Psychopharmacology DOI 10.1007/s00213-013-3314-3
ORIGINAL INVESTIGATION
Effects of stressors on the reinforcing efficacy of nicotine in adolescent and adult rats Sheng Zou & Douglas Funk & Megan J. Shram & A. D. Lê
Received: 24 April 2013 / Accepted: 4 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Rationale and objectives Stress increases drug intake. This depends on the stressor, drug, and aspect of drug seeking assessed. The objectives of these experiments done in adolescent and adult male rats were to (1) examine social defeat effects on acquisition of nicotine self-administration (SA) and the reinforcing efficacy of nicotine and (2) determine the effects of acute exposure to intermittent footshock (FS) or yohimbine on the reinforcing efficacy of nicotine. Methods In experiment 1, rats received four defeat exposures prior to nicotine SA acquisition and progressive ratio (PR) SA sessions (30 μg/kg nicotine/infusion). Exposure to an olfactory cue previously paired with defeat was also tested on responding maintained by nicotine on the PR schedule. In experiments 2 and 3, the effects of FS (5 and 10 min) or yohimbine (0.625 and 1.25 mg/kg, i.p.) on PR responding for nicotine (15, 30, or 60 μg/kg/infusion) were assessed. Adolescents were aged PD34-36 and adults PD81-85 at the beginning of nicotine SA training. Results Defeat did not affect nicotine SA acquisition. Prior exposure to defeat or a defeat-paired olfactory cue did not affect PR responding for nicotine. FS modestly decreased PR responding in adolescents at the middle nicotine infusion S. Zou : D. Funk : A. D. Lê (*) Neurobiology of Alcohol Laboratory, Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada e-mail: [email protected] M. J. Shram : A. D. Lê Department of Pharmacology, University of Toronto, Toronto, Canada A. D. Lê Department of Psychiatry, University of Toronto, Toronto, Canada M. J. Shram INC Research Toronto Inc., Toronto, Canada
dose. Yohimbine increased PR responding independent of nicotine infusion dose and age. Conclusions Together with previous work with other drugs, our data indicate that the effects of stress on the reinforcing efficacy of nicotine are stressor- and drug-dependent. This suggests that there is heterogeneity among stressors on how they affect neuronal systems underlying drug intake. Keywords Adolescents . Adults . Nicotine self-administration . Progressive ratio . Stress . Yohimbine . Footshock . Social defeat Studies in humans show that stressful life events are associated with increased use of, and relapse to, nicotine or other drugs of abuse (Brown et al. 1995; Pomerleau and Pomerleau 1991). The effects of stress on the initiation, maintenance, and reinstatement of drug seeking have been studied extensively using animal models. Using the reinstatement procedure, the effects of stress on relapse have been shown to be reliable and consistent across different laboratories and drug classes. As will be discussed below, there is heterogeneity among stressors in their effects on reinstatement, and these effects can depend on the drug examined (Le and Shaham 2002; Shalev et al. 2010). Despite the fact that stress clearly increases smoking in humans, to our knowledge, there are no studies examining the effects of stress on nicotine self-administration (SA) in laboratory animals. Footshock stress (FS) is one of the most commonly used laboratory stressors. The effects of FS on SA vary as a function of drug class, dose, reinforcement schedule, level of dependence, and interactions among these variables. Although FS has been used extensively to examine the effects of stress on alcohol SA, these studies have produced inconsistent results. The effects of FS may depend on the conditions under which alcohol SA was examined. For
Psychopharmacology
