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Accumulation of glutamic acid decarboxylase in the proximal parts of presumed GABA-ergic neurones after axotomy
Brain Research, 87 (1975) 107-109 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
107
Accumulation of glutamic acid decarboxylase in the proximal parts of presumed GABA-ergic neurones after axotomy
JON STORM-MATHISEN
Norwegian Defence Research Establishment, Division Jor Toxicology, N-2007 Kjelh, r (Norway) (Accepted December 19th, 1974)
Transection of nerve fibres producing acetylcholine or aromatic monoamines results in accumulation of the putative transmitter substances and related enzymes in the proximal parts of the respective neurones 2,v. This is a confirmation that the concomitant losses from the terminal area are due to interruption of the axons, rather than to secondary transsynaptic effects. Such accumulation has hitherto not been demonstrated in ?,-aminobutyric acid (GABA) producing nerve pathways in brain. GABA is probably the transmitter in a striato-nigral pathway 1,3,4,6,s,t°, one important piece of evidence being that the synthesizing enzyme, glutamic acid decarboxylase (GAD), nearly disappears from substantia nigra after surgical interruption of the pathway3,4,6, s. Most workers have concluded that this pathway projects from nucleus caudatus/putamenl,3.6, TM to substantia nigra and globus pallidus, whereas one group has reported evidence that it originates in globus pallidus 4,8. ]n the present paper I report the increase of G A D in nucleus caudatus/putamen of the rat after cutting the connexion with substantia nigra. Hemisections were performed in 200 g male Wistar rats at the level of corpus mamillare 11. At various survival times frontal brain slices were cut by a mechanical chopper in the cold room and nucleus caudatus/putamen and substantia nigra were dissected under the microscope and homogenized in 0.32 M sucrose. GAD, choline acetyltransferase (CHAT), dihydroxyphenylalanine decarboxylase (DOPAD), GABA uptake (at 2 × 10-s M) and protein were measured. The methods are described elsewhere 11. Assay mixtures for enzymes always contained 0.1-0.2~/o Triton X-100 to release full enzyme activity. After the hemisections a maximum increase of G A D in caudatus/putamen (about 50}o above normal) was reached within one week (Fig. 1). Concomitantly G A D in substantia nigra and DOPAD in caudatus/putamen decreased by about 90 or in accordance with the previously demonstrated reciprocity of dopamine-ergic and GABA-ergic fibres in this system s. ChAT did not change, in line with the earlier results showing that cholinergic neurones are intrinsic in caudatus/putamena, 9. Protein/wet wt. was unaltered (93-98~). The values on the control side did not change significantly. The average of the mean values obtained at the various survival
108 '
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SUBSTANTIA NIGRA ,h P"=0,00]~ GAD DIFFERENT FROM P<'OIO2~J CONTROL
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DAYSAFTEROPERATION
Fig. I. Effect of hemisections on GAD, ChAT and DOPAD in nucleus caudatus/putamen, and on GAD in substantia nigra. GAD was assayed at either 1.3 mM (<, 7]) or 6.4 mM(@, I ) f i n a l concentration of L-[l-~C]glutamate. For each experiment the number of animals is indicated by a figure beside the point for GAD. Statistical significance was determined by the two-tailed Student's t-test. The average of the mean activities on the unoperated side through the different experiments was in Hmole/h/g protein ± S.E.M. (number of experiments): GAD in caudatus/putamen, 217 : 12 (4 at 6.4 mMglu) and 102 [~ 17 (3 at 1.3 mMglu); CHAT, 396 ~: 55 (3); DOPAD, 17.0 " 2.0 (5); GAD in substantia nigra, 857 ~ 78 (4 at 6.4 mMglu) and 620 ; 52 (3 at 1.3 mMglu).
