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Gamma-aminobutyric acid in the lower auditory pathway of the guinea pig
370
Brain Research, 69 (1974) 370-374 :(:~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Gamma-aminobutyric acid in the lower auditory pathway of the guinea pig
MASAYOSHI TACHIBANA AND KINYA K U R I Y A M A
Department of Pharmacology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto (Japan) (Accepted December 10th, 1973)
As a result of work in a number of laboratories, it now seems probable that gamma-aminobutyric acid (GABA) is a major inhibitory transmitter in the mammalian central nervous system4, la. However, little is known concerning the distribution of GABA in the auditory system. In this study the detailed regional distribution of GABA, GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) was investigated as a first step to elucidate functional roles of GABA in the lower auditory pathway. Young healthy guinea pigs weighing 200-250 g were used. After decapitation, the temporal bone was immediately frozen with dry ice-ethanol (--78.5 °C) and freeze-dried for 2-3 days. After removal of the bony capsule, the membranous cochlea was dissected into the spiral ligament including vascular stria, outer hair cell layer, inner hair cell layer, spiral ganglion cell layer and modiolar nerve respectively. The dry weight of each sample was then determined by means of the electric balance (minimum detectable change: 500 ng). Meanwhile the brain was immediately frozen by COe gas and the tissue block at the level of the cochlear nucleus was sectioned sagittally (150 #m in thickness) with a microtome in a cryostat. Each section was further divided into pieces (750 #m × 750 #m × 150 #m) in a cold box as described by Aprison and Hancock 1. After determining the location and the size of these sections photographically, each sample was transferred into 5-10 #l of 0.1 N HC1 in a fluorometer tube. After removal of HCI and water by evaporation, GABA content was determined by the microassay method which consisted of a combination of the enzymatic assay for GABA (Scott and Jacoby1~) with the enzymatic cycling procedure (Lowry et al.tl). The maximum sensitivity for GABA was 10-la moles. In some experiments, GABA content in the cerebral cortex, inferior colliculus and cochlear nucleus was assayed with the enzymatic assay method of Scott and Jacoby 15, after extracting GABA from these tissues by the method of Baxter and Roberts `). The GABA-T-SSADH activity in brain sections or in surface specimens o|' the membranous cochlea was visualized histochemically according to the method of van GelderL
371
SHORT COMMUNICATIONS TABLE I DISTRIBUTION OF
GABA
IN THE LOWER AUDITORY PATHWAY
The values in this table represent the mean ~a S.E.M. The numbers in parentheses indicate the number of experiments employed. GABA content was measured by the enzymatic assay procedure (see text).
Region
GABA (mnoDs/mg wet wt.)
Cerebral cortex Inferior colliculus Cochlear nucleus
3.0 :) 0.21 5.5 J: 0.23 3.7 _~ 0.12
(4) (6) (6)
TABLE I1 D I S T R I B U T I O N OF
GABA
I N T H E I N N E R EAR
All results in this table are the average obtained from 3 closely corresponding determinations. GABA content was measured by the microassay method (see text).
Region
GABA (nrnoles/mg dry wt.)
Spiral ligament ÷ vascular stria Outer hair cell layer Inner hair cell layer Spiral ganglion cell layer Modiolar nerve
0* 0* 0* 1.1 0*
* Not detectable by the method employed.
BB > 2 5 fl moles/mm 3 ~-',_', F~,,~ 1 5 - 24
MEDIAL
DORSAL I
1mm
10"'14
POSTERIOR Fig. 1. Three-dimensional presentation of the distribution of GABA in the cochlear nucleus. All results in this figure are the average obtained from 3 closely corresponding determinations. GABA content was measured by the microassay method (see text).
