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ORIGINAL ARTICLE

doi:10.1111/adb.12336

Extended nicotine self-administration increases sensitivity to nicotine, motivation to seek nicotine and the reinforcing properties of nicotine-paired cues. Kelly J. Clemens, Belinda P. P. Lay & Nathan M. Holmes School of Psychology, University of New South Wales, Sydney, Australia

ABSTRACT An array of pharmacological and environmental factors influence the development and maintenance of tobacco addiction. The nature of these influences likely changes across the course of an extended smoking history, during which time drug seeking can become involuntary and uncontrolled. The present study used an animal model to examine the factors that drive nicotine-seeking behavior after either brief (10 days) or extended (40 days) self-administration training. In Experiment 1, extended training increased rats’ sensitivity to nicotine, indicated by a leftward shift in the dose– response curve, and their motivation to work for nicotine, indicated by an increase in the break point achieved under a progressive ratio schedule. In Experiment 2, extended training imbued the nicotine-paired cue with the capacity to maintain responding to the same high level as nicotine itself. However, Experiment 3 showed that the mechanisms involved in responding for nicotine or a nicotine-paired cue are dissociable, as treatment with the partial nicotine receptor agonist, varenicline, suppressed responding for nicotine but potentiated responding for the nicotine-paired cue. Hence, across extended nicotine self-administration, pharmacological and environmental influences over nicotine seeking increase such that nicotine seeking is controlled by multiple sources, and therefore highly resistant to change. Keywords

Cues, dose–response, nicotine, progressive ratio, self-administration, varenicline.

Correspondence to: Kelly J. Clemens School of Psychology, University of New South Wales Sydney, NSW 2052 Australia. Email: [email protected]

INTRODUCTION Drug addiction is a pervasive and debilitating disease. It develops over time, and once established, persists in spite of its adverse health, economic, family and social consequences. (Tiffany, 1990). In the past 40 years, animal models have been increasingly used to study the mechanisms that underlie this loss of control. As a result, much is known about the neurobiological consequences of drug exposure [for recent reviews see Everitt & Robbins (2013), Nestler (2014), & Wise (2004)], the development of phenomena like sensitization and tolerance (DiFranza & Wellman, 2005; Vezina et al., 2007), and the environmental factors implicated in the acquisition and early maintenance of drug-seeking behavior (e.g. Di Ciano & Everitt, 2003, 2004a, 2004b; Ito et al., 2002) [for review see Everitt & Robbins (2005)].

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Much less is known about the changes in associative learning processes that occur across a period of chronic drug exposure. Understanding these processes may be particularly important for drugs such as nicotine, that are generally considered to exhibit only weak reinforcing properties, but nonetheless exhibit very high levels of dependence (Collins et al., 1984; Henningfield & Goldberg, 1983). Exploring this relationship is crucial to the development of effective cognitive and pharmacological therapies that take into account the numerous neurobiological and environmental factors that promote and sustain nicotine dependence. As demonstrated in the cases of cocaine (Zapata et al., 2010) and ethanol seeking in rats (Corbit et al., 2012), we have recently shown that nicotine seeking is initially a goal-directed behavior, mediated by a representation of the drug and its current value (Clemens et al., 2014). However, with additional weeks of training, responding

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Kelly J. Clemens et al.

comes to occur habitually, or independently of this value. These findings have been taken to imply that across extended training, contextual cues antecedent to the drug infusion (e.g. self-administration context) enter into an association with the response. Hence, the very presence of these cues comes to trigger drug seeking automatically or reflexively. The increasing influence of contextual cues across extended training should not be taken to imply that drug seeking becomes insensitive to its consequences. For example, in the case of nicotine, across extended training, tolerance to some aspects of nicotine (Irvine et al., 2001; Szyndler et al., 2001; Zuo et al., 2011) and sensitization to others (Hilario et al., 2012; Lenoir et al., 2013) may alter its perceived value, and therefore, its capacity to reinforce drug seeking. At the same time, responsecontingent cues, which accompany nicotine taking [typically auditory or visual stimuli in the case of intravenous self-administration (IVSA)] can acquire reinforcing properties, thus potentially providing an additional source of support for drug seeking (Caggiula et al., 2001, 2002; Cohen et al., 2005). How these changes then influence later susceptibility to pharmacological manipulation is unclear, but may have consequences for the development and testing of new smokingcessation treatments. Accordingly, the present study used a nicotine IVSA model to examine changes in the factors that control drug seeking across a period of extended training. The first experiment examined changes in sensitivity to, and motivation for, nicotine across extended self-administration training. Within-session patterns of self-administration were also examined as an indirect assay of nicotine regulation (Lau & Sun, 2002; Panlilio et al., 2003). The second experiment examined the importance of drug-associated cues in maintaining drug seeking when the drug itself is not present, and importantly, how this changes across extended self-administration. Finally, a number of pharmacotherapies have been developed to assist with reducing or blocking tobacco use. One of the most widely used medications, varenicline (VAR), robustly suppresses nicotine self-administration and reinstatement of nicotine seeking in rats (George et al., 2011; Le Foll et al., 2012; O’Connor et al., 2009; Wouda et al., 2011). The third experiment investigated the effects of VAR treatment on responding maintained by nicotine alone or the cue alone across extended self-administration.

