- Email: [email protected]
The autoradiographic localization of the gaba-releasing nerve terminals in cerebellar glomeruli
Brain Research, 85 (1975) 255-259
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
255
T H E A U T O R A D I O G R A P H I C L O C A L I Z A T I O N OF T H E G A B A - R E L E A S I N G N E R V E T E R M I N A L S IN C E R E B E L L A R G L O M E R U L I
J. S. KELLY, FABIENNE DICK AND F. SCHON MRC Neurochemical Pharmacology Unit, Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD (Great Britain}
With the possible exception of the long inhibitory pathways originating in the cerebellumZ,4,1a,z° and perhaps those terminating in the substantia nigraS,9,1°, 1~, the identification of the majority o f inhibitory interneurones and their terminals is in all probability not amenable to techniques based on either axonal degeneration or the axonal transport of injected amino acids after their incorporation into proteins within neuronal cell bodies. On the other hand, there is a great deal of evidence to suggest that many of the inhibitory interneurones which operate by releasing 7-aminobutyric acid (GABA) from their terminals, possess a unique high affinity uptake system for the recapture of spent GABA from the synaptic cleft11. Although a number of laboratoriesT, s,12,16 have now taken advantage of this high affinity uptake system for GABA to autoradiographically identify nerve terminals and cell bodies in regions where they are known to be inhibitory on functional grounds, the recent demonstration that GABA is also taken up into glial cells by a high affinity process 17,18 has made electron microscopic autoradiography almost essential for the accurate interpretation of results. Earlier is we suggested that, in time, it may be possible to overcome this difficulty by the use of GABA analogues such as L-2,4-diaminobutyric acid (DABA) which appear to be substrates for the uptake process in nerve terminals but not that into glial cells. In this paper, we would like to draw attention to the ease with which DABA can be used autoradiographically to mark known inhibitory nerve terminals in the cerebellum. The evidence that DABA is preferentially taken up by the same uptake system as that which concentrates GABA in the nerve endings of the cerebral cortex in vitro is outside the scope of this paper (F. Dick and J. S. Kelly in preparation). Rats were anaesthetized with a mixture of halothane, nitrous oxide and oxygen, and received 3 injections of 6.5 × 10-gM [3H]DABA (Amersham; in 2 tzl of artificial CSF containing approximately 75 #Ci of radioactivity). The amino acid was injected under pressure at a depth of about 1 mm into the centre of a single lobule of the cerebellar vermis using a fine glass microelectrode, the tip of which was broken back to approximately 15-20/zm. Ten to 15 min later the animals were killed by perfusion fixation with 5 ~ glutaraldehyde. Subsequently 350/~m thick coronal slices of the lobule were cut on an Oxford 'Vibratome' and prepared for electron microscopic
Fig. I. A and B: autoradiographic localization of [',~HIDABA over glomeruli in the cerebcllar cortex. Clusters of silver grains are selectively concentrated over the small inhibitory Golgi cell axons (indicated by two arrows), which lie adjacent to the large unlabelled excitator5 mossy fibre terminals (MF), both of which form synaptic contacts with the granule cell dendrites. The surrounding gr~l]mle cell bodies are themselves unlabelled (see Table 1). Calibration bat'. 0.5/ml.
257
Fig. 2. Autoradiographic localization of [3H]DABA over granule cell neuropil region. Electron micrograph showing a large granule cell process (gr c) closely apposed by 2 clearly labelled Golgi cell axons (indicated by two arrows) and one unlabelled mossy fibre terminal (MF). Other neuropil structures are devoid of silver grains. Calibration bar, 0.5 l~m.
258 TABLE I DISTRIBUTION OF SILVER GRAINS OVER ELECTRON MICROSCOPIC AUTORADIOGRAPHS OF CEREBELLAR GLOMERULILABELLEDWITH [3H]DABA Means and standard errors from an analysis of 15 different electron micrographs (8 in. x 10 in,) each containing at least one labelled Golgi cell terminal (Go. ax.) and centred on a mossy fibre (M.F.), surrounded by easily identifiable granule cell bodies (gr. c.) and dendrites (gr. d.). An average of 23 individual silver grains could be recognized (on each micrograph) which represented an area of approximately 28 sq. /tm. The area occupied by each structure was determined by placing a transparent sheet bearing a square array of 80 dots over the electron micrographs in two different orientations on separate occasions and counting the dots over each identifiable structure (ef. Haug% Identified structure
gr. d.