example, FS has been shown to enhance alcohol SA under alcohol deprivation conditions (Funk et al. 2004; Vengeliene et al. 2003) but to have weak or inconsistent effects under nondeprivation conditions (Logrip and Zorrilla 2012). In contrast, non-contingent exposure to FS is facilitatory on cocaine SA (Goeders and Guerin 1994; Mantsch and Katz 2007). Similarly, FS increases the oral intake of fentanyl solution under higher demand FR (FR4) and progressive ratio (PR) schedules of reinforcement (Shaham et al. 1993). FS also increased heroin SA under a PR schedule (Shaham and Stewart 1994). Two stressors, FS and yohimbine, an alpha-2 adrenoceptor antagonist, reliably reinstate the seeking of nicotine and other drugs (Anker and Carroll 2010; Buczek et al. 1999; Feltenstein and See 2006; Le et al. 2005; Leao et al. 2009; Shaham et al. 2000; Shepard et al. 2004; Yamada and Bruijnzeel 2011; Zislis et al. 2007). In contrast to the variable effects of FS, we and others have shown that yohimbine also increases alcohol SA (Ayanwuyi et al. 2013; Bertholomey et al. 2013; Le et al. 2005), and we recently reported that it increases nicotine SA in adolescent rats (Li et al. 2012). The effects of yohimbine on the SA of other drugs are not known. Another stressor, social defeat, has a unique pattern of effects on drug-seeking behavior. It produces the most reliable effects on the initiation of drug SA, and importantly, exposure to this stressor can have long-lasting effects on drug SA (Bardo et al. 2013; Miczek et al. 2008). These effects have been systematically studied in cocaine-trained animals. Exposure to social defeat prior to initiation of cocaine SA training increases the rate of acquisition of cocaine SA and increases cocaine intake under FR and PR schedules of reinforcement (Covington et al. 2005; Covington and Miczek 2001a; Tidey and Miczek 1997). In contrast, acute exposure to this stressor may decrease drug intake, possibly due to fear responses such as freezing (Chung et al. 2000). For example, acute exposure to defeat decreases alcohol SA (Funk et al. 2005; van Erp and Miczek 2001). No work has been done on the effects of defeat on any aspect of nicotine seeking. Adolescence is a vulnerable period in the initiation of drug intake (SAMHSA 2011). Stressful events are significantly correlated with smoking and intentions to smoke in adolescents (Booker et al. 2004, 2007, 2008), and adolescents report that stress is a salient precipitant of smoking (DiFranza et al. 2004; Weiss et al. 2008). Stress also enhances the rate of smoking initiation in adolescents (Booker et al. 2007, 2008; Byrne et al. 1995; Byrne and Mazanov 2003). Two possible reasons for this are that adolescence is a stressful developmental period and that adolescents may be more sensitive to the effects of stress than adults (Choi and Kellogg 1996; Novak et al. 2007; Slawecki 2005; Vazquez 1998). In support of these findings, FS was shown to facilitate the development of conditioned place preference (CPP) to nicotine in adolescent rats, suggesting that stress enhances
the rewarding effects of nicotine (Brielmaier et al. 2012). Work with other drugs has demonstrated age-dependent effects of stress on drug seeking. Yohimbine-induced reinstatement of cocaine seeking was shown to be of a higher magnitude in adolescents (Anker and Carroll 2010). Despite these data, no work has been done examining the possibility that there are age differences in sensitivity to the effects of stressors on the reinforcing efficacy of nicotine or whether such differences in stress sensitivity may affect initiation of nicotine SA. The purpose of the present investigation is to examine the effects of three different stressors on nicotine SA and its reinforcing efficacy. In light of the findings of the Miczek laboratory (Covington and Miczek 2001a), the first experiment investigated the effects of exposure to social defeat on acquisition of nicotine SA and then on the reinforcing efficacy of nicotine using a PR schedule of reinforcement, in both adolescent and adult rats. In the second and third experiments, the effects of acute exposure to FS and yohimbine, two stressors that reliably reinstate nicotine seeking, on the reinforcing efficacy of nicotine were determined in adolescent and adult rats previously trained to SA nicotine.
