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antagonist modification of cocaine stimulant effects: implications for cocaine mechanisms
Behavioural Brain Research 132 (2002) 37 – 46 www.elsevier.com/locate/bbr
Research report
5-HT1A agonist/antagonist modification of cocaine stimulant effects: implications for cocaine mechanisms Robert J. Carey *, Gail De Palma, Ernest Damianopoulos VA Medical Center and SUNY Health Science Center, Research and De6elopment 800 Ir6ing A6enue, Syracuse, NY 13210, USA Received 30 June 2001; received in revised form 9 September 2001; accepted 9 September 2001
Abstract The 5-HT1A receptor site has been demonstrated to be an important pharmacological target in the modulation of unconditioned behavioral effects induced by cocaine. In this study, separate groups of rats (n= 7) received a series of the 5-HT1A agonist treatments, 8-OHDPAT (0.2, 0.4 mg/kg) in combination with saline or cocaine (10 mg/kg). Using a crossover design, the treatments were subsequently switched to the 5-HT1A antagonist, WAY 100635 (0.4, 0.8 mg/kg) and then, switched back again to 8-OHDPAT (0.2, 0.4 mg/kg). When the 8-OHDPAT was given in combination with cocaine, locomotion was substantially enhanced but when the treatment was switched to WAY 100635, the cocaine induced locomotion was suppressed. Neither the 8-OHDPAT or WAY 100635 given with saline affected locomotion as compared to saline treated animals. These findings indicated a reciprocal facilitatory/inhibitory influence of 5-HT1A agonists/antagonists upon cocaine induced locomotion. The 8-OHDPAT treatments, however, did not enhance all cocaine behavioral responses. Initially, 8-OHDPAT suppressed cocaine induced rearing and central zone entry, but with repeated treatments, these response suppression effects subsided. As a consequence, the facilitative influence of 8-OHDPAT upon cocaine induced locomotion could not be attributed to response redistribution effects. While WAY 100635 markedly reduced cocaine induced locomotion and rearing to nearly saline response levels, the same WAY 100635 treatments did not modify locomotor stimulant effects induced by caffeine (10 mg/kg). In that caffeine stimulant effects are not directly linked to serotonergic mechanisms, the absence of an influence of WAY 100635 upon caffeine induced locomotor stimulation lent further support to the proposition that the 5-HT1A receptor site contributes to locomotor behavior in situations where the serotonergic system is pharmacologically activated by drugs such as cocaine. These findings point to a potential role for 5-HT1A antagonists in treatment of cocaine abuse. Published by Elsevier Science B.V. Keywords: Cocaine; 8-OHDPAT; WAY 100635; Caffeine; Locomotion; Grooming; Rearing; Rat
1. Introduction Cocaine, as a potent inhibitor of catecholamine and indoleamine transport, can substantially increase extracellular dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NE) [35]. The relationship of these increases in catecholaminergic and serotonergic activity and the behavioral effects of cocaine have proven elusive. While there is considerable evidence to indicate * Corresponding author. Tel.: + 1-315-476-7461x2824; fax: +1315-476-5348. E-mail address: [email protected] (R.J. Carey). 0166-4328/02/$ - see front matter. Published by Elsevier Science B.V. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 3 8 3 - 7
that cocaine induced increases in dopamine in the nucleus accumbens (Nac) may have a critical role in the mediation of cocaine stimulant and reward effects [21,48], recent findings have obscured this connection. It has been [36,44]reported that knock-out mice lacking the DA transporter still self-administered cocaine. In addition, it has been found that selectivity for inhibition of DA transport is not a sufficient mechanism for a drug to induce locomotor stimulation [33,38]. Increasingly, evidence indicates that cocaine effects upon 5-HT transport also contribute to cocaine behavioral effects [1,7,34,47]. Recently, we have shown [4,6] that the 5-HT1A receptor antagonist, WAY 100635 can block
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the locomotor stimulant effects of low dose cocaine and conversely, that the 5-HT1A agonist, 8-OHDPAT can enhance the locomotor stimulant effects of cocaine [5,9]. The further observations [4,6] that the WAY 100635 and 8-OHDPAT treatments did not alter the behavioral baselines for locomotion or modify DA increases induced by cocaine points to a possible contribution of the 5-HT1A receptor site in the mediation of cocaine locomotor stimulant effects. The reciprocity between a 5-HT1A agonist and antagonist in terms of cocaine induced locomotion provides a compelling impetus for implicating the 5-HT1A system in cocaine stimulant effects. When behavior is examined more broadly, however, the interaction between 5-HT1A drug treatments and cocaine is more complex. 8-OHDPAT does not increase all facets of cocaine locomotor stimulant effects rather, it preferentially enhances locomotion at the periphery of an open-field and decreases rearing behaviors [9]. 8-OHDPAT given alone suppresses grooming behavior and increases corticosterone and can have dose related effects upon locomotor behavior [4,20,26,46]. WAY 100635, on the other hand, increases grooming but does not prevent the reduction in grooming behavior induced by cocaine [6]. In view of the important but complex interaction between 5-HT1A selective drug treatments and cocaine, the present study examines the impact of 8-OHDPAT upon cocaine induced behavioral effects in an openfield over a chronic treatment regimen in which multiple behavioral measurements are compiled. To test directly for possible reciprocity between 8-OHDPAT and WAY 100635 in their interaction with cocaine, a substitution test was conducted in which the 8-OHDPAT treatment is replaced with WAY 100635.The outcome of this investigation points to important changes in the 8-OHDPAT– cocaine interaction with chronic treatment and provides additional support for reciprocal effects of the 5-HT1A agonist, 8OHDPAT and antagonist, WAY 100635 on cocaine locomotor stimulant effects.
2. Method
2.1. Animals Naive male Sprague– Dawley rats from Taconic Farms (Germantown, NY) were used. The animals were 4 months old and weighed approximately 400 g at the start of the experiments. Upon arrival, the animals were housed in individual 48×27 ×20 cm clear polycarbonate cages in a climate-controlled room at 22 °C with a 12-h dark/light cycle. During the 1st week after arrival, all animals were handled and weighed daily for 7 days. During the 2nd week the animals received three injections (i.p.) of 0.9% saline (1.0 ml/kg) in order to
acclimate the animals to the injection procedure. All experiments occurred during the 12-h light cycle (06:00–18:00).
2.2. Drugs Cocaine hydrochloride and anhydrous caffeine (Sigma Chemical Co., St. Louis, MO) were dissolved in sterile distilled H2O to a concentration of 10 mg/ml. (9 )-8-hydroxy-dipropylaminotetralin (8-OH DPAT) (RBI/Sigma, Natick, MA) was dissolved in sterile distilled H2O to concentrations of 0.2 or 0.4 mg/ml. N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cycylhexanecarboxaminde maleate (WAY 100635) (RBI/Sigma, Natick, MA) was dissolved in sterile distilled H2O to concentrations of 0.4 and 0.8 mg/ml. All injections were administered I.P.
2.3. Apparatus All of the behavioral tests were conducted in square open-field compartments which were 60× 60×45 cm. Closed-circuit video cameras (RCA TC7011U) were mounted 50 cm above the open-field enclosures. All signals were analyzed by a video tracking system, the Videomex-V from Columbus Instruments (Columbus, OH), and the data imported into a PC compatible computer. The walls of the chamber were white and the floor of the open-field was covered by plain white paper which was changed after each animal. Masking noise (80 dB) was provided by a white noise generator (San Diego Instruments, San Diego, CA) and was turned on immediately prior to placement of the animal in the test chamber and turned off upon removal from the test chamber. Testing was conducted under conditions of red light illumination to avoid the aversive quality of white light and to enhance the contrast between the subject and background as well as to reduce the animal’s shadow. The animal’s head was blackened with a non-toxic marker and the camera only tracked this feature of the rat’s body. A central zone (CZ) comprising one-ninth of the floor area was monitored independently from the rest of the area and is only distinguished by the computer analyzer. During each session, data was collected every 2.5 min by the computer. Dot matrix printers (Epson FX-286e) were placed outside the test rooms and were connected to the image analyzers by a parallel cable and the computer screen tracings of the animal’s movement were printed out every 2.5 min. The complete test procedure was conducted automatically without the presence of the experimenter in the test room. In addition, a VHS VCR was also connected to each camera providing the capability to review and re-imput the video tape signal to the image analyzer in case of a malfunction of either the analyzer or the printer during the experiments. The
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videotapes were also used to score grooming behavior (facial and flank grooming), rearing and tight circling by two experimenters without knowledge of the drug treatments. The circling was initially identified by the presence of 10–20 cm circles on the printout of tracings of an animal’s path (Fig. 1). The circles were confirmed and counted by an experimenter from the videotapes of the test sessions. Videotapes of the initial and final treatment sessions for all treatment protocols were scored. In addition, for the 20 treatments in the initial 8-OHDPAT treatment phase, days 2, 7, 9, 12, 15, 17 and 19 were scored in order to assess possible trend effects.
