Stimulation of the inferior olivary complex alters the distribution of the type 1 corticotropin releasing factor receptor in the adult rat cerebellar cortex

In a previous study, it was shown that populations of climbing fibers, derived from the inferior olivary complex (IOC) contain the peptide corticotropin releasing factor (CRF) and that the expression of this peptide in climbing fibers could be modulated by the level of activity in olivary afferents. The intent of this study was to determine if there was comparable plasticity in the distribution of the type 1 CRF receptor (CRF-R1) in the cerebellum of the rat. Our results indicate that CRF-R1 was localized primarily to Purkinje cell somata and their primary dendrites and granule cells. In addition, scattered immunolabeling was present over the somata of Golgi cells, basket cells and stellate cells, as well as Bergmann glial cells and their processes. IOC stimulation for 30 min at 1 Hz increased CRF-R1 expression in molecular layer interneurons and processes of Bergmann glial cells. Little to no effect on CRF receptor distribution was observed in Purkinje cells, granule cells, or Golgi cells. IOC stimulation at 5 Hz however, increased CRF-R1 expression in the processes of Bergmann glial cells while decreasing its expression in basket, stellate and, to some extent, in Purkinje cells. The present results suggest that there is activity-dependent plasticity in CRF-R1 expression that must be considered in defining the mechanism by which the CRF family of peptides modulates activity in cerebellar circuits. The present results also suggest that the primary targets of CRF released from climbing fibers are Bergmann glial cells and interneurons in the molecular layer. Further, interneurons responded with a decrease in receptor expression following more intense levels of stimulation suggesting the possibility of internalization of the receptor. In contrast, Bergmann glial cells showed an increased expression in receptor expression. These data suggest that CRF released from climbing fibers may modulate the physiological properties of basket and stellate cells as well as having a heretofore unidentified and potentially unique effect on Bergmann glia.     

Ultrastructural analysis of climbing fiber-Purkinje cell synaptogenesis in the rat cerebellum.

Previous reports have described the transient expression of the neuropeptides calcitonin gene-related peptide and neuropeptide Y in selected subsets of rat olivocerebellar compartments during embryonic and postnatal development. Using these neuropeptides as endogenous markers for olivocerebellar fibers, the aim of this electron microscopic analysis was to reveal the synaptogenetic processes occurring between climbing fibers and their target Purkinje cells, from embryonic day 19 to postnatal day 16, the period during which Purkinje cells undergo intense emission and retraction of dendrites, and climbing fibers translocate their synapses along Purkinje cell membrane surfaces.The present findings provide the first direct evidence that climbing fiber synaptogenesis starts on embryonic day 19 and that these first synapses mainly involve the Purkinje cell embryonic dendrite rather than the Purkinje cell soma. At the same age, the presence of unlabeled synapses resembling calcitonin gene-related peptide-labeled synapses in the Purkinje cell plate makes it possible to conclude that climbing fibers form a major synaptic investment on embryonic Purkinje cells, a finding that strongly supports the hypothesis of an early differentiating role of climbing fibers on cerebellar development. Furthermore, during the period of intense dendritic remodeling of Purkinje cells, 'myelin figures' were often detected in Purkinje cell dendrites suggesting that they may at least in part represent real ultrastructural markers of membrane turnover that identifies the sites where Purkinje cell dendritic rearrangement is taking place. Finally the finding that the climbing fiber terminals apposed to degenerating dendrites did not generally show signs of degeneration leads us to suggests that climbing fiber translocation from a perisomatic to a dendritic location may be driven by the Purkinje cell dendritic remodeling.     

Cytodifferentiation of bergmann glia and its relationship with purkinje cells

The Bergmann glia is composed of unipolar protoplasmic astrocytes in the cerebellar cortex. Bergmann glial cells locate their cell bodies around Purkinje cells, and extend radial or Bergmann fibers enwrapping synapses on Purkinje cell dendrites. During development, Bergmann fibers display a tight association with migrating granule cells, from which the concept of glia-guided neuronal migration has been proposed. Thus, it is widely known that the Bergmann glia is associated with granule cells in the developing cerebellum and with Purkinje cells in the adult cerebellum. As the information on how Bergmann glial cells are related structurally and functionally with differentiating Purkinje cells is quite fragmental, this issue has been investigated using cytochemical techniques for Bergmann glial cells. This review classifies the cytodiffer-entiation of Bergmann glial cells into four stages, that is, radial glia, migration, transformation and protoplasmic astrocytes, and then summarizes their structural relationship with Purkinje cells at each stage. The results conclude that the cytodifferentiation of Bergmann glial cells proceeds in correlation with the migration, dendritogenesis, synaptogenesis and maturation of Purkinje cells. Furthermore, morphological and molecular plasticity of this neuroglia appears to be regulated depending on the cytodifferentiation of nearby Purkinje cells. The functional relevance of this intimate neuron-glial relationship is also discussed with reference to recent studies in cell biology, cell ablation and gene knockout.     

