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GABA immunoreactivity in the primary nuclei of the auditory central nervous system
Nearing Research, 23 (1986) 115-121 Elsevier
HRR
115
00788
GABA immunoreactivity
in the primary nuclei of the auditory central nervous system
D. Peyret, M. Geffard
’
and J.-M. Arm
INSERM U 229. L_uhoratoired’Audiologie ExpBnmentale, Univrr.Gti de Bordeaux II. HZpiial Peilegrin. _?XVf, Bordeaux. cmd ’ Insritui de Biochimir Cellulaire et de Neurochimie, CNRS et INSERM, I rue CurniNe Saint-Suens 33077 Bordeuux. France (Received
9 October
1985; accepted
23 January
1986)
Structures containing gamma-ammo butyric acid (GABA) were investigated in the guinea pig cochlear nuclei and superior olivary complexes hy means of an immunohist~hemical procedure using an antibody directed against GABA. lmmunor~activity was observed in cell bodies of the superficial layers of the ventral and dorsal cochlear nuclei, in lateral superior olive, in some neurons of the medial superior olive, in lateral preolivary nuclei and in the lateral nucleus of the trapezoid body. Fibers and profiles exhibiting GABA immunoreactivity were found in almost all regions of the lower auditory pathways. ‘The abundance of GABA in these regions indicates an important role of this inhibitory amino-acid in the auditory brainstem. GABA,
cochlear
nuclei, superior
olives. trapezoid
nuclei, immunohistochemistry.
Introduction Gamma-amino butyric acid (GABA) is one of the main inhibitory trans~tters in vertebrate central nervous system (Krnjenic, 1974; Mugnaini and Oertel, 1985). In the cochlear nucleus (CN), the structure which first receives nervous informations from the cochlea, the presence and action of GABA have been investigated consistently. The inhibitory effect of GABA on spontaneous and tone-evoked activities of neurons from the ventral and dorsal parts of the CN has been reported by iontophoreti~ applications (Caspary et al., 1979). Histological staining for GABA-transaminase, an enzyme directly involved in the catabolism of GABA and supposed to mark GABAceptive neurons, showed enzymatic activity in cells from several parts of the CN, with a predominance in the anteroventral area (Davies, 1975; Tachibana and Kuriyama, 1974). Measurement of the activity of the GABA synthesizing enzyme glutamate decarboxylase (GAD) indicated a relatively high level in the CN (Fex and Wenthold, 1976). Quantitative distribution of GABA among different areas of the CN demonstrated greater concentrations in the dorsal part than in the ventral part of the 037~-5955/86/$03.50
(0 1986 Elsevier Science Publishers
guinea
pig
nucleus (Tachibana and Kuriyama, 1974). especially in the superficial layer (Godfrey et al., 1978). GAD-like immunoreactivity has recently been carefully investigated in the rat CN by Mugnaini (1985). This author describes presumed GABAergic neurons, boutons and fibers in the two superficial layers of the dorsal CN and in small neurons scattered in the superficial layer of the ventral CN. Little is known about the possible involvement of GABA in the superior olivary complex (Caspary et al., 1979). This structure is known to be involved in spatial localization of sound (Phillips and Brugge, 1985). It receives indirect bilateral input from each cochlea (Harrison and Howe. 1974). In the cochlea, GAD-like immunoreactivity has been demonstrated in a few efferent fibers and synaptic regions of the outer hair cells (Fex and Altschuler, 1984). The cells of origin of these fibers could be located in the medial superior olive (MSO) and the trapezoid body (Warr and Guinan. 1979). As GABA appears to be an important neurotrans~tter in the lower auditory pathway, we further investigated the localization of GABAergic structures in the auditory brainstem of guinea pig
B.V.
