The differential effects of alprazolam and oxazepam on methamphetamine self-administration in rats

Drug and Alcohol Dependence 166 (2016) 209–217

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The differential effects of alprazolam and oxazepam on methamphetamine self-administration in rats Allyson L. Spence ∗ , Glenn F. Guerin, Nicholas E. Goeders Department of Pharmacology, Toxicology, & Neuroscience, Louisiana State University Health Sciences Center – Shreveport, Shreveport, LA 71130, United States

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Article history: Received 1 March 2016 Received in revised form 13 July 2016 Accepted 14 July 2016 Available online 26 July 2016 Keywords: Methamphetamine Benzodiazepines Self-administration Polydrug Addiction Reinforcement

a b s t r a c t Background: Methamphetamine is the second most commonly used illicit drug in the world, and despite recent attempts by the Drug Enforcement Administration to combat this epidemic, methamphetamine use is still on the rise. As methamphetamine use increases so does polydrug use, particularly that involving methamphetamine and benzodiazepines. The present study was designed to examine the effects of two benzodiazepines on methamphetamine self-administration. Methods: Five doses of methamphetamine (0.0075, 0.015, 0.03, 0.09, and 0.12 mg/kg/infusion) were tested, producing an inverted U-shaped dose-response curve. Rats were then pretreated with oxazepam, alprazolam, or vehicle prior to methamphetamine self-administration. To determine if the effects of these drugs were due to the GABAA receptor and/or translocator protein (TSPO), we also pretreated rats with an antagonist for the benzodiazepine-binding site on the GABAA receptor (i.e., flumazenil) and a TSPO antagonist (i.e., PK11195) prior to alprazolam or oxazepam administration. Results: Oxazepam significantly reduced methamphetamine self-administration as demonstrated by a downward shift of the dose-response curve. In contrast, alprazolam significantly enhanced methamphetamine self-administration as evidenced by a leftward shift of the dose-response curve. Flumazenil completely blocked the effects of alprazolam on methamphetamine self-administration. When administered individually, both flumazenil and PK11195 partially reversed the effects of oxazepam on methamphetamine self-administration. However, when these two antagonists were combined, the effects of oxazepam were completely reversed. Conclusions: The GABAA receptor is responsible for the alprazolam-induced enhancement of methamphetamine self-administration, while the activation of both the GABAA receptor and TSPO are responsible for the oxazepam-induced reduction of methamphetamine self-administration. Published by Elsevier Ireland Ltd.

1. Introduction Methamphetamine is a highly addictive psychostimulant that is one of the most widely abused drugs in the United States as well as worldwide (Courtney and Ray, 2014; Romanelli and Smith, 2006). Recent studies have demonstrated that methamphetamine use is linked with a diminished quality of life, including violence, impaired everyday functional ability, and risk-taking behaviors (Henry et al., 2010; Costenbader et al., 2007; Sommers et al., 2006; Anderson and Bokor, 1998). Despite the serious negative consequences associated with methamphetamine use, there are still no

∗ Corresponding author at: 1501 Kings Highway, Shreveport, LA 71130, United States. E-mail addresses: [email protected], [email protected] (A.L. Spence). http://dx.doi.org/10.1016/j.drugalcdep.2016.07.015 0376-8716/Published by Elsevier Ireland Ltd.

FDA-approved pharmacological treatments for methamphetamine dependence (Elkashef et al., 2008). Although psychotherapy is the mainstay of treatment for methamphetamine addiction, it is not very effective as evidenced by high relapse rates (Brecht and Herbeck, 2014; McKetin et al., 2012; Rawson et al., 2002). Thus, more effective treatments for methamphetamine dependence are needed. Over the last twenty-five years, our lab has shown a strong relationship between stimulant reinforcement and stress, particularly the activation of the hypothalamic-pituitary-adrenal (HPA) axis (Goeders, 2002, 2004, 2007; Goeders and Guerin, 1994). In that regard, we have investigated the effects of drugs that attenuate HPA axis activity on cocaine self-administration. Benzodiazepine-like drugs represent one class of drugs effective in reducing cocainetaking behavior in rats (Goeders et al., 1993; Goeders and Guerin, 2008). Among the various benzodiazepine receptor agonists tested

