Department of Pharmacology, Medical College of Virginia, Richmond, VA 23298, U.S.A. ... (Lehigh Valley Electronics model 13202) equipped with an exhaust.
Psychopharmacology
Psychopharmacology 68, 283-286 (1980)
9 by Springer-Verlag 1980
Discriminative Stimulus Properties of the Optical Isomers of Nicotine L e o n a r d T. Meltzer, J o h n A. Rosecrans, Mario D. Aceto, and Louis S. Harris Department of Pharmacology, Medical College of Virginia, Richmond, VA 23298, U.S.A.
Abstract. Rats were trained to discriminate 200 or 400 pg/kg (-)nicotine f r o m saline in a two-bar operant paradigm. Dose-response relationships for optically pure (-)- and (+)nicotine as well as antagonistic effects were examined in both groups o f rats. The natural isomer (-)nicotine was approximately nine-times more potent than (+)nicotine. Mecamylamine p r o d u c e d equal blockade o f the (-)- and (+)nicotine cues. H e x a m e t h o n i u m and atropine were without effect. These data demonstrate the possible stereospecificity o f the central nicotinic receptor that mediates the stimulus effect p r o d u c e d by nicotine.
Key
words:
Drug
discrimination
-
Nicotine
Structural changes in the nicotine molecule, such as opening the pyrrolidine ring or increasing the chain length between the pyridine and pyrrolidine rings, decreases the discriminability o f these c o m p o u n d s c o m p a r e d to nicotine. These studies demonstrated one important receptor criterion, namely, the strict structural requirement o f the nicotine receptor in mediating the nicotine cue. To further characterize the central nicotinic receptor, the present study was undertaken to investigate another important receptor criterion, that of pharmacological stereospecificity. Thus, this study tested the ability o f optically pure (+)nicotine, the synthetic isomer, to generalize to the cue p r o d u c e d by optically pure (-)nicotine, the natural isomer.
stereoisomers
Materials and Methods The ability o f nicotine to serve as a discriminative stimulus has been well d o c u m e n t e d (Morrison and Stephenson, 1969; Schechter and Rosecrans, 1971a; Rosecrans and Chance, 1977). The discriminative cue p r o d u c e d by SC nicotine injection depends u p o n the central rather than peripheral effects o f the drug on nicotinic receptors, since h e x a m e t h o n i u m does not alter the cueing potency o f nicotine while m e c a m y l amine blocks the nicotine cue (Schechter and Rosecrans, 1971 a; Hirschhorn and Rosecrans, 1974). The lack o f involvement o f muscarinic receptors in the nicotine cue are demonstrated by the inability of atropine to alter the nicotine cue (Hirschhorn and Rosecrans, 1974) and the lack of generalization to the nicotine cue by the muscarinic agonist arecoline (Schechter and Rosecrans, 1971b). The effects o f structural modifications o f the nicotine nucleus on the nicotine cue were investigated in recent studies in our laboratory (Chance et al., 1978). Offprint requests to: J. A. Rosecrans
Subjects. Male Sprague-Dawley rats (175-200 g) with no previous drug or experimental experience were purchased from Flow Research Animals, Dublin, Virginia, U.S.A. These rats were individually housed in a temperature-controlled environment under a 12-h lightclark cycle. Water was freely available in the home cages and adjusted amounts of commercial rat chow were offered after each experimental session to maintain the animals at 80 % of their expected freefeeding weight. Apparatus. The experimental space was a standard operant test
chamber (Lehigh Valley Electronics model 1417). One wall of the chamber contained two levers with a dipper centered between them for delivery of liquid reinforcement. A force of approximately 15 g was necessary to depress each lever. The experimental chamber was located in a larger sound-insulated and lightproof isolation cubicle (Lehigh Valley Electronics model 13202) equipped with an exhaust fan. Solid state and electromechanical programming equipment were used to control and record the sessions. Equal parts of sugar and nonfat evaporated milk mixed in tap water and delivered by the dipper (0.01 ml) was the reinforcer. Procedure Training. At approximately 90 days of age, food-deprived rats were trained to press first one and then the other lever for liquid reinforcement. After 4 days of responding on a continuous reinforcement (CRF) schedule (2 days on each bar), to insure responding, discrimination training commenced. Nicotine (N) and saline (S) were administered in a double-alternation schedule 15rain prior to the
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Dose-response relationship for (-)- alad (+)nicotine in rats trained to discriminate either 200 (N = 9) or 400 ~tg/kg (N = 7) of (-)nicotine from saline. Data collected during a 2-rain extinction session. Each point is the mean +_ SEM. Nicotine doses expressed as free base
OOSE OF NICOTINE
~g/kg~
session (N, N, S, S, N, N, S, S, etc.). The order of presentation was counterbalanced so that half the rats received nicotine first and the other half S first. One lever was reinforced after the injection of nicotine and the other lever was reinforced after saline injection. For half of the subjects in the experiment, the right lever was reinforced after nicotine injection and the left lever was rewarded after saline injection. These conditions were reversed for the remaining animals in the study. Discrimination training began with placing the rat in the operant chamber, with both levers operative, after receiving a saline or nicotine injection. Only responses on the correct lever were reinforced, incorrect responses were counted but had no consequences. During the first four sessions (one double alternation), each correct bar press was reinforced. During subsequent sessions a variable interval (VI) schedule of reinforcement was imposed. The schedule was increased gradually to a VI 15 s, which was maintained throughout the duration of the studies. After four double alternations, a 2-min period during which no responses were reinforced was imposed at the beginning of each alternation to measure discrimination. A 15 min training session followed this test session. Nine rats were trained to discriminate 200 ~tg/kg (-)nicotine from saline and seven rats were trained to discriminate 400 ~tg/kg (-)nicotine from saline. Discrimination was stable after approximately 1 0 - 1 5 double alternations.