372
SHORT COMMUNICATIONS
Fig. 2. Histochemical visualization of GABA-T-SSADH activity in the cochlear nucleus, A: formazan production due to the GABA-T-SSADH activity m a sagittal section at the level of the cochlear nucleus. Note heavier precipitation on the neurons in the dorsal part than in the ventral part. Magnification, 40. B: the photograph of the dorsal part of the cochlear nucleus with a higher magnification. Note formazan precipitation in the cytoplasm of the neurons, Magnification : 400, Tile results in Table I indicate that the cochlear nucleus and inferio~ colliculus contain G A B A at a higher level than that in the cerebral cortex. In the inner ear the G A B A - T - S S A D H activity was too low to bc visualized histochemically but the microassay for G A B A indicated that the spiral ganglion cell layer contains a considerable a m o u n t of G A B A (Table II). Since spiral ganglion cells are considered to be excitatory in nature and their cell bodies are mostly free from synaptic contacts (Spoendlin16), the functional role o f G A B A in tile spiral ganglion cell layer is unclear at present. However it is possible that some interneurons containing G A B A may exist ill this area. Our result that G A B A was not detected in tile hair cell layer suggests that, contrary to the speculation of Sch~itzle 14, afferent transmitter between the hair cells and the primary afferent fiber is unlikely to be G A B A . This result also suggests that the olivo-cochlear fibers of Rasmussen 12 which terminate on the hair cells with vesiculated nerve endings (Engstr6m a) may not be GABA-nergic. This conclusion is in agreement with previous reports by many authors that the olivo-cochlear fibers are possibly cholinergic a,
SHORT COMMUNICATIONS
373
is shown in Figs. 1 and 2 respectively. The G A B A content is higher in the d o r s a l part o f the cochlear nucleus than in the ventral part. This d i s t r i b u t i o n pattern was constantly observed in each section from the a n t e r i o r to the p o s t e r i o r pole (Fig. 1). The histochemical study also revealed that the dorsal part has the strongest G A B A T - S S A D H activity in the cochlear nucleus (Fig. 2). These results suggest that the dorsal part o f the cochlear nucleus may receive G A B A - n e r g i c neurons. The presence o f centrifugal fibers to the dorsal cochlear nucleus from the inferior colliculus h a d been r e p o r t e d by Lorente de Nd ~° and o u r unpublished studies have suggested that the intimate relationship may be present between these fibers a n d the G A B A system in the dorsal part o f the cochlear nucleus. Recently Whitfield and Comis is have r e p o r t e d that G A B A p r o d u c e s a strong depression in the discharge rate o f neurons in the cochlear nucleus. W a t a n a b e 17 also has r e p o r t e d that picrotoxin disinhibits the ' t w o - t o n e i n h i b i t i o n ' o f a u d i t o r y neurons in the cochlear nucleus. O u r findings together with these d a t a suggest that the G A B A system may play an i m p o r t a n t role in the lower a u d i t o r y pathway. The cellular localization o f G A B A in the spiral ganglion cell laver and a possible involvement o f G A B A - n e r g i c neurons in neuronal inputs to the dorsal part of" the cochlear nucleus remain to be elucidated. This work was s u p p o r t e d in part by a research grant from the Japanese Ministry o f Education ('No. 744023, 1973).
I APRISON, M. l-L, AND HANCOCK, C.J., A controlled humidity constant temperature cold box
for dissection of small frozen tissue sections. In G. A. KERKUT(Ed.), Erperbnents ilz Phys'iologl, and Biochemisto,, Vol. 3, Academic Press, New York, 1970, pp. 39 48. 2 BAXTer, C. F., AND ROBERTS, E., Elevation of ,,,-aminobutyric acid in rat brain with hydroxylamine, Proc. Soe. exp. Biol. (N. Y.), I01 (1959)811-815. 3 BOBBIN, R. P., AND KONISHI, T., Acetylcholine mimics crossed olivo-cochlear bundle stimulation, Naml~ New Biol., 231 (1971) 222-223. 4 CURTIS, O. R., AND JOHNSTON',G. A. R., Amino acid transmitters. In A. LAJTHA(Ed.), Handbook of Neurochemist13,, Vol. 4, Plenum Press, New York, 1970, pp. 115-148. 5 EN(;STR/~)M,H., On the double innervation in the sensory epithelia of the inner ear, Acta oto-lao'ng. (Stoekh.), 49 (1958) 108-118. 6 FEx, J., Efferent inhibition in the cochlear by the olivo-eochlear bundle. In A. V. S. DE REUCK AND J. KNIGHT(Eds.), A Ciba Foundation Symposium, Churchill, London, 1968, p. 169. 