-Intra nucleus accumbens, but not i.p. administration of 5-HT2C receptor antagonist (SB-242084) blocks the expression of ethanol-induced behavioral sensitization in Albino Swiss mice.
Behavioral sensitization to the stimulant effect of ethanol (EtOH) or other drugs, observed in mice as an increase in locomotor activity after repeated drug administration, has been associated with neuroadaptations in the dopaminergic mesolimbic pathway. In the nucleus accumbens (NAc), afferent region of mesolimbic pathway, dopamine release can be modulated by serotonergic 2C receptors (5-HT2CR). The aim of the present study was to evaluate the function of 5-HT2CR in the expression of EtOH-induced behavioral sensitization in Swiss Albino Webster mice with different levels of sensitization to EtOH. In the four experiments performed, mice were treated with saline or 2.2 g/kg EtOH daily for 21 days. According to their locomotion on the 21st day of treatment, EtOH-pretreated mice were classified into two groups: highly-sensitized and low-sensitized to the stimulant effect of EtOH. In each experiment, two weeks after the end of the 21-day treatment (washout period), mice were submitted to four pharmacological challenges, with 2 drug administrations in each one. In experiments 1 and 2, mice received both intraperitoneal (i.p.) administrations and in experiments 3 and 4 they received the first administration intra-NAc and the second one i.p. The four challenges were: saline + saline; saline + EtOH; SB-242084 (5-HT2CR antagonist) i.p. (0.5; 1.0 or 2.0 mg/kg) or intra-NAc (1.0 or 2.0 mg/side) + EtOH; and SB-242084 (0.5; 1.0 or 2.0 mg/kg i.p.) or (1.0 or 2.0 mg/side intra-NAc) + saline. In all doses, i.p. administration of SB-242084 did not block the stimulant effect induced by EtOH in highly-sensitized mice. However, only in the highly-sensitized group, did intra-NAc administration of SB-242084 reduce (1.0 mg/side) or block (2.0mg/side) the expression of EtOH behavioral sensitization. These findings suggest that the expression of behavioral sensitization to the stimulant effect of EtOH depends on the integrity of accumbal 5-HT2CR function.
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Drug addiction is characterized by neurobehavioral adaptations such as craving, seeking and ingestion in spite of negative consequences. Some authors attribute the increase in the frequency of these behaviors to neuroadaptations (Wise and Bozarth, 1987). In addition, this phenomenon has been related to the euphorigenic properties of drugs of abuse in human beings ( Wise and Bozarth, 1987). In animal models, the progressive increase in locomotor activity observed after repeated administration of some psychotropic drugs (known as behavioral sensitization), has been associated with the reinforcing/stimulant properties of these drugs (Masur and Boerngen, 1980, Crabbe et al., 1982, Masur and dos Santos, 1988, Phillips et al., 1994)..The behavioral adaptation underlying sensitization has been associated with neurochemical adaptations that can remain for weeks or months (Pierce and Kalivas, 1997). Repeated EtOH administration induces behavioral sensitization (Masur et al., 1986, Souza-Formigoni et al., 1999, Gonzales et al., 2004). The development and expression of EtOH-induced behavioral sensitization can be influenced by different neurotransmitters , including the serotonergic system. Some authors observed that miaserin, fluoxetine or paroxetine can block the expression of EtOH-induced behavioral sensitization in mice by inhibition of serotonin reuptake transporter (Goeldner et al., 2005, Ferraz and Boerngen-Lacerda, 2008). However, when mice were treated chronically with fluoxetine or paroxetine they presented an increase in the EtOH stimulant effect (Goeldner et al., 2005). These opposite effects suggest that the serotoninergic modulation of behavioral sensitization is a complex phenomenon.
Many studies have highlighted the involvement of 5-HT2 family receptors in drug addiction. The 5-HT2CR subtype is aÂ Gq protein-coupled and mediates excitatory neurotransmission (Berg et al., 2001). Systemic administration of 5-HT2CR antagonists potentiates the locomotor stimulant effects of cocaine or amphetamine (Fletcher et al., 2002, Filip et al., 2006). Indeed, 5-HT2CR knockout mice show higher sensitivity to the locomotor stimulant effect of cocaine and higher cocaine-induced release of dopamine (DA) in the NAc than the wild type mice (Rocha et al., 2002).