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were alprazolam and oxazepam, and we reported that both of these drugs reduced cocaine taking in rats (Goeders et al., 1993; Goeders and Guerin, 2008). We found similar results using oxazepam in a cocaine and methamphetamine discrimination task, whereby oxazepam reduced the apparent discriminative stimulus effects of both cocaine and methamphetamine in rats (Spence et al., 2015). However, we discovered that alprazolam affected cocaine and methamphetamine drug discrimination differently. Surprisingly, while alprazolam did not alter cocaine discrimination, it actually augmented the discriminative stimulus effects of methamphetamine so that less methamphetamine was required to produce similar methamphetamine-appropriate responding as in the absence of alprazolam (Spence et al., 2015). The results of these experiments suggested that these two benzodiazepine receptor agonists could produce seemingly opposite effects on the discriminative stimulus effects of methamphetamine. Since it was not clear whether or not these differential effects were specific for drug discrimination or would carry over to other cocaine- and methamphetamine-related behaviors, we conducted the current study to determine the effects of these benzodiazepines on methamphetamine self-administration. We hypothesized that oxazepam would reduce while alprazolam would enhance methamphetamine self-administration. Although oxazepam and alprazolam have similar affinities for the GABAA receptor, these benzodiazepines have different affinities for the translocator protein (TSPO; Schmoutz et al., 2014). Previous findings in our laboratory have shown that oxazepam binds to and activates the TSPO, while alprazolam does not (Schmoutz, 2013). TSPO activation stimulates the formation of neurosteroids, which have been suggested to play a role in addiction (Costa et al., 1994; Papadopoulos et al., 1992; Doron et al., 2006). Previous experiments have shown that neurosteroids reduce the self-administration of ethanol and cocaine (Besheer et al., 2010; Anker et al., 2010; Doron et al., 2006; Schmoutz, 2013) and can block the rewarding properties of drugs of abuse (Romieu et al., 2003; Russo et al., 2003). To determine if these benzodiazepines’ affinities for the TSPO and/or GABAA receptors could be responsible for their effects on methamphetamine self-administration, we pretreated rats with a TSPO antagonist (i.e., PK11195) and an antagonist for the benzodiazepine-binding site on the GABAA receptor (i.e., flumazenil) prior to alprazolam or oxazepam administration. We hypothesized that flumazenil would block the effects of both oxazepam and alprazolam while PK11195 would only inhibit the effects of oxazepam on methamphetamine self-administration.

2. Materials and methods 2.1. Subjects Adult male Wistar rats (Harlan Sprague Dawley, Indianapolis, IN), 80–100 days old at the start of the experiment were used. Female rats were not included in the present study since our previous data suggested that alprazolam and oxazepam produce similar effects on methamphetamine-related behaviors in both male and female rats (Spence et al., 2015). Rats were maintained at 85–90% of their free-feeding body weights by daily feedings of approximately 14 g of food (Purina Rat Chow) immediately following their self-administration session with free access to water. The average weight for rats at the beginning of the study was 337.4 g, and their average weight upon completion of the study was 336.8 g. Rats were individually housed in cages equipped with a laminar flow unit and air filter in a temperature- and humidity-controlled, AALAC-accredited animal care facility on a reversed 12-h light, 12-h dark cycle (lights on at 18:00). Rats (n = 16) were randomly divided