MATERIALS AND METHODS Subjects Eighty male Sprague Dawley rats (175–200 g; Animal Resources Centre, Perth, Australia) were housed four © 2015 Society for the Study of Addiction

per cage on a 12-h reverse light/dark cycle (lights off at 7 am). Food and water were available ad libitum prior to surgery and during recovery, and thereafter restricted to 20 g/rat/day. All experiments were approved by the University of New South Wales Animal Care and Ethics Committee and were in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th ed, 2013).

Drugs Nicotine tartrate (Sigma, St. Louis, MO, USA) was dissolved in 0.9 percent sterile saline, and concentrations expressed as the base. Varenicline tartrate (Abcam Biochemicals, VIC, AUS) was dissolved in 0.9 percent sterile saline and injected subcutaneously in a volume of 1 ml/kg.

General procedures for self-administration Two weeks after arriving in the laboratory, rats underwent surgery for implantation of a chronic intravenous catheter into the right jugular vein as described previously (Motbey et al., 2013). Training commenced with two habituation sessions during which rats were placed for 1 h/day in the selfadministration chambers (Med Associates, VT, USA) with the nose-pokes covered and house-light on. Selfadministration on a fixed-ratio 1 (FR-1) schedule then began with 1 h daily sessions. A response in the active nose-poke resulted in an infusion of nicotine (30 μg/kg/ 100 μl infusion across 3 s), illumination of the nose-poke cue light (3 s) and extinction of the house light (20s). Responses in the active nose-poke during the 20-s time-out or in the inactive nose-poke were recorded but had no programmed consequences. Rats were considered to have acquired self-administration once they achieved a minimum of six infusions per session, demonstrated a 2:1 preference for the active nose-poke and exhibited stable responding across consecutive sessions (600 s), consistent with the view that responding tended to occur in bouts of clusters that were themselves distributed across the session. In contrast, after extended training, nicotine taking occurred with greater regularity across the session, as evidenced by an increase in the number of moderate inter-infusion intervals (120–240 s) and a decrease in the number of long inter-infusion intervals. The increase in response regularity across training was also evident in the analysis of the cumulative records of active nose pokes on days 9 and 39. Figure 1C and Figure 1D show these records for a sample rat that responded equally at the early and late time points. This illustrates that the pattern of administration changed across training, even when the response rate was similar: at the late time point, rats engaged in significantly fewer nicotine-taking bouts (or clusters) (t19 = 3.20, p < 0.01; Fig. 1E), but each bout lasted longer (t19 = 3.84, p < 0.01; Fig. 1F) and consisted of more nicotine infusions per bout (t19 = 4.80, p < 0.01; Fig. 1G).

the dose–response tests. Training and test data were then compared using Pearson product–moment correlations to examine the degree of linearity in the relationship between the variables. For these comparisons, we used the final ratio achieved by each individual rat on the progressive ratio test (index of motivation), and the maximum slope of each individual rats’ dose–response curve (index of sensitivity to changing nicotine dose). A Bonferroni correction was used to maintain the chance of a type I error at α = .05 across the two comparisons at each time point. All other data were compared using ANOVA, or where appropriate, planned contrasts as described in the text. RESULTS Experiment 1: Dose–response and progressive ratio Five rats were excluded from the analysis (1 failed to acquire, 4 lost catheter patency: 3 brief, 1 extended). The remaining rats successfully acquired self-administration (refer to Table 1 for summary information). Response characteristics across training Figure 1A shows the number of infusions obtained across the acquisition of self-administration, and Fig. 1B shows the distributions of wait times between active nose pokes (excluding time-out responses) in the training sessions on day 9 and day 39. A chi-squared test

