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Effect of nicotine on dopaminergic-cholinergic interaction in the striatum
Brain Research, 567 (1991) 313-316 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 BRES 24963
313
Short Communications
Effect of nicotine on dopaminergic-cholinergic interaction in the striatum Norbert T. Sandor, Tibor Zelles, Janos Kiss, Henry Sershen, Andras Torocsik, Abel Lajtha and E. Sylvester Vizi Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary) and Centerfor Neurochemistry, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962-2210 (U.S.A.) (Accepted 10 September 1991) Key words: Nicotine; Acetylcholinerelease; Rat striatum; Somatodendritic receptor; 6-Hydroxydopamine;Sulpiride
We have investigated the effect of nicotinic receptor stimulation on acetylcholine (ACh) release measured by radioassay in rat striatal slices. Since the release of ACh in the striatum is tonically inhibited by endogenous dopamine and nicotine enhances the release of dopamine, we studied the release of ACh when the dopaminergic input was impaired. We used chemical denervation (6-hydroxydopaminepretreatment) or D2-receptor-bloekade by sulpiride to remove the dopaminergic control of the cholinergic neurons. In our experiments nicotine failed to increase ACh release from striatal slices taken from rats whose dopaminergic-cholinergicinteraction was not impaired but it enhanced the release of ACh from slices dissected from 6-hydroxydopaminepretreated rats or in the presence of sulpiride. Our results provide neurochemical evidence for the existence of nicotinic receptors on striatal cholinergic interneurons. Since the spontaneous release of ACh enhanced by nicotine was inhibited by tetrodotoxin it seems very likely that (-)-nicotine acts on the somatodendritic part of cholinergic interneurons. Nicotine exhibits widespread pharmacological effects in both the central and in the peripheral nervous system. Many of these effects are associated with the ability of nicotine to release various neurotransmitters 2'25. In the central nervous system it was found that nicotinic receptor stimulation enhances the release of acetylcholine (ACh) from the cortex 4'16 and that of noradrenaline 9 and serotonin 11 from the hippocampus, and that it increases the resting release of dopamine from striatal preparations of both synaptosomess'15 and slices 6's'24. The action of nicotine is mediated by specific cholinergic receptors. Several subtypes of nicotinic cholinergic receptors have been distinguished by pharmacological methods 1a'2° and molecular biological techniques 7. Acting on its specific receptors, nicotine may evoke transmitter release from resting neurons or it may stimulate the release evoked by nerve impulses or by other depolarizing stimuli 24. While somatodendritic receptors are considered to enhance the release of transmitters associated with spontaneous neural firing activity, presynaptic nerve terminal receptors are responsible for the stimulatory modulation of the evoked neurotransmitter release 3A2. Although the stimulatory effect of nicotine on the release of dopamine in striatal slices has been known for
a long time s'24 the effect of nicotinic receptor stimulation on the release of other neurotransmitters of the striatum has been ignored. However, the existence of nicotinic receptors on different striatal neurons might have functional importance. Therefore we decided to investigate the effect of nicotinic receptor stimulation on ACh release measured by radioassay in striatal slices. Since the release of ACh is under the tonic control of endogenous dopamine, released either by nicotine or by electrical field stimulation 1A°'1a'21 the dopaminergic-cholinergic interaction was also studied. Evidence was obtained that nicotine is able to enhance, in addition to the release of dopamine, the release of ACh from striatal cholinergic interneurons. This latter effect of nicotine is mediated most likely via somatodendritic nicotinic receptors. All experiments were carried out at 37 °C in modified Krebs solution containing (in mM): NaC1 118, KCI 4.7, CaC12 2.5, KH2PO 4 1.2, MgSO 2 1.2, NaHCO 3 25, and glucose 12.5, continuously saturated with carbogen gas (95% 02 + 5% CO2). The experiments were performed on two groups of male Wistar rats (180-220 g b. wt.): untreated rats and those pretreated by intracerebroventricular (i.c.v.) injection of 6-hydroxydopamine (6OHDA). The dose of 6 - O H D A (2 x 250 btg) was suffi-
Correspondence: E.S. Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, 1450Budapest, Hungary. Fax: (36) (1) 114-1866.
