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.     

Glycoprotein transport in the rat cerebellum

Summary The fate of tritiated fucose in rat cerebellum has been followed using autoradiographic techniques for a period up to one week after intraventricular injection. At intervals of up to three hours after injection, Purkinje and granule cell bodies were found to be most heavily labelled. Subsequently this labelling declined and the highest grain density was observed over the molecular layer. The data is interpreted in terms of fucose incorporation into glycoproteins in nerve cell bodies followed by rapid dendritic transport in the Purkinje cell and fast transport in granule cells along ascending axons and parallel fibres.     

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.     

Brain-derived neurotrophic factor modulates dendritic morphology of cerebellar basket and stellate cells: an in vitro study

The dendrites of cerebellar basket/stellate cells show a highly stereotyped orientation relative to granule cell axons (parallel fibers) and Purkinje cell dendrites. This specific morphology is acquired during the early postnatal phase of cerebellar development, when basket/stellate cells become synaptically integrated with Purkinje neurons and granule cells. In the present study, we used primary cerebellar cultures to test how the spatial arrangement of granule cell axons affects basket/stellate cell dendritic morphology. In addition, we sought to determine whether active signals as might be provided by granule cells, i.e. synaptic input and the neurotrophin, brain-derived neurotrophic factor, affect basket/stellate cell development. Our results confirm the critical role of parallel fiber orientation for basket/stellate dendritic morphogenesis. Moreover, we found that both electrical activity and brain-derived neurotrophic factor increased basket and stellate cell dendritic arborization.Together with previously published findings, our data led to the conclusion that both structural cues and active interneuronal signaling collaborate to bring about the precise morphogenesis of cerebellar basket/stellate cells. The distinct responses of various cerebellar phenotypes towards the morphogenetic effects of brain-derived neurotrophic factor suggest that this neurotrophin, within the developing cerebellum, enhances synaptic connectivity by concerting the formation of appropriate pre- and postsynaptic structures.     

On the development of the cerebellum of the trout, Salmo gairdneri. III. Development of neuronal elements

Summary The differentiation of the cerebellar neurons and of their afferent fibres has been studied in young specimens ofSalmo gairdneri Richardson, 1836. Both light microscopic preparations, stained with haematoxylin-eosin or according to Bodian, Nissl, Klüver-Barrera or Golgi, and electron microscopic preparations were used. The ventricular matrix layer gives rise to the large neurons of the cerebellum,i.e. Purkinje, eurydendroid and Golgi cells; the secondary matrix produces the smaller neurons,i.e. the granule and stellate cells. The afferent fibres of the cerebellum are the mossy and the climbing fibres. The identification of the cell types, originating from either the ventricular matrix or from the secondary matrix, can be made earlier on the basis of the structure of their processes than on the basis of the structure of their somata. The development of the cerebellar neurons in the trout corresponds in many respects to that in higher vertebrates. In general, differentiation is characterized by a decrease in the number of free ribosomes and an increase of the other organelles, particularly of rough endoplasmic reticulum. The ganglionic layer contains, in addition to the Purkinje cells, the eurydendroid cells. The axons of these elements were in some cases observed to leave the cerebellum, whereas the axons of Purkinje cells are mainly confined to the ganglionic layer. In the trout the development of the granule cells shows a varied pattern. The mature shape of the axons of these elements depends on the migration paths followed by their precursors. T-shaped processes occur in all parts of the cerebellum and unbranched processes only in the valvula. The opinion held for mammals that the more superficial a parallel fibre is situated in the molecular layer the later it has been formed, is not valid for the trout. A number of secondary matrix cells performs tangential migration, not in the submeningeal region but deeper in the molecular layer,viz. under bundles of parallel fibres. The granule cells originating from these deeper matrix cells extend their axons at a lower level than the parallel fibres which have been formed previously. Throughout development dark cells are found in osmiumstained material. Their dark aspect is due to the presence of a fine filamentous network and of many free ribosomes in the cytoplasm. These immature cells are considered to be migratory.     

Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata and dendrites: quantitative analysis by immunogold labeling

The immunointensities of calcium-binding proteins parvalbumin (PV) and calbindin D28K were quantified in different parts of Purkinje cells and interneurons (basket cells and stellate cells) of the rat cerebellum. An electron microscopic, postembedding immunogold procedure on Lowicryl K4M-embedded thin sections was applied. Neuronal profiles were identified by double-labeling immunocytochemistry using the combination of the two primary antibodies, mouse monoclonal anti-rat calbindin D28K and rabbit polyclonal anti-rat PV. The secondary antibodies were conjugated with colloidal gold of different sizes (10 and 15 nm diameter). In the cerebellar cortex, double-labeled profiles were identified as Purkinje cells and profiles labeled only with anti-PV were identified as inteneurons. The densities of gold particles were used for statistical comparison of the relative levels of PV and calbindin D28K in somata, dendrites, dendritic spines, axons and axon terminals of Purkinje cells, and interneurons. The axons and axon terminals of Purkinje cells and basket cells had significantly higher levels of PV immunoreactivity than Purkinje cell somata, primary, secondary, and tertiary dendrites, and dendritic spines, as well as interneuron somata. On the other hand, the present study could not determine conclusively whether calbindin D28K was distributed homogeneously throughout soma, dendrites, and axons of Purkinje cells or was also concentrated in Purkinje cell axons. To estimate absolute PV concentrations, we made a series of artificial standard samples which were aldehyde-fixed 10% bovine serum albumin containing given concentrations of PV (0, 12.5, 25, 50, 100, 200, and 400 M, 1 and 2 mM), and calibration curves were deduced from quantitative immunogold analyses of these standard samples. We also analyzed a fast twitch muscle, the superficial part of the gastrocnemius muscle (GCM), whose PV content was previously reported in a biochemical study; the comparison between gold particle densities of GCM and standard samples indicated that these artificial standard samples could be used to estimate the approximate intracellular concentrations of PV. Based on these analyses PV concentrations were estimated as 50-100 M in Purkinje cell somata and dendrites as well as interneuron somata, and as 1 mM or more in axons and axon terminals of Purkinje cells and basket cells.     

A new monoclonal antibody against the GABA-protein conjugate shows immunoreactivity in sensory neurons of the rat.

A monoclonal antibody, 115AD5, was raised against GABA coupled to bovine serum albumin. The monoclonal antibody 115AD5 also reacted with other GABA-protein conjugates. The specificity of the monoclonal antibody was corroborated by enzyme-linked immunoassay, dot-immunobinding experiments and immunostaining of rat cerebellum sections. The monoclonal antibody 115AD5 could successfully be applied on Vibratome and cryostat sections using either indirect immunofluorescence or peroxidase techniques. In rat cerebellar cortex the monoclonal antibody 115AD5 gave an intense immunoreaction in stellate cells, in Golgi neurons, and in basket cells and their processes around Purkinje cell bodies. Purkinje cell dendrites showed GABA immunoreactivity while the cell bodies were non-reactive or only weakly reactive. There was labelling in some nuclei of Purkinje cells. GABA immunoreactivity was also found in dot-like structures in the granular layer. A large population of sensory neurons in rat thoracic and lumbar spinal dorsal root ganglia presented an intense immunoreactivity for the monoclonal antibody 115AD5. Nerve bundles immunoreactive for GABA were also seen in these ganglia. In the trigeminal ganglion, a major population of sensory neurons and some of their processes presented immunoreactivity for GABA. In the sensory nodose ganglion of the vagus nerve, many neuronal cell bodies and some fibres were immunoreactive for GABA. Ligation of the vagus nerve caudal to the ganglion resulted in an increased GABA immunoreactivity in neuronal somata of the ganglion, as well as in nerve fibres on the ganglionic side of the ligature. The present results suggest that in the rat, a population of sensory neurons in thoracic and lumbar spinal dorsal root ganglia, as well as in the trigeminal and nodose ganglia contain GABA. The presence of GABA immunoreactivity in these neurons raises the possibility of a neurotransmitter or modulator role.     

A Golgi study of neurons in the camel cerebellum (Camelus dromedarius)

Neurons in the cerebellar cortex of camels were studied using modified Golgi impregnation methods. Neurons were classified according to their position, morphology of their soma, density and distribution of dendrites, and the course of their axons. Accordingly, eight types of neurons were identified. Three types were found in the molecular layer: upper and lower stellate cells and basket cells, and four types were found in the granular layer: granule cells, Golgi Type II cells, Lugaro cells, and unipolar brush cells. Only the somata of Purkinje cells were found in the Purkinje cell layer. The molecular layer is characterized by the presence of more dendrites, dendritic spines, and transverse fibers. Golgi cells also show extensive dendritic branching and spines. The results illustrate the neuronal features of the camel cerebellum as a large mammal living in harsh environmental conditions. These findings should contribute to advancing our understanding of species-comparative anatomy in achieving better coordination of motor activity.     

