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Sublineage diversification of specific neural cell classes occurs in complex as well as simply organized regions of the central and peripheral nervous systems; the significance of the phenomenon, however, remains insufficiently understood. The unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons that occur at high density in vestibulocerebellum. As they are classified into subsets that differ in chemical phenotypes, intrinsic properties, and lobular distribution, they represent a valuable neuronal model to study subclass diversification. In this study, we show that cerebellar UBCs of adult rats and mice form two subclasses-type I and type II UBCs-defined by somatodendritic expression of calretinin (CR), mGluR1α, phospholipases PLCβ1 and PLCβ4, and diacylglycerol kinase-beta (DGKβ). We demonstrate that PLCβ1 is associated only with the CR(+) type I UBCs, while PLCβ4 and DGKβ are exclusively present in mGluR1α(+) type II UBCs. Notably, all PLCβ4(+) UBCs, representing about 2/3 of entire UBC population, also express mGluR1α. Furthermore, our data show that the sum of CR(+) type I UBCs and mGluR1α(+) type II UBCs accounts for the entire UBC class identified with Tbr2 immunolabeling. The two UBC subtypes also show a very different albeit somehow overlapping topographical distribution as illustrated by detailed cerebellar maps in this study. Our data not only complement and extend the previous knowledge on the diversity and subclass specificity of the chemical phenotypes within the UBC population, but also provide a new angle to the understanding of the signaling networks in type I and type II UBCs.

Unipolar brush cells (UBC) are small, glutamatergic neurons residing in the granular layer of the cerebellar cortex and the granule cell domain of the cochlear nuclear complex. Recent studies indicate that this neuronal class consists of three or more subsets characterized by distinct chemical phenotypes, as well as by intrinsic properties that may shape their synaptic responses and firing patterns. Yet, all UBCs have a unique morphology, as both the dendritic brush and the large endings of the axonal branches participate in the formation of glomeruli. Although UBCs and granule cells may share the same excitatory and inhibitory inputs, the two cell types are distinctively differentiated. Typically, whereas the granule cell has 4-5 dendrites that are innervated by different mossy fibers, and an axon that divides only once to form parallel fibers after ascending to the molecular layer, the UBC has but one short dendrite whose brush engages in synaptic contact with a single mossy fiber terminal, and an axon that branches locally in the granular layer; branches of UBC axons form a non-canonical, cortex-intrinsic category of mossy fibers synapsing with granule cells and other UBCs. This is thought to generate a feed-forward amplification of single mossy fiber afferent signals that would reach the overlying Purkinje cells via ascending granule cell axons and their parallel fibers. In sharp contrast to other classes of cerebellar neurons, UBCs are not distributed homogeneously across cerebellar lobules, and subsets of UBCs also show different, albeit overlapping, distributions. UBCs are conspicuously rare in the expansive lateral cerebellar areas targeted by the cortico-ponto-cerebellar pathway, while they are a constant component of the vermis and the flocculonodular lobe. The presence of UBCs in cerebellar regions involved in the sensorimotor processes that regulate body, head and eye position, as well as in regions of the cochlear nucleus that process sensorimotor information suggests a key role in these critical functions; it also invites further efforts to clarify the cellular biology of the UBCs and their specific functions in the neuronal microcircuits in which they are embedded. High density of UBCs in specific regions of the cerebellar cortex is a feature largely conserved across mammals and suggests an involvement of these neurons in fundamental aspects of the input/output organization as well as in clinical manifestation of focal cerebellar disease., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The espins are Ca(2+)-resistant actin-bundling proteins that are enriched in hair cell stereocilia and sensory cell microvilli. Here, we report a novel localization of espins to a large proportion of rat type I spiral ganglion neurons (SGNs) and their projections to the cochlear nucleus (CN). Moreover, we show that a fraction of these espins is in the nucleus of SGNs owing to the presence of splice-isoforms that contain a functional nuclear localization signal (NLS). Espin antibody labeled approximately 83% of type I SGNs, and the labeling intensity increased dramatically during early postnatal development. Type II SGNs and vestibular ganglion neurons were unlabeled. In the CN, espin-positive auditory nerve fibers showed a projection pattern typical of type I SGNs, with intense labeling in the nerve root region and posteroventral CN (PVCN). The anteroventral CN (AVCN) showed moderate labeling, whereas the dorsal CN showed weak labeling that was restricted to the deep layer. Espin-positive synaptic terminals were enriched around nerve root neurons and octopus cells in the PVCN and were also found on globular bushy cells and multipolar neurons in the PVCN and AVCN. SGNs expressed multiple espin transcripts and proteins, including splice-isoforms that contain a nonapeptide, which is rich in positively charged amino acids and creates a bipartite NLS. The nonapeptide was necessary to target espin isoforms to the nucleus and was sufficient to target an unrelated protein to the nucleus when joined with the upstream di-arginine-containing octapeptide. The presence of cytoplasmic and nuclear espins in SGNs suggests additional roles for espins in auditory neuroscience., (Copyright 2008 Wiley-Liss, Inc.)

