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GABA-like immunoreactivity in the chick vestibular end organs
Brain Research, 418 (1987) 383-387 Elsevier
383
BRE 22443
GABA-like immunoreactivity in the chick vestibular end organs Shin-ichi Usami, Makoto Igarashi and Glenn C. Thompson Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) (Accepted 12 May 1987)
Key words: y-Aminobutyric acid (GABA); Vestibular end organ; Chick
7-Aminobutyric acid (GABA)-like immunoreactivity in the chick vestibular endorgans was examined using an antiserum against GABA coupled with glutaraldehyde to bovine serum albumin. GABA-Iike immunoreactivity was confined to the cytoplasm of the hair cells in both cristae and maculae. GABA-Iike immunoreactive cells were evenly distributed throughout the sensory epithelia, and no difference existed between type I and type II hair cells. The results provide evidence that GABA-Iike immunoreactivity is localized to sensory cells and raises the possibility that GABA may serve as an afferent neurotransmitter in the chick vestibular end organs. Evidence has accumulated that acetylcholine (ACh) may play a role in the efferent transmission from central fibers to hair cells 4'5"12'16A8,but such evidence for a neurotransmitter between the hair cells and afferent fibers is lacking. Several putative substances including G A B A 6'7'9'14"15"17"18, glutamate 1 3,1~ and the catecholamines 22 have been considered as afferent neurotransmitters; among these candidates, G A B A has fulfilled the neurotransmitter criteria. G A B A synthesis from radioactively labeled precursors has been reported in lower vertebrates 9 and chick 14,15, afferent neural responsivity has been inhibited by a G A B A blocker 6'7'9, and high affinity G A B A uptake has been reported in the chick vestibule 17. These data suggest the existence of postsynaptic G A B A receptors in vestibular endorgans. However, there have been no reports describing immunocytochemical localization of G A B A in the vestibular end organs. Accordingly, we have immunohistochemically investigated the chick vestibular end organs. Young chicks (Gallus domesticus) 2, 10 and 20 days old (post-hatch) were used. The animals were anesthetized with an overdose of sodium pentobarbital (Nembutal, 100 mg/kg, i.p.) and perfused intracardially with fixative (4% paraformaldehyde, 0.1%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.2). The vestibular end organs were removed from the bony shell, and postfixed with 4% paraformaldehyde alone (in 0.1 M phosphate buffer, pH 7.2) for 3 h at 4 °C. They were stored at 4 °C in 10% sucrose-phosphate buffer for 3 h and then placed in 30% sucrosephosphate buffer overnight. Serial cryostat sections (15 /~m thick) were cut and placed onto gelatincoated slides. The A B C method was used for immunohistochemical study. Slide-mounted sections were incubated sequentially in: (1) 2% normal goat serum (in PBS with 0.3% Triton X-100, for 30 rain at room temperature); (2) antiserum against G A B A conjugated to bovine serum albumin (Immuno Nuclear Corp., 1/3000-4000, in PBS with 0.3% Triton-X, overnight at 4 °C); (3) biotinylated goat anti-rabbit IgG (Vector Labs., for 30 rain); (4) Vectastain reagent (Vector Labs., for 30 min); and (5) D A B / H 2 0 2, for 12 rain. Three rinses with PBS for 5 rain each were used between incubations. After peroxidase reaction, the sections were counterstained with Cresyl violet and observed with light microscopy. As a control, the primary antiserum was preabsorbed with an excess of G A B A - g l u t a r a l d e h y d e BSA conjugated complex and substituted for anti-
Correspondence: S. Usami. Present address: Department of Otorhinolaryngology, Hirosaki University, School of Medicine. 5 Zaifu Cho, Hirosaki (036), Japan. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