times were significantly different (two-tailed S t u d e n t ' s t-test) for G A D on the o p e r a t e d c o m p a r e d to the u n o p e r a t e d side ( P < 0.00t), a n d for the relative values o f G A D on the o p e r a t e d side c o m p a r e d to those o f C h A T (P -< 0.010). T w o g r o u p s 6,s have previously failed to detect an increase o f G A D in c a u d a t u s p u t a m e n after hemisections. The reason for this is obscure, but M c G e e r a n d cow o r k e r s 4,s used a s o m e w h a t different kind o f t r a n s e c t i o n t m o r e vertical), a n d K a t a o k a e t al. 6 used b a b o o n s . O n the o t h e r hand. H o c k m a n e t al. 5, using cats, f o u n d a subs t a n t i a l increase in some o f the a n i m a l s : out o f 21 cats at survival time 4-16 days 17 r a n g e d 7 5 - 1 2 0 ~ (95 T 3), whereas 4 ranged 140-187~'o (164 -~ 13). The o b l i q u e t r a n s e c t i o n s used in the present study passed in front o f the m o s t d o r s o - c a u d a l p a r t o f the nucleus c a u d a t u s / p u t a m e n (this p a r t c o n s t i t u t e d up to 25 o//o o f the volume, a c o r r e s p o n d i n g p a r t o f the c o n t r a l a t e r a l nucleus was discarded) a n d m a y have injured the d o r s a l tip o f globus pallidus. In a d d i t i o n to the striato-nigral axons, s o m e collaterals were therefore interrupted within the c a u d a t u s / p u t a m e n a n d globus pallidus. The m o r e m a r k e d increase o f G A D activity in the present than in previous studies may possibly be related to this m o r e extensive transection o f the axon tree. T h e m o s t likely i n t e r p r e t a t i o n o f the present results a p p e a r s to be t h a t G A D was piled up in the i n t r a n e o s t r i a t a l p a r t s (perikarya, axons, and terminals of intrinsic collaterals) o f the G A B A - e r g i c striato-nigral n e u r o n e s after transection o f their axons. S p r o u t i n g o f collaterals is less likely, since the lack o f change in G A B A u p l a k e (1 t0 .+
109 12, 109 ± 5 and 104 4- i 0 ~ of unoperated control 4- S.E.M. at 4, 10 and 21 days, respectively, compared to 30-40 ~o in substantia nigra 11) indicates that the intraneostriatal surface of the neurones did not expand. In the experiments at 4 and 21 days survival portions of the homogenates were separated into a pellet (containing the synaptosomes) and a supernatant by centrifugation at 16,000 × g for 60 rain. The pellets contained 75.4 4- 1.9 and 74.6 4- 1 . 5 ~ of the total G A D activity on the operated and control sides, respectively (means 4- S.E.M., for all 5 animals). This shows that the enzyme was not only piled up in perikarya and axons, but was transported to intraneostriatal terminals. The maximum amount of G A D accumulated in the parts of caudatus/putamen anterior to the lesions (50 ~ × 220 nmole/h/mg protein x 3.3 nag protein/nucleus 360 nmole/h/nucleus) was comparable to the total amount of lesion-sensitive G A D in substantia nigra (90 ~ × 860 nmole/h/mg protein × 0.3 mg protein/nucleus ~ 230 nmole/h/nucleus). The surplus may correspond to that destined for cut intraneostriatal collaterals and the dorsal tip of globus pallidus. An additional accumulation presumably occurred in the axons between the transection and caudatus/putamen, i.e. partly within globus pallidus. This region could not be accurately dissected due to its proximity to the lesion.
1 CROSSMAN, A. R., WALKER, R. J., ANO WOODRUFF, G. N., Picrotoxin antagonism of 7-amino-
2 3
4
5
6
7
8
9 10 l [
butyric acid inhibitory responses and synaptic inhibition in the rat substantia nigra, Brit. J. Pharmacol., 49 (1973) 696-698. DAHLSTR()M, A., Axoplasmic transport (with particular respect to adrenergic neurons), Phil. Trans. B, 261 (1971) 325-358. FONNUM, F., GROVOVA,I., R~NVIK, E., STOR~-MATI41SEN, J., AND WALBEgG, F., Origin and distribution of glutamate decarboxylase in substantia nigra of the cat, Bra& Research, 71 (1974) 77-92. HATTORI,T., MCGEER, P. L., FmtGER, H. C., AND 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. HOCKMAN, C. H., LLOYD, K. G., FARLEY, 1. J., AND HORNYKIEWICZ, O., Experimental midbrain lesions: neurochemical comparison between the animal model and Parkinson's disease, Brain Research, 35 (1971) 613-618. KATAOKA, K., ~AK, 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. Bra& Res., 19 (1974) 217-227. LEwfs, P. R., SHUTe, C. C. D., AND SILVER, A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-224. MCGEER, E. G., FIB1GER, H. C., McGEER, P. L., AND BROOKE, S., Temporal changes in amine synthesizing enzymes of rat extrapyramidal structures after hemitransections or 6-hydroxydopamine administration, Brain Research, 52 (1973) 289-300. MCGEER, P. L., MCGEER, E. G., FIBIGER, H. C., AND WICKSON, V., Neostriatal choline acetylase and cholinesterase following selective brain lesions, Brain Research, 35 (1971) 308 314. PRECHT, W., AND YOSH1DA, M., Blockage of caudate-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229-233. STORM-MATHISEN,J., High affinity uptake of GABA in presumed GABA-ergic nerve endings in rat brain, Brain Research, 85 0975) 409 427.
