Withdrawal induces distinct patterns of FosB/∆FosB expression in outbred Swiss mice classified as susceptible and resistant to ethanol-induced locomotor sensitization (2014)

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Pharmacol Biochem Behav. 2014 Feb;117:70-8. doi: 10.1016/j.pbb.2013.12.007. Epub 2013 Dec 16.

De Pauli RF1, Coelhoso CC2, Tesone-Coelho C2, Linardi A3, Mello LE2, Silveira DX1, Santos-Junior JG4.

Abstract

Chronic drug exposure and drug withdrawal induce expressive neuronal plasticity which could be considered as both functional and pathological responses. It is well established that neuronal plasticity in the limbic system plays a pivotal role in relapse as well as in compulsive characteristics of drug addiction. Although increases in FosB/DeltaFosB expression constitute one of the most important forms of neuronal plasticity in drug addiction, 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 in studies involving the ethanol-induced locomotor sensitization paradigm. In the present study we investigated whether sensitized and non-sensitized mice differ in terms of FosB/DeltaFosB expression. Adult male outbred Swiss mice were daily treated with ethanol or saline for 21days. According to the locomotor activity in the acquisition phase, they were classified as sensitized (EtOH_High) or non-sensitized (EtOH_Low). After 18h or 5days, their brains were processed for FosB/DeltaFosB immunohistochemistry. On the 5th day of withdrawal, we could 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. Furthermore, 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.

 


Highlights

  • DeltaFosB expression is an important form of neuronal plasticity in drug addiction

  • However, it is unclear whether it represents functional or pathological plasticity.

  • Here we found differences in DeltaFosB among sensitized and non-sensitized mice.

  • These differences are more related to withdrawal period rather than drug exposure.

  • We suggest that these changes represent both functional and pathological plasticity.


Keywords

  • FosB;
  • DeltaFosB;
  • Locomotor sensitization;
  • Withdrawal;
  • Behavioral variability;
  • Mice

1. Introduction

The challenge of current neurobiological research in drug addiction is to understand the neuronal plasticity mechanisms that mediate the transition from recreational use to the loss of behavioral control over drug seeking and drug taking. One of the most important theories of drug addiction, called “the dark side of addiction”, suggests that there is a progression from impulsivity (related to positive reinforcement) to compulsivity (related to negative reinforcement). This progression, in a collapsed cycle, comprises the following states: preoccupation/anticipation, binge intoxication, and withdrawal/negative affect (Koob and Le Moal, 2005, Koob and Le Moal, 2008 and Koob and Volkow, 2010). From this scenario on, drug addiction studies have been focusing on the neurobiological mechanisms related to negative emotional states emerging from both acute and protracted abstinence. According to the theory of “the dark side of addiction”, there seems to occur long-term and persistent plasticity changes in neural circuits aiming to limit reward. However, these plasticity alterations lead to a negative emotional state that emerges when the access to the drug is prevented. This mechanism provides a strong motivational drive for the establishment of addiction, as well as, for its maintenance (Koob and Le Moal, 2005 and Koob and Le Moal, 2008).

Locomotor sensitization is a useful animal model based on the fact that increases on subjective effects of the drugs along their repeated exposure are similar to increases in the drug-induced stimulant locomotor effects (Vanderschuren and Kalivas, 2000 and Vanderschuren and Pierce, 2010). Although locomotor sensitization does not mimic several behaviors related to drug addiction, its temporal morphological and neurochemical features are in parallel with those leading the transition from recreational use to drug addiction itself (Robinson and Kolb, 1999, Vanderschuren and Kalivas, 2000 and Vanderschuren and Pierce, 2010). Traditionally, locomotor sensitization protocol comprises three phases: acquisition (repeated drug exposure), withdrawal period and challenge (a new contact with the drug after the withdrawal period). Unfortunately, most of the studies using the locomotor sensitization focused only in the acquisition and challenge phase, overlapping the withdrawal period.

It is well established that repeated exposure to drugs of abuse (Perrotti et al., 2008) and chronic stress (Perrotti et al., 2004) increases the expression of the transcription factor fosB/deltafosB in the corticolimbic system. FosB/DeltaFosB accumulation in these regions has been hypothesized to play a central role in the resilience to stress (Berton et al., 2007 and Vialou et al., 2010) and in the rewarding effects of cocaine (Harris et al., 2007 and Muschamp et al., 2012), ethanol (Kaste et al., 2009 and Li et al., 2010), and opioids (Zachariou et al., 2006 and Solecki et al., 2008). Therefore, it is possible that FosB/DeltaFosB modulates some of the neuronal plasticity events related to the ethanol-induced locomotor sensitization, as well as, the withdrawal that ensues the acquisition phase of locomotor sensitization.

