Ethanol-induced behavioral sensitization is associated with dopamine receptor changes in the mouse olfactory tubercle

Physiology & Behavior 96 (2009) 12–17

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Ethanol-induced behavioral sensitization is associated with dopamine receptor changes in the mouse olfactory tubercle☆ Nilza Pereira de Araujo a,1, Daniela Fukue Fukushiro a,1, Christian Grassl a, Débora C. Hipólide b, Maria Lúcia O. Souza-Formigoni b, Sergio Tufik b, Roberto Frussa-Filho a,⁎ a b

Department of Pharmacology, Universidade Federal de São Paulo, R. Botucatu, 862, Ed. Leal Prado, 1° andar, 04023062, São Paulo-SP, Brazil Department of Psychobiology, Universidade Federal de São Paulo, R. Napoleão de Barros, 925, Vl. Clementino, 04024002, São Paulo-SP, Brazil

a r t i c l e

i n f o

Article history: Received 31 March 2008 Received in revised form 29 July 2008 Accepted 30 July 2008 Keywords: Behavioral sensitization D2 binding Ethanol Olfactory tubercle

a b s t r a c t Accumulating evidence points to the mesolimbic and the nigrostriatal dopamine systems as critical to behavioral sensitization induced by several drugs of abuse. In the present study, we analyzed D1 and D2 binding to brain regions related to these dopaminergic systems during the expression of ethanol-induced behavioral sensitization. The first experiment was performed to demonstrate the effectiveness of the ethanol treatment schedule and challenge used to induce the expression of the behavioral sensitization phenomenon. The second experiment was conducted to study D1 and D2 alterations in several brain regions during the expression of this phenomenon. Mice were ip treated with ethanol or saline for 21 consecutive days and 24 h after the last injection they received an ethanol or a saline challenge injection. Five minutes later, the animals were observed in an open-field for locomotion quantification or were sacrificed and their brains were submitted to autoradiographic binding analyses. No differences among the groups were found for D1 binding levels in all the brain regions analyzed. However, ethanol-sensitized mice showed reduced levels of D2 binding in the olfactory tubercle when compared to the other groups. Our data suggest that D2 receptor changes in the olfactory tubercle seem to play an important role in the expression of ethanol-induced behavioral sensitization. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Although many effects elicited by repeated drug treatments exhibit tolerance after some time, the psychomotor and positive reinforcing effects of a number of drugs of abuse often become greater with repeated administration [1–4]. The latter phenomenon is named behavioral sensitization and is usually behaviorally expressed by an increase in drug-induced hyperactivity or stereotypy of rodents [4–7]. Undoubtedly, psychostimulants have been the focus of most of research in the field of the behavioral sensitization phenomenon. Consequently, very little importance has been given to sensitization to the locomotor stimulant effect of ethanol. One possible reason for the lack of attention paid to this drug could be its complex effects on various neurotransmission systems, such as catecholaminergic, opioid, glutamatergic, serotonergic, GABAergic and cholinergic systems. In this respect, the effects of ethanol on dopaminergic neurotransmission seem to be of great relevance to its locomotor stimulant effect as well as to its

☆ Financial support: CNPq, FAPESP, CAPES, AFIP, FADA. ⁎ Corresponding author. Departamento de Farmacologia—UNIFESP, Rua Botucatu, 862-Ed. Leal Prado 1° andar-04023062-São Paulo, SP-Brazil. Tel.: +55 11 5549 4122x219; fax: +55 11 5549 4122x222. E-mail address: [email protected] (R. Frussa-Filho). 1 The first two authors had the same contribution to this study. 0031-9384/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2008.07.029

