GABAA receptor-like immunoreactivity in the goldfish brainstem with emphasis on the Mauthner cell

Pergamon

0306-4522(94)00579-6

Neuroscience Vol. 66, No. 3, pp. 697-706, 1995 Elsevier Science Ltd Copyright 0 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

GABA, RECEPTOR-LIKE IMMUNOREACTIVITY IN THE GOLDFISH BRAINSTEM WITH EMPHASIS ON THE MAUTHNER CELL C. SUR,* R. McKERNANt and A. TRILLER*$ *Laboratoire de Neurobiologie Cellulaire (INSERM U261), mpartment des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France TMerck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, U.K. Abstract-The distribution of the GABA, receptor in the goldfish brainstem and on the Mauthner cell membrane was investigated with both optical and electron microscopy using a polyclonal antibody raised against the intracellular loop of the rat y2 subunit. At the optical level, immunofluorescent dots were detected on small and large neurons belonging to vestibular and reticular nuclei. On the Mauthner cell plasmalemma, a y2-like immunoreactivity was observed predominantly on the tip of the lateral dendrite. Fluorescent parches were intermingled with a more diffuse staining. Immunoreactive spots of weaker intensity were also present on the soma and some were also observed inside and within the periphery of the axon-cap as well. Observations at the electron microscopic level revealed that the peroxidase end-product predominates postsynaptically in front of release sites in the studied nuclei and on the Mauthner cell. On the lateral dendrite of the neuron, numerous immunopositive postsynaptic differentiations were encountered on spines. Stained glial elements were encountered in the different areas studied. These results demonstrate that the GABA, receptor y2 subunit has a precise distribution on neuronal membranes and suggest that it could be involved in the remote dendritic inhibition of the Mauthner cell and in the control of input-output properties of both vestibular and reticular nuclei.

been studied with morphological techniques on the In the Mauthner (M-) cell of goldfish, a large reticulospinal neuron, iontophoretically delivered GABA M-cell. The GABA-gated chloride ion channel (GABA, activates chloride conductance’-” and GABAergic and receptors are present over large areas of this ~ell.‘~~” receptor) is a heteroligomeric receptor4~2’~2s~27~3’ Interestingly, the M-cell is inhibited not only by its subunit composition determines its sensitivity to GABA but also by glycine.‘0,‘9 The distribution of various drugs. 24 For example, it was demonstrated terminals at the M-cell surface containing these that GABA, receptor responsiveness to benzodiazeinhibitory neurotransmitters is well characterized.40s46 pines through allosteric modulation is fully achieved when the y2 subunit is present in the receptor comThe glycinergic boutons are present at both dendritic plex.27 Biochemical studiesi have shown that the and somatic levels of the M-cell,& a pattern which GABA, receptor and its associated benzodiazepine is matched by the distribution of the glycine receptor as deduced from iontophoretic’ and immunohistobinding site are well preserved phylogenically and are present in teleosteen fish.16In addition, electrophysiochemical observations.32,33,45 GABAergic afferents, logical data in the lamprey’ and in the goldfish,8,‘0 in contrast, display a regionalized distribution on where GABA receptor subserved the same inhibitory the M-cell surface.2o,26,46The density of GABAcontaining boutons is highest at the extremity of functions as in mammals, support the existence of a chloride conductance the lateral dendrite and decreases progressively all conserved GABA-dependent along this process to be minimal at the somatic level.& throughout evolution. The distribution of GABA receptors has not yet In this study, we have addressed the question of the cellular distribution of the GABA, receptor. We have used a polyclonal antibody raised against the fTo whom correspondence should be addressed at: intracellular loop between transmembrane segments Laboratoire de Biologie Cellulaire de la Synapse, M3 and M4 of the rat y2 subunit. The pattern CJF 94-10, Ecole Norrnale Su@ieure, 46 Rue cl’Ulm, of GABAA receptor immunoreactivity was studied on 75005 Paris, France. the M-cell surface at both optical and ultrastructural Abbreviations: GAD, glutamate decarboxylase; M-cell, levels. Preliminary results of this work were presented Mauthner cell; PBS, phosphate buffered saline; SVB, small vesicle bouton. in abstract form.38 691

