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Radioautographic identification of [3H]glutamic acid labeled nerve endings in the cat oculomotor nucleus
Brain Research, 231 (1982)433-437
433
Elsevier Biomedical Press
Radioautographic identification of [3H]glutamic acid labeled nerve endings in the cat oculomotor nucleus
D. DEMfEMES and J. RAYMOND Laboratoire de Neurophysiologie Sensorie[le, Universitddes Sciences et Techniques da Languedoc, Phwe E.-Bataillon, 34060 Montpellier Cddex (France)
(Accepted October 8th, 1981) Key words: glutamergic synapses - - oculomotor nucleus - - electron microscopic radioautography
Slices of the cat third oculomotor nucleus were incubated in vitro with [3H]glutamicacid. Electron microscopic radioautographs revealed that glutamate had been taken by small nerve endings distributed on the oculomotor motoneuron distal dendrites. In contrast, there was no uptake in the other types of terminals. The labeled terminals seem to correspond to the excitatory vestibulo-oculomotor nerve endings and different correlations suggest their glutamergic nature. There is now strong evidence from electrophysiological and neurochemical findings that glutamic acid is an excitatory neurotransmitter in the mammalian central nervous system (~,9,14. Furthermore, numerous investigations have shown reductions in glutamate uptake when degeneration of excitatory nerve endings is induced experimentallyS,15,t6, ~3. Recent studies by light- and electron-microscopic radioautography revealed that [3H]glutamic acid, exogenously applied in vitro, is selectively accumulated in slices of hippocampal formation by nerve endings recognized to be excitatory (ref. 24). Electrophysiological experiments provide strong evidence for the existence of excitatory pathways projecting on the oculomotor structures, particularly the third oculomotor nucleus. These fiber systems are: (1) the main vestibulo-oculomotor pathways originating from the median and lateral vestibular nuclei6,13,19,~5,26; (2) other oculomotor afferents, less important, recently investigated, issued from the nucleus abducensl0,12,18 and from the prepositus hypoglossi3, 4. It has recently been reported by Dem~mes and Raymond 7, in an electron-microscopic radioautographic investigation, that the oculomotor motoneuron's synaptic profile seems dominated by inhibitory vestibular endings with nevertheless a few excitatory ones; the latter seem concentrated in the dendritic neuropile. I f the inhibitory transmitter has been demonstrated to be gamma-aminobutyric acid (GABA), in the vestibulo-ocular pathways20; yet, the excitatory transmitter is still unknown. Consequently, it would be a new point of interest, in view of the possible role of glutamic acid as a neurotransmitter substance, to identify morphologically by electron-microscopic radioautography the uptake sites of this exogenously accumu0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
434 lated neurotransmitter in oculomotor nucleus slices. However, it should be mentioned that the glutamic acid uptake may label both glutamate and aspartate nerve endings, since under the conditions used in this experiment the uptake system does not distinguish between these excitatory neurotransmitters. Young cats, 2 or 3 months old, were anesthetized with sodium pentobarbitat and decapitated. The brain was rapidly removed and cooled on ice; serial thin transverse slices (250-300 ~m) were prepared and collected in cold Kreb's phosphate solution (pH - = 7.4). The oculomotor nuclei pieces were dissected out (details of this method have been presented in our former paper 7) and transferred into the identical solution at 25 ~'C, then 5 rain later into Kreb's solution containing 2 #M L-[G-3H]glutamic acid (20-40 Ci/mmol, Radiochemical Centre, Amersham). After a 15 rain incubation period at 25 °C and after rinsing in 2--3 changes of fresh Kreb's phosphate solution, the tissue pieces were fixed in 5 % glutaraldehyde for 3 h at 25 °C, treated with osmium tet~oxide, and embedded in araldite for electron-microscopic radioautography. The ultrathin sections were deposited upon celloidin-covered slides, stained with uranyl acetate and lead citrate, then vaporized with carbon, and were covered with Ilford L4 emulsion (diluted 1:4) by the dipping technique. Following an