- Email: [email protected]
Reduced G-protein coupling to the GABAB receptor in the nucleus accumbens and the medial prefrontal cortex of the rat after chronic treatment with nicotine
Neuroscience Letters 355 (2004) 161–164 www.elsevier.com/locate/neulet
Reduced G-protein coupling to the GABAB receptor in the nucleus accumbens and the medial prefrontal cortex of the rat after chronic treatment with nicotine Diana Amanteaa,*, Michela Tessarib, Norman G. Bowerya a
Department of Pharmacology, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Drug Dependence Behav. Neurochem., Psychiatry-CEDD, GlaxoSmithKline S.p.A., 37135 Verona, Italy Received 23 June 2003; received in revised form 20 October 2003; accepted 20 October 2003
Abstract The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABAB receptor density, affinity and G-protein coupling was investigated in the mesocorticolimbic system of the rat brain. Baclofen-stimulated [35S]GTPgS binding autoradiography revealed that the level of G-protein coupling to GABAB receptors was significantly reduced in the medial prefrontal cortex and the nucleus accumbens of nicotine-treated rats as compared to vehicle-injected controls. By contrast, GABAB receptor density and affinity, as revealed by [3H]GABA saturation binding autoradiography, were not altered by the nicotine exposure in any of the regions examined. Reduced Gprotein coupling to the GABAB receptor may result in disinhibition of mesocorticolimbic dopaminergic neurones, which would contribute to the development of sensitised dopaminergic responses to repeated administration of nicotine. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: GABAB receptor; Nicotine; Drug addiction; Mesocorticolimbic system; G-protein; [35S]GTPgS
Nicotine stimulant and reinforcing effects in rats are primarily mediated by activation of the mesocorticolimbic dopaminergic system of the brain [3,4]. This neuronal pathway consists of dopaminergic neurones projecting from the ventral tegmental area (VTA), located in the midbrain, to forebrain regions, including the medial prefrontal cortex (mPfCx) and the nucleus accumbens core (NAcC) and shell (NAcS) subdivisions. Activation of VTA nicotinic receptors and the resulting release of dopamine (DA) within the mesocorticolimbic terminal regions underlies most of the behavioural effects exerted by nicotine [4,7]. A growing number of preclinical and clinical studies provide evidence for the effectiveness of GABAB receptor agonists in reducing addictive behaviours associated with alcohol and psychostimulant abuse [6], including nicotine self-administration in rats [5]. GABAB receptors are widely distributed throughout the mesocorticolimbic system [2] and their activation may therefore interfere with the mechanisms involved in nicotine * Corresponding author. Tel.: þ44-121-414-4525; fax: þ 44-121-4144509. E-mail address: [email protected] (D. Amantea).
dependence. Interestingly, in naı¨ve rats, GABAB receptor activation inhibits both the firing of VTA dopaminergic cells [11] and the release of DA from isolated VTA (D. Amantea, unpublished observation), as well as from nucleus accumbens [20] and prefrontal cortex [18], as monitored by in vivo microdialysis. Previous studies have also reported that chronic administration of psychostimulants results in altered pharmacological properties of the GABAB receptor. Accordingly, in rats sensitised to cocaine, an increased level of GABAB(1) subunit mRNA was observed in the NAc, the hippocampal CA1 field and the thalamus [24], while receptor downregulation was reported in the dorsolateral septal nucleus [19] and the VTA [13]. Conversely, enhanced GABAB receptor transmission was observed in the VTA of rats 3 days after discontinuing repeated amphetamine administration [9], while altered G-protein coupling to the receptor was detected in the prefrontal cortex and the nucleus accumbens of rats sensitised to the drug [25]. In addition, the decreased GABAB(1) receptor expression found in the hippocampus of rats exposed to nicotine argues for an involvement of the receptor in nicotine-mediated modu-
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.060
162
D. Amantea et al. / Neuroscience Letters 355 (2004) 161–164
lation of cognition [14]. However, to our knowledge, little is known about the effects of repeated administration of nicotine on the pharmacological properties of GABAB receptors in brain reward pathways. Therefore, in the present study we have investigated whether chronic exposure to nicotine alters the density or affinity of GABAB receptors, or affects the coupling of G-proteins to GABAB receptors in mesocorticolimbic regions of the rat brain. Male Wistar rats (180 –200 g) were maintained on a 12-h light/dark cycle, with free access to food and water. Animals, randomly divided into two treatment groups, received daily subcutaneous injections of nicotine (0.4 mg/kg, free base) or vehicle (0.9% NaCl, 1 ml/kg) for 14 consecutive days. Twenty-four hours after the last injection, rats were sacrificed and the brains were dissected and rapidly frozen at 2 80 8C. Successive 12 mm-thick sections were cut in a cryostat, thaw-mounted onto charged microscope slides (BDH Superfrost Plus), and stored at 2 80 8C until use. For baclofen-stimulated [35S]GTPgS binding autoradiography, tissue sections were allowed to thaw-dry at room temperature before being washed for 30 min at 25 8C in assay buffer (50 mM Tris – HCl, 100 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, pH 7.4). Slides were then preincubated for 20 min in assay buffer containing 2 mM GDP, followed by incubation for 2 h at 25 8C in media