Mossy Fiber Rosettes Research Articles

The neurovascular unit of capillary blood vessels in the rat nervous system. A rapid-Golgi electron microscopy study.

We describe a pericapillary organ in the rat forebrain and cerebellar cortex. It consists of a series of tripartite synapses with synaptic extensions enveloped by astrocytic endfeet that are linked to the capillary wall by synaptic extensions. Reciprocal specializations of the pericyte-capillary blood vessel (CBV) with such specialized synapses suggest a mechanoreceptor role. In Golgi-impregnated and 3D reconstructions of the cerebral cortex and thalamus, a series of TSs appear to be sequentially ordered in a common dendrite, paralleled by synaptic outgrowths termed golf club synaptic extensions (GCE) opposed to a longitudinal crest (LC) from the capillary basal lamina (BL). Our results show that, in the cerebellar cortex, afferent fibers and interneurons display microanatomical structures that strongly suggest an interaction with the capillary wall. Afferent mossy fiber (MF) rosettes and ascending granule cell axons and their dendrites define the pericapillary passage interactions that are entangled by endfeet. The presence of mRNA of the mechanosensitive channel Piezo1 in the MF rosettes, together with the surrounding end-feet and the capillary wall form mechanosensory units. The ubiquity of such units to modulate synaptic transmission is also supported by Piezo1 mRNA expressing pyramidal isocortical and thalamic neurons. This scenario suggests that ascending impulses to the cerebellar and cortical targets are presynaptically modulated by the reciprocal interaction with the mechanosensory pericapillary organ that ultimately modulates the vasomotor response.

Differential expression of the c-fos protein and synaptophysin in zebrin II positive and zebrin II negative cerebellar cortical areas in 4-aminopyridine seizures

The present study examined temporal activation patterns of rat cerebellar cortical neurons in 4-aminopyridine induced seizures, using c-fos protein as a marker of neuronal activity. C-fos-containing cells were counted in each cerebellar cortical layer, and cell count was compared between zebrin II positive and zebrin II negative bands of the lobules of the vermis and cerebellar hemispheres. We found significant activation of granule cells and interneurons of the molecular layer in zebrin II positive bands. The Purkinje cells, in contrast, exhibited non-significant, scattered c-fos immunoreactivity across all bands. Fluctuation of synaptophysin expression in the mossy fibre rosettes of the granular layer was determined via light microscopic immunohistochemistry. We detected a transient, significant decrease in synaptophysin staining density following 4-aminopyridine seizures, which may indicate short-term synaptic depression. We also identified different timing of increased c-fos expression in the neurons of the cerebellar cortex in different cortical zones. In particular, the activation pattern of the interneurons of the molecular layer reflected the climbing fibre distribution, reflecting the zonal olivo-cortico-nuclear organization. Seizure-induced activation of the granule cells corresponded with the zebrin II positive zones. This observation raises the possibility that zebrin II positive compartments may be more susceptible to cerebellar convulsions.

Post-natal development of the Reeler mouse cerebellum: An ultrastructural study

Reelin, an extracellular protein promoting neuronal migration in brain areas with a laminar architecture, is missing in the Reeler mouse (reelin(-/-)). Several studies indicate that the protein is also necessary for correct dendritic outgrowth and synapse formation in the adult forebrain. By transmission electron microscopy, we characterize the development and synaptic organization of the cerebellar cortex in Reeler mice and wild type control littermates at birth, postnatal day (P) 5, 7, 10 and 15. Ultrastructural analysis shows deep alterations in cortical architecture and mispositioning of the Purkinje neurons (Pns), which remain deeply embedded in a central cellular mass within the white matter, with highly immature features. Quantitative examination shows that Reeler mice display: (i) a lower density of granule cells and a higher density of Pns, from P10; (ii) a lower density of synaptic contacts between Pn dendrites and parallel or climbing fibers, from P5; (iii) a lower density of synaptic contacts between basket cells and Pns, from P5; and (iv) a lower density of mossy fiber rosettes, from P10. Our results demonstrate that Reelin profoundly affects the structure and synaptic connectivity of post-natal mouse cerebellum.