The mesolimbic DA reward pathway, whose neurons project from ventral tegmental area (VTA) to NAc and prefrontal cortex 5), has a critical role in the reinforcing properties of drugs of abuse and in the development and expression of behavioral sensitization (Di Chiara, 1999). The mRNA and protein for 5-HT2CR are densely expressed in the mesolimbic DA system (Clemett et al., 2000) and exert a critical role on the modulation of the reward system (Higgins and Fletcher, 2003). Specific 5-HT2CR antagonist enhances the firing rate of VTA dopaminergic neurons and DA concentration in the NAc induced by cocaine (Navailles et al., 2004), phencyclidine (Hutson et al., 2000), amphetamine or morphine (Porras et al., 2002). On the other hand, accumbal 5-HT2CR seems to exert the opposite behavioral and neurochemical effect. When microinjected into the NAc, 5-HT2CR, antagonists reduce not only the cocaine-induced hyperactivity , McMahon et al., 2001; Filip and Cunningham, 2002), but also the expression of cocaine-induced sensitization (Zayara et al., 2011) and the accumbal DA efflux (Navailles et al., 2008).
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Although there are some studies on the mechanisms and factors involved in behavioral sensitization to psychostimulants, as regarding EtOH there is a paucity of studies and the data available suggest these mechanisms may be different depending on the drugs. No studies have investigated the role of accumbal 5-HT2CR on behavioral sensitization to EtOH. It is important to consider that behavioral sensitization to EtOH (as well as to other drugs such as amphetamine) does not occur in all animals from the same strain, neither in all strains or species (Piazza et al 1989). Our laboratory has been working with the individual variability to EtOH behavioral sensitization in Swiss Albino Webster mice, an outbred strain. Some studies detected behavioral and neurochemical differences between those mice that have a clear development of EtOH-induced behavioral sensitization and those that do not have or have low levels of sensitization (Masur and dos Santos, 1988, Souza-Formigoni et al., 1999, Quadros et al., 2002). Considering that 5-HT2CR exerts a critical role on the modulation of the reward system, we carried out this study in order to evaluate the effect of systemic and intra-NAc administration of a specific 5-HT2CR antagonist (SB-242084) on the stimulant effect of ethanol in highly-sensitized and low-sensitized mice. We hypothesized that, in mice treated chronically with EtOH, the administration of a specific 5-HT2CR antagonist intra NAc would reduce the expression of EtOH-induced behavioral sensitization only in those mice with clear development of EtOH sensitization.
Materials and methods
In this study we used 107 Swiss Albino Webster male mice derived from the colony provided by the Centro de Desenvolvimento de Modelos Experimentais (CEDEME), Universidade Federal de São Paulo (UNIFESP). The mice were 3 months old in the beginning of each experiment. The animals were housed in groups of 15-20 in plastic cages (44 X 34 X 16 cm), with free access to food and water. After surgical procedures, they were housed in groups of 4-6 in smaller plastic cages (30 X 19 X 13cm). All animals were kept in a temperature-controlled colony room (22 Â± 2 Â°C), with lights on between 07:00 am and 07:00 pm. All procedures were carried out in accordance with the National Institute of Health (NIH) Principles of Laboratory Animal Care (1985) and based on ethical principles in order to reduce the number of animals used and their suffering as much as possible. The project was approved by the Committee of Ethics in Research of the Universidade Federal de São Paulo (CEP 1585/08).
Ethanol (2.2 g/kg, SynthÂ®, Diadema, Brazil) solution was prepared with saline (0.9% w/v NaCl) to a 15% w/v solution. The 5-HT2CR antagonist, SB- 242084 (Tocris, Ellisville, Missouri, USA) named Indoline, was diluted in saline at concentrations targeting a volume of 10ml/kg for intraperitoneal (i.p) administration (0.5; 1.0 and 2.0 mg/kg). For intra-NAc administrations, SB-242084 was diluted in sterile saline (1 Âµg/0.2 Âµl and 2 Âµg/0.2 Âµl per side).
