Embryonic Purkinje Cells Grafted on the Surface of the Cerebellar Cortex Integrate in the Adult Unlesioned Cerebellum

The presence of an injury or the selective degeneration of specific neuronal populations is commonly assumed to be a necessary prerequisite for the survival and the integration of grafted neurons in the recipient brain. In the present study we have placed solid grafts of cerebellar anlage in the fourth ventricle of adult rats, in close contact with the host cerebellar cortex, to assess the capacity of embryonic Purkinje cells to interact with adult neurons and integrate in the unlesioned cerebellar cortex. Numerous grafted Purkinje cells are indeed able to leave the implant and migrate into the host molecular layer, where they develop adult structural features. In addition, such cells are able to elicit the growth of host climbing fibre sprouts which end in newly formed arborizations impinging upon their dendritic trees. Climbing fibre collateral branches also penetrate the implant to innervate Purkinje cells which have not migrated in the host cerebellum. These results show that embryonic Purkinje cells are able to survive and integrate in an adult unlesioned cerebellar cortex. In addition, adult olivary axons respond to the increased size of the target population by expanding their terminal domain to innervate grafted Purkinje cells.     

Organization of Host Afferents to Cerebellar Grafts Implanted into Kainate Lesioned Cerebellum in Adult Rats

This paper examines the organization of host afferents within cerebellar grafts implanted into kainic acid lesioned cerebellum. Our selection of a cerebellum, a prime example of a 'point-to-point' system, permits precise determination of the degree and the specificity of host-graft interactions. One month after a cerebellar injection of kainic acid, the lesion produced can be divided into two concentric regions: (i) a central necrotic zone, totally depleted of neurons (zone 1), and (ii) a peripheral zone which lacks all Purkinje cells but preserves its cortical lamination (zone 2). Two months after the implantation of solid pieces of embryonic cerebellum, the graft has evolved into a minicerebellar structure, occupying most of zone 1. The grafted minicerebellum consists of a highly convoluted trilaminated cortex with a core containing deep nuclear neurons. Purkinje cells are positioned between the molecular and granular layer with their short and irregular dendrites branching within the former. Donor foetal Purkinje cells migrate into the contiguous portion of the molecular layer of the host zone 2. These embryonic neurons set up within the upper three-quarters of the host molecular layer, and develop monoplanar dendritic trees that span the whole width of the layer. The organization of host-graft interactions was studied by autoradiography of anterogradely transported tritiated leucine, injected in the host bulbar region containing the caudal half of the inferior olivary complex (origin of all vermal climbing fibres) and the dorsally adjacent paramedian reticular nucleus (origin of a few mossy fibres). Numerous labelled fibres cross the host-graft interface from the white matter of the host cerebellum, and provide innervation to the minicerebellar structure. The vast majority of these labelled axons terminate in the molecular layer, forming axonal arborizations that follow the shape of the Purkinje cell dendrites. The labelled climbing fibres are organized into uneven sagittally aligned strips, which mimic that of olivocerebellar projections in control rats. Only a small proportion of host labelled fibres end in the donor granular layer, forming typical mossy fibre rosettes. The latter are present in the region of the graft close to the host-graft interface. In addition, labelled axons are observed climbing over the dendritic trees of grafted Purkinje cells that have invaded a portion of the host molecular layer of zone 2. In all regions containing grafted Purkinje cells and labelled climbing fibres, the density of the innervation is close to normal with practically all Purkinje cells receiving a climbing fibre. The extensive integration of the grafted cells into the deficient neuronal networks of the host clearly illustrates the positive neurotropic effect exerted by immature cerebellar neurons on adult extracerebellar afferent fibres. The hodological integration, allowing a possible restoration of the impaired cerebellar circuitry, takes place respecting the specificity and topographic distribution which characterize the 'point-to-point' arrangement of normal cerebellar circuitry.     