Division)
116
using a specific GABA antiserum. It is at present the most sensitive and direct technique. Materials and Methods Antiserum The raising and specificity studies of GABA antiserum have been previously described (Seguela et al., 1984). In brief, conjugates of GABA-glutaraldehyde-protein were injected to rabbits for 4 months. The titer and specificity of the antisera were studied by radioimmunological tests performed in equilibrium dialysis using a radiolabelled ligand mimicking the antigen present in immunogens and tissues. Fixation and tissue sections Nine pigmented female guinea pigs (350-400 g body wt) were used. The animals were anesthetized with a solution of ketamine chlorhydrate (17 mg/kg) and xylazine chlorhydrate (4 mg/kg). Three hundred ml of fixative solution containing 5% glutaraldehyde and 1% sodium metabisulfite in 0.1 M sodium cacodylate, pH 7.4, were perfused for 2 min through the ascending aorta. After removing, the brains were post-fixed in the same solution for 1 h at 4°C and then immersed overnight in 0.1 M sodium cacodylate containing 15% sucrose. Twenty-five pm cryotome sections or 50 pm vibratome sections were cut, washed three times for 15 min with 0.1 M Tris buffer containing 0.9% sodium chloride (TBS) and processed for immunohistochemistry. Immunohistochemical procedure Floating sections were incubated with antiGABA serum diluted l/5000 in TBS containing 0.15% Triton X-100 for 18 h at 4°C under constant shaking, then washed three times for 10 min in TBS, incubated with l/300 biotinylated donkey anti-rabbit immunoglobulins (Amersham) for 1 l/2 h at room temperature, washed three times in TBS and incubated for 30 min with l/400 streptaFig. 1. Coronal cryotome section into the superficial are seen (arrows). Bar = 50 pm.
vidin-peroxidase complex (Amersham). After washing three times in TBS, peroxidase activity was revealed in a TBS solution containing 0.025% diaminobenzidine and 0.01% hydrogen peroxide for 7-10 mm at room temperature. In control sections GABA antiserum was replaced by pre-immune rabbit serum, or immune serum containing different concentrations of immunogen or analogue. Results Control experiments No immunostaining was observed when pre-immune rabbit serum was used instead of GABA antiserum. With GABA antiserum exhausted with 0.1 PM of GABA immunogen, all staining disappeared, whereas no decrease in staining was observed using 10 PM of /3-alanine. Cochlear nucleus In the ventral cochlear nucleus (VCN), a few cell bodies showing GABA-like immunoreactivity (GABA-LI) were seen within the superficial layer (superficial granular layer), from the first anterior sections (Fig. 1). Rarely, GABAergic neurons were observed in the medial border of the VCN, close to the trapezoid body. GABAergic punctiform structures, which may correspond to terminal boutons and dendrites, occurred in high density in the superficial layer of the VCN and more scattered around large cell bodies remaining unstained between branches of cochlear nerve fibers (Fig. 2A). In the deep region of the CN, GABA-LI was often observed in myelinated axons clustered in fascicles (dorsal region) (Fig. 2B) or as isolated fibers into unstained bundles (ventral region). The great majority of GABAergic fibers was included in the upper descending branch of the cochlear nerve. More numerous cell bodies exhibiting GABALI were seen in the dorsal cochlear nucleus (EN). Two types of GABA-positive neurons could be
layer of the very anterior
part of the VCN. Several immunoreactive
cell bodies
Fig. 2. Sagittal cryotome section of the CN. (A) Region of inlet of the cochlear nerve. Unstained neurons surrounded terminal boutons (arrowheads) are observed within the cochlear fibers (CF). (B) Fascicles of GABAergic fibers (arrows) as part of the descending branch. However, ascending fibers (arrowheads) remain unstained. Bars = 50 pm.
with labelled are observed
Fig. 3. Sagittaf cryotome section in the DC??. (A) Small spherical cells (arrowheads) and larger ovoid cells (arrow) exhibiting GABA-LI. Note the large number of immunoreactive profiles in the surrounding tissue. Bar = 50 pm. (B) Density of GABA immunoreactive structures is clearly decreasing from the periphery (granular layer) to the center of the nucleus. Arrowheads indicate GABA immunoreactive cell bodies. Bar = 100 pm.
Fig. 4. Goronai cryotome section observed. Bar = 100 pm” Fig. 5. Coronal Immunoreactive
through
the MSO. Fusiform
cells (arrows)
and spherical
vibratome section into the region of the LSO. Arrowheads fibers are seen in the capsule of the LSO. Bar = tC0 pm.
indicate
cells (arrowbeads)
some
of tbe GABA
showing
positive
GABA-LX
art
cell bodies.