four 8-OHDPAT groups were switched to WAY 100635 as the pretreatment. The 0.2 mg/kg 8-OHDPAT groups were given 0.4 mg/kg WAY 100635 and 0.4 mg/kg 8-OHDPAT groups were given 0.8 mg/kg WAY 100635. The groups received this treatment 5 times over the course of 2 weeks. After the testing was completed, the animals were given their original 8-OHDPAT treatment for five additional treatments spaced over 2 weeks. In the last week of testing, the 8-OHDPAT pretreatment groups were again given the 0.4 and 0.8 mg/kg WAY 100635 pretreatments. This time, however, the animals received saline/caffeine (10 mg/kg) instead of saline/cocaine (10 mg/kg).
2.4. Beha6ioral testing
2.5. Statistical analyses
Initially, all animals underwent 10 days of daily handling including 3 days of saline injections to acclimate the animals to manipulation and injection procedures. Next, all animals were given two 10 min tests in the test environment in order to form groups which were statistically equivalent with respect to the dependent variable of locomotion. Four days after the completion of the matching protocol, the groups received a series of drug treatments. Six treatment groups (n = 7) were used. In the first phase experiment, the effects of 0.2 and 0.4 mg/kg 8-OHDPAT upon cocaine induced locomotor stimulation were measured. All animals received two injections, the first was administered in the homecage 20 min prior to testing in the open-field and the second was administered immediately prior to placement in the open-field. The treatment groups were saline–saline, saline– cocaine (10 mg/kg), 8-OHDPAT (0.2 mg/kg) –saline, 8-OHDPAT (0.2 mg/kg) – cocaine (10 mg/kg), 8-OHDPAT (0.4 mg/kg) – saline and 8OHDPAT (0.4 mg/kg) – cocaine (10 mg/kg). After 20 treatments, (given at the rate of 5 every 2 weeks) the
Two-way Analysis of Variance (ANOVA) was used to evaluate the behavioral data to determine between group effects, as well as the interaction between groups and repeated treatments. Each pretreatment phase (8OHDPAT, WAY 100635, 8-OHDPAT) was evaluated separately. In that the caffeine test was only conducted once, a oneway ANOVA was used to statistically assess between group effects. In order to make specific group comparisons, post-hoc Duncan’s multiple range tests were performed. PB 0.05 was used as the criterion for statistical significance. Only F values for differences which reached the PB 0.05 level of significance are reported.
Fig. 1. Tracing of an animal’s actual path as tracked by the image analyzer. This is an interval in which a circling pattern was suggested. Subsequent observation of a videotape of the test session confirmed the occurrence of tight circling and four circles were recorded. The scale is reduced from 6.67 to 1.
3. Results Fig. 2 presents the locomotion distance scores (upper) and central zone entries (lower) for all treatment groups over the course of the cocaine treatment regimens. As can be seen in Fig. 2 (upper), cocaine increased the locomotion distance scores and the effect was enhanced by 8-OHDPAT (0.2 and 0.4 mg/kg) but subsequently diminished by WAY 100635 (0.4 and 0.8 mg/kg). When the WAY 100635 treatments were switched back to 8-OHDPAT, the cocaine locomotor stimulant effects were again enhanced. While the 8OHDPAT and WAY 100635 pretreatments substantially modulated the locomotor stimulant effects of cocaine, these treatments did not modify baseline locomotion levels as compared to saline controls. The initial 8-OHDPAT pretreatment phase, the WAY 100635 pretreatment phase and the second 8-OHDPAT pretreatment phase were statistically evaluated separately. The F values for the 8-OHDPAT, WAY 100635 and the second 8-OHDPAT pretreatment phases for between group effects were F(5, 36)=13.1, PB0.001, F(5, 36)= 13.4, PB0.001 and F(5, 36)= 10.0, PB 0.001, respectively. None of the group× day interactions were statistically significant, P\0.05.
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Fig. 2. Means and SEMs for locomotion distance scores (upper) and central zone penetrations (lower). The results for six treatment groups (n= 7) are presented. Immediately prior to the 20 min open-field tests, three groups received saline and three groups received cocaine (10 mg/kg). Each saline–cocaine pair also received pretreatments of either saline, 0.2 8-OHDPAT or 0.4 mg/kg 8-OHDPAT 20 min before testing. This 8-OHDPAT pretreatment phase was conducted for 20 sessions. In the WAY 100635 pretreatment phase, the 0.2 and 0.4 mg/kg 8-OHDPAT groups were switched to 0.4 and 0.8 mg/kg WAY 100635, respectively for five sessions. Finally, the WAY 100635 pretreatment groups were switched back for five sessions to their original 8-OHDPAT pretreatment regimen. This is the second 8-OHDPAT phase. See Section 2.5.
Comparisons of mean differences among groups using Duncan’s Multiple Range test indicated that for the initial 8-OHDPAT pretreatment phase, the 0.2 and 0.4 mg/kg 8-OHDPAT groups which received cocaine (10 mg/kg) had higher locomotion distance scores than the saline–cocaine group (P B 0.01) which in turn had higher scores than the three non-cocaine groups (PB 0.01) which did not differ from each other (P \ 0.05). For the WAY 100635 pretreatment phase, the saline– cocaine group had locomotion levels higher than the two WAY 100635-cocaine groups (P B 0.01) which in turn were higher than the three non-cocaine groups (PB 0.05) which did not differ (P \0.05) from each other. When the animals were switched back to the 8-OHDPAT pretreatment regimen, the locomotor dis-
tance effects were similar to those observed during the initial 8-OHDPAT pretreatment phase. As can be seen in the lower half of Fig. 2, the cocaine treatments had substantial effects upon central zone entries. In the initial 8-OHDPAT phase, the WAY 100635 and the second 8-OHDPAT pretreatment phase there were statistically significant between group effects F(5, 36)= 10.2, PB 0.001, F(5, 36)= 6.0, P B 0.001 and F(5, 36)= 7.4, P B 0.001, respectively. Furthermore, the group× day interactions were statistically significant for the initial 8-OHDPAT pretreatment phase and for the second 8-OHDPAT pretreatment phase F(95, 684)= 1.4 PB 0.02 and F(26, 140)= 2.1, PB 0.01, respectively. The interaction effects are evident for the 8-OHDPAT – cocaine groups in Fig. 2 (lower). Ini-
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tially, the 8-OHDPAT pretreatments suppressed entry into the central zone. This effects was particularly evident in the 0.4 mg/kg 8-OHDPAT – cocaine group. In fact, it can be seen that the 0.4 mg/kg 8-OHDPAT – cocaine group on day 2 did not enter the central zone at all even though the locomotion distance for this group was the same as the saline– cocaine group which had a substantial incidence of central zone entries. With repeated treatments, however, the cocaine groups given the 8-OHDPAT pretreatments achieved central zone entry levels comparable to the saline– cocaine group. Fig. 3 presents the results for responses scored from videotapes of the initial and last treatment days across the treatment protocols. For the first 8-OHDPAT pretreatment phase, several intermediate days were scored in order to detect possible progressive changes over the 20 treatment sessions. In this initial 8-OHDPAT pretreatment phase there were statistically significant be-
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tween group effects for each response measure: grooming F(5, 36)= 49.4, P B 0.001, rearing F(5, 36)= 6.2, PB 0.001 and circling F(5, 36)= 5.8, P B0.001. For grooming behavior, the saline group groomed more than all of the other treatment groups (PB0.01) which did not differ from each other (P\ 0.05). As can be seen in Fig. 3 (top), all treatments suppressed grooming as compared to saline controls. In terms of rearing (middle) and circling (bottom), it can be seen in Fig. 3 that cocaine reliably increased rearing and circling and that, initially, both the 0.2 and 0.4 mg/kg 8-OHDPAT pretreatments suppressed these effects of cocaine. With repeated treatments, however, the 0.2 mg/kg 8-OHDPAT plus cocaine group developed levels of rearing and circling which were comparable to the saline– cocaine group. This marked shift over the course of treatments days undoubtedly was the major factor for the statistically significant treatment group×
Fig. 3. Means and SEMs for videotape scoring of grooming (top) rearing (middle) and circling (bottom) obtained in 20 min open-field tests on selected days over the course of a series of saline/cocaine treatments in which the groups received pretreatments of either saline, 0.2 mg/kg 8-OHDPAT or 0.4 mg/kg 8-OHDPAT (days 1, 2, 7, 9, 12, 15, 17, 19, 20), 0.4 or 0.8 mg/kg WAY 100635 (days 21 and 25) and 0.2 or 0.4 mg/kg 8-OHDPAT (days 27 and 30), respectively. See Section 2.5.