Materials and methods Subjects Forty-eight pregnant Long Evans dams (n =16 per experiment; Charles River Laboratories, Quebec, Canada) were used as a source of juvenile rats run in the experiments and were housed singly in plastic cages (51×41×20 cm). Adult male Long Evans rats were purchased from Charles River and were singly housed in plastic cages. On the day of weaning (post-natal day (PD) 21), 92 male pups were group housed by litter. After surgery, all rats were singly housed and fed 20–22 g of rat chow per day. The rats were maintained on a 12/12 h light/dark cycle (lights on at 1900) in a humidity- and temperature-regulated vivarium. The experimental procedures followed the NIH publication “Principles of laboratory animal care” (Eighth edition, 2011) and were approved by animal care and use committee of the Centre for Addiction and Mental Health. Apparatus Nicotine SA was performed in 24 chambers equipped with one active lever and one inactive lever. Appropriate responding on the active lever activated the infusion pump that delivered the intravenous (i.v.) nicotine followed by a 40s timeout. Each infusion was accompanied by a compound light-tone cue (stimulus light illumination for 6 s and a 2,800Hz tone for 1 s). Responding on the inactive lever had no
Psychopharmacology
programmed consequences but was recorded. Sucrose pellet SA (prior to nicotine SA) was conducted in a separate set of 16 similar operant chambers equipped with pellet dispensers. No cues were present during sucrose pellet training. Surgery Rats (adolescents: PD26; adults: PD73-75) were anesthetized using ketamine/xylazine (75 mg/kg ketamine/10 mg/kg xylazine (Shram et al. 2008)). Incision sites were treated with a local anesthetic (0.1 ml bupivacaine, 0.125 %, s.c.). Penicillin (30,000 U, i.m.) was administered as an antibiotic prior to surgery and buprenorphine (0.01 mg/kg, s.c.) as an analgesic after surgery. Catheters were implanted into the right jugular vein as previously described (Corrigall and Coen 1989) and exited between the scapulae and were attached to the modified 22-gauge cannula connected to the fluid swivel system. After recovery, catheters were flushed daily with 0.1 ml of a sterile heparin–saline solution (50 U/ml). Catheter patency was tested after PR testing was completed by i.v. injections of sodium methohexital (0.05 mg/kg). The data from animals that did not show rapid anesthesia following methohexital injection were excluded from analysis. Drugs Solutions of nicotine (Sigma-Aldrich, Oakville, Ontario, Canada) of pH 6.8 to 7.2 were prepared fresh daily. The unit doses for the i.v. nicotine SA were 30 μg/kg/infusion in experiment 1 and 15, 30, or 60 μg/kg/infusion in experiments 2 and 3. Nicotine infusions were made in a volume of 10 μl nicotine solution/100 g body weight over 0.5–1.5 s. Yohimbine solutions (Sigma-Aldrich, Oakville, Ontario, Canada) were prepared fresh daily in a distilled water vehicle and were injected at doses of 0.625 and 1.25 mg/kg i.p., in a volume of 1 ml/kg. Drug doses are expressed as base. Procedures Figure 1 shows the timeline of each of the experiments. Sucrose pellet SA Prior to catheterization surgery, the juvenile (P22) and adult (PD68-70) rats underwent operant training for 45 mg sucrose pellets (Bioserv, Frenchtown, NJ, USA) over 2 days in overnight sessions under an FR1 reinforcement schedule (Li et al. 2012; Shram et al. 2008). Animals spontaneously acquired SA of the sucrose pellets. During these sessions, water was freely available but only the sucrose pellets were available as a source of calories.
Nicotine SA Fixed ratio After sucrose pellet training, surgery, and recovery, rats initiated nicotine SA in 1-h daily sessions at FR1 and then FR2. Progressive ratio After the final FR2 session, the daily 2-h PR sessions were conducted. The PR sequence was determined using the exponential formula (5 × EXP(0.2 × infusion number)−5) (Donny et al. 1999). The number of infusions was recorded, and the session was terminated when no presses were made on the active lever for 20 min.
Stressors Social defeat The social defeat procedure is based on previous studies done on adolescent and adult rats (Burke et al. 2010, 2011; Funk et al. 2005; van Erp and Miczek 2001; van Erp et al. 2001). Animals receiving social defeat (intruders) were introduced into the home cages of larger, dominant male rats (n =12). These “resident” (aggressive) rats were Long Evans males (400–450 g) that had been housed with female rats (n = 12) for 10 days to enhance their territoriality and aggression and were selected based on their display of stable patterns of aggressive behavior (e.g., short latency to induce attacks, threatening postures) towards intruders. After the defeat of the intruders, defined as demonstration of supine posture that typically occurred following one or two attacks by the resident males, a wire screen was inserted into the cage to separate the intruder from the resident for 30 min. This prevented further attacks, but allowed the defeated rat to be exposed to the olfactory, visual, and auditory stimuli of the attacker. To insure consistently robust defeat induction, intruders were exposed to a different resident rat on each of the four defeat episodes. Five minutes prior to and during the defeat sessions, animals were exposed to an olfactory cue (peppermint essence, 1 ml, McCormick, London, Ontario, Canada) on gauze pads in culture dishes placed above the wire mesh of the cages. Control animals not receiving defeat were placed in cages similar to those used for defeat and received exposure to the odor but were not subject to defeat. FS Intermittent FS (5 and 10 min) was administered just prior to the start of the 2-h PR sessions. The shock delivery parameters were as follows: 0.5 s ON, a mean OFF period of 40 s, and a range of 10–70 s between each shock delivery (Le et al. 1998, 2011; Shaham and Stewart 1995). The intensity of FS required to induce vocalization in the adolescents and adults was determined to be 0.6 mA for adolescents and 0.8 mA for adults in a pilot study (data not shown); these intensity values were used for the administration of FS in the present studies.
