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Discriminative stimulus properties of L -phenylisopropyl adenosine: Blockade by caffeine and generalization to 2-chloroadenosine
Life Sciences, Vol. 32, pp. 2329-2333 Printed in the U.S.A.
Pergamon Press
DISCRIMINATIVE STIMULUS PROPERTIES OF L-PHENYLISOPROPYL ADENOSINE: BLOCKADE BY CAFFEINE AND GENERALIZAT]'ON TO 2-CHLOROADENOSINE* David G. Spencer, J r . and Harbans Lal Department of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107 (Received in final form February 15, 1983)
Summary Recent neurochemical data on the effects of activation and blockade of adenosine AI receptors has suggested a direct role of adenosine in neurotransmission. The present research used a drug discrimination procedure to test the hypotheses that Al adenosine receptor activation could serve as a discriminative stimulus and that caffeine, a drug believed to be an A1 receptor antagonist, could block the adenosine discrimination. Food-deprived rats were trained to press one of two levers on an FR 10 schedule of food-pellet delivery. Responses on one lever were reinforced following i . p . injection of N6 - (L-phenylisopropyl) adenosine (f~_IL-PIA); responses on t ~ o t h e r lever were reinforced lowing i.p. injection of saline. L-PIA training dose was increased from 0.064 to O.O~mg/kg_L-PIA in the course of the study. Subjects required an average of 91 sessions to acquire this discrimination. Stimulus control by L-PIA was dose-dependent, with the ED-50 being app~ximately 0.03 mg/kg. 2-Chloroadenosine (2CA) generalized to L-PIA with a tenth the potency. Caffeine blocked T-PIA-induced lever selection. These results indicat-e that 1) rats can be trained to discriminate L-PIA from saline in a twolever food-reinforced tas-s~ and 2) the discriminative stimuli produced by L-PIA are based on i t s agonistic action at the adenosTne AI receptor. Reports of specific CNS adenosine binding sites of markedly heterogenous distribution ( i ) and stimulated i n - v i t r o release of adenosine (2) have aroused interest in characterizing the role of adenosine in CNS-mediated behavior. I t has recently been suggested that the methylxanthines (such as caffeine and theophylline) produce their behavioral effects primarily through antagonism of the A1 h i g h - a f f i n i t y adenosine receptors (3,4,5).
*Portions of this study were presented at the Second International Symposium on Drugs as Discriminative Stimuli, June 29-July 3, 1982, Beerse, Belgium.
0024-3205/83/202329-05503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
2330
Adenosine Discrimintation
Vol. 32, No. 20, 1983
Adenosine agonists have previously been shown to produce sedation (6-12), anti-convulsant effects (8,10), hypotension (8), hypothermia (10), and analgesia (13). Indeed, several reports have noted the antagonistic r e l a t i o n s h i p between the effects of methylxanthines and morphine (14,15). The goal of t h i s experiment was to determine i f the adenosine analog L-PIA ( r e l a t i v e l y specific AI agonist) produces interoceptive stimuli that rats--can be trained to r e l i a b l y discriminate. Methods Sixteen food-deprived male Long-Evans rats served as subjects. Responses on one of two levers were rewarded with food on a fixed r a t i o of I0 (FRIO) schedule when the rat was injected i . p . with L-PIA 15 minutes pre-session, while responses on the other lever were rewarded on the same schedule only when an equivalent volume of saline had been injected i . p . 15 minutes pre-session (for detailed methodology, see 16,17). In order to control for i n i t i a l level preferences, half of the subjects were randomly assigned the l e f t lever as the drug-correct manipulandum and the other h a l f , the r i g h t lever. Lever selection in a session was considered correct i f 9 or fewer responses on the inappropriate lever occurred before completing I0 responses on the correct lever. Provided subjects continued to discriminate c o r r e c t l y , saline and L-PIA were administered in i r r e g u l a r order; otherwise i n j e c t i o n conditions were r-epeated. Since each subject thus followed an unique pattern of drug and saline sessions, odor cues from previous sessions were eliminated. After an acquisition c r i t e r i o n of correct lever selection in i0 consecutive sessions was achieved, generalization and antagonism testing was begun. During t e s t i n g , correct lever selection on three consecutive sessions were required before the next test session. The t r a i n i n g dose of L-PIA was 0.064 mg/kg, which was increased to 0.08 mg/kg p r i o r to the testing ph-ase in order to maintain the d i s c r i m i n a t i o n . During the t e s t i n g phase, 2-chloroadenosine (2CA) was given i . p . 15 minutes before the session, and caffeine was given i . p . 30 minutes pre-session, 15 minutes before an i n j e c t i o n of 0.08 mg/kg L-PIA. Both t r a i n i n g and drug testing sessions were I0 min. Food reward was a ~ i l a b l e during testing sessions, but only for responses on the lever to which the f i r s t i0 responses were emitted.
