Prolonged nicotine exposure does not alter GABAB receptor-mediated regulation of brain reward function

Neuropharmacology 49 (2005) 953e962 www.elsevier.com/locate/neuropharm

Prolonged nicotine exposure does not alter GABAB receptor-mediated regulation of brain reward function Neil E. Paterson a, Adrie W. Bruijnzeel b, Paul J. Kenny a, Cory D. Wright a, Wolfgang Froestl c, Athina Markou a,* a

Department of Molecular and Integrative Neuroscience, CVN-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA b Department of Psychiatry, University of Florida, Gainesville, FL 32610, USA c Novartis Institute for BioMedical Research, Neuroscience Research, Novartis Pharma AG, Basel CH-4002, Switzerland Received 10 December 2004; received in revised form 1 April 2005; accepted 29 April 2005

Abstract g-Aminobutyric acid subtype B (GABAB) receptors play an important role in regulating brain reward function. Accumulating evidence suggests that chronic exposure to drugs of abuse may alter GABAB receptor function. The present studies investigated whether chronic nicotine administration, using a regimen that induces nicotine dependence, increased inhibitory regulation of brain reward function by GABAB receptors, as measured by intracranial self-stimulation (ICSS) thresholds in rats. Such an action of nicotine may contribute to the reward deficit observed during nicotine withdrawal. Nicotine-dependent and control rats received the GABA transaminase inhibitor g-vinyl-GABA or the GABAB receptor agonist CGP44532 according to a within-subjects Latin square design, and ICSS thresholds were assessed post-injection. Systemic administration of the lowest doses of GVG or CGP44532 did not alter reward thresholds in control or nicotine-treated rats, whereas the highest doses of each drug elevated thresholds similarly in both groups. Further, micro-infusion of CGP44532 directly into the ventral tegmental area elevated ICSS thresholds similarly in saline- and nicotine-treated rats. Overall, these data demonstrate that prolonged nicotine exposure did not alter GABAB receptor-mediated regulation of brain reward function, and suggest that alterations in GABAB receptor activity are unlikely to play a role in the brain reward deficits associated with spontaneous nicotine withdrawal. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: GABAB receptors; Intracranial self-stimulation; Reward; Chronic nicotine; Dependence; Ventral tegmental area; GVG; CGP44532; Rat

1. Introduction As one of the major inhibitory neurotransmitters in the central nervous system, g-aminobutyric acid (GABA) has been found to play an inhibitory role in brain reward function. For example, the GABA transaminase inhibitor g-vinyl GABA (GVG), which blocks GABA degradation and results in increased GABA * Corresponding author. Tel.: C1 858 784 7244; fax: C1 858 784 7405. E-mail address: [email protected] (A. Markou). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.04.031

levels throughout the brain (Jung et al., 1977), dosedependently elevated intracranial self-stimulation (ICSS) reward thresholds, and attenuated cocaineinduced lowering of brain stimulation reward thresholds (Kushner et al., 1997). GVG also decreased selfadministration of cocaine (Kushner et al., 1999), ethanol (Stromberg et al., 2001), heroin (Xi and Stein, 2000) and nicotine (Paterson and Markou, 2002), suggesting that GABA also regulates the reinforcing properties of drugs of abuse. Further, GVG dose-dependently attenuated the increased levels of extracellular dopamine in the nucleus accumbens associated with administration of

