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Cortical modulation of cholinergic neurons in the striatum
Life Sciences, Vol. 31, pp. Printed in the U.S.A.
1501-1508
Pergamon
Press
C O R T I C A L M O D U L A T I O N OF C H O L I N E R G I C N E U R O N S
IN THE STRIATUM J. R. Simon Departments of Psychiatry and Biochemistry Institute of Psychiatric Research Indiana University School of Medicine Indianapolis, IN 46223 (Received
in final form July 13, 1982) Summary
Previous reports suggest the existence of a corticostriatal pathway which might use glutamate as the transmitter. In the present study, the possible influence of this pathway on striatal cholinergic neurons was investigated. Two weeks following surgical destruction of the cerebral cortex, the high affinity uptake of glutamate and choline into striatal synaptosomes was significantly reduced whereas GABA uptake was unaffected. In acute experiments (i hour following decortication), only choline uptake was significantly reduced while the uptake of glutamate and GABA were not altered. Acute injection (2 minutes) of kainic acid into the striatum, 1 hour after decortication, reversed the effect of the decortication on choline uptake, perhaps by simulating an excitatory input to the striatum which was presumably removed by the cortical ablation. These observations are consistent with the existence of a cortical input (perhaps glutamatergic) to the striatum and suggest that striat~l cholinergic neurons can be influenced by this cortico-striatal pathway. The striatum is known to receive a substantial innervation from the cerebral cortex (i, 2, 3). This cortico-striatal pathway has been shown by both intracellular and extracellular single cell recordings and cerebral cortex stimulation, to mediate excitation of striatal neurons (4, 5). Furthermore, several lines of evidence suggest that glutamic acid (or aspartic acid) may be the excitatory transmitter release from these cortical afferents to the striatum. For example, excitation of striatal neurons evoked by cortical stimulation can be blocked by the putative glutamate antagonist, glutamate diethylester (6). In addition, destruction of the cortico-striatal tract (cortical ablation or undercutting the cortex) results in a significant reduction in high affinity glutamate uptake when measured 1-6 weeks later in striatal homogenates (7) or synaptosomes (8). Similar deafferentation studies have also been shown to result in a significant decrease in the glutamic acid content of the striatum ipsilateral to the lesioned cortex, with no significant alterations in the content of several other amino acids, including aspartate (9). The striatum
is also known
to contain
a dense population
0024-3205/82/141501-08503.00/0 Copvrl~ht (c) 1982 Pergamon Press Ltd.
of cholinergic
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Chollnerglc
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31,.No.
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1982
i n t e r n e u r o n s (10, ll) w h i c h may receive a variety of inhibitory inputs (12, 13, 14). Recent turnover studies further suggest that these cholinergic i n t e r n e u r o n s may receive an e x c i t a t o r y input from the cerebral c o r t e x (15). C h o l i n e r g i c systems in the brain have been sthdled e x t e n s i v e l y by various techniques. One approach has been the investigation of the s o d i u m - d e p e n d e n t high affinity uptake system for choline, under a variety of c o n d i t i o n s (16; for review). In general, it has been observed that in vivo treatmeDts which increase a c e t y l c h o l i n e turnover result in increased uptake of choline when measured in vitro, and in vivo treatments which reduce acetylcholine turnover result in d e c r e a s e d choline uptake in vitro (12, 18). In the present study, the possible influence of the c o r t i c o - s t r i a t a l pathway in the modulation of stri~tal c h o l i n e r g i c neurons has been examined by studying the effects of s h o r t - t e r m and long-term cortical ablation in high affinity choline uptake in striatal synaptosomes. Materials
and Methods