into two groups (n = 8/group). Six rats did not complete all of the experiments due to complications associated with the experimental procedure, such as catheter failures or the failure to achieve stable baselines of methamphetamine self-administration. The first group of rats was treated with vehicle and alprazolam, and the second group was treated with vehicle and oxazepam. Both experimental groups received similar extinction training. All procedures were carried out in accordance with the “Public Health Services Policy on Humane Care and Use of Laboratory Animals” and the “Guide for the Care and Use of Laboratory Animals” Eighth Edition, 2011 and were approved by the Louisiana State University Health Sciences Center in Shreveport Institutional Animal Care and Use Committee. 2.2. Apparatus Behavioral experiments were conducted in standard Plexiglas and stainless steel, sound-attenuating operant conditioning chambers (Med-Associates, St. Albans, VT). The operant chambers were equipped with a retractable response lever mounted on one wall of the chamber, with a stimulus light located above the lever. The chamber was also equipped with an exhaust fan that supplied ventilation and masked extraneous sounds. Programming and data collection were performed using Med-PC software and interface system and an IBM-compatible computer. 2.3. Surgical procedure Before undergoing any surgical procedures, the rats were allowed at least one week to acclimate to the facility. Then, once the targeted weight was reached (i.e., 85–90% of their free-feeding body weights), the rats were implanted with chronic indwelling jugular catheters (Keller et al., 2013). Prior to this surgery, rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and pretreated with atropine methyl nitrate (10 mg/kg, i.p.) to reduce bronchial secretions and penicillin G procaine suspension (75,000 units, i.m.) to prevent infections. Silicon tubing, Marlex mesh, and a 22-gauge guide cannula were used to construct and secure the catheters. An opening was made in the rat’s neck so that the catheter was inserted into the jugular vein. Once inserted into the vein, the catheter was secured to the vein and continued under the scapula to the back, where the skin was sutured around the guide cannula. 2.4. Methamphetamine self-administration Following all surgical procedures, the rats were allowed a minimum of five days to recover before initiating self-administration training. Rats were trained to self-administer methamphetamine (0.06 mg/kg/infusion) during daily 2-h sessions, Monday-Saturday. Subjects were initially trained under a fixed-ratio of 1 (FR1) schedule of reinforcement, which was gradually increased to FR2 and finally to FR4, so that four depressions of the lever resulted in an infusion (200 ␮l in 5.6 s) of methamphetamine, which traveled through Tygon tubing contained within a spring leash. This leash was attached to a liquid swivel contained within a counterbalanced arm to allow relatively unrestricted movement of the animals. The availability of methamphetamine was indicated by the illumination of a stimulus light located directly above the response lever. During the infusion, the stimulus light was extinguished and remained extinguished for a 20-s time-out period following the infusion. During this period, responses were recorded but did not result in an infusion of methamphetamine. Prior to testing with alprazolam or oxazepam, the rats were exposed to extinction training. During extinction, the methamphetamine syringe was replaced with a saline syringe and responses on the lever only resulted in an infusion of saline.

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2.5. Methamphetamine self-administration + antagonists A new group of adult male Wistar rats was implanted with chronic jugular catheters and trained to self-administer methamphetamine (i.e., 0.0075 and 0.06 mg/kg/infusion) as previously described. The two methamphetamine doses were selected to include our typical training dose for self-administration (i.e., 0.06 mg/kg/infusion) and the lowest dose of methamphetamine tested (i.e., 0.0075 mg/kg/infusion). Importantly, these included a dose from the ascending limb (i.e., 0.0075 mg/kg/infusion) as well as the descending limb (i.e., 0.06 mg/kg/infusion) of the methamphetamine dose-response curve. Once responding stabilized and extinction met the established criteria as described above, rats were pretreated with vehicle (5% emulphor in 0.9% saline; 1.0 ml/kg), flumazenil (10 or 20 mg/kg, ip) or PK11195 (1 or 2 mg/kg, ip) and returned to their home cages. These doses were selected based on our previous data, which demonstrated that they were effective in blocking the binding of alprazolam and/or oxazepam to the GABAA receptor and/or TSPO (Schmoutz et al., 2014; Schmoutz, 2013). Thirty minutes following this pretreatment, rats were administered vehicle, alprazolam (4 mg/kg, ip) or oxazepam (20 mg/kg, ip) and returned to their home cages. Thirty minutes after this second injection, the self-administration session began. 2.6. Drugs Methamphetamine was obtained from the National Institute of Drug Abuse (Research Triangle Park, NC, USA). Methamphetamine was dissolved in 0.9% bacteriostatic saline and delivered intravenously at the indicated doses over 5.6 s. Oxazepam and alprazolam were obtained from Sigma-Aldrich (St. Louis, MO, USA), suspended in 5% emulphor (Alkamuls EL-620, Rhodia, Cranberry, NJ, USA) in 0.9% saline and administered intraperitoneally in a volume of 1.0 ml/kg. 2.7. Statistical analysis All data were plotted and analyzed using SigmaPlot and SigmaStat, respectively. The data collected included the total number of methamphetamine infusions delivered per daily two-hour session at each dose tested following pretreatment with vehicle,

