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Sample records for cell autonomous purkinje

  1. The Purkinje cell; 2008 style

    PubMed Central

    Dun, Wen; Boyden, Penelope A.

    2010-01-01

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

  2. 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

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

    PubMed

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

    2010-12-15

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

  4. 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

  5. THE FINE STRUCTURE OF THE PURKINJE CELL

    PubMed Central

    Herndon, Robert M.

    1963-01-01

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

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    2014-04-01

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

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

    PubMed Central

    Hawkes, Richard

    2014-01-01

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

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

    PubMed

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

    2006-07-15

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

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

    PubMed

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

    2015-07-01

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

  11. 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.

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

    PubMed

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

    2014-06-01

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

  13. Purkinje cell vulnerability to mild traumatic brain injury.

    PubMed

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

    1996-05-01

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

  14. A Signal Processing Analysis of Purkinje Cells in vitro

    PubMed Central

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

    2010-01-01

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

  15. Encoding of whisker input by cerebellar Purkinje cells

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1992-06-15

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

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

    PubMed

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

    2006-03-27

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

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

    PubMed

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

    1997-09-21

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

  1. Motor learning of mice lacking cerebellar Purkinje cells

    PubMed Central

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

    2013-01-01

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

  2. Motor learning of mice lacking cerebellar Purkinje cells.

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1981-12-01

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

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

    PubMed Central

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

    1966-01-01

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

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

    PubMed

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

    2012-07-01

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

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

    PubMed

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

    2010-12-01

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

  8. 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.…

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed

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

    2001-05-15

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

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

    PubMed

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

    2013-06-01

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

  12. 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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2014-05-01

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

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

    PubMed

    Carletti, Barbara; Rossi, Ferdinando

    2005-09-01

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

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

    PubMed

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

    2013-01-01

    Two wild fledgling kestrels exhibited lack of motor coordination, postural reaction deficits, and abnormal propioception. At necropsy, the cerebellum and brainstem were markedly underdeveloped. Microscopically, there was Purkinje cells heterotopy, abnormal circuitry, and hypoplasia with defective foliation. Heterotopic neurons were identified as immature Purkinje cells by their size, location, immunoreactivity for calbindin D-28 K, and ultrastructural features. The authors suggest that this cerebellar abnormality was likely due to a disruption of molecular mechanisms that dictate Purkinje cell migration, placement, and maturation in early embryonic development. The etiology of this condition remains undetermined. Congenital central nervous system disorders have rarely been reported in birds.

  17. Regular Patterns in Cerebellar Purkinje Cell Simple Spike Trains

    PubMed Central

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

    2007-01-01

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

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

    PubMed

    Najac, Marion; Raman, Indira M

    2015-01-14

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

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

    PubMed

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

    2010-10-01

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

  20. 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

  1. 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...

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2007-02-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

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

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

    PubMed

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

    2002-01-01

    nearly complete deafferentation of the lateral aspect of the fastigial nucleus from the cerebellar cortex, in particular the dorsolateral protuberance. Thus, the data point strongly to a cerebellar contribution to audiogenic myoclonus. Single-neuron electrophysiology experiments in monkeys have demonstrated that the evoked activity in the deep cerebellar nuclei occurs too late to initiate the startle response (60) and electromyography of the postischemic myoclonus of rats corroborates this view (see Chapter 31) (20). However, the nearly complete loss of GABAergic terminals in the dorsolateral protuberance after Purkinje cell death would be expected to dramatically increase its tonic firing and the background excitation of the brain-stem structures that it innervates. The fastigial nucleus innervates a large number of autonomic and motor structures in the brainstem and diencephalon, including the ventrolateral nucleus of the thalamus and the gigantocellular reticular nucleus in the medulla--structures that have been implicated in human posthypoxic myoclonus (6, 7). We propose that the posthypoxic myoclonic jerk of rats is, at least in part, due to disinhibition of the fastigial nucleus produced by patterned Purkinje cell death in the vermis. The argument is as follows: the loss of GABAergic inhibition in the fastigial nucleus after ischemia leads to diaschisis of the motor thalamus and reticular formation which, in turn, is responsible for enhanced motor excitability and myoclonus. That the audiogenic myoclonus after global brain ischemia in the rat gradually resolves over a period of 2 to 3 weeks is consistent with this view, as restoration of background excitability after CNS damage in rats has been documented to occur within this time-frame (61). Our view brings together the physiologic finding that posthypoxic myoclonus appears to originate in the sensory-motor cortices and/or reticular formation with the consistent anatomical finding of Purkinje cell loss after

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

    PubMed

    Watanabe, Masahiko

    2008-03-01

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

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

    PubMed

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

    2007-06-05

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

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

    PubMed

    Pascual, Rodrigo; Bustamante, Carlos

    2013-01-01

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

  11. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1986-01-01

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

  15. 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.

  16. Systematic Regional Variations in Purkinje Cell Spiking Patterns

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2012-01-01

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

  18. Climbing fiber synapse elimination in cerebellar Purkinje cells.

    PubMed

    Watanabe, Masahiko; Kano, Masanobu

    2011-11-01

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

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

    PubMed

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

    2010-11-01

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

  20. 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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed Central

    Dusart, Isabelle; Flamant, Frederic

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2006-10-01

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

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

    PubMed Central

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

    1999-01-01

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

  9. 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

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

    PubMed

    Etzion, Y; Grossman, Y

    2001-08-01

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

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

    PubMed

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

    2016-11-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2007-01-01

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

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

    PubMed Central

    Hounsgaard, J; Nicholson, C

    1983-01-01

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

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

    PubMed

    Gianola, Sara; Rossi, Ferdinando

    2004-02-01

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

  16. 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

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

    PubMed Central

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

    2016-01-01

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

  18. 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

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

    PubMed

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

    2008-08-01

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

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

    PubMed

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

    2017-02-13

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

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

    PubMed Central

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

    2008-01-01

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

  2. 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.

  3. 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

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

    PubMed

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

    2012-07-01

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

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

    PubMed

    Pompeiano, O; Andre, P; Manzoni, D

    1997-12-01

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

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

    PubMed

    Yuzaki, Michisuke

    2012-06-01

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

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

    PubMed

    Laure-Kamionowska, Milena; Maślińska, Danuta

    2009-01-01

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

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

    PubMed

    McKay, B E; Turner, R W

    2004-08-01

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

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

    PubMed

    Ibhazehiebo, K; Koibuchi, N

    2012-06-07

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

  10. 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.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2009-02-01

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

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

    PubMed

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

    2013-07-31

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

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

    PubMed

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

    1998-07-01

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

  15. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Llinás, R; Sugimori, M

    1980-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. 3-D confocal laser scanning microscopy used in morphometric analysis of rat Purkinje cell dendritic spines after chronic ethanol consumption.

    PubMed

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

    1998-12-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    1996-01-01

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

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

    SciTech Connect

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

    1988-02-01

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

  3. 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.

  4. 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

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

    PubMed

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

    2003-01-01

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

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

    NASA Astrophysics Data System (ADS)

    de Schutter, Erik; Bower, James M.

    1994-05-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2008-01-01

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

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

    PubMed

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

    2008-02-15

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

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

    PubMed

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

    1998-03-01

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

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

    PubMed

    Grimaldi, Piercesare; Rossi, Ferdinando

    2006-05-01

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

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

    PubMed

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

    2015-09-09

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2008-02-06

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

  15. 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.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2011-04-01

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

  18. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2011-02-01

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

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

    PubMed

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

    2017-03-01

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

  3. 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.

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

    SciTech Connect

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

    1988-07-01

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

  5. 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.

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

    PubMed

    Krasnov, I B

    2008-01-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2005-10-11

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

  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

    2012-12-07

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

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

    PubMed

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

    2008-06-04

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

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

    PubMed

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

    2013-03-01

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

  13. 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.

  14. 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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-03-15

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

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

    PubMed

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

    2010-10-14

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

  18. 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.

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

    PubMed Central

    Alviña, Karina

    2014-01-01

    Although it has been suggested that the cerebellum functions to predict the sensory consequences of motor commands, how such predictions are implemented in cerebellar circuitry remains largely unknown. A detailed and relatively complete account of predictive mechanisms has emerged from studies of cerebellum-like sensory structures in fish, suggesting that comparisons of the cerebellum and cerebellum-like structures may be useful. Here we characterize electrophysiological response properties of Purkinje cells in a region of the cerebellum proper of weakly electric mormyrid fish, the posterior caudal lobe (LCp), which receives the same mossy fiber inputs and projects to the same target structures as the electrosensory lobe (ELL), a well-studied cerebellum-like structure. We describe patterns of simple spike and climbing fiber activation in LCp Purkinje cells in response to motor corollary discharge, electrosensory, and proprioceptive inputs and provide evidence for two functionally distinct Purkinje cell subtypes within LCp. Protocols that induce rapid associative plasticity in ELL fail to induce plasticity in LCp, suggesting differences in the adaptive functions of the two structures. Similarities and differences between LCp and ELL are discussed in light of these results. PMID:24790163

  20. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Han, W; Wang, Z; Nattel, S

    2001-03-01

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

  3. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Micieli, Jonathan A; Margolin, Edward A

    2017-03-01

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

  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. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  11. 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

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2011-01-01

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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-07-09

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

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

    PubMed Central

    Sacco, Tiziana; Tempia, Filippo

    2002-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2006-08-25

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

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

    PubMed

    Spatz, W B

    2003-09-05

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

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

    PubMed

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

    2014-01-01

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

  7. 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

  8. Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

    PubMed

    Tsai, Peter T; Hull, Court; Chu, YunXiang; Greene-Colozzi, Emily; Sadowski, Abbey R; Leech, Jarrett M; Steinberg, Jason; Crawley, Jacqueline N; Regehr, Wade G; Sahin, Mustafa

    2012-08-30

    Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

  9. 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

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

    PubMed Central

    Khilkevich, Andrei; Mauk, Michael D.

    2015-01-01

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

  11. 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.

  12. 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.

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

    PubMed

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

    2009-09-01

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

  14. 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.

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

    PubMed Central

    Rapp, M; Segev, I; Yarom, Y

    1994-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2009-01-01

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

  18. 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

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

    PubMed

    Yang, Zhilai; Wang, Jin-Hui

    2013-12-01

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

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

    PubMed

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

    2007-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-09-26

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

  3. 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

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

    PubMed

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

    2008-04-30

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

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

    PubMed Central

    Roth, Arnd; Häusser, Michael

    2001-01-01

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

  6. 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

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

    PubMed

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

    2009-12-16

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

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

    PubMed Central

    Hanck, D A; Sheets, M F

    1992-01-01

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

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

    PubMed

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

    2016-08-15

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

  10. 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

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

    PubMed

    Rota, Marcello; Vassalle, Mario

    2003-04-01

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

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

    PubMed Central

    Armstrong, D M; Edgley, S A

    1984-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    1988-12-01

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

  15. 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

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

    PubMed

    Coop, A D; Reeke, G N

    2001-01-01

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

  17. Cell Autonomous and Non-autonomous Effects of Senescent Cells in the Skin

    PubMed Central

    Demaria, Marco; Desprez, Pierre Yves; Campisi, Judith; Velarde, Michael C.

    2015-01-01

    Human and mouse skin accumulate senescent cells in both the epidermis and dermis during aging. When chronically present, senescent cells are thought to enhance the age-dependent deterioration of the skin during extrinsic and intrinsic aging. However, when transiently present, senescent cells promote optimal wound healing. Here, we review recent studies on how senescent cells and the senescence-associated secretory phenotype (SASP) contribute to different physiological and pathophysiological conditions in the skin with a focus on some of the cell autonomous and non-autonomous functions of senescent cells in the context of skin aging and wound healing. PMID:25855157

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

    PubMed

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

    2003-01-01

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

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

    PubMed

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

    2003-08-20

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

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

    PubMed Central

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

    2016-01-01

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

  1. Cell-autonomous and non-cell-autonomous toxicity in polyglutamine diseases.

    PubMed

    Sambataro, Fabio; Pennuto, Maria

    2012-05-01

    Polyglutamine diseases are neurodegenerative disorders caused by expansion of polyglutamine tracts in the coding regions of specific genes. One of the most important features of polyglutamine diseases is that, despite the widespread and in some cases ubiquitous expression of the polyglutamine proteins, specific populations of neurons degenerate in each disease. This finding has led to the idea that polyglutamine diseases are cell-autonomous diseases, in which selective neuronal dysfunction and death result from damage caused by the mutant protein within the targeted neuronal population itself. Development of animal models for conditional expression of polyglutamine proteins, along with new pharmacologic manipulation of polyglutamine protein expression and toxicity, has led to a remarkable change of the current view of polyglutamine diseases as cell-autonomous disorders. It is becoming evident that toxicity in the neighboring non-neuronal cells contributes to selective neuronal damage. This observation implies non-cell-autonomous mechanisms of neurodegeneration in polyglutamine diseases. Here, we describe cell-autonomous and non-cell-autonomous mechanisms of polyglutamine disease pathogenesis, including toxicity in neurons, skeletal muscle, glia, germinal cells, and other cell types.

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

    PubMed

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

    2001-10-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

    Xu, Jie; Hoebeek, Freek E.

    2016-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2011-07-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. 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

  10. 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

  11. 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.

  12. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    1997-01-01

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

  15. 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

  16. Do cell-autonomous and non-cell-autonomous effects drive the structure of tumor ecosystems?

    PubMed

    Tissot, Tazzio; Ujvari, Beata; Solary, Eric; Lassus, Patrice; Roche, Benjamin; Thomas, Frédéric

    2016-04-01

    By definition, a driver mutation confers a growth advantage to the cancer cell in which it occurs, while a passenger mutation does not: the former is usually considered as the engine of cancer progression, while the latter is not. Actually, the effects of a given mutation depend on the genetic background of the cell in which it appears, thus can differ in the subclones that form a tumor. In addition to cell-autonomous effects generated by the mutations, non-cell-autonomous effects shape the phenotype of a cancer cell. Here, we review the evidence that a network of biological interactions between subclones drives cancer cell adaptation and amplifies intra-tumor heterogeneity. Integrating the role of mutations in tumor ecosystems generates innovative strategies targeting the tumor ecosystem's weaknesses to improve cancer treatment.

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed Central

    Yamaguchi, Kazuhiko; Itohara, Shigeyoshi; Ito, Masao

    2016-01-01

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

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

    PubMed Central

    Hong, Simon; Optican, Lance M.

    2008-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  1. 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

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

    PubMed Central

    Glitsch, H G; Tappe, A

    1995-01-01

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

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

    PubMed

    Ishikawa, Kinya; Mizusawa, Hidehiro

    2010-10-01

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

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

    PubMed

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

    2008-09-22

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

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

    PubMed

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

    2010-10-22

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

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

    PubMed Central

    Warren, M A; Bedi, K S

    1988-01-01

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

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

    PubMed

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

    2014-01-01

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

  8. Cell Autonomous Shape Changes in Germband Retraction

    NASA Astrophysics Data System (ADS)

    Lynch, Holley; Kim, Elliott; Gish, Robert; Hutson, M. Shane

    2012-02-01

    Germband retraction involves the cohesive movement and regulated cellular mechanics of two tissues on the surface of fruit fly embryos, the germband and the amnioserosa. The germband initially forms a `U' shape, curling from the ventral surface, around the posterior of the embryo, and onto the dorsal surface; the amnioserosa lies between the arms of this `U'. Retraction straightens the germband and leaves it only on the ventral side. During retraction, the germband becomes clearly segmented with deep furrows between segments, and its cells elongate towards the amnioserosa, along what becomes the dorsal-ventral axis. To determine the importance of these changes for the overall movement of the tissues, we observed embryos that did not complete germband retraction due to targeted laser ablation of half the amnioserosa. Without the chemical and mechanical influence of the amnioserosa, germband furrows still formed and germband cells still elongated; however, this elongation was misaligned compared to unablated embryos. Thus, furrow formation and cell elongation in the germband are autonomous, but insufficient to drive proper tissue motion. These results suggest that part of the necessary role of the amnioserosa is proper orientation of germband cell elongation.

  9. Dual functions of cell-autonomous and non-cell-autonomous ADAM10 activity in granulopoiesis.

    PubMed

    Yoda, Masaki; Kimura, Tokuhiro; Tohmonda, Takahide; Uchikawa, Shinichi; Koba, Takeshi; Takito, Jiro; Morioka, Hideo; Matsumoto, Morio; Link, Daniel C; Chiba, Kazuhiro; Okada, Yasunori; Toyama, Yoshiaki; Horiuchi, Keisuke

    2011-12-22

    Previous studies have revealed various extrinsic stimuli and factors involved in the regulation of hematopoiesis. Among these, Notch-mediated signaling has been suggested to be critically involved in this process. Herein, we show that conditional inactivation of ADAM10, a membrane-bound protease with a crucial role in Notch signaling (S2 cleavage), results in myeloproliferative disorder (MPD) highlighted by severe splenomegaly and increased populations of myeloid cells and hematopoietic stem cells. Reciprocal transfer of bone marrow cells between wild-type and ADAM10 mutant mice revealed that ADAM10 activity in both hematopoietic and nonhematopoietic cells is involved in the development of MPD. Notably, we found that MPD caused by lack of ADAM10 in nonhematopoietic cells was mediated by G-CSF, whereas MPD caused by ADAM10-deficient hematopoietic cells was not. Taken together, the present findings reveal previously undescribed nonredundant roles of cell-autonomous and non-cell-autonomous ADAM10 activity in the maintenance of hematopoiesis.

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

    PubMed

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

    2014-01-01

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

  11. Regulation of cell-non-autonomous proteostasis in metazoans

    PubMed Central

    O'Brien, Daniel; van Oosten-Hawle, Patricija

    2016-01-01

    Cells have developed robust adaptation mechanisms to survive environmental conditions that challenge the integrity of their proteome and ensure cellular viability. These are stress signalling pathways that integrate extracellular signals with the ability to detect and efficiently respond to protein-folding perturbations within the cell. Within the context of an organism, the cell-autonomous effects of these signalling mechanisms are superimposed by cell-non-autonomous stress signalling pathways that allow co-ordination of stress responses across tissues. These transcellular stress signalling pathways orchestrate and maintain the cellular proteome at an organismal level. This article focuses on mechanisms in both invertebrate and vertebrate organisms that activate stress responses in a cell-non-autonomous manner. We discuss emerging insights and provide specific examples on how components of the cell-non-autonomous proteostasis network are used in cancer and protein-folding diseases to drive disease progression across tissues. PMID:27744329

  12. Regulation of cell-non-autonomous proteostasis in metazoans.

    PubMed

    O'Brien, Daniel; van Oosten-Hawle, Patricija

    2016-10-15

    Cells have developed robust adaptation mechanisms to survive environmental conditions that challenge the integrity of their proteome and ensure cellular viability. These are stress signalling pathways that integrate extracellular signals with the ability to detect and efficiently respond to protein-folding perturbations within the cell. Within the context of an organism, the cell-autonomous effects of these signalling mechanisms are superimposed by cell-non-autonomous stress signalling pathways that allow co-ordination of stress responses across tissues. These transcellular stress signalling pathways orchestrate and maintain the cellular proteome at an organismal level. This article focuses on mechanisms in both invertebrate and vertebrate organisms that activate stress responses in a cell-non-autonomous manner. We discuss emerging insights and provide specific examples on how components of the cell-non-autonomous proteostasis network are used in cancer and protein-folding diseases to drive disease progression across tissues.