All e x p e r i m e n t s were p e r f o r m e d using male Wistar rats (175-250 gms) o b t a i n e d from Harlan Industries, Indianapolis, IN. The animals were allowed to adapt to our animal quarters on a twelve hour light dark schedule and were given food and water ad libitum. Cortical Ablations. Rats were anesthetized with sodium pentQbarbital (40 mg/kg, i.p.) and mounted in a David Kopf stereotaxic apparatus. Following unilateral removal of the skull overlying the parietal and frontal cortex, the frontal cortex was removed by aspiration. The lesioned area was packed with Gel Foam and the scalp was apposed with wound clips. Kainic Acid Injections. Rats were anesthetized with sodium pent, barbital (40 mg/kg, i.p.) and mounted in a David Kopf stereotaxic apparatus. Kainic acid was d i s s o l v e d in p h o s p h a t e - b u f f e r e d saline (pH 7.4) and 2 bl (I0 nmoles) were injected into the striatum over a period of 2 minutes. The coordinates for intrastriatal injection were 0.5 mm anterior to bregma, 2.6 mm lateral to the midline and 4.2 mm ventral to the dura. Uptake Assays. Uptake of GABA, glutamate and choline in striatal synaptosomes was d e t e r m i n e d e s s e n t i a l l y as d e s c r i b e d previously (17) with minor modifications. The ipsilateral and contralateral striata were weighed and homogenized in 25 vol of 0.32M sucrose. Following c e n t r i f u g a t i o n of the homogenate at i000 x g for i0 minutes, the supernatant was c e n t r i f u g e d at 17,000 x g for 15 minutes. The resulting pellet (crude synaptosomal preparation) was resuspended in 0.32M sucrose to yield a protein c o n c e n t r a t i o n of approximately 1.0 - 1.5 mg/ml. Aliquots of this suspension (50 DI) were placed in 12 X 75 mm d i s p o s a b l e culture tubes which were maintained in an ice-water bath and which contained 0.~5 ml of Krebs-Ringer phosphate media. After the addition of i0 pl of the 3H-substrate (GABA, glutamate or choline) the samples were incubated at 30°C in a shaking water bath. For GABA and glutamate uptake (i DM final concentration), incubation was for 2 minutes; for choline (0.5 HM final concentration), a 4 minute incubation was used. Uptake was terminated by placing the tubes in an ice-water bath, followed immediately by ccntrifugation. Blank values were determined from samples incubated in a sodium-free media. Blank values were subtracted from total uptake and values reported here represent "sodium-dependent" uptake. Following centrifugation, the pellets were s u r f a c e - w a s h e d with ice-cold saline and solubilized in 1.0 ml of TS-I (Research Products International Corp., Elk Grove, IL). After transferral to scintillation vials and the addition of I0 ml of 3a20 counting solution (RPI), radioactivity was determined in a Beckman Model LS-7000 s c i n t i l l a t i o n counter. Under the conditions used, uptakes
Vol.
31, No.
14,
1982
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were linear with both time and tissue. Protein Determination. Protein was determined in aliquots of the crude synaptosomal preparation by the method of Lowry et al. (19) using bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as the standard. Results Two weeks after cortical ablation, high affinity uptake of choline into synaptosomes prepared from the ipsilateral striatum by approximately 35%, whereas GABA uptake was unaltered (Figure observations on GABA and glutamate uptake are in agreement with viously reported (7, 8).
glutamate and was reduced i). These those pre-
900
-
--90
800
-
-- 80
700
-
U,.l ,< I-
-- 70
7"
(5) i 600
-
-I (0
500
-
(D Z
400
-
< e~ < O
300
-
200
--
-- 60
T T
<
100 --
--50
(16)
/ / / /
(5) ,(5"~')
/ / / /
0
--40
/
GLU
FIG.
D LU Z -J O U
--20 --10
/
GABA
--30
uJ v <
0 CHOLINE
1
The uptake of 3H-GABA, -glutamate and -choline was measured in striatal P2-preparations two weeks after unilateral cortical ablation. Final concentrations were 1 HM for GABA and glutamate and 0.5 UM for choline. GABA and glutamate uptake is expressed as pmoles/2 min/mg protein. Choline uptake is expressed as pmoles/4 min/mg protein. Open bars, control; hatched bars, ablated. Values are mean + S.E.M. for the number of determinations indicated in parentheses. *P<0.01 versus contralateral striatum by paired t-test.
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Vol. 31, No. 14, 1982
When similar studies were conducted to investigate the short-term effects of cortical ablation (Figure 21, choline uptake was again found to be significantly reduced by about the same amount as that observed after long-term ablation. On the other hand, high affinity glutamate uptake was unchanged, which is in contrast to the effect seen two weeks after cortical ablation. As with the chronically ablated animals, GABA uptake was not altered 1 hour after cortical ablation.
