Abstract
Serotonin (5-hydroxytryptamine; 5-HT) 5-HT2A receptors have been shown to modulate dopamine (DA) function and a more thorough appreciation of this modulatory interaction between 5-HT2Areceptors 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-HT2Areceptors 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-HT2A receptor antagonist M100907 [R-(+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol] (0.02–2.0 mg/kg), or the 5-HT2 receptor antagonist ketanserin (0.04–4 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 M100907 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. M100907 (0.05–1.6 mg/kg) and ketanserin (0.05–4 mg/kg) evoked a dose-related attenuation of the stimulus effects of cocaine (5 mg/kg, p < 0.05). These results suggest that 5-HT2A receptors play an important role in the behavioral effects of cocaine and that 5-HT2Areceptors should be considered a viable target for analysis in the search for pharmacotherapies useful in the treatment of cocaine dependence.
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 D1- and D2-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 useful in facilitating treatment goals to maintain abstinence in a cocaine abuser.
Of the 14 5-HT receptor subtypes characterized to date, the 5-HT2 receptors (5-HT2Rs), including 5-HT2AR, 5-HT2BR, and 5-HT2CR, are products of different genes and exhibit differential anatomical localization (Hoyer et al., 1994). Stimulation of 5-HT2Rs, which are linked to phospholipase C, results in increased phosphatidyl inositol turnover, increased intracellular levels of Ca2+, and neuronal depolarization (Hoyer et al., 1994). The 5-HT2AR is synonymous with the classical 5-HT2R and has been implicated in hallucinosis, psychosis, and affective disorders (Baxter et al., 1995). The distributions of 5-HT2AR 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-HT2AR 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-HT2AR (Schmidt et al., 1992, 1995; Sorensen et al., 1992, 1993; De Deurwaerdere and Spampinato, 1999; Lucas and Spampinato, 2000), and therefore 5-HT2AR 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-HT2AR in the behavioral effects of cocaine have used ligands that do not effectively differentiate between 5-HT2R subtypes, and frequently possess affinity for other monoamine receptors such as α-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-HT2R antagonist ketanserin (Herges and Taylor, 1998; O'Neill et al., 1999) and the 5-HT2B/2CR antagonist SB 206553 (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-HT2R 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-HT2R in the hypermotive, stimulus and reinforcing effects of cocaine is suggested; however, the identification of roles for specific 5-HT2R subtypes has been hampered by the use of compounds with poor selectivity.
Although only a 2- to 10-fold selectivity of ketanserin for 5-HT2A (Ki = 3.2 nM) over either 5-HT2C(Ki = 28 nM) or α1-adrenergic receptors (Ki = 6.7 nM; Dudley et al., 1988;Yagaloff and Hartig, 1985) is evident, the more recently developed compound M100907 is an antagonist that possesses nanomolar affinity for 5-HT2AR (Ki = 0.85 nM), considerably less affinity for 5-HT2CR (Ki = 88 nM), and α1-adrenergic receptors (Ki = 128 nM), and negligible affinity for most other receptors, including DA D1- and D2-like receptors (>500 nM; Kehne et al., 1996a). Thus, M100907 presents a profile of >100-fold selectivity for the 5-HT2AR versus the 5-HT2CR or α1-adrenergic receptor. The aim of the present study was to more thoroughly analyze the possible role of 5-HT2AR in the in vivo effects of cocaine by directly comparing the abilities of the nonselective 5-HT2R antagonist ketanserin and the selective 5-HT2AR antagonist M100907 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. M100907 [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 (40 × 40 × 40 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 16 × 16 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 M100907 (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 M100907 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 procedure was used to analyze preplanned, pairwise comparisons; all comparisons were conducted with an experimentwise α (αEW) equal to 0.05.
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 water-filled 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 M100907 (0.05–1.6 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'st test for repeated measures was used to compare the percentage of cocaine-appropriate 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 M100907 or ketanserin. Only data from animals that completed the FR 20 during tests were included for analysis. Log-probit analyses were used to estimate the dose of cocaine predicted to elicit 50% cocaine-appropriate responding (ED50), the doses of M100907 or ketanserin to decrease cocaine-appropriate responding by 50% (AD50), and the 95% confidence limits for each (Tallarida and Murray, 1987). All statistical analyses were conducted with αEW = 0.05.
