Neuroscience Letters, 111 (1990) 263-268 Elsevier Scientific Publishers Ireland Ltd.
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Immunohistochemical evidence for GABAergic cell bodies in the medial nucleus of the trapezoid body and in the lateral vestibular nucleus in the guinea pig brainstem J6r6me D u p o n t 1, Michel Geffard 2, Andr6 Calas 3 and Jean-Marie Aran 1 ;Laboratoire d'Audiologie Exp&imentale, I N S E R M U.229 and Universitb de Bordeaux H, H6pital Pellegrin, Bordeaux (France), 21nstitut de Biochimie Cellulaire et de Neurochimie, CNRS, Bordeaux (France) and 3Laboratoire de Physiologic des Interactions Cellulaires, CNRS, Universitk de Bordeaux I, Talence (France) (Received 30 October 1989; Accepted 20 December 1989) Key words." 7-Aminobutyric acid; Medial nucleus of the trapezoid body; Lateral vestibular nucleus; Optical microscopy; Immunohistochemistry; Guinea pig The presence of y-aminobutyric acid (GABA) in two brainstem nuclei is demonstrated by using a preembedding immunohistochemical procedure followed by staining intensification. Firstly, immunoreactivity was found in numerous cell bodies and profiles of the medial nucleus of the trapezoid body (MNTB). Secondly, numerous neurons including giant Deiters' cells, terminals and fibers were strongly labelled within the lateral vestibular nucleus (LVN). These observations suggest that the inhibitory part of the efferent innervation of outer hair cells in the cochlea can originate from the MNTB, and that GABAergic neurons in the LVN may contribute to information processing within this nucleus.
y-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the vertebrate central nervous system [10] and is thought to play a preponderant role in the modulation of chemical neurotransmission involved in the physiological hearing mechanism. Biochemical studies [5, 26] and other investigations using either lesion [15], labelling [19] and retrograde transport of [3H]GABA [11], or microiontophoretic application of GABA [17] have previously revealed the existence of this neurotransmitter within brainstem auditory nuclei. In order to localize GABAergic structures in the guinea pig auditory brainstem, immunohistochemical studies have been conducted using a GABA antiserum [12, 24]. GABAergic cell bodies were observed in the ventral and dorsal cochlear nuclei's suCorrespondence." J. Dupont, Laboratoire d'Audiologie Exp6rimentale, INSERM U.229, H6pital Pellegrin, Place Am61ie Raba L6on, 33076 Bordeaux, France. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
264
perficial layers, in the medial and lateral superior olive (the superior olivary complex is known to be involved in spatial localization of sound [13]), in the lateral preolivary nuclei and in the lateral nucleus of the trapezoid body (LNTB). These observations confirmed by other immunohistochemical studies using antibodies against glutamic acid decarboxylase (GAD) [9, 16], which is the synthesizing enzyme for GABA, have not detected any immunoreactivity in the cell bodies of the medial nucleus of the trapezoid body (MNTB). In the present study we have used a highly specific GABA antiserum [22] to specify, at the optical microscopy level, the localization of this module in the brainstem auditory and vestibular nuclei in the guinea pig. Not only did we observe the same labelled structures as shown in previous studies, but we have also obtained some new results concerning the existence of GABAergic celt bodies and profiles in the MNTB and in the lateral vestibular nucleus (LVN) of the brainstem in guinea pig. Adult guinea pigs were perfused through the ascending aorta with a Karnovsky fixative containing 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). The brainstem was removed and postfixed in the same fixative for 1 h at 4:'C, and then transferred to Tris-buffered saline (TBS) at pH 7.3. Then, 20-30 /~m frontal sections were obtained with a vibratome and shaken overnight in TBS for washing. The floating sections were rinsed three times for 15 rain in TBS, and processed for immunohistochemistry. In order to eliminate the unspecific adsorption of our antiserum, a presaturation step was performed using a non-immune serum (goat) 1:50 for 15 min. Then, sections were incubated with highly specific rabbit