Autolysis of the granular layer of the cerebellar cortex in brain death

Summary In a study of duration of brain death, granular layer autolysis (GLA) of the cerebellar cortex was analyzed in 45 patients who died of acute cerebrovascular diseases (CVDs). Twelve patients who died of causes other than intracranial disease served as controls. Tonsillar herniation occurred in all who died of acute CVDs. More advanced GLA was seen in the central folia adjacent to the central white medullary body of the cerebellum as compared with the peripheral folia. Widespread GLA involving the most of the peripheral folia was found solely in patients in whom brain death had been present over 18 h. Of the 12 control patients, 4 showed GLA only in the central folia. Although GLA of the central folia might develop during immersion fixation of the brain, the alteration of the peripheral folia is assumed to develop in the period of brain death. Widespread GLA extending to the peripheral folia could be a pathological finding characteristic of brain death, where intracranial blood flow could be absent or significantly reduced. Brain death for little less than 1 day would be necessary for GLA to develop.     

The pathogenesis of acute selective necrosis of the granular layer of the human cerebellar cortex

Summary pH determinations by means of a study, spear-electrode inserted in the cerebellar tissue immediately after necropsy were carried out in 60 patients suffering from different disorders and dying in hospital, and from 25 individuals killed instantly by violent accidents. A correlation was found between the presence of NGL and low pH levels in the cerebellum (average pH 5.56); subjects without NGL changes showed an average pH of 6.08. No correlation was found between the pH levels and the time after death, or between pH and any specific disease.The so-called acute selective necrosis of the granular layer of the human cerebellar cortex (NGL), is considered from the present studies to be a phenomenon of postmortem origin. Its relationship to certain specific diseases can be explained by the fact that in these conditions there is a higher postmortem acidity, known to promote autolysis.     

Structure of the olfactory bulb of the hedgehog (Erinaceus europaeus): description of cell types in the granular layer

The cytoarchitecture of the olfactory bulb and the cell types in the granular layer of adult hedgehogs have been studied with the Golgi method. The mitral cell layer does not stand out as a monolayer as in most mammals; it is arranged as a diffuse stratum with mitral cells displaced into the external plexiform layer. The external plexiform layer is exceedingly thick and contains the branches of peripheral processes of granule cells and displaced mitral and tufted cells. The granular layer contains granule cells and varieties of short-axon cells. Among granule cells a type of cell with an elaborate system of protrusions close to the cell body has been found. Four main varieties of short-axon cells are described. These include cells with local or extended axons, according to the branching pattern of their axons inside the granular layer or extending into the external plexiform layer as well. Short-axon cells were also classified as cells with smooth and spinous dendrites. A variety of cell with smooth dendrites and elaborate axonal system reaching the periglomerular zone is described. This type of cell has been found frequently in the olfactory bulb of the hedgehog. In comparison to several other mammals, short-axon cells in the olfactory bulb of the hedgehog have been found to be particularly abundant and to have more complex axonal systems. It is suggested that some of them may represent inhibitory interneurons acting upon granule and periglomerular cells, playing an important role in the centrifugal pathway controlling the olfactory input.     

Maturation of Purkinje cells in mouse cerebellum after neonatal administrations of cytosine arabinoside

Summary ICR-JCL strain mice were injected subcutaneously with 30 mg/kg body weight of cytosine arabinoside at 2, 3, and 4 days of age. This chemical prevented the production of the basket cells, stellate cells, and granule cells in the external granular layer of the cerebellum. Decrease in number of these microneurons affected the normal synaptic connections between the Purkinje cells and the microneurons, thus causing the disarrangement and abnormal arborization of the Purkinje cells. Of the three types of microneurons, the basket and a few stellate cells played a more important role in the disarrangement of the Purkinje cells and abnormal arborization of their primary dendrites than the granule cells did. Abnormal outgrowing directions of other smooth dendrites of the Purkinje cells were caused mainly by the diminution of stellate cells. Although parallel fibers were grossly decreased in number in the treated cerebellums, spines of the spiny dendrites of the Purkinje cells sprouted considerably in the 15-day-old mice, and then their morphological features remained even after 100 days of age.     