314
cient to destroy subtotally the nigrostriatal dopaminergic tract in 5 days as evidenced by the reduction of the dopamine content of the striatum from 7.27 --- 0.49 to 0.51 - 0.13/~g/g tissue. Three days elapsed between the two doses of 6-OHDA. The animals were killed by decapitation two days after the second dose of 6 - O H D A , corpora striata were removed from the skull, cleaned of blood with ice cold saline and sliced into pieces (2 × 0.4 x 1.2 mm) with a tissue chopper. The slices were incubated with [3H]choline chloride 10 #Ci/ml (spec. act.: 78 Ci/mmol) at 37 °C for 40 min in Krebs solution. Then 5 slices were placed into a micro-volume perfusion chamber. After a 60-min preperfusion (flow rate: 0.5 ml/min) the effluent was collected in 3-min samples. During the collection of the 4th and the 16th samples, the slices were
stimulated (S 1 and $2) for 2-min periods of square wave impulses of 2-ms duration at 2 Hz (240 shocks). In some experiments stimulation was not applied. Aliquots of the perfusate and the supernatans of the tissue homogenates were added to scintillation cocktail and assayed for radioactivity by liquid scintillation spectrometry. Radioactivity of the samples was expressed in terms of disintegrations per gram of tissue (Bq/g). High performance liquid chromatography 14 and biochemical separation of labelled choline from ACh 19 indicated that it is mainly [3H]ACh that is responsible for radioactivity released by field stimulation from the striatum, prelabelled with [3H]choline. Therefore the fractional release (FRS) of ACh was calculated as a percentage of the total radioactivity present in the tissue at the beginning of the
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Fig. 1. Effect of (-)-nicotine (100/.tM) on the release of [3H]acetylcholine ([3H]ACh) from striatal slices of the rat. S 1 and S 2 indicate stimulations (2 Hz, 240 shocks). Fractions were collected every 3 min. Vertical lines indicate standard error, if it is not indicated it is less than the size of the symbol. Asterisks indicate significant change in the spontaneous release of ACh compared to control, a: effect of (-)-nicotine (100/tM) on the release of [3H]ACh in control rats. Note that (-)-nicotine (100/~M) failed to affect the release of ACh at rest and in response to field stimulation, b: effect of (-)-nicotine on [aH]ACh release when the dopamine receptor mediated modulation was excluded by D2-receptor antagonist, sulpiride. Nicotine was added 15 rain before the second stimulation. Note that in this case (-)-nicotine enhanced the basal release of [3H]ACh but did not cause change in the FRS2/FRS 1 ratio most likely due to receptor desensitization, c: effect of (-)-nicotine on [3H]ACh release when the dopaminergic input was subtotally destroyed by 6-hydroxydopamine. Note that nicotine (100/tM) added just before the second stimulation enhanced the electric field stimulation evoked release of ACh most likely due to a superposition of the increased spontaneous outflow on the evoked release.
