Antagonism of 5-Hydroxytryptamine 2A Receptors Attenuates the Behavioral Effects of Cocaine in Rats

Transcription

1 /01/ $3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 297, No. 1 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3291/ JPET 297: , 2001 Printed in U.S.A. Antagonism of 5-Hydroxytryptamine 2A Receptors Attenuates the Behavioral Effects of Cocaine in Rats LANCE R. MCMAHON and KATHRYN A. CUNNINGHAM Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas Received September 6, 2000; accepted January 10, 2001 This paper is available online at An understanding of the neuropharmacological actions of cocaine is necessary to guide the development of novel pharmacotherapies for cocaine dependence, which remains a widespread problem in the Unites States. The observations that inhibition of dopamine (DA) reuptake (Koe, 1976) and stimulation of D 1 - and D 2 -like receptors underlie many of the behavioral effects of cocaine in animals (Spealman et al., 1992) have provided a key rationale for the use of dopaminergic compounds in the treatment of cocaine addiction. However, the utility of DA receptor ligands in treatment settings has been limited by adverse side effects (for review, see Klein, 1998). Cocaine also inhibits serotonin (5-hydroxytryptamine; 5-HT) reuptake (Koe, 1976) and stimulation and blockade of 5-HT receptors have been shown to modulate the behavioral effects of cocaine in preclinical studies (for review, see Walsh and Cunningham, 1997; McMahon and Cunningham, 1999). A more thorough appreciation of this modulatory role for 5-HT in the behavioral effects of cocaine may yield insight into how manipulations of 5-HT systems may prove This research was supported by National Institute on Drug Abuse Grants DA 05708, DA (to K.A.C.), and DA (to L.R.M.). Portions of these data were presented as an abstract at the 29th Annual Meeting of the Society for Neuroscience (Miami, 1999). ABSTRACT Serotonin (5-hydroxytryptamine; 5-HT) 5-HT 2A receptors have been shown to modulate dopamine (DA) function and a more thorough appreciation of this modulatory interaction between 5-HT 2A receptors and DA systems may yield insight into novel approaches to treatment of cocaine dependence. The present study examined the effects of two ligands with varying selectivity for 5-HT 2A receptors on the locomotor stimulant and discriminative stimulus effects of cocaine in male rats. Locomotor activity was measured following intraperitoneal injection of vehicle (1 ml/kg), the selective 5-HT 2A receptor antagonist M [R-( )-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol] ( mg/kg), or the 5-HT 2 receptor antagonist ketanserin ( mg/kg) 45 min before administration of saline (1 ml/kg) or cocaine (10 mg/kg); monitoring of activity in photobeam chambers began at once and proceeded for 1 h. Neither M nor ketanserin significantly altered basal locomotor activity, but both drugs attenuated cocaine-induced hyperactivity (p 0.05). In drug discrimination studies, rats were trained to discriminate cocaine (10 mg/kg) from saline (1 ml/kg) in a two-lever, water-reinforced operant task. M ( mg/kg) and ketanserin ( mg/kg) evoked a dose-related attenuation of the stimulus effects of cocaine (5 mg/kg, p 0.05). These results suggest that 5-HT 2A receptors play an important role in the behavioral effects of cocaine and that 5-HT 2A receptors should be considered a viable target for analysis in the search for pharmacotherapies useful in the treatment of cocaine dependence. useful in facilitating treatment goals to maintain abstinence in a cocaine abuser. Of the 14 5-HT receptor subtypes characterized to date, the 5-HT 2 receptors (5-HT 2 Rs), including 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R, are products of different genes and exhibit differential anatomical localization (Hoyer et al., 1994). Stimulation of 5-HT 2 Rs, which are linked to phospholipase C, results in increased phosphatidyl inositol turnover, increased intracellular levels of Ca 2, and neuronal depolarization (Hoyer et al., 1994). The 5-HT 2A R is synonymous with the classical 5-HT 2 R and has been implicated in hallucinosis, psychosis, and affective disorders (Baxter et al., 1995). The distributions of 5-HT 2A R mrna and protein in rat brain closely overlap, with moderate-to-high levels observed in DA cell body and terminal fields of the mesocorticolimbic and nigrostriatal systems, including lamina V of cortex, dorsal striatum, and nucleus accumbens (Lopez-Gimenez et al., 1997; Cornea-Hebert et al., 1999). In fact, 5-HT 2A R protein has been recently localized to DA neurons in the ventral tegmental area of rats (Doherty and Pickel, 2000) and humans (Ikemoto et al., 2000). Dopamine neurotransmission appears to be under afferent regulation by 5-HT 2A R (Schmidt et al., 1992, 1995; Sorensen et al., 1992, 1993; De Deurwaer- ABBREVIATIONS: DA, dopamine; 5-HT, 5-hydroxytryptamine; 5-HT 2 R, 5-hydroxytryptamine 2 receptor; M100907, [R-( )-(2,3-dimethoxyphenyl)- 1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol]; FR, fixed ratio; MDMA, ( )-3,4-methylenedioxymeth-amphetamine; SB , N-3-pyridinyl- 3,5-dihydro-5-methylbenzo(1,2-b:4,5-b )dipyrrole-1(2h)carboxamide. 357

2 358 McMahon and Cunningham dere and Spampinato, 1999; Lucas and Spampinato, 2000), and therefore 5-HT 2A R might be a target through which to treat disorders associated with dysfunctional DA systems, including drug dependence and psychosis. Attempts to elucidate a role for 5-HT 2A R in the behavioral effects of cocaine have used ligands that do not effectively differentiate between 5-HT 2 R subtypes, and frequently possess affinity for other monoamine receptors such as -noradrenergic receptors (e.g., ketanserin; Dudley et al., 1988). Cocaine-evoked hyperactivity has been reported to be both enhanced and attenuated by the nonselective 5-HT 2 R antagonist ketanserin (Herges and Taylor, 1998; O Neill et al., 1999) and the 5-HT 2B/2C R antagonist SB (McCreary and Cunningham, 1999). In drug discrimination studies, ketanserin and ritanserin were reported to attenuate the stimulus effects of cocaine in squirrel monkeys (Schama et al., 1997). However, the stimulus effects of cocaine in rats were reportedly unaltered by nonselective 5-HT 2 R antagonists (Meert and Janssen, 1992; Peltier