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0022-3565/88/2463-0866502.00/0THE JOURNAL OF PHARMACOLOGYND EXPERIMENTALTHERAPEUTICS VoL246, NO.3Copyright 1988 by The American Socie ty for Pharmacology and Experimental Therapeutics Printed in US.A,

D if f e r e n c e s in t h e S t im u lu s P r o p e r t ie s o f 3 ,4 -M e thy lened io xyam phe tam ine and 3 ,4 -M e thy lened io xym e tham phe tam ine in A n im a ls T ra in ed toD is c r im in a t e H a l lu c in o g e n s f r o m S a l in e 1P. M. CALLAHAN and J. B. APPELBehavioralPharmacologyLaboratory,Departmentof Psychology,Universityof SouthCarolina,Columbia,SouthCarolinaAccepted for publication May 19, 1988

ABSTRACTThe stimulus properties of 3,4-methylenedioxyamphetamine and (+)-MDMA as well as (_+)-2,5-dimethoxy-4-methylampheta.(MDA), 3,4-methylenedioxymethamphetamine (MDMA) and sev- mine, LSD, mescaline and psilocybin mimicked the training drug.eral related compounds were compared to those of (+)-Iysergic Neither (+)-arfiphetamine nor cocaine produced mescaline-likeacid diethylamide (LSD) and mescaline (3,4,5-trimethoxyphen- responding; fenfluramine substituted partially for mescaline butylethylamine) in a two-lever, water-reinforced, drug discrimination not LSD. Because all of the phenylisopropylamine enantiomerstask. In animals trained to discriminate LSD (0.08 rog/kg) from mimicked the potent hallucinogen mescaline (10 rog/kg), thesesaline (n = 8), LSD-like responding occurred during substitution results do not support suggestions that similarities in the behav-(generalization) tests with sufficiently high doses of (___)-2,5- ioral effects of "designer" drugs such as MDA and MDMA todimethoxy-4-methylamphetamine, LSD, mescaline, psilocybin those of hallucinogens are limited to (-)-MDA. They also indicateand (-)-MDA; saline appropriate responding occurred after (+)- that, although LSD and mescaline may be pharmacologicallyMDA and both (+)- and (-)-MDMA. In animals trained to discrim- similar (in other assays), these compounds do not have identicalinate mescaline (10 mg/kg; n = 8), (-)-MDA, (+)-MDA, (-)-MDMA stimulus properties.

Interest has increased recently in MDA ("love") and MDMA enantiomers of MDA indicate that only the (-)-isomer has("ecstasy"), at least in part because they appear to have abuse DOM-like stimulus properties (Glennon and Young, 1982)potential (Adler et al., 1985) and are (serotonergic) neurotoxins Given that: 1) diverse hallucinogens are known to be phar-(Schmidt, 1987). The same compounds are also clinically im- macologically similar (Rasmussen and Aghajanian, 1986) andportant because, under some conditions, they may enhance show cross-tolerance in both humans (Isbell et al., 1955) andpsychotherapy (Yensen et al.. 1976L animals (Appel and Freedman. 1968) and 2) there is so much

Because they are phenylatkylamines, both MDA and MDMA structural similarity between substituted amphetamine deny-might be expected to have effects which resemble those of atives and mescaline, the effects of (-)-MDA, (+)-MDA, (-)'stimulants such as (+)-amphetamine (phenylisopropylamine), MDMA, (+)-MDMA and several related compounds were reor hallucinogens such as DOM and mescaline (3,4,5-trimethox- examined systematically in animals trained to discriminateyphenylethylamine). This hypothesis has been supported by either LSD (0.08 rog/kg) or mescaline (10 mg/kg) from saline.behavioral experiments which indicate that (__)-MDA has dis-criminative stimulus properties that mimic those of (+)-am- Methodsphetamine and cocaine as well as DOM and LSD (Glennon et Subjects. Experimentally naive, male albino Sprague-Dawley ratsal., 1982); however, (___)-MDMA has been reported to resemble (Charles River Breeding Laboratories, Wilmington, MA), weighing 400CNS stimulant, but not hallucinogenic drugs (Glennon et al., to 450 g at the beginning of the experiment, were used. Whereas food1982; Nichols et al., 1986). Further studies of the individual was freely available in individual home cages, access to water wasrestricted to the amount received during training sessions, a small