315 stimulation. Basal release was determined during collection periods before and after the stimulation period. We used the R2/R1 ratio to express the effect of nicotine on the resting release of ACh (R 1, percentage of the released radioactivity in 3 samples before and R 2 after nictotine administration). Drugs were applied after the first ($1) but before the second stimulation ($2) so that the fractional release after the S 1 served as an internal control. Drugs were usually applied 15 rain before $2, and maintained until the end of the experiments. When electric field stimulation was not applied, drugs were added to the perfusion Krebs solution from the 10th fraction. In some experiments nicotine was added just before the second stimulation to avoid receptor desensitization. When used, sulpiride was present in the Krebs solution from the preperfusion period throughout the experiments. The effect of drugs on the release of ACh was expressed as the ratio of FRS2 over FRS1 (FRSE/FRS1) or RE/R 1. T h e statistical significance of the results was determined by Student's t-test or two-way analysis of variance with subsequent comparisons by Dunn's test; P < 0.05 was considered significant. Methyl-[3H]choline chloride (78 Ci/mmol) was purchased from Amersham. All other drugs and chemicals were purchased from Sigma Chemical. Drugs were freshly prepared on the day of use. Electrical field stimulation evoked the release of [3H]ACh from striatal slices. The FRS2/FRS1 ratio was 0.84 ± 0.12 in the control experiments (n = 6). (-)Nicotine (100/~M) showed no effect either on FRSE/FRS1 ratio (0.91 ± 0.12; n = 6) or on the basal release of [3H]ACh (Fig. la). However, nicotine enhanced both the spontaneous (data not shown) and the stimulation evoked release of ACh (Fig. lc) from slices dissected from dopamine-deficient rats (FRS2/FRS 1 = 1.34 -+ 0.09; n = 6, control: FRSE/FRS1 = 0.82 ± 0.06; n = 6). If sulpiride was added to the perfusion Krebs solution throughout the experiments nicotine produced a considerable increase in the release of [3H]ACh. Nicotine added just at the onset of the second stimulation, signif-
icantly increased the FRS2/FRS 1 ratio (1.69 - 0.17; n = 4, control: FRS2/FRSI = 0.82 ± 0.1; n = 5). In addition, nicotine added during the resting phase of the experiments enhanced the spontaneous release of ACh (Fig. lb). This latter effect of nicotine was observed also in experiments without field stimulation (R21RI = 1.27 ± 0.1; n = 5, control: R2/R 1 = 0.85 ± 0.09; n = 5). In the presence of tetrodotoxin (1 /~M) nicotine failed to enhance the spontaneous release of [3H]ACh (R2[R 1 = 0.87 ± 0.07; n = 5). In our experiments nicotine failed to increase the release of ACh from striatal slices taken from rats whose dopaminergic-cholinergic interaction was not impaired. It did not affect either the spontaneous or the stimulation evoked release. Since it is known that spontaneous release of D A was effectively increased by nicotine 24 and dopamine released from nigrostriatal terminals is able to inhibit the release of ACh through the stimulation of presynaptic dopaminergic receptors of the D 2 subtype 13A7'22'23it was expected that endogenous dopamine released by nicotine would inhibit the release of ACh, thereby possibly overshadowing the expected stimulatory action of nicotine on ACh release. Indeed, this was the case. Nicotine enhanced the release of ACh from slices dissected from 6 - O H D A pretreated rats or in the presence of the DE-dopamine receptor antagonist sulpiride. Nicotine increased the release of ACh evoked by electrical field stimulation and during resting. Since the spontaneous release of ACh enhanced by nicotine was inhibited by tetrodotoxin, i.e., when the axonal conduction was impaired, it seems very likely that its site of action is on the somatodendritic part of the cholinergic interneurons. The elevation of the FRSE/FRS1 ratio by nicotine when the drug was administered just before the S2 was most likely due to a superposition of the increased spontaneous outflow on the evoked release. In many tissues receptor desensitization occurs within seconds after nicotinic receptor activation. In our experiments the nicotine evoked ACh release from striatal slices apparently showed no desensitization during the first 15 min.
1 Agid, Y., Guyenet, P., Glowinski, J., Beaujouan, J.C. and Jayoy, E, Inhibitory influence of nigrostriatal dopamine system on the striatal cholinergic neurons in rat, Brain Research, 86 (1975) 488-492. 2 Balfour, D.J.K., The effects of nicotine on brain neurotransmitter systems, Pharmacol. Ther., 16 (1982) 269-282. 3 Chesselet, M.F., Presynaptic regulation of neurotransmitter release in the brain: facts and hypothesis, Neuroscience, 12 (1984) 347-375. 4 Chiou, C.Y., Long, J.P., Potrepka, R. and Spratt, J.L., The ability of various nicotinic agents to release acetylcholine from synaptic vesicles, Arch. Int. Pharmacodyn. Ther., 187 (1970) 88-96.