et al., 1994; Callahan and Cunningham, 1995). Ketanserin was also reported to decrease response rates maintained by a second order schedule of cocaine self-administration in squirrel monkeys, at doses that also decreased responding for food (Nader and Barrett, 1990). Failing to alter breakpoints on a progressive ratio schedule of cocaine delivery, ketanserin did modestly reduce cocaine intake in rats (Lacosta and Roberts, 1993). In contrast, ritanserin increased response rates for self-administration of low doses of cocaine (Howell and Byrd, 1995). Thus, a potential role for 5-HT 2 R in the hypermotive, stimulus and reinforcing effects of cocaine is suggested; however, the identification of roles for specific 5-HT 2 R subtypes has been hampered by the use of compounds with poor selectivity. Although only a 2- to 10-fold selectivity of ketanserin for 5-HT 2A (K i 3.2 nm) over either 5-HT 2C (K i 28 nm) or 1 -adrenergic receptors (K i 6.7 nm; Dudley et al., 1988; Yagaloff and Hartig, 1985) is evident, the more recently developed compound M is an antagonist that possesses nanomolar affinity for 5-HT 2A R(K i 0.85 nm), considerably less affinity for 5-HT 2C R(K i 88 nm), and 1 - adrenergic receptors (K i 128 nm), and negligible affinity for most other receptors, including DA D 1 - and D 2 -like receptors ( 500 nm; Kehne et al., 1996a). Thus, M presents a profile of 100-fold selectivity for the 5-HT 2A R versus the 5-HT 2C Ror 1 -adrenergic receptor. The aim of the present study was to more thoroughly analyze the possible role of 5-HT 2A R in the in vivo effects of cocaine by directly comparing the abilities of the nonselective 5-HT 2 R antagonist ketanserin and the selective 5-HT 2A R antagonist M to block the hypermotive and discriminative stimulus effects of cocaine. Materials and Methods Animals Male Sprague-Dawley rats (n 50; Harlan, Houston, TX) weighing between 300 and 350 g at the beginning of the study were used. The rats were housed in pairs in a colony room that was maintained at a constant temperature (21 23 C) and humidity (40 50%); lighting was maintained on a 12-h light/dark cycle (7:00 AM 7:00 PM). For locomotor testing, rats were provided with continuous access to tap water and rodent chow throughout the experiment except during experimental sessions. For drug discrimination testing, food was always available in the home cage but not during experimental sessions. The amount of water each animal received during drug discrimination studies was restricted to that given during operant training sessions, after test sessions (10 15 min), and on weekends (36 h). All experiments were conducted during the light phase (9:00 AM 3:00 PM). Drugs Doses of all drugs refer to the weight of the salt. Cocaine hydrochloride (National Institute on Drug Abuse, Research Triangle Park, NC) was prepared in 0.9% NaCl. Ketanserin tartrate (Sigma/Research Biochemicals International, Natick, MA) was prepared in a solution of 45% 2-hydroxypropyl-cyclodextrin (Sigma/Research Biochemicals International) in sterile distilled water. M [R-( )- (2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol] (Hoechst Marion Roussel, Cincinnati, OH) was prepared in a solution of 1% Tween 80 (Sigma, St. Louis, MO) in sterile distilled water. All drugs were injected in a volume of 1 ml/kg. Measurement of Locomotor Activity Apparatus. Locomotor activity was monitored and quantified using an open field activity system (San Diego Instruments, San Diego, CA). Each clear Plexiglas chamber ( cm) was housed within sound-attenuating enclosures and was surrounded with a 4 4 photobeam matrix located 4 cm from the floor surface. Interruptions of the photobeams resulted in counts of activity in the peripheral and central fields of the chamber. Activity recorded in the inner cm of the open field was counted as central activity, whereas the field bounded by the outer 16-cm band registered peripheral activity. Another horizontal row of 16 photobeams, located 16 cm from the floor surface, provided each chamber with a measurement of vertical activity. Separate counts of peripheral, central, and vertical activity were made by the control software (Photobeam Activity Software; San Diego Instruments) and stored for subsequent statistical evaluation. Peripheral and central activity counts were summed to provide a single measure of total horizontal activity. Video cameras positioned above the chambers permitted continuous observation of behavior without disruption. Behavioral Protocols. All rats were maintained in the colony room for a minimum of 1 week before behavioral testing for acclimation to daily handling procedures. All rats were habituated to the test environment for 3 h/day on each of the 2 days before the start of the experiment. On each of the test days, rats were habituated to the activity monitors for 1 h before administration of drugs. Using a repeated measures design, one group of rats (n 8) was pretreated with an injection of vehicle (1 ml/kg i.p.) or M (0.02, 0.2, or 2 mg/kg i.p.), followed 45 min later by an injection of saline (1 ml/kg i.p.) or cocaine (10 mg/kg i.p.). Using the same design, another group of rats (n 6) was pretreated with an injection of vehicle (1 ml/kg i.p.) or ketanserin (0.04, 0.4, or 4 mg/kg i.p.), followed 45 min later by an injection of saline (1 ml/kg i.p.) or cocaine (10 mg/kg i.p.). Measurement of locomotor activity counts began immediately following injection of saline or cocaine and was divided into 5-min bins for a total of 1 h. Test sessions were conducted every other day, and the order of drug tests was counterbalanced with the caveat that cocaine was administered no more than once per week. Data Analysis. Locomotor activity was totaled across the 60-min session and analyzed as horizontal (peripheral central activity counts) and vertical activity counts. Horizontal and vertical activity was analyzed using a two-way ANOVA for repeated measures for the factors of pretreatment (0, 0.02, 0.2, or 2 mg/kg M or 0, 0.04, 0.4, or 4 mg/kg ketanserin), treatment (0 or 10 mg/kg cocaine), and the pretreatment treatment interaction (SAS for Windows, version 6.12). Horizontal and vertical activity was also divided into four separate 15-min time bins and analyzed with a three-way ANOVA with time as a repeated measures factor to examine the pretreatment treatment time interaction. The Student-Newman-Keuls