  13. 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

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

    PubMed

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

    1998-09-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Kawaguchi, Shin-ya; Hirano, Tomoo

    2013-01-01

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

  17. 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.

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

    PubMed

    Karelina, T V

    2012-01-01

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

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

    PubMed Central

    Hoffman, Jessica F.; McCarthy, Margaret M.

    2016-01-01

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

  20. Salidroside accelerates fracture healing through cell-autonomous and non-autonomous effects on osteoblasts.

    PubMed

    Guo, Xiao Qin; Qi, Lin; Yang, Jing; Wang, Yue; Wang, Chuan; Li, Zong Min; Li, Ling; Qu, Ye; Wang, Dan; Han, Ze Min

    2017-02-01

    Salidroside (SAL), a major active component of Rhodiola rosea L., exhibits diverse pharmacological effects. However, the direct roles of SAL in fracture healing remain largely unknown. Here, we demonstrate that SAL significantly promotes proliferation by altering the cell-cycle distribution of osteoblastic cells. SAL also greatly stimulates osteoblast differentiation and mineralization by inducing the expression of Runx2 and Osterix. In addition to its osteoblast-autonomous effects, SAL can activate the HIF-1α pathway coupling of angiogenesis and osteogenesis through cell-non-autonomous effects. Our in vitro results suggest that SAL significantly up-regulates HIF-1α expression at the mRNA and protein levels. Furthermore, the nuclear translocation and transcriptional activity of HIF-1α and the HIF-responsive gene VEGF increase following SAL treatment. Our mechanistic study revealed that the regulation of osteoblastic proliferation and HIF-1α expression partly involves MAPK/ERK and PI3K/Akt signaling. Our in vivo analysis also demonstrated that SAL can promote angiogenesis within the callus and accelerate fracture healing. Thus, SAL promotes skeletal regeneration in cell-autonomous and cell-non-autonomous ways and might be a potential therapy for accelerating fracture healing.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-10-01

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

  5. Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response

    PubMed Central

    Shao, Li-Wa; Niu, Rong; Liu, Ying

    2016-01-01

    Neurons have a central role in the systemic coordination of mitochondrial unfolded protein response (UPRmt) and the cell non-autonomous modulation of longevity. However, the mechanism by which the nervous system senses mitochondrial stress and communicates to the distal tissues to induce UPRmt remains unclear. Here we employ the tissue-specific CRISPR-Cas9 approach to disrupt mitochondrial function only in the nervous system of Caenorhabditis elegans, and reveal a cell non-autonomous induction of UPRmt in peripheral cells. We further show that a neural sub-circuit composed of three types of sensory neurons, and one interneuron is required for sensing and transducing neuronal mitochondrial stress. In addition, neuropeptide FLP-2 functions in this neural sub-circuit to signal the non-autonomous UPRmt. Taken together, our results suggest a neuropeptide coordination of mitochondrial stress response in the nervous system. PMID:27767096

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

    PubMed Central

    Bower, James M.

    2010-01-01

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

  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. Investigation of autonomous cell dynamics using holographic laser microsurgery

    NASA Astrophysics Data System (ADS)

    Jayasinghe, Aroshan; Hutson, M. Shane

    2012-02-01

    Laser-microsurgery has emerged as a powerful technique for evaluating in vivo tissue mechanics. We extend this technique by using a spatial light modulator (SLM) to diffract a single UV laser pulse to simultaneously ablate multiple points in living tissue. Using this method, we can quickly and cleanly isolate a single cell by destroying all its nearest neighbors. The post-ablation dynamics of such an isolated cell are then largely dependent on autonomous intracellular forces. Here, we use this technique to investigate cell shape pulsations in amnioserosa cells in Drosophila embryos during dorsal closure -- specifically to address the degree to which these pulsations are cell autonomous or driven by the contractions of neighboring cells. When cells are isolated at different points in the pulsation cycle, we find that the post-isolation dynamics are strongly dependent on the pre-isolation pulsation phase: cells in a contractile phase collapse immediately, but cells in an expansionary phase continue to expand -- at least for 20-60 s before collapsing. These results are in conflict with previous pulsation models that place expanding cells under large extensional strain, and instead suggest that even the expansion phase has a significant cell autonomous component.

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

    PubMed

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

    2009-02-01

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

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

    PubMed

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

    2007-02-01

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

  11. Cell-autonomous stress responses in innate immunity.

    PubMed

    Moretti, Julien; Blander, J Magarian

    2017-01-01

    The innate immune response of phagocytes to microbes has long been known to depend on the core signaling cascades downstream of pattern recognition receptors (PRRs), which lead to expression and production of inflammatory cytokines that counteract infection and induce adaptive immunity. Cell-autonomous responses have recently emerged as important mechanisms of innate immunity. Either IFN-inducible or constitutive, these processes aim to guarantee cell homeostasis but have also been shown to modulate innate immune response to microbes and production of inflammatory cytokines. Among these constitutive cell-autonomous responses, autophagy is prominent and its role in innate immunity has been well characterized. Other stress responses, such as metabolic stress, the ER stress/unfolded protein response, mitochondrial stress, or the DNA damage response, seem to also be involved in innate immunity, although the precise mechanisms by which they regulate the innate immune response are not yet defined. Of importance, these distinct constitutive cell-autonomous responses appear to be interconnected and can also be modulated by microbes and PRRs, which add further complexity to the interplay between innate immune signaling and cell-autonomous responses in the mediation of an efficient innate immune response.

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

    PubMed Central

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

    2014-01-01

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

  13. vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis

    PubMed Central

    Herz, Hans-Martin; Chen, Zhihong; Scherr, Heather; Lackey, Melinda; Bolduc, Clare; Bergmann, Andreas

    2008-01-01

    Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, we identified two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, we provide evidence that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer. PMID:16611691

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

    PubMed

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

    2017-04-01

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

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

    PubMed

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

    2016-10-01

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

  16. Calbindin-D28k is a more reliable marker of human Purkinje cells than standard Nissl stains: a stereological experiment.

    PubMed

    Whitney, Elizabeth R; Kemper, Thomas L; Rosene, Douglas L; Bauman, Margaret L; Blatt, Gene J

    2008-02-15

    In a study of human Purkinje cell (PC) number, a striking mismatch between the number of PCs observed with the Nissl stain and the number of PCs immunopositive for calbindin-D28k (CB) was identified in 2 of the 10 brains examined. In the remaining eight brains this mismatch was not observed. Further, in these eight brains, analysis of CB immunostained sections counterstained with the Nissl stain revealed that more than 99% Nissl stained PCs were also immunopositive for CB. In contrast, in the two discordant brains, only 10-20% of CB immunopositive PCs were also identified with the Nissl stain. Although this finding was unexpected, a historical survey of the literature revealed that Spielmeyer [Spielmeyer W. Histopathologie des nervensystems. Julius Springer: Berlin; 1922. p. 56-79] described human cases with PCs that lacked the expected Nissl staining intensity, an important historical finding and critical issue when studying postmortem human brains. The reason for this failure in Nissl staining is not entirely clear, but it may result from premortem circumstances since it is not accounted for by postmortem delay or processing variables. Regardless of the exact cause, these observations suggest that Nissl staining may not be a reliable marker for PCs and that CB is an excellent alternative marker.

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

    PubMed

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

    2007-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Non-cell autonomous and non-catalytic activities of ATX in the developing brain.

    PubMed

    Greenman, Raanan; Gorelik, Anna; Sapir, Tamar; Baumgart, Jan; Zamor, Vanessa; Segal-Salto, Michal; Levin-Zaidman, Smadar; Aidinis, Vassilis; Aoki, Junken; Nitsch, Robert; Vogt, Johannes; Reiner, Orly

    2015-01-01

    The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non-cell autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX) as a non-cell autonomous regulator of neural stem cells. ATX (also known as ENPP2) is best known to catalyze lysophosphatidic acid (LPA) production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA.

  20. Cell-Autonomous and Non-Cell-Autonomous Roles for Irf6 during Development of the Tongue

    PubMed Central

    Goudy, Steven; Angel, Peggi; Jacobs, Britni; Hill, Cynthia; Mainini, Veronica; Smith, Arianna L.; Kousa, Youssef A.; Caprioli, Richard; Prince, Lawrence S.; Baldwin, Scott; Schutte, Brian C.

    2013-01-01

    Interferon regulatory factor 6 (IRF6) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in IRF6 lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the Myf5+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6−/− embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue. PMID:23451037

  1. Cell-autonomous and non-cell-autonomous roles for IRF6 during development of the tongue.

    PubMed

    Goudy, Steven; Angel, Peggi; Jacobs, Britni; Hill, Cynthia; Mainini, Veronica; Smith, Arianna L; Kousa, Youssef A; Caprioli, Richard; Prince, Lawrence S; Baldwin, Scott; Schutte, Brian C

    2013-01-01

    Interferon regulatory factor 6 (IRF6) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in IRF6 lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of Irf6 have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that MCS9.7, an enhancer near Irf6, is active in the tongue, suggesting that Irf6 may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the Myf5+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous Irf6 function with reduced and poorly organized Myf5+ cell lineage in the tongue. Molecular analyses showed that the Irf6-/- embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous Irf6 function with the loss of the inter-molar eminence. Loss of Irf6 function altered Bmp2, Bmp4, Shh, and Fgf10 signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, Irf6 plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.

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

    PubMed

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

    2016-11-01

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

  3. Subversion of Cell-Autonomous Host Defense by Chlamydia Infection.

    PubMed

    Fischer, Annette; Rudel, Thomas

    2016-05-13

    Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

  4. Suppression of the novel ER protein Maxer by mutant ataxin-1 in Bergman glia contributes to non-cell-autonomous toxicity

    PubMed Central

    Shiwaku, Hiroki; Yoshimura, Natsue; Tamura, Takuya; Sone, Masaki; Ogishima, Soichi; Watase, Kei; Tagawa, Kazuhiko; Okazawa, Hitoshi

    2010-01-01

    Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple α-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcription repression in the nucleus. The loss of Maxer eventually induces cell accumulation at G1 phase. It was also shown that mutant Atx1 represses Maxer and inhibits proliferation of Bergmann glia in vitro. Consistently, Bergmann glia are reduced in the cerebellum of mutant Atx1 knockin mice before onset. Glutamate-aspartate transporter reduction in Bergmann glia by mutant Atx1 and vulnerability of Purkinje cell to glutamate are both strengthened by Maxer knockdown in Bergmann glia, whereas Maxer overexpression rescues them. Collectively, these results suggest that the reduction of Maxer mediates functional deficiency of Bergmann glia, and might contribute to the non-cell-autonomous pathology of SCA1. PMID:20531390

  5. Suppression of the novel ER protein Maxer by mutant ataxin-1 in Bergman glia contributes to non-cell-autonomous toxicity.

    PubMed

    Shiwaku, Hiroki; Yoshimura, Natsue; Tamura, Takuya; Sone, Masaki; Ogishima, Soichi; Watase, Kei; Tagawa, Kazuhiko; Okazawa, Hitoshi

    2010-07-21

    Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple alpha-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcription repression in the nucleus. The loss of Maxer eventually induces cell accumulation at G1 phase. It was also shown that mutant Atx1 represses Maxer and inhibits proliferation of Bergmann glia in vitro. Consistently, Bergmann glia are reduced in the cerebellum of mutant Atx1 knockin mice before onset. Glutamate-aspartate transporter reduction in Bergmann glia by mutant Atx1 and vulnerability of Purkinje cell to glutamate are both strengthened by Maxer knockdown in Bergmann glia, whereas Maxer overexpression rescues them. Collectively, these results suggest that the reduction of Maxer mediates functional deficiency of Bergmann glia, and might contribute to the non-cell-autonomous pathology of SCA1.

  6. Autonomous, Retrievable, Deep Sea Microbial Fuel Cell

    NASA Astrophysics Data System (ADS)

    Richter, K.

    2014-12-01

    Microbial fuel cells (MFCs) work by providing bacteria in anaerobic sediments with an electron acceptor (anode) that stimulates metabolism of organic matter. The buried anode is connected via control circuitry to a cathode exposed to oxygen in the overlying water. During metabolism, bacteria release hydrogen ions into the sediment and transfer electrons extra-cellularly to the anode, which eventually reduce dissolved oxygen at the cathode, forming water. The open circuit voltage is approximately 0.8 v. The voltage between electrodes is operationally kept at 0.4 v with a potentiastat. The current is chiefly limited by the rate of microbial metabolism at the anode. The Office of Naval Research has encouraged development of microbial fuel cells in the marine environment at a number of academic and naval institutions. Earlier work in shallow sediments of San Diego Bay showed that the most important environmental parameters that control fuel cell power output in San Diego Bay were total organic carbon in the sediment and seasonal water temperature. Current MFC work at SPAWAR includes extension of microbial fuel cell tests to the deep sea environment (>1000 m) and, in parallel, testing microbial fuel cells in the laboratory under deep sea conditions. One question we are asking is whether MFC power output from deep water sediments repressurized and chilled in the laboratory comparable to those measured in situ. If yes, mapping the power potential of deep sea sediments may be made much easier, requiring sediment grabs and lab tests rather than deployment and retrieval of fuel cells. Another question we are asking is whether in situ temperature and total organic carbon in the deep sea sediment can predict MFC power. If yes, then we can make use of the large collection of publicly available, deep sea oceanographic measurements to make these predictions, foregoing expensive work at sea. These regressions will be compared to those derived from shallow water measurements.

  7. Fibrogenic Lung Injury Induces Non-Cell-Autonomous Fibroblast Invasion.

    PubMed

    Ahluwalia, Neil; Grasberger, Paula E; Mugo, Brian M; Feghali-Bostwick, Carol; Pardo, Annie; Selman, Moisés; Lagares, David; Tager, Andrew M

    2016-06-01

    Pathologic accumulation of fibroblasts in pulmonary fibrosis appears to depend on their invasion through basement membranes and extracellular matrices. Fibroblasts from the fibrotic lungs of patients with idiopathic pulmonary fibrosis (IPF) have been demonstrated to acquire a phenotype characterized by increased cell-autonomous invasion. Here, we investigated whether fibroblast invasion is further stimulated by soluble mediators induced by lung injury. We found that bronchoalveolar lavage fluids from bleomycin-challenged mice or patients with IPF contain mediators that dramatically increase the matrix invasion of primary lung fibroblasts. Further characterization of this non-cell-autonomous fibroblast invasion suggested that the mediators driving this process are produced locally after lung injury and are preferentially produced by fibrogenic (e.g., bleomycin-induced) rather than nonfibrogenic (e.g., LPS-induced) lung injury. Comparison of invasion and migration induced by a series of fibroblast-active mediators indicated that these two forms of fibroblast movement are directed by distinct sets of stimuli. Finally, knockdown of multiple different membrane receptors, including platelet-derived growth factor receptor-β, lysophosphatidic acid 1, epidermal growth factor receptor, and fibroblast growth factor receptor 2, mitigated the non-cell-autonomous fibroblast invasion induced by bronchoalveolar lavage from bleomycin-injured mice, suggesting that multiple different mediators drive fibroblast invasion in pulmonary fibrosis. The magnitude of this mediator-driven fibroblast invasion suggests that its inhibition could be a novel therapeutic strategy for pulmonary fibrosis. Further elaboration of the molecular mechanisms that drive non-cell-autonomous fibroblast invasion consequently may provide a rich set of novel drug targets for the treatment of IPF and other fibrotic lung diseases.

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

    PubMed Central

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

    2016-01-01

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

  9. 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

  10. Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos.

    PubMed

    Yu, Hsiang; Lai, Huey-Jen; Lin, Tai-Wei; Lo, Szecheng J

    2015-04-27

    Generation of DNA fragments is a hallmark of cell apoptosis and is executed within the dying cells (autonomous) or in the engulfing cells (non-autonomous). The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method is used as an in situ assay of apoptosis by labelling DNA fragments generated by caspase-associated DNase (CAD), but not those by the downstream DNase II. In the present study, we report a method of ToLFP (topoisomerase ligation fluorescence probes) for directly visualizing DNA fragments generated by DNase II in Caenorhabditis elegans embryos. ToLFP analysis provided the first demonstration of a cell autonomous mode of DNase II activity in dying cells in ced-1 embryos, which are defective in engulfing apoptotic bodies. Compared with the number of ToLFP signals between ced-1 and wild-type (N2) embryos, a 30% increase in N2 embryos was found, suggesting that the ratio of non-autonomous and autonomous modes of DNase II was ~3-7. Among three DNase II mutant embryos (nuc-1, crn-6 and crn-7), nuc-1 embryos exhibited the least number of ToLFP. The ToLFP results confirmed the previous findings that NUC-1 is the major DNase II for degrading apoptotic DNA. To further elucidate NUC-1's mode of action, nuc-1-rescuing transgenic worms that ectopically express free or membrane-bound forms of NUC-1 fusion proteins were utilized. ToLFP analyses revealed that anteriorly expressed NUC-1 digests apoptotic DNA in posterior blastomeres in a non-autonomous and secretion-dependent manner. Collectively, we demonstrate that the ToLFP method can be used to differentiate the locations of blastomeres where DNase II acts autonomously or non-autonomously in degrading apoptotic DNA.

  11. Mathematical model of bursting in dissociated purkinje neurons.

    PubMed

    Forrest, Michael D

    2013-01-01

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

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

    PubMed

    Forrest, Michael D

    2014-01-01

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

  13. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin.