reinforcing properties [8]. Usually, high doses of ethanol produce sedation and hypnosis, which are subjected to tolerance after repeated treatment. Conversely, low doses produce behavioral stimulation, which is sensitized after repeated injections [9,10]. Behavioral sensitization has been suggested to be a useful animal model to study drug craving in humans [11], since the brain circuitries involved in psychomotor stimulation and in reward seem to be closely related or even identical [12]. Within this context, ample evidence suggests that the mesolimbic and the nigrostriatal dopamine systems mediate most of the neuroadaptations related to behavioral sensitization induced by distinct drugs of abuse [4,13–16]. Furthermore, alterations in brain dopamine receptors, particularly D1 and D2, in the mesolimbic and the nigrostriatal dopamine systems, have been generally associated with chronic treatment with drugs of abuse in humans [17–19] and in animals [20–22]. Specifically concerning ethanol, there are some accounts in the literature showing changes in D1 and/or D2 binding to different brain regions related to reward in ethanol-sensitized animals [23–25]. Notwithstanding, as far as we know, all of them examined dopamine receptor binding when animals were withdrawn from ethanol treatment for different periods of time, and not at the time point of the behavioral expression of sensitization. Importantly, no significant behavioral alteration has been reported in mice withdrawn from repeated treatment with “stimulant doses” of ethanol [26]. Therefore, receptor binding assays

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performed at the moment of the expression of behavioral sensitization (i.e., right after the ethanol challenge injection in ethanol pre-treated animals, when sensitization is behaviorally expressed) would be of special interest for better elucidation of mechanisms underlying the development and the expression of ethanol-induced behavioral sensitization. In the present study, we have examined the pattern of D1 and D2 binding to brain regions belonging to the mesolimbic (ventral tegmental area, nucleus accumbens and olfactory tubercle) and the nigrostriatal (substantia nigra and caudate-putamen) dopamine systems during the expression of behavioral sensitization induced by ethanol in mice. 2. Materials and methods 2.1. Animals Three-month-old Swiss EPM-M1 female mice were used. The animals were housed in groups of 15 in polypropylene cages (41 × 34 × 16.5 cm) containing white pine bedding, with free access to food and water, in a room with controlled temperature (22–23 °C) and under a 12/12 h light/dark cycle (lights on at 07:00 h). Female mice were used mainly because it has been demonstrated that behavioral sensitization is higher in female animals [4]. Animals used in this study were maintained in accordance with the guidelines of the National Institute of Health Guide for Care and Use of Laboratory Animals (NIH Publications N 85-23, revised 1996). In addition, all efforts were made to minimize the number of animals used and their suffering. A total of 60 mice were used in the present study (30 for experiment 1 and 30 for experiment 2). 2.2. Drugs Ethanol (MERCK®) was administered in a 12% w/v solution diluted in saline. Saline was used as control solution. The solutions were given intraperitoneally in the volume of 10 ml/kg body weight. 2.3. Experimental procedure 2.3.1. Experiment 1: Behavioral sensitization induced by ethanol repeated administration in mice Thirty female Swiss mice were randomly allocated to 3 groups of 10 animals each: SAL–SAL, SAL–EtOH and EtOH–EtOH. On days 1–21 animals from the SAL–SAL and SAL–EtOH groups received an intraperitoneal (i.p.) injection of saline (SAL) and animals from the EtOH–EtOH group received an i.p. injection of 1.8 g/kg ethanol (EtOH) once a day. On day 22, the SAL–SAL group received an i.p. challenge injection of SAL whereas the SAL–EtOH and EtOH–EtOH groups received an i.p. challenge injection of 1.8 g/kg EtOH and, 5 min later, they were placed into the open-field for locomotor activity quantification in a 5-min session. During the 5-min period between the challenge injection and behavioral testing, mice were individually placed in separate boxes. During ethanol or saline repeated treatment animals were maintained in optimum housing conditions (see references [27,28]) for the development of ethanol-induced behavioral sensitization. Due to the short-lasting stimulant effect of ethanol on mice’s locomotor activity, the quantification of this behavioral parameter for 5 min has been shown to be effective and sufficient to demonstrate ethanolinduced hyperlocomotion and its sensitization under our laboratory conditions [27–29]. Similarly, the ethanol dose and the withdrawal period between repeated treatment and challenge session (24 h) were chosen on the basis of previous studies of our research group, which succeeded to demonstrate behavioral sensitization to ethanol in mice [26,30].