Testing. Animals continued to receive either 200 or 400 ~tg/kg ( - ) nicotine and saline according to the double-alternation sequence. Generalization to different doses of (-)- and (+)nicotine and antagonists was tested in a 2-min nonreinforced session following day 2 of an alternation (N, N, test, S, S, test ...). This procedure did not disrupt the baseline discrimination. Animals were run for a maximum of 6 consecutive days. At least four training days separated antagonist tests. Testing of drugs and doses was randomized so that approximately half of the test trials followed the nicotine condition and the rest followed the saline condition. Nicotine or saline was administered SC 15 min prior to the test session and antagonists were injected SC 10 min prior to the nicotine or saline injections. The discrimination data are expressed as percent drug bar responding ( ~ DBR), which is the number of responses on the drug-correct lever divided by the total number of responses during the nonreinforced session.
Animals were removed from the chambers after 2min or after five responses were emitted if animals took longer than 2rain to respond. Dose-response relationships were analyzed using linear regression while treatment differences were tested using Student's t-test.
Drugs. Optically pure (-)nicotine di-/-tartrate and (+)nicotine-di-dtartrate were synthesized by Dr. Everette L. May and Dr. I. M. Uwaydah (Aceto et al., 1979). All doses of nicotine are expressed as the base. Mecamylamine HC1 (Merck, Sharp and Dohme, West Point, PA), atropine sulfate (Sigma, St. Louis, MO), and Hexamethonium C1 (Sigma, St. Louis, MO) are expressed as the salt. All drugs were dissolved in 0.9 ~o saline and injected SC in a volume of t ml/kg.
Results F o r r a t s t r a i n e d t o d i s c r i m i n a t e 200 g g / k g ( - ) n i c o t i n e , t h e b a s e l i n e ~ D B R a n d r e s p o n s e r a t e w a s 12.2 + 3.8 % (12.9 + 3.8 r e s p o n s e s / r a i n ) u n d e r t h e s a l i n e c o n d i t i o n a n d 94.7 + 1.9 ~ (10.0 _+ 2.0 r e s p o n s e s / m i n ) u n d e r t h e nicotine condition. For rats trained to discriminate 400 ~tg/kg ( - ) n i c o t i n e , t h e b a s e l i n e ~o D B R a n d res p o n s e r a t e w a s 6.8 _+ 1.8 ~ (15.3 + 2.6 r e s p o n s e s / m i n ) u n d e r t h e s a l i n e c o n d i t i o n a n d 96.7 + 1.3 ~ (20.1 +_ 3.9 responses/min) under the nicotine condition. Figure I shows the dose-response relationship for ( - ) - a n d ( + ) n i c o t i n e in r a t s t r a i n e d a t 2 0 0 a n d 400 g g / k g (-)nicotine. For (+)nicotine an eight-fold increase in the training dose of (-)nicotine was necessary to produce similar ~ DBR. As the dose of(-)- and (+)nicotine was d e c r e a s e d , so d i d t h e ~o D B R . T h e r e w e r e n o differe n c e s i n t h e r e s p o n s e r a t e s u n d e r all c o n d i t i o n s w i t h i n each group. The potency ratios for (-)- and (+)nicotine are s h o w n in T a b l e 1. U s i n g l i n e a r r e g r e s s i o n a n a l y s i s , t h e
L. T. Meltzer et al. : Stimulus Properties of Nicotine Stereoisomers EDso and EDloo for (-)- and (+)nicotine in both groups o f rats were calculated. Using the EDso values to determine the potency ratios resulted in slightly higher values than using the EDloo. The potency ratios derived from comparisons o f the EDs0 and E D l o o was similar in both groups o f rats. Based on these ratios, (-)nicotine is approximately nine-times more potent than (+)nicotine. Mecamylamine (0.5mg/kg in tlhe rats trained at 4 0 0 g g / k g and 0 . 2 5 m g / k g in the rats trained at 200 gg/kg) p r o d u c e d equal blockade o f the generalization p r o d u c e d by equipotent doses o f (-)- and (+)nicotine (Table 2). F o r both training doses, the D B R for (-)- and (+)nicotine following mecamylamine pretreatment were not significantly different ( d f = 15, t = 1.63, P > 0.05). Neither atropine (2 mg/kg) nor h e x a m e t h o n i u m (1 mg/kg) p r o d u c e d any blockade o f the generalization p r o d u c e d by equipotent doses o f ( - ) and (+)nicotine (Table 2). Atropine, hexamethonium, and mecamylamine did not affect the: ~ D B R under the saline condition (Table 2).