    PubMed

    Kim, Seok-Hyung; Speirs, Christina K; Solnica-Krezel, Lilianna; Ess, Kevin C

    2011-03-01

    Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2(vu242) allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2(vu242) is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2(vu242/vu242) homozygous zebrafish, and is moderately increased in tsc2(vu242/+) heterozygotes. Forebrain neurons are poorly organized in tsc2(vu242/vu242) homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2(vu242/vu242) mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

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

    PubMed Central

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

    2007-01-01

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

  15. 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.

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

    PubMed

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

    2009-09-01

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

  17. Cell Non-autonomous Function of Ceramidase in Photoreceptor Homeostasis

    PubMed Central

    Acharya, Jairaj K.; Dasgupta, Ujjaini; Rawat, Satinder S.; Yuan, Changqing; Sanxaridis, Parthena D.; Yonamine, Ikuko; Karim, Pusha; Nagashima, Kunio; Brodsky, Michael H.; Tsunoda, Susan; Acharya, Usha

    2008-01-01

    SUMMARY Neutral Ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila Ceramidase. We show that secreted Ceramidase functions in a cell non-autonomous manner to maintain photoreceptor homeostasis. In the absence of Ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of Rhodopsin, and do not respond to light stimulus. Consistent with a cell non-autonomous function, our studies show that over expression of Ceramidase in a tissue distant from the photoreceptors can suppress photoreceptor degeneration in an Arrestin mutant and facilitate membrane turnover in a Rhodopsin null mutant. Furthermore, our results show that secreted CDase is internalized and localizes to endosomes. Our findings are the first to establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function. PMID:18184565

  18. 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.

  19. 1,2,5,6,9,10-αHexabromocyclododecane (HBCD) impairs thyroid hormone-induced dendrite arborization of Purkinje cells and suppresses thyroid hormone receptor-mediated transcription.

    PubMed

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

    2011-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  1. Neuralized functions cell autonomously to regulate Drosophila sense organ development.

    PubMed

    Yeh, E; Zhou, L; Rudzik, N; Boulianne, G L

    2000-09-01

    Neurogenic genes, including Notch and Delta, are thought to play important roles in regulating cell-cell interactions required for Drosophila sense organ development. To define the requirement of the neurogenic gene neuralized (neu) in this process, two independent neu alleles were used to generate mutant clones. We find that neu is required for determination of cell fates within the proneural cluster and that cells mutant for neu autonomously adopt neural fates when adjacent to wild-type cells. Furthermore, neu is required within the sense organ lineage to determine the fates of daughter cells and accessory cells. To gain insight into the mechanism by which neu functions, we used the GAL4/UAS system to express wild-type and epitope-tagged neu constructs. We show that Neu protein is localized primarily at the plasma membrane. We propose that the function of neu in sense organ development is to affect the ability of cells to receive Notch-Delta signals and thus modulate neurogenic activity that allows for the specification of non-neuronal cell fates in the sense organ.

  2. Interferon-inducible effector mechanisms in cell-autonomous immunity.

    PubMed

    MacMicking, John D

    2012-04-25

    Interferons (IFNs) induce the expression of hundreds of genes as part of an elaborate antimicrobial programme designed to combat infection in all nucleated cells - a process termed cell-autonomous immunity. As described in this Review, recent genomic and subgenomic analyses have begun to assign functional properties to novel IFN-inducible effector proteins that restrict bacteria, protozoa and viruses in different subcellular compartments and at different stages of the pathogen life cycle. Several newly described host defence factors also participate in canonical oxidative and autophagic pathways by spatially coordinating their activities to enhance microbial killing. Together, these IFN-induced effector networks help to confer vertebrate host resistance to a vast and complex microbial world.

  3. Interferon-inducible GTPases in cell autonomous and innate immunity.

    PubMed

    Meunier, Etienne; Broz, Petr

    2016-02-01

    Detection and clearance of invading pathogens requires a coordinated response of the adaptive and innate immune system. Host cell, however, also features different mechanisms that restrict pathogen replication in a cell-intrinsic manner, collectively referred to as cell-autonomous immunity. In immune cells, the ability to unleash those mechanisms strongly depends on the activation state of the cell, which is controlled by cytokines or the detection of pathogen-associated molecular patterns by pattern-recognition receptors. The interferon (IFN) class of cytokines is one of the strongest inducers of antimicrobial effector mechanisms and acts against viral, bacterial and parasitic intracellular pathogens. This has been linked to the upregulation of several hundreds of IFN-stimulated genes, among them the so-called IFN-inducible GTPases. Two subfamilies of IFN-inducible GTPases, the immunity-related GTPases (IRGs) and the guanylate-binding proteins (GBPs), have gained attention due to their exceptional ability to specifically target intracellular vacuolar pathogens and restrict their replication by destroying their vacuolar compartment. Their repertoire has recently been expanded to the regulation of inflammasome complexes, which are cytosolic multi-protein complexes that control an inflammatory cell death called pyroptosis and the release of cytokines like interleukin-1β and interleukin-18. Here we discuss recent advances in understanding the function, the targeting and regulation of IRG and GBP proteins during microbial infections.

  4. 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.

  5. Wnt signaling inhibits adrenal steroidogenesis by cell-autonomous and non-cell-autonomous mechanisms.

    PubMed

    Walczak, Elisabeth M; Kuick, Rork; Finco, Isabella; Bohin, Natacha; Hrycaj, Steven M; Wellik, Deneen M; Hammer, Gary D

    2014-09-01

    Wnt/β-catenin (βcat) signaling is critical for adrenal homeostasis. To elucidate how Wnt/βcat signaling elicits homeostatic maintenance of the adrenal cortex, we characterized the identity of the adrenocortical Wnt-responsive population. We find that Wnt-responsive cells consist of sonic hedgehog (Shh)-producing adrenocortical progenitors and differentiated, steroidogenic cells of the zona glomerulosa, but not the zona fasciculata and rarely cells that are actively proliferating. To determine potential direct inhibitory effects of βcat signaling on zona fasciculata-associated steroidogenesis, we used the mouse ATCL7 adrenocortical cell line that serves as a model system of glucocorticoid-producing fasciculata cells. Stimulation of βcat signaling caused decreased corticosterone release consistent with the observed reduced transcription of steroidogenic genes Cyp11a1, Cyp11b1, Star, and Mc2r. Decreased steroidogenic gene expression was correlated with diminished steroidogenic factor 1 (Sf1; Nr5a1) expression and occupancy on steroidogenic promoters. Additionally, βcat signaling suppressed the ability of Sf1 to transactivate steroidogenic promoters independent of changes in Sf1 expression level. To investigate Sf1-independent effects of βcat on steroidogenesis, we used Affymetrix gene expression profiling of Wnt-responsive cells in vivo and in vitro. One candidate gene identified, Ccdc80, encodes a secreted protein with unknown signaling mechanisms. We report that Ccdc80 is a novel βcat-regulated gene in adrenocortical cells. Treatment of adrenocortical cells with media containing secreted Ccdc80 partially phenocopies βcat-induced suppression of steroidogenesis, albeit through an Sf1-independent mechanism. This study reveals multiple mechanisms of βcat-mediated suppression of steroidogenesis and suggests that Wnt/βcat signaling may regulate adrenal homeostasis by inhibiting fasciculata differentiation and promoting the undifferentiated state of progenitor

  6. Wnt Signaling Inhibits Adrenal Steroidogenesis by Cell-Autonomous and Non–Cell-Autonomous Mechanisms

    PubMed Central

    Walczak, Elisabeth M.; Kuick, Rork; Finco, Isabella; Bohin, Natacha; Hrycaj, Steven M.; Wellik, Deneen M.

    2014-01-01

    Wnt/β-catenin (βcat) signaling is critical for adrenal homeostasis. To elucidate how Wnt/βcat signaling elicits homeostatic maintenance of the adrenal cortex, we characterized the identity of the adrenocortical Wnt-responsive population. We find that Wnt-responsive cells consist of sonic hedgehog (Shh)-producing adrenocortical progenitors and differentiated, steroidogenic cells of the zona glomerulosa, but not the zona fasciculata and rarely cells that are actively proliferating. To determine potential direct inhibitory effects of βcat signaling on zona fasciculata-associated steroidogenesis, we used the mouse ATCL7 adrenocortical cell line that serves as a model system of glucocorticoid-producing fasciculata cells. Stimulation of βcat signaling caused decreased corticosterone release consistent with the observed reduced transcription of steroidogenic genes Cyp11a1, Cyp11b1, Star, and Mc2r. Decreased steroidogenic gene expression was correlated with diminished steroidogenic factor 1 (Sf1; Nr5a1) expression and occupancy on steroidogenic promoters. Additionally, βcat signaling suppressed the ability of Sf1 to transactivate steroidogenic promoters independent of changes in Sf1 expression level. To investigate Sf1-independent effects of βcat on steroidogenesis, we used Affymetrix gene expression profiling of Wnt-responsive cells in vivo and in vitro. One candidate gene identified, Ccdc80, encodes a secreted protein with unknown signaling mechanisms. We report that Ccdc80 is a novel βcat-regulated gene in adrenocortical cells. Treatment of adrenocortical cells with media containing secreted Ccdc80 partially phenocopies βcat-induced suppression of steroidogenesis, albeit through an Sf1-independent mechanism. This study reveals multiple mechanisms of βcat-mediated suppression of steroidogenesis and suggests that Wnt/βcat signaling may regulate adrenal homeostasis by inhibiting fasciculata differentiation and promoting the undifferentiated state of progenitor

  7. 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

  8. Signals and Circuits in the Purkinje Neuron

    PubMed Central

    Abrams, Zéev R.; Zhang, Xiang

    2011-01-01

    Purkinje neurons (PN) in the cerebellum have over 100,000 inputs organized in an orthogonal geometry, and a single output channel. As the sole output of the cerebellar cortex layer, their complex firing pattern has been associated with motor control and learning. As such they have been extensively modeled and measured using tools ranging from electrophysiology and neuroanatomy, to dynamic systems and artificial intelligence methods. However, there is an alternative approach to analyze and describe the neuronal output of these cells using concepts from electrical engineering, particularly signal processing and digital/analog circuits. By viewing the PN as an unknown circuit to be reverse-engineered, we can use the tools that provide the foundations of today’s integrated circuits and communication systems to analyze the Purkinje system at the circuit level. We use Fourier transforms to analyze and isolate the inherent frequency modes in the PN and define three unique frequency ranges associated with the cells’ output. Comparing the PN to a signal generator that can be externally modulated adds an entire level of complexity to the functional role of these neurons both in terms of data analysis and information processing, relying on Fourier analysis methods in place of statistical ones. We also re-describe some of the recent literature in the field, using the nomenclature of signal processing. Furthermore, by comparing the experimental data of the past decade with basic electronic circuitry, we can resolve the outstanding controversy in the field, by recognizing that the PN can act as a multivibrator circuit. PMID:21980311

  9. Homologous Recombination between Autonomously Replicating Plasmids in Mammalian Cells

    PubMed Central

    Ayares, David; Spencer, James; Schwartz, Faina; Morse, Brian; Kucherlapati, Raju

    1985-01-01

    The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neo R colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis. PMID:2996980

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

    PubMed

    Jarius, S; Wildemann, B

    2015-09-17

    Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as 'Medusa-head antibodies' due to their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects and provides a summary and outlook.

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

    PubMed

    Jarius, S; Wildemann, B

    2015-09-17

    Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as 'Medusa head antibodies' due their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects, and provides a summary and outlook.

  12. Cell-autonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis.

    PubMed

    Rivera, Charlene; Yamben, Idella F; Shatadal, Shalini; Waldof, Malinda; Robinson, Michael L; Griep, Anne E

    2009-09-01

    Cell polarity and adhesion are thought to be key determinants in organismal development. In Drosophila, discs large (dlg) has emerged as an important regulator of epithelial cell proliferation, adhesion, and polarity. Herein, we investigated the role of the mouse homolog of dlg (Dlg-1) in the development of the mouse ocular lens. Tissue-specific ablation of Dlg-1 throughout the lens early in lens development led to an expansion and disorganization of the epithelium that correlated with changes in the distribution of adhesion and polarity factors. In the fiber cells, differentiation defects were observed. These included alterations in cell structure and the disposition of cell adhesion/cytoskeletal factors, delay in denucleation, and reduced levels of alpha-catenin, pERK1/2, and MIP26. These fiber cell defects were recapitulated when Dlg-1 was disrupted only in fiber cells. These results suggest that Dlg-1 acts in a cell autonomous manner to regulate epithelial cell structure and fiber cell differentiation.

  13. What cycles the cell? -Robust autonomous cell cycle models.

    PubMed

    Lavi, Orit; Louzoun, Yoram

    2009-12-01

    The cell cycle is one of the best studied cellular mechanisms at the experimental and theoretical levels. Although most of the important biochemical components and reactions of the cell cycle are probably known, the precise way the cell cycle dynamics are driven is still under debate. This phenomenon is not atypical to many other biological systems where the knowledge of the molecular building blocks and the interactions between them does not lead to a coherent picture of the appropriate dynamics. We here propose a methodology to develop plausible models for the driving mechanisms of embryonic and cancerous cell cycles. We first define a key property of the system (a cyclic behaviour in the case of the embryonic cell cycle) and set mathematical constraints on the types of two variable simplified systems robustly reproducing such a cyclic behaviour. We then expand these robust systems to three variables and reiterate the procedure. At each step, we further limit the type of expanded systems to fit the known microbiology until a detailed description of the system is obtained. This methodology produces mathematical descriptions of the required biological systems that are more robust to changes in the precise function and rate constants. This methodology can be extended to practically any type of subcellular mechanism.

  14. Non-autonomous cell proliferation in the mammary gland and cancer.

    PubMed

    Weber, Robert J; Desai, Tejal A; Gartner, Zev J

    2017-03-14

    Cells decide whether to grow and divide by integrating internal and external signals. Non-autonomous cell growth and proliferation occurs when microenvironmental signals from neighboring cells, both physical and secreted, license this decision. Understanding these processes is vital to developing an accurate framework for cell-cell interactions and cellular decision-making, and is useful for advancing new therapeutic strategies to prevent dysregulated growth. Here, we review some recent examples of non-autonomous cell growth in the mammary gland and tumor cell proliferation.

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

    PubMed Central

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

    1982-01-01

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

  16. Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

    PubMed

    Babcock, Daniel T; Ganetzky, Barry

    2015-01-01

    Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

  17. The pacemaker current in cardiac Purkinje myocytes

    PubMed Central

    1995-01-01

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

  18. Poly(ethylene glycol) hydrogels with cell cleavable groups for autonomous cell delivery.

    PubMed

    Kar, Mrityunjoy; Vernon Shih, Yu-Ru; Velez, Daniel Ortiz; Cabrales, Pedro; Varghese, Shyni

    2016-01-01

    Cell-responsive hydrogels hold tremendous potential as cell delivery devices in regenerative medicine. In this study, we developed a hydrogel-based cell delivery vehicle, in which the encapsulated cell cargo control its own release from the vehicle in a protease-independent manner. Specifically, we have synthesized a modified poly(ethylene glycol) (PEG) hydrogel that undergoes degradation responding to cell-secreted molecules by incorporating disulfide moieties onto the backbone of the hydrogel precursor. Our results show the disulfide-modified PEG hydrogels disintegrate seamlessly into solution in presence of cells without any external stimuli. The rate of hydrogel degradation, which ranges from hours to months, is found to be dependent upon the type of encapsulated cells, cell number, and fraction of disulfide moieties present in the hydrogel backbone. The differentiation potential of human mesenchymal stem cells released from the hydrogels is maintained in vitro. The in vivo analysis of these cell-laden hydrogels, through a dorsal window chamber and intramuscular implantation, demonstrated autonomous release of cells to the host environment. The hydrogel-mediated implantation of cells resulted in higher cell retention within the host tissue when compared to that without a biomaterial support. Biomaterials that function as a shield to protect cell cargos and assist their delivery in response to signals from the encapsulated cells could have a wide utility in cell transplantation and could improve the therapeutic outcomes of cell-based therapies.

  19. Poly(ethylene glycol) hydrogels with cell cleavable groups for autonomous cell delivery

    PubMed Central

    Kar, Mrityunjoy; Shih, Yu-Ru Vernon; Velez, Daniel Ortiz; Cabrales, Pedro; Varghese, Shyni

    2015-01-01

    Cell-responsive hydrogels hold tremendous potential as cell delivery devices in regenerative medicine. In this study, we developed a hydrogel-based cell delivery vehicle, in which the encapsulated cell cargo control its own release from the vehicle in a protease-independent manner. Specifically, we have synthesized a modified poly(ethylene glycol) (PEG) hydrogel that undergoes degradation responding to cell-secreted molecules by incorporating disulfide moieties onto the backbone of the hydrogel precursor. Our results show the disulfide-modified PEG hydrogels disintegrate seamlessly into solution in presence of cells without any external stimuli. The rate of hydrogel degradation, which ranges from hours to months, is found to be dependent upon the type of encapsulated cells, cell number, and fraction of disulfide moieties present in the hydrogel backbone. The differentiation potential of human mesenchymal stem cells released from the hydrogels is maintained in vitro. The in vivo analysis of these cell-laden hydrogels, through a dorsal window chamber and intramuscular implantation, demonstrated autonomous release of cells to the host environment. The hydrogel-mediated implantation of cells resulted in higher cell retention within the host tissue when compared to that without a biomaterial support. Biomaterials that function as a shield to protect cell cargos and assist their delivery in response to signals from the encapsulated cells could have a wide utility in cell transplantation and could improve the therapeutic outcomes of cell-based therapies. PMID:26606444

  20. A non-cell autonomous mouse model of CNS haemangioblastoma mediated by mutant KRAS

    PubMed Central

    Bao, Leyuan; Al-Assar, Osama; Drynan, Lesley F.; Arends, Mark J.; Tyers, Pam; Barker, Roger A.; Rabbitts, Terence H.

    2017-01-01

    Haemangioblastoma is a rare malignancy of the CNS where vascular proliferation causes lesions due to endothelial propagation. We found that conditionally expressing mutant Kras, using Rag1-Cre, gave rise to CNS haemangioblastoma in the cortex and cerebellum in mice that present with highly vascular tumours with stromal cells similar to human haemangioblastomas. The aberrant haemangioblastoma endothelial cells do not express mutant Kras but rather the mutant oncogene is expressed in CNS interstitial cells, including neuronal cells and progeny. This demonstrates a non-cell autonomous origin of this disease that is unexpectedly induced via Rag1-Cre expression in CNS interstitial cells. This is the first time that mutant RAS has been shown to stimulate non-cell autonomous proliferation in malignancy and suggests that mutant RAS can control endothelial cell proliferation in neo-vascularisation when expressed in certain cells. PMID:28322325

  1. 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

  2. Both cell-autonomous mechanisms and hormones contribute to sexual development in vertebrates and insects.

    PubMed

    Bear, Ashley; Monteiro, Antónia

    2013-08-01

    The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell-autonomous expression of sex-specific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell-autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell-autonomous versus hormonally guided sexual development in animals.

