Biomedical subjects

E Mugnaini

Publications and source records attributed to E Mugnaini.

At least 37 records · Page 2Linked to original sources

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

Parasolitary nucleus: a source of GABAergic vestibular information to the inferior olive of rat and rabbit.

At least two subnuclei of the inferior olive, the beta-nucleus, and the dorsomedial cell column (dmcc), contain vestibularly responsive neurons that receive a dense descending projection that uses gamma-aminobutyric acid (GABA) as the transmitter. In contrast to the GABAergic innervation of other olivary subnuclei, the terminal boutons that terminate on neurons in the beta-nucleus and the dorsomedial cell column remain intact after cerebellectomy, ruling out both the cerebellum and the cerebellar nuclei as afferent sources. By using both immunohistochemical as well as orthograde and retrograde tracer methods, we have identified the source of the GABAergic pathway to the beta-nucleus and dmcc in both rat and rabbit. Under physiologic recording of single olivary neurons to guide electrode placement, we injected the bidirectional tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the beta-nucleus and dmcc of the inferior olive. These injections retrogradely labeled neurons in the parasolitary nucleus (Psol) near the vestibular complex. Psol neurons were identified as GABAergic with an antibody to glutamic acid decarboxylase (GAD). In the rat, Psol neurons are small (5-7 microm in diameter) and number approximately 1,800. In the rabbit, they are slightly larger (6-9 microm in diameter) and number approximately 2,200. WGA-HRP injections in conjunction with GAD immunohistochemistry double labeled a high percentage of neurons in both the rat and rabbit Psol. Injection of the orthograde tracer Phaseolus vulgaris-leucoagglutinin into the area of the Psol revealed a projection from this region to both the beta-nucleus and dmcc. Subtotal electrolytic lesions of this division of the Psol caused a substantial reduction in GAD-positive synaptic terminals in both the ipsilateral beta-nucleus and dmcc. The location of these GABAergic neurons, bordering both the nucleus solitarius and caudal vestibular complex, emphasizes the importance of the Psol in the processing of both vestibular and autonomic information pertinent to postural control.

Animals↗

PubMed1998

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: neuronal polarity.

Cerebellar Purkinje cells (PC) display a highly distinctive form of polarity. We have cultured murine PCs from dissociated E16 cerebellar anlagen for 1 week to investigate the early stages of neuronal compartmentalization and synaptic interactions, features which are important for the establishment of neuronal polarity. To unequivocally identify the PCs we utilized light and electron microscopic immunocytochemistry with an anti-serum to the cell class-specific marker L7/pcp2 gene product. The PCs typically show a single, long axon, numerous short appendages classified as filopodia and protospines, and a small number of protodendrites. The nucleus is positioned asymmetrically in both the horizontal and vertical axes of the soma. The Golgi apparatus, coated and uncoated vesicles, and mitochondria are prominent ultrastructural features, while the endoplasmic reticulum is highly fragmented. The cell body receives rudimentary synapses on its smooth surfaces and appendages and no consistent morphological differences were detected between these elementary contacts. The axon is clearly identifiable; it emanates from either the cell body or a protodendrite, bifurcates at predominantly right angles, forms beaded collaterals, and terminates with relatively large growth cones. The varicosities of the PC axon contain pleomorphic synaptic vesicles and form rudimentary synapses primarily with the dendritic shafts of immunonegative neurons. The protodendrites are short, quickly tapering and sparsely branched; they emit numerous filopodia and immature spines and terminate with small growth cones. Rudimentary synapses are received on the proximal dendritic shafts and filopodia, and more mature synapses occur frequently on protospines. With few exceptions, PCs lie atop an astrocytic bed layer and glial processes are apposed to the various aspects of the PC body left free by the afferent axons. By contrast, PC processes are largely free of glial sheaths. We conclude that the "stellate stage" of PC development in situ is replicated rather faithfully in culture and that PCs have established polarity and have begun to form intercellular contacts by 1 week in vitro. Moreover, the PCs are already morphologically distinct from other cell types in the 1 week cultures, although they have yet to develop the differentiated features that distinguish mature PCs.

Animals↗

PubMed1998

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: dendritic differentiation, synaptic maturation, and formation of cell-class specific features.

