Sensitization to nicotine significantly decreases expression of GABA transporter GAT-1 in the medial prefrontal cortex

Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 1521–1526

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Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p b p

Sensitization to nicotine significantly decreases expression of GABA transporter GAT-1 in the medial prefrontal cortex Chris Pickering a, Veronica Bergenheim b, Helgi B. Schiöth b, Mia Ericson a,⁎ a b

University of Gothenburg, Institute of Neuroscience and Physiology, Section of Psychiatry and Neurochemistry, PO Box 410, SE-40530 Göteborg, Sweden Uppsala University, Department of Neuroscience, Functional Pharmacology, Box 593, BMC SE-75124 Uppsala, Sweden

A R T I C L E

I N F O

Article history: Received 19 March 2008 Received in revised form 13 May 2008 Accepted 13 May 2008 Available online 20 May 2008 Keywords: GABAA α1 GAT-3 Locomotor activity Schizophrenia SLC6A1 SLC6A11

A B S T R A C T This study investigated GABA signaling following induction of behavioural sensitization to nicotine. Rats were repeatedly injected with saline, nicotine or hexamethonium for 18 days and gene expression was measured with qPCR. Nicotine upregulated GABAA α1 subunit expression in the nucleus accumbens (p b 0.05) while no changes were observed for GABAA α3, α4 or α5. In the medial prefrontal cortex, no change in expression of the GABAA subunits was observed. We found that nicotine significantly decreased expression of the transporter GAT-1/SLC6A1 (p b 0.05) in the medial prefrontal cortex while the expression of the GAT-3/ SLC6A11 (p b 0.05) transporter was increased in the nucleus accumbens. This provides the first evidence of neuroadaptive changes in the GABA system after nicotine sensitization and the first demonstration of an effect on GAT-1 or GAT-3 transporters in the addiction field. The GAT-1 findings also provide evidence for an alternative theory of why most schizophrenic individuals also use tobacco products. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Nicotine and other drugs of abuse activate the mesocorticolimbic dopamine system which originates in the ventral tegmental area (VTA) and projects mainly to the nucleus accumbens (NAc) and prefrontal cortex (PFC) (Koob, 1992). Acute activation of post-synaptic dopamine receptors in the mesolimbic system stimulates locomotor activity in experimental animals while chronic, repeated administration of dependence-producing drugs often results in a progressive enhancement of drug-induced locomotor response (Pierce and Kalivas, 1997). This phenomenon, known as behavioural sensitization, is considered a behavioural marker of persistent underlying pre- and post-synaptic alterations in the mesolimbic dopamine system (Nestler, 1994). In this study, we provide the first evidence for changes in mRNA expression after induction of nicotine sensitization and can, therefore, support the theory that persistent changes in neurotransmission underlie behavioural sensitization.

Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, BLAST, Basic local alignment search tool; GABA, gamma-aminobutyric acid; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GAT, GABA transporter; H3b, histone H3b; nAChR, nicotinic acetylcholine receptor; NMDA, N-methyl-D-aspartate; qPCR, quantitative polymerase chain reaction; RPL19, ribosomal protein L19; SDHA, succinate dehydrogenase complex A subunit A, SLC, solute carrier. ⁎ Corresponding author. Institute of Neuroscience and Physiology, Section of Psychiatry and Neurochemistry, PO Box 410, SE-40530 Göteborg, Sweden. 0278-5846/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2008.05.011

