Withdrawal Induces Distinct Patterns of Fosb/∆Fosb Expression In

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Pharmacology, Biochemistry and Behavior 117 (2014) 70–78

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Withdrawal induces distinct patterns of FosB/ΔFosB expression in outbred Swiss mice classiﬁed as susceptible and resistant to ethanol-induced locomotor sensitization

R.F. De Pauli a,1, C.C. Coelhoso b,1,C.Tesone-Coelhob,A.Linardic,L.E.Mellob, D.X. Silveira a,J.G.Santos-Juniorb,c,⁎ a Department of Psychiatric and Medical Psychology, Federal University of São Paulo, Rua Borges Lagoa, 570, São Paulo, SP 04038-020, Brazil b Laboratory of Neurobiology, Federal University of São Paulo, Rua Pedro de Toledo, 669, São Paulo, SP 04039-032, Brazil c Department of Physiological Science, Faculty of Medical Science of Santa Casa of São Paulo, Rua Cesário Mota Jr, 61, 12 andar, São Paulo, SP 01221-020, Brazil article info abstract

Article history: Chronic drug exposure and drug withdrawal induce expressive neuronal plasticity which could be considered as Received 16 September 2013 both functional and pathological responses. It is well established that neuronal plasticity in the limbic system Received in revised form 12 October 2013 plays a pivotal role in relapse as well as in compulsive characteristics of drug addiction. Although increases in Accepted 6 December 2013 FosB/DeltaFosB expression constitute one of the most important forms of neuronal plasticity in drug addiction, Available online 16 December 2013 it is unclear whether they represent functional or pathological plasticity. It is of noteworthy importance the individual differences in the transition from recreational use to drug addiction. These differences have been reported Keywords: FosB in studies involving the ethanol-induced locomotor sensitization paradigm. In the present study we investigated DeltaFosB whether sensitized and non-sensitized mice differ in terms of FosB/DeltaFosB expression. Adult male outbred Locomotor sensitization Swiss mice were daily treated with ethanol or saline for 21 days. According to the locomotor activity in the acqui- Withdrawal sition phase, they were classiﬁed as sensitized (EtOH_High) or non-sensitized (EtOH_Low). After 18 h or 5 days, Behavioral variability their brains were processed for FosB/DeltaFosB immunohistochemistry. On the 5th day of withdrawal, we could Mice observe increased FosB/DeltaFosB expression in the EtOH_High group (in the motor cortex), in the EtOH_Low group (in the ventral tegmental area), and in both groups (in the striatum). Differences were more consistent in the EtOH_Low group. Therefore, behavioral variability observed in the acquisition phase of ethanol-induced locomotor sensitization was accompanied by differential neuronal plasticity during withdrawal period. Further- more, distinct patterns of FosB/DeltaFosB expression detected in sensitized and non-sensitized mice seem to be more related to withdrawal period rather than to chronic drug exposure. Finally, increases in FosB/DeltaFosB expression during withdrawal period could be considered as being due to both functional and pathological plasticity. © 2013 Elsevier Inc. All rights reserved.

1. Introduction on, drug addiction studies have been focusing on the neurobiological mechanisms related to negative emotional states emerging from both The challenge of current neurobiological research in drug addiction acute and protracted abstinence. According to the theory of “the dark is to understand the neuronal plasticity mechanisms that mediate the side of addiction”, there seems to occur long-term and persistent plas- transition from recreational use to the loss of behavioral control over ticity changes in neural circuits aiming to limit reward. However, drug seeking and drug taking. One of the most important theories of these plasticity alterations lead to a negative emotional state that drug addiction, called “the dark side of addiction”, suggests that there emerges when the access to the drug is prevented. This mechanism pro- is a progression from impulsivity (related to positive reinforcement) vides a strong motivational drive for the establishment of addiction, as to compulsivity (related to negative reinforcement). This progression, well as, for its maintenance (Koob and Le Moal, 2005, 2008). in a collapsed cycle, comprises the following states: preoccupation/an- Locomotor sensitization is a useful animal model based on the fact ticipation, binge intoxication, and withdrawal/negative affect (Koob that increases on subjective effects of the drugs along their repeated ex- and Le Moal, 2005, 2008; Koob and Volkow, 2010). From this scenario posure are similar to increases in the drug-induced stimulant locomotor effects (Vanderschuren and Kalivas, 2000; Vanderschuren and Pierce, 2010). Although locomotor sensitization does not mimic several behav- iors related to drug addiction, its temporal morphological and neuro- ⁎ Corresponding author at: Rua Cesário Mota Jr, 61, 12 andar, São Paulo, SP 01221-020, chemical features are in parallel with those leading the transition from Brazil. Tel./fax: +55 11 33312008. E-mail address: [email protected] (J.G. Santos-Junior). recreational use to drug addiction itself (Robinson and Kolb, 1999; 1 These authors equally participated in the present study. Vanderschuren and Kalivas, 2000; Vanderschuren and Pierce, 2010).