Area as a percentage of the whole 22
i
gr. c.
3
21
M.F.
~ 3
22
Go. ax.
i 2
Silver grains/ 10sq./~m
8.4 ± 0.7
3.0 ± 0.9
3.2 ± 0.6
Relative density
1.02
0.36
0.39
4.5 _+_ 0.4 75 9.15
± 8
Remainder
Total
20.5
100
5.3 ± 1.1
8.2 £ 0.8
0.65
1.0
autoradiography as described previously17. Approximately 200,000 disint./min/g could be recovered from the fixed tissue which was shown by light microscopic autoradiography to be evenly distributed throughout the granular and molecular layers of the injected lobule. Although examination of autoradiographs after only a 10-day exposure period revealed silver grains to be present over small nerve terminals in all layers of the cerebellum, one of the most striking and consistent features of sections taken from the granular layer was the clustering of silver grains in association with the mossy fibre glomeruli. On closer inspection (Figs. 1 and 2), these dense aggregations of silver grains were found to be located almost exclusively over small axo-dendritic synapses in contact with the terminal digits of the granule cell dendrites. These synapses have been identified as Golgi axon terminals on the basis of earlier studies which combined Golgi staining and ultrastructural techniques (cf. reviews by Szenthgothai19; Palay and Chan-Palay15). Silver grains were only rarely found over the other major components of the glomeruli; the mossy fibre terminals and the granule cell bodies and their dendrites. Numerical analysis (Table I) of the electron micrographs shown in Figs. 1 and 2 together with 12 others from nearby glomeruli present on the same section confirmed that the silver grains lay predominantly over the Golgi cell axons which occupied less than 5 ~ of the surface area of the micrographs. Indeed, the silver grain density over these terminals was at least 9 times higher than the average density per micrograph and 20-30 times greater than that over the mossy fibre terminals and the granule cell bodies. Presumably, the high density of silver grains over Golgi cell axons is indicative of their ability to accumulate DABA as well as its naturally occurring analogue,
259 GABA. Likewise the paucity of labelling over the other constituents of the glomeruli is consistent with the view that they are excitatory and thus unlikely to mediate their actions by GABA release. Since there is a wealth of both physiological2 and pharmacological1 evidence to support the view that Golgi cell axons are the only easily identifiable inhibitory input to the glomeruli, these results represent a significant advance in attempts to identify the actual neuronal sites of GABA release. We are indebted to the Wellcome Trust for financial support to F. D. and Mr. David W. Chapman for skilled technical assistance. 1 BISTI, S., IOSIF, G., AND STRATA,P., Suppression of inhibition in the cerebellar cortex by picrotoxin and bicuculline, Brain Research, 28 (1971) 591-593. 2 ECCLES,J. C., ITO, M., AND SZENTAGOTHAI,J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1970. 3 FONNUM, F., STORM-MATHISEN,J., AND WALBERG, F., Glutamate decarboxylase in inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat, Brain Research, 20 (1970) 259-275. 4 FONNUM, F., AND WALBERG, F., An estimation of the concentration of GABA and glutamate decarboxylase in the inhibitory Purkinje axon terminals in the cat, Brain Research, 54 (1973) 115-127. 5 HATTOR1,T,, MCGEER, P. L., FmIGER, H. C., Ant) MCGEER, E. G., On the source of GABAcontaining terminals in the substantia nigra. Electron microscopic autoradiographic and biochemical studies, Brain Research, 54 (1973) 103-114. 6 HAUG, H., Stereological methods in the analysis of neuronal parameters in the central nervous system, J. Microse., 95 (1971) 165-180. 7 HOKFELT, T., AND LJUNGDAHL,/~., Cellular localization of labelled gamma-aminobutyric acid (aH-GABA) in the rat cerebellar cortex: an autoradiographic study, Brain Research, 22 (1970) 391-393. 8 H6KFELT, T., AND LJUNGDArtL, A., Autoradiographic identification of cerebral and cerebellar cortical neurones accumulating labelled gamma-aminobutyric acid (aH-GABA), Exp. Brain Res., 14 (1972) 354-362. 9 KATAOKA,K., BAK, I. J., HASSLER,R., KIM, J. S., AND WAGNER, A., L-Glutamate decarboxylase and choline acetyltransferase activity in the substantia nigra and the striatum after surgical interruption of the strio-nigral fibres of the baboon, Exp. Brain Res., 19 (1974) 217-227. 10 KIM, J. S., BAK, I. J., HASSLER, R., AND OKADA, Y., Role of 7-aminobutyric acid (GABA) in the extrapyramidal motor system. II. Evidence for the existence of GABA-rich strio-nigral neurons, Exp. Brain Res., 14 (1971) 95-104. 