The morphological differentiation of E16 murine Purkinje cells (PCs) in dissociated cerebellar cultures was analyzed by light and electron microscopic immunocytochemistry after 2-5 weeks in vitro (wiv), with particular emphasis on dendritic differentiation, synaptic maturation, and formation of stereotypical fine structural features. This study complements a companion paper on the features of PCs after 1 wiv. After 2 wiv, the PCs have an eccentric nucleus and the cytoplasmic organelles appear immature; the axon has a distinct initial segment and beaded axon collaterals but its boutons still contain sparse synaptic vesicles; dendrites show few bifurcations and tufts of spiny branchlets. After 3 wiv, the PCs display a centered nucleus, an extensive hypolemmal cisternal system, and stacks of up to four cisterns of granular endoplasmic reticulum; there is an increased number of dendritic bifurcations, spiny branchlets, mature spines, and axonal branches; dendritic tips still contain vesicle clusters, suggesting growth, and many synapses and afferent boutons continue to display immature features. After 4 wiv, elaborate perinucleolar coiled body rosettes, subsurface cistern-mitochondrion complexes and large stacks of granular endoplasmic reticulum finally appear within the soma; dendrites show a further increase in the numbers of bifurcations, segments and spines; most spines are synaptic and show mature features; afferent synapses are differentially distributed; PC boutons consistently display mature features and show a considerable degree of target specificity, although naked spines and reduced glial sheaths persist. After 5 wiv, PCs do not show further maturation and some dystrophic features appear. We conclude that under standard conditions and despite the disruption of normal tissue organization, PCs in dissociated cultures differentiate maximally after 4 wiv, at which stage they display many of the light and electron microscopic features that characterize mature PCs in situ. This prolonged developmental time-frame resembles that in the normal cerebellum. In view of the increasing usage of dissociated cerebellar cultures to study aspects of neuronal differentiation, synaptic activation and neuronal-glial interactions, an elucidation of the neurocytology of dissociated cerebellar cultures as presented in this study provides important clues for the interpretation of experimental data.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

Fine structure of the choroidal coat of the avian eye. Vascularization, supporting tissue and innervation.

To clarify further the functional anatomy of the avian choroid, including its innervation, 12 adult White-Leghorn chickens were studied by standard electron microscopy and immunoelectron microscopy with somatostatin antibody. The endothelial cells of the blood vessels in the choriocapillaris have fenestrations only facing the retina, while the nuclei are situated toward the sclera. In addition to tight junctions and zonulae adherentes, adjoining endothelial cells form gap junctions and dense plaques with attached filaments resembling those of smooth muscle cells. The fine structure of arteries and veins is similar to that of the vasculature described in other organs. The supporting tissue is organized in trabeculae, i.e., bridges of cellular and fibrous elements that surround and sustain blood and lymphatic vessels. This tissue consists primarily of a system of fusiform or star-shaped smooth muscle cells, connected to each other and to those in the vessels' walls through macular junctions of the adherent type, less prominent than desmosomes, and perhaps also punctiform gap junctions. Occasionally, trabecular smooth muscle cells approach the lymphatic vessels, which lack a muscular tunica, and abut their endothelium with spinous appendages. This stromal muscle tissue may act as a pump for moving the lymph. The suprachoroidea consists of large lymphatic lacunae and the multilayered membrana fusca. The elongated fuscal cells form adherent junctions, tight junctions, and perhaps also gap junctions, suggesting that the membrana fusca exerts complex functions. Nerves containing myelinated axons reach the choroid and divide into smaller branches, a few of which innervate the membrana fusca. Numerous, thin nerve branches reach both the walls of arteries and veins and the trabeculae, and synaptic terminals abut the outer muscular layers of the vessel's wall and the smooth muscle cells of the supporting tissue. Immunocytochemistry reveals the presence of numerous somatostatin-positive and somatostatin-negative axons and synaptic terminals within both trabeculae and vascular tunica media. The somatostatin-positive axons are presumed to be cholinergic axons of the choroid neurons residing in the ciliary ganglion. Taken together, these observations indicate that the avian choroid is a highly vascularized muscular sheath that may be endowed with degrees of motility and elasticity higher than those of the mammalian choroid and may therefore play an important role in compensation for experimental defocus.

Animals↗

PubMed1997

Cholinergic innervation and receptors in the cerebellum.