Embryonic Purkinje Cells Grafted on the Surface of the Adult Uninjured Rat Cerebellum Migrate in the Host Parenchyma and Induce Sprouting of Intact Climbing Fibres

By grafting solid pieces of cerebellar anlage onto the surface of the adult rat cerebellum, we have investigated the problem of the interactions between embryonic and adult neurons in an intact brain. A few days after grafting, embryonic astrocytic processes crossed the graft-host interface and radiated into the recipient molecular layer. Several grafted Purkinje cells also migrated into the host brain along such processes as well as adult Bergmann glia. Adult climbing fibres, labelled by means of Phaseolus vulgaris leucoagglutinin (PHA-L), sprouted new collateral branches which terminated on embryonic Purkinje cells at both extra- and intraparenchymal levels. No sign of activation of host astroglia or microglia was evident in the host cerebellum in relation to these processes. Embryonic Purkinje cells which migrated into the host cerebellum developed an adult-like morphology. Intraparenchymal grafts of neocortical embryonic tissue induced conspicuous growth of host olivary axons, characterized by a pattern which was different from that observed following cerebellar grafts. By contrast, when neocortical tissue was placed onto the surface of the recipient cerebellum, graft-host interactions were limited and climbing fibre sprouting was rarely seen. These results show that (i) supernumerary Purkinje cells can penetrate and settle in the adult intact cerebellar cortex, (ii) adult climbing fibres are able to innervate these new targets in the absence of any injury or activation of non-neuronal cells of the adult brain, and (iii) in the absence of damage to the adult brain, the plasticity of adult olivary axons is specifically elicited and controlled by embryonic Purkinje cells.     

Rostral Cerebellum Originates from the Caudal Portion of the So-Called 'Mesencephalic' Vesicle: A Study Using Chick/Quail Chimeras

Homotopic and isochronic transplantations of the right dorsal half of the mesencephalic vesicle have been performed between chick and quail embryos at the stage of 10 - 14 somites. Analysis of the extension of the graft, by means of the quail nucleolar marker, combined with cytoarchitectonic analysis has disclosed that the transplanted neuroepithelium gives rise to isthmic nuclei and to a portion of rostral cerebellum, in addition to the optic tectum and mesencephalic dorsal grisea. These results show that the rostral portion of the cerebellar primordium is located in the so-called 'mesencephalic' alar plate, thus considerably more rostrally than previously supposed. This has been confirmed by two other types of chimeric embryos resulting from homotopic transplantation of either: (i) the quail right alar plate of the first rhombencephalic vesicle, which gives rise to caudal but not rostral cerebellum in the operated side, or (ii) the right alar portion of a segment of the quail neural tube including both the caudal third of the mesencephalic vesicle and the rostral half of the first rhombencephalic vesicle, which gives rise to the whole hemicerebellum in the operated side. Moreover, in chimeric embryos with transplants restricted to the mesencephalic alar plate, the grafted portion of the cerebellar primordium gives rise both to deep cerebellar neurons and to all types of cortical neurons. Among the quail cortical neurons, the Purkinje cells, although intermingled with host Purkinje cells, are organized, at E18, in a characteristic longitudinal band which is strongly reminiscent of the longitudinal functional and morphological organization of the cerebellum. Other types of quail cortical neurons, that is, Golgi cells, granule cells, and molecular layer interneurons, are also observed within this sagittal band. In addition, quail granule cells and molecular layer interneurons as well as quail glial cells, extend over a larger territory on both sides of the longitudinal band containing quail Purkinje cells and even cross the midline and invade the contralateral hemicerebellum. In all types of chimeric embryos, the proliferation, migration, and differentiation of quail transplanted neurons, both in the isthmic region and in the cerebellum, evolve asynchronously from the host homologous ones, following a more precocious and faster developmental schedule. This asynchrony in the development of grafted and host isthmic and cerebellar homologous areas confirms and extends previous findings concerning the proliferation and migration of quail tectal cells in chick quail chimeric embryos (Senut and Alvarado-Mallart, 1987).     