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Rats were presented with these extinction probes until stable, reproducible, “extinction-like” behavior was observed. To meet the criteria for extinction, responses on the lever must have been less than 50% of baseline responding without varying more than 10% during at least two consecutive extinction probes. Extinction probes continued to be conducted approximately every two weeks, and these criteria were maintained for every dose of methamphetamine tested. Once responding stabilized (i.e., less than 10% variation for 3 consecutive days) and extinction met the established criteria, rats were tested with vehicle (5% emulphor in 0.9% saline), alprazolam (2 or 4 mg/kg, ip) or oxazepam (10 or 20 mg/kg, ip). These doses were chosen since previous studies demonstrated that they would alter methamphetamine-related behaviors without inducing ataxia or sedation (Spence et al., 2015; Goeders and Guerin, 2008; Goeders et al., 1993). Once testing was complete for the training dose of methamphetamine (i.e., 0.06 mg/kg/infusion), rats were trained to self-administer five other doses (i.e., 0.0075, 0.015, 0.03, 0.09, and 0.12 mg/kg/infusion) in a random order, and responding was allowed to stabilize with each dose. Rats were tested with vehicle, alprazolam (2 or 4 mg/kg, ip) or oxazepam (10 or 20 mg/kg, ip) at each methamphetamine dose. The test drugs and vehicle were administered thirty minutes prior to the start of the selfadministration sessions in a volume of 1.0 ml/kg.

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Methamphetamine (mg/kg/infusion) Fig. 1. Methamphetamine self-administration in rats. (A) Methamphetamine selfadministration in rats produced an inverted U-shaped dose-response curve. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, p < 0.05; n = 5 rats/group.

alprazolam and oxazepam. For extinction probes, the number of saline infusions delivered per daily two-hour session was counted. Data are expressed as means ± SEM. A repeated measures oneway or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, as appropriate, was used to analyze the differences between the experimental groups. For all analyses, alpha was set at P < 0.05. 3. Results 3.1. Methamphetamine self-administration Stable baselines of methamphetamine-reinforced responding were obtained after approximately 20 experimental sessions with 0.06 mg/kg/infusion methamphetamine. The various doses of methamphetamine (0.0075, 0.015, 0.03, 0.09, and 0.12 mg/kg/infusion) produced an inverted U-shaped dose-response curve (Fig. 1), which is typically seen in drug selfadministration studies (Piazza et al., 2000). A repeated measures two-way analysis of variance revealed a significant effect of methamphetamine dose F(5,59) = 103.5, p < 0.05. Importantly, a repeated measures two-way analysis of variance revealed that responding significantly decreased during extinction training, demonstrating that methamphetamine was maintaining responding at all doses except the lowest (i.e., 0.0075 mg/kg/infusion) and highest (i.e., 0.12 mg/kg/infusion) doses tested. 3.2. Methamphetamine self-administration and oxazepam The effects of pretreatment with vehicle or oxazepam on the various doses of methamphetamine self-administration (0.0075, 0.015, 0.03, 0.06, 0.09, and 0.12 mg/kg/infusion) are shown in Fig. 2. A repeated measures two-way analysis of variance revealed a significant effect of oxazepam pretreatment F(2,89) = 261.18, p < 0.05. Pretreatment with both doses of oxazepam (10 and 20 mg/kg, ip) reduced methamphetamine self-administration to levels that were significantly different from vehicle. In fact, both doses of oxazepam reduced methamphetamine self-administration to levels that were not significantly different from extinction conditions. There was also a significant