1200 _
-
90
-
75
-
60
1100 1000 900 800 700 600 -
CABA
CLU
CHOLINE
FIG. 2 3 The uptake of H-GABA, -glutamate and -choline was measured in striatal P2 preparations 1 hour after unilateral cortical ablation. Final concentrations were 1 UM for GABA and glutamate and 0.5 UM for choline. GASA and glutamate uptake is expressed as pmoles/2 min/mg protein. Choline uptake is expressed as pmoles/4 min/mg protein. Open bars, control; hatched bars, ablated. Values are mean + S.E.M. for the number of determinations indicated in parentheses. *P
Vol.
31, No.
14, 1982
Striatal
Cholinergic
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Modulation
The d e c r e a s e in choline uptake observed following l o n g - t e r m (2 weeks) and s h o r t - t e r m (I hour) cortical ablation is consistent with the existence of an e x c i t a t o r y input of cortical origin onto striatal c h o l i n e r g i c interneurons. To further investigate this possibility, kainic acid was locally injected into the striatum 1 hour after removal of the ipsilateral frontal cortex, and the animals were sacrificed 2 minutes after injection. This was done in an attempt to restore the e x c i t a t o r y input to the striatum which was p r e s u m a b l y removed by the cortical ablation. The results are illustrated in Figure 3. Kainic acid injection alone resulted in a slight (12%) increase in choline uptake, however, this did not reach statistical significance. When high affinity choline uptake was measured 1 hour after cortical ablation, it was found to be significantly reduced compared to control as before, however, local injection of kainic acid (i0 nmoles) reversed this effect. Thus, choline uptake into synaptosomes prepared from the s t r i a t u m ipsilateral to the decortication, and treated w i t h an acute injection of kainic acid, was not d i f f e r e n t from control (Figure 3).
I
I CONTROL ~--~ABLATED
~KA 100
--
gO 80-
uJ ,,( Q. D
~ABLATED+KA
T
I(T'
(7)
70-
T
T~71
(4)
(7)
x
60--
M
50-
K
Z
X
.J
0 U
3020100
FIG.
3
The uptake of 3H-choline (0.5 ~M) was measured in striatal P2p r e p a r a t i o n s 2 min. after unilateral intrastriatal injection of kainic acid (KA) or 1 hour after unilateral cortical ablation. For combined treatments, KA was injected one hour after cortical ablation, and animals were sacrificed 2 min. later. KA (i0 nmoles) was injected in a volume of 2 U1 over a period of 2 min. Uptake is expressed as pmoles/4 min/mg protein. Values are mean + S.E.M. for the number of d e t e r m i n a t i o n s ind i c a t e d in parentheses. *P<0.025 versus contralateral striarum by p a i r e d t-test.
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Discussion Interactions among various neurotransmitt~r systems in the striatum has been the focus of several investigations in r e c e n ~ years. It has been shown that interactions exist between glutamatergic and dopaminergic neurons (20, 21, 22), GABAergic and glutamatergic neurons (23) and dopaminergic and cholinergic neurons (12, 24). The results of the present study suggest further, the existence of a modifying influence of cortical origin (presumably glutamate), on striatal cholinergic neurons. The use of choline uptake measurements in vitro has now been established as a valid metho~ for assessing in a qualitative manner, alterations in cholinergic function which exist in vivo (17, 18, 25, 26, 27, 28, 29, 30). In general, treatments which increase cholinergic ac£ivity (increase acetylcholine turnover), result in increased high affinity choline uptake whereas treatments which decrease cholinergic activity (decrease turnover), reduce choline uptake. The decrease in high affinity choline uptake observed in striatal synaptosomes 2 weeks after decortication is suggestive of an underactive cholinergic system induced by removal of the cerebral cortex. The decrease in glutamate uptake, and lack of effect on GABA uptake, is in agreement with previous studies (7, 8) and is indicative of successful interruption of the cortical striatal tract without damaging the striatal interneurons. If the decrease in choline uptake is the result of removal of the excitatory input originating from the cortex, and if this causes a decrease in the activity of the cholinergic interneurons, then a similar effect on choline uptake should also be observed at shorter times after interruption of this input, even though the cortico-striatal neurons have not degenerated. The present finding that striatal high affinity choline uptake is significantly reduced one hour after cortical ablation is consistent with this hypothesis. Further support for this proposed interaction between cortical afferents to the striatum and striatal cholinergic interneurons, derives from the results obtained with kainic acid injection following cortical