Adolescent intermittent ethanol exposure enhances ethanol activation of the nucleus accumbens while blunting the prefrontal cortex responses in adult rat - PubMed (original) (raw)

Adolescent intermittent ethanol exposure enhances ethanol activation of the nucleus accumbens while blunting the prefrontal cortex responses in adult rat

W Liu et al. Neuroscience. 2015.

Abstract

The brain continues to develop through adolescence when excessive alcohol consumption is prevalent in humans. We hypothesized that binge drinking doses of ethanol during adolescence will cause changes in brain ethanol responses that persist into adulthood. To test this hypothesis Wistar rats were treated with an adolescent intermittent ethanol (AIE; 5 g/kg, i.g. 2 days on-2 days off; P25-P54) model of underage drinking followed by 25 days of abstinence during maturation to young adulthood (P80). Using markers of neuronal activation c-Fos, EGR1, and phophorylated extracellar signal regulated kinase (pERK1/2), adult responses to a moderate and binge drinking ethanol challenge, e.g., 2 or 4 g/kg, were determined. Adult rats showed dose dependent increases in neuronal activation markers in multiple brain regions during ethanol challenge. Brain regional responses correlated are consistent with anatomical connections. AIE led to marked decreases in adult ethanol PFC (prefrontal cortex) and blunted responses in the amygdala. Binge drinking doses led to the nucleus accumbens (NAc) activation that correlated with the ventral tegmental area (VTA) activation. In contrast to other brain regions, AIE enhanced the adult NAc response to binge drinking doses. These studies suggest that adolescent alcohol exposure causes long-lasting changes in brain responses to alcohol that persist into adulthood.

Keywords: adolescence; alcohol; development; frontal cortex; networks.

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Figures

Fig. 1

The experimental design (A) and body weight (B), blood ethanol concentration (BEC, B) of Wistar male rats. (A) The timeline of the experimental design. Adolescent intermittent ethanol (AIE) started at postnatal days 25 (P25). Adolescent animals were intermittently administered with either water or ethanol (5 g/kg, 25% ethanol w/v, i.g.) with 2 days on and 2 days off during adolescence (P25-P54). At P80, the ethanol group was subdivided into three groups; two groups of them were separately challenged with 2 or 4 g/kg ethanol (i.g., AIE-challenge), and another group was administered with the same volume water (AIE). Water group also was reassigned to three groups (control, control-challenge 2 and 4 g/kg), and treated as the ethanol group. Animals were handled at least 7 days before the challenge day (P80), and sacrificed 2 h after acute treatment. (B) Body weight was measured every four days during the procedure. A mixed ANOVA with day as the repeated measure revealed a significant day × group interaction [F(13,871) = 2.471, p = 0.003]. There was no difference in the mean of body weight between control and ethanol group depending on body weight match assigned to the experimental groups at P25. The body weight of ethanol group rats significantly decreased at P41 to P62 (16 to 37 days after beginning treatment at P25). *p < 0.05, **p < 0.01 compared with the control. BEC was measured 1 h after treating with ethanol (5 g/kg, i.g.) at P38 and P54, and 2 h after acute ethanol (2 or 4 g/kg, i.g.) at P80. Each point is mean±SEM (n = 32–37/group).

Fig. 2

Brain regions of interest were studied by the histological method. C-Fos- or EGR1-positive cells of shaded areas were counted. Rat atlas panels reprinted from Paxinos and Watson (Paxinos and Watson, 1998). IL, infralimbic cortex; PrL, prelimbic cortex; OFC, orbitalfrontal Cortex; Cg1, cingulate cortex, area 1; Cg2, cingulate cortex, area 2; M2, secondary motor cortex; Pir, piriform cortex; NAc Sh, nucleus accumbens shell; NAc C, nucleus accumbens Core; dBNST, dorsal bed nucleus of stria terminalis; DG, the dentate gyrus; CA2/3, cornu Ammonis 2 and 3; CeA, central nucleus of amygdala; BLA, basolateral nucleus of amygdala; LaA, lateral nucleus of amygdala; Ent, entorhinal cortex; PRh, perirhinal cortex; PVA, paraventricular thalamic nucleus, anterior part; MDL, mediodorsal thalamic nucleus, lateral part; EW, Edinger–Westphal nucleus; aVTA, anterior ventral tegmental area; pVTA posterior ventral tegmental area. We divided VTA in posterior and anterior subdivisions, e.g., pVTA and aVTP respectively. Studies of intracranial self-administration of ethanol find that rats self-administer ethanol into the pVTA, but not the aVTA, (Rodd-Henricks et al., 2000; Rodd et al., 2004; Quertemont et al., 2005) and voluntary ethanol intake by nondependent P rats significantly increased the number of spontaneously active dopamine (DA) neurons in the pVTA (Morzorati et al., 2010) suggesting these subdivisions of VTA differ in ethanol response.