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day interactions, F(40 280)=2.1, PB 0.001 and F(40 280)= 1.6, PB0.05, for rearing and circling, respectively. When the 8-OHDPAT groups were switched to WAY 100635, there was a pronounced increase in grooming behavior in the WAY 100635-saline groups. The treatment effects on grooming were statistically significant F(5, 36)=13.7, P B0.001. The two WAY 100635-saline groups had higher grooming scores than the saline group (P B 0.01) which had higher grooming durations than the three cocaine groups (P B0.01) which did not differ from each other (P \ 0.05). As is evident in Fig. 3 (top), the WAY 100635 pretreatment effect on grooming was the opposite of 8-OHDPAT. During the WAY 100635 pretreatment phase, rearing and circling were also significantly affected by the drug treatments, F(5, 36)=3.6, P B 0.01 and F(5, 36)= 8.4, P \0.001, respectively. For the circling response measure, the three cocaine treatments increased circling relative to the three non-cocaine groups (P B 0.01) and the three cocaine groups did not differ from each other (PB 0.05). In terms of rearing, the saline– cocaine group had more rearing responses than all other groups (PB 0.05). The WAY 100635-cocaine groups did not differ from each other or from the three non-cocaine groups (P\0.05). After the WAY 100635 pretreatments, the groups were switched back to their original 8-OHDPAT pretreatments. Again, the 8-OHDPAT pretreatments exerted statistically significant between group effects upon the three response measures: F(5, 36)= 43.0, P B 0.001, F(5, 36)= 7.3, P B 0.001 and F(5, 36)= 2.7, P B 0.05 for grooming, rearing and circling, respectively. As can be seen in Fig. 3 (top), the 8-OHDPAT pretreatment again suppressed grooming behavior. The group× day interactions were not statistically significant (P \ 0.05). The saline-saline group had higher grooming scores than all other groups (P B0.01). For rearing (middle), the saline–cocaine and 0.2 mg/kg 8-OHDPAT – cocaine group exhibited enhanced rearing compared to the other treatment groups (P B0.05) For circling, the saline – cocaine group exhibited a statistically significant increase in circling as compared to the saline-saline group (P B0.05). None of the other treatment groups differed significantly from the saline-saline group (P\ 0.05). After the completion of the testing with 8-OHDPAT and cocaine, the groups were tested with saline or caffeine (10 mg/kg). The groups which had received the 8-OHDPAT pretreatments were given 0.4 and 0.8 mg/ kg WAY pretreatments. As can be seen in Fig. 4, caffeine enhanced locomotor distance scores and suppressed grooming behavior. Statistical analyses of these results indicated statistically significant between group effects, F(5, 36)= 16.2, P B0.001 and F(5, 36)=11.2, P B0.001, for distance and grooming, respectively. The
Fig. 4. Means and SEMs for distance (upper) and grooming (lower) in which the same six pretreatment groups used in preceding saline/ cocaine tests (n = 7) were treated with saline – caffeine (10 mg/kg). The 8-OHDPAT pretreatment groups received WAY 100635 as pretreatments. The treatment groups were saline-saline, saline-caffeine (10 mg/kg), 0.4 mg/kg WAY 100635 – saline, 0.4 mg/kg WAY 100635 – caffeine (10 mg/kg), 0.8 mg/kg WAY 100635 – saline or 0.8 mg/kg WAY 100635 – caffeine (10 mg/kg) and then tested for 20 min in an open-field. * upper indicates P B0.01 vs all non-caffeine groups. * Lower denotes PB0.01 vs all caffeine groups. ** Indicates PB 0.01 versus the saline – saline and caffeine groups.
WAY 100635 pretreatment did not affect caffeine induced locomotion or saline levels of locomotion. While the WAY 100635 pretreatment did not affect the suppression in grooming behavior induced by caffeine, the WAY pretreatment in combination with saline did increase grooming. This latter observation on grooming is similar to the effect of WAY 100635 on grooming behavior presented in Fig. 2. Thus, the grooming data suggest that the WAY 100635 pretreatment was as effective in the caffeine test as it was in the cocaine phase of the experiment.
4. Discussion In general, the present findings are consistent with a reciprocal facilitatory/inhibitory 5-HT1A agonist/antag-
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onist influence upon cocaine induced locomotor stimulation. This reciprocal relationship appears quite transparent with regard to locomotion distance as the measure of cocaine locomotor stimulant effects but is more complicated for other measures of cocaine effects, such as rearing behavior. In terms of the 5-HT1A agonist facilitation of cocaine locomotor stimulant effects, the two dose levels of 8-OHDPAT had equivalent efficacy in increasing locomotion distance effects and, initially, had similar inhibitory effects in other behavioral aspects of cocaine effects (i.e. central zone activity, circling and rearing). This dual facilitory and inhibitory behavioral actions of 8-OHDPAT on cocaine induced behavioral effects offered a simple behavioral explanation for the 8-OHDPAT enhancement of cocaine induced locomotor stimulation. Namely, 8-OHDPAT suppressed behaviors which competed with and/or interrupted locomotion. That is, the 8-OHDPAT effect upon locomotion appeared to represent a response redistribution effect. Seemingly, it could be argued that the stimulant effect of cocaine was unchanged but its’ behavioral expression was altered by 8-OHDPAT. As the number of treatment sessions increased, however, this account became less tenable. Eventually, the lower dose of 8-OHDPAT given in combination with cocaine generated central zone activity, rearing and circling which matched or exceeded the levels induced by cocaine given alone. While all these response measures increased, the 8-OHDPAT treatment continued to enhance the locomotor distance effect induced by cocaine. Furthermore, the recovery for these response measures was less evident for the higher dose level of 8-OHDPAT but yet the two dose levels remained equally efficacious in enhancing cocaine locomotor distance effects. Altogether, these observations argue against a response redistribution effect of 8-OHDPAT in the enhancement of cocaine induced locomotion. It might be argued, however, that the increase in rearing, circling and central zone activity in the 8-OHDPAT– cocaine treated animals was indicative of the development of tolerance to the 8-OHDPAT. The fact that the 8-OHDPAT treatments continued to enhance cocaine induced locomotion weakens this argument. Furthermore, the 8-OHDPAT treatments maintained suppression of grooming behavior throughout testing indicating a continued pharmacological efficacy. When the 8-OHDPAT treatments were switched to WAY 100635, there were immediate reductions in locomotion distance in the cocaine treated animals which were sustained with repeated treatments. The same WAY 100635 treatments, however, had no effect on locomotion in the saline treated groups. When other behaviors are examined, important differences in the 8-OHDPAT and WAY 100635 were evident. In saline treated animals, the 8-OHDPAT and WAY 100635 had pronounced and opposite effects upon grooming behav-
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ior. Furthermore, 8-OHDPAT initially induced dose related decreases in rearing and central zone activity whereas, the WAY 100635 treatments did not affect these response measures relative to saline treated animals. Despite the pronounced increase in grooming induced by WAY 100635 given prior to saline, when the WAY 100635 was given in combination with cocaine, it did not reliably attenuate the cocaine induced suppression of grooming. Although the WAY 100635 treatment attenuated commonly used indicators of cocaine induced locomotor activation, namely locomotor distance and rearing, the WAY 100635 treatment did not reliably alter the grooming suppression or the circling induced by cocaine. The present findings provide support for 5-HT1A agonist/antagonist facilitory and inhibitory interaction with cocaine in terms of motoric stimulation but, the relationship has substantial complexity. Straightforward behavioral explanations in terms of response redistribution or response competition while having some utility do not adequately account for the present observations. A critical factor evident in the present study is that the interaction between 8-OHDPAT and cocaine can change with repeated treatments. This modification in the interaction can occur without corresponding changes in the response effects of the drugs given separately. Response effects, however, are not the only behaviorally important dimension of these drugs. The stimulus properties of centrally active drugs are another important facet of the behavioral impact of drugs. The stimulus properties of 8-OHDPAT have been extensively studied and documented [11,42,43]. The possible contribution of the stimulus properties of drugs to behavior in studies of drug effects manifested in openfield behavior are not directly observable but yet may be important. This seems particularly likely in the case of multiple drug treatments as in the present study. Not only are the unconditioned response effects of 8-OHDPAT able to alter the unconditioned response effects of cocaine, but the stimulus effects generated by 8-OHDPAT can become linked to the response effects of cocaine. Thus, a possibility that needs to be considered is there is a repeated association of the 8-OHDPAT drug stimuli with the cocaine responses. As a consequence, the 8-OHDPAT drug stimuli, eventually, may acquire cocaine conditioned stimulus properties. Therefore, with the repeated parings of two drug treatments, the interaction is no longer just a summation of 8-OHDPAT and cocaine unconditioned responses but also may come to include the 8-OHDPAT stimuli activating cocaine conditioned responses. Such a process may contribute to the emergence of cocaine— like central zone activity, rearing and circling in the low dose 8-OHDPAT group given the combined 8-OHDPAT – cocaine treatments.