Selective staining of a subset of Purkinje cells in the human cerebellum with monoclonal antibody mabQ113

MabQ113 is a monoclonal antibody raised against rat cerebellum which selectively stains Purkinje cells. Likewise, in mabQ113-immunoperoxidase stained sections of human cerebellum, deposits of reaction product are found only in the Purkinje cells. The dendritic arborizations, cell body, and axonal processes are immunoreactive. In rat, mabQ113 reveals a series of parasagittal antigenic bands which run throughout the cerebellar cortex. The staining distribution in human cerebellar cortex likewise reveals heterogenous staining but the pattern is a complex one and seems to be unlike the parasagittal banding found in the rat. In a number of human diseases Purkinje cell degeneration is not uniform throughout the vermis and cerebellar hemispheres. It is possible that mabQ113⁺ and mabQ113− subsets of Purkinje cells may respond differentially to various pathological conditions.     

Expression of GluR6 kainate receptor subunit in granular layer of weaver mouse cerebellum

The Girk2 ^wv (weaver) mutation impairs migration of cerebellar granule cells from external to internal granular layer and induces neuronal death during the first 2 weeks of postnatal life. Kainate receptors are heteromeric ionotropic receptors of glutamate consisting of five subunits termed GluR5, GluR6, GluR7, KA1 and KA2. In order to investigate whether the weaver gene affects the expression of kainate receptors in weaver cerebellum, we determined mRNA expression levels of GluR6 kainate receptor subunit and [³H]kainic acid specific binding in the developing cerebellum, using in situ hybridization and receptor film autoradiography, respectively. In the weaver postnatal day 10 (P10) cerebellum, our data indicated lower levels of GluR6 mRNA expression and lower [³H]kainic acid specific binding in external granular layer (EGL) compared to normal EGL. Our results are indicative of either down-regulation of kainate receptors or modulation of their functional characteristics in weaver granule cells.     

Partial ablation of the neonatal external granular layer disrupts mossy fiber topography in the adult rat cerebellum

The spinocerebellar projection in the rat is compartmentalized in an array of parasagittal bands of mossy fiber terminals. These bands align reproducibly with bands of Purkinje cells that differentially express zebrin II. To investigate whether this alignment is obligatory, Purkinje cell and mossy fiber compartmentation has been compared in the rat cerebellum where the cytoarchitecture was contorted by neonatal administration of methylazoxymethanol. Methylazoxymethanol ablates many granule cell precursors, leaving a much reduced external granular layer, and adult rats that received a single methylazoxymethanol injection at birth showed varying degrees of abnormal cerebellar foliation. Zebrin II immunocytochemistry nevertheless revealed no fundamental abnormality in the Purkinje cell compartments. However, despite the normal Purkinje cell compartmentation being retained, the spinocerebellar mossy fiber-Purkinje cell topography is disrupted by methylazoxymethanol treatment. The normal precise array of parasagittal mossy fiber terminal fields becomes blurred across the lobule, and the normal clear banding is difficult to follow. These data suggest that, despite the early topography being dependent on the Purkinje cells, the granule cell-mossy fiber interactions also regulate the topography of the spinocerebellar projection. © 1996 Wiley-Liss, Inc.     

Heterotopic cell nests in the developing rat cerebellum

Summary Serial sagittal sections of the postnatally developing cerebellum of Wistar rats were studied to determine the incidence, origin, and relation to other cerebellar structures of nests of heterotopic cells. These nests were found to consist of small, darkly staining cells of either irregular or elongate shape with large, dark nuclei and bore close resemblance to cells of the external granular layer. They were present in every animal from the day of birth to at least day 15 of life and were confined to the uvula, nodulus, and pyramis of the vermis. In addition, the cell nests were found to be perivascular and continuous with the external granular layer via a narrow tongue of cells surrounding capillaries which penetrated to the medullary layer of the cerebellum from the subarachnoid space. The significance of the nests in relation to other studies, and their involution after day 15 of life is briefly considered.     

