Group II Metabotropic and -Amino-3-hydroxy-5-methyl-4- isoxazole Propionate (AMPA)/Kainate Glutamate Receptors Regulate the Deficit in Brain Reward Function Associated with Nicotine Withdrawal in Rats

This study investigated the role of ionotropic and metabotropic glutamate receptors in the deficits in brain reward function, as measured by elevations in intracranial self-stimulation (ICSS) reward thresholds, associated with nicotine withdrawal. The group II metabotropic glutamate (mGluII) receptor agonist LY314582 [a racemic mixture of LY354740 ([ ]-2-aminobicyclo[3.1.0]hexane2,6-dicarboxylic acid])] (2.5–7.5 mg/kg) precipitated withdrawallike elevations in ICSS thresholds, a sensitive measure of reward function, in nicotine-dependent but not control rats. LY314582 did not affect response latencies, a measure of performance in the ICSS paradigm. Bilateral microinfusion of LY314582 (10–100 ng/ side) into the ventral tegmental area likewise precipitated dosedependent threshold elevations in nicotine-dependent rats. Furthermore, a single injection of the mGluII receptor antagonist LY341495 (2S-2-amino-2-[1S,2S-2-carboxycyclopropan-1-yl]-3[xanth-9-yl]propionic acid) (1 mg/kg) attenuated the threshold elevations observed in rats undergoing spontaneous nicotine withdrawal. mGluII receptors are primarily located on glutamatergic terminals throughout the mesocorticolimbic system, where they act as inhibitory autoreceptors. To investigate whether mGluII receptors contributed to nicotine withdrawal by decreasing glutamatergic transmission, we next examined whether direct blockade of postsynaptic glutamate receptors precipitated withdrawallike reward deficits in nicotine-dependent rats. The -amino-3hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX; 0.01–1 mg/kg) precipitated withdrawal-like threshold elevations in nicotine-dependent but not control rats, whereas 6-methyl-2-[phenylethynyl]-pyridine (MPEP; 0.01–3 mg/kg) and dizocilpine (MK-801; 0.01–0.2 mg/kg), antagonists at metabotropic glutamate 5 and N-methyl-D-aspartate receptors, respectively, did not. Overall, these data demonstrate that mGluII receptors play an important role in the reward deficits associated with nicotine withdrawal. Furthermore, it is likely that mGluII receptors generate this reward deficit, at least in part, by decreasing glutamate transmission at AMPA/kainate receptors. There is now compelling evidence that the aversive withdrawal syndrome observed during periods of nicotine abstinence contributes to the persistence of the tobacco habit in smokers (Hughes, 1992; Kenny and Markou, 2001). Nicotine withdrawal was shown to precipitate a deficit in brain reward function, as measured by elevations in intracranial self-stimulation (ICSS) reward thresholds, similar to that observed in rats undergoing withdrawal from other major drugs of abuse (Epping-Jordan et al., 1998). Moreover, avoidance and alleviation of this deficit in brain reward function has been proposed as a major motivational factor contributing to craving, relapse, and continued tobacco consumption in human smokers (Epping-Jordan et al., 1998; Kenny and Markou, 2001). In contrast to the intense investigations into the mechanisms by which acute nicotine produces its rewarding effects, little is known concerning the mechanisms mediating the reward deficits associated with nicotine withdrawal. Most drugs of abuse have been shown to stimulate excitatory glutamatergic transmission throughout brain reward circuitries (Kalivas and Duffy, 1998; Wolf et al., 2000). Increases in glutamatergic transmission have been shown to play an important role in mediating the positive reinforcing actions of addictive drugs (Harris and Aston-Jones, 2003). This research was supported by the Peter F. McManus Charitable Trust (to P.J.K.), the National Institute on Drug Abuse (Grant DA11946 to A.M.), and a Novartis Research Grant (to A.M.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.052027. ABBREVIATIONS: ICSS, intracranial self-stimulation; VTA, ventral tegmental area; mGluII, group II metabotropic glutamate receptor; NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionate; mGlu5, metabotropic glutamate 5 receptors; MK-801, dizocilpine; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline; MPEP, 6-methyl-2-[phenylethynyl]-pyridine; ANOVA, analysis of variance. 