ablation. Thus, intrastriatal injection of kainic acid ipsilateral to the lesioned cortex reverses the effect of the lesion on choline uptake. In these conditions (i hour post ablation; 2 minutes post kainic acid), it is assumed that the terminals of the cortico-striatal pathway are still intact and that the neuroexcitatory rather than the neurotoxic effects of the kainic acid would be manifest. Since kainic acid is such a potent excitant (31, 32), it is conceivable that short-term application in the striatum serves to simulate the excitatory component normally present prior to cortical ablation. Whether this is a direct effect of kainic acid on the cholinergic cells or an indirect effect of kainic acid, e.g., mediated through the release of glutamate from the intact terminals of the cortical afferents (15, 33, 34), remains to be determined. It is of interest to note that in the present study, short-term (2 minutes) application of kainic acid in the striatum, by itself, does not affect high affinity choline uptake, nor does it alter acetylcholine levels or turnover in this region (15). In this regard, and as previously discussed (26, 35), the cholinergic system appears to be more resistant to change in the striatum than in other brain regions. Recent evidence suggest that striatal GABAergic interneurons may be under the control of the cortical-striatal neurons which are presumably glutamatergic. Thus, destruction of the interneuronal population of the striatum with kainic acid results in decreased glutamate binding presumably located on GABA interneurons (36), and surgical interruption of
Vol. 31, No. 14, 1982
Strlatal Chollnerglc Neurons Modulation
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the cortico-striatal pathway results in a decreased turnover rate of GABA in the striatum (15). An additional role proposed for glutamate in the striatum is a direct control of dopamine release (20). If GABA and dopamine both have inhibitory influences in the striatum, then it seems unlikely that the glutamate action on the cholinergic system could be mediated via either of these systems, since one might then expect increased choline uptake. The data available to date are consistent with a direct action of the cortico-striatal path (glutamate) on cholinergic interneurons in the striatum. More complex interactions involving the cholinergic, glutamatergic and other striatal systems no doubt exist; however, further study of such interactions and circuitry will be required to define these more precisely. Acknowledgements This work was supported by PHS Grant NS 15951.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. i0. Ii. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
J.B. CARMAN, W.M. COWAN and T.P.S. POWELL, Brain, 88, 525-562 (1963). J.M. KEMP ar~l '2.P.S. POWELL, Brain, 93, 525-546 (1970). K.E WEBSTER, J. Anat. (Lond.), 95, 532-545 (1961). D.J. BLAKE, P. ZARZECKI and G.G. SOMJEN, J. Neurobiol., ~, 143-156 (1976). N.A. BUCHWALD, D.D. PRICE, L. VERNON and C.D. HULL, Exp. Neurol., 38, 311-323 (1973) . H.J SPENCER, Brain Res., 102, 91-101 (1976). 5. DIVAC, F. FONNUM and J. STORM-MATHISEN, Nature, 266, 377-378 (1977). P.L. McGEER, E.G. McGEER, U. SCHERER and K. SINGH, Brain Res., 12___88,369-373 (1977). J.S KIM, R. HASSLER, P. HUANG and K.S. PAIK, Brain Res., 132, 370-374 (1977) P.L. McGEER, E.G. McGEER, H.C. FIBIGER and V. WICKSON, Brain Res., 35, 308314 (1971). S.G. BUTCHER and L.L. BUTCHER, Brain Res., 71, 161-171 (1974). K.G LLOYD, Essays in Neurochemistry and Neuropharmacology , Vol. 3, Ed: M.B.H. YOUDIM, W. LOVENDERG, D.F. SHARMAN and J.R. LAGNADO, pp. 131-207, Wiley, New York (1978). R. SAMANIN, A. QUATTRONE, G. PERI, H. LADINSKY and S. CONSOLO, Brain Res., 151 73-82 (1978). B. SCATTON and G. BARTHOLINI, Brain Res., 183, 211-216 (1980). P.L. WOOD, F. MORONI, D.L. CHENEY and E. cOSTA, Neurosci. Letters, 12, 349-354 (1979). R.S JOPE, Brain Res. Rev., i, 313-344 (1979). J.R. SIMON, S. ATWEH and M.J. KUHAR, J. Neurochem., 26, 909-922 (1976). S. ATWEH, J.R. SIMON and M.J. KUHAR, Life 5ci. 17, 1535-1544 (1975). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. biol. Chem., 193, 265-275 (1951). M.F. GIORGUIEFF, M.L. KEMEL and J. GLOWINSKI, Neurosci. Letters, 6, 73-78 (1977). G.J. ROWLANDS and P.J. ROBERTS, Eur. J. Pharmacol., 62, 241-242 (1980). P.R. MITCHELL and N.S. DOGGETT, Life Sci., 26, 2073-2081 (1980). R. MITCHELL, Eur. J. Pharmacol., 67, 119-122 (1980). P.G. GUYENET, Y. AGID, F. JAVOY, J.C. BEAUJOUAN, R. ROSSIER and J. GLOWINSKI, Brain Res., 84, 227-244 (1975). J.R. SIMON and M.J. KUHAR, Nature (Lond.), 255, 162-163 (1975). K.A. SHERMAN, M.J. ZIGMOND and I. HANIN, Life Sci., 23, 1863-1870 (1978). R.S. JOPE, J. Neurochem., 33, 487-495 (1979). R. KUCZENSKI, D. SCHMIDT and N. LEITH, Brain Res., 126, 117-129 (1977).
