Preclinical Evidence That 5-HT Receptor Agonists Show Promise

International Journal of Neuropsychopharmacology (2017) 20(8): 644–653

doi:10.1093/ijnp/pyx025 Advance Access Publication: April 22, 2017 Regular Research Article

regular research article

Preclinical Evidence That 5-HT1B Receptor Agonists Show Promise as Medications for Psychostimulant Use Disorders Raul Garcia, BS; Austin R. Cotter, Kenneth Leslie, BS; M. Foster Olive, PhD; Janet L. Neisewander, PhD

School of Life Sciences (Mr Garcia, Mr Cotter, Mr Leslie, and Dr Neisewander), and Psychology Department (Dr Olive), Arizona State University, Tempe, Arizona.

Correspondence: Janet L. Neisewander, PhD, School of Life Sciences, ISTB1, Rm 429, 427 E Tyler Mall, Arizona State University, Tempe, AZ 85287-4501 ([email protected]).

Abstract

Background: 5-HT1B receptor agonists enhance cocaine intake during daily self-administration sessions but decrease cocaine intake when tested after prolonged abstinence. We examined if 5-HT1B receptor agonists produce similar abstinence- dependent effects on methamphetamine intake. Methods: Male rats were trained to self-administer methamphetamine (0.1 mg/kg, i.v.) on low (fixed ratio 5 and variable ratio 5) and high (progressive ratio) effort schedules of reinforcement until intake was stable. Rats were then tested for the effects of the selective 5-HT1B receptor agonist, CP 94,253 (5.6 or 10 mg/kg), or the less selective but clinically available 5-HT1B/1D receptor agonist, zolmitriptan (10 mg/kg), on methamphetamine self-administration both before and after a 21-day forced abstinence period during which the rats remained in their home cages. Results: The inverted U-shaped, methamphetamine dose-response function for intake on the fixed ratio 5 schedule was shifted downward by CP 94,253 both before and after abstinence. The CP 94,253-induced decrease in methamphetamine intake was replicated in rats tested on a variable ratio 5 schedule, and the 5-HT1B receptor antagonist SB 224,289 (10 mg/kg) reversed this effect. CP 94,253 also attenuated methamphetamine intake on a progressive ratio schedule both pre- and postabstinence. Similarly, zolmitriptan attenuated methamphetamine intake on a variable ratio 5 schedule both pre- and postabstinence, and the latter effect was sustained after each of 2 more treatments given every 2 to 3 days prior to daily sessions.

Conclusions: Unlike the abstinence-dependent effect of 5-HT1B receptor agonists on cocaine intake reported previously, both

CP 94,253 and zolmitriptan decreased methamphetamine intake regardless of abstinence. These findings suggest that 5-HT1B receptor agonists may have clinical efficacy for psychostimulant use disorders.

Keywords: methamphetamine, addiction, CP 94,253, zolmitriptan, rodent

Introduction

Psychostimulant addiction remains a prevalent problem that the serotonin1B receptor (5-HT1BR) may be a useful target worldwide (NDIC, 2011; NIDA, 2015), and yet there are still no for medication development for these disorders (Callahan and FDA-approved, effective pharmacological treatments for psy- Cunningham, 1995; Rocha et al., 1997; Miszkiel et al., 2011; chostimulant use disorders. We and others have suggested Neisewander et al., 2014). Advances in medicinal chemistry

Received: December 13, 2016; Revised: March 31, 2017; Accepted: April 18, 2017 © The Author 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 644 provided the original work is properly cited. For commercial re-use, please contact [email protected] Garcia et al. | 645

International Journal of Neuropsychopharmacology (2017) 20(8): 644–653 doi:10.1093/ijnp/pyx025 Significance Statement Advance Access Publication: April 22, 2017 Psychostimulant addiction (e.g., cocaine and methamphetamine) remains a prevalent problem in the United States. Regular Research Article

Prior studies suggest that the serotonin1B receptor (5-HT1BR) modulates cocaine reinforcement and incentive motivation dependent on whether there is a period of abstinence from cocaine in male rats. This study demonstrates that measures

of methamphetamine drug taking in rats are attenuated by pretreatment with 5-HT1BR agonists, including the FDA-

approved agonist zolmitriptan, regardless of abstinence history. Our findings suggest that 5-HT1BR agonists may be useful for treating psychostimulant use disorders.

have discovered drugs with high selectivity for 5-HT1BRs (Koe we examined if acute and intermittent repeated treament with et al., 1992; Selkirk et al., 1998; Murray and Rees, 2009; Rodriguez zolmitriptan affected methamphetamine intake. Since both et al., 2014). The agonist 5-propoxy-3-(1,2,3,6-tetrahydro-4- methamphetamine and cocaine enhance monoaminergic neu- pyridinyl)-1H-pyrrolo[3,2-b]pyridine (CP 94,253) and the antago- rotransmission by an action at monoamine transporters, we nist 1’-methyl-5-[[2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl) hypothesized that CP 94,253 would increase methamphetamine biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro[furo[2,3-f] intake preabstinence and decrease methamphetamine intake

indole-3,4’-piperidine (SB 224,289) have high affinities (Ki = 2 postabstinence similar to that observed with cocaine intake.

