Developmental Psychobiology

Adriana M. Falco Craig G. McDonald Robert F. Smith Department of Psychology George Mason University MSN 3F5, Fairfax, VA, 22030 E-mail: [email protected]

Anxiety Status Affects Nicotine- and Baclofen-Induced Locomotor Activity, Anxiety, and Single-Trial Conditioned Place Preference in Male Adolescent Rats ABSTRACT: Adolescents have an increased vulnerability to nicotine and anxiety may play a role in the development of nicotine abuse. One possible treatment for anxiety disorders and substance abuse is the GABAB agonist, baclofen. The aim of the present study was to determine the effect of anxiety-like behavior on single-trial nicotine conditioned place preference in adolescent rats, and to assess the action of baclofen. Baclofen was shown to have effects on locomotor and anxiety-like behavior in rats divided into high-anxiety and lowanxiety groups. Baclofen decreased locomotor behavior in high-anxiety rats. Baclofen alone failed to produce differences in anxiety-like behavior, but nicotine and baclofen þ nicotine administration were anxiolytic. High- and low-anxiety groups also showed differences in single-trial nicotine-induced place preference. Only high-anxiety rats formed place preference to nicotine, while rats in the low-anxiety group formed no conditioned place preference. These results suggest that among adolescents, high-anxiety individuals are more likely to show preference for nicotine than low-anxiety individuals. ß 2014 Wiley Periodicals, Inc. Dev Psychobiol 56: 1352–1364, 2014. Keywords: nicotine; adolescence; anxiety; conditioned place preference; GABAB receptor; baclofen; rat

INTRODUCTION Tobacco use represents a serious health epidemic, constituting the leading preventable cause of premature death in the United States (US Health & Human Services, 2010). Of interest are the prevention of and/or intervention in

Manuscript Received: 24 June 2013 Manuscript Accepted: 18 March 2014 The authors declared that they have no conflicts of interest. Correspondence to: A.M. Falco The present address of A.M. Falco is University of NebraskaLincoln, 238 Burnett Hall, Lincoln, NE, 68588 Contract grant sponsor: Virginia Healthy Youth Foundation Article first published online in Wiley Online Library (wileyonlinelibrary.com): 9 April 2014 DOI 10.1002/dev.21217  ß 2014 Wiley Periodicals, Inc.

nicotine dependence disorders prior to costly outcomes. Adolescence forms a unique period of vulnerability to nicotine. The majority of smokers begin smoking prior to age 17 and demonstrate a decreased ability to quit smoking compared to smokers who begin smoking later in life (Breslau & Peterson, 1996; Chen & Millar, 1998). Adolescent smokers also report higher levels of tolerance and dependence than adult counterparts (Kandel & Chen, 2000). Research with rodent models demonstrates the vulnerability of adolescents to nicotine. The rewarding effects of nicotine are heightened in adolescent rats, marking a critical period for the development of nicotine dependence (Adriani et al., 2003; Belluzzi, Lee, Oliff, & Leslie, 2004; Brielmaier, McDonald, & Smith, 2007; Shram, Funk, Li, & Leˆ, 2006; Shram & Leˆ, 2010; Torres, Tejeda, Natividad, & O’Dell, 2008).

Developmental Psychobiology

The coexistence of anxiety disorders and substance use disorders is present in numerous populations, including adolescents. However, the direction of causation between anxiety disorders and substance use disorders has yet to be clearly ascertained. Human research has noted that significantly higher percentages of individuals with anxiety disorders will develop substance dependence disorders than those in the general population (Liang, Chikritzhs, & Lenton, 2011). Adolescents who report social fears and social anxiety have a significantly higher risk of using cigarettes, developing nicotine dependence, and tend to have an earlier age of first tobacco use (Henry, Jamner, & Whalen, 2012; Marmorstein, White, Loeber, & Stouthamer-Loeber, 2010; McKenzie, Olsson, Jorm, Romaniuk, & Patton, 2010; Sonntag, Wittchen, Ho¨fler, Kessler, & Stein, 2000). Research with rodent models has investigated the impact of anxiety-like behavior on reward and reinforced behaviors, suggesting that anxiety-like behavior may predict cocaine self-administration and conditioned place preference (Dilleen et al., 2012; Pelloux, Costentin, & Duterte-Boucher, 2009; Schramm-Saptya et al., 2011). Anxiety and substance use disorders may be comorbid, but the underlying pathophysiology that links them has yet to be determined. Dysfunction of the gaminobutyric acid (GABA) system has been implicated in both anxiety (for reviews see Cryan & Kaupmann, 2005; Millan, 2003) and substance use and abuse (Heilig, Goldman, Berrettini, & O’Brien, 2011; Shorter & Kosten, 2011). The metabotropic GABAB receptor has been of research interest in both anxiety disorders (Mombereau et al., 2004; Ong & Kerr, 2005; Partyka et al., 2007) and drug addiction (Bowery, 2006; Cousins, Roberts, & de Wit, 2002; Tyacke, Lingford-Hughes, Reed, & Nutt, 2010). One drug under investigation for both anxiety and substance abuse disorders is baclofen, a GABAB agonist currently approved by the U.S. Food and Drug Administration (FDA) to treat muscle spasticity (US Food and Drug Administration, 2011). Baclofen administration has anxiolytic effects in the elevated plus maze (EPM) in male mice (Amikishieva & Semendyaeva, 2007), though it fails to modify nicotine-induced anxiety-like behavior in mice (Varani & Balerio, 2012). The effects of baclofen on drug addiction have been far better addressed. Work with preclinical samples has shown promise for the use of baclofen as a treatment for drug dependence and addiction. The acute administration of baclofen has been found to suppress responding for alcohol, heroin, and morphine (Colombo et al., 2003; Heinrichs, Leite-Morris, Carey, & Kaplan, 2010; Spano, Fattore, Fratta, & Fadda, 2007). Pretreatment with baclo-