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3.4. Methamphetamine self-administration and flumazenil The effects of pretreatment with vehicle or flumazenil (10 and 20 mg/kg, ip) on two doses of methamphetamine selfadministration (0.0075 and 0.06 mg/kg/infusion) are shown in Figs. 4 and 5. Flumazenil pretreatment did not alter methamphetamine selfadministration (Figs. 4 -A and 5 -A). Flumazenil pretreatment significantly but partially reversed the effects of oxazepam (20 mg/kg, ip) on methamphetamine self-administration (0.06 mg/kg/inf; Fig. 4B). Pretreatment with flumazenil significantly and completely blocked the effects of alprazolam (4 mg/kg, ip) on methamphetamine self-administration (0.0075 and 0.06 mg/kg/inf; Figs. 4 -C and 5 -B). 3.5. Methamphetamine self-administration and PK11195

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A repeated measures two-way analysis of variance revealed a significant effect of alprazolam pretreatment F(2,89) = 82.44, p < 0.05. There was also a significant methamphetamine dose by alprazolam pretreatment interaction F(10,89) = 82.44, p < 0.05. The low dose of alprazolam (2 mg/kg, ip) did not alter methamphetamine self-administration at any dose tested (Fig. 3). However, in contrast to oxazepam, the higher dose of alprazolam (4 mg/kg, ip) significantly increased methamphetamine taking in rats at 0.0075 and 0.015 mg/kg/infusion, as revealed by Tukey’s post hoc analysis. This is illustrated by the leftward shift in the methamphetamine dose-response curve. Responding at 0.03, 0.06 and 0.09 mg/kg/infusion was actually reduced as part of the leftward shift in this dose-response curve. These data suggest that alprazolam actively enhances the reinforcing effects of methamphetamine.

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Methamphetamine (mg/kg/infusion) Fig. 3. Alprazolam pretreatment significantly enhanced methamphetamine selfadministration. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, p < 0.05; n = 5 rats/group.

methamphetamine dose by oxazepam pretreatment interaction F(10,89) = 50.92, p < 0.05. Tukey’s post hoc analysis showed that oxazepam (10 and 20 mg/kg, ip) significantly reduced methamphetamine selfadministration at 0.15, 0.03 and 0.06 mg/kg/infusion (Fig. 2). The high dose of oxazepam also significantly reduced methamphetamine self-administration at 0.09 mg/kg/infusion. This is illustrated by the downward shift in the methamphetamine doseresponse curve to levels similar to extinction. These results suggest that oxazepam reduces the reinforcing effects of methamphetamine. Oxazepam did not produce a left or rightward shift in the methamphetamine dose-response curve, but rather an overall flattening of the curve was observed (Fig. 2).

The effects of pretreatment with vehicle or PK11195 (1 and 2 mg/kg, ip) on two doses of methamphetamine self-administration (0.0075 and 0.06 mg/kg/infusion) are shown in Figs. 6 and 7. PK11195 pretreatment did not alter methamphetamine selfadministration (Figs. 6 -A and 7 -A). PK11195 pretreatment significantly but partially reversed the effects of oxazepam (20 mg/kg, ip) on methamphetamine self-administration (0.06 mg/kg/inf; Fig. 6B). On the other hand, pretreatment with PK11195 did not block the effects of alprazolam (4 mg/kg, ip) on methamphetamine selfadministration (0.0075 and 0.06 mg/kg/inf; Figs. 6 -C and 7 -B). 3.6. Methamphetamine self-administration and flumazenil + PK11195 Pretreatment with flumazenil completely blocked the effects of alprazolam on methamphetamine self-administration. However, neither flumazenil nor PK11195 pretreatment alone completely reversed the effects of oxazepam on methamphetamine selfadministration. Each of these antagonists only partially reversed the effects of oxazepam. Therefore, we pretreated rats with a combination of flumazenil and PK11195 to determine if the combination of these antagonists would completely block the effects of oxazepam on methamphetamine-taking behavior. Pretreatment with the combination of flumazenil (10 mg/kg, ip) and PK11195 (1 mg/kg, ip) did not alter methamphetamine selfadministration (Fig. 8-A ). As hypothesized, flumazenil + PK11195 pretreatment completely blocked the effects of oxazepam (20 mg/kg, ip) so that responding was no longer significantly different from vehicle treatment (Fig. 8-B).