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Behavioral test Five minutes after the ethanol challenge injection, the animals were individually placed in the center of the open-field arena for direct quantification of locomotion frequency for 5 min. The openfield apparatus used in the present study was a circular wooden box (40 cm in diameter and 50 cm high) with an open top and floor divided into 19 squares. Hand-operated counters were used to score locomotion frequency (number of floor units entered) during the 5min session. The observer was always unaware of the experimental design. The animals were used only once. The behavioral test took place between 08:00 a.m. and 11:00 a.m. and a 60-min habituation of the animals to the testing room was allowed before its beginning. Locomotion frequency quantified in the open-field apparatus has been demonstrated to be a very effective method to evaluate behavioral sensitization induced by ethanol [26,27] or other drugs of abuse [6,26,31,32]. In addition, open-field locomotion of rodents has been extensively proven to be a very sensitive behavioral parameter to evaluate the effects of drugs acting on dopamine systems [33–36]. 2.3.2. Experiment 2: D1 and D2 binding analyses during the expression of ethanol-induced behavioral sensitization in mice Other 30 female Swiss mice were used in this experiment, which followed the same protocol of the experiment 1, except that 5 min after the challenge injection, instead of being submitted to the behavioral test, mice were sacrificed by decapitation and had their brains removed for autoradiographic D1 or D2 binding analyses. Autoradiographic binding assays Five minutes after the ethanol challenge injection, mice were sacrificed by decapitation and their brains were immediately removed, frozen over dry ice and stored at −80 °C until cryostat sectioning. Serial 20 μm coronal sections were cut on Leica cryostat at −20 °C, collected onto glass slides and stored at −80 °C until the day of the assays. D1 binding was analyzed with [3H] SCH 23390, as previously described by Quadros et al. [37]. D2 binding analyses were conducted using [3H] Raclopride, as described by SouzaFormigoni et al. [24] earlier. Briefly, slices were brought to room temperature and then pre-incubated in 50 mM Tris–HCl (120 mM NaCl·4 mM MgCl2·1 mM EDTA·1.5 mM CaCl2·2H2O) buffer (pH = 7.4) for 30 (D1) or 15 (D2) minutes. Sections were then incubated in buffer containing 2 nM [3H] SCH 23390 (86.0 Ci/mmol, Perkin Elmer Life Science, Boston, USA) for 90 min at 37 °C (D1) or in buffer containing 2 nM [3H] Raclopride (76.0 Ci/mmol, Perkin Elmer Life Science, Boston, USA) for 2 h at room temperature (D2). Additional sets of slices were incubated in the presence of either 2 μM butaclamol (D1) or 10 μM sulpiride (D2), for determination of nonspecific binding. Sections were then washed once (D1) or twice (D2) in buffer for 5 min, followed by one quick dip rinse of ice-cold distilled water before drying. Slices were exposed to Kodak Biomax MR-1 in tungsten cassettes together with calibrated standards for 4 weeks. All groups were represented in each film. Films were developed and densitometric analyses performed using the MCID system (Imaging Research, St. Catherine's, Ontario, Canada). At least five sections were measured to obtain values for each mouse for each region. Anatomical regions were defined according to the mouse brain atlas of Franklin and Paxinos [38]. 2.4. Statistical analyses For the behavioral test, one-way ANOVA followed by Duncan’s test was used. For autoradiographic binding assays, parametric data were treated using one-way ANOVA followed by Tukey-HSD test and nonparametric data were treated with one-way ANOVA followed by Dunnett-T3 test. A p value less than 0.05 was considered as a statistically significant difference for all comparisons made. Still concerning the

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N.P. de Araujo et al. / Physiology & Behavior 96 (2009) 12–17 Table 1 [3H] SCH-23390 binding to D1 receptors after a saline or an ethanol challenge injection in saline- or ethanol-treated mice

Caudate-putamen Anterior Posterior Dorsomedial Dorsolateral Ventrolateral Nucleus accumbens Core Shell Olfactory tubercle Substantia Nigra Ventral tegmental area