5 de Belleroche, J. and Bradford, H.E, Biochemicalevidence for the presence of presynaptic receptors on dopaminergic nerve terminals, Brain Research, 142 (1978) 53-68. 6 Giorguieff,M.F., Le Floc'h, M.L., Westfall, T.C., Glowinski, J. and Besson, M.J., Nicotinic effect of acetylcholine on the release of newly synthesized [3H]dopaminein rat striatal slices and cat caudate nucleus, Brain Research, 106 (1976) 117-131. 7 Goldman, D., Deneris, E., Luyten, W., Kochhar, A., Patrick, J. and Heinemann, S., Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system, Cell, 48 (1987) 965-973. 8 Goodman, ER., Effects of nicotine on distribution and release of ~4C-norepinephrine and ~4C-dopamine in rat brain striatum
316 and hypothalamus, Neuropharmacology, 3 (1974) 1025-1032. 9 Hall, G.H. and Turner, D.M., Effects of nicotine on the release of 3H-noradrenaline from the hypothalamus, Biochem. Pharmacol., 21 (1972) 1829-1828. 10 Hertting, G., Zumstein, A., Jackisch, R., Hoffmann, I. and Starke, K., Modulation of endogenous dopamine of the release of acetylcholine in the caudate nucleus of the rabbit, Naunyn Schmiedeberg's Arch. Pharmacol., 315 (1980) 111-117. 11 Hery, E, Bourgoin, S., Hamon, N., Ternaux, J.P. and Glowinski, J., Control of the release of newly synthetized 3Hhydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices, Arch. Int. Pharmacodyn. Ther., 296 (1977) 91-97. 12 Lehmann, J. and Langer, S.Z., The striatal cholinergic interneuron: synaptic target of dopaminergic terminals, Neuroscience, 10 (1983) 1105-1120. 13 Miller, J.C. and Friedhoff, A.J., Dopamine receptor-coupled modulation of the K+-depolarized overflow of 3H-acetylcholine from rat striatal slices: alterations after chronic haloperidol and alpha-methyl-p-tyrosine pretreatment, Life Sci., 25 (1979) 12491256. 14 Potter, P.E., Harsing Jr., L.G., Zimanyi, I., Kakucska, I., Gaal, G. and Vizi, E.S., Use of HPLC in characterizing the effect of AF64A, a potential cholinergic neurotoxin. In H. Kalasz and L.S. Ettre (Eds.), Symposium of Liquid Chromatography, Elsevier, Amsterdam, 1986. 15 Rapier, C., Lunt, G.G. and Wonnacott, S., Stereoselective nicotine-induced release of dopamine from striatal synaptosomes: concentration dependence and repetitive stimulation, J, Neurochem., 50 (1988) 1123-1130. 16 Rowell, P.P. and Winkler, D.L., Nicotinic stimulation of [3H]acetylcholine release from mouse cerebral cortical synaptosomes, J. Neurochem., 43 (1984) 1593-1598.
17 Scatton, B., Effect of dopamine receptor agonists and neuroleptic agents on striatal acetylcholine transmission in the rat: evidence against dopamine receptor multiplicity, J. Pharmacol. Exp. Ther., 220 (1982) 197-202. 18 Sershen, H., Reith, M.E.A., Lajtha, A. and Gennaro, J., Noncholinergic saturable binding of (+)-[3H]nicotine to mouse brain, J. Recept. Res., 2 (1981) 1-15. 19 Somogyi, G.T. and Szerb, J.C., Demonstration of acetylcholine release by measuring efflux of labelled choline from cerebral cortical slices, J. Neurochem., 19 (1972) 2667-2671. 20 Todorov, L., Windisch, K., Sershen, H., Lajtha, A., Papasova, M. and Vizi, E.S., Prejunctional nicotinic receptors involved in facilitation of stimulation-evoked noradrenaline release from the vas deferens of the guinea-pig, Br. J. PharmacoL, 102 (1991) 186-190. 21 Vizi, E.S., Harsing Jr., L.G. and Knoll, J., Presynaptic inhibition leading to disinhibition of acetylcholine release from interneurons of the caudate nucleus: effects of dopamine betaendorphin and D-Ala2-ProS-enkephalinamide, Neuroscience, 2 (1977) 953-961. 22 Vizi, E.S., Presynaptic modulation of neurochemical transmission, Prog. NeurobioL, 12 (1979) 181-290. 23 Vizi, E.S., Non-Synaptic Interactions between Neurons, John Wiley, New York, 1984. 24 Westfall, T.C., Effect of nicotine and other drugs on the release of 3H-norepinephrine and 3H-dopamine from rat brain slices, Neuropharmacology, 13 (1974) 693-700. 25 Wonnacott, S., Irons, J., Rapier, C., Thorne, B. and Lunt, G.G., Presynaptic modulation of transmitter release by nicotinic receptors. In A. Nordberg, K. Fuxe, B. Holmstedt and A. Sundwall (Eds.), Progress in Brain Research, Vol. 79, Elsevier, Amsterdam, 1989, pp. 157-163.
