Fine structure of granule cells and related interneurons (termed Golgi cells) in the cochlear nuclear complex of cat, rat and mouse

Summary This paper describes the fine structure of granule cells and granule-associated interneurons (termed Golgi cells) in the cochlear nuclei of cat, rat and mouse. Granule cells and Golgi cells are present in defined regions of ventral and dorsal cochlear nuclei collectively termed cochlear granule cell domain.The granule cells are small neurons with two or three short dendrites that give rise to a few branches with terminal expansions. These participate in glomerular synaptic arrays similar to those of the cerebellar cortex. In the glomeruli the dendrites form short type 1 synapses with a large, centrally-located mossy bouton containing round synaptic vesicles and type 2 synapses with peripherally located, smaller boutons containing pleomorphic vesicles. The granule cell axon is thin and beaded and, on its way to the molecular layer of the DCN, takes a straight course, which in the ventral nucleus is parallel to the pial surface.Neurons of the second category resemble cerebellar Golgi cells and occur everywhere interspersed among the granule cells. They are usually larger than the granule cells and give rise to dendrites which may branch close to and curve around the cell body. The dendrites contain numerous mitochondria and are laden with thin appendages, giving them a hairy appearance. Both the cell body and the stem dendrites participate in glomerular synaptic arrays. Golgi cell glomeruli are distinguishable from the granule cell glomeruli by unique features of the dendritic profiles and by longer, type 1 synaptic junctions with the central mossy bouton. The Golgi cell axon forms a beaded plexus close to the parent cell body.The synaptic vesicle population of the mossy boutons suggests that they are a heterogeneous group and may have multiple origins. Apparently, each of the various classes participates in both granule and Golgi cell glomeruli. The smaller peripheral boutons with pleomorphic vesicles in the two types of glomeruli may represent Golgi cell axons which make synaptic contacts with both granule and Golgi cells. The Golgi cell dendrites, on the other hand, are also contacted by small boutonsen passant with round synaptic vesicles, which may represent granule cell axons. A tentative scheme of the circuitry in the cochlear granule cell domain is presented. The similarity with the cerebellar granule cell layer is striking.     

Cytology and organization of rat cerebellar organ cultures

Roller tube cultures of parasagittal cerebellar slices were taken from young rats aged 9-11 days, and maintained in vitro for 1-2 weeks. Morphological aspects of cell types and synaptic relationships in such organ cultures were examined at light and electron microscopic levels. Some neurons were marked by intracellular injections of horseradish peroxidase for subsequent identification of their connection patterns. Cytoarchitecture of the cerebellar cortex was largely preserved in the organ cultures. Dendritic trees of Purkinje cells exhibited isoplanar organizations that often resembled their orientation at the time of explanation. Other cerebellar neurons, namely granule cells, Golgi cells, basket cells, stellate cells, all differentiated within the organ cultures. In addition, some neurons of the deep cerebellar nuclei remained viable during the period of culture. Mossy fibers most probably of cerebellar nuclear origin were found terminating on the dendrites of granule cells and Golgi cells. Quite unexpected were certain types of direct synapses of afferent fibers on short necked spines arising from Purkinje cell smooth dendrites and somata. Such terminals resembled climbing fibers. They were most likely modified mossy fiber afferents, since the organ cultures did not include neurons of the inferior olive which are well spearated from the cerebellar mass at postnatal stages. These "ascending" mossy fibers presumably occupied postsynaptic surfaces that were either vacated by deafferentation or induced by the afferent fibers themselves. Intracellularly labeled Purkinje cells had widely distributed axonal collateral branches. Labeled axons were distributed within the Purkinje cell layer. Several recurrent Purkinje cell axon collaterals stained with reaction products of horseradish peroxidase tracer were followed at the ultrastructural level. In one case, labeled terminals were examined in an area of approximately 2 mm2. Terminals of Purkinje cell collaterals formed symmetric synapses with somata of basket cells and dendrites of Golgi cells, but not Purkinje cell somata. Some large boutons of serially traced Purkinje cell axon collaterals formed asymmetric contacts with profiles interpreted as Golgi cell dendrites. In contrast to the apparent axonal sprouting in cerebellar organ cultures, maturation of dendritic processes remained static. Astroglia cells of diverse shapes were observed following immunocytochemical staining with antisera to glia filament proteins. The distribution patterns of immunoreactive astrocytes changed dramatically in cerebellar slice cultures maintained for 3-6 weeks in vitro.     