All locomotor activity tests were carried out in Opto-Varimex activity cages [model Opto-M3 (47.5 X 25.7 X 20.5 cm); Columbus Instruments, Columbus-OHIO, USA].
We used a well established model to induce and evaluate the development and expression of EtOH-induced behavioral sensitization in outbred mice, adopted in previous studies (Souza-Formigoni et al., 1999, Quadros et al., 2002, Quadros et al., 2003, Abrahao et al., 2009) Four experiments were carried out, with two drug challenges in each one. Before the drug challenges, all mice were submitted to the same procedures to develop behavioral sensitization, as reported in previous studies (Takahashi et al., 2007, Abrahao et al., 2009).
Development of EtOH-induced behavioral sensitization
Initially, all mice were submitted to a drug-free 15-min test in activity cages for assessment of basal horizontal locomotor activity levels. Mice were stratified according to their locomotor activity basal levels and weight and then allocated to the treatment groups (saline or EtOH, in the proportion of 1 for saline and 3 for EtOH). We adopted this procedure in order to guarantee the homogeneity of these groups and to reduce the influence of novelty reaction. Starting one day after baseline evaluation, mice received daily i.p. administrations of saline or EtOH (2.2 g/kg) for 21 days. Locomotor activity was measured for 15 min immediately after drug injection on days 1, 7, 14 and 21. According to their activity level on the 21st day of treatment, EtOH-treated mice were classified into 3 subgroups as done in previous studies (Takahashi et al., 2007, Abrahao et al., 2009) In the first group, named "highly-sensitized", we allocated mice whose activity scores were in the upper 33% of the locomotion levels distribution and were at least 10 times higher than the upper limit of the 95% confidence interval of the saline group. Those animals whose activity levels were in the lower 33% of the distribution and were at most 4 times higher than the upper limit of the 95% confidence interval of the saline group were classified in the group named "low-sensitized". The group named "intermediate", which was composed by animals classified in the middle 33% of the distribution was not included in the study REESCREVER A FRASE.
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After chronic treatment with ethanol or saline, mice were challenged with i.p. or intra-NAc administrations, as summarized in Figure 1. We performed four challenges in all the experiments, 48 hours apart. In the first one, animals received saline + saline (control). In the second challenge, mice received saline + EtOH to evaluate the levels of expression of EtOH-induced behavioral sensitization. In the third challenge, mice received SB-242084 + EtOH to evaluate the influence of systemic (experiments 1 and 2) or intra-NAc (experiments 3 and 4) blockade of 5-HT2CR on the expression of EtOH-induced behavioral sensitization. In the fourth challenge mice received SB-242084 + saline to evaluate the effect per se of SB-242084.
INSERT FIGURE 1
Experiment 1 and 2: Influence of systemic SB-242084 administration on the expression of EtOH-induced behavioral sensitization
Considering that previous studies (Fletcher et al., 2002, Porras et al., 2002, Fletcher et al., 2006) showed that i.p. administration of 5-HT2CR antagonists could potentiate the reinforcing effect of some drugs, we tested if highly-sensitized or low-sensitized mice could present different profiles of expression of EtOH-induced behavioral sensitization after 5-HT2CR antagonist administration. The doses were based on some studies with rats (Fletcher et al., 2002, Fletcher et al., 2006) and mice (Tecott et al., 1995; Nonogaki et al., 2007).
In Experiment 1, fourteen days after the end of EtOH-induced behavioral sensitization, mice previously treated with saline (n=14) or [EtOH=26 (14 highly- sensitized and 12 low-sensitized)] were submitted to four pharmacological challenges, each other day, in the following order: a) saline + saline; b) saline + EtOH (2.2 g/kg); c) SB-242084 (0.5 mg/kg) + EtOH (2.2 g/kg); d) SB-242084 (0.5 mg/kg) + saline. All substances were i.p. administered. .
Since we aimed at reducing the number of animals, we used the same mice to test SB-242084 at the dose of 1.0 mg/kg. Thus, one week after the last challenge (drug free period), mice entered in a second cycle of challenges in the following order: a) saline + saline; b) saline + EtOH (2.2 g/kg); c) SB-242084 (1.0 mg/kg) + EtOH (2.2 g/kg); d) SB-242084 (1.0 mg/kg) + saline.
