Science.gov

Sample records for purkinje cell clusters

  1. Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge

    PubMed Central

    Chaumont, Joseph; Guyon, Nicolas; Valera, Antoine M.; Dugué, Guillaume P.; Popa, Daniela; Marcaggi, Paikan; Gautheron, Vanessa; Reibel-Foisset, Sophie; Dieudonné, Stéphane; Stephan, Aline; Barrot, Michel; Cassel, Jean-Christophe; Dupont, Jean-Luc; Doussau, Frédéric; Poulain, Bernard; Selimi, Fekrije; Léna, Clément; Isope, Philippe

    2013-01-01

    Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning. PMID:24046366

  2. Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge.

    PubMed

    Chaumont, Joseph; Guyon, Nicolas; Valera, Antoine M; Dugué, Guillaume P; Popa, Daniela; Marcaggi, Paikan; Gautheron, Vanessa; Reibel-Foisset, Sophie; Dieudonné, Stéphane; Stephan, Aline; Barrot, Michel; Cassel, Jean-Christophe; Dupont, Jean-Luc; Doussau, Frédéric; Poulain, Bernard; Selimi, Fekrije; Léna, Clément; Isope, Philippe

    2013-10-01

    Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning. PMID:24046366

  3. 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. PMID:19412945

  4. Mapping the development of cerebellar Purkinje cells in zebrafish.

    PubMed

    Hamling, Kyla R; Tobias, Zachary J C; Weissman, Tamily A

    2015-11-01

    The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. PMID:25655100

  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. Inverse Stochastic Resonance in Cerebellar Purkinje Cells.

    PubMed

    Buchin, Anatoly; Rieubland, Sarah; Häusser, Michael; Gutkin, Boris S; Roth, Arnd

    2016-08-01

    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

  7. Structured variability in Purkinje cell activity during locomotion

    PubMed Central

    Sauerbrei, Britton A.; Lubenov, Evgueniy V.; Siapas, Athanassios G.

    2015-01-01

    Summary The cerebellum is a prominent vertebrate brain structure that is critically involved in sensorimotor function. During locomotion, cerebellar Purkinje cells are rhythmically active, shaping descending signals and coordinating commands from higher brain areas with the step cycle. However, the variation in this activity across steps has not been studied, and its statistical structure, afferent mechanisms, and relationship to behavior remain unknown. Here, using multi-electrode recordings in freely moving rats, we show that behavioral variables systematically influence the shape of the step-locked firing rate. This effect depends strongly on the phase of the step cycle and reveals a functional clustering of Purkinje cells. Furthermore, we find a pronounced disassociation between patterns of variability driven by the parallel and climbing fibers. These results suggest that Purkinje cell activity not only represents step phase within each cycle, but is also shaped by behavior across steps, facilitating control of movement under dynamic conditions. PMID:26291165

  8. 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

  9. Regulation of Purkinje cell alignment by reelin as revealed with CR-50 antibody.

    PubMed

    Miyata, T; Nakajima, K; Mikoshiba, K; Ogawa, M

    1997-05-15

    Cerebellar Purkinje cells are generated in the ventricular zone, migrate outward, and finally form a monolayer in the cortex. In reeler mice, however, most Purkinje cells cluster abnormally in subcortical areas. Reelin, the candidate reeler gene product recognized by the CR-50 monoclonal antibody, is concentrated in a cortical zone along which Purkinje cells are aligned linearly, implying that it may regulate their alignment. We used an in vitro system and a transplantation approach to analyze the function of Reelin. Explant culture for 7 d of cerebella isolated from wild-type and reeler mice at embryonic day 13 (E13) reproduced in a phenotype-dependent manner the two distinct arrangement patterns (linear vs clustered) of Purkinje cells. Extensive CR-50 binding to wild-type explants converted the linear pattern into a reeler-like, clustered pattern. On the other hand, when reeler explants lacking Reelin were crowned with an artificial layer of Reelin+ granule cells, some Reelin molecules were distributed into a superficial zone of the reeler explants, and Purkinje cells formed a linear pattern along the Reelin-rich overlay. This "rescue" effect was also inhibited by CR-50. Hence, Reelin is involved in the Purkinje cell alignment, and the lack of this activity may explain the malformation in reeler cerebella. We further injected Reelin+ granule cells into the fourth ventricle of E12-13 mice. Extensive incorporation of the injected Reelin+ cells into the ventricular zone, but not of Reelin- cells, forced Purkinje cells of the host cerebella to form an aberrant layer, suggesting that premigratory Purkinje cells may already be responsive to Reelin or Reelin-related signals. PMID:9133383

  10. Death and survival of heterozygous Lurcher Purkinje cells in vitro

    PubMed Central

    Zanjani, Hadi; McFarland, Rebecca; Cavelier, Pauline; Blokhin, Andrei; Gautheron, Vanessa; Levenes, Carole; Bambrick, Linda L.; Mariani, Jean; Vogel, Michael W.

    2009-01-01

    The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+ and +/Lc Purkinje cells from postnatal day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2+/+ and GluRδ2+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1-naphthyl acetyl spermine (NASP) increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. PMID:19294643

  11. Purkinje Cell Pathology and Loss in Multiple Sclerosis Cerebellum.

    PubMed

    Redondo, Juliana; Kemp, Kevin; Hares, Kelly; Rice, Claire; Scolding, Neil; Wilkins, Alastair

    2015-11-01

    Cerebellar ataxia commonly occurs in multiple sclerosis, particularly in chronic progressive disease. Previous reports have highlighted both white matter and grey matter pathological changes within the cerebellum; and demyelination and inflammatory cell infiltrates appear commonly. As Purkinje cell axons are the sole output of the cerebellar cortex, understanding pathologic processes within these cells is crucial to develop strategies to prevent their loss and thus reduce ataxia. We studied pathologic changes occurring within Purkinje cells of the cerebellum. Using immunohistochemic techniques, we found changes in neurofilament phosphorylation states within Purkinje cells, including loss of dephosphorylated neurofilament and increased phosphorylated and hyperphosphorylated neurofilament. We also found Purkinje axonal spheroids and Purkinje cell loss, both of which occurred predominantly within areas of leucocortical demyelination within the cerebellar cortex. These changes have important implications for the study of cerebellar involvement in multiple sclerosis and may help design therapies to reduce the burden of ataxia in the condition. PMID:25411024

  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. PMID:16901270

  13. Are Purkinje Cell Pauses Drivers of Classically Conditioned Blink Responses?

    PubMed

    Jirenhed, Dan-Anders; Hesslow, Germund

    2016-08-01

    Several lines of evidence show that classical or Pavlovian conditioning of blink responses depends on the cerebellum. Recordings from cerebellar Purkinje cells that control the eyelid and the conditioned blink show that during training with a conditioning protocol, a Purkinje cell develops a pause response to the conditional stimulus. This conditioned cellular response has many of the properties that characterise the overt blink. The present paper argues that the learned Purkinje cell pause response is the memory trace and main driver of the overt conditioned blink and that it explains many well-known behavioural phenomena. PMID:26400585

  14. Encoding of action by the Purkinje cells of the cerebellum.

    PubMed

    Herzfeld, David J; Kojima, Yoshiko; Soetedjo, Robijanto; Shadmehr, Reza

    2015-10-15

    Execution of accurate eye movements depends critically on the cerebellum, suggesting that the major output neurons of the cerebellum, Purkinje cells, may predict motion of the eye. However, this encoding of action for rapid eye movements (saccades) has remained unclear: Purkinje cells show little consistent modulation with respect to saccade amplitude or direction, and critically, their discharge lasts longer than the duration of a saccade. Here we analysed Purkinje-cell discharge in the oculomotor vermis of behaving rhesus monkeys (Macaca mulatta) and found neurons that increased or decreased their activity during saccades. We estimated the combined effect of these two populations via their projections to the caudal fastigial nucleus, and uncovered a simple-spike population response that precisely predicted the real-time motion of the eye. When we organized the Purkinje cells according to each cell's complex-spike directional tuning, the simple-spike population response predicted both the real-time speed and direction of saccade multiplicatively via a gain field. This suggests that the cerebellum predicts the real-time motion of the eye during saccades via the combined inputs of Purkinje cells onto individual nucleus neurons. A gain-field encoding of simple spikes emerges if the Purkinje cells that project onto a nucleus neuron are not selected at random but share a common complex-spike property. PMID:26469054

  15. Bidirectional Plasticity of Purkinje Cells Matches Temporal Features of Learning

    PubMed Central

    Wetmore, Daniel Z.; Jirenhed, Dan-Anders; Rasmussen, Anders; Johansson, Fredrik; Schnitzer, Mark J.

    2014-01-01

    Many forms of learning require temporally ordered stimuli. In Pavlovian eyeblink conditioning, a conditioned stimulus (CS) must precede the unconditioned stimulus (US) by at least about 100 ms for learning to occur. Conditioned responses are learned and generated by the cerebellum. Recordings from the cerebellar cortex during conditioning have revealed CS-triggered pauses in the firing of Purkinje cells that likely drive the conditioned blinks. The predominant view of the learning mechanism in conditioning is that long-term depression (LTD) at parallel fiber (PF)–Purkinje cell synapses underlies the Purkinje cell pauses. This raises a serious conceptual challenge because LTD is most effectively induced at short CS–US intervals, which do not support acquisition of eyeblinks. To resolve this discrepancy, we recorded Purkinje cells during conditioning with short or long CS–US intervals. Decerebrated ferrets trained with CS–US intervals ≥150 ms reliably developed Purkinje cell pauses, but training with an interval of 50 ms unexpectedly induced increases in CS-evoked spiking. This bidirectional modulation of Purkinje cell activity offers a basis for the requirement of a minimum CS–US interval for conditioning, but we argue that it cannot be fully explained by LTD, even when previous in vitro studies of stimulus-timing-dependent LTD are taken into account. PMID:24478355

  16. Memory trace and timing mechanism localized to cerebellar Purkinje cells.

    PubMed

    Johansson, Fredrik; Jirenhed, Dan-Anders; Rasmussen, Anders; Zucca, Riccardo; Hesslow, Germund

    2014-10-14

    The standard view of the mechanisms underlying learning is that they involve strengthening or weakening synaptic connections. Learned response timing is thought to combine such plasticity with temporally patterned inputs to the neuron. We show here that a cerebellar Purkinje cell in a ferret can learn to respond to a specific input with a temporal pattern of activity consisting of temporally specific increases and decreases in firing over hundreds of milliseconds without a temporally patterned input. Training Purkinje cells with direct stimulation of immediate afferents, the parallel fibers, and pharmacological blocking of interneurons shows that the timing mechanism is intrinsic to the cell itself. Purkinje cells can learn to respond not only with increased or decreased firing but also with an adaptively timed activity pattern. PMID:25267641

  17. Doppel Induces Degeneration of Cerebellar Purkinje Cells Independently of Bax

    PubMed Central

    Dong, Jiaxin; Li, Aimin; Yamaguchi, Naohiro; Sakaguchi, Suehiro; Harris, David A.

    2007-01-01

    Doppel (Dpl) is a prion protein paralog that causes neurodegeneration when expressed ectopically in the brain. To investigate the cellular mechanism underlying this effect, we analyzed Dpl-expressing transgenic mice in which the gene for the proapoptotic protein Bax had been deleted. We found that Bax deletion does not alter either clinical symptoms or Purkinje cell degeneration in Dpl transgenic mice. In addition, we observed that degenerating Purkinje cells in these animals do not display DNA fragmentation or caspase-3 activation. Our results suggest that non-Bax-dependent pathways mediate the toxic effects of Dpl in Purkinje cells, highlighting a possible role for nonapoptotic mechanisms in the death of these neurons. PMID:17569776

  18. Afferent-target cell interactions in the cerebellum: negative effect of granule cells on Purkinje cell development in lurcher mice.

    PubMed

    Doughty, M L; Lohof, A; Selimi, F; Delhaye-Bouchaud, N; Mariani, J

    1999-05-01

    Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor gene that results in a large, constitutive inward current in the cerebellar Purkinje cells of +/Lc mice. +/Lc Purkinje cells fail to differentiate fully and die during postnatal development. In normal mice, interactions with granule cells promote Purkinje cell dendritic differentiation. Partial destruction of the granule cell population in young +/Lc mice by x irradiation resulted in a significant increase in Purkinje cell dendritic growth and improved cytoplasmic structure but did not prevent Purkinje cell death. These results indicate two components to Purkinje cell abnormalities in +/Lc mice: a retardation/blockade of dendritic development that is mediated by interactions with granule cells and the death of the cell. Thus, the normal trophic effects of granule cell interaction on Purkinje cell development are absent in the +/Lc cerebellum, suggesting that granule cells are powerful regulators of Purkinje cell differentiation. PMID:10212305

  19. 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

  20. Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells.

    PubMed

    Fukumitsu, Kansai; Hatsukano, Tetsu; Yoshimura, Azumi; Heuser, John; Fujishima, Kazuto; Kengaku, Mineko

    2016-03-01

    Mitochondria dynamically change their shape by repeated fission and fusion in response to physiological and pathological conditions. Recent studies have uncovered significant roles of mitochondrial fission and fusion in neuronal functions, such as neurotransmission and spine formation. However, the contribution of mitochondrial fission to the development of dendrites remains controversial. We analyzed the function of the mitochondrial fission GTPase Drp1 in dendritic arborization in cerebellar Purkinje cells. Overexpression of a dominant-negative mutant of Drp1 in postmitotic Purkinje cells enlarged and clustered mitochondria, which failed to exit from the soma into the dendrites. The emerging dendrites lacking mitochondrial transport remained short and unstable in culture and in vivo. The dominant-negative Drp1 affected neither the basal respiratory function of mitochondria nor the survival of Purkinje cells. Enhanced ATP supply by creatine treatment, but not reduced ROS production by antioxidant treatment, restored the hypomorphic dendrites caused by inhibition of Drp1 function. Collectively, our results suggest that Drp1 is required for dendritic distribution of mitochondria and thereby regulates energy supply in growing dendritic branches in developing Purkinje cells. PMID:26689905

  1. Autophagy activation and enhanced mitophagy characterize the Purkinje cells of pcd mice prior to neuronal death

    PubMed Central

    Chakrabarti, Lisa; Eng, Jeremiah; Ivanov, Nishi; Garden, Gwenn A; La Spada, Albert R

    2009-01-01

    Purkinje cells are a class of specialized neurons in the cerebellum, and are among the most metabolically active of all neurons, as they receive immense synaptic stimulation, and provide the only efferent output from the cerebellum. Degeneration of Purkinje cells is a common feature of inherited ataxias in humans and mice. To understand Purkinje neuron degeneration, investigators have turned to naturally occurring Purkinje cell degeneration phenotypes in mice to identify key regulatory proteins and cellular pathways. The Purkinje cell degeneration (pcd) mouse is a recessive mutant characterized by complete and dramatic post-natal, cell autonomous Purkinje neuron degeneration and death. As the basis of Purkinje cell death in pcd is unresolved, and contradictory data has emerged for the role of autophagy in Purkinje cell degeneration, we studied the mechanism of Purkinje cell death in pcd mice. BAX null status did not suppress Purkinje neuron death in pcd mice, indicating that classic apoptosis is not responsible for Purkinje cell loss. Interestingly, LC3 Western blot analysis and GFP-LC3 immunostaining of degenerating pcd cerebellum revealed activation of the autophagy pathway. Ultrastructural studies confirmed increased autophagy pathway activity in Purkinje cells, and yielded evidence for mitophagy, in agreement with LC3 immunoblotting of cerebellar fractions. As p62 levels were decreased in pcd cerebellum, our findings suggest that pcd Purkinje cell neurons can execute effective autophagy. However, our results support a role for dysregulated autophagy activation in pcd, and suggest that increased or aberrant mitophagy contributes to the Purkinje cell degeneration in pcd mice. PMID:19640278

  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. PMID:23630472

  3. 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

  4. 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

  5. Cytoplasmic and nuclear polyglutamine aggregates in SCA6 Purkinje cells.

    PubMed

    Ishikawa, K; Owada, K; Ishida, K; Fujigasaki, H; Shun Li, M; Tsunemi, T; Ohkoshi, N; Toru, S; Mizutani, T; Hayashi, M; Arai, N; Hasegawa, K; Kawanami, T; Kato, T; Makifuchi, T; Shoji, S; Tanabe, T; Mizusawa, H

    2001-06-26

    Aggregations of the alpha1A-calcium channel protein have been previously demonstrated in spinocerebellar ataxia type 6 (SCA6). Here the authors show that small aggregates, labeled by a monoclonal antibody 1C2 that preferentially detects expanded polyglutamine larger than that in SCA6 mutation, are present mainly in the cytoplasm but also in the nucleus of Purkinje cells. Although the length of expansion is small in SCA6, the current finding might indicate that SCA6 conforms to the pathogenic mechanism(s) in other polyglutamine diseases. PMID:11425948

  6. β-III spectrin is critical for development of purkinje cell dendritic tree and spine morphogenesis.

    PubMed

    Gao, Yuanzheng; Perkins, Emma M; Clarkson, Yvonne L; Tobia, Steven; Lyndon, Alastair R; Jackson, Mandy; Rothstein, Jeffrey D

    2011-11-16

    Mutations in the gene encoding β-III spectrin give rise to spinocerebellar ataxia type 5, a neurodegenerative disease characterized by progressive thinning of the molecular layer, loss of Purkinje cells and increasing motor deficits. A mouse lacking full-length β-III spectrin (β-III⁻/⁻) displays a similar phenotype. In vitro and in vivo analyses of Purkinje cells lacking β-III spectrin, reveal a critical role for β-III spectrin in Purkinje cell morphological development. Disruption of the normally well ordered dendritic arborization occurs in Purkinje cells from β-III⁻/⁻ mice, specifically showing a loss of monoplanar organization, smaller average dendritic diameter and reduced densities of Purkinje cell spines and synapses. Early morphological defects appear to affect distribution of dendritic, but not axonal, proteins. This study confirms that thinning of the molecular layer associated with disease pathogenesis is a consequence of Purkinje cell dendritic degeneration, as Purkinje cells from 8-month-old β-III⁻/⁻ mice have drastically reduced dendritic volumes, surface areas and total dendritic lengths compared with 5- to 6-week-old β-III⁻/⁻ mice. These findings highlight a critical role of β-III spectrin in dendritic biology and are consistent with an early developmental defect in β-III⁻/⁻ mice, with abnormal Purkinje cell dendritic morphology potentially underlying disease pathogenesis. PMID:22090485

  7. 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.…

  8. The multiple roles of Purkinje cells in sensori-motor calibration: to predict, teach and command

    PubMed Central

    Medina, Javier F

    2014-01-01

    Neurophysiological recordings in the cerebellar cortex of awake-behaving animals are revolutionizing the way we think about the role of Purkinje cells in sensori-motor calibration. Early theorists suggested that if a movement became miscalibrated, Purkinje cell output would be changed to adjust the motor command and restore good performance. The finding that Purkinje cell activity changed in many sensori-motor calibration tasks was taken as strong support for this hypothesis. Based on more recent data, however, it has been suggested that changes in Purkinje cell activity do not contribute to the motor command directly; instead, they are used either as a teaching signal, or to predict the altered kinematics of the movement after calibration has taken place. I will argue that these roles are not mutually exclusive, and that Purkinje cells may contribute to command generation, teaching, and prediction at different times during sensori-motor calibration. PMID:21684147

  9. 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

  10. Cerebellar Globular Cells Receive Monoaminergic Excitation and Monosynaptic Inhibition from Purkinje Cells

    PubMed Central

    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. PMID:22235322

  11. Ultra-rapid axon-axon ephaptic inhibition of cerebellar Purkinje cells by the pinceau.

    PubMed

    Blot, Antonin; Barbour, Boris

    2014-02-01

    Excitatory synaptic activity in the brain is shaped and balanced by inhibition. Because inhibition cannot propagate, it is often recruited with a synaptic delay by incoming excitation. Cerebellar Purkinje cells are driven by long-range excitatory parallel fiber inputs, which also recruit local inhibitory basket cells. The axon initial segment of each Purkinje cell is ensheathed by basket cell axons in a structure called the pinceau, which is largely devoid of chemical synapses. In mice, we found at the single-cell level that the pinceau mediates ephaptic inhibition of Purkinje cell firing at the site of spike initiation. The reduction of firing rate was synchronous with the presynaptic action potential, eliminating a synaptic delay and allowing granule cells to inhibit Purkinje cells without a preceding phase of excitation. Axon-axon ephaptic intercellular signaling can therefore mediate near-instantaneous feedforward and lateral inhibition. PMID:24413696

  12. Early and delayed afterdepolarizations in rabbit heart Purkinje cells viewed by confocal microscopy.

    PubMed

    Cordeiro, J M; Bridge, J H; Spitzer, K W

    2001-05-01

    We investigated action potentials and Ca(2+) transients in rabbit Purkinje myocytes using whole cell patch clamp recordings and a confocal microscope. Purkinje cells were loaded with 5 microM Fluo-3/AM for 30min. Action potentials were elicited by application of a stimulus delivered through the recording pipettes. When Purkinje cells were stimulated in 2.0mM Ca(2+), transverse XT line scans revealed a symmetrical 'U'-shaped Ca(2+) transient demonstrating that the transient was initiated at the cell periphery. When Purkinje cells were superfused with 1 microM isoprenaline, both early and delayed afterdepolarizations were induced. XT line scans of cells exhibiting early afterdepolarizations showed a second symmetrical 'U'-shaped transient. This Ca(2+) transient was initiated at the cell periphery suggesting reactivation of the Ca(2+) current. In contrast, in Purkinje cells exhibiting delayed afterdepolarizations and a corresponding transient inward current, XT line scans revealed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell. Immunofluorescence staining of Purkinje cells with an antibody to ryanodine receptors (RyRs) revealed that RyRs are located at regularly spaced intervals throughout the interior of Purkinje cells. These results suggest that, although RyRs are located throughout Purkinje cells, only peripheral RyRs are activated to produce transients, sparks and early afterdepolarizations. During delayed afterdepolarizations, we observed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell as well as large central increases in Ca(2+). Although the latter may result from central release, we cannot exclude the possibility that it reflects Ca(2+) diffusion from subsarcolemmal sites. PMID:11292386

  13. 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

  14. 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...

  15. Purkinje-cell plasticity and cerebellar motor learning are graded by complex-spike duration.

    PubMed

    Yang, Yan; Lisberger, Stephen G

    2014-06-26

    Behavioural learning is mediated by cellular plasticity, such as changes in the strength of synapses at specific sites in neural circuits. The theory of cerebellar motor learning relies on movement errors signalled by climbing-fibre inputs to cause long-term depression of synapses from parallel fibres to Purkinje cells. However, a recent review has called into question the widely held view that the climbing-fibre input is an 'all-or-none' event. In anaesthetized animals, there is wide variation in the duration of the complex spike (CS) caused in Purkinje cells by a climbing-fibre input. Furthermore, the amount of plasticity in Purkinje cells is graded according to the duration of electrically controlled bursts in climbing fibres. The duration of bursts depends on the 'state' of the inferior olive and therefore may be correlated across climbing fibres. Here we provide a potential functional context for these mechanisms during motor learning in behaving monkeys. The magnitudes of both plasticity and motor learning depend on the duration of the CS responses. Furthermore, the duration of CS responses seems to be a meaningful signal that is correlated across the Purkinje-cell population during motor learning. We suggest that during learning, longer bursts in climbing fibres lead to longer-duration CS responses in Purkinje cells, more calcium entry into Purkinje cells, larger synaptic depression, and stronger learning. The same graded impact of instructive signals for plasticity and learning might occur throughout the nervous system. PMID:24814344

  16. Noradrenergic modulation of the parallel fiber-Purkinje cell synapse in mouse cerebellum.

    PubMed

    Lippiello, Pellegrino; Hoxha, Eriola; Volpicelli, Floriana; Lo Duca, Giuseppina; Tempia, Filippo; Miniaci, Maria Concetta

    2015-02-01

    The signals arriving to Purkinje cells via parallel fibers are essential for all tasks in which the cerebellum is involved, including motor control, learning new motor skills and calibration of reflexes. Since learning also requires the activation of adrenergic receptors, we investigated the effects of adrenergic receptor agonists on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that noradrenaline serves as an endogenous ligand for both α1-and α2-adrenergic receptors to produce synaptic depression between parallel fibers and Purkinje cells. On the contrary, PF-EPSCs were potentiated by the β-adrenergic receptor agonist isoproterenol. This short-term potentiation was postsynaptically expressed, required protein kinase A, and was mimicked by the β2-adrenoceptor agonist clenbuterol, suggesting that the β2-adrenoceptors mediate the noradrenergic facilitation of synaptic transmission between parallel fibers and Purkinje cells. Moreover, β-adrenoceptor activation lowered the threshold for cerebellar long-term potentiation induced by 1 Hz parallel fiber stimulation. The presence of both α and β adrenergic receptors on Purkinje cells suggests the existence of bidirectional mechanisms of regulation allowing the noradrenergic afferents to refine the signals arriving to Purkinje cells at particular arousal states or during learning. PMID:25218865

  17. Differences in ionic currents between canine myocardial and Purkinje cells

    PubMed Central

    Vassalle, Mario; Bocchi, Leonardo

    2013-01-01

    An electrophysiological analysis of canine single ventricular myocardial (VM) and Purkinje (P) cells was carried out by means of whole cell voltage clamp method. The following results in VM versus P cells were obtained. INa3 was present, had a threshold negative to the fast activating–inactivating INa1, its slow inactivation was cut off by INa1, and contributed to Na+ influx at INa1 threshold. INa1 was smaller and had a less negative threshold. There was no comparable slowly inactivating INa2, accounting for the shorter action potential. Slope conductance at resting potential was about double and decreased to a minimum value at the larger and less negative IK1 peak. The negative slope region of I-V relation was smaller during fast ramps and larger during slow ramps than in P cells, occurred in the voltage range of IK1 block by Mg2+, was not affected by a lower Vh and TTX and was eliminated by Ba2+, in contrast to P cells. ICa was larger, peaked at positive potentials and was eliminated by Ni2+. Ito was much smaller, began at more positive values, was abolished by less negative Vh and by 4-aminopyridine, included a sustained current that 4-aminopyridine decreased but did not eliminate. Steeper ramps increased IK1 peak as well as the fall in outward current during repolarization, consistent with a time-dependent block and unblock of IK1 by polyamines. During repolarization, the positive slope region was consistently present and was similar in amplitude to IK1 peak, whereas it was small or altogether missing in P cells. The total outward current at positive potentials comprised a larger IK1 component whereas it included a larger Ito and sustained current in P cells. These and other results provide a better understanding of the mechanisms underlying the action potential of VM and P cells under normal and some abnormal (arrhythmias) conditions. PMID:24062942

  18. Changes in Spontaneous Firing Patterns of Cerebellar Purkinje Cells in p75 Knockout Mice

    PubMed Central

    Tian, Jinbin; Tep, Chhavy; Zhu, Michael X.; Yoon, Sung Ok

    2013-01-01

    The p75 neurotrophin receptor is highly expressed in the developing nervous system and is required for neuronal survival, growth and synaptic transmission. In young mice, p75 is present in both granular cells and Purkinje cells of the cerebellum. Although p75 has been implicated in modulation of neuronal excitability in several neuronal types, whether and how it affects the excitability of cerebellar Purkinje neurons remained unclear. Using extracellular recordings of spontaneous firing of Purkinje neurons in cerebellar slices prepared from wild type and p75 knockout mice, we measured intrinsic firing properties in the presence of fast synaptic blockers of more than 200 Purkinje cells, each for a period of 5 min, for each genotype. We detected a significant increase in the mean firing frequency in p75−/− neurons comparing to the wild type littermates. Upon separating tonically firing from phasically firing cells, i.e. cells with firing pauses of longer than 300 msec, we observed that the change mainly arose from phasic firing cells and can be explained by an increase in the firing/silence ratio and a decrease in the number of long pauses during the 5-min recording period. We conclude that p75 plays an important role in regulating the firing-to-silence transition during the phasic firing period of the spontaneous firing of Purkinje cells. Thus, p75 exerts a modulatory function on Purkinje cell firing patterns, through which it may act as a key player in motor coordination and other cerebellum-regulated activities since Purkinje cells represent the sole neuronal output of the cerebellar cortex. PMID:23307658

  19. Role of Cytosolic Calcium Diffusion in Murine Cardiac Purkinje Cells

    PubMed Central

    Limbu, Bijay; Shah, Kushal; Weinberg, Seth H.; Deo, Makarand

    2016-01-01

    Cardiac Purkinje cells (PCs) are morphologically and electrophysiologically different from ventricular myocytes and, importantly, exhibit distinct calcium (Ca2+) homeostasis. Recent studies suggest that PCs are more susceptible to action potential (AP) abnormalities than ventricular myocytes; however, the exact mechanisms are poorly understood. In this study, we utilized a detailed biophysical mathematical model of a murine PC to systematically examine the role of cytosolic Ca2+ diffusion in shaping the AP in PCs. A biphasic spatiotemporal Ca2+ diffusion process, as recorded experimentally, was implemented in the model. In this study, we investigated the role of cytosolic Ca2+ dynamics on AP and ionic current properties by varying the effective Ca2+ diffusion rate. It was observed that AP morphology, specifically the plateau, was affected due to changes in the intracellular Ca2+ dynamics. Elevated Ca2+ concentration in the sarcolemmal region activated inward sodium–Ca2+ exchanger (NCX) current, resulting in a prolongation of the AP plateau at faster diffusion rates. Artificially clamping the NCX current to control values completely reversed the alterations in the AP plateau, thus confirming the role of NCX in modifying the AP morphology. Our results demonstrate that cytosolic Ca2+ diffusion waves play a significant role in shaping APs of PCs and could provide mechanistic insights in the increased arrhythmogeneity of PCs. PMID:27478391

  20. mTORC1 and mTORC2 have largely distinct functions in Purkinje cells.

    PubMed

    Angliker, Nico; Burri, Michael; Zaichuk, Mariana; Fritschy, Jean-Marc; Rüegg, Markus A

    2015-10-01

    The mammalian target of rapamycin (mTOR) is a key regulator of cellular growth which associates with other proteins to form two multi-protein complexes called mTORC1 and mTORC2. Dysregulation of mTORC1 signalling in brain is implicated in neuropathological conditions such as autism spectrum or neurodegenerative disorders. Accordingly, allosteric mTOR inhibitors are currently in clinical trials for the treatment of such disorders. Here, we ablated either mTORC1 or mTORC2 conditionally in Purkinje cells of the mouse cerebellum to dissect their role in the development, function and survival of these neurons. We find that the two mouse models largely differ from each other by phenotype and cellular responses. Inactivation of mTORC2, but not of mTORC1, led to motor coordination deficits at an early age. This phenotype correlated with developmental deficits in climbing fibre elimination and impaired dendritic self-avoidance in mTORC2-deficient Purkinje cells. In contrast, inactivation of mTORC1, but not of mTORC2, affected social interest of the mice and caused a progressive loss of Purkinje cells due to apoptosis. This cell loss was paralleled by age-dependent motor deficits. Comparison of mTORC1-deficient Purkinje cells with those deficient for the mTORC1 inhibitor TSC1 revealed a striking overlap in Purkinje cell degeneration and death, which included neurofilamentopathy and reactive gliosis. Altogether, our study reveals distinct roles of mTORC1 and mTORC2 in Purkinje cells for mouse behaviour and the survival of neurons. Our study also highlights a convergence between the phenotypes of Purkinje cells lacking mTORC1 activity and those expressing constitutively active mTORC1 due to TSC1 deficiency. PMID:26296489

  1. BK Channels Control Cerebellar Purkinje and Golgi Cell Rhythmicity In Vivo

    PubMed Central

    Cheron, Guy; Sausbier, Matthias; Sausbier, Ulrike; Neuhuber, Winfried; Ruth, Peter; Dan, Bernard; Servais, Laurent

    2009-01-01

    Calcium signaling plays a central role in normal CNS functioning and dysfunction. As cerebellar Purkinje cells express the major regulatory elements of calcium control and represent the sole integrative output of the cerebellar cortex, changes in neural activity- and calcium-mediated membrane properties of these cells are expected to provide important insights into both intrinsic and network physiology of the cerebellum. We studied the electrophysiological behavior of Purkinje cells in genetically engineered alert mice that do not express BK calcium-activated potassium channels and in wild-type mice with pharmacological BK inactivation. We confirmed BK expression in Purkinje cells and also demonstrated it in Golgi cells. We demonstrated that either genetic or pharmacological BK inactivation leads to ataxia and to the emergence of a beta oscillatory field potential in the cerebellar cortex. This oscillation is correlated with enhanced rhythmicity and synchronicity of both Purkinje and Golgi cells. We hypothesize that the temporal coding modification of the spike firing of both Purkinje and Golgi cells leads to the pharmacologically or genetically induced ataxia. PMID:19956720

  2. 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. PMID:25367027

  3. Early-onset Purkinje cell dysfunction underlies cerebellar ataxia in peroxisomal multifunctional protein-2 deficiency.

    PubMed

    De Munter, Stephanie; Verheijden, Simon; Vanderstuyft, Esther; Malheiro, Ana Rita; Brites, Pedro; Gall, David; Schiffmann, Serge N; Baes, Myriam

    2016-10-01

    The cerebellar pathologies in peroxisomal diseases underscore that these organelles are required for the normal development and maintenance of the cerebellum, but the mechanisms have not been resolved. Here we investigated the origins of the early-onset coordination impairment in a mouse model with neural selective deficiency of multifunctional protein-2, the central enzyme of peroxisomal β-oxidation. At the age of 4weeks, Nestin-Mfp2(-/-) mice showed impaired motor learning on the accelerating rotarod and underperformed on the balance beam test. The gross morphology of the cerebellum and Purkinje cell arborization were normal. However, electrophysiology revealed a reduced Purkinje cell firing rate, a decreased excitability and an increased membrane capacitance. The distribution of climbing and parallel fiber synapses on Purkinje cells was immature and was accompanied by an increased spine length. Despite normal myelination, Purkinje cell axon degeneration was evident from the occurrence of axonal swellings containing accumulated organelles. In conclusion, the electrical activity, axonal integrity and wiring of Purkinje cells are exquisitely dependent on intact peroxisomal β-oxidation in neural cells. PMID:27353294

  4. 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. PMID:26510954

  5. P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination

    PubMed Central

    Donald, Sarah; Humby, Trevor; Fyfe, Ian; Segonds-Pichon, Anne; Walker, Simon A.; Andrews, Simon R.; Coadwell, W. John; Emson, Piers; Wilkinson, Lawrence S.; Welch, Heidi C. E.

    2008-01-01

    The small GTPase Rac controls cell morphology, gene expression, and reactive oxygen species formation. Manipulations of Rac activity levels in the cerebellum result in motor coordination defects, but activators of Rac in the cerebellum are unknown. P-Rex family guanine-nucleotide exchange factors activate Rac. We show here that, whereas P-Rex1 expression within the brain is widespread, P-Rex2 is specifically expressed in the Purkinje neurons of the cerebellum. We have generated P-Rex2−/− and P-Rex1−/−/P-Rex2−/− mice, analyzed their Purkinje cell morphology, and assessed their motor functions in behavior tests. The main dendrite is thinned in Purkinje cells of P-Rex2−/− pups and dendrite structure appears disordered in Purkinje cells of adult P-Rex2−/− and P-Rex1−/−/P-Rex2−/− mice. P-Rex2−/− mice show a mild motor coordination defect that progressively worsens with age and is more pronounced in females than in males. P-Rex1−/−/P-Rex2−/− mice are ataxic, with reduced basic motor activity and abnormal posture and gait, as well as impaired motor coordination even at a young age. We conclude that P-Rex1 and P-Rex2 are important regulators of Purkinje cell morphology and cerebellar function. PMID:18334636

  6. Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo

    PubMed Central

    Tsubota, Tadashi; Ohashi, Yohei; Tamura, Keita; Sato, Ayana; Miyashita, Yasushi

    2011-01-01

    Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum

  7. Regional Alterations in Purkinje Cell Density in Patients with Autism

    PubMed Central

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

    2014-01-01

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

  8. 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

  9. Subsurface cistern (SSC) proliferation in Purkinje cells of the rat cerebellum in response to acute and chronic exposure to paint thinner: A light and electron microscopy study.

    PubMed

    Martínez-Alfaro, Minerva; Cárabez-Trejo, Alfonso; Sandoval-Zapata, Francisca; Morales-Tlalpan, Verónica; Palma-Tirado, Lourdes

    2014-09-01

    Intentional inhalation and occupational exposure are two ways humans are exposed to thinner, a widely employed solvent in industry. Inhalation of thinner induces toxic effects in various organs, with the cerebellum being one of the most affected structures of the CNS. The aim of this work was to describe specific structural alterations of cerebellum Purkinje cells in rats following exposure to thinner for 16 weeks. A histological analysis of the cerebellum of solvent-exposed rats revealed swollen Purkinje cell dendrites surrounded by empty space, and electronic microscopy showed an increase in the number of subsurface cisterns (SSCs) within their dendritic processes. After a period of non-exposure, the number of SSCs decreased without reaching normal levels, suggesting a degree of plasticity. Purkinje cell SSCs, which are derived from smooth endoplasmic reticulum, contain inositol trisphosphate receptors (IP3Rs), ryanodine receptors (RR), and a recently identified characteristic cluster of large conductance calcium-activated potassium (BKCa) channels. We found that SSCs in Purkinje cell dendrites were closely associated with mitochondria, and immunofluorescence microscopy showed higher levels of RR and calbindin receptors (CB), in Purkinje cells of exposed than normal rats. These changes are probably related to behavioral manifestations of cerebellar alterations, such as imbalance and ataxia, consistent with the suggested involvement of increases in SSCs in ataxia in rats and humans. This increase in SSCs, taken together with the localization of RR, IP3R and BKCa proteins in this structure, suggests altered intracellular calcium-buffering processes in the Purkinje cells of thinner-exposed rats. PMID:24820124

  10. Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns.