Pharmacological studies indicate a role of the excitatory glutamatergic system in the induction of behavioural sensitization (Vanderschuren and Kalivas, 2000). Firstly, infusion of either the AMPA antagonist CNQX or the NMDA antagonist AP-5 into the VTA blocks nicotine-induced dopamine release in the NAc (Schilstrom et al., 1998). Additionally, coadministration of cocaine with NMDA antagonists MK-801 (Karler et al., 1994; Karler et al., 1989) or CGS-19755 (Li et al., 1999) blocks the induction of sensitization to cocaine. Development of sensitization to nicotine is also blocked via administration of NMDA antagonist MK-801 (Hong et al., 2006; Shim et al., 2002). A further discussion of the role of dopamine and glutamate in the development of sensitization to nicotine is provided in Balfour et al. (1998). Clearly, excitatory neurotransmitters are involved in progression from acute nicotine response to the sensitized state. Little, however, has been studied with respect to GABA, the major inhibitory neurotransmitter of the brain. Two electrophysiological studies have, however, connected nicotine with the GABA system. Administration of nicotinic agonists facilitate the release of GABA in relay neurons from the thalamus (Lena and Changeux, 1997; Lena et al., 1993). Secondly, medium spiny neurons from the dorsal striatum of nicotine-treated mice invoke larger inhibitory potentials compared to saline-treated mice and this effect is mediated by GABAA receptors (Miura et al., 2006). However, the role of GABA in the development of sensitization to nicotine has not been studied. We have previously used the in vivo microdialysis technique to investigate the role of GABA in the effects of ethanol with focus on the nucleus accumbens (Lof et al., 2007a,b). Ethanol-induced dopamine

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release in the nucleus accumbens is prolonged by accumbal infusion of the GABAA antagonist picrotoxin, suggesting a role for GABA in the secondary or declining phase of dopamine release (Lof et al., 2007b). Perfusion of diazepam into the nucleus accumbens decreases basal dopamine levels in this same region but this effect can be reversed by repeated nicotine exposure (sensitization) (Lof et al., 2007a), again suggesting a role of GABAA receptors in the response to nicotine. Based on these findings, we hypothesized that repeated exposure to nicotine and the subsequent induction of behavioural sensitization could persistently alter gene expression of GABAA receptor subunits in key target regions of the mesocorticolimbic dopamine system; namely the nucleus accumbens, prefrontal cortex, medial prefrontal cortex and the caudate putamen. The only previous study of this type is a microarray analysis of gene expression in the mesencephalon of mice after nicotine sensitization in which a significant change in mRNA expression of GABAA receptor subunits δ, α1, α3 and β3 was observed (Saito et al., 2005). We have therefore investigated expression of GABAA α1, α3, α4 and α5 and also the GABA transporters GAT-1 and GAT-3 using a well-validated qPCR method with multiple reference genes (Vandesompele et al., 2002). Changes in GABA transporters are important to provide evidence of alterations in synaptic neurotransmitter release. Additionally, changes in these transporters have not been investigated previously with respect to acute or chronic exposure of any drug of abuse.

(130 mm above the floor level), allowing a computer-based system to register the animal's horizontal and vertical activity, respectively. Activity was defined as interruption of a photocell beam. The software allowed a detailed analysis. Animals subchronically treated with either nicotine or vehicle were placed in the activity boxes for 30 min of habituation after which they were removed, injected with nicotine 1 mg/kg s.c. and put back into the box for another 30 min. In the activity boxes, three variables were recorded: locomotor activity (one count=one breaking of a new beam compared with the previous beam that was broken), corner time (time during which two outer edge beams were broken at the same time) and rearing activity (one count=one breaking of one beam in the upper row). Rearing activity reflects the vertical activity of the rat. 2.4. Neurochemical analysis 2.4.1. Tissue collection Immediately after behavioural testing, the animals were decapitated and brains were dissected according to Heffner et al. (1980) using a brain matrix. The specific areas of prefrontal cortex, medial prefrontal cortex, caudate putamen and nucleus accumbens were analyzed. All samples were frozen on a dry ice block before immersion into RNALater solution (Ambion). Following 1 h of incubation at room temperature to allow complete penetration of the tissue, samples were stored at −20 °C until further processing (within 2 months).

