human adipose-derived mesenchymal stem cells (hASCs) and breast cancer cells (MDA-MB-231) within a collagen gel matrix to show cancer invasion in the cell and extracellular matrix (ECM). Both clusters A (hASC only) and AC (hASC and MDA-MB-231) exhibited different behaviors and expressions of migration and invasion, as observed by the relating markers such as fibronectin, α-SMA, and CXCR4. hASCs showed a protrusive migration from a cluster center, whereas MDA-MB-231 spread out radially followed by hASC migration. Finally, the effect of matrix was further discussed by varying collagen gel densities. The new biomimetic system of 3D cancer clusters developed here has the potential to be utilized for research on migration and invasion of cancer cells in extracellular matrices.

  3. MiR-371-373 cluster acts as a tumor-suppressor-miR and promotes cell cycle arrest in unrestricted somatic stem cells.

    PubMed

    Langroudi, Lida; Jamshidi-Adegani, Fatemeh; Shafiee, Abbas; Rad, Seyed Mohammad Ali Hosseini; Keramati, Farid; Azadmanesh, Kayhan; Arefian, Ehsan; Soleimani, Masoud

    2015-09-01

    Recent advances in small RNA research have implicated microRNAs (miRNAs) as important regulators of proliferation and development. The miR-371-373 cluster is prominently expressed in human embryonic stem cells (ESCs) and rapidly decreases after cell differentiation. MiR-371-373 cluster was investigated as one of the key factors of stem cell maintenance and pluripotency in unrestricted somatic stem cells (USSCs) using a lentivirus system. Gene expression showed a dual effect on proliferation, which revealed a transient cell cycle progression and consequent repression in pluripotency factors and cell cycle genes. Cell proliferation analysis with CFU, MTT, and DNA content assays further confirmed the dual effect of cluster after prolonged exposure. Analyzing the course of action, it seems that miR-371-373 cluster acts as an onco/tumor suppressor-miR. MiR371-373 cluster acts by modulating the function of these factors and limiting the excessive cell cycle propagation upon oncogenic stimuli to protect cells from replicative stress, but also activate CDK inhibitors and transcriptional repressors of the retinoblastoma family to cause