exposure time of 8 weeks, the radioautographs were developed with Kodak Microdol-X. Electron-microscopic radioautographs teveal heavy accumulations of silver grains over some nerve endings identified on the basis of their ultrastructural characteristic features. Among the 5 distinct types of terminals described in the oculomotor nuclei1, 2,7, only one category appears to be labeled. These labeled synaptic boutons are numerous, of small size (1-2/~m in diameter), rarely axosomatic (Figs. 1 and 2); they are mainly axodendritic and are most of the time distributed on the distal part of the dendritic tree of oculomotor motoneurons. The synaptic vesicles of these boutons are numerous, scattered in the presynaptic axoplasm and appear to be clear and spherical; 2-5 mitochondria are also present. No accumulations of silver grains are seen over larger synaptic boutons containing flat vesicles. In addition to the nerve endings, satellite glial cells also present aggregates of silver grains located over cytoplasm and nucleus. On the contrary, oculomotor motoneurons and their proximal dendritic processes are devoid of label. In the presently used method, involving incubation with exogenous glutamic acid in vitro, tissue preservation is far from optimal, but good enough to allow a precise morphological identification of nerve endings. Although glutamic acid is ~eadily metabolized, recent studies have shown that most of the radioactivity present in the slices prior to fixation could be attributed to unchanged [3H]glutamate and not to its labeled metabolites '~4. Although a better preservation of tissue should be obtained by injecting [3H]glutamic acid in vivo, the metabolism of [aH]glutamic acid would present a still more difficult problem in vivo. The uptake of exogenous glutamic acid by the glial cells in the oculomotor nucleus is in clear agreement with other studies 17,zl. It seems indeed that glia may have efficient uptake systems for the transmitter present in the cerebral tissue tI. These labeled synaptic boutons are quite similar in morphological characteristics to the small type lII terminals, described in cat trochlear nuclei after lesion of the
435
-
J
Figs. 1, 2. Electron-microscopic radioautographs of oculomotor nucleus incubated with [3H]glutamic acid. Small labeled nerve endings axodendritically located containing numerous clear synaptic vesicles. Fig. 3. Electron-microscopic radioautograph of oculomotor nucleus following [ZH]proline injection in vestibular nuclei. Labeled axo-dendritic synapse (type II1).
436 vestibulo-trochlear pathway 1,z, as well as in cat o c u l o m o t o r nuclei, where they were identified by anterograde transport o f labeled proteins f r o m the vestibular nuclei 7 (Fig. 3). In relation to their precise vestibular origin, it has been assumed that these type 11I terminals are excitatory in nature. Furthermore, in the present study these [3H]glutamate labeled synaptic boutons have the same localization on the dendritic tree, preferentially distributed on distal dendrites, as the excitatory nerve endings previously described. In the electron-microscopic radioautographs we have examined, glutamic acid uptake is never associated with large terminals (types I and ll) containing flat vesicles and identified as inhibitory synaptic boutons o f vestibular origin, in our previous work. Such a specific distribution o f glutamergic synapses on the o c u l o m o t o r motoneuron is also in accordance with the preferential glutamergic receptive sites on other neuronic systems, such as spinal m o t o n e u r o n s 27 or hippocampal cells zz. Therefore, although the origin of these labeled synapses is unknown, convergent similarities of morphological characteristic features and specific locations, suggest that these glutame~gic b o u t o n s probably have a vestibular origin. Even though other criteria are required for a definitive conclusion, these observations provide some evidence that glutamate would be the excitatory neurotransmitter o f the vestibulo-oculomotor pathway. Surgical interruption of this pathway could give us stronger evidence of the glutamergic nature o f its neurotransmitter. This w o r k was supported by giants from C N R S - A T P ' N e u r o b i o l o g i e du Systbme Nerveux Central' and I N S E R M - A T P 79. l 13.