Dose–response and progressive ratio tests Figure 2A shows that the dose–response function was different at the two stages of training (main effect of

Table 1 Comparison of total active and inactive nose-pokes (SEM) across experiments

Clemens et al. 2014 a (no tests) n = 25 Expt. 1 DR and PR n = 19 Expt. 2 Cue/nicotine extinction n = 19 Expt. 3 varenicline Extinction n = 23 a

Day 10 Before any testing

Day 14 After dose–response in Expt. 1

Day 18 -After PR in Expt. 1Day 21 -After extinction in Expt. 1 and 2

Active

Active

Active

Inactive

Inactive

Inactive

Day 40 Prior to second phase of testing Active

Inactive

18.12 (1.70)

6.80 (0.88)

22.32 (2.20)

6.40 (0.67)

27.20 (2.29)

7.12 (0.87)

29.92 (2.31)

5.68 (0.92)

20.53 (1.97)

6.85 (0.87)

25.47 (1.64)

6.85 (1.09)

27.63 (3.24)

7.55 (1.26)

33.58 (2.72)

13.90 (3.72)

23.55 (2.88)

8.68 (1.31)

30.18 (3.70)

8.63 (1.23)

34.52 (2.35)

8.09 (1.60)

25.90 (2.40)

10.09 (1.29)

26.52 (2.09)

6.52 (1.04)

26.57 (2.26)

10.09 (2.40)

From Clemens et al. (2014), Neuropsychopharmacology Oct;39(11):2584–93.


Addiction Biology

Cue-driven nicotine seeking

Figure 1 Within-session response characteristics following brief or extended nicotine self-administration. (A) Total number of infusions (+ SEM) across acquisition of nicotine self-administration indicating the data (filled circle) used for further analysis and timing of dose response (DR) and progressive ratio (PR) tests. (B) Distributions of the inter-infusion intervals after brief or extended training. (C) Sample record of cumulative infusion record after brief or (D) extended training indicating data used for subsequent change-point analysis. (E) The number of bouts, (F) average bout duration (minutes) and (G) the average number of infusions per bout following brief or extended nicotine self-administration. Bars represent group mean + within-subjects error. Asterisks indicate significant difference when p < 0.01

training: F1,54 = 26.41, p < 0.001; main effect of dose: F3,54 = 16.06, p < 0.001; dose by training interaction: F3,54 = 8.36, p < 0.001). Following brief training, the dose response function was relatively flat, with a shallow peak across the middle doses (15 and 30 μg). In contrast, following extended training, the dose response function shifted upward and to the left, peaking at 7.5 μg and descending uniformly across the higher doses. Responses on the inactive nose-poke did not change overall with training (Fig. 2B); however, a main effect of dose (F3,54 = 6.14, p < 0.001) and a dose by training interaction (F3,54 = 4.90, p < 0.01) shows that inactive responses did fluctuate slightly with changing infusion dose and that this differed depending on the amount of training received. © 2015 Society for the Study of Addiction

During the progressive ratio test, rats worked harder to obtain nicotine following extended training, as reflected in the higher levels of active responses (t18 = 2.88, p < 0.01; Fig. 2C), and break point achieved (t18 = 2.84, p < 0.05; Fig. 2D). Relationships between total infusions and performance on the dose–response and progressive ratio tests at the two test points are described in supplementary materials (Supporting Information Fig. S1). It is worth noting that there was no correlation between measures of performance in each of the two tests (dose–response and progressive ratio) at each of the two time points (r < 0.3), suggesting that testing at the brief time point did not influence testing at the late time point. Addiction Biology


Figure 2 Results from dose–response and progressive ratio tests in Experiment 1. (A) Active and (B) inactive nose-pokes recorded across varying nicotine infusion doses after brief and extended training in the same animals. (C) Active responses and (D) break point achieved during a single session of progressive ratio following either brief or extended training. Data represent group means + within subjects error. Asterisks indicates dose by training interaction when p < 0.05

Experiment 2: the reinforcing properties of nicotine and the nicotine-related cue Five rats were excluded from data analysis (1× failure to acquire, 4× loss of catheter patency: 3 from brief, 1 from extended). The remaining rats successfully acquired self-administration (refer to Table 1 for summary infor mation).

