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Sample records for cerebellar granule cell

  1. Cerebellar granule cells encode the expectation of reward

    PubMed Central

    Wagner, Mark J; Kim, Tony Hyun; Savall, Joan; Schnitzer, Mark J; Luo, Liqun

    2017-01-01

    The human brain contains ~60 billion cerebellar granule cells1, which outnumber all other neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes2–6. Although evidence suggests a role for cerebellum in cognition7–10, granule cells are known to encode only sensory11–13 and motor14 context. Using two-photon calcium imaging in behaving mice, here we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from current understanding of these neurons and dramatically enriches contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum. PMID:28321129

  2. Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors.

    PubMed

    Nakashima, Kie; Umeshima, Hiroki; Kengaku, Mineko

    2015-06-01

    Neurons in the central nervous system (CNS) are generated by symmetric and asymmetric cell division of neural stem cells and their derivative progenitor cells. Cerebellar granule cells are the most abundant neurons in the CNS, and are generated by intensive cell division of granule cell precursors (GCPs) during postnatal development. Dysregulation of GCP cell cycle is causal for some subtypes of medulloblastoma. However, the details and mechanisms underlying neurogenesis from GCPs are not well understood. Using long-term live-cell imaging of proliferating GCPs transfected with a fluorescent newborn-granule cell marker, we found that GCPs underwent predominantly symmetric divisions, generating two GCPs or two neurons, while asymmetric divisions generating a GCP and a neuron were only occasionally observed, in both dissociated culture and within tissues of isolated cerebellar lobules. We found no significant difference in cell cycle length between proliferative and neurogenic divisions, or any consistent changes in cell cycle length during repeated proliferative division. Unlike neural stem cells in the cerebral cortex and spinal cord, which generate many neurons by repeated asymmetric division, cerebellar GCPs produce neurons predominantly by terminal symmetric division. These results indicate diverse mechanisms of neurogenesis in the mammalian brain. © 2015 Wiley Periodicals, Inc.

  3. Segmental identity and cerebellar granule cell induction in rhombomere 1

    PubMed Central

    Eddison, Mark; Toole, Leah; Bell, Esther; Wingate, Richard JT

    2004-01-01

    Background Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment? Results We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2. Conclusions Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a

  4. Stochastic differential equation model for cerebellar granule cell excitability.

    PubMed

    Saarinen, Antti; Linne, Marja-Leena; Yli-Harja, Olli

    2008-02-29

    Neurons in the brain express intrinsic dynamic behavior which is known to be stochastic in nature. A crucial question in building models of neuronal excitability is how to be able to mimic the dynamic behavior of the biological counterpart accurately and how to perform simulations in the fastest possible way. The well-established Hodgkin-Huxley formalism has formed to a large extent the basis for building biophysically and anatomically detailed models of neurons. However, the deterministic Hodgkin-Huxley formalism does not take into account the stochastic behavior of voltage-dependent ion channels. Ion channel stochasticity is shown to be important in adjusting the transmembrane voltage dynamics at or close to the threshold of action potential firing, at the very least in small neurons. In order to achieve a better understanding of the dynamic behavior of a neuron, a new modeling and simulation approach based on stochastic differential equations and Brownian motion is developed. The basis of the work is a deterministic one-compartmental multi-conductance model of the cerebellar granule cell. This model includes six different types of voltage-dependent conductances described by Hodgkin-Huxley formalism and simple calcium dynamics. A new model for the granule cell is developed by incorporating stochasticity inherently present in the ion channel function into the gating variables of conductances. With the new stochastic model, the irregular electrophysiological activity of an in vitro granule cell is reproduced accurately, with the same parameter values for which the membrane potential of the original deterministic model exhibits regular behavior. The irregular electrophysiological activity includes experimentally observed random subthreshold oscillations, occasional spontaneous spikes, and clusters of action potentials. As a conclusion, the new stochastic differential equation model of the cerebellar granule cell excitability is found to expand the range of dynamics

  5. Cerebellar granule cells acquire a widespread predictive feedback signal during motor learning.

    PubMed

    Giovannucci, Andrea; Badura, Aleksandra; Deverett, Ben; Najafi, Farzaneh; Pereira, Talmo D; Gao, Zhenyu; Ozden, Ilker; Kloth, Alexander D; Pnevmatikakis, Eftychios; Paninski, Liam; De Zeeuw, Chris I; Medina, Javier F; Wang, Samuel S-H

    2017-03-20

    Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, as required by forward models of cerebellar control.

  6. Delayed release of neurotransmitter from cerebellar granule cells.

    PubMed

    Atluri, P P; Regehr, W G

    1998-10-15

    At fast chemical synapses the rapid release of neurotransmitter that occurs within a few milliseconds of an action potential is followed by a more sustained elevation of release probability, known as delayed release. Here we characterize the role of calcium in delayed release and test the hypothesis that facilitation and delayed release share a common mechanism. Synapses between cerebellar granule cells and their postsynaptic targets, stellate cells and Purkinje cells, were studied in rat brain slices. Presynaptic calcium transients were measured with calcium-sensitive fluorophores, and delayed release was detected with whole-cell recordings. Calcium influx, presynaptic calcium dynamics, and the number of stimulus pulses were altered to assess their effect on delayed release and facilitation. Following single stimuli, delayed release can be separated into two components: one lasting for tens of milliseconds that is steeply calcium-dependent, the other lasting for hundreds of milliseconds that is driven by low levels of calcium with a nearly linear calcium dependence. The amplitude, calcium dependence, and magnitude of delayed release do not correspond to those of facilitation, indicating that these processes are not simple reflections of a shared mechanism. The steep calcium dependence of delayed release, combined with the large calcium transients observed in these presynaptic terminals, suggests that for physiological conditions delayed release provides a way for cells to influence their postsynaptic targets long after their own action potential activity has subsided.

  7. Cerebellar cortical degeneration with selective granule cell loss in Bavarian mountain dogs.

    PubMed

    Flegel, T; Matiasek, K; Henke, D; Grevel, V

    2007-08-01

    Three Bavarian mountain dogs aged between 18 and 20 months, not related to each other, were presented with chronic signs of cerebellar dysfunction. On sagittal T2-weighted magnetic resonance imaging brain images, the tentative diagnosis of cerebellar hypoplasia was established based on an enlarged cerebrospinal fluid space around the cerebellum and an increased cerebrospinal fluid signal between the folia. Post-mortem examination was performed in one dog and did show an overall reduction of cerebellar size. On histopathologic examination, a selective loss of cerebellar granule cells with sparing of Purkinje cells was evident. Therefore, the Bavarian mountain dog is a breed where cerebellar cortical degeneration caused by the rather exceptional selective granule cell loss can be seen as cause of chronic, slowly progressive cerebellar dysfunction starting at an age of several months.

  8. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

    PubMed Central

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  9. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells

    PubMed Central

    Fu, Zhanyan; Vicini, Stefano

    2009-01-01

    Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2(NLG2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABAA (γ-aminobutyric acid) receptor mediated miniature postsynaptic currents (mISPCs). NLG2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV 7-8). Down-regulation of NLG2 by the isoform specific shRNA-NLG2 resulted in an opposite effect. We and others have shown that the switch of α subunits of GABAA Rs from α2/3 to α1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG2 is due to the recruitment of more α1 containing GABAARs at the synapses, we examined the prolongation of current decay by the zolpidem, which has been shown to preferentially enhance the activity of α1 subunit containing GABA channel. The application of zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG2 over-expressing cells than control cells, suggesting that NLG2 over-expression accelerates synapse maturation by promoting incorporation of the α1 subunit-containing GABAARs at postsynaptic sites in immature cells. In addition, the effect of NLG2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from α1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG2 could be also due to the impacts of NLG2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous

  10. Event-driven simulation of cerebellar granule cells.

    PubMed

    Carrillo, Richard R; Ros, Eduardo; Tolu, Silvia; Nieus, Thierry; D'Angelo, Egidio

    2008-01-01

    Around half of the neurons of a human brain are granule cells (approximately 10(11)granule neurons) [Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science. McGraw-Hill Professional Publishing, New York]. In order to study in detail the functional role of the intrinsic features of this cell we have developed a pre-compiled behavioural model based on the simplified granule-cell model of Bezzi et al. [Bezzi, M., Nieus, T., Arleo, A., D'Angelo, E., Coenen, O.J.-M.D., 2004. Information transfer at the mossy fiber-granule cell synapse of the cerebellum. 34th Annual Meeting. Society for Neuroscience, San Diego, CA, USA]. We can use an efficient event-driven simulation scheme based on lookup tables (EDLUT) [Ros, E., Carrillo, R.R., Ortigosa, E.M., Barbour, B., Ags, R., 2006. Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics. Neural Computation 18 (12), 2959-2993]. For this purpose it is necessary to compile into tables the data obtained through a massive numerical calculation of the simplified cell model. This allows network simulations requiring minimal numerical calculation. There are three major features that are considered functionally relevant in the simplified granule cell model: bursting, subthreshold oscillations and resonance. In this work we describe how the cell model is compiled into tables keeping these key properties of the neuron model.

  11. Integration of quanta in cerebellar granule cells during sensory processing.

    PubMed

    Chadderton, Paul; Margrie, Troy W; Häusser, Michael

    2004-04-22

    To understand the computations performed by the input layers of cortical structures, it is essential to determine the relationship between sensory-evoked synaptic input and the resulting pattern of output spikes. In the cerebellum, granule cells constitute the input layer, translating mossy fibre signals into parallel fibre input to Purkinje cells. Until now, their small size and dense packing have precluded recordings from individual granule cells in vivo. Here we use whole-cell patch-clamp recordings to show the relationship between mossy fibre synaptic currents evoked by somatosensory stimulation and the resulting granule cell output patterns. Granule cells exhibited a low ongoing firing rate, due in part to dampening of excitability by a tonic inhibitory conductance mediated by GABA(A) (gamma-aminobutyric acid type A) receptors. Sensory stimulation produced bursts of mossy fibre excitatory postsynaptic currents (EPSCs) that summate to trigger bursts of spikes. Notably, these spike bursts were evoked by only a few quantal EPSCs, and yet spontaneous mossy fibre inputs triggered spikes only when inhibition was reduced. Our results reveal that the input layer of the cerebellum balances exquisite sensitivity with a high signal-to-noise ratio. Granule cell bursts are optimally suited to trigger glutamate receptor activation and plasticity at parallel fibre synapses, providing a link between input representation and memory storage in the cerebellum.

  12. Diversity and complexity of roles of granule cells in the cerebellar cortex. Editorial.

    PubMed

    Manto, Mario; De Zeeuw, Chris I

    2012-03-01

    The cerebellar granule cell, the most numerous neurons in the brain, forms the main excitatory neuron of the cerebellar cortical circuitry. Granule cells are synaptically connected with both mossy fibers and Golgi cells inside specialized structures called glomeruli, and thereby, they are subject to both feed-forward and feed-back inhibition. Their unique architecture with about four dendrites and a single axon ascending in the cerebellar cortex to bifurcate into two parallel fibers making synapses with Purkinje neurons has attracted numerous scientists. Recent advances show that they are much more than just relays of mossy fibers. They perform diverse and complex transformations in the spatiotemporal domain. This special issue highlights novel avenues in our understanding of the roles of this key neuronal population of the cerebellar cortex, ranging from developmental up to physiological and pathological points of view.

  13. Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors.

    PubMed

    Hanzel, Michalina; Wingate, Richard J T; Butts, Thomas

    2015-12-14

    The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

  14. Culturing of cerebellar granule cells to study neuronal migration: gradient and local perfusion assays.

    PubMed

    Guijarro, Patricia; Jiang, Jian; Yuan, Xiao-bing

    2012-07-01

    Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time-lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed.

  15. Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

    PubMed Central

    Bénard, Magalie; Lebon, Alexis; Komuro, Hitoshi; Vaudry, David; Galas, Ludovic

    2015-01-01

    During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration. PMID:25992599

  16. Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells

    PubMed Central

    Huang, Cheng-Chiu; Sugino, Ken; Shima, Yasuyuki; Guo, Caiying; Bai, Suxia; Mensh, Brett D; Nelson, Sacha B; Hantman, Adam W

    2013-01-01

    Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI: http://dx.doi.org/10.7554/eLife.00400.001 PMID:23467508

  17. Synaptic action of ethanol on cerebellar auditory granule cells reveals acute tolerance

    SciTech Connect

    Huang, C.M.; Liu, G.; Huang, R.H. )

    1991-03-11

    The cerebellum is very sensitive to acute intoxication by ethanol. The authors have recorded electrophysiological responses of granule cells to auditory stimulation from the posterior cerebellar vermis of cats before and after a relatively low dose of ethanol. Auditory responses of granule cells were severely inhibited by ethanol at a transient, peak ethanol concentration of 15-18 mM in the cerebrospinal fluid (CSF). Thereafter, the clearance of ethanol from CSF followed an exponential time course, with 50% of the CSF ethanol being cleared with every passing hour. Auditory responses of granule cells returned to control levels within 60-90 minutes, despite the presence of a DSF ethanol concentration at 8-10mM, indicating acute tolerance. Moreover, a second, identical dose of ethanol, delivered two hours after the first dose produced an attenuated inhibition in the auditory response of cerebellar granule cells. The inhibition took a longer time to be evident but a shorter time to recover than that followed by the first dose of ethanol.

  18. Mitotic Events in Cerebellar Granule Progenitor Cells that Expand Cerebellar Surface Area Are Critical for Normal Cerebellar Cortical Lamination in Mice

    PubMed Central

    Chang, Joshua C.; Leung, Mark; Gokozan, Hamza Numan; Gygli, Patrick Edwin; Catacutan, Fay Patsy; Czeisler, Catherine; Otero, José Javier

    2015-01-01

    Late embryonic and postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a streamlined sampling scheme to generate unbiased estimates of murine cerebellar surface area and volume using stereological principles. We demonstrate that during the proliferative phase of the external granule layer (EGL) and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self-renewal. The topological constraints indicate that during proliferative phases, migration out of the EGL is balanced by self-renewal. Progenitor self-renewal must, therefore, include mitotic events yielding either 2 cells in the same layer to increase surface area (β-events) and mitotic events yielding 2 cells, with 1 cell in a superficial layer and 1 cell in a deeper layer (α-events). As the cerebellum grows, therefore, β-events lie upstream of α-events. Using a mathematical model constrained by the measurements of volume and surface area, we could quantify inter-mitotic times for β-events on a per-cell basis in post-natal mouse cerebellum. Furthermore, we found that loss of CCNA2, which decreases EGL proliferation and secondarily induces cerebellar cortical dyslamination, shows preserved α-type events. Thus, CCNA2-null cerebellar granule progenitor cells are capable of self-renewal of the EGL stem cell niche; this is concordant with prior findings of extensive apoptosis in CCNA2-null mice. Similar methodologies may provide another layer of depth to the interpretation of results from stereological studies. PMID:25668568

  19. Critical role of integrin-linked kinase in granule cell precursor proliferation and cerebellar development.

    PubMed

    Mills, Julia; Niewmierzycka, Agnieszka; Oloumi, Arusha; Rico, Beatriz; St-Arnaud, Rene; Mackenzie, Ian R; Mawji, Nasrin M; Wilson, Jason; Reichardt, Louis F; Dedhar, Shoukat

    2006-01-18

    Integrin-linked kinase (ILK) is a serine/threonine protein kinase that plays an important role in integrin signaling and cell proliferation. We used Cre recombinase (Cre)-loxP technology to study CNS restricted knock-out of the ilk gene by either Nestin-driven or gfap-driven Cre-mediated recombination. Developmental changes in ilk-excised brain regions are similar to those observed in mice lacking the integrin beta1 subunit in the CNS, including defective laminin deposition, abnormal glial morphology, and alterations in granule cell migration. Decreases in 6-bromodeoxyuridine (BrdU) pulse labeling and proliferating cell nuclear antigen expression in the external granule cell layer of the cerebellum demonstrated that proliferation is disrupted in granule cells lacking ILK. Previous studies have shown that laminin-sonic hedgehog (Shh)-induced granule cell precursor (GCP) proliferation is dependent on beta1 integrins, several of which bind laminin and interact with ILK through the beta1 cytoplasmic domain. Both ex vivo deletion of ilk and a small molecule inhibitor of ILK kinase activity decreased laminin-Shh-induced BrdU labeling in cultured GCPs. Together, these results implicate ILK as a critical effector in a signaling pathway necessary for granule cell proliferation and cerebellar development.

  20. Critical Role of Integrin-Linked Kinase in Granule Cell Precursor Proliferation and Cerebellar Development

    PubMed Central

    Mills, Julia; Niewmierzycka, Agnieszka; Oloumi, Arusha; Rico, Beatriz; St-Arnaud, Rene; Mackenzie, Ian R.; Mawji, Nasrin M.; Wilson, Jason; Reichardt, Louis F.; Dedhar, Shoukat

    2009-01-01

    Integrin-linked kinase (ILK) is a serine/threonine protein kinase that plays an important role in integrin signaling and cell proliferation. We used Cre recombinase (Cre)-loxP technology to study CNS restricted knock-out of the ilk gene by either Nestin-driven or gfap-driven Cre-mediated recombination. Developmental changes in ilk-excised brain regions are similar to those observed in mice lacking the integrin β1 subunit in the CNS, including defective laminin deposition, abnormal glial morphology, and alterations in granule cell migration. Decreases in 6-bromodeoxyuridine (BrdU) pulse labeling and proliferating cell nuclear antigen expression in the external granule cell layer of the cerebellum demonstrated that proliferation is disrupted in granule cells lacking ILK. Previous studies have shown that laminin-sonic hedgehog (Shh)-induced granule cell precursor (GCP) proliferation is dependent on β1 integrins, several of which bind laminin and interact with ILK through the β1 cytoplasmic domain. Both ex vivo deletion of ilk and a small molecule inhibitor of ILK kinase activity decreased laminin-Shh-induced BrdU labeling in cultured GCPs. Together, these results implicate ILK as a critical effector in a signaling pathway necessary for granule cell proliferation and cerebellar development. PMID:16421303

  1. Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells

    PubMed Central

    Smith, T Caitlin; Wang, Lu-Yang; Howe, James R

    1999-01-01

    Although glutamate receptors have been shown to be involved in neuronal maturation, a developmental role for kainate-type receptors has not been described. In addition, the single-channel properties of native kainate receptors have not been studied in situ. We have characterized the electrophysiological properties of native kainate receptors of granule cell neurons at two distinct stages in postnatal development, using whole-cell and outside-out patch-clamp recordings in acute cerebellar slices. Kainate-type currents were detected in both immature and mature granule cells. However, noise analysis showed that the apparent unitary conductance of kainate-type channels is significantly higher in proliferating than post-migratory granule cells. The conductance and rectification behaviour of the channels in immature granule cells indicate that they contain unedited GluR5 and GluR6 subunits and are likely to be calcium permeable. Single-channel kainate-type currents were observed in outside-out patches from proliferating granule cells in the external germinal layer. The kinetic behaviour of kainate receptors in immature cells was complex. Openings to multiple conductance levels were observed, although our analysis indicates that the channels spend most of their open time in a 4 pS state. PMID:10226148

  2. Proton sensitivity of rat cerebellar granule cell GABAA receptors: dependence on neuronal development

    PubMed Central

    Krishek, Belinda J; Smart, Trevor G

    2001-01-01

    The effect of GABAA receptor development in culture on the modulation of GABA-induced currents by external H+ was examined in cerebellar granule cells using whole-cell and single-channel recording. Equilibrium concentration-response curves revealed a lower potency for GABA between 11 and 12 days in vitro (DIV) resulting in a shift of the EC50 from 10.7 to 2.4 μM. For granule cells before 11 DIV, the peak GABA-activated current was inhibited at low external pH and enhanced at high pH with a pKa of 6.65. For the steady-state response, low pH was inhibitory with a pKa of 5.56. After 11 DIV, the peak GABA-activated current was largely pH insensitive; however, the steady-state current was potentiated at low pH with a pKa of 6.84. Single GABA-activated ion channels were recorded from outside-out patches of granule cell bodies. At pH 5.4-9.4, single GABA channels exhibited multiple conductance states occurring at 22-26, 16-17 and 12-14 pS. The conductance levels were not significantly altered over the time period of study, nor by changing the external H+ concentration. Two exponential functions were required to fit the open-time frequency histograms at both early (< 11 DIV) and late (> 11 DIV) development times at each H+ concentration. The short and long open time constants were unaffected either by the extracellular H+ concentration or by neuronal development. The distribution of all shut times was fitted by the sum of three exponentials designated as short, intermediate and long. At acidic pH, the long shut time constant decreased with development as did the relative contribution of these components to the overall distribution. This was concurrent with an increase in the mean probability of channel opening. In conclusion, this study demonstrates in cerebellar granule cells that external pH can either reduce, have no effect on, or enhance GABA-activated responses depending on the stage of development, possibly related to the subunit composition of the GABAA receptors

  3. 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.

  4. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  5. Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex.

    PubMed

    Tolbert, D L; Conoyer, B; Ariel, M

    2004-11-01

    The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean +/- SD: 0.84 +/- 0.17 vs 0.64 +/- 0.12 microm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82 +/- 0.34 microm, n = 52) were slightly larger than on parallel fibers (1.43 +/- 0.24 microm, n = 430). In addition, per unit length (100 microm) there were more swellings on ascending axons (11.2 +/- 4.2) than on parallel fibers (9.7 +/- 4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15-20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 microm2) to posteriorly (3090 microm2) and 104% from medially (1690 microm2) to laterally (3450 microm2). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

  6. Model cerebellar granule cells can faithfully transmit modulated firing rate signals

    PubMed Central

    Rössert, Christian; Solinas, Sergio; D'Angelo, Egidio; Dean, Paul; Porrill, John

    2014-01-01

    A crucial assumption of many high-level system models of the cerebellum is that information in the granular layer is encoded in a linear manner. However, granule cells are known for their non-linear and resonant synaptic and intrinsic properties that could potentially impede linear signal transmission. In this modeling study we analyse how electrophysiological granule cell properties and spike sampling influence information coded by firing rate modulation, assuming no signal-related, i.e., uncorrelated inhibitory feedback (open-loop mode). A detailed one-compartment granule cell model was excited in simulation by either direct current or mossy-fiber synaptic inputs. Vestibular signals were represented as tonic inputs to the flocculus modulated at frequencies up to 20 Hz (approximate upper frequency limit of vestibular-ocular reflex, VOR). Model outputs were assessed using estimates of both the transfer function, and the fidelity of input-signal reconstruction measured as variance-accounted-for. The detailed granule cell model with realistic mossy-fiber synaptic inputs could transmit information faithfully and linearly in the frequency range of the vestibular-ocular reflex. This was achieved most simply if the model neurons had a firing rate at least twice the highest required frequency of modulation, but lower rates were also adequate provided a population of neurons was utilized, especially in combination with push-pull coding. The exact number of neurons required for faithful transmission depended on the precise values of firing rate and noise. The model neurons were also able to combine excitatory and inhibitory signals linearly, and could be replaced by a simpler (modified) integrate-and-fire neuron in the case of high tonic firing rates. These findings suggest that granule cells can in principle code modulated firing-rate inputs in a linear manner, and are thus consistent with the high-level adaptive-filter model of the cerebellar microcircuit. PMID:25352777

  7. Regulation of output spike patterns by phasic inhibition in cerebellar granule cells

    PubMed Central

    Nieus, Thierry R.; Mapelli, Lisa; D'Angelo, Egidio

    2014-01-01

    The complex interplay of multiple molecular mechanisms taking part to synaptic integration is hard to disentangle experimentally. Therefore, we developed a biologically realistic computational model based on the rich set of data characterizing the cerebellar glomerulus microcircuit. A specific issue was to determine the relative role of phasic and tonic inhibition in dynamically regulating granule cell firing, which has not been clarified yet. The model comprised the excitatory mossy fiber—granule cell and the inhibitory Golgi cell—granule cell synapses and accounted for vesicular release processes, neurotransmitter diffusion and activation of different receptor subtypes. Phasic inhibition was based on stochastic GABA release and spillover causing activation of two major classes of postsynaptic receptors, α1 and α6, while tonic inhibition was based on steady regulation of a Cl− leakage. The glomerular microcircuit model was validated against experimental responses to mossy fiber bursts while metabotropic receptors were blocked. Simulations showed that phasic inhibition controlled the number of spikes during burst transmission but predicted that it specifically controlled time-related parameters (firing initiation and conclusion and first spike precision) when the relative phase of excitation and inhibition was changed. In all conditions, the overall impact of α6 was larger than that of α1 subunit-containing receptors. However, α1 receptors controlled granule cell responses in a narrow ±10 ms band while α6 receptors showed broader ±50 ms tuning. Tonic inhibition biased these effects without changing their nature substantially. These simulations imply that phasic inhibitory mechanisms can dynamically regulate output spike patterns, as well as calcium influx and NMDA currents, at the mossy fiber—granule cell relay of cerebellum without the intervention of tonic inhibition. PMID:25202237

  8. Multiple extra-synaptic spillover mechanisms regulate prolonged activity in cerebellar Golgi cell–granule cell loops

    PubMed Central

    Holtzman, Tahl; Sivam, Vanessa; Zhao, Tian; Frey, Oivier; van der Wal, Peter Dow; de Rooij, Nico F; Dalley, Jeffrey W; Edgley, Steve A

    2011-01-01

    Abstract Despite a wealth of in vitro and modelling studies it remains unclear how neuronal populations in the cerebellum interact in vivo. We address the issue of how the cerebellar input layer processes sensory information, with particular focus on the granule cells (input relays) and their counterpart inhibitory interneurones, Golgi cells. Based on the textbook view, granule cells excite Golgi cells via glutamate forming a negative feedback loop. However, Golgi cells express inhibitory mGluR2 receptors suggesting an inhibitory role for glutamate. We set out to test this glutamatergic paradox in Golgi cells. Here we show that granule cells and Golgi cells interact through extra-synaptic signalling mechanisms during sensory information processing, as well as synaptic mechanisms. We demonstrate that such interactions depend on granule cell-derived glutamate acting via inhibitory mGluR2 receptors leading causally to the suppression of Golgi cell activity for several hundreds of milliseconds. We further show that granule cell-derived inhibition of Golgi cell activity is regulated by GABA-dependent extra-synaptic Golgi cell inhibition of granule cells, identifying a regulatory loop in which glutamate and GABA may be critical regulators of Golgi cell–granule cell functional activity. Thus, granule cells may promote their own prolonged activity via paradoxical feed-forward inhibition of Golgi cells, thereby enabling information processing over long timescales. PMID:21669981

  9. Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

    PubMed Central

    Toczylowska, Beata; Zieminska, Elzbieta; Goch, Grazyna; Milej, Daniel; Gerega, Anna; Liebert, Adam

    2014-01-01

    The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture. PMID:24688815

  10. FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon

    PubMed Central

    Dover, Katarzyna; Marra, Christopher; Solinas, Sergio; Popovic, Marko; Subramaniyam, Sathyaa; Zecevic, Dejan; D'Angelo, Egidio; Goldfarb, Mitchell

    2016-01-01

    Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell's unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as <1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons. PMID:27666389

  11. Protective Effect of Edaravone in Primary Cerebellar Granule Neurons against Iodoacetic Acid-Induced Cell Injury

    PubMed Central

    Zhou, Xinhua; Zhu, Longjun; Wang, Liang; Guo, Baojian; Zhang, Gaoxiao; Sun, Yewei; Zhang, Zaijun; Lee, Simon Ming-Yuen; Yu, Pei; Wang, Yuqiang

    2015-01-01

    Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal death in vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities. PMID:26557222

  12. Generation and Characterization of an Nse-CreERT2 Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells

    PubMed Central

    Pohlkamp, Theresa; Steller, Laura; May, Petra; Günther, Thomas; Schüle, Roland; Frotscher, Michael

    2014-01-01

    We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo. PMID:24950299

  13. Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

    PubMed Central

    Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

    2015-01-01

    Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

  14. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    PubMed

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  15. LXR agonist rescued the deficit in the proliferation of the cerebellar granule cells induced by dexamethasone

    SciTech Connect

    Bian, Xuting; Zhong, Hongyu; Li, Fen; Cai, Yulong; Li, Xin; Wang, Lian; Fan, Xiaotang

    2016-09-02

    Dexamethasone (DEX) exposure during early postnatal life produces permanent neuromotor and intellectual deficits and stunts cerebellar growth. The liver X receptor (LXR) plays important roles in CNS development. However, the effects of LXR on the DEX-mediated impairment of cerebellar development remain undetermined. Thus, mice were pretreated with LXR agonist TO901317 (TO) and were later exposed to DEX to evaluate its protective effects on DEX-mediated deficit during cerebellar development. The results showed that an acute exposure of DEX on postnatal day 7 resulted in a significant impairment in cerebellar development and decreased the proliferation of granule neuron precursors in the external granule layer of cerebellum. This effect was attenuated by pretreatment with TO. We further found that the decrease in the proliferation caused by DEX occurred via up-regulation of glucocorticoid receptor and p27kip1, which could be partially prevented by LXR agonist pretreatment. Overall, our results suggest that LXR agonist pretreatment could protect against DEX-induced deficits in cerebellar development in postnatal mice and may thus be perspective recruited to counteract such GC side effects.

  16. Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

    PubMed

    Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

    2016-07-19

    Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

  17. Computational study of non-homogeneous distribution of Ca(2+) handling systems in cerebellar granule cells.

    PubMed

    Saftenku, E E

    2009-03-21

    The spatiotemporal distribution of cytosolic free calcium concentration ([Ca(2+)](i)) in cerebellar granule cells (GrCs) is thought to be critical in defining the occurrence and direction of long-term changes in synaptic strength at cerebellar mossy fiber-GrC synapses. Despite this, the mechanisms responsible for shaping Ca(2+) transients in GrCs are not well understood. To investigate the interplay between Ca(2+) entry, extrusion, buffering and dendritic morphology in shaping Ca(2+) elevations in GrCs, we developed a model of Ca(2+) regulation in these cells and examined the requirements for reproducing fluorescence responses to depolarization and synaptic stimulation previously described in the literature. Two conclusions can be drawn from our simulation results. First, a significant progressive decrease in the amplitudes of depolarization-evoked fluorescence transients from the dendritic endings (digits) toward the soma of GrCs, can be reproduced in the model only if the density of Ca(2+) channels is considerably higher or the concentration of endogenous buffers is much lower in the digits than in the parent dendrites. In contrast, heterogeneities in the distribution of Ca(2+) pumps or in cytosolic fractional volume cannot account for the formation of [Ca(2+)](i) gradients in GrCs. Second, much lower amplitudes of fluorescence transients induced by depolarization and synaptic stimulation than expected from typical measurements of Ca(2+) and NMDA receptor-mediated currents can be reconciled with a pronounced slowing of the decay of fluorescence responses in the digits of GrCs after introducing a high-affinity Ca(2+) indicator if a high-capacity immobile Ca(2+) buffer (presumably plasma membrane-associated) is suggested to be present in the soma and apical part of digits. Mitochondria also are likely to modulate synaptically evoked Ca(2+) responses in GrCs. The alternative hypotheses are thoroughly discussed and research avenues for their testing in future

  18. Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells.

    PubMed

    Rampérez, Alberto; Sánchez-Prieto, José; Torres, Magdalena

    2017-03-11

    The recycling of synaptic vesicle (SV) proteins and transmitter release both occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the AP-1/AP-3 complex. As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 minutes after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. This article is protected by copyright. All rights reserved.

  19. β1-Integrins Are Critical for Cerebellar Granule Cell Precursor Proliferation

    PubMed Central

    Blaess, Sandra; Graus-Porta, Diana; Belvindrah, Richard; Radakovits, Randor; Pons, Sebastian; Littlewood-Evans, Amanda; Senften, Mathias; Guo, Huailian; Li, Yuqing; Miner, Jeffrey H.; Reichardt, Louis F.; Müller, Ulrich

    2009-01-01

    We have previously shown that mice with a CNS restricted knock-out of the integrin β1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of β1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence thatβ1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin–Shh induced cell proliferation is dependent on β1 integrin expression in GCPs. Taken together, these data are consistent with a model in which β1 integrin expression in GCPs is required to recruit a laminin–Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation. PMID:15056720

  20. Beta1-integrins are critical for cerebellar granule cell precursor proliferation.

    PubMed

    Blaess, Sandra; Graus-Porta, Diana; Belvindrah, Richard; Radakovits, Randor; Pons, Sebastian; Littlewood-Evans, Amanda; Senften, Mathias; Guo, Huailian; Li, Yuqing; Miner, Jeffrey H; Reichardt, Louis F; Müller, Ulrich

    2004-03-31

    We have previously shown that mice with a CNS restricted knock-out of the integrin beta1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of beta1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence that beta1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin-Shh induced cell proliferation is dependent on beta1 integrin expression in GCPs. Taken together, these data are consistent with a model in which beta1 integrin expression in GCPs is required to recruit a laminin-Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation.

  1. Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis.

    PubMed

    Bobba, A; Casalino, E; Petragallo, V A; Atlante, A

    2014-10-01

    The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.

  2. Expression of NR2B in cerebellar granule cells specifically facilitates effect of motor training on motor learning.

    PubMed

    Jiao, Jianwei; Nakajima, Akira; Janssen, William G M; Bindokas, Vytautas P; Xiong, Xiaoli; Morrison, John H; Brorson, James R; Tang, Ya-Ping

    2008-02-27

    It is believed that gene/environment interaction (GEI) plays a pivotal role in the development of motor skills, which are acquired via practicing or motor training. However, the underlying molecular/neuronal mechanisms are still unclear. Here, we reported that the expression of NR2B, a subunit of NMDA receptors, in cerebellar granule cells specifically enhanced the effect of voluntary motor training on motor learning in the mouse. Moreover, this effect was characterized as motor learning-specific and developmental stage-dependent, because neither emotional/spatial memory was affected nor was the enhanced motor learning observed when the motor training was conducted starting at the age of 3 months old in these transgenic mice. These results indicate that changes in the expression of gene(s) that are involved in regulating synaptic plasticity in cerebellar granule cells may constitute a molecular basis for the cerebellum to be involved in the GEI by facilitating motor skill learning.

  3. Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells.

    PubMed

    Wang, Dong; She, Liang; Sui, Ya-nan; Yuan, Xiao-bing; Wen, Yunqing; Poo, Mu-ming

    2012-12-18

    Directional flow of membrane components has been detected at the leading front of fibroblasts and the growth cone of neuronal processes, but whether there exists global directional flow of plasma membrane components over the entire migrating neuron remains largely unknown. By analyzing the trajectories of antibody-coated single quantum dots (QDs) bound to two membrane proteins, overexpressed myc-tagged synaptic vesicle-associated membrane protein VAMP2 and endogenous neurotrophin receptor TrkB, we found that these two proteins exhibited net forward transport, which is superimposed upon Brownian motion, in both leading and trailing processes of migrating cerebellar granule cells in culture. Furthermore, no net directional transport of membrane proteins was observed in nonmigrating cells with either growing or stalling leading processes. Analysis of the correlation of motion direction between two QDs on the same process in migrating neurons also showed a higher frequency of correlated forward than rearward movements. Such correlated QD movements were markedly reduced in the presence of myosin II inhibitor blebbistatin,suggesting the involvement of myosin II-dependent active transport processes. Thus, a net forward transport of plasma membrane proteins exists in the leading and trailing processes of migrating neurons, in line with the translocation of the soma.

  4. Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells.

    PubMed

    Masoli, Stefano; Rizza, Martina F; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

    2017-01-01

    In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models.

  5. Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells

    PubMed Central

    Masoli, Stefano; Rizza, Martina F.; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

    2017-01-01

    In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models. PMID:28360841

  6. Neurotoxicity of amphetamine derivatives is mediated by caspase pathway activation in rat cerebellar granule cells.

    PubMed

    Jiménez, Andrés; Jordà, Elvira G; Verdaguer, Ester; Pubill, David; Sureda, Francesc X; Canudas, Anna M; Escubedo, Elena; Camarasa, Jordi; Camins, Antoni; Pallàs, Mercè

    2004-04-15

    The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 microM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 microM) but not by 10 microM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using alpha-tocopherol (1-15 microM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors.

  7. Exploring the significance of morphological diversity for cerebellar granule cell excitability

    PubMed Central

    Houston, Catriona M.; Diamanti, Efthymia; Diamantaki, Maria; Kutsarova, Elena; Cook, Anna; Sultan, Fahad; Brickley, Stephen G.

    2017-01-01

    The relatively simple and compact morphology of cerebellar granule cells (CGCs) has led to the view that heterogeneity in CGC shape has negligible impact upon the integration of mossy fibre (MF) information. Following electrophysiological recording, 3D models were constructed from high-resolution imaging data to identify morphological features that could influence the coding of MF input patterns by adult CGCs. Quantification of MF and CGC morphology provided evidence that CGCs could be connected to the multiple rosettes that arise from a single MF input. Predictions from our computational models propose that MF inputs could be more densely encoded within the CGC layer than previous models suggest. Moreover, those MF signals arriving onto the dendrite closest to the axon will generate greater CGC excitation. However, the impact of this morphological variability on MF input selectivity will be attenuated by high levels of CGC inhibition providing further flexibility to the MF → CGC pathway. These features could be particularly important when considering the integration of multimodal MF sensory input by individual CGCs. PMID:28406156

  8. Tactile responses in the granule cell layer of cerebellar folium crus IIa of freely behaving rats

    NASA Technical Reports Server (NTRS)

    Hartmann, M. J.; Bower, J. M.

    2001-01-01

    We recorded activity from the granule cell layer (GCL) of cerebellar folium Crus IIa as freely moving rats engaged in a variety of natural behaviors, including grooming, eating, and free tactile exploration. Multiunit responses in the 1000-4500 Hz range were found to be strongly correlated with tactile stimulation of lip and whisker (perioral) regions. These responses occurred regardless of whether the stimulus was externally or self-generated and during both active and passive touch. In contrast, perioral movements that did not tactually stimulate this region of the face (e.g., chewing) produced no detectable increases in GCL activity. In addition, GCL responses were not correlated with movement extremes. When rats used their lips actively for palpation and exploration, the tactile responses in the GCL were not detectably modulated by ongoing jaw movements. However, active palpation and exploratory behaviors did result in the largest and most continuous bursts of GCL activity: responses were on average 10% larger and 50% longer during palpation and exploration than during grooming or passive stimulation. Although activity levels differed between behaviors, the position and spatial extent of the peripheral receptive field was similar over all behaviors that resulted in tactile input. Overall, our data suggest that the 1000-4500 Hz multiunit responses in the Crus IIa GCL of awake rats are correlated with tactile input rather than with movement or any movement parameter and that these responses are likely to be of particular importance during the acquisition of sensory information by perioral structures.

  9. Tactile responses in the granule cell layer of cerebellar folium crus IIa of freely behaving rats

    NASA Technical Reports Server (NTRS)

    Hartmann, M. J.; Bower, J. M.

    2001-01-01

    We recorded activity from the granule cell layer (GCL) of cerebellar folium Crus IIa as freely moving rats engaged in a variety of natural behaviors, including grooming, eating, and free tactile exploration. Multiunit responses in the 1000-4500 Hz range were found to be strongly correlated with tactile stimulation of lip and whisker (perioral) regions. These responses occurred regardless of whether the stimulus was externally or self-generated and during both active and passive touch. In contrast, perioral movements that did not tactually stimulate this region of the face (e.g., chewing) produced no detectable increases in GCL activity. In addition, GCL responses were not correlated with movement extremes. When rats used their lips actively for palpation and exploration, the tactile responses in the GCL were not detectably modulated by ongoing jaw movements. However, active palpation and exploratory behaviors did result in the largest and most continuous bursts of GCL activity: responses were on average 10% larger and 50% longer during palpation and exploration than during grooming or passive stimulation. Although activity levels differed between behaviors, the position and spatial extent of the peripheral receptive field was similar over all behaviors that resulted in tactile input. Overall, our data suggest that the 1000-4500 Hz multiunit responses in the Crus IIa GCL of awake rats are correlated with tactile input rather than with movement or any movement parameter and that these responses are likely to be of particular importance during the acquisition of sensory information by perioral structures.

  10. Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival.

    PubMed

    Lossi, Laura; Gambino, Graziana; Ferrini, Francesco; Alasia, Silvia; Merighi, Adalberto

    2009-11-01

    Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.

  11. Elements of the nitric oxide/cGMP pathway expressed in cerebellar granule cells: biochemical and functional characterisation.

    PubMed

    Jurado, Sandra; Sánchez-Prieto, José; Torres, Magdalena

    2004-11-01

    It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.

  12. Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism

    PubMed Central

    Hathaway, Hannah A.; Pshenichkin, Sergey; Grajkowska, Ewa; Gelb, Tara; Emery, Andrew C.; Wolfe, Barry B.; Wroblewski, Jarda T.