containing 0.04 nM [35S]GTPgS (Specific activity 1048 Ci/mmol, Amersham) and 2 mM GDP, with or without the selective GABAB agonist (2 )baclofen (100 mM, gift from Novartis), to determine agonist-stimulated and basal binding, respectively. Non-specific binding was determined in the presence of 10 mM unlabelled GTPgS. In some experiments, the selective GABAB antagonist CGP55845A (10 mM, gift from Novartis) was also included. After incubation, slides were washed twice in ice-cold 50 mM Tris – HCl buffer (pH 7) and, when dry, exposed for 72 h to Kodak BioMax MR films (Amersham). After development of the film, the autoradiographic images were digitised and analysed by computer-assisted densitometry (MCID-M4, Imaging Research Inc., Ont., Canada). A mean optical density value across each brain region of interest was obtained by using a variable size cursor, and readings from at least four different sections per brain were taken. Concentration of bound radioactivity was determined from a standard curve generated from film optical densities produced by coexposed [35S]-labelled brain paste standards. Results were expressed as specific [35S]GTPgS binding, calculated by subtracting non-specific binding from basal and baclofenstimulated total binding. [3H]GABA saturation binding was performed on rat brain slices as described by Bowery et al. [2]. Briefly, tissue sections were washed for 90 min in 50 mM Tris –HCl buffer containing 2.5 mM CaCl2 (pH 7.4). A 20-min incubation was performed in buffer containing [3H]GABA (25 – 400 nM, Specific activity 94 Ci/mmol, Amersham) in the
presence of 40 mM isoguvacine, to prevent binding to GABAA sites. Non-specific binding was determined with 100 mM (2 )baclofen. Finally, slides were washed twice in buffer, and, when dry, exposed to 3H-Hyperfilm (Amersham) for 3 weeks along with [3H]-impregnated plastic standards (Amersham). After development of the film, the autoradiographic images were digitised and analysed as described above. Bmax and Kd values were determined by non-linear regression with an interactive curve-fitting package (GraphPad Software, San Diego, CA). Brain regions of interest included mesocorticolimbic areas, namely nucleus accumbens core and shell, ventral tegmental area and medial prefrontal cortex, but also the caudate putamen (CPu) and the parietal cortex (ParCx) were used for comparison. Results were expressed as mean ^ SEM and compared using a two-tailed Student’s t-test. In control rats, baclofen significantly increased [ 35S]GTPgS binding by 54 – 206% of basal binding throughout the brain regions examined, and values returned to basal levels in the presence of CGP55845A, confirming the GABAB receptor specificity of agonist-stimulated [35S]GTPgS binding (Fig. 1). The presence of endogenous GABA may explain the lower levels of binding observed in the presence of CGP55845A as compared to the basal values (Fig. 1). Chronic exposure to nicotine resulted in a significant decrease in baclofen-stimulated [35S]GTPgS binding in the mPfCx, the NAcC and the NAcS, while the reduction found in VTA, CPu and ParCx did not reach statistical significance (Table 1). By contrast, basal [35S]GTPgS binding (data not shown) and GABAB receptor density (Bmax) and affinity (Kd) were not significantly altered by the nicotine treatment in any of the brain regions examined (Fig. 2). Our findings demonstrate that repeated exposure to nicotine results in a decreased functional coupling of GABAB receptors to G-proteins, presumably Gia and Goa [17], in the mPfCx and NAc core and shell. Down-
Fig. 1. Reversal of baclofen-stimulated [35S]GTPgS binding by the selective GABAB antagonist CGP55845A in saline-control rats. Brain sections were incubated with [35S]GTPgS 0.04 nM and GDP 2 mM for the basal binding (open bars), baclofen 100 mM was added to determine agonist-stimulated binding (closed bars), while baclofen 100 mM plus CGP55845A 10 mM were included to the incubation media to determine the effect of GABAB receptor blockade (hatched bars). Values represent mean ^ SEM (n ¼ 3). CPu, caudate-putamen; mPfCx, medial prefrontal cortex; NAcC, nucleus accumbens core; NAcS, nucleus accumbens shell; ParCx, parietal cortex; and VTA, ventral tegmental area.
D. Amantea et al. / Neuroscience Letters 355 (2004) 161–164 Table 1 Specific baclofen-stimulated [35S]GTPgS binding in rats treated with daily subcutaneous injections of nicotine (n ¼ 4) or saline (n ¼ 3) for 14 daysa Brain area
Saline
Nicotine
Nucleus accumbens core Nucleus accumbens shell Medial prefrontal cortex Ventral tegmental area Caudate putamen Parietal cortex
474.5 ^ 22.5 364.2 ^ 17.0 588.7 ^ 14.3 300.4 ^ 8.2 383.9 ^ 23.1 485.6 ^ 26.1
387.8 ^ 16.3* 303.8 ^ 12.9* 406.8 ^ 23.5** 263.7 ^ 15.8 335.5 ^ 11.6 371.7 ^ 61.7
a
Values represent mean ^ SEM in amoles/mg wet tissue. *P , 0.05, **P , 0.01 vs corresponding saline.
regulation of GABAB receptors in these terminal dopaminergic areas may contribute to the sensitised release of DA observed in nucleus accumbens and prefrontal cortex after repeated exposure to nicotine [1,16]. Presynaptic GABAB receptors are located on both dopaminergic and glutamatergic afferents to the nucleus accumbens, and their activation inhibits the release of dopamine and glutamate, respectively, within this region [20,22]. The reduced GABAB receptor coupling to Gproteins that we observed after chronic exposure to nicotine may therefore result in disinhibition of DA and/or glutamate release in the nucleus accumbens, and this is consistent with previous studies reporting a decreased G-protein coupling to GABAB receptors in the nucleus accumbens of rats sensitised to amphetamine [25].