The development of synaptic contacts in the cerebellum of Macaca mulatta

The maturation of various cerebellar cortical cells, the appearance of afferent fibers to the cerebellum, and the development of synaptic contacts in the cerebellar cortex and deep nuclei was investigated in the fetal macaque. Ultrastructural studies were done on cerebellum obtained from fetuses at 75, 100, 125 and 150 days after conception to interrelate the temporal development of these three systems. At 75 days, synaptic contacts were seen on somas and axons of neurons in the deep cerebellar nuclei, and climbing fibers formed pericellular baskets around Purkinje cells. By 100 days the climbing fibers synapsed with somatic spines of the Purkinje cells, and mossy fiber endings were present in the internal granule cell layer. Synaptic contacts were also seen on dendritic processes of neurons in the deep cerebellar nuclei at this time. In the 125 day cerebellum, Golgi cells were identified for the first time and climbing fibers and parallel fibers made synaptic contact with both Purkinje and Golgi cells. At 150 days parallel fibers made synaptic contact with superficial stellate cells and mature cerebellar glomeruli had appeared. At this stage, axosomatic contacts of climbing fibers on the soma of Purkinje cells had disappeared. The relationship of these anatomical observations to possible functional activity is discussed.     

Postsynaptic Targets of Purkinje Cell Terminals in the Cerebellar and Vestibular Nuclei of the Rat

The cerebellar and vestibular nuclei consist of a heterogeneous group of inhibitory and excitatory neurons. A major proportion of the inhibitory neurons provides a GABAergic feedback to the inferior olive, while the excitatory neurons exert more direct effects on motor control via non-olivary structures. At present it is not clear whether Purkinje cells innervate all types of neurons in the cerebellar and vestibular nuclei or whether an individual Purkinje cell axon can innervate different types of neurons. In the present study, we studied the postsynaptic targets of Purkinje cell axons in the rat using a combination of pre-embedding immunolabelling of the Purkinje cell terminals by L7, a Purkinje cell-specific marker, and postembedding GABA and glycine immunocytochemistry. In the cerebellar nuclei, vestibular nuclei and nucleus prepositus hypoglossi Purkinje cell terminals were found apposed to GABAergic and glycinergic neurons as well as to larger non-GABAergic, non-glycinergic neurons. In the cerebellar and vestibular nuclei individual Purkinje cell terminals innervated both the inhibitory and excitatory neurons. Both types of neurons were contacted not only by GABAergic Purkinje cell terminals but also by GABA-containing terminals that were not labelled for L7 and by non-GABAergic, non-glycinergic terminals that formed excitatory synapses. Glycine-containing terminals were relatively scarce (<2% of the GABA-containing terminals) and frequently contacted the larger non-GABAergic, non-glycinergic neurons. To summarize, Purkinje cell axons evoke their effects through different types of neurons present in the cerebellar and vestibular nuclear complex. The observation that individual Purkinje cells can innervate both excitatory and inhibitory neurons suggests that the excitatory cerebellar output system and the inhibitory feedback to the inferior olive are controlled simultaneously.     

Collapsin-1/semaphorin-III/D is regulated developmentally in Purkinje cells and collapses pontocerebellar mossy fiber neuronal growth cones

Most axons in the CNS innervate specific subregions or layers of their target regions and form contacts with specific types of target neurons, but the molecular basis of this process is not well understood. To determine whether collapsin-1/semaphorin-III/D, a molecule known to repel specific axons, might guide afferent axons within their cerebellar targets, we characterized its expression by in situ hybridization and observed its effects on mossy and climbing fiber extension and growth cone size in vitro. In newborn mice sema-D is expressed by cerebellar Purkinje cells in parasagittal bands located medially and in some cells of the cerebellar nuclei. Later, sema-D expression in Purkinje cells broadens such that banded expression is no longer prominent, and expression is detected in progressively more lateral regions. By postnatal day 16, expression is observed throughout the cerebellar mediolateral axis. Collapsin-1 protein, the chick ortholog of sema-D, did not inhibit the extension of neurites from explants of inferior olivary nuclei, the source of climbing fibers that innervate Purkinje cells. In contrast, when it was applied to axons extending from basilar pontine explants, a source of mossy fiber afferents of granule cells, collapsin-1 caused most pontine growth cones to collapse, as evidenced by a reduction in growth cone size of up to 59%. Moreover, 63% of pontine growth cones arrested their extension or retracted. Its effects on mossy fiber extension and its distribution suggest that sema-D prevents mossy fibers from innervating inappropriate cerebellar target regions and cell types.     