Surface and membrane morphology of Bergmann glial cells and their topographic relationships in the cerebellar molecular layer

The cerebellar Bergmann glial cells of Swiss albino mouse, teleost fish and human have been examined by means of freeze-fracture and ethanol-cryofracturing techniques for scanning electron microscopy and freeze-etching and thin sectioning techniques for transmission electron microscopy in order to study the outer surface, membrane morphology and topographic relationship with Purkinje cells, parallel and climbing fibers and stellate axons. Freeze-fracture and ethanol-cryofracturing techniques exposed the somatic outer surface and lamellated processes surrounding the Purkinje cells. Transmission electron microscopy showed a granular substance filling the extracellular space separating Bergmann glial cell cytoplasm from dark Purkinje cells. This substance was absent in the vicinity of clear Purkinje cells. Freeze-etching replicas showed a multilayered ensheathment of Bergmann glial lamellar processes surrounding parallel fibers. The P face of Bergmann glial cell plasma membrane exhibited orthogonal arrays or assemblies of intramembrane particles (IMPs) and high density distribution of round IMPs 6-15 nm in diameter, whereas the E face was characterized by a smooth surface and low density distribution of IMPs. Rod intramembrane particles, 20-30 nm in length, were also found in the plasma membrane P and E faces. The fractured Bergmann glial cell cytoplasm was observed encapsulating the parallel fiber varicosities, or the bundles of non-synaptic segments of parallel fibers and enclosing the climbing fiber and stellate axonal synapses with Purkinje dendrites.     

Postnatal Development of the Central Nervous System: Anomalies in the Formation of Cerebellum Fissures

A natural defect in rat cerebellum postnatal development has been found in the fissura prima, consisting in various complex configurations of the cerebellar layers. We investigated the genesis of fissure malformations through immunoreactions for PCNA, GFAP, GABAA α6, and calbindin to label proliferating cells of the external granular layer (egl), radial glial fibers, mature granule cells, and Purkinje cells, respectively. Results on critical stages of rat postnatal development provided interesting evidences on how the malformation develops in fissures prima and secunda. Early (postnatal day 10) at the site of malformation, the Bergmann radial glia was often retracted and showed distortions and irregular running. The interruption of GFAP‐positive radial glial fibers could fit in with the presence of clusters of PCNA‐unlabeled cells in the sites of fusion of the egl; the clusters of cells are granule cells since their soma is labeled by GABAA α6. Moreover, an altered migration of granule cell precursors to the internal granular layer was evident which, in turn, affected Purkinje cell differentiation and the growth of their dendrites. In summary, the changed relationship among glial fiber morphology, granule cell migration, and Purkinje cell differentiation suggests how the Bergmann glial fibers have a basic role in the foliation process, being the driving physical force in directing migration of the granule cells at the base of fissure. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Properties of kainate receptor/channels on cultured Bergmann glia.

Following the localization, at the electron microscope level, of the immunoreactivity towards a putative kainate receptor on Bergmann glial cells in the chick cerebellar cortex, cultures of Bergmann glia were used to establish the presence of functional kainate receptor/channels and study their properties. Bergmann glia were identified by their fusiform morphology and their ability to bind an anti-kainate binding protein monoclonal antibody, a kainate receptor high affinity ligand--kainyl-bovine serum albumin--and a glial marker--anti-vimentin monoclonal antibody. Membranes prepared from the culture cells displayed, using 25 nM [3H]kainate, the binding of 4.1 pmol of [3H]kainate/mg protein and showed the presence in Western blots of the two polypeptides of 49 and 93 kDa attributed to the kainate binding protein. Kainate, at concentrations above 0.1 mM, was found to increase the influx into cultured Bergmann glia of 22Na+, 86Rb+, 45Ca2+ and 36Cl- ions. The traffic of 22Na+, induced by kainate and glutamate, observed only in the presence of 1 mM ouabain, was blocked by kainate receptor antagonists and by 0.01 mM quisqualate. Analysis of the kinetics of incorporation of 22Na+ and 45Ca2+ ions showed an initial accumulation of 22Na+ and 45Ca2+ ions followed by their total dissipation. The results indicate that the kainate-induced influx of Na+ ions through the kainate receptor/channel causes the reverse transport of Na+ ions, by activation of the Na+/Ca2+ and Na+/H+ exchangers which remove intracellular Na+ ions. Pre-exposure of the cells to 0.5 mM dibutyryl cAMP was found to greatly enhance the kainate-induced 22Na+ ion influx. We propose that the Bergmann glia kainate receptors modulate the efficacy of the glutamatergic synapses between the parallel fibers and Purkinje cell spines and form part of a glial machinery responsible for plastic changes in synaptic transmission.     