3.3. Methamphetamine self-administration and alprazolam 4. Discussion The effects of pretreatment with vehicle or alprazolam on the various doses of methamphetamine self-administration (0.0075, 0.015, 0.03, 0.06, 0.09, and 0.12 mg/kg/infusion) are shown in Fig. 3.

Pretreatment with oxazepam (i.e., 10 and 20 mg/kg, ip) significantly reduced methamphetamine self-administration.This effect

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Flumazenil Dose (mg/kg) + 4 mg/kg Alprazolam Fig. 4. Methamphetamine self-administration (0.06 mg/kg/inf) after flumazenil pretreatment. (A) Flumazenil pretreatment did not alter methamphetamine self-administration. (B) Flumazenil pretreatment partially reversed the effects of oxazepam on methamphetamine self-administration. (C) Flumazenil pretreatment completely blocked the effects of alprazolam on methamphetamine self-administration. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, # indicates a significant difference from oxazepam or alprazolam treatment, p < 0.05; n = 5 rats/group.

Fig. 5. Methamphetamine self-administration (0.0075 mg/kg/inf) after flumazenil pretreatment. (A) Flumazenil pretreatment did not alter methamphetamine self-administration. (B) Flumazenil pretreatment completely blocked the effects of alprazolam on methamphetamine self-administration. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, # indicates a significant difference from alprazolam treatment, p < 0.05; n = 5 rats/group.

is not likely due to non-specific peripheral effects since previous reports have demonstrated that at the doses tested in this experiment (i.e., 10 and 20 mg/kg, ip), oxazepam does not affect food-maintained responding (Goeders and Guerin, 2008). Prior research has also shown that these doses of oxazepam do not significantly alter locomotor behaviors or food-maintained response rates during drug-discrimination testing in rats (Spence et al., 2015). Therefore, these data suggest that the oxazepam-induced decreases in methamphetamine self-administration were specific for methamphetamine reinforcement. In contrast, the higher dose of alprazolam (i.e., 4 mg/kg, ip) enhanced the reinforcing properties of methamphetamine. This dose of alprazolam increased responding up to 7-fold for a dose of methamphetamine that would not maintain lever pressing when administered alone. At the higher doses of methamphetamine (i.e., 0.03, 0.06, and 0.09 mg/kg/infusion), pretreatment with alprazolam reduced drug taking. Since this decrease was seen in the descending limb of the dose-response curve, this shift is presumably produced because alprazolam enhances the reinforcing properties of methamphetamine. In other words, less metham-

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Fig. 7. Methamphetamine self-administration (0.0075 mg/kg/inf) after PK11195 pretreatment. (A) PK11195 pretreatment did not alter methamphetamine selfadministration (B) PK11195 pretreatment did not alter the effects of alprazolam on methamphetamine self-administration. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, p < 0.05; n = 5 rats/group.

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PK11195 Dose (mg/kg) + 4 mg/kg Alprazolam Fig. 6. Methamphetamine self-administration (0.06 mg/kg/inf) after PK11195 pretreatment. (A) PK11195 pretreatment did not alter methamphetamine selfadministration. (B) PK11195 pretreatment partially reversed the effects of oxazepam on methamphetamine self-administration. (C) PK11195 pretreatment did not alter the effects of alprazolam on methamphetamine self-administration. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, # indicates a significant difference from oxazepam or alprazolam treatment, p < 0.05; n = 5 rats/group.

phetamine is required to achieve the same reinforcing effects as in the absence of alprazolam. Notably, previous experiments have demonstrated that alprazolam (i.e., 2 and 4 mg/kg, ip) does not affect food-maintained responding, implying that its ability to enhance methamphetamine self-administration is not due to nonspecific effects (Spence et al., 2015; Goeders et al., 1993). At higher methamphetamine doses, self-administration decreases, forming the descending limb of the dose-response curve (Fig. 1). While the mechanisms underlying the descending limb of the dose-response curve are still in question, one common explanation for this decrease in self-administration is that at high doses, the drug’s aversive qualities counteract the drug’s reinforcing effects (Zernig et al., 2004). Therefore, at the highest dose of methamphetamine tested for self-administration (i.e., 0.12 mg/kg/inf), we did not observe any significant differences in responding compared to extinction. Oxazepam and alprazolam may produce differential effects on methamphetamine self-administration since these drugs have different underlying mechanisms. Previous experiments in our laboratory have shown that both alprazolam and oxazepam produce their anxiolytic effects by activating the GABAA receptor