Cerebellar histamine-H1 receptor distribution: An autoradiographic study of purkinje cell degeneration, staggerer, weaver and reeler mutant mouse strains

The distribution of 3H-mepyramine binding sites in cerebellae of normal mice and Purkinje cell degeneration, staggerer, weaver and reeler mutant mice was studied by light microscopic autoradiography. The binding of 3H-mepyramine to 20 micron coronal sections through the cerebellum and medulla had the characteristics expected of histamine-H1 receptor labeling. In the cerebellar cortex of normal mice, a high density of 3H-mepyramine binding was observed over the molecular layer and an intermediate density over the Purkinje cell layer, while the granule cell layer and white matter were almost devoid of labeling. The deep cerebellar nuclei were labeled to an intermediate density. In the 54 day old Purkinje cell degeneration mutant cerebellum, which is depleted of Purkinje cells, a greatly reduced labeling of the cerebellar cortex was observed. Labeling in the deep cerebellar nuclei was unaffected. In the 27 day old staggerer cerebellum, a mutation characterized by Purkinje cells which are almost devoid of spines and which do not form synaptic contacts with granule cells, a higher than normal grain density was seen over the cerebellar cortex, while normal grain density was observed over the deep cerebellar nuclei. The cerebellar cortex of 81 day old weaver mice, which is almost devoid of granule cells, had a high grain density over medial regions of the cortex, while the portion of the granule cell layer which remained relatively unaffected in the lateral parts of the cerebellum was unlabeled. The deep cerebellar nuclei had grain densities similar to littermate controls. In the 29 day old reeler cerebellae, which contain malpositioned Purkinje cells, high grain density regions corresponding to the heterotopically located Purkinje cells were observed. The present observations suggest that cerebellar cortical histamine-H1 receptors are associated predominantly with Purkinje cells. Furthermore, the expression of these H1 receptors appears not to be adversely affected by several alterations in the Purkinje cell environment, which have previously been shown to dramatically influence Purkinje cell morphology.     

Canine inherited ataxia: ultrastructural observations

Canine Inherited Ataxia is inherited as an autosomal recessive trait in Gordon Setters. This animal model shares features with certain human cerebellar degenerations and offers the opportunity to examine brain tissue at various stages during the evolution of disease. The present investigation focuses on the morphometric and ultrastructural changes of cerebellar neurons. Purkinje and granule cells are the principal intrinsic neurons at risk. The size of Purkinje cells decreases, axonal degeneration is an important feature of the pathology, and synaptic abnormalities occur in the cerebellar glomeruli and deep nuclei of the cerebellum. The sequence and nature of synaptic changes in the molecular layer suggest that the degenerative process begins in Purkinje cells and that granule cells may be secondarily affected.     

Fate of grafted embryonic Purkinje cells in the cerebellum of the adult "Purkinje cell degeneration" mutant mouse. I. Development of reciprocal graft-host interactions

In this paper, we have morphologically studied the developmental events underlying the neuronal replacement, 3-21 days after grafting. Despite their abnormal environment, Purkinje cell progenitors proceed with their proliferation in the grafted neuroepithelium, with a time window similar to that characterizing proliferation of this neuronal class in control mouse embryos. Only postmitotic Purkinje cells leave the grafts and migrate to the host molecular layer following stereotyped pathways. These neurons invade the host molecular layer, either through a tangential migration under the pial basal lamina from the graft/host interface or breaking locally the latter, and passing directly from the lateral swellings of the graft lying on the surface of the host folia. Whatever the pathway for host invasion, the migrating Purkinje cells penetrate radially and/or obliquely into the host molecular layer until their inward-oriented processes attain the molecular/granular layer interface, which occurs about 7 days after grafting. At the end of their migration, the grafted Purkinje cells with bipolar shapes and long and smooth processes begin to build up their ultimate dendritic trees. This dendritogenesis proceeds with constructive and regressive processes, passing through the same three developmental phases described by Ramón y Cajal (Trab. Lab. Invest. Biol. Univ. Madrid 24:215-251, 1926) for control Purkinje cells (phase of the fusiform cell, phase of the stellate cell with disoriented dendrons, and phase of orientation and flattening of the dendrites). In the grafted cerebella, the duration of the second and third phases is somewhat shorter than during normal cerebellar ontogenesis. Synaptogenesis between adult host axons and grafted Purkinje cells starts when the latter attain their second phase of dendritic development. Somatic filopodia emerging from grafted Purkinje cells begin, 10-11 days after grafting, to be synaptically contacted by axonal sprouts of the host climbing fibers resulting, 2 days later, in the formation of pericellular nests. Synaptogenesis between slender dendritic spines and host parallel fibers, together with that of axon terminals from host molecular layer interneurons and the smooth surface of the grafted Purkinje cell somata, begin earlier than in control mouse development, being almost simultaneous with climbing fiber/Purkinje cell synaptogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology and immunohistochemistry of efferent neurons of the goldfish corpus cerebelli