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nicotine (Dewey et al., 1999) or cocaine (Morgan and Dewey, 1998). Such increases in dopaminergic transmission are common effects of drugs of abuse (Koob, 1996). Overall, these observations demonstrate an important role for GABA transmission in negatively regulating brain reward function and the reinforcing properties of drugs of abuse. It is likely that the negative regulation of brain reward function by GABA is mediated by an action at central GABAB receptors. For example, the inhibitory effects of GVG on cocaine-induced increases in nucleus accumbens dopamine were shown to be mediated by GABAB receptors (Ashby et al., 1999). Further, systemic (Macey et al., 2001) or intra-ventral tegmental area (VTA) (Willick and Kokkinidis, 1995; Panagis and Kastellakis, 2002) administration of the GABAB receptor agonists baclofen or CGP44532 (Froestl et al., 1995) elevated ICSS thresholds in drug-naı¨ ve rats. In addition, baclofen and CGP44532 dose-dependently decreased self-administration of various drugs of abuse (Brebner et al., 2000a, 1999; Fattore et al., 2002; Paterson et al., 2004, 2005; Xi and Stein, 1999), an effect also shown to be mediated by GABAB receptors located in the VTA (Corrigall et al., 2000). CGP44532 also attenuated cocaine-induced facilitation of brain stimulation reward (Dobrovitsky et al., 2002). Similarly, administration of GABAB receptor positive modulators, which enhance the actions of endogenous GABA at GABAB receptors, were also shown to decrease cocaine self-administration (Smith et al., 2004), and reverse cocaine-induced facilitation of brain reward function (Slattery et al., in press) in rats. In contrast, effects of the GABAA receptor agonist muscimol on brain reward thresholds have been inconsistent (Panagis and Kastellakis, 2002; Willick and Kokkinidis, 1995). Overall, the above observations suggest that GABA inhibits brain stimulation reward and the reinforcing properties of drugs of abuse by a mechanism that involves activation of GABAB receptors, most likely located in the VTA. Recent studies indicated that chronic psychostimulant administration altered the function of GABAB receptors. Chronic cocaine administration significantly decreased functional coupling of GABAB receptors (Nestler et al., 1990; Striplin and Kalivas, 1992; Kushner and Unterwald, 2001) and attenuated the function of pre-synaptic, but not post-synaptic, GABAB autoreceptors in the dorsolateral septal nucleus in the rat (Shoji et al., 1997). Other studies indicated that chronic amphetamine administration is associated with decreased functional coupling of GABAB receptors in the nucleus accumbens (Zhang et al., 2000; Xi et al., 2003). Giorgetti et al. (2002) showed that chronic amphetamine administration was associated with enhanced function of pre-synaptic GABAB heteroreceptors, located on both dopamine and glutamate terminals

in the VTA. Amantea and Bowery (2004) recently showed that 24 h after the last injection of 14 days of nicotine administration (0.4 mg/kg per day), the inhibitory effect of baclofen on electrically evoked dopamine release in a VTA slice preparation was abolished, indicating reduced sensitivity of VTA GABAB receptors after chronic nicotine administration. In summary, chronic cocaine, amphetamine or nicotine administration is associated with altered GABAB receptor function. Importantly, the observed increase in GABAB heteroreceptor function would be expected to lead to decreased dopamine and glutamate release in the VTA, and may therefore contribute to the reward deficits associated with psychostimulant withdrawal. Here, we hypothesized that chronic nicotine administration may increase GABAB receptor function in the VTA, resulting in excessive inhibition of dopaminergic neurotransmission in the mesolimbic circuit and contributing to the reward deficit associated with nicotine withdrawal. To test this hypothesis, we investigated the effects of GVG and the relatively selective GABAB receptor agonist CGP44532 on ICSS thresholds in nicotine-treated and control rats. Further, we also examined the effects of micro-infusion of CGP44532 directly into the VTA, and 0.5 mm anterior and 2 mm dorsal to the VTA as anatomical control sites, on ICSS thresholds in nicotine-treated and control rats. We predicted that, if chronic nicotine exposure was associated with increased negative regulation of brain reward function by GABAB receptors, nicotine-treated rats would display an increased sensitivity to GVG and CGP44532, indicated by a left-ward shift (i.e. increased sensitivity) in doses of these compounds that elevate ICSS thresholds.

2. Methods 2.1. Subjects The subjects were 61 male Wistar rats from Charles River (Raleigh, NC), weighing 300e350 g at the start of the experiments. Animals were housed two or three per cage in a temperature- and humidity-controlled environment with a reversed 12 h light/dark cycle (lights on at 22:00 h). Food and water were available ad libitum except during testing. All training and testing occurred during the dark phase of the light/dark cycle. For one week after arrival in the vivarium, animals were allowed to habituate to their new environment (rats were handled twice during this week). All subjects were treated in accordance with the National Institutes of Health guidelines regarding the principles of animal care (NIH, 1985). Animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).