1 Bak, 1. J. and Choi, W. B., Electron microscopic investigation of synaptic organization of the trochlear nucleus in cat. I. Normal ultrastructure, Cell Tiss. Res., t50 (1974) 409--423. 2 Bak, I. J., Baker, R., Choi, W. B. and Precht, W., Electron microscopic investigation of the vestibular projection to the cat trochlear nuclei, Neuroseienee, 1 (1976) 477482. 3 Baker, R. and Berthoz, A., Is the prepositus hypoglossi nucleus the source of another vestibuloocular pathway? Brain Research, 86 (1975) 121-127. 4 Baker, R., Delgado-Garcia, J. and Alley, K., Morphological and physiological demonstration that prepositus hypoglossi (pH) neurons terminate on medial rectus motor neurons, Int. Union Physiol. Sci. Abstr., XIII (1977) 46. 5 Baker, R. and Highstein, S. M.. Vestibular projections to medial rectus subdivision of oculomotor nucleus, J. Neurophysiol., 41 (1978) 1629-1646. 6 Curtis, D. R. and Johnston, G. A. R., Amino-acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 7 Demfimes, D. and Raymond. J., Identification des terminaisons vestibulaires clans les noyaux oculomoteurs communs chez le chat par radio-autographic en microscopic 61ectronique. Brain Research, 196 (1980) 331-345. 8 Fonnum, F. and Walaas, I.. The effect of intrahippocampal kainic acid injections and surgical lesions on neurotransmitters in hippocampus and septum, J. Neurochem., 31 (1978) 1173--1181. 9 Freeman, A. R., Polyfunctional role of glutamic acid in excitatory synaptie transmission, Prog. Neurobiol., 3 (1976) 137-153. 10 Grantyn, A., Grantyn, R., Gaunitz, V. and Robin6, K. P., Sources of direct excitatory and inhibitory inputs from the medial rhombencephalic tegrnentum to lateral and medial rectus motoneurons, Exp. Brain Res., 39 (1980] 49-61. 11 Henn, F. A., Neurotransmission and glial cells. A functional relationship? J. Neurosci. Res., 2 (1976) 271-282.
437 12 Highstein, S. M. and Baker, R., Excitatory termination of abducens internuclear neurons on medial rectus motoneurons: relationship to syndrome of internuclear ophthalmoplegia, J. Neurophysiol., 41 (1978) 1647-1661. 13 lto, M., Nisimura, N. and Yamamoto, M., Pathways for the vestibulo-ocular reflex excitation arising from semicircular canals of rabbits, Exp. Brain Res., 24 (1976) 273-283. 14 Johnson, J. L., Glutamic acid as a synaptic transmitter in the nervous system, Brain Research, 37 (1972) I 19. 15 Lund Karlsen, R. A. and Fonnum, F., Evidence for glutamate as a neurotransmitter in corticofugal fibres to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Research, 5 0978) 457-467. 16 McGeer, P. L., McGeer, E. G., Sherer, U. and Singh, K., A glutamergic corticostriataI path? Brain Research, 128 (1977) 369-373. 17 McLennan, H., The autoradiographic localization of L-[3H]g[utamate in rat brain tissue, Brain Research, 115 (1976) 139-144. 18 Nakao, S. and Sasaki, S., Excitatory input from interneurons in the abducens nucleus to medial rectus motoneurons mediating conjugate horizontal nystagmus in the cat, Exp. Brain Res., 39 0980) 23 32. 19 Precht, W. and Baker, R., Synaptic organization of the vestibulo-trochlear pathway, Exp. Brain Res., 14 (1972) 158-184, 20 Precht, W., Baker, R. and Okada, Y,, Evidence for GABA as the synaptic transmitter of the inhibitory vestibulo-ocular pathway, Exp. Brain Res., 18 (1973) 415428. 21 Schon, F. and Kelly, J. S., Autoradiographic localization of [3H]GABA and [3H]glutamate over satellite glia[ cells, Btzlin Research, 66 0974) 275-288. 22 Schwartzkroin, P. A. and Andersen, P., Glutamic acid sensitivity of dendrites in hippocampal slices in vitro. In G. W. Kreutzberg (Ed.), Advances in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 45-51. 23 Storm-Mathisen, J., Glutamic acid and excitatory nerve endings reduction of g[utamic acid uptake after axotomy, Brain Research, 120 (1977) 379-386. 24 Storm-Mathisen, J. and Iversen, L. L., Uptake of [3H]glutamic acid in excitatory nerve endings: light and electronmicroscopic observations in the hippocampal formation of the rat, Neuroscience, 4 (1979) 1237-1253. 25 Uchino, Y., Hirai, H. and Watanabe, S., Vestibulo-ocular reflex from the posterior canal nerve to extra-ocular motoneurons in the cat, Exp. Brain Res., 32 (1978) 377-388. 26 Uchino, Y., Suzuki, S., Miyazawa, T. and Watanabe, S., Horizontal canal input to cat extraocular motoneurons, Brain Research, 177 (1979) 231-240. 27 Zieglg~insberger, W. and Cbampagnat, J., Cat spinal motoneurones exhibit topographic sensitivity to glutamate and glycine, Brain Research, 160 (1979) 95-104.
