Glutamate-like immunoreactivity in the peripheral vestibular system of mammals

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Hearing Research, 46 (1990) 261-270 Elsevier

HEARES 01384

Glutamate-like

immunoreactivity in the peripheral vestibular system of mammals

D. Dememes ‘, R.J. Wenthold 2, B. Moniot ’ INSERM

U-254, Luboratoire de Neurophysiologie

Sensorielle, USTL, Montpellier, NIH, Bethesdq MD, U.S.A.

’

and A. Sans

1

France and 2 Laboratory of Neuro-Otolaryngologv,

(Received 27 September 1989; accepted 28 January 1990)

Using a specific antibody raised against glutamate (Glu) conjugated to bovine serum albumin with glutaraldehyde, the distribution of Glu-like immunoreactivity was studied by postembedding staining in semithin sections of nonosmicated or osmicated tissue through the vestibular sensory epithelia and ganglia of different mammalian species (mouse, rat and cat). Strong immunoreactive staining was found in all ganglion neurons and their peripheral and central nerve processes as well as in the two types of sensory hair cells whereas, in contrast, supporting cells were devoid of immunoreactivity. Glu-like immunoreactivity found in vestibular fibers and ganglion neurons, is in good agreement with the proposition of glutamate as the neurotransmitter involved in vestibular nerve transmission. In sensory hair cells, glutamate, apart from its metabolic function, may play a role in synaptic transmission between the sensory cells and the vestibular afferent fibers. Glutamate; Immunocytochemistry;

Mouse; Rat: Cat; Vestibule

Introduction Both morphological and physiological investigations in the vestibule have demonstrated that synaptic transmission between sensory hair cells and primary afferent nerve fibers is chemically mediated (Wersall, 1956; Ishii et al., 1971; Rossi et al., 1977; Schessel and Highstein, 1981). However, the identity of the afferent transmitter(s) released by hair cells has not been clearly defined. Many substances (Klinke, 1986) have been proposed, especially glutamate (Glu), which is thought to be a major excitatory neurotransmitter in the central nervous system (Cotman et al., 1981; Watkins and Evans, 1981; Fagg and Foster, 1983; Fonnum, 1984). Numerous electrophysiological experiments suggest Glu, or a related substance, as the afferent neurotransmitter of this peripheral synapse in the cochlea (Godfrey et al., 1976;

Correspondence to: D. Demtmes, INSERM U-254, Laboratoire de Neurophysiologie Sensorielle, C.P. 089, U.S.T.L., Place E.Bataillon, 34095 Montpelher, Cedex 5, France. 0378-5955/90/$03.50

Klinke and Oertel, 1977b; Bobbin and Thompson, 1978; Bobbin, 1979; Comis and Leng, 1979; Eybalin and Eujol, 1983; Ryan and Schwartz, 1984; Pujol et al., 1985; Altschuler et al., 1988), vestibule, and other acousticolateralis receptors (Bledsoe et al., 1980; Bobbin et al., 1981; Annoni et al., 1984; Dechesne et al., 1984; Valli et al., 1985; Co&ran et al., 1987; Drescher et al., 1987; Guth et al., 1988). All the previous studies have concerned the labyrinth of lower vertebrates except for a pharmacological study of Dechesne et al. (1984), in the cat vestibule; these authors reported a depolarization of primary afferent fibers after application of Glu in the endolymphatic space and concluded that the Glu action could involve Glu receptors in the vestibular end organ. The only receptors demonstrated until now are the kainic acid receptors, identified by immunocytochemistry in frog vestibule (Dechesne et al., 1988b). Furthermore, we previously demonstrated by a variety of morphological and biochemical approaches the possibility that Glu or aspartate (Asp) are involved in the cat vestibular nerve transmission (Raymond et al., 1988). For these

