Neuroadaptive changes in the mesoaccumbens dopamine system after chronic nicotine self-administration: A microdialysis study

Neuroscience 129 (2004) 415– 424

NEUROADAPTIVE CHANGES IN THE MESOACCUMBENS DOPAMINE SYSTEM AFTER CHRONIC NICOTINE SELF-ADMINISTRATION: A MICRODIALYSIS STUDY S. RAHMAN,a,b* J. ZHANG,a E. A. ENGLEMANc AND W. A. CORRIGALLa1

nicotine exposure. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

a Smoking and Nicotine Dependence Research, Neuroscience Department, Centre for Addiction and Mental Health, University of Toronto, 33 Russell Street, Toronto, Ontario, M5S 2S1 Canada

Key words: Nicotine, dopamine, nucleus accumbens, no-netflux, microdialysis, neuroadaptation.

b Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada

Tobacco use constitutes a global health problem (Peto et al., 1996). Nicotine is the principal psychoactive ingredient in tobacco and possesses powerful rewarding properties (Rose and Corrigall, 1997). Understanding the neurochemical mechanisms that underlie nicotine’s effects will help explain its rewarding properties when it is delivered by tobacco (Balfour, 2003; Corrigall, 1999; Dani, 2003; Koob and Le Moal, 2001). The mesoaccumbens dopamine (DA) system, which originates in the ventral tegmental area (VTA), has been implicated in mediating the reinforcing effects of nicotine (Balfour et al., 1998; Clarke, 1990; Di Chiara, 2000; Picciotto and Corrigall, 2002; Watkins et al., 2000). For example, systemic administration of selective DA receptor antagonists reduces i.v. nicotine self-administration in a dose-dependent manner (Corrigall and Coen, 1991), nicotine self-administration is reduced following application of the DA neurotoxin 6-hydroxy DA into the DA terminal field of the mesoaccumbens system (Corrigall et al., 1992) and, nicotine self-administration is reduced following microinfusion of the high affinity nicotinic acetylcholine receptor (nAChR) antagonist dihydro-␤-erythroidine into the VTA (Corrigall et al., 1994). These and other reports (Balfour et al., 1998; Clarke, 1990; Di Chiara, 2000; Watkins et al., 2000) support the hypothesis that the mesoaccumbens DA system is an important substrate for nicotine reinforcement, and the nAChRs found in the VTA are critical in mediating the reinforcing effects of nicotine. Most previous studies have shown that addictive drugs, including nicotine, typically increase the extracellular DA levels in the mesoaccumbens DA system upon acute systemic administration (Balfour et al., 1998; Brazell et al., 1990; Di Chiara, 2000; Fu et al., 2000; Nisell et al., 1994; Pontieri et al., 1996). In light of these findings, it has been proposed that nicotine-induced enhancement of mesoaccumbens DA transmission is an important mechanism involved in the reinforcing effects of nicotine (Pontieri et al., 1996). On the other hand a number of studies with conflicting reports have indicated that the extracellular DA levels in the nucleus accumbens (ACB) are altered (increase versus decrease) after cessation from chronic exposure with experimenter-administered nicotine (Benwell et al., 1995; Carboni et al., 2000; Hilderbrand et al., 1999). However, there is little evidence to date that shows the mesoaccumbens DA transmission is altered at the neuro-

c

Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202, USA

Abstract—There is little evidence to date to indicate if mesoaccumbens dopamine function at the neurochemical level is altered during early abstinence from chronic i.v. nicotine selfadministration. Thus, a quantitative microdialysis (no-net-flux) approach was used to measure basal extracellular concentrations and extraction fractions of dopamine in the nucleus accumbens (ACB) of rats that self-administered nicotine i.v. for 25 days, as well as in rats serving as yoked comparison groups (yoked nicotine and yoked saline). After 24 – 48 h of the final self-administration session, there was a significant reduction in basal extracellular dopamine levels in the ACB of the self-administration group compared with the yoked saline group (1.35ⴞ0.15 nM versus 3.70ⴞ0.28 nM). The basal extracellular dopamine levels in the yoked nicotine group (1.46ⴞ0.20 nM) were not significantly different compared with the nicotine selfadministration group. The in vivo extraction fraction of dopamine, an indirect measure of dopamine uptake, was significantly increased in the nicotine self-administration (86%) and yoked nicotine (91%) groups compared with the yoked saline group (77%). In addition, a marked reduction in the elevation of extracellular dopamine levels in the ACB occurred after a nicotine challenge as measured by conventional microdialysis in the self-administration (112% of basal) and yoked nicotine (121% of basal) groups as compared with a yoked saline (154% of basal) group. The reduced basal ACB dopamine levels in the nicotine groups during early abstinence appears to be due to increased clearance, suggesting increased dopamine uptake is occurring as a result of the chronic nicotine treatment. The reduced elevation of extracellular dopamine levels in the ACB upon nicotine challenge suggests a functional desensitization or downregulation phenomenon involving acetylcholine receptors (nicotinic nAChRs). Overall, these results provide clear evidence for a neuroadaptive change that alters dopamine transmission in the ACB during abstinence from chronic i.v. 1

Present address: Corrigall Consulting, Thornhill, Ontario, Canada. *Correspondence to: S. Rahman, Smoking and Nicotine Dependence Research, Neuroscience Department, Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada. Tel: ⫹1-416-535-8501; fax: ⫹1-416-595-6922. E-mail address: [email protected] (S. Rahman). Abbreviations: ACB, nucleus accumbens; ACSF, artificial cerebrospinal fluid; CRF, continuous reinforcement schedule; DA, dopamine; FR, fixed ratio; nicotinic nAChRs, acetylcholine receptors; SA, self-administration; TO, time-out; VTA, ventral tegmental area.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.08.010