    PubMed

    Schilling, K; Dickinson, M H; Connor, J A; Morgan, J I

    1991-12-01

    In primary dissociated cultures of mouse cerebellum a number of Purkinje cell-specific marker proteins and characteristic ionic currents appear at the appropriate developmental time. During the first week after plating, Purkinje cell dendrites elongate, but as electrical activity emerges the dendrites stop growing and branch. If endogenous electrical activity is inhibited by chronic tetrodotoxin or high magnesium treatment, dendrites continue to elongate, as if they were still immature. At the time that branching begins, intracellular calcium levels become sensitive to tetrodotoxin, suggesting that this cation may be involved in dendrite growth. Even apparently mature Purkinje cells alter their dendritic growth in response to changes in activity, suggesting long-term plasticity. PMID:1684902

  11. Jun-B expression in Purkinje cells by conjunctive stimulation of climbing fibre and AMPA.

    PubMed

    Yamamori, T; Mikawa, S; Kado, R

    1995-03-27

    Co-application of alpha-amino-3-hydro-5-methyl-4-isoxazole-propionate (AMPA) and 8-bromo cGMP (8-Br-cGMP) which cause long-term desensitization also induces c-Fos and Jun-B expression in Purkinje cells of cerebellar slices [Nakazawa K, Karachot L, Nakabeppu Y et al. NeuroReport 4, 1275-1278 (1993)]. Here, we report an increased local induction of Jun-B immunoreactivity in Purkinje cells in vivo when electrical stimulation of the inferior olive nucleus (IOn) was conjunctively applied with AMPA on the vermis. The present data further supports the idea that conjunctive heterosynaptic inputs to cerebellar Purkinje cells can trigger active gene transcription thus possibly contributing to cerebellar long-term plasticity. They also demonstrate that Jun-B may be a useful transcriptional marker to study cerebellar coincidence phenomena. PMID:7541656

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

    EPA Science Inventory

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

  13. Cell death in the Purkinje cells of the cerebellum of senescence accelerated mouse (SAMP(8)).

    PubMed

    Zhu, Yonghong; Lee, Cleo C L; Lam, W P; Wai, Maria S M; Rudd, John A; Yew, David T

    2007-10-01

    The cerebella of SAMP(8) (accelerated aging mouse) and SAMR(1) controls were analyzed by Western Blotting of tyrosine hydroxylase and choline acetyltransferase, as well as by TUNEL and histological silver staining. Both tyrosine hydroxylase and choline acetyltransferase levels were higher in SAMR(1) than in SAMP(8). There was also an age-related decrease in enzyme levels in SAMP(8), with the reduction of tyrosine hydroxylase being more apparent. Concomitantly, there was an age-related increase of apoptosis in the medial neocerebellum and the vermis as revealed by TUNEL, with changes being significant in the SAMP(8) strain. Histologically, some Purkinje cells appeared to disappear during aging. Taken together, the data suggests that the aging SAMP(8) strain displays differential Purkinje cell death in the medial cerebellum and that some of the dying cells are likely to be catecholaminergic. PMID:17415677

  14. Differential roles of the mevalonate pathway in the development and survival of mouse Purkinje cells in culture.

    PubMed

    Barszczyk, Andrew; Sun, Hong-Shuo; Quan, Yi; Zheng, Wenhua; Charlton, Milton P; Feng, Zhong-Ping

    2015-01-01

    The cerebellum is an important locus for motor learning and higher cognitive functions, and Purkinje cells constitute a key component of its circuit. Biochemically, significant turnover of cholesterol occurs in Purkinje cells, causing the activation of the mevalonate pathway. The mevalonate pathway has important roles in cell survival and development. In this study, we investigated the outcomes of mevalonate inhibition in immature and mature mouse cerebellar Purkinje cells in culture. Specifically, we found that the inhibition of the mevalonate pathway by mevastatin resulted in cell death, and geranylgeranylpyrophosphate (GGPP) supplementation significantly enhanced neuronal survival. The surviving immature Purkinje cells, however, exhibited dendritic developmental deficits. The morphology of mature cells was not affected. The inhibition of squalene synthase by zaragozic acid caused impaired dendritic development, similar to that seen in the GGPP-rescued Purkinje cells. Our results indicate GGPP is required for cell survival and squalene synthase for the cell development of Purkinje cells. Abnormalities in Purkinje cells are linked to motor-behavioral learning disorders such as cerebellar ataxia. Thus, serious caution should be taken when using drugs that inhibit geranylgeranylation or the squalene-cholesterol branch of the pathway in the developing stage. PMID:24973985

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

    PubMed

    Barmack, N H; Yakhnitsa, V

    2015-10-01

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

  16. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

    PubMed

    Jiang, Nan-Wei; Wang, De-Juan; Xie, Ya-Jun; Zhou, Liang; Su, Li-Da; Li, Huashun; Wang, Qin-Wen; Shen, Ying

    2016-06-01

    Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells. PMID:26194056

  17. 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

  18. Number of Purkinje cells and Bergmann astrocytes in rats with CCl4--induced liver disease.

    PubMed

    Diemer, N H

    1977-01-01

    The nuclei of Purkinje cells and Bergmann astrocytes were counted on sagittal sections from cerebellum and the length of stratum gangliosum was measured in rats with CCl4-induced liver disease, using an electronic image analyzer. After 8 weeks of CCl4-administration a reduction was found in the number of Purkinje cells, many of which showed homogenizating changes. Ten weeks after termination of the administration period the number of Purkinje cells was reduced by 12 percent. The number of Bergmann astrocytes remained significantly increased after 8 weeks of CCl4-administration (max. 20 per cent). The changes of Purkinje cell and Bergmann astrocyte density developed during the period of severe liver necrosis, whereas only minor changes were found in the ensuing period of liver "cirrhosis". In the perfusion fixed specimens, the Bergmann astrocyte nuclei increased in volume up to 65 per cent and immersion fixed brains showed typical Alzheimer type II nuclear changes. The impact of the increased plasma ammonia concentration on the astrocytes is discussed. PMID:842279

  19. Coding of stimulus strength via analog calcium signals in Purkinje cell dendrites of awake mice

    PubMed Central

    Najafi, Farzaneh; Giovannucci, Andrea; Wang, Samuel S-H; Medina, Javier F

    2014-01-01

    The climbing fiber input to Purkinje cells acts as a teaching signal by triggering a massive influx of dendritic calcium that marks the occurrence of instructive stimuli during cerebellar learning. Here, we challenge the view that these calcium spikes are all-or-none and only signal whether the instructive stimulus has occurred, without providing parametric information about its features. We imaged ensembles of Purkinje cell dendrites in awake mice and measured their calcium responses to periocular airpuffs that serve as instructive stimuli during cerebellar-dependent eyeblink conditioning. Information about airpuff duration and pressure was encoded probabilistically across repeated trials, and in two additional signals in single trials: the synchrony of calcium spikes in the Purkinje cell population, and the amplitude of the calcium spikes, which was modulated by a non-climbing fiber pathway. These results indicate that calcium-based teaching signals in Purkinje cells contain analog information that encodes the strength of instructive stimuli trial-by-trial. DOI: http://dx.doi.org/10.7554/eLife.03663.001 PMID:25205669

  20. Purkinje cell maturation participates in the control of oligodendrocyte differentiation: role of sonic hedgehog and vitronectin.

    PubMed

    Bouslama-Oueghlani, Lamia; Wehrlé, Rosine; Doulazmi, Mohamed; Chen, Xiao Ru; Jaudon, Fanny; Lemaigre-Dubreuil, Yolande; Rivals, Isabelle; Sotelo, Constantino; Dusart, Isabelle

    2012-01-01

    Oligodendrocyte differentiation is temporally regulated during development by multiple factors. Here, we investigated whether the timing of oligodendrocyte differentiation might be controlled by neuronal differentiation in cerebellar organotypic cultures. In these cultures, the slices taken from newborn mice show very few oligodendrocytes during the first week of culture (immature slices) whereas their number increases importantly during the second week (mature slices). First, we showed that mature cerebellar slices or their conditioned media stimulated oligodendrocyte differentiation in immature slices thus demonstrating the existence of diffusible factors controlling oligodendrocyte differentiation. Using conditioned media from different models of slice culture in which the number of Purkinje cells varies drastically, we showed that the effects of these differentiating factors were proportional to the number of Purkinje cells. To identify these diffusible factors, we first performed a transcriptome analysis with an Affymetrix array for cerebellar cortex and then real-time quantitative PCR on mRNAs extracted from fluorescent flow cytometry sorted (FACS) Purkinje cells of L7-GFP transgenic mice at different ages. These analyses revealed that during postnatal maturation, Purkinje cells down-regulate Sonic Hedgehog and up-regulate vitronectin. Then, we showed that Sonic Hedgehog stimulates the proliferation of oligodendrocyte precursor cells and inhibits their differentiation. In contrast, vitronectin stimulates oligodendrocyte differentiation, whereas its inhibition with blocking antibodies abolishes the conditioned media effects. Altogether, these results suggest that Purkinje cells participate in controlling the timing of oligodendrocyte differentiation in the cerebellum through the developmentally regulated expression of diffusible molecules such as Sonic Hedgehog and vitronectin. PMID:23155445

  1. Purkinje Cell Maturation Participates in the Control of Oligodendrocyte Differentiation: Role of Sonic Hedgehog and Vitronectin

    PubMed Central

    Bouslama-Oueghlani, Lamia; Wehrlé, Rosine; Doulazmi, Mohamed; Chen, Xiao Ru; Jaudon, Fanny; Lemaigre-Dubreuil, Yolande; Rivals, Isabelle; Sotelo, Constantino; Dusart, Isabelle

    2012-01-01

    Oligodendrocyte differentiation is temporally regulated during development by multiple factors. Here, we investigated whether the timing of oligodendrocyte differentiation might be controlled by neuronal differentiation in cerebellar organotypic cultures. In these cultures, the slices taken from newborn mice show very few oligodendrocytes during the first week of culture (immature slices) whereas their number increases importantly during the second week (mature slices). First, we showed that mature cerebellar slices or their conditioned media stimulated oligodendrocyte differentiation in immature slices thus demonstrating the existence of diffusible factors controlling oligodendrocyte differentiation. Using conditioned media from different models of slice culture in which the number of Purkinje cells varies drastically, we showed that the effects of these differentiating factors were proportional to the number of Purkinje cells. To identify these diffusible factors, we first performed a transcriptome analysis with an Affymetrix array for cerebellar cortex and then real-time quantitative PCR on mRNAs extracted from fluorescent flow cytometry sorted (FACS) Purkinje cells of L7-GFP transgenic mice at different ages. These analyses revealed that during postnatal maturation, Purkinje cells down-regulate Sonic Hedgehog and up-regulate vitronectin. Then, we showed that Sonic Hedgehog stimulates the proliferation of oligodendrocyte precursor cells and inhibits their differentiation. In contrast, vitronectin stimulates oligodendrocyte differentiation, whereas its inhibition with blocking antibodies abolishes the conditioned media effects. Altogether, these results suggest that Purkinje cells participate in controlling the timing of oligodendrocyte differentiation in the cerebellum through the developmentally regulated expression of diffusible molecules such as Sonic Hedgehog and vitronectin. PMID:23155445

  2. An active membrane model of the cerebellar Purkinje cell II. Simulation of synaptic responses.

    PubMed

    De Schutter, E; Bower, J M

    1994-01-01

    1. Both excitatory and inhibitory postsynaptic channels were added to a previously described complex compartmental model of a cerebellar Purkinje cell to examine model responses to synaptic inputs. All model parameters remained as described previously, leaving maximum synaptic conductance as the only parameter that was tuned in the studies described in this paper. Under these conditions the model was capable of reproducing physiological recorded responses to each of the major types of synaptic input. 2. When excitatory synapses were activated on the smooth dendrites of the model, the model generated a complex dendritic Ca2+ spike similar to that generated by climbing fiber inputs. Examination of the model showed that activation of P-type Ca2+ channels in both the smooth and spiny dendrites augmented the depolarization during the complex spike and that Ca(2+)-activated K+ channels in the same dendritic regions determined the duration of the spike. When these synapses were activated under simulated current-clamp conditions the model also generated the characteristic dual reversal potential of the complex spike. The shape of the dendritic complex spike could be altered by changing the maximum conductance of the climbing fiber synapse and thus the amount of Ca2+ entering the cell. 3. To explore the background simple spike firing properties of Purkinje cells in vivo we added excitatory "parallel fiber" synapses to the spiny dendritic branches of the model. Continuous asynchronous activation of these granule cell synapses resulted in the generation of spontaneous sodium spikes. However, very low asynchronous input frequencies produced a highly regular, very fast rhythm (80-120 Hz), whereas slightly higher input frequencies resulted in Purkinje cell bursting. Both types of activity are uncharacteristic of in vivo Purkinje cell recordings. 4. Inhibitory synapses of the sort presumably generated by stellate cells were also added to the dendritic tree. When asynchronous

  3. Isolation and characterization of embryonic stem cell-derived cardiac Purkinje cells.

    PubMed

    Maass, Karen; Shekhar, Akshay; Lu, Jia; Kang, Guoxin; See, Fiona; Kim, Eugene E; Delgado, Camila; Shen, Steven; Cohen, Lisa; Fishman, Glenn I

    2015-04-01

    The cardiac Purkinje fiber network is composed of highly specialized cardiomyocytes responsible for the synchronous excitation and contraction of the ventricles. Computational modeling, experimental animal studies, and intracardiac electrical recordings from patients with heritable and acquired forms of heart disease suggest that Purkinje cells (PCs) may also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in isolating and studying this rare population of cells, the precise role of PC in arrhythmogenesis and the underlying molecular mechanisms responsible for their proarrhythmic behavior are not fully characterized. Conceptually, a stem cell-based model system might facilitate studies of PC-dependent arrhythmia mechanisms and serve as a platform to test novel therapeutics. Here, we describe the generation of murine embryonic stem cells (ESC) harboring pan-cardiomyocyte and PC-specific reporter genes. We demonstrate that the dual reporter gene strategy may be used to identify and isolate the rare ESC-derived PC (ESC-PC) from a mixed population of cardiogenic cells. ESC-PC display transcriptional signatures and functional properties, including action potentials, intracellular calcium cycling, and chronotropic behavior comparable to endogenous PC. Our results suggest that stem-cell derived PC are a feasible new platform for studies of developmental biology, disease pathogenesis, and screening for novel antiarrhythmic therapies. PMID:25524238

  4. 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

  5. Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells.

    PubMed

    Narusuye, K; Kawai, F; Matsuzaki, K; Miyachi, E

    2005-02-01

    Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca(2+) imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na(+) current could be fitted by the Hill equation with a half-blocking concentration of 0.56 mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca(2+)](i) response of ORCs, suggesting that linalool suppresses Ca(2+) currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells. PMID:15365786

  6. 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

  7. Purkinje cell compartmentalization in the cerebellum of the spontaneous mutant mouse dreher

    PubMed Central

    Sillitoe, Roy V.; George-Jones, Nicholas A.; Millen, Kathleen J.; Hawkes, Richard

    2014-01-01

    The cerebellar morphological phenotype of the spontaneous neurological mutant mouse dreher (Lmx1adr-J) results from cell fate changes in dorsal midline patterning involving the roof plate and rhombic lip. Positional cloning revealed that the gene Lmx1a, which encodes a LIM homeodomain protein, is mutated in dreher, and is expressed in the developing roof plate and rhombic lip. Loss of Lmx1a causes reduction of the roof plate, an important embryonic signaling center, and abnormal cell fate specification within the embryonic cerebellar rhombic lip. In adult animals, these defects result in variable, medial fusion of the cerebellar vermis and posterior cerebellar vermis hypoplasia. It is unknown whether deleting Lmx1a results in displacement or loss of specific lobules in the vermis. To distinguish between an ectopic and an absent vermis, the expression patterns of two Purkinje cell specific compartmentation antigens, zebrin II/aldolase C and the small heat shock protein HSP25, were analyzed in dreher cerebella. The data reveal that despite the reduction in volume and abnormal foliation of the cerebellum, the transverse zones and parasagittal stripe arrays characteristic of the normal vermis are present in dreher, but may be highly distorted. In dreher mutants with a severe phenotype, zebrin II stripes are fragmented and distributed non-symmetrically about the cerebellar midline. We conclude that although Purkinje cell agenesis or selective Purkinje cell death may contribute to the dreher phenotype, our data suggest that aberrant anlage patterning and granule cell development lead to Purkinje cell ectopia, which ultimately causes abnormal cerebellar architecture in dreher. PMID:23160833

  8. 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. PMID:26074783

  9. 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

  10. 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.

  11. Proteomic Studies of a Single CNS Synapse Type: The Parallel Fiber/Purkinje Cell Synapse

    PubMed Central

    Selimi, Fekrije; Cristea, Ileana M; Heller, Elizabeth; Chait, Brian T; Heintz, Nathaniel

    2009-01-01

    Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKγ, are major unrecognized components of this synapse type. We demonstrate that MRCKγ can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types. PMID:19402746

  12. Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse.

    PubMed

    Selimi, Fekrije; Cristea, Ileana M; Heller, Elizabeth; Chait, Brian T; Heintz, Nathaniel

    2009-04-14

    Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKgamma, are major unrecognized components of this synapse type. We demonstrate that MRCKgamma can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types. PMID:19402746

  13. Ataxia and Purkinje cell degeneration in mice lacking the CAMTA1 transcription factor.

    PubMed

    Long, Chengzu; Grueter, Chad E; Song, Kunhua; Qin, Song; Qi, Xiaoxia; Kong, Y Megan; Shelton, John M; Richardson, James A; Zhang, Chun-Li; Bassel-Duby, Rhonda; Olson, Eric N

    2014-08-01

    Members of the calmodulin-binding transcription activator (CAMTA) family of proteins function as calcium-sensitive regulators of gene expression in multicellular organisms ranging from plants to humans. Here, we show that global or nervous system deletion of CAMTA1 in mice causes severe ataxia with Purkinje cell degeneration and cerebellar atrophy, partially resembling the consequences of haploinsufficiency of the human CAMTA1 locus. Gene-expression analysis identified a large collection of neuronal genes that were dysregulated in the brains of CAMTA1-mutant mice, and elucidation of a consensus sequence for binding of CAMTA proteins to DNA revealed the association of CAMTA-binding sites with many of these genes. We conclude that CAMTA1 plays an essential role in the control of Purkinje cell function and survival. CAMTA1-mutant mice provide a model to study the molecular mechanisms of neurodegenerative diseases and for screening potential therapeutic interventions for such disorders. PMID:25049392

  14. The effect of refeeding after neonatal starvation on Purkinje cell dendritic growth in the rat.

    PubMed

    McConnell, P; Berry, M

    1978-04-15

    Male rats, undernourished from birth until 30 days by restricting access to the lactating dam, were given ad libitum food supplies unitl 80 days. Body weight, whole brain weight and cerebellar remained significantly lower in these animals than in normally fed controls. Significant deficits in the area of the molecular and granular layers persisted at 80 days, although there was some recovery during the refeeding period. At the same time granule and Purkinje cell density declined, suggesting that the areal recovery was due to the expansion of the interneuronal matrix. Granule cell numbers remained unchanged between 30 and 80 days. Network analysis of Golgi-Cox preparations indicated a 28% decrease in overall size of the Purkinje cell dendritic networks, due primarily to a deficit in segment frequency which remained unchanged throughout the rehabilitation period. Segment length did, however, show some recovery; distal segments from 80-day experimental networks were significantly longer than those of 30-day undernourished animals. The results of the topological analysis suggested that dendritic remodelling had taken place during the rehabilitation period. The failure to observe a complete recovery in the size and morphology of the dendritic network may be explained in terms of recent suggestions that dendritic development is influenced both by the metabolism of the neurons themselves, and by the number and density of adjacent afferent axons. It is suggested that refeeding from 30 days leads to the recovery of Purkinje cell metabolism, but is unable to restore the parallel fiber deficit. PMID:632380

  15. 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

  16. Regional Regulation of Purkinje Cell Dendritic Spines by Integrins and Eph/Ephrins

    PubMed Central

    Heintz, Tristan G.; Eva, Richard; Fawcett, James W.

    2016-01-01

    Climbing fibres and parallel fibres compete for dendritic space on Purkinje cells in the cerebellum. Normally, climbing fibres populate the proximal dendrites, where they suppress the multiple small spines typical of parallel fibres, leading to their replacement by the few large spines that contact climbing fibres. Previous work has shown that ephrins acting via EphA4 are a signal for this change in spine type and density. We have used an in vitro culture model in which to investigate the ephrin effect on Purkinje cell dendritic spines and the role of integrins in these changes. We found that integrins α3, α5 and β4 are present in many of the dendritic spines of cultured Purkinje cells. pFAK, the main downstream signalling molecule from integrins, has a similar distribution, although the intenstity of pFAK staining and the percentage of pFAK+ spines was consistently higher in the proximal dendrites. Activating integrins with Mg2+ led to an increase in the intensity of pFAK staining and an increase in the proportion of pFAK+ spines in both the proximal and distal dendrites, but no change in spine length, density or morphology. Blocking integrin binding with an RGD-containing peptide led to a reduction in spine length, with more stubby spines on both proximal and distal dendrites. Treatment of the cultures with ephrinA3-Fc chimera suppressed dendritic spines specifically on the proximal dendrites and there was also a decrease of pFAK in spines on this domain. This effect was blocked by simultaneous activation of integrins with Mn2+. We conclude that Eph/ephrin signaling regulates proximal dendritic spines in Purkinje cells by inactivating integrin downstream signalling. PMID:27518800

  17. Regional Regulation of Purkinje Cell Dendritic Spines by Integrins and Eph/Ephrins.

    PubMed

    Heintz, Tristan G; Eva, Richard; Fawcett, James W

    2016-01-01

    Climbing fibres and parallel fibres compete for dendritic space on Purkinje cells in the cerebellum. Normally, climbing fibres populate the proximal dendrites, where they suppress the multiple small spines typical of parallel fibres, leading to their replacement by the few large spines that contact climbing fibres. Previous work has shown that ephrins acting via EphA4 are a signal for this change in spine type and density. We have used an in vitro culture model in which to investigate the ephrin effect on Purkinje cell dendritic spines and the role of integrins in these changes. We found that integrins α3, α5 and β4 are present in many of the dendritic spines of cultured Purkinje cells. pFAK, the main downstream signalling molecule from integrins, has a similar distribution, although the intenstity of pFAK staining and the percentage of pFAK+ spines was consistently higher in the proximal dendrites. Activating integrins with Mg2+ led to an increase in the intensity of pFAK staining and an increase in the proportion of pFAK+ spines in both the proximal and distal dendrites, but no change in spine length, density or morphology. Blocking integrin binding with an RGD-containing peptide led to a reduction in spine length, with more stubby spines on both proximal and distal dendrites. Treatment of the cultures with ephrinA3-Fc chimera suppressed dendritic spines specifically on the proximal dendrites and there was also a decrease of pFAK in spines on this domain. This effect was blocked by simultaneous activation of integrins with Mn2+. We conclude that Eph/ephrin signaling regulates proximal dendritic spines in Purkinje cells by inactivating integrin downstream signalling. PMID:27518800

  18. Unimpaired trace classical eyeblink conditioning in Purkinje cell degeneration (pcd) mutant mice

    PubMed Central

    Brown, Kevin L.; Agelan, Alexis; Woodruff-Pak, Diana S.

    2009-01-01

    Young adult Purkinje cell degeneration (pcd) mutant mice, with complete loss of cerebellar cortical Purkinje cells, are impaired in delay eyeblink classical conditioning. In the delay paradigm, the conditioned stimulus (CS) overlaps and coterminates with the unconditioned stimulus (US), and the cerebellar cortex supports normal acquisition. The ability of pcd mutant mice to acquire trace eyeblink conditioning in which the CS and US do not overlap has not been explored. Recent evidence suggests that cerebellar cortex may not be necessary for trace eyeblink classical conditioning. Using a 500 ms trace paradigm for which forebrain structures are essential in mice, we assessed the performance of homozygous male pcd mutant mice and their littermates in acquisition and extinction. In contrast to results with delay conditioning, acquisition of trace conditioning was unimpaired in pcd mutant mice. Extinction to the CS alone did not differ between pcd and littermate control mice, and timing of the conditioned response was not altered by the absence of Purkinje cells during acquisition or extinction. The ability of pcd mutant mice to acquire and extinguish trace eyeblink conditioning at levels comparable to controls suggests that the cerebellar cortex is not a critical component of the neural circuitry underlying trace conditioning. Results indicate that the essential neural circuitry for trace eyeblink conditioning involves connectivity that bypasses cerebellar cortex. PMID:19931625

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

    PubMed Central

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

    2010-01-01

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

  20. Bax inactivation in lurcher mutants rescues cerebellar granule cells but not purkinje cells or inferior olivary neurons.

    PubMed

    Selimi, F; Vogel, M W; Mariani, J

    2000-07-15

    Lurcher is a gain-of-function mutation in the delta2 glutamate receptor gene (Grid2) that turns the receptor into a leaky ion channel. The expression of the Lurcher gene in heterozygous (Grid2(Lc/+)) mutants induces the death of almost all Purkinje cells starting from the second postnatal week. Ninety percent of the granule cells and 60-75% of the inferior olivary neurons die because of the loss of their target neurons, the Purkinje cells. The apoptotic nature of the neurodegeneration has been demonstrated previously by the presence of activated caspase-3 and DNA fragmentation. Bax, a pro-apoptotic gene of the Bcl-2 family, has been shown to be involved in developmental neuronal death. To study the role of Bax in Grid2(Lc/+) neurodegeneration, double mutants with Grid2(Lc/)+ mice and Bax knock-out mice (Bax-/-) were generated. Bax deletion had no effect on the death of Purkinje cells and inferior olivary neurons, although a temporary rescue of some Purkinje cells could be detected in P15 Grid2(Lc/)+;Bax-/- animals. From postnatal day 15 (P15) to P60, the number of granule cells in Grid2(Lc/)+;Bax-/-mice did not significantly change and was significantly increased compared with the number found in Grid2(Lc/)+;Bax+/+ mice. Granule cell number in P60 Grid2(Lc/)+;Bax-/- mice corresponded to 70% of the number found in wild-type mice. Our results show that Bax inactivation in Grid2(Lc/+) mice does not rescue intrinsic Purkinje cell death or the target-related cell death of olivary neurons, but Bax inactivation does inhibit persistently target-related cell death in cerebellar granule cells. PMID:10884318

  1. 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. PMID:25962893

  2. Cerebellar transcriptional alterations with Purkinje cell dysfunction and loss in mice lacking PGC-1α.

    PubMed

    Lucas, Elizabeth K; Reid, Courtney S; McMeekin, Laura J; Dougherty, Sarah E; Floyd, Candace L; Cowell, Rita M

    2014-01-01

    Alterations in the expression and activity of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (ppargc1a or PGC-1α) have been reported in multiple movement disorders, yet it is unclear how a lack of PGC-1α impacts transcription and function of the cerebellum, a region with high PGC-1α expression. We show here that mice lacking PGC-1α exhibit ataxia in addition to the previously described deficits in motor coordination. Using q-RT-PCR in cerebellar homogenates from PGC-1α(-/-) mice, we measured expression of 37 microarray-identified transcripts upregulated by PGC-1α in SH-SY5Y neuroblastoma cells with neuroanatomical overlap with PGC-1α or parvalbumin (PV), a calcium buffer highly expressed by Purkinje cells. We found significant reductions in transcripts with synaptic (complexin1, Cplx1; Pacsin2), structural (neurofilament heavy chain, Nefh), and metabolic (isocitrate dehydrogenase 3a, Idh3a; neutral cholesterol ester hydrolase 1, Nceh1; pyruvate dehydrogenase alpha 1, Pdha1; phytanoyl-CoA hydroxylase, Phyh; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1, Uqcrfs1) functions. Using conditional deletion of PGC-1α in PV-positive neurons, we determined that 50% of PGC-1α expression and a reduction in a subset of these transcripts could be explained by its concentration in PV-positive neuronal populations in the cerbellum. To determine whether there were functional consequences associated with these changes, we conducted stereological counts and spike rate analysis in Purkinje cells, a cell type rich in PV, from PGC-1α(-/-) mice. We observed a significant loss of Purkinje cells by 6 weeks of age, and the remaining Purkinje cells exhibited a 50% reduction in spike rate. Together, these data highlight the complexity of PGC-1α's actions in the central nervous system and suggest that dysfunction in multiple cell types contribute to motor deficits in the context of PGC-1α deficiency. PMID:25610371

  3. Mutations in the Microtubule-Associated Protein 1A (Map1a) Gene Cause Purkinje Cell Degeneration

    PubMed Central

    Liu, Ye; Lee, Jeong Woong

    2015-01-01

    The structural microtubule-associated proteins (MAPs) are critical for the organization of neuronal microtubules (MTs). Microtubule-associated protein 1A (MAP1A) is one of the most abundantly expressed MAPs in the mammalian brain. However, its in vivo function remains largely unknown. Here we describe a spontaneous mouse mutation, nm2719, which causes tremors, ataxia, and loss of cerebellar Purkinje neurons in aged homozygous mice. The nm2719 mutation disrupts the Map1a gene. We show that targeted deletion of mouse Map1a gene leads to similar neurodegenerative defects. Before neuron death, Map1a mutant Purkinje cells exhibited abnormal focal swellings of dendritic shafts and disruptions in axon initial segment (AIS) morphology. Furthermore, the MT network was reduced in the somatodendritic and AIS compartments, and both the heavy and light chains of MAP1B, another brain-enriched MAP, was aberrantly distributed in the soma and dendrites of mutant Purkinje cells. MAP1A has been reported to bind to the membrane-associated guanylate kinase (MAGUK) scaffolding proteins, as well as to MTs. Indeed, PSD-93, the MAGUK specifically enriched in Purkinje cells, was reduced in Map1a−/− Purkinje cells. These results demonstrate that MAP1A functions to maintain both the neuronal MT network and the level of PSD-93 in neurons of the mammalian brain. PMID:25788676

  4. 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. PMID:26291161

  5. Clec16a is Critical for Autolysosome Function and Purkinje Cell Survival

    PubMed Central

    Redmann, Veronika; Lamb, Christopher A.; Hwang, Seungmin; Orchard, Robert C.; Kim, Sungsu; Razi, Minoo; Milam, Ashley; Park, Sunmin; Yokoyama, Christine C.; Kambal, Amal; Kreamalmeyer, Darren; Bosch, Marie K.; Xiao, Maolei; Green, Karen; Kim, Jungsu; Pruett-Miller, Shondra M.; Ornitz, David M.; Allen, Paul M.; Beatty, Wandy L.; Schmidt, Robert E.; DiAntonio, Aaron; Tooze, Sharon A.; Virgin, Herbert W.

    2016-01-01

    CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a-mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3, and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival. PMID:26987296

  6. Clec16a is Critical for Autolysosome Function and Purkinje Cell Survival.

    PubMed

    Redmann, Veronika; Lamb, Christopher A; Hwang, Seungmin; Orchard, Robert C; Kim, Sungsu; Razi, Minoo; Milam, Ashley; Park, Sunmin; Yokoyama, Christine C; Kambal, Amal; Kreamalmeyer, Darren; Bosch, Marie K; Xiao, Maolei; Green, Karen; Kim, Jungsu; Pruett-Miller, Shondra M; Ornitz, David M; Allen, Paul M; Beatty, Wandy L; Schmidt, Robert E; DiAntonio, Aaron; Tooze, Sharon A; Virgin, Herbert W

    2016-01-01

    CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a-mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3, and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival. PMID:26987296

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

    PubMed

    O'Hearn, E; Molliver, M E

    2004-01-01

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

  8. 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.

  9. 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

  10. Efficient Generation of Cardiac Purkinje Cells from ESCs by Activating cAMP Signaling

    PubMed Central

    Tsai, Su-Yi; Maass, Karen; Lu, Jia; Fishman, Glenn I.; Chen, Shuibing; Evans, Todd

    2015-01-01

    Summary Dysfunction of the specialized cardiac conduction system (CCS) is associated with life-threatening arrhythmias. Strategies to derive CCS cells, including rare Purkinje cells (PCs), would facilitate models for mechanistic studies and drug discovery and also provide new cellular materials for regenerative therapies. A high-throughput chemical screen using CCS:lacz and Contactin2:egfp (Cntn2:egfp) reporter embryonic stem cell (ESC) lines was used to discover a small molecule, sodium nitroprusside (SN), that efficiently promotes the generation of cardiac cells that express gene profiles and generate action potentials of PC-like cells. Imaging and mechanistic studies suggest that SN promotes the generation of PCs from cardiac progenitors initially expressing cardiac myosin heavy chain and that it does so by activating cyclic AMP signaling. These findings provide a strategy to derive scalable PCs, along with insight into the ontogeny of CCS development. PMID:26028533

  11. Activity-dependent accumulation of calcium in Purkinje cell dendritic spines

    SciTech Connect

    Andrews, S.B.; Leapman, R.D.; Landis, D.M.; Reese, T.S.

    1988-03-01

    The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.

  12. Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells.

    PubMed

    Selimi, Fekrije; Lohof, Ann M; Heitz, Stéphane; Lalouette, Alexis; Jarvis, Christopher I; Bailly, Yannick; Mariani, Jean

    2003-03-01

    The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1. PMID:12628171

  13. Unusual morphological damage of Purkinje cells following postnatal BrdU administration in the cerebellar cortex of mouse.

    PubMed

    Takács, T

    2012-01-01

    Postnatal development of the cerebellum lasts for weeks in rodents and can be disturbed by systemic 5-bromo-2'-deoxyuridine (BrdU) administration. This thymidine analogue incorporates into the DNA of proliferating cells, and result in more or less serious damage or death granule cells, the most actively dividing neuronal population in the developing cerebellar cortex. Further consequences of postnatal BrdU administration are the interrupted postnatal migration and integrations as well as partial loss of cerebellar Purkinje cells. In the present study, C57B16 mice were administered with 50 μg/g body weight BrdU, one sc. injection daily, between P0 and P11 postnatal days, respectively.Large "cavities" appeared in the cytoplasm of a subpopulation of Purkinje cells by P7 in about one-third of administered animals, their number are size of the cavities (and PCs exhibiting unusual morphology) decreased. EM studies revealed that the unusual Purkinje cells received numerous axonal inputs of unknown origin, first of all on their somatic and dendritic spines. The transitory appearance of a subpopulation of Purkinje cells possessing unusual morphology refers to the influence of other (neuronal, glial, or both) cells on their regular differentiation. PMID:22514871

  14. Uniform olivocerebellar conduction time underlies Purkinje cell complex spike synchronicity in the rat cerebellum.

    PubMed Central

    Sugihara, I; Lang, E J; Llinás, R

    1993-01-01

    1. The issue of isochronicity of olivocerebellar fibre conduction time as a basis for synchronizing complex spike activity in cerebellar Purkinje cells has been addressed by latency measurement, multiple-electrode recording and Phaseolus vulgaris leucoagglutinin (PHA-L) tracing of climbing fibres in the adult rat. 2. The conduction time of the olivocerebellar fibres was measured by recording Purkinje cell complex spike (CS) responses from various areas of the cerebellum. The CSs were evoked by stimulating the olivocerebellar fibres near the inferior olive. In spite of a difference in length, as determined directly by light microscopy, the conduction times of different climbing fibres were quite uniform, 3.98 +/- 0.36 ms (mean +/- S.D., n = 660). 3. Multiple-electrode recording of spontaneous Purkinje cell CS activity was employed to study the spatial extent of CS synchronicity in the cerebellar cortex. Recordings of CS were obtained from Purkinje cells located on the surface and along the walls of lobule crus 2a. The rostrocaudal band-like distribution of simultaneous (within 1 ms) CS activity in Purkinje cells extended down the sides of the cerebellar folia to the deepest areas recorded (1.6-2.6 mm deep). As shown in previous experiments, the distribution of simultaneous CS activity did not extend significantly (500 microns) in the mediolateral axis of the cerebellar cortex. 4. In two animals a detailed determination of the length of the olivocerebellar fibre bundles was performed by staining the fibres with PHA-L injected into the contralateral inferior olive. This measurement included fibre bundles terminating in twenty-six different areas, ranging from the tops of the various folia to the bottoms of the fissures in both the hemisphere and the vermis. There was a 47.5% difference between the length of the longest measured fibre bundle (15.8 mm, terminating in lobule 6b, zone A) and the length of the shortest measured fibre bundle (8.3 mm, terminating in the

  15. 'Dark' cell formation under protein malnutrition: process of conversion and concept of 'semi-dark' type Purkinje cells.