Cerebellar macroneurons in microexplant cell culture: Ultrastructural morphology

Microexplant cell cultures of fetal rat cerebellum contain essentially monolayer networks of Purkinje cells, occasional granule cells and neurons from the deep nuclei. The neurons and occasional filament-packed glial cells develop on top of a sheet of flattened, non-neuronal cells. In the absence of extrinsic input to the cerebellum and greatly reduced numbers of granule cells, the Purkinje cells develop a stunted and non-oriented dendritic arbor similar to that observed in agranular cerebella. The Purkinje cell dendritic branches, however, are spine-covered. Although the spines are not enveloped by glia and are only rarely contacted by a presynaptic bouton, most spines display a patch of electron-dense material resembling a postsynaptic membrane specialization. The Purkinje cells develop synaptic interactions among themselves and with granule cells. The ultrastructural morphology of boutons derived from both Purkinje cells and large neurons of the deep nuclei, identified after intracellular injection of horseradish peroxidase, is consistent with that observed in vivo.The present study indicates that cerebellar Purkinje cells survive and differentiate in a culture system in which individual neurons are accessible for electrophysiological and morphological analyses.     

Reconstruction of the defective cerebellar circuitry in adult Purkinje cell degeneration mutant mice by Purkinje cell replacement through transplantation of solid embryonic implants

Solid pieces of cerebellar primordia taken from 12-day-old C57BL embryos were implanted into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice and analysed 2-3 months later. Purkinje cell replacement was followed by means of immunocytochemistry with antisera against either cyclic guanosine monophosphate-dependent protein kinase or vitamin D-dependent calcium-binding protein, which allows the complete staining of these neurons. Although all solid graft implants survived, their fate within the mutant cerebellum varied in three ways: Often, a more or less large fragment of the solid graft remained in the white matter, close to the cortex or even partially replacing it. These remnants contained a few distorted Purkinje cells and a region corresponding to the transplanted deep nuclei, composed of numerous immunostained axons and axon terminals surrounding immunonegative neurons. Less frequently remnants of the graft were extruded to an extracerebellar location, between two adjacent folia. They contained a few Purkinje cells intermixed with granule cells and other neurons. In a few cases corresponding to superficial deposition, the implants developed lobulated and trilaminated minicerebella which were located outside the mutant cerebellum but integrated into it. In all three situations, a large number of grafted Purkinje cells succeeded in moving out of the implants and in invading the host molecular layer. These Purkinje cells develop flattened dendritic trees perpendicular to host bundles of parallel fibres. Ultrastructural examination of the synaptic investment of Purkinje cells which have reached the host molecular layer revealed that they acquire normal synaptic inputs although complex pericellular baskets and pinceau formation do not develop. Axons from molecular layer interneurons synapse on perikaryal and smooth dendritic membranes, climbing fibres synapse on stubby spines emerging from thick dendritic branches, and parallel fibres contact almost exclusively the long-necked spines of the distal spiny branchlets. Finally, Purkinje cells which succeed in migrating to molecular layer regions no further than 0.6 mm from the host deep nuclei are able to grow axons which reach appropriate target areas and establish synaptic connections on nuclear neurons. The results obtained from this series of long-term survival cerebellar transplantations point to the possibility of fulfilling most of the conditions necessary for functional restoration of neural grafts in systems in which neurons are connected in a point-to-point manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

The localisation of urocortin in the adult rat cerebellum: a light and electron microscopic study

Light and electron microscopic immunocytochemistry was used to identify the cellular and subcellular localisation of urocortin in the adult rat cerebellum. Urocortin immunoreactivity (UCN-ir) was visualised throughout the cerebellum, yet predominated in the posterior vermal lobules, especially lobules IX and X, the flocculus, paraflocculus and deep cerebellar nuclei. Cortical immunoreactivity was most evident in the Purkinje cell layer and molecular layer. Reaction product, though sparse, was found in the somata of Purkinje cells, primarily in the region of the Golgi apparatus. Purkinje cell dendritic UCN-ir was compartmentalised, with it being prevalent in proximal regions especially where climbing fibres synapsed, yet absent in distal regions where parallel fibres synapsed. In the Purkinje cell layer, the labelling was also contained in axonal terminals, synapsing directly on Purkinje cell somata. These were identified as axon terminals of basket cells based on their morphology. Terminals of stellate cells in the upper molecular layer also expressed the peptide. Whilst somata of inferior olivary neurones showed intense immunoreactivity, axonal labelling was indistinct, with only the terminals of climbing fibres containing reaction product. UCN-ir in the mossy fibre-parallel fibre system was restricted to mossy fibre rosettes of mainly posterior lobules and the varicose terminals of parallel fibres. Furthermore, labelling also was prevalent in glial perikarya and their sheaths.The current study shows, firstly, that urocortin enjoys a close ligand-receptor symmetry in the cerebellum, probably to a greater degree than corticotropin-releasing factor since corticotropin-releasing factor itself is found exclusively in the two major cerebellar afferent systems. Its congregation in excitatory and inhibitory axonal terminals suggests a significant degree of participation in the synaptic milieu, perhaps in the capacity as a neurotransmitter or effecting the release of co-localised neurotransmitters. Finally, its unique distribution in the Purkinje cell dendrite might serve as an anatomical marker of discrete populations of dendritic spines.     