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chemical level during early abstinence from chronic nicotine self-administration, a drug seeking model for addiction (Corrigall, 1999). Moreover, the measurements of the extracellular DA levels with earlier studies have been examined without considering potential changes in the DA uptake mechanism, which is known to play an important role in controlling the extracellular DA levels in the ACB (OlsonCosford et al., 1996; Smith and Justice, 1994). Therefore, the present study was undertaken to examine the neuroadaptive effects of chronic nicotine exposure on extracellular ACB DA levels in rats during early abstinence after active (response-contingent) self-administration and passive (response-independent) yoked administration using the no-net-flux microdialysis procedure. In addition, we used the conventional microdialysis followed by same nicotine exposure paradigm with a nicotine challenge to examine the neuroadaptive effects on nAChRs regulation in mesoaccumbens DA transmission. The response independent (yoked administration) procedure was used to identify any differential effects resulting from active self-administration versus passive administration of nicotine on the neuroadaptive effects in the mesoaccumbens DA system (Jacobs et al., 2003; Smith et al., 2003; Stefanski et al., 1999). Similar response independent procedures have been used previously with other drugs of abuse including nicotine (Donny et al., 1998, 2000; Smith et al., 2003; Stefanski et al., 1999). The quantitative no-net-flux microdialysis method (Lonnroth et al., 1987; Parsons and Justice, 1994) was used to measure the basal extracellular DA concentrations and DA extraction fractions, an indirect measure of DA uptake, in the ACB of rats after 25 days of nicotine self-administration, yoked nicotine, or yoked saline administration. Recent developments in microdialysis techniques suggest that the no-net-flux method is useful in measuring the true extracellular concentration of DA and also assessing potential changes in DA uptake in the ACB (Smith and Justice, 1994; Yim and Gonzales, 2000). In this method, artificial cerebrospinal fluid (ACSF) containing several different concentrations of DA that bracket the anticipated concentration in the extracellular space are perfused through the dialysis probe and the amount of DA gain or loss from the probe is measured (Chefer and Shippenberg, 2002; OlsonCosford et al., 1996; Smith and Justice, 1994; Smith and Weiss 1999). Information regarding changes in DA release can also be obtained from extracellular DA levels and DA uptake (Chefer and Shippenberg, 2002; Smith and Justice, 1994). Furthermore, extraction fraction (in vivo recovery) is a useful index of extracellular dynamics, and has been shown to reflect changes in DA uptake (Smith and Justice, 1994). As a result, higher rates of DA uptake work to reduce extracellular DA levels.

EXPERIMENTAL PROCEDURES Animals Adult male Long-Evans rats (275–300 g; Charles River, Lachine, PQ, Canada) were used in this study. Rats were singly housed in a reversed light/dark cycle colony room (lights off between 07:00

h and 19:00 h) and maintained in a constant temperature and humidity controlled animal facility with food and water available ad libitum. Animal care and experimental procedures were carried out in compliance with the guidelines of the Canadian Council on Animal Care (compatible with NIH guidelines), and were reviewed and approved by the Institutional Animal Care Committee. All efforts were made to minimize animal suffering, and to reduce the number of animals used in this study.

Nicotine self administration procedure When received from the supplier, rats were first habituated to the colony room for a 1-week period before training procedures were begun. After initial habituation in the animal care facility, animals were single-housed and tagged. Animals were deprived of food for a period of approximately 24 h, and trained to press a lever in a standard two lever operant chamber (Med Associates Inc., St. Albans, VT, USA) on a schedule in which each press resulted in delivery of a 45-mg food pellet (continuous reinforcement schedule, CRF). Training took from 1 to several days depending upon the particular subject. Once trained, rats were no longer maintained in food-deprived condition; they were fed approximately 20 g of chow each day, as single meal, for the duration of the experiment. Since depriving animals of food is known to increase drug self-administration, it is important to point out that subjects in our self-administration experiments, although not allowed ad libitum access to food, were not food deprived. The food ration (20 g) constitutes the daily nutritional requirement for the rat (Canadian Council on Animal Care). Subjects in our studies gained body weight during the months of an experiment with this feeding schedule, but were smaller than animals with free access to food. The objective in our study was to have reasonable a degree of drug-seeking behavior generated by animals that are otherwise normal. A similar approach has extensively been used previously for drug self-administration studies in our laboratory (Corrigall et al., 1992, 1994). All animals were trained to a point whereby they no longer required food deprivation and would in fact obtain 25 or more reinforcements within the 1 h session. Each animal was prepared surgically with a chronic i.v. catheter implanted in the jugular vein; the catheter exited between the scapulae. Catheters were prepared on-site, and were designed to allow repeated connection/ disconnection to/from the drug delivery system in the experimental chambers. Preparation of catheters and techniques for surgery, were similar to those previously described (Corrigall et al., 1992, 1994). Briefly, surgeries were performed in aseptic conditions under anesthesia induced by xylazine (10 mg/kg, i.p.) and ketamine hydrochloride (75 mg/kg, i.p.). Buprenorphine was given for post-operative analgesia (0.01 mg/kg, s.c.), and a single dose of penicillin (30,000 units, i.m.) was administered at the completion of surgical procedures. Animals were allowed to recover for a period of 1 week before the experimental sessions for drug self-administration were begun. Animals were randomly divided based on a yoked design described previously (Donny et al., 1998, 2000). The rats were divided into three groups: nicotine self-administration (n⫽24) yoked nicotine (n⫽24), and yoked saline (n⫽16). The nicotine self-administration group received nicotine (0.03 mg/kg/infusion; dose expressed as free base) contingent upon a single active lever press (except for responding during 1 min time-out [TO] period). Animals in the yoked nicotine group (response independent nicotine) received the same number of nicotine infusions at identical times during each session as compared with their selfadministration partner. The infusions were dependent upon the self-administration partner’s responding and not upon their own lever pressing. The yoked saline group was also yoked to animals in the self-administration group, but received saline infusions instead of nicotine. Experimental sessions were conducted in standard operant chambers as described above. The nicotine selfadministration session lasted for 1 h during which time the animals