Three-Dimensional Imaging of Cerebellar Mossy Fiber Rosettes by Ion-Abrasion Scanning Electron Microscopy

The detailed knowledge of the three-dimensional (3D) organization of the nervous tissue provides essential information on its functional elucidation. We used serial block-face scanning electron microscopy with focused ion beam (FIB) milling to reveal 3D morphologies of the mossy fiber rosettes in the mice cerebellum. Three-week-old C57 black mice were perfused with a fixative of 1% paraformaldehyde/1% glutaraldehyde in phosphate buffer; the cerebellum was osmicated and embedded in the Araldite. The block containing granule cell layer was sliced with FIB and observed by field-emission scanning electron microscopy. The contrast of backscattered electron image of the block-face was similar to that of transmission electron microscopy and processed using 3D visualization software for further analysis. The mossy fiber rosettes on each image were segmented and rendered to visualize the 3D model. The complete 3D characters of the mossy fiber rosette could be browsed on the A-Works, in-house software, and some preliminary quantitative data on synapse of the rosette could be extracted from these models. Thanks to the development of two-beam imaging and optimized software, we could get 3D information on cerebellar mossy fiber rosettes with ease and speedily, which would be an additive choice to explore 3D structures of the nervous systems and their networks.

Extending the bandwidth of long‐term plasticity at the cerebellar input stage

Extending the bandwidth of long‐term plasticity at the cerebellar input stage

Precise spatial relationships between mossy fibers and climbing fibers in rat cerebellar cortical zones.

Classically, mossy fiber and climbing fiber terminals are regarded as having very different spatial distributions in the cerebellar cortex. However, previous anatomical studies have not studied these two major cerebellar inputs with sufficient resolution to confirm this assumption. Here, we examine the detailed pattern of collateralization of both types of cerebellar afferent using small injections of the bidirectional tracer cholera toxin b subunit into the posterior cerebellum. The cortical and zonal location of these injections was characterized by mapping climbing fiber field potentials, the distribution of retrogradely labeled olivary neurons, and the intrinsic zebrin pattern of Purkinje cells. Labeled climbing fiber collaterals were distributed as longitudinal strips and were always accompanied by clusters of labeled mossy fiber rosettes in the subjacent granular layer. Two- and three-dimensional reconstructions and quantitative analysis showed that mossy fibers also collateralized to other stripe-like regions usually below Purkinje cells with the same zebrin-positive or zebrin-negative characteristics as that of the injection site and associated climbing fiber collaterals. The distribution of retrogradely labeled neurons in two major sources of mossy fibers, the lateral reticular and basilar pontine nuclei, revealed interlobular and some interzonal differences. These data indicate that nonadjacent cerebellar zones, sharing the same climbing fiber input and zebrin identity, also share a common mossy fiber input. Other cerebellar cortical regions that receive collaterals from the same mossy fibers usually also have the same zebrin signature. Together with the distribution of neurons in precerebellar centers, the findings suggest a revision of the modular hypothesis for information processing in the cerebellar cortex.

Compartmentation of the reeler cerebellum: Segregation and overlap of spinocerebellar and secondary vestibulocerebellar fibers and their target cells