Early development of the Lurcher cerebellum: Purkinje cell alterations and impairment of synaptogenesis

Summary The postnatal development of the heterozygous Lurcher (Lc/+) mouse cerebellum is characterized by Purkinje cell death with a concomitant reduction in granule cell number. In order to evaluate possible relationships between these two events, this study investigates early morphological abnormalities of the Purkinje cells and possible defects in the formation of their synaptic investment. Cerebella of Lurcher and control age-matched (from P8 to P16) mice were analysed by calbindin immunostaining, silver impregnation and quantitative electron microscopy. Direct signs of Purkinje cell anomaly are obvious from P8, four days before the onset of the necrotic process. These signs include the presence of axonal swellings and perinuclear clumps of chromatin, and a general delayed process of maturation, evidenced in cell bodies (incomplete development of the basal polysomal mass) and in dendritic trees (hyperspinous dendrites, delayed formation of proximal and distal compartments). Also from P8, the external granular layer is reduced in thickness. Despite these abnormalities, the onset of the synaptogenesis between Purkinje cells and their specific inputs (parallel fibres, climbing fibres and basket cell axons) takes place on schedule and, at P8, no defect has been noticed. On and after P10, the rate of parallel fibre synaptogenesis is decreased. Very few climbing fibres translocate from their perisomatic to their peridendritic locations, and basket cell axons fail to develop pinceau formations. All these results suggest that before the death of the Purkinje cell by P12, there is an impaired maturation of these neurons provoked by the Lurcher gene action. The hypoplasia of the external granular layer and the altered synaptic investment of the Purkinje cell after P10 are considered to be consequences of the early Purkinje cell defect.     

Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400.

P400 protein, which is identical to the inositol 1,4,5-trisphosphate receptor protein, is a glycoprotein closely associated with the membranes of Purkinje cells. Three types of monoclonal antibodies against P400 protein were employed for the immunohistochemical detection of Purkinje cells in the cerebellum and brainstem of the normal and reeler mouse. Purkinje cells in both types of mice were immunoreactive against anti-P400 antibodies, and the soma, dendrites, axon and even terminal boutons in the cerebellar and vestibular nuclei could be clearly visualized. In the cerebellum of the reeler mutant, the heterotopic Purkinje cells both within and below the granule cell layer were also immunopositive and could be clearly differentiated from the deep cerebellar nuclei, in which neurons were immunonegative. The molecular layer of the reeler cerebellum varied in thickness and certain parts were completely defective. The dendrites within the molecular layer extended from Purkinje cells whose cell bodies were located in the normal position, abnormally in the granule cell layer, or at the surface of the central mass. Outside the cortex of the cerebellum, ectopic Purkinje cells were demonstrated in 3 cerebellar nuclei, the cerebellar medulla and peduncle, and brainstem of the normal and reeler mouse.     

Anatomical evidence for enkephalin immunoreactive climbing fibres in the cerebellar cortex of the opossum

Summary Enkephalin immunoreactivity is present in the cerebellum of the adult opossum within axonal arbors that resemble mature climbing fibres. In the developing cerebellum, enkephalinergic axons form pericellular nests around the perikarya of Purkinje cells in a manner which resembles developing climbing fibres seen in Golgi impregnations. Serial electron micrographs of adult climbing fibres reveal elongate enkephalin immunoreactive profiles that contain synaptic vesicles and make contact with the thorns and shafts of Purkinje cell dendrites. These results suggest that a peptide, enkephalin or an enkephalin-like substance may mediate synaptic interactions between certain populations of climbing fibres and Purkinje cells in the cerebellum of the opossum. Enkephalin immunoreactive axonal arbors, present in the molecular layer, are localized in restricted areas of vermal lobules II–VIII and X. The intermediate cortex and hemispheres are devoid of enkephalinergic climbing fibres except in restricted areas of the paramedian lobule, paraflocculus and the flocculus. In an attempt to establish the origin of enkephalin axons in the cerebellum, a double labelling technique that combines retrograde labelling of cells with horseradish peroxidase and enkephalin immunohistochemistry has been employed. Enkephalin immunoreactive neurons within specific portions of the medial accessory olive are retrogradely labelled in this paradigm. The presence of enkephalin immunoreactivity in selected climbing fibres provides evidence for chemical heterogeneity within one of the major afferent systems to the cerebellum previously thought to be uniform in its transmitter content.     