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2.2. Apparatus The experimental apparatus consisted of eight Plexiglas chambers measuring 30.5 ! 30 ! 17 cm (Med Associates, St. Albans, VT) each housed in a soundattenuating box (San Diego Instruments, San Diego, CA). Each chamber contained a metal wheel manipulandum (5 cm wide) centered in a side-wall, which required 0.2 N force to rotate it a quarter turn. Brain stimulation was delivered by constant current stimulators (Stimtech 1200, San Diego Instruments, San Diego, CA). Subjects were connected to the stimulation circuit with bipolar leads (Plastics One, Roanoke, VA) attached to gold-swivel commutators (model SL2C, Plastics One, Roanoke, VA). The stimulation parameters, data collection and all test session functions were controlled by a micro-computer.

2.3. Surgical procedures 2.3.1. Intracranial self-stimulation (ICSS) electrode and cannula implantation In all experiments, rats were anesthetized with an isoflurane/oxygen vapor mixture (1e3% isoflurane), and placed in a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA) with the incisor bar set 5.0 mm above the interaural line. Stainless steel bipolar electrodes (model MS303/2 Plastics One, Roanoke, VA) 11 mm in length were implanted in the medial forebrain bundle at the level of the posterior lateral hypothalamus (AP ÿ0.5 mm; ML G 1.7 mm; DV ÿ8.3 mm from dura) (Pellegrino et al., 1979). Work in our laboratory has shown that electrodes implanted with this angle support better self-stimulation than perpendicularly implanted electrodes. In addition to electrodes, in Experiments 3 and 4, rats were prepared with bilateral stainless steel 23 gauge intracerebral cannulae 11 mm in length, prior to electrode implantation. For the cannulae implantation, the incisor bar was set 3.3 mm below the interaural line (flat skull). In Experiment 3, cannulae were implanted 2.5 mm dorsal to the VTA using the coordinates: AP ÿ5.3 mm; ML G 1.0 mm; DV ÿ5.0 mm from dura (Paxinos and Watson, 1998). In Experiment 4, cannulae were implanted 2.5 mm dorsal and 0.5 mm anterior to the VTA target site used in Experiment 3: AP ÿ4.8 mm; ML G 1.0 mm; DV ÿ5.0 mm from dura (Paxinos and Watson, 1998), in order to assess the anatomical specificity of the effects seen in Experiment 3. The electrodes and the intracerebral cannulae were permanently secured to the skull using dental cement anchored with four skull screws. Removable 30 gauge wire stylets were inserted in the cannulae to maintain cannula patency between injections.

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2.3.2. Osmotic minipump implantation and removal Rats were anesthetized with an isoflurane/oxygen vapor mixture (1.0e1.5%), and an osmotic minipump (14-day model 2ML2 in Experiment 1 and 28-day model 2ML4 in Experiments 2, 3 and 4; Alza Corp., Palo Alto, CA) was inserted subcutaneously (back of the animal parallel to the spine) with the flow-moderator directed posteriorly. The wound was stapled and an antibacterial preparation was applied to the incision area. In Experiment 1, on day 14 the pumps were surgically removed under isoflurane anesthesia, and a second minipump was inserted on the other flank of the animal to maintain chronic nicotine infusion. In Experiments 2, 3 and 4, one 28-day minipump was implanted into each subject. 2.4. ICSS behavioral procedure The discrete-trial current-threshold procedure used was a modification of a task initially developed by Kornetsky and Esposito (1979). The rats were initially trained to turn the wheel manipulandum on a fixed ratio 1 (FR1) schedule of reinforcement. Each quarter turn of the wheel resulted in the delivery of a 500 ms train of 0.1 ms cathodal square-wave pulses at a frequency of 100 Hz. After the successful acquisition of responding for stimulation on this FR1 schedule, defined as 100 reinforcements within 10 min, the rats were trained gradually on the discrete-trial current-threshold procedure (Kornetsky and Esposito, 1979; Markou and Koob, 1992). Each trial began with the delivery of a non-contingent electrical stimulus, followed by a 7.5 s response window within which the subject could make a response to receive a second contingent stimulus identical in all parameters to the initial non-contingent stimulus. A response during this time window was labeled a positive response, while the lack of a response was labeled a negative response. During a 2 s period immediately after a positive response, additional responses had no consequence. The inter-trial interval (ITI), which followed either a positive response or the end of the response window (in the case of a negative response), had an average duration of 10 s (ranging from 7.5 s to 12.5 s). Responses that occurred during the ITI were labeled time-out responses and resulted in a further 12.5 s delay of the onset of the next trial. During training on the discrete-trial procedure, the duration of the ITI and delay periods induced by time-out responses were gradually increased until animals performed consistently for a fixed stimulation intensity at standard test parameters. The animals were subsequently tested on the current-threshold procedure in which stimulation intensities were varied according to the classical psychophysical method of limits. A test session consisted of four alternating series of descending and