Psychopharmacology Fig. 1 Procedural timelines of experimental events, including the ages of the subjects (PD, postnatal days) in experiments 1 (a), 2 (b), and 3 (c)
A
Adolescent Adult
Odor cue
Defeat (or no defeat) and odor exposure
Experiment 1 24 73
PD21 PD70
Food training
26 75
28 77
31 32 80 81
39 40 88 89
34 35 36 83 84 85
FR2
FR1
Surgery
47 95
42 43 90 91 PR
FS (min)
B
Experiment 2
Adolescent Adult
24 71
PD21 PD68
Food training
26 73
28 75
34 81
10
45 46 92 93
48 95
PR
FR2
FR1
Surgery
41 42 88 89
37 38 84 85
0 5
Yohimbine (mg/kg)
C
0 1.25 0.625
Experiment 3
Adolescent Adult
PD21 PD68
24 71
Food training
Yohimbine Yohimbine or its vehicle (water) was injected at doses of 0.625 and 1.25 mg/kg (i.p.) 30 min prior to the 2-h PR sessions. The yohimbine doses selected are based on previous work (Le et al. 2011; Li et al. 2012; Marinelli et al. 2007). Statistics and data presentation The dependent measures for SA under FR were the number of infusions and active and inactive lever presses during the 1-h sessions. For the PR sessions, they were the number of infusions and inactive lever presses made before cessation of responding for 20 min. These measures were analyzed separately using ANOVAs or ANCOVAs. Significant interactions (p values
ORIGINAL INVESTIGATION
Effects of stressors on the reinforcing efficacy of nicotine in adolescent and adult rats Sheng Zou & Douglas Funk & Megan J. Shram & A. D. Lê
Received: 24 April 2013 / Accepted: 4 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Rationale and objectives Stress increases drug intake. This depends on the stressor, drug, and aspect of drug seeking assessed. The objectives of these experiments done in adolescent and adult male rats were to (1) examine social defeat effects on acquisition of nicotine self-administration (SA) and the reinforcing efficacy of nicotine and (2) determine the effects of acute exposure to intermittent footshock (FS) or yohimbine on the reinforcing efficacy of nicotine. Methods In experiment 1, rats received four defeat exposures prior to nicotine SA acquisition and progressive ratio (PR) SA sessions (30 μg/kg nicotine/infusion). Exposure to an olfactory cue previously paired with defeat was also tested on responding maintained by nicotine on the PR schedule. In experiments 2 and 3, the effects of FS (5 and 10 min) or yohimbine (0.625 and 1.25 mg/kg, i.p.) on PR responding for nicotine (15, 30, or 60 μg/kg/infusion) were assessed. Adolescents were aged PD34-36 and adults PD81-85 at the beginning of nicotine SA training. Results Defeat did not affect nicotine SA acquisition. Prior exposure to defeat or a defeat-paired olfactory cue did not affect PR responding for nicotine. FS modestly decreased PR responding in adolescents at the middle nicotine infusion S. Zou : D. Funk : A. D. Lê (*) Neurobiology of Alcohol Laboratory, Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada e-mail: [email protected] M. J. Shram : A. D. Lê Department of Pharmacology, University of Toronto, Toronto, Canada A. D. Lê Department of Psychiatry, University of Toronto, Toronto, Canada M. J. Shram INC Research Toronto Inc., Toronto, Canada
dose. Yohimbine increased PR responding independent of nicotine infusion dose and age. Conclusions Together with previous work with other drugs, our data indicate that the effects of stress on the reinforcing efficacy of nicotine are stressor- and drug-dependent. This suggests that there is heterogeneity among stressors on how they affect neuronal systems underlying drug intake. Keywords Adolescents . Adults . Nicotine self-administration . Progressive ratio . Stress . Yohimbine . Footshock . Social defeat Studies in humans show that stressful life events are associated with increased use of, and relapse to, nicotine or other drugs of abuse (Brown et al. 1995; Pomerleau and Pomerleau 1991). The effects of stress on the initiation, maintenance, and reinstatement of drug seeking have been studied extensively using animal models. Using the reinstatement procedure, the effects of stress on relapse have been shown to be reliable and consistent across different laboratories and drug classes. As will be discussed below, there is heterogeneity among stressors in their effects on reinstatement, and these effects can depend on the drug examined (Le and Shaham 2002; Shalev et al. 2010). Despite the fact that stress clearly increases smoking in humans, to our knowledge, there are no studies examining the effects of stress on nicotine self-administration (SA) in laboratory animals. Footshock stress (FS) is one of the most commonly used laboratory stressors. The effects of FS on SA vary as a function of drug class, dose, reinforcement schedule, level of dependence, and interactions among these variables. Although FS has been used extensively to examine the effects of stress on alcohol SA, these studies have produced inconsistent results. The effects of FS may depend on the conditions under which alcohol SA was examined. For