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In terms of neurotransmission, it is well established [35], that cocaine’s high affinity binding to the catecholamine and indoleamine transporters increases catecholaminergic and serotonergic neurotransmission. While the contribution of catecholaminergic effects of cocaine, particularly dopamine, have been well documented [21,48], the behavioral contribution of this increase in serotonergic activity generated by cocaine has also been recognized [24,27,34,36,47]. When cocaine binds to the 5-HT transporter inhibiting the reuptake of 5-HT from the synaptic cleft, this increase in somatic dendritic 5-HT not only stimulates postsynaptic receptors but also activates inhibitory 5-HT1A autoreceptors which decrease 5-HT neuronal activity [23,40]. Blockade of the 5-HT1A receptors with WAY 100635 can attenuate the inhibitory effects of somatodendritically released 5-HT on 5-HT neurons. The combined effect of local 5-HT reuptake blockade in terminal areas by cocaine combined with 5- HT1A antagonism serves to potentiate extracellular 5-HT concentrations in the terminal regions of 5-HT projections. The potentiating effects of WAY 100635 [15,18] and other 5-HT1A antagonists [16,17] on hippocampal 5-HT concentrations induced by SSRI and by cocaine [30] have been reported. While it has yet to be documented, the expectation would be that a 5-HT1A agonist, such as 8-OHDPAT, would have the opposite effect. From this perspective, the 5-HT1A agonist would attenuate and the 5-HT1A antagonist would enhance extracellular 5-HT level increases induced by cocaine. 5-HT1A agonists and antagonists would therefore shift the balance in the effect of cocaine on DA and 5-HT transmission. That is, for 5-HT1A agonists, the cocaine induced increase in DA would occur in a context of a diminished extracellular 5-HT level and, for a 5-HT1A antagonist, the DA–5HT balance would be shifted in the reverse direction. By positing an inhibitory influence for the increases in hippocampal 5-HT induced by cocaine, the modulation of the 5-HT neurons by a 5-HT1A agonist/antagonist provides a possible account for their reciprocal effects of 5-HT1A agonists/antagonists on the locomotor stimulant effects induced by cocaine. Consistent with this proposition, reductions in 5-HT produced by intracerebral injections of the 5-HT neurotoxin 5,7,dihydroxytryptamine [29] enhance cocaine induced locomotor stimulation. Important in this analysis is the assumption that 5-HT1A agonists/antagonists do not directly affect the increase in DA induced by cocaine. For the dose levels, temporal parameters and the behavioral measurement used in the present study, the evidence to date [3,5,30] is that 8-OHDPAT and WAY 100635 do not alter the increase in DA induced by cocaine. At higher dose levels, temporal parameters or different routes of administration (e.g. s.c.), 8-OHDPAT and WAY 100635 undoubtedly may have effects which would alter the baseline for locomotion
behavior [19]. Furthermore, cocaine effects on locomotion behavior are dose related so that the present results must be viewed within the cocaine dose level parameters used in the present experiment. The effects of 5-HT related drug treatments upon locomotor behavior have been studied extensively. With the variety of 5-HT receptor sites [37] and the wide array of pharmaceuticals with different affinities for different receptor sites coupled with the wide range of dose levels of drug used and behavior measures employed, it becomes an understatement to assert that the situation is complex. High levels of 5-HT stimulation and even selective 5-HT1A postsynaptic stimulation can induce profound effects upon certain behaviors [46]. At more modest levels of 5-HT activation, however, the 5-HT system appears to be implicated in arousal, anxiety and stress [8,12,39]. In particular, the 5-HT1A receptors in the hippocampus have been suggested to participate in the development of tolerance to aversive events [14] and may attenuate stress induced behavioral consequences [32]. WAY 100635 and other 5-HT1A receptor antagonists exhibit anxiolytic effects in the plus maze in rats and mice [10,13]. 8-OHDPAT has also been reported to attenuate stress and cocaine induced increases in plasma corticosterone [41]. On the other hand, 5-HT1A receptor agonists are well documented to increase corticosterone levels [20,26], including the 0.2 mg/kg 8-OHDPAT dose level used in the present study [6]. Cocaine also increases stress related hormones, such as corticosterone [2,25,28]. Thus, 5HT1A drug treatment interactions with cocaine may impact upon processes pertinent to anxiety and stress in some yet to be determined manner which is dose and context dependent. While corticosterone measurement is one facet of assessing this component of the interaction, the aspect of open-field behavior which appears to have some relevance to this issue is the location of activity within the open field. Typically, rats prefer to locomote along the walls at the periphery. Anxiolytic drug treatments tend to facilitate entry into the central zone of the open field [22,45]. In the present study, cocaine enhanced entry into the central zone, whereas, 8-OHDPAT decreases central zone entry. As previously noted, however, both of these treatments increase corticosterone concentrations so this hormonal effect cannot directly account for this behavioral difference. In terms of 5-HT, however, the autoreceptor stimulation induced by 8-OHDPAT would be expected to decrease 5-HT whereas, cocaine increases 5-HT. In that WAY 100635 (0.4 mg/kg) in rats potentiates the 5-HT release in hippocampus induced by cocaine [31], the central zone entry may be related to whether the drug treatment increases or decreases 5-HT. Thus, modification in the 5-HT activity induced by cocaine may be an important component of the behavioral activity generated by cocaine. Drug treatments which modify 5-HT
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effects induced by cocaine alter the balance between catecholaminergic and serotonergic effects induced by cocaine. That is, the cocaine effect may become relatively more dopaminergic if 5-HT effects are diminished or more serotonergic if 5-HT effects are potentiated. Such consideration may have relevance to the treatment of cocaine abuse. Pharmacological modulation of the 5-HT1A receptor may be an effective way to shift this neurochemical balance in the direction of 5-HT. In contrast, drugs which directly block the 5-HT transporter (e.g. fluoxetine) may be ineffective in shifting the 5-HT/DA balance toward 5-HT because any further increase in the blockade of 5-HT transport beyond that induced by cocaine would be negligible.