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698 EXPERIMENTAL PROCEDURES

Seven goldfish (Carassius auratus from Paramount Aquarium, Paris) IO-12 cm length were anaesthetized with Tricaine (MS-222, Sandoz) and were perfused through the heart with 2% cold, freshly depolymerized paraformaldehyde in 120mM phosphate buffer at pH 7.4 for 20 min. After dissection, the brains were postfixed for 1 h in the same fixative and immersed overnight in 40% sucrose in phosphate-buffered saline (PBS) at 4°C. The brainstems were freeze sectioned and slices (60 pm thick) were dipped in 0.25% ammonium chloride in PBS to block free aldehyde groups. After washing with cold PBS, tissue samples were incubated with the y2 subunit polyclonal antiserum diluted 1:100 in PBS in the presence of 0.12% gelatin for two days at 4°C. This antiserum was raised against a recombinant protein containing the cytoplasmic loop (between segments M3 and M4) of GABA, receptor y2 subunit of rat.** After extensive rinsing with PBS, slices were treated with a goat anti-rabbit antibody coupled to CY3 (Indocarbocyanine; Jackson ImmunoResearch, West Grove, U.S.A.) diluted 1:200 in PBS-gelatin for 2-3 h. The tissue sections were mounted with Mowiol (Hoechst, Frankfurt) and examined with a Zeiss microscope equipped for fluorescence and a confocal microscope (Molecular Dynamics). The method of analysis with confocal microscopy was similar to that described previously.45 Briefly, digitized series of optical sections which constitute a 3D representation were collected in a host computer and the background noise was reduced by applying a (3 x 3 x 3) Gaussian filter. Surface areas of clusters were computed directIy with the included software, on the graphic station screen on maximum intensity projections visualized with a linear look-up table. For electron microscopy experiments, the slices were incubated with a biotinylated goat anti-rabbit immunoglobulin (1:200 in PBS-gelatin) for 2-3 h. The presence of antibody binding sites was determined with an avidinbiotin-peroxidase complex procedure (Vectastain Elite, Vector Laboratories. Inc.. Burlinaame. CA. U.S.A.). The presence of the complex was revealed by’dipping the sections in a ready to use solution of diaminobenzidine tetrahydrochloride and urea (Sigma) reconstitued in 15 ml of distilled water. The reaction was observed under the microscope and terminated by extensive washing with cold Tris buffer. Subsequently, the sections were washed in PBS, osmicated (1%) for 1h and incubated for 1 h in uranyl acetate (1% in distilled water). After dehydration in graded ethanol and acetone, the slices were infiltrated with Araldite and flat-embedded on plastic films. Regions of interest were re-embedded for sectioning and pale yellow ultrathin sections were observed with a Philips CM 10 electron microscope operating at 80 kV. The stained structures were generally viewed with different tilt angles to avoid misinterpretation of antigen localization (see Fig. 5). In control experiments, omission of the first antibody or incubation with the preimmune serum resulted in the absence of specific staining at the cellular and subcellular levels. RESULTS

The M-cell of the goldfish is a good model in which to study the distribution of neurotransmitter receptors at a single cell level, due mainly to its unique morphological characteristics (Fig. 1). This neuron has two main dendrites, lateral and ventral ones which terminate in vestibular and reticular nuclei, respectively. At the somatic level, it also displays small ventral dendrites which extend ventrally and a particular neuropil, the so-called axon-cap, which surrounds the initial segment of the axon.

Fig. 1. Schematic drawing of the M-cell showing the different cellular compartments mentioned in this study. Distal (dLD) and medial (mLD) lateral dendrite after and before its main bifurcation respectively; S, soma; sVD, small ventral dendrite; AC, axon-cap; ventral dendrite; Ax, axon.

