The cerebellar deficient folia (cdf) gene acts intrinsically in Purkinje cell migrations

Cerebellar deficient folia (cdf) is a recently identified mouse mutation causing ataxia and cerebellar abnormalities including lobulation defects and abnormal placement of a specific subset of Purkinje cells. To understand the etiology of the cerebellar defects in cdf mutant mice, we examined postnatal development of the cdf/cdf cerebellum. Our results demonstrate that Purkinje cell ectopia and foliation defects are apparent at birth, suggesting the cdf mutation disrupts the positioning of many, but not all, Purkinje cells during development. In addition to cerebellar abnormalities, we observed lamination defects in the hippocampus of cdf mutant mice, although neocortical defects were not seen. Furthermore, ectopic Purkinje cells in cdf/cdf mice express an increased level of Dab1 protein, as previously observed in mice with mutations in genes in the reelin signaling pathway. Lastly, analysis of cdf <-->ROSA26 chimeric mice demonstrated that the cdf mutation is intrinsic to Purkinje cells. We suggest that the cdf gene product is required in a subset of Purkinje cells, possibly to respond to Reelin signals.     

Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.     

The direct excitatory effect of IL-1β on cerebellar Purkinje cell

Interleukin (IL)-1β is one of the important proinflammatory cytokines in neural as well as immune systems, and plays a pivotal role in the neuroinflammation. We previously demonstrated that cerebellar IL-1β is involved in kainate-induced ataxia, i.e., IL-1β was activated in the cerebellum with systemic administration of kainate, and its type I receptor (IL-1R) was expressed at a soma of cerebellar Purkinje cells. In this study, we examined the effect of IL-1β on cerebellar Purkinje cell function by recording extracellular neuronal activities in anesthetized mice. Systemic administration of kainate increased the firing rates of cerebellar Purkinje cells in normal mice but showed little effect in IL-1R-knockout (IL-1R-KO) mice. Moreover, microiontophoretic administration of IL-1β to cerebellar Purkinje cells increased the firing rates promptly in response to IL-1β. The present results demonstrate that IL-1 system exerts a direct modulatory effect on cerebellar Purkinje cells.     

Histone Deacetylase 3 Is Necessary for Proper Brain Development

The functional role of histone deacetylase 3 (HDAC3) in the developing brain has yet to be elucidated. We show that mice lacking HDAC3 in neurons and glia of the central nervous system, Nes-Cre/HDAC3 conditional KO mice, show major abnormalities in the cytoarchitecture of the neocortex and cerebellum and die within 24 h of birth. Later-born neurons do not localize properly in the cortex. A similar mislocalization is observed with cerebellar Purkinje neurons. Although the proportion of astrocytes is higher than normal, the numbers of oligodendrocytes are reduced. In contrast, conditional knockout of HDAC3 in neurons of the forebrain and certain other brain regions, using Thy1-Cre and calcium/calmodulin dependent protein kinase II α-Cre for ablation, produces no overt abnormalities in the organization of cells within the cortex or of cerebellar Purkinje neurons at birth. However, both lines of conditional knockout mice suffer from progressive hind limb paralysis and ataxia and die around 6 weeks after birth. The mice display an increase in overall numbers of cells, higher numbers of astrocytes, and Purkinje neuron degeneration. Taken together, our results demonstrate that HDAC3 plays an essential role in regulating brain development, with effects on both neurons and glia in different brain regions.     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

The abnormal cerebellar organization of Weaver and reeler mice does not affect the cellular distribution of three neuronal mRNAs

We used in situ hybridization of 35S-labeled antisense RNAs to study the cellular distribution of three neuronal mRNAs. We compared the expression of these RNAs in cerebellar Purkinje neurons in wild-type (C57Bl-6J) mice and in two mutants (Weaver and reeler) known to have abnormal cerebellar morphologies. In normal mice, GAD mRNA is present in four sets of neurons in the cerebellar cortex while calbindin mRNA is present only in Purkinje neurons. Proenkephalin mRNA is present in Golgi II neurons as well as in a set of neurons in the deep part of the molecular layer. Despite the dramatic differences in structural organization and inputs of Purkinje neurons in the cerebella of adult Weaver and reeler mice, the expression of these RNAs appears unchanged. These results support the hypothesis that Purkinje cell cytodifferentiation proceeds autonomously after its inception in early embryonic life.     