384 G A B A - B S A . The specificity of the G A B A antiserum used in the present study has been tested and no cross-reactivity was found with glutamate, taurine, aspartate, glycine, alanine, tyrosine, enkephalin or substance p~3. F u r t h e r m o r e , we have obtained positive staining of known G A B A e r g i c elements in the guinea pig 19 and squirrel m o n k e y cerebellum, brainstem 2° and cochlea 21 using the same antiserum. In order to identify efferent c o m p o n e n t s of the peripheral vestibular system, some slide-mounted sections were processed for the histochemical local-
A
ization of acetylcholinesterase (ACHE) 2~ G A B A - I i k e immunoreactivity was found in the hair cells of the vestibular epithelium in all chicks. In both cristae and maculae (Figs. t A , C and 2 A - C ) , G A B A - I i k e immunoreactivity was clearly restricted to the cytoplasm of the hair cells while supporting cells were unstained. The distribution of labeled cells was uniform throughout the sensory epithelia of both end organs. No immunoreactivity was observed i n the control specimens (Fig. 1B). The e m i n e n t i a cruciata, which divides the cristae of vertical .semicircu-
B
|
Fig. 1. GABA-Iike immunoreactivit~ and AChE staining in the chick cristae ampullares. A: crista ampuUarisof lateral semicircUlarcanal. GABA-Iike immunoreactivity is confined to the cytoplasm of the hair cells. B: control section from the same crista amputlaris as in A demonstrates no GABA-Iike xmmunoreactwity. A red blood cell [arrowhead) is darkly stained: bar ~ 30 Urn. C: crista ampullaris of superior semicircular canal. No GABA-Iike immunoreactivity is found in the eminentia cruciata (E) whereas hair ceils are clearly posinve: bar = 30 urn. D: AChE-positive staining is found at the base of the hair cells as numerous small puncta: bar = 30am.
385 lar canals, was covered with unlabeled cuboidal cells (Fig. lC). Transitional cells and dark cells showed no GABA-like immunoreactivity. In the macula, each hair cell was positively labeled (Fig. 2A,B) and high magnification allowed us to distinguish flask-shaped type I hair cells from rod-shaped type II hair cells (Fig. 2C). Both types of cells showed no difference in staining intensity between them. AChE-positive staining in the form of numerous small puncta was found at the base of the hair cell layer (Fig. 1D). No positive AChE staining was found in transitional cells, dark cells, or the eminentia cruciata. The distribution of positive AChE staining corresponded to that of efferent nerve endings previously reported 5 and contrasted with the distribution of
A
GABA-like immunoreactivity. These results provide immunocytochemical evidence that G A B A may be an afferent neurotransmitter in the chick vestibular end organs. Flock and Lam suggested that G A B A might be an afferent transmitter between the hair cell and the afferent fiber in lower vertebrates 9. According to their study, the bullfrog basillar papilla, with only afferent innervation, synthesized G A B A from [3H]glutamate, while the lateral line in the toadfish and the skate ampulla, with both afferent and efferent innervation, synthesized both G A B A and ACh. Felix and Ehrenberger supported the proposition 6'7. Micro-iontophoretically applied G A B A and ACh modified the spontaneous firing rate recorded
B 0 M
jiii!i i?
....
~ ! i l ii~ ~!;~!ii!!:¸~! F~:,ilUii~ ~ : : ~ i : i~¸ C ~................ i~i ..... ~. . . . . ~:
ii: #iili;~¸ i
#
Fig. 2. GABA-Iike immunoreactivlty in the chick macula utriculi (A) and macula sacculi (B,C). A,B: GABA-Iike immunoreactivity is contained in hair cells throughout the sensory epithelium. OM, otolith membrane; bar = 30~tm. C: both flask-shaped type I hair cell (I) and rod-shaped type II hair cell (II) are distinguished at high magnification. GABA-like immunoreactivity is found in both types of cells: bar = 10urn.