Results A f t e r 135 sessions, 9 of 16 rats had met the acquisition c r i t e r i o n for the L-PIA-saline d i s c r i m i n a t i o n . The average number of sessions required was 91, ~ i t h a range of 61 to 135. Fig. 1 shows the average course of learning f o r these nine subjects. Since L-PIA generally decreased the FRIO response rate as compared to the precedin-g saline day, the degree of L-PIA-induced rate suppression is also shown. The response rate depression e l i c i t e d by L-PIA decreased over t r a i n i n g sessions to about 90% of control. In order to determine whether the rate-suppressant e f f e c t of L-PIA was d i r e c t l y related to the interoceptive discriminable stimuli (IDS) That L-PIA produced, the discrimination data were reanalyzed using the theory of s~-gnal detection (see 18 for applications to discrimination performance in animals). Using t h i s theory, i t is possible to obtain a r e l a t i v e l y pure estimate of the subjects' s e n s i t i v i t y ( a b i l i t y to detect the IDS) from raw discrimination performance data. Using t h i s analysis, L-PIA-induced IDS s e n s i t i v i t y was compared
Vol. 32, No. 20, 1983
Adenosine Discrimination
2331
with the concurrent L-PIA-induced response-suppression for 7 blocks of 5 sessions after the L-PIA training dose was increased to 0.08 mg/kg, resulting in a non-significant correlation coefficient (r=0.03).
100
"100
] SALINE| PIA
.8o
!J°
'iL=
20,
'20
SUCCESSIVE BLOCKS OF $ llESSlON$
Fig.
1
Acquisition of the L-PIA-saline discrimination. Discrimination accuracy is roughly--proportional to the difference between percent of subjects selecting the L-PIA lever on days in which they received the drug (approprTate lever selection) and percent of subjects choosing the L-PIA lever on saline days (inappropriate lever selection). L-PIA-Tnduced response rate suppression is expressed as the average p~rcent of post-saline response rate for each 5-session block. Following acquisition, dose-response functions were determined for I) stimulus control by~-PIA, 2) generalization of 2CA, and 3) blockade of L-PIA IDS by caffeine. ED-50s were determined by log linear regression analysts. Generalization with different doses of L-PIA showed that stimulus control was dose-dependent with an ED-50 of 0.03 mgTkg (Fig. 2). 2CA dose-dependently generalized with an ED-50 of 0.30 mg/kg. Caffeine blocked the L-PIA IDS almost completely at 2.5 and 5.0 mg/kg (Fig. 3). The ED-50 fo~caffeine antagonism was 1.8.mg/kg. At the time of generalization and blockade testing, the training dose of L-PIA (0.08 mg/kg) continued to significantly reduce response rate by 13% "~elative to response rate after saline pre-treatment (paired t - t e s t , t(7)=3.8, p < .01). Pretreatment with 2CA did not significantly reduce response rate relative to controls although a trend toward reduction was noted at the highest dose tested (0.64 mg/kg). Caffeine at 2.5 and 5.0 mg/kg, when injected prior to L-PIA (0.08), resulted in a 46% and 45% (respectively) decrease relative To control response rate, although only the former was significant (t(7) = 2.93, p < .025).
2332
Adenosine Discrimination
Vol. 32, No. 20, 1983
100.
j
80ru~ -"
20.
0.08
016
DOSE (mg/kg) F i g . 2. Dose-response f u n c t i o n s f o r L-PIA and 2CA-induced drug l e v e r s e l e c t i o n , based on e i g h t animal s--(all of which met the a c q u i s i t i o n criterion). 100.
r-"
u~
IMIM -J--I 60-
=! OO
i| °20.
