Psychopharmacology DOI 10.1007/s00213-013-3404-2

ORIGINAL INVESTIGATION

Effects of inhibitory GABA-active neurosteroids on cocaine seeking and cocaine taking in rats Christopher D. Schmoutz & Scott P Runyon & Nicholas E. Goeders

Received: 26 February 2013 / Accepted: 3 December 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract Rationale Several compounds that potentiate GABA-induced inhibitory currents also decrease stress, anxiety and addictionrelated behaviors. Because of the well-established connection between stress and addiction, compounds that reduce stressinduced responses might be efficacious in treating addiction. Since endogenous neurosteroids such as allopregnanolone may function in a manner similar to benzodiazepines to reduce HPA axis activation and anxiety following stressful stimuli, we hypothesized that exogenously applied neurosteroids would reduce cocaine reinforcement in two animal models. Methods Male Wistar rats were trained to self-administer cocaine and food under a concurrent alternating operant schedule of reinforcement. Two separate groups of rats were trained to self-administer cocaine or food pellets and were then exposed to similar cue-induced reinstatement paradigms. Both groups of rats were pretreated with various doses of neurosteroids. Results Allopregnanolone and 3α-hydroxy-3β-methyl-17βnitro-5α-androstane (R6305-7, a synthetic neurosteroid) were ineffective in selectively decreasing cocaine relative to food self-administration. On the other hand, both allopregnanolone and R6305-7 significantly decreased the cue-induced reinstatement of extinguished cocaine seeking, confirmed by one-way ANOVA.

C. D. Schmoutz (*) : N. E. Goeders Department of Pharmacology, Toxicology & Neuroscience, Louisiana State University Health Sciences Center, 1501 Kings Highway, Box 33932, Shreveport, LA 71130, USA e-mail: [email protected] S. P. Runyon Organic and Medicinal Chemistry, Research Triangle Institute, 3040 Cornwallis Drive, Research Triangle Park, NC 27709, USA

Conclusions These results suggest that neurosteroids may be effective in reducing the relapse to cocaine use without affecting ongoing cocaine self-administration. Keywords Addiction . Cocaine . Drug abuse . Self-administration . GABA receptor . Neuroendocrine axis . Neurosteroid . Psychostimulant . Reinforcement . Relapse

Introduction Although research into cocaine addiction has continued for more than 40 years, there is no FDA-approved medication for the treatment of this condition. Recent evidence suggests that the inhibitory tone of certain brain regions, mediated mainly by gamma-aminobutyric acid (GABA) receptors, may modulate the reinforcing and relapse-promoting properties of cocaine (Kalivas 2007; Sofuoglu and Kosten 2006; Weerts et al. 2005). Preclinical research has also found that increasing GABAergic activity reduces self-administration and relapse in various animal models (Peris 1996; Vocci and Ling 2005; Weerts et al. 2005). Similarly, exposure to stress- or cocainerelated stimuli increases subjective feelings of anxiety and cocaine craving in cocaine-dependent people (Fox et al. 2008; Sinha et al. 2003, 2006). These data suggest that the major inhibitory neurotransmitter GABA (and the GABAA receptor) plays a significant role not only in the phenomenon of cocaine-related interoceptive effects but also in the relapse to cocaine use. Several steroid-derived hormones are able to decrease neuronal excitation by their immediate actions on GABAA receptors. These “neurosteroids” are derived from a branch of the common steroidogenic pathway involving 3-alpha hydroxysteroid dehydrogenase and 5-alpha reductase (Agís-Balboa et al. 2006; Mellon et al. 2001). Inhibitory neurosteroids bind to and potentiate the GABAA receptor complex as positive

Psychopharmacology

allosteric modulators that increase the frequency of the opening of the channel and the decay time of the postsynaptic inhibitory potential (Barker et al. 1987). Given these properties, endogenous inhibitory neurosteroids may produce behavioral effects similar to those produced by benzodiazepines, such as anxiolysis and seizure protection (Reddy and Rogawski 2002). Accordingly, exogenously applied inhibitory neurosteroids act as anxiolytic agents in animal models of anxiety such as the elevated-plus maze (Crawley et al. 1986; Engin and Treit 2007; Finn et al. 2003; Hirani et al. 2005; Ugale et al. 2007). Specifically, a newly developed synthetic neurosteroid, 3α-hydroxy-3β-methyl-17β-nitro-5αandrostane (R6305-7), is a potent modulator of the GABAA complex and reduces anxiety as demonstrated by an increase in the time spent and entries into the open arms of the maze. This anxiolysis is based on the binding of R6305-7 to GABAA receptors (Runyon et al. 2009). Only a few studies have investigated the interaction between cocaine and neurosteroids. Allopregnanolone has been reported to reduce several measures of cocaine-taking and cocaine-seeking behaviors in rodents, especially in female rats. Anker et al. (2009) showed that the subcutaneous injection of allopregnanolone can decrease the cocaine-induced reinstatement of cocaine seeking. Exogenously administered progesterone also decreases cocaine-seeking behavior, but this effect was blocked by pretreatment with finasteride (an inhibitor of 5alpha-reductase, the rate-limiting enzyme in inhibitory neurosteroid biosynthesis), suggesting that the 5α-reduced metabolite, allopregnanolone, is responsible for this effect (Anker et al. 2007, 2009). Similar effects were also observed using the stress-induced reinstatement of extinguished cocaine seeking (Anker and Carroll 2010). These data highlight the emerging role of inhibitory neurosteroids in decreasing cocaine-related behaviors. To this end, we explored the influence of exogenous neurosteroids on cocaine-related behaviors in male rats. Unlike previous research, we used two new modifications to bolster the conclusions drawn from these studies. Primarily, we used food (i.e., sucrose pellets) self-administration in conjunction with cocaine self-administration to assess the behavioral selectivity of our treatments. Food-maintained responding can be analyzed to identify non-selective decreases in operant behavior that may be attributable to sedation or a general decrease in the motivation to respond (Goeders and Guerin 2008). Secondly, we used a cueinduced reinstatement procedure to measure cocaine seeking. Previous research on neurosteroids excluded this animal model of relapse, which we see as a major oversight as it has been shown that conditioned cues (e.g., drug-related imagery or situations) may be a key initiating event in the relapse to cocaine use (Fuchs et al. 2006; See 2002). Our hypothesis that exogenous neurosteroids decrease the cue-induced reinstatement of extinguished cocaine-seeking