This work was supported by U.S. Public Health Service Research Grant R01 amount made available for 10 to 15 rain after each, relatively brief tes!DA02543 from the National Institute on Drug Abuse. Some of these data were session (below), and on weekends (6-12 hr); this procedure maintainedpresentedat the Society forNeuroscienceannual meeting (NewOrleans, 1987). the weight of each animal at 80 to 85% of its free-feeding value. The

ABBREVIATIONS:MDA, 3,4-methylenedioxyamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; DOM, (_+)-2,5-dimethoxy-4-methyar_phetamine; LSD, (+)-lysergic acid diethylamide; CNS. central nervous system; FR, fixed ratio; NIDA, National Institute on Drug Abuse; 5-HT,5'hydroxytryptamineserotonin).866

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1988 MOA and MDMA 867colony in which the animals were housed was maintained at constant animals that completed at least 20 responses during test sessions weretemperature (21-23C), humidity (40-50%) and lighting conditions analyzed.(7:00 A.M.-7:00 P.M.).

Apparatus. The apparatus and general procedure have been de- Resultsscribed in detail elsewhere (Cunningham and Appel, 1987). Eightcommercially available, two-lever chambers (BRS]LVE model 143-25), LSD-saline discrimination. The LSD-saline discrimina-housed in light- and sound-attenuating shells (BRS/LVE model 132- tion was acquired by all rats in an average of 20 sessions (range,04) were located in two adjacent rooms. Each chamber was equipped 8-29). Response rates after LSD (74.4 +_14) were not noticeablywith a dipper, mounted equidistant between two response levers which different from rates after saline (76.8 +_ 3).delivered 0.1 ml of water. Illumination was provided by a 28 V house In dose-response tests, 0.01 to 0.08 mg/kg of LSD producedlight; ventilation and masking noise were supplied by a blower. An a dose-dependent generalization gradient (fig. 1). ResponseApple IIe microcomputer, located in an adjoining room, was used to rates after 0.16 rog/kg of LSD (33.8 +_ 18) were lower thanprogram and record experimental events, those after the training close (0.08 mg/kg) whereas those afterProcedures the other test doses were not different from controls.

Acquisition. One group of animals (n = 8) was trained to discrim- The stimulus properties of LSD (0.08 mg/kg) also generalizedinate 0.08 rog/kg of LSD from an equivalent volume (1 rog/mi) of to sufficiently high doses of DOM, mescaline and psilocybinsaline (0.9% NaCl) whereas a second group (n = 8) was trained to (fig. 1, solid lines); higher doses of each of these hallucinogensdiscriminate 10.0 rog/kg of mescaline from saline. Each of these com- suppressed lever-pressing behavior (fig. 1, broken lines).pounds (LSD, mescaline or saline) was given 15-rain before daily Substitution (generalization) tests with phenylalkylamines(Monday to Friday) experimental sessions. Training began under a indicated that the only enantiomer of these compounds thatschedule of continuous water reinforcement (FR 1) with only the mimics LSD is (-)-MDA, at doses higher than 1 rog/kg (fig. 2,stimulus-appropriate (drug or saline) lever present ("errorless" train- left graph); (+)-MDA, (-)-MDMA and (+)-MDMA engenderedlng). For half of the animals, responding on the left lever was reinforced either saline-appropriate responding or no responding (fig. 2).(with water) under the drug state whereas responding on the right leverwas reinforced after saline; conditions were reversed for the remaining Responding occurred primarily on the saline lever (9% drug-animals. To control for the development of position cues based upon lever responding) after treatment with 1 rog/kg of fenfiuramineolfactory stimuli (Extance and Goudie, 1981), the order of running (data not shown); higher doses of this compound could not beeach day was randomized. Training "stimuli" (drug or saline) were tested because they disrupted lever-pressing too severely topresented in random order with the restriction that neither condition produce reliable choice responding.be in effect for more than three consecutive, 20 rain sessions. As rates Mescaline-saline discrimination. All animals (n = 8)of lever-pressing stabilized, the FR requirement was increased until all acquired the mescaline-saline discrimination within 33 sessionsrats were responding reliably under an FR 20 schedule of reinforcement (range, 11-33). Lever-pressing rates after this drug (37.1 10)after either drug or saline, were considerably lower than rates after saline (84.2 _+ 22).