    PubMed

    James, T J; Sharma, S P; Gupta, S K; Patro, I K

    1992-06-01

    This paper deals with some deleterious effects of protein malnourishment in rat cerebellum. Severe protein deprivation enhanced the formation of 'dark' cells in white rats. It is postulated that abnormal changes in the neuronal contents induced by nutritional stress play a vital role in the formation of the 'dark' cells through an intermediary stage, 'semi-dark' cells. Centrophenoxine a lipofuscinolytic agent, however, seems to interfere with the process of formation of 'dark' cells and/or helps reconversion of the 'dark' cells into the normal or 'light' type Purkinje cells. PMID:1506025

  16. 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. PMID:25697849

  17. Long-term climbing fibre activity induces transcription of microRNAs in cerebellar Purkinje cells.

    PubMed

    Barmack, Neal H; Qian, Zuyuan; Yakhnitsa, Vadim

    2014-09-26

    Synaptic activation of central neurons is often evoked by electrical stimulation leading to post-tetanic potentiation, long-term potentiation or long-term depression. Even a brief electrical tetanus can induce changes in as many as 100 proteins. Since climbing fibre activity is often associated with cerebellar behavioural plasticity, we used horizontal optokinetic stimulation (HOKS) to naturally increase synaptic input to floccular Purkinje cells in mice for hours, not minutes, and investigated how this activity influenced the transcription of microRNAs, small non-coding nucleotides that reduce transcripts of multiple, complementary mRNAs. A single microRNA can reduce the translation of as many as 30 proteins. HOKS evoked increases in 12 microRNA transcripts in floccular Purkinje cells. One of these microRNAs, miR335, increased 18-fold after 24 h of HOKS. After HOKS stopped, miR335 transcripts decayed with a time constant of approximately 2.5 h. HOKS evoked a 28-fold increase in pri-miR335 transcripts compared with an 18-fold increase in mature miR335 transcripts, confirming that climbing fibre-evoked increases in miR335 could be attributed to increases in transcription. We used three screens to identify potential mRNA targets for miR335 transcripts: (i) nucleotide complementarity, (ii) detection of increased mRNAs following microinjection of miR335 inhibitors into the cerebellum, and (iii) detection of decreased mRNAs following HOKS. Two genes, calbindin and 14-3-3-θ, passed these screens. Transfection of N2a cells with miR335 inhibitors or precursors inversely regulated 14-3-3-θ transcripts. Immunoprecipitation of 14-3-3-θ co-immunoprecipitated PKC-γ and GABAAγ2. Knockdown of either 14-3-3-θ or PKC-γ decreased the serine phosphorylation of GABAAγ2, suggesting that 14-3-3-θ and PKC-γ under the control of miR335 homeostatically regulate the phosphorylation and insertion of GABAAγ2 into the Purkinje cell post-synaptic membrane. PMID:25135969

  18. The Cerebellum and SIDS: Disordered Breathing in a Mouse Model of Developmental Cerebellar Purkinje Cell Loss during Recovery from Hypercarbia

    PubMed Central

    Calton, Michele A.; Howard, Jeremy R.; Harper, Ronald M.; Goldowitz, Dan; Mittleman, Guy

    2016-01-01

    The cerebellum assists coordination of somatomotor, respiratory, and autonomic actions. Purkinje cell alterations or loss appear in sudden infant death and sudden death in epilepsy victims, possibly contributing to the fatal event. We evaluated breathing patterns in 12 wild-type (WT) and Lurcher mutant mice with 100% developmental cerebellar Purkinje cell loss under baseline (room air), and recovery from hypercapnia, a concern in sudden death events. Six mutant and six WT mice were exposed to 4-min blocks of increasing CO2 (2, 4, 6, and 8%), separated by 4-min recovery intervals in room air. Breath-by-breath patterns, including depth of breathing and end-expiratory pause (EEP) durations during recovery, were recorded. No baseline genotypic differences emerged. However, during recovery, EEP durations significantly lengthened in mutants, compared to WT mice, following the relatively low levels of CO2 exposure. Additionally, mutant mice exhibited signs of post-sigh disordered breathing during recovery following each exposure. Developmental cerebellar Purkinje cell loss significantly affects compensatory breathing patterns following mild CO2 exposure, possibly by inhibiting recovery from elevated CO2. These data implicate cerebellar Purkinje cells in the ability to recover from hypercarbia, suggesting that neuropathologic changes or loss of these cells contribute to inadequate ventilatory recovery to increased environmental CO2. Multiple disorders, including sudden infant death syndrome (SIDS) and sudden unexpected death in epilepsy (SUDEP), appear to involve both cardiorespiratory failure and loss or injury to cerebellar Purkinje cells; the findings support the concept that such neuropathology may precede and exert a prominent role in these fatal events. PMID:27242661

  19. 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

  20. Effect of diphenylhydantoin on gamma aminobutyric acid (GABA) and succinate activity in rat Purkinje cells.

    PubMed Central

    Hitchcock, E; Gabra-Sanders, T

    1977-01-01

    A study has been made of the effect of diphenylhydantoin (DPH) upon the levels of gamma aminobutyric acid (GABA) and succinic dehydrogenase in rat Purkinje cells. DPH was administered over 26 days in chronic experiments using controls receiving the same injection vehicle without DPH. Animals in this group received daily 1.25 mg/kg body weight, 12.5 mg/kg body weight, and 50 mg/kg body weight DPH. Acute experiments were carried out over the course of not more than four days, three groups of animals receiving 75 mg/kg body weight, 87.5 mg/kg body weight, and 100 mg/kg body weight DPH. No effect upon succinic dehydrogenase could be demonstrated at any dose level. There was a significant progressive loss of GABA with increasing dosage of DPH. Images PMID:903771

  1. 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. PMID:26665171

  2. Precise Control of Movement Kinematics by Optogenetic Inhibition of Purkinje Cell Activity

    PubMed Central

    Heiney, Shane A.; Kim, Jinsook; Augustine, George J.

    2014-01-01

    Purkinje cells (PCs) of the cerebellar cortex are necessary for controlling movement with precision, but a mechanistic explanation of how the activity of these inhibitory neurons regulates motor output is still lacking. We used an optogenetic approach in awake mice to show for the first time that transiently suppressing spontaneous activity in a population of PCs is sufficient to cause discrete movements that can be systematically modulated in size, speed, and timing depending on how much and how long PC firing is suppressed. We further demonstrate that this fine control of movement kinematics is mediated by a graded disinhibition of target neurons in the deep cerebellar nuclei. Our results prove a long-standing model of cerebellar function and provide the first demonstration that suppression of inhibitory signals can act as a powerful mechanism for the precise control of behavior. PMID:24501371

  3. Metabotropic glutamate receptor subtypes modulating neurotransmission at parallel fibre-Purkinje cell synapses in rat cerebellum.

    PubMed

    Neale, S A; Garthwaite, J; Batchelor, A M

    2001-07-01

    The actions of reportedly group-selective metabotropic glutamate (mGlu) receptor agonists and antagonists on neurotransmission at parallel fibre-Purkinje cell synapses in the rat cerebellum have been characterised using sharp microelectrode recording and an in vitro slice preparation. Application of the group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) or the group III selective agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed synaptic transmission in a reversible and concentration-dependent manner (EC(50)=18 and 5 microM, respectively). The depression produced by DHPG was unrelated to the depolarisation observed in some Purkinje cells. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV, 1 microM) had no effect. The effects of DHPG were inhibited by the group I-selective antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), but not by the group II/III antagonist alpha-methyl-4-phosphonophenylglycine (MPPG). The effect of L-AP4 was inhibited by MPPG, but not by the group I/II antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG). By themselves, the antagonists did not affect the EPSPs, suggesting that neither receptor is activated during low frequency neurotransmission. It is concluded that, in addition to the excitatory role for group I receptors described previously, both group I and III (but not group II) mGlu receptors operate at this synapse to inhibit synaptic transmission. The specific receptor subtypes involved are likely to be mGlu1 and mGlu4. PMID:11445184

  4. Single cardiac Purkinje cells: general electrophysiology and voltage-clamp analysis of the pace-maker current.

    PubMed

    Callewaert, G; Carmeliet, E; Vereecke, J

    1984-04-01

    Single Purkinje cells from dog, sheep and cow hearts were isolated by injecting a Ca-free collagenase containing Tyrode solution in the space between the connective tissue sheath and the Purkinje cells. A small proportion of these cells survived the isolation procedure and these cells were used for further investigation. The cells showed electrophysiological properties similar to intact Purkinje fibres as indicated by the following results. Maximum diastolic potentials between -70 and -85 mV and specific membrane resistances of 21-32 k omega cm2 indicated that the single cells were not leaky or hyperpermeable . The action potential showed a rapid upstroke, with a maximum rate of rise, Vmax' between 150 and 750 V/s, and two phases of fast repolarization separated by a plateau phase with a duration of about 200 ms. Each action potential was followed by a spontaneous depolarization with an amplitude between 1 and 10 mV. The upstroke of the action potential could be blocked by tetrodotoxin (TTX) in a dose-dependent manner. The rate of depolarization of the action potential was sensitive to changes in membrane potential; the resulting S-shaped curve showed a half-maximum potential of -65 mV and a steepness of 0.46 mV-1. The duration of the action potential was sensitive to external K concentrations, catecholamines and TTX in a way similar to intact Purkinje fibres. Both application of catecholamines and lowering the external K concentration induced spontaneous activity. The cells were used to study the ionic nature of the pace-maker current under voltage-clamp conditions using the two-micro-electrode technique. This pace-maker current was blocked in a voltage-dependent manner by 1 mM-Cs, and was not affected by 1 mM-Ba. The steady-state activation curve was shifted in the depolarizing direction by application of adrenaline. In contrast to voltage-clamp data obtained on the pace-maker current of intact Purkinje fibres, the pace-maker current in a single cell did not

  5. Short-term modulation of cerebellar Purkinje cell activity after spontaneous climbing fiber input.

    PubMed

    Sato, Y; Miura, A; Fushiki, H; Kawasaki, T

    1992-12-01

    1. There are two opposite points of view concerning the way climbing fiber input in a Purkinje cell modifies simple spike (SS) activity transiently: depression versus enhancement of SS activity. The different groups of investigators favored one effect predominating over the other. In the decerebrate unanesthetized cat, we recorded spontaneous activity of single Purkinje cells and investigated time course of SS activity after the complex spike (CS). 2. In the peri-CS time histogram, there was a SS pause lasting, on average, 10.8 ms after onset of the CS in all of the 316 cells recorded. The pause was followed by a rapid increase in SS activity to a maximum, which was on average 175.6% of a pre-CS control level, and a gradual return to around the control level in the majority of the cells recorded (pause-facilitation type, 71.2%). The increase in SS activity was significant (P < 0.01, t test) during 20-100 ms. The SS activity during the 20-100 ms was, on average, 163.7% of the control level. In some cells (pure-pause type, 25.3%), no significant changes were found (P > 0.01) in the post-pause SS firing. In contrast, only 3.5% of the cells (pause-reduction type) showed a significant (P < 0.01) firing decrease (average 54.0% of the control level) lasting 20-60 ms after the pause period. 3. Analysis of the pre-CS time histogram revealed no significant differences (P > 0.01) in the SS activity between pre-CS periods in all of the cells recorded, suggesting that the SS activity enhancement is not due to a coactivated mossy fiber input just preceding the activation of the climbing fiber input. 4. Analysis of the raster diagram revealed variability of individual SS responses after the CS. The probability of occurrence of the increase in SS number during a post-CS period of 0-100 ms with respect to that during a pre-CS period of -100-0 ms in individual raster traces was high (on average 78.2%), medium (57.3%), and low (36.3%) in the pause-facilitation, pure-pause, and pause

  6. 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.

  7. Methylmercury differentially affects GABAA receptor-mediated spontaneous IPSCs in Purkinje and granule cells of rat cerebellar slices

    PubMed Central

    Yuan, Yukun; Atchison, William D

    2003-01-01

    Using whole-cell recording techniques we compared effects of the environmental cerebellar neurotoxicant methylmercury (MeHg) on spontaneous IPSCs (sIPSCs) of both Purkinje and granule cells in cerebellar slices of the rat. In Purkinje cells, bath application of 10, 20 or 100 μM MeHg initially increased then suppressed the frequency of sIPSCs to zero. In granule cells, the initial increase in frequency was not observed in ≈50 % of cells examined, but suppression of sIPSCs by MeHg occurred in every cell tested. For both cells, time to onset of effects of MeHg was inversely related to the concentration; moreover, the pattern of changes in mIPSCs induced by MeHg in the presence of tetrodotoxin was similar to that in sIPSCs. For each concentration of MeHg, it took 2–3 times longer to block sIPSCs in Purkinje cells than it did in granule cells. MeHg also initially increased then decreased amplitudes of sIPSCs to block in both cells; again the response was more variable in granule cells. In most Purkinje and some granule cells, MeHg induced a giant, slow inward current during the late stages of exposure. Appearance of this current appeared to be MeHg concentration dependent, and the direction of current flow was reversed by changing the holding potentials. Reduction of the [Cl−] in the internal solution caused inwardly directed, but not outwardly directed giant currents to disappear, suggesting that this current is a Cl−-mediated response. However, bicuculline and picrotoxin failed to block it. MeHg apparently acts at both presynaptic and postsynaptic sites to alter GABAA receptor-mediated inhibitory synaptic transmission. GABAA receptors in granule cells appear to be more sensitive to block by MeHg than are those in Purkinje cells, although the general patterns of effects on the two cells are similar. PMID:12879869

  8. Ectopic cerebellar cell migration causes maldevelopment of Purkinje cells and abnormal motor behaviour in Cxcr4 null mice.

    PubMed

    Huang, Guo-Jen; Edwards, Andrew; Tsai, Cheng-Yu; Lee, Yi-Shin; Peng, Lei; Era, Takumi; Hirabayashi, Yoshio; Tsai, Ching-Yen; Nishikawa, Shin-Ichi; Iwakura, Yoichiro; Chen, Shu-Jen; Flint, Jonathan

    2014-01-01

    SDF-1/CXCR4 signalling plays an important role in neuronal cell migration and brain development. However, the impact of CXCR4 deficiency in the postnatal mouse brain is still poorly understood. Here, we demonstrate the importance of CXCR4 on cerebellar development and motor behaviour by conditional inactivation of Cxcr4 in the central nervous system. We found CXCR4 plays a key role in cerebellar development. Its loss leads to defects in Purkinje cell dentritogenesis and axonal projection in vivo but not in cell culture. Transcriptome analysis revealed the most significantly affected pathways in the Cxcr4 deficient developing cerebellum are involved in extra cellular matrix receptor interactions and focal adhesion. Consistent with functional impairment of the cerebellum, Cxcr4 knockout mice have poor coordination and balance performance in skilled motor tests. Together, these results suggest ectopic the migration of granule cells impairs development of Purkinje cells, causes gross cerebellar anatomical disruption and leads to behavioural motor defects in Cxcr4 null mice. PMID:24516532

  9. 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

    2012-12-01

    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. PMID:23042549

  10. 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

  11. 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. PMID:20357073

  12. Purkinje cell activity during classical conditioning with different conditional stimuli explains central tenet of Rescorla–Wagner model

    PubMed Central

    Rasmussen, Anders; Zucca, Riccardo; Johansson, Fredrik; Jirenhed, Dan-Anders; Hesslow, Germund

    2015-01-01

    A central tenet of Rescorla and Wagner’s model of associative learning is that the reinforcement value of a paired trial diminishes as the associative strength between the presented stimuli increases. Despite its fundamental importance to behavioral sciences, the neural mechanisms underlying the model have not been fully explored. Here, we present findings that, taken together, can explain why a stronger association leads to a reduced reinforcement value, within the context of eyeblink conditioning. Specifically, we show that learned pause responses in Purkinje cells, which trigger adaptively timed conditioned eyeblinks, suppress the unconditional stimulus (US) signal in a graded manner. Furthermore, by examining how Purkinje cells respond to two distinct conditional stimuli and to a compound stimulus, we provide evidence that could potentially help explain the somewhat counterintuitive overexpectation phenomenon, which was derived from the Rescorla–Wagner model. PMID:26504227

  13. 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

  14. 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

  15. Lesion-induced and activity-dependent structural plasticity of Purkinje cell dendritic spines in cerebellar vermis and hemisphere.

    PubMed

    Gelfo, Francesca; Florenzano, Fulvio; Foti, Francesca; Burello, Lorena; Petrosini, Laura; De Bartolo, Paola

    2016-09-01

    Neuroplasticity allows the brain to encode experience and learn behaviors, and also to re-acquire lost functions after damage. The cerebellum is a suitable structure to address this topic because of its strong involvement in learning processes and compensation of lesion-induced deficits. This study was aimed to characterize the effects of a hemicerebellectomy (HCb) combined or not with the exposition to environmental enrichment (EE) on dendritic spine density and size in Purkinje cell proximal and distal compartments of cerebellar vermian and hemispherical regions. Male Wistar rats were housed in enriched or standard environments from the 21st post-natal day (pnd) onwards. At the 75th pnd, rats were submitted to HCb or sham lesion. Neurological symptoms and spatial performance in the Morris water maze were evaluated. At the end of testing, morphological analyses assessed dendritic spine density, area, length, and head diameter on vermian and hemispherical Purkinje cells. All hemicerebellectomized (HCbed) rats showed motor compensation, but standard-reared HCbed animals exhibited cognitive impairment that was almost completely compensated in enriched HCbed rats. The standard-reared HCbed rats showed decreased density with augmented size of Purkinje cell spines in the vermis, and augmented both density and size in the hemisphere. Enriched HCbed rats almost completely maintained the spine density and size induced by EE. Both lesion-induced and activity-dependent cerebellar plastic changes may be interpreted as "beneficial" brain reactions, aimed to support behavioral performance rescuing. PMID:26420278

  16. 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. PMID:26538449

  17. Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

    PubMed

    Zhou, Liang; Yang, Dong; Wang, De-Juan; Xie, Ya-Jun; Zhou, Jia-Huan; Zhou, Lin; Huang, Hao; Han, Shuo; Shao, Chong-Yu; Li, Hua-Shun; Zhu, J Julius; Qiu, Meng-Sheng; De Zeeuw, Chris I; Shen, Ying

    2015-12-15

    Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1. PMID:26621723

  18. Treadmill exercise improves motor coordination through ameliorating Purkinje cell loss in amyloid beta23-35-induced Alzheimer's disease rats.

    PubMed

    Lee, Jae-Min; Shin, Mal-Soon; Ji, Eun-Sang; Kim, Tae-Woon; Cho, Han-Sam; Kim, Chang-Ju; Jang, Myung-Soo; Kim, Tae-Wook; Kim, Bo-Kyun; Kim, Dong-Hee

    2014-10-01

    Alzheimer's disease (AD) is a most common age-related neurodegenerative disease. AD is characterized by a progressive loss of neurons causing cognitive dysfunction. The cerebellum is closely associated with integration of movement, including motor coordination, control, and equilibrium. In the present study, we evaluated the effect of tread-mill exercise on the survival of Purkinje neurons in relation with reactive astrocyte in the cerebellum using Aβ25-35-induced AD rats. AD was induced by a bilateral intracerebroventricular (ICV) injection of Aβ25-35. The rats in the exercise groups were forced to run on a motorized treadmill for 30 min once a day for 4 weeks, starting 2 days after Aβ25-35 injection. In the present results, ICV injection of Aβ25-35 deteriorated motor coordination and balance. The number of calbindin-positive cells in the cerebellar vermis was decreased and glial fibrillary acidic protein (GFAP) expression in the cerebellar vermis was increased in the Aβ25-35-induced AD rats. Treadmill exercise improved motor coordination and balance. Treadmill exercise increased the number of Purkinje neurons and suppressed GFAP expression in the cerebellar vermis. The present study demonstrated that treadmill exercises alleviated dysfunction of motor coordination and balance by reduction of Purkinje cell loss through suppressing reactive astrocytes in the cerebellum of AD rats. The present study provides the possibility that treadmill exercise might be an important therapeutic strategy for the symptom improvement of AD patients. PMID:25426461

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26665171

  20. 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

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

    PubMed Central

    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. PMID:22355639

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

    PubMed

    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

  3. Ethanol modulates facial stimulation-evoked outward currents in cerebellar Purkinje cells in vivo in mice.

    PubMed

    Wu, Mao-Cheng; Bing, Yan-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2016-01-01

    Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs. PMID:27489024

  4. Ethanol modulates facial stimulation-evoked outward currents in cerebellar Purkinje cells in vivo in mice

    PubMed Central

    Wu, Mao-Cheng; Bing, Yan-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2016-01-01

    Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs. PMID:27489024

  5. Effects of norepinephrine on spontaneous firing activity of cerebellar Purkinje cells in vivo in mice.

    PubMed

    Guo, Ao; Feng, Jun-Yang; Li, Jia; Ding, Nan; Li, Ying-Jun; Qiu, De-Lai; Piao, Ri-Long; Chu, Chun-Ping

    2016-08-26

    Norepinephrine (NE), from the locus coeruleus (LC), has been supported to affect GABAergic system and parallel fiber (PF)-Purkinje cell (PC) synaptic transmission via adrenoceptor in cerebellum cortex. However, the effects of NE on the spontaneous spike activity of cerebellar PCs in living mouse have not yet been fully understood. We here examined the effects of NE on the spontaneous activity of PC in urethane-anesthetized mice by electrophysiological and pharmacological methods. Cerebellar surface application of NE (2.5-25μM) reduced the PC simple spike (SS) firing rate in a dose-dependent manner. The half-inhibitory concentration (IC50) was 5.97μM. In contrast, NE significantly increased the spontaneous firing rate of molecular layer interneuron (MLI). Application of GABAA receptor antagonist, gabazine (SR95531, 20μM) not only blocked the NE-induced inhibition of PC SS firing but also revealed NE-induced excitation of cerebellar PC. Blocking AMPA receptors activity enhanced NE-induced inhibition of PC spontaneous activity. Moreover, the effects of NE on PC spontaneous activity were abolished by simultaneously blocking GABAA and AMPA receptors activity. These results indicated that NE bidirectional modulated the spontaneous activity of PCs via enhancing both inhibitory inputs from MLIs and excitatory inputs of parallel fibers, but NE-induced enhance of inhibitory inputs overwhelmed the excitatory inputs under in vivo conditions. PMID:27369323

  6. 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

  7. 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

  8. 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. PMID:26535811

  9. 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. PMID:20105240

  10. 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. PMID:27543775

  11. The effect of the timing of ethanol exposure during early postnatal life on total number of Purkinje cells in rat cerebellum

    PubMed Central

    MIKI, TAKANORI; HARRIS, SIMON; WILCE, PETER; TAKEUCHI, YOSHIKI; BEDI, KULDIP S.

    1999-01-01

    We have previously shown that exposing rats to a high dose of ethanol on postnatal d 5 can affect Purkinje cell numbers in the cerebellum whilst similar exposure on d 10 had no such effect. The question arose whether a longer period of ethanol exposure after d 10 could produce loss of Purkinje cells. We have examined this question by exposing young rats to a relatively high dose (∼420–430 mg/dl) of ethanol for 6 d periods between the ages of either 4 and 9 d or 10 and 15 d of age. Exposure was carried out by placing the rats in an ethanol vapour chamber for 3 h per day during the exposure period. Groups of ethanol-treated (ET), separation controls (SC) and mother-reared controls (MRC) were anaesthetised and killed when aged 30 d by perfusion with buffered 2.5% glutaraldehyde. Stereological methods were used to determine the numbers of Purkinje cells in the cerebellum of each rat. MRC, SC and rats treated with ethanol between 10–15 d of age each had, on average, about 254–258 thousand cerebellar Purkinje cells; the differences between these various groups were not statistically significant. However, the rats treated with ethanol vapour between 4–9 d of age had an average of only about 128000±20000 Purkinje cells per cerebellum. This value was significantly different from both the MRC and group-matched SC animals. It is concluded that the period between 4 and 9 d of age is an extremely vulnerable period during which the rat cerebellar Purkinje cells are particularly susceptible to the effects of a high dose of ethanol. However, a similar level and duration of ethanol exposure commencing after 10 d of age has no significant effect on Purkinje cell numbers. PMID:10386779

  12. MMP-2 mediates Purkinje cell morphogenesis and spine development in the mouse cerebellum.

    PubMed

    Verslegers, Mieke; Van Hove, Inge; Dekeyster, Eline; Gantois, Ilse; Hu, Tjing-Tjing; D'Hooge, Rudi; Arckens, Lutgarde; Moons, Lieve

    2015-01-01

    Matrix metalloproteinase-2 (MMP-2) is a highly studied proteolytic enzyme, involved in many detrimental and beneficial functions throughout the body, and also active in the central nervous system (CNS). MMP-2 is profoundly expressed in the developing cerebellum and was recently reported to modulate granule cell proliferation by affecting cell cycle kinetics in cerebella of postnatal day 3 mouse pups. In this report, a two-dimensional difference gel electrophoresis proteomics study was implemented at this postnatal stage and revealed 16 differentially expressed proteins between MMP-2-deficient (MMP-2(-/-)) and wild-type cerebella. Among those, collapsin response mediator protein 1 (CRMP1) could be identified as the most significant differential protein between the two genotypes. Western blot experiments confirmed this finding and further disclosed a significant increase in phosphorylated CRMP1 expression in MMP-2(-/-) cerebella. Strikingly, subsequent immunohistochemical and microscopic analyses revealed an aberrant Purkinje cell (PC) dendritogenesis, possibly related to upregulated (phospho-) CRMP1 levels in these neonatal MMP-2(-/-) animals. Further, detailed morphometric analyses showed persistent PC morphological changes in MMP-2(-/-) mice, from the neonatal stage until adulthood. These were characterized by a reduced growth of PC somata, reduced dendritic tree sizes, and a decreased dendritic arborization. During development, the observed defects were accompanied by a temporarily disturbed parallel fiber and climbing fiber synaptic input on the PCs, while in adult MMP-2(-/-) animals, an increased PC spine density and reduced spine lengths were noted. The observed PC abnormalities might contribute to the mild defects in motor performance, i.e. balance and coordination, detected in adult MMP-2(-/-) mice. Overall, these findings indicate the importance of MMP-2 in CNS development and dendritogenesis, and highlight the importance of a correct developmental wiring

  13. CARTILAGE CELL CLUSTERS

    PubMed Central

    Lotz, Martin K.; Otsuki, Shuhei; Grogan, Shawn P.; Sah, Robert; Terkeltaub, Robert; D’Lima, Darryl

    2010-01-01

    The formation of new cell clusters is a histological hallmark of arthritic cartilage but the biology of clusters and their role in disease are poorly understood. This is the first comprehensive review of clinical and experimental conditions associated with cluster formation. Genes and proteins that are expressed in cluster cells, the cellular origin of the clusters, mechanisms that lead to cluster formation and the role of cluster cells in pathogenesis are discussed. PMID:20506158

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

    PubMed

    Spatz, W B

    2003-09-01

    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. PMID:12914985

  15. Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation.

    PubMed

    Verheijden, Simon; Bottelbergs, Astrid; Krysko, Olga; Krysko, Dmitri V; Beckers, Lien; De Munter, Stephanie; Van Veldhoven, Paul P; Wyns, Sabine; Kulik, Wim; Nave, Klaus-Armin; Ramer, Matt S; Carmeliet, Peter; Kassmann, Celia M; Baes, Myriam

    2013-10-01

    Although peroxisome biogenesis and β-oxidation disorders are well known for their neurodevelopmental defects, patients with these disorders are increasingly diagnosed with neurodegenerative pathologies. In order to investigate the cellular mechanisms of neurodegeneration in these patients, we developed a mouse model lacking multifunctional protein 2 (MFP2, also called D-bifunctional protein), a central enzyme of peroxisomal β-oxidation, in all neural cells (Nestin-Mfp2(-/-)) or in oligodendrocytes (Cnp-Mfp2(-/-)) and compared these models with an already established general Mfp2 knockout. Nestin-Mfp2 but not Cnp-Mfp2 knockout mice develop motor disabilities and ataxia, similar to the general mutant. Deterioration of motor performance correlates with the demise of Purkinje cell axons in the cerebellum, which precedes loss of Purkinje cells and cerebellar atrophy. This closely mimics spinocerebellar ataxias of patients affected with mild peroxisome β-oxidation disorders. However, general knockouts have a much shorter life span than Nestin-Mfp2 knockouts which is paralleled by a disparity in activation of the innate immune system. Whereas in general mutants a strong and chronic proinflammatory reaction proceeds throughout the brain, elimination of MFP2 from neural cells results in minor neuroinflammation. Neither the extent of the inflammatory reaction nor the cerebellar degeneration could be correlated with levels of very long chain fatty acids, substrates of peroxisomal β-oxidation. In conclusion, MFP2 has multiple tasks in the adult brain, including the maintenance of Purkinje cells and the prevention of neuroinflammation but this is not mediated by its activity in oligodendrocytes nor by its role in very long chain fatty acid degradation. PMID:23777740

  16. 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

  17. Purkinje Cell Ataxin-1 Modulates Climbing Fiber Synaptic Input in Developing and Adult Mouse Cerebellum

    PubMed Central

    Ebner, Blake A.; Ingram, Melissa A.; Barnes, Justin; Duvick, Lisa A.; Frisch, Jill L.; Clark, H. Brent; Zoghbi, Huda Y.; Ebner, Timothy J.; Orr, Harry T.

    2013-01-01

    Previous studies indicate that while transgenic mice with ATXN1[30Q]-D776-induced disease share pathological features caused by ATXN1[82Q] having an expanded polyglutamine tract, they fail to manifest the age-related progressive neurodegeneration seen in SCA1. The shared features include morphological alterations in climbing fiber (CF) innervation of Purkinje cells (PCs). To further investigate the ability of ATXN1 to impact CF/PC innervation, this study used morphological and functional approaches to examine CF/PC innervation during postnatal development in ATXN1[30Q]-D776 and ATXN1[82Q] cerebella. Notably, ATXN1[30Q]-D776 induced morphological alterations consistent with the development of the innervation of PCs by CFs being compromised, including a reduction of CF translocation along the PC dendritic tree, and decreased pruning of CF terminals from the PC soma. Like previously shown for ATXN1[82Q], ATXN1[30Q]-D776 must enter the nucleus of PCs to induce these alterations. Experiments using conditional ATXN1[30Q]-D776 mice demonstrate that both the levels and specific timing of mutant ATXN1expression are critical for alteration of the CF-PC synapse. Together these observations suggest that ATXN1, expressed exclusively in PCs, alters expression of a gene(s) in the postsynaptic PC that are critical for its innervation by CFs. To investigate whether ATXN1[30Q]-D776 curbs the progressive disease in ATXN1[82Q]-S776 mice, we crossed ATXN1[30Q]-D776 and ATXN1[82Q]-S776 mice and found that double transgenic mice developed progressive PC atrophy. Thus, the results also show that to develop progressive cerebellar degeneration requires expressing ATXN1 with an expanded polyglutamine tract. PMID:23536093

  18. Elevated mutant dynorphin A causes Purkinje cell loss and motor dysfunction in spinocerebellar ataxia type 23.

    PubMed

    Smeets, Cleo J L M; Jezierska, Justyna; Watanabe, Hiroyuki; Duarri, Anna; Fokkens, Michiel R; Meijer, Michel; Zhou, Qin; Yakovleva, Tania; Boddeke, Erik; den Dunnen, Wilfred; van Deursen, Jan; Bakalkin, Georgy; Kampinga, Harm H; van de Sluis, Bart; Verbeek, Dineke S

    2015-09-01

    Spinocerebellar ataxia type 23 is caused by mutations in PDYN, which encodes the opioid neuropeptide precursor protein, prodynorphin. Prodynorphin is processed into the opioid peptides, α-neoendorphin, and dynorphins A and B, that normally exhibit opioid-receptor mediated actions in pain signalling and addiction. Dynorphin A is likely a mutational hotspot for spinocerebellar ataxia type 23 mutations, and in vitro data suggested that dynorphin A mutations lead to persistently elevated mutant peptide levels that are cytotoxic and may thus play a crucial role in the pathogenesis of spinocerebellar ataxia type 23. To further test this and study spinocerebellar ataxia type 23 in more detail, we generated a mouse carrying the spinocerebellar ataxia type 23 mutation R212W in PDYN. Analysis of peptide levels using a radioimmunoassay shows that these PDYN(R212W) mice display markedly elevated levels of mutant dynorphin A, which are associated with climber fibre retraction and Purkinje cell loss, visualized with immunohistochemical stainings. The PDYN(R212W) mice reproduced many of the clinical features of spinocerebellar ataxia type 23, with gait deficits starting at 3 months of age revealed by footprint pattern analysis, and progressive loss of motor coordination and balance at the age of 12 months demonstrated by declining performances on the accelerating Rotarod. The pathologically elevated mutant dynorphin A levels in the cerebellum coincided with transcriptionally dysregulated ionotropic and metabotropic glutamate receptors and glutamate transporters, and altered neuronal excitability. In conclusion, the PDYN(R212W) mouse is the first animal model of spinocerebellar ataxia type 23 and our work indicates that the elevated mutant dynorphin A peptide levels are likely responsible for the initiation and progression of the disease, affecting glutamatergic signalling, neuronal excitability, and motor performance. Our novel mouse model defines a critical role for opioid

  19. Role of Synchronous Activation of Cerebellar Purkinje Cell Ensembles in Multi-joint Movement Control

    PubMed Central

    Hoogland, Tycho M.; De Gruijl, Jornt R.; Witter, Laurens; Canto, Cathrin B.; De Zeeuw, Chris I.

    2015-01-01

    Summary It is a longstanding question in neuroscience how elaborate multi-joint movements are coordinated coherently. Microzones of cerebellar Purkinje cells (PCs) are thought to mediate this coordination by controlling the timing of particular motor domains. However, it remains to be elucidated to what extent motor coordination deficits can be correlated with abnormalities in coherent activity within these microzones and to what extent artificially evoked synchronous activity within PC ensembles can elicit multi-joint motor behavior. To study PC ensemble correlates of limb, trunk, and tail movements, we developed a transparent disk treadmill that allows quantitative readout of locomotion and posture parameters in head-fixed mice and simultaneous cellular-resolution imaging and/or optogenetic manipulation. We show that PC ensembles in the ataxic and dystonic mouse mutant tottering have a reduced level of complex spike co-activation, which is delayed relative to movement onset and co-occurs with prolonged swing duration and reduced phase coupling of limb movements as well as with enlarged deflections of body-axis and tail movements. Using optogenetics to increase simple spike rate in PC ensembles, we find that preferred locomotion and posture patterns can be elicited or perturbed depending on the behavioral state. At rest, preferred sequences of limb movements can be elicited, whereas during locomotion, preferred gait-inhibition patterns are evoked. Our findings indicate that synchronous activation of PC ensembles can facilitate initiation and coordination of limb and trunk movements, presumably by tuning downstream systems involved in the execution of behavioral patterns. PMID:25843032

  20. 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

  1. Emergence of a 600-Hz buzz UP state Purkinje cell firing in alert mice.

    PubMed

    Cheron, G; Prigogine, C; Cheron, J; Márquez-Ruiz, J; Traub, R D; Dan, B

    2014-03-28

    Purkinje cell (PC) firing represents the sole output from the cerebellar cortex onto the deep cerebellar and vestibular nuclei. Here, we explored the different modes of PC firing in alert mice by extracellular recording. We confirm the existence of a tonic and/or bursting and quiescent modes corresponding to UP and DOWN state, respectively. We demonstrate the existence of a novel 600-Hz buzz UP state of firing characterized by simple spikes (SS) of very small amplitude. Climbing fiber (CF) input is able to switch the 600-Hz buzz to the DOWN state, as for the classical UP-to-DOWN state transition. Conversely, the CF input can initiate a typical SS pattern terminating into 600-Hz buzz. The 600-Hz buzz was transiently suppressed by whisker pad stimulation demonstrating that it remained responsive to peripheral input. It must not be mistaken for a DOWN state or the sign of PC inhibition. Complex spike (CS) frequency was increased during the 600-Hz buzz, indicating that this PC output actively contributes to the cerebello-olivary loop by triggering a disinhibition of the inferior olive. During the 600-Hz buzz, the first depolarizing component of the CS was reduced and the second depolarizing component was suppressed. Consistent with our experimental observations, using a 559-compartment single-PC model - in which PC UP state (of about -43mV) was obtained by the combined action of large tonic AMPA conductances and counterbalancing GABAergic inhibition - removal of this inhibition produced the 600-Hz buzz; the simulated buzz frequency decreased following an artificial CS. PMID:24440752

  2. 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.

  3. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    PubMed Central

    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. PMID:24722417

  4. 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. PMID:24722417

  5. 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. PMID:27003325

  6. Alcohol-induced Purkinje cell loss with a single binge exposure in neonatal rats: a stereological study of temporal windows of vulnerability.

    PubMed

    Goodlett, C R; Eilers, A T

    1997-06-01

    Previous research has shown that the early neonatal period of rats is one of enhanced vulnerability to cerebellar Purkinje cell loss associated with binge-like alcohol exposure, with a prominent sensitive period during the first neonatal week. In this study, an unbiased count of the total number of Purkinje cells was obtained using the stereological optical fractionator, in groups of rats given a single binge-like alcohol exposure either during the most vulnerable neonatal period [postnatal day (PD) 4] or during a later, less vulnerable period (PD 9). Using artificial rearing methods, rats were given 6.6 g/kg of alcohol either on PD 4 or on PD 9, delivered as a 15% (v/v) solution in milk formula on two consecutive feedings of the designated day. Control groups included an artificially reared gastrostomy control and a normally reared suckle control. The mean peak blood alcohol concentrations were not different between the PD 4 and PD 9 alcohol groups, averaging 374 and 347 mg/dl, respectively. The rats were perfused on PD 27. A uniform random sample of sections was obtained from serial frozen sections through the cerebellum, stained with thionin, and Purkinje cells were counted from a uniform random sample of locations on each section with the three-dimensional optical fractionator. The number of Purkinje cells in the suckle control and gastrostomy control groups did not differ from each other, averaging 3.94 (+/- 0.19) and 3.58 (+/- 0.22) x 10(5) cells, respectively. Binge exposure on PD 4 induced significant cell loss (mean of 2.05 +/- 0.20 x (10(5) Purkinje cells), whereas binge exposure on PD 9 did not induce significant Purkinje cell loss (3.70 +/- 0.39 x 10(5) Purkinje cells). These findings confirm that a single neonatal binge alcohol exposure produces pathological Purkinje cell loss, provided that it occurs during the period of enhanced vulnerability coinciding with the early stages of dendritic outgrowth. PMID:9194933

  7. β-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations.

    PubMed

    Clarkson, Yvonne L; Perkins, Emma M; Cairncross, Callum J; Lyndon, Alastair R; Skehel, Paul A; Jackson, Mandy

    2014-07-15

    Beta III spectrin is present throughout the elaborate dendritic tree of cerebellar Purkinje cells and is required for normal neuronal morphology and cell survival. Spinocerebellar ataxia type 5 (SCA5) and spectrin associated autosomal recessive cerebellar ataxia type 1 are human neurodegenerative diseases involving progressive gait ataxia and cerebellar atrophy. Both disorders appear to result from loss of β-III spectrin function. Further elucidation of β-III spectrin function is therefore needed to understand disease mechanisms and identify potential therapeutic options. Here, we report that β-III spectrin is essential for the recruitment and maintenance of ankyrin R at the plasma membrane of Purkinje cell dendrites. Two SCA5-associated mutations of β-III spectrin both reduce ankyrin R levels at the cell membrane. Moreover, a wild-type β-III spectrin/ankyrin-R complex increases sodium channel levels and activity in cell culture, whereas mutant β-III spectrin complexes fail to enhance sodium currents. This suggests impaired ability to form stable complexes between the adaptor protein ankyrin R and its interacting partners in the Purkinje cell dendritic tree is a key mechanism by which mutant forms of β-III spectrin cause ataxia, initially by Purkinje cell dysfunction and exacerbated by subsequent cell death. PMID:24603075

  8. 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

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

    PubMed

    Wang, Kai; Xu, Ruijuan; Schrandt, Jennifer; Shah, Prithvi; Gong, Yong Z; 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

    2015-10-01

    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

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

    PubMed Central

    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” Gi/o-coupled receptor modulation of physiological effectors, including the P-type Ca2+ 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. PMID:18930827

  11. 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

  12. Effect of Gestational Diabetes on Purkinje and Granule Cells Distribution of the Rat Cerebellum in 21 and 28 days of Postnatal Life

    PubMed Central

    Razi, Elahe Mirarab; Ghafari, Soraya; Golalipour, Mohammad Jafar

    2015-01-01

    Introduction: Diabetes mellitus is associated with nervous system alterations in both human and animal models. This study was done to determine the effect of gestational diabetes on the Purkinje and granular cells in the cerebellum of rat offspring. Methods: 10 Wistar rats Dams were randomly allocated in control and diabetic group. The experimental group received 40 mg/kg/body weight of streptozotocin (STZ) at the first day of gestation and control groups received saline injection intraperitoneally (IP). Six male offsprings of gestational diabetic mothers and control dams, at the 21, 28 postnatal days were randomly scarified and coronal sections of cerebellum (6 micrometer) serially collected. The neurons were stained with cresyl violet. Results: The Purkinje cells density in the apex and depth of cerebellum in P21, in the experimental group was reduced 23% and 15% in comparison with the control group (P<0.001). The granular cells density in the experimental group was reduced 19.58% and 18.3% in comparison with the controls (P<0.001). The Purkinje cells density of cerebellum in P28, in the diabetic group reduced to 22.12% and 12.62% in comparison with the control group (P<0.001). The granular cells density in the diabetic group reduced 17.14% and 16.12% in comparison with the control group (P<0.001). Discussion: The Purkinje and granular cells significantly reduced in gestational diabetes rat offspring.