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29. 30. 31. 32. 33. 34. 35. 36.
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A. NORDBERG, Life Sci., 23, 937-944 (1978). L. WECKER, W.D. DETTBARN and D.E. SCHMIDT, Science, 199, 86-87 (1978). H. SHINOZAKI and S. KONISHI, Brain Res., 24, 368-371 (1970). G.A.R. JOHNSTON, D.R. CURTIS, J. DAVIES and R.M. McCULLOCH, Nature (Lond.), 248, 804-805 (1974). K. BIZIERE and J.T. COYLE, Neurosci. Letters, 8, 303-310 (1978). E.G. McGEER, P.L. McGEER and K. SINGH, Brain Res., 139, 381-383 (1978). L. WECKER and W.D. DETTBARN, J. Neurochem., 32, 961-967 (1979). K. BIZIERE, H. THOMPSON and J.T. COYLE, Brain Res., 183, 421-433 (1980).
1501-1508
Pergamon
Press
C O R T I C A L M O D U L A T I O N OF C H O L I N E R G I C N E U R O N S
IN THE STRIATUM J. R. Simon Departments of Psychiatry and Biochemistry Institute of Psychiatric Research Indiana University School of Medicine Indianapolis, IN 46223 (Received
in final form July 13, 1982) Summary
Previous reports suggest the existence of a corticostriatal pathway which might use glutamate as the transmitter. In the present study, the possible influence of this pathway on striatal cholinergic neurons was investigated. Two weeks following surgical destruction of the cerebral cortex, the high affinity uptake of glutamate and choline into striatal synaptosomes was significantly reduced whereas GABA uptake was unaffected. In acute experiments (i hour following decortication), only choline uptake was significantly reduced while the uptake of glutamate and GABA were not altered. Acute injection (2 minutes) of kainic acid into the striatum, 1 hour after decortication, reversed the effect of the decortication on choline uptake, perhaps by simulating an excitatory input to the striatum which was presumably removed by the cortical ablation. These observations are consistent with the existence of a cortical input (perhaps glutamatergic) to the striatum and suggest that striat~l cholinergic neurons can be influenced by this cortico-striatal pathway. The striatum is known to receive a substantial innervation from the cerebral cortex (i, 2, 3). This cortico-striatal pathway has been shown by both intracellular and extracellular single cell recordings and cerebral cortex stimulation, to mediate excitation of striatal neurons (4, 5). Furthermore, several lines of evidence suggest that glutamic acid (or aspartic acid) may be the excitatory transmitter release from these cortical afferents to the striatum. For example, excitation of striatal neurons evoked by cortical stimulation can be blocked by the putative glutamate antagonist, glutamate diethylester (6). In addition, destruction of the cortico-striatal tract (cortical ablation or undercutting the cortex) results in a significant reduction in high affinity glutamate uptake when measured 1-6 weeks later in striatal homogenates (7) or synaptosomes (8). Similar deafferentation studies have also been shown to result in a significant decrease in the glutamic acid content of the striatum ipsilateral to the lesioned cortex, with no significant alterations in the content of several other amino acids, including aspartate (9). The striatum
is also known
to contain
a dense population
0024-3205/82/141501-08503.00/0 Copvrl~ht (c) 1982 Pergamon Press Ltd.
of cholinergic
1502
Striatal
Chollnerglc
Neurons
Modulation
Vol.