386 in the subsynaptic region of the macula sacculi in the cat. G A B A e n h a n c e d the firing rate whereas A C h decreased the firing rate. Such action of G A B A was specifically blocked by the G A B A inhibitors, bicuculline and picrotoxin. M e z a et al. also d e m o n s t r a t e d e n z y m e - m e d i a t e d G A B A synthesis in isolated chick cristae ~4. Using [laC]glutamate as a precursor, glutamic acid decarboxylase ( G A D ) activity was investigated by two different assay methods; [ t a C ] G A B A isolation by p a p e r c h r o m a t o g r a p h y , m e a s u r e m e n t of 14CO2 from [14C]glutamate. F u r t h e r m o r e , in o r d e r to evaluate whether G A D activity is located in the hair cells, streptomycin which results in the d e g e n e r a t i o n of hair cells was used in the guinea pig. G A D decreased in the streptomycin-injected animal, whereas choline acetyltransferase (CHAT), r e m a i n e d constant. They concluded that the reduction in the activity of G A D could be ascribed to the diminution in n u m b e r of hair cells, and thus suggested that the G A B A contained in hair cells may be an afferent transmitter and the A C h in nerve c o m p o n e n t s may be an efferent transmitter. A study using chick embryo showed a differential increase in activity between G A D and C h A T during d e v e l o p m e n t is. Their results also led to the suggestion that each enzyme
This study was partly s u p p o r t e d by G r a n t NS10940 from the N I N C D S and the N A S A ( N A G - 2 289). F o r e x p e r t secretarial help we thank Mrs. Shirley Bonvillain.
1 Akoev, G.N., Andrianov, G.N. and Volpe, N.O., L-Glutamate as possible neurotransmitter in the ampullae of Lorenzini of the skate, Neurosci. Lett., 20 (1980) 307-312. 2 Annoni, J.M., Cochran, S.L. and Precht, W., Synaptic-and amino acid-induced excitation and antagonism at the vestibular hair cell-primary afferent synapse of the frog, Neurosci. Lett., Suppl. 14 (i983) $9. 3 Dechesne, C., Raymond, J. and Sans, A., Action of glutamate in the cat labyrinth, Ann. Otol. Rhinol. Laryngol.. 93 (1984) 163-165. 4 Dememes, D., Raymond, J. and Sans, A., Selective retrograde labelling of vestibular efferent neurons with 3H-choline, Neuroscience, 8 (1983) 285-290, 5 Dohlman, G.F., Farkashidy, J. and Salonna, F., Centrifugal nerve fibers to the sensory epithelium of the vestibular labyrinth, J. Laryngol., 72 (1958) 984-991. 6 Felix, D. and Ehrenberger, K., The action of GABA and acetylcholine in the labyrinth of the cat. In M. Portmann and J.M. Aran (Eds.), Inner Ear Biology, Vol. 68, INSERM, Paris, 1977, pp. 147-154. 7 Felix, D. and Ehrenberger, K., The action of putative neurotransmitter substances in the cat labyrinth, Acta Otolaryngol., 93 (1982) 101-105. 8 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 Research, 366 (1986) 106-117. 9 Flock, A. and Lam, D.M.K., Neurotransmitter synthesis in
inner ear and lateral line sense organs. Nature (Londonl. 249 (1974) 142-144. 10 Guth. S.L. and Norris. C.H.. Pharmacology of the isolated semicircular canal: effect of GABA and picrotoxin. Exp. Brain Res.. 56 (1984) 72-78 11 Higuchi. T.. Nagai, T.. Umekita. S. anti Obara. S.. The afferent neurotransmitter in the ampullary electroreceptors: L-glutamate mimics the natural transmitter, Neurosci. Lett.. Suppl. 4 (1980) $7. 12 lurato. S.. Luciano. L.. Pannese. E. and Reale. E.. Histochemical localization of acetylcholinesterase (ACHE) activity in the inner ear. Acta OtolaryngoL, Suppl. 279 ¢1971) 1-50. 13 Maley, B. and Newton. B.W.. Immunohistochemistry of gamma-aminobutyric acid in the cat nucleus tractus solitarius. Brain Research. 330 (1985) 364-368. 14 Meza. G.. Carabez, A. and Ruiz. M.. GABA synthesis in isolated vestibulary tissue of chick inner ear. Brain Research, 241 (1982) 157-161. 15 Meza. G,, Some characteristics of glutamic acid decarboxylase of chick ampullary cristae. J. Neurochem.. 43 11984) 634-639 16 Meza, G.. Lopez. I. and Ruiz. M.. Possible cholinergic neurotransmission m the cristae ampullares of the chick inner ear. Neurosci. Lett.. 49 (1984) 93-98. 17 Meza. G.. Gonzalez-Viveros. T. and Ruiz, M.. Specific [3H]aminobutyric acid binding to vestibular membranes of the chiek inner ear. Brain Research. 337 (19851 17%183.