Cue and nicotine only test sessions Figure 3 shows the mean (+SEM) levels of responding in each group across the test phases (see Fig. S3 for individual days). Critically, the pattern of responding across the treatment groups differed at the two time points (interaction between training and contrast comparing Group CUE versus the other two groups, F1,16 = 6.90, p < 0.05). After brief training, rats in Groups NIC responded more than rats in Groups CUE and EXT (F1,16 = 78.65, p < 0.001), whereas responding in Groups Cue and EXT was not significantly different (F1,16 = 3.68; Fig. 3A). In contrast, after extended training, rats in Groups NIC and CUE responded more than rats in Group EXT (F1,16 = 10.13, p < 0.05), but did not differ from each other (F < 1; Fig. 3B). Hence, across extended IVSA training, the response-contingent cue acquired the ability to maintain responding to the same degree as presentation of nicotine alone. © 2015 Society for the Study of Addiction

Responses on the inactive nose-poke followed a similar pattern at the brief time point (Fig. 3C), with responding in the EXT and CUE groups significantly lower than rats in the NIC group (F1,16 = 15.56, p < 0.01). However, with extended training, discrimination in the NIC group improved and there no longer any significant group differences. To determine whether the pattern of responding supported by the response contingent cue was the same or different to that supported by nicotine itself, the same measures of within-session IVSA reported in Experiment 1 were used to compare responding in Groups NIC and CUE (Fig. S3). In spite of the fact that Group CUE tended to have shorter wait times between responses than Group NIC, the two groups otherwise performed identically. Hence, after an extended period of nicotine IVSA, presentation of the response contingent cue maintained responding to the same degree as presentation of nicotine alone, and the pattern of this responding was identical to that maintained by nicotine alone. Experiment 3: the effects of varenicline on responding maintained by cues or nicotine alone Nine rats were excluded from data analysis (3 failed to acquire, 6 lost catheter patency: 2 brief, 4 extended). The remaining rats successfully acquired self-administration (refer to Table 1 for summary information). Addiction Biology


Figure 3 The average number of (A) and (B) active and (C) and (D) inactive responses made across the two 6-day test phases after brief (10 days) or extended (40 days) training. Three groups of rats were tested for responding when nose-pokes were of no consequence (EXT), produced the visual cue only, but no nicotine (CUE) or produced nicotine only, but no cues (NIC). Bars represent group mean (+SEM). Asterisks indicate significant difference when p < 0.05

Under initial CUE only or NIC only conditions (day SAL; Fig. 4), responding maintained by nicotine (group NIC) was significantly higher than that maintained by the cue alone (group CUE) (F1,24 = 7.51, p < 0.01). In contrast, after extended training, there was no significant difference in responding maintained by either the CUE only or NIC only (F1,21 = 2.96, p = 0.10). After brief training, the effect of VAR injection on responding in the test phase differed depending on the conditions of testing (main effect of treatment: F1,24 = 4.90, p < 0.05; treatment by group interaction: (F1,24 = 18.51, p < 0.001; Fig. 4): VAR increased responding in Group CUE (F1,13 = 21.57, p < 0.001; Fig. 4A) but had no significant effect on responding in Group NIC (F < 1). After extended training in the same animals, injection of VAR again had contrasting effects on responding depending on the conditions of testing (treatment by group interaction (F1,21 = 12.71, p < 0.01). VAR increased responding in Group CUE (F1,11 = 4.94, p < 0.05; Fig. 4B), but suppressed responding in Group NIC (F1,10 = 8.09, p < 0.05). Responding was highly specific to the active nosepoke, with no effect of VAR treatment on inactive © 2015 Society for the Study of Addiction

Figure 4 The effect of varenicline on responding for nicotine-associated cues (CUE) or nicotine alone (NIC) following brief or extended training. Data represents active (A) and (B) and inactive (C) and (D) responses made on the day immediately prior to test (SAL), and on the day on which rats received a pre-session varenicline injection (VAR). Bars represent group mean (+SEM). Asterisks indicate significant difference when p < 0.05

responses for either NIC or CUE groups at the brief time-point (Fig. 4C). Following extended training, inactive responses decreased slightly in the NIC group with VAR treatment (F1,10 = 5.27, p < 0.05), but had no effect on inactive responding in the CUE group. Injection with VAR also had an impact on locomotor activity. Specifically, this injection increased activity in the CUE, but not NIC, group following both brief (treatment by group interaction: F1,24 = 19.35, p < 0.001) and extended training (treatment by group interaction: F1,24 = 15.53, p < 0.01; data not shown). Therefore, treatment with VAR facilitated responding in Group CUE following both brief and extended training and reduced responding in the Group NIC after extended training only.