2. Methods 2.1. Animals All experiments were approved by the local ethical committee in Gothenburg (Diary# 337/06). Thirty naïve male Wistar rats (Scanbur BK AB, Sollentuna, Sweden) weighing approximately 270 g at the start of the experiment were group-housed (4 per cage) in a climatecontrolled facility (12 h light/dark cycle with lights on at 07.00) with constant temperature (22–23 °C) and 55% humidity. Animals were given 7 days to acclimatize to the local conditions and had access to water and food ad libitum. 2.2. Nicotine sensitization Thirty animals were randomly assigned into 3 treatment groups; saline, nicotine or hexamethonium and were injected daily for 18 days in their homecages according to their assigned treatment group; saline (0.9% NaCl s.c.), nicotine (tartrate salt) (1 mg/kg s.c.), hexamethonium (10 mg/kg i.p.) all from Sigma-Aldrich (Sweden). The different treatment groups were not separated in different home cages but all treatment groups were represented in each home cage. The peripherally-active nicotinic antagonist hexamethonium was included in order to control for possible peripheral actions of nicotine which secondarily affect the brain. In a previous study, we demonstrated that nicotine-induced elevation of ethanol intake can also be mediated via functional blockade of peripheral nAChRs, thus demonstrating the induction of central effects via a peripherally acting agent (Ericson et al., 2000). 2.3. Locomotor activity measurement To demonstrate sensitization to the locomotor stimulatory effects of nicotine, a challenge dose of nicotine was administered to animals subchronically exposed to nicotine or vehicle on day 19 of the experiment. Activity level was measured using photocell detection in eight identical Plexiglas boxes (700 × 700 × 350 mm), which were placed in sound-attenuating enclosures with dim lighting (Kungsbacka Mät-och Reglerteknik, Fjärås, Sweden). The activity meter was equipped with 2 rows of photocell beams, and detectors were placed 100 mm apart at the floor level of the box and at a higher level

2.4.2. RNA extraction and cDNA synthesis Tissue samples were homogenized in 1 ml of TRIzol reagent (Invitrogen, Sweden) using a Branson sonicator and total RNA was extracted according to the manufacturer's protocol (Invitrogen, Sweden). To remove contamination, samples were treated with DNase I (Roche Diagnostics, Sweden) at 37 °C for 3 h followed by 15 min at 75 °C to inactivate the enzyme. To confirm that samples were not contaminated with DNA, a PCR was performed on the RNA sample using reference gene GAPDH as a positive control. If bands were detected after this PCR, DNase I treatment was repeated until no amplification was observed in the RNA. Concentration of RNA was determined with the Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies, Delaware, USA) and cDNA was then synthesized according to the manufacturer's protocol with M-MLV reverse transcriptase (GE Healthcare, Sweden) and random hexamers (GE Healthcare, Sweden). To ensure presence of cDNA in the samples, a PCR was run (again with GAPDH as positive control) but this time a band was expected for each sample. 2.4.3. Primer design Primers were designed using Beacon Designer v4.0 (Premier Biosoft, USA) with parameters of 18–22 nucleotides in length, 70–100 base pair product size and melting point of 55–60 °C. Many of these primers were used in our previous study (Pickering et al., 2007). Specificity was verified via a BLAST search against the rat genome and annealing temperatures of all primers were optimized prior to use. Sequences for references genes were as follows (F denotes forward, R reverse): GAPDH F-acatgccgcctggagaaacct, R-gcccaggatgccctttagtgg; β-actin F-cactgccgcatcctcttcct, R-aacgctcattgccgatagtg; SDHA F-gggagtgccgtggtgtcattg, Rttcgcccatagcccccagtag; RPL19 F-tcgccaatgccaactctcatc, R-agcccgggaatggacagtcac; H3b F-attcgcaagctcccctttcag, R-tggaagcgcaggtctgttttg; β-tubulin F-cggaaggaggcggagagc, R-agggtgcccatgccagagc. Sequences for GABA primers were: GABAA α1 F-tgccagaaattccctcccaaag, R-cagagccgagaacacgaagg; GABAA α3 F-tgctgagaccaagacctacaac, R-tggcaaagagcacagggaag; GABAA α4 F-gcatcttgagaggctggaaacg, R-aggagggcgaggctgacc; GABAA α5 F-caagtctgtggtggtggc, R-tgctggtgctgatgttctc; GAT-1/SLC6A1 F-cagaccagtgcggagagg, R-ataggctgtttgctgttaatgc; GAT-3/SLC6A11 F-gaccgttaatgactgtgagg, R-ggaaggaaggctggagac. Nicotinic receptor primers were: nAChR α4 F-ctcctgtcctccacccaag, R-atgccatcttctgctgcttc; nAChR α7 Fctgctctacattggcttcc, R-aggtgctcatcatgtgttg.