Psychopharmacology
example, FS has been shown to enhance alcohol SA under alcohol deprivation conditions (Funk et al. 2004; Vengeliene et al. 2003) but to have weak or inconsistent effects under nondeprivation conditions (Logrip and Zorrilla 2012). In contrast, non-contingent exposure to FS is facilitatory on cocaine SA (Goeders and Guerin 1994; Mantsch and Katz 2007). Similarly, FS increases the oral intake of fentanyl solution under higher demand FR (FR4) and progressive ratio (PR) schedules of reinforcement (Shaham et al. 1993). FS also increased heroin SA under a PR schedule (Shaham and Stewart 1994). Two stressors, FS and yohimbine, an alpha-2 adrenoceptor antagonist, reliably reinstate the seeking of nicotine and other drugs (Anker and Carroll 2010; Buczek et al. 1999; Feltenstein and See 2006; Le et al. 2005; Leao et al. 2009; Shaham et al. 2000; Shepard et al. 2004; Yamada and Bruijnzeel 2011; Zislis et al. 2007). In contrast to the variable effects of FS, we and others have shown that yohimbine also increases alcohol SA (Ayanwuyi et al. 2013; Bertholomey et al. 2013; Le et al. 2005), and we recently reported that it increases nicotine SA in adolescent rats (Li et al. 2012). The effects of yohimbine on the SA of other drugs are not known. Another stressor, social defeat, has a unique pattern of effects on drug-seeking behavior. It produces the most reliable effects on the initiation of drug SA, and importantly, exposure to this stressor can have long-lasting effects on drug SA (Bardo et al. 2013; Miczek et al. 2008). These effects have been systematically studied in cocaine-trained animals. Exposure to social defeat prior to initiation of cocaine SA training increases the rate of acquisition of cocaine SA and increases cocaine intake under FR and PR schedules of reinforcement (Covington et al. 2005; Covington and Miczek 2001a; Tidey and Miczek 1997). In contrast, acute exposure to this stressor may decrease drug intake, possibly due to fear responses such as freezing (Chung et al. 2000). For example, acute exposure to defeat decreases alcohol SA (Funk et al. 2005; van Erp and Miczek 2001). No work has been done on the effects of defeat on any aspect of nicotine seeking. Adolescence is a vulnerable period in the initiation of drug intake (SAMHSA 2011). Stressful events are significantly correlated with smoking and intentions to smoke in adolescents (Booker et al. 2004, 2007, 2008), and adolescents report that stress is a salient precipitant of smoking (DiFranza et al. 2004; Weiss et al. 2008). Stress also enhances the rate of smoking initiation in adolescents (Booker et al. 2007, 2008; Byrne et al. 1995; Byrne and Mazanov 2003). Two possible reasons for this are that adolescence is a stressful developmental period and that adolescents may be more sensitive to the effects of stress than adults (Choi and Kellogg 1996; Novak et al. 2007; Slawecki 2005; Vazquez 1998). In support of these findings, FS was shown to facilitate the development of conditioned place preference (CPP) to nicotine in adolescent rats, suggesting that stress enhances
the rewarding effects of nicotine (Brielmaier et al. 2012). Work with other drugs has demonstrated age-dependent effects of stress on drug seeking. Yohimbine-induced reinstatement of cocaine seeking was shown to be of a higher magnitude in adolescents (Anker and Carroll 2010). Despite these data, no work has been done examining the possibility that there are age differences in sensitivity to the effects of stressors on the reinforcing efficacy of nicotine or whether such differences in stress sensitivity may affect initiation of nicotine SA. The purpose of the present investigation is to examine the effects of three different stressors on nicotine SA and its reinforcing efficacy. In light of the findings of the Miczek laboratory (Covington and Miczek 2001a), the first experiment investigated the effects of exposure to social defeat on acquisition of nicotine SA and then on the reinforcing efficacy of nicotine using a PR schedule of reinforcement, in both adolescent and adult rats. In the second and third experiments, the effects of acute exposure to FS and yohimbine, two stressors that reliably reinstate nicotine seeking, on the reinforcing efficacy of nicotine were determined in adolescent and adult rats previously trained to SA nicotine.