Selective axonal and terminal degeneration in the chicken brainstem and cerebellum following exposure tobis(1-methylethyl)phosphorofluoridate (DFP)

Utilizing a variation of the Fink-Heimer method, we examined the extent and location of axonal and terminal degeneration within the chicken cervical spinal cord, brainstem and cerebellum resulting from a single subcutaneous dose ofbis(1-methylethyl)phosphorofluoridate (DFP). The effects of DFP on the activities of whole-brain neuropathy target esterase (NTE) and cholinesterase (ChE) were also assessed as were the development and severity of clinical signs characteristic of organophosphorus-induced delayed neuropathy (OPIDN). Both whole brain NTE and ChE activities were maximally inhibited during the first 24 h post-exposure, showing gradual recovery over a period of e weeks. OPIDN clinical signs were not observed at 7 days post-DFP but progressed to severe ataxia by day 14 and paralysis by day 21. There was a relative absence of degeneration at 7 days, a dramatic increase in degeneration density at 14 days, and high density degeneration at both 21 and 28 days. Cervical spinal and medullary tracts containing axonal degeneration included the fasciculus gracilis, dorsal and ventral spinocerebellar tracts, spinal lemniscus, and the intramedullary portions of the glossopharyngeal and vagus nerves. Brainstem nuclei containing terminal degeneration included the lateral cervical, gracile-cuneate, external cuneate, and inferior olivary nuclei, the nucleus tractus solitarius, and the lateral and paragigantocellular lateral reticular nuclei. Mossy fiber degeneration was also present in cerebellar folia I–Vb. These results show that exposure to DFP causes axonal and terminal degeneration in ascending spinal tracts, brainstem nuclei and cerebellar folia associated with the transmission of somatic and visceral sensory information.     

Effects of thyroidectomy and season on the external granular layer of the cerebellum in metamorphosing bullfrog tadpoles (Rana catesbeiana)

Transient formation of the cerebellar external granular layer (EGL) occurs during metamorphosis in frog tadpoles and is dependent on thyroid hormone. Late prometamorphic bullfrog tadpoles at similar stages of development were thyroidectomized and then killed after 1 month and 2 months during the fall and winter. The cerebellum was studied by qualitative and quantitative light microscopy. Thyroidectomy resulted in an inhibition of external metamorphic changes such as hind limb growth. Examination of the cerebellum after thyroidectomy showed the EGL greatly decreased in thickness after 1 month and in some cases was altogether absent after 2 months. Because the disappearance of EGL cells was a result of their inward migration into the IGL, we conclude that thyroid hormone was not necessary for granule cell migration. In addition, metamorphic change normally occurs in the late spring and summer in bullfrog tadpoles, and is inhibited during the fall and winter (metamorphic stasis). During the winter, the volume of the EGL was significantly less compared with tadpoles undergoing active metamorphosis. Although lowered temperature appears to contribute to the large decline in EGL thickness, significant seasonal differences in the EGL volume ratio were still observed in tadpoles maintained under conditions of constant temperature and light. Thyroxine administered to thyroidectomized tadpoles during the winter increased the thickness of the EGL. Therefore, the seasonal decline in the EGL thickness reflects a decrease in thyroid hormone activity, which in turn appears controlled by both internal (hypothalamic) and environmental (e.g., temperature) factors.     

Measuring parallel fiber length in the rat cerebellum

A new approach was devised to obtain the mean length of parallel fibers in Golgi sections of the rat cerebellum. This method was based on the principle that within a given region of the cerebellum the mean length of parallel fibers should be inversely proportional to the likelihood of spotting the ends of parallel fibers. An experimental protocol was designed based on the statistical relationship between the mean fiber length and the number of the ends of parallel fibers for a given total length of parallel fibers examined. Our methodology was simple and could have advantages over other existing methods.     