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2.4.4. qPCR Quantitative PCR was performed in 96-well plates with the MyIQ iCycler real-time detection instrument (Bio-Rad, Sundbyberg, Sweden). SYBR Green I (Invitrogen) dissolved in Tris-EDTA (pH 8) was used as the fluorescent reporter. The 20 µl reaction volume consisted of 2 µl 10× PCR buffer, 20 mM dNTP, 50 mM MgCl2, 0.05 µl forward and reverse primer (100 pmol/µl), 1 µl DMSO, 1:50,000 SYBR Green I, 0.08 µl (5 units/µl) Taq DNA polymerase (Biotools, Spain) and 9.52 µl RNase-free water mixed with 5 µl (5 ng/µl) of cDNA template. For each primer pair, 50 PCR cycles were run with parameters of 95 °C for 15 s, 52–62 °C (depending on primer) annealing for 30 s followed by extension at 72 °C for 30 s. A melting curve analysis was performed after the cycling was completed to ensure that a single PCR product was formed in the reaction. 2.4.5. Normalization and data analysis Data was obtained using the MyIQ software (Bio-Rad) and a normalization factor was calculated using 6 reference genes (β-actin, β-tubulin, cyclophilin, GAPDH, histone H3b, ribosomal protein L19) and the GeNorm method (Vandesompele et al., 2002) as previously used (Pickering et al., 2007). The assumption-free analysis method of

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Table 1 Changes in nicotinic receptor subunits after repeated nicotine or hexamethonium treatment 1-way ANOVA

Bonferroni post-hoc

F

p

Saline vs nicotine

Saline vs hexamethonium

Nucleus accumbens nAChR α4 0.027 nAChR α7 0.31

0.97 0.73

– –

– –

Medial prefrontal cortex nAChR α4 0.0064

0.99

–

–

Prefrontal cortex nAChR α4 0.54 nAChR α7 0.17

0.59 0.85

– –

– –

Caudate putamen nAChR α4 0.15 nAChR α7 0.44

0.86 0.65

– –

– –

Ramakers et al. (2003) was used to calculate primer efficiency and the LinRegPCR method was used to calculate corrected Ct values. To calculate the expression value, the corrected Ct value was divided by the normalization factor obtained for that tissue from GeNorm. Finally, each value was normalized to the mean of the saline group such that all values are relative to this (indicated as % of saline in the figures). To compare expression, a 1-way ANOVA was performed using GraphPad Prism v4 (Graph Pad, US). If the overall ANOVA was significant, a Bonferroni post-hoc test was performed to compare saline vs nicotine and saline vs hexamethonium. The significance level was set to p = 0.05 for all experiments. Locomotor activity data was compared with a 2-way ANOVA with factors Time and Drug. If the overall ANOVA was significant (Factor Drug, in the case of this study), a Bonferroni post-hoc test was used to determine significance at a given time point.

Table 2 Changes in GABA subunits or transporters after repeated nicotine or hexamethonium treatment

Fig. 1. Locomotor activity after repeated injections of saline or nicotine (n = 10 animals per group). A) Nicotine increased locomotor activity. B) Nicotine increased rearing activity. C) Nicotine did not affect corner time.