  3. Sensing the enemy, containing the threat: cell-autonomous immunity to Chlamydia trachomatis.

    PubMed

    Finethy, Ryan; Coers, Jörn

    2016-07-29

    The bacterium Chlamydia trachomatis is the etiological agent of the most common sexually transmitted infection in North America and Europe. Medical complications resulting from genital C. trachomatis infections arise predominantly in women where the initial infections often remain asymptomatic and thus unrecognized. Untreated asymptomatic infections in women can ascend into the upper genital tract and establish persistence, ultimately resulting in extensive scarring of the reproductive organs, pelvic inflammatory disease, infertility and ectopic pregnancies. Previously resolved C. trachomatis infections fail to provide protective immune memory, and no effective vaccine against C. trachomatis is currently available. Critical determinants of the pathogenesis and immunogenicity of genital C. trachomatis infections are cell-autonomous immune responses. Cell-autonomous immunity describes the ability of an individual host cell to launch intrinsic immune circuits that execute the detection, containment and elimination of cell-invading pathogens. As an obligate intracellular pathogen C. trachomatis is constantly under attack by cell-intrinsic host defenses. Accordingly, C. trachomatis evolved to subvert and co-opt cell-autonomous immune pathways. This review will provide a critical summary of our current understanding of cell-autonomous immunity to C. trachomatis and its role in shaping host resistance, inflammation and adaptive immunity to genital C. trachomatis infections.

  4. 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

  5. Autonomous assembly of epithelial structures by subrenal implantation of dissociated embryonic inner-ear cells.

    PubMed

    Wang, Li; Zhang, Kaiqing; Zhu, Helen He; Gao, Wei-Qiang

    2015-05-27

    Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.

  6. Electrogenic sodium extrusion in cardiac Purkinje fibers.

    PubMed

    Gadsby, D C; Cranefield, P F

    1979-06-01

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

  7. RoBlock: a prototype autonomous manufacturing cell

    NASA Astrophysics Data System (ADS)

    Baekdal, Lars K.; Balslev, Ivar; Eriksen, Rene D.; Jensen, Soren P.; Jorgensen, Bo N.; Kirstein, Brian; Kristensen, Bent B.; Olsen, Martin M.; Perram, John W.; Petersen, Henrik G.; Petersen, Morten L.; Ruhoff, Peter T.; Skjolstrup, Carl E.; Sorensen, Anders S.; Wagenaar, Jeroen M.

    2000-10-01

    RoBlock is the first phase of an internally financed project at the Institute aimed at building a system in which two industrial robots suspended from a gantry, as shown below, cooperate to perform a task specified by an external user, in this case, assembling an unstructured collection of colored wooden blocks into a specified 3D pattern. The blocks are identified and localized using computer vision and grasped with a suction cup mechanism. Future phases of the project will involve other processes such as grasping and lifting, as well as other types of robot such as autonomous vehicles or variable geometry trusses. Innovative features of the control software system include: The use of an advanced trajectory planning system which ensures collision avoidance based on a generalization of the method of artificial potential fields, the use of a generic model-based controller which learns the values of parameters, including static and kinetic friction, of a detailed mechanical model of itself by comparing actual with planned movements, the use of fast, flexible, and robust pattern recognition and 3D-interpretation strategies, integration of trajectory planning and control with the sensor systems in a distributed Java application running on a network of PC's attached to the individual physical components. In designing this first stage, the aim was to build in the minimum complexity necessary to make the system non-trivially autonomous and to minimize the technological risks. The aims of this project, which is planned to be operational during 2000, are as follows: To provide a platform for carrying out experimental research in multi-agent systems and autonomous manufacturing systems, to test the interdisciplinary cooperation architecture of the Maersk Institute, in which researchers in the fields of applied mathematics (modeling the physical world), software engineering (modeling the system) and sensor/actuator technology (relating the virtual and real worlds) could

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

    PubMed Central

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

    2013-01-01

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

  9. Non-cell-autonomous Neurotoxicity of α-synuclein Through Microglial Toll-like Receptor 2.

    PubMed

    Kim, Changyoun; Lee, He-Jin; Masliah, Eliezer; Lee, Seung-Jae

    2016-06-01

    Synucleinopathies are a collection of neurological diseases that are characterized by deposition of α-synuclein aggregates in neurons and glia. These diseases include Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. Although it has been increasingly clear that α-synuclein is implicated in the pathogenesis of PD and other synucleinopathies, the precise mechanism underlying the disease process remains to be unraveled. The past studies on how α-synuclein exerts pathogenic actions have focused on its direct, cell-autonomous neurotoxic effects. However, recent findings suggested that there might be indirect, non-cell-autonomous pathways, perhaps through the changes in glial cells, for the pathogenic actions of this protein. Here, we present evidence that α-synuclein can cause neurodegeneration through a non-cell-autonomous manner. We show that α-synuclein can be secreted from neurons and induces inflammatory responses in microglia, which in turn secreted neurotoxic agents into the media causing neurodegeneration. The neurotoxic response of microglia was mediated by activation of toll-like receptor 2 (TLR2), a receptor for neuron-derived α-synuclein. This work suggests that TLR2 is the key molecule that mediates non-cell-autonomous neurotoxic effects of α-synuclein, hence a candidate for the therapeutic target.

  10. Non-autonomous consequences of cell death and other perks of being metazoan

    PubMed Central

    Su, Tin Tin

    2015-01-01

    Drosophila melanogaster remains a foremost genetic model to study basic cell biological processes in the context of multi-cellular development. In such context, the behavior of one cell can influence another. Non-autonomous signaling among cells occurs throughout metazoan development and disease, and is too vast to be covered by a single review. I will focus here on non-autonomous signaling events that occur in response to cell death in the larval epithelia and affect the life-death decision of surviving cells. I will summarize the use of Drosophila to study cell death-induced proliferation, apoptosis-induced apoptosis, and apoptosis-induced survival signaling. Key insights from Drosophila will be discussed in the context of analogous processes in mammalian development and cancer biology. PMID:26069889

  11. Cell autonomous defects in cortical development revealed by two-color chimera analysis.

    PubMed

    Kwiatkowski, Adam V; Garner, Craig C; Nelson, W James; Gertler, Frank B

    2009-05-01

    A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based method to analyze cell autonomy by generating two-color chimeric mouse embryos. Ena/VASP-deficient mutant and control embryonic stem (ES) cells were labeled with different fluorescent chimeric proteins (EGFP and mCherry) that were modified to bind to the plasma membrane. These labeled ES cells were used to generate two-color chimeric embryos possessing two genetically distinct populations of cortical cells, permitting multiple aspects of neuronal morphogenesis to be analyzed and compared between the two cell populations. We observed little difference between the ability of control and Ena/VASP-deficient cells to contribute to cortical organization during development. In contrast, we observed axon fiber tracts originating from control neurons but not Ena/VASP-deficient neurons, indicating that loss of Ena/VASP causes a cell autonomous defect in cortical axon formation. This technique could be applied to determine other cell autonomous functions in different stages of cortical development.

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

    PubMed

    Akemann, Walther; Knöpfel, Thomas

    2006-04-26

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

  13. Towards autonomous lab-on-a-chip devices for cell phone biosensing.

    PubMed

    Comina, Germán; Suska, Anke; Filippini, Daniel

    2016-03-15

    Modern cell phones are a ubiquitous resource with a residual capacity to accommodate chemical sensing and biosensing capabilities. From the different approaches explored to capitalize on such resource, the use of autonomous disposable lab-on-a-chip (LOC) devices-conceived as only accessories to complement cell phones-underscores the possibility to entirely retain cell phones' ubiquity for distributed biosensing. The technology and principles exploited for autonomous LOC devices are here selected and reviewed focusing on their potential to serve cell phone readout configurations. Together with this requirement, the central aspects of cell phones' resources that determine their potential for analytical detection are examined. The conversion of these LOC concepts into universal architectures that are readable on unaccessorized phones is discussed within this context.

  14. Patched1 and Patched2 inhibit Smoothened non-cell autonomously

    PubMed Central

    Roberts, Brock; Casillas, Catalina; Alfaro, Astrid C; Jägers, Carina; Roelink, Henk

    2016-01-01

    Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor. DOI: http://dx.doi.org/10.7554/eLife.17634.001 PMID:27552050

  15. Stabilizing Motifs in Autonomous Boolean Networks and the Yeast Cell Cycle Oscillator

    NASA Astrophysics Data System (ADS)

    Sevim, Volkan; Gong, Xinwei; Socolar, Joshua

    2009-03-01

    Synchronously updated Boolean networks are widely used to model gene regulation. Some properties of these model networks are known to be artifacts of the clocking in the update scheme. Autonomous updating is a less artificial scheme that allows one to introduce small timing perturbations and study stability of the attractors. We argue that the stabilization of a limit cycle in an autonomous Boolean network requires a combination of motifs such as feed-forward loops and auto-repressive links that can correct small fluctuations in the timing of switching events. A recently published model of the transcriptional cell-cycle oscillator in yeast contains the motifs necessary for stability under autonomous updating [1]. [1] D. A. Orlando, et al. Nature (London), 4530 (7197):0 944--947, 2008.

  16. Non-Cell-Autonomous Regulation of Retrograde Motoneuronal Axonal Transport in an SBMA Mouse Model

    PubMed Central

    Halievski, Katherine; Kemp, Michael Q.; Breedlove, S. Marc; Miller, Kyle E.

    2016-01-01

    Abstract Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor (AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique “myogenic” transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons. PMID:27517091

  17. Non-Cell-Autonomous Regulation of Retrograde Motoneuronal Axonal Transport in an SBMA Mouse Model.

    PubMed

    Halievski, Katherine; Kemp, Michael Q; Breedlove, S Marc; Miller, Kyle E; Jordan, Cynthia L

    2016-01-01

    Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor (AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique "myogenic" transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons.

  18. Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

    PubMed

    Raverdeau, Mathilde; Gely-Pernot, Aurore; Féret, Betty; Dennefeld, Christine; Benoit, Gérard; Davidson, Irwin; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

    2012-10-09

    Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

  19. Cell-Autonomous and Non-cell-autonomous Function of Hox Genes Specify Segmental Neuroblast Identity in the Gnathal Region of the Embryonic CNS in Drosophila.

    PubMed

    Becker, Henrike; Renner, Simone; Technau, Gerhard M; Berger, Christian

    2016-03-01

    During central nervous system (CNS) development neural stem cells (Neuroblasts, NBs) have to acquire an identity appropriate to their location. In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of NBs prior to their delamination from the neuroectoderm. Compared to the thoracic, ground state segmental units in the head region are derived to different degrees, and the precise mechanism of segmental specification of NBs in this region is still unclear. We identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. We show that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. We identify the secreted molecule Amalgam (Ama) as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, we provide genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental

  20. Cell-Autonomous and Non-cell-autonomous Function of Hox Genes Specify Segmental Neuroblast Identity in the Gnathal Region of the Embryonic CNS in Drosophila

    PubMed Central

    Becker, Henrike; Renner, Simone; Technau, Gerhard M.; Berger, Christian

    2016-01-01

    During central nervous system (CNS) development neural stem cells (Neuroblasts, NBs) have to acquire an identity appropriate to their location. In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of NBs prior to their delamination from the neuroectoderm. Compared to the thoracic, ground state segmental units in the head region are derived to different degrees, and the precise mechanism of segmental specification of NBs in this region is still unclear. We identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. We show that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. We identify the secreted molecule Amalgam (Ama) as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, we provide genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental

  1. Autonomous proliferation and bcl-2 expression involving haematopoietic cells in patients with myelodysplastic syndrome.

    PubMed Central

    Bincoletto, C.; Saad, S. T.; Soares da Silva, E.; Queiroz, M. L.

    1998-01-01

    In this work, we investigated the autonomous proliferation, bcl-2 expression and number of apoptotic cells in the bone marrow of patients with confirmed diagnosis of myelodysplastic syndromes (MDS). Normal bone marrow cells obtained from donors of the Clinical Hospital of this university were used as a control. The autonomous proliferation, evaluated by clonal culture without exogenous growth factor, and the number of apoptotic cells in bone marrow kept for 10 days in liquid cultures at 37 degrees C and 5% carbon dioxide, were significantly greater in MDS patients than in control subjects (P = 0.001, Wilcoxon). However, bcl-2 expression, measured by immunocytochemistry, was significantly lower in MDS patients than in normal individuals (P = 0.002, Wilcoxon). These results suggest that the high proliferation activity in MDS patients may be counteracted by the high level of medullar cell death, which might be related to the lower bcl-2 expression. PMID:9744502

  2. 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.

  3. Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells.

    PubMed

    Iwahashi, Kazuhiro; Yoshioka, Hirotaka; Low, Eleanor W; McCarrey, John R; Yanagimachi, Ryuzo; Yamazaki, Yukiko

    2007-10-01

    In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.

  4. Purkinje fibers after myocardial ischemia-reperfusion.

    PubMed

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

    2015-07-01

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

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

    PubMed

    Nogami, Akihiko

    2011-05-01

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

  6. Pejvakin, a Candidate Stereociliary Rootlet Protein, Regulates Hair Cell Function in a Cell-Autonomous Manner.

    PubMed

    Kazmierczak, Marcin; Kazmierczak, Piotr; Peng, Anthony W; Harris, Suzan L; Shah, Prahar; Puel, Jean-Luc; Lenoir, Marc; Franco, Santos J; Schwander, Martin

    2017-03-29

    Mutations in the Pejvakin (PJVK) gene are thought to cause auditory neuropathy and hearing loss of cochlear origin by affecting noise-induced peroxisome proliferation in auditory hair cells and neurons. Here we demonstrate that loss of pejvakin in hair cells, but not in neurons, causes profound hearing loss and outer hair cell degeneration in mice. Pejvakin binds to and colocalizes with the rootlet component TRIOBP at the base of stereocilia in injectoporated hair cells, a pattern that is disrupted by deafness-associated PJVK mutations. Hair cells of pejvakin-deficient mice develop normal rootlets, but hair bundle morphology and mechanotransduction are affected before the onset of hearing. Some mechanotransducing shorter row stereocilia are missing, whereas the remaining ones exhibit overextended tips and a greater variability in height and width. Unlike previous studies of Pjvk alleles with neuronal dysfunction, our findings reveal a cell-autonomous role of pejvakin in maintaining stereocilia architecture that is critical for hair cell function.SIGNIFICANCE STATEMENT Two missense mutations in the Pejvakin (PJVK or DFNB59) gene were first identified in patients with audiological hallmarks of auditory neuropathy spectrum disorder, whereas all other PJVK alleles cause hearing loss of cochlear origin. These findings suggest that complex pathogenetic mechanisms underlie human deafness DFNB59. In contrast to recent studies, we demonstrate that pejvakin in auditory neurons is not essential for normal hearing in mice. Moreover, pejvakin localizes to stereociliary rootlets in hair cells and is required for stereocilia maintenance and mechanosensory function of the hair bundle. Delineating the site of the lesion and the mechanisms underlying DFNB59 will allow clinicians to predict the efficacy of different therapeutic approaches, such as determining compatibility for cochlear implants.

  7. Convective exosome-tracing microfluidics for analysis of cell-non-autonomous neurogenesis.

    PubMed

    Oh, Hyun Jeong; Shin, Yoojin; Chung, Seok; Hwang, Do Won; Lee, Dong Soo

    2017-01-01

    The effective role of exosome delivering neurogenic microRNA (miRNA) enables to induce efficient differentiation process during neurogenesis. The microfludic system capable of visualizing the exosomal behavior such as secretion, migration, and uptake of individual exosomes can be used as a robust technique to understand the exosome-mediated change of cellular behavior. Here, we developed the exosome-tracing microfluidic system to visualize exosomal transport carrying the neurogenic miRNA from leading to neighboring cells, and found a new mode of exosome-mediated cell-non-autonomous neurogenesis. The miR-193a facilitated neurogenesis in F11 cells by blocking proliferation-related target genes. In addition to time-lapse live-cell imaging using microfluidics visualized the convective transport of exosomes from differentiated to undifferentiated cells. Individual exosomes containing miR-193a from differentiated donor cells were taken up by undifferentiated cells to lead them to neurogenesis. Induction of anti-miR-193a was sufficient to block neurogenesis in F11 cells. Inhibition of the exosomal production by manumycin-A and treatment of anti-miR-193a in the differentiated donor cells failed to induce neurogenesis in undifferentiated recipient cells. These findings indicate that exosomes of neural progenitors and neurogenic miRNA within these exosomes propagate cell-non-autonomous differentiation to neighboring progenitors, to delineate the roles of exosome mediating neurogenesis of population of homologous neural progenitor cells.

  8. FIP200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells.

    PubMed

    Liu, Fei; Lee, Jae Y; Wei, Huijun; Tanabe, Osamu; Engel, James D; Morrison, Sean J; Guan, Jun-Lin

    2010-12-02

    Little is known about whether autophagic mechanisms are active in hematopoietic stem cells (HSCs) or how they are regulated. FIP200 (200-kDa FAK-family interacting protein) plays important roles in mammalian autophagy and other cellular functions, but its role in hematopoietic cells has not been examined. Here we show that conditional deletion of FIP200 in hematopoietic cells leads to perinatal lethality and severe anemia. FIP200 was cell-autonomously required for the maintenance and function of fetal HSCs. FIP200-deficient HSC were unable to reconstitute lethally irradiated recipients. FIP200 ablation did not result in increased HSC apoptosis, but it did increase the rate of HSC proliferation. Consistent with an essential role for FIP200 in autophagy, FIP200-null fetal HSCs exhibited both increased mitochondrial mass and reactive oxygen species. These data identify FIP200 as a key intrinsic regulator of fetal HSCs and implicate a potential role for autophagy in the maintenance of fetal hematopoiesis and HSCs.

