Nicotine enhancement and reinforcer devaluation: Interaction with opioid receptors

Pharmacology, Biochemistry and Behavior 150–151 (2016) 1–7

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Nicotine enhancement and reinforcer devaluation: Interaction with opioid receptors Ari P. Kirshenbaum ⁎,1, Jesse A. Suhaka 1, Jessie L. Phillips, Maiary Voltolini de Souza Pinto Saint Michael's College, Colchester, VT 05443, United States

a r t i c l e

i n f o

Article history: Received 31 March 2016 Received in revised form 10 August 2016 Accepted 17 August 2016 Available online 19 August 2016 Keywords: Enhancement Naloxone Nicotine Opioid Reinforcement Withdrawal

a b s t r a c t In rats, nicotine enhances responding maintained by non-pharmacological reinforcers, and discontinuation of nicotine devalues those same reinforcers. The goal of this study was to assess the interaction of nicotine and opioid receptors and to evaluate the degree to which nicotine enhancement and nicotine-induced devaluation are related to opioid activation. Nicotine (0.4 mg/kg), or nicotine plus naloxone (0.3 or 3.0 mg/kg), was delivered to rats prior to progressive ratio (PR) schedule sessions in which sucrose was used as a reinforcer. PR-schedule responding was assessed during ten daily sessions of drug delivery, and for three post-dosing days/sessions. Control groups for this investigation included a saline-only condition, and naloxone-only (0.3 or 3.0 mg/kg) conditions. When administered in conjunction with nicotine, both naloxone doses attenuated nicotine enhancement of the sucrose reinforcer, and the combination of the larger dose of naloxone (3.0 mg/kg) with nicotine produced significant impairments in sucrose reinforced responding. When administered alone, neither dose of naloxone (0.3 & 3.0 mg/kg) significantly altered responding in comparison to saline. Furthermore, when dosing was discontinued after ten once-daily doses, all nicotine groups (nicotine-only and nicotine + naloxone combination) demonstrated significant decreases in sucrose reinforcement compared to the saline group. Although opioid antagonism attenuated reinforcement enhancement by nicotine, it did not prevent reinforcer devaluation upon discontinuation of nicotine dosing, and the higher dose of naloxone (3.0 mg/kg) produced decrements upon discontinuation on its own in the absence of nicotine. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Experimental investigations demonstrate that rats will self-administer nicotine (e.g. DeNoble and Mele, 2006; Ismayilova and Shoaib, 2010), and these data have been included as an important part of the scientific canon that nicotine is dependence-producing. However, many researchers agree that direct primary reinforcement by nicotine cannot fully account for the abuse liability of nicotine-containing products (Caggiula et al., 2002, 2009; Dar and Frenk, 2004; Donny et al., 2011; Kirshenbaum et al., 2015; Rupprecht et al., 2015; Weaver et al., 2012). Consequently, additional mechanisms through which nicotine encourages dependence have generated interest. For instance, nicotine's primary reinforcing effects may be complemented by its ability to enhance the value of other reinforcers experienced in its presence, and this reinforcement-enhancing effect of nicotine has been thoroughly researched over the last decade or more (Barret and Bevins, 2013; Cagguila et al., 2002; Cassidy and Dallery, 2014; Chaudhri et al., 2006,

⁎ Corresponding author at: Department of Psychology and PrePharmacy Program, One Winooski Park Ave, Box 193, Colchester, VT 05443, United States. E-mail address: [email protected] (A.P. Kirshenbaum). 1 Contributed equally to the manuscript.

http://dx.doi.org/10.1016/j.pbb.2016.08.003 0091-3057/© 2016 Elsevier Inc. All rights reserved.

