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.

Espins and the actin cytoskeleton of hair cell stereocilia and sensory cell microvilli.

The espins are novel actin-bundling proteins that are produced in multiple isoforms from a single gene. They are present at high concentration in the parallel actin bundle of hair cell stereocilia and are the target of deafness mutations in mice and humans. Espins are also enriched in the microvilli of taste receptor cells, solitary chemoreceptor cells, vomeronasal sensory neurons and Merkel cells, suggesting that espins play important roles in the microvillar projections of vertebrate sensory cells. Espins are potent actin-bundling proteins that are not inhibited by Ca2+. In cells, they efficiently elongate parallel actin bundles and, thereby, help determine the steadystate length of microvilli and stereocilia. Espins bind actin monomer via their WH2 domain and can assemble actin bundles in cells. Certain espin isoforms can also bind phosphatidylinositol 4,5-bisphosphate, profilins or SH3 proteins. These biological activities distinguish espins from other actin-bundling proteins and may make them well-suited to sensory cells.

Time of origin of unipolar brush cells in the rat cerebellum as observed by prenatal bromodeoxyuridine labeling.

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.

The deaf jerker mouse has a mutation in the gene encoding the espin actin-bundling proteins of hair cell stereocilia and lacks espins.

The espins are actin-bundling proteins of brush border microvilli and Sertoli cell-spermatid junctions. We have determined that espins are also present in hair cell stereocilia and have uncovered a connection between the espin gene and jerker, a recessive mutation that causes hair cell degeneration, deafness, and vestibular dysfunction. The espin gene maps to the same region of mouse chromosome 4 as jerker. The tissues of jerker mice do not accumulate espin proteins but contain normal levels of espin mRNAs. The espin gene of jerker mice has a frameshift mutation that affects the espin C-terminal actin-bundling module. These data suggest that jerker mice are, in effect, espin null and that the jerker phenotype results from a mutation in the espin gene.

Domain-restricted expression of two glutamic acid decarboxylase genes in midgestation mouse embryos.

Glutamic acid decarboxylase (GAD) is the biosynthetic enzyme for gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. In addition to the adult CNS, GABA and GAD also have been detected in embryos, although their precise localization and specific functions in embryonic development have not been elucidated. In this paper, the authors studied the cellular distribution of two GAD isoforms, GAD65 and GAD67, in midgestation mouse embryos by in situ hybridization histochemistry. With few exceptions, it was found that GAD65 and GAD67 mRNAs are localized in overlapping cellular domains of the embryonic CNS that later develop into regions with a strong GABAergic contribution. The GAD-expressing cells are situated in the differentiating zone of the embryonic day 10.5 (E10.5) through E11.5 CNS and in the subventricular zone and the mantle zone of the E12.5 CNS, which suggests that they are committed neuronal precursors. By using a specific serum for GABA, a similar pattern of distribution was obtained, indicating that GAD mRNAs are translated efficiently into enzymatically active GAD, which produces embryonic GABA. The expression domains of GAD overlap with those of genes that are known to be involved in the patterning of the embryonic CNS. The two GAD mRNAs also are detected outside of the embryonic CNS in various cell types, mainly those of placodal and neural crest origin. This pattern of expression is consistent with the notion that GAD and its product, GABA, play a signaling role during development.

Ataxia and abnormal cerebellar microorganization in mice with ablated contactin gene expression.

Axon guidance and target recognition depend on neuronal cell surface receptors that recognize and elicit selective growth cone responses to guidance cues in the environment. Contactin, a cell adhesion/recognition molecule of the immunoglobulin gene superfamily, regulates axon growth and fasciculation in vitro, but its role in vivo is unknown. To assess its function in the developing nervous system, we have ablated contactin gene expression in mice. Contactin-/- mutants displayed a severe ataxic phenotype consistent with defects in the cerebellum and survived only until postnatal day 18. Analysis of the contactin-/- mutant cerebellum revealed defects in granule cell axon guidance and in dendritic projections from granule and Golgi cells. These results demonstrate that contactin controls axonal and dendritic interactions of cerebellar interneurons and contributes to cerebellar microorganization.

Using GADlacZ transgenic mice as a marker system for homotopic transplantation.