Discrimination training, Both levers were then presented simul- During dose-response tests, the mescaline cue generalized intaneously. Rats were required to respond on the stimulus-appropriate(correct) lever to obtain water; there were no programmed consequences an orderly, dose-dependent manner to lower doses of the train-for pressing the incorrect lever. This procedure continued until all lng drug (fig. 3), although the percentage of drug-lever respond-animals attained criterion (individual mean performance of at least lng decreased at the highest dose tested (when, however, only85% correct for 10 consecutive sessions), four of the eight animals tested engaged in significant amountsTesting procedure. Substitution (generalization) tests were con- of lever-pressing). Sufficiently high doses of DOM, LSD andducted 1 to 2 times per week with intervening training sessions. During psilocybin also produced drug-appropriate responding and, liketest sessions rats were placed in the chamber as during training except mescaline itself, reduced response rates (fig. 3). More impor-that, once the animal completed 20 responses on either lever or after tantly, the animals responded on the drug-appropriate lever inthe session time (20 min) elapsed, the house light was turned off and a dose-dependent manner after treatment with a//of the iso-the animal was removed from the chamber without reinforcement(extinction}. At the end of each test session, all rats were allowed 10 t o mers of both MDA and MDMA (fig. 4).15 min access to water. Given these results and the fact that (+)-MDA and (_-) -

Drugs. The order of drugs and doses rested was randomized through- MDMA have been reported to have stimulant-like propertiesout the experiment. All compounds were prepared fresh each day in (above), mescaline-trained animals were tested with (+)-am-deionized water and were injected J.p. in a volume of 1.0 mi/kg. Doses phetamine, cocaine and fenfluramine. Neither (+)-ampheta-of the tbllowing drugs refer to the weights of the salt: (+)-amphetamine mine nor cocaine elicited significant amounts of responding onsulfate (Sigma Chemical Co., St. Louis, MO.); cocaine hydrochloride the mescaline-appropriate lever (table 1). Fenfiuramine (1.0(Sigma); (+)-DOM hydrochloride (NIDA, Rockville, MD); fenfiura- mg/kg) substituted partially (53%)for the training drug; highermine hydrochloride (A. H. Robins Co. Richmond, VA); (+)-LSD bitar- doses of this compound disrupted responding too severely totrate (NIDA); (+)-MDA (NIDA); (-)-MDA (NIDA); (+)-MDMA produce interpretable data. Thus, none of the CNS stimulants

, (NIDA); (-)-MDMA (NIDA); and mescaline hydrochloride (Sigma tested mimicked mescaline (10 mg/kg) at doses that did notChemical Co.). Doses of psilocybin (NIDA) refer to the free base. severely disrupt responding._ Data analysis. Drug discrimination performance (accuracy) wasdefined as the percentage of correct responses to total responses before

_ the delivery of the first reinforcer. Performance during test sessions Discussionwas the percentage of drug-appropriate responses to total responses. To the extent that their stimulus properties were found toResponse rate is the total number of responses emitted before either,1) completion of 20 responses on the correct lever (training sessions) generalize to the same hallucinogenic compounds, these resultsor 2) completion of 20 responses on either lever (test sessions), divided agree with a considerable amount of evidence that LSD andby the number of minutes to complete the first FR 20. A test compound mescaline are pharmacologically similar (e.g., Rasmussen andwas said to have substituted completely only when at least 80% of all Aghajanian, 1986); among other things, this suggests (but cer-responses occurred on the drug-appropriate lever; only data from tainly does not prove) that these hallucinogens act at the same