We have studied the source and ultrastructural characteristics of ChAT-immunoreactive fibers in the cerebellum of the rat, and the distribution of muscarinic and nicotinic receptors in the cerebellum of the rat, rabbit, cat and monkey, in order to define which of the cerebellar afferents may use ACh as a neurotransmitter, what target structures are they, and which cholinergic receptor mediate the actions of these pathways. Our data confirm and extend previous observations that cholinergic markers occur at relatively low density in the cerebellum and show not only interspecies variability, but also heterogeneity between cerebellar lobules in the same species. As previously demonstrated by Barmack et al. (1992a,b), the predominant fiber system in the cerebellum that might use ACh as a transmitter or a co-transmitter is formed by mossy fibers originating in the vestibular nuclei and innervating the nodulus and ventral uvula. Our results show that these fibers innervate both granule cells and unipolar brush cells, and that the presumed cholinergic action of these fibers most likely is mediated by nicotinic receptors. In addition to cholinergic mossy fibers, the rat cerebellum is innervated by beaded ChAT-immunoreactive fibers. We have demonstrated that these fibers originate in the pedunculopontine tegmental nucleus (PPTg), the lateral paragigantocellular nucleus (LPGi), and to a lesser extent in various raphe nuclei. In both the cerebellar cortex and the cerebellar nuclei these fibers make asymmetric synaptic junctions with small and medium-sized dendritic profiles. Both muscarinic and nicotinic receptor could mediate the action of these diffuse beaded fibers. In the cerebellar nuclei the beaded cholinergic fibers form a moderately dense network, and could in principle have a significant effect on neuronal activity. For instance, the cholinergic fibers arising in the PPTg may modulate the excitability of the cerebellonuclear neurons in relation to sleep and arousal (e.g. McCormick, 1989). Studies on the distribution of cholinergic markers in the cerebellum have proven valuable besides the issue whether cholinergic mechanism play a role in the cerebellar circuitry, because they illustrate a complexity of the cerebellar anatomy that extends beyond its regular trilaminar and foliar arrangement. For instance, AChE histochemistry has been shown to preferentially stain the borders of white matter compartments (the 'raphes', Voogd, 1967), and therefore is useful in topographical analysis of the cortico-nuclear and olivocerebellar projections (Hess and Voogd, 1986; Tan et al., 1995; Voogd et al., 1996; see Voogd and Ruigrok, 1997, this Volume). ChAT-immunoreactivity, at least in rat, appears to be a good marker to outline the morphological heterogeneity of mossy fibers, and m2-immunocytochemistry could be used to label (subpopulations of) Golgi cells, subsets of mossy fibers and, in the rabbit, a specific subset of Purkinje cells (Jaarsma et al., 1995).

Acetylcholine↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Afferent Pathways↗

PubMed1997

Fine structure of the choroidal coat of the avian eye. Lymphatic vessels.

PURPOSE: To clarify the fine structure of the avian choroid and thus help explain the mechanisms for normal and abnormal eye function and growth. METHODS: Eyes from normal chickens and from experimental chickens subjected to unilateral paracentesis were fixed either by perfusion or in situ, with or without post-fixation by microwave irradiation, and then processed for light and electron microscopic analysis. RESULTS: The avian choroid contains thin-walled lacunae, whose fine structure is identical to that of lymphatic vessels. The lacunae are much smaller toward the anterior chamber and the Schlemm's canal than posteriorly in the eye bulb. Large lacunae are situated primarily in the suprachoroidea, and their blind-ended capillary branches enter the choriocapillaris and the walls of large veins. The walls of the large veins contain villous structures that protrude into their lumina and are penetrated by thin lacunar branches and by side lines of the venous lumen. In normal chickens, the lacunae usually are devoid of blood cells. After paracentesis of the anterior eye chamber, the lacunae become filled with erythrocytes on the side that was operated on, but not on the contralateral side. CONCLUSIONS: The authors propose that the lacunae of the avian choroid represent a system of posterior short lymphatic vessels, which drain intraocular fluids directly into the eye's venous system, and that the villous structures are sites of communication between lacunae and veins. The demonstration of a choroidal lymphatic system opens new insights into the processes of fluid removal, control of intraocular pressure, and regulation of choroidal thickness in the avian eye under normal and experimental conditions.

Animals↗

PubMed1996

Distribution of descending projections from primary auditory neocortex to inferior colliculus mimics the topography of intracollicular projections.