2007; Donny et al., 2003; Kirshenbaum et al., 2015; Liu et al., 2009; Palmatier et al., 2008a & b; Rupprecht et al., 2015; Weaver et al., 2012). Because of its connection to subjective hedonic effects, opioid activation by nicotine may play a critical role in nicotine's ability to produce reinforcer enhancement. In particular, μ opioid receptors have been pinpointed as a mediator of drug reward, with the δ receptor involved on a slightly lesser scale (Berrendero et al., 2012; Hadjiconstantinou and Neff, 2011; Ismayilova and Shoaib, 2010). Opioid antagonists have been shown to counteract nicotine's effects as a primary reinforcer (Ismayilova and Shoaib, 2010; Liu and Jernigan, 2011) but other studies have produced null interactive effects (DeNoble and Mele, 2006; Corrigall and Coen, 1991; Lui et al., 2009). Limited evidence suggests that opioid antagonism can alter nicotine-related associative mechanisms (Liu et al., 2009; Palmatier et al., 2004) which may pertain to reinforcement enhancement. The primary purpose of the present study was to examine opioid system involvement in nicotine-induced reinforcer enhancement. A secondary objective was to examine how reinforced behavior is altered when nicotinic-receptor activation and opioid antagonism are discontinued. These objectives were accomplished by observing the combined effects of the opioid antagonist naloxone and nicotine on sucrose-reinforced behavior. In all experiments, a progressive ratio (PR) schedule was used as a means of testing relative reinforcer efficacy

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(Perkins and Karelitz, 2013; Stafford et al., 1998), and sucrose was used in order to parallel previous investigations of reinforcer enhancement of PR responding (e.g. Palmatier et al., 2008a; Kirshenbaum et al., 2015). Two doses of naloxone were used (0.3 and 3.0 mg/kg) to determine how each would alter sucrose reinforcement when administered in the presence and absence of 0.4 mg/kg nicotine. Experimentation involving the PR schedules occurred over consecutive days to determine the interactive effects of the drugs across (a) 10 days/sessions of drug dosing and (b) upon 3 days of NIC discontinuation. If naloxone disrupts nicotine's effect on sucrose reinforcement during the sessions of drug dosing, then it also may prevent a motivational features typically associated with nicotine discontinuation in rodents, evidenced by diminished PR-schedule responding (Kirshenbaum et al., 2015; LeSage et al., 2006). 2. Materials and methods 2.1. Subjects Fifty-nine naïve Sprague-Dawley rats were involved in the experiment (Rattus norvegicus, obtained from Charles River Laboratories, Montreal, Quebec, Canada at 65 days of age). Rats were pair-housed in an OptiRat™ housing unit (Animal Care Systems, Centennial, Colorado, USA) with 1/8″ corn cob bedding, and a portion of PVC pipe (6″ diameter, 7″ length) was placed into each home cage. Rats received ad lib water and experienced a 12/12 h day/night light cycle and experimentation occurred in the light cycle. All rats received 15 g of rat chow per day (Harlan Industries, 18% protein), and were maintained at or above their initial free-feeding body weight upon delivery. The animal-holding room was temperature and humidity controlled. All experimentation was approved by the Institutional Animal Care and Use Committee of Saint Michael's College. The procedures and animal care practices are in agreement with the ARRIVE guidelines, and conform to those listed in the Guide for the Care and Use of Laboratory Animals. 2.2. Apparatus All behavioral investigation utilized standard rat operant chambers (ENV-008, Med-Associates, Saint Albans, Vermont). Six operant chambers were used to assess liquid-sucrose-reinforced behavior. Each chamber included two nose-poke operant devices located along the same wall, with a liquid dipper equidistant between each nose-poke operant device. The rats were reinforced with sucrose solution (8%, delivered in 0.03 ml per reinforcing event). This concentration of sucrose solution has been demonstrated to be a desirable reinforcer on a progressive-ratio schedule of reinforcement (Kirshenbaum et al., 2015). A desktop personal computer was used to collect and analyze the data through the MED-PC IV platform. 2.3. Drugs Saline (0.9% solution) was administered as a control in 1 ml/kg doses, and was also used as the vehicle for NIC and naloxone (NAL). 0.3 and 3.0 mg/kg doses of NAL were delivered as a base from naloxone hydrochloride (391.87 g per mole) in concentrations that yielded injections of approximately 1 ml/kg. Consistent with prior research, these doses were chosen as representative of a low and moderate dose of NAL (DeNoble and Mele, 2006; Ismayilova and Shoaib, 2010). Furthermore, 0.3 and 3.0 mg/kg of NAL does not appear to reliably reduce the motivation for concentrations of sucrose approaching 10% (Cleary et al., 1996). NIC was delivered as a base from nicotine ditartrate (161.99 g per mole) in a concentration diluted in saline that yielded doses of approximately 1 ml/kg. All drugs were obtained from SigmaAldrich (Sigma Chemical Co., St. Louis, Missouri, USA) and administered subcutaneously.

