Distribution and phenotypes of unipolar brush cells in relation to the granule cell system of the rat cochlear nucleus.

In most mammals the cochlear nuclear complex (CN) contains a distributed system of granule cells (GCS), whose parallel fiber axons innervate the dorsal cochlear nucleus (DCN). Like their counterpart in cerebellum, CN granules are innervated by mossy fibers of various origins. The GCS is complemented by unipolar brush (UBCs) and Golgi cells, and by stellate and cartwheel cells of the DCN. This cerebellum-like microcircuit modulates the activity of the DCN's main projection neurons, the pyramidal, giant and tuberculoventral neurons, and is thought to improve auditory performance by integrating acoustic and proprioceptive information. In this paper, we focus on the rat UBCs, a chemically heterogeneous neuronal population, using antibodies to calretinin, metabotropic glutamate receptor 1alpha (mGluR1alpha), epidermal growth factor substrate 8 (Eps8) and the transcription factor T-box gene Tbr2 (Tbr2). Eps8 and Tbr2 labeled most of the CN's UBCs, if not the entire population, while calretinin and mGluR1alpha distinguished two largely separate subsets with overlapping distributions. By double labeling with antibodies to Tbr2 and the alpha6 GABA receptor A (GABAA) subunit, we found that UBCs populate all regions of the GCS and occur at remarkably high densities in the DCN and subpeduncular corner, but rarely in the lamina. Although GCS subregions likely share the same microcircuitry, their dissimilar UBC densities suggest they may be functionally distinct. UBCs and granules are also present in regions previously not included in the GCS, namely the rostrodorsal magnocellular portions of ventral cochlear nucleus, vestibular nerve root, trapezoid body, spinal tract and sensory and principal nuclei of the trigeminal nerve, and cerebellar peduncles. The UBC's dendritic brush receives AMPA- and NMDA-mediated input from an individual mossy fiber, favoring singularity of input, and its axon most likely forms several mossy fiber-like endings that target numerous granule cells and other UBCs, as in the cerebellum. The UBCs therefore, may amplify afferent signals temporally and spatially, synchronizing pools of target neurons.

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.

Monoclonal antibody Cat-315 detects a glycoform of receptor protein tyrosine phosphatase beta/phosphacan early in CNS development that localizes to extrasynaptic sites prior to synapse formation.

Perineuronal nets (PNs) are lattice-like condensations of the extracellular matrix (ECM) that envelop synapses and decorate the surface of subsets of neurons in the CNS. Previous work has suggested that, despite the fact that PNs themselves are not visualized until later in development, some PN component molecules are expressed in the rodent CNS even before synaptogenesis. In the adult mammalian brain, monoclonal antibody Cat-315 recognizes a glycoform of aggrecan, a major component of PNs. In primary cortical cultures, a Cat-315-reactive chondroitin sulfate proteoglycan (CSPG) is also expressed on neuronal surfaces and is secreted into culture media as early as 24 h after plating. In this study, we show that in primary cortical cultures, the Cat-315 CSPG detected in early neural development is expressed in extrasynaptic sites prior to synapse formation. This suggests that ECM components in the CNS, as in the neuromuscular junction (NMJ), may prepattern neuronal surfaces prior to innervation. We further show that while the Cat-315-reactive carbohydrate decorates aggrecan in the adult, it decorates a different CSPG in the developing CNS. Using receptor protein tyrosine phosphatase beta (RPTPbeta/protein tyrosine phosphatase zeta) knock-out mice and immunoprecipitation techniques, we demonstrate here that in the developing rodent brain Cat-315 recognizes RPTPbeta isoforms. Our further examination of the Cat-315 epitope suggests that it is an O-mannose linked epitope in the HNK-1 family. The presence of the Cat-315 reactive carbohydrate on different PN components--RPTPbeta and aggrecan--at different stages of synapse development suggests a potential role for this neuron-specific carbohydrate motif in synaptogenesis.

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.

Postnatal differentiation of unipolar brush cells and mossy fiber-unipolar brush cell synapses in rat cerebellum.