DISCUSSION This study investigated changes that occur in patterns of nicotine seeking and the factors that influence nicotine seeking, with extended self-administration training. In Experiment 1, rats initially responded for nicotine in distinct bouts or clusters. However, as training progressed, rats responded at regularly spaced intervals, and this change in the pattern of responding occurred independently of the overall increase in response rate (Fig. 1C–F). Response Addiction Biology


regularity has been associated with titration of drug levels in the case of cocaine self-administration (e.g. Lau & Sun, 2002; Panlilio et al., 2003). Thus, the finding that rats came to respond at regularly spaced intervals after an extended history of nicotine self-administration suggests that they became more adept at titrating nicotine levels with increasing amounts of drug exposure. This increased ability to titrate nicotine intake was accompanied by an overall increase in sensitivity to the reinforcing effects of nicotine as indicated by a leftward shift of the dose–response function, and an increase in the break point across progressive ratio tests. Both of these changes correlated with total nicotine intake and are likely to reflect an up-regulation of nicotine acetycholine receptors with extended self-administration (DiFranza & Wellman, 2005; Marks & Collins, 1985; Marks et al., 1992). This sensitization to the reinforcing and motivational properties of nicotine may also contribute to the gradual increase of nicotine intake observed across extended training (Table 1) (Clemens et al., 2014). This contrasts with evidence that tolerance is an important feature of escalating cocaine intake following extended self-administration (Ahmed & Koob, 1999; Morgan et al., 2005), although differences in session length between the two studies, and the marked differences in the addictive profile of these two drugs, may complicate this comparison (Deroche-Gamonet et al., 2002). Experiment 2 showed that the response-contingent cue light also differed in its capacity to maintain responding across extended self-administration training. Consistent with past reports (Caggiula et al., 2001, 2002), after a brief amount of training, rats did not respond for the cue in the absence of nicotine. In contrast, following extended self-administration training, rats responded for the cue alone, and, the level and pattern of responding for the cue was equivalent to that observed among rats responding for nicotine alone (Fig. S3). These findings show that nose-poking was not simply because of frustration induced by the absence of expected nicotine, but instead, suggests that the cue had acquired the capacity to reinforce the drug seeking response (Everitt and Robbins, 2005; Robinson and Berridge, 1993). Notably, this robust ability of cues to maintain responding may be specific to nicotine, as similar studies with cocaine (Deroche-Gamonet et al., 2002) indicate that removal of the drug reward results in the rapid extinction of responding, even if response-contingent cues remain. It is worth noting that removal of the cue had no detectable impact on responding among rats that continued to receive nicotine infusions at either point in training (Group NIC). That is, the change in responding from the last session of normal self-administration training to the first session in which the response contingent cue © 2015 Society for the Study of Addiction

was removed was negligible at both time points (Fig. S2). This contrasts with past nicotine self-administration studies showing that removal of the nicotine-associated cue produces an immediate reduction in responding (Caggiula et al., 2001, 2002). However, this difference may be explained by various procedural differences. For example Caggiula et al. (2001) used an FR-5 schedule, which potentially increases the significance of the cue as it serves to discriminate reinforced from nonreinforced responses. This aspect of the results is instead more consistent with those observed following cocaine self-administration, where removal of responsecontingent cues has little effect on established cocaine self-administration (Deroche-Gamonet et al., 2002). This, in combination with the shift in response patterns observed in Experiment 1, indicates that with extended self-administration, nicotine seeking in rats begins to more closely resemble aspects of established cocaine self-administration. In Experiment 3, a leading smoking cessation drug, VAR, had clearly dissociable effects on responding maintained by either nicotine alone or the responsecontingent cue alone. VAR suppressed responding maintained by nicotine at both time points, which is consistent with its properties as a partial agonist of nicotine receptors (Rollema et al., 2007), and previous findings that VAR reduces nicotine self-administration and blocks drug-primed reinstatement (George et al., 2011; Le Foll et al., 2012; O’Connor et al., 2009; Wouda et al., 2011). In contrast, VAR potentiated responding maintained by the response-contingent cue. This could be because either VAR imbues the cue with additional incentive value, or alternatively, that VAR simply substitutes for nicotine in priming nicotine-seeking behaviors. Whatever the case may be, these results suggest that VAR is effective in reducing nicotine seeking that is driven by the pharmacological actions of nicotine, but is ineffective in reducing responding that has come under the control of drugpaired cues. The increasing influence of environmental stimuli on nicotine-seeking behavior has been related to increased activation of immediate early genes (c-fos) in brain regions including the amygdala, substantia nigra and dorsal striatum (Clemens et al., 2014). This circuitry plays a critical role in the development of stimulus–response habits across the course of extended self-administration of a natural reward (Lingawi & Balleine, 2012) and has also been shown to mediate the conditioned reinforcing properties of discrete drug-paired cues in studies of drug self-administration (Ito et al., 2002; Vanderschuren et al., 2005). Nicotine receptors are highly expressed throughout the striatum and have been shown to play a role in striatal plasticity involved in reward-related learning (Partridge et al., 2002). It remains to be Addiction Biology