S. Rahman et al. / Neuroscience 129 (2004) 415– 424 were connected to a drug delivery swivel system that allowed unrestricted movement in the chamber. Nicotine self-administration and yoked sessions were 1 h in duration and occurred once daily Monday through Friday for 5 weeks unless otherwise stated. Self-administration was initiated on a CRF schedule with a 1-min signaled TO period following nicotine infusion. During the TO, responding was recorded but did not lead to drug delivery. Responding on one of the levers (active lever) resulted in nicotine delivery with infusions of 0.03 mg/kg/infusion delivered in 0.1 ml/kg in ⬍1 s, whereas responding on the other lever was recorded but not reinforced. This dose of nicotine produces maximum responding as previously reported (Corrigall and Coen, 1991; Donny et al., 1998, 2000). Nicotine infusions were paired with a 1-min cue light and followed by a 1-min TO period during which the chamber light was turned off and responding was recorded but not reinforced. This served to minimize toxic effects of nicotine due to rapid and repeated administration as previously described (Donny et al., 1998, 2000). In this study, the response requirement was to the value of fixed ratio 1 (FR1; i.e. one lever press was required for each drug infusion) for week 1 (4 days) and FR2 for week 2 (3 days). After this period, the response requirements were increased to the final value of FR5 for week 3 (8 days); the TO remained at 1 min. There was no limit to the number of infusions that the animals could obtain other than that imposed by the TO and the 1-h duration of the session. Additional nicotine self-administration sessions were allowed under a FR5 schedule for weeks 4 and 5 (10 days) after brain surgery until the no-net-flux or conventional microdialysis procedure (see below).

Surgery Surgeries were performed in aseptic conditions under anesthesia as described above. Animals were placed in a Kopf stereotaxic apparatus. Microdialysis guide cannulae (18 gauge; Plastics One, Roanoke, VA, USA) were implanted in the ACB according to the atlas of Paxinos and Watson (1986) using stereotaxic techniques as previously described (Rahman et al., 2000, 2002). The cannulae were implanted at a 10° angle from the midline using the following coordinates with the incisor bar set at ⫺3.3 mm: AP ⫹1.7 mm from bregma, L ⫹2.3 mm, and D/V ⫺6.3 mm. Rats were allowed to recover for 4 –5 days in their home cages following surgery, during which time they had free access to food and water but without nicotine self-administration. After the recovery period from surgery, animals were allowed to self-administer nicotine for an additional 2 weeks under the FR5 schedule (see above).

Microdialysis probe The loop-style probes were made with dialysis membrane (Spectra/ Por 6 regenerated cellulose dialysis membrane, molecular weight cutoff of 13,000; Medical Industries, Los Angeles, CA, USA) heat shrunk into PE-10 polyethylene tubing (which was fused to PE-20 tubing). Probes were made as previously described (Kohl et al., 1998; Rahman and McBride, 2000, 2002). The loop was oriented in a rostrocaudal direction and extended approximately 500 ␮m. The outside diameter of the dialysis membrane was 220 ␮m. The length of the probe was 2 mm (active dialysis membrane) extending below the guide cannula into the ACB. Similar dialysis membranes have been previously used for no net flux studies (Thielen et al., 2004; Yim and Gonzales, 2000). Loop-style probes were used instead of concentric probes because they provide consistent, higher basal levels of DA, and sample a large portion of the target area (Perry and Fuller, 1992). Comparable loop style probes were also used elsewhere to measure DA levels in the ACB (Benwell and Balfour, 1992). All microdialysis probes were inserted 18 –24 h before the no-net-flux procedure or conventional microdialysis (see below). After probe insertion, rats were placed into the microdialysis chambers (day before dialysis) for an additional 2-h habituation period.