antibodies against GABA at a dilution 1:5000 in TBS containing 0.15% Triton X-100 and 1.5% normal goat serum, for 20 h at 4"C under constant shaking. The floating sections were then washed 3 times, incubated with goat anti-rabbit immunoglobulins (1:100 Immunotech) for l h at room temperature, washed 3 times again in TBS and incubated for 1 h with rabbit peroxidase-antiperoxidase complex (1:1000, Immunotech, Jackson Immuno Research Labs.). After washing 3 times in TBS, the free sections were reacted and intensified using a solution containing 0.03% diaminobenzidine (DAB), 0.3% Nickel (II) sulfate heptahydrate and 0.05% hydrogen peroxide for about 10 min at room temperature and in semi-darkness. Floating sections were then placed on slides, dried and mounted in Eukit for observation with a Leitz photomicroscope. Numerous labelled neurons were visualized throughout the entire inferior collicular nucleus, in the medial superior olive (MSO), lateral superior olive (LSO) (Figs. 1 and 2) and molecular layer of the dorsal cochlear nucleus. Moreover, GABA-posirive neurons were also localized in the medial nucleus of the trapezoid body (MNTB). Indeed, large cell bodies of the M N T B were strongly stained and numerous immunoreactive fibers and scattered punctae were observed (Fig. 3a). In a control experiment, in which primary antibodies were exhausted with 10 5 M GABA, followed by the same immunohistochemical procedure, immunoreactive profiles in the M N T B were not detected (Fig. 3b). In the LVN, (or Deiters' nucleus) (Fig. i), which is the most conspicuous part of the vestibular complex, we observed GABA-positive cell bodies of different types and
265
Fig. 1. Coronal vibratome section of the brainstem showing a general overviewof the differentimmunostained nuclei at a very low magnification. LSO, lateral superior olive; POC, preolivarycomplex;MNTB, medial nucleus of the trapezoid body; LVN, lateral vestibular nucleus; IV, fourth ventricle; VII, facial nerve. Bar = 500/tm. Fig. 2. Frontal vibratome sectionin the inferior part of the brainstem showing numerous immunostained cell bodiesand profileswithin the LSO, medialsuperior olive (MSO), POC and MNTB. Bar = 250/tm.
sizes in diameter, and also numerous labeled fibers and other profiles (Fig. 4c). Indeed this nucleus is characterized by the presence of giant cells measuring up to 60-70 /~m in diameter and of multipolar or oval [14] smaller cells. These Deiters' giant cells and the two categories of smaller cells were strongly stained (Fig. 4a). At a higher magnification ( x 40 microscope objective), we observed a great number of labelled fibers and numerous GABA positive endings surrounding the cell bodies of these different immunostained neurons (Fig. 4c). In the control experiment, all these immunoreactive structures were very weakly coloured (Fig. 4b). We have used the same GABA antiserum [22] as previously used by other authors [21, 23] which clearly demonstrated immunoreactivity in well defined GABAergic structures in the cerebellum. In the present study we have reproduced the same pattern of GABA labelling within brainstem auditory nuclei as described in other studies [12, 24]. Moreover, we have also demonstrated GABA labelling in the M N T B and in the LVN, which to our knowledge has not previously been described. The fact that these GABAergic neurons have not been previously observed might be explained on the one hand by the use of the more sensitive GABA antibody than G A D antibody for the detection of GABAergic structures [23], and on the other by the high sensitivity of our pre-embedding immunohistochemical procedure in which we have systematically used the intensification with Nickel. Indeed, it is well known that to add metal ions to the DAB medium [1, 20] as here, increases the peroxidaseantiperoxidase (PAP) method's sensitivity. It has been proposed that some Deiters' nucleus cells in the LVN in the rabbit, which could be inhibitory, may constitute part of the medial vestibulospinal tract [8]. Furthermore, the existence of GABAergic neurons within the LVN has been proposed because, following extensive lesions of cerebellar afferents (the Purkinje cell projections) 30% of the G A D activity remains. At both light and electron microsco-
266
:i~v i~¸~j!!i~ji¸ !