119
distinguished with regard to their size: small spherical cells (10 pm) located predominantly in the superficial part of the DCN (molecular layer) and large ovoid neurons (17 km) in the same and in a deeper area (Fig. 3A). A small group of medium sized GABAergic neurons was located in the anterior and dorsal region of the DCN, just under the inferior cerebellar peduncle. In all regions where GABA-positive cell bodies were seen, a large number of punctually stained structures could be observed with the higher density in the granular layer (Fig. 3B). Superior
oliuary complex
In the MS0 nucleus. a few neurons exhibiting GABA-LI were observed. They were homogeneously scattered into the nucleus and seemed to be of two morphological categories (Fig. 4). Some of them were fusiform cells with short and thick dendrites at both ends. Their transverse diameters were usually parallel to each other and perpendicular to the dorso-ventral plane of the MSO. The others were more spherical and without any visible extension. Numerous GABAergic cellular bodies were observed in the lateral superior olive (LSO) (Fig. 5). They were more often elongated neurons having sometimes a thick dendrite directed predominantly to the center of the nucleus. Axons exhibiting GABA-LI were found throughout the fibrous capsule of the LSO. GABA immunoreactivity appeared also in some neurons from the lateral preolivary nucleus (LPO), surrounded by fibers of the trapezoid body. The lateral nucleus of the trapezoid body (LTz) appeared very clearly in treated sections because of a very high density of immunoreactive punctiform profiles. Many fibers running within the trapezoid body could also be observed passing through the LTz (Fig. 6). GABA-positive cell bodies were seen in all the nucleus but were more numerous in the posterior part. In other regions of the superior olivary complex, i.e. medial retroolivary region (MRO) and medial nucleus of the trapezoid body (MNTz), no GABAergic cell bodies were seen, except for some Fig. 6. Coronal GABA positive
cryotome section through cell bodies. Bar = 50 pm.
the LTz. Numerous
weakly immunoreactive neurons in the MRO. In contrast, a great number of boutons and fibers without apparent preferential orientation can be observed in these two nuclei. Discussion With the intention of comparing our results with previous works using GABA antibodies from other sources we examined the cerebellum. GABA-LI was observed in Golgi, basket and stellate cells and in terminal boutons around Purkinje cells. This is in very good agreement with former reports (Somogyi et al., 1985; Storm-Mathisen et al., 1983) supporting the results of the biochemical specificity studies. Cochlear
nucleus
In the CN, our results using GABA antiserum are consistent with previous GAD measurements and immunocytochemical studies of GAD and GABA (Godfrey et al., 1978; Mugnaini, 1985; Tachibana and Kuriyama, 1974; Thompson et al., 1985). GABA immunoreactive structures are mainly localized in the superficial part of the nucleus, giving a great density of fibers and boutons. More numerous GABAergic cell bodies were seen in the DCN than in the VCN. In the deep region of the VCN, GABA-LI appeared in terminal boutons surrounding large neurons presumably identified as multipolar cells. previously reported for high GABA-transaminase activity (Davies, 1975). Immunoreactive fibers within the descending cochlear branch had not yet been described by previous authors using GABA antiserum (Ottersen and Storm-Mathisen, 1984; Thompson et al., 1985). The CN is the first relay integrating the information coming from the cochlear nerve. The explanation of the different discharge patterns of the CN neurons requires neuronal interactions between excitatory and inhibitory components (Evans, 1975: Comis and Whitfield, 1968; Comis, 1970; Mast, 1970). DCN neurons receive the most important inhibitory influences within the CN complex, therefore the abundance of GABA containing structures in this GABA
immunoreactive
profiles
can be observed.