Fig. 2. [3H]GABA maximal binding (Bmax) and affinity (Kd) for the GABAB receptor in rats treated with daily subcutaneous injections of nicotine (0.4 mg/kg, n ¼ 3, open bars) or saline (n ¼ 3, closed bars) for 14 days. Values represent mean ^ SEM.
163
In addition to subsensitivity of accumbal GABAB receptors, we have also observed reduced G-protein coupling to the receptor in the mPfCx following chronic exposure to nicotine. Microinjections of baclofen in the prefrontal cortex have been previously shown to reduce the local release of DA by activation of GABAB receptors, presumably located on dopaminergic nerve terminals [18]. This suggests that subsensitivity of prefrontal GABAB receptors may underlie the increased release of dopamine detected in the prefrontal cortex of rats sensitised to nicotine [16]. This phenomenon may be highly significant for the dependence-producing effects of nicotine, although other mechanisms seem to be critical for the development and expression of nicotine addiction. In fact, glutamatergic projections from the prefrontal cortex to the nucleus accumbens and the VTA have also been implicated in stimulant-induced behavioural sensitisation [12,23]. Activation of these excitatory neurones results in increased activity of VTA dopamine cells and enhanced DA release in the nucleus accumbens [15,21]. In addition, the excitation of VTA DA neurones produced by nicotine is attenuated by NMDA receptor antagonists, suggesting that glutamatergic inputs to midbrain neurones may play a critical role in the expression of sensitised responses to nicotine [10]. Interestingly, GABAB heteroreceptors are also located on glutamatergic cortical output neurones, providing inhibition of excitatory efferents to the nucleus accumbens and the VTA [8]. Consequently, the reduced G-protein coupling to the receptor that we have detected in the prefrontal cortex of the rats chronically treated with nicotine may lead to disinhibition of these excitatory projections, resulting in an increased excitatory drive to both the VTA and the nucleus accumbens. Therefore, disrupted GABAB-mediated modulation of excitatory efferents arising from the prefrontal cortex might contribute to the hyperactivity of mesoaccumbens neurones produced by repeated exposure to nicotine [1, 16]. Interestingly, we found no alteration of GABAB receptor coupling to G-proteins in the VTA after nicotine administration. By contrast, chronic cocaine treatment has been reported to decrease the functional GABAB-G-protein coupling in this midbrain region, but not in the terminal fields of the mesocorticolimbic system [13]. Taken together, these findings may account for the higher potency of baclofen, microinjected into the VTA, to reduce nicotine self-administration compared to cocaine [5]. Although reduced G-protein coupling might be expected to decrease agonist binding to the GABAB receptor, we did not detect any difference in [3H]GABA maximal binding in the prefrontal cortex or the nucleus accumbens of the rats treated with nicotine as compared to those injected with saline. Presumably because the reduction in coupling is small, any decrease in receptor binding may be ‘lost’ in the total binding. If, for example, reduced G-protein coupling occurs at presynaptic, but not postsynaptic GABAB receptors, it might produce little or no effect on the total
164
D. Amantea et al. / Neuroscience Letters 355 (2004) 161–164
binding, as the latter is expected to be much greater at the postsynaptic level. However, this issue remains to be clarified as, to date, there is a lack of compounds that selectively discriminate between presynaptic and postsynaptic GABAB sites. In conclusion, in the present study we demonstrate that repeated exposure to nicotine is associated with a functional uncoupling of GABAB receptors from G-proteins in terminal mesocorticolimbic regions, while receptor density and affinity were not affected. Desensitisation of the receptor may result in a reduced ability of GABAB receptors to inhibit dopaminergic and/or glutamatergic neurones in the prefrontal cortex and nucleus accumbens. This, in turn, may contribute to the sensitised activity of the dopaminergic mesolimbic system previously reported in rats chronically exposed to nicotine [1,7,16]. However, further studies on the modulation of dopamine transmission by GABAB receptors in the mesocorticolimbic system of rats sensitised to nicotine are necessary to confirm this hypothesis.
