Molecular Psychiatry (2000) 5, 378–388 2000 Macmillan Publishers Ltd All rights reserved 1359-4184/00 $15.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Selective increase of dopamine D3 receptor gene expression as a common effect of chronic antidepressant treatments C-H Lammers1,2*, J Diaz1*, J-C Schwartz3 and P Sokoloff3 1Laboratoire de Physiologie, Universite´ Rene´ Descartes, 4 Avenue de l’Observatoire, 75006 Paris, France; 3Unite´ de Neurobiologie et Pharmacologie Mole´culaire (U. 109) de l’Institut National de la Sante´ et Recherche Me´dicale, Centre Paul Broca, 2ter rue d’Ale´sia, 75014 Paris, France The mesolimbic dopaminergic system is a neuroanatomical key structure for reward and motivation upon which previous studies indicated that antidepressant drugs exert a stimu- latory influence, via still unknown neurobiological mechanisms. Here we examined the effects of chronic administration of antidepressants of several classes (amitriptyline, desipramine, imipramine, fluoxetine and tranylcypromine) and repeated electroconvulsive shock treatments (ECT) on dopamine D3 receptor expression in the shell of the nucleus accumbens, a major projection area of the mesolimbic dopaminergic system. Short-term drug treatments had vari- able effects on D3 receptor mRNA expression. In contrast, treatments for 21 days (with all drugs except fluoxetine) significantly increased D3 receptor mRNA expression in the shell of nucleus accumbens; D3 receptor binding was also significantly increased by amitriptyline or fluoxetine after a 42-day treatment. ECT for 10 days increased D3 receptor mRNA and binding in the shell of nucleus accumbens. D1 receptor and D2 receptor mRNAs were increased by imipramine and amitriptyline, but not by the other treatments. The time-course of altered D3 receptor expression, in line with the delayed clinical efficiency of antidepressant treatment, and the fact that various antidepressant drugs and ECT treatments eventually produced the same effects, suggest that increased expression of the D3 receptor in the shell of nucleus accumbens is a common neurobiological mechanism of antidepressant treatments, resulting in enhanced responsiveness to the mesolimbic dopaminergic system. Molecular Psychiatry (2000) 5, 378–388. Keywords: depression; antidepressant drug; electroconvulsive shock; mesolimbic dopaminergic system Introduction to an idiopathic manic episode7,8 and the discontinu- ation of such drugs9 or the acute administration of Considerable emphasis has been placed upon the puta- dopamine receptor antagonists can result in a psycho- tive role of nucleus accumbens dopamine systems in pathological state similar to a depressive episode.10 appetitive motivation and positive reinforcement.1,2 Clinical studies in depressed patients using [123I]IBZM- Hence, mesolimbic dopaminergic neurons projecting to SPECT have found increased D receptor binding in the the nucleus accumbens have been suggested to be 2 striatum probably reflecting reduced dopamine func- involved in the neurobiology of depression and the tion in depression.11,12 Furthermore, dopaminergic therapeutic actions of some antidepressant drugs.3–6 drugs such as the dopamine-uptake inhibitors This hypothesis postulates that decreased dopamine amineptine, nomifensin and buproprion have been activity is involved in depression, while increased successfully used for treating major depression.13–15 dopamine function contributes to mania. Accordingly, Chronic treatment with antidepressant drugs pro- dopaminergic drugs (eg amphetamine or cocaine) can duces in rats a variety of changes in dopaminergic produce effects in humans that are remarkably similar neurotransmission, most notably a sensitization of behavioral responses to agonists acting at dopamine D /D receptors within the nucleus accumbens,6,16 an Correspondence: P Sokoloff, Unite´ de Neurobiologie et Pharmaco- 2 3 logie Mole´culaire (U.109) de l’Institut National de la Sante´et increased electrophysiological activity of mesolimbic 17,18 Recherche Me´dicale, Centre Paul Broca, 2ter rue d’Ale´sia, 75014 dopaminergic neurons, an increase in D2/D3 recep- Paris, France tor binding sites19 and an increase in interstitial dopa- See also the Image page, Molecular Psychiatry 2000; 5: 229 mine concentrations in the nucleus accumbens.20,21 *These two authors have contributed equally to this paper 2Present address: Zentrum fur Nervenheilkunde, Klinik fur Neur- Similar results have been obtained after chronic ¨ ¨ 22–24 ologie, Rudolf-Bultmann Str 8, 35039 Marburg, Germany ECT. Received 3 January 2000; revised and accepted 17 March 2000 The D3 receptor is expressed mainly in the limbic Increase of dopamine D3 receptor expression C-H Lammers et al 379 ventral part of the striatal complex, particularly in the wet weight