[11]
[12]
[13]
[14]
[15] [16]
Acknowledgements [17]
This work was supported by NIDA grant DA R01 11457-02 and a VA Merit Review grant. [18]
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[30] Muller CP, Carey RJ, De Souza Silva MA, Jocham G, Huston JP. Cocaine increases serotonergic activity in the hippocampus and nucleus accumbens in vivo: 5-HT1A-receptor antagonism blocks behavioral but potentiates serotonergic activation. Submitted. [31] Muller CP, De Souza Silva MA, Huston JP, DePalma G, Carey RJ. Behavioral but not neurochemical effects of cocaine are blocked by the selective serotonin 1A-receptor antagonist WAY 100635. Submitted. [32] Netto SM, Guimaraes FS. Role of hippocampal 5-HT1A receptors on elevated plus maze exploration after a single restraint experience. Behav Brain Res 1996;77:215 – 8. [33] Newman AH, Allen AC, Izenwasser S, Katz JL. Novel 3a(Diphenylmethoxy)tropane analogs: potent dopamine uptake inhibitors without cocaine-like behavioral profiles. J Med Chem 1994;37:2258 – 61. [34] Reith MEA. 5-HT3 receptor antagonists attenuate cocaine-induced locomotion in mice. Eur J Pharm 1990;186:327 – 30. [35] Ritz MC, Cone EJ, Kuhar MJ. Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: a structure-activity study. Life Sci 1990;46:635 – 45. [36] Rocha BA, Scearce-Levie K, Lucas JJ, Hiro N, Castanon N, Crabbe JC, Nestler EJ, Hen R. Increased vulnerability to cocaine in mice lacking the serotonin-1B receptor. Nature 1998;393:175 – 6. [37] Roth BL. Multiple serotonin receptors: clinical and experimental aspects. Ann Clin Psych 1994;6:67 –78. [38] Rothman RB, Grieg N, Kim A, deCosta BR, Rice KC, Carroll FI, Pert A. Cocaine and GBR 12909 produce equivalent motoric responses at different occupancy of the dopamine transporter. Pharmacol Biochem Behav 1992;43:1135 – 42. [39] Rueter LE, Fornal CA, Jacobs BL. A critical review of 5-HT brain microdialysis and behavior. Rev Neurosci 1997;8:117 –37.
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Research report
5-HT1A agonist/antagonist modification of cocaine stimulant effects: implications for cocaine mechanisms Robert J. Carey *, Gail De Palma, Ernest Damianopoulos VA Medical Center and SUNY Health Science Center, Research and De6elopment 800 Ir6ing A6enue, Syracuse, NY 13210, USA Received 30 June 2001; received in revised form 9 September 2001; accepted 9 September 2001
Abstract The 5-HT1A receptor site has been demonstrated to be an important pharmacological target in the modulation of unconditioned behavioral effects induced by cocaine. In this study, separate groups of rats (n= 7) received a series of the 5-HT1A agonist treatments, 8-OHDPAT (0.2, 0.4 mg/kg) in combination with saline or cocaine (10 mg/kg). Using a crossover design, the treatments were subsequently switched to the 5-HT1A antagonist, WAY 100635 (0.4, 0.8 mg/kg) and then, switched back again to 8-OHDPAT (0.2, 0.4 mg/kg). When the 8-OHDPAT was given in combination with cocaine, locomotion was substantially enhanced but when the treatment was switched to WAY 100635, the cocaine induced locomotion was suppressed. Neither the 8-OHDPAT or WAY 100635 given with saline affected locomotion as compared to saline treated animals. These findings indicated a reciprocal facilitatory/inhibitory influence of 5-HT1A agonists/antagonists upon cocaine induced locomotion. The 8-OHDPAT treatments, however, did not enhance all cocaine behavioral responses. Initially, 8-OHDPAT suppressed cocaine induced rearing and central zone entry, but with repeated treatments, these response suppression effects subsided. As a consequence, the facilitative influence of 8-OHDPAT upon cocaine induced locomotion could not be attributed to response redistribution effects. While WAY 100635 markedly reduced cocaine induced locomotion and rearing to nearly saline response levels, the same WAY 100635 treatments did not modify locomotor stimulant effects induced by caffeine (10 mg/kg). In that caffeine stimulant effects are not directly linked to serotonergic mechanisms, the absence of an influence of WAY 100635 upon caffeine induced locomotor stimulation lent further support to the proposition that the 5-HT1A receptor site contributes to locomotor behavior in situations where the serotonergic system is pharmacologically activated by drugs such as cocaine. These findings point to a potential role for 5-HT1A antagonists in treatment of cocaine abuse. Published by Elsevier Science B.V. Keywords: Cocaine; 8-OHDPAT; WAY 100635; Caffeine; Locomotion; Grooming; Rearing; Rat
1. Introduction Cocaine, as a potent inhibitor of catecholamine and indoleamine transport, can substantially increase extracellular dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NE) [35]. The relationship of these increases in catecholaminergic and serotonergic activity and the behavioral effects of cocaine have proven elusive. While there is considerable evidence to indicate * Corresponding author. Tel.: + 1-315-476-7461x2824; fax: +1315-476-5348. E-mail address: [email protected] (R.J. Carey). 0166-4328/02/$ - see front matter. Published by Elsevier Science B.V. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 3 8 3 - 7
that cocaine induced increases in dopamine in the nucleus accumbens (Nac) may have a critical role in the mediation of cocaine stimulant and reward effects [21,48], recent findings have obscured this connection. It has been [36,44]reported that knock-out mice lacking the DA transporter still self-administered cocaine. In addition, it has been found that selectivity for inhibition of DA transport is not a sufficient mechanism for a drug to induce locomotor stimulation [33,38]. Increasingly, evidence indicates that cocaine effects upon 5-HT transport also contribute to cocaine behavioral effects [1,7,34,47]. Recently, we have shown [4,6] that the 5-HT1A receptor antagonist, WAY 100635 can block
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the locomotor stimulant effects of low dose cocaine and conversely, that the 5-HT1A agonist, 8-OHDPAT can enhance the locomotor stimulant effects of cocaine [5,9]. The further observations [4,6] that the WAY 100635 and 8-OHDPAT treatments did not alter the behavioral baselines for locomotion or modify DA increases induced by cocaine points to a possible contribution of the 5-HT1A receptor site in the mediation of cocaine locomotor stimulant effects. The reciprocity between a 5-HT1A agonist and antagonist in terms of cocaine induced locomotion provides a compelling impetus for implicating the 5-HT1A system in cocaine stimulant effects. When behavior is examined more broadly, however, the interaction between 5-HT1A drug treatments and cocaine is more complex. 8-OHDPAT does not increase all facets of cocaine locomotor stimulant effects rather, it preferentially enhances locomotion at the periphery of an open-field and decreases rearing behaviors [9]. 8-OHDPAT given alone suppresses grooming behavior and increases corticosterone and can have dose related effects upon locomotor behavior [4,20,26,46]. WAY 100635, on the other hand, increases grooming but does not prevent the reduction in grooming behavior induced by cocaine [6]. In view of the important but complex interaction between 5-HT1A selective drug treatments and cocaine, the present study examines the impact of 8-OHDPAT upon cocaine induced behavioral effects in an openfield over a chronic treatment regimen in which multiple behavioral measurements are compiled. To test directly for possible reciprocity between 8-OHDPAT and WAY 100635 in their interaction with cocaine, a substitution test was conducted in which the 8-OHDPAT treatment is replaced with WAY 100635.The outcome of this investigation points to important changes in the 8-OHDPAT– cocaine interaction with chronic treatment and provides additional support for reciprocal effects of the 5-HT1A agonist, 8OHDPAT and antagonist, WAY 100635 on cocaine locomotor stimulant effects.
2. Method
2.1. Animals Naive male Sprague– Dawley rats from Taconic Farms (Germantown, NY) were used. The animals were 4 months old and weighed approximately 400 g at the start of the experiments. Upon arrival, the animals were housed in individual 48×27 ×20 cm clear polycarbonate cages in a climate-controlled room at 22 °C with a 12-h dark/light cycle. During the 1st week after arrival, all animals were handled and weighed daily for 7 days. During the 2nd week the animals received three injections (i.p.) of 0.9% saline (1.0 ml/kg) in order to
acclimate the animals to the injection procedure. All experiments occurred during the 12-h light cycle (06:00–18:00).
2.2. Drugs Cocaine hydrochloride and anhydrous caffeine (Sigma Chemical Co., St. Louis, MO) were dissolved in sterile distilled H2O to a concentration of 10 mg/ml. (9 )-8-hydroxy-dipropylaminotetralin (8-OH DPAT) (RBI/Sigma, Natick, MA) was dissolved in sterile distilled H2O to concentrations of 0.2 or 0.4 mg/ml. N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cycylhexanecarboxaminde maleate (WAY 100635) (RBI/Sigma, Natick, MA) was dissolved in sterile distilled H2O to concentrations of 0.4 and 0.8 mg/ml. All injections were administered I.P.