SAL–SAL n = 10

SAL–EtOH n = 10

EtOH–EtOH n = 10

P

15.6 ± 1.1 14.8 ± 0.4 15.4 ± 0.7 16.6 ± 0.5 16.1 ± 0.3 10.9 ± 0.4 12.8 ± 0.3 9.3 ± 0.5 12.0 ± 0.4 7.5 ± 1.4 1.0 ± 0.2

15.7 ± 0.6 15.0 ± 0.4 15.8 ± 0.5 16.9 ± 0.5 16.7 ± 0.4 11.6 ± 0.7 13.0 ± 1.0 7.6 ± 0.6 13.7 ± 0.4 9.9 ± 0.3 0.7 ± 0.1

15.5 ± 1.8 15.1 ± 0.4 15.6 ± 0.7 16.2 ± 0.8 15.6 ± 0.7 11.2 ± 0.7 13.0 ± 0.6 8.9 ± 0.7 13.4 ± 0.6 10.3 ± 0.3 1.0 ± 0.1

0.998 0.839 0.930 0.745 0.351 0.726 0.979 0.222 0.044 0.042 0.353

Values represent means ± SEM in pmol/g tissue. One-way ANOVA followed by Tukey-HSD or Dunnett-T3 test. Fig. 1. Locomotion frequency of mice which were treated with 1.8 g/kg ethanol (EtOH) or saline (SAL) for 21 consecutive days and received a challenge injection of 1.8 g/kg ethanol (-EtOH) or saline (-SAL) 24 h later. ⁎p b 0.001 compared to the SAL–SAL group. #p b 0.001 compared to the SAL–EtOH group. One-way ANOVA followed by Duncan’s test.

autoradiographic binding assays, in which one-way ANOVA was conducted on each of the 11 regions and the risk of a false positive (type-I errors) was higher, a correction factor (Bonferroni test) was also conducted for statistical significant findings. 3. Results 3.1. Experiment 1: Behavioral sensitization induced by ethanol repeated administration in mice Fig. 1 shows the effects of a challenge injection of 1.8 g/kg ethanol or saline on locomotion frequencies of mice repeatedly treated with

1.8 g/kg ethanol or saline. One-way ANOVA detected significant differences among the three groups in this behavioral parameter [F (2,27) = 13.06, p b 0.001]. Duncan’s post hoc test revealed that the EtOH–EtOH group presented a significant increase in locomotion frequency when compared to the other groups. In addition, the SAL– EtOH group exhibited higher locomotion frequency than that observed for the SAL–SAL group. 3.2. Experiment 2: D1 and D2 binding analyses during the expression of ethanol-induced behavioral sensitization in mice Fig. 2 illustrates the typical appearance of [3H] SCH-23390 and [3H] Raclopride binding at rostrocaudal levels of mice’s brains. Table 1 shows [3H] SCH 23390 binding to D1 receptors in several brain regions after an ethanol or a saline challenge injection in ethanol- or saline-treated mice. One-way ANOVA detected significant differences among the three groups in [3H] SCH 23390 binding to D1 receptors in the olfactory tubercle [F(2,27) = 3.50, p b 0.05) and in the substantia nigra [F(2,27) = 3.65, p b 0.05]. However, according to the Tukey-HSD test for the olfactory tubercle data and the Dunnett-T3 test for the substantia nigra data, no significant differences among groups were observed in these brain regions. As shown in Table 2, one-way ANOVA detected significant differences among the groups in [3H] Raclopride binding to D2 receptors only in the olfactory tubercle [F(2,27) = 4.13, p b 0.05]. The Tukey-HSD test revealed that the ethanol-sensitized group (EtOH– EtOH) presented significant reduced D2 binding values in the olfactory tubercle when compared to all the other groups.