Fig. 3. a: frontal vibratome section in the M N T B and in the POC showing numerous intensely stained GABA-positive cell bodies. Bar = 100 ,urn. b: control experiment using G A B A antiserum preadsorbed with G A B A immunogen. No reaction can be observed through the whole MNTB. B a r = 100 pro. Fig. 4. a: coronal vibratome section in the LVN, GABA-positive magnocellular neurons (stars) can be observed in this part of the vestibular complex. These immunoreactive giant cells are Deiters' neurons of the LVN (or Deiters" nucleus). Other smaller cells are also immunostained, and they are of two morphological types: multipolar (arrows) or oval (arrowheads). Bar = 100 ,urn. b: control experiment with a preadsorbed immunoserum. No evident staining can be observed in the whole Lateral Vestibular Nucleus. B a r = I00 /lm. c: high magnification view within the region of the delimited LVN in Fig. I, showing labelled giant Deiters" neurons and other immunoreactive profiles: immunostained fibers (arrows) and GABA-positive terminals surrounding the cell body (arrowheads). Bar = 25 pro.
267 pical levels, these latter studies suggested that extrinsic and intrinsic GABAergic neurons may contribute to information processing within the LVN [7]. These findings are in agreement with our demonstration of G A B A within Deiters' giant cells and within other smaller cell bodies and terminals of this vestibular nucleus. In the MNTB numerous GABAergic neurons are described here for the first time, and previously there were many arguments in favour of their GABAergic nature. Indeed, several immunohistochemical studies have been made to investigate the distribution of G A D - [3] and GABA-like [4] immunoreactivity in the organ of Corti of guinea pig and rat. Most of the immunostaining was found in the outer hair cells' synaptic region of the third and fourth turns of the cochlea, but this labelling was also seen in the basal turns where scattered clusters of immunoreactivity were found in the outer hair cells' synapses of the first row. It has well been demonstrated by hodological methods [17, 18] that the MNTB, which constitutes in part the source of the large-neurons medial efferent system [6, 25] of the mammalian cochlea, sends projections in these basal turns. The GABAergic nature of some M N T B neurons correlates therefore well with both these hodological data and the immunohistochemistry of G A B A in the guinea pig cochlea. In order to precisely describe the GABAergic immunostaining of neurons within the LVN and the MNTB, it is now necessary to study the intracellular localization of G A B A by adapted ultrastructural procedures. This is presently in progress in our laboratory by observation of preembedding immunolabelled structures at electron microscope level, and by the subcellular detection of G A B A using the postembedding immunocytochemical technique. The authors wish to thank G. Campistron for her help with G A B A antiserum, P. Dubourg for her excellent technical advice and T. Durkin for reviewing the English of the manuscript. This study was supported in part by a M R T grant (Project 88C0558) and the 'Conseil R6gional d'Aquitaine'.
1 Adams, J.C., Heavy metal intensification of DAB-base HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Caspary,D.M., Havey, D.C. and Faingold, C.L., Effectsof microiontophoreticallyapplied glycineand GABA on neural response patterns in the cochlear nuclei, Brain Res., 172 (1979) 179 185. 3 Fex, J. and Altschuler, R.A., Glutamic acid decarboxylaseimmunoreactivityof olivocochlearneurons in the organ of Corti of guinea pig and rat, Hearing Res., 15 (1984) 123-131. 4 Fex, J., Altschuler, R.A., Kachar, B., Wenthold, R.J. and Zempel, J.M., GABA Visualized by Immunocytochemistry in the guinea pig cochlea in axons and endings of efferent neurons, Brain Res., 366 (1986) 106 117. 5 Godfrey, D.A., Carter, J.A., Lowry, O.H. and Matschinsky, F.M., Distribution of gamma aminobutyric acid, glycine,glutamate and aspartate in the cochlearnucleus of the rat, J. Histochem. Cytochem., 26 (1978) 118 126. 6 Guinan, J.J., Warr, W.B. and Norris, B.E., Topographicorganization of the olivocochlearprojections from the lateral and medial zones of the superior olivarycomplex,J. Comp. Neurol., 226 (1984) 21 27. 7 Houser, C.R., Barber, R.P. and Vaughn, J.E., lmmunocytochemicallocalization of glutamic acid decarboxylase in the dorsal lateral vestibular nucleus: evidencefor an intrinsic and extrinsic GABAergic innervation, Neurosci. Lett., 47 (1984) 213-220.
