Damage of purkinje cell axons following chronic phenytoin administration: An animal model of distal axonopathy

Summary An animal model of central distal axonopathy following chronic administration of phenytoin is described. Male C57/BL6J mice received diphenylhydantoin (DPH) in the daily diet (liquid diet Stardit, supplemented with vitamins) over a period of 8 weeks. Control and experimental animals were pair-fed.Twelve mice of both groups were perfused via the left ventricle with glutaraldehyde. Representative samples of the cerebral cortex (area 3), cerebellum (vermis and deep cerebellar nuclei), thalamus, hypothalamus, and liver were embedded in araldite. Semithin sections and electron microscopy of the cerebellar vermis revealed marked dystrophic changes in the Purkinje cell axons. The presynaptic segments of Purkinje cell axons in the deep cerebellar nuclei showed massive enlargement and swelling due to accumulation of spherical particles and tubular structures in the axoplasm. These structures represent a proliferation of the smooth endoplasmic reticulum.Identical changes were found in hepatocytes of treated animals. Because phenytoin induces hepatic microsomal enzymes, we suggest that phenytoin-related Purkinje cell damage may be produced by an induction of Purkinje cell microsomes with proliferation of the smooth endoplasmic reticulum which causes a swelling and enlargement of presynaptic segments of Purkinje cell axons in deep cerebellar nuclei. Chronic phenytoin administration to mice is a new model of phenytoin-induced encephalopathy and of distal axonopathy of cerebellar neurons.Supported by the Deutsche ForschungsgemeinschaftPresented in Part at the Joint Meeting of the German and Scandinavian Neuropathologists, Turku, Finland, June 3–4, 1983     

A study of the synaptogenesis in the cerebellar cortex through chronic treatment and immunocytochemistry of beta-bungarotoxin.

The mechanism of synaptogenesis between dendritic spines of Purkinje cells and parallel fibers in the chicken cerebellum was studied through chronic treatment and immunocytochemistry of beta-Bungarotoxin (beta-BT). Attention was directed to the question of whether the presynaptic specializations (presynaptic vesicular grids composed of presynaptic dense projections and associated vesicles) of the parallel fibers can differentiate in the absence of the Purkinje cells. Normal cerebella from 18-day and 21-day chick embryos, incubated with beta-BT and reacted with HRP-labeled anti beta-BT guinea pig IgG, showed a positive HRP reaction on Purkinje cells but not on external and internal granule cells. Thus, in chicken cerebellum, beta-BT primarily affects Purkinje cells. When beta-BT was applied to chick embryos at 3-day intervals, beginning on the 4th day of incubation, the cerebella were markedly reduced in size and most of the Purkinje cells as well as the nerve fibers in the white matter disappeared between the 18th and the 21st day of incubation. Folia of the 21st day experimental cerebella were irregular in shape and the area of the midsagittal section was one fourth that of the controls. In the 21st day cerebella treated with beta-BT, the majority of Purkinje cells disappear. However, the external granule cells remain intact and showed the usual mitotic activity. The majority of the inner granule cells were normal. Some of the parallel fibers, which display presynaptic vesicular grids, established synaptic contact with stellate cells and the dendritic spines of the few Purkinje cells that survived the treatment. However, vast areas of the molecular layer contained neither dendrites of Purkinje cells, nor parallel fibers displaying presynaptic vesicular grids devoid of their postsynaptic counterpart. In such areas, the molecular layer consisted of parallel fibers of uniform diameter, some of which contained accumulations of vesicles, but displaying no presynaptic dense projections. This suggests that parallel fibers may not be able to completely develop the presynaptic vesicular grids in the absence of their target cells.     

Morphological alterations of Purkinje cell axons and presynaptic terminals in organotypic cerebellar cultures exposed to ionizing irradiation

Summary Ionizing irradiation induced marked morphological alterations in the Purkinje cell axons and axonic terminals in vitro. The myelinated segments demonstrated a very poor development of the myelin sheath. The axoplasm demonstrated various alterations of the mitochondria, and the smooth endoplasmic reticulum and the numerous elongated presynaptic terminals contained large aggregates of tubulovesicular structures and neurofilaments arranged in parallel and reticular array.     