Evidence for an axonal localization of the type 2 corticotropin-releasing factor receptor during postnatal development of the mouse cerebellum

Previous studies have described the embryonic and postnatal development of CRF, as well as the type 1 CRF receptor in the mouse cerebellum. The present immunohistochemical study localizes the cellular distribution of the type 2 CRF receptor (CRF-R2) during postnatal development of the mouse cerebellum. Western blot analysis indicates that the antibody used in this analysis recognizes both a full-length and a truncated isoform of the type 2 receptor. We propose that each isoform has a unique cellular distribution. In the present study, the postnatal (P) development (P0-P14) and cellular localization of CRF-R2 in different cell types was analyzed using PAP and double-label fluorescent immunohistochemistry; cell-specific antibodies were used to identify cells expressing CRF-R2 at different stages of postnatal development. At P0, CRF-R2 immunoreactivity was localized within the somata of Purkinje cells and migrating GABAergic interneurons. CRF-R2 was first observed in the initial axonal segments of some Purkinje cells at P5, and was evident in many Purkinje cell axon hillocks at P8. Punctate immunoreactivity is present in the molecular layer by P5 and is interpreted to be immunolabeled parallel fibers. Between P8 and P14, CRF-R2 immunostaining is present in the initial axonal segments of Golgi cells, within the internal granule cell layer. Finally, CRF-R2 is present in both radial glia in the molecular layer as well as in astrocytes in the white matter and internal granule cell layer from P5 to P14. The present results suggest that CRF-R2, both the truncated and the full-length isoforms, are present in the developing cerebellum, each with a unique cellular distribution. The immunohistochemical evidence indicates that the truncated isoform of the type 2 CRF receptor is in the axons of several different types of cerebellar cortical neurons, and suggests that CRF could play a role in cerebellar development by modulating the release of transmitters from excitatory and/or inhibitory interneurons, which in turn could directly alter the maturation of cerebellar circuits. In contrast, the binding of a ligand to the full-length isoform of CRF-R2 or to CRF-R1, both in a postsynaptic location, may have a more direct effect on regulating the responsiveness of these cells to growth factors or neurotransmitters released from afferent axons by regulating permeability of ion channels or altering second messenger systems.     

Dating the development of human cerebellum

Summary A histological study of normal cerebella in 72 human autopsies allowed defining several readily identifiable landmarks by which cerebellar development was dated for vermis and for hemispheres respectively: Disappearance of the lamina dissecans at 28 and 32 weeks gestation; Purkinje cells first discernible at the same age for both; onset of growth of inner granular layer at 30 and 32 weeks; sharp boundary between inner granular layer and white matter at approximately 36 weeks and about term; onset of marked growth of molecular layer at 30 and 38 weeks; adult thickness of molecular layer at approximately the 8th postnatal month for both; and accelerated involution of outer granular layer between the 2nd and 4th postnatal months, remnants persisting up to 9 and 13 months. Nests of matrix cells in the cerebellar nuclei were found in 28% of the cases less than 4 months old. These cell nests appear to undergo involution by the 4th month—that is, at the same time as the outer granular layer. The significance of these developmental events for the interpretation of pathological alterations in the developing cerebellum is discussed.Supported by US Public Health Grant NS 06239 from the National Institute of Neurological Diseases and Stroke.     

Observations on the cerebellum of normal-reeler mutant mouse chimera

The normal-reeler chimera mouse (+/+ in equilibrium with rl/rl) provides an experimental system in which an analysis of the migration of immature neurons in the cerebellum can be accomplished. In the present study, five chimera mice were produced from embryos of the wild-type control (C57Bl/6N) and the reeler mutant mouse (BALB/c) by the aggregation technique. The isozyme pattern of glucosephosphate isomerase (GPI) revealed that the brain tissue in the chimera contained both isozymes of the BALB/c (reeler) and C57Bl/6N (normal) strains, implying that internal mosaicism of the cerebellum truly existed. We found no abnormality in the cerebellum of the chimera mouse: the neuronal and glial subpopulations revealed no difference from those of the control. Such normalization of the cerebellum in the chimera suggests that the disturbance of neuronal migration in the reeler is attributable to an abnormal cell-to-cell interaction between migrating young neurons and the radial glial cells.     