and 8.2 nM, respectively) for 5-HT1BRs (Koe et al., 1992; Selkirk et al., 1998). The selectivity profiles of CP 94,253 and SB 224,289 Methods have established these drugs as useful tools for studying the

role of 5-HT1BRs in psychostimulant addiction. Another group of Animals

5-HT1BR agonists are tryptamine based, including zolmitriptan (Zomig), which is an FDA-approved drug used to treat migraine Male Sprague Dawley rats (Charles River) weighing 225 to 250 g were single-housed in a climate-controlled environment on a headaches. Although zolmitriptan is not as selective for 5-HT1BRs 14:10 reverse light/dark cycle (lights off at 6:00 am). Rats had as CP 94,253, it has a high affinity (Ki = 5.01 nM) for 5-HT1BRs. ad libitum access to food except for initial self-administration Initial experiments examining the effects of 5-HT1BR agonists found that these drugs facilitated cocaine self-administra- training when they were food restricted to 90% of their ad libi- tion (Parsons et al., 1998). However, we found that the effects of tum weights. The experiments proceeded in accordance with a protocol approved by the Arizona State University Institutional 5-HT1BR agonists vary depending on whether or not animals have undergone abstinence. Specifically, CP 94,253 shifts the cocaine Animal Care and Use Committee. self-administration dose-response curve leftward when given as a pretreatment prior to a daily self-administration session (preabsti- Drugs nence) but produces a downward shift when given as a pretreat- Methamphetamine hydrochloride (Sigma-Aldrich) was dissolved ment prior to resumption of self-administration after prolonged in bacteriostatic saline (Hospira Inc.) and filtered with 0.2-µm (i.e., 21 days) abstinence (Pentkowski et al., 2009; Pentkowski et al., membrane Acrodisc syringe filters (PALL Corporation). CP 94,253 2014). In addition, CP 94,253 pretreatment increases breakpoints hydrochloride (Tocris Bioscience) was dissolved in saline, and and cocaine intake on a progressive ratio (PR) schedule compared SB 224,289 hydrochloride (Tocris Biosciences) was dissolved in with vehicle pretreatment when tested during daily maintenance 10% (2-hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich) in saline sessions. In contrast, following a 21-day period of forced absti- and sonicated for 2 minutes. Zolmitriptan (Sigma-Aldrich) was nence (postabstinence), CP 94,253 decreases cocaine intake and dissolved in 10% dimethyl sulfoxide in saline and sonicated for 5 response rates on the PR schedule. Furthermore, CP 94,253, atten- minutes. CP 94,253, SB 224,289, and zolmitriptan were prepared uates cocaine-seeking behavior in tests of both cue-induced and fresh daily. Vehicle refers to the respective solvent. All drug cocaine-primed reinstatement following a few weeks of extinction injections, with the exception of self-administered metham- training during which the rats were abstinent (Pentkowski et al., phetamine, were injected at a volume of 1 mL/kg body weight. 2009). Cocaine-seeking behavior under these conditions reflects incentive motivational effects produced by the cues and cocaine Surgery priming injections (Markou et al., 1993). Therefore, these results

suggest that preabstinence administration of 5-HT1BR agonists Rats underwent surgery for implantation of chronic indwelling facilitates the reinforcing and motivational properties of cocaine catheters into the jugular vein as detailed previously (Pockros

while postabstinence 5-HT1BR agonists attenuate these effects. et al., 2011). Rats had 6 to 7 days of recovery before commenc- This study examined if CP 94,253 produces a similar absti- ing self-administration training. Catheters were flushed daily nence-dependent decrease in methamphetamine intake. First, with 0.1 mL of either timentin (experiment 1; 66.67 mg/mL; we examined CP 94,253 pretreatment effects on the metham- GlaxoSmithKline) or cefazolin (experiment 2, 3, 4, and 5; 10 mg/ phetamine self-administration dose-response function using mL; WG Critical Care, LLC) mixed with heparin/saline (70 U/mL; low ratio schedules of reinforcement (i.e., fixed and variable APP Pharmaceuticals). In addition, catheter patency was tested ratio 5; FR5 and VR5). Second, we examined CP 94,253 pretreat- periodically by administering 0.05 mL of methohexital sodium ment effects on a PR schedule of methamphetamine reinforce- (16.7 mg/mL; Jones Pharma Inc.), a dose that produces brief loss ment as this more demanding schedule is particularly sensitive of muscle tone when administered i.v. to changes in motivation for a drug. Third, we examined if the effects of CP 94,253 pretreatment on methamphetamine intake Apparatus were 5-HT1BR mediated by administering the 5-HT1BR antagonist SB 224,289 to reverse the agonist effects. Fourth, we examined The operant conditioning chambers (Med Associates) contained if CP 94,253 and SB 224,289 affected locomotor activity. Finally, an active and inactive lever, a cue light, and a tone generator as 646 | International Journal of Neuropsychopharmacology, 2017