268 8 Kawasaki, K., Matsushita, A., Satoh, M. and Takagi, H., The lateral vestibular nucleus is a site for the depressant action of benzodiazepines on the crossed extensor reflex, Brain Res., 461 (1988) 282 289. 9 Moore. J.K. and Moore, R.Y., Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat, J. Comp. Neurol., 260 (1987) 157 174. I(I Mugnaini, E. and Oertel, W.H., An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 4, Elsevier, Amsterdam, 1985, pp. 436 608. 11 Ostapoff, E.M., Morest, D.K. and Potashner, S.J., Retrograde transport of tritiated GABA from the cochlear nucleus of the superior olive in guinea pig, Soc. Neurosci. Abstr., 11 (1985) 1051. 12 Peyret, D., Geffard, M. and Aran, J.M., GABA immunoreactivity in the primary nuclei of the auditory central nervous system, Hearing Res., 23 (1986) 115 121. 13 Phillips, D.P. and Brugge, J.F., Progress in neurophysiology of sound localization, Annu. Rev. Psychol., 36 (1985) 245 274. 14 Pompeiano, O. and Brodal, M., The origin of vestibulospinal fibres in cat. An experimental anatomical study with comments on the descending medial longitudinal fasciculus, Arch. ltal. Biol., 95 (1957) 166 195. 15 Potashner, S.J., Lindberg, N. and Morest, D.K., Uptake and release of GABA in the guinea pig cochlear nucleus after axotomy of cochlear and centrifugal fibers, J. Neurochem., 45 (1985) 1558 1566. 16 Roberts, R.C. and Ribak, C.E., GABAergic neurons and axon terminals in the brainstem auditory nuclei of the gerbil, J. Comp. Neurol., 258 (1987) 267 280. 17 Robertson, D., Brainstem location of efferent neurones projecting to the guinea pig cochlea, Hearing Res.. 20 (1985) 79 84. 18 Robertson, D., Anderson, C.J. and Cole, K.S., Segregation of efferent projections to different turns of the guinea pig cochlea, Hearing Res., 25 (1987) 69 76. 19 Schwartz, I.R., Autoradiographic studies of amino acid labeling of neural elements in the auditory brainstem. In D. Drescher (Ed.), Auditory Biochemistry, Thomas, Springfield, IL, 1985, pp. 258 277. 20 Scopsi, L. and Larsson, 1_..I., Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system, Histochemistry. 84 (1986) 221. 21 Seguela, P., Gamrani, H., Geffard, M., Calas, A. and Le Moal, M., Ultrastructural immunocytochemistry of gamma-aminobutyrate in the cerebral and cerebellar cortex of the rat, Neuroseience, 4 (1985) 865 874. 22 Seguela, P., Geffard, M., Buijs, R.M. and Le Moal, M., Antibodies against gamma aminobutyric acid: specificity studies and immunocytochemical results, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3888 3892. 23 Somogyi, P., Hodgson, A.J., Chubb, 1.W.. Botond, P. and Endei, A., Antisera to gamma-aminobutyric acid. I1. lmmunocytochemical application to the central nervous system, J. Histochem. Cytochem., 33 (1985) 240 248. 24 Thompson, G.C., Cortez, A.M. and Man-Kit Lam, D., Localization of GABA immunoreactivity in the auditory brainstem of the guinea pig, Brain Res., 339 (1985) 119 122. 25 Wart, W.B. and Guinan, J.J. Jr.. Efferent innervation of the organ of Corti: two separate systems, Brain Res., 173 (1979) 152 -155. 26 Wenthold, R.J., Release of endogenous glutamic acid, aspartic acid and GABA from cochlear nucleus slices, Brain Res., 162 (1979)338 343.