  9. The Drosophila melanogaster cinnabar gene is a cell autonomous genetic marker in Aedes aegypti (Diptera: Culicidae).

    PubMed

    Sethuraman, Nagaraja; O'Brochta, David A

    2005-07-01

    The cinnabar gene of Drosophila melanogaster (Meigen) encodes for kynurenine hydroxylase, an enzyme involved in ommochrome biosynthesis. This gene is commonly included as a visible genetic marker in gene vectors used to create transgenic Aedes aegypti (L.) that are homozygous for the khw allele, the mosquito homolog of cinnabar. Unexpectedly, the phenotype of cells expressing kynurenine hydroxylase in transgenic Ae. aegypti is cell autonomous as demonstrated by the recovery of insects heterozygous for the kynurenine hydroxylase transgene with mosaic eye color patterns. In addition, a transgenic gynandromorph was recovered in which one-half of the insect was expressing the kynurenine hydroxylase transgene, including one eye with red pigmentation, whereas the other half of the insect was homozygous khw and included a white eye. The cell autonomous behavior of cinnabar in transgenic Ae. aegypti is unexpected and increases the utility of this genetic marker.

  10. Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.

    PubMed

    Zamir, Evan A; Czirók, András; Cui, Cheng; Little, Charles D; Rongish, Brenda J

    2006-12-26

    Gastrulation is a fundamental process in early development that results in the formation of three primary germ layers. During avian gastrulation, presumptive mesodermal cells in the dorsal epiblast ingress through a furrow called the primitive streak (PS), and subsequently move away from the PS and form adult tissues. The biophysical mechanisms driving mesodermal cell movements during gastrulation in amniotes, notably warm-blooded embryos, are not understood. Until now, a major challenge has been distinguishing local individual cell-autonomous (active) displacements from convective displacements caused by large-scale (bulk) morphogenetic tissue movements. To address this problem, we used multiscale, time-lapse microscopy and a particle image velocimetry method for computing tissue displacement fields. Immunolabeled fibronectin was used as an in situ marker for quantifying tissue displacements. By imaging fluorescently labeled mesodermal cells and surrounding extracellular matrix simultaneously, we were able to separate directly the active and passive components of cell displacement during gastrulation. Our results reveal the following: (i) Convective tissue motion contributes significantly to total cell displacement and must be subtracted to measure true cell-autonomous displacement; (ii) Cell-autonomous displacement decreases gradually after regression from the PS; and (iii) There is an increasing cranial-to-caudal (head-to-tail) cell-autonomous motility gradient, with caudal cells actively moving away from the PS faster than cranial cells. These studies show that, in some regions of the embryo, total mesodermal cell displacements are mostly due to convective tissue movements; thus, the data have profound implications for understanding cell guidance mechanisms and tissue morphogenesis in warm-blooded embryos.

  11. Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

    PubMed Central

    Xu, Tingting; Close, Dan M.; Webb, James D.; Ripp, Steven A.; Sayler, Gary S.

    2013-01-01

    Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion. PMID:24193545

  12. 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.

  13. 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.

  14. The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

    PubMed Central

    Mostowy, Serge; Shenoy, Avinash R.

    2016-01-01

    Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence. PMID:26292640

  15. The cytoskeleton in cell-autonomous immunity: structural determinants of host defence.

    PubMed

    Mostowy, Serge; Shenoy, Avinash R

    2015-09-15

    Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton--actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement--have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence.

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

    PubMed Central

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

    2000-01-01

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

  17. Autonomous Molecular Cascades for Evaluation of Cell Surfaces

    PubMed Central

    Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P.; Rudchenko, Sergei; Stojanovic, Milan N.

    2013-01-01

    Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs1–4. Previously studied nucleic acid-based-automata include game-playing molecular devices (MAYA automata3,5) and finite-state automata for analysis of nucleic acids6 with the latter inspiring circuits for the analysis of RNA species inside cells7,8. Here, we describe automata based on strand-displacement9,10 cascades directed by antibodies that can analyze cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells. PMID:23892986

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

    PubMed Central

    Edgerton, Jeremy R; Reinhart, Peter H

    2003-01-01

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

  19. The Cell Non-Autonomous Nature of Electron Transport Chain-Mediated Longevity

    PubMed Central

    Durieux, Jenni; Wolff, Suzanne; Dillin, Andrew

    2011-01-01

    Summary The life span of C. elegans can be increased via reduced function of the mitochondria; however, the extent to which mitochondrial alteration in a single, distinct tissue may influence aging in the whole organism remains unknown. We addressed this question by asking whether manipulations to ETC function can modulate aging in a cell non-autonomous fashion. We report that the alteration of mitochondrial function in key tissues is essential for establishing and maintaining a pro-longevity cue. We find that regulators of mitochondrial stress responses are essential and specific genetic requirements for the electron transport chain (ETC) longevity pathway. Strikingly, we find that mitochondrial perturbation in one tissue is perceived and acted upon by the mitochondrial stress response pathway in a distal tissue. These results suggest that mitochondria may establish and perpetuate the rate of aging for the whole organism independent of cell-autonomous functions. PMID:21215371

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

    PubMed

    Swensen, Andrew M; Bean, Bruce P

    2003-10-22

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

  1. Evolved Colloidosomes Undergoing Cell-like Autonomous Shape Oscillations with Buckling.

    PubMed

    Tamate, Ryota; Ueki, Takeshi; Yoshida, Ryo

    2016-04-18

    In living systems, there are many autonomous and oscillatory phenomena to sustain life, such as heart contractions and breathing. At the microscopic level, oscillatory shape deformations of cells are often observed in dynamic behaviors during cell migration and morphogenesis. In many cases, oscillatory behaviors of cells are not simplistic but complex with diverse deformations. So far, we have succeeded in developing self-oscillating polymers and gels, but complex oscillatory behaviors mimicking those of living cells have yet to be reproduced. Herein, we report a cell-like hollow sphere composed of self-oscillating microgels, that is, a colloidosome, that exhibits drastic shape oscillation in addition to swelling/deswelling oscillations driven by an oscillatory reaction. The resulting oscillatory profile waveform becomes markedly more complex than a conventional one. Especially for larger colloidosomes, multiple buckling and moving buckling points are observed to be analogous to cells.

  2. Developmental profiling of postnatal dentate gyrus progenitors provides evidence for dynamic cell-autonomous regulation

    PubMed Central

    Gilley, Jennifer A.; Yang, Cui-Ping; Kernie, Steven G.

    2009-01-01

    The dentate gyrus of the hippocampus is one of the most prominent regions in the postnatal mammalian brain where neurogenesis continues throughout life. There is tremendous speculation regarding the potential implications of adult hippocampal neurogenesis, though it remains unclear to what extent this ability becomes attenuated during normal aging, and what genetic changes in the progenitor population ensue over time. Using defined elements of the nestin promoter, we developed a transgenic mouse that reliably labels neural stem and early progenitors with green fluorescent protein (GFP). Using a combination of immunohistochemical and flow cytometry techniques, we characterized the progenitor cells within the dentate gyrus and created a developmental profile from postnatal day 7 (P7) until 6 months of age. In addition, we demonstrate that the proliferative potential of these progenitors is controlled at least in part by cell-autonomous cues. Finally, in order to identify what may underlie these differences, we performed stem cell-specific microarrays on GFP-expressing sorted cells from isolated P7 and postnatal day 28 (P28) dentate gyrus. We identified several differentially expressed genes that may underlie the functional differences that we observe in neurosphere assays from sorted cells and differentiation assays at these different ages. These data suggest that neural progenitors from the dentate gyrus are differentially regulated by cell-autonomous factors that change over time. PMID:20014381

  3. Cadherin-2 Is Required Cell Autonomously for Collective Migration of Facial Branchiomotor Neurons

    PubMed Central

    Rebman, Jane K.; Kirchoff, Kathryn E.

    2016-01-01

    Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs. PMID:27716840

  4. A mechanistic framework for noncell autonomous stem cell induction in Arabidopsis.

    PubMed

    Daum, Gabor; Medzihradszky, Anna; Suzaki, Takuya; Lohmann, Jan U

    2014-10-07

    Cell-cell communication is essential for multicellular development and, consequently, evolution has brought about an array of distinct mechanisms serving this purpose. Consistently, induction and maintenance of stem cell fate by noncell autonomous signals is a feature shared by many organisms and may depend on secreted factors, direct cell-cell contact, matrix interactions, or a combination of these mechanisms. Although many basic cellular processes are well conserved between animals and plants, cell-to-cell signaling is one function where substantial diversity has arisen between the two kingdoms of life. One of the most striking differences is the presence of cytoplasmic bridges, called plasmodesmata, which facilitate the exchange of molecules between neighboring plant cells and provide a unique route for cell-cell communication in the plant lineage. Here, we provide evidence that the stem cell inducing transcription factor WUSCHEL (WUS), expressed in the niche, moves to the stem cells via plasmodesmata in a highly regulated fashion and that this movement is required for WUS function and, thus, stem cell activity in Arabidopsis thaliana. We show that cell context-independent mobility is encoded in the WUS protein sequence and mediated by multiple domains. Finally, we demonstrate that parts of the protein that restrict movement are required for WUS homodimerization, suggesting that formation of WUS dimers might contribute to the regulation of apical stem cell activity.

  5. Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors.

    PubMed

    Gruner, Matthew; Grubbs, Jeremy; McDonagh, Aja; Valdes, Dominic; Winbush, Ari; van der Linden, Alexander M

    2016-08-01

    Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.

  6. Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors

    PubMed Central

    Winbush, Ari; van der Linden, Alexander M.

    2016-01-01

    Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state. PMID:27487365

  7. Autonomous Hedgehog signalling is undetectable in PC-3 prostate cancer cells.

    PubMed

    McCarthy, Frank R K; Brown, Andrew J

    2008-08-15

    The Hedgehog signalling pathway has been implicated in the development of prostate cancer, although this area remains controversial. Some but not all studies have noted relatively high Hedgehog pathway activity in commonly used prostate cancer cell lines. We aimed to evaluate the widely used PC-3 cell line as a model to investigate Hedgehog signalling in a prostate cancer setting. Using a sensitive Hedgehog inducible luciferase reporter assay, we found no evidence of autonomous Hedgehog signalling in PC-3 cells, irrespective of passage number. In addition, manipulations that should either increase (an oxysterol) or decrease (cyclopamine) Hedgehog pathway activity had no effect on reporter activity, and cyclopamine treatment did not affect PC-3 cell viability. Therefore, our findings contradict some earlier reports and caution against the use of PC-3 cells to investigate the Hedgehog pathway in a prostate cancer setting.

  8. Brain-resident memory T cells represent an autonomous cytotoxic barrier to viral infection

    PubMed Central

    Vincenti, Ilena; Wagner, Ingrid; Pinschewer, Daniel

    2016-01-01

    Tissue-resident memory T cells (TRM) persist at sites of prior infection and have been shown to enhance pathogen clearance by recruiting circulating immune cells and providing bystander activation. Here, we characterize the functioning of brain-resident memory T cells (bTRM) in an animal model of viral infection. bTRM were subject to spontaneous homeostatic proliferation and were largely refractory to systemic immune cell depletion. After viral reinfection in mice, bTRM rapidly acquired cytotoxic effector function and prevented fatal brain infection, even in the absence of circulating CD8+ memory T cells. Presentation of cognate antigen on MHC-I was essential for bTRM-mediated protective immunity, which involved perforin- and IFN-γ–dependent effector mechanisms. These findings identify bTRM as an organ-autonomous defense system serving as a paradigm for TRM functioning as a self-sufficient first line of adaptive immunity. PMID:27377586

  9. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes

    PubMed Central

    Eng, George; Lee, Benjamin W.; Protas, Lev; Gagliardi, Mark; Brown, Kristy; Kass, Robert S.; Keller, Gordon; Robinson, Richard B.; Vunjak-Novakovic, Gordana

    2016-01-01

    The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration. PMID:26785135

  10. Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis*

    PubMed Central

    Yin, Yan; Liu, Liren; Yang, Chenyi; Lin, Congxing; Veith, George Michael; Wang, Caihong; Sutovsky, Peter; Zhou, Pengbo; Ma, Liang

    2016-01-01

    CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation of Cul4a in mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4b germ cell-specific conditional knock-out (Cul4bVasa),as well as Cul4b global knock-out (Cul4bSox2) mouse, to investigate its roles in spermatogenesis. Germ cell-specific deletion of Cul4b led to male infertility, despite normal testicular morphology and comparable numbers of spermatozoa. Notably, significantly impaired sperm mobility caused by reduced mitochondrial activity and glycolysis level were observed in the majority of the mutant spermatozoa, manifested by low, if any, sperm ATP production. Furthermore, Cul4bVasa spermatozoa exhibited defective arrangement of axonemal microtubules and flagella outer dense fibers. Our mass spectrometry analysis identified INSL6 as a novel CUL4B substrate in male germ cells, evidenced by its direct polyubiquination and degradation by CUL4B E3 ligase. Nevertheless, Cul4b global knock-out males lost their germ cells in an age-dependent manner, implying failure of maintaining the spermatogonial stem cell niche in somatic cells. Taken together, our results show that CUL4B is indispensable to spermatogenesis, and it functions cell autonomously in male germ cells to ensure spermatozoa motility, whereas it functions non-cell-autonomously in somatic cells to maintain spermatogonial stemness. Thus, CUL4B links two distinct spermatogenetic processes to a single E3 ligase, highlighting the significance of ubiquitin modification during spermatogenesis. PMID:26846852

  11. Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis.

    PubMed

    Yin, Yan; Liu, Liren; Yang, Chenyi; Lin, Congxing; Veith, George Michael; Wang, Caihong; Sutovsky, Peter; Zhou, Pengbo; Ma, Liang

    2016-03-25

    CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation ofCul4ain mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4bgerm cell-specific conditional knock-out (Cul4b(Vasa)),as well asCul4bglobal knock-out (Cul4b(Sox2)) mouse, to investigate its roles in spermatogenesis. Germ cell-specific deletion of Cul4bled to male infertility, despite normal testicular morphology and comparable numbers of spermatozoa. Notably, significantly impaired sperm mobility caused by reduced mitochondrial activity and glycolysis level were observed in the majority of the mutant spermatozoa, manifested by low, if any, sperm ATP production. Furthermore,Cul4b(Vasa)spermatozoa exhibited defective arrangement of axonemal microtubules and flagella outer dense fibers. Our mass spectrometry analysis identified INSL6 as a novel CUL4B substrate in male germ cells, evidenced by its direct polyubiquination and degradation by CUL4B E3 ligase. Nevertheless,Cul4bglobal knock-out males lost their germ cells in an age-dependent manner, implying failure of maintaining the spermatogonial stem cell niche in somatic cells. Taken together, our results show that CUL4B is indispensable to spermatogenesis, and it functions cell autonomously in male germ cells to ensure spermatozoa motility, whereas it functions non-cell-autonomously in somatic cells to maintain spermatogonial stemness. Thus, CUL4B links two distinct spermatogenetic processes to a single E3 ligase, highlighting the significance of ubiquitin modification during spermatogenesis.

  12. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.

    PubMed

    Ramanathan, Chidambaram; Khan, Sanjoy K; Kathale, Nimish D; Xu, Haiyan; Liu, Andrew C

    2012-09-27

    In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host

  13. Power optimization in body sensor networks: the case of an autonomous wireless EMG sensor powered by PV-cells.

    PubMed

    Penders, J; Pop, V; Caballero, L; van de Molengraft, J; van Schaijk, R; Vullers, R; Van Hoof, C

    2010-01-01

    Recent advances in ultra-low-power circuits and energy harvesters are making self-powered body sensor nodes a reality. Power optimization at the system and application level is crucial in achieving ultra-low-power consumption for the entire system. This paper reviews system-level power optimization techniques, and illustrates their impact on the case of autonomous wireless EMG monitoring. The resulting prototype, an Autonomous wireless EMG sensor power by PV-cells, is presented.

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

    PubMed Central

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

    2017-01-01

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

  15. Membrane-Tethered Ligands: Tools for Cell-Autonomous Pharmacological Manipulation of Biological Circuits

    PubMed Central

    Choi, Charles

    2013-01-01

    Detection of secreted signaling molecules by cognate cell surface receptors is a major intercellular communication pathway in cellular circuits that control biological processes. Understanding the biological significance of these connections would allow us to understand how cellular circuits operate as a whole. Membrane-tethered ligands are recombinant transgenes with structural modules that allow them to act on cell-surface receptors and ion channel subtypes with pharmacological specificity in a cell-autonomous manner. Membrane-tethered ligands have been successful in the specific manipulation of ion channels as well as G-protein-coupled receptors, and, in combination with cell-specific promoters, such manipulations have been restricted to genetically defined subpopulations within cellular circuits in vivo to induce specific phenotypes controlled by those circuits. These studies establish the membrane-tethering approach as a generally applicable method for dissecting neural and physiological circuits. PMID:23636262

  16. The cell-autonomous mechanisms underlying the activity of metformin as an anticancer drug.

    PubMed

    Sacco, Francesca; Calderone, Alberto; Castagnoli, Luisa; Cesareni, Gianni

    2016-12-06

    The biguanide drug metformin profoundly affects cell metabolism, causing an impairment of the cell energy balance and triggering a plethora of pleiotropic effects that vary depending on the cellular or environmental context. Interestingly, a decade ago, it was observed that metformin-treated diabetic patients have a significantly lower cancer risk. Although a variety of in vivo and in vitro observations emphasising the role of metformin as anticancer drug have been reported, the underlying mechanisms are still poorly understood. Here, we discuss our current understanding of the molecular mechanisms that are perturbed by metformin treatment and that might be relevant to understand its antitumour activities. We focus on the cell-autonomous mechanisms modulating growth and death of cancer cells.

