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Effect of psychotropic drugs on high affinity choline uptake by excised tissue of rat nucleus caudatus
478
Brain Research, 182 (1980) 478-481 © Elsevier/North-Holland Biomedical Press
Effect of psychotropic drugs on high affinity choline uptake by excised tissue of rat nucleus caudatus
YUKIO TAKANO, YOSHIRO KOHJIMOTO and HIRO-O KAMIYA Department of Pharmacology, School of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814 (Japan)
(Accepted October 4th, 1979)
Key words:
psychotropic drugs - - nucleus caudatus - - choline uptake - - synaptosomes
Recently evidence has been obtained for the existence of a functional linkage between the dopaminergic and cholinergic systems in the striatum z,6,15,22, and dopamine receptor blockers have been shown to increase the turnover of acetylcholine (ACh) a,12,17,24. However, it is not clear whether high affinity choline uptake, the rate limiting event in ACh synthesis, in striatal synaptosomes is changed by blocking dopamine receptorsl,X~,18,19. Therefore, we re-examined the effects of dopamine receptor drugs on high affinity choline uptake in the nucleus caudatus excised from rat brain by a micropuncture technique 7,14. Male Wistar rats (200-250 g) were treated with drugs or saline and then killed by decapitation. The brain was promptly removed from the skull and fresh frontal sections (2 mm thick) were cut in a cold room (4 °C) using a modified Jacobowitz's rat brain slicer 7. The nucleus caudatus (A 8200, according to the atlas of K6nig and Klippel 1°) was punched out from the sections using a hollow stainless steel tube of 1.0 mm internal diameter. For the assay of high affinity choline uptake, the punch from the nucleus caudatus (approximately 1.2 mg wet weight) was used. That is, high affinity choline uptake was measured by a modification of the method of Haga and Noda ~ (spec. act., 84 Ci/mmol) from New England Nuclear. A micropunched tissue was preincubated with 1 ml of the medium (140 mM NaCI, 5 mM KCI, 1.2 mM MgCI2, 0.8 mM CaCI2, 10 mM glucose, 100 # M neostigmine, 20 mM Tris.HCl and 1 mM phosphate buffers, pH 7.4) at 37 °C for 10 min. Blank samples were incubated in sodium-free incubation medium made by replacing sodium salts by an osmotically equivalent amount of sucrose. High affinity choline uptake was defined as the net accumulation of tritium in standard solution minus that in sodium-free solution, and was expressed as pmol/5 min/mg wet weight. A final choline concentration of 5/zM in the medium was obtained by adding unlabeled and labeled (1 /zCi) choline. After 5 min the reaction was
479 stopped by transferring the tubes to an ice-bath and adding 3 ml of ice-cold stopping solution (standard medium containing 10 mM choline), and samples were washed several times with medium of the same ionic composition. Samples were extracted with 0.5 ml of acid ethanol solution (95 ~ ethanol/0.3 ~ acetic acid, 1.5 mM ACh, 100 # M neostigmine) and the extract was mixed with 11 ml toluene-ethanol (10 : 1 by vol.) scintillator containing omunifluor (New England Nuclear). The following drugs were administered intraperitoneally: haloperidol 4 mg/kg (Dainippon Seiyaku), fluphenazine hydrochloride 5 mg/kg (Nippon Roche and Squibb Japan) and apomorphine 10 mg/kg (Mitsui Pharmaceuticals). Control animals were injected with the same volume of saline. Rats were pre-treated with drugs and killed by decapitation 30 min later. Their brains were removed and high affinity choline uptake by tissue of the nucleus caudatus was measured. Administration of haloperidol (4 mg/kg) caused a significant increase in choline uptake (P < 0.001), whereas pretreatment with the dopamine receptor agonist apomorphine (10 mg/kg) resulted in a decrease in choline uptake (P < 0.05) (Table I). The phenothiazine derivatives fluphenazine (5 mg/kg), a dopamine receptor antagonist, had a similar effect as haloperidol causing significant increase in choline uptake by the nucleus caudatus tissue (P < 0.01) (Table I). In studies on the synaptosomal fractionS,la,20, 2a and slices 4,16 of the brain evidence has been obtained that high affinity choline uptake is coupled with the release and synthesis of ACh. Moreover, several recent studies have demonstrated that in the striatum the turnover rate of ACh 3,t2,17,24 and high affinity choline uptake 1,11 are enhanced by blocking inhibitory dopaminergic neurons of the substantia nigra. Some investigators have reported, however, that this increase in ACh turnover in the striatum is not accompanied by increase in high affinity choline uptake by striatal synaptosomes18,19.