previously described (Pentkowski et al., 2010). All chambers had between doses. After again meeting the stability criterion for infusion pumps (Med Associates) that connected to liquid swiv- reinforcement rates across the within-session dose-response els (Instech) fastened to an outlet polyethylene tubing sheltered training days (20–28 sessions), rats were randomly assigned to within a metal leash (PlasticsOne) that attached to the rat’s cathe- receive an injection of either CP 94,253 (5.6 mg/kg, s.c.) or vehi- ter. All operant conditioning chambers were housed within sound cle 15 minutes prior to their daily dose-response session. The attenuating boxes that contained a ventilation fan. dose of CP 94,253 was selected based on our previous research demonstrating that it selectively reduces cocaine intake without General Procedures affecting sucrose intake (Pentkowski et al., 2009). After this first test session, additional training sessions were given until rats Experimental sessions commenced at approximately the same met the stability criterion. Then rats were tested again, receiv- time of day, 6 days/week during the rats’ dark cycle. Rats were ing the treatment opposite from their first treatment (i.e., ani- first trained to self-administer 0.1 mg/kg (i.v.) methampheta- mals that received vehicle first, now received CP 94,253 and vice mine on an FR1 schedule of reinforcement, and they later pro- versa). After this test session, rats underwent abstinence for 21 gressed to either a FR5, VR5, or PR schedule based on individual days, during which they remained in their home cages but con- performance. In all experiments, schedule advancement during tinued to receive daily i.v. administration of heparin/antibiotic training occurred once rats received at least 10 reinforcers/ses- to maintain catheter patency. sion for 2 consecutive sessions, and testing commenced once For the postabstinence tests, rats received CP 94,253 or vehi- rats reached a stability criterion of <15% variability in the num- cle, and 15 minutes later, they were given access to methamphet- ber of infusions obtained across 3 consecutive sessions. The amine using the same within session dose-response procedure training dose was choosen as it has been shown to be effective as used during preabstinence tests. Rats then remained absti- in producing methamphetamine acquisition (Kitamura et al., nent in the home cage for 3 days to allow time for CP 94,253 to 2006; Clemens et al., 2006; Krasnova et al., 2010). The VR5 sched- be eliminated and to reinstate an abstinence period prior to the ule was chosen for its tendency to sustain consistent steady second test. On the second test day, the rats received the treat- response rates (Domjan, 2003). Session length was 2 hours, ment opposite from that given prior to the first postabstinence except for training and testing on the PR schedule for which ses- test. A total of 10 rats ran through the entire experiment and sions were 4 hours long to capture break points in most rats. In their average final weight was 468 ± 4.94 g. the PR schedule, the number of active lever responses required to obtain each subsequent infusion increased exponentially Experiment 2: Effects of CP 94,253 on (e.g., 1, 2, 4, 6, 9, 12, 15, etc.), identical to the exponential equa- Methamphetamine Self-Administration on a tion from Richardson and Roberts (1996). The PR schedule was Progressive Ratio Schedule Pre- and Postabstinence chosen because it is progressively more effortful to obtain reinforcement, reflecting in part how motivated a rat is to work for The timeline for this experiment is outlined in Figure 2. A new methamphetamine (Markou et al., 1993). Therefore, the pattern cohort of experimentally naïve rats was food restricted and of changes across various schedules of reinforcement can help trained to self-administer methamphetamine, progressing from inform how treatments affect behavioral processes. Completion a FR1 to a VR5 schedule during 2-hour sessions (11–13 sessions) of any of the operant schedules activated the tone and light using the same procedure as the previous experiment, with the cue followed 1 second later by an infusion of 0.1 mL metham- exception that food restriction was gradually discontinued once phetamine delivered across 6 seconds (dose is given in the spe- rats were on the VR5 schedule. After meeting the stability crite- cific experiments section). The tone, light, and pump were then rion on the VR5 schedule, rats were trained on the PR schedule turned off, and simultaneously a house-light was activated for of methamphetamine reinforcement during 4-hour sessions 20 seconds to signal a timeout period during which additional until again meeting the stability criterion (10–18 sessions). We lever responses were recorded but had no programmed conse- capped session length to 4 hours to ensure that CP 94,253 would quences. After the timeout period, the house-light turned off remain effective throughout the test (Parsons et al., 1998). We and methamphetamine was available again. Abstinence from also reduced the methamphetamine dose to one-half the train- methamphetamine occurred for 21 or more consecutive days, ing dose (0.05 mg/kg, i.v.) with the intention that more rats during which the rats were maintained in the home cage, han- would reach break point during the 4-hour session. Breakpoint dled, and weighed daily for i.v. administration of timentin or was defined as the last schedule of reinforcement completed cefazolin to maintain catheter patency. prior to a 1-hour period during which the next required ratio failed to be completed, or 4 hours had elapsed, whichever came Experiment 1: Effects of CP 94,253 on the first. After rats met the stability criterion on the PR schedule, Methamphetamine Self-Administration Dose- they were randomly assigned to either a CP 94,253 (10 mg/kg, Response Function Pre- and Postabstinence s.c.; n = 8) or a vehicle group (n = 7), counterbalanced for similar number of total drug infusions during training. The dose of CP The timeline for this experiment is outlined in Figure 1. 94,253 was higher than the previous experiment to sustain CP Beginning 2 days prior to the start of training and continuing 94,253 levels throughout testing, and based on previous research throughout the experiment, rats were food restricted to 90% of this dose selectively decreases cocaine intake while not affect- their initial free-feeding body weight (291 ± 4.6 g). During training sucrose intake (Pentkowski et al., 2009). These groups ing, rats progressed from a FR1 to FR5 schedule (~17–20 sessions) received their respective treatments 15 minutes before test- after reaching a criterion of ≥10 infusions for 2 consecutive ses- ing on the PR schedule. After testing, both groups of rats were sions. Once reinforcement rates met the stability criterion on placed into abstinence for 21 days as described in experiment the FR5, the rats commenced training on a within-session dose- 1. Postabstinence, both groups received their injections, which response procedure. For these sessions, each methamphetamine were identical to those given preabstinence, 15 minutes prior to dose (0.003, 0.01, 0.03, 0.1, and 0.30 mg/kg, i.v.) was available in a test for resumption of methamphetamine self-administration ascending order for 30 minutes with a 5-minute time-out period on the PR schedule. Garcia et al. | 647

Figure 1. Effects of the 5-HT1B receptor agonist, CP 94,253 on infusions (A,D), active lever (B,E), and inactive lever (C,F) responses on a FR5 schedule of methamphetamine (0.1 mg/kg, i.v.) reinforcement during pre- and postabstinence tests (left and right panels, respectively). Data are expressed as the mean (± SEM) during the 30-minute test period for each of the methamphetamine doses tested (0.003–0.30 mg/kg, i.v.). Rats (n = 10) were tested twice, receiving pretreatment with vehicle (1 mL/kg, s.c.; open squares) prior to one test and CP 94,253 (5.6 mg/kg, s.c.; filled triangles) prior to the other test, with order of pretreatment counterbalanced. Insets in A-E show a main effect of CP 94,253 averaged across methamphetamine doses. Asterisks (*) represent a difference from vehicle condition (main effect or Tukey’s posthoc test, P < .05).