Menkes disease: a Golgi and electron microscopic study of the cerebellar cortex

A neuropathological study of a case of Menkes disease is reported, illustrating the involvement of different types of neuronal cells. The cerebellum showed the most striking abnormalities: severe lack of internal granule cells. Purkinje cells with weeping willow pattern, numerous segmental enlargements of dendritic trunks and secondary branches, and presence of numerous eosinphilic spherical bodies in the molecular layer were the most conspicuous features. Using electron microscopy, the dendritic enlargements were observed to be made of both proliferated and enlarged mitochondria, and of saccules of smooth endoplasmic reticulum. The spheroid bodies in the molecular layer were mainly made of concentric lamellar structures which seemed to be proliferated smooth endoplasmic reticulum. The relationship between these morphological abnormalities and the metabolic disorder of Menkes disease is discussed.     

Altered axons and axon terminals in the nucleus gracilis of the dog

Altered axons and axon terminals in the nucleus gracilis o the dogs were followed by means of electron microscopy. The axon terminals with characteristic ultrastructure were accompanied by synapses with altered presynaptic part. The changes of the presynaptic part were expressed by the evident decrease in synaptic vesicle number, hyperplasia of smooth endoplasmic reticulum and by abnormal mitochondrial figures. The altered axons measuring 14,6 micrometers in average contained hyperplastic and quite often dilated smooth endoplasmic reticulum as well as multiplied microtubular profiles. The mitochondria of the altered axons were characterized by the different degree of degeneration. The above mentioned structures in synapses as well as in axoplasma of the nucleus gracilis were found in the dogs of both sexes.     

The development and degeneration of Purkinje cells in pcd mutant mice.

Purkinje cell degeneration (pcd), an autosomal recessive mutation in the mouse, causes the postnatal death of virtually all cerebellar Purkinje cells during the third and fourth postnatal week. We have compared the postnatal development of normal and pcd mutant Purkinje cells. The early deviations from normal development involve primarily the perikaryonal polysomes and endoplasmic reticulum. Many of the mutant Purkinje cells retain abnormally the basal accumulation of polysomes, a finding which permits the identification of affected animals at postnatal day 15, one week prior to the onset of behavioral abnormalities. In addition, the affected Purkinje cells possess unusual configurations of endoplasmic reticulum with associated electron-dense particles similar to but larger than ribosomes, mature and forming intracisternal A particles and nematosomes. Before the pcd Purkinje cells degenerate they appear to receive all their appropriate synaptic contacts. Some disruption, however, of parallel fiber: Purkinje spine synaptogenesis occurs at late stages of development. Some spines lack presynaptic elements, postsynaptic thickenings are present along the dendritic shafts and parallel fibers appear to make synaptic contacts directly onto the shafts. The spectrum of early morphological changes that has been observed in pcd mutant Purkinje cells is thus far unique to this cerebellar abnormality.     

Experimental protein malnutrition in primates: electronmicroscopic observations on the cerebellum of healthy and malnourished squirrel monkeys (Saimiri sciureus).

Eight young adult squirrel monkeys were maintained on a protein deficient (2% calories from proteins) diet for a period of 15 weeks during which the animals lost wieght steadily and developed varying degrees of sensory and motor impairments as evidenced by lack of interest in the surrounding environment and changes in posture and gait. An additional eight animals were maintained on a high protein diet (25% calories from proteins) to serve as controls. Two animals each from the experimental and control groups were sacrificed at 9, 11, 13 and 15 weeks of special feeding schedule. The Purkinje cells of the cerebellum of malnourished animals showed significant changes in the ribosomes, endoplasmic reticulum and Golgi zone indicating greatly disturbed protein metabolism. The granule cells, especially of the pale variety show prominent watery swellings with scant cell organelles. The most prominent change appeared in various types of neurons related to the multiplication of various types of lysosomes, multivesicular lamellar bodies and the lipofuscin pigment bodies. In the animals sacrificed at different intervals, the relatively more malnourished animals showed significantly larger number of lipofuscin pigment in the various types of neurons of the cerebellar cortex. The dendritic changes included honey-comb formation in the lumen. The astrocyte glial fibers were greatly increased. The pericytes of the blood vessels showed irregular outlines and were greatly enlarged with numerous osmiophilic bodies (presumably lipofuscin) in the experimental animals as compared to the controls.     

Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein

Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5' regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-D-aspartate receptors.     

Light and electron microscopic demonstration of guanylate cyclase in rat brain

In the endothelial cells of capillaries in the rat CNS, we have observed abundant, circumferentially oriented, smooth, membrane-bound profiles, found just beneath, and parallel to, the abluminal plasmalemma. These structures are seen particularly well in tissue exposed to potassium ferricyanide-reduced OsO4. We studied these structures cytochemically, using glucose-6-phosphatase as a marker for endoplasmic reticulum, and acid phosphatase as a marker for the Golgi-associated endoplasmic reticulum containing lysosomal enzymes (GERL). We found that they contained glucose-6-phosphate hydrolyzing activity but did not contain acid phosphatase activity. Comparable structures were not seen in the continuous capillaries of skeletal muscle. Based on their morphology and content of glucose-6-phosphate hydrolyzing activity, we conclude that these structures are uniquely oriented smooth endoplasmic reticulum, which is much more abundant in capillaries of the CNS than in other continuous capillaries. The function of this distinctive feature of the CNS capillary is not known.     

An ultrastructural study of sympathetic ganglion satellite cells in the rat 1. Normal and x-ray irradiated satellite cells

Summary The ultrastructure of normal and irradiated satellite cells of the rat superior cervical ganglia was studied. Each neuron is closely and completely invested by satellite cell cytoplasm. The satellite cell cytoplasm usually contains a number of mitochondria, granular endoplasmic reticulum, Golgi apparatus, lysosome-like bodies, filaments, microtubules, lipid droplets, centriole and cilia. The satellite cell also encloses presynaptic nerve endings, and other nerve fibers. X-ray irradiation produces several changes in the satellite cells. These include: (a) increased nuclear density, (b) marked local widening of the perinuclear space, (c) dilation of granular endoplasmic reticulum and loss of attached ribosomes, (d) swelling of mitochondria with disorganization of mitochondrial cristae and loss of matrix, (e) increase in the number of vacuoles, and (f) increase in the number, size and contents of lysosome-like bodies.     

Abundant, uniquely oriented endoplasmic reticulum in capillaries of the CNS: Demonstration using reduced-osmium and glucose-6-phosphatase cytochemistry

In the endothelial cells of capillaries in the rat CNS, we have observed abundant, circumferentially oriented, smooth, membrane-bound profiles, found just beneath, and parallel to, the abluminal plasmalemma. These structures are seen particularly well in tissue exposed to potassium ferricyanide-reduced O_sO₄. We studied these structures cytochemically, using glucose-6-phosphatase as a marker for endoplasmic reticulum, and acid phosphatase as a marker for the Golgi-associated endoplasmic reticulum containing lysosomal enzymes (GERL). We found that they contained glucose-6-phosphate hydrolyzing activity but did not contain acid phosphatase activity. Comparable structures were not seen in the continuous capillaries of skeletal muscle. Based on their morphology and content of glucose-6-phosphate hydrolyzing activity, we conclude that these structures are uniquely oriented smooth endoplasmic reticulum, which is much more abundant in capillaries of the CNS than in other continuous capillaries. The function of this distinctive feature of the CNS capillary is not known.     

Neurons with whorl bodies have increased numbers of synapses

The endoplasmic reticulum in some neurons of the rat hypothalamic arcuate nucleus forms concentric sheets of smooth cisternae which are known as whorl bodies. It has been reported that the number and size of these structures change under varying endocrine conditions. For example, the number of rat arcuate nucleus neurons containing whorl bodies increases after gonadectomy. In studying their relationship to other components of the cell, we found that neuron profiles which contain whorl bodies receive a significantly increased number of axosomatic presynaptic terminals (P less than 0.001). Whorl bodies may mark a subpopulation of endocrine sensitive cells which are characterized by a different pattern of connections and whose response to stimuli includes changes in endoplasmic reticulum organization.     