To ascertain whether the auditory neocortex also innervates the central nucleus of the inferior colliculus (CNIC) and not only its dorsal (DCIC) and external (ECIC) cortices, the anterograde tracers Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran (BD) were injected into the primary auditory neocortex of albino rats (Te1), and labeled corticocollicular fibers were studied via light and electron microscopy. Axons from discrete regions of Te1 form two rostrocaudally oriented laminar plexuses of terminal fibers in the ipsilateral inferior colliculus (IC) and one in the contralateral IC. The first ipsilateral plexus, located in the medial half of the IC, has a dorsomedial to ventrolateral orientation, parallel to the isofrequency planes of the IC; is continuous through the CNIC and DCIC; and extends into the rostral ECIC. The second plexus is located in the deep layers of the lateral ECIC. These two plexuses meet caudally and ventrally, at the border between the CNIC and the lateral ECIC. The plexus in the contralateral IC is less dense and shorter than the two ipsilateral plexuses and is symmetric to the medial plexus. The thickness of the three plexuses is correlated with the size of the injection site, and their mediolateral and dorsoventral positions change as the injection site in Te1 is displaced rostrocaudally, with more caudal injections resulting in more dorsolateral medial plexuses and more dorsomedial lateral plexuses. Furthermore, the ventromedial border of the IC receives nontopographic, convergent projections from wide regions of rostral portions of Te1. The distribution of these corticocollicular plexuses mimics the topography of previously described intracollicular fibers. Electron microscopy shows that, in all three subdivisions of the ipsilateral IC, corticocollicular fibers form small boutons with features generally associated with excitatory transmission; i.e., they contain round synaptic vesicles and form asymmetric synapses with thin dendritic shafts and spines. These results demonstrate that the auditory corticocollicular projections innervate more extensive regions of the IC than were previously observed. Although peripheral regions receive the densest projection, the entire IC appears to be the target of corticofugal input.

Animals↗

PubMed1996

Morphological correlates of bilateral synchrony in the rat cerebellar cortex.

Simultaneous recordings of the left and right crus IIA of the cerebellar cortex in the rat have demonstrated that Purkinje cells of both sides can be activated synchronously by their climbing fibers. Because climbing fibers arise exclusively from the contralateral inferior olive (IO), this physiological finding seems to contradict the anatomy. To define the structural basis responsible for the bilateral synchrony, we examined the possibilities that bilateral common afferent inputs to the IO and interolivary connections form the underlying mechanisms. The bilaterality of the major afferents of the olivary regions that project to crus IIA was studied using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. We found that the excitatory and inhibitory projections from the spinal trigeminal nucleus and dorsolateral hump of the interposed cerebellar nucleus to the transition area between the principal olive and dorsal accessory olive were bilateral. A second possible mechanism for bilateral synchrony, which is the possibility that axons of olivary neurons provide collaterals to the contralateral side, was investigated using biotinylated dextran amine as an anterograde tracer. Labeled axons were traced and reconstructed from the principal olive and dorsal and medial accessory olive up to the entrance of the contralateral restiform body. None of these axons gave rise to collaterals. The possibility that neurons in the left and right IO are electronically coupled via dendrodendritic connections was investigated by examining the midline region of the IO. The neuropil of the left and right IO is continuous in the dorsomedial cell column. Examination of Golgi impregnations of this subdivision demonstrated that (1) many dendrites cross from one side to the other, (2) neurons close to the midline give rise to dendrites that extend into both olives, and (3) dendrites of neurons in the dorsomedial cell column frequently traverse into adjacent olivary subdivisions such as the medial accessory olive and the transition area between the principal olive and dorsal accessory olive. Sections immunostained for dendritic lamellar bodies or GABAergic terminals showed the same pattern: the neuropils of the dorsomedial cell columns on both sides form a continuum with each other as well as with the neuropil of other adjacent olivary subdivisions. Ultrastructural examination of the dorsomedial cell column demonstrated that the midline area includes many complex glomeruli that contain dendritic spines linked by gap junctions. To verify whether the complex spike synchrony observed between left and right crus IIA could indeed be mediated in part through coupled neurons in the dorsomedial cell column, we recorded simultaneously from crus IIA areas and from left and right vermal lobule IX, which receives climbing fibers from the dorsomedial cell column. In these experiments we demonstrated that the climbing fibers of all four areas, i.e., the left and right crus IIA as well as the left and right lobule IX, can fire synchronously. The present results indicate that synchronous climbing fiber activation of the left and right crus IIA in the rat can be explained by (1) bilateral inputs to the transition areas between the principal olive and dorsal accessory olive and (2) dendrodendritic electrotonic coupling between neurons of the left and right dorsomedial cell column and between neurons of the dorsomedial cell column and adjacent olivary subdivisions.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

Glutamate receptor subunits at mossy fiber-unipolar brush cell synapses: light and electron microscopic immunocytochemical study in cerebellar cortex of rat and cat.