3 procedure was used to analyze preplanned, pairwise comparisons; all comparisons were conducted with an experimentwise ( EW ) equal to Drug Discrimination Assays Apparatus. The procedures were conducted in commercially available, two-lever operant chambers (model 80001; Lafayette Instrument, Lafayette, IN). Each chamber was equipped with a waterfilled dispenser mounted equidistant between two response levers on one wall, and was housed in a light- and sound-attenuating shell (model 80015; Lafayette Instrument). Illumination was provided by a 28-V house light; ventilation and masking noise were supplied by a blower. An interface (MedAssociates, St. Albans, VT) connected the chambers to a computer, which controlled and recorded all experimental events. Procedures. Before the initiation of the errorless training phase, the rats were trained to discriminate an injection of cocaine (10 mg/kg i.p.) from saline (1 ml/kg i.p.) given 15 min before daily (Monday through Friday) 30-min sessions. Training began under a schedule of continuous water reinforcement (FR 1) with only the stimulus-appropriate (cocaine or saline) lever present ( errorless training); the schedule of reinforcement was increased until each rat was responding reliably under an FR 20 schedule for each experimental condition. For half of the rats, left-lever responses were reinforced after cocaine administration, whereas right-lever responses were reinforced after saline administration; conditions were reversed for the remaining rats. During this phase of training, cocaine and saline were administered irregularly with the restriction that neither condition prevailed for more than three consecutive sessions. After responding stabilized, both levers were presented simultaneously during 15-min sessions. The rats were required to respond on the stimulus-appropriate (correct) lever to obtain water reinforcement, and there were no programmed consequences for responding on the incorrect lever. This phase of training continued until performance for all rats attained criterion (defined as mean accuracies of at least 80% correct for 10 consecutive sessions). Cocaine dose-response and combination tests were then initiated; test sessions were conducted once or twice per week with training sessions intervening during the remaining days. Rats maintained accuracies of at least 80% correct for the saline and cocaine maintenance sessions, which immediately preceded a test. In dose-response tests, rats were tested for lever selection after the administration of various doses of cocaine. In combination tests, rats were given an injection of a dose of M ( mg/kg i.p.) or ketanserin (0.5 4 mg/kg i.p.) before an injection of 5 mg/kg cocaine. During test sessions, rats were placed in the chamber as during training sessions except that, upon completion of 20 responses on either lever, the animal received a single (water) reinforcer, the house light was turned off, and the rat was removed from the chamber. Sessions were terminated after 15 min if rats did not complete 20 responses on either lever. After returning to the colony, all rats were allowed a 15-min access to water. Data Analysis. Accuracy was defined as the percentage of correct responses to total responses before the delivery of the first reinforcer. During test sessions, performance was expressed as the percentage of drug-lever responses to total responses upon completion of an FR 20 on either lever. The response rate (responses per min) was calculated as the total number of responses emitted before completion of the first FR 20 divided by the number of minutes taken to complete the first ratio. For cocaine dose-response tests, Student s t test for repeated measures was used to compare the percentage of cocaineappropriate responding and response rates during test sessions with the previous cocaine session. For combination tests, Student s t test for repeated measures was used to compare performance following a fixed dose of cocaine (5 mg/kg) and that following the comparable dose of cocaine plus various doses of M or ketanserin. Only data from animals that completed the FR 20 during tests were 5-HT 2A Receptors and Cocaine Behaviors 359 included for analysis. Log-probit analyses were used to estimate the dose of cocaine predicted to elicit 50% cocaine-appropriate responding (ED 50 ), the doses of M or ketanserin to decrease cocaineappropriate responding by 50% (AD 50 ), and the 95% confidence limits for each (Tallarida and Murray, 1987). All statistical analyses were conducted with EW Results Effects of M on Cocaine-Induced Hyperactivity. A significant main effect of pretreatment (F 3, , p 0.001), treatment (F 1, , p 0.001), and a pretreatment treatment interaction (F 3, , p 0.05) were observed for total horizontal activity summed across the 60-min session. Pretreatment with M at each dose significantly attenuated cocaine-induced increases in horizontal activity (p 0.05), with cocaine-induced horizontal activity after 0.2 mg/kg M not differing significantly from activity observed in vehicle-saline controls (p 0.05; Fig. 1A). A significant pretreatment treatment time interaction was observed for horizontal activity divided into four separate 15-min time bins (F 9, , p 0.05). Fig. 1. Horizontal activity following pretreatment with the 5-HT 2A R antagonist M A, mean total horizontal activity (counts/60 min) ( S.E.M.) is depicted following i.p. injection of vehicle (Veh) or M (M100; 0.02, 0.2, or 2 mg/kg) followed by saline or cocaine injection (Coc; 10 mg/kg). *Activity levels that were significantly different from vehiclesaline controls; activity levels that were significantly different from vehicle-cocaine controls based on a preplanned comparison using Student-Newman-Keuls procedure (p 0.05). B, time course of horizontal activity plotted in 15-min bins across the 60-min test session is depicted following i.p. injection of vehicle or M (0.02, 0.2, or 2 mg/kg) followed by saline (open symbols) or cocaine injection (10 mg/kg; closed symbols). **Activity levels that were significantly different from vehiclecocaine controls at the corresponding time point based upon a Student- Newman-Keuls procedure (p 0.05).