    2015-01-01

    The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, β-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function. PMID:25700650

  13. Cannabinoid Type 1 Receptors Transiently Silence Glutamatergic Nerve Terminals of Cultured Cerebellar Granule Cells

    PubMed Central

    Ramírez-Franco, Jorge; Bartolomé-Martín, David; Alonso, Beatris; Torres, Magdalena; Sánchez-Prieto, José

    2014-01-01

    Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1α protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals. PMID:24533119

  14. 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.

  15. The role of calcium and cyclic nucleotide signaling in cerebellar granule cell migration under normal and pathological conditions.

    PubMed

    Komuro, Yutaro; Galas, Ludovic; Lebon, Alexis; Raoult, Emilie; Fahrion, Jennifer K; Tilot, Amanda; Kumada, Tasturo; Ohno, Nobuhiko; Vaudry, David; Komuro, Hitoshi

    2015-04-01

    In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca(2+) and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca(2+) and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca(2+) and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease.

  16. BDNF-Mediated Cerebellar Granule Cell Development is Impaired in Mice Null for CaMKK2 or CaMKIV

    PubMed Central

    Kokubo, Manabu; Nishio, Masahiro; Ribar, Thomas J.; Anderson, Kristin A.; West, Anne E.; Means, Anthony R.

    2009-01-01

    The Ca2+/calmodulin-activated kinases CamKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca2+. To address the biological functions of Ca2+ signaling via these kinases during brain development we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of CREB correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Further, loss of CaMKK2 or CaMKIV results in decreased pCREB, Bdnf exon I and IV-containing mRNAs and BDNF protein in cerebellar granule cell neurons. Re-expression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca2+-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process. PMID:19605628

  17. Group B vitamins protect murine cerebellar granule cells from glutamate/NMDA toxicity.

    PubMed

    Lin, Yanpeng; Desbois, Angele; Jiang, Susan; Hou, Sheng T

    2004-10-05

    The role of B group vitamins in preventing neuronal death against excitotoxicity was investigated. Neuronal death of cultured mouse cerebellar granule neurons (CGNs) caused by glutamate (50 microM) or NMDA (200 microM) was delayed in CGNs that had been treated with riboflavin (B2), folic acid (B9) or cynocobalamin (B12) for 18 h. Such neuroprotection by B2, B9 and B12 was in a dose- and time-dependent manner. In contrast, application of thiamin (B1), nicotinamide (B3), d-pantothenic acid (B5), pyridoxine (B6) or carnitine (BT) did not ameliorate glutamate or NMDA-mediated excitotoxicity to CGCs. These results are the first indication that certain B group vitamins are not only required for the normal brain function, but can also play a protective role against excitotoxicity to the brain.

  18. The chemokine growth-related gene product β protects rat cerebellar granule cells from apoptotic cell death through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors

    PubMed Central

    Limatola, Cristina; Ciotti, Maria Teresa; Mercanti, Delio; Vacca, Fabrizio; Ragozzino, Davide; Giovannelli, Aldo; Santoni, Angela; Eusebi, Fabrizio; Miledi, Ricardo

    2000-01-01

    Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K+. We have found that the growth-related gene product β (GROβ) partially prevents the K+-depletion-induced cell death, and that the neuroprotective action of GROβ on granule cells is mediated through the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GROβ-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-d-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 μM) treatment. Moreover, GROβ-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GROβ is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. PMID:10811878

  19. Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na+ currents through intracellular Ca2+ release

    PubMed Central

    Liu, Dong-Dong; Ren, Zhen; Yang, Guang; Zhao, Qian-Ru; Mei, Yan-Ai

    2014-01-01

    Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa) densities by 62.5%. MT (5 μM) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca2+ level, but it significantly elevated the intracellular Ca2+ level evoked by the high K+ stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca2+ influx-induced Ca2+ release. PMID:24548607

  20. Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na(+) currents through intracellular Ca(2+) release.

    PubMed

    Liu, Dong-Dong; Ren, Zhen; Yang, Guang; Zhao, Qian-Ru; Mei, Yan-Ai

    2014-06-01

    Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa ) densities by 62.5%. MT (5 μM) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca(2+) level, but it significantly elevated the intracellular Ca(2+) level evoked by the high K(+) stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca(2+) influx-induced Ca(2+) release. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  1. AMPK is activated early in cerebellar granule cells undergoing apoptosis and influences VADC1 phosphorylation status and activity.

    PubMed

    Bobba, A; Casalino, E; Amadoro, G; Petragallo, V A; Atlante, A

    2017-06-22

    The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

  2. The brain-specific RasGEF very-KIND is required for normal dendritic growth in cerebellar granule cells and proper motor coordination

    PubMed Central

    Hayashi, Kanehiro; Furuya, Asako; Sakamaki, Yuriko; Akagi, Takumi; Shinoda, Yo; Sadakata, Tetsushi; Hashikawa, Tsutomu; Shimizu, Kazuki; Minami, Haruka; Sano, Yoshitake; Nakayama, Manabu

    2017-01-01

    Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance. PMID:28264072

  3. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats.

    PubMed

    Husmann, K; Faissner, A; Schachner, M

    1992-03-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.

  4. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

    PubMed Central

    1992-01-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats. PMID:1371773

  5. Decreased tonic inhibition in cerebellar granule cells causes motor dysfunction in a mouse model of Angelman syndrome.

    PubMed

    Egawa, Kiyoshi; Kitagawa, Kyoko; Inoue, Koichi; Takayama, Masakazu; Takayama, Chitoshi; Saitoh, Shinji; Kishino, Tatsuya; Kitagawa, Masatoshi; Fukuda, Atsuo

    2012-12-05

    Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the UBE3A gene encoding a ubiquitin E3 ligase. Motor dysfunction is a characteristic feature of Angelman syndrome, but neither the mechanisms of action nor effective therapeutic strategies have yet been elucidated. We report that tonic inhibition is specifically decreased in cerebellar granule cells of Ube3a-deficient mice, a model of Angelman syndrome. As a mechanism underlying this decrease in tonic inhibition, we show that Ube3a controls degradation of γ-aminobutyric acid (GABA) transporter 1 (GAT1) and that deficiency of Ube3a induces a surplus of GAT1 that results in a decrease in GABA concentrations in the extrasynaptic space. Administering low doses of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP), a selective extrasynaptic GABA(A) receptor agonist, improves the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in Ube3a-deficient mice in vivo. These results suggest that pharmacologically increasing tonic inhibition may be a useful strategy for alleviating motor dysfunction in Angelman syndrome.

  6. Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: involvement of MAP kinases.

    PubMed

    Mundy, William R; Freudenrich, Theresa M

    2006-01-01

    Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC(50) of 3 microM. Exposure to MMT, DMT, and MBT at concentrations up to 10 microM had no effect. DBT, however, was very potent, and decreased cell viability with an EC(50) of 0.3 microM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

  7. Casein Kinase 1δ Is an APC/CCdh1 Substrate that Regulates Cerebellar Granule Cell Neurogenesis

    PubMed Central

    Penas, Clara; Govek, Eve-Ellen; Fang, Yin; Ramachandran, Vimal; Daniel, Mark; Wang, Weiping; Maloof, Marie E.; Rahaim, Ronald J.; Bibian, Mathieu; Kawauchi, Daisuke; Finkelstein, David; Han, Jeng-Liang; Long, Jun; Li, Bin; Robbins, David J.; Malumbres, Marcos; Roussel, Martine F.; Roush, William R.; Hatten, Mary E.; Ayad, Nagi G.

    2015-01-01

    SUMMARY Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of central nervous system progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/CCdh1) ubiquitin ligase, and conditional deletion of the APC/CCdh1 activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/CCdh1 also downregulates CK1δ during cell cycle exit. Therefore, we conclude that APC/CCdh1 controls CK1δ levels to balance proliferation and cell cycle exit in the developing central nervous system. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a new therapeutic target. PMID:25843713

  8. Casein kinase 1δ is an APC/C(Cdh1) substrate that regulates cerebellar granule cell neurogenesis.

    PubMed

    Penas, Clara; Govek, Eve-Ellen; Fang, Yin; Ramachandran, Vimal; Daniel, Mark; Wang, Weiping; Maloof, Marie E; Rahaim, Ronald J; Bibian, Mathieu; Kawauchi, Daisuke; Finkelstein, David; Han, Jeng-Liang; Long, Jun; Li, Bin; Robbins, David J; Malumbres, Marcos; Roussel, Martine F; Roush, William R; Hatten, Mary E; Ayad, Nagi G

    2015-04-14

    Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

  9. Ethanol attenuates sensory stimulus-evoked responses in cerebellar granule cells via activation of GABA(A) receptors in vivo in mice.

    PubMed

    Wu, Guang; Liu, Heng; Jin, Juan; Hong, Lan; Lan, Yan; Chu, Chun-Ping; Qiu, De-Lai

    2014-02-21

    Acute alcohol intoxication affects cerebellar motor regulation possibly by altering the transfer and integration of external information in cerebellar cortical neurons, resulting in a dysfunction of cerebellar motor regulation or a cerebellar atexia. However, the synaptic mechanisms of ethanol induced impairments of sensory information processing in cerebellar cortical neurons are not fully understand. In the present study, we used electrophysiological and pharmacological methods to study the effects of ethanol on the sensory stimulation-evoked responses in cerebellar granule cells (GCs) in vivo in urethane anesthetized mice. Air-puff stimulation of the ipsilateral whisker-pad evoked stimulus-on (P1) and stimulus-off responses (P2) in GCs of cerebellar Crus II. Cerebellar surface perfusion of ethanol did not alter the onset latency of the sensory stimulation-evoked responses, but reversible reduced the amplitude of P1 and P2. The ethanol-induced reduction of the GCs sensory responses was concentration-dependent. In the presence of ethanol, the mean half-width, area under curve, rise Tau and decay Tau of P1 were significantly decreased. Blockade of gamma-aminobutyric acid type A (GABA(A)) receptors activity induced an increase in amplitude of P1, and abolished the ethanol induced inhibition of the GCs sensory responses. These results indicate that ethanol inhibits the tactile evoked responses in cerebellar GCs through enhancement of GABA(A) receptors activity.

  10. The effect of gallium nitride on long-term culture induced aging of neuritic function in cerebellar granule cells.

    PubMed

    Chen, Chi-Ruei; Young, Tai-Horng

    2008-04-01

    Gallium nitride (GaN) has been developed for a variety of microelectronic and optical applications due to its unique electric property and chemical stability. In the present study, n-type and p-type GaN were used as substrates to culture cerebellar granule neurons to examine the effect of GaN on cell response for a long-term culture period. It was found that GaN could rapidly induce cultured neurons to exhibit a high phosphorylated Akt level after 20h of incubation. It was assumed that the anti-apoptotic effect of Akt phosphorylation could be correlated with cell survival, neurite growth and neuronal function for up to 35 days of incubation. Morphological studies showed GaN induced larger neuronal aggregates and neurite fasciculation to exhibit a dense fiber network after 8 days of incubation. Western blot analysis and immunocytochemical characterization showed that GaN still exhibited the expression of neurite growth and function, such as high levels of GAP-43, synapsin I and synaptophysin even after 35 days of incubation. In addition, survival of cerebellar granule neurons on GaN was improved by the analysis of lactate dehydrogenase (LDH) release from damaged cells. These results indicated that neuronal connections were formed on GaN by a gradual process from Akt activation and cell aggregation to develop neurite growth, fasciculation and function. Therefore, GaN offers a good model system to identify a well-characterized pattern of neuronal behavior for a long-term culture period, consistent with the development of a neurochip requiring the integration of biological system and semiconductor material.

  11. Endothelin-1 stimulates the release of preloaded ( sup 3 H)D-aspartate from cultured cerebellar granule cells

    SciTech Connect

    Lin, W.W.; Lee, C.Y.; Chuang, D.M. )

    1990-03-16

    We have recently reported that endothelin-1 (ET) induces phosphoinositide hydrolysis in primary cultures of rat cerebellar granule cells. Here we found that ET in a dose-dependent manner (1-30 nM) stimulated the release of preloaded ({sup 3}H)D-aspartate from granule cells. The ET-induced aspartate release was completely blocked in the absence of extracellular Ca{sup 2+}, but was unaffected by 1 mM Co{sup 2+} or 1 microM dihydropyridine derivatives (nisoldipine and nimodipine). At higher concentration (10 microM) of nisoldipine and nimodipine, the release was partially inhibited. Short-term pretreatment of cells with phorbol 12,13-dibutyrate (PDBu) potentiated the ET-induced aspartate release, while long-term pretreatment with PDBu attenuated the release. Long-term exposure of cells to pertussis toxin (PTX), on the other hand, potentiated the ET-induced effects. Our results suggest that ET has a neuromodulatory function in the central nervous system.

  12. Hydroxylated polychlorinated biphenyls increase reactive oxygen species formation and induce cell death in cultured cerebellar granule cells

    SciTech Connect

    Dreiem, Anne Rykken, Sidsel; Lehmler, Hans-Joachim; Robertson, Larry W.; Fonnum, Frode

    2009-10-15

    Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2'-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34, 4'-OH PCB 35, 4-OH PCB 36, 4'-OH PCB 36, 4-OH PCB 39, and 4'-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4'-OH PCB 36 < 4'-OH PCB 68 < 4-OH PCB 39 < 4'-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.

  13. Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.

    PubMed

    Chan, Helen; Zwingmann, Claudia; Pannunzio, Marc; Butterworth, Roger F

    2003-07-01

    Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at

  14. Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells.

    PubMed

    Diaz, Marvin R; Wadleigh, Aya; Hughes, Benjamin A; Woodward, John J; Valenzuela, C Fernando

    2011-01-01

    The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca(2+)-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50-75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABA(A)-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl(-)-channel blockers were also tested: 4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs.

  15. Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

    PubMed Central

    Diaz, Marvin R.; Wadleigh, Aya; Hughes, Benjamin A.; Woodward, John J.; Valenzuela, C. Fernando

    2012-01-01

    The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs. PMID:22275879

  16. Granule cell ascending axon excitatory synapses onto Golgi cells implement a potent feedback circuit in the cerebellar granular layer.

    PubMed

    Cesana, Elisabetta; Pietrajtis, Katarzyna; Bidoret, Céline; Isope, Philippe; D'Angelo, Egidio; Dieudonné, Stéphane; Forti, Lia

    2013-07-24

    The function of inhibitory interneurons within brain microcircuits depends critically on the nature and properties of their excitatory synaptic drive. Golgi cells (GoCs) of the cerebellum inhibit cerebellar granule cells (GrCs) and are driven both by feedforward mossy fiber (mf) and feedback GrC excitation. Here, we have characterized GrC inputs to GoCs in rats and mice. We show that, during sustained mf discharge, synapses from local GrCs contribute equivalent charge to GoCs as mf synapses, arguing for the importance of the feedback inhibition. Previous studies predicted that GrC-GoC synapses occur predominantly between parallel fibers (pfs) and apical GoC dendrites in the molecular layer (ML). By combining EM and Ca(2+) imaging, we now demonstrate the presence of functional synaptic contacts between ascending axons (aa) of GrCs and basolateral dendrites of GoCs in the granular layer (GL). Immunohistochemical quantification estimates these contacts to be ∼400 per GoC. Using Ca(2+) imaging to identify synaptic inputs, we show that EPSCs from aa and mf contacts in basolateral dendrites display similarly fast kinetics, whereas pf inputs in the ML exhibit markedly slower kinetics as they undergo strong filtering by apical dendrites. We estimate that approximately half of the local GrC contacts generate fast EPSCs, indicating their basolateral location in the GL. We conclude that GrCs, through their aa contacts onto proximal GoC dendrites, define a powerful feedback inhibitory circuit in the GL.

  17. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

    PubMed Central

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-01-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  18. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

    PubMed

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-10-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.

  19. TLQP-21, a neuroendocrine VGF-derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation.

    PubMed

    Severini, Cinzia; Ciotti, Maria Teresa; Biondini, Laura; Quaresima, Stefania; Rinaldi, Anna Maria; Levi, Andrea; Frank, Claudio; Possenti, Roberta

    2008-01-01

    Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c-jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.

  20. Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells.

    PubMed

    Canu, Nadia; Tufi, Roberta; Serafino, Anna Lucia; Amadoro, Giuseppina; Ciotti, Maria Teresa; Calissano, Pietro

    2005-03-01

    Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.

  1. Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro.

    PubMed

    Reistad, Trine; Fonnum, Frode; Mariussen, Espen

    2006-11-01

    Polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are compounds used as additive flame retardants in plastics, electronic equipment, and textiles. The aim of the present study was to investigate the in vitro effects of the pentabrominated diphenyl ether mixture, DE-71, and HBCD on cerebellar granule cells (CGC). Both DE-71 and HBCD induced death of CGC in low micromolar concentrations. The NMDA receptor antagonist MK801 (3 microM), and the antioxidant alpha-tocopherol (50 microM) significantly reduced the cell death. Incubation of the compounds together with the rat liver post-mitochondrial (S9) fraction reduced cell death by 58 and 64% for DE-71 and HBCD, respectively. No ROS formation and no elevation in intracellular calcium were observed. We further demonstrated apoptotic morphology (Hoechst straining) after exposure to low levels of the two brominated flame retardants and signs of DNA laddering were found after DE-71 exposure. However, other hallmarks of apoptosis, like caspase activity, were absent indicating an atypical form of apoptosis induced by DE-71. After intraperitoneal injection of the two compounds both DE-71 and HBCD were found in significant amounts in brain (559 +/- 194 and 49 +/- 13 microg/kg, respectively) and liver (4,010 +/- 2,437 and 1,248 +/- 505 microg/kg, respectively) 72 h after injection. Our results indicate that the lower brominated PBDEs have a higher potency of bioaccumulation than HBCD, and that both compounds have a neurotoxic potential in vitro.

  2. Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors.

    PubMed Central

    D'Angelo, E; De Filippi, G; Rossi, P; Taglietti, V

    1995-01-01

    1. Current-clamp recordings were made in whole-cell patch-clamp configuration from ninety-one granule cells in parasagittal cerebellar slices obtained from 21- to 31-day-old rats. Recordings were performed at 30 degrees C. 2. Resting membrane potential was -58 +/- 6 mV (n = 43). The membrane voltage response to step current injection showed inward rectification consistent with increasing input resistance during membrane depolarization. Over -35 +/- 7 mV (n = 14) repetitive firing with little or no adaptation was activated. Spike frequency increased nearly linearly with injected current. 3. Unitary EPSPs obtained by stimulating the mossy fibre bundle had an amplitude of 11.4 +/- 2.1 mV (n = 22, holding potential = -75 mV). Synchronous activation of greater than one to two mossy fibres was needed to elicit action potentials. Antidromic stimulation elicited antidromic spikes and also EPSPs, presumably through a mossy fibre 'axon reflex'. 4. EPSPs were brought about by NMDA and non-NMDA receptor activation, accounting for about 70 and 30%, respectively, of peak amplitude at the holding potential of -70 mV. The EPSP decay conformed to passive membrane discharge after blocking the NMDA receptors. 5. No appreciable correlation was found between the time-to-peak and decay time constant of the EPSPs, consistent with the compact electrotonic structure of these neurons. 6. During membrane depolarization EPSP amplitude increased transiently, due to both a voltage-dependent increase of the NMDA component and inward rectification. In addition, EPSPs slowed down due to a slowdown of the NMDA component. 7. Temporal summation during high-frequency stimulation was sustained by NMDA receptors, whose contribution to depolarization tended to prevail over that of non-NMDA receptors during the trains. A block of the NMDA receptors resulted in reduced depolarization and output spike frequency. 8. This study, as well as extending previous knowledge to the intracellular level in vivo

  3. α6-Containing GABAA Receptors Are the Principal Mediators of Inhibitory Synapse Strengthening by Insulin in Cerebellar Granule Cells.

    PubMed

    Accardi, Michael V; Brown, Patricia M G E; Miraucourt, Loïs S; Orser, Beverley A; Bowie, Derek

    2015-07-01

    Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening. Copyright © 2015 the authors 0270-6474/15/359676-13$15.00/0.

  4. A stereological analysis of the cerebellar granule and Purkinje cells of 30-day-old and adult rats undernourished during early postnatal life.

    PubMed

    Bedi, K S; Hall, R; Davies, C A; Dobbing, J

    1980-10-15

    Male rats undernourished from birth to 30 days of age were nutritionally rehabilitated till 160 days of age. Quantitative stereological procedures at the light microscope level were used to estimate, among other things, the numerical densities of cerebellar granule and Purkinje cells on a "per unit volume of cortex" basis. These were subsequently used to calculate granule-to-Purkinje cell ratios. The 30-day-old undernourished rats had a mean +/- S.E. of 290 +/- 27 granule cells for every Purkinje cell present, compared to 395 +/- 34 for the controls. This was a deficit of about 27% (p < 0.05). At 160 days of age, the previously undernourished rats still showed a persisting deficit of about 25% (p < 0.05) in this ratio, despite the lengthy nutritional rehabilitation. There were no statistically significant age-related changes in this ratio. The numerical density of Purkinje cells, but not that of granule cells, was significantly greater in the previously undernourished rats than in controls, for both age groups, Increasing age caused a fall in the numerical density of both cell types. Granule and Purkinje cell nuclear diameters were unaffected by nutrition. However, Purkinje cell nuclei decreased in size by between 7%--13% with increasing age. These results indicate that undernutrition during early life can cause a permanent distortion of the relative number of the various cell types in the cerebellum.

  5. Adverse effects of 2,4-dichlorophenoxyacetic acid on rat cerebellar granule cell cultures were attenuated by amphetamine.

    PubMed

    Bongiovanni, B; Ferri, A; Brusco, A; Rassetto, M; Lopez, L M; Evangelista de Duffard, A M; Duffard, R

    2011-05-01

    2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress.

  6. Cell Signaling and Neurotoxicity: 3H-Arachidonic acid release (Phospholipase A2) in cerebellar granule neurons

    EPA Science Inventory

    Cell signaling is a complex process which controls basic cellular activities and coordinates actions to maintain normal cellular homeostasis. Alterations in signaling processes have been associated with neurological diseases such as Alzheimer's and cerebellar ataxia, as well as, ...

  7. Cell Signaling and Neurotoxicity: 3H-Arachidonic acid release (Phospholipase A2) in cerebellar granule neurons

    EPA Science Inventory

    Cell signaling is a complex process which controls basic cellular activities and coordinates actions to maintain normal cellular homeostasis. Alterations in signaling processes have been associated with neurological diseases such as Alzheimer's and cerebellar ataxia, as well as, ...

  8. Characterization of muscarinic receptor subtypes in primary cultures of cerebellar granule cells using specific muscarinic receptor antagonists

    SciTech Connect

    McLeskey, S.W.

    1989-01-01

    In cerebellar granule cell cultures, two muscarinic receptor mediated responses were observed: inhibition of adenylate cyclase (M-AC) and stimulation of phosphoinositide hydrolysis (M-PI). These responses were antagonized by three purported specific muscarinic antagonists: pirenzipine and (-)QNX (specific for M-PI) and methoctramine (specific for M-AC). However, the specificity for the three antagonists in blocking these responses is not comparable to the specificity observed in binding studies on these cells or to that quoted in the literature. Two peaks of molecular sizes were found in these cells corresponding to the two molecular sizes of muscarinic receptive proteins reported in the literature. Muscarinic receptive proteins were alkylated with {sup 3}H-propylbenzilylcholine mustard followed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pirenzipine and (-)QNX were able to block alkylation of the high molecular size peak, which corresponds to the receptive protein m{sub 3} reported in the literature. Methoctramine was able to block alkylation of a portion of the lower molecular size peak, possibly corresponding to the m{sub 2} and/or m{sub 4} receptive proteins reported in the literature. Studies attempting to show the presence of receptor reserve for either of the two biochemical responses present in these cells by alkylation of the receptive protein with nonradiolabeled propylbenzilylcholine mustard (PBCM) were confounded by specificity of this agent for the lower molecular weight peak of muscarinic receptive protein. Thus the muscarinic receptive proteins coupled to M-AC were alkylated preferentially over the ones coupled to M-PI.

  9. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

    PubMed Central

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  10. Redox signal regulation via nNOS phosphorylation at Ser847 in PC12 cells and rat cerebellar granule neurons.

    PubMed

    Kasamatsu, Shingo; Watanabe, Yasuo; Sawa, Tomohiro; Akaike, Takaaki; Ihara, Hideshi

    2014-04-15

    Phosphorylation is considered a main mechanism modulating nNOS (neuronal nitric oxide synthase) function to reduce NO production. In the present study, the effects of nNOS phosphorylation on redox signalling, including that of NO, ROS (reactive oxygen species), and 8-nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate), a downstream messenger of redox signalling, were investigated. In vitro experiments revealed that a phosphorylation-mimic mutant of nNOS (Ser847 replaced with aspartic acid, 847D) increased uncoupling to produce a superoxide. In addition, nicotine, which triggers an influx of Ca2+, induced more ROS and 8-nitro-cGMP production in 847D-expressing PC12 cells than WT (wild-type)-expressing cells. Additionally, nicotine-induced phosphorylation of nNOS at Ser847 and increased ROS and 8-nitro-cGMP production in rat CGNs (cerebellar granule neurons). In CGNs, the NOS (nitric oxide synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and superoxide dismutase completely inhibited ROS and 8-nitro-cGMP production, whereas the CaMK (Ca2+/calmodulin-dependent protein kinase) inhibitor KN93 mildly reduced this effect. Nicotine induced HO-1 (haem oxygenase 1) expression in CGNs and showed cytoprotective effects against apoptosis. Moreover, 8-nitro-cGMP treatment showed identical effects that were attenuated by KN93 pre-treatment. The present paper provides the first substantial corroboration for the biological effects of nNOS phosphorylation at Ser847 on redox signalling, including ROS and intracellular 8-nitro-cGMP generation in neurons, which possibly play roles in neuroprotection.

  11. Mefenamic acid bi-directionally modulates the transient outward K{sup +} current in rat cerebellar granule cells

    SciTech Connect

    Zhang Man; Shi Wenjie; Fei Xiaowei; Liu Yarong; Zeng Ximin; Mei Yanai

    2008-02-01

    The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K{sup +} current (I{sub A}) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 {mu}M to 100 {mu}M, mefenamic acid reversibly inhibited I{sub A} in a dose-dependent manner. However, mefenamic acid at a concentration of 1 {mu}M significantly increased the amplitude of I{sub A} to 113 {+-} 1.5% of the control. At more than 10 {mu}M, mefenamic acid inhibited the amplitude of I{sub A} without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I{sub A}, but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I{sub A} channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.

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

    PubMed

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

    2003-01-01

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

  13. Developmental exposure to ethanol increases the neuronal vulnerability to oxygen-glucose deprivation in cerebellar granule cell cultures.

    PubMed

    Le Duc, Diana; Spataru, Ana; Ceanga, Mihai; Zagrean, Leon; Schöneberg, Torsten; Toescu, Emil C; Zagrean, Ana-Maria

    2015-07-21

    Prenatal alcohol exposure is associated with microencephaly, cognitive and behavioral deficits, and growth retardation. Some of the mechanisms of ethanol-induced injury, such as high level oxidative stress and overexpression of pro-apoptotic genes, can increase the sensitivity of fetal neurons towards hypoxic/ischemic stress associated with normal labor. Thus, alcohol-induced sequelae may be the cumulative result of direct ethanol toxicity and increased neuronal vulnerability towards metabolic stressors, including hypoxia. We examined the effects of ethanol exposure on the fetal cerebellar granular neurons' susceptibility to hypoxic/hypoglycemic damage. A chronic ethanol exposure covered the entire prenatal period and 5 days postpartum through breastfeeding, a time interval partially extending into the third-trimester equivalent in humans. After a binge-like alcohol exposure at postnatal day 5, glutamatergic cerebellar granule neurons were cultured and grown for 7 days in vitro, then exposed to a 3-h oxygen-glucose deprivation to mimic a hypoxic/ischemic condition. Cellular viability was monitored by dynamic recording of propidium iodide fluorescence over 20 h reoxygenation. We explored differentially expressed genes on microarray data from a mouse embryonic ethanol-exposure model and validated these by real-time PCR on the present model. In the ethanol-treated cerebellar granule neurons we find an increased expression of genes related to apoptosis (Mapk8 and Bax), but also of genes previously described as neuroprotective (Dhcr24 and Bdnf), which might suggest an actively maintained viability. Our data suggest that neurons exposed to ethanol during development are more vulnerable to in vitro hypoxia/hypoglycemia and have higher intrinsic death susceptibility than unexposed neurons.

  14. L1 modulates PKD1 phosphorylation in cerebellar granule neurons.

    PubMed

    Chen, Shuang-xi; Hu, Cheng-liang; Liao, Yong-hong; Zhao, Wei-jiang

    2015-01-01

    The neural cell adhesion molecule L1 (L1CAM) is crucial for the development of the nervous system, with an essential role in regulating multiple cellular activities. Protein kinase D1 (PKD1) serves as a key kinase given its diverse array of functions within the cell. Here, we investigated various aspects of the functional relationship between L1 and phosphorylated PKD1 (pPKD1) in cerebellar granule neurons. To study the relationship between L1 and PKD1 phosphorylation, human cerebellar tissue microarrays were subject to immunofluorescence staining. We observed a positive correlation between L1 protein levels and PKD1 phosphorylation. In addition, L1 also co-localized with pPKD1. To analyze the regulatory role of L1 on PKD1 phosphorylation, primary mouse cerebellar granule neurons were treated with various concentrations of rL1 for 48 h. Using Western blot, we revealed that L1 significantly increased PKD1 phosphorylation compared with vehicle control, with the maximal effect observed at 5 nM. ERK1/2 phosphorylation was significantly increased by 2.5 nM and 10nM L1, with no apparent change in SRC phosphorylation. However, SRC expression was markedly reduced by 10nM rL1. AKT1 expression and phosphorylation levels were significantly increased by rL1, with the maximal effect observed at 2.5 and 5 nM, respectively. Our combined data revealed a positive relationship between L1 and pPKD1 in both cultured cerebellar neurons and human cerebellar tissue, suggesting that L1 functions in the modulation of PKD1 phosphorylation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. Propofol enhances facial stimulation-evoked responses in the cerebellar granule cell layer via NMDA receptor activation in mice in vivo.

    PubMed

    Jin, Wen-Zhe; Liu, Heng; Wan, Peng; Chu, Chun-Ping; Qiu, De-Lai

    2016-10-05

    We recently reported that propofol depressed facial stimulation-evoked gamma-aminobutyric acid (GABA) transmission at cerebellar molecular layer interneuron-Purkinje cell (PC) synapses in mice in vivo, but facilitated excitatory parallel fiber inputs onto PCs. Here, we examine the effects of propofol on cerebellar granule cell layer (GCL) responses to facial stimulation in urethane-anesthetized mice, using electrophysiological and pharmacological methods. Cerebellar surface perfusion of propofol (50-1000μM) facilitated field potentials evoked in the cerebellar GCL by air-puff stimulation of the ipsilateral whisker pad, shown by increases in the half-width and area under the curve (AUC) of the stimulus onset response (Ron). Propofol also significantly increased the amplitude of the stimulus offset response (Roff) and Roff/Ron ratio. The propofol-induced increase in Ron AUC was dose-dependent, with a 50% effective concentration (EC50) of 242.4µM. Application of the GABAA receptor antagonist gabazine (20μM) significantly increased the amplitude, half-width, rise tau and AUC of Ron, but these parameters were further increased by additional application of propofol (300µM). Notably, application of the N-methyl-d-aspartate (NMDA) receptor blocker D-APV (250µM) significantly attenuated the half-width and AUC of Ron and the amplitude of Roff, without significantly changing the amplitude of Ron. These results indicate that propofol enhanced facial stimulation-evoked responses in the cerebellar GCL via NMDA receptor activation, which resulted in the facilitation of excitatory parallel fiber inputs onto cerebellar PCs in mice in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. The natural scorpion peptide, BmK NT1 activates voltage-gated sodium channels and produces neurotoxicity in primary cultured cerebellar granule cells.

    PubMed

    Zou, Xiaohan; He, Yuwei; Qiao, Jinping; Zhang, Chunlei; Cao, Zhengyu

    2016-01-01

    The scorpion Buthus martensii Karsch has been used in Traditional Chinese Medicine to treat neuronal diseases such as neuropathic pain, paralysis and epilepsy for thousands of years. Studies have demonstrated that scorpion venom is the primary active component. Although scorpion venom can effectively attenuate pain in the clinic, it also produces neurotoxic response. In this study, toxicity guided purification led to identify a mammalian toxin termed BmK NT1 comprising of 65 amino acid residues and an amidated C-terminus, a mature peptide encoded by the nucleotide sequence (GenBank No. AF464898). In contract to the recombinant product of the same nucleotide sequence, BmK AGAP, which displayed analgesic and anti-tumor effect, intravenous injection (i.v.) of BmK NT1 produced acute toxicity in mice with an LD50 value of 1.36 mg/kg. In primary cultured cerebellar granule cells, BmK NT1 produced a concentration-dependent cell death with an IC50 value of 0.65 μM (0.41-1.03 μM, 95% Confidence Intervals, 95% CI) which was abolished by TTX, a voltage-gated sodium channel (VGSC) blocker. We also demonstrated that BmK NT1 produced modest sodium influx in cerebellar granule cell cultures with an EC50 value of 2.19 μM (0.76-6.40 μM, 95% CI), an effect similar to VGSC agonist, veratridine. The sodium influx response was abolished by TTX suggesting that BmK NT1-induced sodium influx is solely through activation of VGSC. Considered these data together, we demonstrated that BmK NT1 activated VGSC and produced neurotoxicity in cerebellar granule cell cultures.

  17. Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

    PubMed

    Fonfría, E; Rodríguez-Farré, E; Suñol, C

    2001-12-01

    Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.

  18. The regulation of synaptic vesicle recycling by cGMP-dependent protein kinase type II in cerebellar granule cells under strong and sustained stimulation.

    PubMed

    Collado-Alsina, Andrea; Ramírez-Franco, Jorge; Sánchez-Prieto, José; Torres, Magdalena

    2014-06-25

    From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, we demonstrate that presynaptic cGK type II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells.

  19. ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.

    EPA Science Inventory

    In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these stu...

  20. YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog - dependent cerebellar granule neuron progenitor cells and medulloblastoma cells

    PubMed Central

    Dey, Abhinav; Robitaille, Mélanie; Remke, Marc; Maier, Caroline; Malhotra, Anshu; Gregorieff, Alex; Wrana, Jeffrey L; Taylor, Michael D; Angers, Stéphane; Kenney, Anna Marie

    2016-01-01

    Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of medulloblastoma (MB), is driven by Sonic Hedgehog (Shh) and Insulin-like Growth Factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is up-regulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh-subgroup of MB in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas. PMID:26725322

  1. Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

    SciTech Connect

    Vendrell, Iolanda; Carrascal, Montserrat; Abian, Joaquin

    2010-01-01

    Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 +- 199, 345 +- 47, and 243 +- 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 +- 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

  2. The taurine uptake inhibitor guanidinoethyl sulphonate is an agonist at gamma-aminobutyric acid(A) receptors in cultured murine cerebellar granule cells.

    PubMed

    Mellor, J R; Gunthorpe, M J; Randall, A D

    2000-05-26

    In patch clamp experiments the beta-amino acid uptake inhibitor guanidinoethyl sulphonate (GES) activated currents in intact cultured murine cerebellar granule neurones. These responses could be attenuated by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonists bicuculline and picrotoxin. With intracellular chloride concentrations of either 20 or 130 mM, GES-induced current responses reversed polarity near the chloride equilibrium potential. When fast applications of agonist were made to excised granule cell macropatches GES responses were dose-dependent and exhibited significant outward rectification. Like taurine (but unlike GABA and beta-alanine) responses, macroscopic desensitisation of GES-induced currents was slow. Our data indicate that care should be exercised when using GES as a taurine uptake inhibitor in systems that also contain GABA(A) receptors.

  3. Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.

    PubMed Central

    Calissano, P; Ciotti, M T; Battistini, L; Zona, C; Angelini, A; Merlo, D; Mercanti, D

    1993-01-01

    Cerebellar granule cells grown in the presence of a serum complex differentiate but are resistant to the lethal action of excitatory amino acids. When these cells are grown also in the presence of insulin-like growth factor I (IGF-I) they become fully susceptible to the toxic, lethal action of glutamate. The glutamate-sensitizing action of IGF-I is dependent on concentration (half-maximal effect at 2-4 ng/ml) and time (half-maximal effect at 2-4 days in vitro) and is paralleled by the appearance of functionally active, glutamate-activated, Ca2+ channels and of voltage-gated Na+ and late K+ channels. IGF-I-induced glutamate sensitivity is rapidly reversible (t1/2 = 30-60 min) after removal of this somatomedin. The action of IGF-I is not mimicked by IGF-II, nerve growth factor, basic or acidic fibroblast growth factor, platelet-derived growth factor, or tumor necrosis factor alpha. We postulate that the constitutive phenotype of cerebellar granule cells is glutamate-resistant and becomes responsive to excitatory amino acids under the action of epigenetic cues among which IGF-I may be one of those operative in vivo. Images Fig. 1 PMID:8104340

  4. YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

    PubMed

    Dey, A; Robitaille, M; Remke, M; Maier, C; Malhotra, A; Gregorieff, A; Wrana, J L; Taylor, M D; Angers, S; Kenney, A M

    2016-08-11

    Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.

  5. Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.

    PubMed

    Babot, Zoila; Cristòfol, Rosa; Suñol, Cristina

    2005-01-01

    Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl

  6. Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development

    PubMed Central

    Zhang, Peter; Ha, Thomas; Larouche, Matt; Swanson, Douglas; Goldowitz, Dan

    2015-01-01

    Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene’s function in neuronal development. PMID:26226504

  7. Anticonvulsive Activity in Audiogenic DBA/2 Mice of 1,4-Benzodiazepines and 1,5-Benzodiazepines with Different Activities at Cerebellar Granule Cell GABAA Receptors.

    PubMed

    Gatta, Elena; Cupello, Aroldo; Di Braccio, Mario; Grossi, Giancarlo; Robello, Mauro; Scicchitano, Francesca; Russo, Emilio; De Sarro, Giovambattista

    2016-12-01

    Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity.

  8. Methylmercury-Dependent Increases in Fluo4 Fluorescence in Neonatal Rat Cerebellar Slices Depend on Granule Cell Migrational Stage and GABAA Receptor Modulation

    PubMed Central

    Bradford, Aaron B.; Mancini, Jayme D.

    2016-01-01

    Methylmercury (MeHg) disrupts cerebellar function, especially during development. Cerebellar granule cells (CGC), which are particularly susceptible to MeHg by unknown mechanisms, migrate during this process. Transient changes in intracellular Ca2+ (Ca2+i) are crucial to proper migration, and MeHg is well known to disrupt CGC Ca2+i regulation. Acutely prepared slices of neonatal rat cerebellum in conjunction with confocal microscopy and fluo4 epifluorescence were used to track changes induced by MeHg in CGC Ca2+i regulation in the external (EGL) and internal granule cell layers (IGL) as well as the molecular layer (ML). MeHg caused no cytotoxicity but did cause a time-dependent increase in fluo4 fluorescence that depended on the stage of CGC development. CGCs in the EGL were most susceptible to MeHg-induced increases in fluo4 fluorescence. MeHg increased fluorescence in CGC processes but only diffusely; Purkinje cells rarely fluoresced in these slices. Neither muscimol nor bicuculline alone altered baseline fluo4 fluorescence in any CGC layer, but each delayed the onset and reduced the magnitude of effect of MeHg on fluo4 fluorescence in the EGL and ML. In the IGL, both muscimol and bicuculline delayed the onset of MeHg-induced increases in fluo4 fluorescence but did not affect fluorescence magnitude. Thus, acute exposure to MeHg causes developmental stage-dependent increases in Ca2+i in CGCs. Effects are most prominent in CGCs during development or early stages of migration. GABAA receptors participate in an as yet unclear manner to MeHg-induced Ca2+i dysregulation of CGCs. PMID:26514794

  9. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  10. Deactivation and desensitization of non-NMDA receptors in patches and the time course of EPSCs in rat cerebellar granule cells.