313
Short Communications
Effect of nicotine on dopaminergic-cholinergic interaction in the striatum Norbert T. Sandor, Tibor Zelles, Janos Kiss, Henry Sershen, Andras Torocsik, Abel Lajtha and E. Sylvester Vizi Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest (Hungary) and Centerfor Neurochemistry, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962-2210 (U.S.A.) (Accepted 10 September 1991) Key words: Nicotine; Acetylcholinerelease; Rat striatum; Somatodendritic receptor; 6-Hydroxydopamine;Sulpiride
We have investigated the effect of nicotinic receptor stimulation on acetylcholine (ACh) release measured by radioassay in rat striatal slices. Since the release of ACh in the striatum is tonically inhibited by endogenous dopamine and nicotine enhances the release of dopamine, we studied the release of ACh when the dopaminergic input was impaired. We used chemical denervation (6-hydroxydopaminepretreatment) or D2-receptor-bloekade by sulpiride to remove the dopaminergic control of the cholinergic neurons. In our experiments nicotine failed to increase ACh release from striatal slices taken from rats whose dopaminergic-cholinergicinteraction was not impaired but it enhanced the release of ACh from slices dissected from 6-hydroxydopaminepretreated rats or in the presence of sulpiride. Our results provide neurochemical evidence for the existence of nicotinic receptors on striatal cholinergic interneurons. Since the spontaneous release of ACh enhanced by nicotine was inhibited by tetrodotoxin it seems very likely that (-)-nicotine acts on the somatodendritic part of cholinergic interneurons. Nicotine exhibits widespread pharmacological effects in both the central and in the peripheral nervous system. Many of these effects are associated with the ability of nicotine to release various neurotransmitters 2'25. In the central nervous system it was found that nicotinic receptor stimulation enhances the release of acetylcholine (ACh) from the cortex 4'16 and that of noradrenaline 9 and serotonin 11 from the hippocampus, and that it increases the resting release of dopamine from striatal preparations of both synaptosomess'15 and slices 6's'24. The action of nicotine is mediated by specific cholinergic receptors. Several subtypes of nicotinic cholinergic receptors have been distinguished by pharmacological methods 1a'2° and molecular biological techniques 7. Acting on its specific receptors, nicotine may evoke transmitter release from resting neurons or it may stimulate the release evoked by nerve impulses or by other depolarizing stimuli 24. While somatodendritic receptors are considered to enhance the release of transmitters associated with spontaneous neural firing activity, presynaptic nerve terminal receptors are responsible for the stimulatory modulation of the evoked neurotransmitter release 3A2. Although the stimulatory effect of nicotine on the release of dopamine in striatal slices has been known for
a long time s'24 the effect of nicotinic receptor stimulation on the release of other neurotransmitters of the striatum has been ignored. However, the existence of nicotinic receptors on different striatal neurons might have functional importance. Therefore we decided to investigate the effect of nicotinic receptor stimulation on ACh release measured by radioassay in striatal slices. Since the release of ACh is under the tonic control of endogenous dopamine, released either by nicotine or by electrical field stimulation 1A°'1a'21 the dopaminergic-cholinergic interaction was also studied. Evidence was obtained that nicotine is able to enhance, in addition to the release of dopamine, the release of ACh from striatal cholinergic interneurons. This latter effect of nicotine is mediated most likely via somatodendritic nicotinic receptors. All experiments were carried out at 37 °C in modified Krebs solution containing (in mM): NaC1 118, KCI 4.7, CaC12 2.5, KH2PO 4 1.2, MgSO 2 1.2, NaHCO 3 25, and glucose 12.5, continuously saturated with carbogen gas (95% 02 + 5% CO2). The experiments were performed on two groups of male Wistar rats (180-220 g b. wt.): untreated rats and those pretreated by intracerebroventricular (i.c.v.) injection of 6-hydroxydopamine (6OHDA). The dose of 6 - O H D A (2 x 250 btg) was suffi-
Correspondence: E.S. Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, 1450Budapest, Hungary. Fax: (36) (1) 114-1866.