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fen has been found to attenuate sensitization and locomotor effects of cocaine (Frankowska, Nowak, & Filip, 2009), amphetamine (Bartoletti, Gubellini, Ricci, & Gaiardi, 2005), morphine (Bartoletti, Ricci, & Gaiardi, 2007), and nicotine (Lobina et al., 2011; Palmatier & Bevins, 2002). Baclofen administration has been found to prevent reinstatement of nicotine self-administration (Fattore et al., 2009) as well as reducing rates of nicotine self-administration (Corrigall, Coen, Adamson, Chow, & Zhang, 2000; Corrigall, Coen, Zhang, & Adamson, 2001; Fattore, Cossu, Martellotta, & Fratta, 2002; Paterson, Froestl, & Markou, 2004). Baclofen pretreatment has also been shown to block expression of nicotine conditioned place preference (CPP) effects (Le Foll, Wertheim, & Goldberg, 2008). The use of baclofen in clinical populations has highlighted a potential role for its use in the treatment of drug addiction and substance use disorders. Baclofen administration has been found to decrease daily alcohol intake among alcoholics (Addolorato et al., 2011), in addition to reducing craving and withdrawal symptoms (Addolorato & Leggio, 2010). Baclofen may be useful in decreasing craving in some cocaine dependent subjects (Haney, Hart, & Foltin, 2006). Baclofen has been found to alter the sensory aspects of smoking, decreasing the enjoyment of cigarettes (Cousins, Stamat, & de Wit, 2001) as well as reducing the number of cigarettes smoked (Franklin et al., 2009). To date, cigarette smoking is a serious problem facing adolescents, but few treatments have been shown to be appropriate for adolescent use. Baclofen may have the potential to modulate both anxiety-like and reward behaviors, but thus far, most work has focused on adults. The present study tested the effects of acute doses of baclofen and nicotine on nicotineinduced locomotion, anxiety-like behavior, and conditioned reinforcement in adolescent male Sprague– Dawley rats. These rats were first split into high anxiety (HA) and low anxiety (LA) groups based on pretesting with a biased conditioned place preference (CPP) apparatus. Nicotine-induced locomotor and anxiety-like behaviors were assessed in the open field (OF) while conditioned reinforcement was measured via single-trial nicotine CPP using a biased methodology. It was hypothesized that administration of baclofen would cause differences in nicotine-induced locomotor and anxiety-like behavior, with HA rats showing larger reductions in the magnitude of anxiety-like behavior due to higher initial anxiety levels. In addition, it was hypothesized that baclofen administration would increase acquisition of single-trial nicotine CPP as a result of potential reinforcing effects due to mitigation of anxiety.

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MATERIALS AND METHODS Animals Male adolescent Sprague–Dawley rats (n ¼ 160) were obtained from Harlan (Indianapolis, IN) at postnatal day (P) 21 and housed in groups of 4 or 5. The rats were acclimatized to the colony for 7 days prior to testing. The rats were housed in a climate-controlled facility with temperatures kept at 22  2˚C on a 12 h light/dark schedule (lights on at 0700). Experiments were conducted during the light phase (from 1000 to 1800). Experiments were conducted using commercially available rats and during the light cycle due to the existing precedent in anxiety-like behavioral research under these conditions (Arrant, Jemal, & Kuhn, 2013; Bouet et al., 2012; Diaz, Chappell, Christian, Anderson, & McCool, 2011; In˜iguez, Warren, & Bolan˜osGuzma´n, 2010; Knoll et al., 2011). Food and water were available ad libitum, with rats being given additional food mash (rat chow mixed with water) at arrival to supplement the diet. All experiments were approved by the George Mason University animal care and use committee and were performed in adherence with George Mason University regulations and the NIH Guide for the Care and Use of Laboratory Animals, with note that the housing of rats in hanging wire cages, as was done during experimental sessions, is considered to be a stressor by NIH. Drugs ()-Nicotine hydrogen tartrate and R (þ) baclofen hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). All drugs were administered at an injection volume of 1 ml/kg body weight. Baclofen and saline were administered intraperitoneally (i.p.), and nicotine and saline were administered subcutaneously (s.c.) between the shoulder blades for both OF and CPP experiments. The doses of .6 mg/kg baclofen and .5 mg/kg nicotine were dissolved in saline solution (.9% NaCl). Dose levels of nicotine are expressed as free base equivalent, and the pH was adjusted to 7.1–7.4. The nicotine dose was selected based on prior work showing that adolescent rats formed single-trial CPP at the given dose (Belluzzi et al., 2004; Brielmaier et al., 2007; Brielmaier, McDonald, & Smith, 2008). The dose of baclofen was selected based on pilot data (not shown) and falls into a range in previous publication (Le Foll et al., 2008). Apparatus