  13. GABA(A) receptor expression and inhibitory post-synaptic currents in cerebellar Purkinje cells in dystrophin-deficient mdx mice.

    PubMed

    Kueh, S L L; Head, S I; Morley, J W

    2008-02-01

    1. Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease and arises as a consequence of an absence or disruption of the protein dystrophin. In addition to wasting of the skeletal musculature, boys with DMD have a significant degree of cognitive impairment. 2. We show here that there is no difference between littermate control and mdx mice (a murine model of DMD) in the overall expression of the GABA(A) receptor a1-subunit, supporting the suggestion that it is the clustering at the synapse that is affected and not the expression of the GABA(A) receptor protein. 3. We report a significant reduction in both the frequency and amplitude of spontaneous inhibitory post-synaptic currents in cerebellar Purkinje cells of mdx mice compared with littermate controls, consistent with the reported reduction in the number and size of GABA(A) receptor clusters immunoreactive for a1- and a2-subunits at the post-synaptic densities. 4. These results may explain some of the behavioural problems and cognitive impairment reported in DMD. PMID:17941889

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

    PubMed

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

    2016-05-01

    Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders. PMID:27152617

  15. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Stahl, John S; Thumser, Zachary C

    2014-11-15

    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

  18. 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

  19. Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures

    PubMed Central

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

    2013-01-01

    Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4–5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis. PMID:24312586

  20. Miglustat improves purkinje cell survival and alters microglial phenotype in feline Niemann-Pick disease type C.

    PubMed

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

    2012-05-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-06-20

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

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

    PubMed

    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

  4. Survival of interneurons and parallel fiber synapses in a cerebellar cortex deprived of Purkinje cells: studies in the double mutant mouse Grid2Lc/+;Bax(-/-).

    PubMed

    Zanjani, S Hadi; Selimi, Fekrije; Vogel, Michael W; Haeberlé, Anne-Marie; Boeuf, Julien; Mariani, Jean; Bailly, Yannick J

    2006-08-01

    The Lurcher mutation in the Grid2 gene causes the cell autonomous death of virtually all cerebellar Purkinje cells and the target-related death of 90% of the granule cells and 60-75% of the olivary neurons. Inactivation of Bax, a pro-apoptotic gene of the Bcl-2 family, in heterozygous Lurcher mutants (Grid2Lc/+) rescues approximately 60% of the granule cells, but does not rescue Purkinje or olivary neurons. Given the larger size of the cerebellar molecular layer in Grid2Lc/+;Bax(-/-) double mutants compared to Grid2Lc/+ mutants, we analyzed the survival of the stellate and basket interneurons as well as the synaptic connectivity of parallel fibers originating from the surviving granule cells in the absence of their Purkinje cell targets in the Grid2Lc/+;Bax(-/-) cerebellum. Quantification showed a significantly higher density of interneurons ( approximately 60%) in the molecular layer of the Grid2Lc/+;Bax(-/-) mice compared to Grid2Lc/+, suggesting that interneurons are subject to a BAX-dependent target-related death in the Lurcher mutants. Furthermore, electron microscopy showed the normal ultrastructural aspect of a number of parallel fibers in the molecular layer of the Grid2Lc/+; Bax(-/-) double mutant mice and preserved their numerous synaptic contacts on interneurons, suggesting that interneurons could play a trophic role for axon terminals of surviving granule cells. Finally, parallel fibers varicosities in the double mutant established "pseudo-synapses" on glia as well as displayed autophagic profiles, suggesting that the connections established by the parallel fibers in the absence of their Purkinje cell targets were subject to a high turnover involving autophagy. PMID:16739195

  5. 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

  6. 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

  7. Optogenetic Control of Motor Coordination by Gi/o Protein-coupled Vertebrate Rhodopsin in Cerebellar Purkinje Cells*

    PubMed Central

    Gutierrez, Davina V.; Mark, Melanie D.; Masseck, Olivia; Maejima, Takashi; Kuckelsberg, Denise; Hyde, Robert A.; Krause, Martin; Kruse, Wolfgang; Herlitze, Stefan

    2011-01-01

    G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific Gi/o protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the Gi/o protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of Gi/o modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar Gi/o-coupled GABAB receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via Gi/o-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner. PMID:21628464

  8. 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

  9. Mutation in archain 1, a subunit of COPI coatomer complex, causes diluted coat color and Purkinje cell degeneration.

    PubMed

    Xu, Xinjie; Kedlaya, Rajendra; Higuchi, Hitoshi; Ikeda, Sakae; Justice, Monica J; Setaluri, Vijayasaradhi; Ikeda, Akihiro

    2010-05-01

    Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)-induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the delta subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration. PMID:20502676

  10. Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebellum

    PubMed Central

    Teigler, Andre; Komljenovic, Dorde; Draguhn, Andreas; Gorgas, Karin; Just, Wilhelm W.

    2009-01-01

    Ether lipids (ELs), particularly plasmalogens, are essential constituents of the mammalian central nervous system. The physiological role of ELs, in vivo, however is still enigmatic. In the present study, we characterized a mouse model carrying a targeted deletion of the peroxisomal dihydroxyacetonephosphate acyltransferase gene that results in the complete lack of ELs. Investigating the cerebellum of these mice, we observed: (i) defects in foliation patterning and delay in precursor granule cell migration, (ii) defects in myelination and concomitant reduction in the level of myelin basic protein, (iii) disturbances in paranode organization by extending the Caspr distribution and disrupting axo-glial septate-like junctions, (iv) impaired innervation of Purkinje cells by both parallel fibers and climbing fibers and (v) formation of axon swellings by the accumulation of inositol-tris-phosphate receptor 1 containing smooth ER-like tubuli. Functionally, conduction velocity of myelinated axons in the corpus callosum was significantly reduced. Most of these phenotypes were already apparent at P20 but still persisted in 1-year-old animals. In summary, these data show that EL deficiency results in severe developmental and lasting structural alterations at the cellular and network level of the cerebellum, and reveal an important role of ELs for proper brain function. Common molecular mechanisms that may underlie these phenotypes are discussed. PMID:19270340

  11. Defects in myelination, paranode organization and Purkinje cell innervation in the ether lipid-deficient mouse cerebellum.

    PubMed

    Teigler, Andre; Komljenovic, Dorde; Draguhn, Andreas; Gorgas, Karin; Just, Wilhelm W

    2009-06-01

    Ether lipids (ELs), particularly plasmalogens, are essential constituents of the mammalian central nervous system. The physiological role of ELs, in vivo, however is still enigmatic. In the present study, we characterized a mouse model carrying a targeted deletion of the peroxisomal dihydroxyacetonephosphate acyltransferase gene that results in the complete lack of ELs. Investigating the cerebellum of these mice, we observed: (i) defects in foliation patterning and delay in precursor granule cell migration, (ii) defects in myelination and concomitant reduction in the level of myelin basic protein, (iii) disturbances in paranode organization by extending the Caspr distribution and disrupting axo-glial septate-like junctions, (iv) impaired innervation of Purkinje cells by both parallel fibers and climbing fibers and (v) formation of axon swellings by the accumulation of inositol-tris-phosphate receptor 1 containing smooth ER-like tubuli. Functionally, conduction velocity of myelinated axons in the corpus callosum was significantly reduced. Most of these phenotypes were already apparent at P20 but still persisted in 1-year-old animals. In summary, these data show that EL deficiency results in severe developmental and lasting structural alterations at the cellular and network level of the cerebellum, and reveal an important role of ELs for proper brain function. Common molecular mechanisms that may underlie these phenotypes are discussed. PMID:19270340

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

    PubMed

    Rahmati, Negah; Vinueza Veloz, Maria Fernanda; Xu, Jie; Barone, Sharon; Rodolfo Ben Hamida, Nahuel; Schonewille, Martijn; Hoebeek, Freek E; Soleimani, Manoocher; De Zeeuw, Chris I

    2016-01-01

    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

  13. Ventricular arrhythmias and the His-Purkinje system.

    PubMed

    Haissaguerre, Michel; Vigmond, Edward; Stuyvers, Bruno; Hocini, Meleze; Bernus, Olivier

    2016-03-01

    Ventricular arrhythmias are a major cause of sudden death, which accounts for approximately half of cardiac mortality. The His-Purkinje system is composed of specialized cells responsible for the synchronous activation of the ventricles. However, experimental studies show that the Purkinje system can be arrhythmogenic during electrolyte imbalance, after exposure to various drugs, and in myocardial ischaemia, during which Purkinje cells can survive in anaerobic conditions. Purkinje cells can generate both automatic and triggered focal rhythms, and their network configuration can accommodate re-entrant circuits. In humans, a variety of monomorphic ventricular tachycardias can be sustained within the architecture of the Purkinje branches. Furthermore, discrete Purkinje sources can serve as critical triggers of ventricular fibrillation in a wide spectrum of patients with structural heart disease or with an apparently normal heart. In drug-resistant cases of monomorphic and polymorphic Purkinje-related ventricular tachycardias, catheter ablation is a very effective treatment. The specific transcriptional signatures and functional properties of Purkinje cells, including their intracellular calcium dynamics, underlie their extreme arrhythmogenicity. However, the identification of vulnerable individuals remains challenging, and the molecular mechanisms of Purkinje-related arrhythmias have to be characterized further to enable the development of interventions to prevent lethal cardiac arrhythmias. PMID:26727298

  14. Treadmill exercise improves motor coordination through ameliorating Purkinje cell loss in amyloid beta23-35-induced Alzheimer’s disease rats

    PubMed Central

    Lee, Jae-Min; Shin, Mal-Soon; Ji, Eun-Sang; Kim, Tae-Woon; Cho, Han-Sam; Kim, Chang-Ju; Jang, Myung-Soo; Kim, Tae-Wook; Kim, Bo-Kyun; Kim, Dong-Hee

    2014-01-01

    Alzheimer’s disease (AD) is a most common age-related neurodegenerative disease. AD is characterized by a progressive loss of neurons causing cognitive dysfunction. The cerebellum is closely associated with integration of movement, including motor coordination, control, and equilibrium. In the present study, we evaluated the effect of tread-mill exercise on the survival of Purkinje neurons in relation with reactive astrocyte in the cerebellum using Aβ25–35–induced AD rats. AD was induced by a bilateral intracerebroventricular (ICV) injection of Aβ25–35. The rats in the exercise groups were forced to run on a motorized treadmill for 30 min once a day for 4 weeks, starting 2 days after Aβ25–35 injection. In the present results, ICV injection of Aβ25–35 deteriorated motor coordination and balance. The number of calbindin-positive cells in the cerebellar vermis was decreased and glial fibrillary acidic protein (GFAP) expression in the cerebellar vermis was increased in the Aβ25–35-induced AD rats. Treadmill exercise improved motor coordination and balance. Treadmill exercise increased the number of Purkinje neurons and suppressed GFAP expression in the cerebellar vermis. The present study demonstrated that treadmill exercises alleviated dysfunction of motor coordination and balance by reduction of Purkinje cell loss through suppressing reactive astrocytes in the cerebellum of AD rats. The present study provides the possibility that treadmill exercise might be an important therapeutic strategy for the symptom improvement of AD patients. PMID:25426461

  15. Distribution of Purkinje cell-specific Zebrin-II/aldolase C immunoreactivity in the mouse, rat, rabbit, and human retina.

    PubMed

    Caffé, A R; Von Schantz, M; Szél, A; Voogd, J; Van Veen, T

    1994-10-01

    The developmental, genetic, and biochemical similarities that have been observed between the cerebellum and retina form the basis for ongoing investigations into retinal expression of cerebellar-specific proteins. We have examined the mouse, rat, rabbit, and human retina for expression of a protein that is present in parasagittal Purkinje cell strips and that is recognized by the antibody Zebrin-II. This protein has recently been identified as a member of the aldolase C isoenzymes. Western blotting and immunocytochemistry have been used. The monoclonal antibody Zebrin-II recognized a prominent 36 kDa protein band on immunoblots of both the cerebellum and the retina of the examined species. Immunocytochemistry showed that, in the three nonhuman species, cells were stained in the ganglion cell layer (GCL). In addition, in the mouse and rabbit, cells in the inner nuclear layer (INL) were also labeled. Except for the visual streak, there were more immunopositive cells in the rabbit GCL and INL than in corresponding areas of the mouse retina. In the human, in contrast to the other species, the photoreceptor cell layer was strongly aldolase C immunoreactive. In all species except for the rat, the photoreceptor inner segments also displayed a weak labeling. The results show that this aldolase C isoenzyme is another protein that is selectively expressed by the cerebellum and retina. Furthermore, the retinal expression is species specific, and this pattern seems to show a good correlation with the oxygenation level of the individual compartments. The indication that this aldolase C isoenzyme has specific developmental functions in the retina provides additional clues for our understanding of cerebellar organization. PMID:7814693

  16. 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. PMID:21054627

  17. 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

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  20. 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. PMID:26819763

  1. Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2.

    PubMed

    Hansen, Stephen T; Meera, Pratap; Otis, Thomas S; Pulst, Stefan M

    2013-01-15

    Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2(Q127)-complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs. PMID:23087021

  2. Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2

    PubMed Central

    Hansen, Stephen T.; Meera, Pratap; Otis, Thomas S.; Pulst, Stefan M.

    2013-01-01

    Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2Q127-complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs. PMID:23087021

  3. 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. PMID:23884927

  4. Diphtheria toxin mutant selectively kills cerebellar Purkinje neurons.

    PubMed Central

    Riedel, C J; Muraszko, K M; Youle, R J

    1990-01-01

    CRM107 (crossreacting material 107), a double point mutant of diphtheria toxin that lacks receptor-binding activity, specifically kills cerebellar Purkinje cells in vivo. After injection into guinea pig cerebrospinal fluid, CRM107 (0.9 micrograms) and CRM107-monoclonal antibody conjugates (10 micrograms) kill up to 90% of the total Purkinje cell population with no detectable toxicity to other neurons. Animals exhibit ataxia, tremor, and abnormalities of posture and tone. Native diphtheria toxin, ricin, and ricin A chain do not cause ataxia and do not reduce the Purkinje cell population after intrathecal injection into guinea pigs at toxic or maximally tolerated doses. However, in rats, which will tolerate higher doses of diphtheria toxin than guinea pigs, Purkinje cells can be killed by both CRM107 and diphtheria toxin. A truncated mutant of diphtheria toxin, called CRM45, can also cause Purkinje cell killing but has additional toxicity not seen with CRM107. Animals treated with intrathecal CRM107 or CRM107 linked to antibodies may serve as models for Purkinje cell loss in a broad spectrum of human diseases and may be used to further study cerebellar physiology. Understanding the basis for the Purkinje cell sensitivity to CRM107 may illuminate other causes of Purkinje cell loss. Images PMID:2367523

  5. The cytoplasmic Purkinje onconeural antigen cdr2 down-regulates c-Myc function: implications for neuronal and tumor cell survival

    PubMed Central

    Okano, Hirotaka J.; Park, Woong-Y.; Corradi, John P.; Darnell, Robert B.

    1999-01-01

    Paraneoplastic cerebellar degeneration (PCD) is a disorder in which breast or ovarian tumors express an onconeural antigen termed cdr2, which normally is expressed in cerebellar Purkinje neurons. This leads to an immune response to cdr2 that is associated with tumor immunity and autoimmune cerebellar degeneration. We have found that cdr2, a cytoplasmic protein harboring a helix–leucine zipper (HLZ) motif, interacts specifically with the HLZ motif of c-Myc. Both proteins colocalize in the cytoplasm of adult cerebellar Purkinje neurons, and coimmunoprecipitate from tumor cell lines and cerebellar extracts. cdr2 down–regulates c-Myc-dependent transcription in cotransfection assays, and redistributes Myc protein in the cytoplasm. Disease antisera from six of six PCD patients specifically blocked the interaction between cdr2 and c-Myc in vitro. These data indicate that cdr2 normally sequesters c-Myc in the neuronal cytoplasm, thereby down-regulating c-Myc activity, and suggest a mechanism whereby inhibition of cdr2 function by autoantibodies in PCD may contribute to Purkinje neuronal death. PMID:10465786

  6. Responses of Purkinje-cells of the cerebellar anterior vermis to stimulation of vestibular and somatosensory receptors.

    PubMed

    Bruschini, L; Andre, P; Pompeiano, O; Manzoni, D

    2006-09-29

    In decerebrate cats, sinusoidal rotation of the forepaw around the wrist modifies the activity of the ipsilateral forelimb extensor triceps brachii (TB) and leads to plastic changes of adaptive nature in the gain of vestibulospinal (VS) reflexes (VSRs). Both effects are depressed by functional inactivation of the cerebellar anterior vermis, which also decreases the gain of VSRs. In order to better understand the mechanisms of these phenomena, the simple spike activity of Purkinje (P-) cells was recorded from the vermal cortex of the cerebellar anterior lobe during individual and/or combined stimulation of somatosensory wrist, neck and vestibular receptors. About one third of the recorded units were affected by sinusoidal rotation of the ipsilateral forepaw around the wrist axis (0.16 Hz, +/-10 degrees ). Most of these neurons ( approximately 60%) increased their activity during ventral flexion of the wrist and decreased it during the oppositely directed movement, with an average phase lag of -141 degrees with respect to the position of maximal dorsiflexion. The remaining cells ( approximately 40%) were excited during dorsiflexion of the wrist, with an average phase lead of 59 degrees with respect to the extreme dorsal flexion. Both populations showed comparable response gains, with an average value of 0.42+/-0.52, S.D., imp/s/deg. About half of the recorded units were also tested during sinusoidal roll tilt of the animal around the longitudinal axis (0.16 Hz, +/-10 degrees ), leading to stimulation of labyrinthine receptors. When both stimuli were applied simultaneously, the responses to combined stimulation usually corresponded to the sum of individual responses. While the phase distribution of somatosensory responses was clearly bimodal, vestibular responses showed phase angle values uniformly scattered between +/-180 degrees and 0 degrees , so that, during combined stimulation, each neuron could be maximally activated by coupling the two stimuli with a

  7. Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

    PubMed Central

    Bielen, F V; Glitsch, H G; Verdonck, F

    1991-01-01

    1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that

  8. 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

  9. G protein-independent neuromodulatory action of adenosine on metabotropic glutamate signalling in mouse cerebellar Purkinje cells

    PubMed Central

    Tabata, Toshihide; Kawakami, Daisuke; Hashimoto, Kouichi; Kassai, Hidetoshi; Yoshida, Takayuki; Hashimotodani, Yuki; Fredholm, Bertil B; Sekino, Yuko; Aiba, Atsu; Kano, Masanobu

    2007-01-01

    Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between Gi/o protein-coupled A1-subtype AR (A1R) and type-1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated-patch voltage-clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1-coupled inward current. A1R agonists also depressed mGluR1-coupled intracellular Ca2+ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist-induced depression persisted after blockade of Gi/o protein. The depression appeared to involve neither the cAMP-protein kinase A cascade downstream of the alpha subunits of Gi/o and Gs proteins, nor cytoplasmic Ca2+ that is suggested to be regulated by the beta-gamma subunit complex of Gi/o protein. Moreover, A1R did not appear to affect Gq protein which mediates the mGluR1-coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R-mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R-mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons. PMID:17379632

  10. mGlu1 receptor mediates homeostatic control of intrinsic excitability through Ih in cerebellar Purkinje cells.

    PubMed

    Shim, Hyun Geun; Jang, Sung-Soo; Jang, Dong Cheol; Jin, Yunju; Chang, Wonseok; Park, Joo Min; Kim, Sang Jeong

    2016-06-01

    Homeostatic intrinsic plasticity is a cellular mechanism for maintaining a stable neuronal activity level in response to developmental or activity-dependent changes. Type 1 metabotropic glutamate receptor (mGlu1 receptor) has been widely known to monitor neuronal activity, which plays a role as a modulator of intrinsic and synaptic plasticity of neurons. Whether mGlu1 receptor contributes to the compensatory adjustment of Purkinje cells (PCs), the sole output of the cerebellar cortex, in response to chronic changes in excitability remains unclear. Here, we demonstrate that the mGlu1 receptor is involved in homeostatic intrinsic plasticity through the upregulation of the hyperpolarization-activated current (Ih) in cerebellar PCs. This plasticity was prevented by inhibiting the mGlu1 receptor with Bay 36-7620, an mGlu1 receptor inverse agonist, but not with CPCCOEt, a neutral antagonist. Chronic inactivation with tetrodotoxin (TTX) increased the components of Ih in the PCs, and ZD 7288, a hyperpolarization-activated cyclic nucleotide-gated channel selective inhibitor, fully restored reduction of firing rates in the deprived neurons. The homeostatic elevation of Ih was also prevented by BAY 36-7620, but not CPCCOEt. Furthermore, KT 5720, a blocker of protein kinase A (PKA), prevented the effect of TTX reducing the evoked firing rates, indicating the reduction in excitability of PCs due to PKA activation. Our study shows that both the mGlu1 receptor and the PKA pathway are involved in the homeostatic intrinsic plasticity of PCs after chronic blockade of the network activity, which provides a novel understanding on how cerebellar PCs can preserve the homeostatic state under activity-deprived conditions. PMID:26912592

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

    PubMed

    Yamaguchi, Kazuhiko; Itohara, Shigeyoshi; Ito, Masao

    2016-09-01

    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

  12. Visualization of Ca2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells.

    PubMed

    Okubo, Yohei; Suzuki, Junji; Kanemaru, Kazunori; Nakamura, Naotoshi; Shibata, Tatsuo; Iino, Masamitsu

    2015-12-01

    The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca(2+) signaling, serving as both a source and sink of Ca(2+), and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca(2+) dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca(2+) dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca(2+) indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca(2+) concentration ([Ca(2+)]ER) in dendrites and spines. In this experimental system, the recovery of [Ca(2+)]ER takes several seconds, and recovery half-time depends on the extent of ER Ca(2+) depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca(2+), rather than Ca(2+) reuptake, is the dominant mechanism for the replenishment of the local [Ca(2+)]ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca(2+) diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca(2+) spikes in PCs, cumulatively increased [Ca(2+)]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca(2+) within the neurons, and as a leaky integrator of Ca(2+) spike-inducing synaptic inputs. PMID:26631466

  13. The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats

    PubMed Central

    Momiyama, Akiko; Silver, R Angus; Häusser, Michael; Notomi, Takuya; Wu, Yue; Shigemoto, Ryuichi; Cull-Candy, Stuart G

    2003-01-01

    We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was ∼300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of ∼5 pS (∼7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 mm), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (∼11 pS). This approach allowed us to obtain a maximum open probability (Po,max) value for the extrasynaptic receptors (Po,max = 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 μm2, measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of ∼900 μm−2 in the postsynaptic membrane. PMID:12665613

  14. 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

  15. Modulation of inhibitory post-synaptic currents (IPSCs) in mouse cerebellar Purkinje and basket cells by snake and scorpion toxin K+ channel blockers

    PubMed Central

    Southan, Andrew P; Robertson, Brian

    1998-01-01

    Using an in vitro mouse cerebellar slice preparation and whole-cell electrophysiological recording techniques we have characterized Purkinje and basket cell inhibitory post-synaptic currents (IPSCs), and examined the effects of a number of selective peptidergic K+ channel blockers.Spontaneous IPSC amplitude ranged from ∼10 pA up to ∼3 nA for both cell types [mean values: Purkinje cells −122.8±20.0 pA (n=24 cells); basket cells −154.8±15.9 pA (n=26 cells)]. Frequency varied from ∼3 up to ∼40 Hz, [mean values: basket cells 14.9±1.7 Hz (n=26 cells); Purkinje cells 17.9±2.2 Hz (n=24 cells)]. 5 μM bicuculline eliminated virtually all spontaneous currents.IPSC rise times were fast (∼0.6 ms) and the decay phase was best fit with the sum of two exponential functions (τ1 and τ2: ∼4 ms and ∼20 ms, n=40; for both cell types).The snake toxins alpha-dendrotoxin (α-DTX) and toxin K greatly enhanced IPSC frequency and amplitude in both cell types; the closely related homologues toxin I and gamma-dendrotoxin (γ-DTX) produced only marginal enhancements (all at 200 nM).Two scorpion toxins, margatoxin (MgTX) and agitoxin-2 (AgTX-2) had only minor effects on IPSC frequency or amplitude (both at 10 nM).Low concentrations of tetraethylammonium (TEA; 200 μM) had no overall effect on cerebellar IPSCs, whilst higher concentrations (10 mM) increased both the frequency and amplitude.The results suggest that native K+ channels, containing Kv1.1 and Kv1.2 channel subunits, play an influential role in controlling GABAergic inhibitory transmission from cerebellar basket cells. PMID:9863670

  16. The inhibitory input to mouse cerebellar Purkinje cells is reciprocally modulated by Bergmann glial P2Y1 and AMPA receptor signaling.

    PubMed

    Rudolph, Ramona; Jahn, Hannah M; Courjaret, Raphael; Messemer, Nanette; Kirchhoff, Frank; Deitmer, Joachim W

    2016-07-01

    Synaptic transmission has been shown to be modulated by glial functions, but the modes of specific glial action may vary in different neural circuits. We have tested the hypothesis, if Bergmann GLIA (BG) are involved in shaping neuronal communication in the mouse cerebellar cortex, using acutely isolated cerebellar slices of wild-type (WT) and of glia-specific receptor knockout mice. Activation of P2Y1 receptors by ADP (100 µM) or glutamatergic receptors by AMPA (0.3 µM) resulted in a robust, reversible and repeatable rise of evoked inhibitory input in Purkinje cells by 80% and 150%, respectively. The ADP-induced response was suppressed by prior application of AMPA, and the AMPA-induced response was suppressed by prior application of ADP. Genetic deletion or pharmacological blockade of either receptor restored the response to the other receptor agonist. Both ADP and AMPA responses were sensitive to Rose Bengal, which blocks vesicular glutamate uptake, and to the NMDA receptor antagonist D-AP5. Our results provide strong evidence that activation of both ADP and AMPA receptors, located on BGs, results in the release of glutamate, which in turn activates inhibitory interneurons via NMDA-type glutamate receptors. This infers that BG cells, by means of metabotropic signaling via their AMPA and P2Y1 receptors, which mutually suppress each other, would interdependently contribute to the fine-tuning of Purkinje cell activity in the cerebellar cortex. GLIA 2016. GLIA 2016;64:1265-1280. PMID:27144942

  17. Saccade-related Purkinje cell activity in the oculomotor vermis during spontaneous eye movements in light and darkness.

    PubMed

    Helmchen, C; Büttner, U

    1995-01-01

    Saccade-related Purkinje cells (PCs) were recorded in the oculomotor vermis (lobules VI, VII) during spontaneous eye movements and fast phases of optokinetic and vestibular nystagmus in the light and darkness, from two macaque monkeys. All neurons (n = 46) were spontaneously active and exhibited a saccade-related change of activity with all saccades and fast phases of nystagmus. Four types of neurons were found: most neurons (n = 31) exhibited a saccade-related burst of activity only (VBN); other units (n = 7) showed a burst of activity with a subsequent pause (VBPN); some of the units (n = 5) paused in relation to the saccadic eye movement (pause units, VPN); a few PCs (n = 3) showed a burst of activity in one direction and a pause of activity in the opposite direction. For all neurons, burst activity varied considerably for similar saccades. There were no activity differences between spontaneous saccades and vestibular or optokinetically elicited fast phases of nystagmus. The activity before, during, and after horizontal saccades was quantitatively analyzed. For 24 burst PCs (VBN, VBPN), the burst started before saccade onset in one horizontal direction (preferred direction), on average by 15.3 ms (range 27-5 ms). For all these neurons, burst activity started later in the opposite (non-preferred) direction, on average 4.9 ms (range 20 to -12 ms, P < 0.01) before saccade onset. The preferred direction could be either with ipsilateral (42% of neurons) or contralateral (58%) saccades. Nine burst PCs had similar latencies and burst patterns in both horizontal directions. The onset of burst activity of a minority of PCs (n = 5) lagged saccade onset in all directions. The pause for VBPN neurons started after the end of the saccade and reached a minimum of activity some 40-50 ms after saccade completion. For all saccades and quick phases of nystagmus, burst duration increased with saccade duration. Peak burst activity was not correlated with saccade amplitude or peak

  18. 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

  19. Administration of memantine during ethanol withdrawal in neonatal rats: effects on long-term ethanol-induced motor incoordination and cerebellar Purkinje cell loss

    PubMed Central

    Idrus, Nirelia M.; McGough, Nancy N.H.; Riley, Edward P.; Thomas, Jennifer D.

    2013-01-01

    Background Alcohol consumption during pregnancy can damage the developing fetus, illustrated by central nervous system dysfunction and deficits in motor and cognitive abilities. Binge drinking has been associated with an increased risk of fetal alcohol spectrum disorders, likely due to increased episodes of ethanol withdrawal. We hypothesized that overactivity of the N-methyl-D-aspartate (NMDA) receptor during ethanol withdrawal leads to excitotoxic cell death in the developing brain. Consistent with this, administration of NMDA receptor antagonists (e.g. MK-801) during withdrawal can attenuate ethanol's teratogenic effects. The aim of this study was to determine if administration of memantine, an NMDA receptor antagonist, during ethanol withdrawal could effectively attenuate ethanol-related deficits, without the adverse side effects associated with other NMDA receptor antagonists. Methods Sprague-Dawley pups were exposed to 6.0 g/kg ethanol or isocaloric maltose solution via intubation on postnatal day 6, a period of brain development equivalent to a portion of the 3rd trimester. Twenty-four and 36 hours after ethanol, subjects were injected with 0, 10 or 15 mg/kg memantine, totaling doses of 0, 20, or 30 mg/kg. Motor coordination was tested on a parallel bar task and the total number of cerebellar Purkinje cells was estimated using unbiased stereology. Results Alcohol exposure induced significant parallel bar motor incoordination and reduced Purkinje cell number. Memantine administration significantly attenuated both ethanol-associated motor deficits and cerebellar cell loss in a dose-dependent manner. Conclusions Memantine was neuroprotective when administered during ethanol withdrawal. These data provide further support that ethanol withdrawal contributes to fetal alcohol spectrum disorders. PMID:21070252

  20. 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. PMID:26093171

  1. Cocaine causes atrial Purkinje fiber damage.

    PubMed

    Gilloteaux, Jacques; Ekwedike, Nelson N

    2010-04-01

    Comparisons of atrial tissues from Syrian hamster offspring born from cocaine-treated mothers during the last days of pregnancy with sham-treated ones demonstrate irreversible focal ischemic damage in the Purkinje myofibers and minor endocardial damages as well as minute cardiomyocyte vacuolization. These defects are consistent with the pharmacotoxicity of cocaine or its metabolites. The damaged Purkinje myocytes apparently remain in contact with adjacent cardiomyocytes but undergo autolytic process similar to that found in autoschizic cell death. Adjacent cell type(s) appear to segregate or engulf the injured cells. Data collected in this report demonstrate why clinical bradyarrhythmias, arrhythmias, or sudden death as cardiac arrest can be found in pre- and postnatal cocaine-abused babies as well as those found in young individuals caused by acute or chronic cocaine abuse. PMID:20192706

  2. AMPA receptor-mediated alterations of intracellular calcium homeostasis in rat cerebellar Purkinje cells in vitro: correlates to dark cell degeneration.

    PubMed

    Strahlendorf, J C; Brandon, T; Miles, R; Strahlendorf, H K

    1998-11-01

    In the rat cerebellar slice preparation in vitro, excessive DL-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-receptor activation elicits a characteristic type of excitotoxicity of Purkinje cells (PCs) known as dark cell degeneration (DCD). DCD models neurotoxicity of PCs and hippocampal pyramidal neurons in vivo following hyperexcitable states. The intent of this study was to: a) determine whether AMPA-induced neurotoxicity of PCs is correlated with temporally and spatially restricted rises in intracellular Ca2+ and b) whether GYKI 52466 and nominal external Ca2+, conditions that reduced expression of AMPA-elicited DCD, altered the induced Ca2+ patterns. Employing the Ca2+-sensitive dye Fluo-3 and a confocal laser scanning microscope, we evaluated changes in intracellular Ca2+ within PCs in a cerebellar slice preparation. AMPA application alone (30 microM for 30 min) caused a significant initial rise in perinuclear and cytoplasmic Ca2+ that returned to control levels during the latter part of the AMPA exposure period. Following removal of AMPA (expression period), perinuclear and cytoplasmic Ca2+ displayed a significant delayed rise peaking transiently 60 min after AMPA removal. The efficacy of GYKI 52466 and nominal external Ca2+ conditions to attenuate AMPA-induced DCD was correlated to reductions in AMPA-induced transient elevations in perinuclear and cytoplasmic Ca2+ levels during the expression phase and to a lesser extent during the exposure period. The present data suggest that during the expression phase, the delayed perinuclear and cytoplasmic Ca2+ transient may be the harbinger of impending loss of Ca2+ homeostasis and cell damage. PMID:9814545

  3. 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

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

    PubMed

    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(-6)M) augmented TR-mediated transcription, but the transcription was suppressed at higher dose (10(-5) to 10(-4)M), 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(-9)M T4 was augmented by low-dose Gd-DTPA-BMA (10(-7)M) but was suppressed by higher dose (10(-5)M). Such augmentation by low-dose Gd-DTPA-BMA was not observed with 10(-9)M 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(-5)M) as seen in T4 treatment. The effect of Gd-DOTA on dendrite arborization was much weaker

  5. High frequency burst firing of granule cells ensures transmission at the parallel fiber to purkinje cell synapse at the cost of temporal coding.

    PubMed

    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

  6. The Importance of Purkinje Activation in Long Duration Ventricular Fibrillation

    PubMed Central

    Huang, Jian; Dosdall, Derek J.; Cheng, Kang‐An; Li, Li; Rogers, Jack M.; Ideker, Raymond E.

    2014-01-01

    Background The mechanisms that maintain long duration ventricular fibrillation (LDVF) are unclear. The difference in distribution of the Purkinje system in dogs and pigs was explored to determine if Purkinje activation propagates to stimulate working myocardium (WM) during LDVF and WM pacing. Methods and Results In‐vivo extracellular recordings were made from 1044 intramural plunge and epicardial plaque electrodes in 6 pig and 6 dog hearts. Sinus activation propagated sequentially from the endocardium to the epicardium in dogs but not pigs. During epicardial pacing, activation propagated along the endocardium and traversed the LV wall almost parallel to the epicardium in dogs, but in pigs propagated away from the pacing site approximately perpendicular to the epicardium. After 1 minute of VF, activation rate near the endocardium was significantly faster than near the epicardium in dogs (P<0.01) but not pigs (P>0.05). From 2 to 10 minutes of LDVF, recordings exhibiting Purkinje activations were near the endocardium in dogs (P<0.01) but were scattered transmurally in pigs, and the WM activation rate in recordings in which Purkinje activations were present was significantly faster than the WM activation rate in recordings in which Purkinje activations were absent (P<0.01). In 10 isolated perfused dog hearts, the LV endocardium was exposed and 2 microelectrodes were inserted into Purkinje and adjacent myocardial cells. After 5 minutes of LDVF, mean Purkinje activation rate was significantly faster than mean WM activation rate (P<0.01). Conclusion These extracellular and intracellular findings about activation support the hypothesis that Purkinje activation propagates to stimulate WM during sinus rhythm, pacing, and LDVF. PMID:24584738

  7. 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

  8. Cbln1 accumulates and colocalizes with Cbln3 and GluRδ2 at parallel fiber-Purkinje cell synapses in the mouse cerebellum

    PubMed Central

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

    2009-01-01

    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 GluRδ2-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 GluRδ2, 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 GluRδ2 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. PMID:19250438

  9. Activity-Dependent Gating of Calcium Spikes by A-type K+ Channels Controls Climbing Fiber Signaling in Purkinje Cell Dendrites

    PubMed Central

    Otsu, Yo; Marcaggi, Païkan; Feltz, Anne; Isope, Philippe; Kollo, Mihaly; Nusser, Zoltan; Mathieu, Benjamin; Kano, Masanobu; Tsujita, Mika; Sakimura, Kenji; Dieudonné, Stéphane

    2014-01-01

    Summary In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules. PMID:25220810

  10. Decreased argyrophilic nucleolar organiser region (AgNOR) expression in Purkinje cells: first signal of neuronal damage in sudden fetal and infant death

    PubMed Central

    Lavezzi, Anna M; Alfonsi, Graziella; Pusiol, Teresa; Matturri, Luigi

    2016-01-01

    Aims The nucleolus is an important cellular component involved in the biogenesis of the ribosome. This study was performed in order to validate the introduction of the argyrophilic nucleolar organiser region (AgNOR) stain technique, specific for the nucleoli detection, in neuropathological studies on sudden fetal and infant death. Methods In a wide set of fetuses and infants, aged from 27 gestational weeks to eight postnatal months and dead from both known and unknown causes, an in-depth neuropathological study usually applied at the Lino Rossi Research Center of the Milan University was implemented by the AgNOR method. Results Peculiar abnormalities of the nucleoli, as partial or total disruption above all in Purkinje cells (PCs), were exclusively found in victims of sudden fetal and infant death, and not in controls. The observed nucleolar alterations were frequently related to nicotine absorption in pregnancy. Conclusions We conclude that these findings represent early hallmarks of PC degeneration, contributing to the pathophysiology of sudden perinatal death. PMID:26567317

  11. Lgr4 protein deficiency induces ataxia-like phenotype in mice and impairs long term depression at cerebellar parallel fiber-Purkinje cell synapses.