In Experiment 2, we used a different group of mice to test the systemic administration of 2.0 mg/kg of SB-242084. This procedure was taken in order to prevent a possible rapid desensitization of 5-HT2CR as described by Berg et al. (2001). Mice previously treated with saline (n=10) or EtOH (13 highly-sensitized and 11 low-sensitized) were submitted to four challenges: a) saline + saline; b) saline + EtOH (2.2 g/kg); c) SB-242084 (2.0 mg/kg) + EtOH (2.2 g/kg); d) SB-242084 (2.0 mg/kg) + saline.
In all challenges of experiments 1 and 2, after the first drug administration mice were placed in plastic cages, remaining there for 30 min. After this period, mice were injected with saline or EtOH and their locomotor activity was measured for 15 min. These procedures were based on previous studies (Fletcher et al., 2002, Filip et al., 2004, 2006).
Experiments 3 and 4: Influence of Intra-NAc SB-242084 administration on the expression of EtOH-induced behavioral sensitization.
These experiments were conducted to evaluate if intra-NAc administration of SB-242084 could block the expression of behavioral sensitization to ethanol in mice with different levels of sensitization.
One day after the 21-day treatment with EtOH or saline, mice underwent the surgical procedure. Before placing the mice in the stereotaxic apparatus (model EFF-333, Insight Ltda., Brazil) we anaesthetized them with xylazine (11 mg/kg in 0.01 ml/g i.p.) and ketamine (8 mg/kg in 0.1 ml/ 10g i.p.). Bilateral stainless-steel guide cannulae (23-gauge, 8.0 mm length) were introduced 2.5 mm above NAc following the coordinates standardized in our laboratory (AP + 1,2 mm, ML Â± 1 mm and DV - 2.1 mm) used in a previous study (Abrahao et al., 2010). The guide cannulae were attached to the skull with a screw and dental cement. To prevent the cannulae's obstruction, at the end of surgery we put wire stylets into the guide cannulae. Mice were allowed to recover for a period of at least seven days. In experiment 3, during the surgical procedure, ten mice died: 3 from the saline group, 4 from the low-sensitized group and 3 from highly-sensitized group. In experiment 4, nine animals died: four from the saline group, two from the low-sensitized group and three from the highly-sensitized group. After surgical procedures, we had the following number of mice, in experiment 3: saline (n=6), EtOH highly-sensitized (n=6) and EtOH low-sensitized (n=6) and in experiment 4: saline (n=5), EtOH highly-sensitized (n=6) and EtOH low-sensitized (n=6).
Habituation to Drug Infusions
One day before the beginning of the challenges, we submitted the mice to a manipulation (removal and re-insertion of the wire stylets into the guide cannulae) with no drug administration. This procedure was taken in order to habituate them to the procedure and to test if the cannulae were not obstructed by cement residues.
One day after habituation to drug infusion, mice were submitted to four pharmacological challenges. The challenges were performed every other day in the following order: a) saline + saline; b) saline + EtOH (2.2 g/kg); c) SB-242084 (1.0 or 2.0 Âµg) + EtOH (2.2 g/kg); d) SB-242084 (1.0 or 2.0 Âµg) + saline as detailed in Figure 1. In all challenges the first drug was administered intra-NAc and the mice returned to their plastic cage and remained there for 15 min. After this period, they received the second drug which was i.p. administered and their locomotor activity was immediately evaluated for 15 min. All drugs were injected bilaterally into the NAc using 10.5mm long injection cannulae that extended an extra 2.5 mm below the guide cannulae. Each injection was administered in a volume of 0.2 Âµl per side at the rate of 0.2Âµl/min. The injectors were linked by polyethylene microtubing to 10 Âµl syringes [model 95RN 5 Âµl (26s/2"/2), Hamilton, USA] and to a micro-drive pump (model EFF-311, Insight Ltda, Brazil). Sixty seconds after the infusion, the injection cannulae were removed.