It is noteworthy that there are individual differences observed during the transition from recreational use to drug addiction (Flagel et al., 2009, George and Koob, 2010 and Swendsen and Le Moal, 2011). For example, DBA/2 J mice are more prone to respond than C57BL/6 J to ethanol-induced locomotor sensitization (Phillips et al., 1997 and Melón and Boehm, 2011a). In outbred Swiss mice, behavioral variability regarding ethanol induced locomotor sensitization was first described by Masur and dos Santos (1988). From then on, other studies have demonstrated important neurochemical features related to behavioral variability in the acquisition of ethanol-induced locomotor sensitization (Souza-Formigoni et al., 1999, Abrahão et al., 2011, Abrahão et al., 2012, Quadros et al., 2002a and Quadros et al., 2002b). However, these studies did not address the impact of behavioral variability during the withdrawal period after the acquisition phase of locomotor sensitization. In a recent study, our laboratory described a significant difference between sensitized and non-sensitized outbred Swiss mice regarding the expression of cannabinoid receptor type 1 (CB1R) throughout withdrawal. In that study, sensitized (but not non sensitized mice) had increased CB1R expression in the prefrontal cortex, ventral tegmental area, amygdala, striatum, and hippocampus (Coelhoso et al., 2013).

Given the well established behavioral variability in outbred Swiss mice regarding ethanol-induced locomotor sensitization, and that this variability is accompanied by distinct neurochemical features during subsequent withdrawal, the present study investigated the expression of FosB/DeltaFosB in sensitized and non sensitized mice at the beginning (18 h) and after 5 days of withdrawal.

2. Material and methods

2.1. Subjects

Male outbred Swiss Webster mice (EPM-1 Colony, São Paulo, SP, Brazil), originally derived from the Albino Swiss Webster line from the Center for the Development of Animal Models in Biology and Medicine at the Universidade Federal de São Paulo, were used. Mice were 12 weeks of age (30–40 g) at the start of testing. Groups of 10 mice were housed in cages (40 × 34 × 17 cm) with woodchip bedding. The temperature (20–22 °C) and humidity (50%) controlled animal colony was maintained on a light/dark cycle (12/12 h), with lights on at 07:00 h, with mouse chow pellets and tap water ad libitum, except during testing. Mice were maintained in these housing conditions for at least 7 days prior to the beginning of drug treatment and behavioral tests. Animal care and experimental procedures were conducted under protocols approved by the Animal Care and Use Ethics Committee of the University (protocol number: 2043/09), according to EU Directive 2010/63/EU for animal experiments (http://ec.europa.eu/environmental/chemicals/lab_animals/legislation_en.htm).

2.2. Locomotor sensitization

The protocol of locomotor sensitization was based on a previous study from our own laboratory (Coelhoso et al., 2013). At the beginning of the protocol, all animals were injected intraperitoneally (i.p.) with saline and immediately tested in an automated activity box (Insight, Brazil) for 15 min to establish basal locomotion. Two days later, animals were daily injected with ethanol (2 g/kg, 15% w/v in 0.9% NaCl, i.p. — EtOH group, N = 40) or saline (similar volume, i.p., — Control group, N = 12), during 21 days. Right after the 1st, the 7th, the 14th, and the 21st injections, animals were placed in the activity cage for 15 min. The horizontal locomotion in each situation was measured by a behavioral analysis system (Pan Lab, Spain). As expected ( Masur and dos Santos, 1988 and Coelhoso et al., 2013), behavioral variability in the locomotor activity at the 21st day of acquisition allows us to distribute the animals of EtOH group in 2 subgroups: EtOH_High (taken from the upper 30% of the distribution) and EtOH_Low (taken from the lower 30% of the distribution). Thus, only the 60% of animals were included in the analysis. This strategy is identical to those used in the studies investigating individual variability within the ethanol sensitization paradigm ( Masur and dos Santos, 1988, Souza-Formigoni et al., 1999, Quadros et al., 2002a, Quadros et al., 2002b, Abrahão et al., 2011, Abrahão et al., 2012 and Coelhoso et al., 2013).