Immunojluorescent staining Light microscopic observations revealed that GABA, receptor immunoreactivity is distributed in a heterogeneous manner over the M-cell membrane, as illustrated in Fig. 2. In all the observed M-cells, a different pattern of labelling was observed between the two main dendrites. The lateral dendrite (Fig. 2A, B) displayed numerous fluorescent patches after (Fig. 2A) and before (Fig. 2B) its main bifurcation. At this magnification, it appeared that the fluorescent spots were present over a more diffuse staining of the M-cell surface. This patchy appearance was most obvious on the distal portion of the lateral dendrite, where immunoreactive dots delineate large circular spaces devoid of staining (Fig. 2B). Observations at the electron microscopy level indicate that the regions free of GABAA receptor immunoreactivity correspond to the zones of contact of large excitatory terminal afferents arising from the vestibular nerve.23 The ventral dendrite also displayed fluorescent patches but they were less intense and less abundant (compare Fig. 2A and C). Most of them were localized after the bifurcation of this process (Fig. 2C). On the soma of the M-cell, the number and intensity of the labelled aggregates were greatly reduced (Fig. 2D). In contrast, the small ventral dendrites issuing from the ventral side of the soma exhibited numerous small, fluorescent dots all along the processes (Fig. 2E). At the axon-cap level, the occurrence of an autofluorescence background27 limited the labelling analysis with conventional fluorescence microscopy. With confocal microscopy, some spots of immunoreactivity were found on structures at the periphery of the axon-cap (Fig. 2F) and less frequently within the central core of the axon-cap (not shown). Using electron microscopy, these dots corresponded to regions on the membrane of ghal processes from cap cells, a category of glia which delinate the outlines of the axon-cap. Rare postsynaptic differentiations were also 1abeIled (not shown). Confocal microscopy analysis of immunoreactivity on the lateral dendrite (Fig. 3A) showed more

Cellular localization of a GABA, receptor subunit

Fig. 2. Regional distribution of y2 subunit immunoreactivity over the M-cell surface. (A, B) Example of a lateral dendrite after (A) and before (B) its bifurcation, displaying a high density of immunofluorescent patches (arrowheads). In B note the presence of large unstained areas (arrow) surrounded by dots of immunoreactivity (arrowheads). (C) A less abundant labelling (arrowheads) was present on the distal portions of the ventral dendrite. (D, E) Fluorescence is present on the somatic membrane (arrows) and on the small ventral dendrite (arrowheads). At higher magnification (E), spots of immunoreactivity (arrowheads) are localized all along the process. (F) Confocal optical section of the axon-cap showing the occurrence of spots of immunoreactivity (arrowheads) outside the central core (delineated by dotted line). Abbreviations are as defined in Fig. 1. Scale bars = 25 pm (A-E); 5 pm (F).

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B M@mArm

SURFACE AREA

= 1.763~1.80 (n= 161)

(pd)

Fig. 3. Size of y2 subunit domains on the lateral dendrite. (A) Digitized fluorescent image of y2 subunit-immunoreactivity patches. Note the heterogeneity in shape and size of fluorescent patches. Scale bar = 5 pm. (B) Histogram of the surface areas (mean rf: S.D.) of y2 subunit-la!Med-domains computed from pictures examplified in A.

accurately the border of the patches of antibody binding sites. These immunoreactive clusters are heterogeneous in shape (round, ellipsoidal) and size (Fig. 3A). A

morphometrical analysis of the receptor domains from three reconstructed distal lateral dendrites (Fig. 3B) indicated that they had a mean surface area (+S.D.) of 1.76 f 1.8 pm’, N = 161. Other structures were also labelled in the brainstem of the goldfish by this antiserum, including cells from the vestibular (Fig. 4A1, A2), the reticular (Fig. 4B, C) and the lateral line (not shown) nuclei. Immunoreactivity was present on vestibular neurons in all nuclei of the complex. As in the case of the M-cell, the staining of reticular and vestibular cells was discontinuous and displayed patches on somatic and dendritic membrane (Fig. 4A-C). Ultrastructural

results

In this study, the preservation of the ultrastructure was limited by the fixation procedure. The antigenicity decreased rapidly when increasing the paraformaldehyde concentration or fixation time, and it was completely abolished by glutaraldehyde. Confirming the observations at the light microscopic level, immunoreactivity was found principally associated with the lateral dendrite membrane facing synaptic contacts (Fig. 5A1, A2) made by small vesicle boutons (SYEs). These endings were identified by their pleiomorphic population of small clear vesicles.23 In addition, dense-core vesicles intermingled with small vesicles were frequently observed (Fig. SA, D). Immunopositive synapses were also encountered on spines (Fig. 5A2, B) which were engulfed in the presynaptic ending (Fig. 5B). With favourable plane of section, the invaginated synapses