  17. The cell-autonomous mechanisms underlying the activity of metformin as an anticancer drug

    PubMed Central

    Sacco, Francesca; Calderone, Alberto; Castagnoli, Luisa; Cesareni, Gianni

    2016-01-01

    The biguanide drug metformin profoundly affects cell metabolism, causing an impairment of the cell energy balance and triggering a plethora of pleiotropic effects that vary depending on the cellular or environmental context. Interestingly, a decade ago, it was observed that metformin-treated diabetic patients have a significantly lower cancer risk. Although a variety of in vivo and in vitro observations emphasising the role of metformin as anticancer drug have been reported, the underlying mechanisms are still poorly understood. Here, we discuss our current understanding of the molecular mechanisms that are perturbed by metformin treatment and that might be relevant to understand its antitumour activities. We focus on the cell-autonomous mechanisms modulating growth and death of cancer cells. PMID:27875520

  18. Non-Cell-Autonomous Postmortem Lignification of Tracheary Elements in Zinnia elegans[W][OA

    PubMed Central

    Pesquet, Edouard; Zhang, Bo; Gorzsás, András; Puhakainen, Tuula; Serk, Henrik; Escamez, Sacha; Barbier, Odile; Gerber, Lorenz; Courtois-Moreau, Charleen; Alatalo, Edward; Paulin, Lars; Kangasjärvi, Jaakko; Sundberg, Björn; Goffner, Deborah; Tuominen, Hannele

    2013-01-01

    Postmortem lignification of xylem tracheary elements (TEs) has been debated for decades. Here, we provide evidence in Zinnia elegans TE cell cultures, using pharmacological inhibitors and in intact Z. elegans plants using Fourier transform infrared microspectroscopy, that TE lignification occurs postmortem (i.e., after TE programmed cell death). In situ RT-PCR verified expression of the lignin monomer biosynthetic cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in not only the lignifying TEs but also in the unlignified non-TE cells of Z. elegans TE cell cultures and in living, parenchymatic xylem cells that surround TEs in stems. These cells were also shown to have the capacity to synthesize and transport lignin monomers and reactive oxygen species to the cell walls of dead TEs. Differential gene expression analysis in Z. elegans TE cell cultures and concomitant functional analysis in Arabidopsis thaliana resulted in identification of several genes that were expressed in the non-TE cells and that affected lignin chemistry on the basis of pyrolysis–gas chromatography/mass spectrometry analysis. These data suggest that living, parenchymatic xylem cells contribute to TE lignification in a non-cell-autonomous manner, thus enabling the postmortem lignification of TEs. PMID:23572543

  19. Uptake of the Fusarium Effector Avr2 by Tomato Is Not a Cell Autonomous Event

    PubMed Central

    Di, Xiaotang; Gomila, Jo; Ma, Lisong; van den Burg, Harrold A.; Takken, Frank L. W.

    2016-01-01

    Pathogens secrete effector proteins to manipulate the host for their own proliferation. Currently it is unclear whether the uptake of effector proteins from extracellular spaces is a host autonomous process. We study this process using the Avr2 effector protein from Fusarium oxysporum f. sp. lycopersici (Fol). Avr2 is an important virulence factor that is secreted into the xylem sap of tomato following infection. Besides that, it is also an avirulence factor triggering immune responses in plants carrying the I-2 resistance gene. Recognition of Avr2 by I-2 occurs inside the plant nucleus. Here, we show that pathogenicity of an Avr2 knockout Fusarium (FolΔAvr2) strain is fully complemented on transgenic tomato lines that express either a secreted (Avr2) or cytosolic Avr2 (ΔspAvr2) protein, indicating that Avr2 exerts its virulence functions inside the host cells. Furthermore, our data imply that secreted Avr2 is taken up from the extracellular spaces in the presence of the fungus. Grafting studies were performed in which scions of I-2 tomato plants were grafted onto either a ΔspAvr2 or on an Avr2 rootstock. Although the Avr2 protein could readily be detected in the xylem sap of the grafted plant tissues, no I-2-mediated immune responses were induced suggesting that I-2-expressing tomato cells cannot autonomously take up the effector protein from the xylem sap. Additionally, ΔspAvr2 and Avr2 plants were crossed with I-2 plants. Whereas ΔspAvr2/I-2 F1 plants showed a constitutive immune response, immunity was not triggered in the Avr2/I-2 plants confirming that Avr2 is not autonomously taken up from the extracellular spaces to trigger I-2. Intriguingly, infiltration of Agrobacterium tumefaciens in leaves of Avr2/I-2 plants triggered I-2 mediated cell death, which indicates that Agrobacterium triggers effector uptake. To test whether, besides Fol, effector uptake could also be induced by other fungal pathogens the ΔspAvr2 and Avr2 transgenic lines were inoculated

  20. Morphological changes in autonomic ganglionic cells of the heart in diabetic patients.

    PubMed

    Tsujimura, T; Nunotani, H; Fushimi, H; Inoue, T

    1986-06-01

    To clarify the histological changes of the cardiac autonomic nervous system in diabetes mellitus, ganglionic cells of the hearts of autopsy cases were examined light microscopically. In 7 severely diabetic patients, the ganglionic neurons showed cellular contraction, cytoplasmic condensation and poor staining of Nissl substance. As neuronal alterations were obvious neither in the mild diabetic patients nor in the non-diabetic patients, these alterations therefore seemed to correlate with diabetes mellitus. The neuronal changes did not seem to correlate with major coronary arterial atherosclerotic narrowing.

  1. Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects.

    PubMed

    Kenner, Lukas; Hoebertz, Astrid; Beil, F Timo; Beil, Timo; Keon, Niamh; Karreth, Florian; Eferl, Robert; Scheuch, Harald; Szremska, Agnieszka; Amling, Michael; Schorpp-Kistner, Marina; Angel, Peter; Wagner, Erwin F

    2004-02-16

    Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunBDelta/Delta mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junBDelta/Delta osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16(INK4a) levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage-osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.

  2. An NAD+ biosynthetic pathway enzyme functions cell non-autonomously in C. elegans development

    PubMed Central

    Crook, Matt; McReynolds, Melanie R.; Wang, Wenqing; Hanna-Rose, Wendy

    2017-01-01

    Background Disruption of cellular metabolite levels can adversely impact development. Specifically, loss-of-function of the C. elegans NAD+ salvage biosynthesis gene PNC-1 results in an array of developmental phenotypes. Intriguingly, PNC-1 and its functional equivalent in vertebrates are secreted, but the contributions of the extracellular enzymes are poorly understood. We sought to study the tissue-specific requirements for PNC-1 expression and to examine the role of the secreted isoform. Results A thorough analysis of PNC-1 expression did not detect expression in tissues that require PNC-1 function. Limited expression of both the secreted and intracellular PNC-1 isoforms provided function at a distance from the tissues with phenotypes. We also find that the secreted isoform contributes to in vivo PNC-1 activity. Furthermore, uv1 cell survival has the most stringent requirements in terms of PNC-1 expression pattern or level. Conclusion Using careful promoter analysis and a restricted expression approach we have shown that both the secreted and the intracellular PNC-1 isoforms function cell non-autonomously, and that the PNC-1a isoform is functionally relevant in vivo. Our work suggests a model where PNC-1 function is provided cell non-autonomously by a mix of intra and extracellular activity, most likely requiring NAD+ salvage metabolite transport between tissues. PMID:24753121

  3. 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

  4. Distance-dependent scaling of AMPARs is cell-autonomous and GluA2 dependent.

    PubMed

    Shipman, Seth L; Herring, Bruce E; Suh, Young Ho; Roche, Katherine W; Nicoll, Roger A

    2013-08-14

    The extensive dendritic arbor of a pyramidal cell introduces considerable complexity to the integration of synaptic potentials. Propagation of dendritic potentials is largely passive, in contrast to regenerative axonal potentials that are maintained by voltage-gated sodium channels, leading to a declination in amplitude as dendritic potentials travel toward the soma in a manner that disproportionally affects distal synaptic inputs. To counteract this amplitude filtering, Schaffer collateral synapses onto CA1 pyramidal cells contain a varying number of AMPA receptors (AMPARs) per synapse that increases with distance from the soma, a phenomenon known as distance-dependent scaling. Here, we undertake an investigation into the molecular mechanisms of distance-dependent scaling. Using dendritic recordings from rat pyramidal neurons, we confirm the basic scaling phenomenon and find that it is expressed and can be manipulated cell autonomously. Finally, we show that it depends on the presence of both a reserve pool of AMPARs and the AMPAR subunit GluA2.

  5. Molecular basis for the autonomous promotion of cell proliferation by angiogenin

    PubMed Central

    Hoang, Trish T.; Raines, Ronald T.

    2017-01-01

    Canonical growth factors act indirectly via receptor-mediated signal transduction pathways. Here, we report on an autonomous pathway in which a growth factor is internalized, has its localization regulated by phosphorylation, and ultimately uses intrinsic catalytic activity to effect epigenetic change. Angiogenin (ANG), a secreted vertebrate ribonuclease, is known to promote cell proliferation, leading to neovascularization as well as neuroprotection in mammals. Upon entering cells, ANG encounters the cytosolic ribonuclease inhibitor protein, which binds with femtomolar affinity. We find that protein kinase C and cyclin-dependent kinase phosphorylate ANG, enabling ANG to evade its inhibitor and enter the nucleus. After migrating to the nucleolus, ANG cleaves promoter-associated RNA, which prevents the recruitment of the nucleolar remodeling complex to the ribosomal DNA promoter. The ensuing derepression of rDNA transcription promotes cell proliferation. The biochemical basis for this unprecedented mechanism of signal transduction suggests new modalities for the treatment of cancers and neurological disorders. PMID:27915233

  6. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity.

    PubMed

    Marusyk, Andriy; Tabassum, Doris P; Altrock, Philipp M; Almendro, Vanessa; Michor, Franziska; Polyak, Kornelia

    2014-10-02

    Cancers arise through a process of somatic evolution that can result in substantial sub-clonal heterogeneity within tumours. The mechanisms responsible for the coexistence of distinct sub-clones and the biological consequences of this coexistence remain poorly understood. Here we used a mouse xenograft model to investigate the impact of sub-clonal heterogeneity on tumour phenotypes and the competitive expansion of individual clones. We found that tumour growth can be driven by a minor cell subpopulation, which enhances the proliferation of all cells within a tumour by overcoming environmental constraints and yet can be outcompeted by faster proliferating competitors, resulting in tumour collapse. We developed a mathematical modelling framework to identify the rules underlying the generation of intra-tumour clonal heterogeneity. We found that non-cell-autonomous driving of tumour growth, together with clonal interference, stabilizes sub-clonal heterogeneity, thereby enabling inter-clonal interactions that can lead to new phenotypic traits.

  7. Thymus-autonomous T cell development in the absence of progenitor import.

    PubMed

    Martins, Vera C; Ruggiero, Eliana; Schlenner, Susan M; Madan, Vikas; Schmidt, Manfred; Fink, Pamela J; von Kalle, Christof; Rodewald, Hans-Reimer

    2012-07-30

    Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

  8. Cytoskeletal turnover and Myosin contractility drive cell autonomous oscillations in a model of Drosophila Dorsal Closure

    NASA Astrophysics Data System (ADS)

    Machado, P. F.; Blanchard, G. B.; Duque, J.; Gorfinkiel, N.

    2014-06-01

    Oscillatory behaviour in force-generating systems is a pervasive phenomenon in cell biology. In this work, we investigate how oscillations in the actomyosin cytoskeleton drive cell shape changes during the process of Dorsal Closure (DC), a morphogenetic event in Drosophila embryo development whereby epidermal continuity is generated through the pulsatile apical area reduction of cells constituting the amnioserosa (AS) tissue. We present a theoretical model of AS cell dynamics by which the oscillatory behaviour arises due to a coupling between active myosin-driven forces, actin turnover and cell deformation. Oscillations in our model are cell-autonomous and are modulated by neighbour coupling, and our model accurately reproduces the oscillatory dynamics of AS cells and their amplitude and frequency evolution. A key prediction arising from our model is that the rate of actin turnover and Myosin contractile force must increase during DC in order to reproduce the decrease in amplitude and period of cell area oscillations observed in vivo. This prediction opens up new ways to think about the molecular underpinnings of AS cell oscillations and their link to net tissue contraction and suggests the form of future experimental measurements.

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

    PubMed

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

    2009-01-01

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

  10. Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis

    PubMed Central

    Xiang, Yan; Stine, Zachary E.; Xia, Jinsong; Lu, Yunqi; O’Connor, Roddy S.; Altman, Brian J.; Hsieh, Annie L.; Gouw, Arvin M.; Thomas, Ajit G.; Gao, Ping; Sun, Linchong; Song, Libing; Yan, Benedict; Slusher, Barbara S.; Zhuo, Jingli; Ooi, London L.; Lee, Caroline G.L.; Mancuso, Anthony; McCallion, Andrew S.; Le, Anne; Milone, Michael C.; Rayport, Stephen; Felsher, Dean W.; Dang, Chi V.

    2015-01-01

    Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell–autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell–autonomous dependence on GLS for cancer therapy. PMID:25915584

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

    PubMed

    Raman, I M; Bean, B P

    1997-06-15

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

  12. 'Medusa head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 3: Anti-Yo/CDR2, anti-Nb/AP3B2, PCA-2, anti-Tr/DNER, other antibodies, diagnostic pitfalls, summary and outlook.

    PubMed

    Jarius, S; Wildemann, B

    2015-09-17

    Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as 'Medusa head antibodies' due to their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects and provides a summary and outlook.

  13. In vivo cell-autonomous transcriptional abnormalities revealed in mice expressing mutant huntingtin in striatal but not cortical neurons.

    PubMed

    Thomas, Elizabeth A; Coppola, Giovanni; Tang, Bin; Kuhn, Alexandre; Kim, SoongHo; Geschwind, Daniel H; Brown, Timothy B; Luthi-Carter, Ruth; Ehrlich, Michelle E

    2011-03-15

    Huntington's disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by abnormal protein aggregates and motor and cognitive dysfunction. Htt protein is ubiquitously expressed, but the striatal medium spiny neuron (MSN) is most susceptible to dysfunction and death. Abnormal gene expression represents a core pathogenic feature of HD, but the relative roles of cell-autonomous and non-cell-autonomous effects on transcription remain unclear. To determine the extent of cell-autonomous dysregulation in the striatum in vivo, we examined genome-wide RNA expression in symptomatic D9-N171-98Q (a.k.a. DE5) transgenic mice in which the forebrain expression of the first 171 amino acids of human Htt with a 98Q repeat expansion is limited to MSNs. Microarray data generated from these mice were compared with those generated on the identical array platform from a pan-neuronal HD mouse model, R6/2, carrying two different CAG repeat lengths, and a relatively high degree of overlap of changes in gene expression was revealed. We further focused on known canonical pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, dopamine signaling and trophic support. While genes related to excitotoxicity, dopamine signaling and trophic support were altered in both DE5 and R6/2 mice, which may be either cell autonomous or non-cell autonomous, genes related to mitochondrial dysfunction, oxidative stress and the peroxisome proliferator-activated receptor are primarily affected in DE5 transgenic mice, indicating cell-autonomous mechanisms. Overall, HD-induced dysregulation of the striatal transcriptome can be largely attributed to intrinsic effects of mutant Htt, in the absence of expression in cortical neurons.

  14. Cell-autonomous activation of Hedgehog signaling inhibits brown adipose tissue development.

    PubMed

    Nosavanh, LaGina; Yu, Da-Hai; Jaehnig, Eric J; Tong, Qiang; Shen, Lanlan; Chen, Miao-Hsueh

    2015-04-21

    Although recent studies have shown that brown adipose tissue (BAT) arises from progenitor cells that also give rise to skeletal muscle, the developmental signals that control the formation of BAT remain largely unknown. Here, we show that brown preadipocytes possess primary cilia and can respond to Hedgehog (Hh) signaling. Furthermore, cell-autonomous activation of Hh signaling blocks early brown-preadipocyte differentiation, inhibits BAT formation in vivo, and results in replacement of neck BAT with poorly differentiated skeletal muscle. Finally, we show that Hh signaling inhibits BAT formation partially through up-regulation of chicken ovalbumin upstream promoter transcription factor II (COUP-TFII). Taken together, our studies uncover a previously unidentified role for Hh as an inhibitor of BAT development.

  15. TREX1 deficiency triggers cell-autonomous immunity in a cGAS-dependent manner.

    PubMed

    Ablasser, Andrea; Hemmerling, Inga; Schmid-Burgk, Jonathan L; Behrendt, Rayk; Roers, Axel; Hornung, Veit

    2014-06-15

    Cytosolic detection of DNA is crucial for the initiation of antiviral immunity but can also cause autoimmunity in the context of endogenous nucleic acids being sensed. Mutations in the human 3' repair exonuclease 1 (TREX1) have been linked to the type I IFN-associated autoimmune disease Aicardi-Goutières syndrome. The exact mechanisms driving unabated type I IFN responses in the absence of TREX1 are only partly understood, but it appears likely that accumulation of endogenous DNA species triggers a cell-autonomous immune response by activating a cytosolic DNA receptor. In this article, we demonstrate that knocking out the DNA sensor cyclic GMP-AMP synthase completely abrogates spontaneous induction of IFN-stimulated genes in TREX1-deficient cells. These findings indicate a key role of cyclic GMP-AMP synthase for the initiation of self-DNA-induced autoimmune disorders, thus providing important implications for novel therapeutic approaches.

  16. Astrocytes and Microglia as Non-cell Autonomous Players in the Pathogenesis of ALS

    PubMed Central

    Hyeon, Seung Jae; Im, Hyeonjoo; Ryu, Hyun; Kim, Yunha

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that leads to a progressive muscle wasting and paralysis. The pathological phenotypes are featured by severe motor neuron death and glial activation in the lumbar spinal cord. Proposed ALS pathogenic mechanisms include glutamate cytotoxicity, inflammatory pathway, oxidative stress, and protein aggregation. However, the exact mechanisms of ALS pathogenesis are not fully understood yet. Recently, a growing body of evidence provides a novel insight on the importance of glial cells in relation to the motor neuronal damage via the non-cell autonomous pathway. Accordingly, the aim of the current paper is to overview the role of astrocytes and microglia in the pathogenesis of ALS and to better understand the disease mechanism of ALS. PMID:27790057

  17. Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish

    PubMed Central

    Lesaffre, Brigitte; Joliot, Alain; Prochiantz, Alain; Volovitch, Michel

    2007-01-01

    Background Modifications in Pax6 homeogene expression produce strong eye phenotypes. This suggested to us that eye development might be an appropriate model to verify if homeoprotein intercellular passage has important functions in early development. Similar to other homeoproteins, Pax6 has two domains that enable secretion and internalization by live cells and, thus, intercellular passage. In principle, a straightforward way to test the hypothesis would be to mutate one of the two sequences to produce a 'cell autonomous only' Pax6. However, this was not possible because these sequences are in the homeodomain and their modification would affect Pax6 transcriptional properties. We have thus developed an approach aimed at blocking Pax6 only in the extracellular milieu of developing zebrafish embryos. Results A first strategy was to inject a one-cell embryo with a mRNA encoding a secreted single-chain anti-Pax6 antibody. A second, complementary, strategy was to inject a Pax6 antibody in the blastula extracellular milieu. In both cases, 'dissymmetric eyes', 'one eye only' and 'no eye' phenotypes were produced. In most cases, lens phenotypes paralleled retina malformations. Although eye phenotypes were analyzed 30 hours post-fertilization, there was a strong correlation between early eye field asymmetry, early asymmetry in Pax6 expression and later-occurring eye malformations. Several controls were introduced, demonstrating that the effect is specific to Pax6 and cannot be explained by intracellular antibody activities. Conclusion This study supports the hypothesis that the Pax6 transcription factor is also a signaling molecule with direct non-cell autonomous activity. PMID:17229313

  18. Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

    PubMed

    Völkening, Bianca; Schönig, Kai; Kronenberg, Golo; Bartsch, Dusan; Weber, Tillmann

    2017-05-01

    Ca(2+) is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Cav 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Cav 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created Tg(GLAST-CreERT2) /Cacna1c(fl/fl) /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Cav 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Cav 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Cav 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion.