was localized in different cell components. O u r results support this notion as the spatial localization of G A B A - I i k e immunoreactivity did not overlap with that of A C h E staining. In the isolated bullfrog semicircular canal p r e p a r a tion, however, the introduction of G A B A had no effect on the e v o k e d response r e c o r d e d from the primary afferent :.m. Also, in the m a m m a l i a n organ of Corti, no evidence of G A B A - l i k e immunoreactivity has been found immunohistochemically in the afferent system, even though it has been found in the efferent system 8"2~. F u r t h e r m o r e , electron microscopic studies have d e m o n s t r a t e d that G A B A - l i k e immunoreactivity is confined to the efferent c o m p o n e n t s 23. Further morphological and electrophysiological experiments are necessary to u n d e r s t a n d these different findings. H o w e v e r , our results support the view that G A B A may be an afferent transmitter at least in the chick vestibular end organs.
387 18 Meza, G., Characterization of GABA-ergic and cholinergic neurotransmission in the chick inner ear. In D.G. Drescher (Ed.), Auditory Biochemistry, Thomas, Springfield, 1985, pp. 80-101. 19 Thompson, G.C., Cortez, A.M. and Lam, D.M.-K., Localization of GABA immunoreactivity in the auditory brainstem of guinea pigs, Brain Research, 339 (1985) 119-122. 20 Thompson, G.C., Igarashi, M. and Cortez, A.M., GABA imbalance in squirrel monkey after unilateral vestibular end-organ ablation, Brain Research, 370 (1986) 182-185. 21 Thompson, G.C., Cortez, A.M. and Igarashi, M., GABA-
like immunoreactivity in the squirrel monkey organ of Corti, Brain Research, 372 (1986) 72-79. 22 Thornhill, R.A., The effect of catecholamine precursors and related drugs on the morphology of the synaptic bars in the vestibular epithelia of the frog, Rana temporaria, Comp. Gen. Pharmacol., 3 (1972) 89-97. 23 Usami, S., Igarashi, M. and Thompson, G.C., Light and electron microscopic study of GABA-like immunoreactivity in the guinea pig organ of Corti, Otorhinolaryngology, submitted.
383
BRE 22443
GABA-like immunoreactivity in the chick vestibular end organs Shin-ichi Usami, Makoto Igarashi and Glenn C. Thompson Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) (Accepted 12 May 1987)
Key words: y-Aminobutyric acid (GABA); Vestibular end organ; Chick
7-Aminobutyric acid (GABA)-like immunoreactivity in the chick vestibular endorgans was examined using an antiserum against GABA coupled with glutaraldehyde to bovine serum albumin. GABA-Iike immunoreactivity was confined to the cytoplasm of the hair cells in both cristae and maculae. GABA-Iike immunoreactive cells were evenly distributed throughout the sensory epithelia, and no difference existed between type I and type II hair cells. The results provide evidence that GABA-Iike immunoreactivity is localized to sensory cells and raises the possibility that GABA may serve as an afferent neurotransmitter in the chick vestibular end organs. Evidence has accumulated that acetylcholine (ACh) may play a role in the efferent transmission from central fibers to hair cells 4'5"12'16A8,but such evidence for a neurotransmitter between the hair cells and afferent fibers is lacking. Several putative substances including G A B A 6'7'9'14"15"17"18, glutamate 1 3,1~ and the catecholamines 22 have been considered as afferent neurotransmitters; among these candidates, G A B A has fulfilled the neurotransmitter criteria. G A B A synthesis from radioactively labeled precursors has been reported in lower vertebrates 9 and chick 14,15, afferent neural responsivity has been inhibited by a G A B A blocker 6'7'9, and high affinity G A B A uptake has been reported in the chick vestibule 17. These data suggest the existence of postsynaptic G A B A receptors in vestibular endorgans. However, there have been no reports describing immunocytochemical localization of G A B A in the vestibular end organs. Accordingly, we have immunohistochemically investigated the chick vestibular end organs. Young chicks (Gallus domesticus) 2, 10 and 20 days old (post-hatch) were used. The animals were anesthetized with an overdose of sodium pentobarbital (Nembutal, 100 mg/kg, i.p.) and perfused intracardially with fixative (4% paraformaldehyde, 0.1%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.2). The vestibular end organs were removed from the bony shell, and postfixed with 4% paraformaldehyde alone (in 0.1 M phosphate buffer, pH 7.2) for 3 h at 4 °C. They were stored at 4 °C in 10% sucrose-phosphate