Neuronal expression of macrophage colony stimulating factor in Purkinje cells and olfactory mitral cells of wild-type and cerebellar-mutant mice

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immuno-histochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.     

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.     

Cellular localization of the Ca2+-binding protein parvalbumin in the developing avian cerebellum

Summary The appearance and distribution of the calciumbinding protein parvalbumin was investigated immunocytochemically at different postnatal developmental stages of the zebra finch cerebellum. Purkinje, basket and stellate, but not granule neurons or glial cells were labeled by an antiserum against chicken parvalbumin. At all developmental stages investigated immunostained Purkinje cells were found in clusters separated by spaces containing unstained large cells, probably Purkinje and Golgi type-II cells, and unstained smaller cells resembling granule neurons. Perisomatic processes, dendrites and spines of Purkinje cells were heavily immunoreactive. Axons of Purkinje cells were observed to be parvalbumin-positive throughout their entire length until developmental stage D 24, i.e., 10 days after hatching. Their immunoreactivity gradually decreased up to adulthood, when only their proximal portions, in addition to a few punctate structures in the internal granular layer and in the deep cerebellar nuclei presumably representing the synaptic terminals, remained immunoreactive. This decrease in immunoreactivity might be related to progressive maturation and/or degree of myelination. The developmental expression of parvalbumin immunoreactivity and its ultrastructural localization in spines, postsynaptic densities and on microtubular elements leads to several suggestions concerning the possible function of parvalbumin in neurons. In outgrowing dendrites and axons the protein might be involved in the regulation of the synthesis of membrane components, their intracellular transport and fusion of new membrane components into the plasmalemma, events that are Ca- and/or Mg-dependent. In spines and postsynaptic densities parvalbumin might be involved in the development and regulation of synaptic activities in Ca-spiking elements such as the inhibitory Purkinje cells, and possibly also in stellate and basket cells. Furthermore, in developing and adult neurons parvalbumin might be involved in the Ca-/Mg-regulation of a variety of enzymatic activities and hence influence the alteration of the intracellular metabolic potential in response to extracellular signals.This work was supported by the Deutsche Forschungsgemeinschaft, SPP Verhaltensontogenie and by the Swiss National Science Foundation, Grant No. 3.185-0.82     

Expression and Electrophysiological Function of Actin in Chick Cerebellar Neurons

Among several monoclonal antibodies obtained by immunizing Balb/c mice with cerebellar synaptic membrane fractions from E20 chick embryos, the antibody, named M35, suppressed Ca-spikes in immature cultured chick cerebellar neurons. M35 immunoprecipitated 43kDa protein from a ¹²⁵I-labeled embryonic crude cerebellar membrane fraction. Immunohistochemically, the M35 antigen was expressed most intensively in Purkinje cells, but its expression was limited to highly motile structures at developmental neuronal remodeling. Electrophysiologically, M35 facilitated current responses to AMPA and inhibited the responses to GABA in cultured cerebellar Purkinje neurons. The several peptides derived from the affinity-purified 43kDa protein were found to have homologous amino acid sequences to non-muscle actins. These results suggest that the antigen recognized by M35 may play an essential role probably as membrane ion channels modulating synaptic functions in not only the development and growth but also the neuronal activity of chick cerebellar Purkinje cells.     