Pannexin expression in the cerebellum

Pannexin1 and pannexin2 are members of the pannexin gene family which are widely expressed in the central nervous system. Here we present an overview of pannexin expression and distribution in the mouse cerebellum. Pannexin1 and pannexin2 are expressed in the Purkinje cells and in some cells of the granule cell layer. Pannexin2 is also expressed in the stellate cells of the molecular layer. A differential expression of pannexin1 and pannexin2 mRNA is observed during cerebellar development. These findings constitute the first indication of the involvement of pannexin molecules in the developing cerebellum. Although the functional relevance of these molecules remains currently unknown, the abundance of pannexins in the Purkinje cells suggests that they may contribute to the generation of cerebellar rhythms.     

Purkinje cell loss and the noradrenergic system in the cerebellum of pcd mutant mice

Purkinje cells in the cerebellum receive inhibitory noradrenergic input from the locus coeruleus. In pcd mutant mice all Purkinje cells degenerate by 45 days of age. The purpose of the present studies was to determine if the loss of these cerebellar neurons affects the amounts of norepinephrine in the cerebellum of mice 25–280 days of age. No significant changes in norepinephrine content were detected during or after Purkinje cell degeneration. However, since degeneration led to a reduction in cerebellar weight, the norepinephrine concentration was increased in pcd mutants. These results indicate that despite the loss of a major postsynaptic target (Purkinje cells), the cerebellar noradrenergic input remains stable.     

A Golgi and electron-microscopic study of cerebellum in methylmercury-poisoned neonatal mice

Summary Neonatal C57BL/6J mice were injected with 5 mg/kg body weight of²⁰³Hg-labeled methylmercuric chloride on postnatal days 3, 4, and 5, totaling 15 mg/kg body weight per animal. The experimental and control animals were sacrificed on postnatal day 15. Whole body radioactivity of²⁰³Hg progressively increased during the 3-day injection period and reached the peak level and remained at peak levels until the time of sacrifice. This indicates a lack of clearance of²⁰³Hg by neonatal mice during the period examined in this study. Golgi preparations of cerebella of MeHg-treated animals revealed significant reduction in dendritic arborization of Purkinje cells. Ultrastructurally, the vascular endothelium showed attenuation with increased electron density and frequent vacuolization of cytoplasm. Marked swelling of perivascular glia was noted in most of the capillaries throughout the cerebella of MeHg-treated animals.This study is supported in parts by USPHS NIEHS Grants 5R01-ES 01722 and ES 01247     

Primary rhabdomyosarcoma of brain and cerebellum

Summary Tumors of the central nervous system (CNS) composed of pure mesenchymal derivatives with both embryonal and mature striated muscle cells devoid of neuroblastic elements should be considered rhabdomyosarcomas. Some 13 cases have been reported, and here we study four additional cases in infancy under 3 years of age which represent 0.82% of 483 intracranial tumors studied by us at the Children's Hospital in the last 12 years. Two cases were localized in the temporal lobes, and two were in the cerebellar vermis. All of them were typical embryonal rhabdomyosarcomas at various stages of differentiation including undifferentiated mesenchymal cells, embryonal cells, and rhabdomyoblasts. Tumor cells achieved a higher degree of differentiation in the cerebellum, as shown by readily detectable immature muscle fibers which were consistently absent in tumors involving the brain. Myoglobin [peroxidase-antiper-oxidase (PAP) techniquel was positive throughout in rhabdomyoblasts and in immature muscle cells, whereas glial fibrillary acidic protein was negative in all four tumors.In spite of the well differentiated appearance of the cerebellar tumors, their behavior was highly malignant with extensive infiltration of brainstem leptomeninges in one case, and all patients survived for only a short time after surgery.These tumors may be observed in the midline structures of the posterior fossa and in the brain, but we suspect their true incidence might be higher if immunohistochemical techniques were applied.Abstract has been previously published in Journal of Neuropath and Exp. Neurology 1983, 42 page 314 with the Proceedings of the 59th Annual Meeting of the American Association of Neuropathologists