314
cient to destroy subtotally the nigrostriatal dopaminergic tract in 5 days as evidenced by the reduction of the dopamine content of the striatum from 7.27 --- 0.49 to 0.51 - 0.13/~g/g tissue. Three days elapsed between the two doses of 6-OHDA. The animals were killed by decapitation two days after the second dose of 6 - O H D A , corpora striata were removed from the skull, cleaned of blood with ice cold saline and sliced into pieces (2 × 0.4 x 1.2 mm) with a tissue chopper. The slices were incubated with [3H]choline chloride 10 #Ci/ml (spec. act.: 78 Ci/mmol) at 37 °C for 40 min in Krebs solution. Then 5 slices were placed into a micro-volume perfusion chamber. After a 60-min preperfusion (flow rate: 0.5 ml/min) the effluent was collected in 3-min samples. During the collection of the 4th and the 16th samples, the slices were
stimulated (S 1 and $2) for 2-min periods of square wave impulses of 2-ms duration at 2 Hz (240 shocks). In some experiments stimulation was not applied. Aliquots of the perfusate and the supernatans of the tissue homogenates were added to scintillation cocktail and assayed for radioactivity by liquid scintillation spectrometry. Radioactivity of the samples was expressed in terms of disintegrations per gram of tissue (Bq/g). High performance liquid chromatography 14 and biochemical separation of labelled choline from ACh 19 indicated that it is mainly [3H]ACh that is responsible for radioactivity released by field stimulation from the striatum, prelabelled with [3H]choline. Therefore the fractional release (FRS) of ACh was calculated as a percentage of the total radioactivity present in the tissue at the beginning of the
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Fig. 1. Effect of (-)-nicotine (100/.tM) on the release of [3H]acetylcholine ([3H]ACh) from striatal slices of the rat. S 1 and S 2 indicate stimulations (2 Hz, 240 shocks). Fractions were collected every 3 min. Vertical lines indicate standard error, if it is not indicated it is less than the size of the symbol. Asterisks indicate significant change in the spontaneous release of ACh compared to control, a: effect of (-)-nicotine (100/tM) on the release of [3H]ACh in control rats. Note that (-)-nicotine (100/~M) failed to affect the release of ACh at rest and in response to field stimulation, b: effect of (-)-nicotine on [aH]ACh release when the dopamine receptor mediated modulation was excluded by D2-receptor antagonist, sulpiride. Nicotine was added 15 rain before the second stimulation. Note that in this case (-)-nicotine enhanced the basal release of [3H]ACh but did not cause change in the FRS2/FRS 1 ratio most likely due to receptor desensitization, c: effect of (-)-nicotine on [3H]ACh release when the dopaminergic input was subtotally destroyed by 6-hydroxydopamine. Note that nicotine (100/tM) added just before the second stimulation enhanced the electric field stimulation evoked release of ACh most likely due to a superposition of the increased spontaneous outflow on the evoked release.