behavior was confirmed in the studies reported below. However, neurosteroids did not selectively block cocaine selfadministration compared to food self-administration, suggesting a non-specific effect during ongoing self-administration.

Methods Subjects and housing conditions Male Wistar rats (∼330 g, 60–90 days old) were housed in individual cages in a temperature- and humidity-controlled animal care facility on a reversed 12-h light–dark cycle for the duration of the experiments. Upon arrival at the facility, rats were weighed and handled daily to acclimate them to the experimental procedures. These rats were maintained at 85 % of their free-feeding body weights by presentation of food pellets (45 mg, Test Diets, Richmond, IN, USA) during the operant behavioral sessions and by supplemental post-session feeding and had free access to water. Previous research has demonstrated that a 15 % reduction in free-feeding body weight increased drug consumption and behavioral motivation for drug-seeking in food-deprived rats (Campbell and Carroll 2001; Carroll and Meisch 1981). Food restriction also stabilized the weights of these rats over the duration of the experiments. All operant training and testing was conducted five consecutive days per week, between 0700 and 1300 hours. All procedures were approved by the LSUHSC-S Institutional Animal Care and Use Committee and were carried out in accordance with the NIH “Principles of laboratory animal care” (NIH publication no. 85–23). Surgical procedures Rats used in the cocaine self-administration and reinstatement experiments were implanted with chronic indwelling jugular catheters under pentobarbital anesthesia (50 mg/kg, IP) with methylatropine nitrate pretreatment (10 mg/kg, IP) according to previously reported procedures (e.g., Goeders and Guerin 2008; Goeders et al. 2009). Catheters, constructed of silastic tubing (0.31 mm ID×0.64 mm OD), were inserted into the right posterior facial vein and continued into the jugular vein, terminating outside the right atrium of the heart. The catheter was attached to the vein with silk thread, guided under the skin around the forelimb and exited posterior to the scapulae through a Marlex® mesh and a 22-gauge guide cannula (Plastics One, Roanoke, VA, USA) assembly that was sutured firmly under the skin. During operant self-administration sessions, this cannula was attached to Tygon tubing threaded through a stainless-steel spring leash attached to a fluid swivel suspended above the experimental chamber. Additional tubing connected the swivel to a 20-ml syringe in a motor-driven pump (Razel Scientific Instruments, Stamford, CT, USA)

Psychopharmacology

located outside the sound-attenuating enclosure of the operant chamber. When the experimental session was completed, the catheters were flushed with a solution of streptokinase (0.0067 mg/0.1 ml) and timentin (6.7 mg/0.1 ml) and a small piece of Tygon tubing, sealed on one end, was placed on the cannula to preserve catheter patency. An IBM-compatible personal computer and interface system (Med-Associates, St. Albans, VT, USA) was used to program the behavioral procedures and collect the experimental data. Cocaine and food self-administration Operant chambers (Med-Associates) contained within soundattenuating enclosures were equipped with two response levers, a stimulus light above each lever and a food pellet dispenser located on the front wall between the two levers. Following 5–7 days of recovery after surgery, rats were trained to respond under a multiple, alternating operant schedule of food (45 mg, Test Diets) and cocaine (0.25 mg/kg/ infusion delivered over 5.6 s) reinforcement (Goeders and Guerin 2008). Training began under a fixed-ratio 1 (FR1) schedule of reinforcement, and the requirement for food and cocaine reinforcement was gradually increased to FR4 over several sessions to increase the number of behavioral responses emitted by each rat. Each 2-h daily selfadministration session was divided into eight 15-min bins during which either food or cocaine was available as indicated by the illumination of stimulus lights above the appropriate levers. A 1-min timeout period followed the completion of each bin. Once stable self-administration (i.e., less than 10 % variability in the number of infusions over three consecutive sessions) was achieved, each rat was exposed to saline substitution (i.e., extinction) testing until reliable extinction-related responding (i.e., responses reduced by at least 80 % for each active lever) was evident. During these extinction sessions, responses on the food-associated lever produced no food pellets. Research from our laboratory has found that introducing these “extinction probe” sessions early and often into operant cocaine self-administration is necessary to prevent the behavioral momentum of lever-pressing behavior (typically associated with “extinction bursts”) from influencing the effects of a proposed treatment. Rats produce variable responding upon the first introduction of saline extinction training, possibly from the loss of the interoceptive effects of the cocaine infusion (Goeders et al. 1997; Peltier et al. 2001). As GABA-active drugs such as neurosteroids and benzodiazepines are known to decrease the discriminative stimulus effects of cocaine (Barrett et al. 2005; Negus et al. 2000; Quinton et al. 2006), we were concerned that pretreatment with neurosteroids would result in extinction-like responding patterns. Following successful extinction training, stable selfadministration (with the re-introduction of responsecontingent food and cocaine presentation) was re-established