Traditionally, locomotor sensitization protocol comprises three phases: 2.2. Locomotor sensitization acquisition (repeated drug exposure), withdrawal period and challenge (a new contact with the drug after the withdrawal period). Unfortu- The protocol of locomotor sensitization was based on a previous nately, most of the studies using the locomotor sensitization focused study from our own laboratory (Coelhoso et al., 2013). At the beginning only in the acquisition and challenge phase, overlapping the withdrawal of the protocol, all animals were injected intraperitoneally (i.p.) with sa- period. line and immediately tested in an automated activity box (Insight, It is well established that repeated exposure to drugs of abuse Brazil) for 15 min to establish basal locomotion. Two days later, animals (Perrotti et al., 2008) and chronic stress (Perrotti et al., 2004) increases were daily injected with ethanol (2 g/kg, 15% w/v in 0.9% NaCl, i.p. — the expression of the transcription factor fosB/deltafosB in the EtOH group, N = 40) or saline (similar volume, i.p., — Control group, corticolimbic system. FosB/DeltaFosB accumulation in these regions N = 12), during 21 days. Right after the 1st, the 7th, the 14th, and the has been hypothesized to play a central role in the resilience to stress 21st injections, animals were placed in the activity cage for 15 min. (Berton et al., 2007; Vialou et al., 2010) and in the rewarding effects of The horizontal locomotion in each situation was measured by a behav- cocaine (Harris et al., 2007; Muschamp et al., 2012), ethanol (Kaste ioral analysis system (Pan Lab, Spain). As expected (Masur and dos et al., 2009; Li et al., 2010), and opioids (Zachariou et al., 2006; Solecki Santos, 1988; Coelhoso et al., 2013), behavioral variability in the loco- et al., 2008). Therefore, it is possible that FosB/DeltaFosB modulates motor activity at the 21st day of acquisition allows us to distribute the some of the neuronal plasticity events related to the ethanol-induced lo- animals of EtOH group in 2 subgroups: EtOH_High (taken from the comotor sensitization, as well as, the withdrawal that ensues the acqui- upper 30% of the distribution) and EtOH_Low (taken from the lower sition phase of locomotor sensitization. 30% of the distribution). Thus, only the 60% of animals were included It is noteworthy that there are individual differences observed dur- in the analysis. This strategy is identical to those used in the studies in- ing the transition from recreational use to drug addiction (Flagel et al., vestigating individual variability within the ethanol sensitization para- 2009; George and Koob, 2010; Swendsen and Le Moal, 2011). For exam- digm (Masur and dos Santos, 1988; Souza-Formigoni et al., 1999; ple, DBA/2 J mice are more prone to respond than C57BL/6 J to ethanol- Quadros et al., 2002a, 2002b; Abrahão et al., 2011, 2012; Coelhoso induced locomotor sensitization (Phillips et al., 1997; Melón and et al., 2013). Boehm, 2011a). In outbred Swiss mice, behavioral variability regarding After the classification defining the experimental groups, we per- ethanol induced locomotor sensitization was first described by Masur formed 2 independent experiments according to the temporal criteria and dos Santos (1988). From then on, other studies have demonstrated of withdrawal period: (i) animals submitted to the acquisition phase important neurochemical features related to behavioral variability in and sacrificed after 18 h of withdrawal and (ii) animals submitted to the acquisition of ethanol-induced locomotor sensitization (Souza- the acquisition phase and sacrificed after 5 days of withdrawal. So, Formigoni et al., 1999; Abrahão et al., 2011, 2012; Quadros et al., this study comprised 3 experimental groups (Control, EtOH_High, and 2002a, 2002b). However, these studies did not address the impact of be- EtOH_Low) that were divided into 2 subgroups (18 h and 5 days of havioral variability during the withdrawal period after the acquisition withdrawal) (N = 6 per subgroup). The choice of these two temporal phase of locomotor sensitization. In a recent study, our laboratory de- marks