0022-3565/03/3063-1068–1076$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 52027/1086499 JPET 306:1068–1076, 2003 Printed in U.S.A. 1068 at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from Indeed, it is thought that nicotine elicits its rewarding actions, at least in part, by activating nicotinic acetylcholine receptors located on glutamate terminals in the ventral tegmental area (VTA), thereby potentiating excitatory glutamatergic transmission in this reward-relevant brain site and increasing mesoaccumbal dopamine transmission (Mansvelder and McGehee, 2000). Accordingly, blockade of glutamatergic transmission reduced nicotine’s stimulatory action on mesoaccumbens dopamine transmission (Schilstrom et al., 1998) and attenuated the rewarding actions of nicotine and other drugs of abuse (Chiamulera et al., 2001; Laviolette and van der Kooy, 2003; Paterson et al., 2003). It has been suggested that the neuroadaptations that occur during prolonged exposure to drugs of abuse, which give rise to the deficits in brain reward function associated with withdrawal, may reside in the same neural elements that mediate the acute rewarding actions of these drugs (Koob and Le Moal, 2001). Indeed, in contrast to nicotine’s acute stimulatory effects, nicotine withdrawal attenuated mesoaccumbens dopamine transmission (Hildebrand et al., 1997), an action likely to contribute to the reward and motivational deficits associated with nicotine withdrawal (Kenny and Markou, 2001). Therefore, because increases in excitatory glutamatergic transmission are believed to play an important role in the reinforcing actions of acute nicotine, we hypothesized that withdrawal from nicotine is associated with decreased glutamatergic transmission in brain reward circuitries, which contributes to the reward deficits observed during withdrawal. To test this hypothesis, the effects of a group II metabotropic glutamate (mGluII) receptor agonist were examined in nicotine-treated and control rats. mGluII receptors, comprising of mGlu2 and mGlu3 receptors, are inhibitory autoreceptors located on glutamate terminals throughout the mesocorticolimbic system, where they act to decrease excitatory glutamatergic transmission (Bonci et al., 1997; Wigmore and Lacey, 1998). Because mGluII receptor agonists decrease glutamatergic transmission in brain reward circuitries (Manzoni and Williams, 1999), we predicted that activation of these receptors would precipitate ICSS threshold elevations in nicotine-dependent rats similar to those observed in rats during spontaneous nicotine withdrawal, whereas blockade of these receptors would reverse the threshold elevations associated with spontaneous nicotine withdrawal. To further investigate the role of glutamatergic transmission in nicotine withdrawal, we also examined whether direct blockade of glutamatergic transmission at postsynaptic NMDA, AMPA/ kainate, and metabotropic glutamate 5 (mGlu5) receptors precipitated withdrawal-like ICSS threshold elevations in nicotine-dependent rats. Materials and Methods Animal Housing. Subjects were 149 male Wistar rats weighing 300 to 320 g at the start of each experiment. Rats were obtained from Charles River Laboratories (Raleigh, NC) and were housed in groups of two or three per cage, with food and water available ad libitum. Animals were maintained in a temperature-controlled vivarium under a 12-h light/dark cycle (lights off at 10:00 AM). Animals were tested during the dark portion of the light/dark cycle, except for the spontaneous nicotine withdrawal experiment when rats were tested at time points according to the experimental design. All animals were treated in accordance with the guidelines of the National Institutes of Health regarding the principles of animal care. Animal facilities and experimental protocols were in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care. Drugs. ( )-Nicotine hydrogen tartrate salt ([ ]-1-methyl-2-[3pyridyl] pyrrolidine) and dizocilpine ([ ]-MK-801 hydrogen maleate; [(5R,10S)-( )-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten5,10-imine hydrogen maleate]) were purchased from Sigma-Aldrich (St. Louis, MO); LY341495 (2S-2-amino-2-[1S,2S-2-carboxycyclopropan-1-yl]-3-[xanth-9-yl]propionic