TABLE I Effects of dopamine receptor antagonists and agonist on high affinity choline uptake by the nucleus caudatus
Rats were killed by decapitation 30 min after administration of haloperidol (4 mg/kg i.p.), fluphenazine (5 mg/kg i.p.), apomorphine (10 mg/kg i.p.) or saline. Values are means ± S.E.M. for the number of determinations shown in parentheses. Treatment
High affinity choline uptake (pmol/5 min/mg wet weight)
Saline Haloperidol Fluphenazine Apomorphine
7.98 ± 0.21 (19) 10.82 4- 0.40 (8)*** 9.45 ± 0.51 (4)** 6.78 ± 0.71 (4)*
% Change from control
+35.6 +11.8 --16.0
* Significantly different from the control value at P < 0.05. ** Significantly different from the control value at P < 0.01. *** Significantly different from the control value at P < 0.001.
480 In a recent study, it was d e m o n s t r a t e d t h a t the activity-related changes in choline u p t a k e u n d e r g o a reversal in p o s t - m o r t e m tissues, a n d the p o s t - m o r t e m reversal seems to be, at least in part, t e m p e r a t u r e - d e p e n d e n t 9. So one o f the reasons for the discrepancies in choline u p t a k e m a y be due to this p o s t - m o r t e m change. In the present w o r k we m e a s u r e d high affinity choline u p t a k e by tissue r e m o v e d from slices o f the nucleus c a u d a t u s by m i c r o p u n c t u r e (A 8200). A m i c r o p u n c h o f tissue o b t a i n e d in this way is considered to be in a m o r e physiological state t h a n synaptosomes. F u r t h e r m o r e , p o s t - m o r t e m changes in m i c r o p u n c h e s o f tissue seem to be small because the process o f this m e t h o d is quicker t h a n t h a t o f s y n a p t o s o m a l fractionation. W e f o u n d that high affinity choline u p t a k e was increased by the d o p a m i n e receptor antagonists h a l o p e r i d o l a n d fluphenazine, a n d decreased by the d o p a m i n e receptor agonist a p o m o r p h i n e (Table I). Therefore, o u r results s u p p o r t the existence o f inhibitory d o p a m i n e r g i c regulation o f striatal cholinergic neurons. The a u t h o r s t h a n k Mr. N. O n o and Mr. K. U c h i m u r a for help with this work.