Experiment 3: Reversing the Attenuating schedule (~12–17 sessions) of methamphetamine (0.1 mg/kg, i.v.) Effects of CP 94,253 on Methamphetamine Self- reinforcement. Rats were food restricted only during acquisition Administration with SB 224,289 of self-administration and all sessions lasted for 2 hours. Once reinforcement rates stabilized under free feeding conditions The timeline for this experiment is outlined in Figure 3. A new (~4–12 sessions), rats were randomly assigned to 1 of 2 groups cohort of experimentally naïve rats was trained to self-admin- (n = 14 and 17, respectively); Group 1 received an i.p. injection ister methamphetamine, progressing from a FR1 to a VR5 of either vehicle or SB 224,289 (10 mg/kg, i.p.) 30 minutes prior 648 | International Journal of Neuropsychopharmacology, 2017

Figure 2. Effects of the 5-HT1B receptor agonist, CP 94,253, on infusions (A), breakpoints (B), active lever (C), and inactive lever (D) responses under a progressive ratio schedule of methamphetamine (0.05 mg/kg, i.v.) reinforcement during pre- and postabstinence tests. Data are expressed as the mean (± SEM) during 4-hour sessions. Rats were pretreated 15 minutes prior to the start of the test sessions with either vehicle (1 mL/kg, s.c.; white bars; n = 7) or CP 94,253 (10 mg/kg, s.c.; black bars; n = 8). Asterisks (*) represent a difference from vehicle at each time point (Tukey’s posthoc test, P < .05). to the first test and the opposite treatment injection 30 min- to record horizontal movement with Topscan software (Clever utes prior to the second test (i.e., rats that received vehicle on Systems). The rats were then tested twice for locomotor activity test 1 received SB 224,289 on test 2 and vice versa). Group 1 also with 3 rest days intervening the 2 test days. They remained in received a vehicle injection 15 minutes prior to both tests. For their home cages during rest days. Thirty minutes prior to the Group 2, identical procedures were followed except that rats first test, rats were pretreated with either vehicle or SB 224,289 received vehicle followed by CP 94,253 (5.6 mg/kg, s.c.) on one (10 mg/kg, i.p.) and 30 minutes prior to the second test the rats test day and SB 224,289 (10 mg/kg, i.p.) followed by CP 94,253 received the opposite treatment as that given on the first test (5.6 mg/kg, s.c.) on the other test day. (i.e., rats that received vehicle on test 1 received SB 224,289 on In addition to testing whether the antagonist would reverse test 2 and vice versa). They were also randomly assigned to 1 of the effects of the agonist in this experiment, we also verified 2 groups. Group 1 (n = 7) received a vehicle injection 15 minutes that these rats showed a CP 94,253-induced decrease in meth- prior to both tests, and Group 2 (n = 7) received CP 94,253 (10 mg/ amphetamine intake postabstinence (not included on time- kg, s.c.) 15 minutes prior to both tests. The tests began by placing line). A subset of rats, randomly selected from both Group 1 and the rat into the test chamber, and distance traveled was meas- Group 2, underwent 21 days of abstinence as described above. ured for 2 hours. They were then assigned to 2 groups, counterbalanced for the number of infusions obtained during training. One group (n = 11) Experiment 5: Effects of Zolmitriptan on received an injection of CP 94,253 (5.6 mg/kg, s.c.) while the other Methamphetamine Self-Administration Pre- and group (n = 11) received an injection of vehicle 15 minutes prior to Postabstinence a test for resumption of methamphetamine self-administration (0.1 mg/kg, i.v.). The timeline for this experiment is outlined in Figure 5. Rats that were used in experiment 3 were tested for the effects of zolmitriptan on methamphetamine after achieving stable SA rates Experiment 4: Effects of 5-HT1BR Drugs on Spontaneous Locomotion on a VR5 schedule (~3–11 sessions) of methamphetamine reinforcement (0.1 mg/kg, i.v.) across 2-hour training sessions. Rats The timeline for this experiment is outlined in Figure 4. Rats were then randomly assigned to either a zolmitriptan (10 mg/ from experiment 2 were used and they had a history of metham- kg, s.c.; n = 9) or a vehicle treatment group (n = 6), counterbal- phetamine self-administration (38 sessions) and had undergone anced for similar number of total drug infusions. Rats received an abstinence period (23 days). On abstinence day 24, rats were their assigned treatment 15 minutes prior to the start of a self- placed into locomotor test chambers for a 60-minute habitua- administration session. Then rats underwent a period of absti- tion period. The test chambers (45.72 x 25.4 x 20.32 cm) were nence for 29 to 36 days followed by a test phase. During the test similar to the home cages and had a camera mounted above phase, the rats again received their assigned treatment of either Garcia et al. | 649

Figure 3. Reversing the attenuating effects of CP 94,253 on methamphetamine Figure 4. Effects of 5-HT receptor drugs on spontaneous locomotion. Rats were (0.1 mg/kg, i.v.) self-administration with SB 224,289 during tests that occurred 1B placed on abstinence from methamphetamine for a total of 29 days following preabstinence. On the first test day, rats (group 1: n = 14, group 2: n = 17) received acquisition and stabilization on a progressive ratio schedule of reinforcement. a pretreatment of either vehicle (Veh, white bar) or SB 224,289 (SB; 10 mg/kg, i.p., Habituation to the locomotor testing chambers occurred on abstinence day 24. gray bar) 30 minutes before the 2-hour session. They then received a treatment On the first test day, rats (group 1: n = 7, group 2: n = 7) received a pretreatment of either vehicle (Veh) or CP 94,253 (CP; 5.6 mg/kg, s.c.; black bar) 15 minutes of either vehicle (Veh, white bar) or SB 224,289 (SB; 10 mg/kg, i.p., gray bar) 30 prior to the test session that commenced under a VR5 schedule of reinforce- minutes before commencement of the session. They then received a treatment ment. Conditions were identical on the second test day for all rats except that of either vehicle (Veh) or CP 94,253 (CP; 10 mg/kg, s.c., black bar) 15 minutes prior the pretreatment given 30 minutes before session start was reversed such that to the same session. Conditions were identical on the second test day for all rats rats that had received vehicle previously were given SB 224,289 and rats that except that the pretreatment given 30 minutes before session start was reversed had received SB 224,289 previously were given vehicle. Data are expressed as the such that rats who had received vehicle previously were given SB 224,289 and mean (+ SEM). Asterisk (*) represents a difference from all other groups (Tukey’s vice versa. All rats underwent 3 days off between tests 1 and 2 while remain- posthoc test, P <.05). ing in abstinence. Data are expressed as the mean (+SEM) distance traveled in