4 360 McMahon and Cunningham Cocaine-induced increases in horizontal activity were significantly attenuated by each dose of M during the first and second 15-min intervals, whereas only 0.2 or 2 mg/kg M significantly attenuated cocaine-induced horizontal activity during the third 15-min interval (p 0.05; Fig. 1B). None of the doses of M significantly altered cocaine-induced horizontal activity during the fourth 15-min interval (p 0.05). M ( mg/kg) did not significantly alter spontaneous activity after saline injection at any time point (p 0.05). A significant main effect of pretreatment (F 3, , p 0.05), treatment (F 1,7 5.62, p 0.05), and a pretreatment treatment interaction (F 3, , p 0.05) was observed for total vertical activity summed across the 60-min session (Fig. 2). Pretreatment with 0.2 or 2 mg/kg M significantly attenuated cocaine-induced increases in vertical activity (p 0.05) to levels that were not significantly different from vehicle-saline controls (p 0.05; Fig. 2). The pretreatment treatment time interaction was not significant for vertical activity divided into four separate 15-min time bins (F 9, , p 0.05; data not shown). Effects of Ketanserin on Cocaine-Induced Hyperactivity. A significant main effect of pretreatment (F 3, , p 0.001), treatment (F 1, , p 0.01), and a pretreatment treatment interaction (F 3, , p 0.01) were observed for total horizontal activity summed across the 60-min session. Pretreatment with 0.4 or 4 mg/kg ketanserin significantly attenuated cocaine-induced increases in horizontal activity (p 0.05), with cocaine-induced horizontal activity after 4 mg/kg ketanserin not differing significantly from activity observed in vehicle-saline controls (p 0.05; Fig. 3A). The pretreatment treatment time interaction was not significant for horizontal activity divided into four separate 15-min time bins (F 9, , p 0.05; data not shown). A significant main effect of pretreatment (F 3, , p 0.05), but not treatment (F 1,5 3.83, p 0.05), was observed for total vertical activity summed across the 60-min session. A significant pretreatment treatment interaction (F 3, , p 0.05) was also observed for total vertical activity. Fig. 2. Vertical activity following pretreatment with the 5-HT 2A R antagonist M Mean total vertical activity (counts/60 min) ( S.E.M.) is depicted following i.p. injection of vehicle (Veh) or M (M100; 0.02, 0.2, or 2 mg/kg) followed by saline or cocaine injection (Coc; 10 mg/kg). *Activity levels that were significantly different from vehicle-saline controls; activity levels that were significantly different from vehicle-cocaine controls based on a preplanned comparison using Student-Newman-Keuls procedure (p 0.05). Fig. 3. Horizontal and vertical activity following pretreatment with the 5-HT 2 R antagonist ketanserin. See legends to Figs. 1A and 2 for explanation of the figures. Pretreatment with 0.4 or 4 mg/kg ketanserin significantly attenuated cocaine-induced increases in vertical activity (p 0.05) to levels that were not significantly different from vehicle-saline controls (p 0.05; Fig. 3B). The pretreatment treatment time interaction was not significant for vertical activity divided into four separate 15-min time bins (F 9, , p 0.05; data not shown). Effects of M or Ketanserin on the Stimulus Effects of Cocaine. The mean number of sessions required to meet the cocaine versus saline discrimination criterion was 13 (range 10 22). During dose-response tests, cocaine ( mg/kg) produced a dose-related increase in cocaine-appropriate responding, whereas saline administration resulted in 10% cocaine-appropriate responding (data not shown). Drug-appropriate responding after cocaine doses of 0.625, 1.25, and 2.5 mg/kg was significantly different from the previous cocaine session (p 0.05; data not shown). The dose of cocaine predicted to elicit 50% cocaine-lever responding (ED 50 ) was 2.2 mg/kg (95% CI, mg/kg). Response rates after cocaine ( mg/kg) did not differ significantly from the previous cocaine maintenance session (p 0.05; data not shown). In combination tests, administration of M ( mg/kg) resulted in a dose-dependent reduction of the percentage of cocaine-appropriate responding produced by a dose of cocaine that completely substituted (97% cocaine-lever responding) for the training dose when tested alone (Fig. 4, left and middle). The dose of M predicted to antagonize