    PubMed

    Guan, Xin; Duan, Yanhong; Zeng, Qingwen; Pan, Hongjie; Qian, Yu; Li, Dali; Cao, Xiaohua; Liu, Mingyao

    2014-09-19

    Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4(-/-) mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4(-/-) mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4(-/-) PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4(-/-) cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia. PMID:25063812

  12. Activity-dependent gating of calcium spikes by A-type K+ channels controls climbing fiber signaling in Purkinje cell dendrites.

    PubMed

    Otsu, Yo; Marcaggi, Païkan; Feltz, Anne; Isope, Philippe; Kollo, Mihaly; Nusser, Zoltan; Mathieu, Benjamin; Kano, Masanobu; Tsujita, Mika; Sakimura, Kenji; Dieudonné, Stéphane

    2014-10-01

    In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules. PMID:25220810

  13. Thyroid hormone triggers the developmental loss of axonal regenerative capacity via thyroid hormone receptor α1 and krüppel-like factor 9 in Purkinje cells

    PubMed Central

    Avci, Hasan X.; Lebrun, Clement; Wehrlé, Rosine; Doulazmi, Mohamed; Chatonnet, Fabrice; Morel, Marie-Pierre; Ema, Masatsugu; Vodjdani, Guilan; Sotelo, Constantino; Flamant, Frédéric; Dusart, Isabelle

    2012-01-01

    Neurons in the CNS of higher vertebrates lose their ability to regenerate their axons at a stage of development that coincides with peak circulating thyroid hormone (T3) levels. Here, we examined whether this peak in T3 is involved in the loss of axonal regenerative capacity in Purkinje cells (PCs). This event occurs at the end of the first postnatal week in mice. Using organotypic culture, we found that the loss of axon regenerative capacity was triggered prematurely by early exposure of mouse PCs to T3, whereas it was delayed in the absence of T3. Analysis of mutant mice showed that this effect was mainly mediated by the T3 receptor α1. Using gain- and loss-of-function approaches, we also showed that Krüppel-like factor 9 was a key mediator of this effect of T3. These results indicate that the sudden physiological increase in T3 during development is involved in the onset of the loss of axon regenerative capacity in PCs. This loss of regenerative capacity might be part of the general program triggered by T3 throughout the body, which adapts the animal to its postnatal environment. PMID:22891348

  14. Electrophysiological and Immunohistochemical Evidence for an Increase in GABAergic Inputs and HCN Channels in Purkinje Cells that Survive Developmental Ethanol Exposure.

    PubMed

    Light, Kim E; Hayar, Abdallah M; Pierce, Dwight R

    2015-08-01

    Ethanol exposures during the early postnatal period of the rat result in significant death of Purkinje cells (PCs). The magnitude, time-course, and lobular specificity of PC death have been well characterized in several studies. Additionally, significant reduction of climbing fiber inputs to the surviving PCs has been characterized. This study investigates whether further alterations to the cerebellar cortical circuits might occur as a result of developmental ethanol exposures. We first examined the firing pattern of PCs in acute slice preparations on postnatal days 13-15. While the basic firing frequency was not significantly altered, PCs from rat pups treated with ethanol on postnatal days 4-6 showed a significantly increased number of inhibitory postsynaptic potentials (IPSCs) and a larger Ih current. We conducted immunofluorescent studies to identify the probable cause of the increased IPSCs. We found a significant 21 % increase in the number of basket cells per PC and a near doubling of the volume of co-localized basket cell axonal membrane with PC. In addition, we identified a significant (~147 %) increase in HCN1 channel volume co-localized to PC volume. Therefore, the cerebellar cortex that survives targeted postnatal ethanol exposure is dramatically altered in development subsequent to PC death. The cerebellar cortical circuit that results is one that operates under a significant degree of increased resting inhibition. The alterations in the development of cerebellar circuitry following ethanol exposure, and the significant loss of PCs, could result in modifications of the structure and function of other brain regions that receive cerebellar inputs. PMID:25667035

  15. MAM-2201, a synthetic cannabinoid drug of abuse, suppresses the synaptic input to cerebellar Purkinje cells via activation of presynaptic CB1 receptors.

    PubMed

    Irie, Tomohiko; Kikura-Hanajiri, Ruri; Usami, Makoto; Uchiyama, Nahoko; Goda, Yukihiro; Sekino, Yuko

    2015-08-01

    Herbal products containing synthetic cannabinoids-initially sold as legal alternatives to marijuana-have become major drugs of abuse. Among the synthetic cannabinoids, [1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)-methanone (MAM-2201) has been recently detected in herbal products and has psychoactive and intoxicating effects in humans, suggesting that MAM-2201 alters brain function. Nevertheless, the pharmacological actions of MAM-2201 on cannabinoid receptor type 1 (CB1R) and neuronal functions have not been elucidated. We found that MAM-2201 acted as an agonist of human CB1Rs expressed in AtT-20 cells. In whole-cell patch-clamp recordings made from Purkinje cells (PCs) in slice preparations of the mouse cerebellum, we also found that MAM-2201 inhibited glutamate release at parallel fiber-PC synapses via activation of presynaptic CB1Rs. MAM-2201 inhibited neurotransmitter release with an inhibitory concentration 50% of 0.36 μM. MAM-2201 caused greater inhibition of neurotransmitter release than Δ(9)-tetrahydrocannabinol within the range of 0.1-30 μM and JWH-018, one of the most popular and potent synthetic cannabinoids detected in the herbal products, within the range of 0.03-3 μM. MAM-2201 caused a concentration-dependent suppression of GABA release onto PCs. Furthermore, MAM-2201 induced suppression of glutamate release at climbing fiber-PC synapses, leading to reduced dendritic Ca(2+) transients in PCs. These results suggest that MAM-2201 is likely to suppress neurotransmitter release at CB1R-expressing synapses in humans. The reduction of neurotransmitter release from CB1R-containing synapses could contribute to some of the symptoms of synthetic cannabinoid intoxication including impairments in cerebellum-dependent motor coordination and motor learning. PMID:25747605

  16. N-methyl-D-Aspartate Receptors Contribute to Complex Spike Signaling in Cerebellar Purkinje Cells: An In vivo Study in Mice

    PubMed Central

    Liu, Heng; Lan, Yan; Bing, Yan-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) are post-synaptically expressed at climbing fiber-Purkinje cell (CF-PC) synapses in cerebellar cortex in adult mice and contributed to CF-PC synaptic transmission under in vitro conditions. In this study, we investigated the role of NMDARs at CF-PC synapses during the spontaneous complex spike (CS) activity in cerebellar cortex in urethane-anesthetized mice, by in vivo whole-cell recording technique and pharmacological methods. Under current-clamp conditions, cerebellar surface application of NMDA (50 μM) induced an increase in the CS-evoked pause of simple spike (SS) firing accompanied with a decrease in the SS firing rate. Under voltage-clamp conditions, application of NMDA enhanced the waveform of CS-evoked inward currents, which expressed increases in the area under curve (AUC) and spikelet number of spontaneous CS. NMDA increased the AUC of spontaneous CS in a concentration-dependent manner. The EC50 of NMDA for increasing AUC of spontaneous CS was 33.4 μM. Moreover, NMDA significantly increased the amplitude, half-width and decay time of CS-evoked after-hyperpolarization (AHP) currents. Blockade of NMDARs with D-(-)-2-amino-5-phosphonopentanoic acid (D-APV, 250 μM) decreased the AUC, spikelet number, and amplitude of AHP currents. In addition, the NMDA-induced enhancement of CS activity could not be observed after α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors were blocked. The results indicated that NMDARs of CF-PC synapses contributed to the spontaneous CS activity by enhancing CS-evoked inward currents and AHP currents. PMID:27445699

  17. Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses

    PubMed Central

    Silver, R Angus; Momiyama, Akiko; Cull-Candy, Stuart G

    1998-01-01

    EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This ‘multiple-probability fluctuation’ (MPF) analysis gave an estimate of 510 ± 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 ± 0.03 nS (n = 6). Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mm Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 ± 0.01 nS and a CV of 0.37 ± 0.01 (n = 4). Similar estimates of q were obtained in 2 mm Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 μm) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (P¯r) was unusually high (0.90 ± 0.03 at 0.033 Hz

  18. The p75 Neurotrophin Receptor Can Induce Autophagy and Death of Cerebellar Purkinje Neurons

    PubMed Central

    Florez-McClure, Maria L.; Linseman, Daniel A.; Chu, Charleen T.; Barker, Phil A.; Bouchard, Ron J.; Le, Shoshona S.; Laessig, Tracey A.; Heidenreich, Kim A.

    2007-01-01

    The cellular mechanisms underlying Purkinje neuron death in various neurodegenerative disorders of the cerebellum are poorly understood. Here we investigate an in vitro model of cerebellar neuronal death. We report that cerebellar Purkinje neurons, deprived of trophic factors, die by a form of programmed cell death distinct from the apoptotic death of neighboring granule neurons. Purkinje neuron death was characterized by excessive autophagic–lysosomal vacuolation. Autophagy and death of Purkinje neurons were inhibited by nerve growth factor (NGF) and were activated by NGF-neutralizing antibodies. Although treatment with antisense oligonucleotides to the p75 neurotrophin receptor (p75ntr) decreased basal survival of cultured cerebellar neurons, p75ntr-antisense decreased autophagy and completely inhibited death of Purkinje neurons induced by trophic factor withdrawal. Moreover, adenoviral expression of a p75ntr mutant lacking the ligand-binding domain induced vacuolation and death of Purkinje neurons. These results suggest that p75ntr is required for Purkinje neuron survival in the presence of trophic support; however, during trophic factor withdrawal, p75ntr contributes to Purkinje neuron autophagy and death. The autophagic morphology resembles that found in neurodegenerative disorders, suggesting a potential role for this pathway in neurological disease. PMID:15140920

  19. Brain-derived neurotrophic factor induces post-lesion transcommissural growth of olivary axons that develop normal climbing fibers on mature Purkinje cells.

    PubMed

    Dixon, Kirsty J; Sherrard, Rachel M

    2006-11-01

    In the adult mammalian central nervous system, reinnervation and recovery from trauma is limited. During development, however, post-lesion plasticity may generate alternate paths providing models to investigate factors that promote reinnervation to appropriate targets. Following unilateral transection of the neonatal rat olivocerebellar pathway, axons from the remaining inferior olive reinnervate the denervated hemicerebellum and develop climbing fiber arbors on Purkinje cells. However, the capacity to recreate this accurate target reinnervation in a mature system remains unknown. In rats lesioned on day 15 (P15) or 30 and treated with intracerebellar injection of brain-derived neurotrophic factor (BDNF) or vehicle 24 h later, the morphology and organisation of transcommissural olivocerebellar reinnervation was examined using neuronal tracing and immunohistochemistry. In all animals BDNF, but not vehicle, induced transcommissural olivocerebellar axonal growth into the denervated hemicerebellum. The distribution of reinnervating climbing fibers was not confined to the injection sites but extended throughout the denervated hemivermis and, less densely, up to 3.5 mm into the hemisphere. Transcommissural olivocerebellar axons were organised into parasagittal microzones that were almost symmetrical to those in the right hemicerebellum. Reinnervating climbing fiber arbors were predominantly normal, but in the P30-lesioned group 10% were either branched within the molecular layer forming a smaller secondary arbor or were less branched, and in the P15 lesion group the reinnervating arbors extended their terminals almost to the pial surface and were larger than control arbors (P < 0.02). These results show that BDNF can induce transcommissural olivocerebellar reinnervation, which resembles developmental neuroplasticity to promote appropriate target reinnervation in a mature environment. PMID:16790241

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

    PubMed

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

    2016-02-23

    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

  1. Loss of MyD88 alters neuroinflammatory response and attenuates early Purkinje cell loss in a spinocerebellar ataxia type 6 mouse model

    PubMed Central

    Aikawa, Tomonori; Mogushi, Kaoru; Iijima-Tsutsui, Kumiko; Ishikawa, Kinya; Sakurai, Miyano; Tanaka, Hiroshi; Mizusawa, Hidehiro; Watase, Kei

    2015-01-01

    Spinocerebellar ataxia type 6 (SCA6) is dominantly inherited neurodegenerative disease, caused by an expansion of CAG repeat encoding a polyglutamine (PolyQ) tract in the Cav2.1 voltage-gated calcium channel. Its key pathological features include selective degeneration of the cerebellar Purkinje cells (PCs), a common target for PolyQ-induced toxicity in various SCAs. Mutant Cav2.1 confers toxicity primarily through a toxic gain-of-function mechanism; however, its molecular basis remains elusive. Here, we studied the cerebellar gene expression patterns of young Sca6-MPI118Q/118Q knockin (KI) mice, which expressed mutant Cav2.1 from an endogenous locus and recapitulated many phenotypic features of human SCA6. Transcriptional signatures in the MPI118Q/118Q mice were distinct from those in the Sca1154Q/2Q mice, a faithful SCA1 KI mouse model. Temporal expression profiles of the candidate genes revealed that the up-regulation of genes associated with microglial activation was initiated before PC degeneration and was augmented as the disease progressed. Histological analysis of the MPI118Q/118Q cerebellum showed the predominance of M1-like pro-inflammatory microglia and it was concomitant with elevated expression levels of tumor necrosis factor, interleukin-6, Toll-like receptor (TLR) 2 and 7. Genetic ablation of MyD88, a major adaptor protein conveying TLR signaling, altered expression patterns of M1/M2 microglial phenotypic markers in the MPI118Q/118Q cerebellum. More importantly, it ameliorated PC loss and partially rescued motor impairments in the early disease phase. These results suggest that early neuroinflammatory response may play an important role in the pathogenesis of SCA6 and its modulation could pave the way for slowing the disease progression during the early stage of the disease. PMID:26034136

  2. 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

  3. Propofol facilitates excitatory inputs of cerebellar Purkinje cells by depressing molecular layer interneuron activity during sensory information processing in vivo in mice.

    PubMed

    He, Yuan-Yuan; Jin, Ri; Jin, Wen-Zhe; Liu, Heng; Chu, Chun-Ping; Qiu, De-Lai

    2015-10-21

    Propofol is a rapid-acting sedative-hypnotic medication that has been widely used for the induction and maintenance of anesthesia; it has specific actions on different areas of the brain, such as sensory information transmission in the somatosensory cortex. However, the effects of propofol on the properties of sensory stimulation-evoked responses in cerebellar Purkinje cells (PCs) are currently unclear. In the present study, we studied the effects of propofol on facial stimulation-evoked responses in cerebellar PCs and molecular level interneurons (MLIs) in urethane-anesthetized mice using electrophysiological and pharmacological methods. Our results showed that cerebellar surface perfusion with propofol induced a decrease in the amplitude of the gamma-aminobutyric acid (GABA)-ergic component (P1) in a dose-dependent manner, but induced a significant increase in the amplitude of the excitatory response (N1). The IC50 of propofol-induced inhibition of P1 was 217.3 μM. In contrast, propofol (100 μM) depressed the spontaneous activity and tactile-evoked responses in MLIs. In addition, blocking GABA(A) receptor activity abolished the propofol (300 μM)-induced inhibition of the tactile-evoked inhibitory response and the increase in the sensory stimulation-evoked spike firing rate of PCs. These results indicated that propofol depressed the tactile stimulation-evoked spike firing of MLIs, resulting in a decrease in the amplitude of the tactile-evoked inhibitory response and an increase in the amplitude of the excitatory response in the cerebellar PCs of mice. Our results suggest that propofol modulates sensory information processing in cerebellar cortical PCs and MLIs through the activation of GABA(A) receptors. PMID:26317477

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

    PubMed Central

    Forrest, Michael D.

    2014-01-01

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

  5. 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.

  6. 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. PMID:24737450

  7. Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons.

    PubMed

    Meera, Pratap; Dodson, Paul D; Karakossian, Movses H; Otis, Thomas S

    2005-11-01

    Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ. PMID:16212990

  8. [Jan Evangelista Purkinje (1787-1869)].

    PubMed

    Kristiansen, K

    1993-12-10

    The author reviews the life and work of the physiologist Jan Evangelista Purkinje. In addition to his remarkable achievements as a scientist, Purkinje played a distinctive role in the struggle to establish national and cultural independence for the Czech people. PMID:8278954

  9. 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.

  10. Transcapillary Trafficking of Clustered Circulating Tumor Cells

    NASA Astrophysics Data System (ADS)

    Storey, Brian; Au, Sam; Chen, Yeng-Long; Sarioglu, Fatih; Javaid, Sarah; Haber, Daniel; Maheswaran, Shyamala; Stott, Shannon; Toner, Mehmet

    2015-11-01

    Aggregates of circulating tumor cells (CTC-clusters) are known to be more metastatic than equal numbers of singlet circulating tumor cells. Yet the mechanisms responsible for CTC-cluster dissemination and tumor seeding are still largely unknown. Without direct experimental evidence, it was assumed that because of their size, CTC-clusters would occlude and rupture capillaries. In this work, we have challenged this assumption by investigating the transit of CTC-clusters through microfluidic capillary constrictions under physiological pressures. Remarkably, cancer cell aggregates containing 2-20 cells were observed to successfully traverse constrictions 5-10 microns with over 90% efficiency. Clusters rapidly and reversibly reorganized into chain-like geometries to pass through constrictions in single file. This observation was verified by computational simulation of clusters modeled with physiological cell-cell interaction energies. Hydrodynamic analysis suggested that CTC-clusters were able to pass narrow constrictions by acting as individual cells in series, not as cohesive units. Upon exiting constrictions, clusters remained viable, proliferative and rapidly returned to `typical' cluster morphologies.

  11. Multiplexed coding by cerebellar Purkinje neurons

    PubMed Central

    Hong, Sungho; Negrello, Mario; Junker, Marc; Smilgin, Aleksandra; Thier, Peter; De Schutter, Erik

    2016-01-01

    Purkinje cells (PC), the sole output neurons of the cerebellar cortex, encode sensorimotor information, but how they do it remains a matter of debate. Here we show that PCs use a multiplexed spike code. Synchrony/spike time and firing rate encode different information in behaving monkeys during saccadic eye motion tasks. Using the local field potential (LFP) as a probe of local network activity, we found that infrequent pause spikes, which initiated or terminated intermittent pauses in simple spike trains, provide a temporally reliable signal for eye motion onset, with strong phase-coupling to the β/γ band LFP. Concurrently, regularly firing, non-pause spikes were weakly correlated with the LFP, but were crucial to linear encoding of eye movement kinematics by firing rate. Therefore, PC spike trains can simultaneously convey information necessary to achieve precision in both timing and continuous control of motion. DOI: http://dx.doi.org/10.7554/eLife.13810.001 PMID:27458803

  12. Cell division, differentiation and dynamic clustering

    NASA Astrophysics Data System (ADS)

    Kaneko, Kunihiko; Yomo, Tetsuya

    1994-08-01

    A novel mechanism for cell differentiation is proposed, based on the dynamic clustering in a globally coupled nonlinear system. A simple model with metabolic reaction, active transport of chemicals from media, and cell division is found to show three successive stages with the growth of the number of cells; coherent growth, dynamic clustering, and fixed cell differentiation. At the last stage, disparity in activities, germ line segregation, somatic cell differentiation, and homeochaotic stability against external perturbation are found. Our results, providing a simple interpretation of the experiments of the preceding paper, imply that cell differentiation can occur without a spatial pattern. From dynamical systems viewpoint, the new concept of “open chaos” is proposed, as a novel and general scenario for systems with growing numbers of elements, also seen in economics and sociology.

  13. 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.

  14. 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

  15. 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

  16. Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

    PubMed Central

    Katayama, E; Funahashi, H; Michikawa, T; Shiraishi, T; Ikemoto, T; Iino, M; Mikoshiba, K

    1996-01-01

    We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule. Images PMID:8890158

  17. 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

  18. Development of an anatomical technique for visualizing the mode of climbing fiber innervation in Purkinje cells and its application to mutant mice lacking GluRδ2 and Ca(v)2.1.

    PubMed

    Miyazaki, Taisuke; Watanabe, Masahiko

    2011-03-01

    In the adult cerebellum, a single climbing fiber (CF) innervates proximal dendrites of Purkinje cells (PCs). This monoinnervation is established by the developmental elimination of surplus CFs through homosynaptic competition among multiply innervating CFs and heterosynaptic competition between CFs and parallel fibers, i.e., granule cell axons innervating distal PC dendrites. Although the developmental process of CF monoinnervation and defects in it in mutant and experimental animal models have been extensively studied by electrophysiological techniques, for quite some time this subject was poorly understood from a morphological perspective due to a lack of neuroanatomical methods that could distinguish CFs with different neuronal origins. Soon after the identification of type 2 vesicular glutamate transporter (VGluT2) that selectively detects CF terminals in the molecular layer, we developed a novel method of combined anterograde tracer labeling and VGluT2 immunohistochemistry. This method enables us to identify the mode (mono vs. multiple) of CF innervation and the site of multiple innervation. Since then, we have applied this method to various kinds of gene-manipulated mice manifesting ataxia and other cerebellar phenotypes. In this review, we summarize experimental procedures for the combined tracer/VGluT2 labeling method, and then introduce what we have learned by applying this method in studies on the role of GluRδ2 and Ca(v)2.1 in CF monoinnervation. This method has provided informative anatomical correlates to electrophysiological data and vice versa, and will extend our knowledge of the molecular and cellular mechanisms for the development, plasticity, degeneration, and repair of the CF-PC projection system. PMID:21153457

  19. Intracellular FGF14 (iFGF14) Is Required for Spontaneous and Evoked Firing in Cerebellar Purkinje Neurons and for Motor Coordination and Balance.

    PubMed

    Bosch, Marie K; Carrasquillo, Yarimar; Ransdell, Joseph L; Kanakamedala, Ajay; Ornitz, David M; Nerbonne, Jeanne M

    2015-04-29

    Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia (SCA27). In addition, mice lacking Fgf14 (Fgf14(-/-)) exhibit an ataxia phenotype resembling SCA27, accompanied by marked changes in the excitability of cerebellar granule and Purkinje neurons. It is not known, however, whether these phenotypes result from defects in neuronal development or if they reflect a physiological requirement for iFGF14 in the adult cerebellum. Here, we demonstrate that the acute and selective Fgf14-targeted short hairpin RNA (shRNA)-mediated in vivo "knock-down" of iFGF14 in adult Purkinje neurons attenuates spontaneous and evoked action potential firing without measurably affecting the expression or localization of voltage-gated Na(+) (Nav) channels at Purkinje neuron axon initial segments. The selective shRNA-mediated in vivo "knock-down" of iFGF14 in adult Purkinje neurons also impairs motor coordination and balance. Repetitive firing can be restored in Fgf14-targeted shRNA-expressing Purkinje neurons, as well as in Fgf14(-/-) Purkinje neurons, by prior membrane hyperpolarization, suggesting that the iFGF14-mediated regulation of the excitability of mature Purkinje neurons depends on membrane potential. Further experiments revealed that the loss of iFGF14 results in a marked hyperpolarizing shift in the voltage dependence of steady-state inactivation of the Nav currents in adult Purkinje neurons. We also show here that expressing iFGF14 selectively in adult Fgf14(-/-) Purkinje neurons rescues spontaneous firing and improves motor performance. Together, these results demonstrate that iFGF14 is required for spontaneous and evoked action potential firing in adult Purkinje neurons, thereby controlling the output of these cells and the regulation of motor coordination and balance. PMID:25926453

  20. 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. PMID:26374457

  1. 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. PMID:21753036

  2. 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.

  3. 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.

  4. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    PubMed

    Cima, Igor; Kong, Say Li; Sengupta, Debarka; Tan, Iain B; Phyo, Wai Min; Lee, Daniel; Hu, Min; Iliescu, Ciprian; Alexander, Irina; Goh, Wei Lin; Rahmani, Mehran; Suhaimi, Nur-Afidah Mohamed; Vo, Jess H; Tai, Joyce A; Tan, Joanna H; Chua, Clarinda; Ten, Rachel; Lim, Wan Jun; Chew, Min Hoe; Hauser, Charlotte A E; van Dam, Rob M; Lim, Wei-Yen; Prabhakar, Shyam; Lim, Bing; Koh, Poh Koon; Robson, Paul; Ying, Jackie Y; Hillmer, Axel M; Tan, Min-Han

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease. PMID:27358499

  5. Analysis of Phosphatidylinositol-4,5-Bisphosphate Signaling in Cerebellar Purkinje Spines

    PubMed Central

    Brown, Sherry-Ann; Morgan, Frank; Watras, James; Loew, Leslie M.

    2008-01-01

    A 3D model was developed and used to explore dynamics of phosphatidylinositol-4,5-bisphosphate (PIP2) signaling in cerebellar Purkinje neurons. Long-term depression in Purkinje neurons depends on coincidence detection of climbing fiber stimulus evoking extracellular calcium flux into the cell and parallel fiber stimulus evoking inositol-1,4,5-trisphosphate (IP3)-meditated calcium release from the endoplasmic reticulum. Experimental evidence shows that large concentrations of IP3 are required for calcium release. This study uses computational analysis to explore how the Purkinje cell provides sufficient PIP2 to produce large amounts of IP3. Results indicate that baseline PIP2 concentration levels in the plasma membrane are inadequate, even if the model allows for PIP2 replenishment by lateral diffusion from neighboring dendrite membrane. Lateral diffusion analysis indicates apparent anomalous diffusion of PIP2 in the spiny dendrite membrane, due to restricted diffusion through spine necks. Stimulated PIP2 synthesis and elevated spine PIP2 mediated by a local sequestering protein were explored as candidate mechanisms to supply sufficient PIP2. Stimulated synthesis can indeed lead to high IP3 amplitude of long duration; local sequestration produces high IP3 amplitude, but of short duration. Simulation results indicate that local sequestration could explain the experimentally observed finely tuned timing between parallel fiber and climbing fiber activation. PMID:18487300

  6. 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

  7. 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

    2016-05-01

    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

  8. 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

  9. Essential Tremor: A Common Disorder of Purkinje Neurons?

    PubMed

    Louis, Elan D

    2016-04-01

    Essential tremor (ET) is one of the most common neurological diseases, with an estimated 7 million affected individuals in the United States. Postmortem studies in the past few years have resulted in new knowledge as well as a new formulation of disease pathophysiology. This new formulation centers on the notion that ET might be a disease of the cerebellum and, more specifically, the Purkinje cell (PC) population. Indeed, several investigators have proposed that ET may be a "Purkinjopathy." Supporting this formulation are data from controlled postmortem studies demonstrating (1) a range of morphological changes in the PC axon, (2) abnormalities in the position and orientation of PC bodies, (3) reduction in the number of PCs in some studies, (4) morphological changes in and pruning of the PC dendritic arbor with loss of dendritic spines, and (5) alterations in both the PC-basket cell interface and the PC-climbing fiber interface in ET cases. This new formulation has engendered some controversy and raised additional questions. Whether the constellation of changes observed in ET differs from that seen in other degenerative disorders of the cerebellum remains to be determined, although initial studies suggest the likely presence of a distinct profile of changes in ET. PMID:26044399

  10. Dynamic maintenance of stochastic molecular clusters on cell membranes

    NASA Astrophysics Data System (ADS)

    Mugler, Andrew; Wehrens, Martijn; Ten Wolde, Pieter Rein

    2015-03-01

    Clustering of molecules on cell membranes is a widely observed phenomenon. A key example is the oncoprotein Ras. Maintenance of Ras clusters has been linked to proper Ras signaling. Yet, the mechanism by which Ras clusters are maintained remains unclear. Recently it was discovered that activated Ras promotes further Ras activation. We show using particle-based simulation that this positive feedback link is sufficient to produce persistent clusters of active Ras molecules via a dynamic nucleation mechanism. The cluster statistics are consistent with experimental observations. Interestingly, our model does not support a Turing regime of macroscopic reaction-diffusion patterning. This means that the clustering we observe is a purely stochastic effect, arising from the coupling of the positive feedback network with the discrete nature of individual molecules. These findings underscore the importance of stochastic and dynamic properties of reaction diffusion systems for biological behavior.

  11. 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.

  12. 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.

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

    PubMed

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

    2014-11-01

    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

  14. 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

  15. 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

  16. The neglect of Purkinje's technique of ophthalmoscopy prior to Helmholtz's invention of the ophthalmoscope.

    PubMed

    Reese, P D

    1986-11-01

    A technique for examining the fundus of the eye was devised by Jan Evangelista Purkinje a generation before Helmholtz invented the ophthalmoscope. Yet, Purkinje's technique of ophthalmoscopy went virtually unnoticed by his contemporaries. This neglect of Purkinje's discovery has never been fully explained and warrants re-examination. PMID:3543788

  17. [Purkinje images in slit lamp videography : Video article].

    PubMed

    Gellrich, M-M; Kandzia, C

    2016-09-01

    Reflexes that accompany every examination with the slit lamp are usually regarded as annoying and therefore do not receive much attention. In the video available online, clinical information "hidden" in the Purkinje images is analyzed according to our concept of slit lamp videography. In the first part of the video, the four Purkinje images which are reflections on the eye's optical surfaces are introduced for the phakic eye. In the pseudophakic eye, however, the refracting surfaces of the intraocular lens (IOL) have excellent optical properties and therefore form Purkinje images 3 and 4 of high quality. Especially the third Purkinje image from the anterior IOL surface, which is usually hardly visible in the phakic eye can be detected deep in the vitreous, enlarged through the eye's own optics like a magnifying glass. Its area of reflection can be used to visualize changes of the anterior segment at high contrast. The third Purkinje image carries valuable information about the anterior curvature and, thus, about the power of the IOL. If the same IOL type is implanted in a patient, often a difference between right and left of 0.5 diopter in its power can be detected by the difference in size of the respective third Purkinje image. In a historical excursion to the "prenatal phase" of the slit lamp in Uppsala, we show that our most important instrument in clinical work was originally designed for catoptric investigations (of specular reflections). Accordingly A. Gullstrand called it an ophthalmometric Nernst lamp. PMID:27558688

  18. Gold cluster/titanium dioxide heterojunction photovoltaic cell

    NASA Astrophysics Data System (ADS)

    Nakamura, Eiji; Kogo, Atsushi; Sakai, Nobuyuki; Tatsuma, Tetsu

    2014-08-01

    Metal clusters have recently been applied as photosensitizers to wet-type photovoltaic cells. However, there are some practical issues including instability of the clusters in a liquid phase and leakage of electrolyte. To address these issues, we fabricated a heterojunction photovoltaic cell with solid-state layers of glutathione-protected Au25 clusters and titanium dioxide (TiO2). The ITO/TiO2/Au25/Au cell responded to visible and near infrared light even at 900 nm. Short-circuit photocurrent was ˜14 μA cm-2 and open-circuit photovoltage was 0.53 V under a xenon lamp (>480 nm, 75 mW cm-2).

  19. Tissue plasminogen activator regulates Purkinje neuron development and survival

    PubMed Central

    Li, Jianxue; Yu, Lili; Gu, Xuesong; Ma, Yinghua; Pasqualini, Renata; Arap, Wadih; Snyder, Evan Y.; Sidman, Richard L.

    2013-01-01

    The cerebellar cortex is centrally involved in motor coordination and learning, and its sole output is provided by Purkinje neurons (PNs). Growth of PN dendrites and their major synaptic input from granule cell parallel fiber axons takes place almost entirely in the first several postnatal weeks. PNs are more vulnerable to cell death than most other neurons, but the mechanisms remain unclear. We find that the homozygous nervous (nr) mutant mouse’s 10-fold–increased cerebellar tissue plasminogen activator (tPA), a part of the tPA/plasmin proteolytic system, influences several different molecular mechanisms, each regulating a key aspect of postnatal PN development, followed by selective PN necrosis, as follows. (i) Excess endogenous or exogenous tPA inhibits dendritic growth in vivo and in vitro by activating protein kinase Cγ and phosphorylation of microtubule-associated protein 2. (ii) tPA/plasmin proteolysis impairs parallel fiber-PN synaptogenesis by blocking brain-derived neurotrophic factor/tyrosine kinase receptor B signaling. (iii) Voltage-dependent anion channel 1 (a mitochondrial and plasma membrane protein) bound with kringle 5 (a peptide derived from the excess plasminogen) promotes pathological enlargement and rounding of PN mitochondria, reduces mitochondrial membrane potential, and damages plasma membranes. These abnormalities culminate in young nr PN necrosis that can be mimicked in wild-type PNs by exogenous tPA injection into cerebellum or prevented by endogenous tPA deletion in nr:tPA-knockout double mutants. In sum, excess tPA/plasmin, through separate downstream molecular mechanisms, regulates postnatal PN dendritogenesis, synaptogenesis, mitochondrial structure and function, and selective PN viability. PMID:23674688

  20. Endocrine Cell Clustering During Human Pancreas Development

    PubMed Central

    Jeon, Jongmin; Correa-Medina, Mayrin; Ricordi, Camillo; Edlund, Helena; Diez, Juan A.

    2009-01-01

    The development of efficient, reproducible protocols for directed in vitro differentiation of human embryonic stem (hES) cells into insulin-producing β cells will benefit greatly from increased knowledge regarding the spatiotemporal expression profile of key instructive factors involved in human endocrine cell generation. Human fetal pancreases 7 to 21 weeks of gestational age, were collected following consent immediately after pregnancy termination and processed for immunostaining, in situ hybridization, and real-time RT-PCR expression analyses. Islet-like structures appear from approximately week 12 and, unlike the mixed architecture observed in adult islets, fetal islets are initially formed predominantly by aggregated insulin- or glucagon-expressing cells. The period studied (7–22 weeks) coincides with a decrease in the proliferation and an increase in the differentiation of the progenitor cells, the initiation of NGN3 expression, and the appearance of differentiated endocrine cells. The present study provides a detailed characterization of islet formation and expression profiles of key intrinsic and extrinsic factors during human pancreas development. This information is beneficial for the development of efficient protocols that will allow guided in vitro differentiation of hES cells into insulin-producing cells. (J Histochem Cytochem 57:811–824, 2009) PMID:19365093

  1. Characterization of the electrogenic sodium pump in cardiac Purkinje fibres

    PubMed Central

    Eisner, D. A.; Lederer, W. J.