Participation of Golgi neuron processes in the cerebellar glomeruli: An electron microscope study

Summary Synaptic relations, within the cerebellar isles, of Golgi II neuron axons and dendrites have been studied in the cat. Golgi axon endings can be identified with some probability in the outer (cortical) zone of the cerebellar glomeruli in normal material. They can well be recognized in the chronically isolated folium in which mossy fibers have completely degenerated. The Golgi axons are very thin preterminal fibers with small enlargements containing synaptic vesicles and contacting the preterminal intraglomerular parts of the granule cell dendrites as well as their terminal spheroid protrusions. The spheroid protrusions of the granule dendrites are the main postsynaptic loci of the granule neuron having their main synapse with the mossy fiber — generally of central position in the glomerulus — and additional synapses, more often on their outer surface, with the Golgi axons. No significant difference is seen between the two contacts, from which one is known to be excitatory (mossy) and the other inhibitory (Golgi ax.). The Golgi cell has also descending dendrites, known from light microscopy to be engaged in the cerebellar isles. By tracing these dendrites from the cell bodies and using their characteristic short finger-like processes as a criterion for their identification, the synapses between mossy endings and Golgi dendrites could be identified under the EM. They are broad contacts between a dendrite passing along one side of the mossy ending, with several synaptic attachment plaques and with small dendritic processes protruding into invaginations of the mossy ending. — The cerebellar glomerulus is thus a complex synaptic apparatus with two different axonal elements (mossy and Golgi endings) and often two dendritic elements (granule and Golgi dendrites) involved. — The possible functional significance of the Golgi cell is discussed in the light of these findings and the new discoveries by Eccles et al. (1964b, 1966) on its inhibitory nature.     

Enrichment of unipolar brush cell-like neurons in primary rat cerebellar cultures

Unipolar brush cells are a distinct class of excitatory interneurons situated in the granular layer of the cerebellar cortex, where they form giant synapses with individual mossy fiber terminals. We have previously shown that primary cerebellar cell cultures from embryonic and postnatal rodents contain neurons displaying morphological and chemical phenotypes characteristic of unipolar brush cells in situ, including intense staining with calretinin antiserum. In cultures from both embryonic and postnatal rats, the large majority of calretinin-positive neurons are unipolar brush cells, while granule cells are usually calretinin-negative. A small percentage of putative Golgi/Lugaro cells also express calretinin. We demonstrate here that the developmental stage of the source tissue, the concentration of potassium in the medium, and treatment with glutamate after differentiation have substantial effects on the density of putative unipolar brush cells in the cultures. In dissociated cultures obtained from embryos at gestation day E18 and E20 and from pups at postnatal day P0, P2, P5, P8, and P10 grown in 25 mM KCl, the percentage calretinin-positive cells progressively decreases from 24% to 0.1% of total cells. In cultures from E20 embryos grown in physiological potassium (5 mM KCl), calretinin-positive cells are enriched to approximately 60% of total cells, while the majority of calretinin-negative cells die. In embryonic cultures exposed to high concentrations of glutamate after 12 days in vitro, calretinin-positive neurons have a survival advantage over calretinin-negative cells and represent up to 83% of total cells.     