31,.No.
14,
1982
i n t e r n e u r o n s (10, ll) w h i c h may receive a variety of inhibitory inputs (12, 13, 14). Recent turnover studies further suggest that these cholinergic i n t e r n e u r o n s may receive an e x c i t a t o r y input from the cerebral c o r t e x (15). C h o l i n e r g i c systems in the brain have been sthdled e x t e n s i v e l y by various techniques. One approach has been the investigation of the s o d i u m - d e p e n d e n t high affinity uptake system for choline, under a variety of c o n d i t i o n s (16; for review). In general, it has been observed that in vivo treatmeDts which increase a c e t y l c h o l i n e turnover result in increased uptake of choline when measured in vitro, and in vivo treatments which reduce acetylcholine turnover result in d e c r e a s e d choline uptake in vitro (12, 18). In the present study, the possible influence of the c o r t i c o - s t r i a t a l pathway in the modulation of stri~tal c h o l i n e r g i c neurons has been examined by studying the effects of s h o r t - t e r m and long-term cortical ablation in high affinity choline uptake in striatal synaptosomes. Materials
and Methods
All e x p e r i m e n t s were p e r f o r m e d using male Wistar rats (175-250 gms) o b t a i n e d from Harlan Industries, Indianapolis, IN. The animals were allowed to adapt to our animal quarters on a twelve hour light dark schedule and were given food and water ad libitum. Cortical Ablations. Rats were anesthetized with sodium pentQbarbital (40 mg/kg, i.p.) and mounted in a David Kopf stereotaxic apparatus. Following unilateral removal of the skull overlying the parietal and frontal cortex, the frontal cortex was removed by aspiration. The lesioned area was packed with Gel Foam and the scalp was apposed with wound clips. Kainic Acid Injections. Rats were anesthetized with sodium pent, barbital (40 mg/kg, i.p.) and mounted in a David Kopf stereotaxic apparatus. Kainic acid was d i s s o l v e d in p h o s p h a t e - b u f f e r e d saline (pH 7.4) and 2 bl (I0 nmoles) were injected into the striatum over a period of 2 minutes. The coordinates for intrastriatal injection were 0.5 mm anterior to bregma, 2.6 mm lateral to the midline and 4.2 mm ventral to the dura. Uptake Assays. Uptake of GABA, glutamate and choline in striatal synaptosomes was d e t e r m i n e d e s s e n t i a l l y as d e s c r i b e d previously (17) with minor modifications. The ipsilateral and contralateral striata were weighed and homogenized in 25 vol of 0.32M sucrose. Following c e n t r i f u g a t i o n of the homogenate at i000 x g for i0 minutes, the supernatant was c e n t r i f u g e d at 17,000 x g for 15 minutes. The resulting pellet (crude synaptosomal preparation) was resuspended in 0.32M sucrose to yield a protein c o n c e n t r a t i o n of approximately 1.0 - 1.5 mg/ml. Aliquots of this suspension (50 DI) were placed in 12 X 75 mm d i s p o s a b l e culture tubes which were maintained in an ice-water bath and which contained 0.~5 ml of Krebs-Ringer phosphate media. After the addition of i0 pl of the 3H-substrate (GABA, glutamate or choline) the samples were incubated at 30°C in a shaking water bath. For GABA and glutamate uptake (i DM final concentration), incubation was for 2 minutes; for choline (0.5 HM final concentration), a 4 minute incubation was used. Uptake was terminated by placing the tubes in an ice-water bath, followed immediately by ccntrifugation. Blank values were determined from samples incubated in a sodium-free media. Blank values were subtracted from total uptake and values reported here represent "sodium-dependent" uptake. Following centrifugation, the pellets were s u r f a c e - w a s h e d with ice-cold saline and solubilized in 1.0 ml of TS-I (Research Products International Corp., Elk Grove, IL). After transferral to scintillation vials and the addition of I0 ml of 3a20 counting solution (RPI), radioactivity was determined in a Beckman Model LS-7000 s c i n t i l l a t i o n counter. Under the conditions used, uptakes
Vol.