1-way ANOVA

Bonferroni post-hoc

F

p

Saline vs nicotine

Saline vs hexamethonium

Nucleus accumbens GABAA α1 4.19 GABAA α3 2.77 GABAA α4 0.63 GABAA α5 0.57 GAT-1 1.47 GAT-3 3.41

0.027 0.081 0.54 0.57 0.25 0.048

*p b 0.05 – – – – *p b 0.05

p N 0.05 – – – – p N 0.05

Medial prefrontal cortex GABAA α1 0.077 GABAA α3 2.33 GABAA α4 1.10 GABAA α5 0.39 GAT-1 4.69 GAT-3 0.49

0.93 0.12 0.35 0.68 0.018 0.62

– – – – *p b 0.05 –

– – – – *p b 0.05 –

Prefrontal cortex GABAA α1 2.20 GABAA α3 1.59 GABAA α4 1.27 GABAA α5 0.042 GAT-1 0.12 GAT-3 0.51

0.13 0.22 0.30 0.96 0.89 0.61

– – – – – –

– – – – – –

Caudate putamen GABAA α1 0.21 GABAA α3 1.53 GABAA α4 1.01 GABAA α5 0.041 GAT-1 1.93 GAT-3 0.58

0.81 0.23 0.38 0.96 0.17 0.57

– – – – – –

– – – – – –

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3. Results

3.3. Effects of nicotine and hexamethonium on nicotinic receptors

3.1. Nicotine sensitization

To test whether repeated nicotine or hexamethonium treatment altered gene expression of nicotinic receptor subunits, qPCR was performed. Results of the 1-way ANOVA analysis (F and p values) are indicated in Table 1 for each region and subunit. No differences in mRNA expression were found for nAChR α4 or α7 subunits in the nucleus accumbens, medial prefrontal cortex, prefrontal cortex or the caudate putamen.

Repeated nicotine injection induced locomotor sensitization in rats when compared to saline-treated control animals (n = 10; Fig. 1). Locomotor Activity (Fig. 1A) was significantly increased after nicotine (Factor Drug: F(1,5) = 29.92; p b 0.0001). Post-hoc analysis found a significant difference 5 min (p b 0.001) and 25 min (p b 0.001) after nicotine and the sum of the total activity after nicotine is provided in the inset of Fig. 1A. Rearing Activity (Fig. 1B) was also significantly increased after nicotine (Factor Drug: F(1,5) = 61.83; p b 0.0001) with post-hoc differences at 5 (p b 0.001), 25 (p b 0.001) and 30 min (p b 0.01). Corner Time (Fig. 1C) did not differ between saline and nicotine-treated animals (Factor Drug: F(1,5) = 0.294; p = 0.59). 3.2. GeNorm analysis of reference genes Six reference genes were analyzed by qPCR for each brain region and the GeNorm method was applied to determine the optimal number of reference genes to achieve a valid estimation of gene expression. For the nucleus accumbens, prefrontal cortex and medial prefrontal cortex, the best estimate was observed with 6 reference genes. Caudate putamen expression was optimal upon inclusion of 5 reference genes.

3.4. Effects of nicotine and hexamethonium on the GABAergic system Gene expression within the GABAergic system was compared for the four brain regions following saline, nicotine or hexamethonium treatment and the results of the ANOVA analyses (F and p values) are indicated in Table 2. For both the prefrontal cortex and caudate putamen, no differences were observed for GABAA subunits α1, α3, α4 or α5 subunits or the two GABA transporters GAT-1 and GAT-3. Repeated exposure to nicotine changed GABAA receptor subunit expression in the nucleus accumbens (Fig. 2A). Nicotine-treated animals had elevated GABAA α1 expression compared to both saline and hexamethonium groups (p b 0.05) but no differences in GABAA α3, α4, or α5. With respect to GABA transporters, no changes were observed for GAT-1 but GAT-3 mRNA expression was significantly increased after nicotine treatment (p b 0.05).