    PubMed Central

    Silver, R A; Colquhoun, D; Cull-Candy, S G; Edmonds, B

    1996-01-01

    1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs were recorded from cerebellar granule cells in slices at approximately 24 and approximately 34 degrees C. The EPSC decay was fitted with the sum of two exponential functions. 2. The time courses of non-NMDA receptor deactivation and desensitization were determined with fast concentration jumps of glutamate onto patches from cultured granule cells. Deactivation (decay time constant tau = 0.6 ms at 24 degrees C) was substantially faster than desensitization (tau = 4 ms). Both processes were fitted by single exponential functions. 3. The decay of the fast component of the spontaneous EPSC (tau EPSCfast = 0.9 ms at 23 degrees C) was marginally slower than deactivation but too fast to be determined by desensitization. Our results suggest that the decay of this component is set by both the rate of decline of transmitter concentration and channel deactivation. 4. A simple diffusion model predicts that the time course of transmitter in the cleft declines slowly during the later stages of its action. The slow phase of transmitter removal could account for the time course of the slow component of the spontaneous EPSC (tau EPSCslow = 8 ms at 23 degrees C). PMID:8735702

  11. Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study

    PubMed Central

    Magistretti, Jacopo; Castelli, Loretta; Forti, Lia; D'Angelo, Egidio

    2006-01-01

    Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ∼−40 mV, showed half-maximal activation at ∼−55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at −35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially

  12. Isovolumetric regulation mechanisms in cultured cerebellar granule neurons.

    PubMed

    Tuz, K; Ordaz, B; Vaca, L; Quesada, O; Pasantes-Morales, H

    2001-10-01

    Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).

  13. Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis.

    PubMed

    Atlante, Anna; de Bari, Lidia; Bobba, Antonella; Marra, Ersilia; Passarella, Salvatore

    2007-11-01

    Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

  14. ZNHIT3 is defective in PEHO syndrome, a severe encephalopathy with cerebellar granule neuron loss.

    PubMed

    Anttonen, Anna-Kaisa; Laari, Anni; Kousi, Maria; Yang, Yawei J; Jääskeläinen, Tiina; Somer, Mirja; Siintola, Eija; Jakkula, Eveliina; Muona, Mikko; Tegelberg, Saara; Lönnqvist, Tuula; Pihko, Helena; Valanne, Leena; Paetau, Anders; Lun, Melody P; Hästbacka, Johanna; Kopra, Outi; Joensuu, Tarja; Katsanis, Nicholas; Lehtinen, Maria K; Palvimo, Jorma J; Lehesjoki, Anna-Elina

    2017-03-01

    Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development.

  15. Down-regulation of CYP1A2 induction during the maturation of mouse cerebellar granule cells in culture: role of nitric oxide accumulation.

    PubMed

    Mulero-Navarro, Sonia; Santiago-Josefat, Belen; Pozo-Guisado, Eulalia; Merino, Jaime M; Fernandez-Salguero, Pedro M

    2003-10-01

    Nitric oxide (NO) is responsible for cytochrome P450 (CYP450) loss during isolation and cytokine treatment of primary rat hepatocytes. As P450s mediate the metabolism of toxic chemicals, their inhibition could compromise the cells competence to eliminate toxins, a condition potentially relevant in neurological diseases involving constitutive activation of nitric oxide synthase (NOS) and NO over-production. Here, we have investigated the correlation between NO accumulation and CYP1A2 down-regulation during maturation of mouse cerebellar granule cells (CGC). As neurons matured in culture, the inducible levels of CYP1A2 protein and catalytic activity decreased to almost undetectable values. In parallel, a significant increase in NO concentration was observed. Neuronal NOS remained constitutively active during maturation, thus contributing to NO accumulation. The NOS inhibitor l-NAME, restored CYP1A2 catalytic activity up to 9 days in vitro, supporting a role for NO in the inhibition process. Maturation was also followed by increased NMDA receptor activity and intracellular Ca2+ concentration. We suggest that maintained NOS activity during CGC maturation could lead to NO accumulation and to decreased CYP1A2 inducibility. Increased NMDA receptor activity and Ca2+ entry could contribute to this process. Thus, neurodegeneration could diminish the induction of specific P450s and impair the metabolism of foreign and/or endogenous chemicals in the CNS.

  16. Neuroprotection comparison of chlorogenic acid and its metabolites against mechanistically distinct cell death-inducing agents in cultured cerebellar granule neurons.

    PubMed

    Taram, Faten; Winter, Aimee N; Linseman, Daniel A

    2016-10-01

    While the number of patients diagnosed with neurodegenerative disorders like Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease is increasing, there are currently no effective treatments that significantly limit the neuronal cell death underlying these diseases. Chlorogenic acid (CGA), a polyphenolic compound found in high concentration in coffee, is known to possess antioxidant and free radical scavenging activity. In this study, we investigated the neuroprotective effects of CGA and its major metabolites in primary cultures of rat cerebellar granule neurons. We show that CGA and caffeic acid displayed a dramatic protective effect against the nitric oxide donor, sodium nitroprusside. In marked contrast, ferulic acid and quinic acid had no protective effect against this nitrosative stress. While CGA and quinic acid had no protective effect against glutamate-induced cell death, caffeic acid and ferulic acid significantly protected neurons from excitotoxicity. Finally, caffeic acid was the only compound to display significant protective activity against hydrogen peroxide, proteasome inhibition, caspase-dependent intrinsic apoptosis, and endoplasmic reticulum stress. These results indicate that caffeic acid displays a much broader profile of neuroprotection against a diverse range of stressors than its parent polyphenol, CGA, or the other major metabolites, ferulic acid and quinic acid. We conclude that caffeic acid is a promising candidate for testing in pre-clinical models of neurodegeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Maturation-dependent modulation of apoptosis in cultured cerebellar granule neurons by cytokines and neurotrophins.

    PubMed

    de Luca, A; Weller, M; Frei, K; Fontana, A

    1996-09-01

    Immature cerebellar granule neurons die by apoptosis within 1 week in vitro unless maintained in depolarizing (high) concentrations of potassium (25 mM K+). Neurons allowed to survive and differentiate in high K+ medium for several days in vitro are still induced to undergo apoptosis when switched back to physiological (low) concentrations of K+ (5 mM). Here we have investigated the effects of various cytokines and growth factors in these two well-defined paradigms of neuronal apoptosis. Tumour necrosis factor-alpha, leukaemia inhibitory factor, ciliary neurotrophic factor, interleukin-10 and interleukin-13 delayed apoptosis and prolonged survival of cerebellar granule neurons maintained in low K+ medium. The effect observed required continuous exposure of the cultures to the cytokines and appeared not to involve modulation of Bcl-2 protein expression. Brain-derived neurotrophic factor accelerated neuronal death in low K+ medium. In contrast, when apoptosis of the neurons was precipitated by switching mature high K+ neurons to low K+ medium, neither tumour necrosis factor-alpha, leukaemia inhibitory factor, ciliary neurotrophic factor, interleukin-10 nor interleukin-13 prevented apoptosis. When testing the cytokines and growth factors for their capacity to alter N-methyl-D-aspartate receptor-mediated excitotoxicity of differentiated cerebellar granule neurons, no significant effect was observed. These data appear to define a maturation-dependent modulation of cerebellar granule cell survival by cytokines and neurotrophic factors that are expressed in a developmental pattern in the mammalian brain.

  18. Protective effect of keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells.

    PubMed

    Shimada, Y; Yokoyama, K; Goto, H; Sekiya, N; Mantani, N; Tahara, E; Hikiami, H; Terasawa, K

    2004-07-01

    Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 microg/ml), Cinnamomi Cortex extract (CCE) (3, 10 and 30 microg/ml), Paeoniae Radix extract (PRE) (100 microg/ml) and Moutan Cortex extract (MCE) (10 and 30 microg/ml) compared with exposure to SNP (30 microM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 microg/ml), CCE (30 and 100 microg/ml), PRE (100 and 300 microg/ml) and MCE (30 and 100 microg/ml) compared with exposure to NOC 18 (100 microM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.

  19. Selective vulnerability of cerebellar granule neuroblasts and their progeny to drugs with abuse liability

    PubMed Central

    Hauser, Kurt F.; Khurdayan, Valeriya K.; Goody, Robin J.; Nath, Avindra; Saria, Alois; Pauly, James R.

    2015-01-01

    Cerebellar development is shaped by the interplay of genetic and numerous environmental factors. Recent evidence suggests that cerebellar maturation is acutely sensitive to drugs with abuse liability including alcohol, opioids, and nicotine. Assuming substance abuse disrupts cerebellar maturation, a central question is to what are the basic mechanisms underlying potential drug-induced developmental defects. Evidence reviewed herein suggests that the maturation of granule neurons and their progeny are intrinsically affected by several classes of substances with abuse liability. Although drug abuse is also likely to target directly other cerebellar neuron and glial types, such as Purkinje cells and Bergmann glia, findings in isolated granule neurons suggest that they are often the principle target for drug actions. Developmental events that are selectively disrupted by drug abuse in granule neurons and/or their neuroblast precursors include proliferation, migration, differentiation (including neurite elaboration and synapse formation), and programmed cell death. Moreover, different classes of drugs act through distinct molecular mechanisms thereby disrupting unique aspects of development. For example, drug-induced perturbations in (i) neurotransmitter biogenesis, (ii) ligand and ion-gated receptor function and their coupling to intracellular effectors, (iii) neurotrophic factor biogenesis and signaling, and (iv) intercellular adhesion are all likely to have significant effects in shaping developmental outcome. In addition to identifying therapeutic strategies for drug abuse intervention, understanding the mechanisms by which drugs affect cellular maturation is likely to provide a better understanding of the neurochemical events that normally shape central nervous system development. PMID:14509568

  20. Weaker control of the electrical properties of cerebellar granule cells by tonically active GABAA receptors in the Ts65Dn mouse model of Down’s syndrome

    PubMed Central

    2013-01-01

    Background Down’s syndrome (DS) is caused by triplication of all or part of human chromosome 21 and is characterized by a decrease in the overall size of the brain. One of the brain regions most affected is the cerebellum, in which the number of granule cells (GCs) is markedly decreased. GCs process sensory information entering the cerebellum via mossy fibres and pass it on to Purkinje cells and inhibitory interneurons. How GCs transform incoming signals depends on their input–output relationship, which is adjusted by tonically active GABAA receptor channels. Results We report that in the Ts65Dn mouse model of DS, in which cerebellar volume and GC number are decreased as in DS, the tonic GABAA receptor current in GCs is smaller than in wild-type mice and is less effective in moderating input resistance and raising the minimum current required for action potential firing. We also find that tonically active GABAA receptors curb the height and broaden the width of action potentials in wild-type GCs but not in Ts65Dn GCs. Single-cell real-time quantitative PCR reveals that these electrical differences are accompanied by decreased expression of the gene encoding the GABAA receptor β3 subunit but not genes coding for some of the other GABAA receptor subunits expressed in GCs (α1, α6, β2 and δ). Conclusions Weaker moderation of excitability and action potential waveform in GCs of the Ts65Dn mouse by tonically active GABAA receptors is likely to contribute to atypical transfer of information through the cerebellum. Similar changes may occur in DS. PMID:23870245

  1. CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

    PubMed

    Alonso, Beatris; Bartolomé-Martín, David; Ferrero, José Javier; Ramírez-Franco, Jorge; Torres, Magdalena; Sánchez-Prieto, José

    2017-08-01

    Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid

  2. Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

    PubMed Central

    Olmos, Gabriel; DeGregorio-Rocasolano, Nuria; Regalado, M Paz; Gasull, Teresa; Boronat, M Assumpció; Trullas, Ramón; Villarroel, Alvaro; Lerma, Juan; García-Sevilla, Jesús A

    1999-01-01

    This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells.Exposure (30 min) of energy deprived cells to L-glutamate (1–100 μM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine).Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L-glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole] (101)>RX821002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors.Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding.In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV.It is concluded that imidazol(ine) drugs and agmatine are

  3. Acute Ethanol Exposure Prevents PMA-mediated Augmentation of N-methyl-d-aspartate Receptor Function in Primary Cultured Cerebellar Granule Cells

    PubMed Central

    Reneau, Jason; Reyland, Mary E.; Popp, R. Lisa

    2011-01-01

    Many intracellular proteins and signaling cascades contribute to the ethanol sensitivity of native N-methyl-d-aspartate receptors (NMDARs). One putative protein is the serine / threonine kinase, Protein kinase C (PKC). The purpose of this study was to assess if PKC modulates the ethanol sensitivity of native NMDARs expressed in primary cultured cerebellar granule cells (CGCs). With the whole-cell patch-clamp technique, we assessed if ethanol inhibition of NMDA-induced currents (INMDA) (100 μM NMDA plus 10 μM glycine) were altered in CGCs in which the novel and classical PKC isoforms were activated by phorbol-12-myristate-13-acetate (PMA). Percent inhibition by 10, 50 or 100 mM ethanol of NMDA-induced steady-state (ISS) or peak current amplitudes (IPk) of NMDARs expressed in CGCs in which PKC was activated by a 12.5 min, 100 nM PMA exposure at 37° C did not differ from currents obtained from receptors contained in control cells. However, PMA-mediated augmentation of IPk in the absence of ethanol was abolished after brief applications of 10 or 1 mM ethanol co-applied with agonists, and this suppression of enhanced receptor function was observed for up to eight minutes post-ethanol exposure. Because we had previously shown that PMA-mediated augmentation of INMDA of NMDARs expressed in these cells is by activation of PKCα, we assessed the effect of ethanol (1, 10, 50 and 100 mM) on PKCα activity. Ethanol decreased PKCα activity by 18% for 1 mM ethanol and activity decreased with increasing ethanol concentrations with a 50% inhibition observed with 100 mM ethanol. The data suggest that ethanol disruption of PMA-mediated augmentation of INMDA may be due to a decrease in PKCα activity by ethanol. However, given the incomplete blockade of PKCα activity and the low concentration of ethanol at which this phenomenon is observed, other ethanol-sensitive signaling cascades must also be involved. PMID:21624785

  4. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  5. Exposure to extremely low-frequency electromagnetic fields modulates Na+ currents in rat cerebellar granule cells through increase of AA/PGE2 and EP receptor-mediated cAMP/PKA pathway.

    PubMed

    He, Yan-Lin; Liu, Dong-Dong; Fang, Yan-Jia; Zhan, Xiao-Qin; Yao, Jin-Jing; Mei, Yan-Ai

    2013-01-01

    Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

  6. Exposure to Extremely Low-Frequency Electromagnetic Fields Modulates Na+ Currents in Rat Cerebellar Granule Cells through Increase of AA/PGE2 and EP Receptor-Mediated cAMP/PKA Pathway

    PubMed Central

    Fang, Yan-Jia; Zhan, Xiao-Qin; Yao, Jin-Jing; Mei, Yan-Ai

    2013-01-01

    Although the modulation of Ca2+ channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na+ channels (Nav). Here, we investigated the effects of ELF-EMF on Nav activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10–60 min significantly increased Nav currents (INa) by 30–125% in a time- and intensity-dependent manner. The Nav channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E2 (PGE2) on INa in cerebellar GCs. Increases in intracellular AA, PGE2 and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the NaV 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of NaV 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE2 receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and INa. In addition, ELF-EMF exposure significantly enhanced the activity of PLA2 in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal INa is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE2 EP receptors PKA signaling pathway. PMID:23349866

  7. COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

    EPA Science Inventory

    A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

  8. COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

    EPA Science Inventory

    A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

  9. Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis.

    PubMed

    Dever, Daniel P; Adham, Zachariah O; Thompson, Bryan; Genestine, Matthieu; Cherry, Jonathan; Olschowka, John A; DiCicco-Bloom, Emanuel; Opanashuk, Lisa A

    2016-05-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

  10. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

    EPA Science Inventory

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

  11. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

    EPA Science Inventory

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

  12. The absence of pleiotrophin modulates gene expression in the hippocampus in vivo and in cerebellar granule cells in vitro.

    PubMed

    González-Castillo, Celia; Ortuño-Sahagún, Daniel; Guzmán-Brambila, Carolina; Márquez-Aguirre, Ana Laura; Raisman-Vozari, Rita; Pallás, Mercé; Rojas-Mayorquín, Argelia E

    2016-09-01

    Pleiotrophin (PTN) is a secreted growth factor recently proposed to act as a neuromodulatory peptide in the Central Nervous System. PTN appears to be involved in neurodegenerative diseases and neural disorders, and it has also been implicated in learning and memory. Specifically, PTN-deficient mice exhibit a lower threshold for LTP induction in the hippocampus, which is attenuated in mice overexpressing PTN. However, there is little information about the signaling systems recruited by PTN to modulate neural activity. To address this issue, the gene expression profile in hippocampus of mice lacking PTN was analyzed using microarrays of 22,000 genes. In addition, we corroborated the effect of the absence of PTN on the expression of these genes by silencing this growth factor in primary neuronal cultures in vitro. The microarray analysis identified 102 genes that are differentially expressed (z-score>3.0) in PTN null mice, and the expression of eight of those modified in the hippocampus of KO mice was also modified in vitro after silencing PTN in cultured neurons with siRNAs. The data obtained indicate that the absence of PTN affects AKT pathway response and modulates the expression of genes related with neuroprotection (Mgst3 and Estrogen receptor 1, Ers 1) and cell differentiation (Caspase 6, Nestin, and Odz4), both in vivo and in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells.

    PubMed

    Borodinsky, Laura N; Coso, Omar A; Fiszman, Mónica L

    2002-03-01

    In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

  14. Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo.

    PubMed

    Kullmann, Jan A; Wickertsheim, Ines; Minnerup, Lara; Costell, Mercedes; Friauf, Eckhard; Rust, Marco B

    2015-01-01

    Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2'-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.

  15. Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken.

    PubMed

    Mathisen, Gro H; Yazdani, Mazyar; Rakkestad, Kirsten E; Aden, Petra K; Bodin, Johanna; Samuelsen, Mari; Nygaard, Unni C; Goverud, Ingeborg L; Gaarder, Mona; Løberg, Else Marit; Bølling, Anette K; Becher, Rune; Paulsen, Ragnhild E

    2013-12-01

    In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum. Copyright © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.

  16. The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

    PubMed Central

    Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

    2014-01-01

    Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

  17. The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells.

    PubMed

    Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

    2013-08-01

    Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity.

  18. WNT3 Inhibits Cerebellar Granule Neuron Progenitor Proliferation and Medulloblastoma Formation via MAPK Activation

    PubMed Central

    Ayrault, Olivier; Kim, Jee Hae; Zhu, Xiaodong; Murphy, David A.; Van Aelst, Linda; Roussel, Martine F.; Hatten, Mary E.

    2013-01-01

    During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical β-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors. PMID:24303070

  19. The Immp2l mutation causes age-dependent degeneration of cerebellar granule neurons prevented by antioxidant treatment.

    PubMed

    Liu, Chunlian; Li, Xue; Lu, Baisong

    2016-02-01

    Reactive oxygen species are implicated in age-associated neurodegeneration, although direct in vivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2-like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age-associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (> 16 months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria-targeted antioxidant SkQ1 from 6 weeks to 21 months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age-matched normal control mice or SkQ1-treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4-hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age-associated neurodegeneration. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  20. Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

    SciTech Connect

    Hogberg, Helena T.; Kinsner-Ovaskainen, Agnieszka; Hartung, Thomas; Coecke, Sandra; Bal-Price, Anna K.

    2009-03-15

    The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.

  1. SUMOylation silences heterodimeric TASK potassium channels containing K2P1 subunits in cerebellar granule neurons.

    PubMed

    Plant, Leigh D; Zuniga, Leandro; Araki, Dan; Marks, Jeremy D; Goldstein, Steve A N

    2012-11-20

    The standing outward K(+) current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. We showed that SUMOylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to SUMOylation, and the TASK (two-P domain, acid-sensitive K(+)) channel subunits K2P3 or K2P9. The heterodimeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that SUMOylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1.

  2. Action of thyroxine on the survival and neurite maintenance of cerebellar granule neurons in culture.

    PubMed

    Oyanagi, Koshi; Negishi, Takayuki; Tashiro, Tomoko

    2015-04-01

    Developmental hypothyroidism causes severe impairments in the cerebellum. To understand the role of thyroid hormones (THs) in cerebellar development, we examined the effect of three different THs, thyroxine (T4), 3,5,3'-triidothyronine (T3), and 3,3',5'-triiodothyronine (reverse T3; rT3), on the survival and morphology of cerebellar granule neurons (CGNs) in culture and found novel actions specific to T4. Rat CGNs obtained at postnatal day 6 were first cultured for 2 days in serum-containing medium with 25 mM K(+) (K25), then switched to serum-free medium with physiological 5 mM K(+) (K5) or with K25 and cultured for an additional 2 or 4 days. CGNs underwent apoptosis in K5 but survived in K25. Addition of T4 at concentrations of 100-200 nM but not T3 or rT3 rescued CGNs from cell death in K5 in a dose-dependent manner. Furthermore, 200 nM T4 was also effective in maintaining the neurites of CGNs in K5. In K5, T4 suppressed tau phosphorylation at two developmentally regulated sites as well as phosphorylation of c-jun N-terminal kinase (JNK) necessary for its activation and localization to axons. These results suggest that, during cerebellar development, T4 exerts its activity in cell survival and neurite maintenance in a manner distinct from the other two thyroid hormones through regulating the activity and localization of JNK.

  3. RNA Granules in Germ Cells

    PubMed Central

    Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo; Nagamori, Ippei

    2011-01-01

    “Germ granules” are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program. PMID:21768607

  4. Assessment of GaN chips for culturing cerebellar granule neurons.

    PubMed

    Young, Tai-Horng; Chen, Chi-Ruei

    2006-06-01

    In this work, the behaviors of cerebellar granule neurons prepared from 7-day-old Wistar rats on gallium nitride (GaN) were investigated. We believe that this is the first time that the GaN has been used as a substrate for neuron cultures to examine its effect on cell response in vitro. The GaN surface structure and its relationship with cells were examined by atomic force microscopy (AFM), metallography microscopy, scanning electron microscopy (SEM), lactate dehydrogenase (LDH) release and Western blot analysis. GaN is a so-called III-V compound semiconductor material with a wide bandgap and a relatively high bandgap voltage. Compared with silicon used for most neural chips, neurons seeded on GaN were able to form an extensive neuritic network and expressed very high levels of GAP-43 coincident with the neurite outgrowth. Therefore, the GaN structure may spatially mediate cellular response that can promote neuronal cell attachment, differentiation and neuritic growth. The favorable biocompatibility characteristics of GaN can be used to measure electric signals from networks of neuronal cells in culture to make it a possible candidate for use in a microelectrode array.

  5. Sumoylation Silences Heterodimeric TASK Potassium Channels Containing K2P1 Subunits in Cerebellar Granule Neurons

    PubMed Central

    Plant, Leigh D.; Zuniga, Leandro; Araki, Dan; Marks, Jeremy D.; Goldstein, Steve A. N.

    2013-01-01

    The standing outward K+ current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. In this study, we showed that sumoylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to sumoylation, and the two P domain, acid-sensitive K+ (TASK) channel subunits, K2P3 or K2P9. The heteromeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that sumoylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1. PMID:23169818

  6. Properties of osmolyte fluxes activated during regulatory volume decrease in cultured cerebellar granule neurons.

    PubMed

    Pasantes-Morales, H; Chacón, E; Murray, R A; Morán, J

    1994-04-15

    Efflux pathways for amino acids, K, and Cl activated during regulatory volume decrease (RVD) were characterized in cultured cerebellar granule neurons exposed to hyposmotic conditions. Results of this study favor diffusion pores (presumably channels) over energy-dependent transporters as the mechanisms responsible for the efflux of these osmolytes. The selectivity of osmolyte pathways activated by RVD was assessed by increasing the extracellular concentrations of cations, anions, and amino acids to such an extent that upon opening of the pathway, a permeable compound will enter the cell and block RVD by reducing the efflux of water carried by the exit of intracellular osmolytes. The cationic pathway was found selective for K (and Rb), whereas the anionic pathway was rather unselective being permeable to Cl, nitrate, iodine, benzoate, thiocyanate, and sulfate but impermeable to gluconate. Glutamate and aspartate as K but not as Na salts were permeable through the anion channel. RVD was slightly inhibited by quinidine but otherwise was insensitive to known K channel blockers. RVD was inhibited by 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS), niflumic acid, and dipyridamole. Gramicidin did not affect cell volume in isosmotic conditions but greatly accelerated RVD, suggesting that cell permeability to Cl is low in isosmotic conditions but increases markedly during RVD making K permeability the rate limit of the process. The permeability pathway for amino acids activated during RVD as permeable to short chain alpha- and beta-amino acids, but excluded glutamine and basic amino acids.

  7. Long-term exposure to dieldrin reduces gamma-aminobutyric acid type A and N-methyl-D-aspartate receptor function in primary cultures of mouse cerebellar granule cells.

    PubMed

    Babot, Zoila; Vilaró, M Teresa; Suñol, Cristina

    2007-12-01

    The organochlorine pesticide dieldrin is a persistent organic pollutant that accumulates in the fatty tissue of living organisms. In mammals, it antagonizes the GABA(A) receptor, producing convulsions after acute exposure. Although accumulation in human brain has been reported, little is known about the effects of long-term exposure to dieldrin in the nervous system. Homeostatic control of the balance between excitation and inhibition has been reported when neuronal activity is chronically altered. We hypothesized that noncytotoxic concentrations of dieldrin could decrease glutamatergic neurotransmission as a consequence of a prolonged reduction in GABA(A) receptor function. Long-term exposure of primary cerebellar granule cell cultures to 3 microM dieldrin reduced the GABA(A) receptor function to 55% of control, as measured by the GABA-induced (36)Cl(-) uptake. This exposure produced a significant reduction (approximately 35%) of the NMDA-induced increase in [Ca(2+)](i) and of the [(3)H]MK-801 binding, which was not accompanied by a reduction in the NMDA receptor subunit NR1, as determined by Western blot. Consistent with the decreased NMDA receptor function, dieldrin-treated cultures were insensitive to an excitotoxic stimulus induced by exposure to high potassium. In summary, we report that the chronic reduction of GABA(A) receptor function induced by dieldrin decreases the number of functional NMDA receptors, which may be attributable to a mechanism of synaptic scaling. These effects could underlie neural mechanisms involved in cognitive impairment produced by low-level exposure to dieldrin. (c) 2007 Wiley-Liss, Inc.

  8. Transcriptional Analysis of Apoptotic Cerebellar Granule Neurons Following Rescue by Gastric Inhibitory Polypeptide

    PubMed Central

    Maino, Barbara; Ciotti, Maria Teresa; Calissano, Pietro; Cavallaro, Sebastiano

    2014-01-01

    Apoptosis triggered by exogenous or endogenous stimuli is a crucial phenomenon to determine the fate of neurons, both in physiological and in pathological conditions. Our previous study established that gastric inhibitory polypeptide (Gip) is a neurotrophic factor capable of preventing apoptosis of cerebellar granule neurons (CGNs), during its pre-commitment phase. In the present study, we conducted whole-genome expression profiling to obtain a comprehensive view of the transcriptional program underlying the rescue effect of Gip in CGNs. By using DNA microarray technology, we identified 65 genes, we named survival related genes, whose expression is significantly de-regulated following Gip treatment. The expression levels of six transcripts were confirmed by real-time quantitative polymerase chain reaction. The proteins encoded by the survival related genes are functionally grouped in the following categories: signal transduction, transcription, cell cycle, chromatin remodeling, cell death, antioxidant activity, ubiquitination, metabolism and cytoskeletal organization. Our data outline that Gip supports CGNs rescue via a molecular framework, orchestrated by a wide spectrum of gene actors, which propagate survival signals and support neuronal viability. PMID:24694544

  9. Differential expression of two-pore domain potassium channels in rat cerebellar granule neurons.

    PubMed

    Burgos, Paulina; Zúñiga, Rafael; Domínguez, Pedro; Delgado-López, Fernando; Plant, Leigh D; Zúñiga, Leandro

    2014-10-31

    Two pore domain potassium (K2P) channels are mostly present in the central nervous system (CNS) where they play important roles in modulating neuronal excitability. K2P channels give rise to background K(+) currents (IKSO) a key component in setting and maintaining the resting membrane potential in excitable cells. Here, we studied the expression and relative abundances of K2P channels in cerebellar granule neurons (CGNs), combining molecular biology, electrophysiology and immunologic techniques. The CGN IKSO was very sensitive to external pH, as previously reported. Quantitative determination of mRNA expression level demonstrated the existence of an accumulation pattern of transcripts in CGN that encode K2P9>K2P1>K2P3>K2P18>K2P2=K2P10>K2P4>K2P5 subunits. The presence of the major K2P subunits expressed was then confirmed by Western blot and immunofluorescence analysis, demonstrating robust expression of K2P1 (TWIK-1), K2P3 (TASK-1), K2P9 (TASK-3) and K2P18 (TRESK) channel protein. Based, on these results, it is concluded that K2P1, -3, -9 and -18 subunits represent the majority component of IKSO current in CGN.

  10. Stromal cell-derived factor-1 promotes migration of cells from the upper rhombic lip in cerebellar development.

    PubMed

    Yu, Tao; Huang, Hai; Li, Hui-Fang

    2010-10-01

    During cerebellar development, the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha) has been shown to play an important role in recruiting cells from the upper rhombic lip (URL) and external granule cell layer (EGL). However, its function in cerebellar development is still poorly understood. Our results have demonstrated that SDF-1 is necessary for EGL development, and URL cells stream to the SDF-1 source in vitro. Results of embryonic URL explant assays and transwell assays indicated that SDF-1 induces neural cell migration from the URL region in chemotactic and chemokinetic responses. The time-lapse results showed that the migration speed of granule cell progenitors out of the URL was accelerated by the addition of recombinant SDF-1 alpha. Collectively, our study shows that SDF-1 increases the motility of URL cells in the absence of a gradient and promotes the migration of granule cell progenitors during cerebellar development.

  11. Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3Δex7/8-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

    PubMed Central

    Finn, Rozzy; Kovács, Attila D.; Pearce, David A.

    2011-01-01

    The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3Δex7/8-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3Δex7/8 CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3Δex7/8 cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3Δex7/8 CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3Δex7/8 mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3Δex7

  12. ROS Produced by NOX2 Controls In Vitro Development of Cerebellar Granule Neurons Development

    PubMed Central

    Olguín-Albuerne, Mauricio

    2015-01-01

    Reactive oxygen species (ROS) act as signaling molecules that regulate nervous system physiology. ROS have been related to neural differentiation, neuritogenesis, and programmed cell death. Nevertheless, little is known about the mechanisms involved in the regulation of ROS during neuronal development. In this study, we evaluated the mechanisms by which ROS are regulated during neuronal development and the implications of these molecules in this process. Primary cultures of cerebellar granule neurons (CGN) were used to address these issues. Our results show that during the first 3 days of CGN development in vitro (days in vitro; DIV), the levels of ROS increased, reaching a peak at 2 and 3 DIV under depolarizing (25 mM KCl) and nondepolarizing (5 mM KCl) conditions. Subsequently, under depolarizing conditions, the ROS levels markedly decreased, but in nondepolarizing conditions, the ROS levels increased gradually. This correlated with the extent of CGN maturation. Also, antioxidants and NADPH-oxidases (NOX) inhibitors reduced the expression of Tau and MAP2. On the other hand, the levels of glutathione markedly increased at 1 DIV. We inferred that the ROS increase at this time is critical for cell survival because glutathione depletion leads to axonal degeneration and CGN death only at 2 DIV. During the first 3 DIV, NOX2 was upregulated and expressed in filopodia and growth cones, which correlated with the hydrogen peroxide (H2O2) distribution in the cell. Finally, NOX2 KO CGN showed shorter neurites than wild-type CGN. Taken together, these results suggest that the regulation of ROS is critical during the early stages of CGN development. PMID:25873309

  13. Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons

    PubMed Central

    Fenster, Catherine; Vullhorst, Detlef; Buonanno, Andres

    2012-01-01

    Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10–12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of activation of synaptic NMDAR with high-glyine/0 Mg2+ on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar

  14. Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons.

    PubMed

    Fenster, Catherine; Vullhorst, Detlef; Buonanno, Andres

    2012-01-04

    Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg(2+) (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar

  15. Subcellular distribution and early signalling events of P2X7 receptors from mouse cerebellar granule neurons.

    PubMed

    Sánchez-Nogueiro, Jesús; Marín-García, Patricia; Bustillo, Diego; Olivos-Oré, Luis Alcides; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2014-12-05

    The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 μM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 μM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.

  16. Actin disruption alters the localization of tau in the growth cones of cerebellar granule neurons.

    PubMed

    Zmuda, J F; Rivas, R J

    2000-08-01

    Cultured cerebellar granule neurons initially extend a single axon, followed by the extension of a second axon to attain a bipolar morphology. Differentiation culminates with the extension of several short dendrites from the cell body. In the present study, we determined the location of the dephosphorylated form of the microtubule-associated protein tau (dtau) within the growth cones of newly forming axons and examined whether this localization was influenced by the actin cytoskeleton. Following elongation of the initial axon at 2-3 days in vitro, dtau immunoreactivity was present along the entire length of the axon, becoming most intense just proximal to the growth cone. Dtau labeling dropped off dramatically along the microtubules of the growth cone and was undetectable along the most distal tips of these microtubules. As the initial axon continued to elongate at 3-4 days in vitro, the actin-rich growth cone peripheral domain characteristically underwent a dramatic reduction in size. Dtau immunoreactivity extended all the way to the most distal tips of the microtubules in the growth cones of these cells. Cytochalasin D and latrunculin A mimicked the effects of this characteristic reduction in growth cone size with regard to dtau localization in the growth cone. Depolymerization of filamentous actin caused the collapse of the peripheral domain and allowed dtau to bind all the way to the most distal tips of microtubules in the axon. Upon removal of the drugs, the peripheral domain of the growth cone rapidly re-formed and dtau was once again excluded from the most distal regions of growth cone microtubules. These findings suggest a novel role for actin in determining the localization of the microtubule-associated protein &tgr; within the growth cones of neurons.

  17. Multiple pathways of Pb(2+) permeation in rat cerebellar granule neurones.

    PubMed

    Mazzolini, M; Traverso, S; Marchetti, C

    2001-10-01

    The pathways of lead (Pb(2+)) uptake were studied in fura-2-loaded cerebellar granule cells from 8-day-old rats. In a nominal Ca-free external bath, Pb(2+) (5-50 microM) determined an increase of the fluorescence emission ratio (R = E(340)/E(380)) even in the absence of any specific stimulus. This rise was dose-dependent, was not significantly affected by mM Mg(2+) or Ca(2+), but it was readily reversed by the membrane-permeant heavy metal chelator tetrakis(2-pyridylmethyl) ethylene-diamine (TPEN, 100 microM), indicating that it was due to Pb(2+) influx. The rate of rise, dR/dt, was increased up to a factor of 5 by depolarizing high-KCl solution, indicating a sizeable permeation through voltage-dependent channels. This effect was neither antagonized by nimodipine, nor enhanced by BayK8644, but it was slackened by omega-agatoxin IVA (200 nM), suggesting an involvement of non-L-type calcium channels. Pb(2+) influx was also stimulated by glutamic acid or NMDA in the presence of 10-30 microM glycine, but only in Mg-free solution, suggesting that glutamate channels of the NMDA type are an additional pathway of Pb(2+) uptake. Pb(2+) caused a time-, dose- and stimulus-dependent saturation of the dye, whose intracellular concentration is approximately 10 microM, indicating that intracellular Pb(2+) can readily reach a concentration in the micromolar range. These results indicate that the particular vulnerability of neurones to Pb(2+) poisoning is linked to the presence of specific transport systems, which mediate the rapid uptake of Pb(2+) into the neurone.

  18. Cerebellar cortical neuron responses evoked from the spinal border cell tract.

    PubMed

    Geborek, Pontus; Spanne, Anton; Bengtsson, Fredrik; Jörntell, Henrik

    2013-01-01

    Spinocerebellar systems are likely to be crucial for cerebellar hallmark functions such as coordination. However, in terms of cerebellar functional analyses, these are perhaps among the least explored systems. The aim of the present study is to achieve activation of a single component of the spinocerebellar systems and to explore to what extent it can influence the spike output of granule cells, Golgi cells, molecular layer (ML) interneurons (stellate and basket cells) and Purkinje cells (PCs). For this purpose, we took advantage of a unique arrangement discovered in neuroanatomical studies, in which the spinal border cell (SBC) component of the ventral spinocerebellar system was found to be the only spinocerebellar tract which ascends in the contralateral lateral funiculus (coLF) and have terminations in sublobulus C1 of the paramedian lobule in the posterior cerebellum. Using electrical stimulation of this tract, we find a subset of the cerebellar cortical neurons in this region to be moderately or powerfully activated. For example, some of our granule cells displayed high intensity responses whereas the majority of the granule cells displayed no response at all. The finding that more than half of the PCs were activated by stimulation of the SBC tract indicated that this system is capable of directly influencing cerebellar cortical output. The implications of these findings for the view of the integrative functions of the cerebellar cortex are discussed.

  19. 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.

  20. Stimulation of kainate toxicity by zinc in cultured cerebellar granule neurons and the role of mitochondria in this process.

    PubMed

    Lozier, Ekaterina R; Stelmashook, Elena V; Uzbekov, Rustem E; Novikova, Svetlana V; Zorov, Savva D; Alieva, Irina B; Arbeille, Brigitte; Zorov, Dmitry B; Isaev, Nickolay K

    2012-01-05

    Zinc chloride (0.01 mM kept for 3h) is not toxic to cultured cerebellar granule neurons (CGNs) while kainate (0.1mM kept for 3h) demonstrates some but very low toxicity towards these cells. Measurements of the relative intraneuronal zinc ion concentration showed that increase in [Zn(2+)](i) under the simultaneous action of ZnCl(2) and kainate was significantly stronger compared to their separate action. Simultaneous treatment of CGNs with kainate and zinc chloride caused the swelling of neuronal mitochondria and consequent intensive neuronal death, which was totally prevented by NBQX (an AMPA/kainate-receptors blocker) or ruthenium red (a mitochondrial Ca(2+) uniporter blocker). These data imply that Zn(2+) synergistically to kainate increase their separate toxic effects on mitochondria leading to rapid neuronal death. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. Exposure to 50 Hz electromagnetic radiation promote early maturation and differentiation in newborn rat cerebellar granule neurons.

    PubMed

    Lisi, A; Ciotti, M T; Ledda, M; Pieri, M; Zona, C; Mercanti, D; Rieti, S; Giuliani, L; Grimaldi, S

    2005-08-01

    The wish of this work is the study of the effect of electromagnetic (EMF) radiations at a frequency of 50 Hz on the development of cerebellar granule neurons (CGN). Granule neurons, prepared from newborn rat cerebellum (8 days after birth), were cultured after plate-seeding in the presence of EMF radiations, with the plan of characterizing their cellular and molecular biochemistry, after exposure to the electromagnetic stimulus. Five days challenge to EMF radiations showed, by the cytotoxic glutamate (Glu) pulse test, a 30% decrease of cells survival, while only 5% of mortality was reported for unexposed sample. Moreover, blocking the glutamate receptor (GluR) with the Glu competitor MK-801, no toxicity effect after CGN challenge to EMF radiations and Glu was detected. By patch-clamp recording technique, the Kainate-induced currents from 6 days old exposed CGN exhibited a significant increase with respect to control cells. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses show that EMF exposure of rats CGN, induces a change in both GluRs proteins and mRNAs expression with respect to control. In addition, the use of monoclonal antibody raised against neurofilament protein (NF-200) reveals an increase in NF-200 synthesis in the exposed CGN. All these results indicate that exposure to non-ionizing radiations contribute to a premature expression of GluRs reducing the life span of CGN, leading to a more rapid cell maturation. (c) 2005 Wiley-Liss, Inc.

  2. Identification of an inhibitory circuit that regulates cerebellar Golgi cell activity.

    PubMed

    Hull, Court; Regehr, Wade G

    2012-01-12

    Here we provide evidence that revises the inhibitory circuit diagram of the cerebellar cortex. It was previously thought that Golgi cells, interneurons that are the sole source of inhibition onto granule cells, were exclusively coupled via gap junctions. Moreover, Golgi cells were believed to receive GABAergic inhibition from molecular layer interneurons (MLIs). Here we challenge these views by optogenetically activating the cerebellar circuitry to determine the timing and pharmacology of inhibition onto Golgi cells and by performing paired recordings to directly assess synaptic connectivity. In contrast to current thought, we find that Golgi cells, not MLIs, make inhibitory GABAergic synapses onto other Golgi cells. As a result, MLI feedback does not regulate the Golgi cell network, and Golgi cells are inhibited approximately 2 ms before Purkinje cells, following a mossy fiber input. Hence, Golgi cells and Purkinje cells receive unique sources of inhibition and can differentially process shared granule cell inputs. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity

    PubMed Central

    Ankri, Lea; Husson, Zoé; Pietrajtis, Katarzyna; Proville, Rémi; Léna, Clément; Yarom, Yosef; Dieudonné, Stéphane; Uusisaari, Marylka Yoe

    2015-01-01

    The cerebellum, a crucial center for motor coordination, is composed of a cortex and several nuclei. The main mode of interaction between these two parts is considered to be formed by the inhibitory control of the nuclei by cortical Purkinje neurons. We now amend this view by showing that inhibitory GABA-glycinergic neurons of the cerebellar nuclei (CN) project profusely into the cerebellar cortex, where they make synaptic contacts on a GABAergic subpopulation of cerebellar Golgi cells. These spontaneously firing Golgi cells are inhibited by optogenetic activation of the inhibitory nucleo-cortical fibers both in vitro and in vivo. Our data suggest that the CN may contribute to the functional recruitment of the cerebellar cortex by decreasing Golgi cell inhibition onto granule cells. DOI: http://dx.doi.org/10.7554/eLife.06262.001 PMID:25965178

  4. NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development.

    PubMed

    Sanchez-Ortiz, Efrain; Cho, Woosung; Nazarenko, Inga; Mo, Wei; Chen, Jian; Parada, Luis F

    2014-11-01

    Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders.