Open Field. Locomotor and anxiety-like behavioral testing was performed in four OF chambers, created from white Plexiglas (42 cm  42 cm  30 cm) and located in a dimly lit (4–6 lx) testing room. The center area of the OF was defined as a square measuring 17.8  17.8 cm2. A camera mounted above the apparatus recorded the 15-min trials and data were acquired in 3  5-min intervals using Videotrack software (Viewpoint, Montreal, QC, Canada). Between each set of rats, each chamber was cleaned with 70% EtOH to eliminate odor cues.

Developmental Psychobiology

Conditioned Place Preference. Testing occurred in a two-chambered apparatus (Med Associates, St. Albans, VT) in the same dimly lit (4–6 lx) room as OF. Each chamber of the apparatus consisted of Plexiglas and had dimensions 21 cm  42 cm  30 cm. One chamber consisted of white walls with a mesh floor over a white paper lining, while the opposite chamber consisted of black walls with a stainless steel rod floor over a black paper lining. A black removable door separated the two chambers. Procedure

Pretesting. Rats were divided into HA and LA groups on the basis of pretesting. Pretesting used the CPP apparatus to determine innate levels of anxiety-like behavior. The apparatus is composed of a black chamber and a white chamber. Rats spend varying amounts of time in the white chamber; those that spend more time in the white chamber were considered LA, those that spend less time in the white chamber were considered HA. This theoretical partition was used due, in part, to work utilizing a paradigm that examines anxiety-like behavior in rats, the light-dark box, or blackwhite box. In this test, rats that prefer a white, more brightly lit chamber are thought to have lower anxiety levels than rats that prefer a black, lower lit chamber (Chaouloff, Durand, & Morme`de, 1997; Enkel, Thomas, & Bartsch, 2013; Miller, Piasecki, & Lonstein, 2011; Slawecki, 2005; Smythe, Murphy, Bhatnagar, Timothy, & Costall, 1996; Timothy, Costall, & Smythe, 1999). This division was performed on the basis of a median split so that that half with higher times in the white chamber were LA, and the half with the lower times in the white chamber were HA. Both experiments (OF and CPP) were run in four replications, and in each replication, HA and LA rats in each drug group were counterbalanced, so that an equal number of HA and LA rats were assigned to each drug group in each replication (Fig. 1). The first day of behavioral testing for all rats consisted of pretesting on P28 in order to divide into HA or LA groups. Rats were given access to both sides of the apparatus for 15 min. After pretesting, rats were divided into HA and LA groups based on median split and used in OF or CPP testing.

Experiment 1: Open Field. Eighty rats from pretesting were divided into the following groups: HA (n ¼ 40) and LA (n ¼ 40). Each group was further split into one of the following four drug treatment groups: saline þ saline (SAL), saline þ nicotine (NIC), baclofen þ saline (BAC), and baclofen þ nicotine (BAC þ NIC). Therefore, each drug treatment group consisted of a HA (n ¼ 10) and a LA (n ¼ 10) component. During the testing period, occurring on P29, rats were housed in individual hanging wire cages and permitted to habituate to the testing room for 20 min. Thirty min prior to OF, rats were injected (i.p.) with either .6 mg/kg baclofen or saline. Immediately before testing, another injection, of either .5 mg/kg nicotine or saline (s.c.) was administered. After drug injections, rats were placed in the center of the OF chamber and testing lasted for 15 min, with data being collected over 3  5 min bins.