buffer for 3 h and then placed in 30% sucrosephosphate buffer overnight. Serial cryostat sections (15 /~m thick) were cut and placed onto gelatincoated slides. The A B C method was used for immunohistochemical study. Slide-mounted sections were incubated sequentially in: (1) 2% normal goat serum (in PBS with 0.3% Triton X-100, for 30 rain at room temperature); (2) antiserum against G A B A conjugated to bovine serum albumin (Immuno Nuclear Corp., 1/3000-4000, in PBS with 0.3% Triton-X, overnight at 4 °C); (3) biotinylated goat anti-rabbit IgG (Vector Labs., for 30 rain); (4) Vectastain reagent (Vector Labs., for 30 min); and (5) D A B / H 2 0 2, for 12 rain. Three rinses with PBS for 5 rain each were used between incubations. After peroxidase reaction, the sections were counterstained with Cresyl violet and observed with light microscopy. As a control, the primary antiserum was preabsorbed with an excess of G A B A - g l u t a r a l d e h y d e BSA conjugated complex and substituted for anti-
Correspondence: S. Usami. Present address: Department of Otorhinolaryngology, Hirosaki University, School of Medicine. 5 Zaifu Cho, Hirosaki (036), Japan. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
384 G A B A - B S A . The specificity of the G A B A antiserum used in the present study has been tested and no cross-reactivity was found with glutamate, taurine, aspartate, glycine, alanine, tyrosine, enkephalin or substance p~3. F u r t h e r m o r e , we have obtained positive staining of known G A B A e r g i c elements in the guinea pig 19 and squirrel m o n k e y cerebellum, brainstem 2° and cochlea 21 using the same antiserum. In order to identify efferent c o m p o n e n t s of the peripheral vestibular system, some slide-mounted sections were processed for the histochemical local-
A
ization of acetylcholinesterase (ACHE) 2~ G A B A - I i k e immunoreactivity was found in the hair cells of the vestibular epithelium in all chicks. In both cristae and maculae (Figs. t A , C and 2 A - C ) , G A B A - I i k e immunoreactivity was clearly restricted to the cytoplasm of the hair cells while supporting cells were unstained. The distribution of labeled cells was uniform throughout the sensory epithelia of both end organs. No immunoreactivity was observed i n the control specimens (Fig. 1B). The e m i n e n t i a cruciata, which divides the cristae of vertical .semicircu-
B
|
Fig. 1. GABA-Iike immunoreactivit~ and AChE staining in the chick cristae ampullares. A: crista ampuUarisof lateral semicircUlarcanal. GABA-Iike immunoreactivity is confined to the cytoplasm of the hair cells. B: control section from the same crista amputlaris as in A demonstrates no GABA-Iike xmmunoreactwity. A red blood cell [arrowhead) is darkly stained: bar ~ 30 Urn. C: crista ampullaris of superior semicircular canal. No GABA-Iike immunoreactivity is found in the eminentia cruciata (E) whereas hair ceils are clearly posinve: bar = 30 urn. D: AChE-positive staining is found at the base of the hair cells as numerous small puncta: bar = 30am.
385 lar canals, was covered with unlabeled cuboidal cells (Fig. lC). Transitional cells and dark cells showed no GABA-like immunoreactivity. In the macula, each hair cell was positively labeled (Fig. 2A,B) and high magnification allowed us to distinguish flask-shaped type I hair cells from rod-shaped type II hair cells (Fig. 2C). Both types of cells showed no difference in staining intensity between them. AChE-positive staining in the form of numerous small puncta was found at the base of the hair cell layer (Fig. 1D). No positive AChE staining was found in transitional cells, dark cells, or the eminentia cruciata. The distribution of positive AChE staining corresponded to that of efferent nerve endings previously reported 5 and contrasted with the distribution of
A
GABA-like immunoreactivity. These results provide immunocytochemical evidence that G A B A may be an afferent neurotransmitter in the chick vestibular end organs. Flock and Lam suggested that G A B A might be an afferent transmitter between the hair cell and the afferent fiber in lower vertebrates 9. According to their study, the bullfrog basillar papilla, with only afferent innervation, synthesized G A B A from [3H]glutamate, while the lateral line in the toadfish and the skate ampulla, with both afferent and efferent innervation, synthesized both G A B A and ACh. Felix and Ehrenberger supported the proposition 6'7. Micro-iontophoretically applied G A B A and ACh modified the spontaneous firing rate recorded
B 0 M
jiii!i i?