The role of thioredoxin reductases in brain development

The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells.     

Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome

Absence of functional FMRP causes Fragile X syndrome. Abnormalities in synaptic processes in the cerebral cortex and hippocampus contribute to cognitive deficits in Fragile X patients. So far, the potential roles of cerebellar deficits have not been investigated. Here, we demonstrate that both global and Purkinje cell-specific knockouts of Fmr1 show deficits in classical delay eye-blink conditioning in that the percentage of conditioned responses as well as their peak amplitude and peak velocity are reduced. Purkinje cells of these mice show elongated spines and enhanced LTD induction at the parallel fiber synapses that innervate these spines. Moreover, Fragile X patients display the same cerebellar deficits in eye-blink conditioning as the mutant mice. These data indicate that a lack of FMRP leads to cerebellar deficits at both the cellular and behavioral levels and raise the possibility that cerebellar dysfunctions can contribute to motor learning deficits in Fragile X patients.     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult cerebellum.     

Neurophysiological mechanisms in the genetics of ethanol sensitivity

Abstract

Cerebellar Purkinje neurons of long‐sleep (LS) mice express a higher sensitivity than do those of short‐sleep (SS) mice to the depressant effects of ethanol in situ, in vitro, and in intraocular cerebellar brain grafts. The ethanol sensitivity of Purkinje neurons is intrinsic to the cerebellum, may be associated with only certain brain areas, and shows a high genetic correlation with the behavioral sensitivity of mice to ethanol‐induced ataxia. Tolerance develops to the depressant effects of ethanol on cerebellar neurons in both lines of mice. However, ethanol‐tolerant LS mice are more sensitive to the electrophysiological effects of ethanol on Purkinje neurons than are ethanol‐tolerant SS mice. In addition, the behavioral sensitivity to this drug probably also involves noncerebellar neurons since neonatally cerebellectomized LS and SS mice retain a different sensitivity to the ataxic effects of ethanol.     

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

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.     

Identification of an inhibitory circuit that regulates cerebellar Golgi cell activity

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.     

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zürich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.     

Expression of FSH and its co-localization with FSH receptor and GnRH receptor in rat cerebellar cortex

The expression of follicle-stimulating hormone (FSH) and its receptor in extrapituitary and non-HPG axis tissues has been demonstrated and their non-reproductive functions in these tissues have been found. However, there have been no reports concerning the expression and function of FSH and its receptor in the cerebellum. In our study, immunofluorescence staining and in situ hybridization were used to detect the expression of FSH, double-labeled immunofluorescence staining was used to detect co-localization of FSH and its receptor and co-localization of FSH and gonadotropin-releasing hormone (GnRH) receptor in the rat cerebellar cortex. Results showed that some cells of the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex showed both FSH immunoreactivity and FSH mRNA positive signals; not only for FSH and FSH receptor, but also for FSH and GnRH receptor co-localized in some cells throughout the Purkinje cell layer, granular layer, and molecular layer of the cerebellar cortex. These suggested that rat cerebellum could express FSH; cerebellum is a target tissue of FSH; FSH may exert certain functions through FSH receptor in a paracrine or autocrine manner; GnRH may regulate FSH positive cells through GnRH receptor in the cerebellum. Our study provides morphological evidence for further functional research on FSH and related hormones in the cerebellum.     

人体小脑神经元发育的定量观察

目的:研究人体小脑神经元的发育过程。方法:应用体视学方法,对18例不同时期人体小脑组织Golgi染色后进行观察,观测小脑皮质分层出现的时间,观测并计算神经元的数密度、体密度和表面积密度。结果:6月龄时,小脑皮质出现较明显的分子层、蒲肯野细胞层和颗粒层;星形细胞、篮状细胞、蒲肯野细胞、颗粒细胞和高尔基细胞的的数密度随月龄/年龄的增长而减少,体密度和表面积密度随月龄/年龄的增长而增加,但这些减小和增大是不等速的,6-8月龄变化最明显。结论:人体小脑神经元的发育呈现快慢交替、不均速发展,6～8月是小脑神经元发育的重要时期。     