Table 1. Potency ratios a for (-)- and (+)nicotine in rats trained to
discriminate either 200 or 400 lag/kg (-)nicotine from saline
EDs0 (+)/EDso @)
200 gg/kg
400 gg/kg
764.4/82.7 = 9.3
1 318.1/128.5 = 10.3
ED100 (+)/ED10o (-) 1 685.1/215.7 = 7.8
3222.9/368.8 = 8.7
" Data obtained from linear regression analysis of dose-response data Table 2 Effects of atropine (Atr), hexamethonium (Hex), and mecamylamine (Mec) on ( ) and (+)nicotine generalization in rats trained to discriminate either 200 or 400 btg/kg (-)nicotine from saline (Sal)
200gg/kg
400gg/kg
285 Discussion
A degree o f stereospecificity o f the central nicotinic receptor was demonstrated using a drug discrimination paradigm. Optically pure (-)nicotine was approximately nine-times more potent than the optically pure (+)nicotine. Both isomers were working t h r o u g h the same central receptor sites as demonstrated by the similar effect o f selective antagonists on (-)- and (+)nicotine generalization. Recently, Aceto et al. (1979) compared the potency o f optically pure (-)- and (+)nicotine in a variety of tests. They f o u n d that the potency ratios o f (-)nicotine, c o m p a r e d to (+)nicotine were 7.4, 16.7, and 52.7 when comparing the LDso in mice, blood pressure in anesthesized rats, and in contraction in the isolated guinea pig ileum, respectively. The potency ratio f o u n d in the present study agrees most closely with the potency ratio for the LDs0 in mice. This potency ratio (approximately 7 - 9 ) m a y best reflect the specificity o f central nicotinic receptors, since both measures (LDs0 in mice and drug discrimination in rats) are central in nature. There is some central c o m p o n e n t to the effects o f nicotine on blood pressure while in the isolated ileum, the effects are mediated by peripheral parasympathetic ganglia. D o m i n o (1965) found an approximately seven-fold potency ratio for effects on shock avoidance in rats using nicotine that was only 87 % optically pure. The difference in the potency ratios may reflect either a true difference in the stereoselectivity o f the different nicotinic receptors or m a y be related to distributional differences. The latter is unlikely since nicotine is a very lipid-soluble molecule which readily crosses nerve membranes. Optical isomers have the
Pretreatment a
Test dose
(N)
~ DBR b
Responses/min
" Sal(lml/kg) Sal (lmi/kg) Atr(2mg/kg) Atr (2mg/kg) Mec(0.25mg/kg) Mec (0.25 mg/kg) Sal (lml/kg) Sal (lmI/kg) Atr(2mg/kg) Atr (2mg/kg) Hex (1 mg/kg) Hex (1 mg/kg) Atr (2 mg/kg) Hex (I mg/kg) Mec(0.5mg/kg) Mec (0.5mg/kg) Mec (0.5 mg/kg)
(-) 200 (+) 1600 (-) 200 (+) 1600 @) 200 (-) 1 600 (-) 400 (+) 3200 (-) 400 (+) 3200 (-) 400 (+) 3200 Sal Sal Sal (-) 400 (+) 3200
9 8 8 6 9 8 7 7 7 7 7 7 7 7 7 7 7
87.1+ 4.2 92.0+- 2.4 89.5_+ 3.4 91.8_+ 3.9 45.4_+11.5 22.7 _+ 6.7 98.9_+ 0.8 96.9+ 3.1 92.8_+ 4.2 96.0+ 2.2 98.2 + 1.8 96.3 _+ 2.0 21.3 +- 14.2 4.3 _+ 2.9 13.5_+ 6.5 20.3 +- 6.8 49.9 _+14.0
13.0_+2.8 13.7+-3.3 10.6+-2.5 11.7_+5.0 13.8+_3.6 9.0 + 2.4 15.i _+3.9 tl.9_+4.3 9.5_+3.0 17.5+2.6 21.3 + 3.9 20.i _+4.8 15.8 + 8.5 30.2 _+9.0 13.9+6.0 21.7 • 4.9 19.3 _+4.5
Pretreatments administered 10min prior to nicotine or saline injection. b Percent drug bar responding. Data obtained during 2-min extinction session. Data is the mean _+ SEM for six to nine rats per treatment
286