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Immunohistochemical evidence for GABAergic cell bodies in the medial nucleus of the trapezoid body and in the lateral vestibular nucleus in the guinea pig brainstem J6r6me D u p o n t 1, Michel Geffard 2, Andr6 Calas 3 and Jean-Marie Aran 1 ;Laboratoire d'Audiologie Exp&imentale, I N S E R M U.229 and Universitb de Bordeaux H, H6pital Pellegrin, Bordeaux (France), 21nstitut de Biochimie Cellulaire et de Neurochimie, CNRS, Bordeaux (France) and 3Laboratoire de Physiologic des Interactions Cellulaires, CNRS, Universitk de Bordeaux I, Talence (France) (Received 30 October 1989; Accepted 20 December 1989) Key words." 7-Aminobutyric acid; Medial nucleus of the trapezoid body; Lateral vestibular nucleus; Optical microscopy; Immunohistochemistry; Guinea pig The presence of y-aminobutyric acid (GABA) in two brainstem nuclei is demonstrated by using a preembedding immunohistochemical procedure followed by staining intensification. Firstly, immunoreactivity was found in numerous cell bodies and profiles of the medial nucleus of the trapezoid body (MNTB). Secondly, numerous neurons including giant Deiters' cells, terminals and fibers were strongly labelled within the lateral vestibular nucleus (LVN). These observations suggest that the inhibitory part of the efferent innervation of outer hair cells in the cochlea can originate from the MNTB, and that GABAergic neurons in the LVN may contribute to information processing within this nucleus.
y-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the vertebrate central nervous system [10] and is thought to play a preponderant role in the modulation of chemical neurotransmission involved in the physiological hearing mechanism. Biochemical studies [5, 26] and other investigations using either lesion [15], labelling [19] and retrograde transport of [3H]GABA [11], or microiontophoretic application of GABA [17] have previously revealed the existence of this neurotransmitter within brainstem auditory nuclei. In order to localize GABAergic structures in the guinea pig auditory brainstem, immunohistochemical studies have been conducted using a GABA antiserum [12, 24]. GABAergic cell bodies were observed in the ventral and dorsal cochlear nuclei's suCorrespondence." J. Dupont, Laboratoire d'Audiologie Exp6rimentale, INSERM U.229, H6pital Pellegrin, Place Am61ie Raba L6on, 33076 Bordeaux, France. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
264
perficial layers, in the medial and lateral superior olive (the superior olivary complex is known to be involved in spatial localization of sound [13]), in the lateral preolivary nuclei and in the lateral nucleus of the trapezoid body (LNTB). These observations confirmed by other immunohistochemical studies using antibodies against glutamic acid decarboxylase (GAD) [9, 16], which is the synthesizing enzyme for GABA, have not detected any immunoreactivity in the cell bodies of the medial nucleus of the trapezoid body (MNTB). In the present study we have used a highly specific GABA antiserum [22] to specify, at the optical microscopy level, the localization of this module in the brainstem auditory and vestibular nuclei in the guinea pig. Not only did we observe the same labelled structures as shown in previous studies, but we have also obtained some new results concerning the existence of GABAergic celt bodies and profiles in the MNTB and in the lateral vestibular nucleus (LVN) of the brainstem in guinea pig. Adult guinea pigs were perfused through the ascending aorta with a Karnovsky fixative containing 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). The brainstem was removed and postfixed in the same fixative for 1 h at 4:'C, and then transferred to Tris-buffered saline (TBS) at pH 7.3. Then, 20-30 /~m frontal sections were obtained with a vibratome and shaken overnight in TBS for washing. The floating sections were rinsed three times for 15 rain in TBS, and processed for immunohistochemistry. In order to eliminate the unspecific adsorption of our antiserum, a presaturation step was performed using a non-immune serum (goat) 1:50 for 15 min. Then, sections were incubated with highly specific rabbit antibodies against GABA at a dilution 1:5000 