Cellular density in the cerebellar molecular layer in essential tremor,spinocerebellar ataxia,and controls

BackgroundIt would be useful to identify additional postmortem markers of Purkinje cell loss in essential tremor (ET). In hereditary cerebellar ataxia, Purkinje cell loss has been reported to result in a secondary increase in the density of the remaining cell populations in the cerebellar molecular layer. However, this phenomenon has not been studied in ET. We quantified cerebellar molecular layer cellular density in 15 ET cases, 15 controls, and 7 spinocerebellar ataxia (SCA) cases (2:2:1 ratio).MethodsA standard neocerebellar tissue block was stained with Luxol fast blue Hematoxylin & Eosin. Within 5 selected fields, cell soma (e.g., stellate, basket, and glial cell bodies) were counted. Cellular density was the number of cells/cm².ResultsThe Purkinje cell count differed across the three groups (p < 0.001), with the highest counts in controls, intermediate counts in ET cases and lowest counts in SCA cases. ET cases and controls had similar molecular layer cellular density (p = 0.79) but SCA cases had higher values than both groups (p < 0.01). A robust inverse correlation between Purkinje cell count and molecular layer cellular density (i.e., brains with more Purkinje cell loss had higher molecular layer cellular density), observed in SCA and controls (r = −0.55, p = 0.008), was not observed in ET cases.DiscussionAlthough Purkinje cell counts were reduced in ET cases compared to controls, an increase in molecular layer cellular density was not evident in ET. The increase in molecular layer cellular density, observed in SCA cases, may require a more marked loss of PCs than occurs in ET.     

The volume of the cerebellar molecular layer predicts attention to novelty in rats

Cerebellum volume was compared with the behavioral measure of attention to novelty in normal rats. The volume of the cerebellar molecular layer significantly predicted individual rats' exploratory tendency. Variations in neuronal process volume may explain part of the interindividual variation for cognitive abilities.     

Cerebellar granular layer aplasia in congenital hydrocephalus

An unusual case of cerebellar granular layer aplasia is reported. A 5‐year‐old boy was born with hydrocephalus and a peritoneal drainage shunt was placed after the delivery. Symptoms of cerebral paralysis, impaired mental function and cerebellar ataxia had developed gradually. Patient's karyotype was 46,XY. Laboratory tests for cytomegalovirus, Herpes simplex virus, Toxoplasma gondii, human immunodeficiency virus, rubella and hepatitis B virus were negative. Further laboratory investigation showed no signs of Tay‐Sachs disease, Niemann‐Pick disease, Gaucher disease, phenylketonuria, galactosemia or glycogen storage disease. No congenital malformations were traced in other family members for three generations. Radiation exposure and infections during the pregnancy were refuted.     

Estimating the number of granule cells in the dentate gyrus with the disector

A practical example is given of how a newly developed stereological estimator of particle number, the disector, can be used to make estimates of neuron number in the dentate gyrus of rats. The estimates are free of biases related to lost caps, overprojection and assumptions about size, shape and orientation of the objects that are counted. The disector principle and the practical considerations relating to histological preparations and sampling are presented.     

Cerebellar abnormalities in the disabled (mdab1–1) mouse

A mouse homolog of the Drosophila Disabled (dab) gene, disabled-1 (mdab1), encodes an adaptor molecule that functions in neural development. Targeted disruption of the mdab1 gene (mdab1–1 mice) leads to anomalies in the development of the cerebrum, hippocampus, and cerebellum. Here we describe a number of histologic abnormalities in the cerebellum of the mdab1–1 mouse. There is a complete absence of foliation, and most Purkinje cells are clumped in central clusters. However, lamination appears to develop normally in areas where the Purkinje cells and external granular layer are closely apposed. The granular layer forms a thin rind over most of the cerebellar surface, but is subdivided by both transverse and parasagittal boundaries. The Purkinje cells, identified by anti-zebrin II in the adult or anti-calbindin in the new born mdab1–1 mutant cerebellum, form a parasagittal banding pattern, similar to but distorted compared with the wild-type design. The data suggest that the development of the mdab1–1 cerebellum parallels the development of reeler. The reeler gene encodes an extracellular protein (Reelin) that is secreted by the external granular layer. Because Reelin expression is retained in the mdab1–1 mutant mouse, mDab1 p80 may act in a parallel pathway or downstream of Reelin, leading to the transformation of embryonic Purkinje cell clusters into the adult parasagittal bands. J. Comp. Neurol. 402:238–251, 1998. © 1998 Wiley-Liss, Inc.     