Distributional pattern of apoptotic cells in rat cerebellar vermis experimentally induced by methylmercury intoxication

This report deals with the distributional pattern of apoptotic granule cells in rat cerebellar vermis following experimental methylmercury chloride (MMC) toxicosis. The rats administered MMC showed clinical signs of hind leg paralysis around day 19 and this developed to moribund by day 26. Pathologic examination of the moribund animals revealed that cerebellar degeneration characterized by apoptosis of the granule cells was not equally distributed in the cerebellar vermis; apoptotic granule cells were observed in the lingula, central lobule, Lobulus ascendens, pyramis vermis and culmen in the order of decreasing frequency. This selective vulnerability in MMC intoxication is similar to the granule cell loss of Minamata disease in humans and suggests that this rat model is a useful pathobiologic model for studying cerebellar degeneration in Minamata disease.     

Temporal development of GABA agonist induced alterations in ultrastructure and GABA receptor expression in cultured cerebellar granule cells

The temporal development of the effect of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) on the ultrastructure composition and GABA receptor expression in cerebellar granule cells was investigated by quantitative electron microscopy (morphometric analysis) and GABA binding assays. It was found that the cytoplasmic density of smooth endoplasmic reticulum was decreased, while the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus, vesicles and coated vesicles was greatly enhanced after exposure of the cells to THIP (150 microM) for only 1 hr. In cerebellar granule cells exposed to THIP (150 microM) for 3 hr low affinity GABA receptors were induced. These findings show that the effect of THIP on the ultrastructure composition and GABA receptor expression in cultured cerebellar granule cells may be interrelated and moreover it is likely that the turn-over of GABA receptors is extremely fast.     

Specialized formations of the endoplasmic reticulum in cultures of murine neuroblastoma cells

Ultrastructural features of cytodifferentiation in monolayer cultures of mouse neuroblastoma cells (clone neuro-2A) was further characterized by the presence of annulate lamellar arrays with up to 10 lamellae. The lamellae were made up of fused smooth surfaced cisternae forming pores or annuli and were surrounded by a dense filamentous to granular material. Stacks of nonfenestrated, parallel, regularly spaced cisternae, designated as lamellar bodies, also appeared in the cytoplasm in association with the endoplasmic reticulum and the nucleus. Such flattened cisternae were also seen to be formed from transformed endoplasmic reticulum. Furthermore a relatively large number of concentric whorled lamellar bodies were presented as well as dense whorled structures reminiscent of the cytoplasmic laminar bodies. All of these structures are derived from the rough endoplasmic reticulum, the tubules of which are coexistent with those of the latter. In addition there is a topographic relationship of these bodies with mitochondria and with the nucleus. There is a relative persistence of most of these organelles in old or atropic cells which show marked changes including loss of endoplasmic reticulum. The significance of all of these cytoplasmic inclusions is discussed.     

GABA influences the ultrastructure composition of cerebellar granule cells during development in culture

The influence of GABA on cerebellar granule cells in culture was followed morphometrically during in vitro development by growing the cells in the absence or presence of 50 μM GABA. The presence of GABA in the culture media increased the number of neurite-extending cells by 50% after 7 days in culture. At the ultrastructural level GABA treatment led to an increased density of neurotubules, rough endoplasmic reticulum, Golgi apparatus, coated vesicles and other vesicles, whereas structures such as mitochondria and smooth endoplasmic reticulum were not affected by GABA. The density of free ribosomes showed a more pronounced tendency to decrease as a function of the culture period (1–7 days) when the cells were grown in the presence of GABA as compared to control cultures. The results strongly indicate that GABA in addition to being an important neurotransmitter serves as a trophic factor in the development of at least certain types of neurons.     