Developmental Psychobiology

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FIGURE 1 A comparison of time (in seconds) spent in the white chamber of the conditioned place preference (CPP) apparatus on the pretesting day. The pretest was 15 min (900 s) in duration. The pretesting data of adolescent male Sprague–Dawley rats that were tested in the open field (OF) is represented in (a) by the drug groups SAL (saline þ saline), BAC (baclofen þ saline), NIC (saline þ nicotine), and BAC þ NIC (baclofen þ nicotine). The pretesting data of adolescent male Sprague–Dawley rats that were tested in CPP is represented in (b) by the drug groups SAL, BAC, NIC, and BAC þ NIC. In both a and b, the median and range is shown. A median split was conducted independently for each behavioral test and rats that spent more time in the white chamber of the apparatus were labeled low anxiety (LA) rats and rats that spent less time in the white chamber of the apparatus were labeled high anxiety (HA) rats. Post hoc comparisons (via Tukey’s test) were conducted and there are no statistical differences between the drug groups in either OF pretesting or CPP pretesting. However, within each drug group, there was a significant difference between the means of the pretest times of HA and LA rats, p  .01 (see figure for means).

Experiment 2: Conditioned Place Preference. Eighty rats from pretesting were split into HA (n ¼ 40) and LA (n ¼ 40) groups and were further designated to SAL, BAC, NIC, or BAC þ NIC groups during CPP testing. All rats underwent single-trial nicotine CPP testing in an apparatus that had previously been found to be biased, so that all rats in this sample spent significantly less than half of the time in the white chamber (Brielmaier et al., 2007, 2008; Brielmaier, McDonald, & Smith, 2012). Therefore, a biased procedure was used, where rats were conditioned with a drug in the non-preferred chamber (white) and saline in the preferred chamber (black). Testing consisted of three aspects: pretest session, conditioning sessions, and test session, occurring from P28 to P31. Each day, rats were placed into individual wire hanging cages during the testing period and permitted to habituate to the testing room for 20 min prior to testing. On the pretest day, each rat was placed in the apparatus and given free access to both chambers for 15 min. Natural, or unconditioned, preference for a chamber was determined by recording the amount of time spent in the white chamber. The definition of time spent in the white chamber was described as when the rat had all four paws completely in the white chamber. All rats were started in the white chamber, facing toward the removable door. These pretesting data were also used to split rats into HA and LA groups.

Rats underwent two conditioning sessions, one to administer drug (or saline in the case of controls), and one where all rats received saline. Rats were counterbalanced so that half received drug on the first conditioning session (and saline in the second), and half received drug in the second conditioning session (and saline in the first). During conditioning sessions, rats were weighed before being placed in the hanging cages and habituated. On drug conditioning days, rats then received an injection of either .6 mg/kg baclofen or saline (i.p.) and waited a period of 30 min. Immediately prior to CPP, rats received an injection of either .5 mg/kg nicotine or saline (s.c.) and were placed in their initially non-preferred chamber, facing away from the door, for 15 min. On the saline conditioning day, all rats received an injection of saline (i.p.) 30 min before CPP, and then an injection of saline (s.c.) immediately prior to CPP testing and were placed in their initially preferred chamber, facing away from the door, for 15 min. On the test day, rats were again given free access to the testing apparatus to determine chamber preference for 15min. All rats were again started in the white chamber facing the removed door. Preference was determined by time spent in the white chamber. Between all trials, both chambers were cleaned with 70% EtOH and paper was changed after each rat to remove odors.

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Statistical Analyses Locomotor and anxiety-like behavioral variables were analyzed in 5-min bins of OF activity using simple regressions with dummy coding in order to take into account the categorical variables. The locomotor variable used was total distance traveled in the OF, while the anxiety-like variables used were distance traveled in the center and time spent in the center of the OF. Drug groups were initially compared to the saline group in order to detect differences from baseline behavior (as represented by the saline group). Additional post hoc comparisons were conducted using Tukey’s test to determine any differences between drug groups. For the CPP experiment, difference scores were calculated for each animal by subtracting time in seconds spent in the initially nonpreferred chamber on the pretest day from time in seconds spent in the initially non-preferred chamber on the test day. Again, simple regressions with dummy coding were conducted to analyze data and take into account the categorical variables. The difference scores of drug groups were compared to the saline group to determine results of CPP. Regressions are reported with b values, to give the slope of the regression line. Analyses were considered significant at p < .05. All analyses were conducted using IBM SPSS 19.0 statistical software.

RESULTS Experiment 1: Open Field Three variables were considered to examine OF behavior, total distance traveled in the arena, distance