Primary degeneration of the granular layer of the cerebellum (Norman type)

Summary Purkinje cells, impregnated with the rapid Golgi method, in a patient with primary degeneration of the granular layer showed abnormal orientation of the perikaryon and dendrites, reduction in size of the dendritic arbor, absence of spiny branchlets, and large numbers of stubby spines and hypertrophic spines on secondary dendritic branches; stubby spines and thorn-like formations were seldom observed on the primary dendrites and perikaryon of some Purkinje cells. These findings are similar to those described in the cerebellum of the homozygous weaver mutant mouse and in the cerebella of experimentally induced agranular phenocopies, thus suggesting that similar plastic changes occur in human and animal Purkinje cells as a result of the absence of parallel fibres input in early developmental stages. In addition, Purkinje cells in this patient showed club-shaped deformities in the distal region of primary dendrites, which were filled with radially oriented, short dendrites covered with stubby spines and hypertrophic spines. These latter structures appear to be fully impregnated asteroid bodies observed in paraffin sections.     

The volume of Purkinje cells decreases in the cerebellum of acrylamide — intoxicated rats,but no cells are lost

The effects of acrylamide intoxication on the numbers of granule and Purkinje cells and the volume of Purkinje cell perikarya have been evaluated with stereological methods. The analysis was carried out in the cerebella of rats that had received a dose of 33.3 mg/kg acrylamide, twice a week, for 7.5 weeks. The total numbers of cerebellar granule and Purkinje cells were estimated using the optical fractionator and the mean volume of the Purkinje cell perikarya was estimated with the vertical rotator technique. The volumes of the molecular layer, the granular cell layer and the white matter were estimated using the Cavalierí principle. The mean weight of the cerebellum of the intoxicated rats was 7% lower than that of the controls rats (2P=0.001). The numbers of the Purkinje cells and granule cells were the same in both groups, but the mean volume of the perikarya of the Purkinje cells in the intoxicated rats was 10.5% less than that of the control group (2P=0.004). The volume of the granular cell layer was reduced by 15% (2P=0.006) but there were no differences in the volumes of the molecular layer and the white matter in the intoxicated and control animals.Supported by grants from Aarhus University Research Foundation and the Danish Medical Research Council     

Granule cell dispersion is restricted across transverse boundaries in mouse chimeras

The granular layer of the developing and adult cerebellum is marked by the presence of several transverse boundaries, revealed in gene expression patterns or as a consequence of genetic mutations. It is unclear whether these boundaries represent fundamental differences between granule cell populations or if they are a secondary response to regional differences in the underlying Purkinje cells. One possibility is that boundaries mark different spatial domains of granule cells in a lineage-dependent fashion. To test this hypothesis, we have analysed a series of murine embryonic stem cell chimeras marked by the constitutive expression of beta-galactosidase in donor granule cells. The chimeras show a consistent spatial restriction boundary, located in the granular layer of lobule VI in the vermis and extending laterally into crus I of the hemispheres. A second boundary was found separating lobules IX and X in the vermis. No correlation was found between the genotypes of molecular layer interneurons and the underlying granule cells, suggesting that they arise independently. No transverse boundaries were observed for the molecular layer interneurons, consistent with the hypothesis that they are not generated from precursors in the external granular layer. These results indicate that the granular layer of the cerebellum comprises cellular domains with different histories separated by consistent spatial restriction boundaries.     