0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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reasons it was of interest to investigate Glu localization in the mammalian labyrinth insofar as it is the most plausible neurotransmitter. Since the recent development of antisera recognizing amino acids fixed in tissue sections, it is now possible to localize specific pathways and neurons that may use them as neurotransmitters (Storm-Mathisen et al., 1983; Ottersen and Storm-Mathisen, 1984). In the present study, we performed an immunocytochemical investigation of the distribution of Glu by postembedding immunostaining procedures in the vestibular receptor epithelia and ganglia of different mammalian vestibules using affinity purified antibodies against Glu. Materials and Methods Young mammalian adult animals (mice, rats and cats) were transcardially perfused under deep anesthesia with a variety of fixatives in 0.1 M phosphate buffer containing different concentrations of paraformaldehyde (l%-4%) and glutaraldehyde (0.2%-3.5%). Two adult cat vestibular epithelia were fixed by the double fixation procedure of Favre and Sans (1983), i.e. simultaneous intra-aortic perfusion and in situ immersion with a mixture of fixative solution consisting of 1’62% formaldehyde (freshly prepared from paraformaldehyde), 1%2% glutaraldehyde, and 05% to 1% dimethylsulfoxide (DMSO) in 0.1 M phosphate buffer (pH 7.4). The vestibular receptors and ganglia were dissected out and postfixed for lh in the same fixative. Except for some samples from cats, they were nonosmicated and classically processed for embedding in epon. Two 14day-old mice were killed by decapitation and the labyrinths were immediately dissected in the fixative (glutaraldehyde 1’63.5 %). Semithin sections (0.5-l pm) were cut and mounted on gelatincoated slides. Removal of the epoxy resin was performed as described by Maxwell (1978), with a solution of 2 g of KOH in 10 ml absolute methyl alcohol and 5 ml propylene oxide. After rinsing, sections were treated for immunoperoxidase by sequential incubations in: (1) 1% normal swine serum and 0.02% Triton x 100 in phosphatebuffered saline (PBS) for 1 h; (2) Glu antiserum diluted 1: 1000 in PBS containing 1% normal swine serum for 15 h at room temperature; (3) swine

anti-rabbit IgG (Dakopatts, 1: 50); (4) rabbit peroxidase-antiperoxidase complex (Biosys, 1 : 100); and (5) diaminobenzidine/hydrogen peroxide. Briefly, antibodies were made in rabbits against Glu conjugated to bovine serum albumin (BSA) with glutaraldehyde. Antibodies were purified by applying antiserum to a column of Glu conjugated to ovalbumin with glutaraldehyde and covalently attached to cyanogen bromide-activated Sepharose. Bound antibodies were eluted with 0.1 M acetic acid, neutralized, and applied to a column of aspartate conjugated to BSA with glutaraldehyde and attached to cyanogen bromide-activated Sepharose. The unretained fraction was used for immunocytochemical analyses. The specificity of the antibodies was tested using conjugates of several amino acids applied to nitrocellulose paper. These studies showed the antibodies to be highly selective for Glu conjugates (Montero and Wenthold, 1989). Controls were carried out by replacing antibodies with non-immune rabbit IgG. Other immunostaining experiments were also performed as supplementary controls with a different but similar antibody. Anti-y-aminobutyric acid (GABA) antibody (from Biosoft, France) made the same way, i.e. conjugated to carrier with glutaraldehyde was tested. Results Glu-like immunoreactive staining was observed in the vestibular hair cells, the vestibular ganglion cells and their peripheral and central nerve processes. Variations in immunostaining intensity were noted and depended on the fixation procedure and the concentration of glutaraldehyde in the fixative solution. In the vestibular ganglia, all neurons were intensely immunoreactive (Fig. lA,B). Their nuclei appeared more strongly labeled than the surrounding cytoplasm. The reaction was weak at glutaraldehyde concentrations < l%, and stronger with increasing concentrations. In some neurons, the periphery of the cytoplasm was more densely stained than the other cytoplasmic areas. The vestibular nerve fibers were strongly labeled, whereas their myelin sheaths (Fig. lB, : inset) and the surrounding Schwann cells were devoid of immunoreactivity (Fig. 1B). A very few fibers of small size and packed in bundles were unlabeled.

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Fig. 1. Light micrographs of semithin (lpm thick) epon-embedded sections showing the localization of Glu-like immunoreactivity in the vestibular ganglia. (A) Rat vestibular ganglion. Neurons and their axonal processes either longitudinally (large arrows) or transversally cut (thin arrows) are strongly stained. Vascular fixation: 3% paraformaldehyde (para) 1% glutaraldehyde (glut); (B) Cat vestibular ganglion. Note the absence of immunoreactivity in the Schwann cells (thin arrows). Double fixation: 2% para, 1% glut, 0.5% DMSO; (Bl): Detail of a bundle of intensely labelled vestibular fibers (cross section). Scale bar = 10 pm.