107
Accumulation of glutamic acid decarboxylase in the proximal parts of presumed GABA-ergic neurones after axotomy
JON STORM-MATHISEN
Norwegian Defence Research Establishment, Division Jor Toxicology, N-2007 Kjelh, r (Norway) (Accepted December 19th, 1974)
Transection of nerve fibres producing acetylcholine or aromatic monoamines results in accumulation of the putative transmitter substances and related enzymes in the proximal parts of the respective neurones 2,v. This is a confirmation that the concomitant losses from the terminal area are due to interruption of the axons, rather than to secondary transsynaptic effects. Such accumulation has hitherto not been demonstrated in ?,-aminobutyric acid (GABA) producing nerve pathways in brain. GABA is probably the transmitter in a striato-nigral pathway 1,3,4,6,s,t°, one important piece of evidence being that the synthesizing enzyme, glutamic acid decarboxylase (GAD), nearly disappears from substantia nigra after surgical interruption of the pathway3,4,6, s. Most workers have concluded that this pathway projects from nucleus caudatus/putamenl,3.6, TM to substantia nigra and globus pallidus, whereas one group has reported evidence that it originates in globus pallidus 4,8. ]n the present paper I report the increase of G A D in nucleus caudatus/putamen of the rat after cutting the connexion with substantia nigra. Hemisections were performed in 200 g male Wistar rats at the level of corpus mamillare 11. At various survival times frontal brain slices were cut by a mechanical chopper in the cold room and nucleus caudatus/putamen and substantia nigra were dissected under the microscope and homogenized in 0.32 M sucrose. GAD, choline acetyltransferase (CHAT), dihydroxyphenylalanine decarboxylase (DOPAD), GABA uptake (at 2 × 10-s M) and protein were measured. The methods are described elsewhere 11. Assay mixtures for enzymes always contained 0.1-0.2~/o Triton X-100 to release full enzyme activity. After the hemisections a maximum increase of G A D in caudatus/putamen (about 50}o above normal) was reached within one week (Fig. 1). Concomitantly G A D in substantia nigra and DOPAD in caudatus/putamen decreased by about 90 or in accordance with the previously demonstrated reciprocity of dopamine-ergic and GABA-ergic fibres in this system s. ChAT did not change, in line with the earlier results showing that cholinergic neurones are intrinsic in caudatus/putamena, 9. Protein/wet wt. was unaltered (93-98~). The values on the control side did not change significantly. The average of the mean values obtained at the various survival
108 '
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'
'
I
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F
.
.
.
.
I
~ - '
J
&3*
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~150o 0
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~ CAVDATUS / PUTAMEN ....DOPADJ
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SUBSTANTIA NIGRA ,h P"=0,00]~ GAD DIFFERENT FROM P<'OIO2~J CONTROL
§
P<005
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GADchATDIFFERENTFROM
LIJ
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5
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. . . . .
15
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20
DAYSAFTEROPERATION
Fig. I. Effect of hemisections on GAD, ChAT and DOPAD in nucleus caudatus/putamen, and on GAD in substantia nigra. GAD was assayed at either 1.3 mM (<, 7]) or 6.4 mM(@, I ) f i n a l concentration of L-[l-~C]glutamate. For each experiment the number of animals is indicated by a figure beside the point for GAD. Statistical significance was determined by the two-tailed Student's t-test. The average of the mean activities on the unoperated side through the different experiments was in Hmole/h/g protein ± S.E.M. (number of experiments): GAD in caudatus/putamen, 217 : 12 (4 at 6.4 mMglu) and 102 [~ 17 (3 at 1.3 mMglu); CHAT, 396 ~: 55 (3); DOPAD, 17.0 " 2.0 (5); GAD in substantia nigra, 857 ~ 78 (4 at 6.4 mMglu) and 620 ; 52 (3 at 1.3 mMglu).