acid) and NBQX disodium (2,3dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline disodium) were purchased from Tocris Cookson (Ballwin, MO). LY314582 (the racemic mixture of LY354740 [( )-2-aminobicyclo(3.1.0)hexane-2,6dicarboxylic acid]) and 6-methyl-2-[phenylethynyl]-pyridine (MPEP) were synthesized by one of the coauthors (F. Gasparini). Drugs were prepared immediately before each administration. For systemic administration, all drugs were dissolved in sterile water and administered by intraperitoneal injection, in a volume of 1 ml/kg body weight, 30 min before the experimental session. For direct intra-VTA administration, LY314582 was dissolved in artificial cerebrospinal fluid of the following composition: 126.6 mM NaCl, 27.4 mM NaHCO3, 2.4 mM KCl, 0.5 mM KH2PO4, 0.89 mM CaCl2, 0.8 mM MgCl2, 0.48 mM Na2HPO4 and 7.1 mM glucose, pH 7.4. Rats received intra-VTA injections immediately before the initiation of the experimental session. Unless otherwise stated, drug doses refer to the salt form. Apparatus. Intracranial self-stimulation training and testing took place in 16 Plexiglas operant chambers (25 31 24 cm) (MED Associates, St. Albans, VT). The floors of the operant chambers were constructed of parallel aluminum rods spaced 1.25 cm apart. One wall contained a metal wheel manipulandum that required 0.2 N force to rotate it one-quarter of a turn. The wheel (5 cm in width) extended out of the wall 3 cm. Each testing chamber was enclosed within a lightand sound-attenuated chamber (62 63 43 cm). Intracranial stimulation was delivered by constant current stimulators (Stimtech model 1200; San Diego Instruments, San Diego, CA). Subjects were connected to the stimulation circuit through flexible bipolar leads (Plastics One, Roanoke, VA) attached to gold-contact swivel commutators (model SL2C; Plastics One) mounted above the chamber. The stimulation parameters, data collection, and all test session functions were controlled by a microcomputer. Placement of Electrodes and Cannulas. Rats were anesthetized by inhalation of 1 to 3% halothane in oxygen and positioned in a stereotaxic frame (Kopf Instruments, Tujunga, CA). The incisor bar was adjusted to 5 mm above the interaural line, and the skull exposed. Stainless steel bipolar electrodes (11 mm in length) were implanted into the posterior lateral hypothalamus (AP 0.5 mm from bregma; ML 1.7 mm; DV 8.3 mm from dura), according to the atlas of Pellegrino et al. (1979). For the VTA infusion experiment, bilateral stainless steel guide cannulas (23-gauge, 14 mm in length) were implanted 3 mm above the VTA (AP 3.2 mm from bregma; ML 1.7 mm; DV 5.3 mm from skull surface; angle of 10 from midline), at the same time that ICSS electrodes were implanted. Four indentations were made in the skull to accommodate screws that together with the application of dental acrylic, held the electrode and cannulas in place. Cannulas were kept patent using 14-mm-long stainless steel stylets (30-gauge). Animals were allowed to recover from surgery for at least 7 days before training in the ICSS paradigm. Osmotic Mini-Pump Surgery. Rats were anesthetized by inhalation of 1 to 3% halothane in oxygen and prepared with Alzet osmotic mini-pumps [model 2ML4 (28 day); Alza, Palo Alto, CA] placed subcutaneously (back of the animal parallel to the spine). Pumps were filled with either sterile water or nicotine salt solution. The concentration of the nicotine salt solution was adjusted according to animal body weight, resulting in delivery of 9 mg/kg/day (3.16 mg/kg, free base). This dose of nicotine maintains stable plasma levels ( 44 ng/ml) comparable with those obtained in human smokers consuming approximately 30 cigarettes per day (Benowitz, 1988). After mini-pump implantation (or removal), the surgical wound was mGluII Receptors and Nicotine Withdrawal 1069 at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from closed with 9-mm stainless steel wound clips (BD Biosciences Primary Care Diagnostics, Sparks, MD) and treated with topical antibiotic (Bacitracin) ointment. ICSS Reward Threshold Procedure. Animals were trained to respond according to a modification of the discrete-trial currentthreshold procedure of Kornetsky and Esposito (1979). Briefly, a trial was initiated by