References [1] M.E.M. Benwell, D.J.K. Balfour, The effects of acute and repeated nicotine treatment on nucleus accumbens dopamine and locomotor activity, Br. J. Pharmacol. 105 (1992) 849–856. [2] N.G. Bowery, A.L. Hudson, G.W. Price, GABAA and GABAB receptor site distribution in the rat central nervous system, Neuroscience 20 (1987) 365–383. [3] P.B.S. Clarke, D.S. Fu, A. Jakubovic, H.C. Fibiger, Evidence that mesolimbic dopaminergic activation underlies the locomotor stimulant action of nicotine in rats, J. Pharmacol. Exp. Ther. 246 (1988) 701–708. [4] W.A. Corrigall, K.M. Coen, K.L. Adamson, Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area, Brain Res. 653 (1994) 278–284. [5] W.A. Corrigall, K.M. Coen, K.L. Adamson, B.L.C. Chow, J. Zhang, Response of nicotine self-administration in the rat to manipulations of mu-opioid and g-aminobutyric acid receptors in the ventral tegmental area, Psychopharmacology 149 (2000) 107–114. [6] M.S. Cousins, D.C.S. Roberts, H. de Wit, GABAB receptor agonists for the treatment of drug addiction: a review of recent findings, Drug Alcohol Depend. 65 (2002) 209–220. [7] G. Di Chiara, Role of dopamine in the behavioural actions of nicotine related to addiction, Eur. J. Pharmacol. 393 (2000) 295–314. [8] M.D. Doherty, A. Gratton, Effects of medial prefrontal cortical injections of GABA receptor agonists and antagonists on the local and nucleus accumbens dopamine responses to stress, Synapse 32 (1999) 288–300. [9] M. Giorgetti, G. Hotsenpiller, W. Froestl, M.E. Wolf, In vivo modulation of ventral tegmental area dopamine and glutamate efflux by local GABAB receptors is altered after repeated amphetamine treatment, Neuroscience 109 (2002) 585–595.
[10] P. Grillner, T. Svensson, Nicotine-induced excitation of midbrain dopamine neurons in vitro involves ionotropic glutamate receptor activation, Synapse 38 (2000) 1–9. [11] S.W. Johnson, R.A. North, Two types of neurones in the rat ventral tegmental area and their synaptic input, J. Physiol. 450 (1992) 455 –468. [12] P.W. Kalivas, Interactions between dopamine and excitatory amino acids in behavioral sensitization to psychostimulants, Drug Alcohol Depend. 37 (1995) 95 –100. [13] S. Kushner, E.M. Unterwald, Chronic cocaine administration decreases the functional coupling of GABAB receptors in the rat ventral tegmental area as measured by baclofen-stimulated 35SGTPgS binding, Life Sci. 69 (2001) 1093–1102. [14] S.P. Li, M.S. Park, J.Y. Bahk, M.O. Kim, Chronic nicotine and smoking exposure decreases GABAB1 receptor expression in the rat hippocampus, Neurosci. Lett. 334 (2002) 135–139. [15] S. Murase, J. Grenhoff, G. Chouvet, F.G. Gonnon, T.H. Svensson, Prefrontal cortex regulates burst firing and transmitter release in rat mesolimbic dopamine neurons studied in vivo, Neurosci. Lett. 157 (1993) 53– 56. [16] M. Nisell, G.G. Nomikos, P. Hertel, G. Panagis, T.H. Svensson, Condition-independent sensitisation of locomotor stimulation and mesocortical dopamine release following chronic nicotine treatment in the rat, Synapse 22 (1996) 369 –381. [17] Y. Odagaki, T. Koyama, Identification of G alpha subtype(s) involved in gamma-aminobutyric acidB receptor-mediated high affinity guanosine triphosphatase activity in rat cerebral cortical membranes, Neurosci. Lett. 297 (2001) 137–141. [18] M. Santiago, A. Machado, J. Cano, Regulation of the prefrontal cortical dopamine release by GABAA and GABAB receptor agonists and antagonists, Brain Res. 630 (1993) 28 –31. [19] S. Shoji, D. Simms, W.C. McDaniel, J.P. Gallagher, Chronic cocaine enhances g-aminobutyric acid and glutamate release by altering presynaptic and not postsynaptic g-aminobutyric acidB receptors within the rat dorsolateral nucleus, J. Pharmacol. Exp. Ther. 280 (1997) 129 –137. [20] I. Smolders, N. De Klippel, S. Sarre, G. Ebinger, Y. Michotte, Tonic GABA-ergic modulation of striatal dopamine release studied by in vivo microdialysis in the freely moving rat, Eur. J. Pharmacol. 284 (1995) 83– 91. [21] M.T. Taber, H.C. Fibiger, Electrical stimulation of the medial prefrontal cortex increases dopamine release in nucleus accumbens of rat: modulation by metabotropic glutamate receptors, J. Neurosci. 15 (1995) 3896–3904. [22] N. Uchimura, R.A. North, Baclofen and adenosine inhibit synaptic potentials mediated by gamma-aminobutyric acid and glutamate release in rat nucleus accumbens, J. Pharmacol. Exp. Ther. 258 (1991) 663 –668. [23] M.E. Wolf, The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants, Prog. Neurobiol. 54 (1998) 679– 720. [24] M. Yamaguchi, T. Suzuki, S. Abe, A. Baba, T. Hori, N. Okado, Repeated cocaine administration increases GABAB(1) subunit mRNA in rat brain, Synapse 43 (2002) 175– 180. [25] K. Zhang, F.I. Tarazi, A. Campbell, R.J. Baldessarini, GABAB receptors: altered coupling to G-proteins in rats sensitized to amphetamine, Neuroscience 101 (2000) 5 –10.