times were significantly different (two-tailed S t u d e n t ' s t-test) for G A D on the o p e r a t e d c o m p a r e d to the u n o p e r a t e d side ( P < 0.00t), a n d for the relative values o f G A D on the o p e r a t e d side c o m p a r e d to those o f C h A T (P -< 0.010). T w o g r o u p s 6,s have previously failed to detect an increase o f G A D in c a u d a t u s p u t a m e n after hemisections. The reason for this is obscure, but M c G e e r a n d cow o r k e r s 4,s used a s o m e w h a t different kind o f t r a n s e c t i o n t m o r e vertical), a n d K a t a o k a e t al. 6 used b a b o o n s . O n the o t h e r hand. H o c k m a n e t al. 5, using cats, f o u n d a subs t a n t i a l increase in some o f the a n i m a l s : out o f 21 cats at survival time 4-16 days 17 r a n g e d 7 5 - 1 2 0 ~ (95 T 3), whereas 4 ranged 140-187~'o (164 -~ 13). The o b l i q u e t r a n s e c t i o n s used in the present study passed in front o f the m o s t d o r s o - c a u d a l p a r t o f the nucleus c a u d a t u s / p u t a m e n (this p a r t c o n s t i t u t e d up to 25 o//o o f the volume, a c o r r e s p o n d i n g p a r t o f the c o n t r a l a t e r a l nucleus was discarded) a n d m a y have injured the d o r s a l tip o f globus pallidus. In a d d i t i o n to the striato-nigral axons, s o m e collaterals were therefore interrupted within the c a u d a t u s / p u t a m e n a n d globus pallidus. The m o r e m a r k e d increase o f G A D activity in the present than in previous studies may possibly be related to this m o r e extensive transection o f the axon tree. T h e m o s t likely i n t e r p r e t a t i o n o f the present results a p p e a r s to be t h a t G A D was piled up in the i n t r a n e o s t r i a t a l p a r t s (perikarya, axons, and terminals of intrinsic collaterals) o f the G A B A - e r g i c striato-nigral n e u r o n e s after transection o f their axons. S p r o u t i n g o f collaterals is less likely, since the lack o f change in G A B A u p l a k e (1 t0 .+
109 12, 109 ± 5 and 104 4- i 0 ~ of unoperated control 4- S.E.M. at 4, 10 and 21 days, respectively, compared to 30-40 ~o in substantia nigra 11) indicates that the intraneostriatal surface of the neurones did not expand. In the experiments at 4 and 21 days survival portions of the homogenates were separated into a pellet (containing the synaptosomes) and a supernatant by centrifugation at 16,000 × g for 60 rain. The pellets contained 75.4 4- 1.9 and 74.6 4- 1 . 5 ~ of the total G A D activity on the operated and control sides, respectively (means 4- S.E.M., for all 5 animals). This shows that the enzyme was not only piled up in perikarya and axons, but was transported to intraneostriatal terminals. The maximum amount of G A D accumulated in the parts of caudatus/putamen anterior to the lesions (50 ~ × 220 nmole/h/mg protein x 3.3 nag protein/nucleus 360 nmole/h/nucleus) was comparable to the total amount of lesion-sensitive G A D in substantia nigra (90 ~ × 860 nmole/h/mg protein × 0.3 mg protein/nucleus ~ 230 nmole/h/nucleus). The surplus may correspond to that destined for cut intraneostriatal collaterals and the dorsal tip of globus pallidus. An additional accumulation presumably occurred in the axons between the transection and caudatus/putamen, i.e. partly within globus pallidus. This region could not be accurately dissected due to its proximity to the lesion.
1 CROSSMAN, A. R., WALKER, R. J., ANO WOODRUFF, G. N., Picrotoxin antagonism of 7-amino-
2 3
4
5
6
7
8
9 10 l [
butyric acid inhibitory responses and synaptic inhibition in the rat substantia nigra, Brit. J. Pharmacol., 49 (1973) 696-698. DAHLSTR()M, A., Axoplasmic transport (with particular respect to adrenergic neurons), Phil. Trans. B, 261 (1971) 325-358. FONNUM, F., GROVOVA,I., R~NVIK, E., STOR~-MATI41SEN, J., AND WALBEgG, F., Origin and distribution of glutamate decarboxylase in substantia nigra of the cat, Bra& Research, 71 (1974) 77-92. HATTORI,T., MCGEER, P. L., FmtGER, H. C., AND 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. HOCKMAN, C. H., LLOYD, K. G., FARLEY, 1. J., AND HORNYKIEWICZ, O., Experimental midbrain lesions: neurochemical comparison between the animal model and Parkinson's disease, Brain Research, 35 (1971) 613-618. KATAOKA, K., ~AK, 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. Bra& Res., 19 (1974) 217-227. LEwfs, P. R., SHUTe, C. C. D., AND SILVER, A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-224. MCGEER, E. G., FIB1GER, H. C., McGEER, P. L., AND BROOKE, S., Temporal changes in amine synthesizing enzymes of rat extrapyramidal structures after hemitransections or 6-hydroxydopamine administration, Brain Research, 52 (1973) 289-300. MCGEER, P. L., MCGEER, E. G., FIBIGER, H. C., AND WICKSON, V., Neostriatal choline acetylase and cholinesterase following selective brain lesions, Brain Research, 35 (1971) 308 314. PRECHT, W., AND YOSH1DA, M., Blockage of caudate-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229-233. STORM-MATHISEN,J., High affinity uptake of GABA in presumed GABA-ergic nerve endings in rat brain, Brain Research, 85 0975) 409 427.