Postsynaptic enrichment of Eps8 at dendritic shaft synapses of unipolar brush cells in rat cerebellum

Epidermal growth factor receptor pathway substrate 8 (Eps8) is a widely expressed multidomain signaling protein that coordinates two disparate GTPase-dependent mechanisms: actin reorganization via Ras/Rac pathways and receptor trafficking via Rab5. Expression of Eps8, the gene encoding the founding member of the Eps8 family of proteins, was found in cerebellum by virtual Northern analysis and in situ hybridization. Because the cerebellum has a well-known cellular architecture and is a favored model to study synaptic plasticity and actin dynamics, we sought to analyze Eps8 localization in rat cerebellar neurons and synapses by light and electron microscopy. Specificity of Eps8-antibody was demonstrated by immunoblots and in brain sections. In cerebellum, unipolar brush cells (UBCs) were densely Eps8 immunopositive and granule cells were moderately immunostained. In both types of neuron immunoreaction product was localized to the somatodendritic and axonal compartments. Postsynaptic immunostained foci were demonstrated in the glomeruli in correspondence of the synapses formed by mossy fiber terminals with granule cell and UBC dendrites. These foci appeared especially evident in the UBC brush, which contains an extraordinary postsynaptic apparatus of actin microfilaments facing synaptic junctions of the long and segmented varieties. Eps8 immunoreactivity was conspicuously absent in Purkinje cells and their actin-rich dendritic spines, in all types of inhibitory interneurons of the cerebellum, cerebellar nuclei neurons, and astrocytes. In conclusion, Eps8 protein in cerebellum is expressed exclusively by excitatory cortical interneurons and is intracellularly compartmentalized in a cell-class specific manner. This is the first demonstration of the presence of a member of the Eps8 protein family in UBCs and its enrichment at postsynaptic sites.     

Cerebellar unipolar brush cells are targets of primary vestibular afferents: an experimental study in the gerbil

The unipolar brush cell (UBC) is an excitatory glutamatergic interneuron, situated in the cerebellar granular layer, that itself receives excitatory synaptic input on its dendritic brush from a single mossy fiber terminal in the form of a giant glutamatergic synapse. The UBC axon branches within the granular layer, giving rise to large terminals that synapse with both granule cell and UBC dendrites within glomeruli and resemble in morphological and functional terms those formed by extrinsic mossy fibers. So far, the only demonstrated extrinsic afferents to the UBC are the choline acetyltransferase (ChAT)-positive mossy fibers, some of which originate from the medial and descending vestibular nuclei. To ascertain whether UBCs are innervated by primary vestibular fibers, we performed a tract-tracing light and electron microscopic study of the vestibulocerebellum in gerbils. Macular and canal vestibular end-organs were individually labeled by injection of biotinylated dextran amine. After an appropriate survival time, gerbils were then processed for light and electron microscopic analysis of central vestibular projections. In the nodulus and uvula, labeled primary vestibular fibers formed mossy terminals synapsing with both granule cells and UBCs in all of the injected gerbils. Thus, innervation of UBCs by extrinsic mossy fibers carrying static and dynamic vestibular signals represents the first synapse of networks that contribute a powerful form of distributed excitation in the granular layer. Electronic Publication     

Quantitative morphology and synaptology of cerebellar glomeruli in the rat

Summary Computer-assisted stereological and quantitative morphological approaches were used to analyse cerebellar glomeruli of the simple type in serial ultrathin sections. It was found that, of the total volume (110–200 m³) of the glomeruli studied, 53% was occupied by granule cell dendrites, 34% by mossy terminal and 13% by Golgi axons. None of the four analysed glomeruli contained Golgi cell dendrites. The mossy terminals that were studied received, on the average, 53 granule cell dendrites. All of the dendrites originated from different granule cells and all made synaptic contacts with mossy terminal. However only about 60% of granule cell dendrites made synapses with Golgi axons. The surface of the mossy terminals occupied by synaptic junctions, was found to be 5.4–5.5%. Each granule cell dendrite emitted 3–5 terminal protrusions (dendritic digits). Each digit receives one or more synaptic contact from either the mossy terminal (67% of all digits), or from Golgi axon varicosities (25%). Only about 8% of all digits were contacted synaptically by both types of axonal terminals. All of the dendritic digits that were observed made synaptic connections. Each digit was, on the average, connected by symmetric attachment plaques to 4 neighbouring digits. Three-dimensional reconstructions of mossy terminal and some of contacting granule cell dendrites demonstrated that the dendrites curved around the central mossy terminal and were much longer than expected from earlier Golgi-impregnation studies. In addition to mossy terminals and Golgi axons, an axon terminal of small calibre that contained large, empty, spheroid vesicles were occasionally observed. These terminals, which are most likely the axonal varicosities of ascending parallel fibers, made synaptic contacts exclusively with granule cell dendrites at the periphery of the glomeruli.The results demonstrate that, in the rat cerebellum, there is a high degree of convergence of granule cells at a glomernius (53 to 1); and that there is a rich inhibitory input to about 60% of all granule cell dendrites. It is also shown that the main postsynaptic targets, for both mossy and Golgi axons, are the dendritic digits. The presence of synaptic contacts between parallel-fiber-like varicosities and granule cell dendrites may be an additional source of excitation within the glomerulus.     