within the withdrawal period was due to the kinetic aspects of scribed a significant difference between sensitized and non-sensitized FosB and DeltaFosB expression after 18 h of withdrawal (as explained outbred Swiss mice regarding the expression of cannabinoid receptor in the discussion section), and after 5 days of withdrawal, based on pre- type 1 (CB1R) throughout withdrawal. In that study, sensitized (but vious studies from our Lab that investigated some neurochemical fea- not non sensitized mice) had increased CB1R expression in the prefron- tures regarding withdrawal period within the locomotor sensitization tal cortex, ventral tegmental area, amygdala, striatum, and hippocam- paradigm (Fallopa et al., 2012; Escosteguy-Neto et al., 2012). Finally, pus (Coelhoso et al., 2013). to perform correlations between locomotor sensitization and FosB/ Given the well established behavioral variability in outbred Swiss DeltaFosB expression, we calculated the score of locomotor sensitiza- mice regarding ethanol-induced locomotor sensitization, and that this tion for each animal, by the formula: score = (Locomotion in the 21st variability is accompanied by distinct neurochemical features during day − Locomotion in the 1st day) *100/Locomotion in the 1st day. subsequent withdrawal, the present study investigated the expression of FosB/DeltaFosB in sensitized and non sensitized mice at the beginning 2.3. Immunohistochemistry (18 h) and after 5 days of withdrawal. After the respective withdrawal period, animals were deeply anes- thetized with a cocktail containing ketamine (75 mg/kg, i.p.) and 2. Material and methods xylazine (25 mg/kg, i.p.). After the loss of corneal reflex, they were per- fused transcardially with 100 ml of phosphate buffer solution 0.1 M 2.1. Subjects [phosphate buffered saline (PBS)], followed by 100 ml of 4% parafor- maldehyde (PFA). The brains were removed immediately after perfu- Male outbred Swiss Webster mice (EPM-1 Colony, São Paulo, SP, sion, stored in PFA for 24 h and then kept in a 30% sucrose/PBS Brazil), originally derived from the Albino Swiss Webster line from the solution for 48 h. Serial coronal sections (30 μm) were cut using a freez- Center for the Development of Animal Models in Biology and Medicine ing microtome and kept inside an anti-freezing solution to be used in at the Universidade Federal de São Paulo, were used. Mice were the immunohistochemistry procedures by free-floating staining. 12 weeks of age (30–40 g) at the start of testing. Groups of 10 mice For immunohistochemistry, a conventional technique of avidin–bio- were housed in cages (40 × 34 × 17 cm) with woodchip bedding. tin–immunoperoxidase was performed. The brain sections of all exper- The temperature (20–22 °C) and humidity (50%) controlled animal col- imental groups were included in the same run, being pretreated with ony was maintained on a light/dark cycle (12/12 h), with lights on at hydrogen peroxidase (3%) for 15 min and then washed with PBS for 07:00 h, with mouse chow pellets and tap water ad libitum, except dur- 30 min. Then, all sections were exposed during 30 min in a PBS-BSA ing testing. Mice were maintained in these housing conditions for at .5% to avoid nonspecific reactions. Thereafter, sections were incubated least 7 days prior to the beginning of drug treatment and behavioral overnight with the primary antibody rabbit anti-FosB/DeltaFosB tests. Animal care and experimental procedures were conducted (1:3,000; Sigma Aldrich, St Louis, MO, USA. no.cat. AV32519) in PBS-T under protocols approved by the Animal Care and Use Ethics Commit- solution (30 ml PBS, 300 μl Triton X-100). Subsequently, sections tee of the University (protocol number: 2043/09), according to EU Di- were incubated for 2 h in a biotinylated goat anti-rabbit IgG secondary rective 2010/63/EU for animal experiments (http://ec.europa.eu/ antibody (1:600; Vector, Burlingame, CA, USA) at room temperature. environmental/chemicals/lab_animals/legislation_en.htm). The sections were then treated with avidin–biotin complex (Vectastain 72 R.F. De Pauli et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 70–78