Developmental Psychobiology

traveled in the center of the arena, and time spent in the center of the arena. The locomotor variable assessed was the total distance traveled in the OF arena. This variable was measured in 3  5 min bins over the 15-min test. Initially, each drug group, BAC, NIC, and BAC þ NIC, was compared to the SAL group to determine statistical differences. Each 5 min bin was analyzed independently of the others. In the HA group, after 5 min, there were decreases in total distance traveled for the BAC (b0 ¼ 1,552.96, b ¼ 309.84, SE b ¼ 112.12, b ¼ .30, p < .05), NIC (b0 ¼ 1,552.96, b ¼ 571.98, SE b ¼ 115.336, b ¼ .53, p < .01), and BAC þ NIC groups (b0 ¼ 1,552.96, b ¼ 1,009.28, SE b ¼ 112.119, b ¼ .97, p < .01) when compared to SAL group (Fig. 2). In the 10 min bin, both the BAC (b0 ¼ 978.86, b ¼ 358.115, SE b ¼ 94.02, b ¼ .60, p < .01) and BAC þ NIC (b0 ¼ 978.86, b ¼ 360.00 SE b ¼ 94.024 b ¼ .60, p < .01) groups showed decreases compared to SAL (p < .05), but NIC did not. In the 15 min bin, none of the groups showed significant differences with respect to SAL. After the planned regression analysis between drug groups and SAL, further posthoc analyses were done between the individual drug groups. At the 5 min timepoint, there was a reduction of the BAC þ NIC group from the BAC group’s value, (Tukey’s test, p < .001). And, at the 5 and 15 min bins, there were differences between NIC and BAC þ NIC groups among HA rats (Tukey’s test, p < .05) with NIC rats traveling longer distances than BAC þ NIC rats.

FIGURE 2 A comparison of BAC (baclofen þ saline), NIC (saline þ nicotine), and BAC þ NIC (baclofen þ nicotine) drug groups with the SAL (saline þ saline) group among high anxiety (HA) and low anxiety (LA) adolescent male Sprague–Dawley rats on the total distance traveled variable. Among HA rats, at the 5 min interval, BAC, NIC, and BAC þ NIC rats traveled significantly less than SAL rats; at the 10 min interval BAC and BAC þ NIC rats still had lower distance traveled scores from SAL; and at the 15 min interval none of the groups differed from SAL. Among LA rats, at 5 min, NIC or BAC þ NIC rats had traveled significantly less distance than SAL rats ( p < .05).

Developmental Psychobiology

Among LA rats, there were also significant differences between drug groups and SAL on total distance traveled (Fig. 2). Rats that received either NIC (b0 ¼ 1,473.88, b ¼ 357.60, SE b ¼ 150.86, b ¼ .38, p < .05) or BAC þNIC (b0 ¼ 1,473.88, b ¼ 759.43, SE b ¼ 150.856, b ¼ .80, p < .01) exhibited reduced locomotor behavior in comparison to SAL rats in the first 5 min. By 10 and 15 min, these differences were no longer significant. There were also differences on total distance traveled among other drug groups in LA rats. In the first 5 min, the BAC þ NIC group showed reduced locomotor behavior in comparison to BAC rats (Tukey’s test, p < .01). There were also reductions from the distances traveled by NIC rats compared to those traveled by BAC þ NIC groups at 5 and 15 min (Tukey’s test, p < .05). The second variable measured was distance traveled in the center. In HA rats, when compared to SAL rats, NIC rats traveled a greater distance in the center of the arena at the 15 min bin, (b0 ¼ 25.39, b ¼ 36.58, SE b ¼ 16.85, b ¼ .39), p < .01. There were no other significant differences on this variable in either HA or LA rats. The third variable evaluated was time spent in the center of the arena, a variable used to gauge anxietylike behavior. Among HA rats, rats that were administered BAC þ NIC spent more time in the center than rats that received SAL at 5 min (b0 ¼ 20.06, b ¼ 29.47, SE b ¼ 12.21, b ¼ .43) and 15 min (b0 ¼ 4.13,

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b ¼ 15.50, SE b ¼ 6.82, b ¼ .41), p < .05 (Fig. 3). Among LA rats, BAC þ NIC rats spent more time in the center than SAL rats at 5 (b0 ¼ 13.23, b ¼ 34.07, SE b ¼ 14.02, b ¼ .45) and 10 min (b0 ¼ 12.97, b ¼ 14.48, SE b ¼ 5.91, b ¼ .45), p < .05, and NIC rats spent more time in the center than SAL rats at 15 min (b0 ¼ 8.20, b ¼ 9.80, SE b ¼ 4.38, b ¼ .41), p < .05 (Fig. 3). In addition, BAC þ NIC rats spent more time in the center than BAC rats at 5 and 10 min (Tukey’s test, p < .05).

Experiment 2: CPP All rats in this sample strongly preferred the black chamber of a biased apparatus upon first exposure to the apparatus (see Fig. 1 for bias). Among HA rats, there were statistically significant differences between rats administered either NIC (b0 ¼ 3.13, b ¼ 102.32, SE b ¼ 36.36, b ¼ .528, p < .01) or BAC þ NIC (b0 ¼ 3.13, b ¼ 98.28, SE b ¼ 35.50, b ¼ .524, p < .01) and rats administered SAL. Both of the NIC and BAC þ NIC groups had a significantly higher difference score than the SAL group (Fig. 4). As a result, HA rats that were administered NIC or BAC þ NIC had a significantly higher positive change in their preference for the white side of the apparatus after the conditioning session in comparison to SAL rats. Among LA rats, there were no significant differences between the SAL group and any of the drug groups (Fig. 4). These findings showed that among rats with high levels of innate anxiety-like

FIGURE 3 A comparison of the BAC (baclofen þ saline), NIC (saline þ nicotine), and BAC þ NIC (baclofen þ nicotine) drug groups with the SAL (saline þ saline) group among high anxiety (HA) and low anxiety (LA) adolescent male Sprague–Dawley rats on the time spent in the center variable. Among HA rats, BAC þ NIC rats spent significantly more time in the center than SAL rats at 5 and 15 min. Among LA rats, BAC þ NIC rats spent significantly more time in the center than SAL rats at 5 and 10 min ( p < .05).