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868 Callahan and Appel VoL246DOM (+) LSD MESCALINE PSILOC YBIN

n 8 6 6 6 4 5 8 8 8 8 7 8 8 7 3 6 7 6I I I IN ? 16 6 ? 6 ? 8 8 8 8 .-_l 8 8 ? ?l 7 ? ?l

' I i /- ' -' T_ I _oo Fig. 1.Resultsof dose-responseand substi-ach

tut!on (generalization)tests with other ha!lu-e ! 80 ? cinogens in rats trained to discriminate LSDJ (0.08 mg/kg) from saline. The compoundsested, numberof animalsresponding(n)and

_ _ '\ 60 _ number of animals tested (N) at dose

_ 6I '\, J are shown at the top of the the figure..,

' _ mean percentageof lever-pressingre-sponses on the drug-appropriate lever (_40 O_4o ! _ S.E.M.; left ordinate);O. mean responserate

? t i '' _ \_i } _ t _ (-+S'E'M';dght Ordinate)' The average de'

, \ gree of drug-appropriate responding (_

_ __ ' '_ \, 20 S.E.M.; e) and response rate (_S.E.M.; O)v 20 ,, ' after salinecontrol injections are shown (at_j '-, C) for comparison.o o_- .... Jo ' ' ' ' '6 ' ' 'o ' ' ' ' '06 12 25 05 I 01 02 04 08 .I 2,5 5 ! 20 12 25 05 10

DOSE OF TEST DRUG (rog/kg)

(-) MDA (+) MDA (-) MDMA (+) MOMAn 8 8 8 445 ? 8 5 0 7 7 5 1 5 5 2 !t_ ' '. J ! I J ! IN 8 8 8788 7 8 88 7 7 7 7 7 7 7 7

100 100

III "3 _ Fig. 2. Results of substitution (generali-z l,,, I/ i60 - - 80 '_ zation)tests with the (+)- and (-)-enantio-a. _ mere of MDA and MDMA in rats trained3Lu to discriminate LSD (0.08 mg/kg) fromrr _ _ T u) saline. Details are the same as those of-_ 40 _ _ 40 figure 1._ 03E _ uJ

20 ! _ 20 t--

_ e o_ o_1_ _ ! I I I I t I I I I IC 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0

DOSE OF TEST DRUG (rog/kg)receptor or, more likely, complex of receptors (Glennon et al.. (Schechter and Rosecrans, 1972), MDA or MDMA (Broadbent1986). et al., 1987). In any case, it seems premature to ar_e that the

Inasmuch as (-)-MDA mimics both LSD and mescaline it is purported hallucinogenic effects of MDA are caused solely byprobably safe to conclude, along with previous investigators its (-)-isomer (Glennon and Young, 1982) or that MDMA is(Glennon et al., 1982; Nichols et al., 1986), that this substituted not hallucinogenic (Glennon et al. , 1982; Nichols et al., 1986).phenylalkylamine has discriminative stimulus and, perhaps Although LSD and mescaline may be pharmacologically sim-other in vivo, effects that resemble those of hallucinogenic ilar, they have different behavioral effects in that their stimulusdrugs, properties do not generalize to the same compounds; thus theyUnfortunately, the remaining results are less easily recon- are not likely to have identical mechanisms of action. In thisciled with the existing literature. Whereas neither (+)-MDA, regard it was suggested some time ago (on the basis of both(-)-MDMA nor (+)-MDMA mimic LSD, all of these sub- electrophysiological and pharmacological evidence) that LSDstances have discriminative stimulus properties that are similar may be a direct (postsynaptic) agonist whereas mescaline actsto those of mescaline (10 rog/kg). This means either that these indirectly, either by facilitating 5-HT release or inhibiting itssubstrates can be hallucinogenic (in humans) or that their reuptake (Haigler and Aghajanian, 1973). Although this idea isability to substitute for mescaline is based on some other somewhat weakened by more recent data indicating that selec-("nonhallucinogenic") effect of these compounds. Whatever tive 5-HT antagonists block the electrophysiological (Rasmus-such an effect might be, it is unlikely to involve pharmacological sen and Aghajanian, 1986) as well as the discriminable (Appelsimilarities among phenylalkylamines as (+)-amphetamine and Callahan, 1987) effects of both hallucinogens, it is sup-(phenylisopropylamine) does not mimic either mescaline ported by the fact that (-)-MDA, which mimics LSD and DOM