ABC Standard kit; Vector, Burlingame, CA, USA) for 90 min and submitted to nickel-intensified diaminobenzidine reaction. Between steps, the sections were rinsed in PBS and agitated on a rotator. Sections were mounted on gelatin-coated slides, dried, dehydrated and coverslipped. The following encephalic regions were analyzed: prefrontal cortex [anterior cingulate cortex (Cg1), prelimbic cortex (PrL) and infralimbic cortex (IL)], motor cortex [primary (M1) and secondary (M2)], dorsal striatum [dorsomedial striatum (DmS) and dorsolateral striatum (DlS)], ventral striatum [nucleus accumbens core (Acbco) and shell (Acbsh), ventral pallidum (VP)], hippocampus [pyramidal layer of Cornus Ammong 1 and 3 (CA1 and CA3, respectively), granular layer of dentate gyrus (DG)], amygdala [basolateral nucleus (BlA), and central nucleus (CeA)], ventromedial nucleus of hypothalamus (VMH) and ventral tegmental area [anterior (VTAA) and posterior (VTAP) portions] (See Fig. 1). A Nikon Eclipse E200 microscope connected to a computer was used to capture images from each section at a ×20 magnification. The images were saved as .tiff archives for posterior analysis of FosB/ DeltaFosB immunoreactivity. The immunoreactive cells were counted using the ImageJ software (NIH Image, Bethesda, MD, USA). The brain regions were delineated on each photograph according to The Stereo- taxic Mouse Brain Atlas (Franklin and Paxinos, 1997). Since photomi- crographies taken by the microscope represents 2.5 × 103 μm2 in a 20× magnification, the quantification of FosB/DeltaFosB labeled cells is expressed as the average of immunostaining cells per 2.5 × 103 μm2. The values obtained in the EtOH groups were normalized to the Control values, and expressed as %. (Control = 100%).