One day after the last challenge, the mice were anaesthetized with chloral hydrate and euthanized by decapitation. The brains were removed, frozen on dry ice and stored at -80Â°C. Brains were then cut in a cryostat into 35Âµm sections, and stained with cresyl violet. Cannulae localizations were settled according to the atlas of Frankin and Paxinos (1997). Only mice with correct localization of both canulae (Figure 2) were included in the statistical analyses for experiments 3 and 4.
INSERT FIGURE 2
Student's t tests were used to compare body weight and locomotor activity baseline levels of mice allocated to saline and EtOH group. After we tested them for normality (Shapiro-Wilk's Test) of the distribution and homogeneity of variance (Levene's Test), locomotor activity levels were analyzed by analysis of variance for repeated measures (ANOVA) considering two factors: group (saline, low-sensitized and highly-sensitized mice) as an independent factor, and tests (1, 7, 14, 21) as repeated measures factor. When significant effects were detected by ANOVA, post hoc analyses were carried out with Newman-Keuls Test for multiple comparisons. The significance level was set at 5% in all analyses. Locomotor activity levels observed after the pharmacological challenges were analyzed by analysis of variance for repeated measures (ANOVA) considering two factors: group (saline, low-sensitized and highly-sensitized mice) as an independent factor and 15 min time as a repeated measures factor.
Experiment 1: Systemic administration of 0.5 and 1.0 mg/kg of SB-242084.
Figure 3 shows the locomotor activity levels of EtOH or saline-treated mice in the tests performed during the 21-day treatment. ANOVA for repeated measures detected significant effects of group (F(2,37)=94.88, p<0.001), test (F(3,111)=31.69, p<0.001) and interaction between group and test factors (F(6,111)=19.94, p<0.001). Highly-sensitized mice presented higher locomotor activity levels than saline and low-sensitized, whose values were higher than those of mice treated with saline on tests 7, 14 and 21. The locomotor activity of the highly-sensitized mice was progressively increased during treatment (Newman-Keuls test, p<0.001) showing a robust behavioral sensitization when compared to the low-sensitized group.
Significant effects of group were detected by ANOVA in both cycles [cycle I: (F(2,37)=6,53, p<0.001)] [cycle II: (F(2,37)=5.38, p<0.05)]. In addition, ANOVA also considered significant the effects of test [(cycle I: (F(3,111)=37.03, p<0.001)] [(cycle II: (F(3,111)=24.92, p<0.001)] and interaction between group and test [(cycle I: (F(6,111)=8.17, p<0.001)] [(cycle II: (F(6,111)=3.75, p<0.001)]. In both cycles, no differences were detected among saline, highly-sensitized and low-sensitized groups in the first challenge, under saline + saline. In the saline + EtOH challenge, post hoc test detected significantly higher locomotor activity levels in highly-sensitized mice than in low-sensitized and saline groups (p<0.05). In addition, the locomotor levels of highly-sensitized mice were higher when compared with their own levels observed in saline + saline challenge. These data confirm the maintenance of the expression of EtOH-induced behavioral sensitization, although at lower levels, only in the highly-sensitized group, even after a two-week drug-free period. When mice were challenged with SB-242084 (at both doses: 0.5 and 1.0 mg/kg) followed by EtOH, the locomotor levels of highly-sensitized mice were higher than those presented by low-sensitized or saline groups (p<0.05), demonstrating that the antagonist did not block the expression of EtOH-induced behavioral sensitization. Finally, the administration of SB-242084 followed by saline did not affect the locomotor activity of mice in both cycles.
INSERT FIGURE 3
Experiment 2: Systemic administration of 2.0 mg/kg of SB-242084.
Figure 4 shows the EtOH-induced behavioral sensitization treatment during 21 days. ANOVA detected significant effects of group (F(2,31)=106.44, p<0.001), tests (F(3,93)=39.4, p<0.001) and interaction between group and test factors (F(6,93)=21.33, p<0.001). Post-hoc analyses showed similar results to those observed in Experiment 1.
When the challenge period was considered, ANOVA detected significant influence of group (F(2,31)=19.39, p<0.001) and challenge factors (F(3,93)=38.61, p<0.001), besides a significant interaction between group and test factors (F(6,93)=12.37, p<0.001). The administration of saline + saline did not affect the levels of locomotor activity. When saline or SB-242084 2.0 mg/kg followed by EtOH were administered, only the highly-sensitized group of mice showed increase of locomotor activity (p<0.05). The administration of SB-242084 + saline did not affect the locomotor activity of any group. All mice showed similar locomotor activity levels to those observed in saline + saline challenge.