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Fig. 8. Methamphetamine self-administration (0.06 mg/kg/inf) after flumazenil + PK11195 pretreatment. (A) Flumazenil + PK11195 pretreatment did not alter methamphetamine self-administration. (B) Flumazenil + PK11195 pretreatment completely blocked the effects of oxazepam on methamphetamine self-administration. Data represent the mean methamphetamine infusions delivered per two-hour session (±SEM). * indicates a significant difference from vehicle responding, # indicates a significant difference from oxazepam treatment, p < 0.05; n = 5 rats/group.

(Schmoutz, 2013). We also found that the anxiolytic effects produced by oxazepam may be at least partially due to its ability to bind to and activate the TSPO, while alprazolam does not appear to have any affinity for the TSPO (Schmoutz et al., 2014; Schmoutz, 2013). Therefore, we hypothesized that the differential effects of these benzodiazepines on methamphetamine-related behaviors may be due to their differing affinities for the TSPO. To determine the role that the GABAA receptor and TSPO play in the effects of oxazepam and alprazolam on methamphetamine self-administration, we pretreated rats with antagonists for these binding sites prior to benzodiazepine pretreatment. Pretreatment with flumazenil partially blocked the effects of oxazepam on methamphetamine self-administration, suggesting that activation of the GABAA receptor may be at least partially responsible for these effects. We saw similar results when rats were pretreated with PK11195, which was also able to only partially reverse the effects of oxazepam on methamphetamine self-administration. Thus, the TSPO also plays a role in the effects

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of oxazepam on methamphetamine self-administration. To determine if a combination of these antagonists would fully block the effects of oxazepam on methamphetamine-taking behavior, we pretreated rats with both flumazenil and PK11195. As hypothesized, the combination of these antagonists fully blocked the effects of oxazepam, implying that activation of both the GABAA receptor and TSPO were responsible for the oxazepam-induced effects on methamphetamine self-administration. Flumazenil pretreatment completely reversed the effects of alprazolam on methamphetamine self-administration (on both the ascending and descending limbs of the dose-response curve), providing evidence that activation of the GABAA receptor is responsible for the ability of alprazolam to enhance methamphetamine selfadministration. As expected, PK11195 pretreatment did not alter the effects of alprazolam on methamphetamine-taking behavior, suggesting that TSPO activation does not play a role in these effects. Since TSPO inhibition only blocked the effects of oxazepam on methamphetamine self-administration, the TSPO may account for the differences seen for oxazepam and alprazolam on methamphetamine self-administration. Agonists of the TSPO have been reported to stimulate the formation of neurosteroids, and research has shown that these neurosteroids may play a role in drugtaking behaviors (Costa et al., 1994; Papadopoulos et al., 1992). Doron et al. (2006) found that the administration of the neurosteroid, dehydroepiandrosterone (DHEA), decreased cocaine self-administration and reinstatement in rats. Motzo et al. (1996) found that neurosteroids reduced basal dopamine content in the medial prefrontal cortex, an area of the brain that is associated with drug reward. Therefore, we hypothesize that oxazepam reduces methamphetamine self-administration partially by activating the TSPO, which produces an increase in neurosteroid levels (Schmoutz et al., 2014), thereby reducing basal dopamine content in the mesocorticolimbic dopaminergic system. While oxazepam may decrease methamphetamine selfadministration by reducing basal dopamine levels, alprazolam may enhance methamphetamine taking by increasing synaptic dopamine. Methamphetamine reinforcement results from its ability to produce a profound increase in dopamine (Cruickshank and Dyer, 2009; Sulzer et al., 2005; Zhang et al., 2001; Fischer and Cho, 1979; Pierce and Kalivas, 1997). Methamphetamine produces an increase in synaptic dopamine through three key mechanisms. Firstly, methamphetamine blocks the reuptake of dopamine in the synapse (Cho and Melega, 2002). Methamphetamine can also reverse the direction of the dopamine transporter (DAT) to stimulate the release of dopamine into the synapse (Cruickshank and Dyer, 2009). Finally, methamphetamine inhibits the key enzyme responsible for dopamine metabolism, thus leading to a further increase in synaptic dopamine (Sulzer et al., 2005). Alprazolam may also produce an increase in synaptic dopamine. Tan et al. (2011) reported that benzodiazepines have a disinhibition mechanism that is similar to that of opioids, thus producing rewarding properties that can lead to addiction. When benzodiazepines interact with the ␣-1 subunit of the GABAA receptor, they decrease inhibition on dopaminergic neurons (i.e., disinhibition), thus leading to an increase in synaptic dopamine in brain regions associated with the mesolimbic reward system, such as the ventral tegmental area (Brown et al., 2010; Riegel and Kalivas, 2010). Importantly, these same brain regions (e.g., ventral tegmental area) are critical for methamphetamine-induced reward (Kousik et al., 2014). Therefore, we hypothesize that when alprazolam activates the GABAA receptor, it produces an increase in synaptic dopamine that enhances the reinforcing properties of methamphetamine. While we found that alprazolam enhanced methamphetamine self-administration, previous findings in our laboratory showed that alprazolam produced the opposite effects on cocaine selfadministration, where, similarly to oxazepam, alprazolam reduced