    1980-01-01

    1. The Na pump is examined in sheep cardiac Purkinje fibres using a two micro-electrode voltage clamp technique. 2. After reducing the external K concentration, [K]o, to zero for 2 min or more, subsequent addition of an `activator cation' (known to activate the Na pump in other preparations) produces a transient increase of outward current. This outward current transient is abolished by 10-5 M-strophanthidin (cf. Gadsby & Cranefield, 1979a). 3. It is concluded that this transient increase of outward current is a result of a transient stimulation of the sodium pump by the raised [Na]i following exposure to 0-Ko. Although this current transient may reflect the activity of an electrogenic Na pump, it is difficult to use K as the activator cation to establish this point. This is due to the extracellular K depletion that occurs during Na pump reactivation and the subsequent change that this K depletion produces in the current—voltage relationship of the Purkinje fibre. 4. Rbo or Cso have been used instead of Ko to reactivate the Na pump when examining the transient increase of outward current. On adding either of these cations after exposing a preparation to a solution without such `activator cations', the outward current transient is relatively voltage independent over a wide range of potentials (-90 to +10 mV). It is concluded that, following the addition of Rbo or Cso, the transient increase of outward current is a direct measure of the transient increase of the electrogenic Na pump current. 5. Increasing [Rb]o or [Cs]o over the range of 0-40 mM increases the rate of decay of the electrogenic Na pump current transient. Using a simple model (cf. Rang & Ritchie, 1968), it is shown that the decay rate constant of the electrogenic Na pump current transient is a good measure of the degree of activation of the external site of the Na pump. At a given concentration of activator cation, Rbo produces a greater activation of the Na pump than does Cso. The K0.5 for Rbo is 6

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

    PubMed

    Sablik, K

    1989-01-01

    The problem of Jan Evangelista Purkinje's succession will be presented according to the results of archival research. The Ministery of Cult and Education in Vienna, and especially Karl Rokitansky, who was the adviser for medical education, in 1867 created a new professorship and Institute for Physiology, beside Purkinje and his Institute. Maximilian Vintschgau was to assist the world-famous 80 years old Purkinje but was not permitted to teach the whole field of physiology and to examine students. The fact that the professors of the Prague Medical Faculty in 1868 started to remove the restrictions for Vintschgau with the argument of academic freedom and in 1869 tried to keep the second institute for the future, is not yet mentioned in the literature. Discussions about the problems of the Czech language and its use in physiological lectures were scarcely mentioned by the Ministery: if one day there should be a Czech-speaking lecturer, the problem would be solved. Unfortunately Purkinje had no genuine pupil in Prague, and after his death, Vintschgau was provisional director of the Institute for half a year. In this situation Rokitansky decided that there should only be one institute for physiology in Prague. The Medical Faculty wanted to have Hermann Helmholtz to succeed Purkinje, but Helmholtz refused to come. Ewald Hering, who was nominated in the second place by the Faculty, accepted the call. Vintschgau had only rank four, third was Conrad Eckhard from Giessen. The Ministery in Vienna, however, made a special decision: The Medical Faculty of Innsbruck was founded in 1869, and there was not professor for physiology at the beginning of 1870. The candidates of the Insbruck Medical Faculty were neglected in favour of Vintschgau, who was considered to be a trustworthy Austrian patriot. Hering and Vintschgau became professors on March 6, 1870, and Hering started his work in Prague in a new institute in the "Wenzelsbad". PMID:2529673

  3. Wnt proteins regulate acetylcholine receptor clustering in muscle cells

    PubMed Central

    2012-01-01

    Background The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. Results In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. Conclusions These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4. PMID:22309736

  4. Hippocampal cells encode places by forming small anatomical clusters

    PubMed Central

    Nakamura, Nozomu H.; Fukunaga, Masaki; Akama, Keith T.; Soga, Tomoko; Ogawa, Sonoko; Pavlides, Constantine

    2010-01-01

    The hippocampus has been hypothesized to function as a “spatial” or “cognitive” map, however, the functional cellular organization of the spatial map remains a mystery. The majority of electrophysiological studies, thus far, have supported the view of a random-type organization in the hippocampus. However, using immediate early genes (IEGs) as an indicator of neuronal activity, we recently observed a cluster-type organization of hippocampal principal cells, whereby a small number (~4) of nearby cells were activated in animals exposed to a restricted part of an environment. To determine the fine structure of these clusters and to provide a 3D image of active hippocampal cells that encode for different parts of an environment, we established a functional mapping of immediate early genes (IEGs) zif268 and Homer1a, using in situ hybridization and 3D-reconstruction imaging methods. We found that, in animals exposed to the same location twice, there were significantly more double IEG-expressing cells, and the clusters of nearby cells were more “tightly” formed, in comparison to animals exposed to two different locations. We propose that spatial encoding recruits specific cell ensembles in the hippocampus and that with repeated exposure to the same place the ensembles become better organized to more accurately represent the “spatial map”. PMID:20060034

  5. Hippocampal cells encode places by forming small anatomical clusters.

    PubMed

    Nakamura, N H; Fukunaga, M; Akama, K T; Soga, T; Ogawa, S; Pavlides, C

    2010-03-31

    The hippocampus has been hypothesized to function as a "spatial" or "cognitive" map, however, the functional cellular organization of the spatial map remains a mystery. The majority of electrophysiological studies, thus far, have supported the view of a random-type organization in the hippocampus. However, using immediate early genes (IEGs) as an indicator of neuronal activity, we recently observed a cluster-type organization of hippocampal principal cells, whereby a small number ( approximately 4) of nearby cells were activated in rats exposed to a restricted part of an environment. To determine the fine structure of these clusters and to provide a 3D image of active hippocampal cells that encode for different parts of an environment, we established a functional mapping of IEGs zif268 and Homer1a, using in situ hybridization and 3D-reconstruction imaging methods. We found that, in rats exposed to the same location twice, there were significantly more double IEG-expressing cells, and the clusters of nearby cells were more "tightly" formed, in comparison to rats exposed to two different locations. We propose that spatial encoding recruits specific cell ensembles in the hippocampus and that with repeated exposure to the same place the ensembles become better organized to more accurately represent the "spatial map." PMID:20060034

  6. The vicissitudes of the pacemaker current I (Kdd) of cardiac purkinje fibers.

    PubMed

    Vassalle, Mario

    2007-11-01

    The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I (Kdd). An alternative proposal is that the hyperpolarization-activated current I (f) underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K(+) depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I (K1). In turn, this decay would be responsible for a "fake" reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba(2+) was used to block the decay of I (K1). In the presence of Ba(2+) the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as -120 mV. In this regard, the distinct possibility needs to be considered that Ba(2+) had blocked I (Kdd) (and not only I (K1)). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba(2+). In the absence of Ba(2+), I (Kdd) was present in the pacemaker potential range and reversed at E (K). In the presence of Ba(2+), I (Kdd) was blocked and I (f) appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I (Kdd) but not with I (f). The fact that I (f) is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I (Kdd) (but not of I

  7. Membrane resistivity estimated for the Purkinje neuron by means of a passive computer model.

    PubMed

    Shelton, D P

    1985-01-01

    A multicompartment passive electrotonic computer model is constructed for the cerebellar Purkinje cell of the guinea-pig. The model has 1089 coupled compartments to accurately represent the morphology of the Purkinje cell. In order that the calculated behavior of the model fit the published electrophysiological observations of somatic and dendritic input conductance, the neural membrane resistivity must be spatially non-uniform. The passive electrical parameter values for which the model best fits the observations of input conductances, pulse attenuation and current-clamp voltage transients are rm,dend = 45,740 omega cm2, rm,soma = 760 omega cm2, ri = 225 omega cm and cm = 1.16 microF/cm2 (the membrane and cytoplasm specific resistivities and membrane specific capacitance, respectively). The model with these parameter values is electrically compact, with electrotonic length X = 0.33 and dendritic dominance ratio p = 0.44. Analysis of the calculated voltage transient of the multicompartment model by the methods of equivalent-cylinder cable theory is shown to result in very different and unreliable conclusions. The significance for neuronal function of the estimated electrical parameter values is discussed. The possible effect of active conductances on these conclusions is assessed. PMID:2579350

  8. Cytosolic PLA2(alpha) activation in Purkinje neurons and its role in AMPA-receptor trafficking.

    PubMed

    Mashimo, Masato; Hirabayashi, Tetsuya; Murayama, Toshihiko; Shimizu, Takao

    2008-09-15

    Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) selectively releases arachidonic acid from membrane phospholipids and has been proposed to be involved in the induction of long-term depression (LTD), a form of synaptic plasticity in the cerebellum. This enzyme requires two events for its full activation: Ca(2+)-dependent translocation from the cytosol to organelle membranes in order to access phospholipids as substrates, and phosphorylation by several kinases. However, the subcellular distribution and activation of cPLA(2)alpha in Purkinje cells and the role of arachidonic acid in cerebellar LTD have not been fully elucidated. In cultured Purkinje cells, stimulation of AMPA receptors, but not metabotropic glutamate receptors, triggered translocation of cPLA(2)alpha to the somatic and dendritic Golgi compartments. This translocation required Ca(2+) influx through P-type Ca(2+) channels. AMPA plus PMA, a chemical method for inducing LTD, released arachidonic acid via phosphorylation of cPLA(2)alpha. AMPA plus PMA induced a decrease in surface GluR2 for more than 2 hours. Interestingly, this reduction was occluded by a cPLA(2)alpha-specific inhibitor. Furthermore, PMA plus arachidonic acid caused the prolonged internalization of GluR2 without activating AMPA receptors. These results suggest that cPLA(2)alpha regulates the persistent decrease in the expression of AMPA receptors, underscoring the role of cPLA(2)alpha in cerebellar LTD. PMID:18713832

  9. Clustering effects on discontinuous gold film NanoCells.

    PubMed

    Seminario, Jorge M; Ma, Yuefei; Agapito, Luis A; Yan, Liuming; Araujo, Roy A; Bingi, Sridhar; Vadlamani, Nagendra S; Chagarlamudi, Krishna; Sudarshan, Tangali S; Myrick, Michael L; Colavita, Paula E; Franzon, Paul D; Nackashi, David P; Cheng, Long; Yao, Yuxing; Tour, James M

    2004-09-01

    Reproducible negative differential resistance (NDR)-like switching behavior is observed in NanoCells. This behavior is attributed to the formation of filaments and clusters between the discontinuous gold films. Control experiments are performed by self-assembly of insulating molecules between the gold islands and conducting molecules on these islands. Additional control experiments are performed by removing the filaments and clusters between islands using a piranha bath. The results are consistent with theoretical predictions and extend the domain of molecular electronics based in organic molecules to include nanosized clusters as active units. This facilitates a scenario where synthetically accessible organic molecules, with defined characteristics, can be adjusted by metallic nanoclusters as an in situ fine-tuning element, able to compensate for the lack of addressing in the nanosize regime. PMID:15570981

  10. 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. PMID:25984697

  11. 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

  12. Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis

    PubMed Central

    Durkin, Charlotte H.; Helaine, Sophie; Boucrot, Emmanuel

    2016-01-01

    Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S. Typhimurium delays epithelial cell turnover in the intestine. PMID:27185791

  13. Response of Purkinje neurons to hypobaric hypoxic exposure as shown by alteration in expression of glutamate receptors, nitric oxide synthases and calcium binding proteins.

    PubMed

    Kaur, C; Sivakumar, V; Singh, G; Singh, J; Ling, E A

    2005-01-01

    Hypobaric hypoxia is known to impair muscular coordination. It is not known whether hypobaric hypoxia causes any damage to the Purkinje neurons which may be responsible for impairment of muscular coordination. Expression of ionotropic glutamate receptors N-methyl-d-aspartate receptor subunit 1, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid GluR2/3, calcium binding proteins and nitric oxide synthases in the Purkinje neurons was examined in rats exposed to hypobaric hypoxia. The mRNA expression of N-methyl-d-aspartate receptor subunit 1, GluR2, GluR3 and nitric oxide synthases [neuronal, endothelial and inducible] was upregulated at 3 h peaking at 24 h after the exposure. This was sustained up to 3 days; thereafter, it was comparable to the controls. Immunohistochemical analysis confirmed a marked expression of N-methyl-d-aspartate receptor subunit 1 and GluR2/3 at the above time intervals. Immunoexpression of calbindin-D28k (calbindin) and parvalbumin was intense in the soma of Purkinje neurons in the control rats. It was, however, drastically downregulated up to 3 days after exposure. At 3 days the neuronal dendrites showed intense expression of calbindin which returned to control levels at 7 days. Expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was markedly upregulated from 3 h to 3 days whereas endothelial nitric oxide synthase expression, localized in the blood vessels and Purkinje neurons, remained elevated up to 24 h after the exposure. A progressive darkening of the Purkinje neuron cell bodies was observed at ultrastructural level up to 3 days but degenerating cells were not observed. A salient alteration was the dilation and stacking of smooth endoplasmic reticulum in the dendrites up to 14 days after the exposure. The present results suggest that hypobaric hypoxia leads to overexpression of N-methyl-d-aspartate receptor subunit 1 and GluR2/3 in Purkinje neurons that may be responsive to altered calcium levels as

  14. 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

  15. 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.

  16. Dendritic Cell-Nerve Clusters Are Sites of T Cell Proliferation in Allergic Airway Inflammation

    PubMed Central

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

    2009-01-01

    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. PMID:19179611

  17. Distinct Cell Clusters Touching Islet Cells Induce Islet Cell Replication in Association with Over-Expression of Regenerating Gene (REG) Protein in Fulminant Type 1 Diabetes

    PubMed Central

    Aida, Kaoru; Saitoh, Sei; Nishida, Yoriko; Yokota, Sadanori; Ohno, Shinichi; Mao, Xiayang; Akiyama, Daiichiro; Tanaka, Shoichiro; Awata, Takuya; Shimada, Akira; Oikawa, Youichi; Shimura, Hiroki; Furuya, Fumihiko; Takizawa, Soichi; Ichijo, Masashi; Ichijo, Sayaka; Itakura, Jun; Fujii, Hideki; Hashiguchi, Akinori; Takasawa, Shin; Endo, Toyoshi; Kobayashi, Tetsuro

    2014-01-01

    Background Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. Procedures The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. Result Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. Conclusion The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells. PMID:24759849

  18. CellPilot: Seamless communication within Cell BE and heterogeneous clusters

    NASA Astrophysics Data System (ADS)

    Girard, N.; Carter, J.; Gardner, W. B.; Grewal, G.

    2010-11-01

    The Pilot library is targeted to novice scientific programmers within High Performance Computing. The CellPilot library extends the Pilot library to the Cell Broadband Engine processor and heterogeneous clusters. Using Pilot's process and channel abstractions, the CellPilot library can create a process on any of the processor types, both PPEs and SPEs, across the cluster. Communication is achieved by creating a channel between any two processes, and using the write/read channel functions in the participating processes. The CellPilot library uses MPI for the inter-node communication and the Cell SDK within a Cell node. All the architecture specific details of Cell communications are hidden from the user.

  19. Variability of Action Potentials Within and Among Cardiac Cell Clusters Derived from Human Embryonic Stem Cells.

    PubMed

    Zhu, Renjun; Millrod, Michal A; Zambidis, Elias T; Tung, Leslie

    2016-01-01

    Electrophysiological variability in cardiomyocytes derived from pluripotent stem cells continues to be an impediment for their scientific and translational applications. We studied the variability of action potentials (APs) recorded from clusters of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using high-resolution optical mapping. Over 23,000 APs were analyzed through four parameters: APD30, APD80, triangulation and fractional repolarization. Although measures were taken to reduce variability due to cell culture conditions and rate-dependency of APs, we still observed significant variability in APs among and within the clusters. However, similar APs were found in spatial locations with close proximity, and in some clusters formed distinct regions having different AP characteristics that were reflected as separate peaks in the AP parameter distributions, suggesting multiple electrophysiological phenotypes. Using a recently developed automated method to group cells based on their entire AP shape, we identified distinct regions of different phenotypes within single clusters and common phenotypes across different clusters when separating APs into 2 or 3 subpopulations. The systematic analysis of the heterogeneity and potential phenotypes of large populations of hESC-CMs can be used to evaluate strategies to improve the quality of pluripotent stem cell-derived cardiomyocytes for use in diagnostic and therapeutic applications and in drug screening. PMID:26729331

  20. The role of the delayed rectifier component IKs in dog ventricular muscle and Purkinje fibre repolarization

    PubMed Central

    Varró, András; Baláti, Beáta; Iost, Norbert; Takács, János; Virág, László; Lathrop, David A; Csaba, Lengyel; Tálosi, László; Papp, Julius Gy

    2000-01-01

    The relative contributions of the rapid and slow components of the delayed rectifier potassium current (IKr and IKs, respectively) to dog cardiac action potential configuration were compared in ventricular myocytes and in multicellular right ventricular papillary muscle and Purkinje fibre preparations. Whole-cell patch-clamp techniques, conventional microelectrode and in vivo ECG measurements were made at 37°C. Action potential duration (APD) was minimally increased (less than 7%) by chromanol 293B (10 μM) and L-735,821 (100 nM), selective blockers of IKs, over a range of pacing cycle lengths (300–5000 ms) in both dog right ventricular papillary muscles and Purkinje fibre strands. D-Sotalol (30 μM) and E-4031 (1 μM), selective blockers of IKr, in the same preparations markedly (20–80%) lengthened APD in a reverse frequency-dependent manner. In vivo ECG recordings in intact anaesthetized dogs indicated no significant chromanol 293B (1 mg kg−1 i.v.) effect on the QTc interval (332.9 ± 16.1 ms before versus 330.5 ± 11.2 ms, n = 6, after chromanol 293B), while D-sotalol (1 mg kg−1 i.v.) significantly increased the QTc interval (323.9 ± 7.3 ms before versus 346.5 ± 6.4 ms, n = 5, after D-sotalol, P < 0.05). The current density estimated during the normal ventricular muscle action potential (i.e. after a 200 ms square pulse to +30 mV or during a 250 ms long ‘action potential-like’ test pulse) indicates that substantially more current is conducted through IKr channels than through IKs channels. However, if the duration of the square test pulse or the ‘action potential-like’ test pulse was lengthened to 500 ms the relative contribution of IKs significantly increased. When APD was pharmacologically prolonged in papillary muscle (1 μM E-4031 and 1 μg ml−1 veratrine), 100 nM L-735,821 and 10 μM chromanol 293B lengthened repolarization substantially by 14.4 ± 3.4 and 18.0 ± 3.4% (n = 8), respectively. We conclude that in this study IKs plays

  1. 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.

  2. Impaired hypercarbic and hypoxic responses from developmental loss of cerebellar Purkinje neurons: Implications for sudden infant death syndrome

    PubMed Central

    Calton, M.; Dickson, P.; Harper, R.M.; Goldowitz, D.; Mittleman, G.

    2014-01-01

    Impaired responsivity to hypercapnia or hypoxia is commonly considered a mechanism of failure in Sudden Infant Death Syndrome (SIDS). The search for deficient brain structures mediating flawed chemosensitivity typically focuses on medullary regions; however, a network that includes Purkinje cells of the cerebellar cortex and its associated cerebellar nuclei also helps mediate responses to CO2 and O2 challenges, and assists integration of cardiovascular and respiratory interactions. Although cerebellar nuclei contributions to chemoreceptor challenges in adult models are well described, Purkinje cell roles in developing models are unclear. We used a model of developmental cerebellar Purkinje cell loss to determine if such loss influenced compensatory ventilatory responses to hypercapnic and hypoxic challenges. Twenty-four Lurcher mutant mice and wildtype controls were sequentially exposed to 2% increases in CO2 (0%-8%), or 2% reductions in O2 (21%-13%) over four minutes, with return to room air (21% O2 / 79% N2 / 0% CO2) between each exposure. Whole-body plethysmography was used to continuously monitor tidal volume (TV) and breath frequency (f). Increased f to hypercapnia was significantly lower in Mutants, slower to initiate, and markedly lower in compensatory periods, except for very high (8%) CO2 levels. The magnitude of TV changes to increasing CO2 appeared smaller in Mutants, but only approached significance. Smaller, but significant differences emerged in response to hypoxia, with Mutants showing smaller TV when initially exposed to reduced O2, and lower f following exposure to 17% O2. Since cerebellar neuropathology appears in SIDS victims, developmental cerebellar neuropathology may contribute to SIDS vulnerability. PMID:25132500

  3. Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization.

    PubMed

    da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

    2013-02-15

    Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. PMID:23117111

  4. G-protein-independent modulation of P-type calcium channels by μ-opioids in Purkinje neurons of rat

    PubMed Central

    Iegorova, Olena; Fisyunov, Alexander; Krishtal, Oleg

    2010-01-01

    P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons since a major part of calcium entering pre-synaptic terminals is delivered via these channels. Using conventional whole-cell patch clamp techniques we have studied the effect of μ-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. The selective μ-opioid agonist DAMGO (10 nM) produced a small, but consistent facilitation of current through P-type calcium channels (10±1%, n=27, p<0.001). The effect of DAMGO was rapid (less than 10 sec) and fully reversible. This effect was both concentration and voltage-dependent. The EC50 for the effect of DAMGO was 1.3±0.4 nM and the saturating concentration was 100 nM. The endogenous selective agonist of μ-opioid receptors, endomorphin-1 demonstrated similar action. Intracellular perfusion of Purkinje neurons with GTPγS (0.5 mM) or GDPβS (0.5 mM), as well as strong depolarizing pre-pulses (+50 mV), did not eliminate facilitatory action of DAMGO on P-channels indicating that this effect is not mediated by G-proteins. Furthermore, the effect of DAMGO was preserved in the presence of a non-specific inhibitor of PKA and PKC, (H7, 10 μM) inside the cell. DAMGO–induced facilitation of P-current was almost completely abolished by the selective μ-opioid antagonist CTOP (100 nM). These observations indicate that μ-type opioid receptors modulate P-type calcium channels in Purkinje neurons via G-protein-independent mechanism. PMID:20541588

  5. Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

    PubMed Central

    Bosch, Marie K.; Nerbonne, Jeanne M.; Ornitz, David M.

    2014-01-01

    Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo. PMID:25093726

  6. Sox17-Mediated Maintenance of Fetal Intra-Aortic Hematopoietic Cell Clusters

    PubMed Central

    Osawa, Mitsujiro; Uemura, Mami; Kishikawa, Yoko; Anani, Maha; Harada, Kaho; Takagi, Haruna; Saito, Kiyoka; Kanai-Azuma, Masami; Kanai, Yoshiakira; Iwama, Atsushi

    2014-01-01

    During mouse development, definitive hematopoiesis is first detected around embryonic day 10.5 (E10.5) in the aorta-gonad-mesonephros (AGM) region, which exhibits intra-aortic cell clusters. These clusters are known to contain hematopoietic stem cells (HSCs). On the other hand, it is not clear how the cells in such clusters maintain their HSC phenotype and how they are triggered to differentiate. Here we found that an endodermal transcription factor marker, Sox17, and other F-group (SoxF) proteins, Sox7 and Sox18, were expressed in E10.5 intra-aortic cell clusters. Forced expression of any of these SoxF proteins, particularly Sox17, in E10.5 AGM CD45low c-Kithigh cells, which are the major component of intra-aortic clusters, led to consistent formation of cell clusters in vitro during several passages of cocultures with stromal cells. Cluster-forming cells with constitutive Sox17 expression retained long-term bone marrow reconstitution activity in vivo. Notably, shutdown of exogenously introduced Sox17 gene expression resulted in immediate hematopoietic differentiation. These results indicate that SoxF proteins, especially Sox17, contribute to the maintenance of cell clusters containing HSCs in the midgestation AGM region. Furthermore, SoxF proteins play a pivotal role in controlling the HSC fate decision between indefinite self-renewal and differentiation during fetal hematopoiesis. PMID:24662049

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

    PubMed

    Alexander, C J; Hammer, J A

    2016-01-01

    We present a simple and efficient method to knock down proteins specifically in Purkinje neurons (PN) present in mixed mouse primary cerebellar cultures. This method utilizes the introduction via nucleofection of a plasmid encoding a specific miRNA downstream of the L7/Pcp2 promoter, which drives PN-specific expression. As proof-of-principle, we used this plasmid to knock down the motor protein myosin Va, which is required for the targeting of smooth endoplasmic reticulum (ER) into PN spines. Consistent with effective knockdown, transfected PNs robustly phenocopied PNs from dilute-lethal (myosin Va-null) mice with regard to the ER targeting defect. Importantly, our plasmid-based approach is less challenging technically and more specific to PNs than several alternative methods (e.g., biolistic- and lentiviral-based introduction of siRNAs). We also present a number of improvements for generating mixed cerebellar cultures that shorten the procedure and improve the total yield of PNs, and of transfected PNs, considerably. Finally, we present a method to rescue cerebellar cultures that develop large cell aggregates, a common problem that otherwise precludes the further use of the culture. PMID:26794514

  8. 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

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

    PubMed Central

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

    2010-01-01

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

  10. Scanning electron microscopy of Purkinje fibres of the pig heart.

    PubMed

    Bytzer, P

    1979-01-01

    Scanning electron microscopy (SEM) of Purkinje fibres (P-fibres) from the septal walls and the septomarginal trabecula was performed on deparaffinized sections, the identification in SEM made possible by comparative light microscopy. The myofibrils in P-fibres from the septal walls were arranged in a cart-wheel fashion, whereas P-fibres from the septomarginal trabecula showed a nearly parallel alignment of the contractile material. Z-line ridges resembling the T-tubules of the myocardial fibres were observed in both kinds of P-fibres. The myofibrillar arrangements are discussed in relation to the expected mechanical stress put upon P-fibres in the 2 locations during systolic-diastolic activity. An adaptive function of the contractile material to the mechanical stress is suggested and the possible need of a T-tubular system is discussed. PMID:507370

  11. Dynamics of the calcium subsystem in cardiac Purkinje fibers

    NASA Astrophysics Data System (ADS)

    Varghese, A.; Winslow, R. L.

    1993-10-01

    A minimal model of the dynamics of internal calcium concentration of the mammalian cardiac Purkinje fiber is examined in order to identify the cause of certain arrhythmias of the heart. The effect of inhibition of the sodium/potassium pump is modeled by an elevated value of internal sodium cocentration. Effects of pump inhibition are examined at different clamp voltages. Such conditions mimic those which have been examined experimentally and which are known to cause oscillatory calcium release [W.J. Lederer, PhD thesis, Yale University, New Haven, CT (1976), 168 pp.; R.S. Kass, W.J. Lederer, R.W. Tsien and R. Weingart, J. Physiol. (London) 281 (1978) 187-208]. System dynamics are investigated using numerical continuation methods. Results of these analyses predict the existence of stable periodic oscillations of internal calcium over a range of voltage-clamp values. The emergence of these oscillations depends on the intracellular sodium concentration.

  12. 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.

  13. Alterations in the intrinsic electrophysiological properties of Purkinje neurons in a rat model of hepatic encephalopathy: Relative preventing effect of PPARγ agonist.

    PubMed

    Aghaei, Iraj; Hajali, Vahid; Dehpour, Ahmadreza; Haghani, Masoud; Sheibani, Vahid; Shabani, Mohammad

    2016-03-01

    Patients suffering from hepatic cirrhosis (HC) have been shown to have motor and cognitive impairments. The cerebellum, which controls coordinated and rapid movements, is a potential target for the deleterious effects of hyperammonemia induced by bile duct ligation. Therefore, the aim of this study was to determine the mechanisms of motor impairments observed in a rat model of HC and second objective of the current study was to evaluate the possible protective effect of pioglitazone (PIO) on these impairments. Male Wistar rats were used in the current study. Bile duct ligation (BDL) surgery was performed and pioglitazone administration was started two weeks after the surgery for the next four weeks. The effects of pioglitazone on BDL-induced electrophysiological changes of the Purkinje cerebellum neurons were evaluated by Whole-cell patch clamp recordings. Purkinje neurons from the BDL group exhibited significant changes in a number of electrophysiological properties and some alterations partially were counteracted by activation of peroxisome proliferator-activated receptor-γ. Purkinje cells from BDL groups showed a significant increase in the spontaneous firing frequency followed by a decrease in the action potential duration of half-amplitude and spike interval. Chronic administration of pioglitazone could contract this effect of BDL on event frequency and interevent interval, though the difference with the sham group was still significant in the duration of action potential. Results of the current study raise the possibility that BDL may profoundly affect the intrinsic membrane properties of the cerebellar Purkinje neurons and PIO administration can counteract some of these effects. PMID:26704786

  14. Cell-laden microengineered pullulan methacrylate hydrogels promote cell proliferation and 3D cluster formation.

    PubMed

    Bae, Hojae; Ahari, Amir F; Shin, Hyeongho; Nichol, Jason W; Hutson, Che B; Masaeli, Mahdokht; Kim, Su-Hwan; Aubin, Hug; Yamanlar, Seda; Khademhosseini, Ali

    2011-01-01

    The ability to encapsulate cells in three-dimensional (3D) environments is potentially of benefit for tissue engineering and regenerative medicine. In this paper, we introduce pullulan methacrylate (PulMA) as a promising hydrogel platform for creating cell-laden microscale tissues. The hydration and mechanical properties of PulMA were demonstrated to be tunable through modulation of the degree of methacrylation and gel concentration. Cells encapsulated in PulMA exhibited excellent viability. Interestingly, while cells did not elongate in PulMA hydrogels, cells proliferated and organized into clusters, the size of which could be controlled by the hydrogel composition. By mixing with gelatin methacrylate (GelMA), the biological properties of PulMA could be enhanced as demonstrated by cells readily attaching to, proliferating, and elongating within the PulMA/GelMA composite hydrogels. These data suggest that PulMA hydrogels could be useful for creating complex, cell-responsive microtissues, especially for applications that require controlled cell clustering and proliferation. PMID:21415929

  15. Cell-laden microengineered pullulan methacrylate hydrogels promote cell proliferation and 3D cluster formation

    PubMed Central

    Bae, Hojae; Ahari, Amir F.; Shin, Hyeongho; Nichol, Jason W.; Hutson, Che B.; Masaeli, Mahdokht; Kim, Su-Hwan; Aubin, Hug; Yamanlar, Seda; Khademhosseini, Ali

    2011-01-01

    The ability to encapsulate cells in three-dimensional (3D) environments is potentially of benefit for tissue engineering and regenerative medicine. In this paper, we introduce pullulan methacrylate (PulMA) as a promising hydrogel platform for creating cell-laden microscale tissues. The hydration and mechanical properties of PulMA were demonstrated to be tunable through modulation of the degree of methacrylation and gel concentration. Cells encapsulated in PulMA exhibited excellent viability. Interestingly, while cells did not elongate in PulMA hydrogels, cells proliferated and organized into clusters, the size of which could be controlled by the hydrogel composition. By mixing with gelatin methacrylate (GelMA), the biological properties of PulMA could be enhanced as demonstrated by cells readily attaching to, proliferating, and elongating within the PulMA/GelMA composite hydrogels. These data suggest that PulMA hydrogels could be useful for creating complex, cell-responsive microtissues, especially for applications that require controlled cell clustering and proliferation. PMID:21415929

  16. 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. PMID:25216490

  17. Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters.

    PubMed

    Cheung, Kevin J; Padmanaban, Veena; Silvestri, Vanesa; Schipper, Koen; Cohen, Joshua D; Fairchild, Amanda N; Gorin, Michael A; Verdone, James E; Pienta, Kenneth J; Bader, Joel S; Ewald, Andrew J

    2016-02-16

    Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14(+) cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14(+) epithelial tumor cell clusters disseminate collectively to colonize distant organs. PMID:26831077

  18. Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

    PubMed Central

    Cheung, Kevin J.; Padmanaban, Veena; Silvestri, Vanesa; Schipper, Koen; Cohen, Joshua D.; Fairchild, Amanda N.; Gorin, Michael A.; Verdone, James E.; Pienta, Kenneth J.; Bader, Joel S.; Ewald, Andrew J.

    2016-01-01

    Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14+ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14+ epithelial tumor cell clusters disseminate collectively to colonize distant organs. PMID:26831077

  19. 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. PMID:26630202

  20. The Role of Purkinje-Myocardial Coupling during Ventricular Arrhythmia: A Modeling Study

    PubMed Central

    Behradfar, Elham; Nygren, Anders; Vigmond, Edward J.

    2014-01-01

    The Purkinje system is the fast conduction network of the heart which couples to the myocardium at discrete sites called Purkinje-Myocyte Junctions (PMJs). However, the distribution and number of PMJs remains elusive, as does whether a particular PMJ is functional. We hypothesized that the Purkinje system plays a role during reentry and that the number of functional PMJs affect reentry dynamics. We used a computer finite element model of rabbit ventricles in which we varied the number of PMJs. Sustained, complex reentry was induced by applying an electric shock and the role of the Purkinje system in maintaining the arrhythmia was assessed by analyzing phase singularities, frequency of activation, and bidirectional propagation at PMJs. For larger junctional resistances, increasing PMJ density increased the mean firing rate in the Purkinje system, the percentage of successful retrograde conduction at PMJs, and the incidence of wave break on the epicardium. However, the mean firing of the ventricles was not affected. Furthermore, increasing PMJ density above 13/ did not alter reentry dynamics. For lower junctional resistances, the trend was not as clear. We conclude that Purkinje system topology affects reentry dynamics and conditions which alter PMJ density can alter reentry dynamics. PMID:24516576

  1. Clustering of neuronal sodium channels requires contact with myelinating Schwann cells.

    PubMed

    Ching, W; Zanazzi, G; Levinson, S R; Salzer, J L

    1999-01-01

    Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the alpha subunit of the sodium channel and against ankyrin(G), a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells. PMID:10739572

  2. Novel spider toxin slows down the activation kinetics of P-type Ca2+ channels in Purkinje neurons of rat.

    PubMed

    Fisyunov, Alexander; Pluzhnikov, Kirill; Molyavka, Anton; Grishin, Eugene; Lozovaya, Natalia; Krishtal, Oleg

    2005-02-01

    We have identified a novel polypeptide toxin (Lsp-1) from the venom of the spider Lycosa (LS). Its effect has been examined on the P-type calcium channels in Purkinje neurons, using whole-cell patch-clamp. This toxin (at saturating concentration 7 nM) produces prominent (four-fold) deceleration of the activation kinetics and partial (71+/-6%) decrease of the amplitude of P-current without affecting either deactivation or inactivation kinetics. These effects are not use-dependent. They are partially reversible within a minute upon the wash-out of the toxin. Intracellular perfusion of Purkinje neurons with 100 microM of GDP or 2 microM of GTPgammaS, as well as strong depolarising pre-pulses (+100 mV), do not eliminate the action of Lsp-1 on P-channels indicating that down-modulation via guanine nucleotide-binding proteins (G-proteins) is not involved in the observed phenomenon. In view of extremely high functional significance of P-channels, the toxin can be suggested as a useful pharmacological tool. PMID:15590128

  3. 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

  4. 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.

  5. Noise-induced dispersion and breakup of clusters in cell cycle dynamics

    PubMed Central

    Gong, Xue; Moses, Gregory; Neiman, Alexander B.; Young, Todd

    2014-01-01

    We study the effects of random perturbations on collective dynamics of a large ensemble of interacting cells in a model of the cell division cycle. We consider a parameter region for which the unperturbed model possesses asymptotically stable two-cluster periodic solutions. Two biologically motivated forms of random perturbations are considered: bounded variations in growth rate and asymmetric division. We compare the effects of these two dispersive mechanisms with additive Gaussian white noise perturbations. We observe three distinct phases of the response to noise in the model. First, for weak noise there is a linear relationship between the applied noise strength and the dispersion of the clusters. Second, for moderate noise strengths the clusters begin to mix, i.e. individual cells move between clusters, yet the population distribution clearly continues to maintain a two-cluster structure. Third, for strong noise the clusters are destroyed and the population is characterized by a uniform distribution. The second and third phases are separated by an order - disorder phase transition that has the characteristics of a Hopf bifurcation. Furthermore, we show that for the cell cycle model studied, the effects of bounded random perturbations are virtually indistinguishable from those induced by additive Gaussian noise, after appropriate scaling of the variance of noise strength. We then use the model to predict the strength of coupling among the cells from experimental data. In particular, we show that coupling must be rather strong to account for the observed clustering of cells given experimentally estimated noise variance. PMID:24694583

  6. Clustering of brain tumor cells: a first step for understanding tumor recurrence

    NASA Astrophysics Data System (ADS)

    Khain, Evgeniy; Nowicki, M. O.; Chiocca, E. A.; Lawler, S. E.; Schneider-Mizell, C. M.; Sander, L. M.

    2012-02-01

    Glioblastoma tumors are highly invasive; therefore the overall prognosis of patients remains poor, despite major improvements in treatment techniques. Cancer cells detach from the inner tumor core and actively migrate away [1]; eventually these invasive cells might form clusters, which can develop to recurrent tumors. In vitro experiments in collagen gel [1] followed the clustering dynamics of different glioma cell lines. Based on the experimental data, we formulated a stochastic model for cell dynamics, which identified two mechanisms of clustering. First, there is a critical value of the strength of adhesion; above the threshold, large clusters grow from a homogeneous suspension of cells; below it, the system remains homogeneous, similarly to the ordinary phase separation. Second, when cells form a cluster, there is evidence that their proliferation rate increases. We confirmed the theoretical predictions in a separate cell migration experiment on a substrate and found that both mechanisms are crucial for cluster formation and growth [2]. In addition to their medical importance, these phenomena present exciting examples of pattern formation and collective cell behavior in intrinsically non-equilibrium systems [3]. [4pt] [1] A. M. Stein et al, Biophys. J., 92, 356 (2007). [0pt] [2] E. Khain et al, EPL 88, 28006 (2009). [0pt] [3] E. Khain et al, Phys. Rev. E. 83, 031920 (2011).

  7. Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

    PubMed

    Maruyama, C L M; Leigh, N J; Nelson, J W; McCall, A D; Mellas, R E; Lei, P; Andreadis, S T; Baker, O J

    2015-11-01

    Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features. PMID:26285810

  8. 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

  9. Regulatory T Cells in Melanoma Revisited by a Computational Clustering of FOXP3+ T Cell Subpopulations

    PubMed Central

    Fujii, Hiroko; Josse, Julie; Tanioka, Miki; Miyachi, Yoshiki; Husson, François

    2016-01-01

    CD4+ T cells that express the transcription factor FOXP3 (FOXP3+ T cells) are commonly regarded as immunosuppressive regulatory T cells (Tregs). FOXP3+ T cells are reported to be increased in tumor-bearing patients or animals and are considered to suppress antitumor immunity, but the evidence is often contradictory. In addition, accumulating evidence indicates that FOXP3 is induced by antigenic stimulation and that some non-Treg FOXP3+ T cells, especially memory-phenotype FOXP3low cells, produce proinflammatory cytokines. Accordingly, the subclassification of FOXP3+ T cells is fundamental for revealing the significance of FOXP3+ T cells in tumor immunity, but the arbitrariness and complexity of manual gating have complicated the issue. In this article, we report a computational method to automatically identify and classify FOXP3+ T cells into subsets using clustering algorithms. By analyzing flow cytometric data of melanoma patients, the proposed method showed that the FOXP3+ subpopulation that had relatively high FOXP3, CD45RO, and CD25 expressions was increased in melanoma patients, whereas manual gating did not produce significant results on the FOXP3+ subpopulations. Interestingly, the computationally identified FOXP3+ subpopulation included not only classical FOXP3high Tregs, but also memory-phenotype FOXP3low cells by manual gating. Furthermore, the proposed method successfully analyzed an independent data set, showing that the same FOXP3+ subpopulation was increased in melanoma patients, validating the method. Collectively, the proposed method successfully captured an important feature of melanoma without relying on the existing criteria of FOXP3+ T cells, revealing a hidden association between the T cell profile and melanoma, and providing new insights into FOXP3+ T cells and Tregs. PMID:26864030

  10. 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

  11. Helicobacter pylori interferes with an embryonic stem cell micro RNA cluster to block cell cycle progression

    PubMed Central

    2011-01-01

    Background MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. Results Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. Conclusions These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections. PMID:22027184

  12. Lithium borate cluster salts as novel redox shuttles for overcharge protection of lithium-ion cells.

    SciTech Connect

    Chen, Z.; Liu, J.; Jansen, A. N.; Casteel, B.; Amine, K.; GirishKumar, G.; Air Products and Chemicals, Inc.

    2010-01-01

    Redox shuttle is a promising mechanism for intrinsic overcharge protection in lithium-ion cells and batteries. Two lithium borate cluster salts are reported to function as both the main salt for a nonaqueous electrolyte and the redox shuttle for overcharge protection. Lithium borate cluster salts with a tunable redox potential are promising candidates for overcharge protection for most positive electrodes in state-of-the-art lithium-ion cells.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. AMPA receptor mediated synaptic excitation drives state-dependent bursting in Purkinje neurons of zebrafish larvae

    PubMed Central

    Sengupta, Mohini; Thirumalai, Vatsala

    2015-01-01

    Purkinje neurons are central to cerebellar function and show membrane bistability when recorded in vitro or in vivo under anesthesia. The existence of bistability in vivo in awake animals is disputed. Here, by recording intracellularly from Purkinje neurons in unanesthetized larval zebrafish (Danio rerio), we unequivocally demonstrate bistability in these neurons. Tonic firing was seen in depolarized regimes and bursting at hyperpolarized membrane potentials. In addition, Purkinje neurons could switch from one state to another spontaneously or with current injection. While GABAAR or NMDAR were not required for bursting, activation of AMPARs by climbing fibers (CFs) was sufficient to trigger bursts. Further, by recording Purkinje neuron membrane potential intracellularly, and motor neuron spikes extracellularly, we show that initiation of motor neuron spiking is correlated with increased incidence of CF EPSPs and membrane depolarization. Developmentally, bistability was observed soon after Purkinje neuron specification and persists at least until late larval stages. DOI: http://dx.doi.org/10.7554/eLife.09158.001 PMID:26416140

  19. 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

  20. 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

  1. Gallic acid induces mitotic catastrophe and inhibits centrosomal clustering in HeLa cells.

    PubMed

    Tan, Si; Guan, Xin; Grün, Christoph; Zhou, Zhiqin; Schepers, Ute; Nick, Peter

    2015-12-25

    Cancer cells divide rapidly, providing medical targets for anticancer agents. The polyphenolic gallic acid (GA) is known to be toxic for certain cancer cells. However, the cellular mode of action has not been elucidated. Therefore, the current study addressed a potential effect of GA on the mitosis of cancer cells. GA inhibited viability of HeLa cells in a dose-dependent and time-dependent manner. We could show, using fluorescence-activated cell sorting (FACS), that this inhibition was accompanied by elevated frequency of cells arrested at the G2/M transition. This cell-cycle arrest was accompanied by mitotic catastrophe, and formation of cells with multiple nuclei. These aberrations were preceded by impaired centrosomal clustering. We arrive at a model of action, where GA inhibits the progression of the cell cycle at the G2/M phase by impairing centrosomal clustering which will stimulate mitotic catastrophe. Thus, GA has potential as compound against cervical cancer. PMID:26368671

  2. 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.