Arrows
indicate
120
region can have a direct functional assuming that GABA is inhibitory.
significance,
Superior olivary complex The main fact in the MS0 is the heterogeneous immunoreactivity within the spindle shaped neurons. These cells have been very well characterized by Kiss and Majorossy (1983) as a homogeneous population receiving a majority of contralateral input from the VCN, and ipsilateral input from the nucleus of the trapezoid body. Our results, and those of Thompson et al. (1985), reveal a subpopulation within fusiform cells of the MSO, not described with GAD immunohistochemistry (Mugnaini and Oertel, 1985) and thus suggest functional differences in this morphological cell category. Although LSO neurons are known to project on central nucleus of inferior colliculus via the lateral lemniscus (Harrison and Howe, 1974), LSO is also the source of the lateral efferent system (Altschuler et al., 1983; Robertson, 1985; Warr and Guinan, 1979). Since part of this innervation is likely to be GABAergic as demonstrated using GAD antiserum (Fex and Altschuler, 1984) it is possible that some of the GABA positive neurons of the LSO seen in this study send projections in the inner spiral bundle. As enkephalin and acetylcholine are already probable neurotransmitters in the LSO (Altschuler et al., 1984), GABA seems to be a third potential candidate. Because GABAergic neurons are homogeneously distributed within LSO and MSO, no correlation between their localization and the tonotopical organization of these nuclei can be made. No immunoreactivity could be observed in cell bodies of the MNTz in this study. This is surprising because (a) the MNTz is supposed to be the main source of efferent innervation of the outer hair cells (Warr and Guinan, 1979) and (b) part of these fibers are presumed to be GABAergic (Fex and Altschuler, 1984) as preliminary observations using GABA antiserum in the cochlea also showed. This data suggest another localization for neurons projecting on synaptic regions of the outer hair cells. In the MRO, MNTz and LTz, many GABA immunoreactive fibers and profiles were observed.
confirming the finding with GAD (Mugnaini and Oertel, 1985). However, optical observations did not allow us to determine whether these structures end or pass through the nuclei. The present work demonstrates that GABA immunoreactivity exists in cell bodies or fibers in all nuclei investigated. The results are in good agreement with previous reports using direct and indirect techniques and confirm that GABA should play an important role in neuronal interactions in the central auditory nervous system, probably as an inhibitory neurotransmitter. References Ahschuler, R.A., Parakkal, M.H. and Fex, J. (1983): Localization of enkephahn-like immunoreactivity in acetylcholinesterase-positive cells in the guinea pig lateral superior olivary complex that project to the cochlea. Neuroscience 9, 621-630. Altschuler, R.A., Fex, J., Parakkal, M.H. and Eckenstein, F. (1984): Colocahzation of enkephalin-like and cholin acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig. J. Histochem. Cytochem. 32, 8399843. Caspary, D.M., Havey, D.C. and Faingold, C.L. (1979): Effects of microiontophoretically applied glycine and GABA on neural response patterns in the cochlear nuclei. Brain Res. 172, 1799185. Comis, SD. (1970): Centrifugal inhibitory processes affecting neurons in the cat co&ear nucleus. J. Physiol. (London) 210, 751-760. Comis, S.D. and Whitfield, 1.C. (1968): Influence of centrifugal pathways on unit activity in the cochlear nucleus. J. Neurophysiol. 31, 62-68. Davies, W.E. (1975): The distribution of GABA transaminasecontaining neurones in the cat cochlear nucleus. Brain Res. 83, 27-33. Evans, E.F. (1975): Co&fear nerve and cochlear nucleus. in: Handbook of Sensory Physiology, vol. 2, pp. l-108: Editors: W.D. Keidel and W.D. Neff. Springer-Verlag, Berlin. Fex, J. and Altschuler, R.A. (1984): Glutamic acid decarboxylase immunoreactivity of the olivocochlear neurons in the organ of Corti of the guinea pig and rat. Hearing Res. 15. 123-131. Fex, J. and Wenthold, R.J. (1976): Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlear nucleus of the guinea pig. Brain Res. 109. 575-585. Godfrey, D.A., Carter, J.A., Lowry, O.H. and Matschinsky. F.M. (1978): Distribution of gamma-aminobutytic acid, glycine, glutamate and aspartate in the co&Lear nucleus of the rat. J. Histochem. Cytochem. 26, 118-126. Harrison, J.M. and Howe, M.E. (1974): Anatomy of the afferent auditory nervous system of mammals. In: Handbook of Sensory Physiology, vol. I, pp. 287-294. Editors: W.D. Keidel and W.D. Neff. Springer-Verlag, Berlin.