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.

The espin actin-bundling proteins, which are the target of deafness mutations, are present in the parallel actin bundles of stereocilia and microvilli and appear to increase their steady-state length. Here, we report a new activity of the espins, one that depends on their enigmatic WH2 domain: the ability to assemble a large actin bundle when targeted to a specific subcellular location. This activity was observed for wild-type espins targeted to the centrosome in transfected neuronal cells and for jerker espins targeted to the nucleolus in a wide variety of transfected cells as a result of the frameshifted peptide introduced into the espin C-terminus by the jerker deafness mutation. This activity, which appears specific to espins, requires two espin F-actin-binding sites and the actin-monomer-binding activity of the espin WH2 domain, but can be mimicked by adding a WH2 domain to an unrelated actin-bundling protein, villin. Espins do not activate the Arp2/3 complex in vitro, and bundle assembly is not indicative of in-vitro nucleation activity. Our results suggest a novel way to build actin bundles at specific sites in cells.

Immediate early gene expression in the cerebellar vermis of cats and squirrel monkeys was stimulated by prolonged whole body rotations. Continuous, earth-horizontal axis rotations that excited only otoliths or high velocity vertical axis rotations that excited only semicircular canals resulted in c-fos immunoreactive nuclei concentrated in the granular layer of lobules X and ventral IX (the nodulus and ventral uvula), which represent the medial parts of the vestibulo-cerebellum. Large clusters of labeled nuclei consisting mainly of granule cells and calretinin-positive unipolar brush cells were present in the granular layer, whereas Purkinje cell nuclei were unlabeled, and labeled basket and stellate cell nuclei were scattered in the molecular layer. In other vermal lobules there was a significant but less dense label than in the nodulus and ventral uvula. Generally, the extent of c-fos labeling of molecular layer interneurons was in relation to nuclear labeling of granular layer neurons: labeling of both basket and stellate cells accompanied nuclear labeling of neurons throughout the depth of the granular layer, whereas only stellate cells were labeled when nuclear labeling was restricted to the superficial granular layer. Yaw horizontal or roll vertical rotations each stimulated c-fos expression in the cat medial vestibulo-cerebellum to approximately the same extent. Low-velocity rotations resulted in much less c-fos expression. Similar, albeit less intense, patterns of c-fos activation were observed in monkeys. Concentrated c-fos expression in the medial vestibulo-cerebellum after exposure to a strong head velocity signal that could originate from either otolith or canal excitation suggests that granule and unipolar brush cells participate in a neuronal network for estimating head velocity, irrespective of the signal source.