,.'~s
2's
s'.o
CAFFEINE IRE-TREATMENT DOSE (mg/kg) Fig. 3 Dose-response f u n c t i o n s f o r c a f f e i n e antagonism of the L-PIA IDS, based on the same e i g h t animals as in F i g . 3 .
Vol. 32, No. 20, 1983
Adenosine Discrimination
2333
Discussion Over half of the subjects trained on the L-PIA-saline discrimination met the acquisition c r i t e r i o n , although extensive Training was required. While many drug discriminations have been reported to be acquired in 15-35 t r a i n i n g sessions, others (such as the haloperidol-saline discrimination) require as much as 80 sessions (19,20). Thus, while L-PIA seems to have furnished a weak discriminative cue in the present study, iT is not unique in t h i s regard. In addition, although L-PIA suppressed response rate, as previously reported (11,12), this effect- was not correlated to accurate detection of i t s IDS. The hypothesis that the L-PIA IDS were in fact a consequence of adenosine AI receptor stimulation was ~pported by the demonstration that another adenosine analog with r e l a t i v e AI agonistic specificity,2CA, generalized to L-PIA, and that caffeine (an AI antagonist) antagonized the_L-PIA IDS. The p~sent finding that 2CA was less potent than L-PIA also agrees with previous studies of effects on response rate (11,12) an-J latency of pentylenetetrazol-induced seizures. Further evidence that the rate-suppressant effects of L-PIA were not related to its IDS was provided by the findings that 1) 2CA dTd not suppress response rate in the dose-range effective in producing generalization to L-PIA, and 2) caffeine did not reverse the L-PIA-induced rate suppression at doseages effective in blocking the L-PIA ID-S. In conclusion, L-PIA has been shown to produce IDS that are l i k e l y to be based on AI receptor--activation and that rats can be trained to discriminate. References 1.
M.E. LEWIS, J. PATEL, S.M. EDLEY and P.J. MARANGOS, Eur. J. Pharmacol. 73:109-110 (1981). 2. D.A. TAYLOR and T.W. STONE, Brain Res. 183:367-376 (1980). 3. T. KATSURAGI and C. SU, J. Pharmacol. Exp. Ther. 220:152-156 (1982). 4. R.E. NICHOLS and E.J. WALASZEK, Fed. Proc. 22:308 (1963). 5. A. SATTIN and T.W. RALL, Mol. Pharmacol. 6:13-23 (1970). 6. E. MARLEY and G. NISTICO, B r i t . J. Pharmacol. 46:619-636 (1972). 7. I. HAULICA, L. ABABEI, D. BRANISTEANU and F. TOPOLICEANU, J. Neurochem. 21:1019-1020 (1973). 8. M. MAITRE, L. CIESIELSKI, A. LEHMANN, E. KEMPF and P. MANDEL, Biochem. Pharmacol. 23:2807-2816 (1974). 9. M. WEINER and J.W. OLSON, Psychopharmacol. 54:61-65 (1977) . i0. T.V. DUNWIDDIE and T. WORTH, J. Pharmacol. Exp. Ther. 220:70-76 (1982). II. J.M. CARNEY and V.L. COFFIN-SlROCHMAN, The Pharmacologist 23:151 (1981). 12. V. SIROCHMANand J.M. CARNEY, Fed. Proc. 40:294 (1981). 13. J.W. DALY, R.F. BRUNS and S.H. SNYDER, Life Sci. 28:2083-2097 (1981). 14. I.K. HO, H.H. LOH and E.L. WAY, J. Pharmacol. Exp. Ther. 185:336-346 (1973). 16. H. LAL, G. GIANUTSOS and S. MIKSIC, Discriminative Stimulus Properties of Drugs, pp. 23-45, Plenum Press, New York (1977). 17. G.T. SHEARMANand H. LAL, Neuropharmac. 19:473-479 (1980). 18. K.S. MILAR, Psychopharmac. 74:383-388 (1981). 19. A. WEISSMAN, in: Stimulus Properties of Drugs: Ten Years of Progress, eds. F.C. Colpaert and J.A. Rosecrans, pp. 99-122, Elsev1~r71~ort-~-h---H-o-ITand Biomedical Press, Amsterdam (1978). 20. S. MIKSIC, G. SHEARMAN, and H. LAL, Psychopharmac. 60: 103-104 (1978).