31, No.
14,
1982
Striatal
Cholinergic
Neurons Modulation
1503
were linear with both time and tissue. Protein Determination. Protein was determined in aliquots of the crude synaptosomal preparation by the method of Lowry et al. (19) using bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as the standard. Results Two weeks after cortical ablation, high affinity uptake of choline into synaptosomes prepared from the ipsilateral striatum by approximately 35%, whereas GABA uptake was unaltered (Figure observations on GABA and glutamate uptake are in agreement with viously reported (7, 8).
glutamate and was reduced i). These those pre-
900
-
--90
800
-
-- 80
700
-
U,.l ,< I-
-- 70
7"
(5) i 600
-
-I (0
500
-
(D Z
400
-
< e~ < O
300
-
200
--
-- 60
T T
<
100 --
--50
(16)
/ / / /
(5) ,(5"~')
/ / / /
0
--40
/
GLU
FIG.
D LU Z -J O U
--20 --10
/
GABA
--30
uJ v <
0 CHOLINE
1
The uptake of 3H-GABA, -glutamate and -choline was measured in striatal P2-preparations two weeks after unilateral cortical ablation. Final concentrations were 1 HM for GABA and glutamate and 0.5 UM for choline. GABA and glutamate uptake is expressed as pmoles/2 min/mg protein. Choline uptake is expressed as pmoles/4 min/mg protein. Open bars, control; hatched bars, ablated. Values are mean + S.E.M. for the number of determinations indicated in parentheses. *P<0.01 versus contralateral striatum by paired t-test.
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Striatal Cholinergic Neurons Modulation
Vol. 31, No. 14, 1982
When similar studies were conducted to investigate the short-term effects of cortical ablation (Figure 21, choline uptake was again found to be significantly reduced by about the same amount as that observed after long-term ablation. On the other hand, high affinity glutamate uptake was unchanged, which is in contrast to the effect seen two weeks after cortical ablation. As with the chronically ablated animals, GABA uptake was not altered 1 hour after cortical ablation.
1200 _
-
90
-
75
-
60
1100 1000 900 800 700 600 -
CABA
CLU
CHOLINE
FIG. 2 3 The uptake of H-GABA, -glutamate and -choline was measured in striatal P2 preparations 1 hour after unilateral cortical ablation. Final concentrations were 1 UM for GABA and glutamate and 0.5 UM for choline. GASA and glutamate uptake is expressed as pmoles/2 min/mg protein. Choline uptake is expressed as pmoles/4 min/mg protein. Open bars, control; hatched bars, ablated. Values are mean + S.E.M. for the number of determinations indicated in parentheses. *P
Vol.
31, No.
14, 1982
Striatal
Cholinergic
Neurons
Modulation
The d e c r e a s e in choline uptake observed following l o n g - t e r m (2 weeks) and s h o r t - t e r m (I hour) cortical ablation is consistent with the existence of an e x c i t a t o r y input of cortical origin onto striatal c h o l i n e r g i c interneurons. To further investigate this possibility, kainic acid was locally injected into the striatum 1 hour after removal of the ipsilateral frontal cortex, and the animals were sacrificed 2 minutes after injection. This was done in an attempt to restore the e x c i t a t o r y input to the striatum which was p r e s u m a b l y removed by the cortical ablation. The results are illustrated in Figure 3. Kainic acid injection alone resulted in a slight (12%) increase in choline uptake, however, this did not reach statistical significance. When high affinity choline uptake was measured 1 hour after cortical ablation, it was found to be significantly reduced compared to control as before, however, local injection of kainic acid (i0 nmoles) reversed this effect. Thus, choline uptake into synaptosomes prepared from the s t r i a t u m ipsilateral to the decortication, and treated w i t h an acute injection of kainic acid, was not d i f f e r e n t from control (Figure 3).
I
I CONTROL ~--~ABLATED
~KA 100
--
gO 80-
uJ ,,( Q. D
~ABLATED+KA
T
I(T'
(7)
70-
T
T~71
(4)
(7)
x
60--
M
50-
K
Z
X
.J
0 U
3020100
FIG.