11 IVERSEN,L. L., The uptake, storage, release and metabolism of GABA in inhibitory nerves. In S. H. SNVDER (Ed.), Perspectives in Neuropharmacology, Oxford Univ. Press, London, 1972, pp. 75-111. 12 IVERSEN,L. L., AND BLOOM,F. E., Studies on the uptake of [aH]GABA and [aH]glycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 13 McGEER, P. L., MCGEER, E. G., WADA, J. A., AND JtJNG, E., Effect of globus pallidus lesions and Parkinson's disease on brain glutamic acid decarboxylase, Brain Research, 32 (1971) 425431. 14 OTSUKA, M., OBATA, K., MIYATA, Y., AND TANAKA, Y., Measurement of y-aminobutyric acid in isolated nerve cells of cat central nervous system, J. Neurochem., 18 (1971) 287-295. 15 PALAY, S. L., AND CHAN-PALAY, V., Cerebellar Cortex. Cytology and Organization, Springer, Berlin, 1974. 16 SCHON, F., AND IVERSEN,L. L., Selective accumulation of [3H]GABA by stellate cells in rat cerebellar cortex in vivo, Brain Research, 42 (1972) 503-507. 17 SCHON, F., AND KELLY, J. S., Autoradiographic localization of [aHIGABA and [all]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 18 SCHON, F., AND KELLY, J. S., The characterization of [aH]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289-300. 19 SZENTA.GOTHAI,J., Structure-function relations in inhibitory synapses, Advanc. Cytopharmacol., 1 (1971) 402417. 20 WALBERG, F., AND JANSEN, J., Cerebellar corticovestibular fibres in the cat, Exp. Neurol, 3 (1971) 32-52.
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
255
T H E A U T O R A D I O G R A P H I C L O C A L I Z A T I O N OF T H E G A B A - R E L E A S I N G N E R V E T E R M I N A L S IN C E R E B E L L A R G L O M E R U L I
J. S. KELLY, FABIENNE DICK AND F. SCHON MRC Neurochemical Pharmacology Unit, Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD (Great Britain}
With the possible exception of the long inhibitory pathways originating in the cerebellumZ,4,1a,z° and perhaps those terminating in the substantia nigraS,9,1°, 1~, the identification of the majority o f inhibitory interneurones and their terminals is in all probability not amenable to techniques based on either axonal degeneration or the axonal transport of injected amino acids after their incorporation into proteins within neuronal cell bodies. On the other hand, there is a great deal of evidence to suggest that many of the inhibitory interneurones which operate by releasing 7-aminobutyric acid (GABA) from their terminals, possess a unique high affinity uptake system for the recapture of spent GABA from the synaptic cleft11. Although a number of laboratoriesT, s,12,16 have now taken advantage of this high affinity uptake system for GABA to autoradiographically identify nerve terminals and cell bodies in regions where they are known to be inhibitory on functional grounds, the recent demonstration that GABA is also taken up into glial cells by a high affinity process 17,18 has made electron microscopic autoradiography almost essential for the accurate interpretation of results. Earlier is we suggested that, in time, it may be possible to overcome this difficulty by the use of GABA analogues such as L-2,4-diaminobutyric acid (DABA) which appear to be substrates for the uptake process in nerve terminals but not that into glial cells. In this paper, we would like to draw attention to the ease with which DABA can be used autoradiographically to mark known inhibitory nerve terminals in the cerebellum. The evidence that DABA is preferentially taken up by the same uptake system as that which concentrates GABA in the nerve endings of the cerebral cortex in vitro is outside the scope of this paper (F. Dick and J. S. Kelly in preparation). Rats were anaesthetized with a mixture of halothane, nitrous oxide and oxygen, and received 3 injections of 6.5 × 10-gM [3H]DABA (Amersham; in 2 tzl of artificial CSF containing approximately 75 #Ci of radioactivity). The amino acid was injected under pressure at a depth of about 1 mm into the centre of a single lobule of the cerebellar vermis using a fine glass microelectrode, the tip of which was broken back to approximately 15-20/zm. Ten to 15 min later the animals were killed by perfusion fixation with 5 ~ glutaraldehyde. Subsequently 350/~m thick coronal slices of the lobule were cut on an Oxford 'Vibratome' and prepared for electron microscopic
Fig. I. A and B: autoradiographic localization of [',~HIDABA over glomeruli in the cerebcllar cortex. Clusters of silver grains are selectively concentrated over the small inhibitory Golgi cell axons (indicated by two arrows), which lie adjacent to the large unlabelled excitator5 mossy fibre terminals (MF), both of which form synaptic contacts with the granule cell dendrites. The surrounding gr~l]mle cell bodies are themselves unlabelled (see Table 1). Calibration bat'. 0.5/ml.