Pergamon Press
DISCRIMINATIVE STIMULUS PROPERTIES OF L-PHENYLISOPROPYL ADENOSINE: BLOCKADE BY CAFFEINE AND GENERALIZAT]'ON TO 2-CHLOROADENOSINE* David G. Spencer, J r . and Harbans Lal Department of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107 (Received in final form February 15, 1983)
Summary Recent neurochemical data on the effects of activation and blockade of adenosine AI receptors has suggested a direct role of adenosine in neurotransmission. The present research used a drug discrimination procedure to test the hypotheses that Al adenosine receptor activation could serve as a discriminative stimulus and that caffeine, a drug believed to be an A1 receptor antagonist, could block the adenosine discrimination. Food-deprived rats were trained to press one of two levers on an FR 10 schedule of food-pellet delivery. Responses on one lever were reinforced following i . p . injection of N6 - (L-phenylisopropyl) adenosine (f~_IL-PIA); responses on t ~ o t h e r lever were reinforced lowing i.p. injection of saline. L-PIA training dose was increased from 0.064 to O.O~mg/kg_L-PIA in the course of the study. Subjects required an average of 91 sessions to acquire this discrimination. Stimulus control by L-PIA was dose-dependent, with the ED-50 being app~ximately 0.03 mg/kg. 2-Chloroadenosine (2CA) generalized to L-PIA with a tenth the potency. Caffeine blocked T-PIA-induced lever selection. These results indicat-e that 1) rats can be trained to discriminate L-PIA from saline in a twolever food-reinforced tas-s~ and 2) the discriminative stimuli produced by L-PIA are based on i t s agonistic action at the adenosTne AI receptor. Reports of specific CNS adenosine binding sites of markedly heterogenous distribution ( i ) and stimulated i n - v i t r o release of adenosine (2) have aroused interest in characterizing the role of adenosine in CNS-mediated behavior. I t has recently been suggested that the methylxanthines (such as caffeine and theophylline) produce their behavioral effects primarily through antagonism of the A1 h i g h - a f f i n i t y adenosine receptors (3,4,5).
*Portions of this study were presented at the Second International Symposium on Drugs as Discriminative Stimuli, June 29-July 3, 1982, Beerse, Belgium.
0024-3205/83/202329-05503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
2330
Adenosine Discrimintation
Vol. 32, No. 20, 1983
Adenosine agonists have previously been shown to produce sedation (6-12), anti-convulsant effects (8,10), hypotension (8), hypothermia (10), and analgesia (13). Indeed, several reports have noted the antagonistic r e l a t i o n s h i p between the effects of methylxanthines and morphine (14,15). The goal of t h i s experiment was to determine i f the adenosine analog L-PIA ( r e l a t i v e l y specific AI agonist) produces interoceptive stimuli that rats--can be trained to r e l i a b l y discriminate. Methods Sixteen food-deprived male Long-Evans rats served as subjects. Responses on one of two levers were rewarded with food on a fixed r a t i o of I0 (FRIO) schedule when the rat was injected i . p . with L-PIA 15 minutes pre-session, while responses on the other lever were rewarded on the same schedule only when an equivalent volume of saline had been injected i . p . 15 minutes pre-session (for detailed methodology, see 16,17). In order to control for i n i t i a l level preferences, half of the subjects were randomly assigned the l e f t lever as the drug-correct manipulandum and the other h a l f , the r i g h t lever. Lever selection in a session was considered correct i f 9 or fewer responses on the inappropriate lever occurred before completing I0 responses on the correct lever. Provided subjects continued to discriminate c o r r e c t l y , saline and L-PIA were administered in i r r e g u l a r order; otherwise i n j e c t i o n conditions were r-epeated. Since each subject thus followed an unique pattern of drug and saline sessions, odor cues from previous sessions were eliminated. After an acquisition c r i t e r i o n of correct lever selection in i0 consecutive sessions was achieved, generalization and antagonism testing was begun. During t e s t i n g , correct lever selection on three consecutive sessions were required before the next test session. The t r a i n i n g dose of L-PIA was 0.064 mg/kg, which was increased to 0.08 mg/kg p r i o r to the testing ph-ase in order to maintain the d i s c r i m i n a t i o n . During the t e s t i n g phase, 2-chloroadenosine (2CA) was given i . p . 15 minutes before the session, and caffeine was given i . p . 30 minutes pre-session, 15 minutes before an i n j e c t i o n of 0.08 mg/kg L-PIA. Both t r a i n i n g and drug testing sessions were I0 min. Food reward was a ~ i l a b l e during testing sessions, but only for responses on the lever to which the f i r s t i0 responses were emitted.