433
Elsevier Biomedical Press
Radioautographic identification of [3H]glutamic acid labeled nerve endings in the cat oculomotor nucleus
D. DEMfEMES and J. RAYMOND Laboratoire de Neurophysiologie Sensorie[le, Universitddes Sciences et Techniques da Languedoc, Phwe E.-Bataillon, 34060 Montpellier Cddex (France)
(Accepted October 8th, 1981) Key words: glutamergic synapses - - oculomotor nucleus - - electron microscopic radioautography
Slices of the cat third oculomotor nucleus were incubated in vitro with [3H]glutamicacid. Electron microscopic radioautographs revealed that glutamate had been taken by small nerve endings distributed on the oculomotor motoneuron distal dendrites. In contrast, there was no uptake in the other types of terminals. The labeled terminals seem to correspond to the excitatory vestibulo-oculomotor nerve endings and different correlations suggest their glutamergic nature. There is now strong evidence from electrophysiological and neurochemical findings that glutamic acid is an excitatory neurotransmitter in the mammalian central nervous system (~,9,14. Furthermore, numerous investigations have shown reductions in glutamate uptake when degeneration of excitatory nerve endings is induced experimentallyS,15,t6, ~3. Recent studies by light- and electron-microscopic radioautography revealed that [3H]glutamic acid, exogenously applied in vitro, is selectively accumulated in slices of hippocampal formation by nerve endings recognized to be excitatory (ref. 24). Electrophysiological experiments provide strong evidence for the existence of excitatory pathways projecting on the oculomotor structures, particularly the third oculomotor nucleus. These fiber systems are: (1) the main vestibulo-oculomotor pathways originating from the median and lateral vestibular nuclei6,13,19,~5,26; (2) other oculomotor afferents, less important, recently investigated, issued from the nucleus abducensl0,12,18 and from the prepositus hypoglossi3, 4. It has recently been reported by Dem~mes and Raymond 7, in an electron-microscopic radioautographic investigation, that the oculomotor motoneuron's synaptic profile seems dominated by inhibitory vestibular endings with nevertheless a few excitatory ones; the latter seem concentrated in the dendritic neuropile. I f the inhibitory transmitter has been demonstrated to be gamma-aminobutyric acid (GABA), in the vestibulo-ocular pathways20; yet, the excitatory transmitter is still unknown. Consequently, it would be a new point of interest, in view of the possible role of glutamic acid as a neurotransmitter substance, to identify morphologically by electron-microscopic radioautography the uptake sites of this exogenously accumu0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
434 lated neurotransmitter in oculomotor nucleus slices. However, it should be mentioned that the glutamic acid uptake may label both glutamate and aspartate nerve endings, since under the conditions used in this experiment the uptake system does not distinguish between these excitatory neurotransmitters. Young cats, 2 or 3 months old, were anesthetized with sodium pentobarbitat and decapitated. The brain was rapidly removed and cooled on ice; serial thin transverse slices (250-300 ~m) were prepared and collected in cold Kreb's phosphate solution (pH - = 7.4). The oculomotor nuclei pieces were dissected out (details of this method have been presented in our former paper 7) and transferred into the identical solution at 25 ~'C, then 5 rain later into Kreb's solution containing 2 #M L-[G-3H]glutamic acid (20-40 Ci/mmol, Radiochemical Centre, Amersham). After a 15 rain incubation period at 25 °C and after rinsing in 2--3 changes of fresh Kreb's phosphate solution, the tissue pieces were fixed in 5 % glutaraldehyde for 3 h at 25 °C, treated with osmium tet~oxide, and embedded in araldite for electron-microscopic radioautography. The ultrathin sections were deposited upon celloidin-covered slides, stained with uranyl acetate and lead citrate, then vaporized with carbon, and were covered with Ilford L4 emulsion (diluted 1:4) by the dipping technique. Following an exposure time of 8 weeks, the radioautographs were developed with Kodak Microdol-X. Electron-microscopic