in TBS containing 0.15% Triton X-100 and 1.5% normal goat serum, for 20 h at 4"C under constant shaking. The floating sections were then washed 3 times, incubated with goat anti-rabbit immunoglobulins (1:100 Immunotech) for l h at room temperature, washed 3 times again in TBS and incubated for 1 h with rabbit peroxidase-antiperoxidase complex (1:1000, Immunotech, Jackson Immuno Research Labs.). After washing 3 times in TBS, the free sections were reacted and intensified using a solution containing 0.03% diaminobenzidine (DAB), 0.3% Nickel (II) sulfate heptahydrate and 0.05% hydrogen peroxide for about 10 min at room temperature and in semi-darkness. Floating sections were then placed on slides, dried and mounted in Eukit for observation with a Leitz photomicroscope. Numerous labelled neurons were visualized throughout the entire inferior collicular nucleus, in the medial superior olive (MSO), lateral superior olive (LSO) (Figs. 1 and 2) and molecular layer of the dorsal cochlear nucleus. Moreover, GABA-posirive neurons were also localized in the medial nucleus of the trapezoid body (MNTB). Indeed, large cell bodies of the M N T B were strongly stained and numerous immunoreactive fibers and scattered punctae were observed (Fig. 3a). In a control experiment, in which primary antibodies were exhausted with 10 5 M GABA, followed by the same immunohistochemical procedure, immunoreactive profiles in the M N T B were not detected (Fig. 3b). In the LVN, (or Deiters' nucleus) (Fig. i), which is the most conspicuous part of the vestibular complex, we observed GABA-positive cell bodies of different types and
265
Fig. 1. Coronal vibratome section of the brainstem showing a general overviewof the differentimmunostained nuclei at a very low magnification. LSO, lateral superior olive; POC, preolivarycomplex;MNTB, medial nucleus of the trapezoid body; LVN, lateral vestibular nucleus; IV, fourth ventricle; VII, facial nerve. Bar = 500/tm. Fig. 2. Frontal vibratome sectionin the inferior part of the brainstem showing numerous immunostained cell bodiesand profileswithin the LSO, medialsuperior olive (MSO), POC and MNTB. Bar = 250/tm.
sizes in diameter, and also numerous labeled fibers and other profiles (Fig. 4c). Indeed this nucleus is characterized by the presence of giant cells measuring up to 60-70 /~m in diameter and of multipolar or oval [14] smaller cells. These Deiters' giant cells and the two categories of smaller cells were strongly stained (Fig. 4a). At a higher magnification ( x 40 microscope objective), we observed a great number of labelled fibers and numerous GABA positive endings surrounding the cell bodies of these different immunostained neurons (Fig. 4c). In the control experiment, all these immunoreactive structures were very weakly coloured (Fig. 4b). We have used the same GABA antiserum [22] as previously used by other authors [21, 23] which clearly demonstrated immunoreactivity in well defined GABAergic structures in the cerebellum. In the present study we have reproduced the same pattern of GABA labelling within brainstem auditory nuclei as described in other studies [12, 24]. Moreover, we have also demonstrated GABA labelling in the M N T B and in the LVN, which to our knowledge has not previously been described. The fact that these GABAergic neurons have not been previously observed might be explained on the one hand by the use of the more sensitive GABA antibody than G A D antibody for the detection of GABAergic structures [23], and on the other by the high sensitivity of our pre-embedding immunohistochemical procedure in which we have systematically used the intensification with Nickel. Indeed, it is well known that to add metal ions to the DAB medium [1, 20] as here, increases the peroxidaseantiperoxidase (PAP) method's sensitivity. It has been proposed that some Deiters' nucleus cells in the LVN in the rabbit, which could be inhibitory, may constitute part of the medial vestibulospinal tract [8]. Furthermore, the existence of GABAergic neurons within the LVN has been proposed because, following extensive lesions of cerebellar afferents (the Purkinje cell projections) 30% of the G A D activity remains. At both light and electron microsco-
266
:i~v i~¸~j!!i~ji¸ !