  5. Igf1 and Pacap rescue cerebellar granule neurons from apoptosis via a common transcriptional program

    PubMed Central

    Maino, B; D’Agata, V; Severini, C; Ciotti, MT; Calissano, P; Copani, A; Chang, Y-C; DeLisi, C; Cavallaro, S

    2015-01-01

    A shift of the delicate balance between apoptosis and survival-inducing signals determines the fate of neurons during the development of the central nervous system and its homeostasis throughout adulthood. Both pathways, promoting or protecting from apoptosis, trigger a transcriptional program. We conducted whole-genome expression profiling to decipher the transcriptional regulatory elements controlling the apoptotic/survival switch in cerebellar granule neurons following the induction of apoptosis by serum and potassium deprivation or their rescue by either insulin-like growth factor-1 (Igf1) or pituitary adenylyl cyclase-activating polypeptide (Pacap). Although depending on different upstream signaling pathways, the survival effects of Igf1 and Pacap converged into common transcriptional cascades, thus suggesting the existence of a general transcriptional program underlying neuronal survival. PMID:26941962

  6. Different Molecular Mechanisms Mediate Direct or Glia-Dependent Prion Protein Fragment 90-231 Neurotoxic Effects in Cerebellar Granule Neurons.

    PubMed

    Thellung, Stefano; Gatta, Elena; Pellistri, Francesca; Villa, Valentina; Corsaro, Alessandro; Nizzari, Mario; Robello, Mauro; Florio, Tullio

    2017-05-25

    Glia over-stimulation associates with amyloid deposition contributing to the progression of central nervous system neurodegenerative disorders. Here we analyze the molecular mechanisms mediating microglia-dependent neurotoxicity induced by prion protein (PrP)90-231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrP(Sc)). PrP90-231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca(++)] i increase. Indeed, while in "pure" cerebellar granule neuron cultures, PrP90-231 causes a delayed intracellular Ca(++) entry mediated by the activation of NMDA receptors; when neuron and glia are co-cultured, a transient increase of [Ca(++)] i occurs within seconds after treatment in both granule neurons and glial cells, then followed by a delayed and sustained [Ca(++)] i raise, associated with the induction of the expression of inducible nitric oxide synthase and phagocytic NADPH oxidase. [Ca(++)] i fast increase in neurons is dependent on the activation of multiple pathways since it is not only inhibited by the blockade of voltage-gated channel activity and NMDA receptors but also prevented by the inhibition of nitric oxide and PGE2 release from glial cells. Thus, Ca(++) homeostasis alteration, directly induced by PrP90-231 in cerebellar granule cells, requires the activation of NMDA receptors, but is greatly enhanced by soluble molecules released by activated glia. In glia-enriched cerebellar granule cultures, the activation of inducible nitric oxide (iNOS) and NADPH oxidase represents the main mechanism of toxicity since their pharmacological inhibition prevented PrP90-231 neurotoxicity, whereas NMDA blockade by D(-)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90-231 neurotoxicity. These data indicate that amyloidogenic peptides

  7. Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture.

    PubMed

    Jiajia, Lin; Shinghung, Mak; Jiacheng, Zheng; Jialing, Wang; Dilin, Xu; Shengquan, Hu; Zaijun, Zhang; Qinwen, Wang; Yifan, Han; Wei, Cui

    2017-05-10

    Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

  8. 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

  9. MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

    PubMed

    Constantin, Lena; Wainwright, Brandon J

    2015-12-01

    MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

  10. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    SciTech Connect

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

  11. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    DOE PAGES

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; ...

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

  12. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons.

    PubMed

    Trivedi, Niraj; Ramahi, Joseph S; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A; Solecki, David J

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. We show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. We propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

  13. Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.

    PubMed

    Holubowska, Anna; Mukherjee, Chaitali; Vadhvani, Mayur; Stegmüller, Judith

    2014-03-17

    Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

  14. Glucose deprivation stimulates Cu(2+) toxicity in cultured cerebellar granule neurons and Cu(2+)-dependent zinc release.

    PubMed

    Isaev, Nickolay K; Genrikhs, Elisaveta E; Aleksandrova, Olga P; Zelenova, Elena A; Stelmashook, Elena V

    2016-05-27

    Copper chloride (0.01mM, 2h) did not have significant influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution. However, CuCl2 caused severe neuronal damage by glucose deprivation (GD). The glutamate NMDA-receptors blocker MK-801 partially and antioxidant N-acetyl-l-cysteine (NAC) or Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) almost entirely protected CGNs from this toxic effect. Measurements of intracellular calcium ions using Fluo-4 AM, or zinc ions with FluoZin-3 AM demonstrated that 1 h-exposure to GD induced intensive increase of Fluo-4 but not FluoZin-3 fluorescence in neurons. The supplementation of solution with CuCl2 caused an increase of FluoZin-3, Fluo-4 and CellROX Green (reactive oxygen species probe) fluorescence by GD. The stimulation of Fluo-4 but not FluoZin-3 fluorescence by copper could be prevented partially by MK-801 and as well as CellROX Green fluorescence by NAC at GD. This data imply that during GD copper ions induce intense displacement zinc ions from intracellular stores, in addition free radical production, glutamate release and Ca(2+) overload of CGNs, that causes death of neurons as a result.

  15. Cerebellar Exposure to Cell-Free Hemoglobin Following Preterm Intraventricular Hemorrhage: Causal in Cerebellar Damage?

    PubMed

    Agyemang, Alex Adusei; Sveinsdóttir, Kristbjörg; Vallius, Suvi; Sveinsdóttir, Snjolaug; Bruschettini, Matteo; Romantsik, Olga; Hellström, Ann; Smith, Lois E H; Ohlsson, Lennart; Holmqvist, Bo; Gram, Magnus; Ley, David

    2017-06-10

    Decreased cerebellar volume is associated with intraventricular hemorrhage (IVH) in very preterm infants and may be a principal component in neurodevelopmental impairment. Cerebellar deposition of blood products from the subarachnoid space has been suggested as a causal mechanism in cerebellar underdevelopment following IVH. Using the preterm rabbit pup IVH model, we evaluated the effects of IVH induced at E29 (3 days prior to term) on cerebellar development at term-equivalent postnatal day 0 (P0), term-equivalent postnatal day 2 (P2), and term-equivalent postnatal day 5 (P5). Furthermore, the presence of cell-free hemoglobin (Hb) in cerebellar tissue was characterized, and cell-free Hb was evaluated as a causal factor in the development of cerebellar damage following preterm IVH. IVH was associated with a decreased proliferative (Ki67-positive) portion of the external granular layer (EGL), delayed Purkinje cell maturation, and activated microglia in the cerebellar white matter. In pups with IVH, immunolabeling of the cerebellum at P0 demonstrated a widespread presence of cell-free Hb, primarily distributed in the white matter and the molecular layer. Intraventricular injection of the Hb scavenger haptoglobin (Hp) resulted in a corresponding distribution of immunolabeled Hp in the cerebellum and a partial reversal of the damaging effects observed following IVH. The results suggest that cell-free Hb is causally involved in cerebellar damage following IVH and that blocking cell-free Hb may have protective effects.

  16. Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    PubMed Central

    Trivedi, Niraj; Stabley, Daniel R.; Cain, Blake; Howell, Danielle; Laumonnerie, Christophe; Ramahi, Joseph S.; Temirov, Jamshid; Kerekes, Ryan A.; Gordon-Weeks, Phillip R.; Solecki, David J.

    2017-01-01

    Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule–actomyosin crosstalk is required for a neuron's ‘two-stroke' nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule–actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbations of drebrin demonstrate that proximal leading process microtubule–actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule–actomyosin interfaces during neuronal differentiation. PMID:28230156

  17. Sigma-1 receptor enhances neurite elongation of cerebellar granule neurons via TrkB signaling.

    PubMed

    Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

    2013-01-01

    Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca(2+) signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB.

  18. Drebrin-mediated microtubule–actomyosin coupling steers cerebellar granule neuron nucleokinesis and migration pathway selection

    DOE PAGES

    Trivedi, Niraj; Stabley, Daniel R.; Cain, Blake; ...

    2017-02-23

    Neuronal migration from a germinal zone to a final laminar position is essential for the morphogenesis of neuronal circuits. While it is hypothesized that microtubule–actomyosin crosstalk is required for a neuron’s ‘two-stroke’ nucleokinesis cycle, the molecular mechanisms controlling such crosstalk are not defined. By using the drebrin microtubule–actin crosslinking protein as an entry point into the cerebellar granule neuron system in combination with super-resolution microscopy, we investigate how these cytoskeletal systems interface during migration. Lattice light-sheet and structured illumination microscopy reveal a proximal leading process nanoscale architecture wherein f-actin and drebrin intervene between microtubules and the plasma membrane. Functional perturbationsmore » of drebrin demonstrate that proximal leading process microtubule–actomyosin coupling steers the direction of centrosome and somal migration, as well as the switch from tangential to radial migration. Finally, the Siah2 E3 ubiquitin ligase antagonizes drebrin function, suggesting a model for control of the microtubule–actomyosin interfaces during neuronal differentiation.« less

  19. Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons.

    PubMed

    Kaasik, Allen; Safiulina, Dzhamilja; Choubey, Vinay; Kuum, Malle; Zharkovsky, Alexander; Veksler, Vladimir

    2007-11-09

    Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.

  20. Cesium chloride protects cerebellar granule neurons from apoptosis induced by low potassium.

    PubMed

    Zhong, Jin; Yao, Weiguo; Lee, Weihua

    2007-10-01

    Neuronal apoptosis plays a critical role in the pathogenesis of neurodegenerative disorders, and neuroprotective agents targeting apoptotic signaling could have therapeutic use. Here we report that cesium chloride, an alternative medicine in treating radiological poison and cancer, has neuroprotective actions. Serum and potassium deprivation induced cerebellar granule neurons to undergo apoptosis, which correlated with the activation of caspase-3. Cesium prevented both the activation of caspase-3 and neuronal apoptosis in a dose-dependent manner. Cesium at 8 mM increased the survival of neurons from 45 +/- 3% to 91 +/- 5% of control. Cesium's neuroprotection was not mediated by PI3/Akt or MAPK signaling pathways, since it was unable to activate either Akt or MAPK by phosphorylation. In addition, specific inhibitors of PI3 kinase and MAP kinase did not block cesium's neuroprotective effects. On the other hand, cesium inactivated GSK3beta by phosphorylation of serine-9 and GSK3beta-specific inhibitor SB415286 prevented neuronal apoptosis. These data indicate that cesium's neuroprotection is likely via inactivating GSK3beta. Furthermore, cesium also prevented H(2)O(2)-induced neuronal death (increased the survival of neurons from 72 +/- 4% to 89 +/- 3% of control). Given its relative safety and good penetration of the brain blood barrier, our findings support the potential therapeutic use of cesium in neurodegenerative diseases.

  1. Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

    PubMed Central

    Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

    2013-01-01

    Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca2+ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB. PMID:24116072

  2. 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.

  3. The cerebellar Golgi cell and spatiotemporal organization of granular layer activity

    PubMed Central

    D'Angelo, Egidio; Solinas, Sergio; Mapelli, Jonathan; Gandolfi, Daniela; Mapelli, Lisa; Prestori, Francesca

    2013-01-01

    The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through both feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of these neurons. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array (MEA) recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain, and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and duration of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research. PMID:23730271

  4. Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

    PubMed

    Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

    2004-09-30

    Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

  5. Voltage-dependent calcium signaling in rat cerebellar unipolar brush cells.

    PubMed

    Birnstiel, S; Slater, N T; McCrimmon, D R; Mugnaini, E; Hartell, N A

    2009-09-01

    Unipolar brush cells (UBCs) are a class of excitatory interneuron found in the granule cell layer of the vestibulocerebellum. Mossy fibers form excitatory inputs on to the paint brush shaped dendrioles in the form of giant, glutamatergic synapses, activation of which results in prolonged bursts of action potentials in the postsynaptic UBC. The axons of UBCs themselves form mossy fiber contacts with other UBCs and granule cells, forming an excitatory, intrinsic cerebellar network that has the capacity to synchronize and amplify mossy fiber inputs to potentially large populations of granule cells. In this paper, we demonstrate that UBCs in rat cerebellar slices express low voltage activated (LVA) fast-inactivating and high voltage activated (HVA) slowly inactivating calcium channels. LVA calcium currents are mediated by T-type calcium channels and they are associated with calcium increases in the dendrites and to a lesser extent the cell soma. HVA currents, mediated by L-type calcium channels, are slowly inactivating and they produce larger overall increases in intracellular calcium but with a similar distribution pattern. We review these observations alongside several recent papers that examine how intrinsic membrane properties influence UBCs firing patterns and we discuss how UBC signaling may affect downstream cerebellar processing.

  6. Hippocampal granule cells opt for early retirement.

    PubMed

    Alme, C B; Buzzetti, R A; Marrone, D F; Leutgeb, J K; Chawla, M K; Schaner, M J; Bohanick, J D; Khoboko, T; Leutgeb, S; Moser, E I; Moser, M-B; McNaughton, B L; Barnes, C A

    2010-10-01

    Increased excitability and plasticity of adult-generated hippocampal granule cells during a critical period suggests that they may "orthogonalize" memories according to time. One version of this "temporal tag" hypothesis suggests that young granule cells are particularly responsive during a specific time period after their genesis, allowing them to play a significant role in sculpting CA3 representations, after which they become much less responsive to any input. An alternative possibility is that the granule cells active during their window of increased plasticity, and excitability become selectively tuned to events that occurred during that time and participate in later reinstatement of those experiences, to the exclusion of other cells. To discriminate between these possibilities, rats were exposed to different environments at different times over many weeks, and cell activation was subsequently assessed during a single session in which all environments were revisited. Dispersing the initial experiences in time did not lead to the increase in total recruitment at reinstatement time predicted by the selective tuning hypothesis. The data indicate that, during a given time frame, only a very small number of granule cells participate in many experiences, with most not participating significantly in any. Based on these and previous data, the small excitable population of granule cells probably correspond to the most recently generated cells. It appears that, rather than contributing to the recollection of long past events, most granule cells, possibly 90-95%, are effectively "retired." If granule cells indeed sculpt CA3 representations (which remains to be shown), then a possible consequence of having a new set of granule cells participate when old memories are reinstated is that new representations of these experiences might be generated in CA3. Whatever the case, the present data may be interpreted to undermine the standard "orthogonalizer" theory of the role of

  7. Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit.

    PubMed

    Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

    2012-04-26

    How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.

  8. Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

    PubMed Central

    Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B.; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C.; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

    2012-01-01

    Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies. PMID:22542184

  9. Ras Protein Activation Is a Key Event in Activity-dependent Survival of Cerebellar Granule Neurons*

    PubMed Central

    Xifró, Xavier; Miñano-Molina, Alfredo J.; Saura, Carlos A.; Rodríguez-Álvarez, José

    2014-01-01

    Neuronal activity promotes the survival of cerebellar granule neurons (CGNs) during the postnatal development of cerebellum. CGNs that fail to receive excitatory inputs will die by apoptosis. This process could be mimicked in culture by exposing CGNs to either a physiological concentration of KCl (5 mm or K5) plus N-methyl-d-aspartate (NMDA) or to 25 mm KCl (K25). We have previously described that a 24-h exposure to NMDA (100 μm) or K25 at 2 days in vitro induced long term survival of CGNs in K5 conditions. Here we have studied the molecular mechanisms activated at 2 days in vitro in these conditions. First we showed that NMDA or K25 addition promoted a rapid stimulation of PI3K and a biphasic phosphorylation on Ser-473 of Akt, a PI3K substrate. Interestingly, we demonstrated that only the first wave of Akt phosphorylation is necessary for the NMDA- and K25-mediated survival. Additionally, we detected that both NMDA and K25 increased ERK activity with a similar time-course. Moreover, our results showed that NMDA-mediated activation of the small G-protein Ras is necessary for PI3K/Akt pathway activation, whereas Rap1 was involved in NMDA phosphorylation of ERK. On the other hand, Ras, but not Rap1, mediates K25 activation of PI3K/Akt and MEK/ERK pathways. Because neuroprotection by NMDA or K25 is mediated by Ras (and not by Rap1) activation, we propose that Ras stimulation is a crucial event in NMDA- and K25-mediated survival of CGNs through the activation of PI3K/Akt and MEK/ERK pathways. PMID:24523415

  10. Melissa officinalis Acidic Fraction Protects Cultured Cerebellar Granule Neurons Against Beta Amyloid-Induced Apoptosis and Oxidative Stress

    PubMed Central

    Soodi, Maliheh; Dashti, Abolfazl; Hajimehdipoor, Homa; Akbari, Shole; Ataei, Nasim

    2017-01-01

    Objective Extracellular deposition of the beta-amyloid (Aβ) peptide, which is the main finding in the pathophysiology of Alzheimer’s disease (AD), leads to oxidative damage and apoptosis in neurons. Melissa officinalis (M. officinalis) is a medicinal plant from the Lamiaceae family that has neuroprotective activity. In the present study we have investigated the protective effect of the acidic fraction of M. officinalis on Aβ-induced oxidative stress and apoptosis in cultured cerebellar granule neurons (CGN). Additionally, we investigated a possible role of the nicotinic receptor. Materials and Methods This study was an in vitro experimental study performed on mice cultured CGNs. CGNs were pre-incubated with different concentrations of the acidic fraction of M. officinalis for 24 hours, followed by incubation with Aβ for an additional 48 hours. CGNs were also pre-incubated with the acidic fraction of M. officinalis and mecamylamin, followed by incubation with Aβ. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay to measure cell viability. Acetylcholinesterase (AChE) activity, reactive oxygen species (ROS) production, lipidperoxidation, and caspase-3 activity were measured after incubation. Hochst/annexin Vfluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was performed to detect apoptotic cells. Results The acidic fraction could protect CGNs from Aβ-induced cytotoxicity. Mecamylamine did not abolish the protective effect of the acidic fraction. AChE activity, ROS production, lipid peroxidation, and caspase-3 activity increased after Aβ incubation. Preincubation with the acidic fraction of M. officinalis ameliorated these factors and decreased the number of apoptotic cells. Conclusion Our results indicated that the protective effect of the acidic fraction of M. officinalis was not mediated through nicotinic receptors. This fraction could protect CGNs through antioxidant and anti-apoptotic activities. PMID

  11. Ectopic Granule Cells of the Rat Dentate Gyrus

    PubMed Central

    Scharfman, Helen; Goodman, Jeffrey; McCloskey, Daniel

    2007-01-01

    Granule cells of the mammalian dentate gyrus normally form a discrete layer, and virtually all granule cells migrate to this location. Exceptional granule cells that are positioned incorrectly, in ‘ectopic’ locations, are rare. Although the characteristics of such ectopic granule cells appear similar in many respects to granule cells located in the granule cell layer, their rare occurrence has limited a full evaluation of their structure and function. More information about ectopic granule cells has been obtained by studying those that develop after experimental manipulations that increase their number. For example, after severe seizures, the number of ectopic granule cells located in the hilus increases dramatically. These experimentally induced ectopic granule cells may not be equivalent to normal ectopic granule cells necessarily, but the vastly increased numbers have allowed much more information to be obtained. Remarkably, the granule cells that are positioned ectopically develop intrinsic properties and an axonal projection that are similar to granule cells that are located normally, i.e., in the granule cell layer. However, dendritic structure and synaptic structure/function appear to differ. These studies have provided new insight into a rare type of granule cell in the dentate gyrus, and the plastic characteristics of dentate granule cells that appear to depend on the location of the cell body. PMID:17148946

  12. Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

    PubMed Central

    Le, Shoshona S.; Loucks, F. Alexandra; Udo, Hiroshi; Richardson-Burns, Sarah; Phelps, Reid A.; Bouchard, Ron J.; Barth, Holger; Aktories, Klaus; Tyler, Kenneth L.; Kandel, Eric R.; Heidenreich, Kim A.; Linseman, Daniel A.

    2008-01-01

    Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGDdependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis. PMID:16092944

  13. Postnatal dendritic morphogenesis of cerebellar basket and stellate cells in vitro.

    PubMed

    Spatkowski, Gabriele; Schilling, Karl

    2003-05-01

    Inhibitory interneurons in the molecular layer of the cerebellar cortex play an essential role in cerebellar physiology by providing feed-forward inhibition to efferent Purkinje cells. Morphologic characteristics have been utilized to classify these cells as either basket cells or stellate cells. Conflicting evidence exists as to whether these cells are of distinct lineage and develop by employing discrete genetic programs, or whether their characteristic morphologic differences result from external cues that they encounter only after they have settled in their final territory in the molecular layer. We used primary dissociated cerebellar cultures established from early postnatal mice to study dendritogenesis of basket/stellate cells, identified by immunostaining for parvalbumin, under experimentally controlled conditions. We find that the radial axonal orientation of stem dendrites is non-random, suggesting a cell-intrinsic component defining this morphologic trait. In contrast, the expanse and complexity of basket/stellate cell dendrites is modulated by the granule cell derived neurotrophin, BDNF. BDNF-induced morphogenetic effects decline with ongoing development. Overall, our data do not provide evidence for a distinct lineage or genetic makeup of cerebellar molecular layer inhibitory interneurons.

  14. The apical complex protein Pals1 is required to maintain cerebellar progenitor cells in a proliferative state.

    PubMed

    Park, Jun Young; Hughes, Lucinda J; Moon, Uk Yeol; Park, Raehee; Kim, Sang-Bae; Tran, Khoi; Lee, Ju-Seog; Cho, Seo-Hee; Kim, Seonhee

    2016-01-01

    Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.

  15. The apical complex protein Pals1 is required to maintain cerebellar progenitor cells in a proliferative state

    PubMed Central

    Park, Jun Young; Hughes, Lucinda J.; Moon, Uk Yeol; Park, Raehee; Kim, Sang-Bae; Tran, Khoi; Lee, Ju-Seog; Cho, Seo-Hee; Kim, Seonhee

    2016-01-01

    Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling. PMID:26657772

  16. Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM dyes.

    PubMed

    Cheung, Giselle; Cousin, Michael A

    2011-11-11

    obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable(11). Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)(12,13).

  17. GDF-15 enhances intracellular Ca2+ by increasing Cav1.3 expression in rat cerebellar granule neurons

    PubMed Central

    Lu, Jun-Mei; Wang, Chang-Ying; Hu, Changlong; Fang, Yan-Jia; Mei, Yan-Ai

    2016-01-01

    GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-β superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K+ currents and Kv2.1 α subunit expression through TβRII (TGF-β receptor II) to activate Src kinase and Akt/mTOR (mammalian target of rapamycin) signalling in rat CGNs (cerebellar granule neurons). In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca2+ concentration ([Ca2+]i) in response to membrane depolarization, as determined by Ca2+ imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca2+ current (ICa) without altering steady-state activation of Ca2+ channels. Treatment with nifedipine, an inhibitor of L-type Ca2+ channels, abrogated GDF-15-induced increases in [Ca2+]i and ICa. The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK (extracellular-signal-regulated kinase) pathways and by pharmacological inhibition of Src-mediated TβRII phosphorylation. Given that Cav1.3 is not only a channel for Ca2+ influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TβRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons. PMID:27114559

  18. Acute neonatal glucocorticoid exposure produces selective and rapid cerebellar neural progenitor cell apoptotic death.

    PubMed

    Noguchi, K K; Walls, K C; Wozniak, D F; Olney, J W; Roth, K A; Farber, N B

    2008-10-01

    There has been a growing controversy regarding the continued use of glucocorticoid therapy to treat respiratory dysfunction associated with prematurity, as mounting clinical evidence has shown neonatal exposure produces permanent neuromotor and cognitive deficits. Here we report that, during a selective neonatal window of vulnerability, a single glucocorticoid injection in the mouse produces rapid and selective apoptotic cell death of the proliferating neural progenitor cells in the cerebellar external granule layer and permanent reductions in neuronal cell counts of their progeny, the cerebellar internal granule layer neurons. Our estimates suggest that this mouse window of vulnerability would correspond in the human to a period extending from approximately 20 weeks gestation to 6.5 weeks after birth. This death pathway is critically regulated by the proapoptotic Bcl-2 family member Puma and is independent of p53 expression. These rodent data indicate that there exists a previously unknown window of vulnerability during which a single glucocorticoid exposure at clinically relevant doses can produce neural progenitor cell apoptosis and permanent cerebellar pathology that may be responsible for some of the iatrogenically induced neurodevelopmental abnormalities seen in children exposed to this drug. This vulnerability may be related to the physiological role of glucocorticoids in regulating programmed cell death in the mammalian cerebellum.

  19. The Cellular State Determines the Effect of Melatonin on the Survival of Mixed Cerebellar Cell Culture

    PubMed Central

    Franco, Daiane Gil; Markus, Regina P.

    2014-01-01

    The constitutive activation of nuclear factor-κB (NF-κB), a key transcription factor involved in neuroinflammation, is essential for the survival of neurons in situ and of cerebellar granule cells in culture. Melatonin is known to inhibit the activation of NF-κB and has a cytoprotective function. In this study, we evaluated whether the cytoprotective effect of melatonin depends on the state of activation of a mixed cerebellar culture that is composed predominantly of granule cells; we tested the effect of melatonin on cultured rat cerebellar cells stimulated or not with lipopolysaccharide (LPS). The addition of melatonin (0.1 nM–1 µM) reduced the survival of naïve cells while inhibiting LPS-induced cell death. Melatonin (100 nM) transiently (15 min) inhibited the nuclear translocation of both NF-κB dimers (p50/p50, p50/RelA) and, after 60 min, increased the activation of p50/RelA. Melatonin-induced p50/RelA activity in naïve cells resulted in the transcription of inducible nitric oxide synthase (iNOS) and the production of NO. Otherwise, in cultures treated with LPS, melatonin blocked the LPS-induced activation of p50/RelA and the reduction in p50/p50 levels and inhibited iNOS expression and NO synthesis. Therefore, melatonin in vehicle-treated cells induces cell death, while it protects against LPS-induced cytotoxicity. In summary, we confirmed that melatonin is a neuroprotective drug when cerebellar cells are challenged; however, melatonin can also lead to cell death when the normal balance of the NF-κB pathway is disturbed. Our data provide a mechanistic basis for understanding the influence of cell context on the final output response of melatonin. PMID:25184316

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

    PubMed

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

    2017-02-01

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

  1. Reactive oxygen species are related to ionic fluxes and volume decrease in apoptotic cerebellar granule neurons: role of NOX enzymes.

    PubMed

    Hernández-Enríquez, Berenice; Guemez-Gamboa, Alicia; Morán, Julio

    2011-05-01

    Reactive oxygen species (ROS) are produced early during apoptosis of cerebellar granule neurons induced by low potassium (K5) and staurosporine (Sts). In addition, K5 and Sts activate NADPH oxidases (NOX). Recently, we described that K5 and Sts induce apoptotic volume decrease (AVD) at a time when ROS generation and NOX activity occur. In the present study, we evaluated the relationship between ROS generation and ionic fluxes during AVD. Here, we showed that K5- and Sts-induced AVD was inhibited by antioxidants and that direct ROS production induced AVD. Moreover, NOX inhibitors eliminated AVD induced by both K5 and Sts. Sts, but not K5, failed to induce AVD in cerebellar granule neurons from NOX2 knockout mice. These findings suggest that K5- and Sts-induced AVD is largely mediated by ROS produced by NOX. On the other hand, we also found that the blockage of ionic fluxes involved in AVD inhibited both ROS generation and NOX activity. These findings suggest that ROS generation and NOX activity are involved in ionic fluxes activation, which in turn could maintain ROS generation by activating NOX, leading to a self-amplifying cycle. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  2. Role of glutamate receptors in tetrabrominated diphenyl ether (BDE-47) neurotoxicity in mouse cerebellar granule neurons

    PubMed Central

    Costa, Lucio G.; Tagliaferri, Sara; Roqué, Pamela J.; Pellacani, Claudia

    2015-01-01

    The polybrominated diphenyl ether (PBDE) flame retardants are developmental neurotoxicants, as evidenced by numerous in vitro, animal and human studies. PBDEs can alter the homeostasis of thyroid hormone and directly interact with brain cells. Induction of oxidative stress, leading to DNA damage and apoptotic cell death is a prominent mechanism of PBDE neurotoxicity, though other mechanisms have also been suggested. In the present study we investigated the potential role played by glutamate receptors in the in vitro neurotoxicity of the tetrabromodiphenyl ether BDE-47, one of the most abundant PBDE congeners. Toxicity of BDE-47 in mouse cerebellar neurons was diminished by antagonists of glutamate ionotropic receptors, but not by antagonists of glutamate metabotropic receptors. Antagonists of NMDA and AMPA/Kainate receptors also inhibited BDE-47-induced oxidative stress and increases in intracellular calcium. The calcium chelator BAPTA-AM also inhibited BDE-47 cytotoxicity and oxidative stress. BDE-47 caused a rapid increase of extracellular glutamate levels, which was not antagonized by any of the compounds tested. The results suggest that BDE-47, by still unknown mechanisms, increases extracellular glutamate which in turn activates ionotropic glutamate receptors leading to increased calcium levels, oxidative stress, and ultimately cell death. PMID:26640238

  3. Low in situ expression of antioxidative enzymes in rat cerebellar granular cells susceptible to methylmercury.

    PubMed

    Fujimura, M; Usuki, F

    2014-01-01

    Methylmercury (MeHg), an environmental neurotoxicant, induces site-specific toxicity in the brain. Although oxidative stress has been demonstrated with MeHg toxicity, the site-specific toxicity is not completely understood. Among the cerebellar neurons, cerebellar granule cells (CGCs) appear vulnerable to MeHg, whereas Purkinje cells and molecular layer neurons are resistant. Here, we use a MeHg-intoxicated rat model to investigate these cerebellar neurons for the different causes of susceptibility to MeHg. Rats were exposed to 20 ppm MeHg for 4 weeks and subsequently exhibited neuropathological changes in the cerebellum that were similar to those observed in humans. We first isolated the three cerebellar neuron types using a microdissection system and then performed real-time PCR analyses for antioxidative enzymes. We observed that expression of manganese-superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GPx1), and thioredoxin reductase 1 (TRxR1) was significantly higher in Purkinje cells and molecular layer neurons than in CGCs. Finally, we performed immunohistochemical analyses on the cerebellum. Immunohistochemistry showed increased expression of Mn-SOD, GPx1, and TRxR1 in Purkinje cells and molecular layer neurons, which was coincident with the mRNA expression patterns. Considering Mn-SOD, GPx1, and TRxR1 are critical for protecting cells against MeHg intoxication, the results indicate that low expression of these antioxidative enzymes increases CGCs vulnerability to MeHg toxicity.

  4. Activity deprivation-dependent induction of the proapoptotic BH3-only protein Bim is independent of JNK/c-Jun activation during apoptosis in cerebellar granule neurons.

    PubMed

    Shi, Leyu; Gong, Shoufang; Yuan, Zhongmin; Ma, Chi; Liu, Yanling; Wang, Chuanfu; Li, Wenming; Pi, Rongbiao; Huang, Shoujian; Chen, Ruzhu; Han, Yifan; Mao, Zixu; Li, Mingtao

    2005-02-25

    Bcl-2-interacting mediator of cell death (Bim), a proapoptotic BH3-only protein, plays a critical role in neuronal apoptosis. Cerebellar granule neurons (CGNs) depend on activity for their survival and undergo apoptosis when deprived of depolarizing concentration of KCl. While it has been proposed that the activation of c-Jun NH2-terminal protein kinase (JNK)/c-Jun pathway contributes to the upregulation of bim gene in neurons subjected to survival signaling withdrawal, here we show that neither inhibition of JNK activity nor expression of dominant-negative c-Jun suppresses the expression of bim gene induced by activity deprivation in CGNs. We conclude that induction of bim gene is independent of the activation of JNK/c-Jun signaling pathway by activity deprivation during apoptosis of CGNs.

  5. Stimulation of high affinity gamma-aminobutyric acidB receptors potentiates the depolarization-induced increase of intraneuronal ionized calcium content in cerebellar granule neurons.

    PubMed

    De Erausquin, G; Brooker, G; Costa, E; Wojcik, W J

    1992-09-01

    In the treatment of spasticity, the therapeutic cerebrospinal fluid levels of (+/-)-baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, are below 1 microM. However, the mechanism of the therapeutic action of (+/-)-baclofen remains unknown, because, for the most part, the action of (+/-)-baclofen on GABAB receptors requires micromolar concentrations. Using fura-2 fluorescence microscopy, intracellular ionized calcium was measured in cerebellar granule neurons. Stimulation of a high affinity GABAB receptor potentiated by 2-3-fold the rise in intracellular calcium observed after depolarization of the cell with a Krebs Ringer's buffered solution containing 40 mM K+. Both GABA (100 nM) and (+/-)-baclofen (10-100 nM) stimulated this high affinity receptor. The potentiation of the depolarization-induced rise in intracellular calcium by (+/-)-baclofen (100 nM) was completely blocked by the GABAB receptor antagonist CGP 35348 (200 microM). Also, the intracellular calcium response induced by the activation of high affinity GABAB receptors was prevented by dantrolene (10 microM). The cerebellar granule neurons contained calcium-induced calcium release (CICR) stores. Caffeine (3 mM) and ryanodine (100 microM) potentiated the depolarization-induced rise in intracellular calcium, and this response to both drugs was blocked by dantrolene (10 microM). Because dantrolene does not prevent the rise in intracellular calcium after cell depolarization (this calcium originated from the influx of extracellular calcium), (+/-)-baclofen acting via the high affinity GABAB receptor indirectly activates the CICR stores, allowing the influx of extracellular calcium to trigger the release of calcium from these dantrolene-sensitive CICR stores. Thus, this high affinity GABAB receptor might become activated during persistent depolarization caused by pathological states and could be a mechanism to be studied for the therapeutic action of (+/-)-baclofen in spasticity.

  6. Vaccine adjuvants: Tailor-made mast-cell granules

    NASA Astrophysics Data System (ADS)

    Gunzer, Matthias

    2012-03-01

    Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.

  7. Developmental hypothyroxinemia and hypothyroidism reduce proliferation of cerebellar granule neuron precursors in rat offspring by downregulation of the sonic hedgehog signaling pathway.

    PubMed

    Wang, Yuan; Wang, Yi; Dong, Jing; Wei, Wei; Song, Binbin; Min, Hui; Yu, Ye; Lei, Xibing; Zhao, Ming; Teng, Weiping; Chen, Jie

    2014-06-01

    Iodine deficiency (ID)-induced hypothyroxinemia and hypothyroidism during development result in dysfunction of the central nervous system, affecting psychomotor and motor function, although the underlying mechanisms causing these alterations are still unclear. Therefore, our aim is to study the effects of developmental hypothyroxinemia, caused by mild ID, and developmental hypothyroidism, caused by severe ID or methimazole (MMZ), on the proliferation of cerebellar granule neuron precursors (CGNPs), an excellent experimental model of cerebellar development and function. The sonic hedgehog (Shh) signaling pathway is essential for CGNP proliferation, and as such, its activation is also investigated here. A maternal hypothyroxinemia model was established in Wistar rats by administrating a mild ID diet, and two maternal hypothyroidism models were developed either by administrating a severe ID diet or MMZ water. Our results showed that hypothyroxinemia and hypothyroidism reduced proliferation of CGNPs on postnatal day (PN) 7, PN14, and PN21. Accordingly, the mean intensity of proliferating cell nuclear antigen and Ki67 nuclear antigen immunofluorescence was reduced in the mild ID, severe ID, and MMZ groups. Moreover, maternal hypothyroxinemia and hypothyroidism reduced expression of the Shh signaling pathway on PN7, PN14, and PN21. Our study supports the hypothesis that developmental hypothyroxinemia induced by mild ID, and hypothyroidism induced by severe ID or MMZ, reduce the proliferation of CGNPs, which may be ascribed to the downregulation of the Shh signaling pathway.

  8. The compartmental restriction of cerebellar interneurons

    PubMed Central

    Consalez, G. Giacomo; Hawkes, Richard

    2013-01-01

    The Purkinje cells (PC's) of the cerebellar cortex are subdivided into multiple different molecular phenotypes that form an elaborate array of parasagittal stripes. This array serves as a scaffold around which afferent topography is organized. The ways in which cerebellar interneurons may be restricted by this scaffolding are less well-understood. This review begins with a brief survey of cerebellar topography. Next, it reviews the development of stripes in the cerebellum with a particular emphasis on the embryological origins of cerebellar interneurons. These data serve as a foundation to discuss the hypothesis that cerebellar compartment boundaries also restrict cerebellar interneurons, both excitatory [granule cells, unipolar brush cells (UBCs)] and inhibitory (e.g., Golgi cells, basket cells). Finally, it is proposed that the same PC scaffold that restricts afferent terminal fields to stripes may also act to organize cerebellar interneurons. PMID:23346049

  9. Changes in mitogen-activated protein kinase in cerebellar granule neurons by polybrominated diphenyl ethers and polychlorinated biphenyls

    SciTech Connect

    Fan Chunyang; Besas, Jonathan

    2010-05-15

    Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants and have been detected in human blood, adipose tissue, and breast milk. Both in vitro and in vivo studies have shown that the effects of PBDEs are similar to the known human developmental neurotoxicants such as polychlorinated biphenyls (PCBs) on a molar basis. Previously, we reported that PBDE mixtures and congeners, perturbed calcium homeostasis which is critical for the development and function of the nervous system. In the present study, we tested whether environmentally relevant PBDE/PCB mixtures and congeners affected mitogen-activated protein kinase (MAPK) pathways, which are down-stream events of calcium signaling in cerebellar granule neuronal cultures. In this study, phosphorylated extracellular signal-regulated kinase (pERK)1/2, a widely studied MAPK cascade and known to be involved in learning and memory, levels were quantitated using western blot technique with phospho-specific antibodies. Glutamate (a positive control) increased pERK1/2 in a time- and concentration-dependent manner reaching maximum activation at 5-30 min of exposure and at doses >= 10 muM. Both Aroclor 1254 (a commercial penta PCB mixture) and DE-71 (a commercial penta PBDE mixture) elevated phospho-ERK1/2, producing maximum stimulation at 30 min and at concentrations >= 3 mug/ml; Aroclor 1254 was more efficacious than DE-71. DE-79 (an octabrominated diphenyl ether mixture) also elevated phospho-ERK1/2, but to a lesser extent than that of DE-71. PBDE congeners 47, 77, 99, and 153 also increased phospo-ERK1/2 in a concentration-dependent manner. The data indicated that PBDE congeners are more potent than the commercial mixtures. PCB 47 also increased phospho-ERK1/2 like its structural analog PBDE 47, but to a lesser extent, suggesting that these chemicals affect similar pathways. Cytotoxicity, measured as %LDH release, data showed that higher concentrations (> 30 muM) and longer exposures (> 30 min) are

  10. Ethanol impairs Rho GTPase signaling and differentiation of cerebellar granule neurons in a rodent model of fetal alcohol syndrome.

    PubMed

    Joshi, S; Guleria, R S; Pan, J; Bayless, K J; Davis, G E; Dipette, D; Singh, U S

    2006-12-01

    Developmental exposure to ethanol impairs fetal brain development and causes fetal alcohol syndrome. Although the cerebellum is one of the most alcohol-sensitive brain areas, signaling mechanisms underlying the deleterious effects of ethanol on developing cerebellar granule neurons (CGNs) are largely unknown. Here we describe the effects of in vivo ethanol exposure on neurite formation in CGNs and on the activation of Rho GTPases (RhoA and Rac1), regulators of neurite formation. Exposure of 7-day-old rat pups to ethanol for 3 h moderately increased blood alcohol concentration (BAC) ( approximately 40 mM) and inhibited neurite formation and Rac1 activation in CGNs. Longer exposure to ethanol for 5 h resulted in higher BAC ( approximately 80 mM), induced apoptosis, inhibited Rac1, and activated RhoA. Studies demonstrated a regulatory role of Rho GTPases in differentiation of cerebellar neurons, and indicated that ethanol-associated impairment of Rho GTPase signaling might contribute to brain defects observed in fetal alcohol syndrome.