Subcellular localization of a putative kainate receptor in Bergmann glial cells using a monoclonal antibody in the chick and fish cerebellar cortex

A monoclonal antibody, IX-50, that was raised against a kainate binding protein (Mr = 49,000) from chicken cerebellum, was used in light and electron microscopic immunocytochemical studies to localize putative kainate receptors. Pre- and postembedding immunoperoxidase and immunogold methods were used in the cerebellar cortices of one to 26-day old chickens and adult rainbow trout. Immunoreactivity was detected only in association with Golgi epithelial/Bergmann glial cells. Intracellular immunoreactivity was present in the granular and agranular endoplasmic reticulum, Golgi apparatus and in lysosomes, representing the sites of synthesis, glycosylation and degradation of the protein. In the fish the granular endoplasmic reticulum was not immunoreactive. Extracellular immunoreactivity was associated with the plasma membrane. In the fish it was established that the epitope is on the outer surface of the membrane. The protein seems to be uniformly distributed along the membrane including the somata, the radial stem processes and the leafy lamellae surrounding Purkinje cell dendrites. Areas of the glial membrane in contact with other glial cells were also immunopositive. High-resolution light microscopy demonstrated all the Bergmann glial plasma membrane in the cortex, providing a "negative" image of Purkinje cell dendrites. It is apparent that Bergmann glial processes selectively outline the dendrites of the Purkinje cells by surrounding the parallel fibre terminal/Purkinje cell spine synaptic complexes. The parallel fiber terminals were highly immunoreactive for glutamate, as shown by an immunogold procedure. The association of Bergmann glial processes, carrying the Mr = 49,000 kainate binding protein, with the Purkinje cell dendrites and spine synapses could provide a basis for neuronal signalling to the Bergmann glia, possibly by glutamate.     

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.     

Interactions between astroglia and ectopic granule cells in the cerebellar cortex of normal adult rats: a morphological and cytochemical study

Summary The cytology and organization of astroglial cells associated with ectopic granule cells (EGCs) have been studied in the cerebellar cortex of normal adult rats, using Golgi preparations, immunohistochemistry against the glial fibrillary acidic protein (GFAP) and ultrastructural analysis. Elongated perikarya of EGCs scattered in the molecular layer were usually attached to radial Bergmann fibers, which exhibited a normal morphology. A second configuration of EGCs consisted of intrafissural colonies of ectopic neurons. The molecular layer that surrounded these ectopic colonies showed disorientation of both Bergmann fibers and parallel fibers. Within the ectopic tissue, typical velate astrocytes were commonly observed. They formed homologous associations with EGC perikarya and cerebellar glomeruli. However, a restricted astroglial plasticity was detected in the form of heterologous interactions between astrocytes and Purkinje cell dendrites, including their associated synapses with parallel fibers. In spite of this astroglial plasticity, our results suggest that in the physiological systems of granule cell ectopia studied here, the different specific interactions of Bergmann glia and astrocytes with neurons tend to be conserved.     

Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport

Non-neuronal cells may be pivotal in neurodegenerative disease, but the mechanistic basis of this effect remains ill-defined. In the polyglutamine disease spinocerebellar ataxia type 7 (SCA7), Purkinje cells undergo non-cell-autonomous degeneration in transgenic mice. We considered the possibility that glial dysfunction leads to Purkinje cell degeneration, and generated mice that express ataxin-7 in Bergmann glia of the cerebellum with the Gfa2 promoter. Bergmann glia-specific expression of mutant ataxin-7 was sufficient to produce ataxia and neurodegeneration. Expression of the Bergmann glia-specific glutamate transporter GLAST was reduced in Gfa2-SCA7 mice and was associated with impaired glutamate transport in cultured Bergmann glia, cerebellar slices and cerebellar synaptosomes. Ultrastructural analysis of Purkinje cells revealed findings of dark cell degeneration consistent with excitotoxic injury. Our studies indicate that impairment of glutamate transport secondary to glial dysfunction contributes to SCA7 neurodegeneration, and suggest a similar role for glial dysfunction in other polyglutamine diseases and SCAs.     