appeared to be connected to the M-cell by a stalk (Fig. 5A2), and in some instances the presynz+ptic active zone could extend over a large proportion of the head of the spine. The advantage of viewing the same stained structure with di@erent tilt angles is dramatically exempliiid in Fig. 561 and A2: the observation of Fig. 5A1 alone suggested a presynaptic staining in SVBl, but close examination under different tilt angles confirmed that the electrondense precipitate was associated with the postsynaptic membrane (Fig. 5A2) of a spine. A weak background peroxidase precipitate is also frequently seen in the cytosol of the lateral dendrite tirating cjrtoskeletal elements (Fig. 5A1, A2). However, at this level, numerous SVBs apposed to unstained postsynaptic differentiations (Fig. 5C) were also encountered. At the somatic level, electron-dense product was usually found postsynapticaliy in front of synaptic endings on the small ventral dendrite (Fig. SD), in agreement with the regional distribution noted at the optical level. Inside the axon-cap, rare synaptic differentiations (not shown) were immunoreactive and labelling was mainly encountered in association with cap cell processes (Fig. 5E), thus con&ming observations of fluorescent elements in this region. In the vestibular (Fig. 6A, B) and reticular (Fig. 6C, D) nuclei, the perotidase end-product predominates, postsynaptica#y, at the synaptic complex present on dendritic elements. The presynaptic apposed terminal endings usually contained a pleiomorphic population of small vesicies. Reaction end-product deposition was also observed on the cytoplasmic component of the spine (Fig. 5B), on mitochondrial membranes (Fig. 6Af and on cytoskeletal elements (e.g., Fig. 63, D).

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701

Fig. 4. Presence of y2 subunit immunoreactivity on vestibular (Al, A2) and reticular (B, C) neurons. (Al, A2) Same neuron at different focal planes showing the discontinuous lahelling (arrowheads) on the soma (Al) and proximal dendrite (A2). (B, C) Examples of reticular cells revealing that the staining (arrows) on the soma and dendrite appears as small dots of fluorescence Scale bars = 25 pm.

DISCUSSION

The conditions of fixation and the subsequent treatment of tissue samples (see Experimental Procedures) used here to preserve the antigenicity allowed a limited preservation of the structural organization of the tissue. As a consequence, we may have lost some binding sites. However, at both the cellular and subcellular levels, we were able to observe significant immunocytochemical staining and to show that the GABA, receptor y2 subunit forms clusters at the M-cell neuronal surface and exhibits a somatodendritic distribution, in agreement with the known GABAergic innervation.20~26~46 Evidence for benzodiazepine goldfish brain

binding

sites

in the

The presence of a y2 subunit-like immunoreactivity in goldfish brainstem confirms the observation that benzodiazepine binding sites are present in this phylum.6,7,29 In radioligand binding studies,7 the

fish GABA benzodiazepine receptor has the characteristics of a benzodiazepine type 2 receptor. The density of binding sites, the affinity constants for [3H]muscimol and [‘Hjflunitrazepam, the kinetics and the pharmacological profile are all consistent with a receptor of this type observed in mammals.’ A homooligomeric structure has been suggested for the teleost GABA, receptor.’ However, there are likely to be multiple GABA, receptor subunits in fish, as in other lower species (e.g., Lymnaea stagnalis;15,” Drosophila ‘**13)and mammals!Z5,27 In the mollusc two complementary DNA Lymnaea stagnalis, sequences have been isolated that encodes polypeptides /Iis and [,I’ which exhibit around 50% and 35% identity to the vertebrate GABA, receptor jl subunit. Interestingly, the molluscan /I subunit can replace the vertebrate fl subunit in co-expression experiments with the bovine GABA, receptor ctl subunit,15 and the positions of introns in the c subunit gene were found in similar positions of those of vertebrate GABA, receptor genes.” These results

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Fig. 5. Ultrastructural localization of y2 subunit on different regions of the M-cell. (Al, A2) Same region of the lateral dendrite viewed with two different tilted angles (0” in Al; 45” in A2). (Al) SVBl makes a stained synapse (arrows in Al and A2) with the M-cell. In this micrograph, labelling is also present in the apposition zone between SVBl and SVB2 (crossed arrows). (A2) This view clearly shows that SVB2 in fact synapses positively with the head of a spine (arrowheads). Note the presence of dense-core vesicles (open arrows) within SVBl. (B) An immunoreactive spine (arrows) engulfed in a terminal containing pleiomorphic small vesicles on the lateral dendrite. (C) An immunonegative synapse (arrowheads) on the lateral dendrite taken from a region close to boutons shown in Al and A2. (D) A labelled synapse (arrows) established on a small ventral dendrite by a terminal filled with pleiomorphic clear vesicles and dense-core vesicles (arrowheads). (E) Evidence of glial staining within the axon-cap. The positive glial element (arrows) is in the vicinity of a cap cell somata (asterisk). MC, M-cell cytoplasm. Scale bars = 0.5 nm.