3
The uptake of 3H-choline (0.5 ~M) was measured in striatal P2p r e p a r a t i o n s 2 min. after unilateral intrastriatal injection of kainic acid (KA) or 1 hour after unilateral cortical ablation. For combined treatments, KA was injected one hour after cortical ablation, and animals were sacrificed 2 min. later. KA (i0 nmoles) was injected in a volume of 2 U1 over a period of 2 min. Uptake is expressed as pmoles/4 min/mg protein. Values are mean + S.E.M. for the number of d e t e r m i n a t i o n s ind i c a t e d in parentheses. *P<0.025 versus contralateral striarum by p a i r e d t-test.
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Chollnergic
Neurons Modulation
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Discussion Interactions among various neurotransmitt~r systems in the striatum has been the focus of several investigations in r e c e n ~ years. It has been shown that interactions exist between glutamatergic and dopaminergic neurons (20, 21, 22), GABAergic and glutamatergic neurons (23) and dopaminergic and cholinergic neurons (12, 24). The results of the present study suggest further, the existence of a modifying influence of cortical origin (presumably glutamate), on striatal cholinergic neurons. The use of choline uptake measurements in vitro has now been established as a valid metho~ for assessing in a qualitative manner, alterations in cholinergic function which exist in vivo (17, 18, 25, 26, 27, 28, 29, 30). In general, treatments which increase cholinergic ac£ivity (increase acetylcholine turnover), result in increased high affinity choline uptake whereas treatments which decrease cholinergic activity (decrease turnover), reduce choline uptake. The decrease in high affinity choline uptake observed in striatal synaptosomes 2 weeks after decortication is suggestive of an underactive cholinergic system induced by removal of the cerebral cortex. The decrease in glutamate uptake, and lack of effect on GABA uptake, is in agreement with previous studies (7, 8) and is indicative of successful interruption of the cortical striatal tract without damaging the striatal interneurons. If the decrease in choline uptake is the result of removal of the excitatory input originating from the cortex, and if this causes a decrease in the activity of the cholinergic interneurons, then a similar effect on choline uptake should also be observed at shorter times after interruption of this input, even though the cortico-striatal neurons have not degenerated. The present finding that striatal high affinity choline uptake is significantly reduced one hour after cortical ablation is consistent with this hypothesis. Further support for this proposed interaction between cortical afferents to the striatum and striatal cholinergic interneurons, derives from the results obtained with kainic acid injection following cortical ablation. Thus, intrastriatal injection of kainic acid ipsilateral to the lesioned cortex reverses the effect of the lesion on choline uptake. In these conditions (i hour post ablation; 2 minutes post kainic acid), it is assumed that the terminals of the cortico-striatal pathway are still intact and that the neuroexcitatory rather than the neurotoxic effects of the kainic acid would be manifest. Since kainic acid is such a potent excitant (31, 32), it is conceivable that short-term application in the striatum serves to simulate the excitatory component normally present prior to cortical ablation. Whether this is a direct effect of kainic acid on the cholinergic cells or an indirect effect of kainic acid, e.g., mediated through the release of glutamate from the intact terminals of the cortical afferents (15, 33, 34), remains to be determined. It is of interest to note that in the present study, short-term (2 minutes) application of kainic acid in the striatum, by itself, does not affect high affinity choline uptake, nor does it alter acetylcholine levels or turnover in this region (15). In this regard, and as previously discussed (26, 35), the cholinergic system appears to be more resistant to change in the striatum than in other brain regions. Recent evidence suggest that striatal GABAergic interneurons may be under the control of the cortical-striatal neurons which are presumably glutamatergic. Thus, destruction of the interneuronal population of the striatum with kainic acid results in decreased glutamate binding presumably located on GABA interneurons (36), and surgical interruption of
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the cortico-striatal pathway results in a decreased turnover rate of GABA in the striatum (15). An additional role proposed for glutamate in the striatum is a direct control of dopamine release (20). If GABA and dopamine both have inhibitory influences in the striatum, then it seems unlikely that the glutamate action on the cholinergic system could be mediated via either of these systems, since one might then expect increased choline uptake. The data available to date are consistent with a direct action of the cortico-striatal path (glutamate) on cholinergic interneurons in the striatum. More complex interactions involving the cholinergic, glutamatergic and other striatal systems no doubt exist; however, further study of such interactions and circuitry will be required to define these more precisely. Acknowledgements This work was supported by PHS Grant NS 15951.
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