Correlative microscopy of cerebellar Bergmann glial cells

Double fluorescent labelling of rat cerebellar cortex using antibody to glial fibrillary acidic protein (GFAP) and Alexa fluor conjugates for secondary detection for confocal laser scanning microscope (CLSM), field emission scanning electron microscopy (FESEM) of Rhesus monkey cerebellar cortex, ultrathin sectioning and freeze-etching replica method for transmission electron microscopy of mouse cerebellar cortex have been examined in an attempt to obtain a new and more accurate view of three-dimensional image of Bergmann glial cells (BGC) and their topographic relations in the molecular layer. Intense immunopositive GFAP green staining was observed in the BGC and glial limiting layer. Secondary antibody conjugated with Alexa fluor 488 and Alexa fluor 668-1B4 stained in red capillary endothelial cells and microglial cells. BGC morphology revealed the existence of several cell types or subpopulations of BGC. Bergmann glial fibers, in palisade arrangement, branch and rebranch forming a complex glial network in the molecular layer. Field emission SEM and freeze-fracture SEM method show the SE-I image of high mass dense Bergmann glial cytoplasm ensheathing like a veil the Purkinje cell (PC) soma and dendritric arborization. Bergmann glial fibers appeared completely surrounding individual parallel fibers or parallel fiber bundles, terminal climbing fiber collaterals, basket and stellate cells and capillaries. Freeze-etching direct replicas showed the typical orthogonal arrangement of intramembrane particles, corresponding to the large repertoire of BGC receptors. The study reveals three-dimensional Bergmann glial cells heterogeneity and the complex network formed by Bergmann glial cells in the molecular layer.     

小鼠放射状胶质细胞和小脑皮质的发育

目的探讨小鼠小脑皮质发育过程中放射状胶质细胞的分化。方法应用免疫荧光及5-溴脱氧尿嘧啶核苷(BrdU)检测技术,标记小鼠胚胎8d至生后180d小脑(57例,分为19组,每组3只)的神经干细胞、放射状胶质细胞、普肯耶细胞及颗粒细胞。结果放射状胶质细胞于胚胎13d的神经上皮出现,尔后该细胞分化为各种神经元和贝格曼胶质细胞,并在小脑皮质层状结构的形成中起着重要作用。结论放射状胶质细胞来源于神经上皮细胞,是神经细胞和神经胶质细胞的前体细胞。在小脑皮质的发育过程中,放射状胶质细胞能分化为普肯耶细胞和颗粒细胞,并为神经细胞的迁移提供路径和支架。     

Peptide localization in the mouse inferior olive

The inferior olivary complex is the sole source of climbing fibers to the cerebellar cortex. Physiologically these afferents have been shown to have a powerful excitatory effect on their target neurons, namely Purkinje cells. Thus, any modulation of olivary firing rate or responsiveness will alter Purkinje cell firing and ultimately cerebellar function. Neuropeptides have been shown to modulate neuronal activity in several systems. The intent of the present study is to determine the olivary distribution of five peptides previously shown to be present and functional in cerebellar circuitry including cholecystokinin, calcitonin gene-related peptide, corticotropin releasing factor, enkephalin and substance P. These studies were carried out in the adult C57BL/6J mouse using the peroxidase anti-peroxidase immunohistochemical technique. All five peptides labeled varicosities of varying sizes. Varicosities labeled for cholecystokinin, calcitonin gene-related peptide and corticotropin releasing factor were densely distributed throughout the inferior olive. In contrast, varicosities immunostained for substance P and enkephalin, were more restricted in their distribution. The overlap in the distribution of these peptides suggests that they may be colocalized with each other as well as with excitatory or inhibitory amino acids known to be present in afferents to the inferior olive. Because of the extensive distribution of the peptides, it is likely that they are derived from multiple brainstem sources. These findings serve as baseline data for future physiological studies designed to address the functional role of peptides in olivary circuitry.     