2.4. Procedure Rats were randomly assigned to one of six experimental groups: (i) SAL control, (ii) 0.4 mg/kg NIC only, (iii) 0.3 mg/kg NAL + 0.4 mg/kg NIC, (iv) 3.0 mg/kg NAL + 0.4 mg/kg NIC, (v) 0.3 mg/kg NAL only, and (vi) 3.0 mg/kg NAL only. For all groups n = 10, with the exception of group vi (n = 9). 2.4.1. Operant shaping Rats were shaped to respond for sucrose solution using two 30-min sessions of variable time (VT) 30-s presentations of sucrose to create the association between the sound of the liquid-dipper being raised and the availability of sucrose reinforcement. The VT sessions occurred in the dark without any visual cues. Photobeam sensors in the liquid-dipper well detected rat's responses to reinforcement, and the sucrose solution remained available for 5 s following the detection of a head entry. Fixed-ratio (FR) schedule shaping sessions followed the two days (one session per day) of VT 30-s training. During all of the operant-response sessions, green illumination LED of the right nose-poke device (LEDg) signaled the start of the training period. Upon completion of the response requirement (e.g. 2 responses under the FR2 requirement), LEDg was terminated, and red illumination of the right nosepoke device (LEDr) occurred simultaneously with the raising of the liquid dipper to signify that the reinforcer was available. Rats were allowed 5-s to retrieve their reward, and a photobeam interruption detected a head-entry into the liquid dipper chamber. LEDr and the availability of the liquid dipper terminated simultaneously, and the LEDg light was reactivated to signal the availability of reinforcement. The first session of reinforced responding occurred using an FR1 schedule that terminated after 50 reinforcers. The FR schedule was increased on a per-subject basis from an FR1 to an FR2 following the receipt of all 50 reinforcers. The transition from an FR1 to an FR2 occurred over three consecutive days, and on the third day of reinforced responding, all subjects had met the FR2 requirement. All subjects then experienced four consecutive once-daily sessions of the FR3 schedule maintained by sucrose. 2.4.2. Progressive ratio The PR-schedule is a sequentially increasing schedule of reinforcement. As the response requirement for sugar increases (1, 2, 4, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, 737, 901), the actual amount of sugar being delivered never changes. The PR schedule is a standard means of assessing motivation in behavioral pharmacological research because of the large response costs and diminishing returns (for a review, see: Bradshaw and Killeen, 2012; Richardson and Roberts, 1996; Stafford et al., 1998). The present study used a formula for PR-schedule escalations derived from a critique by Arnold and Roberts (1997), and used by Kirshenbaum et al. (2015) and Palmatier et al. (2008a) for sucrose reinforcement and NIC pre-session administration. The reinforcer concentration (8%), magnitude (0.3 ml) and the stimulus conditions (i.e. LEDg and LEDr) surrounding the delivery of sucrose solution were identical to the FR schedule sessions in operant shaping. The PR-schedule sessions expired after 40 min, or when the rat's inter-response times or post-reinforcement pauses exceeded 2.5 min. The primary dependent variable used for the present experiment was the session duration, or the amount of time (min) that the rats continuously worked to obtain reinforcement under these parameters, and the use of session durations is consistent with previous research (Kirshenbaum et al., 2015). The baseline phase of PR schedule responding concluded when all groups displayed no monotonic increases or decreases in session durations, with a maximum of 15 consecutive baseline-phase sessions per subject. 2.4.3. Drug dosing protocol Each dosing day consisted of a single PR session combined with drug treatment, and the conditions of this phase persisted for a total of 10 consecutive days. All rats received two subcutaneous injections (s.c.)