257
Fig. 2. Autoradiographic localization of [3H]DABA over granule cell neuropil region. Electron micrograph showing a large granule cell process (gr c) closely apposed by 2 clearly labelled Golgi cell axons (indicated by two arrows) and one unlabelled mossy fibre terminal (MF). Other neuropil structures are devoid of silver grains. Calibration bar, 0.5 l~m.
258 TABLE I DISTRIBUTION OF SILVER GRAINS OVER ELECTRON MICROSCOPIC AUTORADIOGRAPHS OF CEREBELLAR GLOMERULILABELLEDWITH [3H]DABA Means and standard errors from an analysis of 15 different electron micrographs (8 in. x 10 in,) each containing at least one labelled Golgi cell terminal (Go. ax.) and centred on a mossy fibre (M.F.), surrounded by easily identifiable granule cell bodies (gr. c.) and dendrites (gr. d.). An average of 23 individual silver grains could be recognized (on each micrograph) which represented an area of approximately 28 sq. /tm. The area occupied by each structure was determined by placing a transparent sheet bearing a square array of 80 dots over the electron micrographs in two different orientations on separate occasions and counting the dots over each identifiable structure (ef. Haug% Identified structure
gr. d.
Area as a percentage of the whole 22
i
gr. c.
3
21
M.F.
~ 3
22
Go. ax.
i 2
Silver grains/ 10sq./~m
8.4 ± 0.7
3.0 ± 0.9
3.2 ± 0.6
Relative density
1.02
0.36
0.39
4.5 _+_ 0.4 75 9.15
± 8
Remainder
Total
20.5
100
5.3 ± 1.1
8.2 £ 0.8
0.65
1.0
autoradiography as described previously17. Approximately 200,000 disint./min/g could be recovered from the fixed tissue which was shown by light microscopic autoradiography to be evenly distributed throughout the granular and molecular layers of the injected lobule. Although examination of autoradiographs after only a 10-day exposure period revealed silver grains to be present over small nerve terminals in all layers of the cerebellum, one of the most striking and consistent features of sections taken from the granular layer was the clustering of silver grains in association with the mossy fibre glomeruli. On closer inspection (Figs. 1 and 2), these dense aggregations of silver grains were found to be located almost exclusively over small axo-dendritic synapses in contact with the terminal digits of the granule cell dendrites. These synapses have been identified as Golgi axon terminals on the basis of earlier studies which combined Golgi staining and ultrastructural techniques (cf. reviews by Szenthgothai19; Palay and Chan-Palay15). Silver grains were only rarely found over the other major components of the glomeruli; the mossy fibre terminals and the granule cell bodies and their dendrites. Numerical analysis (Table I) of the electron micrographs shown in Figs. 1 and 2 together with 12 others from nearby glomeruli present on the same section confirmed that the silver grains lay predominantly over the Golgi cell axons which occupied less than 5 ~ of the surface area of the micrographs. Indeed, the silver grain density over these terminals was at least 9 times higher than the average density per micrograph and 20-30 times greater than that over the mossy fibre terminals and the granule cell bodies. Presumably, the high density of silver grains over Golgi cell axons is indicative of their ability to accumulate DABA as well as its naturally occurring analogue,