5 Fig. 4. Results of combination tests with the 5-HT 2A R antagonist M or the 5-HT 2 R antagonist ketanserin plus 5 mg/kg cocaine. Closed symbols denote the mean percentage of cocaine-appropriate responses ( S.E.M.; left ordinate); open symbols denote the mean response rate per minute ( S.E.M.; right ordinate). M data points represent the mean values from 13/13 or 14/14 rats; ketanserin data points represent the mean values from 8/8 rats [n/n, number of subjects (n) completing at least 20 responses out of the number of subjects tested (N)]. *Performances during test sessions that were significantly different from cocaine (5 mg/kg) administered alone (p 0.05). For comparison, mean percentage of cocaine-appropriate responses ( S.E.M.; f) and mean response rate per minute ( S.E.M.; ) are depicted after cocaine (5 mg/kg) alone. the cocaine response by 50% (AD 50 ) was 0.25 mg/kg (95% CI, mg/kg). Doses of 0.8 or 1.6 mg/kg of M in combination with cocaine (5 mg/kg) significantly suppressed response rates compared with cocaine (5 mg/kg) alone (p 0.05). Similarly, ketanserin dose dependently reduced the percentage of cocaine-appropriate responding produced by 5 mg/kg cocaine (Fig. 4, right). The AD 50 for ketanserin was 1.48 mg/kg (95% CI, mg/kg). Each dose of ketanserin administered in combination with cocaine (5 mg/kg) significantly suppressed response rates compared with cocaine (5 mg/kg) alone (p 0.05). Discussion The 5-HT 2A R antagonist M and nonselective 5-HT 2 R antagonist ketanserin significantly attenuated hyperactivity induced by cocaine, in keeping with previous observations (Herges and Taylor, 1998; O Neill et al., 1999). Because ketanserin discriminates poorly among 5-HT 2A, 5-HT 2C, and 1 -adrenergic receptors (Yagaloff and Hartig, 1985; Dudley et al., 1988), the reduction of cocaine-induced hyperactivity by ketanserin may reflect actions at any of these receptor subtypes. Suppression of cocaine-induced hyperactivity has been observed following pretreatment with the 5-HT 2B/2C R antagonist SB (McCreary and Cunningham, 1999) and the 1 -adrenergic receptor antagonist prazosin (Snoddy and Tessel, 1985); however, the efficacious blockade afforded by low doses of M100907, which has a 100-fold higher affinity for 5-HT 2A over 5-HT 2C and 1 -adrenergic receptors, supports the hypothesis that antagonism of 5-HT 2A R may account in whole (M100907) or part (ketanserin) for the attenuation of the motor stimulant properties of cocaine. This argument is further reinforced by the finding that M100907, at doses up to 30 times higher than those used here, did not antagonize the behavioral effects of 5-HT 2C R, D 2 -like, and 1 -adrenergic agonists (Kehne et al., 1996a; Dekeyne et al., 1999). 5-HT 2A Receptors and Cocaine Behaviors 361 This effective antagonism of cocaine-stimulated hyperactivity afforded by M and ketanserin occurred in the absence of altered basal activity seen after the same doses of M or ketanserin administered alone. Because activity measurements were obtained from rats well habituated to the test environment, the argument can be made that the resultant low levels of spontaneous activity may render this baseline insensitive to potential behavioral suppression due to administration of M or ketanserin alone, and thus, raise suspicion concerning the specificity of the antagonism. However, behaviorally suppressive effects of M (1 mg/ kg) have not been noted in unhabituated rats tested in our activity monitors (P. S. Frankel and K. A. Cunningham, unpublished data); this finding is consistent with observations that appreciable sedation is seen only at much higher doses of M ( 8 mg/kg; Sorensen et al., 1993; Kehne et al., 1996a,b; but see Gleason and Shannon, 1997). Similarly, we noted no statistically significant effect of ketanserin on basal activity, although a disruptive effect of this compound at 3 mg/kg has been observed in mice with little prior exposure to the activity monitor (Gleason and Shannon, 1997), but not in acclimatized rats (Wing et al., 1990). Thus, although modest behaviorally suppressant effects of M and ketanserin may be associated with systemic injection, 5-HT 2A R antagonism appears to evoke significantly less sedation than that associated with DA or 1 -adrenergic receptor antagonism (Jackson et al., 1994; Mathe et al., 1996). The present study revealed that M (ED mg/kg) and ketanserin (ED mg/kg) effectively antagonized the stimulus properties of cocaine. This finding is in keeping with the observation that ketanserin (and a similar nonselective 5-HT 2 R antagonist, ritanserin) attenuated the cocaine cue in squirrel monkeys (Schama et al., 1997), although ritanserin did not alter the stimulus properties of cocaine in rats (Meert and Janssen, 1992; Peltier et al., 1994). The inconsistencies in the ability of 5-HT 2 antagonists to block the stimulus effects of cocaine are likely to be related to the varying affinity profiles of these ligands for monoamine receptors, particularly given the difficulty in predicting the exact profile of receptor occupancy after systemic administration. However, the efficacy of M to block the stimulus effects of cocaine in the face of its selective receptor affinity profile and the failure of this ligand to antagonize the behavioral effects of 5-HT 2C,D 2 -like, and 1 - adrenergic receptor agonists (Kehne et al., 1996a; Dekeyne et al., 1999) provide the most compelling evidence to