Fig. 2. GABAergic gene expression after saline, nicotine or hexamethonium treatment (n = 10 all groups). A) GABAA channel subunits α1, and GABA transporters GAT-1 and GAT-3 in the nucleus accumbens. B) GABAA channel subunits α1 and GABA transporters GAT-1 and GAT-3 in the medial prefrontal cortex.

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In the medial prefrontal cortex (Fig. 2B), no nicotine or hexamethonium-induced changes in GABAA α1, α3, α4, or α5 receptor subunit mRNA expression were observed. However, mRNA expression of GABA transporter GAT-1 was significantly decreased after nicotine (p b 0.05) or hexamethonium (p b 0.05) compared to saline-treated animals. 4. Discussion Repeated injection of nicotine over 18 consecutive days changed gene expression in the GABAergic system and this suggests a possible role of adaptation in GABA release as a consequence of behavioural sensitization to nicotine. This study also represents the first report of a change in GAT-1 (medial prefrontal cortex) or GAT-3 (nucleus accumbens) mRNA expression after exposure to a drug of abuse. Our current results indicate a compensatory upregulation of GABAA receptor subunit α1 in the nucleus accumbens after nicotine treatment, an effect not observed for hexamethonium. This change could be secondary to the increase in GAT-3 which would increase reuptake of GABA from the synapse, thus producing a similar net effect as reduction in GABA release that we have discussed previously (Lof et al., 2007a). Both GAT-1 and GAT-3 GABA transporters are thought to regulate the tonic inhibition of pyramidal neurons in the cortex (Kinney, 2005) and changes in the expression of these transporters have been observed in several studies. For example, induction of ischemia increases GAT-3 expression in the cortex of rats (Melone et al., 2003). A clinical study of patients with schizophrenia observed a significantly lower expression of GAT-1 mRNA in the prefrontal cortex compared to healthy controls (Volk et al., 2001) and a second study also reports decreases in GAT-1 (Schleimer et al., 2004). Further investigation of these changes showed that about 25% of chandelier neurons (which project in synapses to pyramidal neurons) have undetectable levels of both GAT-1 and GAD67 (Volk and Lewis, 2002). Interestingly, the smoking rate among schizophrenic patients is higher than that observed for healthy subjects (Kumari and Postma, 2005) and this observation has led to the ‘self-medication’ hypothesis of schizophrenia. This hypothesis suggests a use of nicotine by patients in order to stimulate dopamine release in the frontal cortex to counteract anhedonia or use to alleviate negative symptoms induced by antipsychotic treatment (Kumari and Postma, 2005). In our study, however, decreased GAT-1 expression in the prefrontal cortex was a persistent consequence of nicotine sensitization. This similar expression pattern in schizophrenic patients could therefore offer a new perspective on the frequent tobacco use in this group. The downregulation of GAT-1 may mimic a nicotine-dependent brain state with respect to GABAergic neurotransmission which could ultimately motivate craving for nicotine or drug-seeking behaviour. While it is too early to say whether this theory is true, it does suggest a need for further investigation of the GAT-1 and GAT-3 transporters. In particular, studies of patients with drug dependence, depression and other psychiatric conditions could help to understand elevated prevalence of smoking among these populations. A change in mRNA expression was only observed for the GABAA α1 subunit but not α3, α4 or α5 after nicotine treatment. The α1 subunit is abundantly expressed throughout the brain (Wisden et al., 1992) and is important for GABAergic neurotransmission in general (Ortinski et al., 2006). The α1 subunit is also thought to mediate the sedative component of benzodiazepines (Crestani et al., 2002; Rudolph et al., 1999). By changing expression of the most abundant GABAA receptor, we can speculate that nicotine-induced sensitization significantly affects the GABAergic system. Additionally, hexamethonium did not change expression of the GABAA α1 subunit while this peripherally acting drug did affect GAT-1 and GAT-3 expression. Since both nicotine and hexamethonium previously were demonstrated to exert behavioural effects interpreted to be the result of rapid desensitization of peripheral nAChRs with central consequences (Ericson et al., 2000), our findings suggest that change in the transport of GABA could be an