meters across the 2-hour session. Pretreatment with 5-HT1B receptor drugs pro- zolmitriptan or vehicle 15 minutes prior to each of 3 self-admin- duced no significant differences in spontaneous locomotion. istration sessions with 2 to 3 self-administration sessions without treatment between each of the treatment test days. active lever responses [F (4, 36) = 8.40, P < .05]. Posthoc tests indicated that the 0.01- and 0.03-mg/kg doses produced higher values Data Analysis than the lowest dose (0.003 mg/kg) and the highest dose (0.30 mg/ kg) produced lower values than all doses except 0.10 mg/kg (Tukey’s Statistical analyses were conducted with IBM SPSS Statistics comparisons, P < .05) (Figure 1A,B). There were also main effects of v. 23. Descriptive statistics are reported as the mean ± SEM. treatment, which indicated that averaged across methampheta- Self-administration data, including active and inactive lever mine dose, rats exhibited lower infusion and active lever response responses, and infusions obtained were analyzed by either rates when pretreated with CP 94,253 than when pretreated with repeated-measures or a mixed-design ANOVA with drug vehicle (Figure 1A,B,insets). There were no significant effects for pretreatment(s) and dose of methamphetamine as between- inactive lever responses during preabstinence tests (Figure 1C). subject or within-subject factors depending on the experimen- Analysis of postabstinence infusions showed main effects of tal design. In addition, breakpoint and total distance travelled treatment [F (1, 9) = 30.74, P < .05] and methamphetamine dose were analyzed as described above for experiments 2 and 4, [F (4, 36) = 35.27, P < .05], as well as a treatment by metham- respectively. All sources of significant effects were further ana- phetamine dose interaction [F (4, 36) = 2.87, P < .05]. CP 94,253 lyzed by Tukey’s posthoc tests. There was some attrition in each pretreatment decreased infusions at the 3 lowest doses of meth- experiment due to catheter failure or failure to acquire SA. amphetamine compared with vehicle pretreatment (Tukey’s comparisons, P < .05) (Figure 1D). The analysis of postabstinence Results active lever responses also revealed main effects for treatment [F (1, 9) = 10.38, P < .05] and methamphetamine dose [F (4, Experiment 1 36) = 22.20, P < .05], but no treatment by methamphetamine dose interaction. The main effect of methamphetamine dose was due Methamphetamine produced an inverted U-shaped dose-effect to the inverted U-shaped dose-response function similar to pre- function, and CP 94,253 decreased methamphetamine infusions abstinence. The main effect of treatment shows that averaged and active lever responses both pre- and postabstinence (Figure 1). across methamphetamine doses, rats exhibited lower infusions For the preabstinence tests, there were main effects of metham- and active lever response rates when pretreated with CP 94,253 phetamine dose for both infusions [F (4, 36) = 17.67, P < .05] and than when pretreated with vehicle (Figure 1D,E, insets). There 650 | International Journal of Neuropsychopharmacology, 2017

The rats in this experiment underwent 21 days of abstinence and were then tested for the effects of CP 94,253 (5.6 mg/kg, s.c.) on resumption of methamphetamine self-administration (0.1 mg/kg, i.v.). Similar to the results from experiment 1, pretreatment with a 5.6-mg/kg dose of CP 94,253 decreased the number of infusions and active lever responses compared with pretreatment with vehicle during the postabstinence tests (data not shown). The mean number of methamphetamine infusions obtained in the vehicle vs CP 94,253 pretreatment groups was 12.40 ± 0.88 and 8.90 ± 0.71, respectively [t (9) = 7.72, P < .05]. The mean number of active lever responses in the vehicle vs CP 94,253 pretreatment groups was 70.30 ± 20.81 and 49.20 ± 17.89, respectively [t (9) = 2.62, P < .05]. There was no group difference for inactive lever responses.

Experiment 4

Neither CP 94,253 or SB 224,289 pretreatment altered locomotor activity (Figure 4). There were no main or interaction effects between treatment and the total distance traveled by rats.