1 Atweh, S., Simon, J. R. and Kuhar, M. J., Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo, Life ScL, 17 (1975) 15351544. 2 Consolo, S., Ladinsky, H., Bianchi, S. and Ghezzi, D., Apparent lack of a dopaminergic-cholinergic link in the rat nucleus accumbens septi-tuberculum olfactorium, Brain Research, 135 (1977) 255-263. 3 Guyenet, P. G., Agid, Y., Javoy, F., Beaujouan, J.C., Rossier, J. and Glowinski, J., Effects of dopaminergic receptor agonists and antagonists on the activity of the neo-striatal cholinergic system, Brain Research, 84 (1975) 227-244. 4 Hadh~izy, P. and Szerb, J. C., The effect of cholinergic drugs on [aH]acetylcholine release from slices of rat hippocampus, striatum and cortex, Brain Research, 123 (1977) 311-322. 5 Haga, T. and Noda, H., Choline uptake systems of rat brain synaptosomes, Biochim. biophys. Acta (Amst.), 291 (1973) 564-575. 6 Hattori, T., Singh, V. K., McGeer, E. G. and McGeer, P. L., Immunohistochemical localization of choline acetyltransferase containing neostriatal neurons and their relationship with dopaminergic synapses, Brain Research, 102 (1976) 164-173. 7 Jacobowitz, D. M., Removal of discrete fresh regions of the rat brain, Brain Research, 80 (1974) 635-637. 8 Jenden, D. J., Jope, R. S. and Weiler, M. H., Regulation of acetylcholine synthesis: does cytoplasmic acetylcholine control high affinity choline uptake?, Science, 194 (1976) 635-637. 9 Klemm, N. and Kuhar, M. J., Post-mortem changes in high affinity choline uptake, J. Neurochem., 32 (1979) 1487-1494. 10 Ktinig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 11 Kuczenski, R., Schmidt, D. and Leith, N., Amphetamine-haloperidol interactions in rat striatum: failure to correlate behavioral effects with dopaminergic and cholinergic dynamics, Brain Research, 126 (1977) 117-129. 12 Ladinsky, H., Consolo, S., Bianchi, S., Samanin, R. and Ghezzi, D., Cholinergic-dopaminergic interaction in the striatum: the effect of 6-hydroxydopamine or pimozide treatment on the increased striatal acetylcholine levels induced by apomorphine, piribedil and o-amphetamine, Brain Research, 84 (1975) 221-226. 13 Murrin, L. C., DeHaven, R. N. and Kuhar, M. J., On the relationship between all-choline uptake activation and aH-acetylcholine release, J. Neurochem., 29 (1977) 681-687. 14 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450.
481 15 P6rez de la Mora, M. and Fuxe, K., Brain GABA, dopamine and acetylcholine interaction, studies with oxotremorine, Brain Research, 135 (1977) 107-122. 16 Polak, R. L., Molenaar, P. C. and van Gelder, M., Acetylcholine metabolism and choline uptake in cortical slices, J. Neurochem., 29 (1977) 477-485. 17 Racagni, G., Cheney, D. L., Trabucchi, M. and Costa, E., In vivo actions of clozapine and haloperidol on the turnover rate of acetylcholine in rat striatum, J. Pharmacol. exp. Ther., 196 (1976) 323-332. 18 Sherman, K. A., Hannin, I. and Zigmond, M. J., The effect of neuroleptics on acetylcholine concentration and choline uptake in striatum: implications for regulation of acetylcholine metabolism, J. PharmacoL exp. Ther., 206 (1978) 677-686. 19 Sherman, K. A., Zigmond, M. J. and Hannin, I., High affinity choline uptake in striatum and hippocampus. Differential effects of treatments which release actylcholine, Life Sci., 23 (1978) 1863-1870. 20 Simon, J. R. and Kuhar, M. J., Impulse-flow regulation of high affinity choline uptake in brain cholinergic nerve terminals, Nature (Lond.), 255 (1975) 162-163. 21 Simon, J. R., Atweh, S. and Kuhar, M. J., Sodium-dependent high affinity choline uptake: a regulatory step in the synthesis of acetylcholine, J. Neurochem., 26 (1976) 909-922. 22 Stadler, H., Lloyd, K. G., Gadea-Ciria, M. and Bartholini, G., Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine, Brain Research, 55 (1973) 476-480. 23 Takano, Y., Kohjimoto, Y. and Kamiya, H., Studies on the high affinity choline uptake s)stem with respect to the muscarinic receptors using a synaptosomal fraction abundant in presynaptic membranes, Jap. J. Pharmacol., in press. 24 Trabucchi, M., Cheney, D., Racagni, G. and Costa, E., Involvement of brain cholinergic mechanisms in the action of chlorpromazine, Nature (Lond.), 289 (1974) 664-666.