Fig. 3

Effects of adolescent intermittent ethanol (AIE, 5 g/kg, i.g.) on ethanol (2 or 4 g/kg, i.g.)-induced c-Fos expression in the prelimbic (PrL), orbitofrontal (OFC) and infralimbic (IL) cortex of the adult rat brain. Figures on the left side: there was a significant dose × group interaction [F(2,35) = 8.77, p = 0.001] in the PrL and [F(2,36) = 18.98, p < 0.001] in the OFC. Ethanol ( formula image 2 or 4 g/kg, i.g., control-challenge) significantly induced the increase of c-Fos expression in the PrL, OFC and IL of rat brain at P80. **p < 0.05, **p < 0.01, ***p < 0.001 compared with control-challenge to AIE-challenge group at 0.0 g/kg point (, control or AIE) in group respectively. Adolescent intermittent ethanol (AIE) blunted the increase by acute ethanol ( formula image 2 or 4 g/kg, i.g., AIE-challenge). #p < 0.05, ###p < 0.001 compared separately with the water–ethanol (control-challenge) group at either 2.0 or 4.0 g/kg point. Data were expressed as the numbers of c-Fos-positive cells, each point is mean±S.E.M. per mm2 (n = 5–8/group). Right panels: less c-Fos-positive cells expressed for the control group in the PrL (PrL-A, photomicrography from brain section of control-challenge group at 0.0 g/kg point, see marker “A” in the left top figure; “ formula image ” control) and OFC (OFC-D, photomicrography from brain section of control-challenge group at 0.0 g/kg point, see marker “D” in the left middle figure; “” control),). Ethanol induced the increase of c-Fos expression in both the regions (PrL-B, photomicrography from brain section of control-challenge group at 4.0 g/kg point, see marker “B” in the left top figure and OFC-E, see marker “E” in the left middle figure; “ formula image ” control-challenge), and the increases were blocked by AIE (PrL-C, photomicrography from brain section of AIE-challenge group at 4.0 g/kg point, see marker “C” in the left top figure and OFC-F, see marker “F” in the left middle figure; “” AIE-challenge). Scale bar = 50 μm.

Fig. 4

Expression of the transcription factor protein Tbr1 and c-Fos in the Prelimbic cortex (PrL) of adult rat brain. Figure on the left side: 13.23±1.33% (control) and 18.88±1.08% (AIE) of Tbr1+ IR were c-Fos+ IR, ethanol (4 g/kg, i.g., control-challenge) resulted in 42.74±8.18% of increase of c-Fos+ IR in the Tbr1+ IR cells. AIE treatment blocked the increase (30.40±7.45% AIE-challenge). Right side: photomicrography of confocal imagines in the PrL, c-Fos (red) and Tbr1 (green), were shown in right panel (original magnification × 80).

Fig. 5

Effects of adolescent intermittent ethanol (AIE, 5 g/kg, i.g.) on ethanol (4 g/kg, i.g)-induced EGR1 expression in the prelimbic (PrL), orbitofrontal (OFC) and infralimbic (IL) cortex of adult rat brain. Ethanol (4 g/kg, i.g., control-challenge) significantly induced the increase of EGR1 expression in the PrL, OFC and IL cortex at P80. ***p < 0.001 compared with control group. AIE blunted the increase by acute ethanol (4 g/kg, i.g., AIE-challenge). ##p < 0.01, ###p < 0.001 compared with the control-challenge group (left side). Middle panels show the presence of EGR1-positive cells in the PrL (Immunohistochemical staining, Scale bar = 50 μm), and co-labeling with c-Fos-positive cells (right side: immunofluorescence staining, original magnification × 20). There was about 31.94±3.57% c-Fos (green) and EGR1 (red) co-labeling in the c-Fos-positive cells of the PrL area in the control group. Ethanol (4 g/kg, i.g., control-challenge) increased the co-label cells to 64.68±3.09% (p < 0.001). AIE blocked the increase of the co-label cells (47.59±4.52%, p < 0.05). Each point is mean±SEM (n = 5–7/group).

Fig. 6

Effects of adolescent intermittent ethanol (AIE, 5 g/kg, i.g.) on ethanol (4 g/kg, i.g)-induced pERK1/2 expression in the prelimbic (PrL), orbitofrontal (OFC) and infralimbic (IL) cortex of the adult rat brain. Ethanol (4 g/kg, i.g., control-challenge) significantly induced the decrease of pERK1/2 expression in the PrL (73±4%), OFC (83±7%) and IL (78±4%) at P80. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group. AIE blunted the decrease by acute ethanol (4 g/kg, i.g., AIE-challenge) in the PrL and IL. #p < 0.05, ##p < 0.01 compared with control-challenge group (left side). Right panels show the presence of pERK1/2-positive cells in the PrL (Immunohistochemical staining, Scale bar = 50 μm), each point is mean±SEM (n = 5–7/group).