the delivery of a noncontingent electrical stimulus. This electrical reinforcer had a train duration of 500 ms and consisted of 0.1-ms rectangular cathodal pulses that were delivered at a frequency of 50 to 100 Hz. The frequency of the stimulation was selected for individual animals so that current-intensity thresholds of each subject were within 85 to 160 A, and thus allowed both threshold elevations and lowerings to be detected. This frequency was held constant throughout the experiment. A one-quarter turn of the wheel manipulandum within 7.5 s of the delivery of the noncontingent electrical stimulation resulted in the delivery of an electrical stimulus identical in all parameters to the noncontingent stimulus that initiated the trial. After a variable intertrial interval (7.5–12.5 s, average of 10 s), another trial was initiated with the delivery of a noncontingent electrical stimulus. Failure to respond to the noncontingent stimulus within 7.5 s resulted in the onset of the intertrial interval. Responding during the intertrial interval delayed the onset of the next trial by 12.5 s. Current levels were varied in alternating descending and ascending series. A set of three trials was presented for each current intensity. Current intensities were altered in 5A steps. In each testing session, four alternating descending and ascending series were presented. The threshold for each series was defined as the midpoint between two consecutive current intensities that yielded “positive scores” (animals responded for at least two of the three trials) and two consecutive current intensities that yielded “negative scores” (animals did not respond for two or more of the three trials). The overall threshold of the session was defined as the mean of the thresholds for the four individual series. Each testing session was 30 min in duration. The time between the onset of the noncontingent stimulus and a positive response was recorded as the response latency. The response latency for each test session was defined as the mean response latency of all trials during which a positive response occurred. After establishment of stable ICSS reward thresholds, rats were tested in the ICSS procedure once daily except for the spontaneous nicotine withdrawal experiment when rats were tested at time points according to the experimental design. Intracerebral Injection Procedure. All injections were administered bilaterally in a volume of 0.5 l/side given over 66 s through 17-mm injectors. The injectors were connected to calibrated polyethylene-10 tubing preloaded with drug solution and protruded 3 mm below the ends of the cannulas into the VTA. After infusion, the injectors were kept in place for an additional 60 s to allow for drug diffusion and to minimize diffusion along the injection tract when pulling out the injector. Injectors were then removed and replaced with 14-mm wire stylets, and the animals were placed directly into the ICSS testing apparatus. Injections were made using a microinfusion pump (model 975; Harvard Apparatus Inc., Holliston, MA). Systemic Drug Administration Experiments. These experiments investigated whether nicotine withdrawal, as measured by elevations in ICSS thresholds, could be precipitated in nicotinetreated rats by systemic administration of an agonist at mGluII receptors (LY314582), or antagonists at mGlu5 (MPEP), NMDA (dizocilpine), or AMPA/kainate (NBQX) glutamate receptors. For each drug tested, rats were trained in the ICSS paradigm until stable baseline responding was achieved, defined as 10% variation in thresholds for three consecutive days and requiring approximately 14 days of daily testing. In each case, drug-naı̈ve rats were then assigned to two separate groups such that there was no difference in mean baseline ICSS thresholds or body weight between groups. One group was then prepared with subcutaneous osmotic mini-pumps delivering vehicle and the second group with mini-pumps delivering 9 mg/kg/day nicotine hydrogen tartrate (3.16 mg/kg/day nicotine free base). There was a minimum 7-day interval after mini-pump implantation, during which ICSS reward thresholds continued to be measured daily, before the effect of any systemically administered drug on reward thresholds was evaluated. This time period was