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iii

1988 MDA and MDMA 869DOM (+) LSO MESCALINE PSILOCYBIN

n S 6 5 4 8 8 ? 7 8 e 8 4 I 4 6 ? 3--4 t i i i i ._ ',N 8 6 5 6 8 8 8 8 8 8 e 8 5 6 ? 5/ ooI T: 5 Fig. 3. Results of dose-response and sub-i/// so '_

,_, _ stitution (generalization) tests with otherhallucinogens in rats trained to discriminate

l\ _ mescaline (10 mg/kg) from saline. Detailso _ a re th e s a m e a s th o s e o f figu re 1.(3z _ J I CO/ t4J20, cro-L oLe 112 ' OI I ' 012 I I 116 ........25 5 10 01 04 .OS 2.5 5 I0 20 12 25 05 I0

DOSE OF TEST DRUG (mE/kg)(-) MOA (+) MOA (-) MDMA (+) MOMA

n 8 8 ? 5 8 8 7 2 8 6 6 5 6 681_ I I I_ lB 7 71 lB 8 B 8 6 6 5 S 6

I 100 tOO

(,gW 80 8B _)Z IO Ico Fig. 4. Results of substitution (generalization)so 6o '_ tests with the (+}- and (-)-enantiomers ofE MDA and MDMA in rats trained to discrimi-

. _ nate mescaline (10 rog/kg) from saline. De-_' 40 40 o tails are the same as those of figure 1.2 ', a.Ltl / ,,,, ttJ

20 / ,. vO tlJco _W n'aeo _ _ o--l-- I I J k I I L I I I i I I

C 0.5 _0 2 0 0.5 l0 20 05 LO 2.0 25 0,5 10 2.0DOSE OF TEST DRUG (mg/kg)

TABLE 1 ReferencesResults of substitution tests with various CNS stimulants in rats ADLER.J,, ABHAMSON.P..KATZ.S.AND]"IAGLER.._1.; Getting High on"Ecstasy,"trained to discriminate mescaline (10 rog/kg J.p.) from saline Newsweek,April 15.p. 96, 1985.All drugs were given 15 rain before behavioral testing, APPEL, J. B. AND CALLAHAN, P. M.; Involvement et' 5-HT Receptor Subtypes inthe Discriminative Stimulus Properties of Mescaline, Paper presented at meet-

% Drug-Lever Rate lng of the Society for Neuroscience, Satellite Session of the Society for theCompound Dose Responses Responses/rain n/N' Stimulus Properties of Drugs, New Orleans, 1987.

(S,E.M.} (_+S.E.M,) APPEL, J. B. AND FREEDMAN, D. X.: Tolerance and cross-tolerance amongpsychotomimetrlc drugs. Psychopharmacoiogia 13: 267-274, 1968.

rog/kg BROADBENT, J., MICHAEL, E. K., RICKER, J. H. AND APPEL, J. B.: Comparisond-Amphetamine 0,5 15 +_15 23 +_12 5/5 of the discriminative stimuli of (+) and (-)-3,4-methylenedioxyamphetamine1,0 24 _+ 15 29 _+ 11 8/8 (MDA) with those of hallucinogenic and stimulant drugs. Soc. Neurosci. Abstr.2,0 40 _+40 9 +' 7 2/7 13: 1720, 1987.