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ascending current intensities starting with a descending series. Blocks of three trials were presented to the subject at a given stimulation intensity, and the intensity changed by steps of 5 mA between blocks of trials. The initial stimulus intensity was set at approximately 40 mA above the baseline current-threshold for each animal. Each test session typically lasted 30e40 min and provided two dependent variables for behavioral assessment: threshold and response latency. 2.4.1. Threshold The current-threshold for each descending series was defined as the stimulus intensity between a successful completion of a set of trials (positive responses during two or more of the three trials) and the stimulus intensity for the first set of trials, of two consecutive sets, during which the animal failed to respond positively on two or more of the three trials. 2.4.2. Response latency The time interval between the beginning of the noncontingent stimulus and a positive response was recorded as the response latency. The response latency for each session was defined as the mean response latency on all trials during which a positive response occurred. 2.5. Drugs Nicotine bitartrate and g-vinyl GABA (GVG) were purchased from Sigma-Aldrich. The GABAB receptor agonist CGP44532 was kindly donated by Novartis Pharma AG. All drugs were dissolved in 0.9% saline and all doses are reported as salt, with the exception of nicotine which is reported as base. 2.6. Experimental procedures Rats were surgically prepared with ICSS electrodes and/or cannulae and trained in the discrete-trial ICSS procedure until establishment of stable baseline thresholds (less than 10% variation during five consecutive days). In all experiments, animals were then assigned to one of two groups, counterbalanced for body weight and baseline current intensity thresholds, and prepared with osmotic minipumps delivering saline (n Z 10, n Z 5, n Z 6 and n Z 9 in Experiments 1, 2, 3 and 4, respectively) or nicotine (n Z 9, n Z 5, n Z 8 and n Z 9 in Experiments 1, 2, 3 and 4, respectively; 9.0 mg/kg/day salt; equivalent to 3.16 mg/kg/day free base). ICSS behavior was assessed daily. In Experiment 1, GVG (0, 75, 150 and 300 mg/kg) was administered intraperitoneally in a volume of 1 ml/kg, with a pre-treatment time of 3 h (Paterson and Markou, 2002). In Experiment 2, the GABAB agonist CGP44532 (0, 0.065, 0.125, 0.25 and 0.5 mg/kg) was administered subcutaneously in

a volume of 1 ml/kg with a pre-treatment time of 30 min. In Experiments 3 and 4, CGP44532 (0, 0.5, 1, 2 and 4 ng total bilateral dose; 0, 2, 4, 8 and 16 ng total bilateral dose, respectively) was administered in a volume of 0.5 ml/side, over 66 s; the injectors were left in place for 15 s after drug injection to minimize drug diffusion along the injector and cannula tracks. In Experiment 3, after the completion of the Latin square, all rats received an anatomical control injection of 4 ng total bilateral dose, 2 mm dorsal to the target site. In all experiments, GVG or CGP44532 were administered according to a within-subjects Latin square design, only when subjects had demonstrated stable brain reward thresholds during the preceding three days (i.e. GVG or CGP44532 was administered every 4e5 days). In Experiment 1, GVG was tested as soon as stable brain reward thresholds were established after the minipump surgery (a minimum of 3 days after pump implantation). In Experiments 2, 3 and 4, CGP44532 was tested after an initial 6e7 day period of nicotine or saline infusion elapsed. 2.7. Histologies At the completion of the experiments, rats were euthanized with sodium pentobarbital (80 mg, given intraperitoneally) and underwent transcardial perfusion with physiological saline (100 ml) followed by 10% formaldehyde (100 ml). Brains were post-fixed for 24 h, then cryoprotected in 10% sucrose solution for 48 h. Sections were cut in a cryostat to 50 mm thickness then mounted on slides and stained with cresyl violet. Injection sites were verified using light microscopy and a stereotaxic rat brain atlas (Paxinos and Watson, 1998). Histological reconstruction of bilateral injection sites are shown in Fig. 1 (see below). One rat (a nicotine-treated subject in Experiment 3) was excluded from statistical analyses because the cannulae placements could not be confirmed. 2.8. Statistical analyses All threshold and response latency data obtained during drug testing were expressed as a percentage of the mean of the last three values prior to each test day (preinjection baseline). Data were analyzed using separate two-way ANOVAs for each experiment with Pump (2 levels) defined as the between-subjects factor, and GVG/CGP44532 Dose (4/5 levels, respectively) defined as the within-subjects factor. Based on our a priori hypotheses, follow-up one-way ANOVAs were used to analyze the effects of GVG/CGP44532 within nicotineand saline-treated groups of rats. Significant main effects in one-way ANOVAs were followed by NewmaneKeuls post hoc comparisons (Winer, 1971). The level of significance was set at 0.05. The effects of CGP44532