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Developmental Psychobiology

FIGURE 4 The difference scores representing the conditioned place preference (CPP) behavior of the SAL (saline þ saline), BAC (baclofen þ saline), NIC (saline þ nicotine), and BAC þ NIC (baclofen þ nicotine) drug groups among high anxiety (HA) and low anxiety (LA) adolescent male Sprague–Dawley rats. The difference score is calculated by subtracting time spent on the white side of chamber on the pretest day from time spent on the white side of the chamber on the test (or postconditioning) day. Among HA rats, the NIC and BAC þ NIC groups showed significant increases in the time spent on the white side of the chamber, where nicotine was delivered, compared to the SAL group;  p < .05. Among LA rats, there were no significant differences between any of the groups.

behavior that were dosed with nicotine formed CPP, while no dosed group among rats with low levels of innate anxiety-like behavior formed CPP.

DISCUSSION Adolescent male Sprague–Dawley rats were separated into HA and LA groups using a median split analysis based on time spent in the white chamber of a biased CPP apparatus during the first exposure to the apparatus. This method was utilized because pilot testing found that pretesting with the EPM prevented early adolescents from forming single-trial nicotine CPP in a biased apparatus (unpublished pilot data). However, the biased CPP chamber employed is similar to the lightdark box, a common method utilized to ascertain anxiety-like behavior, in that rats with higher levels of anxiety-like behavior prefer the black side of a black and white chamber (Chaouloff et al., 1997; Enkel et al., 2013; Miller et al., 2011; Slawecki, 2005; Smythe et al., 1996; Timothy et al., 1999), so it is likely that the CPP apparatus is also capable of providing a measure of anxiety-like behavior. However, there are significant differences in the lighting in the light-dark box, the white side tends to be more brightly

lit while the black side is dim, which were not as clearly present in this CPP methodology. Use of this methodology alleviates the issues of EPM blocking the ability to achieve single-trial nicotine CPP and the inability to use the center preference behavior in the OF as a pretest of anxiety-like behavior due to its inclusion later in the protocol. In addition, other tests of emotion-like behavior which could be used as predictors, such as the forced swim test, are known to be stressors which clearly affect CPP behavior (Attarzadeh-Yazdi, Karimi, Azizi, Yazdi-Ravandi, Hesam, & Haghparast, 2013; Brielmaier et al., 2012; Lea˜o, Cruz, & Planeta, 2009; Schindler, Li, & Chavkin, 2010). However, the use of the CPP apparatus to collect pretesting data makes anxiety-like status a confound of CPP behavior in that it is a second behavior that is dependent on the CPP apparatus. It may be more methodologically sound to use the lightdark box, or determine another low-stress emotion-like test, to delineate between HA and LA rats. Testing using the OF found differences in locomotor and anxiety-like behavior in rats dependent on HA/LA status post-baclofen and/or nicotine administration. Notably, BAC administration significantly decreased locomotor behavior from SAL levels in HA rats, but did not do so in LA rats. In both HA and LA groups,

Developmental Psychobiology

BAC þ NIC co-administration significantly decreased locomotor behavior from locomotor activity levels in NIC rats. In these findings, nicotine seems to play a central role. However, the role of acute nicotine on locomotor activity in adolescents is unclear. While this study found depressant effects on locomotor activity in the NIC and BAC þ NIC groups, other studies have found either no effects of acute nicotine on locomotor activity or have found motor-activating effects of nicotine in adolescents (Belluzzi et al., 2004; Schochet, Kelley, & Landry, 2004). However, these differences could be entirely due to differences in the paradigms used to measure locomotor activity or the dose of nicotine used. One particularly notable finding of this study is that HA rats showed a reduction in locomotor behavior between those in the BAC group and controls (SAL group). This relationship was not statistically significant in the LA group. Administration of baclofen is known to reduce locomotion in rats, though often at higher doses (Frankowska et al., 2009; Le Foll et al., 2008; Palmatier & Bevins, 2002). This finding suggests that adolescents may be more susceptible to the sedating effects of baclofen. However, the R (þ) baclofen isoform is used here and is a purer isoform than the commonly used RS-baclofen. Research suggests that when the R-isoform is used, about half the dose is needed of the S-isoform to get similar plasma and cerebrospinal fluid concentrations (Mandema, Heijligers-Feigen, Tukker, De Boer, & Danhof, 1992). Attempts were made to take this relationship into account during pilot testing of the dose, but as this work was done in adult rats, it may be that adolescents metabolize the drug differently. This current study also found, that in adolescents, innate anxiety-like behavior is a variable that determines reaction to the locomotor effects of baclofen. It is possible that these findings are only applicable to adolescents, as adolescents are known to exhibit higher levels of anxiety-like behavior than adults in numerous paradigms (Lynn & Brown, 2010); clearly these results would have to be replicated in adults. Another interesting finding is that in both HA and LA groups, dosing with baclofen þ nicotine significantly reduced the locomotor behavior in comparison to rats dosed only with nicotine (see Fig. 2). This is supported by literature which shows that baclofen administration reduces the activity levels of adult rodents dosed with nicotine (Lobina et al., 2011; Palmatier & Bevins, 2002) and cocaine (Frankowska et al., 2009). The means of rats in the NIC group and rats in the BAC þ NIC group are significantly lower than SAL rats at several time points. It appears that the GABAB activation in the rats in the BAC þ NIC group is playing a role in the further