date in support for a modulatory role of 5-HT 2A R in the discriminative stimulus properties of cocaine. Furthermore, in contrast to the overt behavioral suppression evident at doses of the classical DA antagonists that block the stimulus effects of cocaine (Callahan et al., 1991), M effectively attenuated the cocaine cue in the absence of robust changes in response rates. Thus, selective antagonism of 5-HT 2A R with M appears to be an effective strategy for attenuating the behavioral effects of cocaine in the absence of prominent sedative effects. The lack of overt changes in basal behavior upon administration of M may reflect the relative absence of a tonic regulatory influence of 5-HT 2A R on DA function, a hypothesis supported by other neuropharmacological investigations. For example, systemic administration of 5-HT 2A R antagonists did not alter basal levels of DA cell firing (Sorensen et al.,

6 362 McMahon and Cunningham 1993) or striatal (Schmidt et al., 1992) and accumbal DA efflux (De Deurwaerdere and Spampinato, 1999). Furthermore, striatal DA efflux was not evoked by coperfusion of the 5-HT 2A/2B/2C R agonist 1-(2,5-dimethoxy-4-iodo)-2-aminopropane (DOI) and the 5-HT 2B/2C R antagonist SB , supporting the concept that sole stimulation of 5-HT 2A Risnot sufficient to enhance DA efflux (Lucas and Spampinato, 2000). However, under conditions of DA stimulation, the 5-HT 2A R does appear to positively modulate DA outflow. For example, antagonism of 5-HT 2A R has been shown to attenuate striatal DA efflux stimulated by systemic administration of ( )-3,4-methylenedioxymethamphetamine (MDMA; Schmidt et al., 1992), blockade of D 2 -like autoreceptors with haloperidol (Lucas and Spampinato, 2000), and electrical stimulation of the dorsal raphe nucleus (De Deurwaerdere and Spampinato, 1999). Despite the evidence to suggest that the 5-HT 2A R exercises little tonic control over DA function under normal conditions (see above), basal 5-HT concentrations may provide sufficient tone on 5-HT 2A R such that, under conditions of stimulated DA function, antagonism of 5-HT 2A R triggers functional mechanisms to compensate for the overactivation of DA neurons. Such a mechanism might help to explain why M blocks the in vivo consequences of drugs thought to predominantly enhance DA efflux, such as amphetamine (Sorensen et al., 1993; Moser et al., 1996) and the DA reuptake inhibitor GBR (Carlsson, 1995). On the other hand, further increases in interstitial levels of 5-HT, such as the case following cocaine (present results) or the 5-HT- and DA-releaser MDMA (Schmidt et al., 1992; Kehne et al., 1996b), may be required to uncover a 5-HT 2A R control of stimulated DA neurotransmission. In this case, one could postulate that amphetamine and GBR might alter interstitial levels of 5-HT, possibly below the limits of detectability in some microdialysis experiments, that might contribute to the ability of 5-HT 2A R to control DA function. To complicate matters further, in addition to 5-HT-evoked increases in DA efflux (Benloucif and Galloway, 1991; Steward et al., 1996; De Deurwaerdere and Spampinato, 1999; Lucas and Spampinato, 2000), DA has also been shown to increase 5-HT release (Matsumoto et al., 1996) and these effects appear to be mediated, at least in part, by stimulation of specific 5-HT and DA receptors, respectively. The mechanism of action for M to control DA function has been suggested to involve blockade of 5-HT 2A R, which putatively controls DA synthesis under conditions of stimulated DA neurotransmission (Schmidt et al., 1992, 1995; Lucas and Spampinato, 2000). This hypothesis is based upon the fact that the DA precursor l-dihydroxyphenylalanine reversed the inhibitory actions of M on amphetamine-evoked suppression of DA cell firing (Sorensen et al., 1992) and MDMA-induced DA synthesis (Schmidt et al., 1995) and is further strengthened by the reported ineffectiveness of M to reduce hyperactivity induced by direct stimulation of postsynaptic DA receptors (Carlsson, 1995; O Neill et al., 1999). However, this hypothesis is most likely too simplistic to encompass the ability of M to block cocaine-evoked behavioral effects because cocaine inhibits DA reuptake (Koe, 1976) and decreases DA synthesis for a duration encompassing the behavioral effects of cocaine reported here, presumably via increased DA acting upon D 2 - like synthesis-modulating autoreceptors (Galloway, 1990). This also appears to be the case for GBR (Nissbrandt et al., 1991). Thus, blockade of a facilitatory role of 5-HT 2A R on DA synthesis is unlikely to account for the suppressive effect of M on cocaine- (and GBR 12909)-induced behavior. Despite this incongruity, intracranial microinjection studies from our laboratory show that the ventral tegmental area, but not the nucleus accumbens shell, is a critical locus of action for M to block the systemic effects of cocaine (L. R. McMahon, M. Filip, and K. A. Cunningham, submitted). Thus, we propose that other mechanisms of action, in addition to the control of DA synthesis ascribed to 5-HT 2A R, contribute to 5-HT 2A R-mediated control of DA neurotransmission and that actions at the level of the DA cell body are particularly critical to the interaction between 5-HT 2A R and DA systems. In summary, the present study is the first to demonstrate an effective antagonism of the hypermotive and discriminative stimulus effects of cocaine by the 5-HT 2A R antagonist M100907; similar results were obtained with the 5-HT 2 R antagonist ketanserin. M appeared to selectively attenuate cocaine-induced behavior at doses that did not elicit overt changes in basal behavior. These results suggest that the 5-HT 2A R plays an important role in the behavioral effects of cocaine and that 5-HT 2A R should be considered a viable target for analysis in the search for pharmacotherapies for the treatment of cocaine dependence, particularly in light of a potentially more acceptable side effect profile presented by M than DA receptor antagonists. Acknowledgments We thank Lauren Lively for providing technical assistance and Michael Bankson for helpful editorial comments on this manuscript. References Baxter G, Kennett G, Blaney F and Blackburn T (1995) 5-HT 2 receptor subtypes: A family re-united? Trends Pharmacol Sci 16: Benloucif S and Galloway MP (1991) Facilitation of dopamine release in vivo by serotonin agonists: Studies with microdialysis. Eur J Pharmacol 200:1 8. Callahan PM, Appel JB and Cunningham KA (1991) Dopamine D-1 and D-2 mediation of the discriminative stimulus properties of d-amphetamine and cocaine. Psychopharmacology 103: Callahan PM and Cunningham KA (1995) Modulation of the discriminative stimulus properties of cocaine by 5-HT 1B and 5-HT 2C receptors. J Pharmacol Exp Ther 274: Carlsson ML (1995) The selective 5-HT 2A receptor antagonist MDL counteracts the psychomotor stimulation ensuing manipulations with monoaminergic, glutamatergic, or muscarinic neurotransmission in the mouse implications for psychosis. J Neural Transm 100: Cornea-Hebert V, Riad M, Wu C, Singh SK and Descarries L (1999) Cellular and subcellular distribution of the serotonin 5-HT 2A receptor in the central nervous system of adult rat. J Comp Neurol 409: De Deurwaerdere P and Spampinato U (1999) Role of serotonin 2A and serotonin 2C/2B receptor subtypes in the control of accumbal and striatal dopamine release elicited in vivo by dorsal raphe nucleus electrical stimulation. J Neurochem 73: Dekeyne A, Girardon S and Millan MJ (1999) Discriminative stimulus properties of the novel serotonin (5-HT) 5-HT2C receptor agonist, RO : A pharmacological analysis. Neuropharmacology 38: Doherty MD and Pickel VM (2000) Ultrastructural localization of the serotonin 2A receptor in dopaminergic neurons in the ventral tegmental area. Brain Res 864: Dudley MW, Wiech NL, Miller FP, Carr AA, Cheng HC, Roebel LE, Doherty NS, Yamamura HI, Ursillo RC and Palfreyman MG (1988) Pharmacological effects of MDL 11,939: A selective, centrally acting antagonist of 5-HT 2 receptors. Drug Dev Res 13: Galloway MP (1990) Regulation of dopamine and serotonin synthesis by acute administration of cocaine. Synapse 6: Gleason SD and Shannon HE (1997) Blockade of phencyclidine-induced hyperlocomotion by olanzapine, clozapine, and serotonin receptor subtype selective antagonists in mice. Psychopharmacology 129: Herges S and Taylor DA (1998) Involvement of serotonin in the modulation of cocaine-induced locomotor activity in the rat. Pharmacol Biochem Behav 59: Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylechrane EJ, Sazena PR and Humphrey PPA (1994) VII. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol Rev 46: Howell LL and Byrd LD (1995) Serotonergic modulation of the behavioral effects of cocaine in the squirrel monkey. J Pharmacol Exp Ther 275:

7 5-HT 2A Receptors and Cocaine Behaviors 363 Ikemoto K, Nishimura A, Okado N, Mikuni M, Nishi K and Nagatsu I (2000) Human midbrain dopamine neurons express serotonin 2A receptor: An immunohistochemical demonstration. Brain Res 853: Jackson DM, Johansson C, Lindgren L-M and Bengtsson A (1994) Dopamine receptor antagonists block amphetamine and phencyclidine-induced motor stimulation in rats. Pharmacol Biochem Behav 48: Kehne JH, Baron BM, Carr AA, Chaney SF, Elands J, Feldman DJ, Frank RA, van Giersbergen PLM, McCloskey TC, Johnson MP, et al. (1996a) Preclinical characterization of the potential of the putative atypical antipsychotic MDL as a potent 5-HT 2A antagonist with a favorable CNS safety profile. J Pharmacol Exp Ther 277: Kehne JH, Ketteler HJ, McCloskey TC, Sullivan CK, Dudley MW and Schmidt CJ (1996b) Effects of the selective 5-HT 2A antagonist MDL on MDMA-induced locomotor stimulation in rats. Neuropsychopharmacology 15: Klein M (1998) Research issues