Jacqueline Raymond

Evidence for glutamate as a neurotransmitter in the cat vestibular nerve: radioautographic and biochemical studies

SummaryEvidence that glutamate acts as a neuro-transmitter in vestibular nerve fibers was sought (1) by electron microscope radioautographic identification of the uptake sites of [3H]-glutamic acid after incubation of slices of cat vestibular nuclei, and (2) by measuring changes in sodium-dependent high affinity glutamate uptake in nerve endings containing homogenates from normal and deafferented vestibular nuclei 8 to 11 days after unilateral vestibular nerve lesion. Electron microscopic radioautography revealed that glutamate had been taken up by numerous nerve endings projecting over the whole vestibular nuclear complex. The biochemical approach indicated that after section of the vestibular nerve, a significant decrease in high affinity glutamate uptake occurred in the vestibular nuclei, which lost their exclusively ipsilateral projection. This decrease varied from one area of the deafferented vestibular nuclei to another, reaching −58% in the lateral area of the central part corresponding to the ventral lateral vestibular nucleus and the rostral part of the descending vestibular nucleus. It is concluded that glutamate (or aspartate) is used by the vestibular nerve fibers as a neurotransmitter in the vestibular nuclei.

Chapter 3 Neurotransmitters in vestibular pathways

Labyrinthic neurotransmitters. Afferent synapses: A Glu-like component may act as the endogenous transmitter of the vestibular hair-cell-afferent-fibre synapse. The proof of the synaptic origin of this component requires the identification of the adequate Glu synaptic receptors using more specific agonists or antagonists effects. A GABA-like component may also act as a modulator of the excitability of the postsy-naptic neurons. Efferent synapses: ACh is the major efferent transmitter but the possibility remains that other additional transmitters or possibly cotransmit-ters are involved in the vestibular efferent pathways. Central vestibular neurotransmission. Neurotransmitters of the vestibular nerve: Evidence from a variety of approaches supports the possibility that Glu or Asp are involved in the vestibular nerve transmission. These experiments include: (1) the localization of AAT, an enzyme interconverting Glu and Asp, in the vestibular ganglion neurons, (2) the selective retrograde labelling of vestibular ganglion neurons after high affinity uptake of D-[ 3 H]Asp by the vestibular nerve terminals, (3) the selective labelling of vestibular nerve terminals following high-affinity uptake of [ 3 H]Glu, and (4) biochemicals measurements of the high-affinity glutamate uptake in normal and deaffe-rented vestibular nuclei. Nonlabyrinthic pathways neurotransmitters: Only a part of the neurotransmitters involved in the complex intrication of synapses devoted to each specific vestibular connection has been identified. For the commissural connections which are mainly inhibitory, GABA and Gly have been proposed from different data. In the frog, the excitatory commissural action involves an excitatory amino acid as transmitter. GABA and Glu have been proposed repectively as inhibitory and excitatory transmitters in vestibulo-ocular pathways. In the ce-rebello-vestibular interactions, one of the well-documented actions is the inhibitory GABAergic effect of the Purkinje cell terminals on the large Deiters neurons. In the vestibulospinal pathways, it has been demonstrated that some direct vestibulospinal projections are cholinergic. Nevertheless, further studies are necessary to reveal the specific role of neuroactive substances which have been recently identified both in the vestibular nuclei and in other structures involved in the pathways.

Identification of neuron subpopulations in the rat vestibular ganglion by calbindin-D 28K, calretinin and neurofilament proteins immunoreactivity

Immunocytochemical and morphometric analyses were combined to demonstrate the presence of neuron subpopulations in the rat vestibular ganglion. Monoclonal antibodies reacting with neurofilament proteins (NF), calbindin-D 28K (CaBP) and calretinin (CaR) were used. Three subpopulations were identified: (1) CaBP- and CaR-positive neurons were the largest neurons (16%) and they were also highly NF-immunoreactive; (2) exclusively NF-positive neurons; (3) unlabelled neurons, representing about two-thirds of the population.

Carbohydrates detected by lectins in the vestibular organ.

In the vestibular organ the presence of carbohydrates in the cupula and otoconia of young (6-day-old) and adult rats was investigated using fluorescent lectins. The following sugars have been identified in both young and adult rats: N-acetyl-glucosamine, galactose, mannose and fucose. In contrast, N-acetyl-galactosamine was not detected. In order to demonstrate the specificity of the reaction, control experiments were performed after preincubation of the lectin with its specific inhibitory sugar. The same sugars were identified in calcified (i.e. otoconia) and non-calcified (i.e. cupula) structures. The role of these sugars in the mineralization and fusion process of otoconia is discussed.