After the classification defining the experimental groups, we performed 2 independent experiments according to the temporal criteria of withdrawal period: (i) animals submitted to the acquisition phase and sacrificed after 18 h of withdrawal and (ii) animals submitted to the acquisition phase and sacrificed after 5 days of withdrawal. So, this study comprised 3 experimental groups (Control, EtOH_High, and EtOH_Low) that were divided into 2 subgroups (18 h and 5 days of withdrawal) (N = 6 per subgroup). The choice of these two temporal marks within the withdrawal period was due to the kinetic aspects of FosB and DeltaFosB expression after 18 h of withdrawal (as explained in the discussion section), and after 5 days of withdrawal, based on previous studies from our Lab that investigated some neurochemical features regarding withdrawal period within the locomotor sensitization paradigm ( Fallopa et al., 2012 and Escosteguy-Neto et al., 2012). Finally, to perform correlations between locomotor sensitization and FosB/DeltaFosB expression, we calculated the score of locomotor sensitization for each animal, by the formula: score = (Locomotion in the 21st day − Locomotion in the 1st day) *100/Locomotion in the 1st day.

2.3. Immunohistochemistry

After the respective withdrawal period, animals were deeply anesthetized with a cocktail containing ketamine (75 mg/kg, i.p.) and xylazine (25 mg/kg, i.p.). After the loss of corneal reflex, they were perfused transcardially with 100 ml of phosphate buffer solution 0.1 M [phosphate buffered saline (PBS)], followed by 100 ml of 4% paraformaldehyde (PFA). The brains were removed immediately after perfusion, stored in PFA for 24 h and then kept in a 30% sucrose/PBS solution for 48 h. Serial coronal sections (30 μm) were cut using a freezing microtome and kept inside an anti-freezing solution to be used in the immunohistochemistry procedures by free-floating staining.

For immunohistochemistry, a conventional technique of avidin–biotin–immunoperoxidase was performed. The brain sections of all experimental groups were included in the same run, being pretreated with hydrogen peroxidase (3%) for 15 min and then washed with PBS for 30 min. Then, all sections were exposed during 30 min in a PBS-BSA .5% to avoid nonspecific reactions. Thereafter, sections were incubated overnight with the primary antibody rabbit anti-FosB/DeltaFosB (1:3,000; Sigma Aldrich, St Louis, MO, USA. no.cat. AV32519) in PBS-T solution (30 ml PBS, 300 μl Triton X-100). Subsequently, sections were incubated for 2 h in a biotinylated goat anti-rabbit IgG secondary antibody (1:600; Vector, Burlingame, CA, USA) at room temperature. The sections were then treated with avidin–biotin complex (Vectastain 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 Stereotaxic Mouse Brain Atlas (Franklin and Paxinos, 1997). Since photomicrographies 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%).

  •  
  • 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 ventral tegmental area, VTAP = posterior portion of ventral tegmental area.

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 histological results were analyzed by two-way ANOVA, considering as factors: period of withdrawal (18 h and 5 days) and experimental group (Control, EtOH_High and EtOH_Low). The nonparametric variables were standardized 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 significance level was set at 5% (p < 0.05).

3. Results

3.1. Locomotor sensitization

ANOVA for repeated measures detected significant differences in the group factor [F(2,32) = 68.33, p < 0.001], in the period of protocol [F(4,128) = 9.13, p < 0.001], and the interaction between them [F(8,128) = 13.34, p < 0.001]. There were no differences in the basal locomotion, and both EtOH groups had similar increases in locomotion in the first day of acquisition, when compared to the Control group (p < 0.01). However, EtOH_High (but not EtOH_Low) presented a progressive increase in locomotor activity throughout acquisition phase (p < 0.01, in relation to Control and EtOH_Low groups, in the last day of acquisition; p < 0.01 in relation to its locomotor activity in the first day of acquisition) ( Fig. 2). These data corroborated the results from the original study ( Masur and dos Santos, 1988) and from our previous report ( Coelhoso et al., 2013) concerning behavioral variability in outbred Swiss mice submitted to ethanol-induced locomotor sensitization.

  • Ethanol promotes a gradual and robust increase of locomotion throughout chronic ...
  • Fig. 2.  