Results
Effects of M100907 on Cocaine-Induced Hyperactivity.
A significant main effect of pretreatment (F3,21 = 8.61, p < 0.001), treatment (F1,7 = 289.58,p < 0.001), and a pretreatment × treatment interaction (F3,21 = 4.06,p < 0.05) were observed for total horizontal activity summed across the 60-min session. Pretreatment with M100907 at each dose significantly attenuated cocaine-induced increases in horizontal activity (p < 0.05), with cocaine-induced horizontal activity after 0.2 mg/kg M100907 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 (F9,63 = 2.06, p < 0.05). Cocaine-induced increases in horizontal activity were significantly attenuated by each dose of M100907 during the first and second 15-min intervals, whereas only 0.2 or 2 mg/kg M100907 significantly attenuated cocaine-induced horizontal activity during the third 15-min interval (p < 0.05; Fig. 1B). None of the doses of M100907 significantly altered cocaine-induced horizontal activity during the fourth 15-min interval (p > 0.05). M100907 (0.02–2 mg/kg) did not significantly alter spontaneous activity after saline injection at any time point (p > 0.05).
A significant main effect of pretreatment (F3,21 = 4.22, p < 0.05), treatment (F1,7 = 5.62,p < 0.05), and a pretreatment × treatment interaction (F3,21 = 3.22,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 M100907 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 (F9,63 = 1.36, p > 0.05; data not shown).
Effects of Ketanserin on Cocaine-Induced Hyperactivity.
A significant main effect of pretreatment (F3,15 = 8.28, p < 0.001), treatment (F1,5 = 21.05,p < 0.01), and a pretreatment × treatment interaction (F3,15 = 6.00,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 (F9,45 = 1.59,p > 0.05; data not shown).
A significant main effect of pretreatment (F3,15 = 8.28, p < 0.05), but not treatment (F1,5 = 3.83,p > 0.05), was observed for total vertical activity summed across the 60-min session. A significant pretreatment × treatment interaction (F3,15 = 3.42,p < 0.05) was also observed for total vertical activity. 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 (F9,45 = 0.97,p > 0.05; data not shown).
Effects of M100907 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 (0.625–10 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 (ED50) was 2.2 mg/kg (95% CI, 0.8–6.1 mg/kg). Response rates after cocaine (0.625–10 mg/kg) did not differ significantly from the previous cocaine maintenance session (p > 0.05; data not shown).
In combination tests, administration of M100907 (0.05–1.6 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 M100907 predicted to antagonize the cocaine response by 50% (AD50) was 0.25 mg/kg (95% CI, 0.14–0.43 mg/kg). Doses of 0.8 or 1.6 mg/kg of M100907 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 AD50 for ketanserin was 1.48 mg/kg (95% CI, 0.87–2.51 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-HT2AR antagonist M100907 and nonselective 5-HT2R 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-HT2A, 5-HT2C, and α1-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-HT2B/2CR antagonist SB 206553 (McCreary and Cunningham, 1999) and the α1-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-HT2A over 5-HT2C and α1-adrenergic receptors, supports the hypothesis that antagonism of 5-HT2AR 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-HT2CR, D2-like, and α1-adrenergic agonists (Kehne et al., 1996a;Dekeyne et al., 1999).
This effective antagonism of cocaine-stimulated hyperactivity afforded by M100907 and ketanserin occurred in the absence of altered basal activity seen after the same doses of M100907 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 M100907 or ketanserin alone, and thus, raise suspicion concerning the specificity of the antagonism. However, behaviorally suppressive effects of M100907 (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 M100907 (>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 M100907 and ketanserin may be associated with systemic injection, 5-HT2AR antagonism appears to evoke significantly less sedation than that associated with DA or α1-adrenergic receptor antagonism (Jackson et al., 1994; Mathe et al., 1996).