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No-net-flux microdialysis procedure The no-net-flux microdialysis experiment and data analysis were conducted as described previously (Parsons and Justice, 1994; Smith and Justice, 1994; Smith and Weiss, 1999; Thielen et al., 2004). The no-net-flux method is a useful technique to determine quantitatively the true extracellular DA concentration and also examine the potential changes in DA uptake (Cheffer and Shippenberg, 2002; Yim and Gonzales, 2000). Thus, the no-net-flux procedure provides an in vivo estimation of basal DA release and uptake (Cheffer and Shippenberg, 2002). Accordingly, changes in DA release can be obtained from extracellular DA levels and uptake. In addition, the ‘extraction fraction’ is used to examine changes in DA uptake (Smith and Justice, 1994). As a result, higher extraction fraction would indicate higher DA uptake and would therefore tend to lower extracellular DA levels. In this study, we employed this procedure to characterize the true basal DA release and uptake. The method requires the perfusion of multiple concentrations of DA over a period of several hours after a washout period and an establishment of a baseline. Therefore, the no-net-flux method is better suited in observing neuroadaptive effects of chronic drug treatments on neurotransmitter systems (Thielen et al., 2004) in the absence of acute drug (i.e. nicotine) or behavioral (i.e. self-administration) effects. No-net-flux microdialysis started 24 or 48 h after the last nicotine self-administration session. On the day of the experiment, the rats were placed in clear Plexiglas microdialysis chambers (25⫻44⫻38 cm). ACSF (in mM: 145 NaCl, 2.7 KCl, 1.0 MgCl2, 1.2 CaCl2, 2 Na2HPO4, 0.2 ascorbate) filtered through a 0.2 ␮m sterile filter, was perfused through the probe at a flow rate of 1 ␮l/min for 2 h prior to collection of the baseline samples. After this equilibration period, three baseline samples were collected into polyethylene microfuge tubes containing 5 ␮l of 0.1 N perchloric acid every 20 min for an additional 60 min. After collection of baseline samples, probes were perfused with ACSF containing one of three DA concentrations (5, 10, or 20 nM). These concentrations were chosen because the expected extracellular concentration of DA falls within this range, allowing determination of extracellular DA concentrations from interpolation. After 20 min of perfusion with the new concentration of DA, four 20 min samples were collected at each concentration of DA. Dialysate samples were collected 20 min after switching the perfusate to allow equilibration of the perfusate. At the end of this perfusion period, the microdialysis solution was switched to ACSF containing a different concentration of DA, as described above. The order of perfusion of the DA concentrations was randomized. After the rats were perfused with all three DA concentrations, ACSF alone was perfused again and an additional three 20 min samples were collected. Samples were immediately frozen on dry ice and stored at ⫺70 °C until assayed for DA content. The entire sample was used to ensure that the 20 ␮l injection loop was completely filled.

Conventional microdialysis procedure Conventional microdialysis experiments were conducted as described previously (Rahman and McBride, 2002; Rahman et al., 2003). This dialysis procedure was also begun 24 or 48 h after the last nicotine self-administration session (see above). Microdialysis experiments were done in clear Plexiglas chambers (25⫻44⫻38 cm). ACSF (in mM: 145 NaCl, 2.7 KCl, 1.0 MgCl2, 1.2 CaCl2; pH adjusted to 7.3–7.4 with 2 mM sodium phosphate buffer; filtered through a 0.2 ␮m sterile filter) was perfused through the probe at a flow rate of 1 ␮l/min for 2 h prior to collection of the baseline samples. After this equilibration period, three baseline samples were collected into polyethylene microfuge tubes containing 5 ␮l of 0.1 N perchloric acid every 20 min for an additional 60 min. After collection of baseline samples, each rat was exposed through an i.v. catheter to a saline or nicotine (0.065 mg/kg, dissolved in saline) challenge. The total volume for saline or

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nicotine challenge was 0.1 ml and infusion time was 60 s (each delivered at a constant rate by an infusion pump). The nicotine concentration used in this study was similar to other microdialysis studies that determined the DA levels in the ACB (Fu et al., 2000). Dialysis samples were collected every 20 min for an additional 2 h after nicotine challenge. Samples were either analyzed directly or immediately frozen on dry ice and stored at ⫺70 °C for later analysis. At the end of the experiments, a 1% Bromphenol Blue solution was perfused through the probes to verify the placements. Animals were anesthetized deeply with pentobarbital and transcardially perfused with isotonic saline followed by 10% formalin. Brains were removed and fixed for subsequent sectioning to determine the location of the microdialysis probe. Probe placements were evaluated according to the atlas of Paxinos and Watson (1986). Only data from animals with correct probe placements in the ACB were used.

HPLC analysis of DA content Samples were analyzed for DA levels using an HPLC-EC (ESA Chelmsford, MA, USA) as previously described (Rahman et al., 2003). The system consists of a solvent delivery system, a Coulochem II 5200A electrochemical detector equipped with an ESA 5011 analytical cell and 5020 guard cells. The guard cell was set at 225 mV, electrode 1 at ⫺50 mV, and electrode 2 at 175 mV with the gain at 10 nA. The mobile phase was composed of 75 mM NaH2PO4, 1.7 mM 1-octanesulfonic acid, 25 ␮M EDTA, 100 ␮l/l triethylamine and 10% acetonitrile; pH 3.0 adjusted with phosphoric acid, and pumped through the system at 0.4 ml/min. Samples were loaded into a 20 ␮l sample loop and injected onto an analytical column (BetaBasic-18 column, 150 mm⫻3 mm; Keystone Scientific, PA, USA). Chromatograms were integrated, compared with standard curve constructed from analysis of the DA solutions used in this experiment, and analyzed using an ESA Chromatography DATA station.

Drugs and solutions The following agents were used: [⫺]-nicotine hydrogen tartrate and DA HCl (Sigma-Aldrich, St. Louis, USA). Nicotine was dissolved in sterile saline for nicotine self-administration. The pH was adjusted to 7.0 –7.2 for all agents prior to application. Nicotine doses are expressed as the free base.