Distribution and light microscopic features of granule cells in the cochlear nuclei of cat, rat, and mouse

In the present study the cytology and the topography of the cochlear granule cell domain (a comprehensive term introduced here for all granule cell-containing regions of the cochlear nuclear complex) have been studied light microscopically in Nissl, Bielschowsky, and Golgi-Del Rio-Hortega material of cats, rats, and mice; in Golig rapid material of 0-14-day-old kittens; and in sections of 6-week-old kittens following HRP injections in the superficial dorsal cochlear nucleus (DCN). The domain has been parcellated in seven subdivisions which, in spite of some species' differences, are easily identifiable in all of the included animals. The cochlear granule cells are considered as a particular class of neuron, which is slightly different from, but nevertheless principally similar to the cerebellar granule cells in both shape and mode of neuronal connections. The digitiform terminals of the cochlear granule cells differentiate after the first two weeks of extrauterine life. In several respects these cells show larger variation among species than do the cerebellar granules, the similarity between the two classes of granule cells being most conspicuous in the rodent. The silver, Golgi rapid, and HRP material suggest that all, or at least the majority, of the granule cell axons project to the molecular layer of the DCN, forming parallel fibers similar to those of the cerebellar cortex. Also, the cochlear parallel fibers traverse the spiny apical dendrites of principal neurons (the pyramidal cells) and the smoother dendrites of molecular layer stellate cells.     

Cytoplasmic tubular inclusion in ganglioneuroma

Summary Unusual tubular inclusions were observed in the cytoplasm of ganglion cells of a mediastinal ganglioneuroma in a boy of 7 years. The inclusions consisted of an aggregation of about 100-nm-sized tubular structures resembling honeycomb-like tubular structures in the axoplasm of rats and mice and suggested to be derived from dilated rough endoplasmic reticulum. These structures differ from profiles reported in normal and pathologic conditions and seem to be related to the neoplastic character of the ganglion cells.     

Degeneration of the synaptic boutons of parallel fibers in rats treated with methylmercury: Chronological observations

We investigated the changes in the cerebellar cortex of adult rats after oral administration of 10mg/kg per day methylmercury (MeHg) for 10 consecutive days. Using a light microscope, darkly stained granule cells were shown to first appear on day 4 after the final dose, and increased in number thereafter. On day 11, many granule cells became degenerated, whereas the Purkinje cells were well preserved. Ultrastructural investigation revealed swelling of the parallel fiber synaptic boutons (the axonal terminals of granule cells), as early as on day 2, although the cell bodies and parallel fibers showed no obvious change. From day 8, in addition to the swollen boutons, many electron-dense boutons were observed. The dendritic spines of the Purkinje cells (i.e. the postsynaptic structures of parallel fibers), showed no obvious change at any stage. Immunohistochemistry for synaptic vesicle-specific protein (SVP) demonstrated a decrease of granular labeling in the molecular layer from day 8 onwards. Immunohistochemical expression of inositol 1,4,5-trisphosphate 3-kinase (IP3K), which is known to be associated with the spine, was constant throughout. It is considered that the SVP and IP3K profiles correspond well to the ultrastructural features of the pre-and postsynaptic structures, respectively. In conclusion, this study shows that MeHg intoxication of the granule cells in adult rats initially induces morphological change in the synaptic boutons, the degeneration of which does not involve the dendritic spines of the Purkinje cells transneuronally at least for the survival observed.     

Experimental reorganization of the cerebellar cortex. VII. Effects of late x-irradiation schedules that interfere with cell acquisition after stellate cells are formed.

In Long-Evans rats the area of the cerebellum was irradiated with multiple doses of low-level X-ray beginning on day 12 after the bulk of stellate cells were acquired. The treatment spared basket, stellate and early-forming granule cells but led to a substantial reduction in the total granule cell population and a correlated miniaturization of the cerebellar cortex. Nevertheless most Purkinje cells had normally shaped planar dendritic arbors, with an upward directed stem dendrite, several smooth branches and a multitude of spiny branchlets. The frequency piling up of spiny branchlets near the surface was attributed to the truncation of the bed of parallel fibers by this radiation schedule. In this last paper of the series the accumulated results are summarized and evaluated. The hypothesis is offered that while the growth of the Purkinje cell perikaryon is an autonomous process, the oriented perpendicular growth of a single stem dendrite depends on the presence of basket cell axons, the outgrowth of smooth branches on the presence of stellate cell axons, and the proliferation of spiny branchlets on interaction with parallel fibers. The parallel fibers are responsible for the orthogonal, planar growth of the dendritic arbor and a hypothesis is offered about the mechanisms involved.