259 GABA. Likewise the paucity of labelling over the other constituents of the glomeruli is consistent with the view that they are excitatory and thus unlikely to mediate their actions by GABA release. Since there is a wealth of both physiological2 and pharmacological1 evidence to support the view that Golgi cell axons are the only easily identifiable inhibitory input to the glomeruli, these results represent a significant advance in attempts to identify the actual neuronal sites of GABA release. We are indebted to the Wellcome Trust for financial support to F. D. and Mr. David W. Chapman for skilled technical assistance. 1 BISTI, S., IOSIF, G., AND STRATA,P., Suppression of inhibition in the cerebellar cortex by picrotoxin and bicuculline, Brain Research, 28 (1971) 591-593. 2 ECCLES,J. C., ITO, M., AND SZENTAGOTHAI,J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1970. 3 FONNUM, F., STORM-MATHISEN,J., AND WALBERG, F., Glutamate decarboxylase in inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat, Brain Research, 20 (1970) 259-275. 4 FONNUM, F., AND WALBERG, F., An estimation of the concentration of GABA and glutamate decarboxylase in the inhibitory Purkinje axon terminals in the cat, Brain Research, 54 (1973) 115-127. 5 HATTOR1,T,, MCGEER, P. L., FmIGER, H. C., Ant) MCGEER, E. G., On the source of GABAcontaining terminals in the substantia nigra. Electron microscopic autoradiographic and biochemical studies, Brain Research, 54 (1973) 103-114. 6 HAUG, H., Stereological methods in the analysis of neuronal parameters in the central nervous system, J. Microse., 95 (1971) 165-180. 7 HOKFELT, T., AND LJUNGDAHL,/~., Cellular localization of labelled gamma-aminobutyric acid (aH-GABA) in the rat cerebellar cortex: an autoradiographic study, Brain Research, 22 (1970) 391-393. 8 H6KFELT, T., AND LJUNGDArtL, A., Autoradiographic identification of cerebral and cerebellar cortical neurones accumulating labelled gamma-aminobutyric acid (aH-GABA), Exp. Brain Res., 14 (1972) 354-362. 9 KATAOKA,K., BAK, I. J., HASSLER,R., KIM, J. S., AND WAGNER, A., L-Glutamate decarboxylase and choline acetyltransferase activity in the substantia nigra and the striatum after surgical interruption of the strio-nigral fibres of the baboon, Exp. Brain Res., 19 (1974) 217-227. 10 KIM, J. S., BAK, I. J., HASSLER, R., AND OKADA, Y., Role of 7-aminobutyric acid (GABA) in the extrapyramidal motor system. II. Evidence for the existence of GABA-rich strio-nigral neurons, Exp. Brain Res., 14 (1971) 95-104. 11 IVERSEN,L. L., The uptake, storage, release and metabolism of GABA in inhibitory nerves. In S. H. SNVDER (Ed.), Perspectives in Neuropharmacology, Oxford Univ. Press, London, 1972, pp. 75-111. 12 IVERSEN,L. L., AND BLOOM,F. E., Studies on the uptake of [aH]GABA and [aH]glycine in slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 13 McGEER, P. L., MCGEER, E. G., WADA, J. A., AND JtJNG, E., Effect of globus pallidus lesions and Parkinson's disease on brain glutamic acid decarboxylase, Brain Research, 32 (1971) 425431. 14 OTSUKA, M., OBATA, K., MIYATA, Y., AND TANAKA, Y., Measurement of y-aminobutyric acid in isolated nerve cells of cat central nervous system, J. Neurochem., 18 (1971) 287-295. 15 PALAY, S. L., AND CHAN-PALAY, V., Cerebellar Cortex. Cytology and Organization, Springer, Berlin, 1974. 16 SCHON, F., AND IVERSEN,L. L., Selective accumulation of [3H]GABA by stellate cells in rat cerebellar cortex in vivo, Brain Research, 42 (1972) 503-507. 17 SCHON, F., AND KELLY, J. S., Autoradiographic localization of [aHIGABA and [all]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 18 SCHON, F., AND KELLY, J. S., The characterization of [aH]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289-300. 19 SZENTA.GOTHAI,J., Structure-function relations in inhibitory synapses, Advanc. Cytopharmacol., 1 (1971) 402417. 20 WALBERG, F., AND JANSEN, J., Cerebellar corticovestibular fibres in the cat, Exp. Neurol, 3 (1971) 32-52.