Morphological development and neurochemical differentiation of cerebellar inhibitory interneurons in microexplant cultures

The cerebellar cortex comprises a rather limited variety of interneurons, prominently among them inhibitory basket and stellate cells and Golgi neurons. To identify mechanisms subserving the positioning, morphogenesis, and neurochemical maturation of these inhibitory interneurons, we analyzed their development in primary microexplant cultures of the early postnatal cerebellar cortex. These provide a well-defined, patterned lattice within which the development of individual cells is readily accessible to experimental manipulation and observation. Pax-2-positive precursors of inhibitory interneurons were found to effectively segregate from granule cell perikarya. They emigrate from the core explant and avoid the vicinity of granule cells, which also emigrate and aggregate into small clusters around the explant proper. This contrasts with the behavior of Purkinje neurons, which remain within the explant proper. During migration, a subset of Pax-2-positive cells gradually acquires a GABAergic phenotype, and subsequently also expresses the type 2 metabotropic receptor for glutamate, or parvalbumin, markers for Golgi neurons and basket or stellate cells, respectively. The latter eventually orient their dendrites such that they take a preferentially perpendicular orientation relative to granule cell axons. Both the neurochemical maturation of basket/stellate cells and the specific orientation of their dendrites are independent of their continuous contact with radially oriented glia or Purkinje cell dendrites projecting from the core explant. Numbers of parvalbumin-positive basket/stellate cells and the prevalence of glutamate-positive neurites, which form a dense network preferentially within cell clusters containing granule cell perikarya and their dendrites, are subject to regulation by chronic depolarization. In contrast, brain-derived neurotrophic factor results in a drastic decrease of numbers of basket/stellate cells. These findings document that granule cell axons (parallel fibers) are the major determinant of basket/stellate cell dendritic orientation. They also show that the neurochemical maturation of cerebellar interneurons is sensitive to regulation by activity and neurotrophic factors.     

Location of profilin at presynaptic sites in the cerebellar cortex; implication for the regulation of the actin-polymerization state during axonal elongation and synaptogenesis

Summary Profilin is a 15 kDa protein that binds actin monomers and inhibits their polymerizationin vitro. The actin-profilin complex can be rapidly dissociatedin vitro by phosphatidylinositol-4,5-bis-phosphate, providing a mechanism for regulating actin assembly-disassembly cycles during cell motile events. We have used a polyclonal antibody to calf spleen profilin to analyse the developmental expression and cellular distribution of profilin in the rat cerebellum and cultured cortical neurons. Immature neurons contain large amount of profilin both in vivo and in vitro. Immunofluorescence showed it to be present in developing neurites and growth cones but not in the filopodia of cortical neurons in culture. Profilin immunoreactivity was intense in the parallel fibres, the granule cell axons of the cerebellar cortex, at the time when they are elongating. Purkinje cell dendrites were not labelled. Profilin immunostaining was present in presynaptic varicosities, but not in dendritic spines within the molecular layer of juvenile and adult rats. The profilin concentration was higher in synaptosomes than in the total cerebellum during the second and third postnatal weeks, a period of intense synaptogenesis. Thus, profilin may help regulate actin polymerization and depolymerization during axonal elongation and synaptogenesis. Its restriction to the presynaptic site in the adult suggests that it may also be involved in the regulation of the release of synaptic vesicles.     