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prior to the start of each session. The first injection occurred 30 min before the second injection, and during this 30-min interval rats were placed back into their home cages. The second s.c. injection occurred at the end of the 30-min interval, and rats were immediately placed into their respective operant chambers for a 5 min black-out period in which no stimuli were presented and operant responses were not reinforced. The 30-min interval between the first and second injection mimicked the procedures used in previous research (Lynch and Burns, 1990; Ngai et al., 1976; Ismayilova and Shoaib, 2010; Sirohi et al., 2007), and allowed NAL time for full circulatory distribution. Sequentially, the first and second s.c. injections (first/s), per group (i–vi), were as follows (all doses in mg/kg): i. SAL/SAL; ii. SAL/NIC; iii. 0.3 NAL/NIC; iv. 3.0 NAL/ NIC; v. 0.3 NAL/SAL; and vi. 3.0 NAL/SAL. 2.4.4. Discontinuation of drug dosing This phase of discontinuation testing followed the same methods described in the drug dosing phase, except all groups received two sequential s.c. injections of saline, only (SAL/SAL). The discontinuation phase lasted for 3 consecutive days with one session per day to assess how prior exposure to the various drug combinations altered PR schedule session durations. 2.5. Dependent measures and statistical analyses Session duration was the primary dependent variable of behavior in these experiments in order to make them comparable to previous report on nicotine-induced reinforcer devaluation (Kirshenbaum et al., 2015). Session duration is not an uncommon variable for studies involving PR-schedule performance (Stafford et al., 1998) and they provide an index of prolonged, uninterrupted responding given the current parameters which define session length (i.e. sessions terminated when interresponse times or post-reinforcement pauses exceeded 2.5 min). The number of reinforcing events per session, a.k.a. the number of ratio requirements completed, was used as a secondary dependent variable. Although one might expect reinforcing events to parallel session durations, as rats accrue reinforcers on the PR schedule, there is less freedom for this measure to vary at the upper limits. Other secondary dependent variables included response rate and adjunctive-response rate on the inactive nose-hole poke, and these were calculated as: total responses per session/session duration, in min. Response rate on the active nose-poke operant can be used to identify rate-enhancing effects of the drugs on schedule-controlled behavior, and response rate on the inactive operant may be indicative of global locomotor stimulant effects. In order to determine whether groups differed in regard to PRschedule controlled responses prior to the delivery of drugs, one-way ANOVA was performed across six groups on session durations and all secondary dependent variables (reinforcing events, response rate, adjunctive response rate) using means from the last three baseline sessions, per rat. To examine whether nicotine increased operant responding and whether naloxone attenuated that increase, general between-group differences were assessed using one-way ANOVAs performed at the beginning (first 3-day means) and end (last 3-day means) of the dosing phase. These one-way ANOVAs also allowed for post-hoc multiple comparisons at the beginning and end of dosing to examine specific between-group differences resulting from NAL, NIC, and NAL + NIC drug exposure. Between-group comparisons to the saline control served as the primary indicator of drug-induced changes in schedule-controlled behavior. In order to evaluate whether nicotine discontinuation resulted in a devaluation of the sucrose reinforcer, a between-group one-way ANOVA was performed again on each separate dependent variable, per group, in the 3-days following the termination of drug dosing. This one-way ANOVA, and the multiple comparisons which followed, allowed for the detection of whether naloxone was able to prevent the devaluative effect of nicotine; furthermore, these comparisons