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3. Results 3.1. Experiment 1: the effects of systemic administration of the GABA transaminase inhibitor GVG on brain reward function during continuous nicotine or saline infusion Mean (GSEM) absolute reward thresholds prior to treatment with GVG for saline- and nicotine-treated rats were 128.5 G 21.4 and 131.7 G 11.3 mA, respectively. Mean (GSEM) absolute response latencies prior to treatment with GVG for saline- and nicotine-treated rats were 3.42 G 0.19 and 3.52 G 0.1 s, respectively. A twoway ANOVA indicated that GVG administration elevated brain reward thresholds in both treatment groups [GVG Dose: F(3,51) Z 10.52, p ! 0.001; Fig. 2], regardless of whether nicotine or saline was infused via the minipumps [Pump: F(1,17) Z 0.35, n.s.; Pump ! GVG Dose: F(3,51) Z 0.01, n.s.]. Simple oneway ANOVAs (GVG Dose, 4 levels) indicated that GVG significantly elevated brain reward thresholds in both nicotine- [F(3,24) Z 6.02, p ! 0.05] and saline-treated [F(3,27) Z 4.77, p ! 0.05] subjects. GVG had no effect on response latencies at any dose tested [Pump: F(1,17) Z 1.2, n.s.; GVG Dose: F(3,51) Z 0.83, n.s.; Pump ! GVG Dose: F(3,51) Z 1.14, n.s.; data not shown]. 3.2. Experiment 2: the effects of systemic administration of the GABAB receptor agonist CGP44532 on brain reward function during continuous nicotine or saline infusion Mean (GSEM) absolute reward thresholds prior to treatment with the relatively selective GABAB receptor

Fig. 1. The locations of the tips of injection cannulae, as determined from histological examinations, in Experiment 3 (Panel A) where CGP44532-induced threshold elevations were seen, and Experiment 4 (Panel B) where no CGP44532-induced threshold elevations were seen. The plus signs (C) indicate the injection tip locations in the nicotine group (n Z 7; n Z 9, respectively), and the solid circles () indicate the injector tip locations in the saline group (n Z 6; n Z 9, respectively).

administered within the VTA or 2 mm dorsal to the VTA on ICSS thresholds were compared using a twoway ANOVA where Pump (2 levels) was defined as the between-subjects factor and Site (2 levels) was defined as the within-subjects factor.

Fig. 2. The effects of GVG administration on brain reward thresholds during chronic nicotine/saline exposure. Brain reward thresholds are expressed as a percent of baseline thresholds (mean G SEM). Asterisks (*p ! 0.01) indicate significant differences from vehicle administration within nicotine- and saline-treated groups.