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Fig. 6. Subcellular localization of y2 subunit immunoreactivity within vestibular (A, B) and reticular (C, D) nuclei. (A, B) Two examples of immunoreaction at synaptic differentiations (arrows) occurring on dendrite profiles. Note that the reaction end-product does not extend beyond the limits of the synaptic complex, even where two synapses are close to each other (A). (C) A terminal containing dense-core vesicles (arrowhead) making a positive synapse (arrows) with a dendritic shaft in the reticular formation.

(D) An immunoreactive synapse (arrows) established on a dendritic profile located in the reticular formation. Scale bars = 0.5 pm (A, B); 0.2 pm (C, D).

suggested that invertebrate GABA, receptors are present in vivo as hetero-oligomeric complexes. However, the epitopes recognized by this antiserum, located in the intracellular loop between transmembrane segments M3 and M4 of the y2 subunit, seem to be phylogenetically preserved and able to detect the y2 subunit in rat brain,28,39human brain (Quirk and McKernan, unpublished observations) and goldfish brain (this study). In addition, behavourial studies on the fathead minnows2’ (Pimephales promelas) have demonstrated that central benzodiazepine receptors can modulate behaviour in anxiety-like states as in mammals. Although molecular proof of the existence of a 72 subunit in goldfish brain are still lacking, the specificity of the antiserum used here is substantiated by the following points: (i) it did not detect either y 1 and y3 subunits by western blot,28 (ii) the distribution of y Zlike immunoreactivity in the different regions of the M-cell fits well with that of presynaptic GABAergic terminals determined by glutamate decarboxy-

lase (GAD)26940and GABA& immunocytochemistry, (iii) in the M-cell, stained postsynaptic differentiations were found in front of SVBs which are Furthermore, most of these inhibitory terminals. 23~40,46 boutons exhibited large dense-core vesicles that are preferentially located within GABAergic40,46 and not glycinergic endings apposed to the M-cell. Topographical relationship between GABA, and GABAergic afferent innervation

receptor

The characteristic morphology of the M-cell provides a favourable model to study the cellular distribution of neurotransmitter receptors in relation to afferent innervations.32x33,46The immunofluorescence clearly showed a regionalization of the y2-like immunoreactivity over the surface of the M-cell. The highest density of GABA, receptors on the distal part of the lateral dendrite and its progressive decrease towards the soma are consistent with the reported proportion of GABA& and GAD20326 containing boutons synapsing on these regions. This pattern