    Ethanol promotes a gradual and robust increase of locomotion throughout chronic treatment in EtOH_High, but not in EtOH_Low group. The data were expressed as mean ± S.E.M. N = 12 for Control, EtOH_High and EtOH_Low groups. ⁎⁎P < 0.01 in relation to Control group, at the same period. ##P < 0.01 in relation to EtOH_Low group, at the same period. ‡‡P < 0.01 in relation to basal locomotor activity, within the same group. ¥¥P < 0.01 in relation to locomotor activity on the 1st day of acquisition, within the same group.

3.2. FosB/DeltaFosB expression

The illustrative photomicrographics of FosB/DeltaFosB immunoreactivity are depicted in Fig. 3 and the normalized values are shown in Fig. 4, Fig. 5, Fig. 6 and Fig. 7. Two-way ANOVA detected significant differences in the M1, M2, DmS, DlS, Acbco, Acbsh, VP and VTA (for non-normalized values of FosB/DeltaFosB immunoreactivity and statistical analyses of all structures, see Table Suppl1 and Table 1, respectively). In the structures where statistical differences could be observed, there were four different patterns of FosB/DeltaFosB expression. In the first one, observed in M1 and M2, there was an increase in FosB/DeltaFosB expression in the fifth day of ethanol withdrawal only in the EtOH_High group (compared to EtOH_High values at 18 h of withdrawal, as well as, to the Control and EtOH_Low groups at 5 days of withdrawal) (see Fig. 4). In the second pattern, observed in the VTAA, FosB/DeltaFosB expression increased at 5 days of ethanol withdrawal only in the EtOH_Low group (compared to EtOH_Low values at 18 h of withdrawal, as well as, to the Control group at 5 days of withdrawal) (see Fig. 5). In the third pattern, observed in the DmS, Acbco, and Acbsh, FosB/DeltaFosB expression increased at 5 days of ethanol withdrawal in both EtOH_High and EtOH_Low groups (compared to their respective values at 18 h of withdrawal), however, only EtOH_Low group differed from Control group (see Fig. 6). Finally, in the fourth pattern, observed in DlS and VP, FosB/DeltaFosB expression increased at 5 days of ethanol withdrawal in both EtOH_High and EtOH_Low groups (compared to their respective values at 18 h of withdrawal), although this increase was statistically more expressive in EtOH_Low than in EtOH_High group, and only EtOH_Low group differed from Control group (see Fig. 7).

  • Illustrative photomicrography of FosB/DeltaFosB immunoreactivity at ×20 of ...
  • 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.

  •  
  • Fig. 4.  

    Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in EtOH_High and EtOH_Low groups in the M1 and M2. 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 < 0.01 in relation to its respective Control group; ## P < 0.01, in relation to its respective value at 18 hours of withdrawal. ‡‡ P < 0.01, in relation to EtOH_Low group within the same period. M1 = primary motor cortex, M2 = secondary motor cortex.

  • Expression of FosB/DeltaFosB at 18h and 5days of withdrawal period in EtOH_High ...
  • Fig. 5.  

    Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in EtOH_High and EtOH_Low groups in the VTA. 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 < 0.01 in relation to its respective Control group; ## P < 0.01, in relation to its respective value at 18 h of withdrawal. VTA = ventral tegmental area.

  • Expression of FosB/DeltaFosB at 18h and 5days of withdrawal period in EtOH_High ...
  • 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 < 0.05 ** P < 0.01, in relation to its respective Control group; ## P < 0.01, in relation to its respective value at 18 h of withdrawal. Acbco = nucleus accumbens core, Acbsh = nucleus accumbens shell, DmS = dorsomedial striatum.

  • Expression of FosB/DeltaFosB at 18h and 5days of withdrawal period in EtOH_High ...
  • Fig. 7.  

    Expression of FosB/DeltaFosB at 18 h and 5 days of withdrawal period in EtOH_High and EtOH_Low groups in the VP and DlS. 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 < 0.01 in relation to its respective Control group; # P < 0.05 ## P < 0.01, in relation to its respective value at 18 h of withdrawal. ‡‡ P < 0.01, in relation to EtOH_Low group within the same period. VP = ventral pallidum, DlS = dorsolateral striatum.

  • Table 1. 

    Statistical parameters obtained in the two-way ANOVA regarding the analysis of FosB/DeltaFosB expression.