radioautographs teveal heavy accumulations of silver grains over some nerve endings identified on the basis of their ultrastructural characteristic features. Among the 5 distinct types of terminals described in the oculomotor nuclei1, 2,7, only one category appears to be labeled. These labeled synaptic boutons are numerous, of small size (1-2/~m in diameter), rarely axosomatic (Figs. 1 and 2); they are mainly axodendritic and are most of the time distributed on the distal part of the dendritic tree of oculomotor motoneurons. The synaptic vesicles of these boutons are numerous, scattered in the presynaptic axoplasm and appear to be clear and spherical; 2-5 mitochondria are also present. No accumulations of silver grains are seen over larger synaptic boutons containing flat vesicles. In addition to the nerve endings, satellite glial cells also present aggregates of silver grains located over cytoplasm and nucleus. On the contrary, oculomotor motoneurons and their proximal dendritic processes are devoid of label. In the presently used method, involving incubation with exogenous glutamic acid in vitro, tissue preservation is far from optimal, but good enough to allow a precise morphological identification of nerve endings. Although glutamic acid is ~eadily metabolized, recent studies have shown that most of the radioactivity present in the slices prior to fixation could be attributed to unchanged [3H]glutamate and not to its labeled metabolites '~4. Although a better preservation of tissue should be obtained by injecting [3H]glutamic acid in vivo, the metabolism of [aH]glutamic acid would present a still more difficult problem in vivo. The uptake of exogenous glutamic acid by the glial cells in the oculomotor nucleus is in clear agreement with other studies 17,zl. It seems indeed that glia may have efficient uptake systems for the transmitter present in the cerebral tissue tI. These labeled synaptic boutons are quite similar in morphological characteristics to the small type lII terminals, described in cat trochlear nuclei after lesion of the
435
-
J
Figs. 1, 2. Electron-microscopic radioautographs of oculomotor nucleus incubated with [3H]glutamic acid. Small labeled nerve endings axodendritically located containing numerous clear synaptic vesicles. Fig. 3. Electron-microscopic radioautograph of oculomotor nucleus following [ZH]proline injection in vestibular nuclei. Labeled axo-dendritic synapse (type II1).
436 vestibulo-trochlear pathway 1,z, as well as in cat o c u l o m o t o r nuclei, where they were identified by anterograde transport o f labeled proteins f r o m the vestibular nuclei 7 (Fig. 3). In relation to their precise vestibular origin, it has been assumed that these type 11I terminals are excitatory in nature. Furthermore, in the present study these [3H]glutamate labeled synaptic boutons have the same localization on the dendritic tree, preferentially distributed on distal dendrites, as the excitatory nerve endings previously described. In the electron-microscopic radioautographs we have examined, glutamic acid uptake is never associated with large terminals (types I and ll) containing flat vesicles and identified as inhibitory synaptic boutons o f vestibular origin, in our previous work. Such a specific distribution o f glutamergic synapses on the o c u l o m o t o r motoneuron is also in accordance with the preferential glutamergic receptive sites on other neuronic systems, such as spinal m o t o n e u r o n s 27 or hippocampal cells zz. Therefore, although the origin of these labeled synapses is unknown, convergent similarities of morphological characteristic features and specific locations, suggest that these glutame~gic b o u t o n s probably have a vestibular origin. Even though other criteria are required for a definitive conclusion, these observations provide some evidence that glutamate would be the excitatory neurotransmitter o f the vestibulo-oculomotor pathway. Surgical interruption of this pathway could give us stronger evidence of the glutamergic nature o f its neurotransmitter. This w o r k was supported by giants from C N R S - A T P ' N e u r o b i o l o g i e du Systbme Nerveux Central' and I N S E R M - A T P 79. l 13.