Statistical analysis For no-net-flux studies, the extracellular DA concentrations and the DA extraction fraction (in vivo recovery) were obtained from plots of net gain or loss of DA versus concentration of DA added to perfusion media. A difference score of the concentration of DA perfused through the probe minus the concentration of DA recovered out of the probe (DAin⫺DAout) was calculated for each sample. These differences between inflow and outflow concentrations of DA were plotted against their inflow concentrations and multiple linear regression analysis was used to compare the point of no-net-flux (x-intercept, the extracellular DA concentration) and the DA extraction fraction (slope) of the different experimental groups. The significance level was set at P⬍0.05. Additional statistical analyses are included in the figure legends. For conventional microdialysis, values were not corrected for in vitro probe recovery efficiency, which is approximately 15% and in close agreement with published values (Kohl et al., 1998; Rahman and McBride, 2002; Thielen et al., 2004). Due to a significant difference in basal DA levels, the original data in nM concentrations for individual experiments were used without percentage conversion. Data were analyzed by two-way repeated measures ANOVA using SPSS (Chicago, IL, USA), followed by

post hoc Tukey HSD test for multiple comparisons unless otherwise stated. When a significant within group main effect of time was found, paired t-tests were used to compare levels at specific time points with mean basal levels. The significance level was set at P⬍0.05. The details of the statistical analysis are contained in the figure legends.

RESULTS Nicotine infusions in different sessions Five to six nicotine infusions in a given session were considered the criteria for acquisition of nicotine self-administration in this study (Corrigall et al., 1992, 1994). In fact, very few animals (one to two) failed to meet these criteria. Therefore, only those rats that acquired self-administration and had correct probe placements (see below) were included in this study. The number of infusions/session (mean⫾S.E.M.) in nicotine self-administration (n⫽20) and yoked nicotine (n⫽12) groups were 22.2⫾2 and 21.2⫾3, respectively, in weeks 1 and 2. Similarly, the total infusions/session were 19.1⫾2 and 19.2⫾3 in nicotine self-administration and yoked groups, respectively, in week 3 (before brain surgery). On the other hand, the mean number of infusions/session was 21.0⫾2 and 21⫾4 in the self-administration and yoked nicotine groups, respectively, in weeks 4 and 5 (after brain surgery for microdialysis). The average of nicotine infusions/ session was not significantly different (P⬎0.05) before and after brain surgery. Microdialysis probe placements Only data from animals that had probes correctly implanted in the ACB were included in this study. Therefore our data from nicotine self-administration (n⫽9), yoked nicotine (n⫽6) and yoked saline (n⫽6) are included in this study for final analysis for no-net-flux studies. Similarly, data from the nicotine self-administration (n⫽11), yoked nicotine (n⫽6) and yoked saline (n⫽6) groups are included in the final analysis of the conventional microdialysis studies (see below). Thus, in addition to inaccurate probe placements, i.v. catheter leaking or blockage was also responsible for reducing the rat numbers in yoked nicotine and saline groups. Fig. 1 shows representative placements in the ACB; overlapping probe placements are not shown. Therefore, this figure is qualitative distribution and not a complete quantitative representation of the distribution of probe placements. Within the ACB, almost all of the probes (active dialysis membranes) were in the medial ACB to varying degrees with portions of most probes in both shell and core. A few probes had small portions (no more than 10% of the active dialysis membrane) in the striatum and there were few probes with tips located close to the olfactory tubercle. Based upon the relative length of the active portion of the dialysis membrane, any contribution to DA levels in the dialysis sample from the striatum or olfactory tubercle is likely to be small (Kohl et al., 1998; Rahman and McBride, 2000; Thielen et al., 2004; Yim and Gonzales, 2000). Furthermore, the highly efficient uptake process for DA in the tissue surrounding the probe reduces its ability to diffuse to the probe from a

S. Rahman et al. / Neuroscience 129 (2004) 415– 424

Nicotine SA

Yoked nicotine

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Yoked saline

Core Shell Fig. 1. Representative locations of microdialysis probe placements in the ACB. Overlapping placements are not shown. Each line represents a probe placement for each of the three groups [nicotine self-administration (nicotine self-administration), yoked nicotine, and yoked saline] shown separately. The active dialysis membrane was 2 mm in length. Most probe placements in the ACB sampled both the shell and core regions. Numbers to the right indicate distance in (mm) from bregma (Paxinos and Watson, 1986).

long distance (Yim and Gonzales, 2000; Chefer and Shippenberg, 2002). Therefore, it is likely that DA detected in the dialysis sample is mainly from the ACB. Basal extracellular DA levels and extraction fractions after chronic nicotine exposure A comparison of the basal extracellular DA levels and extraction fractions of no-net-flux studies in animals tested 24 (n⫽11) and 48 (n⫽10) hours after the last nicotine self-administration session using multiple linear regression revealed no main effect of day F(1, 15)⫽0.6426, P⬎0.05 and no day⫻group interaction F(2, 15)⫽0.8849, P⬎0.05 on extracellular DA levels, and no main effect of day F(1, 15)⫽0.6443, P⬎0.05 and no day⫻group interaction F(2, 15)⫽0.8842, P⬎0.05 on extraction fraction values. Given the lack of effect of day on either the extracellular DA concentration or extraction fraction in the ACB, values from these two time points were combined for all further analyses of treatment effects. The mean (⫾S.E.M.) basal extracellular concentration and in vivo extraction fraction of DA in the yoked saline (n⫽6), yoked nicotine (n⫽6) and nicotine self-administration (n⫽9) groups were 3.7⫾0.3 nM and 77⫾4%, 1.5⫾0.2 nM and 91⫾3%, and 1.34⫾0.2 nM and 86⫾3%, respectively. For the x-intercept (extracellular DA concentration) the multiple linear regression analysis revealed a significant effect and post hoc analysis showed that the yoked saline group was significantly different from the yoked nicotine and the chronic nicotine self-administration group for both statistical comparisons (Figs. 2 and 3). There was no significant difference between the nicotine self-administration and yoked nicotine groups. For the in vivo recovery or extraction fraction, there was a