Fig. 3. a: frontal vibratome section in the M N T B and in the POC showing numerous intensely stained GABA-positive cell bodies. Bar = 100 ,urn. b: control experiment using G A B A antiserum preadsorbed with G A B A immunogen. No reaction can be observed through the whole MNTB. B a r = 100 pro. Fig. 4. a: coronal vibratome section in the LVN, GABA-positive magnocellular neurons (stars) can be observed in this part of the vestibular complex. These immunoreactive giant cells are Deiters' neurons of the LVN (or Deiters" nucleus). Other smaller cells are also immunostained, and they are of two morphological types: multipolar (arrows) or oval (arrowheads). Bar = 100 ,urn. b: control experiment with a preadsorbed immunoserum. No evident staining can be observed in the whole Lateral Vestibular Nucleus. B a r = I00 /lm. c: high magnification view within the region of the delimited LVN in Fig. I, showing labelled giant Deiters" neurons and other immunoreactive profiles: immunostained fibers (arrows) and GABA-positive terminals surrounding the cell body (arrowheads). Bar = 25 pro.
267 pical levels, these latter studies suggested that extrinsic and intrinsic GABAergic neurons may contribute to information processing within the LVN [7]. These findings are in agreement with our demonstration of G A B A within Deiters' giant cells and within other smaller cell bodies and terminals of this vestibular nucleus. In the MNTB numerous GABAergic neurons are described here for the first time, and previously there were many arguments in favour of their GABAergic nature. Indeed, several immunohistochemical studies have been made to investigate the distribution of G A D - [3] and GABA-like [4] immunoreactivity in the organ of Corti of guinea pig and rat. Most of the immunostaining was found in the outer hair cells' synaptic region of the third and fourth turns of the cochlea, but this labelling was also seen in the basal turns where scattered clusters of immunoreactivity were found in the outer hair cells' synapses of the first row. It has well been demonstrated by hodological methods [17, 18] that the MNTB, which constitutes in part the source of the large-neurons medial efferent system [6, 25] of the mammalian cochlea, sends projections in these basal turns. The GABAergic nature of some M N T B neurons correlates therefore well with both these hodological data and the immunohistochemistry of G A B A in the guinea pig cochlea. In order to precisely describe the GABAergic immunostaining of neurons within the LVN and the MNTB, it is now necessary to study the intracellular localization of G A B A by adapted ultrastructural procedures. This is presently in progress in our laboratory by observation of preembedding immunolabelled structures at electron microscope level, and by the subcellular detection of G A B A using the postembedding immunocytochemical technique. The authors wish to thank G. Campistron for her help with G A B A antiserum, P. Dubourg for her excellent technical advice and T. Durkin for reviewing the English of the manuscript. This study was supported in part by a M R T grant (Project 88C0558) and the 'Conseil R6gional d'Aquitaine'.
1 Adams, J.C., Heavy metal intensification of DAB-base HRP reaction product, J. Histochem. Cytochem., 29 (1981) 775. 2 Caspary,D.M., Havey, D.C. and Faingold, C.L., Effectsof microiontophoreticallyapplied glycineand GABA on neural response patterns in the cochlear nuclei, Brain Res., 172 (1979) 179 185. 3 Fex, J. and Altschuler, R.A., Glutamic acid decarboxylaseimmunoreactivityof olivocochlearneurons in the organ of Corti of guinea pig and rat, Hearing Res., 15 (1984) 123-131. 4 Fex, J., Altschuler, R.A., Kachar, B., Wenthold, R.J. and Zempel, J.M., GABA Visualized by Immunocytochemistry in the guinea pig cochlea in axons and endings of efferent neurons, Brain Res., 366 (1986) 106 117. 5 Godfrey, D.A., Carter, J.A., Lowry, O.H. and Matschinsky, F.M., Distribution of gamma aminobutyric acid, glycine,glutamate and aspartate in the cochlearnucleus of the rat, J. Histochem. Cytochem., 26 (1978) 118 126. 6 Guinan, J.J., Warr, W.B. and Norris, B.E., Topographicorganization of the olivocochlearprojections from the lateral and medial zones of the superior olivarycomplex,J. Comp. Neurol., 226 (1984) 21 27. 7 Houser, C.R., Barber, R.P. and Vaughn, J.E., lmmunocytochemicallocalization of glutamic acid decarboxylase in the dorsal lateral vestibular nucleus: evidencefor an intrinsic and extrinsic GABAergic innervation, Neurosci. Lett., 47 (1984) 213-220.