related to development of medications for treatment of cocaine addiction. Ann NY Acad Sci 844: Koe BK (1976) Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J Pharmacol Exp Ther 199: Lacosta S and Roberts DCS (1993) MDL 72222, ketanserin, and methysergide pretreatments fail to alter breaking points on a progressive ratio schedule reinforced by intravenous cocaine. Pharmacol Biochem Behav 44: Lopez-Gimenez JF, Mengod G, Palacios JM and Vilaro MT (1997) Selective visualization of rat brain 5-HT 2A receptors by autoradiography with [ 3 H]MDL Naunyn-Schmiedeberg s Arch Pharmacol 356: Lucas G and Spampinato U (2000) Role of striatal serotonin 2A and serotonin 2C receptor subtypes in the control of in vivo dopamine outflow in the rat striatum. J Neurochem 74: Mathe JM, Nomikos GG, Hildebrand BE, Hertel P and Svensson TH (1996) Prazosin inhibits MK-801-induced hyperlocomotion and dopamine release in the nucleus accumbens. Eur J Pharmacol 309:1 11. Matsumoto M, Yoshioka M, Togashi H, Ikeda T and Saito H (1996) Functional regulation by dopamine receptors of serotonin release from the rat hippocampus: In vivo microdialysis study. Naunyn-Schmiedeberg s Arch Pharmacol 353: McCreary AC and Cunningham KA (1999) Effects of the 5-HT 2C/2B antagonist SB on hyperactivity induced by cocaine. Neuropsychopharmacology 20: McMahon LR and Cunningham KA (1999) Antagonism of 5-hydroxytryptamine 4 receptors attenuates hyperactivity induced by cocaine: Putative role for 5- hydroxytryptamine 4 receptors in the nucleus accumbens shell. J Pharmacol Exp Ther 291: Meert TF and Janssen PAJ (1992) Ritanserin, a new therapeutic approach for drug abuse. Part 2: Effects on cocaine. Drug Dev Res 25: Moser PC, Moran PM, Frank RA and Kehne JH (1996) Reversal of amphetamineinduced behaviours by MDL 100,907, a selective 5-HT 2A antagonist. Behav Brain Res 73: Nader MA and Barrett JE (1990) Effects of chlordiazepoxide, buspirone, and serotonin receptor agonists and antagonists on responses of squirrel monkeys maintained under second-order schedules of intramuscular cocaine injection or food presentation. Drug Dev Res 20:5 17. Nissbrandt H, Engberg G and Pileblad E (1991) The effects of GBR 12909, a dopamine re-uptake inhibitor, on monoaminergic neurotransmission in rat striatum, limbic forebrain, cortical hemispheres and substantia nigra. Naunyn- Schmiedeberg s Arch Pharmacol 344: O Neill MF, Heron-Maxwell CL and Shaw G (1999) 5-HT 2 receptor antagonism reduces hyperactivity induced by amphetamine, cocaine, and MK-801 but not D 1 agonist C-APB. Pharmacol Biochem Behav 63: Peltier RL, Emmett-Oglesby MW, Thomas WH and Schenk S (1994) Failure of ritanserin to block the discriminative or reinforcing stimulus effects of cocaine. Pharmacol Biochem Behav 48: Schama KF, Howell LL and Byrd LD (1997) Serotonergic modulation of the discriminative-stimulus effects of cocaine in squirrel monkeys. Psychopharmacology 132: Schmidt CJ, Fadayel GM, Sullivan CK and Taylor VL (1992) 5-HT 2 receptors exert a state-dependent regulation of dopaminergic function: Studies with MDL and the amphetamine analogue, 3,4-methylenedioxymethamphetamine. Eur J Pharmacol 223: Schmidt CJ, Sorensen SM, Kehne JH, Carr AA and Palfreyman MG (1995) The role of 5-HT 2A receptors in antipsychotic activity. Life Sci 56: Snoddy AM and Tessel RE (1985) Prazosin: Effect on psychomotor-stimulant cues and locomotor activity in mice. Eur J Pharmacol 116: Sorensen SM, Humphreys TM, Taylor VL and Schmidt CJ (1992) 5-HT 2 antagonists reverse amphetamine-induced slowing of dopaminergic neurons by interfering with stimulated dopamine synthesis. J Pharmacol Exp Ther 260: Sorensen SM, Kehne JH, Fadayel GM, Humphreys TM, Ketteler HJ, Sullivan CK, Taylor VL and Schmidt CJ (1993) Characterization of the 5-HT 2 receptor antagonist MDL as a putative atypical antipsychotic: Behavioral, electrophysiological, and neurochemical studies. J Pharmacol Exp Ther 266: Spealman RD, Bergman J, Madras BK, Kamien JB and Melia KF (1992) Role of D 1 and D 2 dopamine receptors in the behavioral effects of cocaine. Neurochem Int 20:S147 S152. Steward, L.J., et al. (1996) Ability of 5-HT 4 receptor ligands to modulate rat striatal dopamine release in vitro and in vivo. Br J Pharmacol 117: Tallarida RJ and Murray RB (1987) Manual of Pharmacological Calculations with Computer Programs. Springer Verlag, New York. Walsh SL and Cunningham KA (1997) Serotonergic mechanisms involved in the discriminative stimulus, reinforcing and subjective effects of cocaine. Psychopharmacology 130: Wing LL, Tapson GS and Geyer MA (1990) 5-HT-2 mediation of acute behavioral effects of hallucinogens in rats. Psychopharmacology 100: Yagaloff KA and Hartig PR (1985) 125 I-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells. J Neurosci 5: Send reprint requests to: Kathryn A. Cunningham, Ph.D., Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX cunningham@utmb.edu