Fig. 7

Effects of adolescent intermittent ethanol (AIE, 5 g/kg, i.g.) on ethanol (2 or 4 g/kg, i.g.)-induced c-Fos expression in the amygdala of the adult rat brain. There was a significant region × dose × group interaction [F(4,64)=3.640, _p_=0.010] between basolateral (BLA), central (CeA) and lateral (LaA) amygdala. There were dose × group interaction [F(2,39)=6.935, _p_=0.003] in the BLA (left top), and [F(2,39)=6.195, _p_=0.005] in the LaA (left below). Ethanol ( formula image 2 or 4 g/kg, i.g., control-challenge) significantly induced the increase of c-Fos expression in the BLA, CeA and LaA at P80. *p<0.05, **p<0.01, ***p<0.001 compared with the control-challenge to the AIE-challenge group at 0.0-g/kg point (, control or AIE) in group respectively. AIE blunted the increase by acute ethanol ( formula image 2 or 4 g/kg, i.g., AIE-challenge). #p<0.05, ##p<0.01 and ###p<0.001 compared with control-challenge group at either 2- or 4-g/kg point (control-challenge). Each point is mean±SEM per mm2 (_n_=6–9/group). Right panels: less c-Fos-positive cells were expressed in the control group in the BLA (BLA-A, photomicrograph from the brain section of the control-challenge group at 0.0-g/kg point, see marker “A” in the left top figure; “ formula image ” control); the expression of c-Fos was increased by acute ethanol (BLA-B, photomicrograph from the brain section of the control-challenge group at 4-g/kg point, see marker “B”; “” control-challenge). Adolescent AIE blocked the increase (BLA-C: Photomicrograph from the brain section of AIE-challenge group at 4-g/kg point, see marker “C”; “ formula image ” AIE-challenge). Scale bar=50 μm.

Fig. 8

Effects of adolescent intermittent ethanol (AIE, 5 g/kg, i.g.) on ethanol (2 or 4 g/kg, i.g.)-induced c-Fos expression in the nucleus accumbens (NAc) shell and core of the adult rat brain. There were a significant dose × group interaction [F(2,40)=4.181, _p_=0.022] in the nucleus accumbens shell (top left) and [F(2,42)=3.467, _p_=0.040] in the core (top right). Ethanol ( formula image 2.0 or 4.0 g/kg, i.g., control-challenge) significantly induced the increase of c-Fos expression in both the core and shell (The bottom photomicrograph, Scale bar=50 μm). **p<0.01, ***p<0.001 compared with control-challenge to AIE-challenge group at 0.0 g/kg point (, control or AIE). AIE augmented ethanol-induced c-Fos expression with 4 g/kg dose ( formula image , AIE-challenge). #p<0.05 compared with control-challenge group at 4.0 g/kg point. Each point is mean±SEM per mm2 (_n_=6–9/group).

Fig. 9

Effects of adolescent intermittent ethanol (AIE, 5 g/kg, i.g.) on adjacent cell numbers of c-Fos+ with Iba1+ co-labeling in the nucleus accumbens (NAc). There were 11.92±1.11% c-Fos+ cells adjacent Iba1+ in control, 17.65±1.35% in AIE-challenge (4 g/kg, 48% increase, p<0.05) in Iba1+ IR (A). Photomicrography of confocal imagines in the NAc, c-Fos (green) and Iba1 (red), were shown in the middle panel (original magnification × 160). There was a correlation between c-Fos+ IR and c-Fos+/Iba+ adjacent cell numbers in the control group [F(1,4)=13.664, _p_=0.034], but not in the AIE-challenge group [F(1,4)=4.637, _p_=0.120] (C).

Fig. 10

Correlations of c-Fos increase across brain regions. Brain regions with significant Pearson correlations of ethanol-induced neuronal activation c-Fos responses across anatomically connected brain regions. Only statistically significant correlations (p < 0.05 or p < 0.01) are shown. A and B Moderate Ethanol challenge: control-challenge (2 g/kg, i.g., blue solid line) and, AIE-challenge (2 g/kg, red-dashed line). C High Ethanol Challenge, Control-challenge (4 g/kg; i.g., green solid line) and AIE-challenge (4 g/kg; i.g., purple-dashed line). Note how AIE markedly altered the moderate ethanol challenge thalamic correlations. Also with the high ethanol challenge the only significant correlations found are VTA and NAc, two reward-related brain regions.

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Adolescent intermittent ethanol exposure enhances ethanol activation of the nucleus accumbens while blunting the prefrontal cortex responses in adult rat - PubMed (original) (raw)