Results A f t e r 135 sessions, 9 of 16 rats had met the acquisition c r i t e r i o n for the L-PIA-saline d i s c r i m i n a t i o n . The average number of sessions required was 91, ~ i t h a range of 61 to 135. Fig. 1 shows the average course of learning f o r these nine subjects. Since L-PIA generally decreased the FRIO response rate as compared to the precedin-g saline day, the degree of L-PIA-induced rate suppression is also shown. The response rate depression e l i c i t e d by L-PIA decreased over t r a i n i n g sessions to about 90% of control. In order to determine whether the rate-suppressant e f f e c t of L-PIA was d i r e c t l y related to the interoceptive discriminable stimuli (IDS) That L-PIA produced, the discrimination data were reanalyzed using the theory of s~-gnal detection (see 18 for applications to discrimination performance in animals). Using t h i s theory, i t is possible to obtain a r e l a t i v e l y pure estimate of the subjects' s e n s i t i v i t y ( a b i l i t y to detect the IDS) from raw discrimination performance data. Using t h i s analysis, L-PIA-induced IDS s e n s i t i v i t y was compared
Vol. 32, No. 20, 1983
Adenosine Discrimination
2331
with the concurrent L-PIA-induced response-suppression for 7 blocks of 5 sessions after the L-PIA training dose was increased to 0.08 mg/kg, resulting in a non-significant correlation coefficient (r=0.03).
100
"100
] SALINE| PIA
.8o
!J°
'iL=
20,
'20
SUCCESSIVE BLOCKS OF $ llESSlON$
Fig.
1
Acquisition of the L-PIA-saline discrimination. Discrimination accuracy is roughly--proportional to the difference between percent of subjects selecting the L-PIA lever on days in which they received the drug (approprTate lever selection) and percent of subjects choosing the L-PIA lever on saline days (inappropriate lever selection). L-PIA-Tnduced response rate suppression is expressed as the average p~rcent of post-saline response rate for each 5-session block. Following acquisition, dose-response functions were determined for I) stimulus control by~-PIA, 2) generalization of 2CA, and 3) blockade of L-PIA IDS by caffeine. ED-50s were determined by log linear regression analysts. Generalization with different doses of L-PIA showed that stimulus control was dose-dependent with an ED-50 of 0.03 mgTkg (Fig. 2). 2CA dose-dependently generalized with an ED-50 of 0.30 mg/kg. Caffeine blocked the L-PIA IDS almost completely at 2.5 and 5.0 mg/kg (Fig. 3). The ED-50 fo~caffeine antagonism was 1.8.mg/kg. At the time of generalization and blockade testing, the training dose of L-PIA (0.08 mg/kg) continued to significantly reduce response rate by 13% "~elative to response rate after saline pre-treatment (paired t - t e s t , t(7)=3.8, p < .01). Pretreatment with 2CA did not significantly reduce response rate relative to controls although a trend toward reduction was noted at the highest dose tested (0.64 mg/kg). Caffeine at 2.5 and 5.0 mg/kg, when injected prior to L-PIA (0.08), resulted in a 46% and 45% (respectively) decrease relative To control response rate, although only the former was significant (t(7) = 2.93, p < .025).
2332
Adenosine Discrimination
Vol. 32, No. 20, 1983
100.
j
80ru~ -"
20.
0.08
016
DOSE (mg/kg) F i g . 2. Dose-response f u n c t i o n s f o r L-PIA and 2CA-induced drug l e v e r s e l e c t i o n , based on e i g h t animal s--(all of which met the a c q u i s i t i o n criterion). 100.
r-"
u~
IMIM -J--I 60-
=! OO
i| °20.