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made it possible to determine whether naloxone dosing alone disrupted session durations and reinforcing events relative to a saline-only control group upon discontinuation of drug dosing. 3. Results One rat from the nicotine group (n = 10) failed to complete the experiment; dental complications prevented the rat from eating and interfered with consumption of the sucrose reinforcer, resulting in an n = 9 for this group, and an overall N = 58. One-way ANOVA performed on baseline (pre-dosing) session durations failed to demonstrate significant differences between the six groups; F (5, 57) = 1.05, P N 0.05, see Fig. 1. In fact, all groups failed to differ with regard to any secondary dependent variable at baseline (reinforcing events, response rate, and adjunctive-response rate); all F′s (5, 57) b 1.80, all P's N 0.05. To compare groups in terms of session duration at the initial period of dosing, a one-way ANOVA was performed using the means of the first three sessions of drug exposure; F (5, 57) = 2.90, P b 0.05, partial-η2 = 0.29. Post-hoc multiple LSD comparisons demonstrated the NIC group differed from saline/control group, the 3.0 NAL + NIC, and the 0.3 NAL only groups (all P′s b 0.5), and the 3.0 NAL + NIC group differed from the 0.3 NAL + NIC group (P b 0.05), but no other post-hoc differences were discovered. See Fig. 1 for a comparison of session durations. Between-group differences were also discovered for reinforcing events; F (5, 57) = 3.73, P b 0.01, partial-η2 = 0.26. LSD Post-hoc multiple comparisons revealed that the NIC group differed from saline and the 3.0 NAL + NIC groups (P′s b 0.05), and 3.0 NAL + NIC group differed from all other groups (all P′s b 0.05), but no other significant differences were found. Between group differences were not found for either response-rate measure at this initial portion of the dosing phase; F's (5, 57) b 1.02, P's N 0.05. To determine whether groups differed at the end of the dosing phase, a one-way ANOVA was performed on the average of session durations for the final three days of dosing, resulting in significant between-group differences; F (5, 57) = 5.55, P b 0.001, partial-η2 = 0.35. Post-hoc multiple comparisons (LSD) demonstrated that the NIC group's session durations were significantly greater from the saline, 3.0 NAL, and 3.0 NAL + NIC groups (P's b 0.05). The 3.0 NAL + NIC group differed from all other groups (P's b 0.05) in that they were lower. Between-group differences were also discovered for reinforcing events (F (5, 57) = 8.93, P b 0.001, partial-η2 = 0.46) and response rate on the active nose-poke (F (5, 57) = 5.41, P b 0.001, partial-η2 = 0.34). LSD post-hoc multiple comparisons revealed that the 3.0 NAL + NIC group differed from all others (all P′s b 0.01) for both of

Fig. 1. Mean session durations and standard errors for (i) the last three sessions of baseline, (ii) the first three sessions of dosing, and (iii) the last three sessions of dosing; ⁎P′s b 0.05 and ⁎⁎P b 0.01 in comparison to the saline-control group.

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A.P. Kirshenbaum et al. / Pharmacology, Biochemistry and Behavior 150–151 (2016) 1–7

Table 1 Baseline means and standard errors are not listed in the table below given that no significant differences in any dependent variable were discovered at baseline for any group. Differences from the saline group are presented below in the table as: ⁎p's b 0.05, ⁎⁎p's b 0.01. Dependent variables

Groups

Dosing days 1–3

Dosing days 8–10

Post-dosing

Saline Nicotine (0.4 mg/kg) 0.3 mg/kg Naloxone + Nicotine 3.0 mg/kg Naloxone + Nicotine 0.3 mg/kg Naloxone 3.0 mg/kg Naloxone

11.20 +/− 1.00 *13.93 +/− 0.96 11.73 +/− 1.16 *7.83 +/− 1.40 11.43 +/− 0.73 12.11 +/− 0.65

10.23 +/− 1.12 11.76 +/− 0.58 10.28 +/− 1.14 *4.23 +/− 1.05 10.93 +/− 0.64 10.52 +/− 0.67

11.02 +/− 0.50 **8.22 +/− 0.68 *8.85 +/− 0.77 9.65 +/− 0.77 11.10 +/− 0.72 10.04 +/− 0.60

Saline Nicotine (0.4 mg/kg) 0.3 mg/kg Naloxone + Nicotine 3.0 mg/kg Naloxone + Nicotine 0.3 mg/kg Naloxone 3.0 mg/kg Naloxone

13.91 +/− 1.00 13.93 ±+/− 0.96 14.57 +/− 2.31 10.87 +/− 3.19 12.71 +/− 1.73 12.06 +/− 1.48

14.00 +/− 2.31 12.30 ±+/− 1.73 12.86 +/− 2.46 **2.05 +/− 0.51 12.76 +/− 2.04 13.80 +/− 2.17

16.03 +/− 2.01 *11.89 ±+/− 2.32 12.36 +/− 1.79 **9.47 +/− 1.12 16.62 +/− 1.90 15.50 +/− 2.42

Saline Nicotine (0.4 mg/kg) 0.3 mg/kg Naloxone + Nicotine 3.0 mg/kg Naloxone + Nicotine 0.3 mg/kg Naloxone 3.0 mg/kg Naloxone

0.07 +/− 0.03 0.07 +/− 0.02 0.06 +/− 0.02 0.07 +/− 0.03 0.05 +/− 0.02 0.07 +/− 0.01