121
Kiss. A. and Majorossy, K. (1983): Neuron morphology and synaptic architecture in the medial superior olivary nucleus. Exp. Brain Res. 52, 315-327. KrnJemc. D. (1974): Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54, 418-540. Mast. ‘1.E. (1970): Binaural interaction and contralateral inhibition in dorsal cochlear nucleus of the chinchilla. J. Neurophysiol. 33. 108~115. Mugnaini, E. (1985): GABA neurons in the superficial layer of the rat dorsal cochlear nucleus: light and electron microscopic immunocytochemistry. J. Comp. Neurol. 235, 61-X1. Mugnaini. E. and Gertel. W.H. (1985): An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In: Handbook of Chemical Neuroanatomy, vol. 4, pp. 4366608. Editors: A. Bjorklund and T. Hokfelt. Elsevier Science Publishers, Amsterdam. Ottersen, O.P. and Storm-Mathisen, J. (1984): Neurons contaming or accumulating transmitter amino acids. In: Handbook of Chemical Neuroanatomy. vol. 3. pp. 141-246. Editors: A. Bjorklund and T. Hokfeh. Elsevier Science Publishers. Amsterdam. Phillips. D.P. and Brugge. J.F. (1985): Progress in neurophysiology of sound localization. Annu. Rev. Psychol. 36. 245 214.
Robertson, D. (1985): Brainstem location of efferent neurones projectmg to the guinea pig cochlea. Hearing Res. 20. 79984. Seguela, P.. Geffard, M.. Buijs. R.M. and Le Moal. M. (1984): Antibodies against gamma-aminobutyric acid: spectficity studies and immunocytochemical results. Proc. Natl. Acad. Sci. USA 81. 3888-3892. Somogyi. P.. Hodgson. A.J., Chubb, I.W., Botond. P. and Erdei. A. (1985): Antisera to gamma-aminobutyrtc acid. II. lmmunocytochemical application to the central nervous system. J. Histochem. Cytochem. 33, 240-248. Storm-Mathisen, J.. Leknes. A.K.. Bore, A.T., Vaaland, J.L.. Edminson, P., Haug. F.-MS. and Ottersen. G.P. (1983): First visualisation of glutamate and GABA in neuronea by immunocytochemistry. Nature 301. 517.520. Tachibana. M. and Kuriyama, K. (1974): Gamma-aminobutyric acid in the lower auditory pathway of the guinea pig. Brain Res. 69, 370-374. Thompson. G.C., Cortez. A.M. and Man-Kit Lam, D. (19X5): Localization of GABA immunoreactiwty in the auditory brainstem of the guinea pig. Brain Res. 339. 119~122. Warr, W.B. and Guinan. J.J. Jr. (1979): Efferent innervation of the organ of Corti: two separate systems. Bratn Rea. 173. 152-155.
HRR
115
00788
GABA immunoreactivity
in the primary nuclei of the auditory central nervous system
D. Peyret, M. Geffard
’
and J.-M. Arm
INSERM U 229. L_uhoratoired’Audiologie ExpBnmentale, Univrr.Gti de Bordeaux II. HZpiial Peilegrin. _?XVf, Bordeaux. cmd ’ Insritui de Biochimir Cellulaire et de Neurochimie, CNRS et INSERM, I rue CurniNe Saint-Suens 33077 Bordeuux. France (Received
9 October
1985; accepted
23 January
1986)
Structures containing gamma-ammo butyric acid (GABA) were investigated in the guinea pig cochlear nuclei and superior olivary complexes hy means of an immunohist~hemical procedure using an antibody directed against GABA. lmmunor~activity was observed in cell bodies of the superficial layers of the ventral and dorsal cochlear nuclei, in lateral superior olive, in some neurons of the medial superior olive, in lateral preolivary nuclei and in the lateral nucleus of the trapezoid body. Fibers and profiles exhibiting GABA immunoreactivity were found in almost all regions of the lower auditory pathways. ‘The abundance of GABA in these regions indicates an important role of this inhibitory amino-acid in the auditory brainstem. GABA,
cochlear
nuclei, superior
olives. trapezoid
nuclei, immunohistochemistry.