and the endogenous neurosteroid allopregnanolone (5– 40 mg/kg, IP), the synthetic neurosteroid R6305-7 (5– 40 mg/kg, IP), or vehicle were subsequently administered in a random-dose order 30 min prior to the start of a selfadministration session. These doses of allopregnanolone and R6305-7 have been shown to be behaviorally active in rodent models of anxiety (Bitran et al. 1991; Rodgers and Johnson 1998; Runyon et al. 2009; Wieland et al. 1991). Each subject was presented with four doses of a single neurosteroid and vehicle, with each test session separated by at least two stable self-administration sessions. Reinstatement of cocaine-seeking behavior A separate group of rats were trained to self-administer cocaine (0.25 mg/kg/infusion over 5.6 s) under an operant FR1 schedule of reinforcement. This requirement was gradually increased to FR4 as described above. During training, each infusion was paired with the illumination of a house light and the sounding of a tone (66 dB). Following at least 10 days of stable FR4 self-administration, each animal was subjected to extinction training whereby operant responses on either lever produced no programmed consequences. When the criteria for extinction (i.e., responses reduced by at least 80 % for the active lever over two consecutive sessions), rats were exposed to a single reinstatement session whereby each response on the previously cocaine-associated lever was recorded and resulted in the presentation of the compound conditioned stimulus of the house light and tone, but no cocaine was delivered. Responses on the inactive lever were also recorded but resulted in no programmed consequences. Thirty minutes prior to the reinstatement test session, each animal was pretreated with allopregnanolone (2.5–40 mg/kg, IP), R6305-7 (5–40 mg/kg, IP) or vehicle in a random-dose order. Following the reinstatement session, each rat was retrained on the FR4 schedule of cocaine self-administration until stable responding (as defined above) was again observed. Self-administration behavior was extinguished and reinstated four times per rat using three different doses of the test steroid and one vehiclepretreatment session. This procedure allowed each animal to serve as its own control and decreased the total number of animals used in the experiments (Goeders et al. 2009; Keller et al. 2013). Reinstatement of food-seeking behavior A separate group of rats was used for a control experiment and were trained to self-administer food pellets (45 mg) according to the same operant schedule and training as described above for cocaine seeking. Starting under an FR1 schedule, responses on the food-associated lever resulted in the presentation of a food pellet and the same compound stimulus as above (i.e., activation of the house light and a 66 dB tone). The

Psychopharmacology

response requirement was gradually increased to FR4 and following 10 days of stable FR4 responding, each rat was exposed to extinction and reinstatement testing. These procedures were precisely the same as described above for the reinstatement of cocaine-seeking behavior except that the alternative food reinforcer was used for training rather than cocaine infusions. Statistical analyses All operant experiments employed a within-subjects design where possible and data were analyzed using a two-way analysis of variance (ANOVA) with repeated measures analysis for the factor of dose. Dunnett’s multiple comparison tests were employed to determine significant differences among treatment groups and doses. Data are presented as average reinforcer presentations (cocaine infusions and/or food pellets) per daily session. Raw reinstatement responses were analyzed by one-way repeated measures ANOVA and subsequently analyzed using Tukey’s HSD tests to determine significance among treatment groups.

Results Cocaine and food self-administration Rats successfully acquired cocaine and food selfadministration under a multiple, concurrent FR4 operant schedule of reinforcement over an average of 5 weeks (24.7 ±3.4 sessions). Following successful extinction training and vehicle treatment (12.3±2.6 sessions), administration of the neurosteroids allopregnanolone and R6305-7 was well tolerated with no significant outward signs of aversion or toxicity. Extinction training resulted in significantly decreased cocaineand food-reinforced responding (p

Effects of inhibitory GABA-active neurosteroids on cocaine seeking and cocaine taking in rats.

Several compounds that potentiate GABA-induced inhibitory currents also decrease stress, anxiety and addiction-related behaviors. Because of the well-...
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