significant concentration⫻group interaction and post hoc analysis revealed differences between the yoked saline, yoked nicotine, and nicotine self-administration groups (Fig. 3). The yoked saline group was significantly different from the nicotine self-administration and yoked nicotine groups, but the nicotine self-administration group was not significantly different from the yoked nicotine group. Effects of nicotine challenge after chronic nicotine exposure Twenty-four hours to 48 h after the last nicotine self-administration (see Experimental Procedures), a saline and nicotine (0.065 mg/kg, i.v.) challenge produced an increase of extracellular DA levels in the ACB to a peak of approximately 106% and 154% of basal, respectively, in the yoked saline group. Nicotine challenge produced a slow rise in DA levels that returned to the baseline over time. Notable behavioral activation was not observed during or after nicotine challenge. The same saline and nicotine challenge produced peak increases in extracellular DA levels of approximately 105% and 112% of basal, respectively, in rats that had selfadministered nicotine and 106% and 121% of basal, respectively, in the yoked nicotine group. No significant elevation was observed in the nicotine self-administration group at any time point. The systemic administration of saline had no effect on extracellular DA levels. The average basal DA level in the yoked saline group (1.9⫾0.3) was higher than the yoked nicotine (1.0⫾0.1) and the nicotine self-administration (0.8⫾0.02) groups. There was no significant difference in basal DA levels between 24 and 48 h time points. Therefore, both time points were combined for all analysis and treatment effects.

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DA no net flux in the ACB 4

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Fig. 2. Mean (⫾S.E.M.) gain or loss of DA to or from the rat brain as a function of the perfusate DA concentration in rats that self-administered nicotine (nicotine self-administration; n⫽9), or were yoked nicotine (n⫽6) or yoked saline (n⫽6) control groups. A positive number on the y axis indicates diffusion to the brain, whereas a negative number indicates diffusion from the brain. The zero point on the y axis represents the extracellular DA concentration. The slope of the line is a measure of the in vivo recovery for DA. To correct for DA degradation during the experiment, and thus provide accurate levels of [DA]in a separate experiment was conducted in which all DA concentrations were prepared and perfused through all of the tubing up to the probe in a manner identical to the conditions of the experiment. DA aliquots were collected and immediately analyzed for DA content and the data were corrected accordingly. DA degraded approximately 24%, 27%, and 27% for the 5 nM, 10 nM, and 20 nM concentrations, respectively.

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Yoked nicotine

Nicotine SA

DISCUSSION The important findings in this study are i) the basal extracellular DA levels in the ACB are reduced after abstinence from nicotine self-administration (Figs. 2 and 3A); ii) the extracellular DA levels and in vivo extraction fractions in the yoked nicotine groups are not significantly different from the nicotine self-administration group (Figs. 2 and 3B). Similarly, a reduction in basal DA levels was also evident in the difference in baseline levels observed in the conventional microdialysis experiment (Fig. 4). The reduced basal extracellular DA levels in the self-administration and yoked nicotine groups (nicotine groups) appear to be due, at least in part, to increased clearance, suggesting increased DA uptake is occurring as a result of the chronic nicotine treatment. The lack of elevation in extracellular DA levels in the ACB in the nicotine selfadministration group upon nicotine challenge suggests that a desensitization phenomenon is occurring involving nAChRs. These findings indicate a neuroadaptive change that alters DA function and clearance in the ACB after i.v. active and passive intermittent nicotine exposure. Thus, our data suggest a pharmacological neuroadaptive effect after chronic nicotine administration on DA neurotransmission in the ACB, but does not indicate an additional effect of self-administration behavior on these parameters. Since the neurochemical values were measured 24 – 48 h after the last drug infusion, it may not be surprising that no effect of self-administration behavior was observed. It is noteworthy to mention that the effects of acute nicotine exposure during self-administration

Fig. 3. Mean (⫾S.E.M.) (A) extracellular DA concentrations and (B) in vivo extraction fractions of DA in rats in the nicotine self-administered (nicotine self-administration [SA]), yoked nicotine and yoked saline groups. For the x-intercept (no-net-flux DA levels) the multiple linear regression revealed a highly significant effect F(2, 245)⫽67.53 and post hoc analysis showed that the yoked saline group was significantly different from the yoked nicotine and the nicotine SA groups (P⬍0.0001) for both comparisons. There was no difference between the yoked nicotine and nicotine SA groups (P⫽0.2116). For the extraction fraction, the multiple linear regression model revealed a significant (concentration⫻group) interaction F(2, 243)⫽6.69, P⬍0.002) and post hoc analysis revealed differences between yoked saline and the two nicotine groups. Yoked saline was different from the nicotine SA (P⫽0.017) and yoked nicotine (P⫽0.0004) groups, but nicotine SA group was not different from the yoked nicotine group (P⫽0.1124).

were not addressed in the present study. Rather, the focus of the current study was to examine the neuroadaptive response in the mesoaccumbens DA system associated with early abstinence from chronic nicotine exposure for 25 days. In the present study, we found a marked reduction in basal extracellular DA levels in the nicotine self-administration and yoked nicotine groups, indicating that chronic nicotine alters the active processes associated with maintaining extracellular DA levels. How long this effect lasts is not known, nor is it known if a different period of exposure results in additional change of the mesoaccumbens DA system. Nevertheless, the reduction in basal extracellular DA levels in both nicotine groups observed in the present study is consistent with previous studies, where a marked reduction in extracellular DA levels in the ACB has been found after absti-