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suppression of locomotor activity due to the fact that the addition of baclofen to nicotine may have an additive effect. The OF was also used to examine potential differences in anxiety-like behavior. The BAC group in both the HA and LA groups failed to produce any differences in anxiety-like behavior, but the NIC group and BAC þ NIC group had slightly different patterns of effects on anxiety-like behavior in HA and LA rats, though administration of both drugs was anxiolytic. It was expected that the BAC and BAC þ NIC groups would have a more significant impact on anxiety-like behavior. This hypothesis was driven by several studies suggesting a link for the GABAB receptor in anxiety-like behavior. Genetic work has shown that GABAB(1)/ mice, which lack functional GABAB(1) receptors, were more anxious than wildtype littermates in the light-dark box and staircase test (Mombereau et al., 2004). Studies with baclofen have shown that baclofen administration has anxiolytic effects on the EPM in male mice (Amikishieva & Semendyaeva, 2007), however, it has also been shown that baclofen administration was unable to alter the dose-dependent anxiety-like behavior produced by nicotine in male mice (Varani & Balerio, 2012). It is possible that baclofen had little effect on anxiety-like behavior in this study because the subjects were adolescents, whereas most previous work has been done with adults, or due to biological differences between rats and mice. It is difficult to determine if nicotine is the agent driving anxiolytic behaviors in both the NIC and BAC þ NIC groups, or if baclofen, when added to nicotine, has an additive effect. The behaviors displayed in the NIC and BAC þ NIC groups, where the majority of the behavioral effects are seen in the first 5 or 10 min bin, are consistent with the pharmacokinetics of both nicotine and baclofen. Nicotine reaches its highest brain concentration within 20 min of s.c. administration (.4 mg/kg) and then drop by approximately 1/3 over the next 40 min (Rosecrans & Schechter, 1972). This effect is supported by behavioral data which shows peak locomotor behaviors at the dose of .4 mg/ kg between 0 and 20 min (Clarke & Kumar, 1983; Ksir, Hakan, Hall, & Kellar, 1985). These findings mirror the locomotor results reported here. Rats were placed into the testing arenas 30 min after baclofen administration, approximately when both blood and brain concentrations of the drug reach their peaks before decreasing (Mandema et al., 1992). It is also possible that these effects are due to habituation to the OF chamber. On all three measures, SAL animals decrease markedly between 5 and 15 min. In most cases, the drug-treated animals are almost indistinguishable from SAL animals at the 15 min marks. It is likely that all animals have

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habituated to a point where floor effects were reached and there was no room to distinguish between cohorts. Though GABA is the main inhibitory neurotransmitter in adults, it actually serves as an excitatory neurotransmitter in early postnatal development (BenAri, Khazipov, Leinekugel, Caillard, & Gaiarsa, 1997) and studies have shown that during early adolescence, GABA neurons respond more weakly to GABA agonists due to immaturity of the neurons (Cohen, Lin, & Coulter, 2000). Many areas of the GABA system are changing during adolescence. The GABA system, including the GABAB-containing areas of the brain, are in flux at approximately this point in development (see Kilb, 2012 for review). Some areas containing GABAB receptors and neurons are developing before reaching adult levels at approximately P35-40. In addition, the GABAA areas of the brain, which are known to play a role in innate anxiety levels, are in flux until about P60. It may be that the results seen here are different than expected because adolescents have a different GABAB receptor sensitivity or different numbers of receptors than adult populations. Similar profiles of the dopamine system exist in adolescent rats (Andersen, Thompson, Rutstein, Hostetter, & Teicher, 2000). It is possible that an interaction between the dopamine and GABA system could also be responsible for these behaviors during this period of adolescence. Therefore, it is entirely possible that baclofen can be impacting locomotor or anxiety-like behavior in this study via GABAB receptors and neurons, or that the GABAA areas of the brain that may be regulating differences between the HA and LA rats are playing a role on locomotor or anxiety-like behavior. This study used a two-chamber biased apparatus and a biased assignment procedure for the CPP portion of the study. The research surrounding these topics in CPP is extensive, but pertinent to the results of this study. Drug research tends to support the use of a threechambered apparatus which has been found to produce larger effects with heroin, amphetamine, and cocaine (Bardo, Rowlett, & Harris, 1995). This may be because the separation of the drug and saline context are more distinct than in the two-chambered apparatus. Other positives of the three-chambered apparatus are that the middle chamber can be used either as a starting point or a novel chamber to assess drug preference to novelty (Bardo & Bevins, 2000; Tzschentke, 1998). However, the two-chambered design is quite prevalent in CPP research and may be useful when a biased design is preferential. A biased design, seems to work well with cocaine, but also may be necessary for achieving nicotine CPP (Bardo et al., 1995; Le Foll & Goldberg, 2005). Studies that have compared biased to unbiased design in nicotine CPP have found that