  11. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

    PubMed

    LaSarge, Candi L; Santos, Victor R; Danzer, Steve C

    2015-03-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

    PubMed Central

    LaSarge, Candi L.; Santos, Victor R; Danzer, Steve C.

    2015-01-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offset the reduction in boutons per axon length. These morphological changes predicts a net increase in granule cell >> CA3 innervation. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell >> CA3 communication. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. PMID:25600212

  13. Single Granule Cells Excite Golgi Cells and Evoke Feedback Inhibition in the Cochlear Nucleus

    PubMed Central

    Yaeger, Daniel B.

    2015-01-01

    In cerebellum-like circuits, synapses from thousands of granule cells converge onto principal cells. This fact, combined with theoretical considerations, has led to the concept that granule cells encode afferent input as a population and that spiking in individual granule cells is relatively unimportant. However, granule cells also provide excitatory input to Golgi cells, each of which provide inhibition to hundreds of granule cells. We investigated whether spiking in individual granule cells could recruit Golgi cells and thereby trigger widespread inhibition in slices of mouse cochlear nucleus. Using paired whole-cell patch-clamp recordings, trains of action potentials at 100 Hz in single granule cells was sufficient to evoke spikes in Golgi cells in ∼40% of paired granule-to-Golgi cell recordings. High-frequency spiking in single granule cells evoked IPSCs in ∼5% of neighboring granule cells, indicating that bursts of activity in single granule cells can recruit feedback inhibition from Golgi cells. Moreover, IPSPs mediated by single Golgi cell action potentials paused granule cell firing, suggesting that inhibitory events recruited by activity in single granule cells were able to control granule cell firing. These results suggest a previously unappreciated relationship between population coding and bursting in single granule cells by which spiking in a small number of granule cells may have an impact on the activity of a much larger number of granule cells. PMID:25788690

  14. Transplantation and Stem Cell Therapy for Cerebellar Degenerations.

    PubMed

    Cendelin, Jan

    2016-02-01

    Stem cell-based and regenerative therapy may become a hopeful treatment for neurodegenerative diseases including hereditary cerebellar degenerations. Neurotransplantation therapy mainly aims to substitute lost cells, but potential effects might include various mechanisms including nonspecific trophic effects and stimulation of endogenous regenerative processes and neural plasticity. Nevertheless, currently, there remain serious limitations. There is a wide spectrum of human hereditary cerebellar degenerations as well as numerous cerebellar mutant mouse strains that serve as models for the development of effective therapy. By now, transplantation has been shown to ameliorate cerebellar function, e.g. in Purkinje cell degeneration mice, Lurcher mutant mice and mouse models of spinocerebellar ataxia type 1 and type 2 and Niemann-Pick disease type C. Despite the lack of direct comparative studies, it appears that there might be differences in graft development and functioning between various types of cerebellar degeneration. Investigation of the relation of graft development to specific morphological, microvascular or biochemical features of the diseased host tissue in various cerebellar degenerations may help to identify factors determining the fate of grafted cells and potential of their functional integration.

  15. Cell death and neurodegeneration in the postnatal development of cerebellar vermis in normal and Reeler mice.

    PubMed

    Castagna, Claudia; Merighi, Adalberto; Lossi, Laura

    2016-09-01

    Programmed cell death (PCD) was demonstrated in neurons and glia in normal brain development, plasticity, and aging, but also in neurodegeneration. (Macro)autophagy, characterized by cytoplasmic vacuolization and activation of lysosomal hydrolases, and apoptosis, typically entailing cell shrinkage, chromatin and nuclear condensation, are the two more common forms of PCD. Their underlying intracellular pathways are partly shared and neurons can die following both modalities, according to the type of death-triggering stimulus. Reelin is an extracellular protein necessary for proper neuronal migration and brain lamination. In the mutant Reeler mouse, its absence causes neuronal mispositioning, with a notable degree of cerebellar hypoplasia that was tentatively related to an increase in PCD. We have carried out an ultrastructural analysis on the occurrence and type of postnatal PCD affecting the cerebellar neurons in normal and Reeler mice. In the forming cerebellar cortex, PCD took the form of apoptosis or autophagy and mainly affected the cerebellar granule cells (CGCs). Densities of apoptotic CGCs were comparable in both mouse strains at P0-P10, while, in mutants, they increased to become significantly higher at P15. In WT mice the density of autophagic neurons did not display statistically significant differences in the time interval examined in this study, whereas it was reduced in Reeler in the P0-P10 interval, but increased at P15. Besides CGCs, the Purkinje neurons also displayed autophagic features in both WT and Reeler mice. Therefore, cerebellar neurons undergo different types of PCD and a Reelin deficiency affects the type and degree of neuronal death during postnatal development of the cerebellum.

  16. Hyperpolarization induces a long-term increase in the spontaneous firing rate of cerebellar Golgi cells.

    PubMed

    Hull, Court A; Chu, YunXiang; Thanawala, Monica; Regehr, Wade G

    2013-04-03

    Golgi cells (GoCs) are inhibitory interneurons that influence the cerebellar cortical response to sensory input by regulating the excitability of the granule cell layer. While GoC inhibition is essential for normal motor coordination, little is known about the circuit dynamics that govern the activity of these cells. In particular, although GoC spontaneous spiking influences the extent of inhibition and gain throughout the granule cell layer, it is not known whether this spontaneous activity can be modulated in a long-term manner. Here we describe a form of long-term plasticity that regulates the spontaneous firing rate of GoCs in the rat cerebellar cortex. We find that membrane hyperpolarization, either by mGluR2 activation of potassium channels, or by somatic current injection, induces a long-lasting increase in GoC spontaneous firing. This spike rate plasticity appears to result from a strong reduction in the spike after hyperpolarization. Pharmacological manipulations suggest the involvement of calcium-calmodulin-dependent kinase II and calcium-activated potassium channels in mediating these firing rate increases. As a consequence of this plasticity, GoC spontaneous spiking is selectively enhanced, but the gain of evoked spiking is unaffected. Hence, this plasticity is well suited for selectively regulating the tonic output of GoCs rather than their sensory-evoked responses.

  17. Hyperpolarization induces a long-term increase in the spontaneous firing rate of cerebellar Golgi cells

    PubMed Central

    Hull, Court; Chu, YunXiang; Thanawala, Monica; Regehr, Wade G.

    2013-01-01

    Golgi cells (GoCs) are inhibitory interneurons that influence the cerebellar cortical response to sensory input by regulating the excitability of the granule cell layer. While GoC inhibition is essential for normal motor coordination, little is known about the circuit dynamics that govern the activity of these cells. In particular, while GoC spontaneous spiking influences the extent of inhibition and gain throughout the granule cell layer, it is not known whether this spontaneous activity can be modulated in a long-term manner. Here we describe a form of long-term plasticity that regulates the spontaneous firing rate of GoCs in the rat cerebellar cortex. We find that membrane hyperpolarization, either by mGluR2 activation of potassium channels, or by somatic current injection, induces a long-lasting increase in GoC spontaneous firing. This spike rate plasticity appears to result from a strong reduction in the spike afterhyperpolarization (AHP). Pharmacological manipulations suggest the involvement of calcium-calmodulin dependent kinase II (CaMKII) and calcium-activated potassium channels in mediating these firing rate increases. As a consequence of this plasticity, GoC spontaneous spiking is selectively enhanced, but the gain of evoked spiking is unaffected. Hence this plasticity is well-suited for selectively regulating the tonic output of GoCs rather than their sensory-evoked responses. PMID:23554471

  18. Early degeneration of the cerebellar cortex, particularly the granular cells.

    PubMed

    Bugiani, O; Berio, A; Di Stefano, A; Mangiante, G; Mancardi, G L; Leonardi, A

    1978-12-07

    An 8 month old infant, who died of severe gastroenteritis, presented a degeneration of the cerebellar cortex involving cells arising from the outer granular layer as well as Purkinje and Golgi II cells. Residual Purkinje cells showed vacuolar change of the cell body and dendritic abnormalities. Related lesions were atrophy of the inferior olives and degeneration of the mossy fibers.

  19. Rapid Signaling Actions of Environmental Estrogens in Developing Granule Cell Neurons Are Mediated by Estrogen Receptor β

    PubMed Central

    Le, Hoa H.; Belcher, Scott M.

    2010-01-01

    Estrogenic endocrine disrupting chemicals (EDCs) constitute a diverse group of man-made chemicals and natural compounds derived from plants and microbial metabolism. Estrogen-like actions are mediated via the nuclear hormone receptor activity of estrogen receptor (ER)α and ERβ and rapid regulation of intracellular signaling cascades. Previous study defined cerebellar granule cell neurons as estrogen responsive and that granule cell precursor viability was developmentally sensitive to estrogens. In this study experiments using Western blot analysis and pharmacological approaches have characterized the receptor and signaling modes of action of selective and nonselective estrogen ligands in developing cerebellar granule cells. Estrogen treatments were found to briefly increase ERK1/2-phosphorylation and then cause prolonged depression of ERK1/2 activity. The sensitivity of granule cell precursors to estrogen-induced cell death was found to require the integrated activation of membrane and intracellular ER signaling pathways. The sensitivity of granule cells to selective and nonselective ER agonists and a variety of estrogenic and nonestrogenic EDCs was also examined. The ERβ selective agonist DPN, but not the ERα selective agonist 4,4′,4′-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol or other ERα-specific ligands, stimulated cell death. Only EDCs with selective or nonselective ERβ activities like daidzein, equol, diethylstilbestrol, and bisphenol A were observed to induce E2-like neurotoxicity supporting the conclusion that estrogen sensitivity in granule cells is mediated via ERβ. The presented results also demonstrate the utility of estrogen sensitive developing granule cells as an in vitro assay for elucidating rapid estrogen-signaling mechanisms and to detect EDCs that act at ERβ to rapidly regulate intracellular signaling. PMID:20926581

  20. Robustness effect of gap junctions between Golgi cells on cerebellar cortex oscillations

    PubMed Central

    2011-01-01

    Background Previous one-dimensional network modeling of the cerebellar granular layer has been successfully linked with a range of cerebellar cortex oscillations observed in vivo. However, the recent discovery of gap junctions between Golgi cells (GoCs), which may cause oscillations by themselves, has raised the question of how gap-junction coupling affects GoC and granular-layer oscillations. To investigate this question, we developed a novel two-dimensional computational model of the GoC-granule cell (GC) circuit with and without gap junctions between GoCs. Results Isolated GoCs coupled by gap junctions had a strong tendency to generate spontaneous oscillations without affecting their mean firing frequencies in response to distributed mossy fiber input. Conversely, when GoCs were synaptically connected in the granular layer, gap junctions increased the power of the oscillations, but the oscillations were primarily driven by the synaptic feedback loop between GoCs and GCs, and the gap junctions did not change oscillation frequency or the mean firing rate of either GoCs or GCs. Conclusion Our modeling results suggest that gap junctions between GoCs increase the robustness of cerebellar cortex oscillations that are primarily driven by the feedback loop between GoCs and GCs. The robustness effect of gap junctions on synaptically driven oscillations observed in our model may be a general mechanism, also present in other regions of the brain. PMID:22330240

  1. 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

  2. Reactive Oxygen Species Evoked by Potassium Deprivation and Staurosporine Inactivate Akt and Induce the Expression of TXNIP in Cerebellar Granule Neurons

    PubMed Central

    2017-01-01

    The reactive oxygen species (ROS) play a critical role in neuronal apoptosis; however, the mechanisms are not well understood. It has been shown that thioredoxin-interacting protein (TXNIP) overexpression renders cells more susceptible to oxidative stress and promotes apoptosis and that the activation of PI3K/Akt pathway leads to a downregulation of TXNIP. Here, we evaluated the role of ROS in the regulation of Akt activity and the subsequent regulation of the TXNIP expression in a model of apoptotic death of cerebellar granule neurons (CGN). We observed that two apoptotic conditions that generate ROS at short times led to an increase in the expression of TXNIP in a time-dependent manner; antioxidants significantly reduced this expression. Also, H2O2 caused an increase in TXNIP expression. Moreover, apoptotic conditions induced inactivation of Akt in a time-dependent manner similar to TXNIP expression and H2O2 treatment led to Akt inactivation. Besides, the pharmacological inhibition of Akt increases TXNIP expression and induces CGN cell death. Together, these results suggest that ROS promote neuronal apoptosis through the Akt-TXNIP signaling pathway, supporting the idea that the PI3K/Akt pathway regulates the TXNIP expression. This study highlights the potential importance of this mechanism in neuronal death. PMID:28367274

  3. Increased excitability and altered action potential waveform in cerebellar granule neurons of the Ts65Dn mouse model of Down syndrome

    PubMed Central

    Usowicz, Maria M.; Garden, Claire L.P.

    2012-01-01

    Down syndrome (DS) is characterized by intellectual disability and impaired motor control. Lack of coordinated movement, poor balance, and unclear speech imply dysfunction of the cerebellum, which is known to be reduced in volume in DS. The principal cause of the smaller cerebellum is a diminished number of granule cells (GCs). These neurons form the ‘input layer’ of the cerebellar cortex, where sensorimotor information carried by incoming mossy fibers is transformed before it is conveyed to Purkinje cells and inhibitory interneurons. However, it is not known how processing of this information is affected in the hypogranular cerebellum that characterizes DS. Here we explore the possibility that the electrical properties of the surviving GCs are changed. We find that in the Ts65Dn mouse model of DS, GCs have a higher input resistance at voltages approaching the threshold for firing, which causes them to be more excitable. In addition, they fire narrower and larger amplitude action potentials. These subtly modified electrical properties may result in atypical transfer of information at the input layer of the cerebellum. PMID:22627164

  4. 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.

  5. 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

  6. Context-dependent toxicity of amyloid-β peptides on mouse cerebellar cells.

    PubMed

    Alasia, Silvia; Aimar, Patrizia; Merighi, Adalberto; Lossi, Laura

    2012-01-01

    Alzheimer's disease (AD) is the major cause of dementia in old people. AD pathology is characterized by amyloid-β (Aβ) deposits in several regions of the brain, and links have been hypothesized between Aβ toxicity and apoptosis. Cerebellar granule cells (CGCs) have been widely used as in vitro tools for molecular studies correlating apoptosis with AD, although the cerebellum is a relatively spared area of the brain in vivo. We have used mixed neuronal-glial cerebellar cultures (NGCCs) and organotypic cerebellar cultures (OCCs) obtained from postnatal mice to assess the toxic effect of the Aβ oligomer 1-40 (Aβ1-40) after propidium iodide uptake in vitro. Our results demonstrate that NGCCs, which are primarily composed of CGCs, are resistant to Aβ1-40 challenge (5-10 μM) when cultured in physiological (5 mM) extracellular KCl ([K+]e) concentrations, i.e., in a condition in which CGCs undergo full maturation. Conversely, when 10 μM Aβ1-40 is given to NGCCs cultured in elevated (25 mM) [K+]e (and thus maintained in an immature state), there is a statistically significant increase in cell death. Cell death is by apoptosis, as demonstrated by ultrastructural examination. OCCs are resistant to Aβ challenge in any of the conditions tested (variation of [K+]e, presence or absence of serum, or addition of the neprilysin blocker phosphoramidon). Altogether these observations lead us to conclude that cerebellar cells in an organotypic context may be less susceptible to damage by Aβ, raising the question whether isolated CGCs are a reliable assay in drug discovery studies of AD.

  7. NMDA-Receptors Are Involved in Cu2+/Paraquat-Induced Death of Cultured Cerebellar Granule Neurons.

    PubMed

    Stelmashook, E V; Genrikhs, E E; Aleksandrova, O P; Amelkina, G A; Zelenova, E A; Isaev, N K

    2016-08-01

    Rat cultured cerebellar granule neurons (CGNs) were not sensitive to CuCl2 (1-10 µM, 24 h), whereas paraquat (150 µM) decreased neuronal survival to 79 ± 3% of control level. Simultaneous treatment of CGNs with paraquat and CuCl2 (2, 5, or 10 µM Cu2+/paraquat) caused significant copper dose-dependent death, lowering their survival to 56 ± 4, 37 ± 3, or 16 ± 2%, respectively, and stimulating elevated production of free radicals in CGNs. Introduction of vitamin E, a non-competitive antagonist of NMDA subtype of glutamate receptors (MK-801), and also removal of glutamine from the incubation medium decreased toxicity of Cu2+/paraquat mixture. However, addition of Cu2+ into the incubation medium did not affect CGNs death caused by glutamate. These data emphasize that excessive copper in the brain may trigger oxidative stress, which in turn results in release of glutamate, overstimulation of glutamate receptors, and neuronal death.

  8. Growth hormone-releasing peptide-6 inhibits cerebellar cell death in aged rats.

    PubMed

    Pañeda, Covadonga; Arroba, Ana I; Frago, Laura M; Holm, Anne Mette; Rømer, John; Argente, Jesús; Chowen, Julie A

    2003-08-26

    Insulin-like growth factor (IGF)-I is essential for cerebellar granule neuron survival and a decline in IGF-I is implicated in various age-dependent processes. Here we show that IGF-I mRNA levels are decreased in the cerebellum of old rats compared with young rats and this was associated with increased cell death and activation of caspases 3 and 9. Growth hormone-releasing peptide (GHRP)-6, a synthetic ligand for the ghrelin receptor, increased IGF-I mRNA levels, decreased cell death and inhibited caspase 3 and 9 activation in the cerebellum of aged rats. These results suggest that increasing IGF-I expression in the cerebellum can decrease cell death in aged rats via inhibition of caspase 3 and 9 activation.

  9. Role of the actin-binding protein profilin1 in radial migration and glial cell adhesion of granule neurons in the cerebellum

    PubMed Central

    Rust, Marco B.; Kullmann, Jan A.; Witke, Walter

    2012-01-01

    Profilins are small G-actin-binding proteins essential for cytoskeletal dynamics. Of the four mammalian profilin isoforms, profilin1 shows a broad expression pattern, profilin2 is abundant in the brain, and profilin3 and profilin4 are restricted to the testis. In vitro studies on cancer and epithelial cell lines suggested a role for profilins in cell migration and cell-cell adhesion. Genetic studies in mice revealed the importance of profilin1 in neuronal migration, while profilin2 has apparently acquired a specific function in synaptic physiology. We recently reported a mouse mutant line lacking profilin1 in the brain; animals display morphological defects that are typical for impaired neuronal migration. We found that during cerebellar development, profilin1 is specifically required for radial migration and glial cell adhesion of granule neurons. Profilin1 mutants showed cerebellar hypoplasia and aberrant organization of cerebellar cortex layers, with ectopically arranged granule neurons. In this commentary, we briefly introduce the profilin family and summarize the current knowledge on profilin activity in cell migration and adhesion. Employing cerebellar granule cells as a model, we shed some light on the mechanisms by which profilin1 may control radial migration and glial cell adhesion. Finally, a potential implication of profilin1 in human developmental neuropathies is discussed. PMID:22647936

  10. Whole-Cell Properties of Cerebellar Nuclei Neurons In Vivo.

    PubMed

    Canto, Cathrin B; Witter, Laurens; De Zeeuw, Chris I

    2016-01-01

    Cerebellar nuclei neurons integrate sensorimotor information and form the final output of the cerebellum, projecting to premotor brainstem targets. This implies that, in contrast to specialized neurons and interneurons in cortical regions, neurons within the nuclei encode and integrate complex information that is most likely reflected in a large variation of intrinsic membrane properties and integrative capacities of individual neurons. Yet, whether this large variation in properties is reflected in a heterogeneous physiological cell population of cerebellar nuclei neurons with well or poorly defined cell types remains to be determined. Indeed, the cell electrophysiological properties of cerebellar nuclei neurons have been identified in vitro in young rodents, but whether these properties are similar to the in vivo adult situation has not been shown. In this comprehensive study we present and compare the in vivo properties of 144 cerebellar nuclei neurons in adult ketamine-xylazine anesthetized mice. We found regularly firing (N = 88) and spontaneously bursting (N = 56) neurons. Membrane-resistance, capacitance, spike half-width and firing frequency all widely varied as a continuum, ranging from 9.63 to 3352.1 MΩ, from 6.7 to 772.57 pF, from 0.178 to 1.98 ms, and from 0 to 176.6 Hz, respectively. At the same time, several of these parameters were correlated with each other. Capacitance decreased with membrane resistance (R2 = 0.12, P<0.001), intensity of rebound spiking increased with membrane resistance (for 100 ms duration R2 = 0.1503, P = 0.0011), membrane resistance decreased with membrane time constant (R2 = 0.045, P = 0.031) and increased with spike half-width (R2 = 0.023, P<0.001), while capacitance increased with firing frequency (R2 = 0.29, P<0.001). However, classes of neuron subtypes could not be identified using merely k-clustering of their intrinsic firing properties and/or integrative properties following activation of their Purkinje cell input

  11. Whole-Cell Properties of Cerebellar Nuclei Neurons In Vivo

    PubMed Central

    De Zeeuw, Chris I.

    2016-01-01

    Cerebellar nuclei neurons integrate sensorimotor information and form the final output of the cerebellum, projecting to premotor brainstem targets. This implies that, in contrast to specialized neurons and interneurons in cortical regions, neurons within the nuclei encode and integrate complex information that is most likely reflected in a large variation of intrinsic membrane properties and integrative capacities of individual neurons. Yet, whether this large variation in properties is reflected in a heterogeneous physiological cell population of cerebellar nuclei neurons with well or poorly defined cell types remains to be determined. Indeed, the cell electrophysiological properties of cerebellar nuclei neurons have been identified in vitro in young rodents, but whether these properties are similar to the in vivo adult situation has not been shown. In this comprehensive study we present and compare the in vivo properties of 144 cerebellar nuclei neurons in adult ketamine-xylazine anesthetized mice. We found regularly firing (N = 88) and spontaneously bursting (N = 56) neurons. Membrane-resistance, capacitance, spike half-width and firing frequency all widely varied as a continuum, ranging from 9.63 to 3352.1 MΩ, from 6.7 to 772.57 pF, from 0.178 to 1.98 ms, and from 0 to 176.6 Hz, respectively. At the same time, several of these parameters were correlated with each other. Capacitance decreased with membrane resistance (R2 = 0.12, P<0.001), intensity of rebound spiking increased with membrane resistance (for 100 ms duration R2 = 0.1503, P = 0.0011), membrane resistance decreased with membrane time constant (R2 = 0.045, P = 0.031) and increased with spike half-width (R2 = 0.023, P<0.001), while capacitance increased with firing frequency (R2 = 0.29, P<0.001). However, classes of neuron subtypes could not be identified using merely k-clustering of their intrinsic firing properties and/or integrative properties following activation of their Purkinje cell input

  12. Monoaminergic modulation of GABAergic transmission onto cerebellar globular cells.

    PubMed

    Hirono, Moritoshi; Nagao, Soichi; Yanagawa, Yuchio; Konishi, Shiro

    2017-03-11

    Cerebellar Purkinje cells (PCs) project their axon collaterals to underneath of the PC layer and make GABAergic synaptic contacts with globular cells, a subgroup of Lugaro cells. GABAergic transmission derived from the PC axon collaterals is so powerful that it could inhibit globular cells and regulate their firing patterns. However, the physiological properties and implications of the GABAergic synapses on globular cells remain unknown. Using whole-cell patch-clamp recordings from globular cells in the mouse cerebellum, we examined the monoaminergic modulation of GABAergic inputs to these cells. Application of either serotonin (5-HT) or noradrenaline (NA) excited globular cells, thereby leading to their firing. The 5-HT- and NA-induced firing was temporally confined and attenuated by GABAergic transmission, although 5-HT and NA exerted an inhibitory effect on the release of GABA from presynaptic terminals of PC axon collaterals. Agonists for 5-HT1B receptors and α2-adrenoceptors mimicked the 5-HT- and NA-induced suppression of GABAergic activity. Through their differential modulatory actions on the cerebellar inhibitory neural circuits, 5-HT facilitated PC firing, whereas NA suppressed it. These results indicate that 5-HT and NA regulate the membrane excitability of globular cells and PCs through their differential modulation of not only the membrane potential but also GABAergic synaptic circuits. Monoaminergic modulation of the neural connections between globular cells and PCs could play a role in cerebellar motor coordination.

  13. WNT/β-catenin pathway activation in Myc immortalised cerebellar progenitor cells inhibits neuronal differentiation and generates tumours resembling medulloblastoma.

    PubMed

    Rogers, H A; Sousa, S; Salto, C; Arenas, E; Coyle, B; Grundy, R G

    2012-09-25

    Medulloblastoma is the most common malignant childhood brain tumour. Aberrant activation of the WNT/β-catenin pathway occurs in approximately 25% of medulloblastomas. However, its role in medulloblastoma pathogenesis is not understood. We have developed a model of WNT/β-catenin pathway-activated medulloblastoma. Pathway activation was induced in a Myc immortalised cerebellar progenitor cell line through stable expression of Wnt1. In vitro and in vivo analysis was undertaken to understand the effect of pathway activation and identify the potential cell of origin. Tumours that histologically resembled classical medulloblastoma formed in vivo using cells overexpressing Wnt1, but not with the control cell line. Wnt1 overexpression inhibited neuronal differentiation in vitro, suggesting WNT/β-catenin pathway activation prevents cells terminally differentiating, maintaining them in a more 'stem-like' state. Analysis of cerebellar progenitor cell markers demonstrated the cell line resembled cells from the cerebellar ventricular zone. We have developed a cell line with the means of orthotopically modelling WNT/β-catenin pathway-activated medulloblastoma. We provide evidence of the role pathway activation is playing in tumour pathogenesis and suggest medulloblastomas can arise from cells other than granule cell progenitors. This cell line is a valuable resource to further understand the role of pathway activation in tumorigenesis and for investigation of targeted therapies.

  14. BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis

    PubMed Central

    Pediaditakis, Iosif; Kourgiantaki, Alexandra; Prousis, Kyriakos C.; Potamitis, Constantinos; Xanthopoulos, Kleanthis P.; Zervou, Maria; Calogeropoulou, Theodora; Charalampopoulos, Ioannis; Gravanis, Achille

    2016-01-01

    Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma. PMID:28082899

  15. 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.

  16. 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.

  17. Brain-derived neurotrophic factor (BDNF) ameliorates the suppression of thyroid hormone-induced granule cell neurite extension by hexabromocyclododecane (HBCD).

    PubMed

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

    2011-04-08

    Thyroid hormone (TH) plays an essential role in growth and differentiation of the central nervous system. Deficiency of TH during perinatal period results in abnormal brain development known as cretinism in human. We recently reported that an environmental chemical 1,2,5,6,9,10-α-hexabromocyclododecane (HBCD) suppressed TH receptor (TR)-mediated transcription. To examine the effect of HBCD on cerebellar granule cells, we used purified rat cerebellar granule cells in reaggregate culture. Low dose HBCD (10(-10)M) significantly suppressed TH-induced neurite extension of granule cell aggregate. To clarify further the mechanisms of such suppression, we added brain-derived neurotrophic factor (BDNF) into culture medium, since BDNF plays a critical role in promoting granule cell development and is regulated by TH. BDNF completely rescued HBCD-induced suppression of granule cell neurite extension in the presence of T3. These results indicate that HBCD may disrupt TH-mediated brain development at least in part due to a disruption of the T3 stimulated increase in BDNF and BDNF may possess ability to ameliorate the effect of HBCD in granule cells.

  18. Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells

    PubMed Central

    Ferretti, Elisabetta; De Smaele, Enrico; Miele, Evelina; Laneve, Pietro; Po, Agnese; Pelloni, Marianna; Paganelli, Arianna; Di Marcotullio, Lucia; Caffarelli, Elisa; Screpanti, Isabella; Bozzoni, Irene; Gulino, Alberto

    2008-01-01

    MicroRNAs (miRNA) are crucial post-transcriptional regulators of gene expression and control cell differentiation and proliferation. However, little is known about their targeting of specific developmental pathways. Hedgehog (Hh) signalling controls cerebellar granule cell progenitor development and a subversion of this pathway leads to neoplastic transformation into medulloblastoma (MB). Using a miRNA high-throughput profile screening, we identify here a downregulated miRNA signature in human MBs with high Hh signalling. Specifically, we identify miR-125b and miR-326 as suppressors of the pathway activator Smoothened together with miR-324-5p, which also targets the downstream transcription factor Gli1. Downregulation of these miRNAs allows high levels of Hh-dependent gene expression leading to tumour cell proliferation. Interestingly, the downregulation of miR-324-5p is genetically determined by MB-associated deletion of chromosome 17p. We also report that whereas miRNA expression is downregulated in cerebellar neuronal progenitors, it increases alongside differentiation, thereby allowing cell maturation and growth inhibition. These findings identify a novel regulatory circuitry of the Hh signalling and suggest that misregulation of specific miRNAs, leading to its aberrant activation, sustain cancer development. PMID:18756266

  19. Mechanisms of synchronous activity in cerebellar Purkinje cells

    PubMed Central

    Wise, Andrew K; Cerminara, Nadia L; Marple-Horvat, Dilwyn E; Apps, Richard

    2010-01-01

    Complex spike synchrony is thought to be a key feature of how inferior olive climbing fibre afferents make their vital contribution to cerebellar function. However, little is known about whether the other major cerebellar input, the mossy fibres (which generate simple spikes within Purkinje cells, PCs), exhibit a similar synchrony in impulse timing. We have used a multi-microelectrode system to record simultaneously from two or more PCs in the posterior lobe of the ketamine/xylazine-anaesthetized rat to examine the relationship between complex spike and simple spike synchrony in PC pairs located mainly in the A2 and C1 zones in crus II and the paramedian lobule. PC pairs displaying correlations in the occurrence of their complex spikes (coupled PCs) were usually located in the same zone and were also more likely to exhibit correlations in the timing of their spontaneous simple spikes and associated pauses in activity. In coupled PCs, synchrony in both complex spike and simple spike activity was enhanced and the relative timing in the occurrence of complex spikes could be altered by peripheral stimulation. We conclude that the functional coupling between PC pairs in their complex spike and simple spike activity can be significantly modified by sensory inputs, and that mechanisms besides electrotonic coupling are involved in generating PC synchrony. Synchronous activity in multiple PCs converging onto the same cerebellar nuclear cells is likely to have a significant impact on cerebellar output that could form important timing signals to orchestrate coordinated movements. PMID:20442262

  20. Functional role of RNA polymerase II and P70 S6 kinase in KCl withdrawal-induced cerebellar granule neuron apoptosis.

    PubMed

    Padmanabhan, Jaya; Brown, Kristy R; Padilla, Amelia; Shelanski, Michael L

    2015-02-27

    KCl withdrawal-induced apoptosis in cerebellar granule neurons is associated with aberrant cell cycle activation, and treatment with cyclin-dependent kinase (Cdk) inhibitors protects cells from undergoing apoptosis. Because the Cdk inhibitor flavopiridol is known to inhibit RNA polymerase II (Pol II)-dependent transcription elongation by inhibiting the positive transcription elongation factor b (P-TEFb, a complex of CDK9 and cyclin T), we examined whether inhibition of RNA Pol II protects neurons from apoptosis. Treatment of neurons with 5, 6-dichloro-1-β-D-ribobenzimidazole (DRB), an RNA Pol II-dependent transcription elongation inhibitor, and flavopiridol inhibited phosphorylation and activation of Pol II and protected neurons from undergoing apoptosis. In addition to Pol II, neurons subjected to KCl withdrawal showed increased phosphorylation and activation of p70 S6 kinase, which was inhibited by both DRB and flavopiridol. Immunostaining analysis of the neurons deprived of KCl showed increased nuclear levels of phospho-p70 S6 kinase, and neurons protected with DRB and flavopiridol showed accumulation of the kinase into large spliceosome assembly factor-positive speckle domains within the nuclei. The formation of these foci corresponded with cell survival, and removal of the inhibitors resulted in dispersal of the speckles into smaller foci with subsequent apoptosis induction. Because p70 S6 kinase is known to induce translation of mRNAs containing a 5'-terminal oligopyrimidine tract, our data suggest that transcription and translation of this subset of mRNAs may contribute to KCl withdrawal-induced apoptosis in neurons.

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

    PubMed

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

    2015-11-01

    The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. © 2015 Wiley Periodicals, Inc.

  2. Mitigation of cerebellar neuropathy in globoid cell leukodystrophy mice by AAV-mediated gene therapy.

    PubMed

    Lin, Dar-Shong; Hsiao, Chung-Der; Lee, Allan Yueh-Luen; Ho, Che-Sheng; Liu, Hsuan-Liang; Wang, Tuen-Jen; Jian, Yuan-Ren; Hsu, Jui-Cheng; Huang, Zon-Darr; Lee, Tsung-Han; Chiang, Ming-Fu

    2015-10-15

    Globoid cell leukodystrophy (GLD) is an autosomal recessive, lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). The absence of GALC activity leads to the accumulation of the toxic substance psychosine and the preferential loss of myelinating cells in the central and peripheral nervous systems. Profound demyelination, astrogliosis and axonopathy are the hallmarks of the pathogenesis of GLD, and cerebellar ataxia is one of the dominant manifestations in adolescents and adults affected with GLD. To date, studies regarding cerebellar degeneration in GLD are limited. In this study, the efficacy of cerebellum-targeted gene therapy on the cerebellar neuropathology in twitcher mice (a murine model of GLD) has been validated. We observed degeneration of Purkinje cells, Bergmann glia, and granule cells in addition to astrocytosis and demyelination in the cerebellum of the twitcher mice. Ultrastructural analysis revealed dark cell degeneration and disintegration of the cellular composition of Purkinje cells in untreated twitcher mice. In addition, the expressions of neurotrophic factors CNTF, GDNF and IGF-I were up-regulated and the expression of BDNF was down-regulated. Intracerebellar-mediated gene therapy efficiently corrected enzymatic deficiency by direct transduction to Purkinje cells and cross-correction in other cell types in the cerebellum, leading to the amelioration of both neuroinflammation and demyelination. The population, dendritic territory, and axonal processes of Purkinje cells remained normal in the cerebellum of treated twitcher mice, where radial fibers of Bergmann glia spanned the molecular layer and collateral branches ensheathed the dendritic processes of Purkinje cells. Moreover, the aberrant expressions of neurotrophic factors were mitigated in the cerebellum of treated twitcher mice, indicating the preservation of cellular function in addition to maintaining the neuronal architecture. The life span of the

  3. Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1) in normal and transformed cerebellar cells

    PubMed Central

    Glassmann, Alexander; Molly, Sabine; Surchev, Lachezar; Nazwar, Tommy A; Holst, Martin; Hartmann, Wolfgang; Baader, Stephan L; Oberdick, John; Pietsch, Torsten; Schilling, Karl

    2007-01-01

    Background Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. Results During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. Conclusion Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons. PMID:17925019

  4. Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons

    PubMed Central

    Reddy, C E; Albanito, L; De Marco, P; Aiello, D; Maggiolini, M; Napoli, A; Musti, A M

    2013-01-01

    Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a

  5. Sonic hedgehog patterning during cerebellar development.

    PubMed

    De Luca, Annarita; Cerrato, Valentina; Fucà, Elisa; Parmigiani, Elena; Buffo, Annalisa; Leto, Ketty

    2016-01-01

    The morphogenic factor sonic hedgehog (Shh) actively orchestrates many aspects of cerebellar development and maturation. During embryogenesis, Shh signaling is active in the ventricular germinal zone (VZ) and represents an essential signal for proliferation of VZ-derived progenitors. Later, Shh secreted by Purkinje cells sustains the amplification of postnatal neurogenic niches: the external granular layer and the prospective white matter, where excitatory granule cells and inhibitory interneurons are produced, respectively. Moreover, Shh signaling affects Bergmann glial differentiation and promotes cerebellar foliation during development. Here we review the most relevant functions of Shh during cerebellar ontogenesis, underlying its role in physiological and pathological conditions.

  6. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control

    PubMed Central

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections). PMID:25983678

  7. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control.

    PubMed

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections).

  8. Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats

    PubMed Central

    González-Reyes, Susana; Guzmán-Beltrán, Silvia; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

    2013-01-01

    Curcumin is a bifunctional antioxidant derived from Curcuma longa. This study identifies curcumin as a neuroprotectant against hemin-induced damage in primary cultures of cerebellar granule neurons (CGNs) of rats. Hemin, the oxidized form of heme, is a highly reactive compound that induces cellular injury. Pretreatment of CGNs with 5–30 μM curcumin effectively increased by 2.3–4.9 fold heme oxygenase-1 (HO-1) expression and by 5.6–14.3-fold glutathione (GSH) levels. Moreover, 15 μM curcumin attenuated by 55% the increase in reactive oxygen species (ROS) production, by 94% the reduction of GSH/glutathione disulfide (GSSG) ratio, and by 49% the cell death induced by hemin. The inhibition of heme oxygenase system or GSH synthesis with tin mesoporphyrin and buthionine sulfoximine, respectively, suppressed the protective effect of curcumin against hemin-induced toxicity. These data strongly suggest that HO-1 and GSH play a major role in the protective effect of curcumin. Furthermore, it was found that 24 h of incubation with curcumin increases by 1.4-, 2.3-, and 5.2-fold the activity of glutathione reductase, glutathione S-transferase and superoxide dismutase, respectively. Additionally, it was found that curcumin was capable of inducing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation into the nucleus. These data suggest that the pretreatment with curcumin induces Nrf2 and an antioxidant response that may play an important role in the protective effect of this antioxidant against hemin-induced neuronal death. PMID:24454990

  9. The role of CD4-dependent signaling in interleukin-16 induced c-Fos expression and facilitation of neurite outgrowth in cerebellar granule neurons.

    PubMed

    Fenster, Catherine P; Chisnell, Hope K; Fry, Carl R; Fenster, Steven D

    2010-11-26

    Neuronal interleukin 16 (NIL-16) is the larger neural-specific splice variant of the interleukin-16 (IL16) gene and shows restricted expression to post-mitotic neurons of the mammalian hippocampus and cerebellum. Although the N-terminus of NIL-16 is unique to the neuronal variant, the C-terminus is identical to pro-IL-16, the IL-16 precursor expressed primarily in T-cells. IL-16 was originally described as a proinflammatory cytokine and has diverse immunoregulatory effects which involve signaling through CD4. NIL-16-expressing neurons can secrete IL-16 and may express CD4; moreover, treatment of cultured cerebellar granule neurons (CGCs) with IL-16 increases the expression of c-Fos, an immediate-early gene which transcriptionally regulates genes directing survival, proliferation, and growth. Taken together, we hypothesize that IL-16 functions as a neuroregulatory cytokine which signals through neuronal CD4 receptors. In this study, we investigated the role of CD4 in IL-16-induced c-Fos expression in CGCs, as well as the effects of IL-16 on neuronal survival and growth. We detected components involved in IL-16-signaling in lymphocytes, including CD4 and the associated tyrosine kinase p56(lck), in CGCs using qRT-PCR and immunoblotting. We also show that IL-16 induces c-Fos expression in wild-type CGCs, but not CD4-deficient CGCs or following inhibition of p56(lck). Finally, treatment of CGCs with IL-16 enhanced neurite outgrowth, an effect also observed in CD4-deficient CGCs. Taken together, our results indicate that IL-16-signaling affects neuronal gene expression and growth through CD4-dependent and independent pathways. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  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. 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. Neuroglobin Plays a Protective Role in Arsenite-Induced Cytotoxicity by Inhibition of Cdc42 and Rac1GTPases in Rat Cerebellar Granule Neurons.

    PubMed

    Liu, Xiaona; Gao, Yanhui; An, Yuan; Fu, Xiaoyan; Li, Yuanyuan; Sun, Dianjun; Wang, Jing

    2015-01-01

    We have previously shown that neuroglobin (Ngb) expression can be regulated by sodium arsenite (NaAsO2) exposure in rat cerebellar granule neurons (CGNs). However, the precise molecular mechanisms of Ngb action are largely unknown. Ras homolog (Rho) guanosine triphosphatases (Rho GTPases) are involved in the regulation of a number of cellular processes, including cell cytotoxicity. It has been reported that Ngb can act as a guanine nucleotide dissociation inhibitior (GDI) role to inactivate Rho GTPases. Therefore, we investigated Rho GTPases activation induced by NaAsO2 exposure in rat CGNs and effects of Rho GTPases activation on the cells. We also investigated the role of Ngb in this process. Primary cultures of CGNs were prepared from 7-day-old Wistar rat pups. The cytotoxic effects of NaAsO2 on CGNs were evaluated using the Cell Counting Kit-8 assay and TUNEL staining. RNA interference technology was used to silence Ngb, and the subsequent effects were evaluated by quantitative RT-PCR and Western blot. Cdc42 and Rac1 activation were measured by pull-down assay and Western blot. NaAsO2 induced cytotoxicity in rat CGNs, increased GTP-bound form of Cdc42 and Rac1 GTPases in the cells. Furthermore, inhibition of Cdc42 or Rac1 activity using the inhibitor ZCL278 or NSC23766 decreased apoptosis and increased cell viability in the cells exposed to NaAsO2. Using siRNA-mediated knockdown, we show that NaAsO2-induced cytotoxicity was exacerbated, activation of Cdc42 (GTP-Cdc42) and Rac1 (GTP-Rac1) was increased in Ngb RNA silencing cells. cytotoxic effects of NaAsO2 on rat CGNs is induced at least partly by Cdc42 and Rac1 activation, and Ngb can inhibit Cdc42 and Rac1 activation to play protective role in rat CGNs exposed to NaAsO2. © 2015 S. Karger AG, Basel.