INSERT FIGURE 4
Experiment 3: Intra NAc administration of 1.0 Âµg/side of SB-242084.
Considering the four tests performed during the development of EtOH-induced behavioral sensitization (see Figure 5), ANOVA detected significant effect of factors group (F(2,15)=32.17, p<0.001), test (F(3,45)=1.78, p<0.05) and interaction group and test (F(6,45)=4.70, p<0.001). Post-hoc analyses showed similar results to those in the previous experiments.
For the challenges, ANOVA detected significant effect of factors group (F(2,15)=14.67, p<0.001), test (F(3,45)=10.12, p<0.001) and interaction between group and test (F(6,45)=2.54, p<0.001). Post-hoc analyses detected difference between highly-sensitized and low-sensitized in challenges 2 (saline + EtOH) and 3 (SB-242084 + EtOH) but not in challenges 1 (saline + saline) and 4 (SB-242084 + saline). Interestingly, highly-sensitized mice showed lower significant locomotor activity levels compared with themselves (p<0.001) in the previous challenge (saline + EtOH). These data, taken together, indicate that 1.0 Âµg/side of SB-242084 reduced the expression of EtOH-induced behavioral sensitization. ( O REVISOR QUER QUE ARRUME AS COMPARAÇÕESâ€¦)
INSERT FIGURE 5
Experiment 4: Intra NAc administration of 2.0 Âµg/side of SB-242084.
Figure 6 shows the development of behavioral sensitization to EtOH in the other group of mice. As in the previous experiments (1-3), ANOVA detected significant effect of factors group (F(2,14)=44.35, p<0.001), test (F(3,42)=5.68, p<0.05) and the interaction between group and test (F(6,42)=4.53, p<0.001). Similar results to those reported in the previous experiments were also observed.
Regarding the challenges, (Figure 6) ANOVA detected significant influence of factors test (F(3,42)=20.96, p<0.001) and group (F(2,14)=4.16, p<0.05), and also a significant interaction between test and group (F(6,42)=3.06, p<0.01). The post hoc tests detected differences between highly-sensitized and low-sensitized mice only after intra-NAc administration of saline followed by EtOH (challenge 2). With regards to third challenge, highly-sensitized mice showed a robust decrease of locomotor activity levels when compared with the saline + EtOH challenge. No differences were detected when animals received saline + saline (challenge 1) and SB-242084 + saline (challenge 4).
INSERT FIGURE 6
Although there are a number studies on the mechanisms and factors involved in behavioral sensitization to psychostimulants, there is a paucity of studies as regards EtOH-induced sensitization. To the best of our knowledge, this was the first study to describe the blockade of the expression of EtOH-induced behavioral sensitization by a specific antagonist of 5-HT2CR (SB-242084) administered systemically as well as intra-NAc. On the other hand, in this study we also showed that systemic (i.p.) administration of SB-242084 did not affect the expression of behavioral sensitization, at least in the doses tested in our experiments.
The administration of SB-242084 reduced the expression of EtOH-induced behavioral sensitization at 1.0 Âµg and a complete blockade at 2.0Âµg. These results indicate an important role of the serotonergic system on the modulation of behavioral sensitization. Our data are consistent with those reported by McMahon et al. (2001), Filip and Cunningham (2002) in which microinjection of a specific 5-HT2C antagonist (RS-102221) into the NAc attenuated cocaine-induced hyperactivity in rats. In addition, intra NAc administration of SB-242084 blocked the expression of cocaine-induced sensitization (Zayara et al., 2011).
The inhibition of the expression of EtOH-induced sensitization by the intra-NAc SB-242084 administration could be due to the fact that in the NAc the 5-HT2CR exerts a negative modulation on GABAergic neurons which project to VTA. These neurons maintain an inhibitory control on the dopaminergic neurons which release DA in NAc. Some authors reported that the blockage of 5-HT2CR leads to the activation of these GABAergic neurons and, as a consequence, could reduce the dopaminergic firing rate in VTA [Gobert et al., 2000; Porras et al., 2002; De Deurwaerdère et al. 2004; Navailles et al., 2004; Theile et al. 2009 - for a detail review see Bubar and Cunningham (2008) and Alex and Pehek (2007)].