Brain Research, 182 (1980) 478-481 © Elsevier/North-Holland Biomedical Press
Effect of psychotropic drugs on high affinity choline uptake by excised tissue of rat nucleus caudatus
YUKIO TAKANO, YOSHIRO KOHJIMOTO and HIRO-O KAMIYA Department of Pharmacology, School of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814 (Japan)
(Accepted October 4th, 1979)
Key words:
psychotropic drugs - - nucleus caudatus - - choline uptake - - synaptosomes
Recently evidence has been obtained for the existence of a functional linkage between the dopaminergic and cholinergic systems in the striatum z,6,15,22, and dopamine receptor blockers have been shown to increase the turnover of acetylcholine (ACh) a,12,17,24. However, it is not clear whether high affinity choline uptake, the rate limiting event in ACh synthesis, in striatal synaptosomes is changed by blocking dopamine receptorsl,X~,18,19. Therefore, we re-examined the effects of dopamine receptor drugs on high affinity choline uptake in the nucleus caudatus excised from rat brain by a micropuncture technique 7,14. Male Wistar rats (200-250 g) were treated with drugs or saline and then killed by decapitation. The brain was promptly removed from the skull and fresh frontal sections (2 mm thick) were cut in a cold room (4 °C) using a modified Jacobowitz's rat brain slicer 7. The nucleus caudatus (A 8200, according to the atlas of K6nig and Klippel 1°) was punched out from the sections using a hollow stainless steel tube of 1.0 mm internal diameter. For the assay of high affinity choline uptake, the punch from the nucleus caudatus (approximately 1.2 mg wet weight) was used. That is, high affinity choline uptake was measured by a modification of the method of Haga and Noda ~ (spec. act., 84 Ci/mmol) from New England Nuclear. A micropunched tissue was preincubated with 1 ml of the medium (140 mM NaCI, 5 mM KCI, 1.2 mM MgCI2, 0.8 mM CaCI2, 10 mM glucose, 100 # M neostigmine, 20 mM Tris.HCl and 1 mM phosphate buffers, pH 7.4) at 37 °C for 10 min. Blank samples were incubated in sodium-free incubation medium made by replacing sodium salts by an osmotically equivalent amount of sucrose. High affinity choline uptake was defined as the net accumulation of tritium in standard solution minus that in sodium-free solution, and was expressed as pmol/5 min/mg wet weight. A final choline concentration of 5/zM in the medium was obtained by adding unlabeled and labeled (1 /zCi) choline. After 5 min the reaction was
479 stopped by transferring the tubes to an ice-bath and adding 3 ml of ice-cold stopping solution (standard medium containing 10 mM choline), and samples were washed several times with medium of the same ionic composition. Samples were extracted with 0.5 ml of acid ethanol solution (95 ~ ethanol/0.3 ~ acetic acid, 1.5 mM ACh, 100 # M neostigmine) and the extract was mixed with 11 ml toluene-ethanol (10 : 1 by vol.) scintillator containing omunifluor (New England Nuclear). The following drugs were administered intraperitoneally: haloperidol 4 mg/kg (Dainippon Seiyaku), fluphenazine hydrochloride 5 mg/kg (Nippon Roche and Squibb Japan) and apomorphine 10 mg/kg (Mitsui Pharmaceuticals). Control animals were injected with the same volume of saline. Rats were pre-treated with drugs and killed by decapitation 30 min later. Their brains were removed and high affinity choline uptake by tissue of the nucleus caudatus was measured. Administration of haloperidol (4 mg/kg) caused a significant increase in choline uptake (P < 0.001), whereas pretreatment with the dopamine receptor agonist apomorphine (10 mg/kg) resulted in a decrease in choline uptake (P < 0.05) (Table I). The phenothiazine derivatives fluphenazine (5 mg/kg), a dopamine receptor antagonist, had a similar effect as haloperidol causing significant increase in choline uptake by the nucleus caudatus tissue (P < 0.01) (Table I). In studies on the synaptosomal fractionS,la,20, 2a and slices 4,16 of the brain evidence has been obtained that high affinity choline uptake is coupled with the release and synthesis of ACh. Moreover, several recent studies have demonstrated that in the striatum the turnover rate of ACh 3,t2,17,24 and high affinity choline uptake 1,11 are enhanced by blocking inhibitory dopaminergic neurons of the substantia nigra. Some investigators have reported, however, that this increase in ACh turnover in the striatum is not accompanied by increase in high affinity choline uptake by striatal synaptosomes18,19.