Glucocorticoids and the hippocampus

When threatened, the rapid induction of fear and anxiety responses is adaptive. This article summarizes the current knowledge of the neurobiological development of behavioral inhibition, a prominent response occurring in fear and anxiety-provoking situations. In the rat, behavioral inhibition as exemplified by freezing first appears near the end of the second postnatal week. This emergence of freezing coincides with the developmental period marked by the rapid increase in plasma concentrations of glucocorticoids. Studies show that removal of glucocorticoids at this time severely impairs the age-dependent appearance of freezing. This behavioral impairment produced by adrenalectomy, however, is prevented by exogenous glucocorticoid administration. The effectiveness of glucocorticoids in facilitating the development of freezing appears to be caused by its actions in the hippocampus. In particular, glucocorticoids appear to play a vital role in the postnatal cellular development of the hippocampal dentate gyrus. Doses of glucocorticoids shown to reverse the behavioral inhibitory deficits occurring after adrenalectomy are ineffective when hippocampal dentate granule neurons are destroyed by neurotoxins. Notably, site-specific administration of glucocorticoids to the dorsal hippocampus is successful in promoting the occurrence of freezing in the adrenalectomized rat pup. It is hypothesized that glucocorticoids exert their behavioral inhibitory effects by influencing the development of the septohippocampal cholinergic system. Support for this hypothesis is derived from work demonstrating the importance of glucocorticoids on nerve growth factor systems that play a critical role in septohippocampal cholinergic survival.     

Optimization of Single-Cell Electroporation Protocol for Forced Gene Expression in Primary Neuronal Cultures

The development and function of the central nervous system (CNS) are realized through interactions between many neurons. To investigate cellular and molecular mechanisms of the development and function of the CNS, it is thus crucial to be able to manipulate the gene expression of single neurons in a complex cell population. We recently developed a technique for gene silencing by introducing small interfering RNA into single neurons in primary CNS cultures using single-cell electroporation. However, we had not succeeded in forced gene expression by introducing expression plasmids using single-cell electroporation. In the present study, we optimized the experimental conditions to enable the forced expression of green fluorescent protein (GFP) in cultured cerebellar Purkinje neurons using single-cell electroporation. We succeeded in strong GFP expression in Purkinje neurons by increasing the inside diameter of micropipettes or by making the size of the original plasmid smaller by digestion and cyclizing it by ligation. Strong GFP expression in Purkinje neurons electroporated under the optimal conditions continued to be observed for more than 25 days after electroporation. Thus, this technique could be used for forced gene expression in single neurons to investigate cellular and molecular mechanisms of the development, function, and disease of the CNS.     

Distinct Modes of Neuritic Growth in Purkinje Neurons at Different Developmental Stages: Axonal Morphogenesis and Cellular Regulatory Mechanisms

Background

During development, neurons modify their axon growth mode switching from an elongating phase, in which the main axon stem reaches the target territory through growth cone-driven extension, to an arborising phase, when the terminal arbour is formed to establish synaptic connections. To investigate the relative contribution of cell-autonomous factors and environmental signals in the control of these distinct axon growth patterns, we examined the neuritogenesis of Purkinje neurons in cerebellar cultures prepared at elongating (embryonic day 17) or arborising (postnatal day zero) stages of Purkinje axon maturation.

Methodology/Principal Findings

When placed in vitro, Purkinje cells of both ages undergo an initial phase of neurite elongation followed by the development of terminal ramifications. Nevertheless, elongation of the main axon stem prevails in embryonic Purkinje axons, and many of these neurons are totally unable to form terminal branches. On the contrary, all postnatal neurites switch to arbour growth within a few days in culture and spread extensive terminal trees. Regardless of their elongating or arborising pattern, defined growth features (e.g. growth rate and tree extension) of embryonic Purkinje axons remain distinct from those of postnatal neurites. Thus, Purkinje neurons of different ages are endowed with intrinsic stage-specific competence for neuritic growth. Such competence, however, can be modified by environmental cues. Indeed, while exposure to the postnatal environment stimulates the growth of embryonic axons without modifying their phenotype, contact-mediated signals derived from granule cells specifically induce arborising growth and modulate the dynamics of neuritic elongation.

Conclusions/Significance

Cultured Purkinje cells recapitulate an intrinsically coded neuritogenic program, involving initial navigation of the axon towards the target field and subsequent expansion of the terminal arborisation. The execution of this program is regulated by environmental signals that modify the growth competence of Purkinje cells, so to adapt their endogenous properties to the different phases of neuritic morphogenesis.     