Intrinsic determinants of synaptic phenotype: an experimental study of abducens internuclear neurons connecting with anomalous targets

The present experiments investigate the role of postsynaptic neurons in the morphological differentiation of presynaptic terminals that are formed de novo in the adult CNS. Abducens internuclear neurons in the adult cat were chosen as the experimental model. These neurons project onto the contralateral medial rectus motoneurons of the oculomotor nucleus. Abducens internuclear axon terminals were identified by their anterograde labeling with biocytin and analyzed at the electron microscopic level. To promote the formation of new synapses, two different experimental approaches were used. First, after the selective ablation of medial rectus motoneurons with ricin, abducens internuclear neurons reinnervated the neighboring oculomotor internuclear neurons. Second, after axotomy followed by embryonic cerebellar grafting, abducens internuclear axons invaded the implanted tissue and established synaptic connections in both the molecular and granule cell layer. Boutons contacting the oculomotor internuclear neurons developed ultrastructural characteristics that resembled the control synapses on medial rectus motoneurons. In the grafted cerebellar tissue, abducens internuclear axons and terminals did not resemble climbing or mossy fibers but showed similarities with control boutons. However, labeled boutons analyzed in the granule cell layer established a higher number of synaptic contacts than controls. This could reflect a trend towards the mossy fiber phenotype, although labeled boutons significantly differed in every measured parameter with the mossy fiber rosettes found in the graft.We conclude that at least for the abducens internuclear neurons, the ultrastructural differentiation of axon terminals reinnervating novel targets in the adult brain seems to be mainly under intrinsic control, with little influence by postsynaptic cells.     

Moving up or moving down? Malpositioned cerebellar unipolar brush cells in reeler mouse

Cerebellar morphogenesis occurs through a complex interplay of cell proliferation and migration that in mouse and rat begins about midgestation and ends in the third postnatal week. Cerebellar cells derive from germinative matrices in the ventricular zone and the external granular layer. Like granule cells, unipolar brush cells (UBCs) are excitatory interneurons situated in the granular layer of the cortex and innervated by mossy fibers. While granule cells are produced from the external granular layer, the generation of UBCs is still controversial. We utilized the reeler mutant mouse, which has widespread misplacement of neurons due to lack of Reelin protein, to ascertain the origin of UBCs. In the reeler cerebellum, which is small and lacks foliation, Purkinje cells are greatly reduced in number and in large part are located ectopically in deep cerebellar masses. Granule cells are also reduced in number and form an irregular granule cell layer. In this study we demonstrate that the reeler mutation influences the positioning of UBCs and also significantly reduces their number. Both subsets of UBCs identified in normal mouse, the calretinin-positive and the metabotropic glutamate receptor 1alpha-positive subsets, are affected in the reeler. About 40% of the calretinin-positive UBCs are ectopically situated in the deep cerebellar regions and the immediate vicinity of the ependyma of the fourth ventricle. Ectopic UBCs have discrete, although somewhat looser brushes than granular layer UBCs, but form synaptic junctions with complex axon terminals, possibly belonging to mossy fibers and UBC axons, like their normally situated counterpart. The observed displacement of UBCs in the reeler suggests that they originate from the ventricular zone.     