using 14Cor3H standard stripes shell of the nucleus accumbens,25,26 thought to be (Amersham). involved in reward, emotional, and cognitive pro- cesses.27 A recent study indicates that antidepressant Statistical analysis ± drugs administered repeatedly enhance D3 receptor Values reported are means SEM. Differences between binding.28 groups of animals treated with saline or antidepressant In this study we have examined the changes in cer- drugs were analyzed by one-way ANOVA followed by ebral expression of dopamine receptors, as well as a Dunnett’s multiple comparison post-hoc test when P Ͻ those in dynorphin and substance P, two neuropep- 0.05. Analysis of variations of D3 receptor mRNA tides whose expression is regulated by dopamine, in with treatment duration was performed by two-way rats receiving various kinds of antidepressant treat- ANOVA followed by a Least Significant Difference test. ment, including monoamine uptake inhibitors, a Effects of electroconvulsive shocks were analysed by monoamineoxidase inhibitor and ECT. Since responses the Mann–Whitney U test. to antidepressant drugs emerge in humans after several weeks of treatment, we have compared the effects of Results these drugs produced by either acute or repeated administrations. A daily regimen of antidepressant drugs of various chemical classes with different pharmacological pro- files (see Table 1) was administered for 21 days, and Materials and methods dopamine D1,D2 and D3 receptor mRNA levels meas- ured by quantitative in situ hybridization studies 24 h Drug treatments after the last injection. Hybridization signals were ana- Male Wistar rats (IFFA Credo, l’Arbresles, France (180– lyzed in several brain regions as specified in Figure 1e. 200 g)) were housed in groups of five in a temperature- As exemplified with amitriptyline (Figure 1a, b), D controlled room on a 12 h : 12 h light : dark schedule, 3 receptor mRNA level increased prominently in the light starting at 7.00 am, with free access to food and shell part of nucleus accumbens (particularly in the water. They received daily intraperitoneal injections ventral part) and ventromedial part of the striatum, (between 4.00 and 6.00 pm) of either desimipramine − − whereas lesser effects were produced in the core of (15 mg kg 1) or imipramine (15 mg kg 1), amitriptyline − − nucleus accumbens. Analysis of in situ hybridization (15 mg kg 1), fluoxetine (15 mg kg 1) or tranylcyprom- − signals at high magnification (Figure 1c, d) indicated ine (7.5 mg kg 1) for 1, 5, 10, 21 or 42 days. Rats were an increased D receptor mRNA per cell and not an killed by decapitation 24 h after the last injection and 3 increased number of D receptor mRNA-expressing the brain rapidly removed and frozen by dipping in 3 cells. isopentane maintained at −30°C. After 21-day treatments, other mixed norepinephrine/ serotonin uptake inhibitors, ie desip- Electroconvulsive shocks ramine and imipramine, as well as the monoaminoxid- Electroconvulsive treatment (85 V, 50 Hz, 0.05 s) was ase inhibitor tranylcypromine all increased, like ami- administered using ear clip electrodes in non anes- triptyline, D3 receptor mRNA by 35–54% in the shell thetised rats once a day for 10 days. Each treatment of nucleus accumbens, but fluoxetine produced an caused a typical tonic clonic convulsion for 20–35 s. opposite effect (Table 1). In addition, some compounds Sham treatment consisted in application of electrodes also increased D3 receptor mRNA in some other without current. Rats were killed 24 h after the last regions, ie desipramine in the fronto-parietal cortex, treatment and their brain rapidly dissected and frozen. septum and olfactory tubercle and islands of Calleja. No changes were observed in the dorso-lateral In situ hybridization and receptor autoradiography striatum. Coronal tissue sections (10 ␮m in striatal region and On the contrary, when imipramine, amitriptyline 20 ␮m in mesencephalic region) were cut on a cryostat and tranylcypromine were administered only once, and thaw-mounted on RNAse-free slides. Sections they elicited a decrease in the D3 receptor mRNA level were used either unfixed (in receptor autoradiography in the shell of nucleus accumbens; imipramine also experiments) or fixed (in in situ hybridization decreased D3 receptor mRNA in the islands of Calleja experiments) in 4% paraformaldehyde. In situ (Table 2). Analysis of the changes produced during hybridization experiments were performed with spe- prolonged treatment by amitriptyline (Figure 2) indi- 33 cific [ P] cRNA probes for D1,D2 and D3 receptor, cated that after an initial reduction, D3 receptor mRNA dynorphin or substance P mRNAs as described.25,29 in the shell of nucleus accumbens was rapidly nor- D3 receptor binding autoradiography was performed malized within