,.'~s
2's
s'.o
CAFFEINE IRE-TREATMENT DOSE (mg/kg) Fig. 3 Dose-response f u n c t i o n s f o r c a f f e i n e antagonism of the L-PIA IDS, based on the same e i g h t animals as in F i g . 3 .
Vol. 32, No. 20, 1983
Adenosine Discrimination
2333
Discussion Over half of the subjects trained on the L-PIA-saline discrimination met the acquisition c r i t e r i o n , although extensive Training was required. While many drug discriminations have been reported to be acquired in 15-35 t r a i n i n g sessions, others (such as the haloperidol-saline discrimination) require as much as 80 sessions (19,20). Thus, while L-PIA seems to have furnished a weak discriminative cue in the present study, iT is not unique in t h i s regard. In addition, although L-PIA suppressed response rate, as previously reported (11,12), this effect- was not correlated to accurate detection of i t s IDS. The hypothesis that the L-PIA IDS were in fact a consequence of adenosine AI receptor stimulation was ~pported by the demonstration that another adenosine analog with r e l a t i v e AI agonistic specificity,2CA, generalized to L-PIA, and that caffeine (an AI antagonist) antagonized the_L-PIA IDS. The p~sent finding that 2CA was less potent than L-PIA also agrees with previous studies of effects on response rate (11,12) an-J latency of pentylenetetrazol-induced seizures. Further evidence that the rate-suppressant effects of L-PIA were not related to its IDS was provided by the findings that 1) 2CA dTd not suppress response rate in the dose-range effective in producing generalization to L-PIA, and 2) caffeine did not reverse the L-PIA-induced rate suppression at doseages effective in blocking the L-PIA ID-S. In conclusion, L-PIA has been shown to produce IDS that are l i k e l y to be based on AI receptor--activation and that rats can be trained to discriminate. References 1.
M.E. LEWIS, J. PATEL, S.M. EDLEY and P.J. MARANGOS, Eur. J. Pharmacol. 73:109-110 (1981). 2. D.A. TAYLOR and T.W. STONE, Brain Res. 183:367-376 (1980). 3. T. KATSURAGI and C. SU, J. Pharmacol. Exp. Ther. 220:152-156 (1982). 4. R.E. NICHOLS and E.J. WALASZEK, Fed. Proc. 22:308 (1963). 5. A. SATTIN and T.W. RALL, Mol. Pharmacol. 6:13-23 (1970). 6. E. MARLEY and G. NISTICO, B r i t . J. Pharmacol. 46:619-636 (1972). 7. I. HAULICA, L. ABABEI, D. BRANISTEANU and F. TOPOLICEANU, J. Neurochem. 21:1019-1020 (1973). 8. M. MAITRE, L. CIESIELSKI, A. LEHMANN, E. KEMPF and P. MANDEL, Biochem. Pharmacol. 23:2807-2816 (1974). 9. M. WEINER and J.W. OLSON, Psychopharmacol. 54:61-65 (1977) . i0. T.V. DUNWIDDIE and T. WORTH, J. Pharmacol. Exp. Ther. 220:70-76 (1982). II. J.M. CARNEY and V.L. COFFIN-SlROCHMAN, The Pharmacologist 23:151 (1981). 12. V. SIROCHMANand J.M. CARNEY, Fed. Proc. 40:294 (1981). 13. J.W. DALY, R.F. BRUNS and S.H. SNYDER, Life Sci. 28:2083-2097 (1981). 14. I.K. HO, H.H. LOH and E.L. WAY, J. Pharmacol. Exp. Ther. 185:336-346 (1973). 16. H. LAL, G. GIANUTSOS and S. MIKSIC, Discriminative Stimulus Properties of Drugs, pp. 23-45, Plenum Press, New York (1977). 17. G.T. SHEARMANand H. LAL, Neuropharmac. 19:473-479 (1980). 18. K.S. MILAR, Psychopharmac. 74:383-388 (1981). 19. A. WEISSMAN, in: Stimulus Properties of Drugs: Ten Years of Progress, eds. F.C. Colpaert and J.A. Rosecrans, pp. 99-122, Elsev1~r71~ort-~-h---H-o-ITand Biomedical Press, Amsterdam (1978). 20. S. MIKSIC, G. SHEARMAN, and H. LAL, Psychopharmac. 60: 103-104 (1978).