268 8 Kawasaki, K., Matsushita, A., Satoh, M. and Takagi, H., The lateral vestibular nucleus is a site for the depressant action of benzodiazepines on the crossed extensor reflex, Brain Res., 461 (1988) 282 289. 9 Moore. J.K. and Moore, R.Y., Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat, J. Comp. Neurol., 260 (1987) 157 174. I(I Mugnaini, E. and Oertel, W.H., An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 4, Elsevier, Amsterdam, 1985, pp. 436 608. 11 Ostapoff, E.M., Morest, D.K. and Potashner, S.J., Retrograde transport of tritiated GABA from the cochlear nucleus of the superior olive in guinea pig, Soc. Neurosci. Abstr., 11 (1985) 1051. 12 Peyret, D., Geffard, M. and Aran, J.M., GABA immunoreactivity in the primary nuclei of the auditory central nervous system, Hearing Res., 23 (1986) 115 121. 13 Phillips, D.P. and Brugge, J.F., Progress in neurophysiology of sound localization, Annu. Rev. Psychol., 36 (1985) 245 274. 14 Pompeiano, O. and Brodal, M., The origin of vestibulospinal fibres in cat. An experimental anatomical study with comments on the descending medial longitudinal fasciculus, Arch. ltal. Biol., 95 (1957) 166 195. 15 Potashner, S.J., Lindberg, N. and Morest, D.K., Uptake and release of GABA in the guinea pig cochlear nucleus after axotomy of cochlear and centrifugal fibers, J. Neurochem., 45 (1985) 1558 1566. 16 Roberts, R.C. and Ribak, C.E., GABAergic neurons and axon terminals in the brainstem auditory nuclei of the gerbil, J. Comp. Neurol., 258 (1987) 267 280. 17 Robertson, D., Brainstem location of efferent neurones projecting to the guinea pig cochlea, Hearing Res.. 20 (1985) 79 84. 18 Robertson, D., Anderson, C.J. and Cole, K.S., Segregation of efferent projections to different turns of the guinea pig cochlea, Hearing Res., 25 (1987) 69 76. 19 Schwartz, I.R., Autoradiographic studies of amino acid labeling of neural elements in the auditory brainstem. In D. Drescher (Ed.), Auditory Biochemistry, Thomas, Springfield, IL, 1985, pp. 258 277. 20 Scopsi, L. and Larsson, 1_..I., Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system, Histochemistry. 84 (1986) 221. 21 Seguela, P., Gamrani, H., Geffard, M., Calas, A. and Le Moal, M., Ultrastructural immunocytochemistry of gamma-aminobutyrate in the cerebral and cerebellar cortex of the rat, Neuroseience, 4 (1985) 865 874. 22 Seguela, P., Geffard, M., Buijs, R.M. and Le Moal, M., Antibodies against gamma aminobutyric acid: specificity studies and immunocytochemical results, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3888 3892. 23 Somogyi, P., Hodgson, A.J., Chubb, 1.W.. Botond, P. and Endei, A., Antisera to gamma-aminobutyric acid. I1. lmmunocytochemical application to the central nervous system, J. Histochem. Cytochem., 33 (1985) 240 248. 24 Thompson, G.C., Cortez, A.M. and Man-Kit Lam, D., Localization of GABA immunoreactivity in the auditory brainstem of the guinea pig, Brain Res., 339 (1985) 119 122. 25 Wart, W.B. and Guinan, J.J. Jr.. Efferent innervation of the organ of Corti: two separate systems, Brain Res., 173 (1979) 152 -155. 26 Wenthold, R.J., Release of endogenous glutamic acid, aspartic acid and GABA from cochlear nucleus slices, Brain Res., 162 (1979)338 343.