  3. Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms

    PubMed Central

    Stoodley, Paul; Wilson, Suzanne; Hall-Stoodley, Luanne; Boyle, John D.; Lappin-Scott, Hilary M.; Costerton, J. W.

    2001-01-01

    Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment. PMID:11722913

  4. 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. PMID:26855123

  5. Improved computing scheme for measuring eye alignment with Purkinje images I and IV.

    PubMed

    Barry, J C; Backes, A; Pongs, U M; Kirschkamp, T; Dunne, M C

    1997-09-01

    This study introduces an improved computing scheme for determining eye rotation from Purkinje images I and IV. The original computing scheme systematically underestimated eye rotation. Paraxial raytracing calculations revealed that this error resulted from failure to account for the fact that Purkinje images I and IV fall at different distances behind the cornea. The error could be overcome with a correction factor derived from paraxial raytracing calculations. A series of experiments were carried out to test the validity of this correlation factor, involving exact raytracing calculations as well as measurements on physical model eyes and human eyes. The influence on the correction factor of ocular surface asphericity, accommodation, age and ocular component variations were examined. The new method was also compared to Hirschberg's technique, which makes use of Purkinje image I alone, as a means of screening for strabismus. PMID:9390370

  6. 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. PMID:19276060

  7. Photothermolysis by laser-induced microbubbles generated around gold nanorod clusters selectively formed in leukemia cells

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitri; Lukianova-Hleb, Ekaterina; Zhdanok, Sergei; Rostro, Betty; Simonette, Rebecca; Hafner, Jason; Konopleva, Marina; Andreeff, Michael; Conjusteau, Andre; Oraevsky, Alexander

    2008-02-01

    In an effort of developing clinical LANTCET (laser-activated nano-thermolysis as cell elimination technology) we achieved selective destruction of individual tumor cells through laser generation of vapor microbubbles around clusters of light absorbing gold nanorods (GNR) selectively formed in target tumor cells. Among all gold nanoparticles, nanorods offer the highest optical absorption in the near-infrared. We applied covalent conjugates of gold nanorods with targeting vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia), while GNR conjugates with polyethylene-glycol (PEG) were used as nonspecific targeting control. GNR clusters were formed inside the tumor cells at 37 °C due to endocytosis of large concentration of nanorods accumulated on the surface of tumor cells targeted at 4 °C. Formation of GNR clusters significantly reduces the threshold of tumor cell damage making LANTCET safe for normal cells. Appearance of GNR clusters was verified directly with optical resonance scattering microscopy. LANTCET was performed in vitro with living cells of (1) model myeloid K562 cells (CD33 positive), (2) primary human bone marrow CD33-positive blast cells from patients diagnosed with acute myeloid leukemia. Laser-induced microbubbles were generated and detected with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser. GNT cluster formation caused a 100-fold decrease in the threshold optical fluence for laser microbubble generation in tumor cells compared with that in normal cells under the same targeting and irradiation conditions. Combining imaging based on resonance optical scattering with photothermal imaging of microbubbles, we developed a method for detection, image-guided treatment and monitoring of LANTCET. Pilot experiments were performed in flow mode bringing LANTCET closer to reality of clinical procedure of purging tumor cells from bone marrow grafts.

  8. High throughput assembly of spatially controlled 3D cell clusters on a micro/nanoplatform.

    PubMed

    Gallego-Perez, Daniel; Higuita-Castro, Natalia; Sharma, Sadhana; Reen, Rashmeet K; Palmer, Andre F; Gooch, Keith J; Lee, L James; Lannutti, John J; Hansford, Derek J

    2010-03-21

    Guided assembly of microscale tissue subunits (i.e. 3D cell clusters/aggregates) has found applications in cell therapy/tissue engineering, cell and developmental biology, and drug discovery. As cluster size and geometry are known to influence cellular responses, the ability to spatially control cluster formation in a high throughput manner could be advantageous for many biomedical applications. In this work, a micro- and nanofabricated platform was developed for this purpose, consisting of a soft-lithographically fabricated array of through-thickness microwells structurally bonded to a sheet of electrospun fibers. The microwells and fibers were manufactured from several polymers of biomedical interest. Human hepatocytes were used as model cells to demonstrate the ability of the platform to allow controlled cluster formation. In addition, the ability of the device to support studies on semi-controlled heterotypic interactions was demonstrated by co-culturing hepatocytes and fibroblasts. Preliminary experiments with other cells of interest (pancreatic cells, embryonic stem cells, and cardiomyocytes) were also conducted. Our platform possesses several advantages over previously developed microwell arrays: a more in vivo-like topographical stimulation of cells; better nutrient/waste exchange through the underlying nanofiber mat; and easy integration into standard two-chamber cell culture well systems. PMID:20221567

  9. 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.

  10. Influence of the Purkinje-muscle junction on transmural repolarization heterogeneity

    PubMed Central

    Walton, Richard D.; Martinez, Marine E.; Bishop, Martin J.; Hocini, Mélèze; Haïssaguerre, Michel; Plank, Gernot; Bernus, Olivier; Vigmond, Edward J.

    2014-01-01

    Aims To elucidate the properties of the PMJ and myocardium underlying these effects. Transmural heterogeneity of action potential duration (APD) is known to play an important role in arrhythmogenesis. Regions of non-uniformities of APD gradients often overlap considerably with the location of Purkinje-muscle junctions (PMJs). We therefore hypothesized that such junctions are novel sources of local endocardial and transmural heterogeneity of repolarization, and that remodelling due to heart failure modulates this response. Methods and results Spatial gradients of endocardial APD in left ventricular wedge preparations from healthy sheep (n = 5) were correlated with locations of PMJs identified through Purkinje stimulation under optical mapping. APD prolongation was dependent on proximity of the PMJ to the imaged surface, whereby shallow PMJs significantly modulated local APD when stimulating either Purkinje (P = 0.0116) or endocardium (P = 0.0123). In addition, we model a PMJ in 5 × 5× 10 mm transmural tissue wedges using healthy and novel failing human ventricular and Purkinje ionic models. Short distances of the PMJ to cut surfaces (<0.875 mm) revealed that APD maxima were localized to the PMJ in healthy myocardium, whereas APD minima were observed in failing myocardium. Amplitudes and spatial gradients of APD were prominent at functional PMJs and quiescent PMJs. Furthermore, increasing the extent of Purkinje fibre branching or decreasing tissue conductivity augmented local APD prolongation in both failing and non-failing models. Conclusions The Purkinje network has the potential to influence myocardial AP morphology and rate-dependent behaviour, and furthermore to underlie enhanced transmural APD heterogeneities and spatial gradients of APD in non-failing and failing myocardium. PMID:24997066

  11. 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

  12. 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.

  13. Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

    PubMed Central

    Avilés, Evelyn C.; Pinto, Cristina; Hanna, Patricia; Ojeda, Jorge; Pérez, Viviana; De Ferrari, Giancarlo V.; Zamorano, Pedro; Albistur, Miguel; Sandoval, Daniel; Henríquez, Juan P.

    2014-01-01

    Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ. PMID:24860427

  14. 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. PMID:26887857

  15. 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.

  16. 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.

  17. Involvement of gene methylation changes in the differentiation of human amniotic epithelial cells into islet-like cell clusters.

    PubMed

    Peng, Lin; Wang, Jian; Lu, Guangxiu

    2014-09-01

    Insulin-dependent diabetes results from destruction of the insulin-producing β-cells of the pancreas. Islet cell transplantation is a promising cure for diabetes. Here, we induced human amniotic epithelial cells (hAECs) to differentiate into islet-like cell clusters by nicotinamide plus betacellulin in vitro, and further investigated the DNA methylation status by a Nimble MeDIP microarray before and after cell differentiation to shed light on the molecular mechanisms of this differentiation. In addition, 5-Aza-2'-deoxycytidine was used to investigate whether the differentiation of hAECs into islet-like cells occurred through demethylation. Purified hAECs (CK18(+)/E-cadherin(+)/CD29(+)/CD90(-)/CD34(-)/CD45(-)) were isolated from human amnia. After induction, hAECs were found to be insulin positive and sensitive to glucose, indicating successful induction to islet-like cells. The methylation status of cell cytoskeleton-related genes was down-regulated and that of negative regulation of cell adhesion-related genes was up-regulated. The methylation status of pancreas development-related genes such as HNF1α and DGAT1 was decreased in hAECs after induction. After brief demethylation, INS gene expression was up-regulated in islet-like cell clusters, suggesting that DNA methylation changes were associated with the differentiation of hAECs into islet-like cell clusters. PMID:24945458

  18. New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay*

    PubMed Central

    Li, Xue-Wen; Rees, Johanna S.; Xue, Peng; Zhang, Hong; Hamaia, Samir W.; Sanderson, Bailey; Funk, Phillip E.; Farndale, Richard W.; Lilley, Kathryn S.; Perrett, Sarah; Jackson, Antony P.

    2014-01-01

    In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30–40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics. PMID:24706754

  19. 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

  20. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium

    PubMed Central

    Wilson, Calum; Saunter, Christopher D.; Girkin, John M.; McCarron, John G.

    2016-01-01

    Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca2+ concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca2+ signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca2+ signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.—Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium. PMID:26873937

  1. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

    PubMed

    Wilson, Calum; Saunter, Christopher D; Girkin, John M; McCarron, John G

    2016-05-01

    Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium. PMID:26873937

  2. 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.

  3. 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. PMID:9614967

  4. 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.

  5. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    PubMed Central

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-01-01

    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. PMID:27353000

  6. 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

  7. Positive feedback can lead to dynamic nanometer-scale clustering on cell membranes

    NASA Astrophysics Data System (ADS)

    Wehrens, Martijn; ten Wolde, Pieter Rein; Mugler, Andrew

    2014-11-01

    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.

  8. 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.

  9. Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje neurons to Bergmann glia

    PubMed Central

    Sawada, Yusuke; Konno, Ayumu; Nagaoka, Jun; Hirai, Hirokazu

    2016-01-01

    Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs. PMID:27291422

  10. Micro RNA-17-92 cluster mediates interleukin-4-suppressed IL-10 expression in B cells

    PubMed Central

    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. PMID:27347339

  11. Pre-Clustering of the B Cell Antigen Receptor Demonstrated by Mathematically Extended Electron Microscopy

    PubMed Central

    Fiala, Gina J.; Kaschek, Daniel; Blumenthal, Britta; Reth, Michael; Timmer, Jens; Schamel, Wolfgang W. A.

    2013-01-01

    The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation. PMID:24367367

  12. miR-23∼27∼24 clusters control effector T cell differentiation and function.

    PubMed

    Cho, Sunglim; Wu, Cheng-Jang; Yasuda, Tomoharu; Cruz, Leilani O; Khan, Aly Azeem; Lin, Ling-Li; Nguyen, Duc T; Miller, Marina; Lee, Hyang-Mi; Kuo, Ming-Ling; Broide, David H; Rajewsky, Klaus; Rudensky, Alexander Y; Lu, Li-Fan

    2016-02-01

    Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, we show that the miR-23∼27∼24 clusters regulate multiple aspects of T cell biology, particularly helper T (Th) 2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23∼27∼24 clusters revealed that miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, although overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17, and induced regulatory T cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest that tight regulation of miR-23∼27∼24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses. PMID:26834155

  13. Label-free Electrophysiological Cytometry for Stem Cell-Derived Cardiomyocyte Clusters

    PubMed Central

    Myers, Frank B.; Abilez, Oscar J.; Zarins, Christopher K.; Lee, Luke P.

    2012-01-01

    Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However, a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here, we describe a non-genetic, label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes, neurons, smooth muscle cells), this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 μVrms. Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals, the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry, in which a cell’s functionality is assessed rather than its expression profile or physical characteristics. PMID:23207961

  14. The microRNA-212/132 cluster regulates B cell development by targeting Sox4

    PubMed Central

    Mehta, Arnav; Mann, Mati; Zhao, Jimmy L.; Marinov, Georgi K.; Majumdar, Devdoot; Garcia-Flores, Yvette; Du, Xiaomi; Erikci, Erdem; Chowdhury, Kamal

    2015-01-01

    MicroRNAs have emerged as key regulators of B cell fate decisions and immune function. Deregulation of several microRNAs in B cells leads to the development of autoimmune disease and cancer in mice. We demonstrate that the microRNA-212/132 cluster (miR-212/132) is induced in B cells in response to B cell receptor signaling. Enforced expression of miR-132 results in a block in early B cell development at the prepro–B cell to pro–B cell transition and induces apoptosis in primary bone marrow B cells. Importantly, loss of miR-212/132 results in accelerated B cell recovery after antibody-mediated B cell depletion. We find that Sox4 is a target of miR-132 in B cells. Co-expression of SOX4 with miR-132 rescues the defect in B cell development from overexpression of miR-132 alone, thus suggesting that miR-132 may regulate B lymphopoiesis through Sox4. In addition, we show that the expression of miR-132 can inhibit cancer development in cells that are prone to B cell cancers, such as B cells expressing the c-Myc oncogene. We have thus uncovered miR-132 as a novel contributor to B cell development. PMID:26371188

  15. Sodium-dependent control of intracellular pH in Purkinje fibres of sheep heart.

    PubMed Central

    Ellis, D; MacLeod, K T

    1985-01-01

    Intracellular pH (pHi) of Purkinje fibres from sheep heart was recorded with pH-sensitive glass micro-electrodes. The cells were acidified by one of three methods: (1) exposure to and subsequent removal of NH4Cl, (2) exposure to solutions containing 5% CO2 or (3) exposure to an acidic Tyrode solution. The pHi recovery from these acidifications was studied. The time constant of recovery from an acidification induced by NH4Cl was almost twice as long as that from one induced by CO2 or acid extracellular pH. Following an acidification induced by exposure to CO2 the time constant of pHi recovery was not changed when the cell was depolarized to -40 mV (by replacement of some Na+ by K+). An intracellular acidification was produced when extracellular Na+ was removed and replaced by quaternary ammonium ions or K+. Such Na+-free solutions also inhibited pHi recovery from an acidification. A 50% inhibition of the rate of recovery was produced by lowering the [Na+]o to 8 mM. When used as a Na+ substitute, Li+ could permit recovery. Tris (22 mM) changed pHi in the alkaline direction. Amiloride (1 mM) or a decrease in temperature slowed the recovery from an acidification (Q10 = 2.65). There was no effect of SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid disodium salt; 100 microM) on the recovery. Na+-sensitive glass micro-electrodes were used to measure the intracellular Na+ activity when [Na+]o was lowered to levels used in our pHi recovery experiments. From these data we have calculated the apparent Na+ electrochemical gradient at different values of [Na+]o. If this gradient is responsible for H+ efflux from the cell then, by applying thermodynamic considerations, it can be shown that only low concentrations (1-2mM) of extracellular Na+ are required. Solutions containing a very low [Ca2+]o (less than 10(-8) M, buffered with EGTA) were used to prevent large rises of [Ca2+]i which may occur on removal of external Na+. Under these conditions pHi recovery is

  16. 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.

  17. 3D imaging of Sox2 enhancer clusters in embryonic stem cells

    PubMed Central

    Liu, Zhe; Legant, Wesley R; Chen, Bi-Chang; Li, Li; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert

    2014-01-01

    Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription. DOI: http://dx.doi.org/10.7554/eLife.04236.001 PMID:25537195

  18. Combined function of HoxA and HoxB clusters in neural crest cells.

    PubMed

    Vieux-Rochas, Maxence; Mascrez, Bénédicte; Krumlauf, Robb; Duboule, Denis

    2013-10-01

    The evolution of chordates was accompanied by critical anatomical innovations in craniofacial development, along with the emergence of neural crest cells. The potential of these cells to implement a craniofacial program in part depends upon the (non-)expression of Hox genes. For instance, the development of jaws requires the inhibition of Hox genes function in the first pharyngeal arch. In contrast, Hox gene products induce craniofacial structures in more caudal territories. To further investigate which Hox gene clusters are involved in this latter role, we generated HoxA;HoxB cluster double mutant animals in cranial neural crest cells. We observed the appearance of a supernumerary dentary-like bone with an endochondral ossification around a neo-Meckel's cartilage matrix and an attachment of neo-muscle demonstrating that HoxB genes enhance the phenotype induced by the deletion of the HoxA cluster alone. In addition, a cervical and hypertrophic thymus was associated with the supernumerary dentary-like bone, which may reflect its ancestral position near the filtrating system. Altogether these results show that the HoxA and HoxB clusters cooperated during evolution to lead to present craniofacial diversity. PMID:23850771

  19. Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

    PubMed Central

    2010-01-01

    Background Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells. Results Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes. Conclusions These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both. PMID:20843318

  20. 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.

  1. Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

    PubMed Central

    2010-01-01

    Background We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. Results Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. Conclusions This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies. PMID:20579360

  2. 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

  3. 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

  4. Evaluation of intraocular lens implant location in the eyeball basing on the Purkinje images

    NASA Astrophysics Data System (ADS)

    Jóźwik, A.; Siedlecki, D.; Zajac, M.

    2012-01-01

    Intraocular lens (IOL) is an artificial implant substituting natural crystalline lens which is non-transparent due to cataract. Incorrect location of the IOL in the eyeball (e.g. its shift or tilt) causes significant deterioration of patient's vision. The analysis of Purkinje images (i.e. reflections from successive refracting surfaces in the eye) enables to determine the real IOL location and thus helps in evaluating the retinal image quality. The experimental setup for Purkinje images recording consists of illuminator, composed of a number of infrared LEDs, telecentric lens and detector (CCD camera). Analysis of mutual position of particular reflections enables to evaluate the lens location in respect to the corneal axis. The actual measurements are realized on artificial eye model, what allows to estimate the precision of the algorithm applied in the calculations. In the future the experimental set-up will be adapted to measure the eyes of real patients.

  5. Cluster headache: incorporation of (1-14C)oleic acid into phosphatidylserine in polymorphonuclear cells.

    PubMed

    Fragoso, Y D; Stovner, L J; Bjerve, K S; Sjaastad, O

    1989-09-01

    As recently demonstrated by our group, polymorphonuclear cells (PMNs) from cluster headache patients have an increased ability to incorporate arachidonic acid (AA) and L-serine into phosphatidylserine (PS). To evaluate whether there is an increased incorporation into PS also from fatty acids not involved in eicosanoid metabolism, PMNs from controls (n = 14) and cluster headache patients (n = 12) were incubated with (1-14C)oleic acid. After 1 h 2.7% +/- 1.1 (mean value +/- SD) of the glycerophospholipid radioactivity was found in PS in controls, whereas 4.2% +/- 1.2 was found in cluster headache patients (p less than 0.005). For phosphatidylcholine (PC) the corresponding figures were 74.2 +/- 5.4 in controls and 66.7 +/- 7.6 in cluster headache patients (p less than 0.01). The results suggest that the de novo biosynthesis of PS is increased and the biosynthesis of PC is decreased in cluster headache. The results may have an effect on the role of PS as an obligate protein kinase C activator. PMID:2507162

  6. 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.

  7. 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

  8. The Magea gene cluster regulates male germ cell apoptosis without affecting the fertility in mice.

    PubMed

    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

  9. 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

  10. Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer.

    PubMed

    Servín-González, Luis Steven; Granados-López, Angelica Judith; López, Jesús Adrián

    2015-01-01

    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

  11. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid.

    PubMed

    Kashyap, Vasundhra; Gudas, Lorraine J; Brenet, Fabienne; Funk, Patricia; Viale, Agnes; Scandura, Joseph M

    2011-02-01

    Retinoic acid (RA) regulates clustered Hox gene expression during embryogenesis and is required to establish the anterior-posterior body plan. Using mutant embryonic stem cell lines deficient in the RA receptor γ (RARγ) or Hoxa1 3'-RA-responsive element, we studied the kinetics of transcriptional and epigenomic patterning responses to RA. RARγ is essential for RA-induced Hox transcriptional activation, and deletion of its binding site in the Hoxa1 enhancer attenuates transcriptional and epigenomic activation of both Hoxa and Hoxb gene clusters. The kinetics of epigenomic reorganization demonstrate that complete erasure of the polycomb repressive mark H3K27me3 is not necessary to initiate Hox transcription. RARγ is not required to establish the bivalent character of Hox clusters, but RA/RARγ signaling is necessary to erase H3K27me3 from activated Hox genes during embryonic stem cell differentiation. Highly coordinated, long range epigenetic Hox cluster reorganization is closely linked to transcriptional activation and is triggered by RARγ located at the Hoxa1 3'-RA-responsive element. PMID:21087926

  12. Clustering of voltage-dependent sodium channels on axons depends on Schwann cell contact.

    PubMed

    Joe, E H; Angelides, K

    1992-03-26

    In myelinated nerves, segregation of voltage-dependent sodium channels to nodes of Ranvier is crucial for saltatory conduction along axons. As sodium channels associate and colocalize with ankyrin at nodes of Ranvier, one possibility is that sodium channels are recruited and immobilized at axonal sites which are specified by the subaxolemmal cytoskeleton, independent of glial cell contact. Alternatively, segregation of channels at distinct sites along the axon may depend on glial cell contact. To resolve this question, we have examined the distribution of sodium channels, ankyrin and spectrin in myelination-competent cocultures of sensory neurons and Schwann cells by immunofluorescence, using sodium channel-, ankyrin- and spectrin-specific antibodies. In the absence of Schwann cells, sodium channels, ankyrin and spectrin are homogeneously distributed on sensory axons. When Schwann cells are introduced into these cultures, the distribution of sodium channels dramatically changes so that channel clusters on axons are abundant, but ankyrin and spectrin remain homogeneously distributed. Addition of latex beads or Schwann cell membranes does not induce channel clustering. Our results suggest that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to organize and maintain channel distribution during differentiation of myelinated axons. PMID:1312680

  13. 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.

  14. PDGF‑stimulated dispersal of cell clusters and disruption of fibronectin matrix on three-dimensional collagen matrices requires matrix metalloproteinase-2

    PubMed Central

    da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

    2015-01-01

    Formation of cell clusters is a common morphogenic cell behavior observed during tissue and organ development and homeostasis, as well as during pathological disorders. Dynamic regulation of cell clustering depends on the balance between contraction of cells into clusters and migration of cells as dispersed individuals. Previously we reported that under procontractile culture conditions, fibronectin fibrillar matrix assembly by human fibroblasts functioned as a nucleation center for cell clustering on three-dimensional collagen matrices. Here we report that switching preformed cell clusters from procontractile to promigratory culture conditions results in cell dispersal out of clusters and disruption of FN matrix. Experiments using small interfering RNA silencing and pharmacological inhibition demonstrated that matrix metalloproteinase activity involving MMP-2 was necessary for fibronectin matrix disruption and dispersal of cell clusters. PMID:25589674

  15. 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

  16. 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

  17. Slow cluster formation of purified human or rhesus T cells requires protein kinase C and LFA-1.

    PubMed

    Eylar, E H; Molina, C; Báez, I; Kessler, M

    1996-03-01

    Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74% of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26%. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more

  18. Pea (Pisum sativum) cells arrested in G2 have nascent DNA with breaks between replicons and replication clusters

    SciTech Connect

    Van't Hof, J.

    1980-01-01

    DNA fiber autoradiography and alkaline sucrose sedimentation of DNA of cultured pea-root cells (Pisum sativum) arrested in G2 by carbohydrate starvation demonstrated that nascent DNA molecules of replicon (16 to 27 x 10/sup 6/D) and apparent cluster (approx. 330 x 10/sup 6/D) size were not joined. That the arrested cells were in G2 was confirmed by single-cell autoradiography and cytophotometry. In pea there are about 18 replicons per average cluster, 4.2 x 10/sup 3/ clusters, and 7.7 x 10/sup 4/ replicons per genome.

  19. 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.

  20. 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

  1. Effect of particle clustering of silver nanoparticles on ultrathin silicon solar cell

    NASA Astrophysics Data System (ADS)

    Shokeen, Poonam; Jain, Amit; Kapoor, Avinashi; Gupta, Vinay

    2016-07-01

    Particle clustering is a major concern for uniform dispersal of nanoparticles in various deposition procedures. Well separated uniform distribution of metal nanoparticles is essential for effective coupling of surface plasmons. This work experimentally and theoretically, discusses the effect of nanoparticle clustering on the light trapping efficiency of silver nanoparticles. Pulsed laser deposition system has been used for deposition of silver nanoparticles, and substrate heating has been used to promote uniform distribution of nanoparticles. Pre-heated substrate depositions are compared with corresponding post-annealed samples. XRD, FESEM, Photoluminescence and UV-visible spectroscopy have been used to study the variations in their structural and optical properties. Mono-dispersal of silver nanoparticles for pre-heated substrates results in sharper surface plasmon resonance in comparison to post-annealed samples. Mie theory is used to estimate the particle size of the nanoparticles and findings are in accordance with quantitative analysis of FESEM images. Finite-difference time domain technique is used to discuss the effect of particle distribution on an ultrathin film silicon solar cell. Device degradation is observed as a result of clustering of silver nanoparticles. Hence, mono-dispersal of plasmonic nanostructures is important for required results and pre-heated deposition of metal nanoparticles by pulsed laser deposition can effectively solve the problem of particle clustering.

  2. Role of lattice defects in catalytic activities of graphene clusters for fuel cells.

    PubMed

    Zhang, Lipeng; Xu, Quan; Niu, Jianbing; Xia, Zhenhai

    2015-07-14

    Defects are common but important in graphene, which could significantly tailor the electronic structures and physical and chemical properties. In this study, the density functional theory (DFT) method was applied to study the electronic structure and catalytic properties of graphene clusters containing various point and line defects. The electron transfer processes in oxygen reduction reaction (ORR) on perfect and defective graphene clusters in fuel cells was simulated, and the free energy and reaction energy barrier of the elementary reactions were calculated to determine the reaction pathways. It was found that the graphene cluster with the point defect having pentagon rings at the zigzag edge, or line defects (grain boundaries) consisting of pentagon-pentagon-octagon or pentagon-heptagon chains also at the edges, shows the electrocatalytic capability for ORR. Four-electron and two-electron transfer processes could occur simultaneously on graphene clusters with certain types of defects. The energy barriers of the reactions are comparable to that of platinum(111). The catalytic active sites were determined on the defective graphene. PMID:26033301

  3. 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

    NASA Astrophysics Data System (ADS)

    Roy, Subhasish; Baral, Abhishek; Bhattacharjee, Rameswar; Jana, Batakrishna; Datta, Ayan; Ghosh, Surajit; Banerjee, Arindam

    2015-01-01

    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.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

  4. 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.

  5. Identification of Vaccine-Altered Circulating B Cell Phenotypes Using Mass Cytometry and a Two-Step Clustering Analysis.

    PubMed

    Pejoski, David; Tchitchek, Nicolas; Rodriguez Pozo, André; Elhmouzi-Younes, Jamila; Yousfi-Bogniaho, Rahima; Rogez-Kreuz, Christine; Clayette, Pascal; Dereuddre-Bosquet, Nathalie; Lévy, Yves; Cosma, Antonio; Le Grand, Roger; Beignon, Anne-Sophie

    2016-06-01

    Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20(+) B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27(hi)CD21(lo) activated memory and CD27(+)CD21(+) resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery. PMID:27183591

  6. 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. PMID:20944843

  7. Cross-linked Glucose Oxidase Clusters for Biofuel Cell Anode Catalysts

    PubMed Central

    Dudzik, Jonathan; Chang, Wen-Chi; Kannan, A.M.; Filipek, Slawomir; Viswanathan, Sowmya; Li, Pingzuo; Renugopalakrishnan, V.; Audette, Gerald F.

    2013-01-01

    The efficient localization of increased levels of active enzymes onto conducting scaffolds is important for the development of enzyme-based biofuel cells. Crosslinked enzyme clusters (CEC) of glucose oxidase (GOx) constrained to functionalized carbon nanotubes (CEC-CNTs) were generated in order to evaluate the potential of using CECs for developing GOx-based bioanodes functioning via direct electron transfer from the GOx active site to the CNT scaffold. CEC-CNTs generated from several weight-to-weight ratios of GOx:CNT were examined for comparable catalytic activity to free GOx in solution, with CEC-CNTs generated from a 100% GOx solution displaying the greatest enzymatic activity. STEM analysis of CEC-CNTs generated from 100% GOx to CNT (wt/wt) ratios revealed CEC clusters of ~78 μm2 localized that the CNT surface. Electrochemical analysis indicates that the enzyme is engaging in direct electron transfer, and BFCs generated using GOx CEC-CNT bioanodes were observed to have a peak power density of ~180 μW·cm−2. These data indicate that the generation of nano-to-micro sized active enzyme clusters is an attractive option for the design of enzyme-specific biofuel cell powered implantable devices. PMID:23880606

  8. A coupled 3D-1D numerical monodomain solver for cardiac electrical activation in the myocardium with detailed Purkinje network

    NASA Astrophysics Data System (ADS)

    Vergara, Christian; Lange, Matthias; Palamara, Simone; Lassila, Toni; Frangi, Alejandro F.; Quarteroni, Alfio

    2016-03-01

    We present a model for the electrophysiology in the heart to handle the electrical propagation through the Purkinje system and in the myocardium, with two-way coupling at the Purkinje-muscle junctions. In both the subproblems the monodomain model is considered, whereas at the junctions a resistor element is included that induces an orthodromic propagation delay from the Purkinje network towards the heart muscle. We prove a sufficient condition for convergence of a fixed-point iterative algorithm to the numerical solution of the coupled problem. Numerical comparison of activation patterns is made with two different combinations of models for the coupled Purkinje network/myocardium system, the eikonal/eikonal and the monodomain/monodomain models. Test cases are investigated for both physiological and pathological activation of a model left ventricle. Finally, we prove the reliability of the monodomain/monodomain coupling on a realistic scenario. Our results underlie the importance of using physiologically realistic Purkinje-trees with propagation solved using the monodomain model for simulating cardiac activation.

  9. Evolutionary conservation and function of the human embryonic stem cell specific miR-302/367 cluster.

    PubMed

    Chen, Liang; Heikkinen, Liisa; Emily Knott, K; Liang, Yanchun; Wong, Garry

    2015-12-01

    miRNA clusters define a group of related miRNAs closely localized in the genome with an evolution that remains poorly understood. The miR-302/367 cluster represents a single polycistronic transcript that produces five precursor miRNAs. The cluster is highly expressed and essential for maintenance of human embryonic stem cells. We found the cluster to be highly conserved and present in most mammals. In primates, seed sequence and miRNA structure are conserved, but inter-precursor sequences are evolving. Insertions of new miRNAs, deletions of individual miRNAs, and a cluster duplication observed in different species suggest an actively evolving cluster. Core transcriptional machinery consisting of NANOG and OCT-4 transcription factors that define stem cells are present upstream of the miR-302/367 cluster. Interestingly, we found the miR-302/367 cluster flanking region to be enriched as a target site of other miRNAs suggesting a mechanism for feedback control. Analysis of miR-302 and miR-367 targets demonstrated concordance of gene set enrichment groups at high gene ontology levels. This cluster also expresses isomiRs providing another means of establishing sequence diversity. Finally, using three different kidney tumor datasets, we observed consistent expression of miR-302 family members in normal tissue while adjacent tumor tissue showed a significant lack of expression. Clustering expression levels of miR-302 validated target genes showed a significant correlation between miR-302/367 cluster miRNAs and a subset of validated gene targets in healthy and adjacent tumor tissues. Taken together, our data show a highly conserved and still evolving miRNA cluster that may have additional unrecognized functions. PMID:26363379

  10. Identification of microRNAs expressed highly in pancreatic islet-like cell clusters differentiated from human embryonic stem cells.

    PubMed

    Chen, Bo-Zhi; Yu, Sung-Liang; Singh, Sher; Kao, Li-Pin; Tsai, Zong-Yun; Yang, Pan-Chyr; Chen, Bai-Hsiun; Shoei-Lung Li, Steven

    2011-01-01

    Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self-renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES-T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet-like cell clusters derived from T3 cells), which expressed pancreatic islet cell-specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES-T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein-coding mRNAs. The T3pi showed very high expression of microRNAs, miR-186, miR-199a and miR-339, which down-regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics. PMID:20735361

  11. Cluster synchronization and spatio-temporal dynamics in networks of oscillatory and excitable Luo-Rudy cells

    NASA Astrophysics Data System (ADS)

    Kanakov, O. I.; Osipov, G. V.; Chan, C.-K.; Kurths, J.

    2007-03-01

    We study collective phenomena in nonhomogeneous cardiac cell culture models, including one- and two-dimensional lattices of oscillatory cells and mixtures of oscillatory and excitable cells. Individual cell dynamics is described by a modified Luo-Rudy model with depolarizing current. We focus on the transition from incoherent behavior to global synchronization via cluster synchronization regimes as coupling strength is increased. These regimes are characterized qualitatively by space-time plots and quantitatively by profiles of local frequencies and distributions of cluster sizes in dependence upon coupling strength. We describe spatio-temporal patterns arising during this transition, including pacemakers, spiral waves, and complicated irregular activity.

  12. CD82 Restrains Angiogenesis by Altering Lipid Raft Clustering and CD44 Trafficking in Endothelial Cells

    PubMed Central

    Wei, Quan; Zhang, Feng; Richardson, Mekel M.; Roy, Nathan H.; Rodgers, William; Liu, Yuechueng; Zhao, Wenyuan; Fu, Chenying; Ding, Yingjun; Huang, Chao; Chen, Yuanjian; Sun, Yao; Ding, Lexi; Hu, Yang; Ma, Jianxing; Boulton, Michael E.; Pasula, Satish; Wren, Jonathan D.; Tanaka, Satoshi; Huang, Xiaolin; Thali, Markus; Hämmerling, Günter J.; Zhang, Xin A.

    2014-01-01

    Background Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Methods and Results Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes of Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules (CAMs) such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Conclusions Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, lipid raft clustering and CAM trafficking modulate angiogenic potential, and the perturbation of CD82-ganglioside-CD44 signaling attenuates angiogenesis. PMID:25149363

  13. Enhanced performance of anion exchange membranes via crosslinking of ion cluster regions for fuel cells

    NASA Astrophysics Data System (ADS)

    Lai, Ao Nan; Guo, Dong; Lin, Chen Xiao; Zhang, Qiu Gen; Zhu, Ai Mei; Ye, Mei Ling; Liu, Qing Lin

    2016-09-01

    Development of anion exchange membranes (AEMs) with high hydroxide conductivity, good dimensional and alkaline stabilities is still a challenge for the practical application of AEM fuel cells. In this study, we report a new strategy to prepare high-performance AEMs with crosslinked ionic regions. A series of phenolphthalein-containing poly(arylene ether sulfone)s crosslinked AEMs was synthesized by grafting ion groups selectively and densely on the phenolphthalein units to form ion clusters that are further crosslinked to generate the hydrophilic ionic regions. The crosslinking reaction not only improved the dimensional stability of the AEMs, but also increased the aggregation of the ion clusters leading to the formation of hydrophilic/hydrophobic phase-separated morphology and ion-conducting channels. As a result, enhancements in both ion conductivity and dimensional stability can be achieved. The crosslinked AEMs showed high hydroxide conductivities in the range of 52.2-143.4 mS cm-1 from 30 to 80 °C and a superb ratio of relative conductivity to relative swelling at 80 °C. Furthermore, the crosslinked AEMs also exhibited good mechanical properties, thermal and alkaline stabilities and desirable single cell performance. This work presents a promising strategy for the synthesis of high-performance AEMs for fuel cells.

  14. MicroRNA-183-96-182 Cluster Regulates Bovine Granulosa Cell Proliferation and Cell Cycle Transition by Coordinately Targeting FOXO1.