2.3. Apparatus All of the behavioral tests were conducted in square open-field compartments which were 60× 60×45 cm. Closed-circuit video cameras (RCA TC7011U) were mounted 50 cm above the open-field enclosures. All signals were analyzed by a video tracking system, the Videomex-V from Columbus Instruments (Columbus, OH), and the data imported into a PC compatible computer. The walls of the chamber were white and the floor of the open-field was covered by plain white paper which was changed after each animal. Masking noise (80 dB) was provided by a white noise generator (San Diego Instruments, San Diego, CA) and was turned on immediately prior to placement of the animal in the test chamber and turned off upon removal from the test chamber. Testing was conducted under conditions of red light illumination to avoid the aversive quality of white light and to enhance the contrast between the subject and background as well as to reduce the animal’s shadow. The animal’s head was blackened with a non-toxic marker and the camera only tracked this feature of the rat’s body. A central zone (CZ) comprising one-ninth of the floor area was monitored independently from the rest of the area and is only distinguished by the computer analyzer. During each session, data was collected every 2.5 min by the computer. Dot matrix printers (Epson FX-286e) were placed outside the test rooms and were connected to the image analyzers by a parallel cable and the computer screen tracings of the animal’s movement were printed out every 2.5 min. The complete test procedure was conducted automatically without the presence of the experimenter in the test room. In addition, a VHS VCR was also connected to each camera providing the capability to review and re-imput the video tape signal to the image analyzer in case of a malfunction of either the analyzer or the printer during the experiments. The
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videotapes were also used to score grooming behavior (facial and flank grooming), rearing and tight circling by two experimenters without knowledge of the drug treatments. The circling was initially identified by the presence of 10–20 cm circles on the printout of tracings of an animal’s path (Fig. 1). The circles were confirmed and counted by an experimenter from the videotapes of the test sessions. Videotapes of the initial and final treatment sessions for all treatment protocols were scored. In addition, for the 20 treatments in the initial 8-OHDPAT treatment phase, days 2, 7, 9, 12, 15, 17 and 19 were scored in order to assess possible trend effects.
four 8-OHDPAT groups were switched to WAY 100635 as the pretreatment. The 0.2 mg/kg 8-OHDPAT groups were given 0.4 mg/kg WAY 100635 and 0.4 mg/kg 8-OHDPAT groups were given 0.8 mg/kg WAY 100635. The groups received this treatment 5 times over the course of 2 weeks. After the testing was completed, the animals were given their original 8-OHDPAT treatment for five additional treatments spaced over 2 weeks. In the last week of testing, the 8-OHDPAT pretreatment groups were again given the 0.4 and 0.8 mg/kg WAY 100635 pretreatments. This time, however, the animals received saline/caffeine (10 mg/kg) instead of saline/cocaine (10 mg/kg).
2.4. Beha6ioral testing
2.5. Statistical analyses
Initially, all animals underwent 10 days of daily handling including 3 days of saline injections to acclimate the animals to manipulation and injection procedures. Next, all animals were given two 10 min tests in the test environment in order to form groups which were statistically equivalent with respect to the dependent variable of locomotion. Four days after the completion of the matching protocol, the groups received a series of drug treatments. Six treatment groups (n = 7) were used. In the first phase experiment, the effects of 0.2 and 0.4 mg/kg 8-OHDPAT upon cocaine induced locomotor stimulation were measured. All animals received two injections, the first was administered in the homecage 20 min prior to testing in the open-field and the second was administered immediately prior to placement in the open-field. The treatment groups were saline–saline, saline– cocaine (10 mg/kg), 8-OHDPAT (0.2 mg/kg) –saline, 8-OHDPAT (0.2 mg/kg) – cocaine (10 mg/kg), 8-OHDPAT (0.4 mg/kg) – saline and 8OHDPAT (0.4 mg/kg) – cocaine (10 mg/kg). After 20 treatments, (given at the rate of 5 every 2 weeks) the
Two-way Analysis of Variance (ANOVA) was used to evaluate the behavioral data to determine between group effects, as well as the interaction between groups and repeated treatments. Each pretreatment phase (8OHDPAT, WAY 100635, 8-OHDPAT) was evaluated separately. In that the caffeine test was only conducted once, a oneway ANOVA was used to statistically assess between group effects. In order to make specific group comparisons, post-hoc Duncan’s multiple range tests were performed. PB 0.05 was used as the criterion for statistical significance. Only F values for differences which reached the PB 0.05 level of significance are reported.
Fig. 1. Tracing of an animal’s actual path as tracked by the image analyzer. This is an interval in which a circling pattern was suggested. Subsequent observation of a videotape of the test session confirmed the occurrence of tight circling and four circles were recorded. The scale is reduced from 6.67 to 1.
3. Results Fig. 2 presents the locomotion distance scores (upper) and central zone entries (lower) for all treatment groups over the course of the cocaine treatment regimens. As can be seen in Fig. 2 (upper), cocaine increased the locomotion distance scores and the effect was enhanced by 8-OHDPAT (0.2 and 0.4 mg/kg) but subsequently diminished by WAY 100635 (0.4 and 0.8 mg/kg). When the WAY 100635 treatments were switched back to 8-OHDPAT, the cocaine locomotor stimulant effects were again enhanced. While the 8OHDPAT and WAY 100635 pretreatments substantially modulated the locomotor stimulant effects of cocaine, these treatments did not modify baseline locomotion levels as compared to saline controls. The initial 8-OHDPAT pretreatment phase, the WAY 100635 pretreatment phase and the second 8-OHDPAT pretreatment phase were statistically evaluated separately. The F values for the 8-OHDPAT, WAY 100635 and the second 8-OHDPAT pretreatment phases for between group effects were F(5, 36)=13.1, PB0.001, F(5, 36)= 13.4, PB0.001 and F(5, 36)= 10.0, PB 0.001, respectively. None of the group× day interactions were statistically significant, P\0.05.
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Fig. 2. Means and SEMs for locomotion distance scores (upper) and central zone penetrations (lower). The results for six treatment groups (n= 7) are presented. Immediately prior to the 20 min open-field tests, three groups received saline and three groups received cocaine (10 mg/kg). Each saline–cocaine pair also received pretreatments of either saline, 0.2 8-OHDPAT or 0.4 mg/kg 8-OHDPAT 20 min before testing. This 8-OHDPAT pretreatment phase was conducted for 20 sessions. In the WAY 100635 pretreatment phase, the 0.2 and 0.4 mg/kg 8-OHDPAT groups were switched to 0.4 and 0.8 mg/kg WAY 100635, respectively for five sessions. Finally, the WAY 100635 pretreatment groups were switched back for five sessions to their original 8-OHDPAT pretreatment regimen. This is the second 8-OHDPAT phase. See Section 2.5.
Comparisons of mean differences among groups using Duncan’s Multiple Range test indicated that for the initial 8-OHDPAT pretreatment phase, the 0.2 and 0.4 mg/kg 8-OHDPAT groups which received cocaine (10 mg/kg) had higher locomotion distance scores than the saline–cocaine group (P B 0.01) which in turn had higher scores than the three non-cocaine groups (PB 0.01) which did not differ from each other (P \ 0.05). For the WAY 100635 pretreatment phase, the saline– cocaine group had locomotion levels higher than the two WAY 100635-cocaine groups (P B 0.01) which in turn were higher than the three non-cocaine groups (PB 0.05) which did not differ (P \0.05) from each other. When the animals were switched back to the 8-OHDPAT pretreatment regimen, the locomotor dis-
tance effects were similar to those observed during the initial 8-OHDPAT pretreatment phase. As can be seen in the lower half of Fig. 2, the cocaine treatments had substantial effects upon central zone entries. In the initial 8-OHDPAT phase, the WAY 100635 and the second 8-OHDPAT pretreatment phase there were statistically significant between group effects F(5, 36)= 10.2, PB 0.001, F(5, 36)= 6.0, P B 0.001 and F(5, 36)= 7.4, P B 0.001, respectively. Furthermore, the group× day interactions were statistically significant for the initial 8-OHDPAT pretreatment phase and for the second 8-OHDPAT pretreatment phase F(95, 684)= 1.4 PB 0.02 and F(26, 140)= 2.1, PB 0.01, respectively. The interaction effects are evident for the 8-OHDPAT – cocaine groups in Fig. 2 (lower). Ini-
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tially, the 8-OHDPAT pretreatments suppressed entry into the central zone. This effects was particularly evident in the 0.4 mg/kg 8-OHDPAT – cocaine group. In fact, it can be seen that the 0.4 mg/kg 8-OHDPAT – cocaine group on day 2 did not enter the central zone at all even though the locomotion distance for this group was the same as the saline– cocaine group which had a substantial incidence of central zone entries. With repeated treatments, however, the cocaine groups given the 8-OHDPAT pretreatments achieved central zone entry levels comparable to the saline– cocaine group. Fig. 3 presents the results for responses scored from videotapes of the initial and last treatment days across the treatment protocols. For the first 8-OHDPAT pretreatment phase, several intermediate days were scored in order to detect possible progressive changes over the 20 treatment sessions. In this initial 8-OHDPAT pretreatment phase there were statistically significant be-
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tween group effects for each response measure: grooming F(5, 36)= 49.4, P B 0.001, rearing F(5, 36)= 6.2, PB 0.001 and circling F(5, 36)= 5.8, P B0.001. For grooming behavior, the saline group groomed more than all of the other treatment groups (PB0.01) which did not differ from each other (P\ 0.05). As can be seen in Fig. 3 (top), all treatments suppressed grooming as compared to saline controls. In terms of rearing (middle) and circling (bottom), it can be seen in Fig. 3 that cocaine reliably increased rearing and circling and that, initially, both the 0.2 and 0.4 mg/kg 8-OHDPAT pretreatments suppressed these effects of cocaine. With repeated treatments, however, the 0.2 mg/kg 8-OHDPAT plus cocaine group developed levels of rearing and circling which were comparable to the saline– cocaine group. This marked shift over the course of treatments days undoubtedly was the major factor for the statistically significant treatment group×
Fig. 3. Means and SEMs for videotape scoring of grooming (top) rearing (middle) and circling (bottom) obtained in 20 min open-field tests on selected days over the course of a series of saline/cocaine treatments in which the groups received pretreatments of either saline, 0.2 mg/kg 8-OHDPAT or 0.4 mg/kg 8-OHDPAT (days 1, 2, 7, 9, 12, 15, 17, 19, 20), 0.4 or 0.8 mg/kg WAY 100635 (days 21 and 25) and 0.2 or 0.4 mg/kg 8-OHDPAT (days 27 and 30), respectively. See Section 2.5.