Introduction Gamma-amino butyric acid (GABA) is one of the main inhibitory trans~tters in vertebrate central nervous system (Krnjenic, 1974; Mugnaini and Oertel, 1985). In the cochlear nucleus (CN), the structure which first receives nervous informations from the cochlea, the presence and action of GABA have been investigated consistently. The inhibitory effect of GABA on spontaneous and tone-evoked activities of neurons from the ventral and dorsal parts of the CN has been reported by iontophoreti~ applications (Caspary et al., 1979). Histological staining for GABA-transaminase, an enzyme directly involved in the catabolism of GABA and supposed to mark GABAceptive neurons, showed enzymatic activity in cells from several parts of the CN, with a predominance in the anteroventral area (Davies, 1975; Tachibana and Kuriyama, 1974). Measurement of the activity of the GABA synthesizing enzyme glutamate decarboxylase (GAD) indicated a relatively high level in the CN (Fex and Wenthold, 1976). Quantitative distribution of GABA among different areas of the CN demonstrated greater concentrations in the dorsal part than in the ventral part of the 037~-5955/86/$03.50
(0 1986 Elsevier Science Publishers
guinea
pig
nucleus (Tachibana and Kuriyama, 1974). especially in the superficial layer (Godfrey et al., 1978). GAD-like immunoreactivity has recently been carefully investigated in the rat CN by Mugnaini (1985). This author describes presumed GABAergic neurons, boutons and fibers in the two superficial layers of the dorsal CN and in small neurons scattered in the superficial layer of the ventral CN. Little is known about the possible involvement of GABA in the superior olivary complex (Caspary et al., 1979). This structure is known to be involved in spatial localization of sound (Phillips and Brugge, 1985). It receives indirect bilateral input from each cochlea (Harrison and Howe. 1974). In the cochlea, GAD-like immunoreactivity has been demonstrated in a few efferent fibers and synaptic regions of the outer hair cells (Fex and Altschuler, 1984). The cells of origin of these fibers could be located in the medial superior olive (MSO) and the trapezoid body (Warr and Guinan. 1979). As GABA appears to be an important neurotrans~tter in the lower auditory pathway, we further investigated the localization of GABAergic structures in the auditory brainstem of guinea pig
B.V.
Division)
116
using a specific GABA antiserum. It is at present the most sensitive and direct technique. Materials and Methods Antiserum The raising and specificity studies of GABA antiserum have been previously described (Seguela et al., 1984). In brief, conjugates of GABA-glutaraldehyde-protein were injected to rabbits for 4 months. The titer and specificity of the antisera were studied by radioimmunological tests performed in equilibrium dialysis using a radiolabelled ligand mimicking the antigen present in immunogens and tissues. Fixation and tissue sections Nine pigmented female guinea pigs (350-400 g body wt) were used. The animals were anesthetized with a solution of ketamine chlorhydrate (17 mg/kg) and xylazine chlorhydrate (4 mg/kg). Three hundred ml of fixative solution containing 5% glutaraldehyde and 1% sodium metabisulfite in 0.1 M sodium cacodylate, pH 7.4, were perfused for 2 min through the ascending aorta. After removing, the brains were post-fixed in the same solution for 1 h at 4°C and then immersed overnight in 0.1 M sodium cacodylate containing 15% sucrose. Twenty-five pm cryotome sections or 50 pm vibratome sections were cut, washed three times for 15 min with 0.1 M Tris buffer containing 0.9% sodium chloride (TBS) and processed for immunohistochemistry. Immunohistochemical procedure Floating sections were incubated with antiGABA serum diluted l/5000 in TBS containing 0.15% Triton X-100 for 18 h at 4°C under constant shaking, then washed three times for 10 min in TBS, incubated with l/300 biotinylated donkey anti-rabbit immunoglobulins (Amersham) for 1 l/2 h at room temperature, washed three times in TBS and incubated for 30 min with l/400 streptaFig. 1. Coronal cryotome section into the superficial are seen (arrows). Bar = 50 pm.