The cerebellum of the reeler mutant mouse has an abnormal organization; its single lobule is composed of a severely hypogranular cortex and a central cerebellar mass (CCM) consisting of Purkinje cell clusters intermixing with the cerebellar nuclei. As such the reeler represents an excellent model in which to examine the effect of the abnormal distribution of cerebellar cells on afferent-target relationships. To this effect we studied the organization of the spinocerebellar and secondary vestibulocerebellar afferent projections in homozygous reeler mice (rl/rl) using anterograde tracing techniques. Spinal cord injections resulted in labeled spinocerebellar mossy fiber rosettes in specific anterior and posterior regions of the cerebellar cortex. Some vestiges of parasagittal organization may be present in the anterior projection area. Within the CCM, labeled fibers appeared to terminate on distinct groups of Purkinje cells. Thus, the spinocerebellar mossy fibers seem to form both normal and heterologous synapses in the reeler cerebellum. Secondary vestibular injections resulted in both retrograde and anterograde labeling. Retrograde labeling was seen in clusters of Purkinje cells and cerebellar nuclear cells; anterograde labeling was distributed in the white matter and in specific regions of the anterior and posterior cortex of the cerebellum. The labeled spinocerebellar and secondary vestibulocerebellar afferents overlapped in the anterior region but in the posterior region the vestibulocerebellar termination area was ventral to the spinocerebellar area. An area devoid of labeled terminals was also observed ventral to the posterior secondary vestibulocerebellar termination field. Using calretinin immunostaining it was determined that this area contains unipolar brush cells, a cell type found primarily in the vestibulocerebellum of normal mice. Our data indicate that despite of the lack of known landmarks (fissures, lobules) the spinocerebellar and vestibulocerebellar afferent projections in the reeler cerebellum do not distribute randomly but have specific target regions, and the position of these regions, relative to each other, appears to be conserved. Two caveats to this were the finding of overlapping terminal fields of these afferents in the anterior region, and a posteroventral region that contains unipolar brush cells yet is devoid of secondary vestibulocerebellar afferents. The distribution of Purkinje cells and cerebellar nuclear cells is not random either; those that give rise to cerebellovestibular efferents form distinct groups within the central cerebellar mass.

Localization of synapsin-I and PSD-95 in developing postnatal rat cerebellar cortex

The comparative localization of two prominent synaptic proteins, synapsin-I (Syn-I) and PSD-95, was investigated in slices of developing (P3–P21) rat cerebellar cortex using double- or triple-label fluorescence immunohistochemistry and confocal microscopy. During the first postnatal week, Syn-I and PSD-95 immunoreactive (IR) puncta were strongly concentrated in the Purkinje cell layer (PCL) where they circumscribed irregularly shaped PC somata, forming pericellular nests that likely correspond to early climbing fiber synapses. PSD95 and Syn-I puncta also were found along the shafts and at the tips of growing PC dendrite branches labeled with calbindin. During the second postnatal week, synaptic puncta were lost from the PC layer, while many new puncta were added to the molecular layer (ML). At P10, about half of the PCs were circumscribed by PSD-95 or Syn-I puncta, whereas at P14 no PCs were circumscribed. By P14, PSD95 and Syn-I became most strongly localized to many small puncta in the ML and to large clusters at mossy fiber rosettes in the glomerular layer (GL) where PSD-95 often encircled Syn-I clusters. Some large clusters in the GL contained only PSD-95 or Syn-I, but not both, suggesting differential growth or remodeling of pre- and post-synaptic structures. No PSD-95 staining of pre-synaptic terminal pinceau was observed during the first 3 weeks of postnatal development. Thus, in relation to PCs, there is a developmental shift in PSD-95 localization whereby, first, it is concentrated on PC cell bodies and short dendrites (P3–P7), then it is lost on PC cell bodies (P7–14) and becomes localized almost exclusively to PC dendrites (P14–P21).

Collateralization of climbing and mossy fibers projecting to the nodulus and flocculus of the rat cerebellum

Collateralization of mossy and climbing fibers was investigated using cortical injections of cholera toxin b-subunit in the rat vestibulocerebellum. Injections were characterized by their retrograde labeling within the inferior olive. Collateral labeling was plotted using color-coded density profiles of the whole cerebellar cortex. Injections in the medial part of the nodulus resulted in olivary labeling that was restricted to the rostral part of the dorsal cap. Climbing fiber collaterals were found in medial and lateral nodular zones as well as in the ventral paraflocculus and adjacent flocculus. Injections in the intermediate part of the nodulus resulted in olivary labeling of the beta-subnucleus but could also involve the ventrolateral outgrowth. In the latter case, climbing fiber collaterals were found in the two floccular zones and in a small region in the lateral-most part of crus I. All nodular injections showed a bilaterally symmetric distribution of collateral mossy fiber rosettes that was mostly confined to the vestibulocerebellum and originated predominantly from the vestibular nuclei. Injections in the flocculus labeled the caudal part of the dorsal cap and/or the ventrolateral outgrowth. Mossy fiber rosettes were observed throughout the vestibulocerebellum but also included other regions of the cerebellar cortex in a bilaterally symmetric pattern corresponding with a more widespread precerebellar origin. Climbing fibers originating in the rostral dorsal cap, labeled from an injection in the ventral paraflocculus, collateralize to a medial and lateral zone in the nodulus. Climbing fiber collaterals were usually accompanied by subjacent labeling of mossy fiber rosettes. These results demonstrate that some nodular and floccular zones are related and, at least partially, share a common input.