Bromodeoxyuridine (BrdU) is broadly used in neuroscience to study embryonic development and adult neurogenesis. The potential toxicity of this halogenated pyrimidine analogue is frequently neglected. In this study, we administered BrdU in small doses by the progressively delayed cumulative labeling method to immunocytochemically tag different cerebellar cell types with antibodies to specific markers and BrdU in the same section. The well-known structure of the cerebellum made it possible to ascertain several toxic effects of the treatment. Time-pregnant rats were given five or six injections of 5 or 6 mg of BrdU ( approximately 12-20 mg/kg) at 8-hour intervals over 2 successive days between day 11 and 21 of pregnancy (E11-E12 to E20-E21), and the adult progeny was processed by immunocytochemistry. We demonstrate that this treatment effectively labeled distinct cerebellar cell populations but produced striking defects in the proliferation, migration, and settling of the Purkinje cells; reduced the size of the cerebellar cortex and nuclei; produced defects in the patterning of foliation; and also affected litter size, body weight, and mortality of the offspring. The observed toxic effects were consistent within individual treatment groups but varied between different treatment groups. Treatment with BrdU at the peak of neurogenesis of cerebellar projection neurons (E14) produced the most severe malformations. We observed no overt effects on the timing of neurogenesis for cerebellar neurons and glia across experimental groups. In conclusion, BrdU is a useful tool to study neural development, but its cytotoxicity represents a serious pitfall particularly when multiple doses are used to label cells., (Copyright 2004 Wiley-Liss, Inc.)

The vertebrate olfactory system has long been an attractive model for studying neuronal regeneration and adaptive plasticity due to the continuous neurogenesis and synaptic remodelling throughout adult life in primary and secondary olfactory centres, its precisely ordered synaptic network and accessibility for manipulation. After homotopic transplantation of fetal olfactory bulbs in bulbectomized neonatal rodents, newly regenerated olfactory neurons form glomeruli within the graft, and the efferent mitral/tufted cells of the transplant innervate the host brain, terminating in higher olfactory centres. However, the synaptic connections of the transplanted relay neurons within the graft and/or host's olfactory centres could not be characterized mainly because of lack of suitable cell-specific markers for these neurons. In this study, we have used olfactory bulbs from transgenic fetuses, in which the majority of the mitral/tufted cells express the bacterial enzyme beta-galactosidase, for homotopic olfactory bulb transplantation following complete unilateral bulbectomy. In the transplants, the cell bodies and terminals of the donor mitral/tufted cells were identified by beta-galactosidase histochemistry and immunocytochemistry at both light and electron microscope levels. We demonstrate that transplanted relay neurons re-establish specific synaptic connections with host neurons of the periphery, source of the primary signal and central nervous system, thereby providing the basis for a functional recovery in the lesioned olfactory system.

Unipolar brush cells (UBCs) are a class of excitatory, glutamatergic interneurons occurring at high density in the granular layer of the vestibulo-cerebellum. UBCs are intermediate in size between granule cells, which in rat originate postnatally from precursors in the external granular layer, and Golgi cells, which are generated prenatally and postnatally from precursors in the ventricular zone that continue to divide while they migrate toward the cortex. The origin of the UBCs is still poorly understood. In this study, we set forth to ascertain the possible prenatal origin of UBCs, taking advantage of the immunocytochemical 5-bromo-2′-deoxyuridine (BrdU) method to label dividing cells in combination with antisera to cell population markers, that distinguish UBCs from granule and Golgi cells. Pregnant rat dams received six i.p. injections of BrdU (total 36 mg/animal) over 2 successive days at different stages of prenatal development (embryonic day [E]14/15–E20/21). Adult offspring were analyzed for histology. Using antibodies against the ionotropic glutamate receptor GluR2 and the calcium binding protein calretinin we found two populations of UBCs. A subset of about 30% of UBCs was calretinin and GluR2 positive, while the majority of the UBCs were calretinin negative and GluR2 positive. Results indicate that UBCs originate from precursors proliferating between E16 and E21. However, UBCs defined by calretinin immunoreactivity were primarily born in a narrow time window at E17–18. UBCs immunostained with antiserum to GluR2, but not labeled with calretinin were generated later, from E19 to E21. Our data also indicate that a part of GluR2 positive UBCs are born around and after E22. The subset of later born, calretinin negative UBCs may coincide with the pale cells, a group of cerebellar interneurons previously identified by [3H]thymidine labeling. [Copyright &y& Elsevier]