Toll样受体4基因缺失导致小鼠运动平衡障碍

目的分析Toll样受体4（TLR4）基因缺失对小鼠运动平衡能力的影响。 方法通过运动平衡能力测试相关的行为学测试,检测并比较TLR4基因缺失（TLR4^-/-）小鼠和其同窝对照的野生型（TLR4^+/+）小鼠在运动平衡能力测试相关的行为学中的表现差异,并进一步分析与运动平衡能力密切相关的小脑结构的变化。 结果TLR4^-/-小鼠在足迹实验中的表现与野生型小鼠比较,差异无统计学意义（P>0.05）,但在加速转棒实验中的表现则明显优于野生型小鼠（P<0.05）,而在梯踏步实验中的表现则较野生型小鼠更差（P<0.05）。H&E染色结果显示,TLR4^-/-小鼠的小脑结构并未发生明显改变,但小脑分子层的厚度较野生型小鼠却有所减少（P<0.05）。 结论TLR4基因缺失可导致小鼠运动平衡能力受损。     

A note on the distribution of glial cells in the molecular layer of the dentate gyrus

KISHI, K., B. B. STANFIELD AND W. M. COWAN. A note on the distribution of glial cells in the molecular layer of thedentate gyrus. BRAIN RES. BULL. 4(1) 35–41, 1979.—The distribution of the perikarya of astrocytes and other glial cells in the molecular layer of the dentate gyrus has been studied in gold chloride-sublimate preparations of rats and of normal and reeler mice, and in plastic embedded material from young adult rats. Contrary to previous reports (Rose et al. [7]), we have found no evidence for a distinct “line” or “band” of astrocytic cell bodies along the interface between the zones of termination of the entorhinal and hippocampal afferents to the dentate gyrus. Indeed, apart from a conspicuous accumulation of astrocytes immediately beneath the pial surface and hippocampal fissure, the distribution of glial cells across the extent of the molecular layer appears to be more or less random. In view of this it is difficult to ascribe a critical role to the astrocytes in either determining the normal distribution of afferent fibers to the dentate gyrus or in promoting their re-organization following its partial deafferentation.     

Regionalization defects in the weaver mouse cerebellum

The mammalian cerebellum consists of parasagittal bands and transverse zones that are laid down early in development. When the adult cerebellum is immunostained for the Purkinje cell-specific antigen zebrin II (i.e., aldolase C), compartmentation is reflected in alternating zebrin II⁺ (P⁺) and zebrin II⁻ bands (P⁻). The zebrin II phenotype is Purkinje cell autonomous; thus, disruptions in the zebrin pattern may reflect early problems in pattern formation. Zebrin II expression has been examined in the weaver (wv) mouse cerebellum. Both zebrin II⁺ and zebrin II⁻ Purkinje cells are present in the homozygous weaver (wv/wv) mouse, but they are not distributed normally. In the posterior vermis, although the zebrin II⁺ bands are wider and multilaminate, the standard compartmentation is present. However, a large zebrin II⁺ cell mass is absent from the central vermis, and analysis of the anterior lobe reveals several missing zebrin II⁺ bands. The cytoarchitectonic defects in wv mice are not simply related to the Purkinje cell abnormalities. Instead, serial reconstruction reveals two transverse boundaries—one rostrally in lobule VI and the other caudally in lobule IX—that delineate cytoarchitectonic transverse zones important in cerebellar development. The abnormal zebrin expression pattern in wv/wv mice may be secondary to the deletion of a transverse zone. This is the first demonstration that Purkinje cell compartmentation can be altered by mutation; therefore, the wv mutation should prove valuable in understanding cerebellar regionalization. J. Comp. Neurol. 394:431–444, 1998. © 1998 Wiley-Liss, Inc.     