0.06 +/− 0.02 0.16 +/− 0.04 0.21 +/− 0.07 0.16 +/− 0.08 0.06 +/− 0.02 0.06 +/− 0.02

0.05 +/− 0.02 0.06 +/− 0.02 0.07 +/− 0.04 0.12 +/− 0.04 0.03 +/− 0.01 0.04 +/− 0.01

Reinforcing events

Response Rate (per min)

Adjunctive Response Rate (per min)

these two measures, again in that the groups means were significantly lower than other groups. No other between-group differences were found for reinforcing events or response rate. Adjunctive-responserate differences were not apparent at the standard threshold for significance (F (5, 57) = 2.30, P = 0.07). For between-group differences in the aforementioned primary and secondary dependent variables, see Fig. 1 and Table 1, respectively. To determine whether discontinuation of drug dosing produced decrements in PR-schedule session durations, a one-way ANOVA was performed on the average of the three post-dosing days across groups; F (5, 57) = 2.75, P b 0.05, partial-η2 = 0.22. Post-hoc multiple comparisons (LSD) revealed that saline differed from all groups (P's b 0.05) with the exception of the 0.3 NAL group (P N 0.05, see Fig. 2). The NIC group failed to differ from any group (P's N 0.05) other than saline. The 0.3 NAL group failed to differ from any group (P's N 0.05). The 3.0 NAL group failed to differ from any group (P's N 0.05) other than saline. The combination of either dose of NAL (0.3 or 3.0 mg/kg) with NIC produced significant impairments in session durations upon discontinuation compared to the saline-control group (P's b 0.05), but these groups do not differ from NIC-only or either of the NAL-only groups

(P's N 0.05). Therefore, relative to the saline-control, discontinuation of the larger dose of 3.0 mg/kg NAL, 0.4 mg/kg NIC, or the combination of NIC with either NAL dose (0.3 or 3.0 mg/kg) produced impairments in session durations in comparison to saline, and the degree of sucrose devaluation post-dosing appears to be quite similar across these particular dosing conditions (see Fig. 2). Also for the post-dosing phase, the secondary dependent measures of reinforcing events and response rates both demonstrated significant differences; F′s (5, 57) = 2.94 & 2.85, P's b 0.05, partial-η2 = 0.22 & 0.21, respectively. LSD multiple comparisons of reinforcing events revealed the NIC group differed from the saline and the 0.3 NAL-only groups (P′s b 0.05), and this result is in alignment with session-duration data. Neither NAL-only group differed from saline (Table 1), they did not differ from one another, nor did either differ from the 3.0 NAL + NIC group (P′s N 0.05). The 0.3 NAL + NIC group differed from both saline and 0.3 NAL-only groups (P′s b 0.05), but the 3.0 NAL + NIC group failed to differ from any group (P′s N 0.05). In terms of response rates, both the NIC-only and 3.0 NAL + NIC differed from saline (Table 1) and they also differed from both NAL-only groups (P′s b 0.05). The NAL-only groups failed to differ from each other, and neither differed from saline (P′ s N 0.05). The 0.3 NAL + NIC group failed to differ from any group (P′ s N 0.05). Taken together, the two secondary dependent variables of PR-schedule performance (reinforcing events and response rates) demonstrate that the delivery of nicotine during the dosing phase is a necessary, but not sufficient, precursor for post-dosing decrements to be evident for these particular measures of PR performance. Furthermore, the data demonstrate that NAL, by itself, is not a precursor to decrements in reinforcement on these two measures, although the results from the secondary and primary (session duration) dependent variables are somewhat inconsistent in regards to the larger dose of NAL (3.0 mg/ kg). No significant between-group differences were revealed for adjunctive response rate; F (5, 57) = 2.20, P = 0.08. 4. Discussion

Fig. 2. Means and standard errors for session durations during the last three PR schedule sessions; ⁎P′s b 0.05, ⁎⁎P b 0.01, and ⁎⁎⁎P b 0.001 compared to the saline-control group.