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.

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.

Topological relationship between corticotropin-releasing factor-immunoreactive cerebellar afferents and tyrosine hydroxylase-immunoreactive purkinje cells in a hereditary ataxic mutant, rolling mouse nagoya

Using immunohistochemistry we examined the distribution of corticotropin-releasing factor-positive cerebellar afferents and the topological relationship between their projections and the distribution of tyrosine hydroxylase-positive Purkinje cells in an ataxic mutant, rolling mouse Nagoya. In the mutants, some climbing fibers were more intensely stained for corticotropin-releasing factor, but their zonal distribution remained the same as in non-ataxic littermates (control mice). These climbing fibers arose from the dorsal accessory nucleus, the ventral lamella of principal nucleus, the dorsomedial cell group, the subnucleus A, the β subnucleus and the ventrolateral protrusion of the inferior olive, since perikarya in these olivary subdivisions were more intensely stained for corticotropin-releasing factor than in controls. Some mossy fiber rosettes in the vermal lobules, the simple lobule, the crus I of ansiform lobule, the copula pyramidis and the flocculus also exhibited corticotropin-releasing factor immunoreactivity and were more densely stained in the mutants than in controls. Double immunostaining for corticotropin-releasing factor and tyrosine hydroxylase in the mutant cerebellum revealed that the distribution of tyrosine hydroxylase-positive Purkinje cells corresponded to terminal fields of corticotropin-releasing factor-positive climbing fibers but not corticotropin-releasing factor-positive mossy fibers. This study indicated an increased corticotropin-releasing factor immunoreactivity in some climbing or mossy fibers in the cerebellum of rolling mouse Nagoya. We also found that the distribution of tyrosine hydroxylase-positive Purkinje cells corresponded to terminal fields of corticotropin-releasing factor-positive climbing fibers in the mutant cerebellum. As the transcription of the tyrosine hydroxylase gene is facilitated by Ca 2+, abnormal tyrosine hydroxylase expression in the mutant Purkinje cells may indicate functional abnormality by alterations in intracellular Ca 2+ concentrations. Therefore, we suggest that an increased level of corticotropin-releasing factor in a specific population of climbing fibers may alter the function of their target Purkinje cells.

Postsynaptic actin filaments at the giant mossy fiber-unipolar brush cell synapse

The unipolar brush cell (UBC), a small interneuron occurring at high density in the granular layer of the mammalian vestibulocerebellum, receives a giant glutamatergic synapse from a single mossy fiber (MF) rosette, usually on a brush of dendritic branchlets. MF stimulation produces a current in the UBC several orders of magnitude greater in duration than at other glutamatergic synapses. We assumed that the cytoskeleton would have a special role in plasticity of the MF-UBC synapse. Neurofilaments and microtubules are enriched in the UBC somatodendritic compartment but are conspicuously absent in close proximity to the giant synapse, where standard electron microscopy reveals a granulo-flocculent material. Because osmium tetroxide fixation during sample preparation for standard electron microscopy destabilizes actin filaments, we hypothesized that this subsynaptic granulo-flocculent material is actin-based. After actin stabilization, we observed prominent, but loosely organized, bundles of microfilaments at the subsynaptic region of the MF-UBC synapse that linked the postsynaptic density with the cytoskeletal core of the dendritic branchlets. Confocal fluorescence microscopy and pre- and postembedding immunogold labeling with phalloidin and actin antibodies showed that these microfilaments consist of f-actin and contain little beta-actin. This extraordinary postsynaptic actin apparatus is ideally situated to form a dynamic framework for glutamate receptors and other postsynaptic molecules, and to mediate activity-dependent plastic rearrangements of the giant synapse.