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day interactions, F(40 280)=2.1, PB 0.001 and F(40 280)= 1.6, PB0.05, for rearing and circling, respectively. When the 8-OHDPAT groups were switched to WAY 100635, there was a pronounced increase in grooming behavior in the WAY 100635-saline groups. The treatment effects on grooming were statistically significant F(5, 36)=13.7, P B0.001. The two WAY 100635-saline groups had higher grooming scores than the saline group (P B 0.01) which had higher grooming durations than the three cocaine groups (P B0.01) which did not differ from each other (P \ 0.05). As is evident in Fig. 3 (top), the WAY 100635 pretreatment effect on grooming was the opposite of 8-OHDPAT. During the WAY 100635 pretreatment phase, rearing and circling were also significantly affected by the drug treatments, F(5, 36)=3.6, P B 0.01 and F(5, 36)= 8.4, P \0.001, respectively. For the circling response measure, the three cocaine treatments increased circling relative to the three non-cocaine groups (P B 0.01) and the three cocaine groups did not differ from each other (PB 0.05). In terms of rearing, the saline– cocaine group had more rearing responses than all other groups (PB 0.05). The WAY 100635-cocaine groups did not differ from each other or from the three non-cocaine groups (P\0.05). After the WAY 100635 pretreatments, the groups were switched back to their original 8-OHDPAT pretreatments. Again, the 8-OHDPAT pretreatments exerted statistically significant between group effects upon the three response measures: F(5, 36)= 43.0, P B 0.001, F(5, 36)= 7.3, P B 0.001 and F(5, 36)= 2.7, P B 0.05 for grooming, rearing and circling, respectively. As can be seen in Fig. 3 (top), the 8-OHDPAT pretreatment again suppressed grooming behavior. The group× day interactions were not statistically significant (P \ 0.05). The saline-saline group had higher grooming scores than all other groups (P B0.01). For rearing (middle), the saline–cocaine and 0.2 mg/kg 8-OHDPAT – cocaine group exhibited enhanced rearing compared to the other treatment groups (P B0.05) For circling, the saline – cocaine group exhibited a statistically significant increase in circling as compared to the saline-saline group (P B0.05). None of the other treatment groups differed significantly from the saline-saline group (P\ 0.05). After the completion of the testing with 8-OHDPAT and cocaine, the groups were tested with saline or caffeine (10 mg/kg). The groups which had received the 8-OHDPAT pretreatments were given 0.4 and 0.8 mg/ kg WAY pretreatments. As can be seen in Fig. 4, caffeine enhanced locomotor distance scores and suppressed grooming behavior. Statistical analyses of these results indicated statistically significant between group effects, F(5, 36)= 16.2, P B0.001 and F(5, 36)=11.2, P B0.001, for distance and grooming, respectively. The
Fig. 4. Means and SEMs for distance (upper) and grooming (lower) in which the same six pretreatment groups used in preceding saline/ cocaine tests (n = 7) were treated with saline – caffeine (10 mg/kg). The 8-OHDPAT pretreatment groups received WAY 100635 as pretreatments. The treatment groups were saline-saline, saline-caffeine (10 mg/kg), 0.4 mg/kg WAY 100635 – saline, 0.4 mg/kg WAY 100635 – caffeine (10 mg/kg), 0.8 mg/kg WAY 100635 – saline or 0.8 mg/kg WAY 100635 – caffeine (10 mg/kg) and then tested for 20 min in an open-field. * upper indicates P B0.01 vs all non-caffeine groups. * Lower denotes PB0.01 vs all caffeine groups. ** Indicates PB 0.01 versus the saline – saline and caffeine groups.
WAY 100635 pretreatment did not affect caffeine induced locomotion or saline levels of locomotion. While the WAY 100635 pretreatment did not affect the suppression in grooming behavior induced by caffeine, the WAY pretreatment in combination with saline did increase grooming. This latter observation on grooming is similar to the effect of WAY 100635 on grooming behavior presented in Fig. 2. Thus, the grooming data suggest that the WAY 100635 pretreatment was as effective in the caffeine test as it was in the cocaine phase of the experiment.
4. Discussion In general, the present findings are consistent with a reciprocal facilitatory/inhibitory 5-HT1A agonist/antag-
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onist influence upon cocaine induced locomotor stimulation. This reciprocal relationship appears quite transparent with regard to locomotion distance as the measure of cocaine locomotor stimulant effects but is more complicated for other measures of cocaine effects, such as rearing behavior. In terms of the 5-HT1A agonist facilitation of cocaine locomotor stimulant effects, the two dose levels of 8-OHDPAT had equivalent efficacy in increasing locomotion distance effects and, initially, had similar inhibitory effects in other behavioral aspects of cocaine effects (i.e. central zone activity, circling and rearing). This dual facilitory and inhibitory behavioral actions of 8-OHDPAT on cocaine induced behavioral effects offered a simple behavioral explanation for the 8-OHDPAT enhancement of cocaine induced locomotor stimulation. Namely, 8-OHDPAT suppressed behaviors which competed with and/or interrupted locomotion. That is, the 8-OHDPAT effect upon locomotion appeared to represent a response redistribution effect. Seemingly, it could be argued that the stimulant effect of cocaine was unchanged but its’ behavioral expression was altered by 8-OHDPAT. As the number of treatment sessions increased, however, this account became less tenable. Eventually, the lower dose of 8-OHDPAT given in combination with cocaine generated central zone activity, rearing and circling which matched or exceeded the levels induced by cocaine given alone. While all these response measures increased, the 8-OHDPAT treatment continued to enhance the locomotor distance effect induced by cocaine. Furthermore, the recovery for these response measures was less evident for the higher dose level of 8-OHDPAT but yet the two dose levels remained equally efficacious in enhancing cocaine locomotor distance effects. Altogether, these observations argue against a response redistribution effect of 8-OHDPAT in the enhancement of cocaine induced locomotion. It might be argued, however, that the increase in rearing, circling and central zone activity in the 8-OHDPAT– cocaine treated animals was indicative of the development of tolerance to the 8-OHDPAT. The fact that the 8-OHDPAT treatments continued to enhance cocaine induced locomotion weakens this argument. Furthermore, the 8-OHDPAT treatments maintained suppression of grooming behavior throughout testing indicating a continued pharmacological efficacy. When the 8-OHDPAT treatments were switched to WAY 100635, there were immediate reductions in locomotion distance in the cocaine treated animals which were sustained with repeated treatments. The same WAY 100635 treatments, however, had no effect on locomotion in the saline treated groups. When other behaviors are examined, important differences in the 8-OHDPAT and WAY 100635 were evident. In saline treated animals, the 8-OHDPAT and WAY 100635 had pronounced and opposite effects upon grooming behav-
43
ior. Furthermore, 8-OHDPAT initially induced dose related decreases in rearing and central zone activity whereas, the WAY 100635 treatments did not affect these response measures relative to saline treated animals. Despite the pronounced increase in grooming induced by WAY 100635 given prior to saline, when the WAY 100635 was given in combination with cocaine, it did not reliably attenuate the cocaine induced suppression of grooming. Although the WAY 100635 treatment attenuated commonly used indicators of cocaine induced locomotor activation, namely locomotor distance and rearing, the WAY 100635 treatment did not reliably alter the grooming suppression or the circling induced by cocaine. The present findings provide support for 5-HT1A agonist/antagonist facilitory and inhibitory interaction with cocaine in terms of motoric stimulation but, the relationship has substantial complexity. Straightforward behavioral explanations in terms of response redistribution or response competition while having some utility do not adequately account for the present observations. A critical factor evident in the present study is that the interaction between 8-OHDPAT and cocaine can change with repeated treatments. This modification in the interaction can occur without corresponding changes in the response effects of the drugs given separately. Response effects, however, are not the only behaviorally important dimension of these drugs. The stimulus properties of centrally active drugs are another important facet of the behavioral impact of drugs. The stimulus properties of 8-OHDPAT have been extensively studied and documented [11,42,43]. The possible contribution of the stimulus properties of drugs to behavior in studies of drug effects manifested in openfield behavior are not directly observable but yet may be important. This seems particularly likely in the case of multiple drug treatments as in the present study. Not only are the unconditioned response effects of 8-OHDPAT able to alter the unconditioned response effects of cocaine, but the stimulus effects generated by 8-OHDPAT can become linked to the response effects of cocaine. Thus, a possibility that needs to be considered is there is a repeated association of the 8-OHDPAT drug stimuli with the cocaine responses. As a consequence, the 8-OHDPAT drug stimuli, eventually, may acquire cocaine conditioned stimulus properties. Therefore, with the repeated parings of two drug treatments, the interaction is no longer just a summation of 8-OHDPAT and cocaine unconditioned responses but also may come to include the 8-OHDPAT stimuli activating cocaine conditioned responses. Such a process may contribute to the emergence of cocaine— like central zone activity, rearing and circling in the low dose 8-OHDPAT group given the combined 8-OHDPAT – cocaine treatments.