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agonist CGP44532 for saline- and nicotine-treated rats were 157.4 G 10.6 and 154.0 G 2.3 mA, respectively. Mean (GSEM) absolute response latencies prior to treatment with the selective GABAB receptor agonist CGP44532 for saline- and nicotine-treated rats were 3.12 G 0.08 and 3.20 G 0.19 s, respectively. A two-way ANOVA indicated that CGP44532 administration elevated brain reward thresholds in both groups [CGP44532 Dose: F(4,32) Z 16.62, p ! 0.001; Fig. 3], regardless of whether nicotine or saline was infused via the minipumps [Pump: F(1,17) Z 0.35, n.s.; Pump ! CGP44532 Dose: F(4,32) Z 0.05, n.s.]. Simple one-way ANOVAs (CGP44532 Dose, 5 levels) indicated that CGP44532 significantly elevated brain reward thresholds in nicotine- [F(3,24) Z 6.02, p ! 0.05] and saline-treated [F(3,27) Z 4.77, p ! 0.05] subjects. CGP44532 had no effect on response latencies at any dose tested [Pump: F(1,8) Z 1.1, n.s.; CGP Dose: F(4,32) Z 0.6, n.s.; Pump ! CGP44532 Dose: F(4,32) Z 0.78, n.s.; data not shown].

thresholds in both treatment groups [CGP44532 Dose: F(4,44) Z 7.52, p ! 0.001; Fig. 4], regardless of whether nicotine or saline was infused via the minipumps [Pump: F(1,11) Z 1.36, n.s.; Pump ! CGP44532 Dose: F(4,44) Z 0.69, n.s.]. Simple one-way ANOVAs (CGP44532 Dose, 5 levels) indicated that intra-VTA CGP44532 significantly elevated brain reward thresholds in nicotine- [F(4,24) Z 6.11, p ! 0.05] and saline-treated [F(3,27) Z 4.77, p ! 0.05] subjects. CGP44532 had no effect on response latencies at any dose tested [Pump: F(1,11) Z 0.14, n.s.; CGP44532 Dose: F(4,44) Z 1.34, n.s.; Pump ! CGP44532 Dose: F(4,44) Z 1.75, n.s.; data not shown]. ICSS thresholds after administration of the anatomical control injection of 4 ng total bilateral dose 2 mm dorsal to the intra-VTA target site were 111.28 G 2.42 and 118.98 G 6.51 percent of baseline thresholds in the nicotine- and saline-treated rats, respectively (see

3.3. Experiment 3: the effects of intra-VTA CGP44532 administration on brain reward function during continuous nicotine or saline infusion Mean (GSEM) absolute reward thresholds prior to treatment with CGP44542 for saline- and nicotine-treated rats were 101.67 G 18.25 and 123.80 G 16.26 mA, respectively. Mean (G SEM) absolute response latencies prior to treatment with CGP44542 for saline- and nicotine-treated rats were 3.12 G 0.18 and 3.26 G 0.2 s, respectively. A two-way ANOVA indicated that intraVTA CGP44532 administration elevated brain reward

Fig. 3. The effects of administration of the GABAB receptor agonist CGP44532 on brain reward thresholds during chronic nicotine/saline exposure. Brain reward thresholds are expressed as a percent of baseline thresholds (mean G SEM). Asterisks (*p ! 0.01) indicate significant differences from vehicle administration within nicotine- and saline-treated groups.

Fig. 4. The effects of micro-infusion of CGP44532 administration on brain reward function during chronic nicotine/saline exposure. Panel A depicts the effects of intra-VTA administration of CGP44532 on brain reward function. Panel B depicts the effects of CGP44532 administration 2 mm anterior to the VTA. Brain reward thresholds are expressed as a percent of baseline thresholds (mean G SEM). Asterisks (*p ! 0.05) indicate significant differences from vehicle administration within nicotine- and saline-treated groups.