Materials and methods Subjects Forty-eight pregnant Long Evans dams (n =16 per experiment; Charles River Laboratories, Quebec, Canada) were used as a source of juvenile rats run in the experiments and were housed singly in plastic cages (51×41×20 cm). Adult male Long Evans rats were purchased from Charles River and were singly housed in plastic cages. On the day of weaning (post-natal day (PD) 21), 92 male pups were group housed by litter. After surgery, all rats were singly housed and fed 20–22 g of rat chow per day. The rats were maintained on a 12/12 h light/dark cycle (lights on at 1900) in a humidity- and temperature-regulated vivarium. The experimental procedures followed the NIH publication “Principles of laboratory animal care” (Eighth edition, 2011) and were approved by animal care and use committee of the Centre for Addiction and Mental Health. Apparatus Nicotine SA was performed in 24 chambers equipped with one active lever and one inactive lever. Appropriate responding on the active lever activated the infusion pump that delivered the intravenous (i.v.) nicotine followed by a 40s timeout. Each infusion was accompanied by a compound light-tone cue (stimulus light illumination for 6 s and a 2,800Hz tone for 1 s). Responding on the inactive lever had no
Psychopharmacology
programmed consequences but was recorded. Sucrose pellet SA (prior to nicotine SA) was conducted in a separate set of 16 similar operant chambers equipped with pellet dispensers. No cues were present during sucrose pellet training. Surgery Rats (adolescents: PD26; adults: PD73-75) were anesthetized using ketamine/xylazine (75 mg/kg ketamine/10 mg/kg xylazine (Shram et al. 2008)). Incision sites were treated with a local anesthetic (0.1 ml bupivacaine, 0.125 %, s.c.). Penicillin (30,000 U, i.m.) was administered as an antibiotic prior to surgery and buprenorphine (0.01 mg/kg, s.c.) as an analgesic after surgery. Catheters were implanted into the right jugular vein as previously described (Corrigall and Coen 1989) and exited between the scapulae and were attached to the modified 22-gauge cannula connected to the fluid swivel system. After recovery, catheters were flushed daily with 0.1 ml of a sterile heparin–saline solution (50 U/ml). Catheter patency was tested after PR testing was completed by i.v. injections of sodium methohexital (0.05 mg/kg). The data from animals that did not show rapid anesthesia following methohexital injection were excluded from analysis. Drugs Solutions of nicotine (Sigma-Aldrich, Oakville, Ontario, Canada) of pH 6.8 to 7.2 were prepared fresh daily. The unit doses for the i.v. nicotine SA were 30 μg/kg/infusion in experiment 1 and 15, 30, or 60 μg/kg/infusion in experiments 2 and 3. Nicotine infusions were made in a volume of 10 μl nicotine solution/100 g body weight over 0.5–1.5 s. Yohimbine solutions (Sigma-Aldrich, Oakville, Ontario, Canada) were prepared fresh daily in a distilled water vehicle and were injected at doses of 0.625 and 1.25 mg/kg i.p., in a volume of 1 ml/kg. Drug doses are expressed as base. Procedures Figure 1 shows the timeline of each of the experiments. Sucrose pellet SA Prior to catheterization surgery, the juvenile (P22) and adult (PD68-70) rats underwent operant training for 45 mg sucrose pellets (Bioserv, Frenchtown, NJ, USA) over 2 days in overnight sessions under an FR1 reinforcement schedule (Li et al. 2012; Shram et al. 2008). Animals spontaneously acquired SA of the sucrose pellets. During these sessions, water was freely available but only the sucrose pellets were available as a source of calories.