Cocaine 5,0 20 _+20 34 _+12 5/5 CHENG,H. P., LONG,J. P., NICHOLS,D. E. ANOBARFKNECHT,C. F.: Effects of10.0 37 + 23 15 --. 3 4/5 psychotomimetics on vascular strips: Studies of methoxylated amphetamines

Fenfiuramine 1,0 53 _+ 17 20 _+12 8/8 and optical isomers of 2,5-dimethoxy-methylamphetamine and 2,4-dJmethoxy-

Saline 4 _ 4 84 --. 22 8/8 4-bromoamphetamine. J. Pharmacol. Exp. Ther. 188: 114-123, 1974.CUNNINGHAM, K. A. AND APPEL, J. B. Neuropharmacological reassessment of' n, number of animalsresponding;N, numberof animals tested, the discriminative stimulus properties of d-lysergic acid diethylamide (LSD).Psychopharmacology91: 67-73, 1987.EXTANCE, K. AND GOUDIE, A. J.: In_er-animal olfactory cues in operant drug

as well as mescaline, has been reported to have direct receptor discrimination procedures in rats. Psychopharmacology 73: 363-371, 1981.GLENNON, R. A., TITELER, M. AND YOUNG, R.: Structure-activity relationshipsactions (Cheng et al., 1974) whereas (+)-MDA and both isomers and mechanisms of action of hallucinogenic agents based on drug diecrimina-of MDMA, which substitute completely for mescaline but not tion and radiollgand binding studies. Psychopharmacol. Bull. 22: 953-958,1986 .LSD or DOM, also facilitate the release and inhibit the uptake GLENNON, R. A. ANDYOUNG R.: Further investigation of the discriminativeof 5-HT (Nichols et al., 1982). stimulus properties of MDA. Pharmacol. Biochem. Behav. 20: 501-505, 1982.

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370 Callahan and Appel Vol. 246GLENNON, R. A., YOUNG, R., ROSECRANS, J. A. AND ANDERSON, G. M.: Dis- RASMUSSEN, K. AND AGHAJANIAN, G. K. Eff ects o f ha llucinogens on spontaneouscriminative stimulus properties of MDA analogs. Biol. Psychiatry 17: 807- and sensory-evoked locus coeruleus unit activity in the rat: Reversal by selective814, 1982. 5-HT_ antagonists. Brain Res. 385: 395-400, 1986.

HAIGLER, H. J. AND AGHAJANIAN, G. K.: Mescaline and LSD: Direct and indirect SCHECHTER, M. D. AND ROSECRANS, J. A.: d-Amphetamine as a discriminativeeffects on serotonin-containing neurons. Eur. J. Pharmacol. 21: 53-60, 1973. cue: Drugs with similar stimulus properties. Eur J. Pharmacol. 21: 212-216,1972.ISBELL, H., FRASER, H. F., WIKLER, A. AND BELLEVILLE, R. E.: Tolerance to SCHMIDT, C. J.: Neurotoxicity of the psychedelic amphetamine methylenedi-diethylamide of lysergic acid. Fed. Proc. 14: 354, 1965. oxymethamphetamine. J. Pharmacol. Exp. Ther. 240: 1-7, 1987.NICHOLS, D. E., HOFFMAN, A. J., OBERLENDER, R. A., JACOB, P. AND SHULGIN, YENSEN R., DILEO, F. B., RHEAD, J. C., RICHARDS, W. A., SOSKIN, R. A.,A. T.: Derivatives of 1-(1,3-benzodioxol-5-yl)-2-butanamine: Representatives ATUREK,B. AND KURLAND, A. A.: MDA~assisted psychotherapy with neuroticof a novel therapeutic class. J. Med. Chem. 29: 2009-2015, 1986. outpatients: A pilot study. J. Nerv. Ment. Dis. 163: 233-245, 1976.NICHOLS,D. E., LLOYD,D. H., HOFFMAN,A. J., NICHOLS, M. B. ANDYIM, G.

K. W.: Effects of certain hallucinogenic amphetamine analogs on the release Send reprint requests to: James B. Appel, Ph.D., Department ofPsychology,of [3H]serotonin from rat brain synaptosomes. J. Med. Chem. 25: 530-535, University of South Carolina, Columbia, SC 29208.1982.