7 GELDER, N. M. VAN, The histochemical demonstration of 7-aminobutyric acid metabolism by reduction o f tetrazolium salt, J. Neurochem., 12 (1965) 231-244. 8 JASSER, A., AND GUTH, P. S., The synthesis of acetylcholine by the olivocochlear bundle, J. Neurochem., 20 (1973) 45-53. 9 KoNls~II, T., Action of tubocurarine and atropine on the crossed olivocochlear bundles, Acta oto-larj,ng. (Stockh.), 74 (1972) 252-264. 10 LORENTE DE No, R., Anatomy of the eighth nerve, 111, Laryngoscope (St. Louis), 63 (19331 327-350. [ I LOWRY,O. H., PASSONEAU,J. V., SCHULZ, D. W., AND ROCK, M. K., The meastlrement of pyridine nucleotides by enzymatic cycling, J. biol. Chem., 236 (1961) 2746-2775. 12~RAs~tussEN, G. L., The olivary peduncle and other fiber projections of the superior olivary complex, J. comp. Nemvl., 84 (1946) 141-219. 13 RoRerTS, E., and KurrvAMA, K., Biochemical-physiological correlations in studies of the 7aminobutyric acid system, Brain Research, 8 (1968) 1-35. 14 SCU~TZL~, W., Azetylcho[in und Erregungsi.ibertragung. In Histochemie des bmenohres, Urban and Schwarzenberg, Munich, 1971, p. 118.
374
SHORT COMMUNICATIONS
15 SCOTT, E. M., AND JACOBY, W. B., Soluble y-aminobutyric-glutamate transaminase from Pseudomonas fluorescens, J. biol. Chem., 234 (1959) 932-936. 16 SPOENDI.lY, H., Degenerative behavior of the cochlear nerve, Arch. klin. exp. Ohr.-. Nas.-, u. Kehlk.-Heilk., 200 (1971 ) 275-291. 17 WXrAr~ABE, T., Effect of picrotoxin on two-tone inhibition of auditory neurons in the cochlear nucleus, Brain Research, 28 (1971) 586-590. 18 WHLrFIELD, 1. C., AriD COMIS, S. D., The interaction of centrifugal and centripetal stimulation on the cochlear nucleus, USAF Final Rep., 2 (1966).
Brain Research, 69 (1974) 370-374 :(:~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Gamma-aminobutyric acid in the lower auditory pathway of the guinea pig
MASAYOSHI TACHIBANA AND KINYA K U R I Y A M A
Department of Pharmacology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto (Japan) (Accepted December 10th, 1973)
As a result of work in a number of laboratories, it now seems probable that gamma-aminobutyric acid (GABA) is a major inhibitory transmitter in the mammalian central nervous system4, la. However, little is known concerning the distribution of GABA in the auditory system. In this study the detailed regional distribution of GABA, GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) was investigated as a first step to elucidate functional roles of GABA in the lower auditory pathway. Young healthy guinea pigs weighing 200-250 g were used. After decapitation, the temporal bone was immediately frozen with dry ice-ethanol (--78.5 °C) and freeze-dried for 2-3 days. After removal of the bony capsule, the membranous cochlea was dissected into the spiral ligament including vascular stria, outer hair cell layer, inner hair cell layer, spiral ganglion cell layer and modiolar nerve respectively. The dry weight of each sample was then determined by means of the electric balance (minimum detectable change: 500 ng). Meanwhile the brain was immediately frozen by COe gas and the tissue block at the level of the cochlear nucleus was sectioned sagittally (150 #m in thickness) with a microtome in a cryostat. Each section was further divided into pieces (750 #m × 750 #m × 150 #m) in a cold box as described by Aprison and Hancock 1. After determining the location and the size of these sections photographically, each sample was transferred into 5-10 #l of 0.1 N HC1 in a fluorometer tube. After removal of HCI and water by evaporation, GABA content was determined by the microassay method which consisted of a combination of the enzymatic assay for GABA (Scott and Jacoby1~) with the enzymatic cycling procedure (Lowry et al.tl). The maximum sensitivity for GABA was 10-la moles. In some experiments, GABA content in the cerebral cortex, inferior colliculus and cochlear nucleus was assayed with the enzymatic assay method of Scott and Jacoby 15, after extracting GABA from these tissues by the method of Baxter and Roberts `). The GABA-T-SSADH activity in brain sections or in surface specimens o|' the membranous cochlea was visualized histochemically according to the method of van GelderL
371
SHORT COMMUNICATIONS TABLE I DISTRIBUTION OF
GABA
IN THE LOWER AUDITORY PATHWAY
The values in this table represent the mean ~a S.E.M. The numbers in parentheses indicate the number of experiments employed. GABA content was measured by the enzymatic assay procedure (see text).