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the reinforcing properties of cocaine (Goeders et al., 1993). We recently reported that the discriminative stimulus effects of methamphetamine and cocaine were also differentially affected by alprazolam (Spence et al., 2015). One question that presented from these data was why alprazolam would differentially affect the behavioral properties of two related CNS stimulants. While both of these drugs produce an increase in synaptic dopamine, methamphetamine and cocaine interact differently with the dopamine transporter (DAT; Kahlig and Galli, 2003). Methamphetamine reverses the transport of dopamine through DAT, leading to a reduction in DAT surface expression and a reduction in dopamine transport capacity (Fischer and Cho, 1979; Pierce and Kalivas, 1997; Zhang et al., 2006; Kahlig and Galli, 2003). Cocaine only blocks the DAT, thereby leading to an increase in DAT expression and enhanced dopamine transport capacity (Ritz et al., 1987; Little et al., 2002; Kahlig and Galli, 2003). Although alprazolam produces an increase in synaptic dopamine in the presence of both of these stimulants, the enhanced DAT surface expression from cocaine prompts a rapid uptake of extracellular dopamine, thus rapidly reducing any alprazolam-induced effects (Kahlig and Galli, 2003; Brown et al., 2010; Riegel and Kalivas, 2010). However, we hypothesize that in the presence of methamphetamine, the diminished DAT cell surface expression allows extracellular dopamine levels to remain elevated after exposure to alprazolam, thus enhancing the discriminative stimulus and reinforcing effects of methamphetamine (Cervinski et al., 2005; Fleckenstein et al., 1999; Kokoshka et al., 1998). However, we did not measure DAT surface expression in these experiments, which presents a potential caveat in this theory. Therefore, future experiments will be necessary to measure DAT surface expression following exposure to methamphetamine self-administration, both alone and following pretreatment with alprazolam or oxazepam. To the best of our knowledge, these are the first experiments to demonstrate that alprazolam enhances methamphetaminetaking behavior by activating the GABAA receptor while a different benzodiazepine, oxazepam, reduces methamphetamine self-administration by activating both the GABAA receptor and the TSPO.

Conflict of interest No conflict declared.

Role of funding source Nothing declared.

Contributors Participated in research design: Spence, Guerin, and Goeders. Conducted experiments: Spence. Contributed new reagents or analytic tools: Guerin. Performed data analysis: Spence. Wrote or contributed to the writing of the manuscript: Spence, Guerin, and Goeders.

Acknowledgements This research was funded by the Department of Pharmacology, Toxicology & Neuroscience at Louisiana State University Health Sciences Center in Shreveport. We thank Sarah Harold for her assistance in conducting these experiments.

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