1 Bak, 1. J. and Choi, W. B., Electron microscopic investigation of synaptic organization of the trochlear nucleus in cat. I. Normal ultrastructure, Cell Tiss. Res., t50 (1974) 409--423. 2 Bak, I. J., Baker, R., Choi, W. B. and Precht, W., Electron microscopic investigation of the vestibular projection to the cat trochlear nuclei, Neuroseienee, 1 (1976) 477482. 3 Baker, R. and Berthoz, A., Is the prepositus hypoglossi nucleus the source of another vestibuloocular pathway? Brain Research, 86 (1975) 121-127. 4 Baker, R., Delgado-Garcia, J. and Alley, K., Morphological and physiological demonstration that prepositus hypoglossi (pH) neurons terminate on medial rectus motor neurons, Int. Union Physiol. Sci. Abstr., XIII (1977) 46. 5 Baker, R. and Highstein, S. M.. Vestibular projections to medial rectus subdivision of oculomotor nucleus, J. Neurophysiol., 41 (1978) 1629-1646. 6 Curtis, D. R. and Johnston, G. A. R., Amino-acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 97-188. 7 Demfimes, D. and Raymond. J., Identification des terminaisons vestibulaires clans les noyaux oculomoteurs communs chez le chat par radio-autographic en microscopic 61ectronique. Brain Research, 196 (1980) 331-345. 8 Fonnum, F. and Walaas, I.. The effect of intrahippocampal kainic acid injections and surgical lesions on neurotransmitters in hippocampus and septum, J. Neurochem., 31 (1978) 1173--1181. 9 Freeman, A. R., Polyfunctional role of glutamic acid in excitatory synaptie transmission, Prog. Neurobiol., 3 (1976) 137-153. 10 Grantyn, A., Grantyn, R., Gaunitz, V. and Robin6, K. P., Sources of direct excitatory and inhibitory inputs from the medial rhombencephalic tegrnentum to lateral and medial rectus motoneurons, Exp. Brain Res., 39 (1980] 49-61. 11 Henn, F. A., Neurotransmission and glial cells. A functional relationship? J. Neurosci. Res., 2 (1976) 271-282.
437 12 Highstein, S. M. and Baker, R., Excitatory termination of abducens internuclear neurons on medial rectus motoneurons: relationship to syndrome of internuclear ophthalmoplegia, J. Neurophysiol., 41 (1978) 1647-1661. 13 lto, M., Nisimura, N. and Yamamoto, M., Pathways for the vestibulo-ocular reflex excitation arising from semicircular canals of rabbits, Exp. Brain Res., 24 (1976) 273-283. 14 Johnson, J. L., Glutamic acid as a synaptic transmitter in the nervous system, Brain Research, 37 (1972) I 19. 15 Lund Karlsen, R. A. and Fonnum, F., Evidence for glutamate as a neurotransmitter in corticofugal fibres to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Research, 5 0978) 457-467. 16 McGeer, P. L., McGeer, E. G., Sherer, U. and Singh, K., A glutamergic corticostriataI path? Brain Research, 128 (1977) 369-373. 17 McLennan, H., The autoradiographic localization of L-[3H]g[utamate in rat brain tissue, Brain Research, 115 (1976) 139-144. 18 Nakao, S. and Sasaki, S., Excitatory input from interneurons in the abducens nucleus to medial rectus motoneurons mediating conjugate horizontal nystagmus in the cat, Exp. Brain Res., 39 0980) 23 32. 19 Precht, W. and Baker, R., Synaptic organization of the vestibulo-trochlear pathway, Exp. Brain Res., 14 (1972) 158-184, 20 Precht, W., Baker, R. and Okada, Y,, Evidence for GABA as the synaptic transmitter of the inhibitory vestibulo-ocular pathway, Exp. Brain Res., 18 (1973) 415428. 21 Schon, F. and Kelly, J. S., Autoradiographic localization of [3H]GABA and [3H]glutamate over satellite glia[ cells, Btzlin Research, 66 0974) 275-288. 22 Schwartzkroin, P. A. and Andersen, P., Glutamic acid sensitivity of dendrites in hippocampal slices in vitro. In G. W. Kreutzberg (Ed.), Advances in Neurology, Vol. 12, Raven Press, New York, 1975, pp. 45-51. 23 Storm-Mathisen, J., Glutamic acid and excitatory nerve endings reduction of g[utamic acid uptake after axotomy, Brain Research, 120 (1977) 379-386. 24 Storm-Mathisen, J. and Iversen, L. L., Uptake of [3H]glutamic acid in excitatory nerve endings: light and electronmicroscopic observations in the hippocampal formation of the rat, Neuroscience, 4 (1979) 1237-1253. 25 Uchino, Y., Hirai, H. and Watanabe, S., Vestibulo-ocular reflex from the posterior canal nerve to extra-ocular motoneurons in the cat, Exp. Brain Res., 32 (1978) 377-388. 26 Uchino, Y., Suzuki, S., Miyazawa, T. and Watanabe, S., Horizontal canal input to cat extraocular motoneurons, Brain Research, 177 (1979) 231-240. 27 Zieglg~insberger, W. and Cbampagnat, J., Cat spinal motoneurones exhibit topographic sensitivity to glutamate and glycine, Brain Research, 160 (1979) 95-104.