same physical properties, so dispositional factors should not play a role in the potency difference. Various studies have shown that (-)nicotine enters the brain very rapidly following either IV or SC administration (Schmiterlow et al., 1967; Rosecrans, 1972; Hirschhorn and Rosecrans, 1974). A differential metabolism is unlikely since in the mouse LDso determinations all animals died within 3 min (Aceto et al., 1979). Abood et at. (1978) reported a potency difference of at least 100-fold following intraventricnlar (IVT) administration of (-)- and ( +)nicotine that was approximately 98~o pure. They were able to demonstrate pronounced changes in behavior and hippocampal electrical activity following 2 - 1 0 IJg (-)nicotine IVT. These effects were not blocked by IVT decamethonium, however, other nicotinic receptor blockers were not administered. Rosecrans and Chance (1977) and Chance et al. (1978) found rats relatively insensitive to the effects of IVT nicotine. It was necessary to administer 16-321ag 9 8 ~ pure (-)nicotine IVT to obtain generalization equal to that produced by 200 pg/kg SC. These sensitivity differences may be attributable to tolerance due to the chronic administration of nicotine in the discrimination paradigm, as welt as a difference in anatomical location (proximity to ventricles) of receptors that mediate the acute behavioral syndrome seen by Abood et al. (1978), and the receptors that mediate the nicotine cue. Acknowledgements. Supported by Council for Tobacco Research grant 1157 and National Institute of Drug Abuse grant DA-07027 and DA-0040.
Psychopharmacoiogy 68 (1980) noncholinergic, stereospecific site for nicotine in rat brain. J. Neurosci. Res. 3, 327-333 (1978) Aceto, M. D., Martin, B. R., Uwaydah, I. M., May, E. L., Harris, L. S., Izazola-Conde, C., Dewey, W. L., Bradshaw, T. J., Vincek, W. C. : Optically pure (+)nicotine from (_+)nicotine and biological comparisons with (-)nicotine. J. Med. Chem. 22, 174-177 (1979) Chance, W. T., Kallman, M. D., Rosecrans, J. A., Spencer, R. M. : A comparison of nicotine and structurally related compounds as discriminative stimuli. Br. J. Pharmacol. 63, 609-616 (1978) Domino, E. F. : Some comparative pharmacological actions of (-)nicotine, its optical isomer, and related compounds. In: Tobacco Tobacco alkaloids and related compounds, Wenner-Gren center international symposimn series, vol. 4, U.S. yon Euler, ed., pp. 303-314. New York, London: Pergamon 1965 Hirschhorn, I. D., Rosecrans, J. A. : Studies on the time course and the effect of cholinergic and adrenergic receptor blockers on the stimulus effect of nicotine. Psychopharmacologia 40, 109-120 (1974) Morrison, C. F., Stephenson, J. A.: Nicotine injections as the conditioned stimulus in discrimination learning. Psychopharmacologia 15, 3 5 1 - 360 (.1969) Rosecrans, J. A. : Brain area nicotine levels in male and female rats with different levels of spontaneous activity. Neuropharmacology 11, 863-870 (1972) Rosecrans, J. A., Chance, W. T. : Cholinergic and non-cholinergic aspects of the discriminative stimulus properties of nicotine. In: Discriminative stimulus properties of drugs, H. Lal, ed., pp. 155-185. New York: Plenum 1977 Schechter, M. D., Rosecrans, J. A.: CNS effect of nicotine as the discriminative stimulus for the rat in a T-maze. Life Sci. 10, 821 - 832 (1971 a) Schechter, M. D., Rosecrans, J. A. : Behavioral evidence for two types of cholinergic receptors in the CNS. Eur. J. Pharmacol. 15, 375-378 (1971b) Schmiterlow, G. E., Hansson, E., Andersson, G., Appelgren, L. E., Hoffman, P. C. : Distribution of nicotine in the central nervous system. Ann. N.Y. Acad. Sci. 142, 2 - 1 4 (1967)
References Abood, L. G., Lowy, K., Tometsko, A., Booth, H.: Electrophysiological, behavioral and chemical evidence for a
Received July 12, 1979; Final Version January 21, 1980