Table 2 [3H]Raclopride binding to D2 receptors after a saline or an ethanol challenge injection in saline- or ethanol-treated mice SAL–SAL n = 10 SAL–EtOH n = 10 EtOH–EtOH n = 10 P

Fig. 2. Distribution of [3H]SCH 23390 (D1) and [3H]Raclopride (D2) binding at comparable rostrocaudal levels in normal mice’s brains. Acb = accumbens; CPu DL = caudate-putamen dorsolateral; CPu VL = caudate-putamen ventrolateral; CPu P = caudate-putamen posterior; Tu = olfactory tubercle; SN = substantia nigra.

Caudate-putamen Anterior Posterior Dorsomedial Dorsolateral Ventrolateral Nucleus accumbens Core Shell Olfactory tubercle Substantia Nigra Ventral tegmental area

5.9 ± 0.2 6.4 ± 0.3 5.5 ± 0.2 7.8 ± 0.2 7.8 ± 0.3 4.1 ± 0.2 4.6 ± 0.2 3.5 ± 0.2 3.8 ± 0.2 1.9 ± 0.1 0.6 ± 0.1

5.7 ± 0.2 6.4 ± 0.2 5.5 ± 0.2 7.7 ± 0.3 7.9 ± 0.3 3.9 ± 0.2 4.0 ± 0.2 3.1 ± 0.3 3.7 ± 0.2 1.7 ± 0.1 0.7 ± 0.1

5.7 ± 0.4 6.2 ± 0.3 5.5 ± 0.2 7.4 ± 0.2 7.6 ± 0.2 3.7 ± 0.2 4.1 ± 0.2 2.9 ± 0.3 3.1 ± 0.2 1.7 ± 0.1 0.7 ± 0.1

Values represent means ± SEM in pmol/g tissue. One-way ANOVA followed by Tukey-HSD test. ⁎ p b 0.05 compared to the SAL–SAL and SAL–EtOH groups.

0.840 0.841 0.965 0.412 0.686 0.365 0.116 0.290 0.030⁎ 0.455 0.557

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4. Discussion The most important finding of this study was that mice which received an ethanol challenge injection after repeated treatment with this drug showed lower levels of D2 binding in the olfactory tubercle. As far as we know, this is the first paper examining the pattern of D1 and D2 binding in different brain regions at the time point that ethanol-treated (i.e., previously “sensitized”) animals received an ethanol challenge injection. This methodological issue is very relevant since it allowed us to study D1 and D2 receptor alterations during the expression of ethanol-induced behavioral sensitization. This phenomenon was behaviorally characterized in our study by an increase in locomotor activity of ethanol-treated animals in response to a challenge injection of this drug when compared to ethanol-challenged mice previously treated with saline (Fig. 1). Using this experimental protocol we found that D2 binding levels in the olfactory tubercle are decreased during the expression of ethanol-induced behavioral sensitization. Concerning a methodological issue, one could argue that the behavioral response analyzed (i.e., an increase in locomotion of ethanoltreated and -challenged mice, observed 5 min after ethanol injection in a 5-min session) would be in fact a reflection of the differences in anxietyrelated behaviors of animals withdrawn from ethanol versus animals not withdrawn from ethanol. However, this possibility seems unlikely since in our previous report [28], using the same subjects and experimental protocol, we have demonstrated that ethanol- and saline-treated mice presented similar locomotion frequencies in response to a saline challenge injection. We should state here that a group consisting of ethanol-treated mice challenged with saline (EtOH–SAL) was not included in the present study due to ethical purposes, since we have already demonstrated that under these same experimental conditions previous treatment with ethanol did not modify spontaneous locomotion of mice [28]. Thus, it seems unlikely that under our experimental conditions ethanol-treated mice developed a conditioned locomotor response to injection or an enhanced stress response to injection as a consequence of ethanol withdrawal. The use of female mice without concurrent estrous cycle tracking in our study could also raise the question whether neurochemical changes over the course of the estrous cycle might have contributed to the results obtained. Within this context, the pharmacokinetics of and motivation to self-administer ethanol are known to vary over the course of the rodent estrous cycle [39,40]. Despite evidence in the literature on the participation of estrous cycle in ethanol-induced behaviors, we have not tracked it during the experiments because stress induced by estrous cycle tracking would insert a confounding variable on our data. Within this context, it has been extensively demonstrated that different types of stress can potentiate the behavioral sensitization phenomenon [41–43]. Within this respect, since female rodents tend to develop more robust sensitization than male rats [44,45], female animals are frequently used (without estrous cycle tracking) to study the behavioral sensitization phenomenon, particularly ethanol-induced behavioral sensitization [46–49]. An interesting possibility to explain the reliability of such studies using female rodents may be the fact that the ovarian cycles of female rodents become synchronized when they live together, as do the cycles of many other mammals [50]. Concerning a methodological issue, it should be noted that since we used a single concentration of radiolabeled ligand, it is not possible to interpret our binding results as a change in receptor number or affinity. This concern notwithstanding, autoradiographic studies generally use only one concentration of the ligand, chosen from pharmacologic characterization in previous studies using homogenates. This chosen concentration leads to occupation of a high percentage of receptors (generally three times the Kd value). Although evaluation of receptor function was not performed in the current