In teleosts, cerebellar efferent neurons, known as eurydendroid cells, are dispersed within the cerebellar cortex rather than coalescing into deep cerebellar nuclei. To clarify their morphology, eurydendroid cells were labeled retrogradely by biotinylated dextran amine injection into the base of the corpus cerebelli. Labeling allowed the cells to be classified into three types-fusiform, polygonal, and monopolar-depending on their somal shapes and numbers of primary dendrites. The fusiform and polygonal type cells were distributed not only in the Purkinje cell layer but also in the molecular and granule cell layers. The monopolar type cells were distributed predominantly in the Purkinje cell layer of the ventrocaudal portion of the corpus cerebelli. These results suggest that there are some functional differences between these eurydendroid cell types. The eurydendroid cells were double-labeled by retrograde labeling and immunohistochemistry using specific antibodies against GABA, aspartate, and zebrin II. No GABA-like immunoreactivity was detected in the retrogradely labeled eurydendroid cells. About half of retrogradely labeled cells were immunoreactive to the anti-aspartate antibody, suggesting that some eurydendroid cells utilize aspartate as a neurotransmitter. Zebrin II reacts with cerebellar Purkinje cells but left all retrogradely labeled neurons nonreactive, although some of these were surrounded by immunopositive fibers. This relationship between the eurydendroid and Purkinje cells is similar to that between the deep cerebellar nuclei and Purkinje cells in mammals.     

Cyto- and chemoarchitecture of the cerebellum of the short-beaked echidna (Tachyglossus aculeatus)

The monotremes (echidnas and platypus) have been claimed by some authors to show 'avian' or 'reptilian' features in the gross morphology and microscopic anatomy of the cerebellum. We have used Nissl staining in conjunction with enzyme histochemistry to acetylcholinesterase and cytochrome oxidase and immunohistochemistry to non-phosphorylated neurofilament protein (SMI-32 antibody), calcium binding proteins (parvalbumin, calbindin and calretinin) and tyrosine hydroxylase to examine the cyto- and chemoarchitecture of the cerebellar cortex and deep cerebellar nuclei in the short-beaked echidna. Immunoreactivity for non-phosphorylated neurofilament (SMI-32 antibody) was found in the deep cerebellar nuclei and in Purkinje cells of most regions except the nodule. Purkinje cells identified with SMI-32 immunoreactivity were clearly mammalian in morphology. Parvalbumin and calbindin immunoreactivity was found in Purkinje cells with some regional variation in staining intensity and in Purkinje cell axons traversing cerebellar white matter or terminating on Lugaro cells. Calbindin immunoreactivity was also present in inferior olivary complex neurons. Calretinin immunoreactivity was found in pontocerebellar fibers and small cells in the deep granule cell layer of the ansiform lobule. We found that, although the deep cerebellar nuclei were much less clearly demarcated than in the rodent cerebellum, it was possible to distinguish medial, interposed and lateral nuclear components in the echidna. As far as we can determine from our techniques, the cerebellum of the echidna shows all the gross and cytological features familiar from the cerebellum of therian mammals.     

Differentiation of cerebellar anlage heterotopically transplanted to adult rat brain: A light and electron microscopic study