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 mGluR1alpha 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 mGluR1alpha 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 mGluR1alpha 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 mGluR1alpha 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.

Chapter 8 The unipolar brush cells of the mammalian cerebellum and cochlear nucleus: cytology and microcircuitry

The unipolar brush cell (UBC) is a novel type of small neuron that is characterized by sets of morphological and chemical phenotypes. UBCs occur in the granular layer of the mammalian cerebellar cortex, particularly in folia of the vestibulocerebellum, and in the granule cell domains of the dorsal cochlear nucleus. The UBC is characterized by a single dendrite that terminates with a brush-like tip of dendrioles. The soma, the dendritic stem, and especially the dendrioles emit short, non-synaptic appendages. The dendrioles represent the main synaptic apparatus of the UBC and articulate tightly with a single mossy fiber rosette forming a glomerular array characterized by an extraordinarily extensive synaptic contact. Electron microscopic and electrophysiological observations indicate that the unusual synaptic ultrastructure may produce entrapment of neurotransmitter in the synaptic cleft. While ionotropic glutamate receptors are enriched in correspondence of the postsynaptic density, metabotropic glutamate receptors are situated extrasynaptically and are particularly enriched at the appendages, which usually do not bear synaptic junctions. Some of the UBCs receive their input from choline acetyltransferase-positive mossy rosettes originating from the vestibular nuclei, suggesting that ACh and glutamate are co-released at these synapses. The UBC brush occupies a glomerulus where granule cell dendrites are intermixed with the UBC dendrioles, both of which receive synapses from the same mossy fiber rosette and portions of the Golgi axonal plexus. In addition, the dendrioles are presynaptic to granule cell dendrites, forming dendrodendritic contacts that display features of excitatory synapses. Branches of the UBC axon in the granular layer bear large endings resembling mossy fibers. The UBCs may represent an extraordinary device for feedforward, excitatory links along the mossy fiber pathways of cerebellum and dorsal cochlear nucleus.

Localization of the insulin-like growth factor I receptor in the cerebellum and hypothalamus of adult rats: an electron microscopic study.

Insulin-like growth factor I (IGF-I) is an important modulator of cell growth and plasticity in the CNS. Expression of the IGF-I receptor mRNA in brain peaks at times of active cell development perinatally and remains detectable, albeit at lower levels, in the adult. While both autoradiographic and in situ hybridization studies show a wide and specific distribution of IGF-I receptor throughout the adult rat brain, nothing is yet known about its subcellular localization, a critical issue that will help clarify the biological role of this trophic factor in the adult brain. The present study describes the subcellular localization of IGF-I receptor immunoreactivity in the cerebellar cortex and the hypothalamic arcuate nucleus by using electron microscopic immunocytochemistry. In the cerebellum, IGF-I receptor immunoreactivity is present postsynaptically in the dendrites and soma of the Purkinje cell and presynaptically in axon terminals impinging upon the Purkinje cell soma, as well as in mossy fibre rosettes in the cerebellar glomeruli. Neurons in the mediobasal hypothalamus also contain IGF-I receptors located pre- and postsynaptically. Endothelial cells, astroglial end-feet surrounding micro vessels thoughout all the brain parenchyma, tanycytes of the third ventricle and oligodendrocytes in the cerebellar white matter are also rich in IGF-I receptors. These results strongly support previous observations that IGF-I is a neuromodulator in the adult brain, probably acting as both a pre- and a postsynaptic messenger. They also suggest that glial cells may be involved in the actions of IGF-I in the adult brain.

Fine structure of the cell clusters in the cochlear nerve root: stellate, granule, and mitt cells offer insights into the synaptic organization of local circuit neurons.