TABLE I Effects of dopamine receptor antagonists and agonist on high affinity choline uptake by the nucleus caudatus
Rats were killed by decapitation 30 min after administration of haloperidol (4 mg/kg i.p.), fluphenazine (5 mg/kg i.p.), apomorphine (10 mg/kg i.p.) or saline. Values are means ± S.E.M. for the number of determinations shown in parentheses. Treatment
High affinity choline uptake (pmol/5 min/mg wet weight)
Saline Haloperidol Fluphenazine Apomorphine
7.98 ± 0.21 (19) 10.82 4- 0.40 (8)*** 9.45 ± 0.51 (4)** 6.78 ± 0.71 (4)*
% Change from control
+35.6 +11.8 --16.0
* Significantly different from the control value at P < 0.05. ** Significantly different from the control value at P < 0.01. *** Significantly different from the control value at P < 0.001.
480 In a recent study, it was d e m o n s t r a t e d t h a t the activity-related changes in choline u p t a k e u n d e r g o a reversal in p o s t - m o r t e m tissues, a n d the p o s t - m o r t e m reversal seems to be, at least in part, t e m p e r a t u r e - d e p e n d e n t 9. So one o f the reasons for the discrepancies in choline u p t a k e m a y be due to this p o s t - m o r t e m change. In the present w o r k we m e a s u r e d high affinity choline u p t a k e by tissue r e m o v e d from slices o f the nucleus c a u d a t u s by m i c r o p u n c t u r e (A 8200). A m i c r o p u n c h o f tissue o b t a i n e d in this way is considered to be in a m o r e physiological state t h a n synaptosomes. F u r t h e r m o r e , p o s t - m o r t e m changes in m i c r o p u n c h e s o f tissue seem to be small because the process o f this m e t h o d is quicker t h a n t h a t o f s y n a p t o s o m a l fractionation. W e f o u n d that high affinity choline u p t a k e was increased by the d o p a m i n e receptor antagonists h a l o p e r i d o l a n d fluphenazine, a n d decreased by the d o p a m i n e receptor agonist a p o m o r p h i n e (Table I). Therefore, o u r results s u p p o r t the existence o f inhibitory d o p a m i n e r g i c regulation o f striatal cholinergic neurons. The a u t h o r s t h a n k Mr. N. O n o and Mr. K. U c h i m u r a for help with this work.