  • Nucleus Period factor Treatment factor Period*Treatment
    M1 F(1,30) = 5.61, P = 0.025 F(2,30) = 3.21, P = 0.055 F(2,30) = 2.61, P = 0.089
    M2 F(1,30) = 4.72, P = 0.038 F(2,30) = 1.53, P = 0.233 F(2,30) = 3.45, P = 0.045
    CG1 F(1,30) = 11.08 P = 0.002 F(2,30) = 0.95, P = 0.398 F(2,30) = 3.31, P = 0.050
    PrL F(1,30) = 8.53, P = 0.007 F(2,30) = 1.72, P = 0.197 F(2,30) = 2.74, P = 0.081
    IL F(1,30) = 3.77, P = 0.062 F(2,30) = 1.91, P = 0.167 F(2,30) = 0.98, P = 0.389
    Acbco F(1,30) = 22.23 P < 0.001 F(2,30) = 2.63, P = 0.089 F(2,30) = 5.68, P = 0.008
    Acbsh F(1,30) = 50.44 P < 0.001 F(2,30) = 4.27, P = 0.023 F(2,30) = 13.18, P < 0.000
    VP F(1,30) = 38.01 P < 0.001 F(2,30) = 5.07, P = 0.013 F(2,30) = 10.93, P < 0.000
    DmS F(1,30) = 28.89 P < 0.001 F(2,30) = 3.75, P = 0.035 F(2,30) = 7.71, P = 0.002
    DlS F(1,30) = 13.58 P = 0.001 F(2,30) = 5.41, P = 0.011 F(2,30) = 4.72, P = 0.017
    CA1 F(1,30) = 4.81, P = 0.036 F(2,30) = 7.37, P = 0.002 F(2,30) = 1.62, P = 0.215
    CA3 F(1,30) = 14.92 P = 0.001 F(2,30) = 2.46, P = 0.102 F(2,30) = 3.81, P = 0.034
    DG F(1,30) = 0.59, P = 0.447 F(2,30) = 1.49, P = 0.241 F(2,30) = 0.24, P = 0.785
    BlA F(1,30) = 6.47, P = 0.016 F(2,30) = 0.12, P = 0.884 F(2,30) = 1.71, P = 0.199
    CeA F(1,30) = 2.55, P = 0.121 F(2,30) = 0.22, P = 0.801 F(2,30) = 0.71, P = 0.501
    VmH F(1,30) = 6.51, P = 0.016 F(2,30) = 0.71, P = 0.503 F(2,30) = 1.75, P = 0.192
    VTAA F(1,30) = 9.64, P = 0.004 F(2,30) = 3.76, P = 0.035 F(2,30) = 2.65, P = 0.087
    VTAP F(1,30) = 6.05, P = 0.021 F(2,30) = 1.79, P = 0.184 F(2,30) = 1.64, P = 0.211
  • 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 ventral tegmental area; VTAP = posterior portion of central tegmental area.

To confirm that changes in FosB/DeltaFosB expression were due to withdrawal, and not to ethanol exposure, we performed correlations between the score of locomotor sensitization and the FosB/DeltaFosB immunolabelled cells at the 5th day of withdrawal in the nuclei above mentioned (M1, M2, Acbco, Acbsh, DmS, DlS, VP, VTAA). As expected, there were no significant correlations for any of these nuclei (M1 — r2 = 0.027862, p = 0.987156; M2 — r2 = 0.048538, p = 0.196646; Acbco — r2 = 0.001920, p = 0.799669; Acbsh — r2 = 0.006743, p = 0.633991; DmS — r2 = 0.015880, p = 0.463960; DlS — r2 = 0.023991, p = 0.914182; VP — r2 = 0.002210, p = 0.785443; VTAA — r2 = 0.001482, p = 0.823630).

4. Discussion

The results observed in the present study suggest that the increased expression of FosB/DeltaFosB observed in the ethanol-induced locomotor sensitization paradigm is likely to be related to withdrawal rather than to chronic drug exposure. However, the behavioral variability in the development locomotor sensitization was accompanied by distinct patterns of FosB/DeltaFosB expression during withdrawal. The role of motor cortex, ventral tegmental area and striatum in the acquisition and expression of locomotor sensitization paradigm is well established (Vanderschuren and Pierce, 2010). Furthermore, deregulation of mesolimbic pathway is one of the central neurobiological features of withdrawal period, together with the emergence of extended amygdala (Koob and Le Moal, 2005 and Koob and Le Moal, 2008). However, only few studies explored the withdrawal period of the locomotor sensitization paradigm. Our results encountered interesting changes in FosB/DeltaFosB expression in the motor cortex, ventral tegmental area, and striatum within this period.