sufficient to produce robust elevations in thresholds in nicotine-treated but not vehicle-treated rats upon abrupt removal of mini-pumps (i.e., spontaneous withdrawal) or administration of nicotinic receptor antagonists (i.e., precipitated withdrawal; Epping-Jordan et al., 1998). Separate groups of nicotine-treated rats and their corresponding nicotine-naive control group were then injected intraperitoneally with the mGluII receptor agonist LY314582 (0, 2.5, 0.5, and 7.5 mg/kg; n 9 nicotine, n 11 control), the mGlu5 receptor antagonist MPEP (0, 0.01, 0.05, and 0.1 mg/kg; n 8 nicotine, n 7 vehicle or 0, 0.5, 1, 2, and 3 mg/kg; n 13 nicotine, n 13 vehicle), the NMDA receptor antagonist dizocilpine (0, 0.01, 0.05, 0.1, 0.175, and 0.2 mg/kg; n 10 nicotine, n 9 control), or the AMPA/kainate receptor antagonist NBQX (0, 0.01, 0.025, 0.05, 0.075, 0.1, 0.5, and 1 mg/kg; n 10 nicotine, n 12 control) according to within-subjects Latinsquare designs and ICSS thresholds were evaluated 30 min later. A minimum of 48 h was allowed between each injection in the Latinsquare design, during which ICSS thresholds continued to be measured daily, to ensure that ICSS thresholds returned to baseline levels before the next drug administration. The doses of LY314582 and MPEP were chosen based on a previous study demonstrating that 10 mg/kg LY314582 and 3 mg/kg MPEP elevated ICSS thresholds in drug-naı̈ve rats (Harrison et al., 2002). For the potential demonstration of statistical interaction effects, it was important to include doses of the test drugs that did not alter thresholds under baseline conditions. Intraventral Tegmental Area Administration Experiment. After stable baseline ICSS responding was achieved ( 10% variation in threshold for three consecutive days), rats (n 15) with bilateral cannulas directed toward the VTA were allocated to two groups such that there were no differences in mean baseline reward thresholds or body weight between groups. One group was then prepared with subcutaneous osmotic mini-pumps delivering vehicle and a second group with mini-pumps delivering nicotine (3.16 mg/kg/day nicotine freebase). Animals again were tested in the ICSS paradigm each day for 7 days before drug treatment. Both groups of rats were then injected directly into the VTA, as described above, with LY314582 (0, 10, 50, and 100 ng/side; n 7 nicotine, n 8 control) according to a within-subjects Latin square design, and ICSS reward thresholds were evaluated immediately postinjection. There was a minimum 48-h interval between each injection, during which ICSS thresholds continued to be measured, to allow thresholds to return to baseline levels before further drug tests. At the conclusion of the experiment, all animals were anesthetized and their brains removed and immediately placed on ice. The brains were cut in 50m sections, and placements of the injectors and the electrodes were examined (Fig. 1 for histological verification of injection sites). Only those rats with injection tips located within the VTA were included in statistical analyses. Spontaneous Nicotine Withdrawal Experiment. Osmotic mini-pumps were surgically removed from nicotine-treated rats (n 15) (defined as rats having been prepared with mini-pumps delivering 3.16 mg/kg/day nicotine free-base for at least 7 days) or corresponding control rats (n 17; rats prepared with vehicle-containing mini-pumps). All rats were then tested in the ICSS procedure at 12, 18, 24, 36, 48, and 72 h after the removal of osmotic mini-pumps. These time points were chosen based on the time course of threshold elevations previously observed during spontaneous nicotine withdrawal after removal of nicotine-delivering osmotic mini-pumps (Harrison et al., 2001). Based on the ICSS reward thresholds obtained at the 12-h time point, nicotine-withdrawing rats were allocated to two groups such that there was no difference in the magnitude of reward threshold elevations between each group (117.67 3.1%, n 8; 119.93 3.5%, n 7). Similarly, control rats were allocated to two groups such that there was no difference in mean reward thresholds between these groups (106.45 5.2%, n 7; 103.63 3.6%, n 10). Thirty min before being tested at the 18-h 1070 Kenny et al. at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from