Developmental Iodine Deficiency and Hypothyroidism Impair Neural Development, Upregulate Caveolin-1, and Downregulate Synaptotagmin-1 in the Rat Cerebellum

Adequate thyroid hormone is critical for cerebellar development. Developmental hypothyroidism induced by iodine deficiency during gestation and postnatal period results in permanent impairments of cerebellar development with an unclear mechanism. In the present study, we implicate cerebellar caveolin-1 and synaptotagmin-1, the two important molecules involved in neuronal development, in developmental iodine deficiency, and in developmental hypothyroidism. Two developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil (PTU, 5 or 15?ppm)-added drinking water from gestational day?6 till postnatal day (PN) 28. Nissl staining and the levels of caveolin-1 and synaptotagmin-1 in cerebella were assessed on PN28 and PN42. The results show that the numbers of Purkinje cells were reduced in the iodine-deficient and PTU-treated rats. The upregulation of caveolin-1 and the downregulation of synaptotagmin-1 were observed in both iodine-deficient and PTU-treated rats. These findings may implicate decreases in the number of Purkinje cells and the alterations in the levels of caveolin-1 and synaptotagmin-1 in the impairments of cerebellar development induced by developmental iodine deficiency and hypothyroidism.     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

Culture condition and embryonic stage dependent silence of glucocorticoid receptor expression in hippocampal neurons

Glucocorticoid (GC) plays a key role in controlling numerous cellular processes during embryogenesis and fetal development. The actions of glucocorticoids are mediated by interaction with their receptors. We previously reported that hippocampal neurons from embryonic day 18 (E18) rats showed silence of glucocorticoid receptor (GR) expression when cultured in serum-free condition. In this study, using western blot, immunofluorescence staining and real-time RT-PCR, we found that while this silence occurred in hippocampal neurons isolated from E16 and E18 rats, it did not happen in those from E20 and neonatal (P0) rats. And when cultured under serum-containing condition, none of them showed GR silence anymore. Corticosterone could not rescue the expression of GR in E16 and E18 neurons in serum-free condition, whereas adding of serum could induce the re-expression of the silenced GR. The absence of GR silence in P0 neurons was not due to the perturbation during parturition. Moreover, the unique expression profile of GR in protein and mRNA level was well reflected in the changes of GR function. These results suggested that under in vitro condition, serum was critical for the maintaining of GR expression in hippocampal neurons of early embryonic stages but less important in later developmental stages. Thus, our data implied that at different developmental stages, the expression of GR in hippocampal neurons might have different susceptibilities to environment changes and there might be a critical time window for the switching of such characteristics during development.     

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

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

A novel inhibitor rescues cerebellar defects in a zebrafish model of Down syndrome–associated kinase Dyrk1A overexpression

The highly conserved dual-specificity tyrosine phosphorylation–regulated kinase 1A (Dyrk1A) plays crucial roles during central nervous system development and homeostasis. Furthermore, its hyperactivity is considered responsible for some neurological defects in individuals with Down syndrome. We set out to establish a zebrafish model expressing human Dyrk1A that could be further used to characterize the interaction between Dyrk1A and neurological phenotypes. First, we revealed the prominent expression of dyrk1a homologs in cerebellar neurons in the zebrafish larval and adult brains. Overexpression of human dyrk1a in postmitotic cerebellar Purkinje neurons resulted in a structural misorganization of the Purkinje cells in cerebellar hemispheres and a compaction of this cell population. This impaired Purkinje cell organization was progressive, leading to an age-dependent dispersal of Purkinje neurons throughout the cerebellar molecular layer with larval swim deficits resulting in miscoordination of swimming and reduced exploratory behavior in aged adults. We also found that the structural misorganization of the larval Purkinje cell layer could be rescued by pharmacological treatment with Dyrk1A inhibitors. We further reveal the in vivo efficiency of a novel selective Dyrk1A inhibitor, KuFal194. These findings demonstrate that the zebrafish is a well-suited vertebrate organism to genetically model severe neurological diseases with single cell type specificity. Such models can be used to relate molecular malfunction to cellular deficits, impaired tissue formation, and organismal behavior and can also be used for pharmacological compound testing and validation.     