D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor

D-serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.     

Developmental changes of chick cerebellar cortex revealed by monoclonal antibodies

Immunohistochemistry studies of the embryonic and newly hatched chick cerebellum were performed with 13 monoclonal antibodies (MAbs) raised against the embryonic chick optic nerve and a MAb which binds to cell nuclei. Neural MAbs differentially stained Purkinje cells, the external granular layer, molecular layer, internal granular layer, climbing fibers, basket cell axons, Bergmann glia and Ramón y Cajal's ansiform fibers. At the different developmental stages each component responded to MAbs differently. For example, staining of Purkinje cells with MAbs 23C10, 82E10 and 94C2 appeared on day 11 of incubation and disappeared sequentially after day 18. These results reveal molecular heterogeneity not only in cerebellar neurons but also at various developmental stages.     

Destruction of meningeal cells over the newborn hamster cerebellum with 6-hydroxydopamine prevents foliation and lamination in the rostral cerebellum

Intracisternal injection of 30 micrograms 6-hydroxydopamine was used to destroy meningeal cells in the newborn hamster. After 20 or 30 days the cerebella of treated animals showed severe morphological alterations including: an absence of distinct folia anterior to the primary fissure; a disruption of lamination in the same region by the displacement of both Purkinje cells and cerebellar interneurons; a reduction in size and frequency of branching of the medullary tree with anomalous anterobasal branches and splaying; reductions in the area of the molecular layer, the total area occupied by granule cells, the length of the pial surface and the length of the Purkinje cell layer of 29, 21, 57 and 27%, respectively; disorganization of the radially organized glial scaffold by outgrowth of Bergmann glial fibers and displacement of their cell bodies, the Golgi epithelial cells, and anomalous orientation, polarity, size and branching frequency of Purkinje cell dendritic trees. These findings support our earlier hypothesis that the initial destruction of meningeal cells destabilizes the cerebellar surface (basal lamina and glia limitans superficialis) and disorganizes the glial scaffold, while the neuronal cerebellar malformations are secondary to this glial defect.     

Chimeric analysis of the site of Lurcher gene action with respect to glial enzyme expression

Immunocytochemical analysis of aggregation chimeras made using either of the mutants, Lurcher or Purkinje cell degeneration, previously showed that only Bergmann glia close to surviving Purkinje cells expressed an apparently normal level of the enzyme, glycerol-3-phosphate dehydrogenase (GPDH) (Fisher and Mullen, 1988). In the present study, aggregation chimeras were made using embryos from a B6D2 hybrid mouse strain carrying the Lurcher (Lc/+) mutation and homozygous for an allele specifying a high level of beta-glucuronidase activity and embryos from a wild-type C3H strain with low beta-glucuronidase activity. Chimeric cerebella were analyzed immunocytochemically and histochemically to determine whether Lurcher mutant Bergmann glia could express a normal, high level of GPDH. Comparisons between pairs of alternately stained, 2 microns thick serial sections showed that Bergmann glia with high level of beta-glucuronidase (i.e., Lurcher) expressed normal GPDH immunoreactivity only when close to surviving, wild-type Purkinje cells. Interestingly, in Purkinje cell-free areas of cerebellar cortex that were sufficiently large that GPDH expression was diminished, Bergmann glia also showed a reduced level of histochemically detectable beta-glucuronidase activity, as do Bergmann glia in adult Lc/+ mice. The results indicate that Lc/+ mutant Bergmann glia are not intrinsically defective with regard to expression of the enzymes, GPDH and beta-glucuronidase, but rather, expression of these enzymes depends on glial interaction with wild-type Purkinje cells.     

Characterization and developmental evolution of a high-affinity binding site for calcitonin gene-related peptide on chick skeletal muscle membrane.