Reduced G-protein coupling to the GABAB receptor in the nucleus accumbens and the medial prefrontal cortex of the rat after chronic treatment with nicotine Diana Amanteaa,*, Michela Tessarib, Norman G. Bowerya a
Department of Pharmacology, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Drug Dependence Behav. Neurochem., Psychiatry-CEDD, GlaxoSmithKline S.p.A., 37135 Verona, Italy Received 23 June 2003; received in revised form 20 October 2003; accepted 20 October 2003
Abstract The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABAB receptor density, affinity and G-protein coupling was investigated in the mesocorticolimbic system of the rat brain. Baclofen-stimulated [35S]GTPgS binding autoradiography revealed that the level of G-protein coupling to GABAB receptors was significantly reduced in the medial prefrontal cortex and the nucleus accumbens of nicotine-treated rats as compared to vehicle-injected controls. By contrast, GABAB receptor density and affinity, as revealed by [3H]GABA saturation binding autoradiography, were not altered by the nicotine exposure in any of the regions examined. Reduced Gprotein coupling to the GABAB receptor may result in disinhibition of mesocorticolimbic dopaminergic neurones, which would contribute to the development of sensitised dopaminergic responses to repeated administration of nicotine. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: GABAB receptor; Nicotine; Drug addiction; Mesocorticolimbic system; G-protein; [35S]GTPgS
Nicotine stimulant and reinforcing effects in rats are primarily mediated by activation of the mesocorticolimbic dopaminergic system of the brain [3,4]. This neuronal pathway consists of dopaminergic neurones projecting from the ventral tegmental area (VTA), located in the midbrain, to forebrain regions, including the medial prefrontal cortex (mPfCx) and the nucleus accumbens core (NAcC) and shell (NAcS) subdivisions. Activation of VTA nicotinic receptors and the resulting release of dopamine (DA) within the mesocorticolimbic terminal regions underlies most of the behavioural effects exerted by nicotine [4,7]. A growing number of preclinical and clinical studies provide evidence for the effectiveness of GABAB receptor agonists in reducing addictive behaviours associated with alcohol and psychostimulant abuse [6], including nicotine self-administration in rats [5]. GABAB receptors are widely distributed throughout the mesocorticolimbic system [2] and their activation may therefore interfere with the mechanisms involved in nicotine * Corresponding author. Tel.: þ44-121-414-4525; fax: þ 44-121-4144509. E-mail address: [email protected] (D. Amantea).
dependence. Interestingly, in naı¨ve rats, GABAB receptor activation inhibits both the firing of VTA dopaminergic cells [11] and the release of DA from isolated VTA (D. Amantea, unpublished observation), as well as from nucleus accumbens [20] and prefrontal cortex [18], as monitored by in vivo microdialysis. Previous studies have also reported that chronic administration of psychostimulants results in altered pharmacological properties of the GABAB receptor. Accordingly, in rats sensitised to cocaine, an increased level of GABAB(1) subunit mRNA was observed in the NAc, the hippocampal CA1 field and the thalamus [24], while receptor downregulation was reported in the dorsolateral septal nucleus [19] and the VTA [13]. Conversely, enhanced GABAB receptor transmission was observed in the VTA of rats 3 days after discontinuing repeated amphetamine administration [9], while altered G-protein coupling to the receptor was detected in the prefrontal cortex and the nucleus accumbens of rats sensitised to the drug [25]. In addition, the decreased GABAB(1) receptor expression found in the hippocampus of rats exposed to nicotine argues for an involvement of the receptor in nicotine-mediated modu-
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.10.060
162
D. Amantea et al. / Neuroscience Letters 355 (2004) 161–164
lation of cognition [14]. However, to our knowledge, little is known about the effects of repeated administration of nicotine on the pharmacological properties of GABAB receptors in brain reward pathways. Therefore, in the present study we have investigated whether chronic exposure to nicotine alters the density or affinity of GABAB receptors, or affects the coupling of G-proteins to GABAB receptors in mesocorticolimbic regions of the rat brain. Male Wistar rats (180 –200 g) were maintained on a 12-h light/dark cycle, with free access to food and water. Animals, randomly divided into two treatment groups, received daily subcutaneous injections of nicotine (0.4 mg/kg, free base) or vehicle (0.9% NaCl, 1 ml/kg) for 14 consecutive days. Twenty-four hours after the last injection, rats were sacrificed and the brains were dissected and rapidly frozen at 2 80 8C. Successive 12 mm-thick sections were cut in a cryostat, thaw-mounted onto charged microscope slides (BDH Superfrost Plus), and stored at 2 80 8C until use. For baclofen-stimulated [35S]GTPgS binding autoradiography, tissue sections were allowed to thaw-dry at room temperature before being washed for 30 min at 25 8C in assay buffer (50 mM Tris – HCl, 100 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, pH 7.4). Slides were then preincubated for 20 min in assay buffer containing 2 mM GDP, followed by incubation for 2 h at 25 8C in media containing 0.04 nM [35S]GTPgS (Specific activity 1048 Ci/mmol, Amersham) and 2 mM GDP, with or without the selective GABAB agonist (2 )baclofen (100 mM, gift from Novartis), to determine agonist-stimulated and basal binding, respectively. Non-specific binding was determined in the presence of 10 mM unlabelled GTPgS. In some experiments, the selective GABAB antagonist CGP55845A (10 mM, gift from Novartis) was also included. After incubation, slides were washed twice in ice-cold 50 mM Tris – HCl buffer (pH 7) and, when dry, exposed for 72 h to Kodak BioMax MR films (Amersham). After development of the film, the autoradiographic images were digitised and analysed by computer-assisted densitometry (MCID-M4, Imaging Research Inc., Ont., Canada). A mean optical density value across each brain region of interest was obtained by using a variable size cursor, and readings from at least four different sections per brain were taken. Concentration of bound radioactivity was determined from a standard curve generated from film optical densities produced by coexposed [35S]-labelled brain paste standards. Results were expressed as specific [35S]GTPgS binding, calculated by subtracting non-specific binding from basal and baclofenstimulated total binding. [3H]GABA saturation binding was performed on rat brain slices as described by Bowery et al. [2]. Briefly, tissue sections were washed for 90 min in 50 mM Tris –HCl buffer containing 2.5 mM CaCl2 (pH 7.4). A 20-min incubation was performed in buffer containing [3H]GABA (25 – 400 nM, Specific activity 94 Ci/mmol, Amersham) in the
presence of 40 mM isoguvacine, to prevent binding to GABAA sites. Non-specific binding was determined with 100 mM (2 )baclofen. Finally, slides were washed twice in buffer, and, when dry, exposed to 3H-Hyperfilm (Amersham) for 3 weeks along with [3H]-impregnated plastic standards (Amersham). After development of the film, the autoradiographic images were digitised and analysed as described above. Bmax and Kd values were determined by non-linear regression with an interactive curve-fitting package (GraphPad Software, San Diego, CA). Brain regions of interest included mesocorticolimbic areas, namely nucleus accumbens core and shell, ventral tegmental area and medial prefrontal cortex, but also the caudate putamen (CPu) and the parietal cortex (ParCx) were used for comparison. Results were expressed as mean ^ SEM and compared using a two-tailed Student’s t-test. In control rats, baclofen significantly increased [ 35S]GTPgS binding by 54 – 206% of basal binding throughout the brain regions examined, and values returned to basal levels in the presence of CGP55845A, confirming the GABAB receptor specificity of agonist-stimulated [35S]GTPgS binding (Fig. 1). The presence of endogenous GABA may explain the lower levels of binding observed in the presence of CGP55845A as compared to the basal values (Fig. 1). Chronic exposure to nicotine resulted in a significant decrease in baclofen-stimulated [35S]GTPgS binding in the mPfCx, the NAcC and the NAcS, while the reduction found in VTA, CPu and ParCx did not reach statistical significance (Table 1). By contrast, basal [35S]GTPgS binding (data not shown) and GABAB receptor density (Bmax) and affinity (Kd) were not significantly altered by the nicotine treatment in any of the brain regions examined (Fig. 2). Our findings demonstrate that repeated exposure to nicotine results in a decreased functional coupling of GABAB receptors to G-proteins, presumably Gia and Goa [17], in the mPfCx and NAc core and shell. Down-
Fig. 1. Reversal of baclofen-stimulated [35S]GTPgS binding by the selective GABAB antagonist CGP55845A in saline-control rats. Brain sections were incubated with [35S]GTPgS 0.04 nM and GDP 2 mM for the basal binding (open bars), baclofen 100 mM was added to determine agonist-stimulated binding (closed bars), while baclofen 100 mM plus CGP55845A 10 mM were included to the incubation media to determine the effect of GABAB receptor blockade (hatched bars). Values represent mean ^ SEM (n ¼ 3). CPu, caudate-putamen; mPfCx, medial prefrontal cortex; NAcC, nucleus accumbens core; NAcS, nucleus accumbens shell; ParCx, parietal cortex; and VTA, ventral tegmental area.
D. Amantea et al. / Neuroscience Letters 355 (2004) 161–164 Table 1 Specific baclofen-stimulated [35S]GTPgS binding in rats treated with daily subcutaneous injections of nicotine (n ¼ 4) or saline (n ¼ 3) for 14 daysa Brain area
Saline
Nicotine
Nucleus accumbens core Nucleus accumbens shell Medial prefrontal cortex Ventral tegmental area Caudate putamen Parietal cortex
474.5 ^ 22.5 364.2 ^ 17.0 588.7 ^ 14.3 300.4 ^ 8.2 383.9 ^ 23.1 485.6 ^ 26.1
387.8 ^ 16.3* 303.8 ^ 12.9* 406.8 ^ 23.5** 263.7 ^ 15.8 335.5 ^ 11.6 371.7 ^ 61.7
a
Values represent mean ^ SEM in amoles/mg wet tissue. *P , 0.05, **P , 0.01 vs corresponding saline.
regulation of GABAB receptors in these terminal dopaminergic areas may contribute to the sensitised release of DA observed in nucleus accumbens and prefrontal cortex after repeated exposure to nicotine [1,16]. Presynaptic GABAB receptors are located on both dopaminergic and glutamatergic afferents to the nucleus accumbens, and their activation inhibits the release of dopamine and glutamate, respectively, within this region [20,22]. The reduced GABAB receptor coupling to Gproteins that we observed after chronic exposure to nicotine may therefore result in disinhibition of DA and/or glutamate release in the nucleus accumbens, and this is consistent with previous studies reporting a decreased G-protein coupling to GABAB receptors in the nucleus accumbens of rats sensitised to amphetamine [25].