Olfactory bulb (OB) transplantation is a well characterized model that has been widely used for studying neuronal plasticity and regeneration [G. Sekerková, Z. Katarova, E. Mugnaini, F. Joó, J.R. Wolff, S. Prodan, G. Szabó, Intrinsically labeled relay neurons of homotopic olfactory bulb transplants establish proper afferent and afferent synaptic connections with host neurons, Neuroscience, 80 (1997) 973-979 [10]; G. Sekerková, Z. Malatová, J. Orendácová, T. Zigová, Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study, Restor. Neurol. Neurosci., 6 (1993) 1-8 [11]; E. Raceková, I. Vanický, T. Zigová, Correlation of functional alteration with lesion extent after olfactory bulbectomy in rats, Int. J. Neurosci., 79 (1994) 13-20 [12]; T. Zigová, P.P.C. Graziadei, A.G. Monti Graziadei, Olfactory bulb transplantation into the olfactory bulb of neonatal rats: an autoradiographic study, Brain Res., 539 (1991) 51-58 [13]]. In previous studies, the OB grafts have been routinely labeled by tritiated thymidine [S.M. Onifer, L.A. White, S.R. Whittemore, V.R. Holets, In vitro labelling strategies for identifying primary neural tissue and neuronal cell line after transplantation in the CNS, Cell Transplant., 2 (1993) 131-149 [7]; [13]] allowing distinction of graft from the surrounding tissue by the presence of silver grains over the cell nuclei of the transplant. However, this approach has some disadvantages, namely: partial or insufficient labeling of a defined neuronal subclasses due to the length of the period of their generation, variation in the number of labeled cells due to differences in the gestation stage between individual embryos at the time of i.p. injection of tritiated thymidine, inability to follow the dendritic arborization and axonal outgrowth of the transplanted neurons or to detect directly their actual synaptic contacts, and finally, the need to work with radioactive isotopes. In this paper, we describe an alternative approach, in which the donor OBs in a homotopic OB transplantation were derived from transgenic mice carrying the bacterial gene lacZ under control from the regulatory region of GAD67 gene. In these mice, beta-galactosidase (beta-gal), encoded by lacZ is stably, ectopically expressed in the vast majority of mitral/tufted (M/T) cells of the OB and served as their intrinsic cellular marker in the OB transplant. By using a simple histochemical reaction for beta-gal or immunocytochemistry with anti-beta-gal antibody, we could detect the cell bodies and processes of the donor M/T cells and their synaptic contacts with host neurons after long-term survival using both light and electron microscopy. Given the great number of existing transgenic mouse lines that express in the nervous system, this approach may have an even wider application in neural transplantation.

Regulation of cell-type specific expression of lacZ by the 5'-flanking region of mouse GAD67 gene in the central nervous system of transgenic mice.

The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by beta-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5'-upstream regulatory region of GAD67 and E. coli lacZ. No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers. In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites. The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5'-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region. Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects. Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system.

The intermediate filament protein peripherin is a marker for cerebellar climbing fibres.

Immunocytochemical staining with antibodies to the class III intermediate filament protein peripherin reveals discrete subpopulations of neurons and nerve fibres throughout the rat central nervous system. Some of these fibres enter the cerebellar granular and molecular layers. Here we use light and electron microscopic immunocytochemistry and confocal fluorescence microscopy to identify the peripherin positive fibres in the molecular layer of the cerebella of various mammals. (1) The peripherin positive fibres in the molecular layer have morphological attributes of climbing fibres, and peripherin positive fibres are also detected in the olivo-cerebellar tract. Furthermore peripherin positive neurons can be seen in the inferior olive, from which climbing fibres originate. (2) The peripherin positive molecular layer fibres rapidly degenerate in rats treated with 3-acetylpyridine (3-AP), a reagent which destroys neurons in the inferior olive, and the time course of degeneration of these mirrors that previously described for 3-AP induced destruction of climbing fibres. (3) Cerebella of other mammal species tested (mouse, rabbit, pig, cow and human) revealed a similar peripherin staining pattern in the cerebellum, including fibres in the molecular layer with the morphology of climbing fibres. (4) We also noted peripherin positive spinocerebellar and vestibulocerebellar mossy fibres in the cerebellar granular layer of folia known to receive these inputs. (5) A subset of perivascular nerve fibres are also peripherin positive. These results show that peripherin is a useful marker for mammalian cerebellar climbing fibres, and that a subset of morphologically distinct cerebellar mossy fibres are also peripherin positive.

Beta-galactosidase-labelled relay neurons of homotopic olfactory bulb transplants establish proper afferent and efferent synaptic connections with host neurons.

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.

Visualization of beta-galactosidase by enzyme and immunohistochemistry in the olfactory bulb of transgenic mice carrying the LacZ transgene.

In the olfactory bulb (OB) of a transgenic mouse line that carries the bacterial LacZ gene under the control of the 5'-regulatory region of the GAD67 gene, expression of the beta-galactosidase was confined almost exclusively to the non-GABAergic mitral and tufted cells. By light microscopy, enzyme histochemistry showed strong staining in the cell bodies and faint diffuse staining in the axons and dendrites. With immunohistochemistry for beta-galactosidase the entire cytoplasm, including the axons and dendrites, was strongly stained. By electron microscopy, beta-galactosidase enzyme histochemistry resulted in a submicroscopic reaction product that was diffusely distributed in the cytoplasm of neurons. In addition, large deposits of the reaction product were also seen attached to the cytoplasmic side of the membranes. In contrast, when the intracellular localization of beta-galactosidase was determined by immunohistochemistry, homogeneous cytoplasmic staining was obtained that filled the entire cytoplasm including the terminal dendrites and fine axons. Therefore, synaptic contacts of the beta-galactosidase-positive output neurons with other beta-galactosidase-negative neuronal cells were readily recognized in the OB. As we demonstrated, transgenic mouse lines expressing the LacZ reporter gene in a well-defined neuronal subpopulation can be used to follow beta-galactosidase-positive neurons and to directly identify their synaptic connections.