The small cell shell of the cochlear nucleus contains a complex integrative machinery which can be used to study the roles of interneurons in sensory processing. The cell clusters in the cochlear nerve root of the chinchilla provide the simplest example of this structure. Reported here are the neuronal architecture and synaptic organization of the three principal cell types and the three distinctive neuropil structures that could be characterized with the Nissl and Golgi methods and electron microscopy. Granule cells were characterized by several dendrites with claw-like terminals that received synaptic contacts from multiple excitatory mossy fiber rosettes. Given their relatively large number and their prolific parallel fiber synapses, the granule cells provide a suitable substrate for a tangential spread of excitatory activity, which could build to considerable proportions. The mitt cells had a thickened, single dendrite, its terminal branches arranged in a shape reminiscent of a baseball catcher's mitt. The dendritic mitt enclosed an enormous, convoluted mossy fiber rosette forming many excitatory synapses on just one cell. This could provide for a discrete, comparatively fast input-output relay of signals. Small stellate cells had longer, radiating dendrites that engaged the synaptic nests. These nests were strung in long strands, containing heterogeneous synapses from putative excitatory and inhibitory inputs. Given the prevalence of the synaptic nests, the small stellate cells appear to have the greatest integrative capacity. They provide the main output of the synaptic nests.

Ultrastructural localization of the plasmalemmal calcium pump in cerebellar neurons.

In a previous study, fluorescence labeling of a plasmalemmal ATPase protein with the 5F10 monoclonal antibody revealed prominent antigen in the cerebellar molecular layer surrounding the somata and dendrites of Purkinje cells. In the present study, this antibody labeled with silver enhanced nano-sized gold particles on semithin plastic sections revealed a clearly demarcated plasma membrane outlining the somata and entire dendritic arbors of Purkinje cells including their spines. Ultrastructural analysis of horseradish peroxidase preparations showed reaction product along the plasmalemma and extending on to the sub-plasmalemmal endoplasmic reticulum. In the granular layer, somata of granule cells were reactive, as were their dendritic extensions into glomeruli where reactive claws surrounded voids formed by mossy fiber rosettes. Somata and dendrites of cerebellar nuclear cells also had reactive zones that were limited to the plasma membrane and a narrow zone of the sub-plasmalemmal endoplasmic reticulum. Comparative labeling of this protein and P channel protein revealed similar plasmalemmal locations. This study shows that a specific calcium ATPase pump protein is located on the plasmalemma of certain types of cerebellar neurons. The ultrastructural distribution of calcium pump and P channel antibodies occurred in punctate sites along the plasma membrane of dendrites and spines of Purkinje cells. The close association between P-type calcium channels and the plasma membrane calcium pump is consistent with rapid extrusion of intracellular calcium from neurons endowed with large numbers of voltage-gated calcium channels.

Cerebellar choline acetyltransferase positive mossy fibres and their granule and unipolar brush cell targets: a model for central cholinergic nicotinic neurotransmission.

A subset of cerebellar mossy fibres is rich in choline acetyltransferase, the rate-limiting enzyme for the synthesis of acetylcholine. These choline acetyltransferase-positive mossy fibres are concentrated in the vestibulocerebellum and originate predominantly from the medial vestibular nucleus. The granular layer of the vestibulocerebellum is also enriched in unipolar brush cells, an unusual type of small neuron that form giant synapses with mossy fibres. In this immunocytochemical light and electron microscopic study, we explored whether choline acetyltransferase-positive mossy fibres innervate unipolar brush cells of the rat cerebellum. We utilized monoclonal antibodies to rat choline acetyltransferase of proven specificity, and immunoperoxidase procedures with 3,3'-diaminobenzidine tetrahydrochloride as the chromogen. A high density of choline acetyltransferase-positive fibres occurred in the nodulus and ventral uvula, where they showed an uneven, zonal distribution. Immunostained mossy fibre rosettes contained high densities of round synaptic vesicles and mitochondria. They formed asymmetric synaptic junctions with dendritic profiles of both granule cells and unipolar brush cells. The synaptic contacts between choline acetyltransferase-immunoreactive mossy fibres and unipolar brush cells were very extensive, and did not differ from synapses of choline acetyltransferase-negative mossy fibres with unipolar brush cells. Analysis of a total area of 1.25 mm2 of the nodulus from three rats revealed that 14.2% of choline acetyltransferase-immunoreactive mossy fibre rosettes formed synapses with unipolar brush cells profiles. Choline acetyltransferase-positive rosettes accounted for 21.7% of the rosettes forming synapses with unipolar brush cells. Thus, the present data demonstrate that unipolar brush cells are innervated by a heterogeneous population of mossy fibres, and that some unipolar brush cells receive cholinergic synaptic input from the medial vestibular nucleus. The ultrastructure of these synapses is compatible with the possibility that choline acetyltransferase-positive mossy fibres co-release acetylcholine and glutamate. As the granular layer of the vestibulocerebellum contains nicotinic binding sites, the choline acetyltransferase-positive mossy fibres may be a model for studying nicotinic neurotransmission in the CNS.