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study, this specific procedure provides a reliable evaluation of receptor binding [24,37]. Our result showing that D2 binding in the olfactory tubercle diminished in ethanol-sensitized animals suggests that changes in dopamine receptors in this region in response to ethanol treatment may play an important role in behavioral sensitization to this drug. In the last years, a great deal of importance has been given to D1 and D2 dopaminergic receptors, relating them to mechanisms surrounding behavioral sensitization to drugs of abuse, especially psychostimulants. However, most, if not all, studies have focused on D1 and/or D2 receptor alterations in structures such as the nucleus accumbens and the caudate-putamen. For instance, several authors have demonstrated that the development of a D1 functional supersensitivity in the nucleus accumbens seems to be a molecular marker for psychostimulants-induced behavioral sensitization [51–54]. In addition, Henry et al. [55] demonstrated that the D1 functional supersensitivity in the nucleus accumbens is accompanied and potentiated by the development of a functional subsensitivity of D2 autoreceptors in the ventral tegmental area. Regarding ethanol and its effects on D1 and D2 receptors, data from literature are contradictory. Thus, although ethanol increases the firing of dopamine neurons in the ventral tegmental area [56–58] and elevates extracellular dopamine concentration in the nucleus accumbens [59], elevation, reduction or no alteration in D1 and D2 binding have been reported after chronic treatment with this drug in several strains of rats and mice [21,24,37, 60–66]. More specifically, Souza-Formigoni et al. [24] observed an increase in D2 binding to the anterior and ventrolateral caudate-putamen of ethanol-sensitized Swiss mice 24 h after an ethanol challenge injection, i.e., 24 h after the expression of behavioral sensitization to ethanol (or during withdrawal from chronic treatment with ethanol). No alteration in D1 binding was found in any of the brain regions analyzed (caudateputamen, nucleus accumbens, olfactory tubercle, substantia nigra and others) in Swiss mice by Quadros et al. [37] using this same protocol of experiments. Likewise, Bailey et al. [23] found no alterations in D1 or D2 binding in the striatum of TO mice withdrawn from chronic ethanol consumption for 6 or 21 days. Nevertheless, Vasconcelos et al. [25] found decreases in D1 and D2 binding levels in the striatum of Wistar rats at a 48-h withdrawal from treatment with a high dose of ethanol. In this same study, withdrawal from the treatment with a lower dose of ethanol diminished only striatum D2 binding levels. The contradictory results obtained by these studies may be due to different subjects or strains of rodents used or, more likely, to different periods of withdrawal applied, which therefore might have accounted for the development of distinct neuroadaptations in D1 and D2 receptors. In spite of the contradictory findings about the effects of withdrawal from ethanol treatment on D1 and D2 receptors, our data suggest that repeated treatment and subsequent challenge with this drug do not modify D1 binding and specifically decrease D2 binding in the olfactory tubercle. It should be noted that although in previous studies [24,37] we have demonstrated an increase in D2 binding in the caudate-putamen and no alterations in D1 binding in any brain region using the same chronic ethanol administration protocol, those changes seem to be more closely related to abstinence from ethanol. Conversely, the different results obtained in the present study (a decrease in D2 binding in the olfactory tubercle) seem to be more closely associated with the expression of ethanol-induced behavioral sensitization. The present result emphasizes the importance of the olfactory tubercle, a usually neglected structure, on addiction processes and the behavioral sensitization phenomenon at least related to ethanol repeated administration. In fact, although both the nucleus accumbens and the olfactory tubercle receive efferences from dopamine neurons of the ventral tegmental area [67], only the former is usually considered a critical structure involved in the reinforcing effects of