  13. Regulation of Tlx3 by Pax6 is required for the restricted expression of Chrnα3 in Cerebellar Granule Neuron progenitors during development

    PubMed Central

    Divya, Thulasi Sheela; Lalitha, Soundararajan; Parvathy, Surendran; Subashini, Chandramohan; Sanalkumar, Rajendran; Dhanesh, Sivadasan Bindu; Rasheed, Vazhanthodi Abdul; Divya, Mundackal Sivaraman; Tole, Shubha; James, Jackson

    2016-01-01

    Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6−/−Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD). PMID:27452274

  14. 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.

  15. Gestational lead exposure induces developmental abnormalities and up-regulates apoptosis of fetal cerebellar cells in rats.

    PubMed

    Mousa, Alyaa M; Al-Fadhli, Ameera S; Rao, Muddanna S; Kilarkaje, Narayana

    2015-01-01

    Lead (Pb), a known environmental toxicant, adversely affects almost all organ systems. In this study, we investigated the effects of maternal lead exposure on fetal rat cerebellum. Female Sprague-Dawley rats were given lead nitrate in drinking water (0, 0.5, and 1%) for two weeks before conception, and during pregnancy. Fetuses were collected by caesarian section on gestational day 21 and observed for developmental abnormalities. The fetal cerebellar sections from control and 1% lead group were stained with cresyl violet. Immunohistochemical expressions of p53, Bax, Bcl-2, and caspase 3 were quantified by AnalySIS image analyzer (Life Science, Germany). Lead exposure induced developmental abnormalities of eyes, ear, limbs, neck and ventral abdominal wall; however, these abnormalities were commonly seen in the 1% lead-treated group. In addition, lead also caused fetal mortality and reduced body growth in both dose groups and reduced brain weight in the 1% lead-treated group. The fetal cerebella from the 1% lead-treated group showed unorganized cerebellar cortical layers, and degenerative changes in granule and Purkinje cells such as the formation of clumps of Nissl granules. An increase in Bax and caspase 3, and a decrease in Bcl-2 (p < 0.05), but not in p53, showed apoptosis of the neurons. In conclusion, gestational lead exposure in rats induces fetal toxicity and developmental abnormalities. The lead exposure also impairs development of cerebellar layers, induces structural changes, and apoptosis in the fetal cerebellar cortex. These results suggest that lead exposure during gestation is extremely toxic to developing cerebellum in rats.

  16. 2', 3'-cyclic nucleotide 3'-phosphodiesterase is expressed in dissociated rat cerebellar cells and included in the postsynaptic density fraction.

    PubMed

    Cho, Sun-Jung; Jung, Jae Seob; Jin, IngNyol; Moon, Il Soo

    2003-08-31

    We have shown by protein sequencing that the phosphotyrosine-containing 48 kDa protein band of the rat cerebellar postsynaptic density fraction (CBL-PSD) is 2', 3'-cyclic nucleotide 3'-phosphodiesterase 2 (CNP2). Immunoblot analysis indicated that both CNP1 and CNP2 isoforms are present in the CBL-PSD fraction, whereas there is little CNP2 in the forebrain (FB)-PSD fraction. Both isoforms in the CBL-PSD fraction were tyrosine-phosphorylated to a basal extent. They were efficiently dissociated from the complexes in the PSD fraction by salt, but not by non-ionic detergents such as n-octyl glucoside (OG) and Triton X-100. Immunocytochemistry of dissociated cerebellar cultures revealed patchy CNP staining in oligodendrocytes (OLs), Purkinje cells (PCs), and unidentified PSD95-positive cells, but no staining in granule cells (GCs). Our results indicate that both CNP1 and CNP2 are expressed in cerian populations of cerebellar cells in addition to OL, and that they are associated with complexes that are co-isolated with the PSD.

  17. Cerebellar Output in Zebrafish: An Analysis of Spatial Patterns and Topography in Eurydendroid Cell Projections

    PubMed Central

    Heap, Lucy A.; Goh, Chi Ching; Kassahn, Karin S.; Scott, Ethan K.

    2013-01-01

    The cerebellum is a brain region responsible for motor coordination and for refining motor programs. While a great deal is known about the structure and connectivity of the mammalian cerebellum, fundamental questions regarding its function in behavior remain unanswered. Recently, the zebrafish has emerged as a useful model organism for cerebellar studies, owing in part to the similarity in cerebellar circuits between zebrafish and mammals. While the cell types composing their cerebellar cortical circuits are generally conserved with mammals, zebrafish lack deep cerebellar nuclei, and instead a majority of cerebellar output comes from a single type of neuron: the eurydendroid cell. To describe spatial patterns of cerebellar output in zebrafish, we have used genetic techniques to label and trace eurydendroid cells individually and en masse. We have found that cerebellar output targets the thalamus and optic tectum, and have confirmed the presence of pre-synaptic terminals from eurydendroid cells in these structures using a synaptically targeted GFP. By observing individual eurydendroid cells, we have shown that different medial-lateral regions of the cerebellum have eurydendroid cells projecting to different targets. Finally, we found topographic organization in the connectivity between the cerebellum and the optic tectum, where more medial eurydendroid cells project to the rostral tectum while lateral cells project to the caudal tectum. These findings indicate that there is spatial logic underpinning cerebellar output in zebrafish with likely implications for cerebellar function. PMID:23554587

  18. Platelet Granule Exocytosis: A Comparison with Chromaffin Cells

    PubMed Central

    Fitch-Tewfik, Jennifer L.; Flaumenhaft, Robert

    2013-01-01

    The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli. PMID:23805129

  19. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development.

    PubMed

    Dengler, Christopher G; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A

    2017-02-20

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses.

  20. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development

    PubMed Central

    Dengler, Christopher G.; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A.

    2017-01-01

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses. PMID:28218241

  1. Resistance to kynurenic acid of the NMDA receptor-dependent toxicity of 3-nitropropionic acid and cyanide in cerebellar granule neurons.

    PubMed

    Fatokun, Amos A; Smith, Robert A; Stone, Trevor W

    2008-06-18

    During cerebral hypoxia or ischaemia, mitochondrial dysfunction is induced which can lead to free radical production and cell death. This phenomenon is mimicked by the acute administration of mitochondrial poisons such as 3-nitropropionic acid (3-NPA) and potassium cyanide (KCN), with the production of reactive molecular species secondary to the activation of glutamate receptors. Also during ischaemia, the kynurenine pathway of tryptophan metabolism is activated, leading to the production of quinolinic acid and kynurenic acid which can modulate N-methyl-D-aspartate (NMDA) receptors as agonist and antagonist respectively. Since kynurenic acid is known to be neuroprotective, we have now examined its ability to prevent the neurotoxic effects of mitochondrial dysfunction in primary cultures of postnatal rat cerebellar granule neurons. Viability was quantified using the Alamar Blue (AB) assay and by direct morphological examination. Both 3-NPA and KCN (10 microM-1 mM) reduced neuronal viability in a concentration-dependent manner. The NMDA receptor antagonists 2-amino-5-phosphonopentanoic acid (D-AP5) at a concentration of 50 microM, and a 10 microM dose of (+)-5-Methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801) prevented cell death, although the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at a concentration of 10 microM did not. The antioxidant enzymes catalase and superoxide dismutase, and the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) afforded partial protection. Kynurenic acid, a glutamate antagonist with preference for the glycine site of the NMDA receptors, had no protective effect at all against 3-NPA or KCN toxicity at concentrations up to 1 mM. Although these data confirm a major role for NMDA receptors and oxidative stress in the neurotoxic effects of mitochondrial inhibitors, they reveal a resistance to kynurenic acid which suggests a non

  2. Characterization of mast cell secretory granules and their cell biology.

    PubMed

    Azouz, Nurit Pereg; Hammel, Ilan; Sagi-Eisenberg, Ronit

    2014-10-01

    Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition.

  3. Primary cerebellar extramedullary myeloid cell tumor mimicking oligodendroglioma.

    PubMed

    Ho, D M; Wong, T T; Guo, W Y; Chang, K P; Yen, S H

    1997-10-01

    Extramedullary myeloid cell tumors (EMCTs) are tumors consisting of immature cells of the myeloid series that occur outside the bone marrow. Most of them are associated with acute myelogenous leukemia or other myeloproliferative disorders, and a small number occur as primary lesions, i.e., are not associated with hematological disorders. Occurrence inside the cranium is rare, and there has been only one case of primary EMCT involving the cerebellum reported in the literature. The case we report here is a blastic EMCT occurring in the cerebellum of a 3-year-old boy who had no signs of leukemia or any hematological disorder throughout the entire course. The cerebellar tumor was at first misdiagnosed as an "oligodendroglioma" because of the uniformity and "fried egg" artifact of the tumor cells. The tumor disappeared during chemotherapy consisting of 12 treatments. However, it recurred and metastasized to the cerebrospinal fluid (CSF) shortly after the therapy was completed. A diagnosis of EMCT was suspected because of the presence of immature myeloid cells in the CSF, and was confirmed by anti-myeloperoxidase and anti-lysozyme immunoreactivity of the cerebellar tumor. The patient succumbed 1 year and 3 months after the first presentation of the disease.

  4. Excitatory tonus is required for the survival of granule cell precursors during postnatal development within the cerebellum.

    PubMed

    Kanungo, A K; Liadis, N; Robertson, J; Woo, M; Henderson, J T

    2009-02-18

    In addition to protective effects within the adult central nervous system (CNS), in vivo application of N-methyl-d-aspartate inhibitors such as (+) MK-801 have been shown to induce neurodegeneration in neonatal rats over a specific developmental period. We have systematically mapped the nature and extent of MK-801-induced neurodegeneration throughout the neonatal murine brain in order to genetically dissect the mechanism of these effects. Highest levels of MK-801-induced neurodegeneration are seen in the cerebellar external germinal layer; while mature neurons of the internal granule layer are unaffected by MK-801 treatment. Examination of external germinal layer neurons by electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and bromodeoxyuridine (BrdU) labeling, and caspase-3 activation demonstrate that these neurons die through the process of programmed cell death soon after they exit from the cell cycle. Significantly, ablation of caspase-3 activity completely inhibited the MK-801-induced (and developmental) programmed cell death of external germinal layer neurons. Similar to caspase-3, inactivation of muscarinic acetylcholine receptors in vivo using scopolamine inhibited MK-801-induced programmed cell death. By contrast, the GABAergic agonist diazepam, either alone or in combination with MK-801, enhanced programmed cell death within external germinal layer neurons. These data demonstrate that, in vivo, cerebellar granule neurons undergo a dramatic change in intracellular signaling in response to molecules present in the local cellular milieu during their first 24 h following exit from the cell cycle.

  5. 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.

  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. 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

  8. Ionic mechanisms of autorhythmic firing in rat cerebellar Golgi cells

    PubMed Central

    Elisabetta Cesana, Lia Forti; Mapelli, Jonathan; D'Angelo, Egidio

    2006-01-01

    Although Golgi cells (GoCs), the main type of inhibitory interneuron in the cerebellar granular layer (GL), are thought to play a central role in cerebellar network function, their excitable properties have remained unexplored. GoCs fire rhythmically in vivo and in slices, but it was unclear whether this activity originated from pacemaker ionic mechanisms. We explored this issue in acute cerebellar slices from 3-week-old rats by combining loose cell-attached (LCA) and whole-cell (WC) recordings. GoCs displayed spontaneous firing at 1–10 Hz (room temperature) and 2–20 Hz (35–37°C), which persisted in the presence of blockers of fast synaptic receptors and mGluR and GABAB receptors, thus behaving, in our conditions, as pacemaker neurons. ZD 7288 (20 μm), a potent hyperpolarization-activated current (Ih) blocker, slowed down pacemaker frequency. The role of subthreshold Na+ currents (INa,sub) could not be tested directly, but we observed a robust TTX-sensitive, non-inactivating Na+ current in the subthreshold voltage range. When studying repolarizing currents, we found that retigabine (5 μm), an activator of KCNQ K+ channels generating neuronal M-type K+ (IM) currents, reduced GoC excitability in the threshold region. The KCNQ channel antagonist XE991 (5 μm) did not modify firing, suggesting that GoC IM has low XE991 sensitivity. Spike repolarization was followed by an after-hyperpolarization (AHP) supported by apamin-sensitive Ca2+-dependent K+ currents (Iapa). Block of Iapa decreased pacemaker precision without altering average frequency. We propose that feed-forward depolarization is sustained by Ih and INa,sub, and that delayed repolarizing feedback involves an IM-like current whose properties remain to be characterized. The multiple ionic mechanisms shown here to contribute to GoC pacemaking should provide the substrate for fine regulation of firing frequency and precision, thus influencing the cyclic inhibition exerted by GoCs onto the cerebellar GL

  9. 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

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

    ERIC Educational Resources Information Center

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

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

  11. 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.…

  12. Development and specification of cerebellar stem and progenitor cells in zebrafish: from embryo to adult

    PubMed Central

    2013-01-01

    Background Teleost fish display widespread post-embryonic neurogenesis originating from many different proliferative niches that are distributed along the brain axis. During the development of the central nervous system (CNS) different cell types are produced in a strict temporal order from increasingly committed progenitors. However, it is not known whether diverse neural stem and progenitor cell types with restricted potential or stem cells with broad potential are maintained in the teleost fish brain. Results To study the diversity and output of neural stem and progenitor cell populations in the zebrafish brain the cerebellum was used as a model brain region, because of its well-known architecture and development. Transgenic zebrafish lines, in vivo imaging and molecular markers were used to follow and quantify how the proliferative activity and output of cerebellar progenitor populations progress. This analysis revealed that the proliferative activity and progenitor marker expression declines in juvenile zebrafish before they reach sexual maturity. Furthermore, this correlated with the diminished repertoire of cell types produced in the adult. The stem and progenitor cells derived from the upper rhombic lip were maintained into adulthood and they actively produced granule cells. Ventricular zone derived progenitor cells were largely quiescent in the adult cerebellum and produced a very limited number of glia and inhibitory inter-neurons. No Purkinje or Eurydendroid cells were produced in fish older than 3 months. This suggests that cerebellar cell types are produced in a strict temporal order from distinct pools of increasingly committed stem and progenitor cells. Conclusions Our results in the zebrafish cerebellum show that neural stem and progenitor cell types are specified and they produce distinct cell lineages and sub-types of brain cells. We propose that only specific subtypes of brain cells are continuously produced throughout life in the teleost fish

  13. Development and specification of cerebellar stem and progenitor cells in zebrafish: from embryo to adult.

    PubMed

    Kaslin, Jan; Kroehne, Volker; Benato, Francesca; Argenton, Francesco; Brand, Michael

    2013-05-04

    Teleost fish display widespread post-embryonic neurogenesis originating from many different proliferative niches that are distributed along the brain axis. During the development of the central nervous system (CNS) different cell types are produced in a strict temporal order from increasingly committed progenitors. However, it is not known whether diverse neural stem and progenitor cell types with restricted potential or stem cells with broad potential are maintained in the teleost fish brain. To study the diversity and output of neural stem and progenitor cell populations in the zebrafish brain the cerebellum was used as a model brain region, because of its well-known architecture and development. Transgenic zebrafish lines, in vivo imaging and molecular markers were used to follow and quantify how the proliferative activity and output of cerebellar progenitor populations progress. This analysis revealed that the proliferative activity and progenitor marker expression declines in juvenile zebrafish before they reach sexual maturity. Furthermore, this correlated with the diminished repertoire of cell types produced in the adult. The stem and progenitor cells derived from the upper rhombic lip were maintained into adulthood and they actively produced granule cells. Ventricular zone derived progenitor cells were largely quiescent in the adult cerebellum and produced a very limited number of glia and inhibitory inter-neurons. No Purkinje or Eurydendroid cells were produced in fish older than 3 months. This suggests that cerebellar cell types are produced in a strict temporal order from distinct pools of increasingly committed stem and progenitor cells. Our results in the zebrafish cerebellum show that neural stem and progenitor cell types are specified and they produce distinct cell lineages and sub-types of brain cells. We propose that only specific subtypes of brain cells are continuously produced throughout life in the teleost fish brain. This implies that the post

  14. Optogenetic manipulation of cerebellar Purkinje cell activity in vivo.

    PubMed

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

    2011-01-01

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

  15. Neocortical networks entrain neuronal circuits in cerebellar cortex

    PubMed Central

    Roš, Hana; Sachdev, Robert N. S.; Yu, Yuguo; Šestan, Nenad; McCormick, David A.

    2011-01-01

    Activity in neocortex is often characterized by synchronized oscillations of neurons and networks, resulting in the generation of a local field potential and electroencephalogram. Do the neuronal networks of the cerebellum also generate synchronized oscillations and are they under the influence of those in the neocortex? Here we show that in the absence of any overt external stimulus, the cerebellar cortex generates a slow oscillation that is correlated with that of the neocortex. Disruption of the neocortical slow oscillation abolishes the cerebellar slow oscillation, whereas blocking cerebellar activity has no overt effect on the neocortex. We provide evidence that the cerebellar slow oscillation results in part from the activation of granule, Golgi, and Purkinje neurons. In particular, we show that granule and Golgi cells discharge trains of single spikes, and Purkinje cells generate complex spikes, during the Up state of the slow oscillation. Purkinje cell simple spiking is weakly related to the cerebellar and neocortical slow oscillation in a minority of cells. Our results indicate that the cerebellum generates rhythmic network activity that can be recorded as an LFP in the anesthetized animal, which is driven by synchronized oscillations of the neocortex. Furthermore, we show that correlations between neocortical and cerebellar LFPs persist in the awake animal, indicating that neocortical circuits modulate cerebellar neurons in a similar fashion in natural behavioral states. Thus, the projection neurons of the neocortex collectively exert a driving and modulatory influence on cerebellar network activity. PMID:19692605

  16. 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

  17. Excitation of Rat Cerebellar Golgi Cells by Ethanol: Further Characterization of the Mechanism

    PubMed Central

    Botta, Paolo; de Souza, Fabio M. Simões; Sangrey, Thomas; De Schutter, Erik; Valenzuela, C. Fernando

    2012-01-01

    Background Studies with rodents suggest that acute ethanol exposure impairs information flow through the cerebellar cortex, in part, by increasing GABAergic input to granule cells. Experiments suggest that an increase in the excitability of specialized GABAergic interneurons that regulate granule cell activity (i.e. Golgi cells, GoCs) contributes to this effect. In GoCs, ethanol increases spontaneous action potential firing frequency, decreased the afterhyperpolarization amplitude, and depolarized the membrane potential. Studies suggest that these effects could be mediated by inhibition of the Na+/K+ ATPase. The purpose of this study was to characterize the potential role of other GoC conductances in the mechanism of action of ethanol. Methods Computer modeling techniques and patch-clamp electrophysiological recordings with acute slices from rat cerebella were used for these studies. Results Computer modeling suggested that modulation of subthreshold Na+ channels, hyperpolarization activated currents and several K+ conductances could explain some but not all actions of ethanol on GoCs. Electrophysiological studies did not find evidence consistent with a contribution of these conductances. Quinidine, a non-selective blocker of several types of channels (including several K+ channels) that also antagonizes the Na+/K+ ATPase, reduced the effect of ethanol on GoC firing. Conclusions These findings lend further support to the conclusion that ethanol increases GoC excitability via modulation of the Na+/K+ ATPase, and suggest that a quinidine-sensitive K+ channel may also play a role in the mechanism of action of ethanol. PMID:22004123

  18. Long-Term Potentiation at the Mossy Fiber-Granule Cell Relay Invokes Postsynaptic Second-Messenger Regulation of Kv4 Channels.

    PubMed

    Rizwan, Arsalan P; Zhan, Xiaoqin; Zamponi, Gerald W; Turner, Ray W

    2016-11-02

    Mossy fiber afferents to cerebellar granule cells form the primary synaptic relay into cerebellum, providing an ideal site to process signal inputs differentially. Mossy fiber input is known to exhibit a long-term potentiation (LTP) of synaptic efficacy through a combination of presynaptic and postsynaptic mechanisms. However, the specific postsynaptic mechanisms contributing to LTP of mossy fiber input is unknown. The current study tested the hypothesis that LTP induces a change in intrinsic membrane excitability of rat cerebellar granule cells through modification of Kv4 A-type potassium channels. We found that theta-burst stimulation of mossy fiber input in lobule 9 granule cells lowered the current threshold to spike and increases the gain of spike firing by 2- to 3-fold. The change in postsynaptic excitability was traced to hyperpolarizing shifts in both the half-inactivation and half-activation potentials of Kv4 that occurred upon coactivating NMDAR and group I metabotropic glutamatergic receptors. The effects of theta-burst stimulation on Kv4 channel control of the gain of spike firing depended on a signaling cascade leading to extracellular signal-related kinase activation. Under physiological conditions, LTP of synaptically evoked spike output was expressed preferentially for short bursts characteristic of sensory input, helping to shape signal processing at the mossy fiber-granule cell relay.

  19. 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.

  20. Optogenetics in the cerebellum: Purkinje cell-specific approaches for understanding local cerebellar functions.

    PubMed

    Tsubota, Tadashi; Ohashi, Yohei; Tamura, Keita

    2013-10-15

    The cerebellum consists of the cerebellar cortex and the cerebellar nuclei. Although the basic neuronal circuitry of the cerebellar cortex is uniform everywhere, anatomical data demonstrate that the input and output relationships of the cortex are spatially segregated between different cortical areas, which suggests that there are functional distinctions between these different areas. Perturbation of cerebellar cortical functions in a spatially restricted fashion is thus essential for investigating the distinctions among different cortical areas. In the cerebellar cortex, Purkinje cells are the sole output neurons that send information to downstream cerebellar and vestibular nuclei. Therefore, selective manipulation of Purkinje cell activities, without disturbing other neuronal types and passing fibers within the cortex, is a direct approach to spatially restrict the effects of perturbations. Although this type of approach has for many years been technically difficult, recent advances in optogenetics now enable selective activation or inhibition of Purkinje cell activities, with high temporal resolution. Here we discuss the effectiveness of using Purkinje cell-specific optogenetic approaches to elucidate the functions of local cerebellar cortex regions. We also discuss what improvements to current methods are necessary for future investigations of cerebellar functions to provide further advances. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Isolating stromal stem cells from periodontal granulation tissues.

    PubMed

    Hung, Tzu-Yuan; Lin, Hsiang-Chun; Chan, Ying-Jen; Yuan, Kuo

    2012-08-01

    Stem cell therapy is a promising area in regenerative medicine. Periodontal granulation tissues are often discarded during conventional surgery. If stromal stem cells can be isolated from these tissues, they can be used for subsequent surgery on the same patient. Fifteen human periodontal granulation tissue samples were obtained from intrabony defects during surgery. Immunohistochemistry (IHC) was carried out on five of the samples to identify STRO-1, a marker of mesenchymal stem cells. Five samples underwent flow cytometry analysis for the same marker. The remaining five samples were characterized by "colony formation unit-fibroblast" (CFU-f) assay and selected for proliferation assay, flow cytometry of stem cell markers, immunocytochemistry (ICC), multipotent differentiation assays, and repairing critical-size defects in mice. The ratio of STRO-1(+) cells detected by IHC was 5.91 ± 1.50%. The analysis of flow cytometry for STRO-1 was 6.70 ± 0.81%. Approximately two thirds of the CFU-f colonies had a strong reaction to STRO-1 in ICC staining. The cells were multipotent both in vitro and in vivo. Mice given bone grafts and stem cells showed significantly better bone healing than those without stem cells. Multipotent stromal stem cells can be isolated from human periodontal granulation tissues. These cells improve new bone formation when transplanted in mouse calvarial defects. Isolating stem cells from relatively accessible sites without extra procedures is clinically advantageous. This study demonstrated that human periodontal granulation tissues contain isolatable multipotent stem cells. The cells may be a good source for autotransplantation in subsequent treatment.

  2. Dynamic functions of GABA signaling during granule cell maturation

    PubMed Central

    Dieni, Cristina V.; Chancey, Jessica H.; Overstreet-Wadiche, Linda S.

    2013-01-01

    The dentate gyrus is one of the few areas of the brain where new neurons are generated throughout life. Neural activity influences multiple stages of neurogenesis, thereby allowing experience to regulate the production of new neurons. It is now well established that GABAA receptor-mediated signaling plays a pivotal role in mediating activity-dependent regulation of adult neurogenesis. GABA first acts as a trophic signal that depolarizes progenitors and early post mitotic granule cells, enabling network activity to control molecular cascades essential for proliferation, survival and growth. Following the development of glutamatergic synaptic inputs, GABA signaling switches from excitatory to inhibitory. Thereafter robust synaptic inhibition enforces low spiking probability of granule cells in response to cortical excitatory inputs and maintains the sparse activity patterns characteristic of this brain region. Here we review these dynamic functions of GABA across granule cell maturation, focusing on the potential role of specific interneuron circuits at progressive developmental stages. We further highlight questions that remain unanswered about GABA signaling in granule cell development and excitability. PMID:23316139

  3. Alterations in cerebellar germinal cell division induced by graft-versus-host disease

    SciTech Connect

    Griffin, W.S.; Crom, E.N.; Head, J.R.

    1981-11-20

    A systemic immunological syndrome, graft-versus-host disease (GVHD), which does not cause inflammation or cell death in the cerebellum, is shown to retard granule cell production by decreasing the rate of DNA synthesis (S phase) and prolonging mitosis (M), at metaphase. The rate of cell production in diseased animals at postnatal day 14, quantitated by analysis of the rate of labeling of DNA with 3H-thymidine (3H-Tdr), revealed decreased ability to synthesize new DNA. The number of cells taking up 3H-Tdr label per mm2, as detected by autoradiography, was similar in 14-day-old GVHD and control tissue as was the area of the germinal matrix zone and the number of mitotically active germinal cells per mm2 in sagittal sections near the midline. However, because the total volume of the cerebellum was less, the total number of mitotically active cells in the whole cerebellum of 11-, 14-, and 17-day-old diseased animals was less than in littermate controls. Furthermore, DNA synthesis per mitotically active germinal cell was less in diseased animals at each age examined. The mitotic index was unaffected until late in the disease (day 17), suggesting that a prolongation of the cell cycle was responsible for this GVHD-induced decrease in DNA synthesis. Consistent with a prolongation of the cell cycle was the finding that the mitotic figures in 14-day-old GVHD cerebella were mostly metaphase figures, whereas those in control cerebella were, as predicted, mostly prophase. Prolongation of the cerebellar cell cycle in 11- and 14-day-old diseased animals may explain the dramatic decrease in the mitotic index, the thickness of the germinal matrix zone, and the number of germinal cells at postnatal day 17.

  4. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    PubMed Central

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  5. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  6. 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.

  7. Rapid Feedforward Inhibition and Asynchronous Excitation Regulate Granule Cell Activity in the Mammalian Main Olfactory Bulb

    PubMed Central

    Burton, Shawn D.

    2015-01-01

    Granule cell-mediated inhibition is critical to patterning principal neuron activity in the olfactory bulb, and perturbation of synaptic input to granule cells significantly alters olfactory-guided behavior. Despite the critical role of granule cells in olfaction, little is known about how sensory input recruits granule cells. Here, we combined whole-cell patch-clamp electrophysiology in acute mouse olfactory bulb slices with biophysical multicompartmental modeling to investigate the synaptic basis of granule cell recruitment. Physiological activation of sensory afferents within single glomeruli evoked diverse modes of granule cell activity, including subthreshold depolarization, spikelets, and suprathreshold responses with widely distributed spike latencies. The generation of these diverse activity modes depended, in part, on the asynchronous time course of synaptic excitation onto granule cells, which lasted several hundred milliseconds. In addition to asynchronous excitation, each granule cell also received synchronous feedforward inhibition. This inhibition targeted both proximal somatodendritic and distal apical dendritic domains of granule cells, was reliably recruited across sniff rhythms, and scaled in strength with excitation as more glomeruli were activated. Feedforward inhibition onto granule cells originated from deep short-axon cells, which responded to glomerular activation with highly reliable, short-latency firing consistent with tufted cell-mediated excitation. Simulations showed that feedforward inhibition interacts with asynchronous excitation to broaden granule cell spike latency distributions and significantly attenuates granule cell depolarization within local subcellular compartments. Collectively, our results thus identify feedforward inhibition onto granule cells as a core feature of olfactory bulb circuitry and establish asynchronous excitation and feedforward inhibition as critical regulators of granule cell activity. SIGNIFICANCE

  8. Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules.

    PubMed

    Rao, Tejeshwar C; Santana Rodriguez, Zuleirys; Bradberry, Mazdak M; Ranski, Alexandra H; Dahl, Peter J; Schmidtke, Michael W; Jenkins, Paul M; Axelrod, Daniel; Chapman, Edwin R; Giovannucci, David R; Anantharam, Arun

    2017-08-07

    Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K(+), we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca(2+) channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca(2+) channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca(2+) Upon introduction of Ca(2+) into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca(2+) levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca(2+) for activation. At Ca(2+) concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional

  9. Newborn granule cells in the ageing dentate gyrus

    PubMed Central

    Morgenstern, Nicolás A; Lombardi, Gabriela; Schinder, Alejandro F

    2008-01-01

    The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm−1 and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells. PMID:18565998

  10. Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

    PubMed Central

    Schnell, Eric; Bensen, AeSoon L.; Washburn, Eric K.; Westbrook, Gary L.

    2012-01-01

    Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons. PMID:23110172

  11. 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.

  12. Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum

    PubMed Central

    Legué, Emilie; Riedel, Elyn; Joyner, Alexandra L.

    2015-01-01

    The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium. PMID:25834018

  13. Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum.

    PubMed

    Legué, Emilie; Riedel, Elyn; Joyner, Alexandra L

    2015-05-01

    The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium.

  14. FIB/SEM cell sectioning for intracellular metal granules characterization

    NASA Astrophysics Data System (ADS)

    Milani, Marziale; Brundu, Claudia; Santisi, Grazia; Savoia, Claudio; Tatti, Francesco

    2009-05-01

    Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron dissection of cells and subcellular structures. In combination with Scanning Electron Microscope (SEM), FIB provides complementary morphological information, that can be further completed by EDX (Energy Dispersive X-ray Spectroscopy). This study focus onto intracellular microstructures, particularly onto metal granules (typically Zn, Cu and Fe) and on the possibility of sectioning digestive gland cells of the terrestrial isopod P. scaber making the granules available for a compositional analysis with EDX. Qualitative and quantitative analysis of metal granules size, amount and distribution are performed. Information is made available of the cellular storing pattern and, indirectly, metal metabolism. The extension to human level is of utmost interest since some pathologies of relevance are metal related. Apart from the common metal-overload-diseases (hereditary hemochromatosis, Wilson's and Menkes disease) it has been demonstrated that metal in excess can influence carcinogenesis in liver, kidney and breast. Therefore protocols will be established for the observation of mammal cells to improve our knowledge about the intracellular metal amount and distribution both in healthy cells and in those affected by primary or secondary metal overload or depletion.

  15. Determinants of action potential propagation in cerebellar Purkinje cell axons.

    PubMed

    Monsivais, Pablo; Clark, Beverley A; Roth, Arnd; Häusser, Michael

    2005-01-12

    Axons have traditionally been viewed as highly faithful transmitters of action potentials. Recently, however, experimental evidence has accumulated to support the idea that under some circumstances axonal propagation may fail. Cerebellar Purkinje neurons fire highfrequency simple spikes, as well as bursts of spikes in response to climbing fiber activation (the "complex spike"). Here we have visualized the axon of individual Purkinje cells to directly investigate the relationship between somatic spikes and axonal spikes using simultaneous somatic whole-cell and cell-attached axonal patch-clamp recordings at 200-800 microm from the soma. We demonstrate that sodium action potentials propagate at frequencies up to approximately 260 Hz, higher than simple spike rates normally observed in vivo. Complex spikes, however, did not propagate reliably, with usually only the first and last spikes in the complex spike waveform being propagated. On average, only 1.7 +/- 0.2 spikes in the complex spike were propagated during resting firing, with propagation limited to interspike intervals above approximately 4 msec. Hyperpolarization improved propagation efficacy without affecting total axonal spike number, whereas strong depolarization could abolish propagation of the complex spike. These findings indicate that the complex spike waveform is not faithfully transmitted to downstream synapses and that propagation of the climbing fiber response may be modulated by background activity.

  16. Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

    PubMed

    Santodomingo, Jaime; Vay, Laura; Camacho, Marcial; Hernández-Sanmiguel, Esther; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2008-10-01

    The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

  17. Cr (VI) induced oxidative stress and toxicity in cultured cerebellar granule neurons at different stages of development and protective effect of Rosmarinic acid.

    PubMed

    Dashti, Abolfazl; Soodi, Maliheh; Amani, Nahid

    2016-03-01

    Chromium (Cr) is a widespread metal ion in the workplace, industrial effluent, and water. The toxicity of chromium (VI) on various organs including the liver, kidneys, and lung were studied, but little is known about neurotoxicity. In this study, neurotoxic effects of Cr (VI) have been investigated by cultured cerebellar granule neurons (CGNs). Immature and mature neurons were exposed to different concentrations of potassium dichromate for 24 h and cytotoxicity was measured by MTT assay. In addition, immature neurons were exposed for 5 days as regards cytotoxic effect in development stages. The reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the protective effect of Rosmarinic acid on mature and immature neurons exposed to potassium dichromate, were measured. Furthermore, lipid peroxidation, glutathione peroxidase (GPx), and acetylcholinesterase activity in mature neurons were assessed following exposure to potassium dichromate. The results indicate that toxicity of Cr (VI) dependent on maturation steps. Cr (VI) was less toxic for immature neurons. Also, Cr (VI) induced MMP reduction and ROS production in both immature and mature neurons. In Cr (VI) treated neurons, increased lipid peroxidation and GPx activity but not acetylcholinesterase activity was observed. Interestingly, Rosmarinic acid, as a natural antioxidant, could protect mature but not immature neurons against Cr (VI) induced toxicity. Our findings revealed vulnerability of mature neurons to Cr (VI) induced toxicity and oxidative stress.

  18. The Role of Kalirin9 in p75/Nogo Receptor-mediated RhoA Activation in Cerebellar Granule Neurons*S⃞

    PubMed Central

    Harrington, Anthony W.; Li, Qi Ming; Tep, Chhavy; Park, Jong Bae; He, Zhigang; Yoon, Sung Ok

    2008-01-01

    p75 and the Nogo receptor form a signaling unit for myelin inhibitory molecules, with p75 being responsible for RhoA activation. Because p75 lacks the GDP/GTP exchange factor domain, it has remained unclear how p75 activates RhoA. Here, we report that Kalirin9, a dual RhoGEF, binds p75 directly and regulates p75-Nogo receptor-dependent RhoA activation and neurite inhibition in response to myelin-associated glycoprotein. The region of p75 that Kalirin9 binds includes its mastoparan-like fifth helix, which was shown to recruit RhoGDI-RhoA. As predicted from the presence of a shared binding site, we found that Kalirin9 competes with RhoGDI for p75 binding in a dose-dependent manner in vitro. In line with these data, myelin-associated glycoprotein addition to cerebellar granule neurons resulted in a reduction in the association of Kalirin9 with p75, and a simultaneous increase in the binding of RhoGDI to p75. These results reveal a mechanism by which the fifth helix of p75 regulates RhoA activation. PMID:18625710

  19. Functional Properties of Dendritic Gap Junctions in Cerebellar Golgi Cells.

    PubMed

    Szoboszlay, Miklos; Lőrincz, Andrea; Lanore, Frederic; Vervaeke, Koen; Silver, R Angus; Nusser, Zoltan

    2016-06-01

    The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Single granule cells reliably discharge targets in the hippocampal CA3 network in vivo.

    PubMed

    Henze, Darrell A; Wittner, Lucia; Buzsáki, György

    2002-08-01

    Processing of neuronal information depends on interactions between the anatomical connectivity and cellular properties of single cells. We examined how these computational building blocks work together in the intact rat hippocampus. Single spikes in dentate granule cells, controlled intracellularly, generally failed to discharge either interneurons or CA3 pyramidal cells. In contrast, trains of spikes effectively discharged both CA3 cell types. Increasing the discharge rate of the granule cell increased the discharge probability of its target neuron and decreased the delay between the onset of a granule cell train and evoked firing in postsynaptic targets. Thus, we conclude that the granule cell to CA3 synapses are 'conditional detonators,' dependent on granule cell firing pattern. In addition, we suggest that information in single granule cells is converted into a temporal delay code in target CA3 pyramidal cells and interneurons. These data demonstrate how a neural circuit of the CNS may process information.

  1. 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.

  2. 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.

  3. 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.

  4. Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

    PubMed Central

    Strasser, Markus J; Mackenzie, Natalia C; Dumstrei, Karin; Nakkrasae, La-Iad; Stebler, Jürg; Raz, Erez

    2008-01-01

    Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network. PMID:18507824

  5. Linking oscillations in cerebellar circuits

    PubMed Central

    Courtemanche, Richard; Robinson, Jennifer C.; Aponte, Daniel I.

    2013-01-01

    In many neuroscience fields, the study of local and global rhythmicity has been receiving increasing attention. These network influences could directly impact on how neuronal groups interact together, organizing for different contexts. The cerebellar cortex harbors a variety of such local circuit rhythms, from the rhythms in the cerebellar cortex per se, or those dictated from important afferents. We present here certain cerebellar oscillatory phenomena that have been recorded in rodents and primates. Those take place in a range of frequencies: from the more known oscillations in the 4–25 Hz band, such as the olivocerebellar oscillatory activity and the granule cell layer oscillations, to the more recently reported slow (<1 Hz oscillations), and the fast (>150 Hz) activity in the Purkinje cell layer. Many of these oscillations appear spontaneously in the circuits, and are modulated by behavioral imperatives. We review here how those oscillations are recorded, some of their modulatory mechanisms, and also identify some of the cerebellar nodes where they could interact. A particular emphasis has been placed on how these oscillations could be modulated by movement and certain neuropathological manifestations. Many of those oscillations could have a definite impact on the way information is processed in the cerebellum and how it interacts with other structures in a variety of contexts. PMID:23908606

  6. Role of nitric oxide produced by iNOS through NF-κB pathway in migration of cerebellar granule neurons induced by Lipopolysaccharide.

    PubMed

    Arias-Salvatierra, Daniela; Silbergeld, Ellen K; Acosta-Saavedra, Leonor C; Calderon-Aranda, Emma S

    2011-02-01

    Inflammatory stimulus during development increases the risk for adverse neurologic outcome. One possible mechanism is disrupting neuronal migration. Using lipopolysaccharide (LPS)-treatment to assess inflammatory stimulus on neuronal migration of cerebellar granule neurons, we previously found that LPS-activation increased the neuronal migration. The precise mechanisms behind these effects have not been investigated. Independently, it was shown that nitric oxide (NO(•-)) regulates neuronal migration during development, that NO(•-) is produced by inducible nitric oxide synthase (iNOS) in response to LPS through the activation of nuclear factor (NF)-κB, and that LPS induce the expression of genes under the transcriptional control of NF-κB in primary cultures from developing mouse cerebellum. To investigate the relationship between these events, we used this culture model to study the role of NO(•-) produced by iNOS through NF-κB signaling pathway, in the effect of LPS on neuron migration. LPS increased NO(•-) production, iNOS protein levels and NF-κB nuclear levels; concomitantly with NO(•-) production, LPS increased the neuronal migration as compared to non stimulated cultures. The necessary roles of the NO(•-) and iNOS were demonstrated by chelating of NO(•-) with hemoglobin and the inhibition of iNOS by 1400W. Each of these treatments reduced neuronal migration induced by LPS. The role of NF-κB was showed by using the inhibitor JSH-23, which decreased NO(•-) production and neuronal migration in LPS activated cultures. These results suggest that neuronal migration during development is susceptible to be modified by pro-inflammatory stimulus such as LPS through intracellular pathways associated with their receptors.