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Table 1). Comparison of ICSS thresholds obtained during the three day pre-injection baseline period and thresholds obtained after CGP44532 administration indicated a significant effect of CGP44532 administration [F(1,11) Z 23.53, p ! 0.01], regardless of pump treatment [Pump: F(1,11) Z 0.57, n.s.; Pump ! CGP44532 Dose: F(1,11) Z 0.06, n.s.]. Nonetheless, comparison of ICSS thresholds (expressed as a percentage of pre-injection baseline) obtained after dorsal- and intra-VTA administration of 4 ng CGP44532 indicated a significant effect of Site [F(1,11) Z 7.69, p ! 0.05], regardless of pump treatment [Pump: F(1,11) Z 0.73, n.s.; Pump ! Site: F(1,11) Z 0.14, n.s.]. Fig. 1A shows the location of the injectors. 3.4. Experiment 4: the effects of CGP44532 administration in areas anterior to the VTA on brain reward function during continuous nicotine or saline infusion Mean (GSEM) absolute reward thresholds prior to treatment with CGP44542 for saline- and nicotine-treated rats were 117.86 G 10.67 and 119.71 G 10.58 mA, respectively. Mean (GSEM) absolute response latencies prior to treatment with CGP44542 for saline- and nicotine-treated rats were 3.20 G 0.12 and 3.08 G 0.11 s, respectively. A two-way ANOVA indicated that CGP44532 administration 0.5 mm anterior to the VTA did not elevate brain reward thresholds in either treatment group [CGP44532 Dose: F(4,64) Z 0.68, n.s.], regardless of whether nicotine or saline was infused via the minipumps [Pump: F(1,16) Z 1.16, n.s.; Pump ! CGP44532 Dose: F(4,64) Z 0.42, n.s.]. CGP44532 had no effect on response latencies at any dose tested (Pump: F(1,16)Z 0.46, n.s.; CGP44532 Dose: F(4,64) Z 1.5, n.s.; Pump ! CGP44532 Dose: F(4,64) Z 0.75, n.s.; see Fig. 4B). Fig. 1B shows the location of the injectors.

4. Discussion In confirmation of previous studies (Kushner et al., 1997; Macey et al., 2001), the present data indicated that administration of GVG or the GABAB receptor agonist CGP44532 elevated brain reward thresholds in drugnaı¨ ve rats. In addition, the present study demonstrated

that GABAB receptor regulation of brain reward function was unchanged in rats chronically exposed to nicotine compared to rats chronically exposed to saline. The lack of differences between the nicotine- and salinetreated animals was seen after systemic administration of GVG, which increases GABA levels throughout the brain, after systemic administration of the relatively selective GABAB receptor agonist CGP44532, and after intra-VTA administration of CGP44532. Administration of CGP44532 anterior to the VTA target site had no effect on either ICSS thresholds or response latencies. Administration of CGP44532 dorsal to the VTA significantly elevated ICSS thresholds, although this effect was significantly less than the effect of the same CGP44532 dose delivered intra-VTA. Given the anatomical specificity demonstrated by administration of CGP44532 anterior to the VTA, it is most likely that the effects seen after CGP44532 was administered dorsal to the VTA were due to some diffusion of the drug solution down the injection track and into the VTA. In summary, the present data suggest that chronic nicotine infusion does not induce significant changes in GABAB receptormediated regulation of brain reward function, as measured with the ICSS procedure and in the continued presence of nicotine. The effect of intra-VTA CGP44532 administration in both saline- and nicotine-treated rats was consistent with the previously demonstrated role of intra-VTA GABAB receptors on brain reward function (Panagis and Kastellakis, 2002; Willick and Kokkinidis, 1995). Inhibitory GABAB receptors are present on dopaminergic and glutamatergic neurons in the VTA (Bowery et al., 1987; Wirtshafter and Sheppard, 2001). Accordingly, GABAB receptor activation in the VTA reduced extracellular dopamine levels in the nucleus accumbens (Westerink et al., 1996). Furthermore, VTA GABAB receptors have been implicated in the mediation of the effects of systemic baclofen administration on cocaine (Brebner et al., 2000b; Shoaib et al., 1998), heroin (Xi and Stein, 1999) and nicotine self-administration (Corrigall et al., 2000), drugs which enhance dopaminergic neurotransmission in brain reward pathways (Koob, 1996). Although GABAB receptors are present also in the nucleus accumbens (Bowery et al., 1987), higher doses of baclofen were required in the nucleus accumbens than the VTA (Brebner et al., 2000b) to

Table 1 ICSS thresholds (mean G SEM) after CGP44532 administration, either anterior or dorsal to the VTA target site Anatomical location relative to VTA site

CGP44532 dose (ng, total bilateral dose)