Developmental Psychobiology

nicotine CPP occurs only in the biased design in adult populations (Calcagnetti & Schechter, 1994; Le Foll & Goldberg, 2005). However, research with adolescent has shown mixed results with biased designs. Two studies using adolescents in multiple nicotine session biased designs failed to produce CPP (Perna, Henderson, Bruner, & Brown, 2011; Torrella, Badanich, Philpot, Kirstein, & Wecker, 2004). However, the study described in the current paper uses a biased, single conditioning trial design which has been shown to be effective in creating nicotine-induced CPP in adolescents (Belluzzi et al., 2004; Brielmaier et al., 2007, 2008, 2012). There may also be a benefit to single trial CPP as fewer sessions tend to produce stronger effects with psychostimulants (Bardo et al., 1995; Tzschentke, 1998). The present study found that HA rats that were in the NIC or BAC þ NIC group were able to form single-trial nicotine CPP, while no group among LA rats were able to achieve CPP. This indicates that chamber preference on the pretest day is a predictor of strength of nicotine CPP, and suggests that the pretest measure, possibly analogous to the measure provided by light-dark box, may be a valid indicator of anxiety. Previous work with cocaine has suggested that high anxiety rats achieve higher rates of cocaine CPP (Pelloux et al., 2009), in addition to higher rates of cocaine self-administration (Dilleen et al., 2012; Schramm-Saptya et al., 2011), though this relationship has not been seen with alcohol (Langen & Fink, 2004). Among the HA rats, it seems likely that nicotine is driving the CPP effect among rats BAC þ NIC group, as there was no significant alterations in the difference scores between the NIC and BAC þ NIC groups. Previous studies using baclofen have shown that administration of 3 mg/kg of R (þ) baclofen, though neither .3 or 1 mg/kg of baclofen, blocked expression of nicotine CPP (Le Foll et al., 2008). In addition, administration of baclofen was capable of preventing reinstatement of nicotine CPP in mice (Fattore et al., 2009). It may be that baclofen has an ability to block acquisition of nicotine CPP, but only at high doses. However, it would seem that, at least in adolescents, the sedative effects at such a high dose may be problematic, though in this study the baclofen is received during CPP conditioning, not on the test day, so the sedative effect may have negligible effect. The administration of the current dose of baclofen did not have a long-lasting locomotor impact that impaired CPP, as is demonstrated by the fact the BAC þ NIC and NIC groups acquired comparable single-trial nicotine CPP. It is also noteworthy, that during OF testing, nicotine dosing did not alter anxiety-like behavior in either

Developmental Psychobiology

HA or LA groups, supporting the concept that the anxiety-like behavioral difference here is innate and not drug-induced. Unfortunately, using HA and LA groups in a CPP paradigm with drugs that have been shown to be anxiolytic does not provide any final word on reward, only on conditioned reinforcement, as CPP can be caused by anxiolysis (File, 1985; Schechter & Calcagnetti, 1993). It is entirely possible that the HA rats find the NIC and BAC þ NIC administrations reinforcing partly because they are anxiolytic and not completely rewarding per se. In conclusion, our results found motor suppressant and anxiolytic effects of nicotine administration and nicotine and baclofen co-administration in male, adolescent, Sprague–Dawley rats that underwent a median split into high and low anxiety groups. In addition, anxiety status served as a predictor status for developing single-trial nicotine CPP, with only HA rats developing CPP, though baclofen administration did nothing to alter this effect. This study questions if baclofen is feasible as a nicotine treatment at all points in the addiction process or in all populations, but elucidates the coexistence of adolescence and high anxiety as dual roles in forming nicotine-reinforced behaviors.

NOTES This research was funded in part by the Virginia Foundation for Healthy Youth (VFHY). Thank you to C.J. Blanchard for assistance in collecting pilot data.

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