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important marker of the central response to the peripheral actions of the drugs and the change in GABAA subunit could be a marker of specific effects only observed by nicotine acting centrally. In line with previous studies (Buisson and Bertrand, 2002; Fenster et al., 1999a) no change in nicotinic receptor subunit α4 or α7 expression was observed after repeated nicotine, which served to validate our qPCR method for determination of nicotine-specific effects. Nicotinic receptors rapidly desensitize upon agonist exposure and chronic smokers have an upregulation of nicotinic binding sites compared to controls (Perry et al., 1999). This upregulation, however, does not involve transcriptional changes in mRNA since the same upregulation is observed when receptors are expressed in Xenopus oocytes (Fenster et al., 1999b), a model system lacking ability to transcriptionally regulate these genes. In fact, upregulation of nicotinic receptors was also intact after blockade of protein synthesis by cycloheximide (Buisson and Bertrand, 2001), thus indicating upregulation via conformational change to a high-conductance form of channel or the transport of high-conductance channels from intracellular stores (Buisson and Bertrand, 2002). Locomotor sensitization to nicotine (Fig. 1) in this study was modest but was specific to nicotine since animals injected with saline for 18 days prior to the challenge dose of nicotine on day 19 moved significantly less than nicotine-treated animals. The increase in rearing activity just prior to nicotine injection (Fig. 1B) is likely due to anticipation or some type of drug-seeking behaviour. Nicotineconditioned locomotor activity develops shortly after behavioural sensitization (Kosowski and Liljequist, 2005) and both are ensured by the prolonged, 18-day treatment. Corner time, however, did not differ between groups so no conclusions can be made regarding time in the centre of the open field or possible anxiolytic-like effects of nicotine. 5. Conclusions In conclusion, we have provided evidence for nicotine-induced increases in GABAA α1 subunit and GAT-3 transporter expression in the nucleus accumbens and decreases in GAT-1 transporter expression in the medial prefrontal cortex. These changes in the mesolimbic system provide the first markers of a persistent neurochemical change after induction of behavioural sensitization to nicotine. This study also suggests that regulation of GABA transporters may explain the heavy use of tobacco in schizophrenic patients. It is also possible that GABA transporters may be involved in the self-administration of other abused drugs such as alcohol. Acknowledgments The authors wish to thank the Council for Medical Tobacco Research — Swedish Match, the Swedish Medical Research Council (Dnr 2006-4988 and 2006-6385) the Swedish Labor Market Insurance (AFA) support for biomedical alcohol research, governmental LUA/ALF support, the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly and Gunnar och Märta Bergendahls Stiftelse. References Balfour DJ, Benwell ME, Birrell CE, Kelly RJ, Al-Aloul M. Sensitization of the mesoaccumbens dopamine response to nicotine. Pharmacol Biochem Behav 1998;59:1021–30. Buisson B, Bertrand D. Chronic exposure to nicotine upregulates the human (alpha)4 ((beta)2 nicotinic acetylcholine receptor function. J Neurosci 2001;21:1819–29. Buisson B, Bertrand D. Nicotine addiction: the possible role of functional upregulation. Trends Pharmacol Sci 2002;23:130–6. Crestani F, Assandri R, Tauber M, Martin JR, Rudolph U. Contribution of the alpha1-GABA (A) receptor subtype to the pharmacological actions of benzodiazepine site inverse agonists. Neuropharmacology 2002;43:679–84. Ericson M, Engel JA, Soderpalm B. Peripheral involvement in nicotine-induced enhancement of ethanol intake. Alcohol 2000;21:37–47. Fenster CP, Hicks JH, Beckman ML, Covernton PJ, Quick MW, Lester RA. Desensitization of nicotinic receptors in the central nervous system. Ann N Y Acad Sci 1999a;868:620–3.

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