vidin-peroxidase complex (Amersham). After washing three times in TBS, peroxidase activity was revealed in a TBS solution containing 0.025% diaminobenzidine and 0.01% hydrogen peroxide for 7-10 mm at room temperature. In control sections GABA antiserum was replaced by pre-immune rabbit serum, or immune serum containing different concentrations of immunogen or analogue. Results Control experiments No immunostaining was observed when pre-immune rabbit serum was used instead of GABA antiserum. With GABA antiserum exhausted with 0.1 PM of GABA immunogen, all staining disappeared, whereas no decrease in staining was observed using 10 PM of /3-alanine. Cochlear nucleus In the ventral cochlear nucleus (VCN), a few cell bodies showing GABA-like immunoreactivity (GABA-LI) were seen within the superficial layer (superficial granular layer), from the first anterior sections (Fig. 1). Rarely, GABAergic neurons were observed in the medial border of the VCN, close to the trapezoid body. GABAergic punctiform structures, which may correspond to terminal boutons and dendrites, occurred in high density in the superficial layer of the VCN and more scattered around large cell bodies remaining unstained between branches of cochlear nerve fibers (Fig. 2A). In the deep region of the CN, GABA-LI was often observed in myelinated axons clustered in fascicles (dorsal region) (Fig. 2B) or as isolated fibers into unstained bundles (ventral region). The great majority of GABAergic fibers was included in the upper descending branch of the cochlear nerve. More numerous cell bodies exhibiting GABALI were seen in the dorsal cochlear nucleus (EN). Two types of GABA-positive neurons could be
layer of the very anterior
part of the VCN. Several immunoreactive
cell bodies
Fig. 2. Sagittal cryotome section of the CN. (A) Region of inlet of the cochlear nerve. Unstained neurons surrounded terminal boutons (arrowheads) are observed within the cochlear fibers (CF). (B) Fascicles of GABAergic fibers (arrows) as part of the descending branch. However, ascending fibers (arrowheads) remain unstained. Bars = 50 pm.
with labelled are observed
Fig. 3. Sagittaf cryotome section in the DC??. (A) Small spherical cells (arrowheads) and larger ovoid cells (arrow) exhibiting GABA-LI. Note the large number of immunoreactive profiles in the surrounding tissue. Bar = 50 pm. (B) Density of GABA immunoreactive structures is clearly decreasing from the periphery (granular layer) to the center of the nucleus. Arrowheads indicate GABA immunoreactive cell bodies. Bar = 100 pm.
Fig. 4. Goronai cryotome section observed. Bar = 100 pm” Fig. 5. Coronal Immunoreactive
through
the MSO. Fusiform
cells (arrows)
and spherical
vibratome section into the region of the LSO. Arrowheads fibers are seen in the capsule of the LSO. Bar = tC0 pm.
indicate
cells (arrowbeads)
some
of tbe GABA
showing
positive
GABA-LX
art
cell bodies.
119
distinguished with regard to their size: small spherical cells (10 pm) located predominantly in the superficial part of the DCN (molecular layer) and large ovoid neurons (17 km) in the same and in a deeper area (Fig. 3A). A small group of medium sized GABAergic neurons was located in the anterior and dorsal region of the DCN, just under the inferior cerebellar peduncle. In all regions where GABA-positive cell bodies were seen, a large number of punctually stained structures could be observed with the higher density in the granular layer (Fig. 3B). Superior
oliuary complex
In the MS0 nucleus. a few neurons exhibiting GABA-LI were observed. They were homogeneously scattered into the nucleus and seemed to be of two morphological categories (Fig. 4). Some of them were fusiform cells with short and thick dendrites at both ends. Their transverse diameters were usually parallel to each other and perpendicular to the dorso-ventral plane of the MSO. The others were more spherical and without any visible extension. Numerous GABAergic cellular bodies were observed in the lateral superior olive (LSO) (Fig. 5). They were more often elongated neurons having sometimes a thick dendrite directed predominantly to the center of the nucleus. Axons exhibiting GABA-LI were found throughout the fibrous capsule of the LSO. GABA immunoreactivity appeared also in some neurons from the lateral preolivary nucleus (LPO), surrounded by fibers of the trapezoid body. The lateral nucleus of the trapezoid body (LTz) appeared very clearly in treated sections because of a very high density of immunoreactive punctiform profiles. Many fibers running within the trapezoid body could also be observed passing through the LTz (Fig. 6). GABA-positive cell bodies were seen in all the nucleus but were more numerous in the posterior part. In other regions of the superior olivary complex, i.e. medial retroolivary region (MRO) and medial nucleus of the trapezoid body (MNTz), no GABAergic cell bodies were seen, except for some Fig. 6. Coronal GABA positive
cryotome section through cell bodies. Bar = 50 pm.