....
~ ! i l ii~ ~!;~!ii!!:¸~! F~:,ilUii~ ~ : : ~ i : i~¸ C ~................ i~i ..... ~. . . . . ~:
ii: #iili;~¸ i
#
Fig. 2. GABA-Iike immunoreactivlty in the chick macula utriculi (A) and macula sacculi (B,C). A,B: GABA-Iike immunoreactivity is contained in hair cells throughout the sensory epithelium. OM, otolith membrane; bar = 30~tm. C: both flask-shaped type I hair cell (I) and rod-shaped type II hair cell (II) are distinguished at high magnification. GABA-like immunoreactivity is found in both types of cells: bar = 10urn.
386 in the subsynaptic region of the macula sacculi in the cat. G A B A e n h a n c e d the firing rate whereas A C h decreased the firing rate. Such action of G A B A was specifically blocked by the G A B A inhibitors, bicuculline and picrotoxin. M e z a et al. also d e m o n s t r a t e d e n z y m e - m e d i a t e d G A B A synthesis in isolated chick cristae ~4. Using [laC]glutamate as a precursor, glutamic acid decarboxylase ( G A D ) activity was investigated by two different assay methods; [ t a C ] G A B A isolation by p a p e r c h r o m a t o g r a p h y , m e a s u r e m e n t of 14CO2 from [14C]glutamate. F u r t h e r m o r e , in o r d e r to evaluate whether G A D activity is located in the hair cells, streptomycin which results in the d e g e n e r a t i o n of hair cells was used in the guinea pig. G A D decreased in the streptomycin-injected animal, whereas choline acetyltransferase (CHAT), r e m a i n e d constant. They concluded that the reduction in the activity of G A D could be ascribed to the diminution in n u m b e r of hair cells, and thus suggested that the G A B A contained in hair cells may be an afferent transmitter and the A C h in nerve c o m p o n e n t s may be an efferent transmitter. A study using chick embryo showed a differential increase in activity between G A D and C h A T during d e v e l o p m e n t is. Their results also led to the suggestion that each enzyme
This study was partly s u p p o r t e d by G r a n t NS10940 from the N I N C D S and the N A S A ( N A G - 2 289). F o r e x p e r t secretarial help we thank Mrs. Shirley Bonvillain.
1 Akoev, G.N., Andrianov, G.N. and Volpe, N.O., L-Glutamate as possible neurotransmitter in the ampullae of Lorenzini of the skate, Neurosci. Lett., 20 (1980) 307-312. 2 Annoni, J.M., Cochran, S.L. and Precht, W., Synaptic-and amino acid-induced excitation and antagonism at the vestibular hair cell-primary afferent synapse of the frog, Neurosci. Lett., Suppl. 14 (i983) $9. 3 Dechesne, C., Raymond, J. and Sans, A., Action of glutamate in the cat labyrinth, Ann. Otol. Rhinol. Laryngol.. 93 (1984) 163-165. 4 Dememes, D., Raymond, J. and Sans, A., Selective retrograde labelling of vestibular efferent neurons with 3H-choline, Neuroscience, 8 (1983) 285-290, 5 Dohlman, G.F., Farkashidy, J. and Salonna, F., Centrifugal nerve fibers to the sensory epithelium of the vestibular labyrinth, J. Laryngol., 72 (1958) 984-991. 6 Felix, D. and Ehrenberger, K., The action of GABA and acetylcholine in the labyrinth of the cat. In M. Portmann and J.M. Aran (Eds.), Inner Ear Biology, Vol. 68, INSERM, Paris, 1977, pp. 147-154. 7 Felix, D. and Ehrenberger, K., The action of putative neurotransmitter substances in the cat labyrinth, Acta Otolaryngol., 93 (1982) 101-105. 8 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 Research, 366 (1986) 106-117. 9 Flock, A. and Lam, D.M.K., Neurotransmitter synthesis in