  7. Exposure to 50 Hz magnetic field modulates GABAA currents in cerebellar granule neurons through an EP receptor-mediated PKC pathway

    PubMed Central

    Yang, Guang; Ren, Zhen; Mei, Yan-Ai

    2015-01-01

    Previous work from both our lab and others have indicated that exposure to 50 Hz magnetic fields (ELF-MF) was able to modify ion channel functions. However, very few studies have investigated the effects of MF on γ-aminobutyric acid (GABA) type A receptors (GABAARs) channel functioning, which are fundamental to overall neuronal excitability. Here, our major goal is to reveal the potential effects of ELF-MF on GABAARs activity in rat cerebellar granule neurons (CGNs). Our results indicated that exposing CGNs to 1 mT ELF-MF for 60 min. significantly increased GABAAR currents without modifying sensitivity to GABA. However, activation of PKA by db-cAMP failed to do so, but led to a slight decrease instead. On the other hand, PKC activation or inhibition by PMA or Bis and Docosahexaenoic acid (DHA) mimicked or eliminated the field-induced-increase of GABAAR currents. Western blot analysis indicated that the intracellular levels of phosphorylated PKC (pPKC) were significantly elevated after 60 min. of ELF-MF exposure, which was subsequently blocked by application of DHA or EP1 receptor-specific (prostaglandin E receptor 1) antagonist (SC19220), but not by EP2-EP4 receptor-specific antagonists. SC19220 also significantly inhibited the ELF-MF-induced elevation on GABAAR currents. Together, these data obviously demonstrated for the first time that neuronal GABAA currents are significantly increased by ELF-MF exposure, and also suggest that these effects are mediated via an EP1 receptor-mediated PKC pathway. Future work will focus on a more comprehensive analysis of the physiological and/or pathological consequences of these effects. PMID:26176998

  8. 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

  9. A marked paucity of granule cells in the developing cerebellum of the Npc1−/− mouse is corrected by a single injection of hydroxypropyl-β-cyclodextrin

    PubMed Central

    Nusca, S.; Canterini, S.; Palladino, G.; Bruno, F.; Mangia, F.; Erickson, R.P.; Fiorenza, M.T.

    2014-01-01

    In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1−/− mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%–25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8–PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1−/− mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1−/− mice. A single injection of hydroxypropyl-β-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum. PMID:24969023

  10. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

  11. Automated insulin granule segmentation from electron photomicrographs of rat pancreatic β-cells

    NASA Astrophysics Data System (ADS)

    McClanahan, Timothy P.; Straub, Susanne G.; Sharp, Geoffrey W. G.; Loew, Murray

    2005-04-01

    Increased blood glucose stimulates pancreatic β-cells and induces an exocytotic release of insulin. The β-cell, which contains ~10^4 insulin-containing granules, releases only a few percent of the granules during a given stimulus such as a meal. The temporal response function to a square wave increase in the concentration of glucose is characteristically biphasic. It is not known whether the granules exhibit random or directed migration patterns as a function of phase. Directed migration would suggest the development of an intracellular gradient directing the path and velocity of insulin granule movement. Our ongoing research investigates this process using manual morphometric analysis of electron micrographs of rat pancreatic β-cells. This is a tedious and time-consuming stereological process. Consequently, we have developed an automated algorithm for accurately segmenting and deriving granule counts, areas, and measuring distance to the plasma membrane. The method is a data-driven image processing approach that implements Mahalanobis classifiers to hierarchically classify pixel candidates and subsequently pixel aggregates as insulin granules. Granule cores and halos are classified independently and fused by intersecting the convex difference of granule halos with core candidates. Once fused, total and individual granule areas and distance metrics to the β-cell plasma membrane are obtained. This algorithm provides a rapid and accurate method for the determination of granule numbers, location, and potential gradients in the pancreatic β-cell under different experimental conditions.

  12. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    SciTech Connect

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  13. Toluene decreases Purkinje cell output by enhancing inhibitory synaptic transmission in the cerebellar cortex.

    PubMed

    Gmaz, Jimmie M; McKay, Bruce E

    2014-02-07

    Toluene belongs to a class of psychoactive drugs known as inhalants. Found in common household products such as adhesives, paint products, and aerosols, toluene is inhaled for its intoxicating and euphoric properties. Additionally, exposure to toluene disrupts motor behaviors in a manner consistent with impairments to cerebellar function. Previous work has suggested a role of GABA in mediating toluene's neurobehavioral effects, but how this manifests in the cerebellar cortex is not yet understood. In the present study, we examined the effects of toluene on cerebellar Purkinje cell action potential output and inhibitory synaptic transmission onto Purkinje cells using patch clamp electrophysiology in acute rat cerebellar slices. Toluene (1mM) reduced the frequency of Purkinje cell action potential output without affecting input resistance. Furthermore, toluene dose-dependently enhanced inhibitory synaptic transmission onto Purkinje cells, increasing the amplitude and frequency of inhibitory postsynaptic currents; no change in the frequency of action potentials from molecular layer interneurons was noted. The observed decreases in Purkinje cell action potential output could contribute to toluene-evoked impairments in cerebellar and motor functions. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Differentiation of apical and basal dendrites in pyramidal cells and granule cells in dissociated hippocampal cultures.

    PubMed

    Wu, You Kure; Fujishima, Kazuto; Kengaku, Mineko

    2015-01-01

    Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.

  15. Using human induced pluripotent stem cells to model cerebellar disease: hope and hype.

    PubMed

    Wiethoff, Sarah; Arber, Charles; Li, Abi; Wray, Selina; Houlden, Henry; Patani, Rickie

    2015-01-01

    The cerebellum forms a highly ordered and indispensible component of motor function within the adult neuraxis, consisting of several distinct cellular subtypes. Cerebellar disease, through a variety of genetic and acquired causes, results in the loss of function of defined subclasses of neurons, and remains a significant and untreatable health care burden. The scarcity of therapies in this arena can partially be explained by unresolved disease mechanisms due to inaccessibility of human cerebellar neurons in a relevant experimental context where initiating disease mechanisms could be functionally elucidated, or drug screens conducted. In this review we discuss the potential promise of human induced pluripotent stem cells (hiPSCs) for regenerative neurology, with a particular emphasis on in vitro modelling of cerebellar degeneration. We discuss progress made thus far using hiPSC-based models of neurodegeneration, noting the relatively slower pace of discovery made in modelling cerebellar dysfunction. We conclude by speculating how strategies attempting cerebellar differentiation from hiPSCs can be refined to allow the generation of accurate disease models. This in turn will permit a greater understanding of cerebellar pathophysiology to inform mechanistically rationalised therapies, which are desperately needed in this field.

  16. Postnatal Migration of Cerebellar Interneurons

    PubMed Central

    Galas, Ludovic; Bénard, Magalie; Lebon, Alexis; Komuro, Yutaro; Schapman, Damien; Vaudry, Hubert; Vaudry, David; Komuro, Hitoshi

    2017-01-01

    Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders. PMID:28587295

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

    SciTech Connect

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

    2007-08-01

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

  18. Only a Minority of the Inhibitory Inputs to Cerebellar Golgi Cells Originates from Local GABAergic Cells123

    PubMed Central

    2016-01-01

    Abstract Cerebellar Golgi cells (GoCs) efficiently control the spiking activity of granule cells through GABAA receptor-mediated tonic and phasic inhibition. Recent experiments provided compelling evidence for the extensive interconnection of GoCs through electrical synapses, but their chemical inhibitory synaptic inputs are debated. Here, we investigated the GABAergic synaptic inputs of GoCs using in vitro electrophysiology and quantitative light microscopy (LM) and electron microscopy (EM). We characterized GABAA receptor-mediated IPSCs in GoCs and Lugaro cells (LuCs), and found that IPSCs in GoCs have lower frequencies, smaller amplitudes, and much slower decay kinetics. Pharmacological and LM immunolocalization experiments revealed that GoCs express α3, whereas LuCs express α1 subunit-containing GABAA receptors. The selective expression and clustered distribution of the α3 subunit in GoCs allowed the quantitative analysis of GABAergic synapses on their dendrites in the molecular layer (ML). EM and LM experiments in rats, and wild-type and GlyT2-GFP transgenic mice revealed that only one third of axon terminals establishing GABAergic synapses on GoC dendrites contain GlyT2, ruling out LuCs, globular cells, and any noncortical glycinergic inputs as major inhibitory sources. We also show that axon terminals of stellate/basket cells very rarely innervate GlyT2-GFP-expressing GoCs, indicating that only a minority of the inhibitory inputs to GoCs in the ML originates from local interneurons, and the majority of their inhibitory inputs exclusively releases GABA. PMID:27257627

  19. 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

  20. Thrombopoietin-induced Dami cells as a model for alpha-granule biogenesis.

    PubMed

    Briquet-Laugier, Véronique; El Golli, Nargès; Nurden, Paquita; Lavenu-Bombled, Cécile; Dubart-Kupperschmitt, Anne; Nurden, Alan; Rosa, Jean-Philippe

    2004-09-01

    Megakaryocytic alpha-granules contain secretory proteins relevant to megakaryocyte and platelet functions. Understanding alpha-granule biogenesis is hampered because human primary megakaryocytes are difficult to manipulate. Existing promegakaryocytic cell lines do not spontaneously exhibit mature alpha-granules. Dami cells, transfected with the megakaryocytic platelet factor 4, fused to GFP (PF4-GFP), were induced in the presence of thrombopoietin (TPO), a megakaryocyte cytokine and PMA. Using confocal microscopy, PF4-GFP colocalized with von Willebrand Factor (vWF) in newly formed storage granules. Immunoelectron microscopy demonstrated alpha-granule-like features, including a dense core or parallel tubules and colocalization of PF4-GFP and vWF. Hence, TPO-treated Dami cells are a suitable model to study alpha-granules and their biogenesis.

  1. Regionalization of the isthmic and cerebellar primordia.

    PubMed

    Narboux-Nême, Nicolas; Louvi, Angeliki; Alexandre, Paula; Wassef, Marion

    2005-01-01

    The complex migrations of neurons born in the dorsal neural tube of the isthmic and rhombomere l (rl) domains complicate the delineation of the cerebellar primordium. We show that Purkinje cells (P) are likely generated over a wide territory before gathering in the future cerebellar primordium under the developing external granular layer. Later expansion of the cerebellum over a restricted ependymal domain could rely on mutual interations between P cells and granule cell progenitors (GCP). P are attracted by GCP and in turn stimulate their proliferation, increasing the surface of the developing cortex. At later stages, regionalization of the developing and adult cerebellar cortex can be detected through regional variations in the distribution of several P cell markers. Whether and how the developmental and adult P subtypes are related is still unknown and it is unclear if they delineate the same sets of cerebellar subdivisions. We provide evidence that the early P regionalization is involved in intrinsic patterning of the cerebellar primordium, in particular it relate to the organization of the corticonuclear connection. We propose that the early P regionalization provides a scaffold to the mature P regionalization but that the development of functional afferent connections induces a period of P plasticity during which the early regional identity of P could be remodeled.

  2. 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

  3. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo

    PubMed Central

    McDole, Brittnee; Isgor, Ceylan; Pare, Christopher; Guthrie, Kathleen

    2015-01-01

    Olfactory bulb granule cells are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on granule cell spines. These contacts are established in the distal apical dendritic compartment, while granule cell basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong granule cell neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb granule cell spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF produces a marked increase in granule cell spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on granule cells, suggesting a role for this factor in modulating granule cell functional connectivity within adult olfactory circuitry. PMID:26211445

  4. 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.

  5. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    SciTech Connect

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-03-01

    Incubation of (/sup 35/S)heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of /sup 35/S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of (/sup 35/S)heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular (/sup 35/S)heparin proteoglycan after 24 hours and the appearance of free (/sup 35/S)sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading (/sup 35/S)heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule.

  6. Three-Dimensional Tracking of Single Granules in Living PC-12 Cells Employing TIRFM and WFFM.

    PubMed

    Xiong, Jun; Li, Dongdong; Zhu, Dan; Qu, Anlian

    2005-01-01

    A comparative study was carried out on evaluating the performance of total internal reflection fluorescence microscopy (TIRFM) and deconvolution wide-field fluorescence microscopy (WFFM) in tracking single secretory granules. Both techniques have been applied to follow the three-dimensional mobility of single secretory granules in living neuroendocrine PC-12 cells. Both techniques return the similar result that most acridine orange-labeled granules were found to travel in random and caged diffusion, and only a small fraction of granules traveled in directed diffusion. Furthermore, the size and 3-D diffusion coefficient of secretory granules, obtained by these two imaging techniques, yield the same value. Together, our results demonstrate the potential of the combination TIRFM and WFFM in tracking long-termed motion of granules throughout live whole cells.

  7. Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation

    PubMed Central

    Bender, R. A.; Lauterborn, J. C.; Gall, C. M.; Cariaga, W.; Baram, T. Z.

    2011-01-01

    Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network. PMID:11207803

  8. Synthesis of Prostaglandins and Eicosanoids by the Mast Cell Secretory Granule

    DTIC Science & Technology

    1988-01-01

    various lipid-derived mediators during exocytosis. MATERIALS AND METHODS The procedure for granule preparation is similar to that which has been described...Press, Inc. Printed in U.S.A. SYNTHESIS OF PROSTACLANDINS AND KICOSANOIDS BY THE M&ST CELL SECRETORY GRANULE Stephen P. Chock and Elsa A. Schmauder-Chock...SCISNTIFIC XeiOT Received September 30, 1988 SR88-32 The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of

  9. Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

    PubMed Central

    Chen, Huan-Yuan; Chiang, Dapi Meng-Lin; Lin, Zi-Jing; Hsieh, Chia-Chun; Yin, Gung-Chian; Weng, I.-Chun; Guttermann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Lai, Lee-Jene; Liu, Fu-Tong

    2016-01-01

    Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level. PMID:27748356

  10. Distorted secretory granule composition in mast cells with multiple protease deficiency.

    PubMed

    Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

    2013-10-01

    Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

  11. Sparse activity of identified dentate granule cells during spatial exploration

    PubMed Central

    Diamantaki, Maria; Frey, Markus; Berens, Philipp; Preston-Ferrer, Patricia; Burgalossi, Andrea

    2016-01-01

    In the dentate gyrus – a key component of spatial memory circuits – granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure–function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population. DOI: http://dx.doi.org/10.7554/eLife.20252.001 PMID:27692065

  12. Giant cytoplasmic granules in Langerhans cells of Chediak-Higashi syndrome.

    PubMed

    Carrillo-Farga, J; Gutiérrez-Palomera, G; Ruiz-Maldonado, R; Rondán, A; Antuna, S

    1990-02-01

    Giant membrane-bound cytoplasmic granules were found in the epidermal Langerhans cells of a patient with the Chediak-Higashi syndrome. These cells also contained normal-appearing Birbeck granules. The giant granules had a granular or sometimes globular internal structure; they are believed to derive from fusion of lysosomes or some portion of Birbeck granules. It is unclear whether this morphologic change in Langerhans cell interferes with their antigen-presenting function; it may be, in part, responsible for the frequent infections seen in patients with Chediak-Higashi syndrome that are otherwise more clearly related to the abnormalities in neutrophils and lymphocytes. The Langerhans cell is another cellular type in Chediak-Higashi syndrome in which giant cytoplasmic granules are found.

  13. Differentiated cytoplasmic granule formation in quiescent and non-quiescent cells upon chronological aging

    PubMed Central

    Lee, Hsin-Yi; Cheng, Kuo-Yu; Chao, Jung-Chi; Leu, Jun-Yi

    2016-01-01

    Stationary phase cultures represent a complicated cell population comprising at least two different cell types, quiescent (Q) and non-quiescent (NQ) cells. Q and NQ cells have different lifespans and cell physiologies. However, less is known about the organization of cytosolic protein structures in these two cell types. In this study, we examined Q and NQ cells for the formation of several stationary phase-prevalent granule structures including actin bodies, proteasome storage granules, stress granules, P-bodies, the compartment for unconventional protein secretion (CUPS), and Hsp42-associated stationary phase granules (Hsp42-SPGs). Most of these structures preferentially form in NQ cells, except for Hsp42-SPGs, which are enriched in Q cells. When nutrients are provided, NQ cells enter mitosis less efficiently than Q cells, likely due to the time requirement for reorganizing some granule structures. We observed that heat shock-induced misfolded proteins often colocalize to Hsp42-SPGs, and Q cells clear these protein aggregates more efficiently, suggesting that Hsp42-SPGs may play an important role in the stress resistance of Q cells. Finally, we show that the cell fate of NQ cells is largely irreversible even if they are allowed to reenter mitosis. Our results reveal that the formation of different granule structures may represent the early stage of cell type differentiation in yeast stationary phase cultures. PMID:28357341

  14. Structural reappraisal of dendritic tree of cerebellar Purkinje cell for novel functional modeling of elementary sensorimotor adaptive processes

    NASA Astrophysics Data System (ADS)

    Simon, Laszlo; Garab, Sandor; Noszek, Annamaria

    2005-08-01

    Both compound spike generating process revealed in the (cat's) cerebellar Purkinje cell [6] and the separating dendritic areas, identified by structural analysis of ramification pattern [9] indicate a phase-sensitive, nonlinear integration of input information. This sophisticated paradigm could be the basic tool of novel cerebellar functional theories and models.

  15. The stealthy nano-machine behind mast cell granule size distribution.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2015-01-01

    The classical model of mast cell secretory granule formation suggests that newly synthesized secretory mediators, transported from the rough endoplasmic reticulum to the Golgi complex, undergo post-transitional modification and are packaged for secretion by condensation within membrane-bound granules of unit size. These unit granules may fuse with other granules to form larger granules that reside in the cytoplasm until secreted. A novel stochastic model for mast cell granule growth and elimination (G&E) as well as inventory management is presented. Resorting to a statistical mechanics approach in which SNAP (Soluble NSF Attachment Protein) REceptor (SNARE) components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation that can perform granule growth and secretion. Granule stock is maintained as a buffer to meet uncertainty in demand by the extracellular environment and to serve as source of supply during the lead time to produce granules of adaptive content. Experimental work, mathematical calculations, statistical modeling and a rationale for the emergence of nearly last-in, first out inventory management, are discussed.

  16. Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells

    PubMed Central

    1983-01-01

    The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep- etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low- Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule- microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules. PMID:6841448

  17. Time‐invariant feed‐forward inhibition of Purkinje cells in the cerebellar cortex in vivo

    PubMed Central

    Blot, Antonin; de Solages, Camille; Ostojic, Srdjan; Szapiro, German; Hakim, Vincent; Léna, Clément

    2016-01-01

    Key points We performed extracellular recording of pairs of interneuron–Purkinje cells in vivo.A single interneuron produces a substantial, short‐lasting, inhibition of Purkinje cells.Feed‐forward inhibition is associated with characteristic asymmetric cross‐correlograms. In vivo, Purkinje cell spikes only depend on the most recent synaptic activity. Abstract Cerebellar molecular layer interneurons are considered to control the firing rate and spike timing of Purkinje cells. However, interactions between these cell types are largely unexplored in vivo. Using tetrodes, we performed simultaneous extracellular recordings of neighbouring Purkinje cells and molecular layer interneurons, presumably basket cells, in adult rats in vivo. The high levels of afferent synaptic activity encountered in vivo yield irregular spiking and reveal discharge patterns characteristic of feed‐forward inhibition, thus suggesting an overlap of the afferent excitatory inputs between Purkinje cells and basket cells. Under conditions of intense background synaptic inputs, interneuron spikes exert a short‐lasting inhibitory effect, delaying the following Purkinje cell spike by an amount remarkably independent of the Purkinje cell firing cycle. This effect can be explained by the short memory time of the Purkinje cell potential as a result of the intense incoming synaptic activity. Finally, we found little evidence for any involvement of the interneurons that we recorded with the cerebellar high‐frequency oscillations promoting Purkinje cell synchrony. The rapid interactions between interneurons and Purkinje cells might be of particular importance in fine motor control because the inhibitory action of interneurons on Purkinje cells leads to deep cerebellar nuclear disinhibition and hence increased cerebellar output. PMID:26918702

  18. Time-invariant feed-forward inhibition of Purkinje cells in the cerebellar cortex in vivo.

    PubMed

    Blot, Antonin; de Solages, Camille; Ostojic, Srdjan; Szapiro, German; Hakim, Vincent; Léna, Clément

    2016-05-15

    We performed extracellular recording of pairs of interneuron-Purkinje cells in vivo. A single interneuron produces a substantial, short-lasting, inhibition of Purkinje cells. Feed-forward inhibition is associated with characteristic asymmetric cross-correlograms. In vivo, Purkinje cell spikes only depend on the most recent synaptic activity. Cerebellar molecular layer interneurons are considered to control the firing rate and spike timing of Purkinje cells. However, interactions between these cell types are largely unexplored in vivo. Using tetrodes, we performed simultaneous extracellular recordings of neighbouring Purkinje cells and molecular layer interneurons, presumably basket cells, in adult rats in vivo. The high levels of afferent synaptic activity encountered in vivo yield irregular spiking and reveal discharge patterns characteristic of feed-forward inhibition, thus suggesting an overlap of the afferent excitatory inputs between Purkinje cells and basket cells. Under conditions of intense background synaptic inputs, interneuron spikes exert a short-lasting inhibitory effect, delaying the following Purkinje cell spike by an amount remarkably independent of the Purkinje cell firing cycle. This effect can be explained by the short memory time of the Purkinje cell potential as a result of the intense incoming synaptic activity. Finally, we found little evidence for any involvement of the interneurons that we recorded with the cerebellar high-frequency oscillations promoting Purkinje cell synchrony. The rapid interactions between interneurons and Purkinje cells might be of particular importance in fine motor control because the inhibitory action of interneurons on Purkinje cells leads to deep cerebellar nuclear disinhibition and hence increased cerebellar output. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  19. Analysis of Spine Motility of Newborn Granule Cells in Acute Brain Slices.

    PubMed

    Tashiro, Ayumu; Zhao, Chunmei; Suh, Hoonkyo; Gage, Fred H

    2015-10-01

    In this protocol, acute brain slices are prepared from mice in which newborn granule cells have been labeled using retroviral vector technology. Using a live-cell imaging stage and confocal microscopy coupled to imaging software, dendritic spines are analyzed.

  20. Studies on the pH gradient and histamine uptake of isolated mast cell granules

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1986-05-01

    A purified preparation of mast cell granules with intact perigranular membranes was obtained using a method involving probe sonication of rat serosal mast cells followed by differential centrifugation and Percoll gradient separation of the granules. Purification was assessed with histamine and mast cell granule protease assays. Granule integrity was demonstrated by light and electron microscopy and quantitated with a ruthenium red binding assay. The low yield of granules (20 ..mu..g protein/4 rats) necessitated the development of two microanalytical techniques to demonstrate the existence of a pH gradient across the membrane: 9-aminoacridine fluorescence studies in a cuvet with 50 ..mu..l capacity and /sup 14/C-methylamine distribution studies on microgram quantities of granule protein. Quantitation of results from isotope studies were confounded by the presence of oil used for separating granules from the aqueous phase. Nonetheless, an extrapolation procedure calibrated by external pH yielded an internal pH value of 5.46 +/- .03 (n = 4), consistent with values observed in granules obtained from other secretory cells. Collapse of the pH gradient by NH/sub 4//sup +/ or nigericin/KCl was demonstrated using either technique. Addition of histamine depressed intragranular pH, suggesting that histamine transport may utilize the ..delta..pH as a driving force.

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

    PubMed

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

    2010-10-01

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

  2. Purkinje Cell Death After Uptake of Anti-Yo Antibodies in Cerebellar Slice Cultures

    PubMed Central

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

    2010-01-01

    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. PMID:20838245

  3. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus

    PubMed Central

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7⁎nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7⁎nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2–3 week-old Wistar rats, and 2–9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7⁎nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7⁎nicotinic receptor modulator, which were blocked by a specific α7⁎nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7⁎nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7⁎nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  4. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus.

    PubMed

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-03-19

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7*nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7*nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7*nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7*nicotinic receptor modulator, which were blocked by a specific α7*nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7*nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7*nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain.

  5. Calcium Imaging Reveals Coordinated Simple Spike Pauses in Populations of Cerebellar Purkinje Cells.

    PubMed

    Ramirez, Jorge E; Stell, Brandon M

    2016-12-20

    The brain's control of movement is thought to involve coordinated activity between cerebellar Purkinje cells. The results reported here demonstrate that somatic Ca(2+) imaging is a faithful reporter of Na(+)-dependent "simple spike" pauses and enables us to optically record changes in firing rates in populations of Purkinje cells in brain slices and in vivo. This simultaneous calcium imaging of populations of Purkinje cells reveals a striking spatial organization of pauses in Purkinje cell activity between neighboring cells. The source of this organization is shown to be the presynaptic gamma-Aminobutyric acid producing (GABAergic) network, and blocking ionotropic gamma-Aminobutyric acid receptor (GABAARs) abolishes the synchrony. These data suggest that presynaptic interneurons synchronize (in)activity between neighboring Purkinje cells, and thereby maximize their effect on downstream targets in the deep cerebellar nuclei. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Traveling waves in developing cerebellar cortex mediated by asymmetrical Purkinje cell connectivity.

    PubMed

    Watt, Alanna J; Cuntz, Hermann; Mori, Masahiro; Nusser, Zoltan; Sjöström, P Jesper; Häusser, Michael

    2009-04-01

    Correlated network activity is important in the development of many neural circuits. Purkinje cells are among the first neurons to populate the cerebellar cortex, where they sprout exuberant axon collaterals. We used multiple patch-clamp recordings targeted with two-photon microscopy to characterize monosynaptic connections between the Purkinje cells of juvenile mice. We found that Purkinje cell axon collaterals projected asymmetrically in the sagittal plane, directed away from the lobule apex. On the basis of our anatomical and physiological characterization of this connection, we constructed a network model that robustly generated waves of activity that traveled along chains of connected Purkinje cells. Consistent with the model, we observed traveling waves of activity in Purkinje cells in sagittal slices from young mice that require GABA(A) receptor-mediated transmission and intact Purkinje cell axon collaterals. These traveling waves are absent in adult mice, suggesting they have a developmental role in wiring the cerebellar cortical microcircuit.

  7. Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

    PubMed Central

    Brunello, Cecilia A.; Yan, Xu; Huttunen, Henri J.

    2016-01-01

    Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer’s disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology. PMID:27460788

  8. Three-dimensional tracking of single secretory granules in live PC12 cells.

    PubMed

    Li, Dongdong; Xiong, Jun; Qu, Anlian; Xu, Tao

    2004-09-01

    Deconvolution wide-field fluorescence microscopy and single-particle tracking were used to study the three-dimensional mobility of single secretory granules in live PC12 cells. Acridine orange-labeled granules were found to travel primarily in random and caged diffusion, whereas only a small fraction of granules traveled in directed fashion. High K(+) stimulation increased significantly the percentage of granules traveling in directed fashion. By dividing granules into the near-membrane group (within 1 microm from the plasma membrane) and cytosolic group, we have revealed significant differences between these two groups of granules in their mobility. The mobility of these two groups of granules is also differentially affected by disruption of F-actin, suggesting different mechanisms are involved in the motion of the two groups of granules. Our results demonstrate that combined deconvolution and single-particle tracking may find its application in three-dimensional tracking of long-term motion of granules and elucidating the underlying mechanisms.

  9. 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.

  10. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    PubMed

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  11. Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

    SciTech Connect

    Gianotti, A.J.; Clark, D.T.; Dash, J. )

    1991-04-01

    A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent 'sphaerocrystals' located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

  12. Somatic translocation: a novel mechanism of granule cell dendritic dysmorphogenesis and dispersion

    PubMed Central

    Murphy, Brian L.; Danzer, Steve C.

    2011-01-01

    Pronounced neuronal remodeling is a hallmark of temporal lobe epilepsy. Here, we use real-time confocal imaging of tissue from mouse brain to demonstrate that remodeling can involve fully-differentiated granule cells following translocation of the soma into an existing apical dendrite. Somatic translocation converts dendritic branches into primary dendrites and shifts adjacent apical dendrites to the basal pole of the cell. Moreover, somatic translocation contributes to the dispersion of the granule cell body layer in vitro, and when granule cell dispersion is induced in vivo, the dispersed cells exhibit virtually identical derangements of their dendritic structures. Together, these findings identify novel forms of neuronal plasticity which contribute to granule cell dysmorphogenesis in the epileptic brain. PMID:21414917

  13. Potassium currents in acutely isolated human hippocampal dentate granule cells.

    PubMed Central

    Beck, H; Clusmann, H; Kral, T; Schramm, J; Heinemann, U; Elger, C E

    1997-01-01

    1. Properties of voltage- and Ca(2+)-dependent K+ currents were investigated in thirty-four dentate granule cells acutely isolated from the resected hippocampus of eleven patients with therapy-refractory temporal lobe epilepsy (TLE). 2. When intracellular Ca2+ was strongly buffered with 11.5 mM EGTA-1 mM Ca2+ in the recording pipette, K+ currents (IK) with a slow activation and biexponential time-dependent decay could be elicited, which showed a threshold for activation around -30 mV. 3. A contribution of Ca(2+)-dependent K+ currents became apparent with intracellular solution containing 1 mM BAPTA-0.1 mM Ca2+. Superfusion of low-Ca2+ extracellular solution blocked 43% of outward currents in this recording configuration. Outward current components could also be blocked by substituting 5 mM Ba2+ for extracellular Ca2+ (78%), or by application of 100 microM Cd2+ (25%). 4. The Ca(2+)-dependent K+ currents could be pharmacologically subdivided into two components. One component was sensitive to 500 microM tetraethylammmonium (TEA; 41%) and 10 nM charybdotoxin (CTX; 47.2%). The blocking effects of 10 nM CTX and 500 microM TEA were not additive, suggesting that both agents block the same conductance. A second, smaller outward current component was blocked by 50 nM apamin (13%). 5. A transient A-type K+ current could be observed in six neurones and showed a fast monoexponential time-dependent inactivation with a steady-state voltage dependence that was distinct from that of IK. The A-type current was blocked by 4-aminopyridine (4-AP) but not by TEA or low-Ca2+ solution. 6. We conclude that outward currents in human hippocampal dentate granule cells can be separated into at least four types by their kinetic and pharmacological properties. These include at least one voltage-dependent current similar to those observed in mammalian hippocampal neurones, and two Ca(2+)-dependent K+ currents that most probably correspond to SK- and BK-type currents. A classical A-type current

  14. Emerging Roles of Granule Recycling in Mast Cell Plasticity and Homeostasis.

    PubMed

    Flores, Juan A; Balseiro-Gómez, Santiago; Ales, Eva

    2016-01-01

    Secretory granules (SGs) of mast cells (MCs) release their contents to mediate many biological events and a variety of inflammatory diseases and have important protective roles in innate host defense and pathological functions in allergic reactions and anaphylaxis. There are two modes of MC degranulation during the release of granule contents to the extracellular environment. Anaphylactic degranulation (AND) after IgE-mediated activation is characterized by a rapid swelling and fusion of MC granules as well as abrupt mediators release. Piecemeal degranulation (PMD) is a slow and selective secretion of distinct granule mediators by vesicles shuttling from the granule compartment to the plasma membrane, and it is associated with several chronic diseases. Following degranulation, endocytosis is a fundamental biological event that is necessary to recycle granules and maintain the secretory response during repetitive stimulation. Rapid endocytosis through transient fusion (kiss-and-run, cavicapture and compound exo-endocytosis) has been described in MCs and can also result in the selective release of granule contents. In summary, several possible exo-endocytic mechanisms control the kinetics and magnitude of transmitter release, and each mechanism is associated with a different impact on granule replenishment, cell recovery, and consequently MC function under both normal and pathological conditions.

  15. Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells

    PubMed Central

    Linster, Christiane

    2015-01-01

    Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and afterpotentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of afterdepolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function. PMID:26334007

  16. Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells.

    PubMed

    Li, Guoshi; Linster, Christiane; Cleland, Thomas A

    2015-12-01

    Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and afterpotentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of afterdepolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function.

  17. Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish.

    PubMed

    Lacy, E R; Reale, E

    1989-07-01

    Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinacea), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphyrna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish.

  18. Comparison of the effects of barbiturate and ethanol given to neonates on the cerebellar morphology.

    PubMed

    Yanai, J; Waknin, S

    1985-01-01

    Previous studies from different laboratories have suggested that neonatal exposure to barbiturate and ethanol induces long-term changes in cerebellar morphology. The present study was designed to compare in similar conditions the effect of neonatal exposure to maximal doses of barbiturate or ethanol on cerebellar morphology. Phenobarbital was administered via daily injections of 50 mg/kg on neonatal days 2-21 (B group). Ethanol was similarly administered in doses of 3 g/kg (E3g) and the submaximal dose of 2 g/kg (E2g). At age 50 days, the cerebella of treated and control offspring were subjected to histological analysis. The sagittal areas of the cerebellar layers were similarly reduced compared to controls in both B and E3g groups. In addition, B and E3g groups exhibited a similar deficit in the number of the cerebellar Purkinje and granule neurons. As barbiturate and E3g, a submaximal dose of ethanol (B2g) induced a deficit in the number of cerebellar Purkinje cells. However, it did not affect the granule cells and the area of the cerebellar layers. The results suggest that under standardized conditions, barbiturate and ethanol have a similar potent neurotoxic effect on the cerebellum. That is, they both impair the development of the cerebellar layers to a similar extent and destroy neurons even after they have already formed.

  19. Formation of tRNA granules in the nucleus of heat-induced human cells

    SciTech Connect

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  20. Comparative study of cerebellar degeneration in canine neuroaxonal dystrophy, cerebellar cortical abiotrophy, and neuronal ceroid-lipofuscinosis.

    PubMed

    Nibe, Kazumi; Nakayama, Hiroyuki; Uchida, Kazuyuki

    2010-11-01

    The cerebellar lesions of three dogs with canine neuroaxonal dystrophy (NAD), one dog with cerebellar cortical abiotrophy (CCA), and 4 dogs with neuronal ceroid-lipofuscinosis (NCL) were examined to understand their pathogeneses. Purkinje cell loss was most severe in the vermis of a dog with CCA, and granule cell loss was most prominent in the cerebellar hemisphere of dogs with NCL. Immunohistochemically, CD3-and HLA-DR-positive cells were most frequent in the dogs with NCL, and moderate in dogs with NAD, but not in a dog with CCA. The number of cleaved caspase 3-positive cells was prominent in a dog with CCA, but no significant in the dogs with NAD. The results indicate different pathway of neuronal loss of these canine neuronal disorders.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-04

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

  3. 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

  4. Consensus Paper: Neuroimmune Mechanisms of Cerebellar Ataxias.

    PubMed

    Mitoma, Hiroshi; Adhikari, Keya; Aeschlimann, Daniel; Chattopadhyay, Partha; Hadjivassiliou, Marios; Hampe, Christiane S; Honnorat, Jérôme; Joubert, Bastien; Kakei, Shinji; Lee, Jongho; Manto, Mario; Matsunaga, Akiko; Mizusawa, Hidehiro; Nanri, Kazunori; Shanmugarajah, Priya; Yoneda, Makoto; Yuki, Nobuhiro

    2016-04-01

    In the last few years, a lot of publications suggested that disabling cerebellar ataxias may develop through immune-mediated mechanisms. In this consensus paper, we discuss the clinical features of the main described immune-mediated cerebellar ataxias and address their presumed pathogenesis. Immune-mediated cerebellar ataxias include cerebellar ataxia associated with anti-GAD antibodies, the cerebellar type of Hashimoto's encephalopathy, primary autoimmune cerebellar ataxia, gluten ataxia, Miller Fisher syndrome, ataxia associated with systemic lupus erythematosus, and paraneoplastic cerebellar degeneration. Humoral mechanisms, cell-mediated immunity, inflammation, and vascular injuries contribute to the cerebellar deficits in immune-mediated cerebellar ataxias.

  5. 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.

  6. 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

  7. Prox1 postmitotically defines dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus.

    PubMed

    Iwano, Tomohiko; Masuda, Aki; Kiyonari, Hiroshi; Enomoto, Hideki; Matsuzaki, Fumio

    2012-08-01

    The brain is composed of diverse types of neurons that fulfill distinct roles in neuronal circuits, as manifested by the hippocampus, where pyramidal neurons and granule cells constitute functionally distinct domains: cornu ammonis (CA) and dentate gyrus (DG), respectively. Little is known about how these two types of neuron differentiate during hippocampal development, although a set of transcription factors that is expressed in progenitor cells is known to be required for the survival of granule cells. Here, we demonstrate in mice that Prox1, a transcription factor constitutively expressed in the granule cell lineage, postmitotically functions to specify DG granule cell identity. Postmitotic elimination of Prox1 caused immature DG neurons to lose the granule cell identity and in turn terminally differentiate into the pyramidal cell type manifesting CA3 neuronal identity. By contrast, Prox1 overexpression caused opposing effects on presumptive hippocampal pyramidal cells. These results indicate that the immature DG cell has the potential to become a granule cell or a pyramidal cell, and Prox1 defines the granule cell identity. This bi-potency is lost in mature DG cells, although Prox1 is still required for correct gene expression in DG granule cells. Thus, our data indicate that Prox1 acts as a postmitotic cell fate determinant for DG granule cells over the CA3 pyramidal cell fate and is crucial for maintenance of the granule cell identity throughout the life.

  8. Hippocampal granule cell pathology in epilepsy - a possible structural basis for comorbidities of epilepsy?

    PubMed

    Hester, Michael S; Danzer, Steve C

    2014-09-01

    Temporal lobe epilepsy in both animals and humans is characterized by abnormally integrated hippocampal dentate granule cells. Among other abnormalities, these cells make axonal connections with inappropriate targets, grow dendrites in the wrong direction, and migrate to ectopic locations. These changes promote the formation of recurrent excitatory circuits, leading to the appealing hypothesis that these abnormal cells may by epileptogenic. While this hypothesis has been the subject of intense study, less attention has been paid to the possibility that abnormal granule cells in the epileptic brain may also contribute to comorbidities associated with the disease. Epilepsy is associated with a variety of general findings, such as memory disturbances and cognitive dysfunction, and is often comorbid with a number of other conditions, including schizophrenia and autism. Interestingly, recent studies implicate disruption of common genes and gene pathways in all three diseases. Moreover, while neuropsychiatric conditions are associated with changes in a variety of brain regions, granule cell abnormalities in temporal lobe epilepsy appear to be phenocopies of granule cell deficits produced by genetic mouse models of autism and schizophrenia, suggesting that granule cell dysmorphogenesis may be a common factor uniting these seemingly diverse diseases. Disruption of common signaling pathways regulating granule cell neurogenesis may begin to provide mechanistic insight into the cooccurrence of temporal lobe epilepsy and cognitive and behavioral disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Diffuse large B-cell lymphoma of stomach presenting with paraneoplastic cerebellar degeneration syndrome.

    PubMed

    Nomani, Ali Zohair; Wazir, Marina; Kashmir, Saba Binte; Qureshi, Muhammad Saleem

    2014-03-01

    Paraneoplastic syndromes are most often diagnosed in the setting of a known malignancy. It is not uncommon for a paraneoplastic disorder to develop before a cancer is identified. While syndrome of cerebellar degeneration has been identified as a paraneoplastic manifestation of Hodgkin's lymphoma, thymoma, lung and breast cancer, ovarian and testicular tumors, melanoma, renal cell carcinoma, follicular lymphoma and adenocarcinoma of stomach, its association with non-Hodgkin's lymphoma and particularly diffuse large B-cell lymphoma has not been established previously. This case report describes the primary presentation with signs of paraneoplastic cerebellar degeneration as the only manifestation of an underlying diffuse large B-cell lymphoma making it the first of its kind to be formally reported. Furthermore, it also includes the identification of associated paraneoplastic antibodies for this particular syndrome.

  10. Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy.