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In terms of neurotransmission, it is well established [35], that cocaine’s high affinity binding to the catecholamine and indoleamine transporters increases catecholaminergic and serotonergic neurotransmission. While the contribution of catecholaminergic effects of cocaine, particularly dopamine, have been well documented [21,48], the behavioral contribution of this increase in serotonergic activity generated by cocaine has also been recognized [24,27,34,36,47]. When cocaine binds to the 5-HT transporter inhibiting the reuptake of 5-HT from the synaptic cleft, this increase in somatic dendritic 5-HT not only stimulates postsynaptic receptors but also activates inhibitory 5-HT1A autoreceptors which decrease 5-HT neuronal activity [23,40]. Blockade of the 5-HT1A receptors with WAY 100635 can attenuate the inhibitory effects of somatodendritically released 5-HT on 5-HT neurons. The combined effect of local 5-HT reuptake blockade in terminal areas by cocaine combined with 5- HT1A antagonism serves to potentiate extracellular 5-HT concentrations in the terminal regions of 5-HT projections. The potentiating effects of WAY 100635 [15,18] and other 5-HT1A antagonists [16,17] on hippocampal 5-HT concentrations induced by SSRI and by cocaine [30] have been reported. While it has yet to be documented, the expectation would be that a 5-HT1A agonist, such as 8-OHDPAT, would have the opposite effect. From this perspective, the 5-HT1A agonist would attenuate and the 5-HT1A antagonist would enhance extracellular 5-HT level increases induced by cocaine. 5-HT1A agonists and antagonists would therefore shift the balance in the effect of cocaine on DA and 5-HT transmission. That is, for 5-HT1A agonists, the cocaine induced increase in DA would occur in a context of a diminished extracellular 5-HT level and, for a 5-HT1A antagonist, the DA–5HT balance would be shifted in the reverse direction. By positing an inhibitory influence for the increases in hippocampal 5-HT induced by cocaine, the modulation of the 5-HT neurons by a 5-HT1A agonist/antagonist provides a possible account for their reciprocal effects of 5-HT1A agonists/antagonists on the locomotor stimulant effects induced by cocaine. Consistent with this proposition, reductions in 5-HT produced by intracerebral injections of the 5-HT neurotoxin 5,7,dihydroxytryptamine [29] enhance cocaine induced locomotor stimulation. Important in this analysis is the assumption that 5-HT1A agonists/antagonists do not directly affect the increase in DA induced by cocaine. For the dose levels, temporal parameters and the behavioral measurement used in the present study, the evidence to date [3,5,30] is that 8-OHDPAT and WAY 100635 do not alter the increase in DA induced by cocaine. At higher dose levels, temporal parameters or different routes of administration (e.g. s.c.), 8-OHDPAT and WAY 100635 undoubtedly may have effects which would alter the baseline for locomotion
behavior [19]. Furthermore, cocaine effects on locomotion behavior are dose related so that the present results must be viewed within the cocaine dose level parameters used in the present experiment. The effects of 5-HT related drug treatments upon locomotor behavior have been studied extensively. With the variety of 5-HT receptor sites [37] and the wide array of pharmaceuticals with different affinities for different receptor sites coupled with the wide range of dose levels of drug used and behavior measures employed, it becomes an understatement to assert that the situation is complex. High levels of 5-HT stimulation and even selective 5-HT1A postsynaptic stimulation can induce profound effects upon certain behaviors [46]. At more modest levels of 5-HT activation, however, the 5-HT system appears to be implicated in arousal, anxiety and stress [8,12,39]. In particular, the 5-HT1A receptors in the hippocampus have been suggested to participate in the development of tolerance to aversive events [14] and may attenuate stress induced behavioral consequences [32]. WAY 100635 and other 5-HT1A receptor antagonists exhibit anxiolytic effects in the plus maze in rats and mice [10,13]. 8-OHDPAT has also been reported to attenuate stress and cocaine induced increases in plasma corticosterone [41]. On the other hand, 5-HT1A receptor agonists are well documented to increase corticosterone levels [20,26], including the 0.2 mg/kg 8-OHDPAT dose level used in the present study [6]. Cocaine also increases stress related hormones, such as corticosterone [2,25,28]. Thus, 5HT1A drug treatment interactions with cocaine may impact upon processes pertinent to anxiety and stress in some yet to be determined manner which is dose and context dependent. While corticosterone measurement is one facet of assessing this component of the interaction, the aspect of open-field behavior which appears to have some relevance to this issue is the location of activity within the open field. Typically, rats prefer to locomote along the walls at the periphery. Anxiolytic drug treatments tend to facilitate entry into the central zone of the open field [22,45]. In the present study, cocaine enhanced entry into the central zone, whereas, 8-OHDPAT decreases central zone entry. As previously noted, however, both of these treatments increase corticosterone concentrations so this hormonal effect cannot directly account for this behavioral difference. In terms of 5-HT, however, the autoreceptor stimulation induced by 8-OHDPAT would be expected to decrease 5-HT whereas, cocaine increases 5-HT. In that WAY 100635 (0.4 mg/kg) in rats potentiates the 5-HT release in hippocampus induced by cocaine [31], the central zone entry may be related to whether the drug treatment increases or decreases 5-HT. Thus, modification in the 5-HT activity induced by cocaine may be an important component of the behavioral activity generated by cocaine. Drug treatments which modify 5-HT
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effects induced by cocaine alter the balance between catecholaminergic and serotonergic effects induced by cocaine. That is, the cocaine effect may become relatively more dopaminergic if 5-HT effects are diminished or more serotonergic if 5-HT effects are potentiated. Such consideration may have relevance to the treatment of cocaine abuse. Pharmacological modulation of the 5-HT1A receptor may be an effective way to shift this neurochemical balance in the direction of 5-HT. In contrast, drugs which directly block the 5-HT transporter (e.g. fluoxetine) may be ineffective in shifting the 5-HT/DA balance toward 5-HT because any further increase in the blockade of 5-HT transport beyond that induced by cocaine would be negligible.
[11]
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Acknowledgements [17]
This work was supported by NIDA grant DA R01 11457-02 and a VA Merit Review grant. [18]
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