The present study was designed to examine the relationship between nicotine and the opioid system during repeated daily drug exposure and subsequent discontinuation. This was achieved by administering subcutaneous injections of nicotine prior to sucrose reinforcement on a progressive-ratio (PR) schedule. Subcutaneous injections of nicotine on the first days of dosing produced an increase in the motivation to obtain sucrose compared to rats injected with saline,

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and this was evidenced in terms of both session durations (Fig. 1) and reinforcing events (Table 1). After ten days of dosing, the nicotine group continued to show elevated session durations compared to saline. When nicotine dosing was discontinued, the NIC-group rats demonstrated significantly diminished session durations compared to the saline control (Fig. 2). The secondary dependent variables of reinforcing events and response rates paralleled the findings for diminished reinforcement (Table 1), and this result is congruent with previous research on nicotine discontinuation and PR-schedule responding (Kirshenbaum et al., 2015; Lesage et al., 2006). Moreover, the reinforcer devaluation effect of nicotine seems congruent with other findings that interruption of nicotine dosing results in reward desensitization (Pergadia et al., 2014) and a reduction in brain-reward sensitivity (i.e. intracranial-self stimulation threshold increases: Bruijinzeel et al., 2012; Cryan et al., 2003; Epping-Jordan et al., 1998; Markou and Kenny, 2002). When naloxone was given alone during the drug dosing phase, neither dose (0.3 nor 3.0 mg/kg; Fig. 1) produced differences in session durations compared to the saline-only control group. Although opioid antagonism may be predicted to interfere with sucrose reinforcement, the inability of naloxone to alter sucrose reinforcement in the present study was anticipated because responding for a sucrose solution above concentrations of 5% seems unaffected by opioid antagonism in the represented range of dosages used (Cleary et al., 1996). Opioid antagonism can reduce both consumption and operant responding for sweet solutions and ad lib food, but this relationship is mediated by concentration of the sucrose-solution, level of food deprivation/satiety, and naloxone dosage (Cleary et al., 1996; Grimm et al., 2007; Hayward and Low, 2001; Hayward et al., 2006; Levine et al., 1995; O'Hare et al., 1997; Rudski et al., 1994; Sharpe and Samson, 2001). The low-dose naloxone group (0.3 mg/kg) failed to differ from the saline group during the post dosing phase with regard to any PR-schedule measure, and differed from the nicotine group in terms of reinforcing events and response rates. Therefore, 0.3 mg/kg naloxone was insufficient to alter PR-schedule behavior during any phase of the study. Although the larger of the two naloxone doses used in this study (3.0 mg/kg NAL) failed to alter sucrose reinforcement during the ten days of dosing, it provoked reinforcer devaluation upon discontinuation in terms of session durations in comparison to saline (Fig. 2), but not the secondary measures of PR-schedule responding (Table 1). The inconsistency across primary and secondary measures of PR-schedule performance in the post-dosing phase prevents strong conclusions from being drawn about the high dose of naloxone during post-dosing. Decrements in session durations resulting from 3.0 mg/kg naloxone discontinuation appears consistent with other reports suggesting that antagonism of opioid receptors in rodents may be linked to the withdrawal syndromes associated with a variety of recreational drugs (Malin et al., 1982). Alternatively, one may have predicted that a history of opioid antagonism would engender an increased hedonic value of sucrose due to the potential of up-regulation of opioid receptors (Lynch & Burns, 1990; Sirohi et al. 2007), and in turn, increase the efficacy of the reinforcer upon discontinuation. Further research is necessary to determine how reinforced responding by sucrose may be altered when prolonged opioid antagonism is terminated. One objective of the present investigation was to determine the how opioid antagonism interferes with nicotine-induced reinforcement enhancement. Previous investigations have illuminated the role of the opioid system in nicotine-mediated associative learning (Palmatier et al., 2004) and reinforcement enhancement (Liu and Jernigan, 2011; Liu et al., 2009). The co-administration of both naloxone doses (0.3 and 3.0 mg/kg) attenuated reinforcement enhancement of sucrose by 0.4 mg/kg nicotine. This is an important finding because neither dose of naloxone administered by itself produced impairments in responding during dosing. On all measures of PR schedule performance, the combined dosing group (0.3 NAL + NIC) failed to differ from, and closely resembles, the performance of the saline-control during the dosing phase (Fig. 1, Table 1). A caveat to this clear effect is that although this low