2.4. Statistical analysis

Initially, Shapiro–Wilk was used to verify the normality of distribution of all variables. The behavioral results were analyzed by one-way ANOVA for repeated measure considering as factor the 5 periods of locomotor sensitization: basal, day 1, day 7, day 14, and day 21. The histolog- ical results were analyzed by two-way ANOVA, considering as factors: period of withdrawal (18 h and 5 days) and experimental group (Con- trol, EtOH_High and EtOH_Low). The nonparametric variables were stan- dardized into Z scores in order to decrease the dispersion of data, and subsequently applied in the two-way ANOVA, as previously described. Newman Keuls post-hoc was used when necessary. Finally, we investigated possible correlations between FosB/DeltaFosB positive cells and the scores of locomotor sensitization. These correlations were calculated only for the nuclei where statistical differences between experimental groups had been found. Because these differences were restricted to the 5 days of withdrawal (See results section), the FosB/DeltaFosB values considered in these correlations refer to this specific period of time of withdrawal. Because these differences were restricted to the 5 days of withdrawal (See results section), the FosB/DeltaFosB values considered in these correlation refer to this specific time of withdrawal. The signifi- cance level was set at 5% (p b0.05).

3. Results

3.1. Locomotor sensitization

ANOVA for repeated measures detected signiﬁcant differences in the

group factor [F(2,32) =68.33,p b 0.001], in the period of protocol

Fig. 1. Schematic representation of the brain regions sampled. Schematic drawing of mice brain coronal sections indicating areas sampled (adapted from Franklin and Paxinos, 1997). M1 = primary motor cortex; M2 = secondary motor cortex, CG1 = anterior cingulate cortex, PrL = prelimbic cortex, IL = infralimbic cortex, Acbco = nucleus accumbens core, Acbsh = nucleus accumbens shell, VP = ventral pallidum DmS = dorsomedial striatum, DlS = dorsolateral striatum, CA1 = Cornus Ammonis 1, CA3 = Cornus Ammonis 3; DG = granular layer of dentate gyrus, BlA = basolateral nucleus of amygdala, CeA = central nucleus of amygdala, VmH = ventromedial hypothalamic nucleus,VTAA = anterior portion of ventraltegmental area, VTAP = posterior portion of ventral tegmental area. R.F. De Pauli et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 70–78 73

[F(4,128) =9.13, p b 0.001], and the interaction between them To confirm that changes in FosB/DeltaFosB expression were due [F(8,128) =13.34,p b 0.001]. There were no differences in the basal lo- to withdrawal, and not to ethanol exposure, we performed correla- comotion, and both EtOH groups had similar increases in locomotion in tions between the score of locomotor sensitization and the FosB/ the first day of acquisition, when compared to the Control group DeltaFosB immunolabelled cells at the 5th day of withdrawal in the (p b 0.01). However, EtOH_High (but not EtOH_Low) presented a pro- nuclei above mentioned (M1, M2, Acbco, Acbsh, DmS, DlS, VP, gressive increase in locomotor activity throughout acquisition phase VTAA). As expected, there were no significant correlations for any (p b 0.01, in relation to Control and EtOH_Low groups, in the last day of these nuclei (M1 — r2 = 0.027862, p = 0.987156; M2 — of acquisition; p b 0.01 in relation to its locomotor activity in the first r2 = 0.048538, p = 0.196646; Acbco — r2 = 0.001920, p = day of acquisition) (Fig. 2). These data corroborated the results from 0.799669; Acbsh — r2 = 0.006743, p = 0.633991; DmS — r2 = the original study (Masur and dos Santos, 1988) and from our previous 0.015880, p = 0.463960; DlS — r2 = 0.023991, p = 0.914182; VP — report (Coelhoso et al., 2013) concerning behavioral variability in out- r2 = 0.002210, p = 0.785443; VTAA — r2 = 0.001482, p = 0.823630). bred Swiss mice submitted to ethanol-induced locomotor sensitization. 4. Discussion