Region
GABA (mnoDs/mg wet wt.)
Cerebral cortex Inferior colliculus Cochlear nucleus
3.0 :) 0.21 5.5 J: 0.23 3.7 _~ 0.12
(4) (6) (6)
TABLE I1 D I S T R I B U T I O N OF
GABA
I N T H E I N N E R EAR
All results in this table are the average obtained from 3 closely corresponding determinations. GABA content was measured by the microassay method (see text).
Region
GABA (nrnoles/mg dry wt.)
Spiral ligament ÷ vascular stria Outer hair cell layer Inner hair cell layer Spiral ganglion cell layer Modiolar nerve
0* 0* 0* 1.1 0*
* Not detectable by the method employed.
BB > 2 5 fl moles/mm 3 ~-',_', F~,,~ 1 5 - 24
MEDIAL
DORSAL I
1mm
10"'14
POSTERIOR Fig. 1. Three-dimensional presentation of the distribution of GABA in the cochlear nucleus. All results in this figure are the average obtained from 3 closely corresponding determinations. GABA content was measured by the microassay method (see text).
372
SHORT COMMUNICATIONS
Fig. 2. Histochemical visualization of GABA-T-SSADH activity in the cochlear nucleus, A: formazan production due to the GABA-T-SSADH activity m a sagittal section at the level of the cochlear nucleus. Note heavier precipitation on the neurons in the dorsal part than in the ventral part. Magnification, 40. B: the photograph of the dorsal part of the cochlear nucleus with a higher magnification. Note formazan precipitation in the cytoplasm of the neurons, Magnification : 400, Tile results in Table I indicate that the cochlear nucleus and inferio~ colliculus contain G A B A at a higher level than that in the cerebral cortex. In the inner ear the G A B A - T - S S A D H activity was too low to bc visualized histochemically but the microassay for G A B A indicated that the spiral ganglion cell layer contains a considerable a m o u n t of G A B A (Table II). Since spiral ganglion cells are considered to be excitatory in nature and their cell bodies are mostly free from synaptic contacts (Spoendlin16), the functional role o f G A B A in tile spiral ganglion cell layer is unclear at present. However it is possible that some interneurons containing G A B A may exist ill this area. Our result that G A B A was not detected in tile hair cell layer suggests that, contrary to the speculation of Sch~itzle 14, afferent transmitter between the hair cells and the primary afferent fiber is unlikely to be G A B A . This result also suggests that the olivo-cochlear fibers of Rasmussen 12 which terminate on the hair cells with vesiculated nerve endings (Engstr6m a) may not be GABA-nergic. This conclusion is in agreement with previous reports by many authors that the olivo-cochlear fibers are possibly cholinergic a,
SHORT COMMUNICATIONS
373
is shown in Figs. 1 and 2 respectively. The G A B A content is higher in the d o r s a l part o f the cochlear nucleus than in the ventral part. This d i s t r i b u t i o n pattern was constantly observed in each section from the a n t e r i o r to the p o s t e r i o r pole (Fig. 1). The histochemical study also revealed that the dorsal part has the strongest G A B A T - S S A D H activity in the cochlear nucleus (Fig. 2). These results suggest that the dorsal part o f the cochlear nucleus may receive G A B A - n e r g i c neurons. The presence o f centrifugal fibers to the dorsal cochlear nucleus from the inferior colliculus h a d been r e p o r t e d by Lorente de Nd ~° and o u r unpublished studies have suggested that the intimate relationship may be present between these fibers a n d the G A B A system in the dorsal part o f the cochlear nucleus. Recently Whitfield and Comis is have r e p o r t e d that G A B A p r o d u c e s a strong depression in the discharge rate o f neurons in the cochlear nucleus. W a t a n a b e 17 also has r e p o r t e d that picrotoxin disinhibits the ' t w o - t o n e i n h i b i t i o n ' o f a u d i t o r y neurons in the cochlear nucleus. O u r findings together with these d a t a suggest that the G A B A system may play an i m p o r t a n t role in the lower a u d i t o r y pathway. The cellular localization o f G A B A in the spiral ganglion cell laver and a possible involvement o f G A B A - n e r g i c neurons in neuronal inputs to the dorsal part of" the cochlear nucleus remain to be elucidated. This work was s u p p o r t e d in part by a research grant from the Japanese Ministry o f Education ('No. 744023, 1973).