Antigenic compartmentation in the mouse cerebellar cortex: Zebrin and HNK-1 reveal a complex,overlapping molecular topography

Two monoclonal antibodies-anti-zebrin I and anti-HNK-1-have been used to study the compartmentation of the mouse cerebellar cortex. As in other species, the pattern of localization of the Purkinje cell specific antigen zebrin I is confined to a subset of Purkinje cells that are organized into parasagittal bands. The basic pattern consists of two abutting paramedian bands (P1+) and up to three additional vermal bands on either side (P2⁺? P4⁺) This patern is altered in the vermal regions of lobules X and VI-VII where all Purkinje cells are immunoreactive. In the hemisphere there are three additional bands present (P5⁺? P7⁺) plus two shorter bands in the paravermal area (P4b⁺ and P5a⁺) that extend from the paramedian lobule through the lobulus simplex. This pattern is very similar, but perhaps not identical, to that previously described for the rat. These results suggest a common mammalian plan for the expression and localization of zebrin I. By using a monoclonal antibody to an epitope associated with HNK-1, we have now identified a novel pattern of compartmentation in mouse cerebellum. The HNK-1 epitope is expressed most notably on Purkinje cells and Golgi cells. The molecular layer immunoreactivity associated with the Purkinje cell dendrites varies in intensity in a systematic and reproducible fashion. This reveals a novel cerebellar compartmentation that is sometimes complementary, sometimes overlapping, to that revealed by anti-zebrin. As a result, it is now possible to subdivide the cerebellar cortex into a still finer mosaic of antigenic patches and bands than was possible by using zebrins alone.© 1993 Wiley-Liss, Inc.     

Compartmentation of the mouse cerebellar cortex by sphingosine kinase

Classic cerebellar anatomy is based on the characteristic array of lobes and lobules. However, there is substantial evidence to suggest that more fundamental architecture is built around arrays of parasagittal stripes, which encompass both the inputs and outputs of the Purkinje cells (PCs). Sphingosine kinase (SPHK) is an enzyme that converts sphingosine (Sph) into sphingosine-1-phosphate (S1P). Recent reports have indicated that ceramide, Sph, and S1P play a role in cell survival, growth, and differentiation in several cell types, including neurons. In this study, we examined the localization of SPHK in the mouse cerebellum by using immunohistochemistry. Anti-SPHK immunoreactivity appeared in the cerebellar molecular layer and the PC membranes. The staining pattern is striped. In the molecular layer, the staining pattern probably reflects dendritic spines and dendrites. By electron microscopy, peroxidase reaction product was deposited within dendrites especially along the plasma membranes near spines. Seen at higher magnification, the staining was in and near the postsynaptic complexes. By double immunostaining, the striped pattern of SPHK expression was shown to be identical to that revealed by anti-zebrin II, although the subcellular distribution within PC's is not. This is the first demonstration of the cerebellar compartmentation of an enzyme related to lipid metabolism, and as such, it provides an insight into the roles of SPHK and formation of S1P. The selective expression of SPHK in the zebrin II-immunoreactive PCs may explain their resistance to cell death when ceramide metabolism is disrupted, as in the acid sphingomyelinase knockout model of Niemann-Pick type A/B disease.     

Cerebellar injury due to phenytoin. Identification and evolution of Purkinje cell axonal swellings in deep cerebellar nuclei of mice