Pieces of cerebellar primordia from (days 14 or 15 of gestation) E14 or E15 rat embryos were dissected out and transplanted into a cavity of the occipital cortex and underlying hippocampus, over the superior colliculus of 2-month-old rats. The host animals were allowed to survive for 2 to 3 months. The cytoarchitectonic and the synaptic organizations were analyzed in 16 of such transplants. Only 4 of the implants established connections with the host brain through several thin peduncles composed of myelinated fibers. The remaining 12 implants survived in an extraparenchymal situation. Independently of its partial linking to the host brain, the graft grew and developed a cerebellar structure composed of nuclear and cortical regions. The latter exhibited normal lamination and foliation, and contained the five categories of neurons which characterize normal cerebellar cortex. Electron microscopic examination disclosed that the synaptic connections normally present in the cerebellar cortex were also formed in the implants with the exception of climbing fibers, which were absent. The cerebellar interneurons kept their normal topographic distribution and gave origin to numerous synapses which maintained their own specificity. Some mossy fibers were present in the granule cell layer at the center of typical glomeruli. However, abnormal synaptic arrangements were also observed within the neuropil of this granule cell layer. They consisted of pseudo-glomerular formations composed of clusters of tightly packed small axon terminals covered by granule cell dendrites. The origin of these boutons was not established. Since they did not correspond to the classes of presynaptic elements normally synapsing on these dendrites, they constitute a new example of cerebellar heterologous synapses. Their presence could be related to changes in the cellular environment due to the rarity of mossy afferents. HRP tracing experiments, carried out in extraparenchymal transplants, have allowed us to determine that the corticonucleocortical loop of normal cerebellum is also developed in the implants. Nuclear neurons are at the origin of the mossy fibers involved in glomerular formations, whereas Purkinje cells project to the nuclear region. The establishment of these reciprocal connections could determine the functional stabilization of both kinds of cerebellar neurons and thus the long survival of extraparenchymal grafts. These results allow the conclusion that the presence of extracerebellar afferents is not necessary for the organotypic and synaptotypic differentiation of cerebellar anlage.     

Cerebellar benzodiazepine receptors: cellular localization and consequences of neurological mutations in mice

The distribution of cerebellar [3H]flunitrazepam binding sites was studied autoradiographically in Purkinje cell degeneration (pcd/pcd), weaver (wv/wv), staggerer (sg/sg) and reeler (rl/rl) mutant mice. In the normal 78-day-old C57BL/6J mouse cerebellum, the highest concentration of [3H]flunitrazepam binding sites was observed over the molecular layer. Intermediate grain density was present over the Purkinje cell layer and intermediate to high density over the deep cerebellar nuclei. Low labeling was observed over the granule cell layer. Negligible concentrations of binding sites were seen in the white matter. In 45-49-day-old Purkinje cell degeneration mutants, where essentially all Purkinje cells have disappeared by day 45, there was a small decrease in grain density over the cerebellar cortex. Concomitantly, a substantial increase in grain density was observed over the deep cerebellar nuclei of the pcd/pcd mutants when compared to littermate controls. A significant increase in [3H]flunitrazepam labeling was observed over the cerebellar cortex of 81-86-day-old wv/wv mutants; this was most pronounced in the vermis where the granule cell loss was greatest. Over the hemispheres, where fewer granule cells degenerate, a lower density of binding sites was seen. Grain density over the wv/wv deep cerebellar nuclei was comparable to that of littermates. Substantially lower [3H]flunitrazepam labeling was detected over the cerebellar cortex of 25-27-day-old sg/sg mutants in which the number of granule, Purkinje and Golgi cells is greatly reduced; the labeling over the deep nuclei, however, was significantly increased. In 27-29-day-old rl/rl mutant cerebella, where all classes of cells are malpositioned, labeling density over all areas of the cerebellar cortex, including the Purkinje cell masses, was increased. Our autoradiographic data suggest that a proportion of cerebellar cortical benzodiazepine receptors are associated with Purkinje cells; we propose that the remainder of the receptors are localized on Golgi cells, while granule cells are devoid of receptors. In the deep cerebellar nuclei, the observed increase in benzodiazepine receptors in the pcd/pcd and sg/sg mutants may be a manifestation of denervation supersensitivity subsequent to the loss of innervation by Purkinje cell axon terminals. The finding of a high receptor density in the Purkinje cell masses of the rl/rl mutant, where Purkinje cells are devoid of afferent basket cell input, suggests that benzodiazepine receptors are expressed and maintained in the absence of a full complement of GABAergic afferents.     