On the development of the cerebellum of the trout, Salmo gairdneri. IV. Development of the pattern of connectivity

Summary Synaptogenesis has been studied in the corpus cerebelli of the troutSalmo gairdneri, Richardson, 1836. The first synapses are observed in hatchlings and occur between parallel fibres and the shafts of Purkinje dendrites. Subsequently the axosomatic synapses of Purkinje axon collaterals on the neurons of the ganglionic layer appear, and finally the synapses made by climbing fibres and mossy fibres, and by stellate cell axons develop. Young synapses in the cerebellum of the trout resemble the mature structures so closely that the criteria for the identification of the latter can also be applied to the former. The number of parallel fibre synapses and of Purkinje axon collateral synapses increases considerably during development. Eurydendroid cells, the axons of which leave the cerebellum, receive an abundance of Purkinje axon collaterals on their somata and main dendritic trunks. Mossy fibre synapses are numerous in the granular layer. Climbing fibre contacts and synapses of stellate cell axons, both with Purkinje cells, are found occasionally. the following pattern of connectivity is proposed. The main input-output system is formed by the mossy fibres, the granule cells, the Purkinje cells and the eurydendroid cells. Additional pathways are formed by (1) the mossy fibres, granule cells and eurydendroid cells, and (2) the climbing fibres, Purkinje cells and eurydendroid cells. The afferent-efferent systems, mentioned above, are influenced by a number of internuncial elements: (1) The Golgi cells receive their input from the parallel fibres and contact with their axon collaterals the dendrites of granule cells. (2) Axon collaterals of Purkinje cells are in synaptic relation with Golgi cells. (3) Axon collaterals of Purkinje cells impinge upon the somata and main dendrites of other Purkinje cells. (4) Stellate cells, which derive their input from the parallel fibres, synapse with the dendrites and somata of Purkinje cells. The possible functional roles of all of these neuronal elements are discussed.     

The synapse en marron between Golgi II neurons and mossy fibers in the rat's cerebellar cortex

Summary Mossy fibers engage in synapses en marron with the somata of some Golgi II cells. These synapses resemble in all particulars the synapses en marron made by climbing fibers except for the distinctive characteristics of the presynaptic terminal. The mossy fiber, with its axial stream of neurofilaments and mitochondria and its loose aggregations of round synaptic vesicles, makes an extensive contact with the wrinkled surface of the Golgi II perikaryon. Synaptic complexes are confined to the depths and sides of the furrows in the Golgi cell. The free side of the mossy fiber terminal often articulates with large numbers of granule cell dendrites, an arrangement similar to that found in ordinary glomeruli.These synaptic connections may be interpreted in the light of the physiological evidence that Golgi II cells inhibit granule cells that are not strongly activated by mossy fibers. Since each granule cell receives four to six mossy fibers, strong activation may require either a selected frequency pattern or synchrony of several inputs. The collateral inhibition indirectly evoked by the same mossy fiber via Golgi II cells would suppress those granule cells not receiving concurrent excitation from other mossy fibers or the favored pattern of excitation. In contrast, granule cells simultaneously activated by other mossy fibers would not be inhibited but would send impulses to the molecular layer. Thus, the glomerulus would behave as a filter that increases the signal-to-noise ratio of the excitatory input to the Purkinje cells.Supported by U.S. Public Health Service Grants NS03659 and NS05591 from the National Institute of Neurological Diseases and Stroke.     