    PubMed

    Kobayashi, Masayuki; Buckmaster, Paul S

    2003-03-15

    Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were <40% of controls, and the frequency of GABA(A) receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was <50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin- and somatostatin-immunoreactive interneuron numbers and the frequency of both fast- and slow-rising GABA(A) receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

  11. Minocycline fails to protect cerebellar granular cell cultures against malonate-induced cell death.

    PubMed

    Fernandez-Gomez, F J; Gomez-Lazaro, M; Pastor, D; Calvo, S; Aguirre, N; Galindo, M F; Jordán, J

    2005-11-01

    Experimental and clinical studies support the view that the semisynthetic tetracycline minocycline exhibits neuroprotective roles in several models of neurodegenerative diseases, including ischemia, Huntington, Parkinson diseases, and amyotrophic lateral sclerosis. However, recent evidence indicates that minocycline does not always present beneficial actions. For instance, in an in vivo model of Huntington's disease, it fails to afford protection after malonate intrastriatal injection. Moreover, it reverses the neuroprotective effect of creatine in nigrostriatal dopaminergic neurons. This apparent contradiction prompted us to analyze the effect of this antibiotic on malonate-induced cell death. We show that, in rat cerebellar granular cells, the succinate dehydrogenase inhibitor malonate induces cell death in a concentration-dependent manner. By using DFCA, monochlorobimane and 10-N-nonyl-Acridin Orange to measure, respectively, H2O2-derived oxidant species and reduced forms of GSH and cardiolipin, we observed that malonate induced reactive oxygen species (ROS) production to an extent that surpasses the antioxidant defense capacity of the cells, resulting in GSH depletion and cardiolipin oxidation. The pre-treatment for 4 h with minocycline (10-100 microM) did not present cytoprotective actions. Moreover, minocycline failed to block ROS production and to abrogate malonate-induced oxidation of GSH and cardiolipin. Additional experiments revealed that minocycline was also unsuccessful to prevent the mitochondrial swelling induced by malonate. Furthermore, malonate did not induce the expression of the iNOS, caspase-3, -8, and -9 genes which have been shown to be up-regulated in several models where minocycline resulted cytoprotective. In addition, malonate-induced down-regulation of the antiapoptotic gene Bcl-2 was not prevented by minocycline, controversially the mechanism previously proposed to explain minocycline protective action. These results suggest that the

  12. Late-Onset Langerhans Cell Histiocytosis with Cerebellar Ataxia as an Initial Symptom

    PubMed Central

    Pyun, Jung-Min; Park, Hyeyoung; Moon, Kyung Chul; Jeon, Beomseok

    2016-01-01

    Late-onset progressive cerebellar ataxia is a diagnostic challenge because of a poor correlation between genotype and phenotype, and a broad range of secondary causes that extend beyond the neurological field. We report the case of a 45-year-old woman admitted after 2 years of slowly progressing cerebellar ataxia, dysarthria, and emotional instability. Notably, she was diagnosed with diabetes insipidus at the age of 35. As ‘idiopathic cerebellar ataxia’ was suspected, diagnostic tests, including genetic testing as well as serum and cerebrospinal fluid analyses, and brain magnetic resonance imaging (MRI) were performed. All results were normal except those of MRI, performed 9 months prior to admission, which showed multiple dot-like white matter lesions with unclear cause. On a repeated brain MRI, a new lesion presenting as a 1.5-cm-sized highly enhancing mass attached to the right frontal skull was found. A sharply marginated lytic skull defect was also evident on skull X-ray, which corresponded to the lesion mass. Given these new radiological findings, a systemic review of the patient's medical history for rare secondary causes of cerebellar ataxia was performed, with particular attention to her past ‘diabetes insipidus’. The mass, lytic lesion of the skull, white matter lesion, diabetes insipidus, and cerebellar ataxia all suggested a final diagnosis of Langerhans cell histiocytosis (LCH), which was confirmed histopathologically. This is a rare case of late-onset LCH with an unusual initial symptom which underlines the importance of carefully reviewing the patient's medical history and broadening the search for etiologies beyond the nervous system. PMID:27920713

  13. Control of a simulated arm using a novel combination of Cerebellar learning mechanisms

    NASA Technical Reports Server (NTRS)

    Assad, C.; Hartmann, M.; Paulin, M. G.

    2001-01-01

    We present a model of cerebellar cortex that combines two types of learning: feedforward predicitve association based on local Hebbian-type learning between granule cell ascending branch and parallel fiber inputs, and reinforcement learning with feedback error correction based on climbing fiber activity.

  14. Control of a simulated arm using a novel combination of Cerebellar learning mechanisms

    NASA Technical Reports Server (NTRS)

    Assad, C.; Hartmann, M.; Paulin, M. G.

    2001-01-01

    We present a model of cerebellar cortex that combines two types of learning: feedforward predicitve association based on local Hebbian-type learning between granule cell ascending branch and parallel fiber inputs, and reinforcement learning with feedback error correction based on climbing fiber activity.

  15. Two modes of lytic granule fusion during degranulation by natural killer cells.

    PubMed

    Liu, Dongfang; Martina, Jose A; Wu, Xufeng S; Hammer, John A; Long, Eric O

    2011-08-01

    Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor-ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

  16. [A case of paraneoplastic cerebellar degeneration associated with small cell lung cancer].

    PubMed

    Nakashima, N; Takayama, K; Nakanishi, Y; Ishihara, S; Tanaka, T; Kaneko, Y; Takano, K; Inoue, K; Hara, N

    1999-02-01

    Paraneoplastic cerebellar degeneration (PCD) is a clinical syndrome and known to be occasionally associated with small cell carcinoma of the lung (SCLC). PCD usually affects patients before the cancer is evident. The disorder evolves subacutely, and causes severe pancerebellar dysfunction. In this paper, we report a case of PCD associated with SCLC. A 65-year-old man presenting with 2 weeks of progressive vertigo, gait ataxia, and speech disturbance, was readmitted to our hospital. He had earlier been given a diagnosis of SCLC, oat cell carcinoma, and had undergone high-dose chemotherapy with peripheral blood stem cell transplantation during his first admission. Following that treatment regimen, the tumor disappeared completely and the patient had been in remission. Based on neurological findings and the presence of anti-neuronal antibodies a diagnosis of PCD was made. Although cyclophosphamide (500 mg/m2) was administered, the patient experienced no relief of his cerebellar ataxia. Six months afer readmission, he died of cardiac tamponade due to malignant pericarditis. A histological examination at autopsy found few Purkinje cells and a proliferation of Bergmann's astrocytes in the cerebellar cortex. These findings were consistent with the diagnosis of PCD.

  17. 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

  18. Cerebellar Dysfunction in a Patient with HIV.

    PubMed

    Gonzalez-Ibarra, Fernando; Abdul, Waheed; Eivaz-Mohammadi, Sahar; Foscue, Christopher; Gongireddy, Srinivas; Syed, Amer

    2014-01-01

    A 50-year-old AIDS patient with a CD4 T-cell count of 114/mm(3) was admitted with cerebellar symptoms of left CN XI weakness, wide-based gait with left-sided dysmetria, abnormal heel-knee-shin test, and dysdiadochokinesia. MRI showed region of hyperintensity in the left inferior cerebellar hemisphere involving the cortex and underlying white matter. Serological tests for HSV1, HSV2, and syphilis were negative. Her CSF contained high protein content and a WBC of 71/mm(3), predominantly lymphocytes. The CSF was also negative for cryptococcal antigen and VDRL. CSF culture did not grow microbes. CSF PCR assay was negative for HSV1 and HSV2 but was positive for JC virus (1,276 copies). The most likely diagnosis is granule cell neuronopathy (GCN), which can only be definitively confirmed with biopsy and immunohistochemistry.

  19. Secretory granules of mast cells accumulate mature and immature MHC class II molecules.

    PubMed

    Vincent-Schneider, H; Théry, C; Mazzeo, D; Tenza, D; Raposo, G; Bonnerot, C

    2001-01-01

    Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.

  20. Bone marrow derived mesenchymal stem cell transplantation in cerebellar degeneration: a behavioral study.

    PubMed

    Edalatmanesh, Mohammad Amin; Bahrami, Ahmad Reza; Hosseini, Ebrahim; Hosseini, Mahmoud; Khatamsaz, Saeid

    2011-11-20

    In addition to its key role in complex motor function, the cerebellum is increasingly recognized to have a role in cognition. Thus, motor and cognitive deficits can be associated with cerebellar degeneration. After unilateral lesion in cerebellum (folia VI) was caused by Quinolinic acid, CM-DiI labeled mesenchymal stem cells (MSCs), which were isolated and purified from bone marrow, were transplanted into the damaged folium. Motor function was assessed using the cylinder test, rotarod, hanging wire and beam balance during 6 weeks after transplantation. Cognitive function was assessed using the Morris water maze learning paradigm in 3 weeks after transplantation. Six weeks after transplantation surviving MSCs were detectable in QA-treated animals. The MSC-transplanted group showed markedly improved functional performance in spatial memory, motor learning, locomotor asymmetry, dysmetria, abnormality in neuromuscular strength and equilibrium 2-6 weeks compared with the controls. We found that cerebellar lesions produced deficits (folia VI) in motor and cognitive aspects of a spatial task. The results indicate that transplantation of MSCs can significantly reduce the behavioral abnormalities of these animals during six weeks after engraftment. According to results of this assay, cell therapy by means of bone marrow derived adult stem cells promises for treatment of cerebellar diseases.

  1. EFFECTS OF ORGANOTINS ON RACTIVE OXYGEN SPECIES AND INTRACELLULAR CALCIUM IN CEREBELLAR GRANULE CELLS IN CULTURE.

    EPA Science Inventory

    Use of organotins has increased drastically in the past decade, including their use as stabilizers in polyvinylchloride pipes. Monomethyl- (MMT), dimethyl- (DMT), monobutyl- (MBT), and dibutyltin (DBT) have been found in home water samples and in human blood at concentrations up...

  2. EFFECTS OF ORGANOTINS ON RACTIVE OXYGEN SPECIES AND INTRACELLULAR CALCIUM IN CEREBELLAR GRANULE CELLS IN CULTURE.

    EPA Science Inventory

    Use of organotins has increased drastically in the past decade, including their use as stabilizers in polyvinylchloride pipes. Monomethyl- (MMT), dimethyl- (DMT), monobutyl- (MBT), and dibutyltin (DBT) have been found in home water samples and in human blood at concentrations up...

  3. Excitatory Cerebellar Nucleocortical Circuit Provides Internal Amplification during Associative Conditioning

    PubMed Central

    Gao, Zhenyu; Proietti-Onori, Martina; Lin, Zhanmin; ten Brinke, Michiel M.; Boele, Henk-Jan; Potters, Jan-Willem; Ruigrok, Tom J.H.; Hoebeek, Freek E.; De Zeeuw, Chris I.

    2016-01-01

    Summary Closed-loop circuitries between cortical and subcortical regions can facilitate precision of output patterns, but the role of such networks in the cerebellum remains to be elucidated. Here, we characterize the role of internal feedback from the cerebellar nuclei to the cerebellar cortex in classical eyeblink conditioning. We find that excitatory output neurons in the interposed nucleus provide efference-copy signals via mossy fibers to the cerebellar cortical zones that belong to the same module, triggering monosynaptic responses in granule and Golgi cells and indirectly inhibiting Purkinje cells. Upon conditioning, the local density of nucleocortical mossy fiber terminals significantly increases. Optogenetic activation and inhibition of nucleocortical fibers in conditioned animals increases and decreases the amplitude of learned eyeblink responses, respectively. Our data show that the excitatory nucleocortical closed-loop circuitry of the cerebellum relays a corollary discharge of premotor signals and suggests an amplifying role of this circuitry in controlling associative motor learning. PMID:26844836

  4. Common pediatric cerebellar tumors: correlation between cell densities and apparent diffusion coefficient metrics.

    PubMed

    Koral, Korgün; Mathis, Derek; Gimi, Barjor; Gargan, Lynn; Weprin, Bradley; Bowers, Daniel C; Margraf, Linda

    2013-08-01

    To test whether there is correlation between cell densities and apparent diffusion coefficient (ADC) metrics of common pediatric cerebellar tumors. This study was reviewed for issues of patient safety and confidentiality and was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center and was compliant with HIPAA. The need for informed consent was waived. Ninety-five patients who had preoperative magnetic resonance imaging and surgical pathologic findings available between January 2003 and June 2011 were included. There were 37 pilocytic astrocytomas, 34 medulloblastomas (23 classic, eight desmoplastic-nodular, two large cell, one anaplastic), 17 ependymomas (13 World Health Organization [WHO] grade II, four WHO grade III), and seven atypical teratoid rhabdoid tumors. ADCs of solid tumor components and normal cerebellum were measured. Tumor-to-normal brain ADC ratios (hereafter, ADC ratio) were calculated. The medulloblastomas and ependymomas were subcategorized according to the latest WHO classification, and tumor cellularity was calculated. Correlation was sought between cell densities and mean tumor ADCs, minimum tumor ADCs, and ADC ratio. When all tumors were considered together, negative correlation was found between cellularity and mean tumor ADCs (ρ = -0.737, P < .05) and minimum tumor ADCs (ρ = -0.736, P < .05) of common pediatric cerebellar tumors. There was no correlation between cellularity and ADC ratio. Negative correlation was found between cellularity and minimum tumor ADC in atypical teratoid rhabdoid tumors (ρ = -0.786, P < .05). In atypical teratoid rhabdoid tumors, no correlation was found between cellularity and mean tumor ADC and ADC ratio. There was no correlation between the ADC metrics and cellularity of the pilocytic astrocytomas, medulloblastomas, and ependymomas. Negative correlation was found between cellularity and ADC metrics of common pediatric cerebellar tumors. Although ADC metrics are

  5. Ablation of Newly Generated Hippocampal Granule Cells Has Disease-Modifying Effects in Epilepsy

    PubMed Central

    Hosford, Bethany E.; Liska, John P.

    2016-01-01

    Hippocampal granule cells generated in the weeks before and after an epileptogenic brain injury can integrate abnormally into the dentate gyrus, potentially mediating temporal lobe epileptogenesis. Previous studies have demonstrated that inhibiting granule cell production before an epileptogenic brain insult can mitigate epileptogenesis. Here, we extend upon these findings by ablating newly generated cells after the epileptogenic insult using a conditional, inducible diphtheria-toxin receptor expression strategy in mice. Diphtheria-toxin receptor expression was induced among granule cells born up to 5 weeks before pilocarpine-induced status epilepticus and these cells were then eliminated beginning 3 d after the epileptogenic injury. This treatment produced a 50% reduction in seizure frequency, but also a 20% increase in seizure duration, when the animals were examined 2 months later. These findings provide the first proof-of-concept data demonstrating that granule cell ablation therapy applied at a clinically relevant time point after injury can have disease-modifying effects in epilepsy. SIGNIFICANCE STATEMENT These findings support the long-standing hypothesis that newly generated dentate granule cells are pro-epileptogenic and contribute to the occurrence of seizures. This work also provides the first evidence that ablation of newly generated granule cells can be an effective therapy when begun at a clinically relevant time point after an epileptogenic insult. The present study also demonstrates that granule cell ablation, while reducing seizure frequency, paradoxically increases seizure duration. This paradoxical effect may reflect a disruption of homeostatic mechanisms that normally act to reduce seizure duration, but only when seizures occur frequently. PMID:27798182

  6. Ablation of Newly Generated Hippocampal Granule Cells Has Disease-Modifying Effects in Epilepsy.

    PubMed

    Hosford, Bethany E; Liska, John P; Danzer, Steve C

    2016-10-26

    Hippocampal granule cells generated in the weeks before and after an epileptogenic brain injury can integrate abnormally into the dentate gyrus, potentially mediating temporal lobe epileptogenesis. Previous studies have demonstrated that inhibiting granule cell production before an epileptogenic brain insult can mitigate epileptogenesis. Here, we extend upon these findings by ablating newly generated cells after the epileptogenic insult using a conditional, inducible diphtheria-toxin receptor expression strategy in mice. Diphtheria-toxin receptor expression was induced among granule cells born up to 5 weeks before pilocarpine-induced status epilepticus and these cells were then eliminated beginning 3 d after the epileptogenic injury. This treatment produced a 50% reduction in seizure frequency, but also a 20% increase in seizure duration, when the animals were examined 2 months later. These findings provide the first proof-of-concept data demonstrating that granule cell ablation therapy applied at a clinically relevant time point after injury can have disease-modifying effects in epilepsy. These findings support the long-standing hypothesis that newly generated dentate granule cells are pro-epileptogenic and contribute to the occurrence of seizures. This work also provides the first evidence that ablation of newly generated granule cells can be an effective therapy when begun at a clinically relevant time point after an epileptogenic insult. The present study also demonstrates that granule cell ablation, while reducing seizure frequency, paradoxically increases seizure duration. This paradoxical effect may reflect a disruption of homeostatic mechanisms that normally act to reduce seizure duration, but only when seizures occur frequently. Copyright © 2016 the authors 0270-6474/16/3611013-11$15.00/0.

  7. Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

    PubMed

    Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A

    2008-04-01

    Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

  8. Cerebellar Ataxia.

    PubMed

    Perlman

    2000-05-01

    urinary tract infections, aspiration, occult respiratory failure, and obstructive sleep apnea, all of which can be life threatening. Depression in the patient and family members is common. Although no cures exist for most of the causes of cerebellar ataxia and there are as yet no proven ways to protect neurons from premature cell death or to restore neuronal populations that have been lost, symptomatic treatment can greatly improve the quality of life of these patients and prevent complications that could hasten death. Supportive interventions should always be offered-- education about the disease itself, genetic counseling, individual and family counseling, referral to support groups and advocacy groups, and guidance to online resources. Misinformation, fear, depression, hopelessness, isolation, and financial and interpersonal stress can often cause more harm to the patient and caregiver than the ataxia itself.

  9. Quantification of Cerebellar Hemispheric Purkinje Cell Linear Density: 32 ET Cases vs. 16 Controls

    PubMed Central

    Louis, Elan D.; Babij, Rachel; Lee, Michelle; Cortés, Etty; Vonsattel, Jean-Paul G.

    2013-01-01

    Background Although essential tremor (ET) is among the most prevalent neurological diseases, its precise pathogenesis is not understood. Purkinje cell loss has been observed in some studies and is the focus of interest and debate. Expressing these data as Purkinje cells/layer length allows one to adjust for the inherent curved nature of the cerebellar folia. Capitalizing on the Essential Tremor Centralized Brain Repository, we quantified Purkinje cell linear density in cases vs. controls. Methods Free-floating, 100 μm, parasagittal cerebellar hemispheric sections were subjected to rabbit polyclonal anti-Calbindin D28k antibody, and 10 random fields/brain were selected for quantification of Purkinje cells/mm−1 Purkinje cell layer. Results Purkinje cell linear density was lower in 32 ET cases than 16 controls (1.14 ± 0.32 vs. 1.35 ± 0.31 per mm−1, p = 0.03). Purkinje cell linear density was inversely associated with torpedo count (r = −0.38, p = 0.028). Discussion The current sample of ET cases demonstrates a reduction in Purkinje cell number relative to that of controls. Greater Purkinje cell axonal remodeling (torpedoes) was found in individuals who had the most Purkinje cell drop out. The role of Purkinje cell loss in the pathogenesis of this disorder merits additional study. PMID:23925732

  10. Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1987-04-01

    The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and (/sup 14/C)methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca/sup 2 +/ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.

  11. Purkinje cells in posterior cerebellar vermis encode motion in an inertial reference frame.

    PubMed

    Yakusheva, Tatyana A; Shaikh, Aasef G; Green, Andrea M; Blazquez, Pablo M; Dickman, J David; Angelaki, Dora E

    2007-06-21

    The ability to orient and navigate through the terrestrial environment represents a computational challenge common to all vertebrates. It arises because motion sensors in the inner ear, the otolith organs, and the semicircular canals transduce self-motion in an egocentric reference frame. As a result, vestibular afferent information reaching the brain is inappropriate for coding our own motion and orientation relative to the outside world. Here we show that cerebellar cortical neuron activity in vermal lobules 9 and 10 reflects the critical computations of transforming head-centered vestibular afferent information into earth-referenced self-motion and spatial orientation signals. Unlike vestibular and deep cerebellar nuclei neurons, where a mixture of responses was observed, Purkinje cells represent a homogeneous population that encodes inertial motion. They carry the earth-horizontal component of a spatially transformed and temporally integrated rotation signal from the semicircular canals, which is critical for computing head attitude, thus isolating inertial linear accelerations during navigation.

  12. Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

    PubMed

    Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

    2000-01-01

    The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

  13. 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.

  14. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed Central

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-01-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells. PMID:8751945

  15. Ultrastructural pathology of human peritumoural oedematous cerebellar cortex.

    PubMed

    Castejón, O J

    2016-01-01

    Cerebellar cortical biopsies of the peritumoural region of seven patients with cerebellar haemangioma, mesencephalic meningioma, cerebellopontine astrocytoma, cerebellopontine meningioma, and medulloblastoma of cerebellar vermis were examined by means of conventional transmission electron microscopy. Granule cells showed oedematous cytoplasm and mitochondria. Swollen Golgi cells exhibited lipofuscin granules and intranuclear inclusions. Both neuron cell types displayed swollen dendritic digits synapsing with afferent mossy fibre endings. Degenerated myelinated axons corresponding to afferent mossy and climbing fibres and efferent Purkinje cell axons were observed at the granular layer. Dense and clear ischaemic Purkinje cells established degenerated synapses with swollen parallel fibre synaptic varicosities. Degenerated Purkinje cell recurrent axonal collaterals were found at the molecular layer. Swollen and clear Bergmann glial cell cytoplasm was observed closely applied to the oedematous clear and dark Purkinje cell body, dendritic trunk, secondary and tertiary dendritic branches. Swollen climbing fibre endings featured by numerous microtubules and neurofilaments, and a decreased number of synaptic vesicles were observed making degenerated axo-spinodendritic synapses with clear and swollen dendritic spines from Purkinje, Golgi, basket and stellate cell dendrites. Swollen stellate neurons showed oedematous mitochondria. Lipofuscin-rich astrocytes and reactive phagocytic astrocytes were observed. The latter appeared engulfing haematogenous proteinaceous oedema fluid. All cerebellar neurons showed stress endoplasmic reticulum dysfunction featured by focal dilated cisterns and detachment of associated ribosomes. Myelin sheath degeneration was related with oligodendrocyte degenerating hydropic changes. The peritumoural ischaemic cerebellar nerve and glial cell abnormalities were related with neurobehavioral changes, tremor, nystagmus, dismetry and gait disturbance

  16. Disrupted Dentate Granule Cell Chloride Regulation Enhances Synaptic Excitability during Development of Temporal Lobe Epilepsy

    PubMed Central

    Pathak, Hemal R.; Weissinger, Florian; Terunuma, Miho; Carlson, Gregory C.; Hsu, Fu-Chun; Moss, Stephen J.; Coulter, Douglas A.

    2008-01-01

    GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl− concentration ([Cl−]in ) at low levels. In neurons in the developing CNS, [Cl−]in is elevated, EGABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1–60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that EGABA was positively shifted in granule cells. This shift in EGABA altered synaptic integration, increased granule cell excitability, and resulted in compromised “gate” function of the dentate gyrus. EGABA recovered to control values at longer latencies post-STEP (2–8 weeks), when animals had developed epilepsy. During this period of shifted EGABA, expression of the Cl− extruding K+/Cl− cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked EGABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on EGABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy. PMID:18094240

  17. Depletion of primary cilia from mature dentate granule cells impairs hippocampus-dependent contextual memory

    PubMed Central

    Rhee, Soyoung; Kirschen, Gregory W.; Gu, Yan; Ge, Shaoyu

    2016-01-01

    The primary cilium, a sensory organelle, regulates cell proliferation and neuronal development of dentate granule cells in the hippocampus. However, its role in the function of mature dentate granule cells remains unknown. Here we specifically depleted and disrupted ciliary proteins IFT20 and Kif3A (respectively) in mature dentate granule cells and investigated hippocampus-dependent contextual memory and long-term plasticity at mossy fiber synapses. We found that depletion of IFT20 in these cells significantly impaired context-dependent fear-related memory. Furthermore, we tested synaptic plasticity of mossy fiber synapses in area CA3 and found increased long-term potentiation upon depletion of IFT20 or disruption of Kif3A. Our findings suggest a role of primary cilia in the memory function of mature dentate granule cells, which may result from abnormal mossy fiber synaptic plasticity. A direct link between the primary cilia of mature dentate granule cells and behavior will require further investigation using independent approaches to manipulate primary cilia. PMID:27678193

  18. Otolith organ or semicircular canal stimulation induces c-fos expression in unipolar brush cells and granule cells of cat and squirrel monkey.

    PubMed

    Sekerková, Gabriella; Ilijic, Ema; Mugnaini, Enrico; Baker, James F

    2005-07-01

    Immediate early gene expression in the cerebellar vermis of cats and squirrel monkeys was stimulated by prolonged whole body rotations. Continuous, earth-horizontal axis rotations that excited only otoliths or high velocity vertical axis rotations that excited only semicircular canals resulted in c-fos immunoreactive nuclei concentrated in the granular layer of lobules X and ventral IX (the nodulus and ventral uvula), which represent the medial parts of the vestibulo-cerebellum. Large clusters of labeled nuclei consisting mainly of granule cells and calretinin-positive unipolar brush cells were present in the granular layer, whereas Purkinje cell nuclei were unlabeled, and labeled basket and stellate cell nuclei were scattered in the molecular layer. In other vermal lobules there was a significant but less dense label than in the nodulus and ventral uvula. Generally, the extent of c-fos labeling of molecular layer interneurons was in relation to nuclear labeling of granular layer neurons: labeling of both basket and stellate cells accompanied nuclear labeling of neurons throughout the depth of the granular layer, whereas only stellate cells were labeled when nuclear labeling was restricted to the superficial granular layer. Yaw horizontal or roll vertical rotations each stimulated c-fos expression in the cat medial vestibulo-cerebellum to approximately the same extent. Low-velocity rotations resulted in much less c-fos expression. Similar, albeit less intense, patterns of c-fos activation were observed in monkeys. Concentrated c-fos expression in the medial vestibulo-cerebellum after exposure to a strong head velocity signal that could originate from either otolith or canal excitation suggests that granule and unipolar brush cells participate in a neuronal network for estimating head velocity, irrespective of the signal source.

  19. FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

    SciTech Connect

    Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki

    2015-08-07

    The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.

  20. Altered morphology of hippocampal dentate granule cell presynaptic and postsynaptic terminals following conditional deletion of TrkB.

    PubMed

    Danzer, Steve C; Kotloski, Robert J; Walter, Cynthia; Hughes, Maya; McNamara, James O

    2008-01-01

    Dentate granule cells play a critical role in the function of the entorhinal-hippocampal circuitry in health and disease. Dentate granule cells are situated to regulate the flow of information into the hippocampus, a structure required for normal learning and memory. Correspondingly, impaired granule cell function leads to memory deficits, and, interestingly, altered granule cell connectivity may contribute to the hyperexcitability of limbic epilepsy. It is important, therefore, to understand the molecular determinants of synaptic connectivity of these neurons. Brain-derived neurotrophic factor and its receptor TrkB are expressed at high levels in the dentate gyrus (DG) of the hippocampus, and are implicated in regulating neuronal development, neuronal plasticity, learning, and the development of epilepsy. Whether and how TrkB regulates granule cell structure, however, is incompletely understood. To begin to elucidate the role of TrkB in regulating granule cell morphology, here we examine conditional TrkB knockout mice crossed to mice expressing green fluorescent protein in subsets of dentate granule cells. In stratum lucidum, where granule cell mossy fiber axons project, the density of giant mossy fiber boutons was unchanged, suggesting similar output to CA3 pyramidal cell targets. However, filopodial extensions of giant boutons, which contact inhibitory interneurons, were increased in number in TrkB knockout mice relative to wildtype controls, predicting enhanced feedforward inhibition of CA3 pyramidal cells. In knockout animals, dentate granule cells possessed fewer primary dendrites and enlarged dendritic spines, indicative of disrupted excitatory synaptic input to the granule cells. Together, these findings demonstrate that TrkB is required for development and/or maintenance of normal synaptic connectivity of the granule cells, thereby implying an important role for TrkB in the function of the granule cells and hippocampal circuitry.

  1. Clumsiness and disturbed cerebellar development: insights from animal experiments.

    PubMed

    Gramsbergen, Albert

    2003-01-01

    Cerebellar functioning has been implied in the fine adjustments of muscle tone, in the coordination and the feed-forward control of movements and posture, as well as in the establishment and performance of motor skills. The cerebellar cortex in mammals develops late in neuro-ontogeny and an extrapolation from experimental results indicates that in the human the proliferation of the granule cells and the development of circuitry in the cerebellar cortex starts only in the last trimester of pregnancy and lasts until beyond the first birthday. This late development makes the cerebellar development particularly vulnerable to situations like an insufficient supply of nutrients, which may follow placental dysfunction, or to side effects of pharmacological treatments like the administration of corticosteroids in the postnatal period. We studied whether such situations might also lead to motor impairments. In rats, the effects of undernutrition during the brain growth spurt were investigated as well as those of corticosteroids administered in a period that is analogous to the 7th to 8th month of pregnancy in the human. Both these interferences affect cerebellar development and our results in rats indicate that they also lead to retardations in the emergence of certain reflexes, as well as to longer lasting motor impairments during locomotion. Extrapolation of these results strongly suggests that a disturbed cerebellar development should be considered as an important etiological factor in clumsiness in human children.

  2. Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado-Joseph disease mice.

    PubMed

    Mendonça, Liliana S; Nóbrega, Clévio; Hirai, Hirokazu; Kaspar, Brian K; Pereira de Almeida, Luís

    2015-02-01

    Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.

  3. An LRRTM4-HSPG complex mediates excitatory synapse development on dentate gyrus granule cells.

    PubMed

    Siddiqui, Tabrez J; Tari, Parisa Karimi; Connor, Steven A; Zhang, Peng; Dobie, Frederick A; She, Kevin; Kawabe, Hiroshi; Wang, Yu Tian; Brose, Nils; Craig, Ann Marie

    2013-08-21

    Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.

  4. Olfactory experiences dynamically regulate plasticity of dendritic spines in granule cells of Xenopus tadpoles in vivo

    PubMed Central

    Zhang, Li; Huang, Yubin; Hu, Bing

    2016-01-01

    Granule cells, rich in dendrites with densely punctated dendritic spines, are the most abundant inhibitory interneurons in the olfactory bulb. The dendritic spines of granule cells undergo remodeling during the development of the nervous system. The morphological plasticity of the spines’ response to different olfactory experiences in vivo is not fully known. In initial studies, a single granule cell in Xenopus tadpoles was labeled with GFP plasmids via cell electroporation; then, morphologic changes of the granule cell spines were visualized by in vivo confocal time-lapse imaging. With the help of long-term imaging, the total spine density, dynamics, and stability of four types of dendritic spines (mushroom, stubby, thin and filopodia) were obtained. Morphological analysis demonstrated that odor enrichment produced a remarkable increase in the spine density and stability of large mushroom spine. Then, with the help of short-term imaging, we analyzed the morphological transitions among different spines. We found that transitions between small spines (thin and filopodia) were more easily influenced by odor stimulation or olfactory deprivation. These results indicate that different olfactory experiences can regulate the morphological plasticity of different dendritic spines in the granule cell. PMID:27713557

  5. Perinuclear P granules are the principal sites of mRNA export in adult C. elegans germ cells

    PubMed Central

    Sheth, Ujwal; Pitt, Jason; Dennis, Shannon; Priess, James R.

    2010-01-01

    Germline-specific granules of unknown function are found in a wide variety of organisms, including C. elegans, where they are called P granules. P granules are cytoplasmic bodies in oocytes and early embryos. Throughout most of the C. elegans life cycle, however, P granules are associated with clusters of nuclear pore complexes (NPCs) on germ cell nuclei. We show that perinuclear P granules differ from cytoplasmic P granules in many respects, including structure, stability and response to metabolic changes. Our results suggest that nuclear-associated P granules provide a perinuclear compartment where newly exported mRNAs are collected prior to their release to the general cytoplasm. First, we show that mRNA export factors are highly enriched at the NPCs associated with P granules. Second, we discovered that the expression of high-copy transgenes could be induced in a subset of germ cells, and used this system to demonstrate that nascent mRNA traffics directly to P granules. P granules appear to sequester large amounts of mRNA in quiescent germ cells, presumably preventing translation of that mRNA. However, we did not find evidence that P granules normally sequester aberrant mRNAs, or mRNAs targeted for destruction by the RNAi pathway. PMID:20223759

  6. Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase.

    PubMed

    Bembenek, Joshua N; Richie, Christopher T; Squirrell, Jayne M; Campbell, Jay M; Eliceiri, Kevin W; Poteryaev, Dmitry; Spang, Anne; Golden, Andy; White, John G

    2007-11-01

    In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

  7. Cadherins in cerebellar development: translation of embryonic patterning into mature functional compartmentalization.

    PubMed

    Redies, Christoph; Neudert, Franziska; Lin, Juntang

    2011-09-01

    Cadherins are cell adhesion molecules with multiple morphogenic functions in brain development, for example, in neuroblast migration and aggregation, axon navigation, neural circuit formation, and synaptogenesis. More than 100 members of the cadherin superfamily are expressed in the developing and mature brain. Most of the cadherins investigated, in particular classic cadherins and δ-protocadherins, are expressed in the cerebellum. For several cadherin subtypes, expression begins at early embryonic stages and persists until mature stages of cerebellar development. At intermediate stages, distinct Purkinje cell clusters exhibit unique rostrocaudal and mediolateral expression profiles for each cadherin. In the chicken, mouse, and other species, the Purkinje cell clusters are separated by intervening raphes of migrating granule cells. This pattern of Purkinje cell clusters/raphes is, at least in part, continuous with the parasagittal striping pattern that is apparent in the mature cerebellar cortex, for example, for zebrin II/aldolase C. Moreover, subregions of the deep cerebellar nuclei, vestibular nuclei and the olivary complex also express cadherins differentially. Neuroanatomical evidence suggests that the nuclear subregions and cortical domains that express the same cadherin subtype are connected to each other, to form neural subcircuits of the cerebellar system. Cadherins thus provide a molecular code that specifies not only embryonic structures but also functional cerebellar compartmentalization. By following the implementation of this code, it can be revealed how mature functional architecture emerges from embryonic patterning during cerebellar development. Dysfunction of some cadherins is associated with psychiatric diseases and developmental impairments and may also affect cerebellar function.

  8. Monitoring Performance Degradation of Cerebellar Functions Using Computational Neuroscience Methods: Implications on Neurological Diseases

    PubMed Central

    Nawrocki, Robert A.; Shaalan, Majid; Shaheen, Sean E.; Lorenzon, Nancy M.

    2012-01-01

    Neurodegeneration is a major cause of human disease. Within the cerebellum, neuronal degeneration and/or dysfunction has been associated with many diseases, including several forms of cerebellar ataxia, since normal cerebellar function is paramount for proper motor coordination, balance, and motor learning. The cerebellum represents a well-established neural circuit. Determining the effects of neuronal loss is of great importance for understanding the fundamental workings of the cerebellum and disease-associated dysfunctions. This paper presents computational modeling of cerebellar function in relation to neurodegeneration either affecting a specific cerebellar cell type, such as granule cells or Purkinje cells, or more generally affecting cerebellar cells and the implications on effects in relation to performance degradation throughout the progression of cell death. The results of the models show that the overall number of cells, as a percentage of the total cell number in the model, of a particular type and, primarily, their proximity to the circuit output, and not the neuronal convergence due to the relative number of cells of a particular type, is the main indicator of the gravity of the functional deficit caused by the degradation of that cell type. Specifically, the greater the percentage loss of neurons of a specific type and the closer proximity of those cells to the deep cerebellar neurons, the greater the deficit caused by the neuronal cell loss. These findings contribute to the understanding of the functional consequences of neurodegeneration and the functional importance of specific connectivity within a neuronal circuit. PMID:23029114

  9. The role of serotonin in cerebellar development.

    PubMed

    Oostland, M; van Hooft, J A

    2013-09-17

    In adult animals, the cerebellum is richly innervated by serotonin: serotonergic fibres are the third main afferent fibres into the cerebellum. However, the physiology of the serotonergic system and its functional significance are not fully known during development in the cerebellum. In this review we will focus on the serotonergic regulation of the cerebellum during postnatal development. We hypothesize a powerful role for serotonin in the physiology of the developing cerebellum. A presumably tonic activation of serotonin receptors by binding of serotonin becomes specific by temporally and spatially restricted expression of different serotonin receptors, each with their own (sometimes antagonizing) functions. During the first postnatal week, activation of 5-HT₁ receptors expressed by both granule cells and Purkinje cells stimulates dendritic growth and synapse formation. Later, activation of 5-HT₃ receptors expressed by granule cells limits dendritic growth of Purkinje cells via mediating the secretion of reelin, influences physiological maturation of Purkinje cells, modulates synaptic plasticity at parallel fibre-Purkinje cell synapses and thereby affects competition with the climbing fibres on Purkinje cell dendrites resulting in proper climbing fibre elimination. Last, activation of 5-HT₂ receptors expressed by granule cells and Purkinje cells both during late postnatal development and in the mature cerebellum promotes the stability of synaptic activity. Thus, we propose that serotonin controls cerebellar development in three phases: (1) stimulation of dendritic growth and formation of synapses, (2) hard-wiring of neuronal connections with limits to dendritic growth but ensuring synaptic plasticity, and (3) stabilization of synapses. Taken together, serotonin receptors expressed by different cells in the cerebellum have a specialized role during postnatal development, but with some similar main effects. Distinct spatial and temporal expression of these

  10. Role of LAT in the Granule-Mediated Cytotoxicity of CD8 T Cells

    PubMed Central

    Ou-Yang, Chih-wen; Zhu, Minghua; Fuller, Deirdre M.; Sullivan, Sarah A.; Chuck, Mariana I.; Ogden, Sarah

    2012-01-01

    Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is essential to bridge T cell receptor (TCR) engagement to downstream signaling events. The indispensable role of LAT in thymocyte development and T cell activation has been well characterized; however, the function of LAT in cytotoxic-T-lymphocyte (CTL) cytotoxicity remains unknown. We show here that LAT-deficient CTLs failed to upregulate FasL and produce gamma interferon after engagement with target cells and had impaired granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT deficiency led to altered synapse formation, subsequently causing unstable T cell–antigen-presenting cell (APC) conjugates. Microtubule organizing center polarization and granule reorientation were also impaired by LAT deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and phospholipase C (PLC) activity. Our data demonstrated that LAT-mediated signaling intricately regulates CTL cytotoxicity at multiple steps. PMID:22566687

  11. Ganglioside GD3 monoclonal antibody-induced paxillin tyrosine phosphorylation and filamentous actin assembly in cerebellar growth cones.

    PubMed

    Yuyama, Kohei; Sekino-Suzuki, Naoko; Yamamoto, Naomasa; Kasahara, Kohji

    2011-03-01

    We have demonstrated that antibody to ganglioside GD3 (R24) immunoprecipitates src-family tyrosine kinase Lyn from primary cerebellar granule cells and R24 treatment of the intact cells induces Lyn activation and rapid tyrosine phosphorylation of several substrates, suggesting the functional association of ganglioside GD3 with Lyn. In this study, R24 treatment of primary cerebellar granule cells enhances phosphorylation of paxillin at tyrosine residue 118 and induces filamentous actin assembly and neurite outgrowth. R24 treatment of cerebellar growth cone membrane fraction induces prominent tyrosine phosphorylation of 68 kDa protein which comigrates with phosphopaxillin at tyrosine residue 118. Tyrosine phosphorylation of paxillin is known to regulate actin cytoskeleton-dependent changes in cell morphology. Signal transduction by ganglioside GD3 is involved in growth cone morphology via tyrosine phosphorylation of paxillin. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  12. 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

  13. Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

    PubMed

    Shah, Khyati H; Nostramo, Regina; Zhang, Bo; Varia, Sapna N; Klett, Bethany M; Herman, Paul K

    2014-12-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival. Copyright © 2014 by the Genetics Society of America.

  14. 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.

  15. A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice

    PubMed Central

    Becker, Esther B. E.; Oliver, Peter L.; Glitsch, Maike D.; Banks, Gareth T.; Achilli, Francesca; Hardy, Andrea; Nolan, Patrick M.; Fisher, Elizabeth M. C.; Davies, Kay E.

    2009-01-01

    The hereditary ataxias are a complex group of neurological disorders characterized by the degeneration of the cerebellum and its associated connections. The molecular mechanisms that trigger the loss of Purkinje cells in this group of diseases remain incompletely understood. Here, we report a previously undescribed dominant mouse model of cerebellar ataxia, moonwalker (Mwk), that displays motor and coordination defects and loss of cerebellar Purkinje cells. Mwk mice harbor a gain-of-function mutation (T635A) in the Trpc3 gene encoding the nonselective transient receptor potential cation channel, type C3 (TRPC3), resulting in altered TRPC3 channel gating. TRPC3 is highly expressed in Purkinje cells during the phase of dendritogenesis. Interestingly, growth and differentiation of Purkinje cell dendritic arbors are profoundly impaired in Mwk mice. Our findings define a previously unknown role for TRPC3 in both dendritic development and survival of Purkinje cells, and provide a unique mechanism underlying cerebellar ataxia. PMID:19351902

  16. Decrease in tonic inhibition contributes to increase in dentate semilunar granule cell excitability after brain injury.

    PubMed

    Gupta, Akshay; Elgammal, Fatima S; Proddutur, Archana; Shah, Samik; Santhakumar, Vijayalakshmi

    2012-02-15

    Brain injury is an etiological factor for temporal lobe epilepsy and can lead to memory and cognitive impairments. A recently characterized excitatory neuronal class in the dentate molecular layer, semilunar granule cell (SGC), has been proposed to regulate dentate network activity patterns and working memory formation. Although SGCs, like granule cells, project to CA3, their typical sustained firing and associational axon collaterals suggest that they are functionally distinct from granule cells. We find that brain injury results in an enhancement of SGC excitability associated with an increase in input resista