Calcitonin gene-related peptide is a putative neurotransmitter of central and peripheral nervous systems which coexists with acetylcholine in motor nerve terminals and exerts multiple effects on skeletal muscle, suggesting a trophic role for this neuropeptide. Using radiolabeled calcitonin gene-related peptide as a probe in a specific binding assay, we have characterized calcitonin gene-related peptide binding sites on chick skeletal muscle membranes. Binding is time-dependent, saturable and reversible. Scatchard analyses revealed two classes of sites: high-affinity sites with a KD value of 62 pM, and low-affinity sites with a KD value of 3.3 nM. The maximal number of sites is, respectively, 22 and 155 fmol/mg protein for high- and low-affinity binding sites. Specific binding was not affected by the presence, in excess, of other neuropeptides such as salmon calcitonin or somatostatin or vasoactive intestinal polypeptide. Affinity of the binding site for calcitonin gene-related peptide was decreased in the presence of 5'-guanylyl-imidodiphosphate, suggesting a physiological coupling of calcitonin gene-related peptide receptor to a GTP binding protein. In a developmental study of chick muscle, we found the highest activity of calcitonin gene-related peptide binding sites in 11-14 day embryos, following a pattern of evolution similar to that of acetylcholine receptors (constant ratio of 12 acetylcholine receptors per calcitonin gene-related peptide binding site). However, both receptors appear differentially regulated: while the number of acetylcholine receptors increases 5-16-fold after denervation, calcitonin gene-related peptide binding sites slightly diminish in number. These results are discussed in terms of the physiological significance of calcitonin gene-related peptide binding sites on chick skeletal muscle membrane.     

Harmaline-induced climbing fiber activation causes amino acid and peptide release in the rodent cerebellar cortex and a unique temporal pattern of Fos expression in the olivo-cerebellar pathway

Cerebellar climbing fibers have a unique relationship with the dendritic tree of cerebellar Purkinje cells and have been proposed as a key input in establishing long-term plastic changes in the cerebellar cortex. Although both glutamate and aspartate and a number of neuropeptides have been implicated as climbing fiber-released neurotransmitters/neuromodulators, the in vivo release of these substances during climbing fiber stimulation remains to be demonstrated. In the present study, climbing fibers were activated with harmaline and rats or mice were implanted with a microdialysis probe or a microperfusion probe, respectively, to measure amino acid or peptide release. Additional rats were euthanized at various timepoints post-harmaline injection and Fos immunocytochemistry was used to visualize the activation pattern of the inferior olive, cerebellar cortex and deep nuclei over time. Fos expression was first detected in the inferior olive at 15 min post-harmaline injection followed by expression in the deep cerebellar nuclei (30 min) and then in the cerebellar cortex (1 h). Between 2 and 6 h Purkinje cells expressing Fos were found in variable numbers in both the vermal and paravermal regions and there was a distinct parasagittal-banding pattern in the vermal region. Of several amino acids measured following harmaline administration only glutamate and aspartate levels increased significantly in the first dialysate sample compared to preharmaline levels and their release was blocked by prior lesion of the inferior olive. Citrulline also increased following climbing fiber stimulation, but this occurred in the second and third dialysate samples and may reflect nitric oxide production. Four peptides were examined in cerebellar microperfusates following climbing fiber stimulation. Only corticotropin releasing factor (CRF), calcitonin gene related peptide (CGRP) and bradykinin were significantly increased compared to pre-harmaline levels. These results suggest that glutamate, aspartate, CRF and CGRP are released from climbing fibers during activation of the olivocerebellar system.     

Overexpression of HGF attenuates the degeneration of Purkinje cells and Bergmann glia in a knockin mouse model of spinocerebellar ataxia type 7

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder associated with cerebellar neurodegeneration caused by expansion of a CAG repeat in the ataxin-7 gene. Hepatocyte growth factor (HGF), a pleiotrophic growth factor, displays highly potent neurotrophic activities on cerebellar neurons. A mutant c-met/HGF receptor knockin mouse model has revealed a role for HGF in the postnatal development of the cerebellum. The present study was designed to elucidate the effect of HGF on cerebellar neurodegeneration in a knockin mouse model of SCA7 (SCA7-KI mouse). SCA7-KI mice were crossed with transgenic mice overexpressing HGF (HGF-Tg mice) to produce SCA7-KI/HGF-Tg mice that were used to examine the phenotypic differences following HGF overexpression. The Purkinje cellular degeneration is thought to occur via cell-autonomous and non-cell autonomous mechanisms mediated by a reduction of glutamate transporter levels in Bergmann glia. The Purkinje cellular degeneration and reduced expression of glutamate transporters in the cerebellum of SCA7-KI mice were largely attenuated in the SCA7-KI/HGF-Tg mice. Moreover, phenotypic impairments exhibited by SCA7-KI mice during rotarod tests were alleviated in SCA7-KI/HGF-Tg mice. The bifunctional nature of HGF on both Purkinje cells and Bergmann glia highlight the potential therapeutic utility of this molecule for the treatment of SCA7 and related disorders.