Neovascularization of the dorsal root ganglia transplanted into the olfactory bulb of neonatal rats.

Dorsal root ganglia (DRG), whose vessels are permeable to blood-born proteins, were syngenically transplanted into 2-5 days old Wistar albino rats after partial unilateral bulbectomy. Our aim was to follow survival and vascularization of the DRG, transplanted into a new environment, the developing olfactory bulb (OB). Three months after grafting the DRG graft was found fused with the spared portion of the OB. Only a subpopulation of the transplanted neurons survived the transplantation. Cholinesterase histochemistry showed BuChE positive vessels of host origin around the surviving DRG neurons. The majority of the vessels was impermeable to i.v. applied fluorescent dyes (Evans blue and lucifer yellow) and only few (1-3 vessel profiles/section) of them were labeled at the graft surface. By lanthanum nitrate tracing at the ultrastructural level, tight junctions were seen in the majority of the blood vessels of the graft. Our study shows that during the neovascularization the transplanted DRG is invaded by the host-derived blood vessels which possess blood brain barrier properties-they are impermeable to applied micro-and macromolecules. The newly formed/reestablished circulation appeared to be sufficient for maintaining a subpopulation of the transplanted sensory neurons.

Distribution of growth associated protein (B-50/GAP-43) and glial fibrillary acidic protein (GFAP) immunoreactivity in rat homotopic olfactory bulb transplants.

Recent studies on olfactory bulb homotopic transplantation after partial or subtotal bulb ablation have shown that regenerated olfactory axon are able to form glomeruli-like structures either in the transplant or in the spared olfactory bulb. To investigate the maturation of olfactory axons and their terminal connections in the transplant and remnant olfactory bulb we performed immunohistochemistry utilizing as markers B-50/GAP43 for neurite outgrowth and glial fibrillary acidic protein (GFAP) for the glial response to the surgery. Radioactively prelabeled olfactory bulbs (E 18) were homotopically transplanted in unilaterally bulbectomized neonatal rats (P6). Two to four months after transplantation in the partially bulbectomized rats the laminar organization of the olfactory bulb remnant depended on the extent of lesion. The transplant was disorganized showing irregularly distributed glomeruli. In a few cases pseudo-laminar organization was observed resembling that of the normal olfactory bulb. In 2 month-old transplants, outgrowing axons had a newly formed glomeruli displayed prominent B-50/GAP43 immunoreactivity. Four months after the operation B-50/GAP43 immunoreactivity was still present in the regrowing axons and some glomeruli in the transplant were B-50/GAP43 positive, while other glomeruli revealed a patchy pattern. The same B-50/GAP43 immunostained patchy structure were present in remnants of the lesioned olfactory bulb. Distinct increase of GFAP activity was observed along the olfactory axons and in the glomeruli-like structures. The persisting B-50/GAP43 immunoreactivity in the glomeruli of the transplant and in the remnants of lesioned olfactory bulb suggests that maturation of the newly formed glomeruli was delayed in comparison to the intact control olfactory bulb. Furthermore, the decrease of B-50/GAP43 immunoreactivity and the patchy distribution may indicate that synaptogenesis occurred in the glomeruli of the transplant, in spite of the altered topography. Together with previous findings on reinnervation of the piriform cortex from projection neurons situated in olfactory bulb transplants, our immunohistochemical data support the notion that olfactory bulb transplantation may reestablish to some degree the neuronal circuits affected by the experimental surgery.

Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study.

We have transplanted encapsulated dorsal root ganglia (DRG) from adult Wistar albino rats unilaterally into partially bulbectomized (n = 20) neonatal (P3-5) rats of the same strain. Three months postoperatively the animals were perfused and their brains processed by direct thiocholine method for cholinesterases (Ch), specific acetylcholinesterase (AChE) and nonspecific butyrylcholinesterase (BuChE) or stained by Cresyl violet. Selected sections were immunohistochemically stained for olfactory marker protein (OMP). In 17 cases we found surviving transplanted DRG. Fifteen transplants were well integrated with the spared portion of the olfactory bulb (OB) as clearly demonstrated by AChE and BuChE histochemistry, while two did not integrate. Regenerated OMP positive olfactory axons originating from neuroepithelium and AChE positive fibres from OB remnant penetrated into the transplants. In one case, fibers connected with BuChE positive Schwann cells grew from the transplanted DRG into the host OB. Individual sensory neurons of the transplants revealed variable intensity of the AChE staining, thus resembling the pattern of AChE activity in normal DRG. BuChE activity was mostly localized on the surface of sensory neurons in the ring of satellite cells. Some BuChE positive blood vessels penetrated into the DRG, and were observed around sensory neurons. The results showed a considerable viability and adaptability of the sensory neurons in the new environment after a long-term transplantation.