Light-microscopic distribution and parasagittal organisation of muscarinic receptors in rabbit cerebellar cortex

Recent studies on the effects of intrafloccular injections of muscarinic agonists and antagonists on compensatory eye movements in rabbit, indicate that muscarinic receptors may play a modulatory role in the rabbit cerebellar circuitry. It was previously demonstrated by Neustadt et al. (1988), that muscarinic receptors in rabbit cerebellar cortex are distributed into alternating longitudinal zones of very high and very low receptor density. In the present study, the zonal and cellular distribution of muscarinic receptors in the rabbit cerebellar cortex is investigated in detail using in vitro ligand autoradiography with the non-selective high-affinity antagonist [ 3H]quinuclidinyl benzilate (QNB), and the M2-specific antagonist [ 3H]AF-DX384, and immunocytochemistry with a monoclonal antibody specific for the cloned m2 muscarinic receptor protein. [ 3H]QNB and [ 3H]AF-DX384 binding sites and m2-immunoreactivity had similar overall distributions: dense labeling occurred in the dendritec arbors of a subset of Purkinje cells that are organized into parasagittal bands. A high level of muscarinic receptor labeling was also observed in a thin substratum of the molecular layer immediately above the Purkinje cell layer of the vestibulo-cerebellar lobules, i.e. the nodulus, the ventral uvula and the flocculus. Labeling in this stratum was associated with densely packed fibres, which were putatively identified as parallel fibres. Also Golgi cells, which were localized in part in the molecular layer, and a subset of mossy fibre rosettes, primarily concentrated in lobule VI, were immunoreactive for the m2 receptor. The parasagittal bands of labeled Purkinje cell dendrites were most prominent in the anterior lobe (lobules I–V), in crus 1 and 2, in the flocculus, the ventral paraflocculus and the rostral folium of the nodulus. In other lobules, only infrequent Purkinje cells contained muscarinic receptors. The parasagittal organisation of muscarinic receptors differed from that of zebrin I, a Purkinje cell-specific protein which is often used as a marker of parasagittal parcelation of the cerebellar cortex. In the anterior lobe, however, there was a partial correspondence between muscarinic receptor and zebrin I bands. In the flocculus the distribution of muscarinic-receptor-positive Purkinje cells was related to the distinct white matter compartments as revealed with acetylcholinesterase (AChE) histochemistry. Muscarinic receptor-containing Purkinje cells were located primarily in the floccular zone 1, which is implicated in the control of eye movements about a horizontal axis. In order to relate the distribution of muscarinic receptor labeling to that of cholinergic nerve terminals, [ 3H]QNB binding sites and sodium-dependent [ 3H]hemicholinium-3 binding were compared. Sodium-dependent [ 3H]hemicholinium-3 binding sites mainly occurred in the granule cell layer of the vestibulo-cerebellum, which corresponds well with the distribution of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT). However, sodium-dependent [ 3H]hemicholinium binding complemented, rather than co-localized with, muscarinic receptors which were primarily distributed in the molecular layer of the lobules of the vestibulo-cerebellar lobules. Their functional significance is puzzling, since their distribution does not correspond to that of markers of cholinergic innervation.