Selective retrograde labeling of neurons of the cat vestibular ganglion with [3H]d-aspartate

D-[2,3-3H]Aspartate [( 3H]D-Asp) was injected in the cat vestibular nuclei. Labeling patterns resulting from retrograde axonal transport by the vestibular nerve fibers were observed in the vestibular ganglion neurons and also in the nerve fibers. The selectivity of such labeling, related to the neurotransmitters specificity, is strongly indicated.

In vitro electrophysiological study of spontaneous activity in neonatal mouse vestibular ganglion neurons during development

Spontaneous discharges from vestibular ganglion neurons have been studied in mice between 0 and 10 days in freshly isolated in vitro preparations. The vestibular ganglion neurons were electrophysiologically active at birth with irregular activities. From the 3rd day. the activities could be divided into irregular and regular discharging units based on the coefficient of variation of their interspike intervals. This study provides two criteria for maturation of vestibular activities: a decreasing number of irregular units with age and an increase of the spontaneous discharge frequencies.

Different Calcium—Binding Proteins Identify Subpopulations of Vestibular Ganglion Neurons in the Rat

Vestibular neurons were studied by cytochrome oxidase (CO) histochemistry and by immunocytochemistry using antibodies against parvalbumin (PV), calbindin (CaBP), calretinin (CaR) and 160 KD neurofilament protein (NF). All the neurons present a high level of CO activity and a high content of PV. CaBP and CaR are restricted to a specific population of about 16% of the neurons and are among the largest ones. The latter neurons also have a high density of NF 160 KD protein. In conclusion the biochemical characteristics of the vestibular ganglion neurons are discussed in relation to their morphological and physiological properties.

Development of the rat efferent vestibular system on the ground and in microgravity

We investigated whether plastic changes occurred in the organization of the vestibular efferent network in the rat utricle during a 17-day episode of microgravity, from postnatal (PN) day 8 to PN23, and on return to earth on PN25. We also determined the normal pattern of efferent development from birth to PN25. Immunofluorescence experiments were performed with a specific biochemical marker of the efferent system, the calcitonin gene-related peptide (CGRP), and vibratome sections of the utricles were analyzed by laser scanning confocal microscopy. At birth, a few efferent fibers were detected beneath the sensory epithelium. These then massively invaded the epithelium between PN2 and PN4. At the time of launch, PN8, most fiber paths in the utricular epithelium, after following transient courses (towards the epithelial surface for example) returned to the base and were stabilized in the lower part of the epithelium, in which they established synaptic contacts with sensory cells, except at a few immature locations. The main difference between this stage (on PN8) and subsequent more mature stages was the lower density of fibers and synapses in the utricle. The maturation of the vestibular efferent system was similar in microgravity and on the ground. Thus, maturation of the efferent system between PN8 and PN23 was not sensitive to a change in gravitational environment. These results suggest that periods of microgravity at earlier stages are required to identify critical periods in peripheral vestibular system development.

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH. In cochlear and vestibular ganglia, calretinin mRNA was localized in subpopulations of neurons with patterns of distribution similar to those shown by immunocytochemistry. By contrast, the observations in the sensory epithelia differed with the two techniques, ISH revealing less positive structures than immunocytochemistry. Rat inner hair cells and guinea pig inner hair cells, Hensens cells and Deiters cells, which had been described strongly immunoreactive, appeared positive with radioactive but not with non-radioactive ISH. On the other hand, rat vestibular type II hair cells and guinea pig interdental cells of the spiral limbus which were faintly immunoreactive were not positive with both ISH techniques.

Quantitative autoradiographic characterization of l-[3H] glutamate binding sites in rat vestibular nuclei

SummaryQuantitative autoradiography has been used to characterize l-[3H] glutamate binding sites and to describe their distribution in frozen sections of rat vestibular nuclei. Scatchard plots and Hill coefficients of glutamate binding suggest that glutamate interacts with a single population of sites having a KD of about 126 nM and a capacity of 2.5 pmol/mg of protein. Although the level of glutamate binding was not very high compared to the highest levels described for some other brain regions, it was nonetheless substantial. The sites were distributed unevenly in the four vestibular nuclei and their distribution correlated well with the projection areas of the vestibular nerve, which has been described as a glutamate-mediated pathway. The highest numbers of glutamate binding sites were observed in the medial vestibular nuclei. This technique provides a very sensitive assay for characterizing the pharmacological subtypes of glutamate binding in the vestibular nuclei and for analyzing changes in these sites during development or after deafferentation of the vestibular nuclei.