FosB cDNA encodes the expression of 33, 35, and 37 kDa proteins. Acute stimuli exposure leads strong 33- and discrete 35- and 37- kDa Fos protein induction. As a consequence, under acute activation, the predominant FosB expression is related to 33 kDa (McClung et al., 2004 and Nestler, 2008). There is another remarkable difference between these proteins: only 35–37 kDa proteins are highly stable isoforms. Because of this high stability, these truncated forms of FosB, also called DeltaFosB, accumulate in the brain and are highly expressed in response to chronic stimuli, such as psychotropic drug treatments, chronic electroconvulsive seizures, and stress (Kelz and Nestler, 2000, Nestler et al., 2001 and McClung et al., 2004). As a consequence, DeltaFosB have been viewed as a sustained molecular switch to mediate forms of 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 stimulus (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 and García-Pérez et al., 2012). As a consequence, besides these experimental limitations, we will discuss our results considering the role of DeltaFosB in neuronal plasticity.

It is well established that chronic drug exposure increases FosB/DeltaFosB expression in several regions of brain (Nestler et al., 2001 and Perrotti et al., 2008). Curiously, in the present study neither ethanol sensitized nor ethanol non-sensitized mice differed from chronic saline treated mice regarding FosB/DeltaFosB expression 18 h after acquisition phase. Furthermore, there were no significant correlations between FosB/DeltaFosB expression and the scores of locomotor sensitization. This divergence could be explained, at least partially, by the differences found in the experimental protocol. For example, considering the ethanol exposure, in two studies the two bottle free choice paradigm was used in 15 intermittent drinking sessions (Li et al., 2010) or nutritionally complete liquid diet auto-administered during 17 days (where animals consume ethanol at doses ranging from 8 to 12 g/kg/day) (Perrotti et al., 2008). In another study, although the authors refer to chronic treatment, the protocol consisted in only 4 ethanol exposures (Ryabinin and Wang, 1998). So, protocols used elsewhere are totally distinct from the one used here, which consisted of 21 days of treatment where daily ethanol injections were administered by an experimenter. Despite these differences, there are several studies involving intraperitoneal injections reporting increases in FosB/DeltaFosB expression after protocols of locomotor sensitization induced by psychostimulants (Brenhouse and Stellar, 2006, Conversi et al., 2008 and Vialou et al., 2012) and opioids (Kaplan et al., 2011). However, the protocols of locomotor sensitization in those studies involve much less than 21 drug exposures, and in some of them, drug was administered in an intermittent way. In contrast, our protocol used the same treatment described in previous studies involving 21 daily ethanol injections (Masur and dos Santos, 1988, Souza-Formigoni et al., 1999, Quadros et al., 2002a, Quadros et al., 2002b, Abrahão et al., 2011 and Abrahão et al., 2012). There is evidence that although chronic cocaine administration promotes accumulation of DeltaFosB expression in the nucleus accumbens, it also promotes tolerance to DeltaFosB mRNA induction in both ventral and dorsal striatum (Larson et al., 2010). Therefore, we hypothesized that the lack of differences in our experimental groups in the acquisition phase might be due to a tolerance regarding FosB/DeltaFosB induction, since in the present protocol there was a larger period of acquisition phase compared to periods used for psychostimulant and opioids in other studies.