S. Rahman et al. / Neuroscience 129 (2004) 415– 424

Dialysate DA (nM)

4.0

3.5

3.0

Yoked saline Yoked nicotine Nicotine SA

2.5

2.0

1.5

1.0

---------------------------------------------------

0.5

0.0

Basal

Saline

-40 -20 0

Nicotine

20 40 60 80 100 120 140 160 180

Time(min) Fig. 4. Time course and acute effects of a saline or nicotine (0.065 mg/kg, i.v.) challenge on extracellular DA levels in the ACB of rats that had self-administered nicotine for 25 days (see Experimental Procedures) and, yoked comparison groups (yoked nicotine and yoked saline). Conventional microdialysis was carried out 24 – 48 h after the last self-administration session. On the test day, after establishment of stable baselines, the animals were perfused with a saline and a nicotine challenge as indicated by the arrows. The baseline was the mean of the three time points immediately preceding acute nicotine administration. Since basal DA levels were different in the nicotine groups versus the yoked saline group, no percentage conversion was done. Data are the mean⫾S.E.M. of six to 11 animals included in this study. The overall two-way repeated measures ANOVA revealed significant main effects of time [F(11,220)⫽12.57, P⬍0.05], and treatment [F(2,20)⫽5.98, P⬍0.05], with a time⫻treatment interaction [F(22,220)⫽3.03, P⬍0.05]. A post hoc Tukey’s HSD test revealed a significant difference (P⬍0.05) in the DA levels between the yoked saline and nicotine self-administration groups after acute nicotine challenge, but the difference did not reach significance between the yoked saline and the yoked nicotine groups (P⫽0.07). The systemic administration of saline showed no significant main effect of time [F(5,100)⫽1.873, P⬎0.05], nor a treatment by time interaction [F(10,100)⫽1.251, P⬎0.05]. The average basal extracellular level of DA in the yoked saline, yoked nicotine, and nicotine self-administration groups was 1.9⫾0.02, 1.0⫾0.1, and 0.8⫾0.03 nM, respectively.

nence from cocaine, ethanol, heroin, morphine, and amphetamine (Gerrits et al., 2002; Rossetti et al., 1992; Weiss et al., 1992, 1996). Similarly, Fung et al. (1996) observed a reduction in total tissue DA content in the ACB 24 h after terminating nicotine infusion. Thus the reduction in ACB DA levels following nicotine exposure observed in the present study may be due to, in part, decrease the responsiveness of nAChRs (see below) in the mesoaccumbens DA system (Ochoa et al., 1990). Taken together these findings support the conclusion that in addition to the important role on the rewarding effects of nicotine, the mesoaccumbens DA transmission is associated with a neuroadaptive change (Koob and Le Moal, 2001) during nicotine abstinence. Consistent with this notion, it has been suggested that cessation of prolonged nicotine administration or precipitation with a nicotine antagonist reduces extracellular DA levels in the ACB (Hilderbrand et al., 1999). Since activation of the VTA DA neurons is believed to contribute to DA release in the ACB (Balfour et al., 1998; Nisell et al., 1994), the reduced firing rates of the VTA DA neurons after abstinence from nicotine treatment may account, in part, for the reduction of overall DA output in the ACB. Taken together, it is possible that altered

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DA transmission in the mesoaccumbens DA system observed in this study is associated with nicotine abstinence. Epping-Jordan et al. (1998) demonstrated a reduction of brain reward function as measured by elevations in brain reward thresholds during nicotine abstinence from chronic nicotine infusion. Additionally, the influence of nicotine dose and pattern of nicotine exposure also influences the severity of nicotine abstinence (Skjei and Markou, 2003). In the present study, we have taken neurochemical measurements 24 – 48 h after the last nicotine (intermittent exposure) infusion. Although basal DA levels were not significantly different between these two time points (see results), additional studies are necessary to determine the effects in earlier time periods [i.e. 2–12 h of nicotine abstinence (Epping-Jordan et al., 1998)] on basal DA levels in the ACB as well as acute effects of nicotine on varying time points during abstinence. It is not known whether abstinence in general or physical aspects of nicotine abstinence (Malin et al., 1992) is associated with altered DA transmission in the ACB. However, there are no data supporting a clear role of the mesoaccumbens DA system in the mediation of the somatic nicotine abstinence syndrome (see Kenny and Markou, 2001). Moreover, based on the effects of opiates on mesoaccumbal DA activity (Diana et al., 1999), it is suggested that the reduced DA output in the ACB observed during abstinence is likely related to reward deficits, but not somatic signs of abstinence (Kenny and Markou, 2001). Another factor that could have influenced the current data is the possibility of anticipation of reward in the rats receiving nicotine. In this regard, it is not likely a major factor influencing the observed DA levels for the following reasons. i) The self-administration boxes and the dialysis chambers are very different apparatuses; the self-administration chambers are enclosed in sound-attenuated chambers whereas the dialysis chambers are clear and open air chambers, so none of the cues (levers, lights) available in the self-administration chambers that predict nicotine administration are available in the dialysis chambers. ii) The rats were in the dialysis chambers for 2 h prior to the first baseline sample collection, so any effect of anticipation resulting from the movement from the home cage to the dialysis chambers would likely have dissipated by the time sampling began for DA. Thus it is likely that the pharmacological effects of nicotine rather than the anticipation of nicotine are responsible for the observed effects on the ACB DA system. It is well known that once released, DA is immediately removed from the synaptic cleft by a high affinity DA uptake mechanism. Accordingly, DA uptake by means of the DA transporter is the major clearance mechanism for synaptic DA in the ACB (Jones et al., 1996). The in vivo extraction fraction in no-net-flux microdialysis experiments depends predominantly on changes in reuptake mechanisms and not on neuronal release or metabolism (Smith and Justice, 1994; Olson-Cosford et al., 1996). Therefore, higher rates of neurotransmitter uptake may be responsible, in part, for the lower levels of extracellular neurotransmitter. In the present study, we report a significant increase in the in vivo DA extraction fraction (in vivo DA recovery) in both nicotine groups (Fig. 3B), suggesting an increase in