In the vestibular epithelia of the cristae ampulhues and utricular maculae, type I and type II sensory hair cells of all the species (Fig. 2) were intensely stained except the weakly labelled type I in samples from one cat (Fig. 2c), whereas their nuclei were unlabeled. The afferent nerves calyces surrounding the type I cells, the afferent fibers and the afferent boutons contacting the type II cells were also stained and often more intensely labeled than the sensory cells. The apical portion of the calyces in particular (Fig. 2c) was slightly more immunoreactive. Under the basal epithelial membrane, the afferent fibers were also strongly labeled. On tissues fixed with a low concentration (0.2%) of glutaraldehyde (not illustrated), a faint immunoreactive signal appeared in the hair cells,

whereas the afferent calyces and afferent fibers beneath the basal membrane were always significantly stained. In contrast, the cytoplasm of supporting cells was immunonegative. In control sections with normal rabbit sera instead of anti-Glu antisera, no immunoreactivity was found in ganglion neurons or in sensory cells. With anti-GABA antisera, the vestibular ganglia and epithelia of the three species examined (mouse, rat and cat) are devoid of GABA-like immunoreactivity (Fig. 3) whereas positive GABA immunolabeling of small neurons and axon terminals is found in cat lateral vestibular nucleus. This vestibular neuronal population is used as a positive control because it is well known as the site of intrinsic GABAergic neurons and GABAergic innervation.

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Fig. 2. Light micrographs of semithin sections showing the distribution of Glu-like immunoreactivity in the vestibular sensory epithelia. (A) Glu-irmmmoreactive sensory hair cells in the mouse utricular macula. Type I (I) with their afferent nerve calyces and type II hair cells {IX)am well visible. Afferent nerve fibers in epithelium and beneath the basal membrane are also intensely labelled (arrows). In contrast, supporting cells (s) am unlabelled. Fixation by immersion: 3,.5 4; glut. (B) General view of a cat crista ampullaris showing Glu-like immunoreactivity in the two types of hair cells and the afferent fibers (arrows). Double fixation: 1% para, 2% glut, 0.5% DMSO. Postfixation: 1% osmium tetroxide. (C) Crista ampullaris of the cat showing inununoreactive afferent fibers in cross sections and afferent boutons surrounding immunoreactive type II hair cells (thin arrows). Type I hair cells are weakly labelled or unlabelled. Intensely labelkl nerve calyces show a strong imrnunoreactivity in their apical portion (arrowheads). Doubie fixation: 2% para, 1% glut, 1% DMSO. Osmic acid postfiiation. (D) Mutation of a detail on a seriated section of the same cat crista ampullaris in (B) illustrating immunoreactive hair cells (types I and II), strongly immunoreactive afferent nerve caIyces, afferent boutons (thin arrows) and afferent nerve fibers. Scale bar = 10 pm.

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Discussion

One approach toward visualizing amino acid neurotransmitters in neurons and pathways is the recent use of antisera raised against amino acids (Storm-Mathisen et al., 1983; Ottersen and Storm-Mathisen, 1984). However, immunocytochemical localization of a substance in certain neuron populations does not necessarily imply that it is releasable as a transmitter, especially in the case of Glu which has multiple functions in general metabolism apart from its role as a transmitter (Hertz et al., 1983). Consequently, immunostaining reflects the total amount of Glu, i.e. the transmitter pool plus the metabolic pool. Nevertheless, it can be expected that the highest levels of Glu will be concentrated in various populations of neurons that, on the basis of experimental evidence other than immunocytochemistry, are believed to use Glu as a ne~otr~s~tter (StormMathisen et al, 1983; Ottersen and Storm-Mathisen, 1985; Somogyi et al., 1986 ; Conti et al., 1987).. In all the species studied here, Glu-like immunoreactivity occurred in vestibular ganglion neurons, their peripheral and central processes, and in the two types of sensory hair cells, The ~unost~~g intensity of hair cells was more sensitive to the fixation conditions than the ganglion neurons and required optimal, rapid fixation of the antigenic sites, i.e. in situ fixation or fixation by direct immersion. Routine vascular perfusion gave satisfactory results in mice and rats. In