  19. Autonomous and Autonomic Swarms

    NASA Technical Reports Server (NTRS)

    Hinchey, Michael G.; Rash, James L.; Truszkowski, Walter F.; Rouff, Christopher A.; Sterritt, Roy

    2005-01-01

    A watershed in systems engineering is represented by the advent of swarm-based systems that accomplish missions through cooperative action by a (large) group of autonomous individuals each having simple capabilities and no global knowledge of the group s objective. Such systems, with individuals capable of surviving in hostile environments, pose unprecedented challenges to system developers. Design and testing and verification at much higher levels will be required, together with the corresponding tools, to bring such systems to fruition. Concepts for possible future NASA space exploration missions include autonomous, autonomic swarms. Engineering swarm-based missions begins with understanding autonomy and autonomicity and how to design, test, and verify systems that have those properties and, simultaneously, the capability to accomplish prescribed mission goals. Formal methods-based technologies, both projected and in development, are described in terms of their potential utility to swarm-based system developers.

  20. Prkci is required for a non-autonomous signal that coordinates cell polarity during cavitation.

    PubMed

    Mah, In Kyoung; Soloff, Rachel; Izuhara, Audrey K; Lakeland, Daniel L; Wang, Charles; Mariani, Francesca V

    2016-08-01

    Polarized epithelia define boundaries, spaces, and cavities within organisms. Cavitation, a process by which multicellular hollow balls or tubes are produced, is typically associated with the formation of organized epithelia. In order for these epithelial layers to form, cells must ultimately establish a distinct apical-basal polarity. Atypical PKCs have been proposed to be required for apical-basal polarity in diverse species. Here we show that while cells null for the Prkci isozyme exhibit some polarity characteristics, they fail to properly segregate apical-basal proteins, form a coordinated ectodermal epithelium, or participate in normal cavitation. A failure to cavitate could be due to an overgrowth of interior cells or to an inability of interior cells to die. Null cells however, do not have a marked change in proliferation rate and are still capable of undergoing cell death, suggesting that alterations in these processes are not the predominant cause of the failed cavitation. Overexpression of BMP4 or EZRIN can partially rescue the phenotype possibly by promoting cell death, polarity, and differentiation. However, neither is sufficient to provide the required cues to generate a polarized epithelium and fully rescue cavitation. Interestingly, when wildtype and Prkci(-/-) ES cells are mixed together, a polarized ectodermal epithelium forms and cavitation is rescued, likely due to the ability of wildtype cells to produce non-autonomous polarity cues. We conclude that Prkci is not required for cells to respond to these cues, though it is required to produce them. Together these findings indicate that environmental cues can facilitate the formation of polarized epithelia and that cavitation requires the proper coordination of multiple basic cellular processes including proliferation, differentiation, cell death, and apical-basal polarization.

  1. Differential cell autonomous responses determine the outcome of coxsackievirus infections in murine pancreatic α and β cells

    PubMed Central

    Marroqui, Laura; Lopes, Miguel; dos Santos, Reinaldo S; Grieco, Fabio A; Roivainen, Merja; Richardson, Sarah J; Morgan, Noel G; Op de beeck, Anne; Eizirik, Decio L

    2015-01-01

    Type 1 diabetes (T1D) is an autoimmune disease caused by loss of pancreatic β cells via apoptosis while neighboring α cells are preserved. Viral infections by coxsackieviruses (CVB) may contribute to trigger autoimmunity in T1D. Cellular permissiveness to viral infection is modulated by innate antiviral responses, which vary among different cell types. We presently describe that global gene expression is similar in cytokine-treated and virus-infected human islet cells, with up-regulation of gene networks involved in cell autonomous immune responses. Comparison between the responses of rat pancreatic α and β cells to infection by CVB5 and 4 indicate that α cells trigger a more efficient antiviral response than β cells, including higher basal and induced expression of STAT1-regulated genes, and are thus better able to clear viral infections than β cells. These differences may explain why pancreatic β cells, but not α cells, are targeted by an autoimmune response during T1D. DOI: http://dx.doi.org/10.7554/eLife.06990.001 PMID:26061776

  2. Advanced Modular "All in One" Battery System with Intelligent Autonomous Cell Balancing Management

    NASA Astrophysics Data System (ADS)

    Petitdidier, X.; Pasquier, E.; Defer, M.; Koch, M.; Knorr, W.

    2008-09-01

    A new generation of energy storage systems based on Li-ion technology emerged at the end of the last century.To perform the first tests in safe conditions, Saft designed a simple electronic.Today, all Li-ion batteries for autonomous applications such as drones, launchers, missiles, torpedoes and "human" applications such as cellular, laptop, hybrid vehicle and nearly sub-marines need a Battery Management System.The minimum in terms of functions is the overcharge and over-discharge protections.For a battery made of 2 cells connected in series or more, a balancing system is added to maintain the available energy during all the life of the battery. For stringent/demanding applications, the state of charge and state of health are calculated by one or more computers.It is now time to take benefit of the past 10 years of Saft's experience in the domain to re-evaluate the constraints of Li-ion batteries and provide customers with improved products by optimizing the battery management.Benefits of electronic for satellite applications:• Full control over battery.• Confidence whatever the possible change of conditions in environment.• The battery system can resist long exposure to gradient conditions with mitigated and stabilized impact on performances.• The balancing function allow to use all the energy of all the cells: optimize of installed energy (compact design, mass saving). It started out with the basic fact that electrochemists are not intended to be space rated electronic experts and vice versa, even if Saft has a good heritage in the electronic battery management system. Consequently, considering heritage and expertise in their respective core businesses, Saft and ASP teamed up.It became necessary to provide an "all in one" modular energy storage system with intelligent autonomous cell balancing management.

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

    PubMed

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

    2004-10-15

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

  4. Deciphering Human Cell-Autonomous Anti-HSV-1 Immunity in the Central Nervous System.

    PubMed

    Lafaille, Fabien G; Ciancanelli, Michael J; Studer, Lorenz; Smith, Gregory; Notarangelo, Luigi; Casanova, Jean-Laurent; Zhang, Shen-Ying

    2015-01-01

    Herpes simplex virus 1 (HSV-1) is a common virus that can rarely invade the human central nervous system (CNS), causing devastating encephalitis. The permissiveness to HSV-1 of the various relevant cell types of the CNS, neurons, astrocytes, oligodendrocytes, and microglia cells, as well as their response to viral infection, has been extensively studied in humans and other animals. Nevertheless, human CNS cell-based models of anti-HSV-1 immunity are of particular importance, as responses to any given neurotropic virus may differ between humans and other animals. Human CNS neuron cell lines as well as primary human CNS neurons, astrocytes, and microglia cells cultured/isolated from embryos or cadavers, have enabled the study of cell-autonomous anti-HSV-1 immunity in vitro. However, the paucity of biological samples and their lack of purity have hindered progress in the field, which furthermore suffers from the absence of testable primary human oligodendrocytes. Recently, the authors have established a human induced pluripotent stem cells (hiPSCs)-based model of anti-HSV-1 immunity in neurons, oligodendrocyte precursor cells, astrocytes, and neural stem cells, which has widened the scope of possible in vitro studies while permitting in-depth explorations. This mini-review summarizes the available data on human primary and iPSC-derived CNS cells for anti-HSV-1 immunity. The hiPSC-mediated study of anti-viral immunity in both healthy individuals and patients with viral encephalitis will be a powerful tool in dissecting the disease pathogenesis of CNS infections with HSV-1 and other neurotropic viruses.

  5. 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.

  6. Cell-Autonomous Gβ Signaling Defines Neuron-Specific Steady State Serotonin Synthesis in Caenorhabditis elegans

    PubMed Central

    Xu, Lu; Choi, Sunju; Xie, Yusu; Sze, Ji Ying

    2015-01-01

    Heterotrimeric G proteins regulate a vast array of cellular functions via specific intracellular effectors. Accumulating pharmacological and biochemical studies implicate Gβ subunits as signaling molecules interacting directly with a wide range of effectors to modulate downstream cellular responses, in addition to their role in regulating Gα subunit activities. However, the native biological roles of Gβ-mediated signaling pathways in vivo have been characterized only in a few cases. Here, we identified a Gβ GPB-1 signaling pathway operating in specific serotonergic neurons to the define steady state serotonin (5-HT) synthesis, through a genetic screen for 5-HT synthesis mutants in Caenorhabditis elegans. We found that signaling through cell autonomous GPB-1 to the OCR-2 TRPV channel defines the baseline expression of 5-HT synthesis enzyme tryptophan hydroxylase tph-1 in ADF chemosensory neurons. This Gβ signaling pathway is not essential for establishing the serotonergic cell fates and is mechanistically separated from stress-induced tph-1 upregulation. We identified that ADF-produced 5-HT controls specific innate rhythmic behaviors. These results revealed a Gβ-mediated signaling operating in differentiated cells to specify intrinsic functional properties, and indicate that baseline TPH expression is not a default generic serotonergic fate, but is programmed in a cell-specific manner in the mature nervous system. Cell-specific regulation of TPH expression could be a general principle for tailored steady state 5-HT synthesis in functionally distinct neurons and their regulation of innate behavior. PMID:26402365

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

    PubMed Central

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

    2014-01-01

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

  8. Multiresolution motion planning for autonomous agents via wavelet-based cell decompositions.

    PubMed

    Cowlagi, Raghvendra V; Tsiotras, Panagiotis

    2012-10-01

    We present a path- and motion-planning scheme that is "multiresolution" both in the sense of representing the environment with high accuracy only locally and in the sense of addressing the vehicle kinematic and dynamic constraints only locally. The proposed scheme uses rectangular multiresolution cell decompositions, efficiently generated using the wavelet transform. The wavelet transform is widely used in signal and image processing, with emerging applications in autonomous sensing and perception systems. The proposed motion planner enables the simultaneous use of the wavelet transform in both the perception and in the motion-planning layers of vehicle autonomy, thus potentially reducing online computations. We rigorously prove the completeness of the proposed path-planning scheme, and we provide numerical simulation results to illustrate its efficacy.

  9. Pulse-transmission Oscillators: Autonomous Boolean Models and the Yeast Cell Cycle

    NASA Astrophysics Data System (ADS)

    Sevim, Volkan; Gong, Xinwei; Socolar, Joshua

    2010-03-01

    Models of oscillatory gene expression typically involve a constitutively expressed or positively autoregulated gene which is repressed by a negative feedback loop. In Boolean representations of such systems, which include the repressilator and relaxation oscillators, dynamical stability stems from the impossibility of satisfying all of the Boolean rules at once. We consider a different class of networks, in which oscillations are due to the transmission of a pulse of gene activation around a ring. Using autonomous Boolean modeling methods, we show how the circulating pulse can be stabilized by decoration of the ring with certain feedback and feed-forward motifs. We then discuss the relation of these models to ODE models of transcriptional networks, emphasizing the role of explicit time delays. Finally, we show that a network recently proposed as a generator of cell cycle oscillations in yeast contains the motifs required to support stable transmission oscillations.

  10. Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.

    PubMed

    Albiero, Mattia; Poncina, Nicol; Tjwa, Marc; Ciciliot, Stefano; Menegazzo, Lisa; Ceolotto, Giulio; Vigili de Kreutzenberg, Saula; Moura, Rute; Giorgio, Marco; Pelicci, Piergiuseppe; Avogaro, Angelo; Fadini, Gian Paolo

    2014-04-01

    Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.

  11. Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.

    PubMed

    He, Lan; Hamm, J Austin; Reddy, Alex; Sams, David; Peliciari-Garcia, Rodrigo A; McGinnis, Graham R; Bailey, Shannon M; Chow, Chi-Wing; Rowe, Glenn C; Chatham, John C; Young, Martin E

    2016-06-01

    Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCβ, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

  12. Paraneoplastic cerebellar degeneration with a circulating antibody against neurons and non-neuronal cells.

    PubMed

    Tomimoto, H; Brengman, J M; Yanagihara, T

    1993-01-01

    We describe a woman with paraneoplastic cerebellar degeneration associated with para-ovarian adenocarcinoma, who had a circulating antibody with a corresponding antigen not only in cerebellar Purkinje cells but also in neurons located in the molecular layer of the human and rat cerebellum. The antigen was also present in neurons in the cerebral cortex, brain stem, anterior horn cells, dorsal root ganglia, intestinal autonomic neurons, retinal ganglion cells, Schwann cells of the peripheral nerve and epithelial cells of the renal glomerulus in rats. Immunoelectron microscopy revealed immunoprecipitates in the smooth and rough endoplasmic reticulum and polyribosomes in human and rat cerebellar Purkinje cells and other neuronal cell bodies as well as Schwann cells of the peripheral nerve. Even though patients with this disorder manifest primarily with cerebellar and some extracerebellar signs, the antigen also exists in many neurons other than cerebellar Purkinje cells and even in non-neuronal cells. The clinicopathologic significance of the observed immunologic reaction in diverse neurons remains to be determined.

  13. Neural crest cell-autonomous roles of fibronectin in cardiovascular development

    PubMed Central

    Wang, Xia; Astrof, Sophie

    2016-01-01

    The chemical and mechanical properties of extracellular matrices (ECMs) modulate diverse aspects of cellular fates; however, how regional heterogeneity in ECM composition regulates developmental programs is not well understood. We discovered that fibronectin 1 (Fn1) is expressed in strikingly non-uniform patterns during mouse development, suggesting that regionalized synthesis of the ECM plays cell-specific regulatory roles during embryogenesis. To test this hypothesis, we ablated Fn1 in the neural crest (NC), a population of multi-potent progenitors expressing high levels of Fn1. We found that Fn1 synthesized by the NC mediated morphogenesis of the aortic arch artery and differentiation of NC cells into vascular smooth muscle cells (VSMCs) by regulating Notch signaling. We show that NC Fn1 signals in an NC cell-autonomous manner through integrin α5β1 expressed by the NC, leading to activation of Notch and differentiation of VSMCs. Our data demonstrate an essential role of the localized synthesis of Fn1 in cardiovascular development and spatial regulation of Notch signaling. PMID:26552887

  14. The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

    PubMed Central

    Brown, J A; Holmes, S G; Smith, M M

    1991-01-01

    The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure. Images PMID:1922046

  15. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    PubMed Central

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. PMID:20805991

  16. Crim1 has cell-autonomous and paracrine roles during embryonic heart development

    PubMed Central

    Iyer, Swati; Chou, Fang Yu; Wang, Richard; Chiu, Han Sheng; Raju, Vinay K. Sundar; Little, Melissa H.; Thomas, Walter G.; Piper, Michael; Pennisi, David J.

    2016-01-01

    The epicardium has a critical role during embryonic development, contributing epicardium-derived lineages to the heart, as well as providing regulatory and trophic signals necessary for myocardial development. Crim1 is a unique trans-membrane protein expressed by epicardial and epicardially-derived cells but its role in cardiogenesis is unknown. Using knockout mouse models, we observe that loss of Crim1 leads to congenital heart defects including epicardial defects and hypoplastic ventricular compact myocardium. Epicardium-restricted deletion of Crim1 results in increased epithelial-to-mesenchymal transition and invasion of the myocardium in vivo, and an increased migration of primary epicardial cells. Furthermore, Crim1 appears to be necessary for the proliferation of epicardium-derived cells (EPDCs) and for their subsequent differentiation into cardiac fibroblasts. It is also required for normal levels of cardiomyocyte proliferation and apoptosis, consistent with a role in regulating epicardium-derived trophic factors that act on the myocardium. Mechanistically, Crim1 may also modulate key developmentally expressed growth factors such as TGFβs, as changes in the downstream effectors phospho-SMAD2 and phospho-ERK1/2 are observed in the absence of Crim1. Collectively, our data demonstrates that Crim1 is essential for cell-autonomous and paracrine aspects of heart development. PMID:26821812

  17. Impulse responses of automaticity in the Purkinje fiber.

    PubMed

    Chay, T R; Lee, Y S

    1984-04-01

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

  18. Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases.

    PubMed

    Nussbaum-Krammer, Carmen I; Morimoto, Richard I

    2014-01-01

    Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.

  19. hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature

    PubMed Central

    Acevedo, Lisette M.; Lindquist, Jeffrey N.; Walsh, Breda M.; Sia, Peik; Cimadamore, Flavio; Chen, Connie; Denzel, Martin; Pernia, Cameron D.; Ranscht, Barbara; Terskikh, Alexey; Snyder, Evan Y.; Cheresh, David A.

    2015-01-01

    Summary To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning. PMID:26004631

  20. Cell-autonomous inactivation of the Reelin pathway impairs adult neurogenesis in the hippocampus

    PubMed Central

    Teixeira, Catia M.; Kron, Michelle M.; Masachs, Nuria; Zhang, Helen; Lagace, Diane C.; Martinez, Albert; Reillo, Isabel; Duan, Xin; Bosch, Carles; Pujadas, Lluis; Brunso, Lucas; Song, Hongjun; Eisch, Amelia J.; Borrell, Victor; Howell, Brian W.; Parent, Jack M.; Soriano, Eduardo

    2012-01-01

    Adult hippocampal neurogenesis is thought to be essential for learning and memory and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of disabled-1 (Dab1), an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain- and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adult-generated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders. PMID:22933789

  1. Guanylate-binding protein 1 (Gbp1) contributes to cell-autonomous immunity against Toxoplasma gondii.

    PubMed

    Selleck, Elizabeth M; Fentress, Sarah J; Beatty, Wandy L; Degrandi, Daniel; Pfeffer, Klaus; Virgin, Herbert W; Macmicking, John D; Sibley, L David

    2013-01-01

    IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1(-/-) cells, and decreased virulence of this mutant was compensated in Gbp1(-/-) mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.