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this absence of extrasynaptic staining associated with the M-cell membrane. A recent subcellular analysis of GABAA receptor subunit distribution in the cerebellum3 revealed that the different isotypes may have different Iocalizations with respect to release sites. For example, a6 subwits are restricted to synaptic differentiations and al and fl2j3 have a broader distribution. An explanation for the difference in sub cellular localization depending on the subunit considered is not fully satisfactory since we have also observed patches of extrasynaptic y2 subunit immunoreactivity at the neuronal surface in the rat spinal cord.39 The restricted postsynaptic localization observedhere may result from the gentle fixation procedures (see Experimental Comparison of y2 subunit and glycine receptor organProcedures) necessary to observe immunoreactivity. ization of the Mauthner-cell These technical constraints would not favour a preservation of neuronal membranes, particularly y2 antigenic sites predominate on the lateral extrasynaptic ones, which are not stabilized by dendrite, whereas glycine receptor clusters have been observed to be present all over the M-ce11.32,45 the underlying cytoskeleton. The occurrence of y2In a quantitative confocal microscopy study of gly- positive synapses on spines is comparable to that observed for glycinergic synapses on the ventral tine receptor clusters on this neuron4j it has been dendrite of the M-ce11.r2This organization may allow shown that the surface area of the receptor matrix GABAergic a&rents to exert their electrical effects increases according to a somatodendritic gradient. without a great disturbance of the chemical compoThe value computed for surface area of y2 clusters sition of the target cell.r* Another interesting obser(1.76 + 1.8 pm*) is close to that reported for glycine vation is the presence of the y2 labelling in small receptor aggregates (1.70 + 1.13pm2)in the same ventral dendrites, a region also enriched in gIycine distal region of the lateral dendrite!j However, the receptors32*45and contacted by boutons inside which surface areas of both GABA* and glycine receptor GABA and glycine neurotransmitters co-exist.46 complexes evaluated with confocal microscopy This partieular distribution suggests that at this level were systematically larger that the apposed presynof the M-cell, both GABA, and glycine receptors aptic release site measured with the electron micromight be co-localized within the same postsynaptic ~cope.~*%~’ This discrepancy may have several origins: differentiation. (i) the limit of precision of the confocal microscope The presence of diaminobenzidine deposit on due to the non-reducible size of pixels,14 (ii) on the microtubules and on mitochondrial membranes lateral dendrite, GAD-positive terminals and corresponding synaptic complexes are closely packed.26 may have various origins. First, it may be due to the These two points may lead to an apparent fusion of well established diffusion of reaction end-product. adjacent individual clusters of receptors. As for the Second, the staining on microtubules could correglycine receptor,32s43,44the y2 subunits are mainly spond to genuine localization of GABAA receptors localized on the postsynaptic membrane facing prebeing transported toward the plasmalemma. In adsynaptic release sites. Thus, the equality of the size of dition, a recent report by Whatley and co-workers4’ glycine and GABAA y2 subunit fluorescent clusters suggests that GABA* receptors may interact with agrees well with the ultrastructural measurements on microtubules. this process, showing that GABAergic and glycinPotential roles of GABA, receptor in relation to its ergic presynaptic active zones have the same mean cellular localization surface area.37 of distribution on the target cell is also consistent with previous mapping of GABA receptors using iontophoretically applied GABA,&” except in the axon-cap, where positive synapses and GABAcontaining boutons4” were detected only rarely. The presence of y2 subunit staining in neurons within reticular and vestibular nuclei also matches well with the high density of GAw” and GABA (Sur and Triller, personnal observations) terminals impinging on these cells. The close relationship between the y2 subunit and GABAergic input distribution patterns on the M-cell and within brainstem nuclei favours the specificity of the antiserum used in this work.

The presence of pleiomorphic vesicles within boutons apposed to immunoreactive postsynaptic differentiation is compatible with an inhibitory action.23*47Furthermore, dense-core vesicles found in these SVBs were shown in other studies40.46to be predominantly present in GABAergic endings. In this study, we have not observed extrasynaptic staining on the M-cell membrane with the electron microscope, as suspected from the presence of a diffuse immunofluorescent labelling. Other immunohistochemical studies30s3’36have shown that GABA, receptors are diffusely distributed over non-synaptic membranes. Several explanations may account for

On the lateral dendrite, the GABA, receptor localization, around the area of contact of large vestibular nerve afferents, appears to be well suited to shunt these powerful excitatory inputs. As a matter of fact, the eighth nerve afferents contain mixed electrical and chemical synapsesz3 whose activation triggers the M-cell spiking. In cooperation with glycine receptors, they would therefore participate in the remote dendritic inhibition.” The presence of the y2 subunit on cap cells, the astrocytes which delimit the region surrounding the initial segment of the M-cell axon,49 raises the question of the physiological relevance of such a

Cellular localization of a GABA, receptor subunit

distribution. GABA could theoretically modulate indirectly the M-cell output through these glial cells by regulating their capacity to buffer external potassium.2~22 In the brainstem nuclei analysed here, the localization of the y2-positive synapses suggest that GABA controls the output properties of vestibular and reticular neurons, some of them being involved in networks which themselves control M-cell excitability. 5,19,41,42.50

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unit immunoreactivity corresponds well to that of the GABA-containing terminal. This observation suggests that the afferent innervation plays a key role in the neuronal organization of receptors at the cell surface. Furthermore, the strategic localization of GABA and glycine receptors, close to primary vestibular inputs, may allow an accurate adjustment of inhibition by different sets of neurons under various excitatory stimulation conditions. Acknowledgements-We thank P. Caramelle for his technical assistance with micrographs. C. S. is a recipient of a fellowship from the Ministbre de la Recherche et de la

CONCLUSION

The heterogeneous distribution on the M-cell lateral dendrite of the GABA, receptor ~2 sub-

Technoldgie. This work was supported by grants (no. 92-058) from DRET and from Association Fraqaise contre les Myopathies.

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