the cat, the best results were obtained by in situ fixation in anesthetized animals. Under these conditions, the tissues are not affected by ischemia, which, as shown in rat hippocampus by intracerebral microdialysis, is associated with an increased efflux of Glu (Benveniste et al., 1984) and which may thus result in a dramatic loss of Glu-like ~unoreacti~ty in neurons. In the central nervous system, postembedding staining technique of thin sections is usually performed on nonosmicated tissue, because osmication may alter or almost completely suppress antigenicity, especially when osmic acid postfixation follows the primary aldehydic fixation. Nevertheless a variety of antigens can survive postfixation with osmium tetroxide, for example Glu, as previously observed by Somogyi et al. (1986), Altschuler et al. (1989). In our study in the cat, the combination of in situ glutaraldehydic fixation and osmic acid postf~ation provides excellent results. The strong Glu-like immunoreactivity in vestibular ganglion neurons and vestibular fibers is in good agreement with the proposition of Glu or Asp as the neurotransmitter involved in vestibular nerve transmission. A variety of experimental approaches other than immunocytochemistry strongly support the suggested transmitter role of Glu in lower vertebrates (Bledsoe et al., 1980; Bobbin et al., 1981; Amroni et al., 1984; Valli et al, 1985; Cochran et al., 1987; Drescher et al,, 1987; Bledsoe et al., 1988; Guth et al., 1988). Furthermore, in the cat, it has been shown that D

Fig. 3. Light micrographs illustrating sections processed with GABA antiserum. The cat vestibular ganglion (A) and epithelium (B), apart from the peroxydase reaction in the red blood cells (B), are devoid of GABA-like immunoreactivity. Scale bar = 10 pm.

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(3H) Asp undergoes selective retrograde transport through the vestibular nerve until ganglion neurons after high-affinity uptake by the vestibular nerve terminals (DemEmes et al., 1984). Therefore, selective labeling of vestibular nerve terminals was observed here by electron microscopic radioautography following the same high-affinity uptake mechanisms of (3H) Glu in the vestibular nuclei (Raymond et al., 1984). Biochemical measurements of high-affinity Glu uptake in normal and deafferented vestibular nuclei have confirmed that the vestibular input is glutamatergic (Raymond et al., 1984). The presence of strong Glu immunoreactivity in vestibular neurons and their nerve processes provides additional support for the candidacy of Glu as the transmitter of the vestibular nerve. The presence of Glu-like immunoreactivity in the afferent calyces and in the afferent boutons, which was stronger than in the sensory hair cells, even under less than optimal fixation conditions, could reflect a selective retention of nerve terminal Glu. It has been demonstrated that the Glu-like immunoreactivity remaining in immersion-fixed rat hippocampus slices after a loss of Glu-immunoreactivity due to in vitro conditions represents a selective retention of nerve terminal Giu that is part of a transmitter pool (Ottersen and Storm-Mathisen, 1985). Scarfone et al. (1988) have recently reported that the apical portions of these afferent nerve calyces are densely populated by microvesicles that are morphologically similar to typical small presynaptic vesicles, and are strongly immunoreactive with Synapsin I and Synaptophysin, the two major components of small synaptic vesicle membranes. Consequently, these authors hypothesized that these sensory calyces may act as nerve terminals and modulate the function of type I hair cells via the secretion of neurotransmitter-like substances. If Glu is stored in vesicles as a neurotransmitter, it can be expected that the ~n~ntration of Glu is higher in vesicles than in cytoplasm. Indeed, using a procedure allowing vesicle isolation, it was recently found that the Glu concentration in highly purified vesicles is, at least 10 times higher than in rat brain cortex (Riveros et al., 1986). Thus, the numerous small microvesicles with clear content in these vestibular nerve calyces assumed to be

involved in the release of neurotransmitter-like substances may contain Glu. In the vestibular epithelia, Glu-immunoreactivity was mostly concentrated in the sensory cells. The absence or the very low levels of immunoreactivity in the supporting cells is in agreement with other data in different brain structures where a qu~tification of immunogold labeling showed a very low level of Glu-like immunoreactivity in glial cell processes (Somogyi et al., 1986; Montero and Wenthold, 1989). Faint staining has been also reported by Altschuler et al. (1988) in the supporting cells of the guinea pig cochlea. This low labeling density in supporting cells probably reflects Glu levels in the metabolic pool. Our observations revealed significantly high levels of Glu in the two types of sensory hair cells in the species examined, with variations in labeling intensity among cells of the same receptor or the same section. These data suggest the possibility of glutamatergic transmission between hair cells and afferent fibers. Several pharmacological investigations have suggested that the hair cell transmitter in vestibular and lateral line systems may be Glu, but the site of action of Glu and the kind of receptors involved in transmission remain to be clarified. The recent study of Dechesne et al. (1988b), using monoclonal and polyclonal antibodies in the frog labyrinth has shown the presence of kainic acid receptors at the postsynaptic site of the synapse. Most of these studies do not concern the mammalian vestibular labyrinth. The sole exception, to our knowledge, is the in vivo investigations of Glu action in the cat labyrinth, where the authors (Dechesne et al., 1984) concluded that L-Glu or kainic acid applied in the endolymphatic space mimics the excitatory transmitter substance at the synaptic junction, thereby increasing the spontaneous discharge of primary afferents. Raymond et al. (1985, 1988) using the neurotoxic effects of excitatory amino acid agonists on in vitro preparations of mouse otic vesicles, have reported the existence of Glu receptors in the mouse vestibular epithelium. Furthermore, the evidence for Glu as a hair celI transmitter has been the subject of much controversy. GABA has also been proposed and opposed as a candidate for neurotransmission at this excitatory synapse in the vertebrate vestibular sys-