1 Atweh, S., Simon, J. R. and Kuhar, M. J., Utilization of sodium-dependent high affinity choline uptake in vitro as a measure of the activity of cholinergic neurons in vivo, Life ScL, 17 (1975) 15351544. 2 Consolo, S., Ladinsky, H., Bianchi, S. and Ghezzi, D., Apparent lack of a dopaminergic-cholinergic link in the rat nucleus accumbens septi-tuberculum olfactorium, Brain Research, 135 (1977) 255-263. 3 Guyenet, P. G., Agid, Y., Javoy, F., Beaujouan, J.C., Rossier, J. and Glowinski, J., Effects of dopaminergic receptor agonists and antagonists on the activity of the neo-striatal cholinergic system, Brain Research, 84 (1975) 227-244. 4 Hadh~izy, P. and Szerb, J. C., The effect of cholinergic drugs on [aH]acetylcholine release from slices of rat hippocampus, striatum and cortex, Brain Research, 123 (1977) 311-322. 5 Haga, T. and Noda, H., Choline uptake systems of rat brain synaptosomes, Biochim. biophys. Acta (Amst.), 291 (1973) 564-575. 6 Hattori, T., Singh, V. K., McGeer, E. G. and McGeer, P. L., Immunohistochemical localization of choline acetyltransferase containing neostriatal neurons and their relationship with dopaminergic synapses, Brain Research, 102 (1976) 164-173. 7 Jacobowitz, D. M., Removal of discrete fresh regions of the rat brain, Brain Research, 80 (1974) 635-637. 8 Jenden, D. J., Jope, R. S. and Weiler, M. H., Regulation of acetylcholine synthesis: does cytoplasmic acetylcholine control high affinity choline uptake?, Science, 194 (1976) 635-637. 9 Klemm, N. and Kuhar, M. J., Post-mortem changes in high affinity choline uptake, J. Neurochem., 32 (1979) 1487-1494. 10 Ktinig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 11 Kuczenski, R., Schmidt, D. and Leith, N., Amphetamine-haloperidol interactions in rat striatum: failure to correlate behavioral effects with dopaminergic and cholinergic dynamics, Brain Research, 126 (1977) 117-129. 12 Ladinsky, H., Consolo, S., Bianchi, S., Samanin, R. and Ghezzi, D., Cholinergic-dopaminergic interaction in the striatum: the effect of 6-hydroxydopamine or pimozide treatment on the increased striatal acetylcholine levels induced by apomorphine, piribedil and o-amphetamine, Brain Research, 84 (1975) 221-226. 13 Murrin, L. C., DeHaven, R. N. and Kuhar, M. J., On the relationship between all-choline uptake activation and aH-acetylcholine release, J. Neurochem., 29 (1977) 681-687. 14 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450.
481 15 P6rez de la Mora, M. and Fuxe, K., Brain GABA, dopamine and acetylcholine interaction, studies with oxotremorine, Brain Research, 135 (1977) 107-122. 16 Polak, R. L., Molenaar, P. C. and van Gelder, M., Acetylcholine metabolism and choline uptake in cortical slices, J. Neurochem., 29 (1977) 477-485. 17 Racagni, G., Cheney, D. L., Trabucchi, M. and Costa, E., In vivo actions of clozapine and haloperidol on the turnover rate of acetylcholine in rat striatum, J. Pharmacol. exp. Ther., 196 (1976) 323-332. 18 Sherman, K. A., Hannin, I. and Zigmond, M. J., The effect of neuroleptics on acetylcholine concentration and choline uptake in striatum: implications for regulation of acetylcholine metabolism, J. PharmacoL exp. Ther., 206 (1978) 677-686. 19 Sherman, K. A., Zigmond, M. J. and Hannin, I., High affinity choline uptake in striatum and hippocampus. Differential effects of treatments which release actylcholine, Life Sci., 23 (1978) 1863-1870. 20 Simon, J. R. and Kuhar, M. J., Impulse-flow regulation of high affinity choline uptake in brain cholinergic nerve terminals, Nature (Lond.), 255 (1975) 162-163. 21 Simon, J. R., Atweh, S. and Kuhar, M. J., Sodium-dependent high affinity choline uptake: a regulatory step in the synthesis of acetylcholine, J. Neurochem., 26 (1976) 909-922. 22 Stadler, H., Lloyd, K. G., Gadea-Ciria, M. and Bartholini, G., Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine, Brain Research, 55 (1973) 476-480. 23 Takano, Y., Kohjimoto, Y. and Kamiya, H., Studies on the high affinity choline uptake s)stem with respect to the muscarinic receptors using a synaptosomal fraction abundant in presynaptic membranes, Jap. J. Pharmacol., in press. 24 Trabucchi, M., Cheney, D., Racagni, G. and Costa, E., Involvement of brain cholinergic mechanisms in the action of chlorpromazine, Nature (Lond.), 289 (1974) 664-666.