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drugs of abuse [see reference [68] for review] and behavioral sensitization processes [55]. Notwithstanding, studies conducted by Ikemoto's group have demonstrated that both the nucleus accumbens and the olfactory tubercle may play important roles in the rewarding effects of some drugs and in addiction processes [69,70]. In those studies, it was verified that naïve rats quickly learned to selfadminister amphetamine or high doses of cocaine infused into the medial accumbens shell or the medial olfactory tubercle. Conversely, another paper from Ikemoto's group [71] has demonstrated that, for MDMA, only the medial accumbens shell appears to support its selfadministration in naïve rats. Future studies involving repeated and/or challenge local injections of ethanol into the olfactory tubercle may clarify the specific role of this brain region in the ethanol-induced behavioral sensitization phenomenon. Some data from the literature have also demonstrated the importance of the olfactory tubercle in the locomotion of rodents, which is closely related to the behavioral sensitization phenomenon and, therefore, to addiction. In this regard, microinjections of amphetamine, dopamine or other direct dopamine agonists into the olfactory tubercle can facilitate locomotion [72]. Likewise, injections of mixtures of the D1 and D2 agonists SKF 38393 and quinpirole, or cocaine, into the olfactory tubercle (particularly the medial portion) induced vigorous locomotion in rats [73] and Cools [74] has demonstrated that injections of dopamine or the dopamine agonist apomorphine into the olfactory tubercle, but not into the nucleus accumbens, consistently increased rats’ locomotor activity in a familiar environment. Those data seem to be contradictory to the decrease in D2 receptor binding herein observed in the olfactory tubercle of ethanol-“sensitized” mice (i.e. with increased locomotor activity). However, the possibility may be raised that such a decrease in D2 binding is related to D2 dopamine autoreceptors, which is an interesting working hypothesis. To the extent that the observed receptor change in the olfactory tubercle may be relevant to ethanol-induced behavioral sensitization, our data would suggest that changes in the nucleus accumbens would not be critically involved in this specific process. This would be in line with the accumulating evidence suggesting that perhaps alterations in the nucleus accumbens would not be necessary to the development of ethanol addiction. In this regard, while rats self-administer ethanol in the ventral tegmental area [75], studies of 6-OHDA lesions clearly demonstrate that denervation of the nucleus accumbens does not interfere with ethanol consumption [76]. Likewise, in rats, the elevation of dopamine levels in the nucleus accumbens induced by a selective inhibitor of dopamine re-uptake does not modify ethanol self-administration [77]. According to Weiss and Porrino [78], these results indicate that ethanol self-administration is not dependent on dopamine activation in the nucleus accumbens. In summary, our results demonstrated reduced levels of D2 binding in the olfactory tubercle of mice presenting behavioral sensitization to ethanol. It is not clear how important those changes are for locomotion or behavioral sensitization to ethanol, requiring further investigation. Indeed, an association between changes in dopamine D2 receptor expression and a change in behavioral response does not prove a cause-and-effect relationship. This concern notwithstanding, our data emphasize that more studies of ethanol addiction should focus in other brain regions rather than the nucleus accumbens. Acknowledgements This research was supported by fellowships from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), from the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), from the Fundo de Apoio ao Docente e Aluno (FADA), and from the Associação Fundo de Pesquisa em Psicobiologia (AFIP). The authors would like to thank Ms. Teotila R. R. Amaral, Mr. Cleomar S. Ferreira and Mr. Antônio R. Santos for capable technical assistance.

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