3.2. FosB/DeltaFosB expression The results observed in the present study suggest that the increased expression of FosB/DeltaFosB observed in the ethanol-induced locomo- The illustrative photomicrographics of FosB/DeltaFosB immunoreac- tor sensitization paradigm is likely to be related to withdrawal rather tivity are depicted in Fig. 3 and the normalized values are shown in than to chronic drug exposure. However, the behavioral variability in Figs. 4, 5, 6 and 7. Two-way ANOVA detected significant differences in the development locomotor sensitization was accompanied by distinct the M1, M2, DmS, DlS, Acbco, Acbsh, VP and VTA (for non-normalized patterns of FosB/DeltaFosB expression during withdrawal. The role of values of FosB/DeltaFosB immunoreactivity and statistical analyses of motor cortex, ventral tegmental area and striatum in the acquisition all structures, see Table Suppl1 and Table 1, respectively). In the struc- and expression of locomotor sensitization paradigm is well established tures where statistical differences could be observed, there were four (Vanderschuren and Pierce, 2010). Furthermore, deregulation of different patterns of FosB/DeltaFosB expression. In the first one, ob- mesolimbic pathway is one of the central neurobiological features of served in M1 and M2, there was an increase in FosB/DeltaFosB expres- withdrawal period, together with the emergence of extended amygdala sion in the fifth day of ethanol withdrawal only in the EtOH_High (Koob and Le Moal, 2005, 2008). However, only few studies explored group (compared to EtOH_High values at 18 h of withdrawal, as well the withdrawal period of the locomotor sensitization paradigm. Our re- as, to the Control and EtOH_Low groups at 5 days of withdrawal) (see sults encountered interesting changes in FosB/DeltaFosB expression in Fig. 4). In the second pattern, observed in the VTAA, FosB/DeltaFosB ex- the motor cortex, ventral tegmental area, and striatum within this pression increased at 5 days of ethanol withdrawal only in the period. EtOH_Low group (compared to EtOH_Low values at 18 h of withdrawal, FosB cDNA encodes the expression of 33, 35, and 37 kDa proteins. as well as, to the Control group at 5 days of withdrawal) (see Fig. 5). In Acute stimuli exposure leads strong 33- and discrete 35- and 37- kDa the third pattern, observed in the DmS, Acbco, and Acbsh, FosB/ Fos protein induction. As a consequence, under acute activation, the DeltaFosB expression increased at 5 days of ethanol withdrawal in predominant FosB expression is related to 33 kDa (McClung et al., both EtOH_High and EtOH_Low groups (compared to their respective 2004; Nestler, 2008). There is another remarkable difference between values at 18 h of withdrawal), however, only EtOH_Low group differed these proteins: only 35–37 kDa proteins are highly stable isoforms. Be- from Control group (see Fig. 6). Finally, in the fourth pattern, observed cause of this high stability, these truncated forms of FosB, also called in DlS and VP, FosB/DeltaFosB expression increased at 5 days of ethanol DeltaFosB, accumulate in the brain and are highly expressed in response withdrawal in both EtOH_High and EtOH_Low groups (compared to to chronic stimuli, such as psychotropic drug treatments, chronic elec- their respective values at 18 h of withdrawal), although this increase troconvulsive seizures, and stress (Kelz and Nestler, 2000; Nestler was statistically more expressive in EtOH_Low than in EtOH_High et al., 2001; McClung et al., 2004). As a consequence, DeltaFosB have group, and only EtOH_Low group differed from Control group (see been viewed as a sustained molecular switch to mediate forms of Fig. 7). long-lasting neural and behavioral plasticity. Interestingly, an elegant study using mouse lines expressing differentially FosB and DeltaFosB showed that FosB is essential for the enhancement of stress tolerance and also neutralizes the correlation between psychostimulant-induced locomotor sensitization and accumulation of DeltaFosB in the striatum (Ohnishi et al., 2011). Therefore, both proteins could play important roles in the experimental protocol used in the present study. It is noteworthy that the FosB antibody used recognizes both FosB and DeltaFosB. Since FosB diminishes to baseline levels within 6 h after an acute stim- ulus (Nestler et al., 2001) and DeltaFosB accumulate after repeated stimuli exposures, we decided to sacrifice the animals 18 h after the acquisition phase, to avoid possible biases of ethanol treatment over FosB expression. Nonetheless, to be technically precise, we will refer in the present study as FosB/DeltaFosB expression. It is important to note that this strategy has been used in others studies, including those that used the same primary antibody described here (Conversi et al., 2008; Li et al., 2010; Flak et al., 2012; García-Pérez et al., 2012). As a consequence, besides these experimental limitations, we will discuss our re- Fig. 2. Ethanol promotes a gradual and robust increase of locomotion throughout chronic sults considering the role of DeltaFosB in neuronal plasticity. treatment in EtOH_High, but not in EtOH_Low group. The data were expressed as It is well established that chronic drug exposure increases FosB/ ⁎⁎ mean ± S.E.M. N = 12 for Control, EtOH_High and EtOH_Low groups. P b 0.01 in rela- DeltaFosB expression in several regions of brain (Nestler et al., 2001; tion to Control group, at the same period. ## P b 0.01 in relation to EtOH_Low group, at ‡‡ Perrotti et al., 2008). Curiously, in the present study neither ethanol sen- the same period. P b 0.01 in relation to basal locomotor activity, within the same group. ¥¥ P b 0.01 in relation to locomotor activity on the 1st day of acquisition, within sitized nor ethanol non-sensitized mice differed from chronic saline the same group. treated mice regarding FosB/DeltaFosB expression 18 h after acquisition 74 R.F. De Pauli et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 70–78