Besides, Navailles et al (2006) reported that microinfusions of SB-242084 into the NAc reduced dopamine release and the increased accumbal dopamine efflux induced by cocaine (Navailles et al., 2008). In a similar way, the stimulant effect of ethanol can be reduced or blocked, as a consequence of the reduction in dopamine levels. In the present study we observed a reduction (by 1.0 Âµg) or blockage (by 2.0 Âµg) of the stimulant effect of ethanol by SB-242084 administration into the NAc.
Most of the studies evaluating the role of 5-HT2CR on behavioral sensitization were carried with repeated administration of psychostimulants such as cocaine and amphetamine and not with ethanol. Considering these drugs affect mainly the cathecolaminergic and serotonergic systems, and that ethanol affects many other systems (such as GABAergic and glutamatergic), one can hypothesize that the lack of effect of SB-242084 i.p. administered could be due to the influence of the antagonism of 5HT2CR on other systems or brain areas.
Ferraz and Boerngen-Lacerda (2008) showed that i.p. administration of mianserin, a non-selective 5-HT2CR antagonist, blocked the development and expression of EtOH-induced behavioral sensitization. Although apparently these data are different to those observed in the present paper, it is important to consider that mianserin is not a very specific antagonist of 5-HT2CR. Mianserin also presents some affinity with 5-HT2AR, whose activation is associated with a reduction in DA efflux (De Deurwaerdere and Spampinato, 1999, McMahon et al., 2001). Other authors demonstrated that the i.p. administration of specific 5-HT2AR antagonists not only decreases the efflux of DA induced by psychostimulants (Auclair et al., 2004; Broderick et al., 2004) but also the psychostimulant-induced sensitization (McMahon et al., 2001, Fletcher et al., 2002, Filip et al., 2004). Thus, the results observed by Ferraz and Boerngen-Lacerda (2008) could be more linked with the 5-HT2AR than with the 5-HT2CR antagonism.
In the present study, SB-242084 blocked the EtOH stimulant effect observed in highly-sensitized mice, but did not affect the locomotor activity of saline or low-sensitized mice. In previous studies we detected neurochemical differences between low-sensitized and highly-sensitized mice. Highly-sensitized mice presented higher D2 receptor binding in the anterior caudate-putamen area (Souza-Formigoni et al., 1999) than control and low-sensitized mice. On the other hand, low-sensitized mice presented higher NMDA receptor binding in the NAc and in the prefrontal cortex (Quadros et al., 2002). In a previous study in our laboratory, (Quadros et al., 2008) we observed, in VTA, higher binding levels to the gabaergic BDZ receptors in highly-sensitized than in low-sensitized mice and a cross-sensitization between ethanol and diazepam. These results could partially explain the difference in the intensity of effect of 5-HT2C antagonism in highly-sensitized and low-sensitized mice. If highly-sensitized mice present a higher number of gabaergic receptors in VTA and the 5-HT2C antagonist administered intra-NAc increases the firing rate of GABAergic neurons onto the VTA, one could expect a higher intensity of GABA inhibition of dopamine release in VTA. As a consequence, a proportionally higher reduction of locomotor activity could be observed in highly-sensitized than in low-sensitized mice. Alternatively, the lack of effect of 5-HT2C antagonism in low-sensitized mice could be explained by a "floor effect", since those mice presented similar locomotor activity levels as those that received saline.
In summary, we confirmed our hypothesis that, in mice treated chronically with EtOH, the administration of a specific 5-HT2CR antagonist intra-NAc would reduce the expression of EtOH-induced behavioral sensitization only in highly-sensitized mice. However, we did not observe the same effect when the 5-HT2CR antagonist was i.p. administered, probably due to the influence of other neurotransmission systems. Our data suggest that due to the involvement of other neurotransmission systems, the sensitization to the stimulant effect of ethanol is a more complex phenomenon than the sensitization to classical psychostimulants, such as cocaine and amphetamine