The unipolar brush cells are excitatory, cerebellar granular layer interneurons that receive mossy fiber input on their dendritic brushes in the form of a giant glutamatergic synapse. We investigated the postnatal development of the brush of the unipolar brush cell in lobules IX and X by light microscopy and defined the maturation of mossy fiber-unipolar brush cell synapses and mossy fiber-granule cell synapses by electron microscopy using calretinin immunocytochemistry to identify unipolar brush cells. During the first postnatal week, unipolar brush cells possessed one or two short, branched dendrites. The brush differentiated primarily during the successive 21 postnatal (P) days, during which it underwent progressive maturation. This developmental process was subdivided into stages 1-4, which were descriptively termed protodendritic unipolar brush cell (P2-12), filopodial brush (P12-16), intermediate brush (P16-21), and dendriolar brush (P21-28) stages. Electron microscopic measurements of individual mossy fiber-unipolar brush cell and mossy fiber-granule cell synaptic junctions were made at P12, 16, 21, and 28. While the average length of mossy fiber-unipolar brush cell synapses increased during development, that of mossy fiber-granule cell synapses decreased. Comparisons of the lengths of mossy fiber-unipolar brush cell and mossy fiber-granule cell synapses demonstrated that mossy fiber-unipolar brush cell synapses were longer on average than mossy fiber-granule cell synapses for all ages. Frequency distribution histograms also showed that the percentage of mossy fiber-unipolar brush cell synapses longer than 0.5 microm was lower in the pooled P12-P16 groups than in the pooled P21-P28 groups (8 versus 20%). In contrast, mossy fiber-granule cell synapses longer than 0.5 microm were a small minority at P12, 16, and 21, and occurred rarely at P28. The present study indicates that mossy fiber-unipolar brush cell synapses increase in length with the differentiation of the brush dendrioles, while that of mossy fiber-granule cell synapses decrease with differentiation of the granule cell dendritic claws. The finding that mossy fiber-unipolar brush cell synapses were generally longer than mossy fiber-granule cell synapses may indicate that the properties of the postsynaptic targets play a major role in shaping synaptic appositions within cerebellar glomeruli.

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.

Unipolar brush cell: a potential feedforward excitatory interneuron of the cerebellum.

Unipolar brush cells are a class of interneurons in the granular layer of the mammalian cerebellum that receives excitatory mossy fiber synaptic input in the form of a giant glutamatergic synapse. Previously, it was shown that the unipolar brush cell axon branches within the granular layer, giving rise to large terminals. Single mossy fiber stimuli evoke a prolonged burst of firing in unipolar brush cells, which would be distributed to postsynaptic targets within the granular layer. Knowledge of the ultrastructure of the unipolar brush cell terminals and of the cellular identity of its postsynaptic targets is required to understand how unipolar brush cells contribute to information processing in the cerebellar circuit. To investigate the unipolar brush cell axon and its targets, unipolar brush cells were patch-clamped in fresh parasagittal slices from rat cerebellar vermis with electrodes filled with Lucifer Yellow and Biocytin, and examined by confocal fluorescence and electron microscopy. Biocytin was localized with diaminobenzidine chromogen or gold-conjugated, silver-intensified avidin. Light microscopic examination revealed a single thin axon emanating from the unipolar brush cell soma that gave rise to 2-3 axon collaterals terminating in mossy fiber-like rosettes in the granular layer, typically within a few hundred microm of the soma. In some cases, axon collaterals crossed the white matter within the same folium before terminating in the adjacent granular layer. Electron microscopic examination of serial ultrathin sections revealed that proximal unipolar brush cell axons and axon collaterals were unmyelinated and devoid of synaptic contacts. However, the rosette-shaped enlargements of each collateral formed the central component of glomeruli where they were surrounded by dendrites of granule cells and/or other unipolar brush cells, with which they formed asymmetric synaptic contacts. A long-latency repetitive burst of polysynaptic activity was observed in granule cells in this cerebellar region following white matter stimulation. The unipolar brush cell axons, therefore, form a system of cortex-intrinsic mossy fibers. The results indicate that synaptic excitation of unipolar brush cells by mossy fibers will drive a large population of granule cells, and thus will contribute a powerful form of distributed excitation within the basic circuit of the cerebellar cortex.

Distribution of unipolar brush cells and other calretinin immunoreactive components in the mammalian cerebellar cortex.

We have compared the distribution of unipolar brush cells (UBCs) in the cerebellum of Brazilian opossum (Monodelphis domestica), mouse, guinea pig, rabbit, cat, and Rhesus monkey, using an antiserum to calretinin which is present in those cells. The morphology and calretinin staining intensity of the UBCs remains constant across species. As a general trend, in all species studied, UBCs are particularly enriched in the vestibulocerebellum. Interspecies differences, however, were noted in the distribution of UBCs across other regions of the cerebellar cortex. A major variation involves the extent of the UBC-rich region of the ventral portion of the paraflocculus. The distribution of UBCs in non-vestibular vermal folia also varies substantially. UBCs are deployed in more or less distinct parasagittal zones in the vermis of the opossum, rabbit, cat, and macaque. The density of UBCs decreases progressively from medial to lateral portions of the same folium and is lowest in the lateral, posterior portions of the cerebellar hemispheres (crus II) and in the dorsal portion of the paraflocculus. In cat and macaque, the decrease in the density of UBCs across the intermediate cortex is more gradual than in the other species. The data indicate that the UBCs play a more prominent role in the modulation of sensorimotor transformations in carnivores and primates than in smaller mammals and should not be considered a vestigial form of neuron. In addition to the UBCs, calretinin antibody distinctly stains the following neurons in different species: granule cells and parallel fibers in all species except rabbit and cat; Golgi cells, especially in rat and macaque; Lugaro-like cells, especially in mouse, rat, and macaque; basket cells in macaque; subsets of mossy fibers in all species; and subsets of climbing fibers in all species but guinea pig. Usually, the distribution of UBCs is related to that of calretinin stained granule cells and mossy fibers.

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.

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.

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.