Experiment 5

Figure 5. Effects of zolmitriptan, a 5-HT receptor agonist, on infusions 1B/1D Acute zolmitriptan treatment decreased methamphetamine (A,C), and active lever (B,C) responses on a VR5 schedule of methamphetamine infusions and active lever responses during preabstinence tests (0.1 mg/kg, i.v.) reinforcement during pre- and postabstinence tests (left and right panels, respectively). Data are expressed as the mean (±SEM). Rats were (Figure 5A,B). Comparisons between vehicle and zolmitriptan pretreated 15 minutes prior to the start of the 2-hour sessions with either vehi- showed a difference in infusions [t (10) = 3.50, P < .05] and active cle (white bar/open circles; n = 6) or zolmitriptan (10 mg/kg, s.c.; black bar/filled lever responses [t (10) = 2.90, P < .05]. There were no effects on squares; n = 9). Asterisks (*) represent a difference from vehicle condition (t test inactive lever responses after vehicle or zolmitriptan treatment or ANOVA main effect, P < .05). with means of 39.18 ± 16.37 and 16.55 ± 6.41, respectively. Zolmitriptan pretreatment given intermittently across 3 were no significant effects for inactive lever responses during postabstinence tests consistently decreased infusions and the postabstinence tests (Figure 1F). active lever responses (Figure 5C,D). The ANOVA showed a main effect of treatment group for infusions [F (1, 11) = 21.92, P < .05] but no effect of treatment day or treatment group by treatment Experiment 2 day interaction. For active lever responses, there were significant main effects of treatment group [F (1, 10) = 21.17, P < .05] CP 94,253 decreased methamphetamine infusions, breakpoint, and treatment day [F (2, 20) = 6.08, P < .05], but no treatment and active lever responses (Figure 2, A-C). Most rats reached group by treatment day interaction. Posthoc tests for treatment breakpoint within the 4-hour test during both the preabstinence day showed that zolmitriptan treatment produced lower active (80%) and postabstinence (73%) tests. The analyses showed main lever response rates on day 2 compared with treatment days 1 effects of pretreatment across pre- and postabstinence tests for and 3. There were no effects on inactive lever responses after infusions [F (1, 13) = 9.86, P < .05], breakpoints [F (1, 13) = 6.46, vehicle or zolmitriptan treatment with means of 7.89 ± 3.68 and P < .05], and active lever responses [F (1, 12) = 11.09, P < .05]. 3.56 ± 0.68, respectively. In each case, CP 94,253 pretreatment decreased these measures compared with the vehicle pretreatment (Tukey’s comparisons, P < .05). There were no main effects for inactive lever responses Discussion (Figure 2D) and no pretreatment by time interaction effects for any of the measures. Unlike the abstinence-dependent modulatory role of 5-HT1BR agonists on cocaine intake that we observed previously (Pentkowski et al., 2012, 2014), this study found that 5-HT R ago- Experiment 3 1B nists attenuated methamphetamine intake when given either CP 94,253 decreased methamphetamine infusions, and this pre- or postabstinence. Specifically, a moderate dose of CP 94,253 effect was blocked by SB 224,289 (Figure 3). Data analysis showed (5.6 mg/kg, s.c.) decreased methamphetamine intake and active a main effect of the first pretreatment (i.e., vehicle vs SB 224,289) lever response averaged across methamphetamine dose avail- [F (1, 29) = 6.86, P < .05] and an interaction between the first and able both when administered during maintenance of daily self- second (vehicle vs CP 94,253) pretreatments [F (1, 29) = 12.52, P < administration sessions and following a period of abstinence .05]. Posthoc tests showed that pretreatment with vehicle + CP (main effect of pretreatment, Figure 1, insets). After abstinence, 94,253 decreased methamphetamine infusions compared with the effect of CP 94,253 was more pronounced at lower doses of all other pretreatment conditions, including SB 224,289 + CP methamphetamine, primarily because intake appeared higher 94,253 (Tukey’s comparisons, P < .05). This finding indicates that under the vehicle pretreatment condition postabstinence com- administration of the antagonist SB 224,289 blocked the attenu- pared with preabstinence. This enhancement of cocaine intake ating effect of CP 94,253 on methamphetamine intake, suggest- postabstinence is consistent with sensitized cocaine self-admin- ing this effect was mediated by 5-HT1BRs. There were no effects istration reported previously (Schenk and Partridge, 1997). Thus, for active or inactive lever responses (data not shown). the findings suggest that CP 94,253 attenuates expression of the Garcia et al. | 651

abstinence-induced, enhanced sensitivity to methampheta- 5-HT1BR overexpression by viral gene transfer attenuate cocaine mine observed with vehicle pretreatment. CP 94,253 (10 mg/ self-administration following a period of abstinence, as well kg, s.c.) also decreased methamphetamine intake (0.05 mg/kg, as reduce cocaine-seeking behavior (Pentkowski et al., 2009, i.v.) under a PR schedule of reinforcement both pre- and postab- 2012, 2014). Furthermore, these agonist effects are reversed by stinence (Figure 2), further suggesting attenuation of metham- 5-HT1BR antagonists, including SB 224,289, supporting a 5-HT1BR phetamine reinforcing and/or motivational effects. Importantly, mechanism. administration of the 5-HT1BR antagonist, SB 224,289 (10 mg/ We were surprised that CP 94,253 attenuated metham- kg, i.p.), blocked the attenuating effects of CP 94,253 (5.6 mg/kg, phetamine intake prior to any abstinence given that this s.c.) on methamphetamine intake in rats tested during mainte- same treatment enhances cocaine intake prior to abstinence nance of self-administration on a VR5 schedule (Figure 3), sug- (Pentkowski et al., 2009, 2014). However, others have shown gesting that the effects of the agonist are mediated by 5-HT1BRs. that 5-HT1BR agonists attenuate d-amphetamine intake with- Finally, we report that zolmitriptan (10 mg/kg, s.c.) also attenu- out prolonged abstinence, as well as attenuate d-ampheta- ated methamphetamine intake on a VR5 schedule. Zolmitriptan mine-induced responding for conditioned reward (Fletcher treatment given acutely during maintenance, as well as given and Korth, 1999; Fletcher et al., 2002; Miszkiel et al., 2012; intermittently following abstinence, decreased methampheta- Miszkiel and Przegalinski, 2013). Therefore, the 5-HT1BR ago- mine intake and active lever responses (Figure 5). nist enhancement of cocaine intake prior to abstinence may