Voltage-dependent calcium signaling in rat cerebellar unipolar brush cells

Unipolar brush cells (UBCs) are a class of excitatory interneuron found in the granule cell layer of the vestibulocerebellum. Mossy fibers form excitatory inputs on to the paint brush shaped dendrioles in the form of giant, glutamatergic synapses, activation of which results in prolonged bursts of action potentials in the postsynaptic UBC. The axons of UBCs themselves form mossy fiber contacts with other UBCs and granule cells, forming an excitatory, intrinsic cerebellar network that has the capacity to synchronize and amplify mossy fiber inputs to potentially large populations of granule cells. In this paper, we demonstrate that UBCs in rat cerebellar slices express low voltage activated (LVA) fast-inactivating and high voltage activated (HVA) slowly inactivating calcium channels. LVA calcium currents are mediated by T-type calcium channels and they are associated with calcium increases in the dendrites and to a lesser extent the cell soma. HVA currents, mediated by L-type calcium channels, are slowly inactivating and they produce larger overall increases in intracellular calcium but with a similar distribution pattern. We review these observations alongside several recent papers that examine how intrinsic membrane properties influence UBCs firing patterns and we discuss how UBC signaling may affect downstream cerebellar processing.     

Formation of presynaptic dendrites in the rat cerebellum following neonatal X-irradiation

The ultrastructure of the cerebellum was studied after X-irradiation of the whole head of newborn rats. The neuronal elements forming the cerebellar circuitry in rats 30–60 days after irradiation were limited to climbing fibers, mossy fibers, and Purkinje and Golgi cells. Under these conditions the perikarya and dendrites of the Golgi neurons develop presynaptic vesicular grids. These unconventional presynaptic elements establish numerous synaptic contacts with spines and occasionally with shafts of Purkinje cell dendrites.The results indicate that interference with normal cerebellar development, such that granule, basket and stellate cells are absent, generates new types of cellular interactions during synaptogenesis which allow Golgi cells to express their latent potentiality to form presynaptic perikarya and dendrites. It is concluded that this latent potentiality is repressed during development of the normal cerebellum by the presence of the other interneurons.     

The timing of granule cell differentiation and mossy fiber morphogenesis in the opossum

Summary The timing of developmental events may be important for the orderly formation of neuronal interconnections. In the present study, the timing of granule cell migration is compared with the arrival and maturation of mossy fiber projections. The opossum was chosen as the experimental animal because its protracted postnatal development enables the examination of developmental sequences not as easily recognized in other more commonly used mammalian species. It is shown that all areas that project to the cerebellum as mossy fibers in the adult opossum do so by postnatal day (PD) 30. Their major target, the granule cells begin inward migration from the external germinal layer (EGL) prior to PD 30, but do not form a distinct internal granular layer (IGL) until PD 35. Migrating granule cells penetrate into the IGL deep to granule cells that have begun dendritic differentiation. By PD 50, Golgi impregnations reveal that many granule cells have numerous immature processes, somal spines and dendritic growth cones. After this age these structures are rare and the vast majority of granule cells exhibit short dendrites with digiform endings. Dendritic differentiation subsequent to PD 54 involves an increase in the length of the shaft and the further maturation of terminal digits. Also from Golgi material, immature mossy fiber endings can be identified in the IGL by PD 35 and exhibit mature characteristics at PD 73. Thus, the formation and maturation of granule cell dendrites and their afferents (mossy fibers) occur over an extended period of time (PD 35–73). Moreover, granule cells exhibit a sequence of development similar to that of Purkinje cells: 1) early arrival of their primary afferent projections in the cerebellar anlage: 2) a period of exuberant dendritic growth; and 3) a protracted and overlapping period for dendritic and synaptic maturation.     

Differentiation of granule cells in relation to GABAergic neurons in the rat fascia dentata

Summary Golgi impregnation was used to study the dendritic differentiation of granule cells in the rat fascia dentata. The impregnated granule cells were gold-toned allowing for a fine structural study of the same identified neurons and of the input synapses onto their cell bodies and dendrites. Due to the long postnatal formation of these cells it was possible to describe a sequence of maturational stages coexisting on the same postnatal day (P5). Characteristic features of the dendritic development of granule cells were i) occurrence of varicose swellings along the dendrites, ii) growth cones on dendritic tips, iii) transient formation of basal dendrites, and iv) progressive development of dendritic spines. Incoming synapses on the differentiating granule cells were mainly found on dendritic shafts. Their membrane specializations were symmetric. At least some of these symmetric synapses were GABAergic because immunostaining of Vibratome sections from the same postnatal stage (P5) demonstrated a well-developed GABAergic axon plexus in the fascia dentata (antibodies against glutamate decarboxylase (GAD), the GABA synthesizing enzyme). Electron microscopy of the immunostained axon plexus revealed numerous GABAergic terminals that formed symmetric synaptic contacts, mainly on shafts of differentiating dendrites but also on cell bodies of granule cells. Our results thus indicate that the plexus of inhibitory GABAergic axons is already well developed at a stage when the target neurons, the granule cells, are still being formed.     