Localization of the type 1 corticotropin releasing factor receptor (CRF-R1) in the embryonic mouse cerebellum

Corticotropin releasing factor (CRF) is present in the adult, as well as in the embryonic and postnatal rodent cerebellum. Further, the distribution of the type 1 CRF receptor has been described in adult and postnatal animals. The focus of the present study is to determine the distribution and cellular relationships of the type 1 CRF receptor (CRF-R1) during embryonic development of the cerebellum. Between embryonic day (E)11 and E12, CRF-R1 immunoreactive puncta are uniformly distributed in the ventricular zone, the site of origin of Purkinje cells, nuclear neurons, and GABAergic interneurons, as well as the germinal trigone, the birthplace of the precursors of granule cells. Between E13 and 18, the distribution of immunolabeled puncta decreases in both the ventricular zone and the germinal trigone and increases in the intermediate zone, as well as in the dorsal aspect of the cerebellar plate. Between E14 and 18, antibodies that label specific populations of cerebellar neurons were combined with the antibody for the receptor to determine the cellular elements that expressed CRF-R1. At E14, CRF-R1 immunoreactivity is co-localized in neurons immunolabeled with PAX-2, an antibody that is specific for GABAergic interneurons. These neurons continue to express CRF-R1 as they migrate dorsally toward the cerebellar surface. Between E16 and 18, Purkinje cells, immunolabeled with calbindin, near the dorsal surface of the cerebellum express CRF-R1 in their cell bodies and apical processes. CRF has been shown to have a depolarizing effect on adult and postnatal Purkinje cells. Further, CRF has been shown to contribute to excitability of hippocampal neurons during embryonic development by binding to CRF-R1; depolarization induced excitability appears to be critical for cell survival. The location of the type one CRF receptor and the presence of its primary ligand, CRF, in the germinal zones of the cerebellum and in migrating neurons suggest that this receptor/ligand interaction could be important in the regulation of neuronal survival through cellular mechanisms that lead to depolarization of embryonic cerebellar neurons.     

Ceramide and Its Interconvertible Metabolite Sphingosine Function as Indispensable Lipid Factors Involved in Survival and Dendritic Differentiation of Cerebellar Purkinje Cells

Abstract: Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell-permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP-1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell-permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.     

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 502–516, 1997     

Sphingolipid Biosynthesis Is Necessary for Dendrite Growth and Survival of Cerebellar Purkinje Cells in Culture

Abstract: The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 µM fumonisin B₁, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [³H]galactose/glucosamine and [¹⁴C]serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell-enriched sphingolipids, including GD1α, 9-O-acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B₁, dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B₁ also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B₁. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B₁ were reversed, but not completely, by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]caproyl]sphingosine (C₆-NBD-ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.     

The intrinsic mechanisms underlying the maturation of programming sequential spikes at cerebellar Purkinje cells

Cerebellum is involved in the motion coordination and working memory, to which the programming of sequential spikes at Purkinje cells is essential. It is not clear about the intrinsic mechanisms underlying spike capacity and timing precision as well as their postnatal maturation. We investigated the programming and intrinsic property of sequential spikes at Purkinje neurons during postnatal development by whole-cell recording in cerebellar slices. Cerebellar Purkinje neurons demonstrate the increasing of spike capacity and timing precision, as well as the lowering of refractory periods and threshold potentials during the postnatal maturation. In addition, the correlation between spike parameters and intrinsic properties converts to be more linear. This postnatal plasticity of neuronal intrinsic properties improves the timing precision and capacity of spike programming at cerebellar Purkinje neurons.