小熊猫小脑皮层的组织结构及RT-97、KGF和Bax蛋白在小脑皮层的表达

为了解小熊猫(Ailurus fulgens)小脑皮层的结构特征,观察神经丝蛋白抗体RT-97、角质细胞生长因子(KGF)及Bax蛋白在小脑皮层中的表达,利用组织学方法和免疫组织化学方法观察了小熊猫小脑皮层的显微结构,检测了RT-97、KGF和Bax蛋白的表达.结果表明,小脑皮层从外向内依次可分为分子层、Purkinje细胞层、颗粒层3层.RT-97在小熊猫小脑皮层Purkinje细胞层、颗粒层中神经细胞的轴突、分子层中颗粒细胞的轴突及小脑髓质中有阳性表达;KGF在小脑皮层分子层、Purkinje细胞层和颗粒细胞层及髓质中均有阳性表达;Bax蛋白在小脑皮层分子层、Purkinje细胞层和颗粒细胞层中有阳性表达.RT-97、KGF和Bax蛋白在小脑皮层神经结构的构筑中可能发挥着不同的功能.     

A relationship between cerebellar Purkinje cells and spatial working memory demonstrated in a lurcher/chimera mouse model system

New emphasis has been placed upon cerebellar research because of recent reports demonstrating involvement of the cerebellum in non-motor cognitive behaviors. Included in the growing list of cognitive functions associated with cerebellar activation is working memory. In this study, we explore the potential role of the cerebellum in spatial working memory using a mouse model of Purkinje cell loss. Specifically, we make aggregation chimeras between heterozygous lurcher (Lc/+) mutant embryos and +/+ (wildtype) embryos and tested them in the delayed matching-to-position (DMTP) task. Lc/+ mice lose 100% of their Purkinje cells postnatally due to a cell-intrinsic gain-of-function mutation. Lc/+<->+/+ chimeras therefore have Purkinje cells ranging from 0 to normal numbers. Through histological examination of chimeric mice and observations of motor ability, we showed that ataxia is dependent upon both the number and distribution of Purkinje cells in the cerebellum. In addition, we found that Lc/+ mice, with a complete loss of Purkinje cells, have a generalized deficit in DMTP performance that is probably associated with their motor impairment. Finally, we found that Lc/+<->+/+ chimeric mice, as a group, did not differ from control mice in this task. Rather, surprisingly, analysis of their total Purkinje cells and performance in the DMTP task revealed a significant negative relationship between these two variables. Together, these findings indicate that the cerebellum plays a minor or indirect role in spatial working memory.     

Cerebellar development: afferent organization and Purkinje cell heterogeneity.

Olivo- and spinocerebellar maps in the adult cerebellum of small rodents are discontinuous, with sharp boundaries. Cortical Purkinje cells constitute a heterogeneous population, organized into parasagittal, mutually exclusive compartments. The boundaries of the intrinsic cortical compartments and those of the projectional maps are congruent. During development; (i) The incoming olivary fibres, once they penetrate in the cerebellar parenchyma, are attracted toward their ultimate terminal fields, without passing through a stage of random dispersion. (ii) Migrating Purkinje cells and inferior olivary neurons begin, asynchronously, to express cellular markers in an independent manner, giving rise to a transient compartmentation of the cerebellar cortex and the inferior olivary complex respectively. In both instances, the biochemical heterogeneity disappears during the first postnatal week, simultaneously with the acquisition of adult-like cerebellar maps. (iii) The formation of the maps is an early event, prior to the establishment of the synaptology of the cerebellar cortical circuitry. Moreover, the organization of the spinocerebellar projection in adult mutant mice does not depend on the presence of granule cells (staggerer) but on the presence of normal Purkinje cells (weaver), indicating that synaptogenesis with their target neurons is not involved in the process of map formation. The matching of region specific chemical labels between incoming afferent fibres and heterogeneous sets of Purkinje cells is the most appealing mechanism for the formation of cerebellar maps.     

Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy

Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus gamma-aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.