Nicotine-treated rats

Saline-treated rats

0.5 mm 0.5 mm 0.5 mm 0.5 mm 0.5 mm 2.0 mm

0 2 4 8 16 4

106.63 G 3.73 99.52 G 3.69 107.8 G 4.03 101.72 G 4.81 102.38 G 3.67 111.28 G 2.42

104.28 G 4.57 98.68 G 2.72 97.47 G 8.97 98.11 G 3.27 101.32 G 4.41 118.98 G 6.51

anterior anterior anterior anterior anterior dorsal

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decrease cocaine self-administration (but see Shoaib et al., 1998). Further, intra-VTA, but not intraaccumbens, baclofen decreased the reinforcing effects of heroin (Xi and Stein, 1999). The above data, including the present study, comprises compelling evidence for a role for VTA GABAB receptors in the regulation of brain reward function. Nonetheless, the present data suggest that chronic nicotine administration does not alter the function of GABAB receptors in the VTA that regulate brain reward function. Previously, decreased GABAB1 receptor expression was found in the rat hippocampus immediately after chronic exposure to nicotine or cigarette smoke (Li et al., 2002). Further, Amantea et al. (2004) demonstrated reduced G-protein coupling to GABAB receptors in the nucleus accumbens and medial prefrontal cortex in rat brain 24 h after the last injection of 14 days of daily nicotine administration, but no changes in GABAB receptor density or affinity were seen in either of these two sub-regions of the mesocorticolimbic system. An identical nicotine treatment regimen was associated with attenuation of the inhibitory effect of baclofen on electrically evoked dopamine release in a VTA in vitro slice preparation (Amantea and Bowery, 2004). These previous studies (Amantea et al., 2004; Amantea and Bowery, 2004) administered 0.4 mg/kg nicotine daily, subcutaneously for 14 days, prior to testing 24 h after the last nicotine injection. In contrast, the present study utilized a much higher daily dose of nicotine (3.16 mg/kg/day) administered continuously via subcutaneous minipump, for 7 days prior to the initiation of testing, and over a total of 28 days. Further, the assessment of the function of GABAB receptors was done in the presence of nicotine in the studies reported here, while in previous work by Amantea and colleagues assessments were made after cessation of nicotine administration. Numerous previous studies indicated changes in GABAB receptor function during psychostimulant withdrawal (Striplin and Kalivas, 1992, 1993; Shoji et al., 1997; Zhang et al., 2000; Kushner and Unterwald, 2001; Giorgetti et al., 2002; Xi et al., 2003) although these studies were restricted to cocaine and amphetamine, often used different doses and administration schedules, and investigated GABAB receptor function in different brain areas. Most importantly, all of the above studies examining GABAB receptor function in the mesolimbic system used treatment regimens that induced sensitization to the locomotor activating effects of nicotine, cocaine or amphetamine rather than the continuous nicotine exposure regimen used in the present study that induces nicotine ‘withdrawal’ as measured by elevations in brain reward thresholds and somatic signs of nicotine withdrawal upon discontinuation of nicotine administration or administration of nicotinic acetylcholine receptor antagonists (e.g., Malin et al., 1992; Epping-

Jordan et al., 1998). It would be interesting to determine whether the reported changes in GABAB receptor function associated with locomotor sensitization (e.g., Giorgetti et al., 2002; Kushner and Unterwald, 2001) are also associated with decreased brain reward function observed during withdrawal from higher doses of amphetamine (e.g., Paterson et al., 2000) or cocaine (Markou and Koob, 1991). In addition, unlike the present study, the previous studies examined GABAB receptor number or function in the absence of the drug of abuse. Nonetheless, nicotine-induced alterations in glutamate-mediated regulation of brain reward function have been successfully demonstrated using an approach identical to the present study (Kenny et al., 2003), suggesting that the present approach can lead to the detection of nicotine dependence-induced alterations in GABAB receptor-mediated regulation of brain reward function. In conclusion, the present results suggest that chronic nicotine infusion does not induce significant changes in GABAB receptor-mediated regulation of brain reward function as measured with the ICSS procedure and in the continued presence of nicotine.

Acknowledgements This is publication number 15787-NP from The Scripps Research Institute. The authors would like to thank Rahasson Ager, Randy Ayres and Robert Lintz for technical assistance, and Mike Arends for editorial assistance. This work was supported by National Institute on Drug Abuse grant R01 DA11946, and a National Institute of Mental Health and National Institute on Drug Abuse grant U01 MH69062 to AM.

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