Fig. 2. [3H]GABA maximal binding (Bmax) and affinity (Kd) for the GABAB receptor in rats treated with daily subcutaneous injections of nicotine (0.4 mg/kg, n ¼ 3, open bars) or saline (n ¼ 3, closed bars) for 14 days. Values represent mean ^ SEM.
163
In addition to subsensitivity of accumbal GABAB receptors, we have also observed reduced G-protein coupling to the receptor in the mPfCx following chronic exposure to nicotine. Microinjections of baclofen in the prefrontal cortex have been previously shown to reduce the local release of DA by activation of GABAB receptors, presumably located on dopaminergic nerve terminals [18]. This suggests that subsensitivity of prefrontal GABAB receptors may underlie the increased release of dopamine detected in the prefrontal cortex of rats sensitised to nicotine [16]. This phenomenon may be highly significant for the dependence-producing effects of nicotine, although other mechanisms seem to be critical for the development and expression of nicotine addiction. In fact, glutamatergic projections from the prefrontal cortex to the nucleus accumbens and the VTA have also been implicated in stimulant-induced behavioural sensitisation [12,23]. Activation of these excitatory neurones results in increased activity of VTA dopamine cells and enhanced DA release in the nucleus accumbens [15,21]. In addition, the excitation of VTA DA neurones produced by nicotine is attenuated by NMDA receptor antagonists, suggesting that glutamatergic inputs to midbrain neurones may play a critical role in the expression of sensitised responses to nicotine [10]. Interestingly, GABAB heteroreceptors are also located on glutamatergic cortical output neurones, providing inhibition of excitatory efferents to the nucleus accumbens and the VTA [8]. Consequently, the reduced G-protein coupling to the receptor that we have detected in the prefrontal cortex of the rats chronically treated with nicotine may lead to disinhibition of these excitatory projections, resulting in an increased excitatory drive to both the VTA and the nucleus accumbens. Therefore, disrupted GABAB-mediated modulation of excitatory efferents arising from the prefrontal cortex might contribute to the hyperactivity of mesoaccumbens neurones produced by repeated exposure to nicotine [1, 16]. Interestingly, we found no alteration of GABAB receptor coupling to G-proteins in the VTA after nicotine administration. By contrast, chronic cocaine treatment has been reported to decrease the functional GABAB-G-protein coupling in this midbrain region, but not in the terminal fields of the mesocorticolimbic system [13]. Taken together, these findings may account for the higher potency of baclofen, microinjected into the VTA, to reduce nicotine self-administration compared to cocaine [5]. Although reduced G-protein coupling might be expected to decrease agonist binding to the GABAB receptor, we did not detect any difference in [3H]GABA maximal binding in the prefrontal cortex or the nucleus accumbens of the rats treated with nicotine as compared to those injected with saline. Presumably because the reduction in coupling is small, any decrease in receptor binding may be ‘lost’ in the total binding. If, for example, reduced G-protein coupling occurs at presynaptic, but not postsynaptic GABAB receptors, it might produce little or no effect on the total
164
D. Amantea et al. / Neuroscience Letters 355 (2004) 161–164
binding, as the latter is expected to be much greater at the postsynaptic level. However, this issue remains to be clarified as, to date, there is a lack of compounds that selectively discriminate between presynaptic and postsynaptic GABAB sites. In conclusion, in the present study we demonstrate that repeated exposure to nicotine is associated with a functional uncoupling of GABAB receptors from G-proteins in terminal mesocorticolimbic regions, while receptor density and affinity were not affected. Desensitisation of the receptor may result in a reduced ability of GABAB receptors to inhibit dopaminergic and/or glutamatergic neurones in the prefrontal cortex and nucleus accumbens. This, in turn, may contribute to the sensitised activity of the dopaminergic mesolimbic system previously reported in rats chronically exposed to nicotine [1,7,16]. However, further studies on the modulation of dopamine transmission by GABAB receptors in the mesocorticolimbic system of rats sensitised to nicotine are necessary to confirm this hypothesis.