  2. Miniature Piezoelectric Shaker Mechanism for Autonomous Distribution of Unconsolidated Sample to Instrument Cells

    NASA Technical Reports Server (NTRS)

    Sherrit, Stewart; Frankovich, Kent; Bao, Xiaoqi; Tucker, Curtis

    2009-01-01

    To perform in-situ measurements on Mars or other planetary bodies many instruments require powder produced using some sampling technique (drilling/coring) or sample processing technique (core crushing) to be placed in measurement cells. This usually requires filling a small sample cell using an inlet funnel. In order to minimize cross contamination with future samples and ensure the sample is transferred from the funnel to the test cell with minimal residual powder the funnel is shaken. The shaking assists gravity by fluidizing the powder and restoring flow of the material. In order to counter cross contamination or potential clogging due to settling during autonomous handling a piezoelectric shaking mechanism was designed for the deposition of sample fines in instrument inlet funnels. This device was designed to be lightweight, consume low power and demonstrated to be a resilient solid state actuator that can be mechanically and electrically tuned to shake the inlet funnel. In the final design configuration tested under nominal Mars Ambient conditions the funnel mechanism is driven by three symmetrically mounted piezoelectric flexure actuators that are out of the funnel support load path. The frequency of the actuation can be electrically controlled and monitored and mechanically tuned by the addition of tuning mass on the free end of the actuator. Unlike conventional electromagnetic motors these devices are solid state and can be designed with no macroscopically moving parts. This paper will discuss the design and testing results of these shaking mechanisms.

  3. Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae

    PubMed Central

    Arlia-Ciommo, Anthony; Leonov, Anna; Piano, Amanda; Svistkova, Veronika; Titorenko, Vladimir I.

    2014-01-01

    A body of evidence supports the view that the signaling pathways governing cellular aging - as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae. Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific ʺmaster regulatorʺ proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest), the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast. PMID:28357241

  4. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    PubMed Central

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2015-01-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety. PMID:26516295

  5. Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

    NASA Astrophysics Data System (ADS)

    Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

    2013-05-01

    Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

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

    PubMed

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

    2014-01-01

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

  7. Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion

    PubMed Central

    Perrin, Laurent; Loizides-Mangold, Ursula; Skarupelova, Svetlana; Pulimeno, Pamela; Chanon, Stephanie; Robert, Maud; Bouzakri, Karim; Modoux, Christine; Roux-Lombard, Pascale; Vidal, Hubert; Lefai, Etienne; Dibner, Charna

    2015-01-01

    Objective Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. Methods We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. Results Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. Conclusions Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes. PMID:26629407

  8. NF-κB signaling regulates cell-autonomous regulation of CXCL10 in breast cancer 4T1 cells

    PubMed Central

    Jin, Won Jong; Kim, Bongjun; Kim, Darong; Park Choo, Hea-Young; Kim, Hong-Hee; Ha, Hyunil; Lee, Zang Hee

    2017-01-01

    The chemokine CXCL10 and its receptor CXCR3 play a role in breast cancer metastasis to bone and osteoclast activation. However, the mechanism of CXCL10/CXCR3-induced intracellular signaling has not been fully investigated. To evaluate CXCL10-induced cellular events in the mouse breast cancer cell line 4T1, we developed a new synthetic CXCR3 antagonist JN-2. In this study, we observed that secretion of CXCL10 in the supernatant of 4T1 cells was gradually increased during cell growth. JN-2 inhibited basal and CXCL10-induced CXCL10 expression and cell motility in 4T1 cells. Treatment of 4T1 cells with CXCL10 increased the expression of P65, a subunit of the NF-κB pathway, via activation of the NF-κB transcriptional activity. Ectopic overexpression of P65 increased CXCL10 secretion and blunted JN-2-induced suppression of CXCL10 secretion, whereas overexpression of IκBα suppressed CXCL10 secretion. These results indicate that the CXCL10/CXCR3 axis creates a positive feedback loop through the canonical NF-κB signaling pathway in 4T1 cells. In addition, treatment of osteoblasts with conditioned medium from JN-2-treated 4T1 cells inhibited the expression of RANKL, a crucial cytokine for osteoclast differentiation, which resulted in an inhibitory effect on osteoclast differentiation in the co-culture system of bone marrow-derived macrophages and osteoblasts. Direct intrafemoral injection of 4T1 cells induced severe bone destruction; however, this effect was suppressed by the CXCR3 antagonist via downregulation of P65 expression in an animal model. Collectively, these results suggest that the CXCL10/CXCR3-mediated NF-κB signaling pathway plays a role in the control of autonomous regulation of CXCL10 and malignant tumor properties in breast cancer 4T1 cells. PMID:28209986

  9. Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

    PubMed Central

    Ariga, H; Itani, T; Iguchi-Ariga, S M

    1987-01-01

    We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Atk- cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences. Images PMID:3031448

  10. Cell-Autonomous Regulation of Dendritic Spine Density by PirB.

    PubMed

    Vidal, George S; Djurisic, Maja; Brown, Kiana; Sapp, Richard W; Shatz, Carla J

    2016-01-01

    Synapse density on cortical pyramidal neurons is modulated by experience. This process is highest during developmental critical periods, when mechanisms of synaptic plasticity are fully engaged. In mouse visual cortex, the critical period for ocular dominance (OD) plasticity coincides with the developmental pruning of synapses. At this time, mice lacking paired Ig-like receptor B (PirB) have excess numbers of dendritic spines on L5 neurons; these spines persist and are thought to underlie the juvenile-like OD plasticity observed in adulthood. Here we examine whether PirB is required specifically in excitatory neurons to exert its effect on dendritic spine and synapse density during the critical period. In mice with a conditional allele of PirB (PirB(fl/fl)), PirB was deleted only from L2/3 cortical pyramidal neurons in vivo by timed in utero electroporation of Cre recombinase. Sparse mosaic expression of Cre produced neurons lacking PirB in a sea of wild-type neurons and glia. These neurons had significantly elevated dendritic spine density, as well as increased frequency of miniature EPSCs, suggesting that they receive a greater number of synaptic inputs relative to Cre(-) neighbors. The effect of cell-specific PirB deletion on dendritic spine density was not accompanied by changes in dendritic branching complexity or axonal bouton density. Together, results imply a neuron-specific, cell-autonomous action of PirB on synaptic density in L2/3 pyramidal cells of visual cortex. Moreover, they are consistent with the idea that PirB functions normally to corepress spine density and synaptic plasticity, thereby maintaining headroom for cells to encode ongoing experience-dependent structural change throughout life.

  11. Cell-Autonomous Regulation of Dendritic Spine Density by PirB

    PubMed Central

    2016-01-01

    Synapse density on cortical pyramidal neurons is modulated by experience. This process is highest during developmental critical periods, when mechanisms of synaptic plasticity are fully engaged. In mouse visual cortex, the critical period for ocular dominance (OD) plasticity coincides with the developmental pruning of synapses. At this time, mice lacking paired Ig-like receptor B (PirB) have excess numbers of dendritic spines on L5 neurons; these spines persist and are thought to underlie the juvenile-like OD plasticity observed in adulthood. Here we examine whether PirB is required specifically in excitatory neurons to exert its effect on dendritic spine and synapse density during the critical period. In mice with a conditional allele of PirB (PirBfl/fl), PirB was deleted only from L2/3 cortical pyramidal neurons in vivo by timed in utero electroporation of Cre recombinase. Sparse mosaic expression of Cre produced neurons lacking PirB in a sea of wild-type neurons and glia. These neurons had significantly elevated dendritic spine density, as well as increased frequency of miniature EPSCs, suggesting that they receive a greater number of synaptic inputs relative to Cre– neighbors. The effect of cell-specific PirB deletion on dendritic spine density was not accompanied by changes in dendritic branching complexity or axonal bouton density. Together, results imply a neuron-specific, cell-autonomous action of PirB on synaptic density in L2/3 pyramidal cells of visual cortex. Moreover, they are consistent with the idea that PirB functions normally to corepress spine density and synaptic plasticity, thereby maintaining headroom for cells to encode ongoing experience-dependent structural change throughout life. PMID:27752542

  12. How the Purkinje System Determines the Ventricular Activation Sequence

    DTIC Science & Technology

    2007-11-02

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

  13. Cell-Autonomous Progeroid Changes in Conditional Mouse Models for Repair Endonuclease XPG Deficiency

    PubMed Central

    Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; Sarker, Altaf H.; Jaspers, Nicolaas G. J.; van der Horst, Gijsbertus T. J.; Cooper, Priscilla K.; Hoeijmakers, Jan H. J.; van der Pluijm, Ingrid

    2014-01-01

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg−/− mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg−/− mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging. PMID:25299392

  14. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency

    SciTech Connect

    Barnhoorn, Sander; Uittenboogaard, Lieneke M.; Jaarsma, Dick; Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; Sarker, Altaf H.; Jaspers, Nicolaas G. J.; van der Horst, Gijsbertus T. J.; Cooper, Priscilla K.; Hoeijmakers, Jan H. J.; van der Pluijm, Ingrid; Niedernhofer, Laura J.

    2014-10-09

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which—in a C57BL6/FVB F1 hybrid genetic background—displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

  15. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency

    DOE PAGES

    Barnhoorn, Sander; Uittenboogaard, Lieneke M.; Jaarsma, Dick; ...

    2014-10-09

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which—in a C57BL6/FVB F1 hybrid genetic background—displays manymore » progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.« less

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

    PubMed

    Haghani, M; Shabani, M; Moazzami, K

    2013-10-10

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

  17. Autologous Adipose Stem Cell Therapy for Autonomic Nervous System Dysfunction in Two Young Patients

    PubMed Central

    Kamdar, Ankur; Young, Jane; Butler, Ian. J.

    2017-01-01

    Postural orthostatic tachycardia syndrome and neurocardiogenic syncope are clinical manifestations of autonomic nervous system dysfunction (dysautonomia) that can lead to impaired daily functions. We report two young patients presenting with dysautonomia and autoimmune disease who both received autologous adipose stem cells (ASCs) infusions. This report is the first description of ASCs therapy for patients with combined dysautonomia and autoimmune disease. Case 1: A 21-year-old female presented at 12 years of age with escalating severe dysautonomia with weight loss and gastrointestinal symptoms. She had elevated autoantibodies and cytokines and received multiple immune modulation therapies. Her dysautonomia was treated by volume expanders, vasoconstrictors, and beta blockers with mild improvement. She received ASCs about 2 years before this report with dramatic improvement in her dysautonomia and autoimmune symptoms with a 10 kg weight gain. Case 2: A 7-year-old boy presented at 2 years of age with polyarthritis. At 5 years of age, he manifested orthostatic intolerance. He received immune modulatory therapies with mild improvement. He received ASCs and showed marked improvement of his dysautonomia and immune symptoms. Dysautonomia symptoms of these two patients improved significantly after modulation of autoimmune components by ASC therapy. Favorable clinical responses of these two cases warrant further case–control studies. PMID:27959743

  18. Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology

    PubMed Central

    Grice, Stuart J.; Sleigh, James N.; Motley, William W.; Liu, Ji-Long; Burgess, Robert W.; Talbot, Kevin; Cader, M. Zameel

    2015-01-01

    Charcot–Marie–Tooth (CMT) neuropathies are collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D. This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Here, using novel Drosophila models, we show a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene. PMID:25972375

  19. MeCP2 Affects Skeletal Muscle Growth and Morphology through Non Cell-Autonomous Mechanisms.

    PubMed

    Conti, Valentina; Gandaglia, Anna; Galli, Francesco; Tirone, Mario; Bellini, Elisa; Campana, Lara; Kilstrup-Nielsen, Charlotte; Rovere-Querini, Patrizia; Brunelli, Silvia; Landsberger, Nicoletta

    2015-01-01

    Rett syndrome (RTT) is an autism spectrum disorder mainly caused by mutations in the X-linked MECP2 gene and affecting roughly 1 out of 10.000 born girls. Symptoms range in severity and include stereotypical movement, lack of spoken language, seizures, ataxia and severe intellectual disability. Notably, muscle tone is generally abnormal in RTT girls and women and the Mecp2-null mouse model constitutively reflects this disease feature. We hypothesized that MeCP2 in muscle might physiologically contribute to its development and/or homeostasis, and conversely its defects in RTT might alter the tissue integrity or function. We show here that a disorganized architecture, with hypotrophic fibres and tissue fibrosis, characterizes skeletal muscles retrieved from Mecp2-null mice. Alterations of the IGF-1/Akt/mTOR pathway accompany the muscle phenotype. A conditional mouse model selectively depleted of Mecp2 in skeletal muscles is characterized by healthy muscles that are morphologically and molecularly indistinguishable from those of wild-type mice raising the possibility that hypotonia in RTT is mainly, if not exclusively, mediated by non-cell autonomous effects. Our results suggest that defects in paracrine/endocrine signaling and, in particular, in the GH/IGF axis appear as the major cause of the observed muscular defects. Remarkably, this is the first study describing the selective deletion of Mecp2 outside the brain. Similar future studies will permit to unambiguously define the direct impact of MeCP2 on tissue dysfunctions.

  20. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

    PubMed Central

    Lakhal-Littleton, Samira; Wolna, Magda; Chung, Yu Jin; Christian, Helen C; Heather, Lisa C; Brescia, Marcella; Ball, Vicky; Diaz, Rebeca; Santos, Ana; Biggs, Daniel; Clarke, Kieran; Davies, Benjamin; Robbins, Peter A

    2016-01-01

    Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain. DOI: http://dx.doi.org/10.7554/eLife.19804.001 PMID:27897970

  1. The cell-autonomous role of excitatory synaptic transmission in the regulation of neuronal structure and function.

    PubMed

    Lu, Wei; Bushong, Eric A; Shih, Tiffany P; Ellisman, Mark H; Nicoll, Roger A

    2013-05-08

    The cell-autonomous role of synaptic transmission in the regulation of neuronal structural and electrical properties is unclear. We have now employed a genetic approach to eliminate glutamatergic synaptic transmission onto individual CA1 pyramidal neurons in a mosaic fashion in vivo. Surprisingly, while electrical properties are profoundly affected in these neurons, as well as inhibitory synaptic transmission, we found little perturbation of neuronal morphology, demonstrating a functional segregation of excitatory synaptic transmission from neuronal morphological development.

  2. Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

    PubMed

    Yu, Vionnie W C; Yusuf, Rushdia Z; Oki, Toshihiko; Wu, Juwell; Saez, Borja; Wang, Xin; Cook, Colleen; Baryawno, Ninib; Ziller, Michael J; Lee, Eunjung; Gu, Hongcang; Meissner, Alexander; Lin, Charles P; Kharchenko, Peter V; Scadden, David T

    2016-11-17

    Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.

  3. Evolution of Cell-Autonomous Effector Mechanisms in Macrophages versus Non-Immune Cells

    PubMed Central

    Gaudet, Ryan G.; Bradfield, Clinton J.; MacMicking, John D.

    2016-01-01

    Summary Specialized adaptations for killing microbes is synonymous with phagocytic cells including macrophages, monocytes, inflammatory neutrophils and eosinophils. Recent genome sequencing of extant species, however, reveals analogous antimicrobial machineries exist in certain non-immune cells and also within species that ostensibly lack a well-defined immune system. Here we probe the evolutionary record for clues about the ancient and diverse phylogenetic origins of macrophage killing mechanisms and how some of their properties are shared with cells outside the traditional bounds of immunity in higher vertebrates such as mammals. PMID:28087931

  4. Cell-autonomous and environmental contributions to the interstitial migration of T cells

    PubMed Central

    Petravic, Janka; Davenport, Miles P.

    2010-01-01

    A key to understanding the functioning of the immune system is to define the mechanisms that facilitate directed lymphocyte migration to and within tissues. The recent development of improved imaging technologies, most prominently multi-photon microscopy, has enabled the dynamic visualization of immune cells in real-time directly within intact tissues. Intravital imaging approaches have revealed high spontaneous migratory activity of T cells in secondary lymphoid organs and inflamed tissues. Experimental evidence points towards both environmental and cell-intrinsic cues involved in the regulation of lymphocyte motility in the interstitial space. Based on these data, several conceptually distinct models have been proposed in order to explain the coordination of lymphocyte migration both at the single cell and population level. These range from “stochastic” models, where chance is the major driving force, to “deterministic” models, where the architecture of the microenvironment dictates the migratory trajectory of cells. In this review, we focus on recent advances in understanding naïve and effector T cell migration in vivo. In addition, we discuss some of the contradictory experimental findings in the context of theoretical models of migrating leukocytes. PMID:20623124

  5. Cell-autonomous and environmental contributions to the interstitial migration of T cells.

    PubMed

    Mrass, Paulus; Petravic, Janka; Davenport, Miles P; Weninger, Wolfgang

    2010-09-01

    A key to understanding the functioning of the immune system is to define the mechanisms that facilitate directed lymphocyte migration to and within tissues. The recent development of improved imaging technologies, most prominently multi-photon microscopy, has enabled the dynamic visualization of immune cells in real-time directly within intact tissues. Intravital imaging approaches have revealed high spontaneous migratory activity of T cells in secondary lymphoid organs and inflamed tissues. Experimental evidence points towards both environmental and cell-intrinsic cues involved in the regulation of lymphocyte motility in the interstitial space. Based on these data, several conceptually distinct models have been proposed in order to explain the coordination of lymphocyte migration both at the single cell and population level. These range from "stochastic" models, where chance is the major driving force, to "deterministic" models, where the architecture of the microenvironment dictates the migratory trajectory of cells. In this review, we focus on recent advances in understanding naïve and effector T cell migration in vivo. In addition, we discuss some of the contradictory experimental findings in the context of theoretical models of migrating leukocytes.

  6. HIF-1α regulates the response of primary sarcomas to radiation therapy through a cell autonomous mechanism

    PubMed Central

    Zhang, Minsi; Qiu, Qiong; Li, Zhizhong; Sachdeva, Mohit; Min, Hooney; Cardona, Diana M.; DeLaney, Thomas F.; Han, Tracy; Ma, Yan; Luo, Lixia; Ilkayeva, Olga R.; Lui, Ki; Nichols, Amanda G.; Newgard, Christopher B.; Kastan, Michael B.; Rathmell, Jeffrey C.; Dewhirst, Mark W.; Kirsch, David G.

    2016-01-01

    Hypoxia is a major cause of radiation resistance, which may predispose to local recurrence after radiation therapy (RT). While hypoxia increases tumor cell survival after RT because there is less oxygen to oxidize damaged DNA, whether signaling pathways triggered by hypoxia contribute to radiation resistance is poorly understood. For example, intratumoral hypoxia can increase hypoxia inducible factor 1 alpha (HIF-1α), which may regulate pathways that contribute to radiation sensitization or radiation resistance. To clarify the role of HIF-1α in regulating tumor response to radiation therapy, we generated a novel genetically engineered mouse model of soft tissue sarcoma with an intact or deleted HIF-1α. Deletion of HIF-1α sensitized primary sarcomas to RT in vivo.