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dose of naloxone (0.3 mg/kg) attenuated nicotine enhancement relative to the saline-control, the results from this combined dosing group also failed to statistically differ from the nicotine-only group for any measure of PR-schedule responding during the dosing phase. Therefore, one can conclude that 0.3 mg/kg naloxone attenuates, albeit imperfectly, the enhancing effects of 0.4 mg/kg nicotine. The opioid antagonism of 3.0 mg/kg naloxone might be expected to block the action of nicotine during drug dosing, but an unanticipated finding in this study was that the combined 3.0 mg/kg NAL + NIC dose resulted in a large and significant disruption in session durations (Fig. 1), reinforcing events, and response rates (Table 1). The decrements reported here as a result of combined 3.0 NAL + NIC dosing are difficult to interpret because, to the best of our knowledge, similar findings have not been reported in the preclinical literature. The behavioral depression witnessed in this group during combined 3.0 mg/kg NAL + NIC dosing could be due to potential sedative, depressant or GI effects. Naltrexone as a treatment for tobacco dependence has been shown to produce nausea and decreased appetite (King et al., 2013b), and others have found that the combination with nicotine replacement therapy can result in nausea for some patients as well as attenuating post-cessation weight gain (O'Malley et al., 2006). The combination of naltrexone and nicotine replacement can also result in significant but small increases in depressed mood (Toll et al., 2010), but some research has shown that naltrexone alone does not appear to produce sedation or negative affect (Jayaram-Lindström et al., 2004; Marks et al., 2014). The 3.0 mg/kg NAL + NIC dose may have disrupted appetitive responses due to a gastrointestinal effect; an examination of ad lib feeding behaviors in the hour following drug dosing, and a direct comparison of the present results to operant responses maintained by a visual stimulus would have been helpful to address this question. Further research using a variety of preclinical behavioral assays, that include but are not limited to reinforced responding, is needed to carefully address the combined effects of a large dose of naloxone and nicotine. Opioid antagonists, such as naloxone and naltrexone, have been shown to reduce overall cigarette consumption (Epstein and King, 2004; Karras and Kane, 1980; King and Meyer, 2000), and this suggests that both antagonists may have a related pharmacological interaction with nicotine (Balerio et al., 2005; Ismayilova and Shoaib, 2010). In a recent meta-analysis, David et al. (2013) determined that naltrexone does not significantly alter smoking abstinence compared to control groups utilized in various clinical trials. However, some evidence in humans smokers suggests that opioid antagonism can lower the subjective desire to smoke, cravings, and total cigarettes smoked (Epstein and King, 2004; King and Meyer, 2000). Naltrexone reduces the relative-reinforcement of nicotine in tobacco cigarettes (Rukstalis et al., 2005), but when added to nicotine-replacement therapy, can produce negative affect (Brauer et al., 1999; Toll et al., 2010). The apparent contradictions present in clinical literature warrants further inquiry into the pharmacological interaction of nicotine and opioid antagonists. Although naltrexone facilitates smoking cessation (King et al., 2012) and can be used as to augment nicotine-replacement therapy (e.g. O'Malley et al., 2006), many of the beneficial effects of opioid blockade abate upon discontinuation of naltrexone treatment (King et al., 2012). These particular results may lead to speculation about the interacting roles of opioid antagonism and reinforcer enhancement in the maintenance of habitual tobacco smoking. Human evidence of reinforcer enhancement is still quite limited (Perkins and Karelitz, 2013, Perkins, Karelitz, and Michael, 2015), and many questions remain regarding the extent to which reinforcement enhancement by nicotine is clinically relevant. The results reported here indicate that naloxone's ability to attenuate nicotine's enhancement effect is dose dependent. Furthermore, the combination of naloxone with nicotine disrupted sucrose reinforcement during the dosing phase and upon discontinuation, and this may have some relevance to the findings that opioid antagonism mediates weight gain during tobacco cessation in the clinical treatment literature (King et al., 2006, 2012, 2013a; Langleben et al., 2012; Toll et al., 2010).

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Acknowledgements This work was made possible by internal grants from the Vice President of Academic Affairs office at Saint Michael's College. The authors are grateful for the animal-care support from Dr. Jim Nichols, DVM, and for the devoted research technicians in their diligent work on this project: Courtney Pinto, Allison Shea, Jason Stone, and Sarah Thompson. Also worth noting are the exceptional critiques by the reviewers of the original manuscript.

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