Cellular localization of corticotropin releasing factor receptors in the adult mouse cerebellum

Corticotropin releasing factor is a 41 amino acid peptide that is present in afferent systems that project to the cerebellum. In the adult, this peptide modulates the activity of Purkinje cells by enhancing their responsiveness to excitatory amino acids. Two different types of corticotropin releasing factor receptors, designated type 1 and type 2, have been identified. The purpose of this study is to use immunohistochemistry to identify which corticotropin releasing factor receptors are present in the cerebellum of the adult mouse and to determine their cellular distribution. Receptor type 1 immunostaining is present throughout all lobules of the cerebellar cortex. Distinct labeling is present over the somas of most, if not all, Purkinje cells as well as the primary dendrites of Purkinje cells located at the base of vermal folia. In vermal lobules V, VI, VIII and IX numerous glial fibrillary acidic protein immunoreactive processes, oriented radially in the molecular layer, also are immunoreactive for receptor type 1. In the granule cell layer, scattered type 1 immunoreactive puncta are present throughout most cerebellar lobules. Receptor type 2 immunoreactive puncta are present throughout the molecular layer in all lobules. In addition, scattered basket and/or stellate cells, identified with a GABA antibody, are immunopositive for the type 2 receptor. In the Purkinje cell layer, the type 2 receptor immunolabeling is confined to the basal pole of the Purkinje cell including the initial axonal segment. In the granule cell layer, labeling is present over large cell bodies, and their initial axonal segments. These are likely to be Golgi cells, based on their co-staining with GABA. Finally, numerous elongated processes within the white matter, which are likely to be axons, also are type 2 immunoreactive.These data indicate that both types of corticotropin releasing factor receptor are present in the mouse cerebellum. However, the unique distribution of the two types of receptor strongly suggests a differential role for corticotropin releasing factor in modulating the activity of neurons, axons and glial cells via cell-specific ligand-receptor interactions.     

Immunohistochemical localization of glial cell line-derived neurotrophic factor in the human central nervous system

Glial cell line-derived neurotrophic factor, initially purified from the rat glial cell line B49, has the ability to promote the survival and differentiation of various types of neurons in the central and peripheral nervous systems. In the present study, to evaluate the physiological role of glial cell line-derived neurotrophic factor in the central nervous system, we investigated the cellular and regional distribution of glial cell line-derived neurotrophic factor immunoreactivity in autopsied control human brains and spinal cords using a polyclonal glial cell line-derived neurotrophic factor-specific antibody. On western blot analysis, the antibody reacted with recombinant human glial cell line-derived neurotrophic factor, and recognized a single band at a molecular weight of approximately 34,000 in human brain homogenates. Glial cell line-derived neurotrophic factor immunoreactivity was observed mainly in the neuronal somata, dendrites and axons. In the telencephalon, diencephalon and brainstem, the cell bodies and proximal processes of several neuronal subtypes were immunostained with punctate dots. Furthermore, immunopositive nerve fibers were also observed, and numerous axons were intensely immunolabeled in the internal segment of the globus pallidus and the pars reticulata of the substantia nigra. In the cerebellum, the most conspicuous immunostaining was found in the Purkinje cells, in which the somata and dendrites were strongly immunolabeled. Intense immunoreactivity was also detected in the posterior horn of the spinal cord. In addition to the neuronal elements, immunopositive glial cell bodies and processes were observed in various regions.

Our results suggest that glial cell line-derived neurotrophic factor is widely localized, but can be found selectively in certain neuronal subpopulations of the human central nervous system. Glial cell line-derived neurotrophic factor may regulate the maintenance of neuronal functions under normal circumstances.     

Distribution and subcellular localization of calmodulin in adult and developing brain tissue

The distribution and subcellular localization of calmodulin in adult and developing cerebellum was studied in rats by immunocytochemistry. Calmodulin immunoreactivity was found both in neurons and in glial cells. Within neurons the staining was particularly intense in the cell nucleus and in dendrites, the cytoplasm of the cell body was more lightly stained than the nucleus, and light immunoreactivity was observed in axons. Electron microscopic analysis confirmed the association of calmodulin with the nuclear chromatin, while the nucleolus remained unstained. The reaction product was also found overlying the membranes of several organelles, in postsynaptic densities and decorating both dendritic and axonal microtubules. In developing Purkinje cells, calmodulin immunoreactivity was found as early as 5 days after birth. During the initial phases of dendritic development (5-10 days post-natal), the reaction product was associated with the organelles of the apical cone, while little or no staining was observed in the elongating dendrites or in the cell nucleus. Later in development, calmodulin was found in primary and secondary dendrites, and by 20 days after birth immunoreactivity appeared in the cell nucleus, and in the postsynaptic densities of immature spines located in dendrites. The presence of calmodulin in the apical cone suggests the possibility that this protein may participate in the regulation of microtubule formation during the initial stages of dendritic development. Its presence in dendrites at later stages (during the period of synaptogenesis) may indicate that it also participates in the formation of synapses between the parallel fibres and dendritic spines.