Corticosterone replacement restores normal morphological features to the hippocampal dendrites, axons and synapses of adrenalectomized rats

A thorough evaluation of hippocampal dendrites, axons and synaptic contacts has not been undertaken following prolonged periods of absence of corticosteroids despite the marked granule cell loss which occurs in the dentate gyrus of adrenalectomized rats. Thus, we have applied morphometric techniques to analyse the dendrites of granule and pyramidal cells, the mossy fiber system, and the number and morphology of synapses between the mossy fibers and the excrescences of CA3 pyramidal cells in rats submitted to different periods of adrenalectomy. In addition, to search for the presence of neuritic reorganisation in the hippocampal formation once normal corticosteroid levels were re-established, we incorporated in this study a group of rats replaced with corticosterone one month after adrenalectomy. The results obtained in adrenalectomized rats showed a striking impoverishment of the dendrites of surviving granule cells, subtle alterations in the apical dendritic arborization of CA3 pyramidal cells and no changes in the apical dendrites of CA1 pyramidal cells. In addition, in adrenalectomized rats there was a progressive reduction in the total number of synapses established between mossy fibers and CA3 pyramids, as a consequence of a reduction in the volume of the suprapyramidal part of the mossy fiber system, and profound changes in the morphology of mossy fiber terminals and CA3 dendritic excrescences. A remarkable reorganisation of neurites was found to occur following the administration of low doses of corticosterone, completely reversing the adrenalectomy-induced synaptic loss and partially restoring the morphology of hippocampal axons and dendrites. These plastic mechanisms provide a sound structural basis for the reversibility of cognitive deficits observed after corticosterone administration to adrenalectomized rats.     

Postnatal development of the mouse dentate gyrus in organotypic cultures of the hippocampal formation

The hippocampal formation of newborn mice was explanted and maintained in Maximow culture assemblies for up to 35 days. At the time of explantation, only the suprapyramidal limb of the dentate gyrus was cytoarchitectonically distinct, and electron microscopy of newborn hippocampus revealed no definitive synapses. Histogenesis, as indicated by the development of the infrapyramidal limb of the dentate gyrus, and synaptogenesis, as indicated by the in vitro formation of mossy fiber synapses on the dendrites of hippocampal pyramidal neurons were studied by light and electron microscopy. At 12 days and thereafter in culture, mossy fiber terminals were found in synapsis with dendritic spines probably belonging to pyramidal cells of the hippocampal zone CA4. Near dentate granule cell somata a few axosomatic and many axospinous and axodendritic synapses were found. The data indicate that granule cells of the developing dentate gyrus are capable of differentiation in vitro into a structure essentially equivalent to that developed in vivo. The granule cells may become arranged into a recognizable granule cell layer, and develop dendritic processes which receive synapses virtually identical to those found in the intact organ. The differentiation of these features proceeds in the absence of the extrinsic afferents from the septum or from the contralateral hippocampal formation.     