References [1] M.E.M. Benwell, D.J.K. Balfour, The effects of acute and repeated nicotine treatment on nucleus accumbens dopamine and locomotor activity, Br. J. Pharmacol. 105 (1992) 849–856. [2] N.G. Bowery, A.L. Hudson, G.W. Price, GABAA and GABAB receptor site distribution in the rat central nervous system, Neuroscience 20 (1987) 365–383. [3] P.B.S. Clarke, D.S. Fu, A. Jakubovic, H.C. Fibiger, Evidence that mesolimbic dopaminergic activation underlies the locomotor stimulant action of nicotine in rats, J. Pharmacol. Exp. Ther. 246 (1988) 701–708. [4] W.A. Corrigall, K.M. Coen, K.L. Adamson, Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area, Brain Res. 653 (1994) 278–284. [5] W.A. Corrigall, K.M. Coen, K.L. Adamson, B.L.C. Chow, J. Zhang, Response of nicotine self-administration in the rat to manipulations of mu-opioid and g-aminobutyric acid receptors in the ventral tegmental area, Psychopharmacology 149 (2000) 107–114. [6] M.S. Cousins, D.C.S. Roberts, H. de Wit, GABAB receptor agonists for the treatment of drug addiction: a review of recent findings, Drug Alcohol Depend. 65 (2002) 209–220. [7] G. Di Chiara, Role of dopamine in the behavioural actions of nicotine related to addiction, Eur. J. Pharmacol. 393 (2000) 295–314. [8] M.D. Doherty, A. Gratton, Effects of medial prefrontal cortical injections of GABA receptor agonists and antagonists on the local and nucleus accumbens dopamine responses to stress, Synapse 32 (1999) 288–300. [9] M. Giorgetti, G. Hotsenpiller, W. Froestl, M.E. Wolf, In vivo modulation of ventral tegmental area dopamine and glutamate efflux by local GABAB receptors is altered after repeated amphetamine treatment, Neuroscience 109 (2002) 585–595.
[10] P. Grillner, T. Svensson, Nicotine-induced excitation of midbrain dopamine neurons in vitro involves ionotropic glutamate receptor activation, Synapse 38 (2000) 1–9. [11] S.W. Johnson, R.A. North, Two types of neurones in the rat ventral tegmental area and their synaptic input, J. Physiol. 450 (1992) 455 –468. [12] P.W. Kalivas, Interactions between dopamine and excitatory amino acids in behavioral sensitization to psychostimulants, Drug Alcohol Depend. 37 (1995) 95 –100. [13] S. Kushner, E.M. Unterwald, Chronic cocaine administration decreases the functional coupling of GABAB receptors in the rat ventral tegmental area as measured by baclofen-stimulated 35SGTPgS binding, Life Sci. 69 (2001) 1093–1102. [14] S.P. Li, M.S. Park, J.Y. Bahk, M.O. Kim, Chronic nicotine and smoking exposure decreases GABAB1 receptor expression in the rat hippocampus, Neurosci. Lett. 334 (2002) 135–139. [15] S. Murase, J. Grenhoff, G. Chouvet, F.G. Gonnon, T.H. Svensson, Prefrontal cortex regulates burst firing and transmitter release in rat mesolimbic dopamine neurons studied in vivo, Neurosci. Lett. 157 (1993) 53– 56. [16] M. Nisell, G.G. Nomikos, P. Hertel, G. Panagis, T.H. Svensson, Condition-independent sensitisation of locomotor stimulation and mesocortical dopamine release following chronic nicotine treatment in the rat, Synapse 22 (1996) 369 –381. [17] Y. Odagaki, T. Koyama, Identification of G alpha subtype(s) involved in gamma-aminobutyric acidB receptor-mediated high affinity guanosine triphosphatase activity in rat cerebral cortical membranes, Neurosci. Lett. 297 (2001) 137–141. [18] M. Santiago, A. Machado, J. Cano, Regulation of the prefrontal cortical dopamine release by GABAA and GABAB receptor agonists and antagonists, Brain Res. 630 (1993) 28 –31. [19] S. Shoji, D. Simms, W.C. McDaniel, J.P. Gallagher, Chronic cocaine enhances g-aminobutyric acid and glutamate release by altering presynaptic and not postsynaptic g-aminobutyric acidB receptors within the rat dorsolateral nucleus, J. Pharmacol. Exp. Ther. 280 (1997) 129 –137. [20] I. Smolders, N. De Klippel, S. Sarre, G. Ebinger, Y. Michotte, Tonic GABA-ergic modulation of striatal dopamine release studied by in vivo microdialysis in the freely moving rat, Eur. J. Pharmacol. 284 (1995) 83– 91. [21] M.T. Taber, H.C. Fibiger, Electrical stimulation of the medial prefrontal cortex increases dopamine release in nucleus accumbens of rat: modulation by metabotropic glutamate receptors, J. Neurosci. 15 (1995) 3896–3904. [22] N. Uchimura, R.A. North, Baclofen and adenosine inhibit synaptic potentials mediated by gamma-aminobutyric acid and glutamate release in rat nucleus accumbens, J. Pharmacol. Exp. Ther. 258 (1991) 663 –668. [23] M.E. Wolf, The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants, Prog. Neurobiol. 54 (1998) 679– 720. [24] M. Yamaguchi, T. Suzuki, S. Abe, A. Baba, T. Hori, N. Okado, Repeated cocaine administration increases GABAB(1) subunit mRNA in rat brain, Synapse 43 (2002) 175– 180. [25] K. Zhang, F.I. Tarazi, A. Campbell, R.J. Baldessarini, GABAB receptors: altered coupling to G-proteins in rats sensitized to amphetamine, Neuroscience 101 (2000) 5 –10.