Summary The present study describes the identification and the ultrastructural and numerical evolution of Purkinje cell axonal swellings induced by phenytion. Thirty male C57BI/6J mice received phenytion orally in doses up to 100 mg/kg daily and were killed after 3, 6, 10, 14, and 48 days of treatment. Light and electron microscopic investigations as well as morphometric analysis of cut surface area and numerical density of axonal swellings were performed. The swellings appeared as early as 6 days after initiation of treatment and gradually increased in size and frequency. Use of an anti-lymphocyte monoclonal antibody (CD 3), specifically cross-reacting with Purkinje cells, identified the swellings as dystrophic Purkinje cell axons. On grounds of their ultrastructural appearance they were classified into three distinct types occurring at different time intervals after phenytoin exposure. At 6 days, most axonal swellings contained loosely aggregated membranous vesicles and tubules in a finely granulated matrix (type 1). At 14 days, larger axonal swellings appeared characterized by the presence of three-dimensional networks of branched and anastomosing membranous tubules (type 2). At 48 days, even larger axons contained bodies of highly condensed membranous material of sometimes paracrystalline appearance (type 3). It is suggested that phenytoin-induced axonal pathology of Purkinje cells is a dynamic process characterized by the progressive accumulation of proliferating membranous material arranged in an increasingly complex fashion.Supported by the Deutsche Forschungsgemeinschaft, grant VO 272, 5-1     

Postsynaptic membrane domains in the molecular layer of the cerebellum: a correlation between presynaptic inputs and postsynaptic plasma membrane organization

The intramembrane particle (IMP) content of Purkinje, basket, stellate and Golgi cell plasma membrane was quantitatively assessed in freeze-fracture replicas of the cerebellum of normal rats and Weaver mutant mice. This analysis showed that, irrespective of the cell type innervated (i.e. Purkinje, stellate, basket or Golgi cells) postsynaptic membranes for parallel fibers had a relatively low IMP content in their cytoplasmic P-face (≈ 750 IMP/μm², while postsynaptic membranes for climbing, basket and stellate axons were characterized by a significantly higher IMP content (≈ 1400 IMP/μm²). This difference of IMP content between the targets for parallel fibers and those for climbing, basket and stellate axons was restricted to 3IMP smaller than 10 nm and appeared progressively during the development of the molecular layer, suggesting a correlation between the formation of synaptic contacts and the segregation of the postsynaptic membrane in these two different domains. In addition, the study of the Weaver mice cerebellum, which is deprived of parallel fibers, but yet shows a normal IMP content in the postsynaptic membrane for the missing fibers, indicated that this characteristic IMP content is established before or during the afferent's reaching its target, and independently of whether the contact ultimately occurs.     

Distribution of Cutaneous Nociceptive and Tactile Climbing Fibre Input to Sagittal Zones in Cat Cerebellar Anterior Lobe

Climbing fibres projecting to the cerebellar C3 zone (and the related C1 and Y zones) receive spatially well organized tactile and nociceptive inputs from the skin. In the present study, cutaneous tactile and nociceptive input to climbing fibres projecting to the X, B, C2 and D1 zones in lobule V were investigated in pentobarbitone-anaesthetized cats. From the present results and previous studies, it is concluded that the X, C1, CX, C3 and Y zones receive cutaneous nociceptive climbing fibre input. By contrast, climbing fibres to the B, C2 and D1 zones lack cutaneous nociceptive input. Tactile input was found in all zones. The spatial organization of receptive fields of climbing fibres projecting to the X and D1 zones was similar to that in the C3 zone. They were located on the ipsilateral forelimb, mainly its lateral and distal parts, and their proximal borders were located close to joints. In the B zone, more than half of the receptive fields of climbing fibres were confined to the ipsilateral hind- or forelimb. However, frequently more than one limb and parts of the trunk were included. In the C2 zone, the majority of climbing fibres had distal ipsi- or bilateral receptive fields on the forelimbs, often also including the head/face. Some of the bilateral forelimb receptive fields additionally included the hindlimbs ipsi- or bilaterally. The results indicate that each zone has a characteristic set of climbing fibre receptive fields, which is probably related to its efferent control functions.     

Development of Purkinje cell somatic spines in the weaver mouse

Summary The cerebellar cortices of weaver mice and their normal littermates ranging in age from six to thirteen days after birth were examined with the electron microscope. The initial development of spines, both somatic and dendritic, are the same in both the weaver and its normal littermates despite the extreme paucity of parallel fibers in the former.