Selective innervation of embryonic hippocampal transplants by adult host dentate granule cell axons

Fragments containing different cytoarchitectonic fields were dissected out of late embryonic rat hippocampal primordia and transplanted into the hippocampus or septum of adult syngeneic hosts. Field CA3 transplants contained clusters of large, angular (pyramidal) cell bodies surrounded by a radiating corona of dendrites. These cells stained selectively with our monoclonal antibody Py, and a proportion were labelled by [3H]thymidine administered on the 15th day of embryonic life. Field CA1 transplants contained smaller, angular, Py-negative cells, which formed elongated laminae rather than globular clusters. The ability of the host dentate granule cells to project to the transplants was examined by (1) the Timm stain for mossy fibres, (2) electron microscopy of Golgi-impregnated CA3 pyramidal neurons in the transplants, and (3) quantitative electron microscopic assessment of the proportions of large mossy fibre terminals in the synaptic population of the transplants. The Timm stain showed that CA3 transplants received a projection from host dentate granule cells when the transplants were placed in direct contact with the axons in the host mossy fibre pathway. As in the normal host field CA3, the ingrowing mossy fibres terminated selectively on the juxtacellular regions of the dendritic tree and ignored the major part of the dendrites in the radiating corona. The electron micrographs showed that within this territory the host mossy fibres formed synaptic terminals with all the complex features typical of normal mossy fibres, and were presynaptic to complex spines arising from the juxtacellular region of Golgi-impregnated donor CA3 pyramidal cells. The quantitative electron microscopic study demonstrated that the mossy fibre-innervated juxtacellular regions of the field CA3 transplants had up to 20% of the normal density of mossy fibre synapses found in the stratum lucidum of field CA3 in situ. CA3 transplants which were placed in the septum, remote from the host mossy fibres, had either trivial numbers of mossy fibre synapses or none. This confirmed that the abundant mossy fibre terminals in the intrahippocampal CA3 transplants were of host origin, and not due to donor dentate granule cells inadvertently included in the grafts. The selectivity of the host dentate projection for field CA3 transplants was demonstrated by the observation that CA1 transplants in the same locations received only slight mossy fibre projections in the Timm stain, and in electron micrographs their synaptic population had only insignificant numbers of large mossy fibre terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabotropic glutamate receptors are associated with non-synaptic appendages of unipolar brush cells in rat cerebellar cortex and cochlear nuclear complex

Unipolar brush cells (UBCs) are a class of small neurons that are densely concentrated in the granular layers of the vestibulocerebellar cortex and dorsal cochlear nucleus. The UBCs form giant synapses with individual mossy fibre rosettes on the dendrioles which make up their brush formations and are provided with numerous, unusual non-synaptic appendages. In accord with the glutamatergic nature of mossy fibres, our previous post-embedding immunocytochemical studies indicated that various ionotropic glutamate receptor subunits are localized at the post-synaptic densities of the giant synapses, whereas the non-synaptic appendages are immunonegative. On the contrary, the metabotropic glutamate receptors mGluR1 and mGluR2/3 are situated at the non-synaptic appendages and are lacking at the post-synaptic densities. Other authors, however, have shown that antibodies to these metabotropic receptors stain both appendages and post-synaptic densities. In the present study, we have re-evaluated the distribution of metabotropic glutamate receptors in the UBCs of the cerebellum and the cochlear nuclear complex by light and electron microscopic pre-embedding immunocytochemistry with subtype-specific antibodies. We confirm that UBCs dendritic brushes are densely immunostained by antibody to mGluR1 particularly in the cerebellum and that antibody to mGluR2/3 labels at least a percentage of the UBC brushes in both the cerebellum and cochlear nuclei. At the ultrastructural level, it appears that mGluR1 and mGluR2/3 immunoreactivities are not associated with the post-synaptic densities of the giant mossy fibre–UBC synapses, but instead are concentrated on the non-synaptic appendages of the cerebellar UBCs. The non-synaptic appendages, therefore, may be an important avenue for regulating the excitability of UBCs and mediating glutamate effects on their still unknown intracellular signal transduction cascades. We also show that the pre-synaptic densities of UBC dendrodendritic junctions are mGluR2/3 positive. As previously demonstrated, antibodies to mGluR1 and mGluR2/3 label subsets of Golgi cells. Antibody to mGluR5 does not stain UBCs in the cerebellum and cochlear nucleus and reveals the somatodendritic compartment of Golgi cells situated in the core of the cerebellar granular layer, whilst cochlear nucleus Golgi cells are mGluR5 negative.