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tern (Flock and Lam, 1974; Felix and Ehrenberger, 1977; Klinke and Oertel, 1977a; Usami et al., 1977a,b; Meza et al., 1982; Guth and Norris, 1984; Vega et al., 1987). The only pharmacological work concerning the mammalian labyrinth is that of Felix and Ehrenberger (1977,1982), who applied GABA iontophoretically to demonstrate its excitatory effect on afferent nerve fibers in the saccule of the cat; they suggested that GABA may play a role in sensory transduction and some of the GABA may play a modulator role. In opposition to our negative results in mouse, rat and cat vestibular epithelia, GABA has been imunocytochemically demonstrated in the hair cells of the chick (Usami et al., 1987b), whereas it was present in the efferents of the squirrel monkey (Usami et al., 1987a) and very recently in the vestibular sensory cells and the afferent calyces of the guinea pig (Lopez et al., 1989). In these species, GABAmediated interactions between hair cells and afferent synapses have to be determined and the transmitter function of GABA remains highly controversial in this system. It has been suggested that calbindin D-28K plays a protective role against Glu-induced neurotoxicity in neuron cultures (Baimbridge and Kao, 1988), and it is interesting to note that the vestibular hair cells and ganglion neurons with high Glulike immunoreactivity, also contain this calcium binding protein (Dechesne et al., 1988a). Relatively large quantities of calbindin D-28K have been detected by radioimmunoassay and immunocytochemistry in electron microscopy in the peripheral vestibular system, sensory epithelia, and ganglion neurons (Sam et al., 1986). The present results show that all sensory hair cells contain high levels of Glu-immunoreactivity, which supports the hypothesis that apart from its metabolic function, Glu may play a role in transmission between hair cells and vestibular afferent fibers. Its presence in the vestibular neurons and the central and peripheral nerve fibers also suggests a role as a neurotransmitter possibly acting as well in the apical portion of the afferent calyces. Acknowledgements The authors wish to thank B. Arnaud for his photographic work and J. Boyer for her secretarial

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269 tween the vestibular type I hair ceil and its primary afferent chemical? Ann. New-York Acad. SC. 314,21-214. Somogyi, K., HaIasy, J., Storm-Math&n, J. and Ottersen, O.P. (1986) Quantification of immunogold labe.IIing reveals enrichment of glutamate in mossy and parallel fibre terminals in cat cerebellum. Neuroscience 19,1045-1050. Storm-Math&n, J., Leknes, A.K., Bore, A.T., VaaIand, J.L., Edminson, P., Haug, F.-M.S. and Ottersen, O.P. (1983) Fit visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301, 517-520 Usami, S., Igarashi, M. and Thompson, G.C. (1987a) GABAIike immunoreactivity in the squirrel monkey vestibular end organs. Brain Res. 417, 367-370. Usami, S., Igarasbi, M. and Thompson, G.C. (1987b) GABA-

Iike immtmoreactivity in the chick vestibtdar end organs. Brain Res. 418, 383-387. VaBi, P., Zucca, G., Prigioni, I., Botta, L., CaseIla, C. and Guth, P. (1985) The effect of glutamate on the frog semicircular canal Brain Res. 330, l-9. Vega, R., Soto, E., Budelli, R. and Gonzalez-Estrada, M.T. (1987) Is GABA an afferent transmitter in the vestibular system? Hear. Res. 29, 163-167. Watkins, J.C. and Evans, R.H. (1981) Excitatory amino acid transmitters. Armu. Rev. Pharmacol. Toxicol 21, 165-204. WersaB, J. (1956) Studies on the structure and innervation of the sensory epitheIium of the cristae ampuIIares in the guinea pig. A light and electronmicroscopic investigation. Acta Otolaryngol. 126, l-85.