Localization of serotonin immunoreactivity in the opossum cerebellum

We have used the indirect antibody peroxidase-antiperoxidase technique to analyze the course of serotonin (5-hydroxytryptamine; 5HT) fibers to the deep cerebellar nuclei; the distribution of serotonin within the nuclei; the continued course of 5HT fibers to the cerebellar cortex; and the lobular and laminar distribution of this indoleamine in the cerebellar cortex. Only rarely are fibers found in either the restiform body or the brachium pontis. However, a distinct bundle of serotoninergic axons is present in the medial aspect of the brachium conjunctivum. Axons arise from this bundle and course dorsally into the neuropil of the deep cerebellar nuclei. The densest immunostaining is present in posterior and ventral regions of all four cerebellar nuclei. Within the nuclei large (24% of total) and small (76% of total) varicosities are present. The average distance between varicosities on individual axons is 3.85 micron (S.D. = 1.2). The innervation of the cerebellar cortex is derived primarily from fibers that course through the deep nuclei. At levels caudal to the deep nuclei a single midsagittal band courses into lobules VIII and IX. In the cerebellar cortex, serotoninergic axons and varicosities are present in all lobules; however, the fiber density is not uniform. The densest distribution is present in vermal lobule VIII and the dorsal folia of lobule IX. Within the granule cell layer of lobules VIII and IX, immunoreactive elements form a midsagittal band, and to a lesser degree, two parasagittal bands. Beaded serotoninergic fibers course through the deep and middle portion of the granule cell layer and give rise to a plexus at the border between the Purkinje cell and granule cell layers. Within this plexus axons extend long distances in the transverse and sagittal planes. Long beaded axons oriented in the transverse plane of the folia are also present in the deep molecular layer. A few radial serotoninergic fibers ascend to the pial surface and give rise to very short tangential branches. In all three cortical layers, both large (19% of total) and small (81% of total) varicosities are present. The average distance between varicosities on individual fibers is 5.3 micron (S.D. = 2.2).(ABSTRACT TRUNCATED AT 400 WORDS)     

Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. I. Light microscopic and electrophysiological studies

Granuloprival cerebellar cultures were transplanted after 9 or 16 days in vitro with cerebellar explants that had been exposed to kainic acid. The latter contained granule cells and differentiated glia, elements lacking in granuloprival cultures. Changes induced by transplantation observed by light microscopy included interposition of granule cells among the large cortical neurons of host explants; a reduction of the excess neurites of the Purkinje cell axon collateral system that is characteristic of granuloprival explants; and the appearance of myelinated fibers in previously unmyeli-nated cultures. The most notable electrophysiologic consequence of transplantation was the disappearance of inhibition of cortical spontaneous activity in response to antidromic stimulation of Purkinje cell axons, correlating with the disappearance of excess neurites, and suggesting that Purkinje cell recurrent collateral inhibition was no longer the dominant mode of cortical inhibition. Restoration of missing elements in granuloprival cultures incited development of structural and functional characteristics resembling those of normal cerebellar explants.     

Tottering mouse motor dysfunction is abolished on the Purkinje cell degeneration (pcd) mutant background

Tottering (tg) mice inherit a recessive mutation of the calcium channel alpha 1A subunit gene, which encodes the pore-forming protein of P/Q-type voltage-sensitive calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons. The phenotypic consequences of the tottering mutation include ataxia, polyspike discharges, and an intermittent motor dysfunction best described as paroxysmal dystonia. These dystonic episodes induce c-fos mRNA expression in the cerebellar circuitry, including cerebellar granule and Purkinje neurons, deep cerebellar nuclei, and the postsynaptic targets of the deep nuclei. Cellular abnormalities associated with the mutation include hyperarborization of brainstem nucleus locus ceruleus axons and abnormal expression of L-type calcium channels in cerebellar Purkinje cells. Here, the role of these two distinct neural pathways in the expression of tottering mouse intermittent dystonia was assessed. Lesion of locus ceruleus axons with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine (DSP-4) did not affect the frequency of tottering mouse dystonic episodes. In contrast, removal of cerebellar Purkinje cells with the Purkinje cell degeneration (pcd) mutation by generation of tg/tg; pcd/pcd double mutant mice completely eliminated tottering mouse dystonia. Further, the c-fos expression pattern of tg/tg; pcd/pcd double mutants following restraint was indistinguishable from that of wild-type mice, suggesting that the pcd lesion eliminated an essential link in this abnormal neural network. These data suggest that the cerebellar cortex, where the mutant gene is abundantly expressed, contributes to the expression of tottering mouse dystonic episodes.     