I APRISON, M. l-L, AND HANCOCK, C.J., A controlled humidity constant temperature cold box
for dissection of small frozen tissue sections. In G. A. KERKUT(Ed.), Erperbnents ilz Phys'iologl, and Biochemisto,, Vol. 3, Academic Press, New York, 1970, pp. 39 48. 2 BAXTer, C. F., AND ROBERTS, E., Elevation of ,,,-aminobutyric acid in rat brain with hydroxylamine, Proc. Soe. exp. Biol. (N. Y.), I01 (1959)811-815. 3 BOBBIN, R. P., AND KONISHI, T., Acetylcholine mimics crossed olivo-cochlear bundle stimulation, Naml~ New Biol., 231 (1971) 222-223. 4 CURTIS, O. R., AND JOHNSTON',G. A. R., Amino acid transmitters. In A. LAJTHA(Ed.), Handbook of Neurochemist13,, Vol. 4, Plenum Press, New York, 1970, pp. 115-148. 5 EN(;STR/~)M,H., On the double innervation in the sensory epithelia of the inner ear, Acta oto-lao'ng. (Stoekh.), 49 (1958) 108-118. 6 FEx, J., Efferent inhibition in the cochlear by the olivo-eochlear bundle. In A. V. S. DE REUCK AND J. KNIGHT(Eds.), A Ciba Foundation Symposium, Churchill, London, 1968, p. 169. 7 GELDER, N. M. VAN, The histochemical demonstration of 7-aminobutyric acid metabolism by reduction o f tetrazolium salt, J. Neurochem., 12 (1965) 231-244. 8 JASSER, A., AND GUTH, P. S., The synthesis of acetylcholine by the olivocochlear bundle, J. Neurochem., 20 (1973) 45-53. 9 KoNls~II, T., Action of tubocurarine and atropine on the crossed olivocochlear bundles, Acta oto-larj,ng. (Stockh.), 74 (1972) 252-264. 10 LORENTE DE No, R., Anatomy of the eighth nerve, 111, Laryngoscope (St. Louis), 63 (19331 327-350. [ I LOWRY,O. H., PASSONEAU,J. V., SCHULZ, D. W., AND ROCK, M. K., The meastlrement of pyridine nucleotides by enzymatic cycling, J. biol. Chem., 236 (1961) 2746-2775. 12~RAs~tussEN, G. L., The olivary peduncle and other fiber projections of the superior olivary complex, J. comp. Nemvl., 84 (1946) 141-219. 13 RoRerTS, E., and KurrvAMA, K., Biochemical-physiological correlations in studies of the 7aminobutyric acid system, Brain Research, 8 (1968) 1-35. 14 SCU~TZL~, W., Azetylcho[in und Erregungsi.ibertragung. In Histochemie des bmenohres, Urban and Schwarzenberg, Munich, 1971, p. 118.
374
SHORT COMMUNICATIONS
15 SCOTT, E. M., AND JACOBY, W. B., Soluble y-aminobutyric-glutamate transaminase from Pseudomonas fluorescens, J. biol. Chem., 234 (1959) 932-936. 16 SPOENDI.lY, H., Degenerative behavior of the cochlear nerve, Arch. klin. exp. Ohr.-. Nas.-, u. Kehlk.-Heilk., 200 (1971 ) 275-291. 17 WXrAr~ABE, T., Effect of picrotoxin on two-tone inhibition of auditory neurons in the cochlear nucleus, Brain Research, 28 (1971) 586-590. 18 WHLrFIELD, 1. C., AriD COMIS, S. D., The interaction of centrifugal and centripetal stimulation on the cochlear nucleus, USAF Final Rep., 2 (1966).