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DA uptake in the ACB after nicotine treatment. Similar effects of systemic nicotine have been reported elsewhere on DA clearance in the ACB (Hart and Ksir, 1996), suggesting nAChRs in the mesoaccumbens DA system modulate DA transporter function. Thus, future studies are necessary to examine the in vivo DA transporter function in the ACB after chronic nicotine exposure. Based on the probe locations (see Fig. 1), it is important to indicate that no distinction can be made between the core and shell regions of the ACB with the probes used in the present study. Recently, several reports have indicated a difference in DA transmission in the core versus shell region of the ACB with respect to innervation patterns (Heimer et al., 1991; Zahm, 1999), DA transporter density (Jones et al., 1996), pharmacological characteristics (Deutch and Cameron, 1992) and sensitivity of drugs of abuse (David et al., 1998). Moreover, studies with acute i.v. administration of the drug suggest that nicotine produces greater increases in extracellular DA levels in the shell than in the core region of the ACB (Pontieri et al., 1996). It is not surprising with our current mixed probe placements that we were unable to distinguish between the core and shell subregion of the ACB. Using smaller and/or concentric microdialysis probes, future studies should focus for a possible difference in DA regulation in the core versus shell of the ACB associated with chronic nicotine exposure. In the present study, we found that an acute i.v. nicotine challenge produces an increase in extracellular DA levels in the ACB of yoked saline rats (Fig. 4). The acute nicotineinduced enhancement in extracellular DA levels is consistent with previous data in nicotine naive animals (Fu et al., 2000). However, our nicotine challenge produced little or no increase in the yoked nicotine and self-administration groups, respectively (Fig. 4), suggesting a desensitization phenomenon is occurring in the mesoaccumbens DA transmission. It is widely established that nAChRs are found at several levels in the mesoaccumbens DA system. However, as reported (see introduction), nAChRs found in the VTA are critical in mediating the reinforcing effects of nicotine obtained by self-administration (Corrigall et al., 1994) and therefore, activation of nicotinic receptors in the VTA is a trigger for DA release in the ACB (Nisell et al., 1994). However, several studies have indicated that chronic experimenter-administered nicotine exposure causes desensitization of nAChRs in the VTA (Marks et al., 1985; Schwartz and Kellar, 1985; Pidoplichko et al., 1997), which in turn reduces DA release in the ACB (Benwell et al., 1995). This is particularly important because there is evidence that the nAChRs, through which nicotine evokes this effect, are desensitized after exposure to the drug at concentrations lower than those found in the plasma of rats that have self-administered nicotine for 1 h (Benwell et al., 1995; Shoaib and Stolerman, 1999). Taken together, these data suggest that desensitization may occur in both nicotine groups due to chronic nicotine exposure, which likely plays a role in reducing the extracellular DA levels in the ACB seen in the present study. Although it remains to be determined how the receptor desensitization phenomenon is associated with the underlying molecular mechanisms of neuroadaptive change, i.e. tolerance (Ochoa et al., 1990) seen in this study.

It is possible that nicotine-induced desensitization of DA release could be due to changes in phosphorylation of specific synaptic-vesicle proteins involved in the exocytotic process leading to DA release (see Ochoa et al., 1990). Thus desensitization of nAChRs in the mesoaccumbens DA system may be responsible for the observed neuroadaptive change. Consistent with this idea, it has been suggested that prolonged nicotine exposure to nicotine may result in a reduced Ca2⫹ influx, thereby promoting the dephosphorylated state of nicotinic receptors (Fenster et al., 1999; Ochoa and O’Shea, 1994). In summary, using a quantitative microdialysis procedure with the nicotine self-administration paradigm, the present report provides clear evidence for reduced basal extracellular DA levels in the ACB accompanied by an increase in DA uptake (as indicated by extraction fraction) during early abstinence from chronic nicotine self-administration. In addition, using conventional microdialysis, our data suggest that upon nicotine challenge, nAChRs in the mesoaccumbens DA system undergo desensitization during early abstinence from chronic nicotine self-administration. Taken together, these data provide new information about neuroadaptive changes in the mesoaccumbens DA neurotransmission during early abstinence from chronic i.v. nicotine exposure. The marked reduction in extracellular DA levels in the ACB may play a role in the maintenance of nicotine self-administration as stated in a recent proposed theory of allostasis (Koob and Le Moal, 2001). Since neuroadaptations can manifest as classic symptoms of drug addiction, including nicotine-taking behavior, these neuroadaptive changes observed in mesoaccumbens DA transmission may play an important role in the maintenance of nicotine self-administration and/or craving. Furthermore, these data also have broader implications for understanding the neurochemical basis of drug use and abuse in humans. Acknowledgments—The authors thank Dr. William J. McBride for his advice and critical comments on the manuscript and Dr. Richard J. Thielen for help in statistical analysis. This research was supported by NIH/NIDA grant DA 09577.

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(Accepted 13 August 2004) (Available online 29 September 2004)