    PubMed

    Gebremedhn, Samuel; Salilew-Wondim, Dessie; Hoelker, Michael; Rings, Franca; Neuhoff, Christiane; Tholen, Ernst; Schellander, Karl; Tesfaye, Dawit

    2016-06-01

    Large-scale expression profiling of micro-RNAs (miRNAs) in bovine granulosa cells from dominant and subordinate follicles on Day 19 of the estrous cycle revealed enriched micro-RNA-183-96-182 cluster miRNAs in preovulatory dominant follicles that coordinately regulate the forkhead box protein O1 (FOXO1) gene. However, little is known about the role of this cluster in bovine granulosa cell function. We used an in vitro granulosa cell culture model to investigate this role. Granulosa cells aspirated from small growing follicles (3-5 mm in diameter) were cultured in Dulbecco modified Eagle medium/F-12 medium supplemented with fetal bovine serum and transfected with locked nucleic acid-based miRNA mimics, inhibitors, and corresponding negative controls. Overexpression of the miRNA cluster resulted in suppression of FOXO1 mRNA and protein, whereas inhibition of the cluster increased expression of FOXO1 mRNA. Overexpression also increased the relative rate of cell proliferation, whereas inhibition slowed it down. Similarly, the proportion of cells under G0/G1 arrest declined, whereas the ratio of cells in S phase increased in response to miR-183-96-182 overexpression. Selective knockdown of FOXO1 mRNA using anti-FOXO1 small interfering RNA increased the rate of granulosa cell proliferation, decreased the proportion of cells under G0/G1 arrest, and increased the proportion of cells in the S phase of cell cycle. Our data suggest that miR-183-96-182 cluster miRNAs promote proliferation and G1/S transition of bovine granulosa cells by coordinately targeting FOXO1, suggesting a critical role in granulosa cell function. MicroRNA-183-96-182 cluster regulates bovine granulosa cell function by targeting FOXO1 gene. PMID:27122636

  15. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  16. Glutathione Depletion and Carbon Ion Radiation Potentiate Clustered DNA Lesions, Cell Death and Prevent Chromosomal Changes in Cancer Cells Progeny

    PubMed Central

    Hanot, Maïté; Boivin, Anthony; Malésys, Céline; Beuve, Michaël; Colliaux, Anthony; Foray, Nicolas; Douki, Thierry; Ardail, Dominique; Rodriguez-Lafrasse, Claire

    2012-01-01

    Poor local control and tumor escape are of major concern in head-and-neck cancers treated by conventional radiotherapy or hadrontherapy. Reduced glutathione (GSH) is suspected of playing an important role in mechanisms leading to radioresistance, and its depletion should enable oxidative stress insult, thereby modifying the nature of DNA lesions and the subsequent chromosomal changes that potentially lead to tumor escape. This study aimed to highlight the impact of a GSH-depletion strategy (dimethylfumarate, and l-buthionine sulfoximine association) combined with carbon ion or X-ray irradiation on types of DNA lesions (sparse or clustered) and the subsequent transmission of chromosomal changes to the progeny in a radioresistant cell line (SQ20B) expressing a high endogenous GSH content. Results are compared with those of a radiosensitive cell line (SCC61) displaying a low endogenous GSH level. DNA damage measurements (γH2AX/comet assay) demonstrated that a transient GSH depletion in resistant SQ20B cells potentiated the effects of irradiation by initially increasing sparse DNA breaks and oxidative lesions after X-ray irradiation, while carbon ion irradiation enhanced the complexity of clustered oxidative damage. Moreover, residual DNA double-strand breaks were measured whatever the radiation qualities. The nature of the initial DNA lesions and amount of residual DNA damage were similar to those observed in sensitive SCC61 cells after both types of irradiation. Misrepaired or unrepaired lesions may lead to chromosomal changes, estimated in cell progeny by the cytome assay. Both types of irradiation induced aberrations in nondepleted resistant SQ20B and sensitive SCC61 cells. The GSH-depletion strategy prevented the transmission of aberrations (complex rearrangements and chromosome break or loss) in radioresistant SQ20B only when associated with carbon ion irradiation. A GSH-depleting strategy combined with hadrontherapy may thus have considerable advantage in the

  17. Notch-Jagged signalling can give rise to clusters of cells exhibiting a hybrid epithelial/mesenchymal phenotype.

    PubMed

    Boareto, Marcelo; Jolly, Mohit Kumar; Goldman, Aaron; Pietilä, Mika; Mani, Sendurai A; Sengupta, Shiladitya; Ben-Jacob, Eshel; Levine, Herbert; Onuchic, Jose' N

    2016-05-01

    Metastasis can involve repeated cycles of epithelial-to-mesenchymal transition (EMT) and its reverse mesenchymal-to-epithelial transition. Cells can also undergo partial transitions to attain a hybrid epithelial/mesenchymal (E/M) phenotype that allows the migration of adhering cells to form a cluster of circulating tumour cells. These clusters can be apoptosis-resistant and possess an increased metastatic propensity as compared to the cells that undergo a complete EMT (mesenchymal cells). Hence, identifying the key players that can regulate the formation and maintenance of such clusters may inform anti-metastasis strategies. Here, we devise a mechanism-based theoretical model that links cell-cell communication via Notch-Delta-Jagged signalling with the regulation of EMT. We demonstrate that while both Notch-Delta and Notch-Jagged signalling can induce EMT in a population of cells, only Jagged-dominated Notch signalling, but not Delta-dominated signalling, can lead to the formation of clusters containing hybrid E/M cells. Our results offer possible mechanistic insights into the role of Jagged in tumour progression, and offer a framework to investigate the effects of other microenvironmental signals during metastasis. PMID:27170649

  18. Divergence and transcriptional analysis of the division cell wall (dcw) gene cluster in Neisseria spp.

    PubMed

    Snyder, Lori A S; Shafer, William M; Saunders, Nigel J

    2003-01-01

    Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3' of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox-like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5' of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw-associated CREE, the first demonstration of active promoter generation at the 5' end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components. PMID:12519193

  19. Gangliosides GM1 and GM3 in the Living Cell Membrane Form Clusters Susceptible to Cholesterol Depletion and Chilling

    PubMed Central

    Fujita, Akikazu; Cheng, Jinglei; Hirakawa, Minako; Furukawa, Koichi; Kusunoki, Susumu

    2007-01-01

    Presence of microdomains has been postulated in the cell membrane, but two-dimensional distribution of lipid molecules has been difficult to determine in the submicrometer scale. In the present paper, we examined the distribution of gangliosides GM1 and GM3, putative raft molecules in the cell membrane, by immunoelectron microscopy using quick-frozen and freeze-fractured specimens. This method physically immobilized molecules in situ and thus minimized the possibility of artifactual perturbation. By point pattern analysis of immunogold labeling, GM1 was shown to make clusters of <100 nm in diameter in normal mouse fibroblasts. GM1-null fibroblasts were not labeled, but developed a similar clustered pattern when GM1 was administered. On cholesterol depletion or chilling, the clustering of both endogenous and exogenously-loaded GM1 decreased significantly, but the distribution showed marked regional heterogeneity in the cells. GM3 also showed cholesterol-dependent clustering, and although clusters of GM1 and GM3 were found to occasionally coincide, these aggregates were separated in most cases, suggesting the presence of heterogeneous microdomains. The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling. PMID:17392511

  20. A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.

    PubMed

    Zhou, Harry Y; Katsman, Yulia; Dhaliwal, Navroop K; Davidson, Scott; Macpherson, Neil N; Sakthidevi, Moorthy; Collura, Felicia; Mitchell, Jennifer A

    2014-12-15

    The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells. PMID:25512558

  1. PKS and NRPS gene clusters from microbial symbiont cells of marine sponges by whole genome amplification.

    PubMed

    Siegl, Alexander; Hentschel, Ute

    2010-08-01

    Whole genome amplification (WGA) approaches provide genomic information on single microbial cells and hold great promise for the field of environmental microbiology. Here, the microbial consortia of the marine sponge Aplysina aerophoba were sorted by fluorescence-activated cell sorting (FACS) and then subjected to WGA. A cosmid library was constructed from the WGA product of a sample containing two bacterial cells, one a member of the candidate phylum Poribacteria and one of a sponge-specific clade of Chloroflexi. Library screening led to the genomic characterization of three cosmid clones, encoding a polyketide synthase (PKS), a non-ribosomal peptide synthetase (NRPS) and the Chloroflexi 16S rRNA gene. PCR screening of WGA products from additional, FACS-sorted single bacterial symbiont cells supports the assignment of the Sup-PKS gene to the Poribacteria and the novel NRPS gene to the Chloroflexi. This promising single-cell genomics approach has permitted cloning of entire gene clusters from single microbial cells of known phylogenetic origin and thus provides a sought-after link between phylogeny and function. PMID:23766222

  2. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging

    NASA Astrophysics Data System (ADS)

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-01

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes.Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the

  3. The Effects of Cell Compressibility, Motility and Contact Inhibition on the Growth of Tumor Cell Clusters using the Cellular Potts Model

    PubMed Central

    Li, Jonathan F.; Lowengrub, John

    2014-01-01

    There are numerous biological examples where genes associated with migratory ability of cells also confer the cells with an increased fitness even though these genes may not have any known effect on the cell mitosis rates. Here, we provide insight into these observations by analyzing the effects of cell migration, compression, and contact inhibition on the growth of tumor cell clusters using the Cellular Potts Model (CPM) in a monolayer geometry. This is a follow-up of a previous study (Thalhauser et al., Biol. Direct, 2010, 5:21) in which a Moran-type model was used to study the interaction of cell proliferation, migratory potential and death on the emergence of invasive phenotypes. Here, we extend the study to include the effects of cell size and shape. In particular, we investigate the interplay between cell motility and compressibility within the CPM and find that the CPM predicts that increased cell motility leads to smaller cells. This is an artifact in the CPM. An analysis of the CPM reveals an explicit inverse-relationship between the cell stiffness and motility parameters. We use this relationship to compensate for motility-induced changes in cell size in the CPM so that in the corrected CPM, cell size is independent of the cell motility. We find that subject to comparable levels of compression, clusters of motile cells grow faster than clusters of less motile cells, in qualitative agreement with biological observations and our previous study. Increasing compression tends to reduce growth rates. Contact inhibition penalizes clumped cells by halting their growth and gives motile cells an even greater advantage. Finally, our model predicts cell size distributions that are consistent with those observed in clusters of neuroblastoma cells cultured in low and high density conditions. PMID:24211749

  4. Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules.

    PubMed

    Ghosh, R N; Webb, W W

    1994-05-01

    We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces. PMID:8061186

  5. Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules.

    PubMed Central

    Ghosh, R N; Webb, W W

    1994-01-01

    We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces. Images FIGURE 1 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 PMID:8061186

  6. Notch-Jagged signalling can give rise to clusters of cells exhibiting a hybrid epithelial/mesenchymal phenotype

    PubMed Central

    Jolly, Mohit Kumar; Goldman, Aaron; Pietilä, Mika; Mani, Sendurai A.; Sengupta, Shiladitya; Ben-Jacob, Eshel; Levine, Herbert; Onuchic, Jose’ N.

    2016-01-01

    Metastasis can involve repeated cycles of epithelial-to-mesenchymal transition (EMT) and its reverse mesenchymal-to-epithelial transition. Cells can also undergo partial transitions to attain a hybrid epithelial/mesenchymal (E/M) phenotype that allows the migration of adhering cells to form a cluster of circulating tumour cells. These clusters can be apoptosis-resistant and possess an increased metastatic propensity as compared to the cells that undergo a complete EMT (mesenchymal cells). Hence, identifying the key players that can regulate the formation and maintenance of such clusters may inform anti-metastasis strategies. Here, we devise a mechanism-based theoretical model that links cell–cell communication via Notch-Delta-Jagged signalling with the regulation of EMT. We demonstrate that while both Notch-Delta and Notch-Jagged signalling can induce EMT in a population of cells, only Jagged-dominated Notch signalling, but not Delta-dominated signalling, can lead to the formation of clusters containing hybrid E/M cells. Our results offer possible mechanistic insights into the role of Jagged in tumour progression, and offer a framework to investigate the effects of other microenvironmental signals during metastasis. PMID:27170649

  7. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    NASA Technical Reports Server (NTRS)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  8. Purkinje Activation Precedes Myocardial Activation After Defibrillation Following Long Duration Ventricular Fibrillation

    PubMed Central

    Dosdall, Derek J.; Osorio, Jose; Robichaux, Robert P.; Huang, Jian; Li, Li; Ideker, Raymond E.

    2010-01-01

    Background While reentry within the ventricular myocardium (VM) is responsible for the maintenance of short duration ventricular fibrillation (SDVF, VF duration < 1 min), Purkinje fibers (PFs) are important in the maintenance of long duration ventricular fibrillation (LDVF, VF duration > 1 min). Objective We hypothesized that the mechanisms of defibrillation may also be different for SDVF and LDVF. Methods A multielectrode basket catheter was deployed in the left ventricle of 8 beagles. External defibrillation shocks were delivered with a ramp-up protocol following SDVF (20 s) and LDVF (150 s). Earliest VM and PF activations were identified following the highest energy shock that failed to terminate VF and the successful shock. Results Defibrillation was successful after 36±12 s and 181±14 s for SDVF and LDVF, respectively. The time after shock delivery until earliest activation was detected for failed shocks and was significantly longer following LDVF (138.7±24.1 ms) than SDVF (75.6±8.7 ms). Earliest postshock activation following SDVF typically initiated in the VM (14 of 16 episodes) while it always initiated in the PF (16 of 16 episodes) following LDVF. Sites of earliest activity during sinus rhythm correlated with sites of earliest postshock activation for PF-led cycles but not VM-led cycles. Conclusion Earliest recorded postshock activation is in the Purkinje system following LDVF but not SDVF. This difference raises the possibility that the optimal defibrillation strategy is different for SDVF and LDVF. PMID:20061187

  9. T Cell Epitope Clustering in the Highly Immunogenic BZLF1 Antigen of Epstein-Barr Virus

    PubMed Central

    Rist, Melissa J.; Neller, Michelle A.; Burrows, Jacqueline M.

    2014-01-01

    ABSTRACT Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8+ T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8+ T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8+ T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8+ T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating “immune blind spots” through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by

  10. Desmoplastic small round cell tumor with sphere-like clusters mimicking adenocarcinoma.

    PubMed

    Hattori, Yukinori; Yoshida, Akihiko; Sasaki, Naoshi; Shibuki, Yasuo; Tamura, Kenji; Tsuta, Koji

    2015-03-01

    Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive neoplasm that predominantly affects young men. DSRCT often presents as multiple nodules on the serosal surface and is histologically categorized as a small round cell tumor. However, the cytological spectrum of DSRCT is not fully understood because of its rarity. Here, we report an unusual case of DSRCT that showed spheres of cells without stromal cores in pleural fluid cytology material, a finding that is typically associated with metastatic adenocarcinoma and mesothelioma. The specimen from a simultaneous needle biopsy showed the classic histology of DSRCT, comprising nests of small round cells set in desmoplasia. The diagnosis of DSRCT was further supported by immunohistochemical coexpression of cytokeratin and desmin, as well as Ewing sarcoma breakpoint region 1 gene rearrangement, which was determined by fluorescence in situ hybridization. The unusual cytological finding in this case illustrates a potential pitfall of the cytological diagnosis of pleural fluid or ascites. DSRCT should not be excluded from the differential diagnosis when sphere-like round cell clusters are observed in pleural or abdominal effusion, particularly in young male patients. PMID:24819999

  11. Action of HMGB1 on miR-221/222 cluster in neuroblastoma cell lines

    PubMed Central

    Mari, Emanuela; Zicari, Alessandra; Fico, Flavia; Massimi, Isabella; Martina, Lolli; Mardente, Stefania

    2016-01-01

    microRNA (miR/miRNA) are small non-coding RNAs that control gene expression at the post-transcriptional level by targeting mRNAs. Aberrant expression of miRNAs is often observed in different types of cancer. Specific miRNAs function as tumor suppressors or oncogenes and interfere with various aspects of carcinogenesis, including differentiation, proliferation and invasion. Upregulation of miRNAs 221 and 222 has been shown to induce a malignant phenotype in numerous human cancers via inhibition of phosphatase and tensin homolog (PTEN) expression. Neuroblastoma is the most common extracranial solid malignancy in children, which is characterized by cellular heterogeneity that corresponds to different clinical outcomes. The different cellular phenotypes are associated with different gene mutations and miRs that control genetic and epigenetic factors. For this reason miRs are considered a potential therapeutic target in neuroblastoma. The aim of the present study was to investigate the mechanisms by which extracellular high mobility group box 1 (HMGB1) promotes cell growth in neuroblastoma. SK-N-BE(2) and SH-SY5Y neuroblastoma derived cell lines were transfected with the antisense oligonucleotides, anti-miR-221 and −222, followed by treatment with HMGB1 to investigate the expression of the oncosuppressor PTEN. In this study, it was demonstrated that HMGB1, which is released by damaged cells and tumor cells, upregulates miR-221/222 oncogenic clusters in the two human neuroblastoma derived cell lines. The results revealed that the oncogenic cluster miRs 221/222 were more highly expressed by the most undifferentiated cell line [SK-N-BE(2)] compared with the the less tumorigenic cell line (SH-SY5Y) and that exogenous HMGB1 increases this expression. In addition, HMGB1 modulates PTEN expression via miR-221/222, as demonstrated by transiently blocking miR-221/222 with anti-sense oligonucleotides. These results may lead to the development of novel therapeutic strategies for

  12. Performance of genetically-colorblind individuals on a rapid dark adaptation test based on the Purkinje shift.

    PubMed

    Kim, V; Solomons, N W

    1983-02-01

    An experiment was conducted to determine whether or not genetic colorblindness would limit performance on a rapid dark adaptation test (RDAT) which is based on the Purkinje shift in retinal sensitivity to lower wavelengths of light energy under mesopic/scotopic conditions of illumination. No differences in RDAT performance between age-equivalent colorblind and non-colorblind subjects was observed. PMID:6601793

  13. Clustering and synchronization of lightning flashes in adjacent thunderstorm cells from lightning location networks data

    NASA Astrophysics Data System (ADS)

    Yair, Yoav Y.; Aviv, Reuven; Ravid, Gilad

    2009-05-01

    We analyzed sequences of lightning flashes in several thunderstorms on the basis of data from various ground-based lightning location systems. We identified patterns of clustering and synchronicity of flashes in separate thunderstorm cells, distanced by tens to hundreds of kilometers from each other. This is in-line with our early findings of lightning synchronicity based on space shuttle images (Yair et al., 2006), hinting at a possible mutual electromagnetic coupling of remote thunderstorms. We developed a theoretical model that is based on the leaky integrate-and-fire concept commonly used in models of neural activity, in order to simulate the flashing behavior of a coupled network of thunderstorm cells. In this type of network, the intensity of the electric field Ei within a specific region of thunderstorm (i) grows with time until it reaches the critical breakdown value and generates a lightning flash while its electric field drops to zero, simultaneously adding a delta E to the intensity of the internal electric field in all thundercloud cells (Ej,k,l…) that are linked to it. The value of ΔE is inversely proportional to the distance between the "firing" cell i and its neighbors j, k, l; we assumed that thunderstorm cells are not identical and occupy a grid with random spacing and organization. Several topologies of the thunderstorm network were tested with varying degrees of coupling, assuming a predetermined probability of links between active cells. The results suggest that when the group coupling in the network is higher than a certain threshold value, all thunderstorm cells will flash in a synchronized manner.

  14. New Alzheimer amyloid beta responsive genes identified in human neuroblastoma cells by hierarchical clustering.

    PubMed

    Uhrig, Markus; Ittrich, Carina; Wiedmann, Verena; Knyazev, Yuri; Weninger, Annette; Riemenschneider, Matthias; Hartmann, Tobias

    2009-01-01

    Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Abeta(42), in contrast to Abeta(40), is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Abeta(40) and Abeta(42) levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Abeta(40) and Abeta(42) levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Abeta(42)/Abeta(40) ratio. Importantly however, an increased Abeta(42)/Abeta(40) ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Abeta(42)/Abeta(40) ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Abeta(42)/Abeta(40) ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes. PMID:19707560

  15. New Alzheimer Amyloid β Responsive Genes Identified in Human Neuroblastoma Cells by Hierarchical Clustering

    PubMed Central

    Uhrig, Markus; Ittrich, Carina; Wiedmann, Verena; Knyazev, Yuri; Weninger, Annette; Riemenschneider, Matthias; Hartmann, Tobias

    2009-01-01

    Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Aβ42, in contrast to Aβ40, is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Aβ40 and Aβ42 levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Aβ40 and Aβ42 levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Aβ42/Aβ40 ratio. Importantly however, an increased Aβ42/Aβ40 ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Aβ42/Aβ40 ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Aβ42/Aβ40 ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes. PMID:19707560

  16. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging.

    PubMed

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-14

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes. PMID:27362510

  17. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

    PubMed Central

    Brust, M.; Aouane, O.; Thiébaud, M.; Flormann, D.; Verdier, C.; Kaestner, L.; Laschke, M. W.; Selmi, H.; Benyoussef, A.; Podgorski, T.; Coupier, G.; Misbah, C.; Wagner, C.

    2014-01-01

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects. PMID:24614613

  18. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

    NASA Astrophysics Data System (ADS)

    Brust, M.; Aouane, O.; Thiébaud, M.; Flormann, D.; Verdier, C.; Kaestner, L.; Laschke, M. W.; Selmi, H.; Benyoussef, A.; Podgorski, T.; Coupier, G.; Misbah, C.; Wagner, C.

    2014-03-01

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.

  19. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows.

    PubMed

    Brust, M; Aouane, O; Thiébaud, M; Flormann, D; Verdier, C; Kaestner, L; Laschke, M W; Selmi, H; Benyoussef, A; Podgorski, T; Coupier, G; Misbah, C; Wagner, C

    2014-01-01

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects. PMID:24614613

  20. Microarray Analysis of Siberian Ginseng Cyclic Somatic Embryogenesis Culture Systems Provides Insight into Molecular Mechanisms of Embryogenic Cell Cluster Generation

    PubMed Central

    Zhou, Chenguang; Liu, Likun; Li, Chenghao

    2014-01-01

    Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters. PMID:24743225

  1. Nanoscale Obstacle Arrays Frustrate Transport of EphA2 – Ephrin-A1 Clusters in Cancer Cell Lines

    PubMed Central

    Lohmüller, Theobald; Xu, Qian

    2013-01-01

    Juxtacrine signaling interactions between the EphA2 receptor tyrosine kinase and its ephrin-A1 ligand contribute to healthy tissue maintenance and misregulation of this system is observed in at least 40% of human breast cancer. Hybrid live cell – supported membrane experiments, in which membrane-linked ephrin-A1 displayed in supported membranes interacts with EphA2 in living cells, have revealed large scale clustering of EphA2:ephrin-A1 complexes as well as their lateral transport across the cell surface during triggering. Here, we utilize 100nm spaced hexagonally ordered arrays of gold nanodots embedded within supported membranes to present defined obstacles to the movement and assembly of EphA2 clusters. By functionalizing both the supported membrane and the nanodots with ephrin-A1, we perform a type of affinity chromatography on EphA2 signaling clusters in live cell membranes. Analysis of ten different breast cancer cell lines reveals that EphA2 transport is most frustrated by nanodot arrays in the most diseased cell lines. These observations suggest that strong physical association among EphA2 receptors, as well as their assembly into larger clusters, correlates with and may contribute to the pathological misregulation of the EphA2:ephrin-A1 pathway in breast cancer. PMID:23668885

  2. The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective Migration of Corneal Fibroblasts in Different ECM Environments

    PubMed Central

    Miron-Mendoza, Miguel; Graham, Eric; Kivanany, Pouriska; Quiring, Jonathan; Petroll, W. Matthew

    2015-01-01

    Purpose. We previously reported that extracellular matrix composition (fibrin versus collagen) modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study, we investigate the role of thrombin and cell contractility in mediating these differences in cell behavior. Methods. To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs were cultured in serum-free media containing platelet-derived growth factor (PDGF), with or without thrombin, the Rho kinase inhibitor Y-27632, and/or the myosin II inhibitor blebbistatin. We used 3-dimensional and 4-dimensional imaging to assess cell mechanical behavior, connectivity and cytoskeletal organization. Results. Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also induced Rho kinase–dependent clustering of cells plated on top of compliant collagen matrices, but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when Rho kinase was inhibited. In nested matrices, cells always migrated independently through collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but cells continued to migrate collectively. Conclusions. The results suggest that while thrombin-induced actomyosin contraction can induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is not required for clustering or collective migration of corneal fibroblasts interacting with fibin. PMID:25736789

  3. Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

    PubMed

    Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

    2013-08-01

    Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. PMID:23665237

  4. Micro-ultrasonic cleaving of cell clusters by laser-generated focused ultrasound and its mechanisms

    PubMed Central

    Baac, Hyoung Won; Lee, Taehwa; Guo, L. Jay

    2013-01-01

    Laser-generated focused ultrasound (LGFU) is a unique modality that can produce single-pulsed cavitation and strong local disturbances on a tight focal spot (<100 μm). We utilize LGFU as a non-contact, non-thermal, high-precision tool to fractionate and cleave cell clusters cultured on glass substrates. Fractionation processes are investigated in detail, which confirms distinct cell behaviors in the focal center and the periphery of LGFU spot. For better understanding of local disturbances under LGFU, we use a high-speed laser-flash shadowgraphy technique and then fully visualize instantaneous microscopic processes from the ultrasound wave focusing to the micro-bubble collapse. Based on these visual evidences, we discuss possible mechanisms responsible for the focal and peripheral disruptions, such as a liquid jet-induced wall shear stress and shock emissions due to bubble collapse. The ultrasonic micro-fractionation is readily available for in vitro cell patterning and harvesting. Moreover, it is significant as a preliminary step towards high-precision surgery applications in future. PMID:24010006

  5. B Cell Super-Enhancers and Regulatory Clusters Recruit AID Tumorigenic Activity

    PubMed Central

    Qian, Jason; Wang, Qiao; Dose, Marei; Pruett, Nathanael; Kieffer-Kwon, Kyong-Rim; Resch, Wolfgang; Liang, Genqing; Tang, Zhonghui; Mathé, Ewy; Benner, Christopher; Dubois, Wendy; Nelson, Steevenson; Vian, Laura; Oliveira, Thiago Y.; Jankovic, Mila; Hakim, Ofir; Gazumyan, Anna; Pavri, Rushad; Awasthi, Parirokh; Song, Bin; Liu, Geng; Chen, Longyun; Zhu, Shida; Feigenbaum, Lionel; Staudt, Louis; Murre, Cornelis; Ruan, Yijun; Robbiani, Davide F.; Pan-Hammarström, Qiang; Nussenzweig, Michel C.; Casellas, Rafael

    2014-01-01

    SUMMARY The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA+ enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment. PMID:25483777

  6. An automatic measure for classifying clusters of suspected spikes into single cells versus multiunits

    NASA Astrophysics Data System (ADS)

    Tankus, Ariel; Yeshurun, Yehezkel; Fried, Itzhak

    2009-10-01

    While automatic spike sorting has been investigated for decades, little attention has been allotted to consistent evaluation criteria that will automatically determine whether a cluster of spikes represents the activity of a single cell or a multiunit. Consequently, the main tool for evaluation has remained visual inspection by a human. This paper quantifies the visual inspection process. The results are well-defined criteria for evaluation, which are mainly based on visual features of the spike waveform, and an automatic adaptive algorithm that learns the classification by a given human and can apply similar visual characteristics for classification of new data. To evaluate the suggested criteria, we recorded the activity of 1652 units (single cells and multiunits) from the cerebrum of 12 human patients undergoing evaluation for epilepsy surgery requiring implantation of chronic intracranial depth electrodes. The proposed method performed similar to human classifiers and obtained significantly higher accuracy than two existing methods (three variants of each). Evaluation on two synthetic datasets is also provided. The criteria are suggested as a standard for evaluation of the quality of separation that will allow comparison between different studies. The proposed algorithm is suitable for real-time operation and as such may allow brain-computer interfaces to treat single cells differently than multiunits.

  7. Nonblack patients with sickle cell disease have African. beta. sup s gene cluster haplotypes

    SciTech Connect

    Rogers, Z.R.; Powars, D.R.; Williams, W.D. ); Kinney, T.R. ); Schroeder, W.A. )

    1989-05-26

    Of 18 nonblack patients with sickle cell disease, 14 had sickle cell anemia, 2 had hemoglobin SC disease, and 2 had hemoglobin S-{beta}{sup o}-thalassemia. The {beta}{sup s} gene cluster haplotypes that were determined in 7 patients were of African origin and were identified as Central African Republic, Central African Republic minor II, Benin, and Senegal. The haplotype Central African Republic minor II was present on the {beta}{sup o}-thalassemia chromosome in 2 patients. None of 10 patients whose {alpha}-gene status was determined had {alpha}-thalassemia-2. These data strongly support the concept that the {beta}{sup s} gene on chromosome 11 of these individuals is of African origin and that the {alpha}-gene locus on chromosome 16 is of white or native American origin. The clinical severity of the disease in these nonblack patients is appropriate to their haplotype without {alpha}-thalassemia-2 and is comparable with that of black patients. All persons with congenital hemolytic anemia should be examined for the presence of sickle cell disease regardless of physical appearance or ethnic background.

  8. An automatic measure for classifying clusters of suspected spikes into single cells versus multiunits

    PubMed Central

    Tankus, Ariel; Yeshurun, Yehezkel; Fried, Itzhak

    2010-01-01

    While automatic spike sorting has been investigated for decades, little attention has been allotted to consistent evaluation criteria that will automatically determine whether a cluster of spikes represents the activity of a single cell or a multiunit. Consequently, the main tool for evaluation has remained visual inspection by a human. This paper quantifies the visual inspection process. The results are well-defined criteria for evaluation, which are mainly based on visual features of the spike waveform, and an automatic adaptive algorithm that learns the classification by a given human and can apply similar visual characteristics for classification of new data. To evaluate the suggested criteria, we recorded the activity of 1652 units (single cells and multiunits) from the cerebrum of 12 human patients undergoing evaluation for epilepsy surgery requiring implantation of chronic intracranial depth electrodes. The proposed method performed similar to human classifiers and obtained significantly higher accuracy than two existing methods (three variants of each). Evaluation on two synthetic datasets is also provided. The criteria are suggested as a standard for evaluation of the quality of separation that will allow comparison between different studies. The proposed algorithm is suitable for real-time operation and as such may allow brain–computer interfaces to treat single cells differently than multiunits. PMID:19667458

  9. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells.

    PubMed

    Stern, Michael D; Maltseva, Larissa A; Juhaszova, Magdalena; Sollott, Steven J; Lakatta, Edward G; Maltsev, Victor A

    2014-05-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating

  10. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells

    PubMed Central

    Maltseva, Larissa A.; Juhaszova, Magdalena; Sollott, Steven J.; Lakatta, Edward G.; Maltsev, Victor A.

    2014-01-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This “calcium clock” takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic–mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in

  11. Adaptive Mesh Refinement and Adaptive Time Integration for Electrical Wave Propagation on the Purkinje System

    PubMed Central

    Ying, Wenjun; Henriquez, Craig S.

    2015-01-01

    A both space and time adaptive algorithm is presented for simulating electrical wave propagation in the Purkinje system of the heart. The equations governing the distribution of electric potential over the system are solved in time with the method of lines. At each timestep, by an operator splitting technique, the space-dependent but linear diffusion part and the nonlinear but space-independent reactions part in the partial differential equations are integrated separately with implicit schemes, which have better stability and allow larger timesteps than explicit ones. The linear diffusion equation on each edge of the system is spatially discretized with the continuous piecewise linear finite element method. The adaptive algorithm can automatically recognize when and where the electrical wave starts to leave or enter the computational domain due to external current/voltage stimulation, self-excitation, or local change of membrane properties. Numerical examples demonstrating efficiency and accuracy of the adaptive algorithm are presented. PMID:26581455

  12. Dark Adaptation and Purkinje Shift: A Laboratory Exercise in Perceptual Neuroscience

    PubMed Central

    Wolfe, Uta; Ali, Nasim

    2015-01-01

    The systematic measurement of luminance thresholds during dark adaptation usually requires advanced optical equipment not available in most undergraduate classes. Here we describe an easy, inexpensive alternative that uses a printed grayscale to measure visual thresholds. Adaptation curves found with this method are comparable to those found with the technologically advanced tools in the standard literature and even show the shift from cone to rod vision at around 4–8 minutes. The exercise can furthermore be easily combined with a demonstration of the Purkinje shift (the different spectral sensitivity of the rod and cone systems) and of multi-sensory integration across vision, touch and proprioception. The lab allows students to collect, graph and analyze both qualitative and quantitative data. Student ratings of the activity are highly positive, even when compared to other visual neuroscience labs. The activity provides an effective and accessible tool for teaching several important neuroscience concepts, including retinal circuitry, spectral sensitivity, and multi-sensory integration. PMID:25838803

  13. Partial characterization of genes whose transcripts accumulate preferentially in cell clusters at the earliest stage of carrot somatic embryogenesis.

    PubMed

    Yasuda, H; Nakajima, M; Ito, T; Ohwada, T; Masuda, H

    2001-04-01

    We attempted to identify genes that are preferentially expressed immediately after somatic cells divide to form cell clusters at the earliest stage of carrot somatic embryogenesis when they are not or barely expressed in non-embryogenic suspension-cultured cells in the presence of 2,4-D. Using the differential display technique, we isolated three cDNA clones, designated No. 43, No. 87 and No. 93. The No. 43 transcript was preferentially expressed in the earliest cell clusters, its level decreased drastically at the globular and heart-shaped and torpedo-shaped stages, and it was not detected in non-embryogenic suspension-cultured cells. No. 43 cDNA encoded a protein with homology to thaumatin-like proteins and the deduced positions of seven cysteine residues in the 63 amino acid sequence from the carboxyl terminus were identical to those in thaumatin-like proteins. The full-length nucleotide sequence of No. 93 cDNA was determined and its product was about 80% homologous to precursor of the 14 kDa proline-rich DC 2.15 protein of carrot at the amino acid level. However, the deduced amino acid sequence lacked the characteristic core of repeating Pro-X motifs found in DC 2.15. The No. 93 transcript accumulated preferentially in the earliest cell clusters but it was also detected at a low level in non-embryogenic suspension-cultured cells, unlike DC 2.15 transcripts that begin to accumulate in heart-shaped embryos before their level falls in torpedo-shaped embryos. No. 87 transcripts were expressed preferentially in the earliest cell clusters that has been incubated with 2,4-D but were also detected at a low level in suspension-cultured cells subcultured in the continued presence of 2,4-D. The No. 87 cDNA exhibited no significant homology to any sequences in databases. PMID:11430432

  14. Costimulation with anti-cluster of differentiation 3 and anti-cluster of differentiation 28 reduces the activity of mucin 1-stimulated human mononuclear cells

    PubMed Central

    WRIGHT, STEPHEN E.; REWERS-FELKINS, KATHLEEN A.; QUINLIN, IMELDA; ZOHRA, FATEMA; AHMED, JEWEL

    2016-01-01

    Cytotoxic T-lymphocyte activation and extension of the cell life span is necessary in order to enable immunotherapy to perform effectively against cancer. In the present study, mucin 1 (MUC1)-stimulated human mononuclear cells (M1SHMCs) were costimulated with bead-attached monoclonal antibodies specific for cluster of differentiation (CD)3 and CD28 receptors. The study was undertaken to determine whether costimulation was capable of enhancing the killing of cancer cells in vitro and of protecting non-obese diabetic severe combined immunodeficient mice from tumor development. Lysis of MCF-7 tumor cells by M1SHMCs was reduced following costimulation with anti-CD3 and anti-CD28. Furthermore, costimulation with anti-CD3 and anti-CD28 eliminated the protective effects of M1SHMCs on MCF-7 breast cancer cell growth in the non-obese diabetic severe combined immunodeficient mice. The present study suggested that costimulation with anti-CD3 and anti-CD28 is not advisable following antigen activation of lymphocytes under the conditions used here. Using a lower anti-CD3/CD28 bead to T-cell ratio may prevent immune suppression, however, further studies are required to support this hypothesis. PMID:26870234

  15. Dimensionality and size scaling of coordinated Ca(2+) dynamics in MIN6 β-cell clusters.

    PubMed

    Hraha, Thomas H; Bernard, Abigail B; Nguyen, Linda M; Anseth, Kristi S; Benninger, Richard K P

    2014-01-01

    Pancreatic islets of Langerhans regulate blood glucose homeostasis by the secretion of the hormone insulin. Like many neuroendocrine cells, the coupling between insulin-secreting β-cells in the islet is critical for the dynamics of hormone secretion. We have examined how this coupling architecture regulates the electrical dynamics that underlie insulin secretion by utilizing a microwell-based aggregation method to generate clusters of a β-cell line with defined sizes and dimensions. We measured the dynamics of free-calcium activity ([Ca(2+)]i) and insulin secretion and compared these measurements with a percolating network model. We observed that the coupling dimension was critical for regulating [Ca(2+)]i dynamics and insulin secretion. Three-dimensional coupling led to size-invariant suppression of [Ca(2+)]i at low glucose and robust synchronized [Ca(2+)]i oscillations at elevated glucose, whereas two-dimensional coupling showed poor suppression and less robust synchronization, with significant size-dependence. The dimension- and size-scaling of [Ca(2+)]i at high and low glucose could be accurately described with the percolating network model, using similar network connectivity. As such this could explain the fundamentally different behavior and size-scaling observed under each coupling dimension. This study highlights the dependence of proper β-cell function on the coupling architecture that will be important for developing therapeutic treatments for diabetes such as islet transplantation techniques. Furthermore, this will be vital to gain a better understanding of the general features by which cellular interactions regulate coupled multicellular systems. PMID:24411262

  16. Global optimization and oxygen dissociation on polyicosahedral Ag32Cu6 core-shell cluster for alkaline fuel cells

    NASA Astrophysics Data System (ADS)

    Zhang, N.; Chen, F. Y.; Wu, X. Q.

    2015-07-01

    The structure of 38 atoms Ag-Cu cluster is studied by using a combination of a genetic algorithm global optimization technique and density functional theory (DFT) calculations. It is demonstrated that the truncated octahedral (TO) Ag32Cu6 core-shell cluster is less stable than the polyicosahedral (pIh) Ag32Cu6 core-shell cluster from the atomistic models and the DFT calculation shows an agreeable result, so the newfound pIh Ag32Cu6 core-shell cluster is further investigated for potential application for O2 dissociation in oxygen reduction reaction (ORR). The activation energy barrier for the O2 dissociation on pIh Ag32Cu6 core-shell cluster is 0.715 eV, where the d-band center is -3.395 eV and the density of states at the Fermi energy level is maximal for the favorable absorption site, indicating that the catalytic activity is attributed to a maximal charge transfer between an oxygen molecule and the pIh Ag32Cu6 core-shell cluster. This work revises the earlier idea that Ag32Cu6 core-shell nanoparticles are not suitable as ORR catalysts and confirms that Ag-Cu nanoalloy is a potential candidate to substitute noble Pt-based catalyst in alkaline fuel cells.

  17. Global optimization and oxygen dissociation on polyicosahedral Ag32Cu6 core-shell cluster for alkaline fuel cells

    PubMed Central

    Zhang, N.; Chen, F. Y.; Wu, X.Q.

    2015-01-01

    The structure of 38 atoms Ag-Cu cluster is studied by using a combination of a genetic algorithm global optimization technique and density functional theory (DFT) calculations. It is demonstrated that the truncated octahedral (TO) Ag32Cu6 core-shell cluster is less stable than the polyicosahedral (pIh) Ag32Cu6 core-shell cluster from the atomistic models and the DFT calculation shows an agreeable result, so the newfound pIh Ag32Cu6 core-shell cluster is further investigated for potential application for O2 dissociation in oxygen reduction reaction (ORR). The activation energy barrier for the O2 dissociation on pIh Ag32Cu6 core-shell cluster is 0.715 eV, where the d-band center is −3.395 eV and the density of states at the Fermi energy level is maximal for the favorable absorption site, indicating that the catalytic activity is attributed to a maximal charge transfer between an oxygen molecule and the pIh Ag32Cu6 core-shell cluster. This work revises the earlier idea that Ag32Cu6 core-shell nanoparticles are not suitable as ORR catalysts and confirms that Ag-Cu nanoalloy is a potential candidate to substitute noble Pt-based catalyst in alkaline fuel cells. PMID:26148904

  18. Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells

    PubMed Central

    De Kumar, Bony; Parrish, Mark E.; Slaughter, Brian D.; Unruh, Jay R.; Gogol, Madelaine; Seidel, Christopher; Paulson, Ariel; Li, Hua; Gaudenz, Karin; Peak, Allison; McDowell, William; Fleharty, Brian; Ahn, Youngwook; Lin, Chengqi; Smith, Edwin; Shilatifard, Ali; Krumlauf, Robb

    2015-01-01

    The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development. PMID:26025802

  19. Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.

    PubMed

    De Kumar, Bony; Parrish, Mark E; Slaughter, Brian D; Unruh, Jay R; Gogol, Madelaine; Seidel, Christopher; Paulson, Ariel; Li, Hua; Gaudenz, Karin; Peak, Allison; McDowell, William; Fleharty, Brian; Ahn, Youngwook; Lin, Chengqi; Smith, Edwin; Shilatifard, Ali; Krumlauf, Robb

    2015-08-01

    The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their ass