the LTz. Numerous
weakly immunoreactive neurons in the MRO. In contrast, a great number of boutons and fibers without apparent preferential orientation can be observed in these two nuclei. Discussion With the intention of comparing our results with previous works using GABA antibodies from other sources we examined the cerebellum. GABA-LI was observed in Golgi, basket and stellate cells and in terminal boutons around Purkinje cells. This is in very good agreement with former reports (Somogyi et al., 1985; Storm-Mathisen et al., 1983) supporting the results of the biochemical specificity studies. Cochlear
nucleus
In the CN, our results using GABA antiserum are consistent with previous GAD measurements and immunocytochemical studies of GAD and GABA (Godfrey et al., 1978; Mugnaini, 1985; Tachibana and Kuriyama, 1974; Thompson et al., 1985). GABA immunoreactive structures are mainly localized in the superficial part of the nucleus, giving a great density of fibers and boutons. More numerous GABAergic cell bodies were seen in the DCN than in the VCN. In the deep region of the VCN, GABA-LI appeared in terminal boutons surrounding large neurons presumably identified as multipolar cells. previously reported for high GABA-transaminase activity (Davies, 1975). Immunoreactive fibers within the descending cochlear branch had not yet been described by previous authors using GABA antiserum (Ottersen and Storm-Mathisen, 1984; Thompson et al., 1985). The CN is the first relay integrating the information coming from the cochlear nerve. The explanation of the different discharge patterns of the CN neurons requires neuronal interactions between excitatory and inhibitory components (Evans, 1975: Comis and Whitfield, 1968; Comis, 1970; Mast, 1970). DCN neurons receive the most important inhibitory influences within the CN complex, therefore the abundance of GABA containing structures in this GABA
immunoreactive
profiles
can be observed.
Arrows
indicate
120
region can have a direct functional assuming that GABA is inhibitory.
significance,
Superior olivary complex The main fact in the MS0 is the heterogeneous immunoreactivity within the spindle shaped neurons. These cells have been very well characterized by Kiss and Majorossy (1983) as a homogeneous population receiving a majority of contralateral input from the VCN, and ipsilateral input from the nucleus of the trapezoid body. Our results, and those of Thompson et al. (1985), reveal a subpopulation within fusiform cells of the MSO, not described with GAD immunohistochemistry (Mugnaini and Oertel, 1985) and thus suggest functional differences in this morphological cell category. Although LSO neurons are known to project on central nucleus of inferior colliculus via the lateral lemniscus (Harrison and Howe, 1974), LSO is also the source of the lateral efferent system (Altschuler et al., 1983; Robertson, 1985; Warr and Guinan, 1979). Since part of this innervation is likely to be GABAergic as demonstrated using GAD antiserum (Fex and Altschuler, 1984) it is possible that some of the GABA positive neurons of the LSO seen in this study send projections in the inner spiral bundle. As enkephalin and acetylcholine are already probable neurotransmitters in the LSO (Altschuler et al., 1984), GABA seems to be a third potential candidate. Because GABAergic neurons are homogeneously distributed within LSO and MSO, no correlation between their localization and the tonotopical organization of these nuclei can be made. No immunoreactivity could be observed in cell bodies of the MNTz in this study. This is surprising because (a) the MNTz is supposed to be the main source of efferent innervation of the outer hair cells (Warr and Guinan, 1979) and (b) part of these fibers are presumed to be GABAergic (Fex and Altschuler, 1984) as preliminary observations using GABA antiserum in the cochlea also showed. This data suggest another localization for neurons projecting on synaptic regions of the outer hair cells. In the MRO, MNTz and LTz, many GABA immunoreactive fibers and profiles were observed.
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