Immunohistochemical localization of cannabinoid CB<Subscript>1</Subscript> receptor in inhibitory interneurons in the cerebellum

Inhibitory interneurons terminating on Purkinje cell dendrites contribute to cannabinoid-mediated cerebellar plasticity, consistent with the intense expression of cannabinoid CB1 receptor protein in the cerebellar molecular layer. CB1 labelling in the molecular layer has been attributed to parallel fibers originating from granule cells, climbing fibers originating in the inferior olive, and inhibitory interneurons in the deep molecular layer (basket cells). However, the cellular distribution of CB1 in the cerebellar molecular layer has remained poorly understood. We used double fluorescence labelling to test for co-localization of nuclei with CB1 receptor protein. Labelling was intense surrounding nuclei in the deep and superficial molecular layer; consistent with basket cell and stellate cell inhibitory interneurons that regulate depolarization-induced suppression of inhibition (DSI) of Purkinje cells.     

Purkinje cell neuroaxonal dystrophy similar to nervous mutant mice phenotype in two sibling kittens

Three 4-month-old kittens from the same litter were presented, two of which were exhibiting cerebellar signs. Euthanasia was requested. No cerebellum atrophy was disclosed on necropsy. General cerebellar anatomy was normal, including the thickness of the cortical layers, myelination, and neurons of the deep cerebellar nuclei. In the ataxic cat vermis, Purkinje cells were lacking along broad parasagittal bands symmetrically disposed relative to the midline. Many Purkinje cells were also lacking in the hemispheres. The nodulus and the flocculus were normal. Surviving Purkinje cells had frequent main dendrite swellings visible with anti-calbindin and anti-microtubule associated protein. In affected regions, calbindin and phosphorylated neurofilaments immunesera stained numerous axonal torpedoes located in the granular layer and the folial white matter. They were also present in processes of the deep cerebellar nuclei and lateral vestibular nucleus. Loss of synaptic endings onto the neurons of these nuclei was evident. Hypertrophied Purkinje cell recurrent axons and enhanced retrograde synaptic endings were present in the granular layer. Bergmann glia was strongly labeled by anti-GFAP, but no abnormal supplementary fibers were seen. None of these alterations were present in the normal sister. However, abnormal vacuolation of the Purkinje cell main dendrites was evident in all three cats, but not in six unrelated control cats that were 3–6 months old. The inferior olive and pontine nuclei were also normal. The two ataxic cats had a primary Purkinje cell degeneration that shared many common features with the abnormal Purkinje cells of the nervous mutant mouse.     

On the cellular localization of cerebellar muscarinic receptors: An autoradiographic analysis of weaver, reeler, purkinje cell degeneration and staggerer mice

Light microscopic autoradiography of [3H]quinuclidinyl benzilate binding sites was used to study the distribution of muscarinic cholinergic receptors in mouse mutants which have abnormalities affecting specific cerebellar cell types. In the normal C57BL/6J mouse, binding sites were distributed throughout the cerebellar cortex, with the highest levels in the granule cell layer and deep cerebellar nuclei. Normal binding site density was observed in the cerebellum of the weaver mutant in which the majority of granule cells had degenerated. The density of [3H]quinuclidinyl benzilate binding sites was elevated in the cortex of the reeler, despite a reduction in the number of granule cells. The concentration of binding sites was also high over the Purkinje cell masses where granule cells were largely absent. No significant reduction in cortical [3H]quinuclidinyl benzilate binding site density was detected in the Purkinje cell degeneration mutant, in which essentially all Purkinje cells had degenerated. In contrast, receptor binding in the deep cerebellar nuclei of this mutant was significantly increased. A substantial increase in labeling was observed in the cortex and deep nuclei of the staggerer cerebellum in which a large fraction of Golgi II cells, Purkinje cells, granule cells and mossy fibers have degenerated. We discuss the possibility that the persistence of [3H]quinuclidinyl benzilate binding sites in all four mutants may imply a non-neuronal localization for a large proportion of muscarinic receptors in the mouse cerebellar cortex.