There are a number of possible reasons for the 5-HT1BR ago- be due to differences in the pharmacological actions of cocaine nist-induced decreases in methamphetamine self-administra- vs amphetamines. While both amphetamines and cocaine tion, including an effect on methamphetamine reinforcement inhibit and downregulate 5-HT, dopamine, and norepineph- value and/or incentive motivation, an effect on anxiety, or an rine transporters (Azzaro and Rutledge, 1973; Ritz, Cone, and effect on motor capability. The decrease in methamphetamine Kuhar, 1990), they interact differently with the transporters. intake is unlikely due to impairments in motor capability, as Amphetamines, including methamphetamine, not only inhibit treatment with the 5-HT1BR ligands did not alter spontaneous monoamine transport, but also redistribute intracellular mon- locomotion (Figure 4) or inactive lever responses at the doses oamines by acting at the vesicular monoamine transporter used in the present study. Furthermore, previous research from causing release of monoamines into the cytosol while at the our laboratory has shown that CP 94,253 (0.3–10.0 mg/kg, s.c.) same time reversing monoamine transport across the plasma has no effect on sucrose reinforcement (Pentkowski et al., 2009). membrane, resulting in monoamine release (Sulzer et al., Although the antagonist SB 224,289 decreases cocaine-induced 1995; Sager and Torres, 2011; Panenka et al., 2013). Cocaine and locomotion in drug naïve rats (Hoplight et al., 2005), it has no amphetamines also interact at different sites on the dopamine effect on locomotion in rats with a history of cocaine self- transporter and produce differential effects on the releas- administration (Pentkowski et al., 2009, 2014). Thus, it seems able vesicular pool and on regulation of vesicular monoamine unlikely that CP 94,253 or SB 224,289 altered methamphetamine transporter (Thomsen et al., 2009). The latter effects may result intake by impairing motor capability. in differences in the releasable pool of dopamine after cocaine We cannot rule out the possibility that CP 94,253 may vs methamphetamine following acute or subchronic adminis- have influenced methamphetamine intake nonspecifically by tration (Brown et al., 2001). increasing anxiety-like behaviors rather than attenuating rein- Although the specific mechanisms responsible for the atten- forcement per se. Indeed, previous studies have found that uating effect of 5-HT1BR agonists on cocaine and methampheta- either 5-HT1BR agonists or antagonists can increase baseline and mine self-administration are unclear, we hypothesize that such cocaine-induced anxiety-like behaviors (Lin and Parsons, 2002; mechanisms may involve a dysregulation of 5-HT1BR functions

Hoplight et al., 2005; Pentkowski et al., 2009). It is important to (Neisewander et al., 2014). 5-HT1BRs are widely distributed in note that the rats in these previous studies were naïve to the the brain (Bruinvels et al., 1994; Lanfumey and Hamon, 2004; experimental procedures used to assess anxiety-like behaviors, Varnas et al., 2005; Clark et al., 2006) and are expressed as either which would likely maximize any potential drug effect. In con- 5-HT terminal autoreceptors or heteroreceptors on terminals of trast, rats tested for CP 94,253 effects on methamphetamine non-5-HTergic cells. In both cases, these receptors negatively self-administration in the present study were well habituated to couple to adenylyl cyclase activity via G-proteins and func- the testing environment, which would likely minimize potential tion to inhibit neurotransmitter release (Hen 1992; Sari, 2004; anxiogenic effects. Furthermore, animals with increased expo- McDevitt and Neumaier, 2011; Barnes and Neumaier, 2011). sure to stress, such as foot-shock, often increase rather than Several manipulations in the mesolimbic system have provided decrease drug intake (Goeders and Guerin, 1994; Ahmed and evidence for a modulatory role of 5-HT1BRs in psychostimulant

Koob, 1997; Piazza and Le Moal, 1998; Logrip et al., 2012), mitigat- addiction; specifically, overexpression of 5-HT1BRs in the nucleus ing the idea that CP 94,253 may have induced anxiety-like states accumbens of rats facilitates the rewarding and reinforcing that interfered with responding. Finally, it seems that potential effects of cocaine (Neumaier et al., 2002; Pentkowski et al., 2012). anxiogenic effects of CP 94,253 on reinforcement would mani- Furthermore, local activation of 5-HT1BRs in the ventral tegmen- fest as a decrease in both sucrose and psychostimulant intake, tal area alters cocaine-induced increases in dopamine levels yet CP 94,253 has been shown to selectively decrease cocaine in the nucleus accumbens and cocaine-induced decreases in intake (Pentkowski et al., 2009). gamma-aminobutyric acid levels (Parsons et al., 1999; O’Dell and

We suggest that the most likely explanation for the agonist- Parsons, 2004). Similarily, activation of 5-HT1BRs in the nucleus induced decreases in methamphetamine intake in the present accumbens dose-dependently decreases the rewarding and study is that 5-HT1BRs modulate psychostimulant reinforce- reinforcing effects of amphetamine (Fletcher and Korth, 1999; ment and/or incentive motivation (Pentkowski et al., 2012, Fletcher, 2002).

2014). The decrease of methamphetamine intake in response The present findings suggest that 5-HT1BRs are potential to 5-HT1BR stimulation following abstinence may result from targets for developing pharmacotherapies for psychostimu- an attenuation of the expression of enhanced sensitivity to the lant addiction. Here we show that the clinically available reinforcing effects of methamphetamine. This explanation is anti-migraine medication zolmitriptan, which is a 5-HT1B/1DR consistent with our previous findings that 5-HT1BR agonists or agonist, attenuated methamphetamine intake. The attenuation 652 | International Journal of Neuropsychopharmacology, 2017

of methamphetamine intake was likely mediated, at least in Azzaro AJ, Rutledge CO (1973) Selectivity of release of norepi- part, by stimulation of 5-HT1BRs, although it is possible that nephrine, dopamine, and 5-hydroxytryptamine by amphet-

5-HT1DRs may have also contributed to the attenuation effect. amine in various regions of rat brain. Biochem Pharmacol

Zolmitriptan, unlike CP 94,253, has a higher affinity for 5-HT1DRs 22:2801–2813.

(Ki = 0.63 nM) than for 5-HT1BRs (Ki = 5.01 nM; Murray, et al., Barnes NM, Neumaier JF (2011) Neuronal 5-HT receptors and

2011). CP 94,253 also has affinity for 5-HT1DRs (Ki = 49 nM; Koe SERT. Tocris Bioscience Review 1–16. et al., 1992), and therefore 5-HT1DRs may also contribute to its Brown JM, Hanson GR, Fleckenstein AE (2001) Regulation of the effects on methamphetamine self-administration. The effects vesicular monoamine transporter-2: a novel mechanism of zolmitriptan on methamphetamine self-administration were for cocaine and other psychostimulants. J Pharm Exp Ther not likely due to a decrease in general activity, as we did not 296:762–767 observe any differences in inactive lever responses in our treat- Bruinvels AT, Landwehrmeyer B, Gustafson EL, Durkin MM, Men- ment groups. Furthermore, previous research found that zolmi- god G, Branchek TA, Hoyer D, Palacios JM (1994) Localization triptan (1–30 mg/kg, i.p.) attenuates alcohol-induced aggression of 5-HT1B, 5-HT1Da, 5-HT1E, and 5-HT1F receptor messenger in mice but has no effect on locomotion (de Almeida et al., 2001). RNA in rodent and primate brain. Neuropharmacol 33:367– In conclusion, this study provides evidence that the selective 386.