The present study revealed that M100907 (ED50 = 0.25 mg/kg) and ketanserin (ED50 = 1.48 mg/kg) effectively antagonized the stimulus properties of cocaine. This finding is in keeping with the observation that ketanserin (and a similar nonselective 5-HT2R 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-HT2antagonists 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 M100907 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-HT2C, D2-like, and α1-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-HT2AR 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), M100907 effectively attenuated the cocaine cue in the absence of robust changes in response rates. Thus, selective antagonism of 5-HT2AR with M100907 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 M100907 may reflect the relative absence of a tonic regulatory influence of 5-HT2AR on DA function, a hypothesis supported by other neuropharmacological investigations. For example, systemic administration of 5-HT2AR antagonists did not alter basal levels of DA cell firing (Sorensen et al., 1993) or striatal (Schmidt et al., 1992) and accumbal DA efflux (De Deurwaerdere and Spampinato, 1999). Furthermore, striatal DA efflux wasnot evoked by coperfusion of the 5-HT2A/2B/2CR agonist 1-(2,5-dimethoxy-4-iodo)-2-aminopropane (DOI) and the 5-HT2B/2CR antagonist SB 206553, supporting the concept that sole stimulation of 5-HT2AR is not sufficient to enhance DA efflux (Lucas and Spampinato, 2000). However, under conditions of DA stimulation, the 5-HT2AR does appear to positively modulate DA outflow. For example, antagonism of 5-HT2AR has been shown to attenuate striatal DA efflux stimulated by systemic administration of (±)-3,4-methylenedioxymethamphetamine (MDMA; Schmidt et al., 1992), blockade of D2-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-HT2AR exercises little tonic control over DA function under normal conditions (see above), basal 5-HT concentrations may provide sufficient tone on 5-HT2AR such that, under conditions of stimulated DA function, antagonism of 5-HT2AR triggers functional mechanisms to compensate for the overactivation of DA neurons. Such a mechanism might help to explain why M100907 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 12909 (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-HT2AR control of stimulated DA neurotransmission. In this case, one could postulate that amphetamine and GBR 12909 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-HT2AR 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 M100907 to control DA function has been suggested to involve blockade of 5-HT2AR, 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 precursorl-dihydroxyphenylalanine reversed the inhibitory actions of M100907 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 M100907 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 M100907 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 D2-like synthesis-modulating autoreceptors (Galloway, 1990). This also appears to be the case for GBR 12909 (Nissbrandt et al., 1991). Thus, blockade of a facilitatory role of 5-HT2AR on DA synthesis is unlikely to account for the suppressive effect of M100907 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 M100907 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-HT2AR, contribute to 5-HT2AR-mediated control of DA neurotransmission and that actions at the level of the DA cell body are particularly critical to the interaction between 5-HT2AR 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-HT2AR antagonist M100907; similar results were obtained with the 5-HT2R antagonist ketanserin. M100907 appeared to selectively attenuate cocaine-induced behavior at doses that did not elicit overt changes in basal behavior. These results suggest that the 5-HT2AR plays an important role in the behavioral effects of cocaine and that 5-HT2AR 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 M100907 than DA receptor antagonists.
Acknowledgments
We thank Lauren Lively for providing technical assistance and Michael Bankson for helpful editorial comments on this manuscript.
Footnotes
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Send reprint requests to: Kathryn A. Cunningham, Ph.D., Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX 77555-1031. E-mail: cunningham{at}utmb.edu
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This research was supported by National Institute on Drug Abuse Grants DA 05708, DA 06511 (to K.A.C.), and DA 05879 (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).
- Abbreviations:
- DA
- dopamine
- 5-HT
- 5-hydroxytryptamine
- 5-HT2R
- 5-hydroxytryptamine2 receptor
- M100907
- [R-(+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol]
- FR
- fixed ratio
- MDMA
- (±)-3,4-methylenedioxymeth-amphetamine
- SB 206553
- N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b′)dipyrrole-1(2H)carboxamide
- Received September 6, 2000.
- Accepted January 10, 2001.
- The American Society for Pharmacology and Experimental Therapeutics