determined whether changes in mid-brain nicotine receptor expression across extended training contribute to the evolving role of antecedent (Clemens et al., 2014) and response-contingent (current study) cues in sustaining the nicotine-seeking response. The current study used a within-subjects design to examine the effect of extended self-administration training on distinct aspects of nicotine seeking in rats. While this design allows the opportunity to track the behavior of the same animal across time, it raises the possibility that testing at the brief time-point (particularly the progressive ratio schedule) may interfere with testing at the later time point. However, in this respect, there were no significant correlations between performance on the dose– response and progressive ratio tests after brief and extended training. Furthermore, the progression of selfadministration after the early period of testing was indistinguishable from that observed across experiments in the current study, and with other published studies where rats were trained with the same parameters for the same amount of time, but in the absence of any testing (refer to Table 1; Clemens et al., 2014). Hence, we are confident that testing at the early time point had minimal (if any) impact on subsequent self-administration and testing. In summary, across extended nicotine selfadministration training, patterns of drug-seeking change, sensitization to the reinforcing properties of nicotine increases, and motivation to work for nicotine increases (Experiment 1). Concomitant with these changes, the self-administration context acquires the capacity to trigger nicotine seeking automatically or reflexively (Clemens et al., 2014), cues which accompany delivery of nicotine acquire greater control over drug seeking (Experiment 2), and almost paradoxically, systemic treatment with VAR increases responding for response-contingent cues presented in the absence of nicotine (Experiment 3). The combination of these factors may encourage persistence of nicotine seeking after a history of chronic smoking, and precipitate relapse after periods of successful abstinence. It will be important for future work to identify the neural circuitries and neurotransmitter systems that mediate these effects, and how treatments like VAR interact with these systems to influence smoking behavior and its cessation.

Acknowledgements The authors would like to thank Angela Cantarella and Anne Rowan for animal care, and Justine Fam for assistance in carrying out behavioral experiments. Dr Clemens’ work has been funded by an Australian Research Council Discovery Early Career Researcher Award (DECRA). The authors declare no conflict of interest. © 2015 Society for the Study of Addiction

Author Contribution KC and NH were responsible were responsible for the study concept and design. KC and BL contributed to the acquisition of animal data. KC and NH analyzed data and interpreted findings. KC and NH wrote the manuscript. All authors critically reviewed content and approved final version for publication.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Correlations between nicotine intake and performance on dose–response and progressive ratio tests in Experiment 1. Scatter plots of the relationships between total number of infusions made across the preceding nine days of self-administration after brief or extended training with either the maximum slope of the dose–response curve as an index of the sensitivity to changing dose (A and B) or the maximum ratio achieved under a Addiction Biology


progressive ratio schedule as an index of motivation to seek nicotine (C and D). Data points represent individual animals. Figure S2. Active responses (A, B), inactive responses (C, D) and locomotor activity (E, F) across self-administration immediately prior to (d5-10 or d35-40), during (T1–T6), and immediately after (d17-22 or d47-52) the two test phases in Experiment 2. Three groups of rats were tested for responding when nose-pokes were of no consequence (standard extinction), produced the visual cue only (CUE), but no nicotine (CUE) or produced nicotine only, but no cues [nicotine only (NIC)] at two time points, after brief (10 days) or extended (40 days) or training. Data points represent group mean (+SEM).


Figure S3. Within-session response characteristics following brief or extended nicotine self-administration in rats nose-poking for the CUE or NIC. (A) Distributions of the inter-infusion intervals after brief or extended training. (B) The number of bouts, (C) average bout duration (minutes) and (D) the average number of infusions per bout following brief or extended nicotine self-administration. CUE represents conditions where responses resulted in presentation of the visual stimulus, but not nicotine. NIC indicates availability of nicotine, but no response-contingent cues. The extinction group is not included in this analysis as responses were of no consequence. Bars represent group mean + withinsubjects error.

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