315 stimulation. Basal release was determined during collection periods before and after the stimulation period. We used the R2/R1 ratio to express the effect of nicotine on the resting release of ACh (R 1, percentage of the released radioactivity in 3 samples before and R 2 after nictotine administration). Drugs were applied after the first ($1) but before the second stimulation ($2) so that the fractional release after the S 1 served as an internal control. Drugs were usually applied 15 rain before $2, and maintained until the end of the experiments. When electric field stimulation was not applied, drugs were added to the perfusion Krebs solution from the 10th fraction. In some experiments nicotine was added just before the second stimulation to avoid receptor desensitization. When used, sulpiride was present in the Krebs solution from the preperfusion period throughout the experiments. The effect of drugs on the release of ACh was expressed as the ratio of FRS2 over FRS1 (FRSE/FRS1) or RE/R 1. T h e statistical significance of the results was determined by Student's t-test or two-way analysis of variance with subsequent comparisons by Dunn's test; P < 0.05 was considered significant. Methyl-[3H]choline chloride (78 Ci/mmol) was purchased from Amersham. All other drugs and chemicals were purchased from Sigma Chemical. Drugs were freshly prepared on the day of use. Electrical field stimulation evoked the release of [3H]ACh from striatal slices. The FRS2/FRS1 ratio was 0.84 ± 0.12 in the control experiments (n = 6). (-)Nicotine (100/~M) showed no effect either on FRSE/FRS1 ratio (0.91 ± 0.12; n = 6) or on the basal release of [3H]ACh (Fig. la). However, nicotine enhanced both the spontaneous (data not shown) and the stimulation evoked release of ACh (Fig. lc) from slices dissected from dopamine-deficient rats (FRS2/FRS 1 = 1.34 -+ 0.09; n = 6, control: FRSE/FRS1 = 0.82 ± 0.06; n = 6). If sulpiride was added to the perfusion Krebs solution throughout the experiments nicotine produced a considerable increase in the release of [3H]ACh. Nicotine added just at the onset of the second stimulation, signif-
icantly increased the FRS2/FRS 1 ratio (1.69 - 0.17; n = 4, control: FRS2/FRSI = 0.82 ± 0.1; n = 5). In addition, nicotine added during the resting phase of the experiments enhanced the spontaneous release of ACh (Fig. lb). This latter effect of nicotine was observed also in experiments without field stimulation (R21RI = 1.27 ± 0.1; n = 5, control: R2/R 1 = 0.85 ± 0.09; n = 5). In the presence of tetrodotoxin (1 /~M) nicotine failed to enhance the spontaneous release of [3H]ACh (R2[R 1 = 0.87 ± 0.07; n = 5). In our experiments nicotine failed to increase the release of ACh from striatal slices taken from rats whose dopaminergic-cholinergic interaction was not impaired. It did not affect either the spontaneous or the stimulation evoked release. Since it is known that spontaneous release of D A was effectively increased by nicotine 24 and dopamine released from nigrostriatal terminals is able to inhibit the release of ACh through the stimulation of presynaptic dopaminergic receptors of the D 2 subtype 13A7'22'23it was expected that endogenous dopamine released by nicotine would inhibit the release of ACh, thereby possibly overshadowing the expected stimulatory action of nicotine on ACh release. Indeed, this was the case. Nicotine enhanced the release of ACh from slices dissected from 6 - O H D A pretreated rats or in the presence of the DE-dopamine receptor antagonist sulpiride. Nicotine increased the release of ACh evoked by electrical field stimulation and during resting. Since the spontaneous release of ACh enhanced by nicotine was inhibited by tetrodotoxin, i.e., when the axonal conduction was impaired, it seems very likely that its site of action is on the somatodendritic part of the cholinergic interneurons. The elevation of the FRSE/FRS1 ratio by nicotine when the drug was administered just before the S2 was most likely due to a superposition of the increased spontaneous outflow on the evoked release. In many tissues receptor desensitization occurs within seconds after nicotinic receptor activation. In our experiments the nicotine evoked ACh release from striatal slices apparently showed no desensitization during the first 15 min.
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