inner ear and lateral line sense organs. Nature (Londonl. 249 (1974) 142-144. 10 Guth. S.L. and Norris. C.H.. Pharmacology of the isolated semicircular canal: effect of GABA and picrotoxin. Exp. Brain Res.. 56 (1984) 72-78 11 Higuchi. T.. Nagai, T.. Umekita. S. anti Obara. S.. The afferent neurotransmitter in the ampullary electroreceptors: L-glutamate mimics the natural transmitter, Neurosci. Lett.. Suppl. 4 (1980) $7. 12 lurato. S.. Luciano. L.. Pannese. E. and Reale. E.. Histochemical localization of acetylcholinesterase (ACHE) activity in the inner ear. Acta OtolaryngoL, Suppl. 279 ¢1971) 1-50. 13 Maley, B. and Newton. B.W.. Immunohistochemistry of gamma-aminobutyric acid in the cat nucleus tractus solitarius. Brain Research. 330 (1985) 364-368. 14 Meza. G.. Carabez, A. and Ruiz. M.. GABA synthesis in isolated vestibulary tissue of chick inner ear. Brain Research, 241 (1982) 157-161. 15 Meza. G,, Some characteristics of glutamic acid decarboxylase of chick ampullary cristae. J. Neurochem.. 43 11984) 634-639 16 Meza, G.. Lopez. I. and Ruiz. M.. Possible cholinergic neurotransmission m the cristae ampullares of the chick inner ear. Neurosci. Lett.. 49 (1984) 93-98. 17 Meza. G.. Gonzalez-Viveros. T. and Ruiz, M.. Specific [3H]aminobutyric acid binding to vestibular membranes of the chiek inner ear. Brain Research. 337 (19851 17%183.
was localized in different cell components. O u r results support this notion as the spatial localization of G A B A - I i k e immunoreactivity did not overlap with that of A C h E staining. In the isolated bullfrog semicircular canal p r e p a r a tion, however, the introduction of G A B A had no effect on the e v o k e d response r e c o r d e d from the primary afferent :.m. Also, in the m a m m a l i a n organ of Corti, no evidence of G A B A - l i k e immunoreactivity has been found immunohistochemically in the afferent system, even though it has been found in the efferent system 8"2~. F u r t h e r m o r e , electron microscopic studies have d e m o n s t r a t e d that G A B A - l i k e immunoreactivity is confined to the efferent c o m p o n e n t s 23. Further morphological and electrophysiological experiments are necessary to u n d e r s t a n d these different findings. H o w e v e r , our results support the view that G A B A may be an afferent transmitter at least in the chick vestibular end organs.
387 18 Meza, G., Characterization of GABA-ergic and cholinergic neurotransmission in the chick inner ear. In D.G. Drescher (Ed.), Auditory Biochemistry, Thomas, Springfield, 1985, pp. 80-101. 19 Thompson, G.C., Cortez, A.M. and Lam, D.M.-K., Localization of GABA immunoreactivity in the auditory brainstem of guinea pigs, Brain Research, 339 (1985) 119-122. 20 Thompson, G.C., Igarashi, M. and Cortez, A.M., GABA imbalance in squirrel monkey after unilateral vestibular end-organ ablation, Brain Research, 370 (1986) 182-185. 21 Thompson, G.C., Cortez, A.M. and Igarashi, M., GABA-
like immunoreactivity in the squirrel monkey organ of Corti, Brain Research, 372 (1986) 72-79. 22 Thornhill, R.A., The effect of catecholamine precursors and related drugs on the morphology of the synaptic bars in the vestibular epithelia of the frog, Rana temporaria, Comp. Gen. Pharmacol., 3 (1972) 89-97. 23 Usami, S., Igarashi, M. and Thompson, G.C., Light and electron microscopic study of GABA-like immunoreactivity in the guinea pig organ of Corti, Otorhinolaryngology, submitted.