Nicotine SA Fixed ratio After sucrose pellet training, surgery, and recovery, rats initiated nicotine SA in 1-h daily sessions at FR1 and then FR2. Progressive ratio After the final FR2 session, the daily 2-h PR sessions were conducted. The PR sequence was determined using the exponential formula (5 × EXP(0.2 × infusion number)−5) (Donny et al. 1999). The number of infusions was recorded, and the session was terminated when no presses were made on the active lever for 20 min.
Stressors Social defeat The social defeat procedure is based on previous studies done on adolescent and adult rats (Burke et al. 2010, 2011; Funk et al. 2005; van Erp and Miczek 2001; van Erp et al. 2001). Animals receiving social defeat (intruders) were introduced into the home cages of larger, dominant male rats (n =12). These “resident” (aggressive) rats were Long Evans males (400–450 g) that had been housed with female rats (n = 12) for 10 days to enhance their territoriality and aggression and were selected based on their display of stable patterns of aggressive behavior (e.g., short latency to induce attacks, threatening postures) towards intruders. After the defeat of the intruders, defined as demonstration of supine posture that typically occurred following one or two attacks by the resident males, a wire screen was inserted into the cage to separate the intruder from the resident for 30 min. This prevented further attacks, but allowed the defeated rat to be exposed to the olfactory, visual, and auditory stimuli of the attacker. To insure consistently robust defeat induction, intruders were exposed to a different resident rat on each of the four defeat episodes. Five minutes prior to and during the defeat sessions, animals were exposed to an olfactory cue (peppermint essence, 1 ml, McCormick, London, Ontario, Canada) on gauze pads in culture dishes placed above the wire mesh of the cages. Control animals not receiving defeat were placed in cages similar to those used for defeat and received exposure to the odor but were not subject to defeat. FS Intermittent FS (5 and 10 min) was administered just prior to the start of the 2-h PR sessions. The shock delivery parameters were as follows: 0.5 s ON, a mean OFF period of 40 s, and a range of 10–70 s between each shock delivery (Le et al. 1998, 2011; Shaham and Stewart 1995). The intensity of FS required to induce vocalization in the adolescents and adults was determined to be 0.6 mA for adolescents and 0.8 mA for adults in a pilot study (data not shown); these intensity values were used for the administration of FS in the present studies.
Psychopharmacology Fig. 1 Procedural timelines of experimental events, including the ages of the subjects (PD, postnatal days) in experiments 1 (a), 2 (b), and 3 (c)
A
Adolescent Adult
Odor cue
Defeat (or no defeat) and odor exposure
Experiment 1 24 73
PD21 PD70
Food training
26 75
28 77
31 32 80 81
39 40 88 89
34 35 36 83 84 85
FR2
FR1
Surgery
47 95
42 43 90 91 PR
FS (min)
B
Experiment 2
Adolescent Adult
24 71
PD21 PD68
Food training
26 73
28 75
34 81
10
45 46 92 93
48 95
PR
FR2
FR1
Surgery
41 42 88 89
37 38 84 85
0 5
Yohimbine (mg/kg)
C
0 1.25 0.625
Experiment 3
Adolescent Adult
PD21 PD68
24 71
Food training
Yohimbine Yohimbine or its vehicle (water) was injected at doses of 0.625 and 1.25 mg/kg (i.p.) 30 min prior to the 2-h PR sessions. The yohimbine doses selected are based on previous work (Le et al. 2011; Li et al. 2012; Marinelli et al. 2007). Statistics and data presentation The dependent measures for SA under FR were the number of infusions and active and inactive lever presses during the 1-h sessions. For the PR sessions, they were the number of infusions and inactive lever presses made before cessation of responding for 20 min. These measures were analyzed separately using ANOVAs or ANCOVAs. Significant interactions (p values