Fig. 3. Illustrative photomicrography of FosB/DeltaFosB immunoreactivity at ×20 of magniﬁcation. DmS = dorsomedial striatum; DlS = dorsolateral striatum; Acbco = nucleus accumbens core; Acbsh = nucleus accumbens shell; VP = ventral pallidum; VTAa = anterior portion of ventral tegmental area. phase. Furthermore, there were no signiﬁcant correlations between consume ethanol at doses ranging from 8 to 12 g/kg/day) (Perrotti FosB/DeltaFosB expression and the scores of locomotor sensitization. et al., 2008). In another study, although the authors refer to chronic This divergence could be explained, at least partially, by the differences treatment, the protocol consisted in only 4 ethanol exposures found in the experimental protocol. For example, considering the etha- (Ryabinin and Wang, 1998). So, protocols used elsewhere are totally nol exposure, in two studies the two bottle free choice paradigm was distinct from the one used here, which consisted of 21 days of treat- used in 15 intermittent drinking sessions (Li et al., 2010) or nutritionally ment where daily ethanol injections were administered by an experi- complete liquid diet auto-administered during 17 days (where animals menter. Despite these differences, there are several studies involving R.F. De Pauli et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 70–78 75

Studies using knockout and transgenic mice showed that FosB mutant mice have enhanced behavioral response to cocaine, such as stimulant locomotor effects and conditioned place preference. Furthermore, the expression of both basal and cocaine-inducible DeltaFosB is absent in this mutant mice (Hiroi et al., 1997). In contrast, transgenic mice with inducible overexpression of DeltaFosB show increased sensitivity to the rewarding effects of cocaine and morphine (Muschamp et al., 2012). These results provided direct evidence of close correlation between DeltaFosB and the rewarding process. Besides repeated drug exposures, chronic stress also increases DeltaFosB expression in corticolimbic circuits (Perrotti et al., 2004). Interestingly, transgenic mice overexpressing DeltaFosB are less sensitive to the pro-depressive effects of kappa-opioid agonist, known to induce dysphoria and stress-like effects in rodents (Muschamp et al., 2012). So, besides reward process, DeltaFosB also plays a pivotal role in the emotional aspects of the phenomena. In this scenario, withdrawal could also trigger FosB/DeltaFosB expression, since stress is a key component of drug's withdrawal. This perspective is in accordance to our results, because there were no correlations between FosB/DeltaFosB expression and the scores of sensitization, and furthermore the increase in FosB/ DeltaFosB expression was observed only on the ﬁfth day of withdrawal. Interestingly, in some structures, FosB/DeltaFosB increases were Fig. 4. Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in seen in both EtOH_High and EtOH_Low group, although more expres- EtOH_High and EtOH_Low groups in the M1 and M2. The data were expressed as sive in the former group, suggesting that these increases could have dif- mean ± S.E.M. and represent the normalized data according to the values of Control ferent functional consequences, according to their intensity. This — groups (dotted line considered as 100%). Grey bars = 18 h of ethanol withdrawal; hypothesis could be explained by several distinct functional roles of Black bars = 5 days of ethanol withdrawal. ** P b 0.01 in relation to its respective Control group; ## P b 0.01, in relation to its respective value at 18 hours of withdrawal. ‡‡ FosB/DeltaFosB. For example, rats chronically exposed to cocaine had P b 0.01, in relation to EtOH_Low group within the same period. M1 = primary motor increased DeltaFosB expression in the nucleus accumbens during with- cortex, M2 = secondary motor cortex. drawal period, an effect positively correlated with cocaine preference, but negatively with novelty preference. Furthermore, stress during intraperitoneal injections reporting increases in FosB/DeltaFosB expres- withdrawal increases the behavioral response to psychostimulants by sion after protocols of locomotor sensitization induced by psycho- increasing DeltaFosB expression in corticolimbic neurons (Nikulina stimulants (Brenhouse and Stellar, 2006; Conversi et al., 2008; Vialou et al., 2012). So, DeltaFosB could predict the dysregulation of hedonic et al., 2012) and opioids (Kaplan et al., 2011). However, the protocols processing that occurs during protracted withdrawal (Marttila et al., of locomotor sensitization in those studies involve much less than 21 2007). On the other hand, both resilience to stress and antidepressant drug exposures, and in some of them, drug was administered in an in- responses are related to higher DeltaFosB expression in striatum termittent way. In contrast, our protocol used the same treatment de- (Vialou et al., 2010). Therefore, we speculate that increased FosB/ scribed in previous studies involving 21 daily ethanol injections DeltaFosB on striatum in the EtOH_High could have enhanced the re- (Masur and Dos Santos, 1988; Souza-Formigoni et al., 1999; Quadros warding effects of ethanol, conferring a higher susceptibility to subse- et al., 2002a, 2002b; Abrahão et al., 2011, 2012). There is evidence quent drug exposures. On the other side, a more intense increase in that although chronic cocaine administration promotes accumulation FosB/DeltaFosB seen in the EtOH_Low group could have decreased the of DeltaFosB expression in the nucleus accumbens, it also promotes tol- sensitivity to both dysphoria and stress effects, minimizing negative re- erance to DeltaFosB mRNA induction in both ventral and dorsal stria- inforcement effects of subsequent drug exposure and, as a consequence, tum (Larson et al., 2010). Therefore, we hypothesized that the lack of explaining a higher resistance in this group. Interestingly, this paradox differences in our experimental groups in the acquisition phase might had a neurochemical basis. For example, transgenic mice overexpress- be due to a tolerance regarding FosB/DeltaFosB induction, since in the ing FosB in medium spine GABAergic neurons of nucleus accumbens present protocol there was a larger period of acquisition phase com- had increased levels of both mu- and kappa- opioid receptors (Sim- pared to periods used for psychostimulant and opioids in other studies. Selley et al., 2011), and those receptors respectively increase and inhibit mesolimbic tone (Manzanares et al., 1991; Devine et al., 1993). Further- more, the cell type expression could also drastically change the functional consequences of the increased FosB/DeltaFosB. In an elegant study using mice overexpressing DeltaFosB in D1- or D2- expressing neurons in the nucleus accumbens revealed that DeltaFosB in the D1- (but not in the D2-) neurons enhances behavioral responses to cocaine (Grueter et al., 2013). Curiously, regarding the motor cortex, there was an increase in FosB/ DeltaFosB expression only in EtOH_High group, and it was restricted to the 5th day of withdrawal. The lack of increase at 18 h of withdrawal could be explained by a possible tolerance mechanism in the FosB/ DeltaFosB expression in this region after chronic ethanol exposure. Fur- thermore, our results suggest that there are active neurochemical Fig. 5. Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in changes in the motor cortex during withdrawal period, despite the EtOH_High and EtOH_Low groups in the VTA. The data were expressed as mean ± S.E.M. fact that the animals were not manipulated during this period. This is in- and represent the normalized data according to the values of Control groups (dotted line — considered as 100%). Grey bars = 18 h of ethanol withdrawal; Black bars = 5 days of teresting, because this plasticity could play a role, at least partially, in ethanol withdrawal. ** P b 0.01 in relation to its respective Control group; ## P b 0.01, in the maintenance of locomotor sensitization. Although the sustained relation to its respective value at 18 h of withdrawal. VTA = ventral tegmental area. hyperlocomotion after several days of withdrawal was not studied 76 R.F. De Pauli et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 70–78