5-HT1BR agonist, CP 94,253, attenuates methamphetamine self- Callahan PM, Cunningham KA (1995) Modulation of the discrim- administration pre- and postabstinence under several schedules inative stimulus properties of cocaine by 5-HT1B and 5-HT2C of reinforcement and in an antagonist-reversible manner. These receptors. J Pharm Exp Ther 274: 1414–1424. findings build upon previous research demonstrating similar Clark MS, McDevitt RA, Neaumaier JF (2006) Quantitative map- effects of 5-HT1BR agonists on d-amphetamine self-adminis- ping of tryptophan hydroxlase-2, 5-HT1A, 5-HT1B, and sero- tration (Fletcher and Korth, 1999; Fletcher et al., 2002; Miszkiel tonin transporter expression across the anteroposterior axis et al., 2012; Miszkiel and Przegalinski, 2013), and together the of the rat dorsal and median raphe nuclei. J Comp Neurol effect of the agonists on self-administration of amphetamines 498:611–623. contrasts with the enhanced cocaine self-administration that Clemens KJ, Cornish JL, Hunt GE, McGregor IS (2006) Intrave- has been observed prior to any abstinence (Pentkowski et al., nous methamphetamine self-administration in rats: effects

2009, 2014). These results suggest that 5-HT1BR agonists may of intravenous or intraperitoneal MDMA co-administration. differentially modulate cocaine and methamphetamine self- Pharmacol Biochem Behav 85:454–463 administration initially, but that after a period of abstinence, de Almeida RM, Nikulina EM, Faccidomo S, Fish EW, Miczek KA the agonists inhibit the reinforcing effects of both psychostim- (2001) Zolmitriptan-a 5-HT1B/D agonist, alcohol, and aggres- ulants. The latter findings suggest that 5-HT1BR agonists may sion in mice. Psychopharmacology (Berl) 157:131–141. have potential therapeutic effects for psychostimulant abuse. Domjan M (2003) The essentials of learning and conditioning. In addition, we have provided evidence that the FDA-approved Belmont, CA: Wadsworth/Thomson Learning:123

5-HT1D/1BR agonist, zolmitriptan, also attenuates methampheta- Fletcher PJ, Azampanah A, Korth KM (2002) Activation of 5-HT1B mine self-administration both pre- and postabstinence. Our receptors in the nucleus accumbens reduces self-administra- findings suggest that 5-HT1BR agonists warrant further investi- tion of amphetamine on a progresive ratio schedule. Pharma- gation as putative treatments of psychostimulant use disorders. col Biochem Behav 71:717–725. Important future directions include determining the neural Fletcher PJ, Korth KM (1999) Activation of 5-HT1B receptors in circuitry involved in the agonist effects, whether the effects the nucleus accumbens reduces amphetamine-induced are also observed in female rats, and whether the effects are enhancement of responding for conditioned reward. Psy- observed in rats given more extensive access to the stimulants chopharmacology (Berl) 142:165–174. and more extensive abstinence. Goeders NE, Guerin GF (1994) Non-contingent electric footshock facilitates the acquisition of intravenous cocaine self-admin- Acknowledgments istration in rats. Psychopharmacology (Berl) 114(1):63–70. Hen R (1992) Of mice and flies: commonalities among 5-HT We thank Nathan Pentkowski for his input on this study and receptors. Trends Pharmacol Sci 13:160–165. Juliette Venault, Allegra Campagna, Katelin Ennis, Thomas Hoplight BJ, Vincow ES, Neumaier JF (2005) The effects of SB Benson, Jennifer Taylor, and JP Bonadonna for their technical 224289 on anxiety and cocaine-related behaviors in a novel assistance. object task. Physiol Behav 84:707–714. This work was supported by the National Institute on Drug Kitamura O, Wee S, Specio SE, Koob GF, Pulvirenti L (2006) Esca- Abuse (DA011064 to J.L.N. and DA025606 to M.F.O.), and the lation of methamphetamine self-administration in rats: a National Institutes of General Medical Sciences for ASU Post- dose-effect function. Psychopharmacology 186:48–53 baccalaureate Research Education Program for Biomedical Koe KB, Nielsen JA, Macor JE, Heym J (1992) Biochemical and Research and Initiative to Maximize Student Development behavioral studies of the 5-HT1B receptor agonist, CP-94,253. (GM071798 and GM099650 to R.G.). Drug Develop Res 26(3):241–250. Krasnova IN, Justinova Z, Ladenheim B, Jayanthi S, McCoy MT, Statement of Interest Barnes C, Warner JE, Goldberg SR, Cadet JL (2010) Metham- petamine self-administration is associated with persistent None. biochemical alterations in striatal and cortical dopaminergic terminals in the rat. PLos One 5:e8790 Lanfumey L, Hamon M (2004) 5-HT1 receptors. CNS Neurol Dis- References ord Drug Targets 3:1–10. Ahmed SH, Koob GF (1997) Cocaine- but not food-seeking Lin D, Parsons LH (2002) Anxiogenic-like effect of serotonin1B behavior is reinstated by stress after extinction. Pharmacol receptor stimulation in the rat elevated plus-maze. Pharma- 132:289–295. col Biochem Behav 74:581–587. Garcia et al. | 653

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