The present study provides a survey of the immunolocalization of ionotropic glutamate receptor subunits throughout the rat and cat cerebellar cortex, with emphasis on the unipolar brush cell (UBC), a hitherto neglected cerebellar cell that is densely concentrated in the granular layer of the vestibulocerebellum and that forms giant synapses with mossy fibers. An array of nine previously characterized antibodies has been used, each of which stained a characteristic profile of cerebellar cells. The UBCs of both rat and cat were strongly immunostained by an antibody against the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor subunits, GluR2 and GluR3; were moderately immunostained by a monoclonal antibody to kainate receptor subunits, GluR5/6/7; were weakly immunostained by antibodies to NR1 subunits; and were not stained by antibodies to GluR1, GluR4, GluR6/7, KA-2, and NR2A/B. Postsynaptic densities of the giant mossy fiber-UBC synapses were GluR2/3, GluR5/6/7, and NR1 immunoreactive. The other cerebellar neurons were all immunolabeled to some extent with the GluR2/3 and NR1 antibodies. In addition, Purkinje cells were immunopositive for GluR1 and GluR5/6/7; granule cells were immunopositive for GluR5/6/7, GluR6/7, KA-2, and NR2A/B. The Golgi-Bergmann glia was densely stained by GluR1 and GluR4 antibodies, whereas astrocytes of the granular layer were stained by the GluR4 antiserum. Data provided herein may guide further electrophysiological and pharmacological studies of cerebellar cells in general and the UBCs in particular.

Afferent Pathways↗

PubMed1995

Properties of transmission at a giant glutamatergic synapse in cerebellum: the mossy fiber-unipolar brush cell synapse.

1. The synaptic activation by mossy fibers (MFs) of unipolar brush cells (UBCs) in the vestibular cerebellum (nodulus and uvula) was examined using patch-clamp recording methods in thin, rat cerebellar slices with Lucifer yellow-filled pipettes for subsequent fluorescence microscopic verification of the cell morphology. 2. UBCs were distinguished from adjacent granule cells in thin cerebellar slices in the uvula and nodulus regions by their larger soma diameters and short dendritic brush, greater whole-cell capacitance, and a prolonged, biphasic excitatory postsynaptic current (EPSC) to stimulation of MFs. 3. Thin-section transmission electron micrographs of the MF-UBC synapse displayed an unusually extensive area of synaptic apposition estimated to measure 12-40 microns2. The majority of UBCs was innervated by a single MF. At high magnification, individual clusters of presynaptic vesicles could be discerned, separated by regions of presynaptic membrane lacking vesicles, but apposed to continuous regions of postsynaptic density. Thus, after release, transmitter diffusion from the synaptic cleft must traverse considerable stretches of postsynaptic membrane before escape into extracellular space. In contrast, MF-granule cell synapses in these cerebellar regions resembled glutamate synapses in other brain regions in that the total synaptic area measured < or = 4 microns2. These synaptic junctions were flanked by short stretches of unspecialized plasma membrane, providing a short (0.5 micron) diffusional path from the site of neurotransmitter release to a branch point of the extracellular space. 4. The MF-evoked EPSC in UBCs was composed of a fast (10-90% rise time: 0.70 ms) and slow (10-90% rise time: 395 ms; 10-90% decay time: 3.1 s) component. The fast component was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and displayed linear current-voltage (I-V) relations in the presence or absence of external magnesium. 5. The slow EPSC was also mediated by glutamate receptors, but in most neurons both AMPA/KA and N-methyl-D-aspartate (NMDA) receptors contributed to the slow EPSC, with the contribution of NMDA receptors predominating in the majority of cells. Consequently, although all cells displayed linear I-V relations in Mg(2+)-free saline, cells in which the slow EPSC was predominently mediated by NMDA receptors exhibited voltage-dependent rectification in the presence of external Mg2+ (1 mM). 6. With increasing postnatal age (10-30 d), the contribution made to the slow EPSC by NMDA receptors declined, with a reciprocal increase in the contribution being made by AMPA/KA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

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