Fig. 6. Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in EtOH_High and EtOH_Low groups in the Acbco, Acbsh and DmS. The data were expressed as mean ± S.E.M. and represent the normalized data according to the values of Control groups (dotted line — considered as 100%). Grey bars = 18 h of ethanol withdrawal; Black bars = 5 days of ethanol withdrawal. * P b 0.05 ** P b 0.01, in relation to its respective Control group; ## P b 0.01, in relation to its respective value at 18 h of withdrawal. Acbco = nucleus accumbens core, Acbsh = nucleus accumbens shell, DmS = dorsomedial striatum. here, there are several studies, including previous ones from our Lab, projections and neurochemical profiles, and their participation in the re- showing that sensitized mice (but not non-sensitized) had enhanced lo- ward process depends on several factors (Ikemoto, 2007). For example, comotion when challenged with ethanol after a given withdrawal peri- rats' self-administration of ethanol is related to the posterior, but not to od (Masur and dos Santos, 1988; Souza-Formigoni et al., 1999; Quadros the ventral portion of the ventral tegmental area (Rood-Henricks et al., et al., 2002a,2002b; Abrahão et al., 2011, 2012; Fallopa et al., 2012; 2000; Rood et al., 2004). Furthermore, the endocannabinoid system, as Coelhoso et al., 2013). well as GABA-A, dopaminergic D1-D3, and serotoninergic 5HT3 recep- Finally, it is noteworthy that only the EtOH_Low group displayed an tors, plays an important role in ethanol seeking behavior (Linsenbardt increased FosB/DeltaFosB expression in the anterior (but not posterior) and Boehm, 2009; Rood et al., 2010; Melón and Boehm, 2011b; Hauser portion of the ventral tegmental area. These portions have distinct et al., 2011). However, GABA-B in the anterior portion of ventral tegmental area is important in terms of the rewarding (Moore and Boehm, 2009) and stimulant locomotor effects (Boehm et al., 2002) of ethanol. Furthermore, cholinergic nicotinic receptors in the anterior portion are involved in the increased accumbal dopamine levels induced by ethanol (Ericson et al., 2008). Therefore, regardless the distinct profile of these portions, it is possible that changes seen in the EtOH_Low group in the anterior portions could be related to the rewarding process. Chronic cocaine but not chronic morphine or chronic stress exposure increases DeltaFosB in ventral tegmental area, specifically in a gamma-aminobutyric acid (GABA) cell population (Perrotti et al., 2005). This fact could explain the normal levels of FosB/DeltaFosB throughout withdrawal encountered in ventral tegmental area of EtOH_High mice, regardless the putative high stress experience in this period. Furthermore, this data corroborates, at least partially, the hypothesis that the increase of FosB/DeltaFosB expression throughout withdrawal in EtOH_Low could be characterized as being an adaptive response. Individual differences observed during the transition from recreational use to drug addiction are remarkable (Flagel et al., 2009; George and Koob, 2010; Swendsen and Le Moal, 2011). As a consequence, it is imperative to study the neurobiological features related to individual variability. Behavioral sensitization is an animal model commonly used to investigate the neurobiological features of drug addiction. The

Fig. 7. Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in basis of this model is that the subjective effects of the drugs increase EtOH_High and EtOH_Low groups in the VP and DlS. The data were expressed as along their repeated exposure. Once acquired, locomotor sensitization mean ± S.E.M. and represent the normalized data according to the values of Control is long lasting and is in direct temporal relation with morphological groups (dotted line — considered as 100%). Grey bars = 18 h of ethanol withdrawal; and neurochemical changes in the mesolimbic pathway and several Black bars = 5 days of ethanol withdrawal. ** P b 0.01 in relation to its respective Control encephalic nuclei related to emotionality and motor behavior group; # P b 0.05 ## P b 0.01, in relation to its respective value at 18 h of withdrawal. ‡‡ P b 0.01, in relation to EtOH_Low group within the same period. VP = ventral pallidum, (Robinson and Kolb, 1999; Vanderschuren and Pierce, 2010). A pioneer DlS = dorsolateral striatum. study conducted by Masur and dos Santos (1988) demonstrated that R.F. De Pauli et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 70–78 77

Table 1 Acknowledgments Statistical parameters obtained in the two-way ANOVA regarding the analysis of FosB/ DeltaFosB expression. R.F.P. and C.C.C. received master fellowship from CAPES and FAPESP, Nucleus Period factor Treatment factor Period*Treatment respectively. C.T.C., L.E.M., D.X.S. and J.G.S.J. are granted by FAPESP and CNPq. M1 F(1,30) = 5.61, F(2,30) =3.21, F(2,30) =2.61, P =0.025 P =0.055 P = 0.089

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DG F(1,30) = 0.59, F(2,30) =1.49, F(2,30) =0.24, ventro-medial caudate underlies the induction but not the expression of behavioral P =0.447 P =0.241 P = 0.785 sensitization by both repeated amphetamine and stress. Eur J Neurosci 2008;27: 191–201. BlA F(1,30) = 6.47, F(2,30) =0.12, F(2,30) =1.71, P =0.016 P =0.884 P = 0.199 Devine DP, Leone P, Wise RA. Mesolimbic dopamine neurotransmission is increased by administration of mu-opioid receptor antagonists. Eur J Pharmacol 1993;243:55–64. CeA F = 2.55, F =0.22, F =0.71, (1,30) (2,30) (2,30) Ericson M, Löf E, Stomberg R, Chau P, Söderpalm B. Nicotinic acetylcholine receptors in P =0.121 P =0.801 P = 0.501 the anterior, but not posterior, ventral tegmental area mediate ethanol-induced ele- VmH F = 6.51, F =0.71, F =1.75, (1,30) (2,30) (2,30) vation of accumbal dopamine levels. J Pharmacol Exp Ther 2008;326:76–82. P =0.016 P =0.503 P = 0.192 Escosteguy-Neto JC, Fallopa P, Varela P, Filev R, Tabosa A, Santos-Junior JG. 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