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 seen in both EtOH_High and EtOH_Low group, although more expressive in the former group, suggesting that these increases could have different functional consequences, according to their intensity. This hypothesis could be explained by several distinct functional roles of FosB/DeltaFosB. For example, rats chronically exposed to cocaine had increased DeltaFosB expression in the nucleus accumbens during withdrawal period, an effect positively correlated with cocaine preference, but negatively with novelty preference. Furthermore, stress during withdrawal increases the behavioral response to psychostimulants by increasing DeltaFosB expression in corticolimbic neurons (Nikulina et al., 2012). So, DeltaFosB could predict the dysregulation of hedonic processing that occurs during protracted withdrawal (Marttila et al., 2007). On the other hand, both resilience to stress and antidepressant responses are related to higher DeltaFosB expression in striatum (Vialou et al., 2010). Therefore, we speculate that increased FosB/DeltaFosB on striatum in the EtOH_High could have enhanced the rewarding effects of ethanol, conferring a higher susceptibility to subsequent drug exposures. On the other side, a more intense increase in FosB/DeltaFosB seen in the EtOH_Low group could have decreased the sensitivity to both dysphoria and stress effects, minimizing negative reinforcement effects of subsequent drug exposure and, as a consequence, explaining a higher resistance in this group. Interestingly, this paradox had a neurochemical basis. For example, transgenic mice overexpressing FosB in medium spine GABAergic neurons of nucleus accumbens had increased levels of both mu- and kappa- opioid receptors (Sim-Selley et al., 2011), and those receptors respectively increase and inhibit mesolimbic tone (Manzanares et al., 1991 and Devine et al., 1993). Furthermore, 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. Furthermore, our results suggest that there are active neurochemical changes in the motor cortex during withdrawal period, despite the fact that the animals were not manipulated during this period. This is interesting, because this plasticity could play a role, at least partially, in the maintenance of locomotor sensitization. Although the sustained hyperlocomotion after several days of withdrawal was not studied here, there are several studies, including previous ones from our Lab, showing that sensitized mice (but not non-sensitized) had enhanced locomotion when challenged with ethanol after a given withdrawal period (Masur and dos Santos, 1988, Souza-Formigoni et al., 1999, Quadros et al., 2002a, Quadros et al., 2002b, Abrahão et al., 2011, Abrahão et al., 2012, Fallopa et al., 2012 and Coelhoso et al., 2013).

Finally, it is noteworthy that only the EtOH_Low group displayed an increased FosB/DeltaFosB expression in the anterior (but not posterior) portion of the ventral tegmental area. These portions have distinct projections and neurochemical profiles, and their participation in the reward process depends on several factors (Ikemoto, 2007). For example, rats' self-administration of ethanol is related to the posterior, but not to the ventral portion of the ventral tegmental area (Rodd-Henricks et al., 2000 and Rodd et al., 2004). Furthermore, the endocannabinoid system, as well as GABA-A, dopaminergic D1-D3, and serotoninergic 5HT3 receptors, plays an important role in ethanol seeking behavior (Linsenbardt and Boehm, 2009, Rodd et al., 2010, Melón and Boehm, 2011b and Hauser 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 and 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 basis of this model is that the subjective effects of the drugs increase along their repeated exposure. Once acquired, locomotor sensitization is long lasting and is in direct temporal relation with morphological and neurochemical changes in the mesolimbic pathway and several encephalic nuclei related to emotionality and motor behavior (Robinson and Kolb, 1999 and Vanderschuren and Pierce, 2010). A pioneer study conducted by Masur and dos Santos (1988) demonstrated that there is a large behavioral variability in outbred Swiss mice regarding ethanol-induced locomotor sensitization. From then on other studies have demonstrated an important correlation between neurochemical features and behavioral variability, mainly those related to the dopaminergic (Abrahão et al., 2011, Abrahão et al., 2012 and Souza-Formigoni et al., 1999) and the glutamatergic systems (Quadros et al., 2002a and Quadros et al., 2002b). Furthermore, a previous study from our laboratory using the ethanol-induced locomotor sensitization paradigm showed that sensitized (but not non-sensitized) mice presented a remarkable increase on cannabinoid receptor type 1 (CB1R) during the withdrawal period (Coelhoso et al., 2013). Here we identified different patterns of FosB/DeltaFosB expression during withdrawal between EtOH_High and EtOH_Low groups.

To summarize, behavioral variability observed in acquisition phase of ethanol induced locomotor sensitization is accompanied by distinct neuronal plasticity during withdrawal period. Interestingly, our results suggest that different patterns of FosB/DeltaFosB expression detected in sensitized and non-sensitized mice are more related to withdrawal period rather than to the chronic drug exposure, probably due to the tolerance of drug-induced FosB/DeltaFosB transcription.

The following is the supplementary data related to this article.

Acknowledgments

R.F.P. and C.C.C. received master fellowship from CAPES and FAPESP, respectively. C.T.C., L.E.M., D.X.S. and J.G.S.J. are granted by FAPESP and CNPq.

References

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  • Corresponding author at: Rua Cesário Mota Jr, 61, 12 andar, São Paulo, SP 01221-020, Brazil. Tel./fax: + 55 11 33312008.
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  • These authors equally participated in the present study.

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