Adolescent binge drinking alters adult brain neurotransmitter gene expression, behavior, brain regional volumes, and neurochemistry in mice.

Experimental Design included adolescent treatment groups (A) and adult treatment groups (B) followed with assessments 40-50 days after treatment. (A) Adolescent mice (P28) were treated with either water or ethanol (5g/kg i.g.) once daily for ten days during adolescence (P28-37). One group underwent behavioral testing in adulthood (P60-72) and were sacrificed one week after the end of behavioral testing for postmortem brain magnetic resonance imaging (MRI) and immunohistochemistry (IHC) (N=10 Control, 8 Ethanol). A second group of adolescent mice was treated with either water or ethanol (5g/kg i.g.) once daily for ten days (P28-37)and assessed for neurotransmitter gene expression 24 hours after ethanol treatment (P38; N=6 Control, 6 Ethanol) as well as after a 50 day period of maturation to adulthood (P88; N=6 Control, 6 Ethanol). (B) Young adult (P88) treatment with either water or ethanol (5g/kg i.g.) once daily for ten days (P88-97). Mice were sacrificed either 24 hours (P98; N=6 Control, 6 Ethanol) or 50 days later (P148; N=5 Control, 6 Ethanol). The Neurotransmitter Receptor and Regulator Gene Superarray™ was used to measure gene expression 24 hours after ethanol treatment in both adolescent (P38) and young adult groups (P98). Similarly following a 50 day period of maturation the Neurotransmitter Receptor and Regulator Gene Superarray™ determined expression in young adult (P88) and mature adult (P148) groups.

Adolescent Binge Ethanol Treatment Reduces Choline Acetyltransferase (ChAT) gene expression and ChAT-immunoreactivity. Acetylcholine neurons were identified by immunohistochemistry for choline acetyltransferase (ChAT). (A) ChAT mRNA levels changed significantly across development. ChAT mRNA in adolescent mice (P38 – 100%) decreased during maturation to P88 (64% reduction, *p<0.05) in control animals. ChAT mRNA expression 24 hours following adolescent ethanol binge treatment showed a 55% reduction (P38). Fifty days after the adolescent ethanol binge ChAT mRNA levels were reduced 85% (**p<0.01 vs P38 control). (B) Reduced ChAT-immunoreactive (ChAT+IR) neurons in adults. ChAT+IR neurons were counted in the posterior regions of the basal forebrain (bregma 0.14mm to −0.22mm), where the greatest volume changes were observed. This included the horizontal diagonal band, substantia innominata, ventral pallidum, and magnocellular preoptic nucleus. Significant reductions in the number of ChAT+IR neurons per area were observed (7.5% reduction, *p<0.05, N=10 Control, 8 Ethanol). Representative images from an adult controls (C) and adults following adolescent binge ethanol treatment (D) at the same bregma (0.14mm) showing differences in ChAT+IR.

Additional details are within the methods. Adolescent ethanol (AE) binge treatment, but not adult ethanol binge, robustly alters the developmental trajectory of neurotransmitter-specific gene expression. Mice received water or ethanol (5g/kg, i.g.) once a day for ten days during either adolescence (P28-37) or adulthood (P88-97). Neurotransmitter-specific gene expression was assessed using a gene SuperArray either 24 hours (P38 or P88) or 50 days (P88 or P148) after the last alcohol treatment. Expression for each gene is compared to control expression levels 24 hours after treatment (P38 or P88). (A) Developmental changes in gene expression in young control mice. A developmental reduction in neurotransmitter receptor and regulator gene expression was observed across the entire array (Average of 56% reduction, 2-way ANOVA, ***p<0.001) with 36 genes reaching a p<0.05. Genes that showed either a potential increase in expression or no reduction at all are labeled. (B) Developmental changes in gene expression in adult control mice. A developmental reduction in neurotransmitter receptor and regulator gene expression was observed across all the genes tested (2-way ANOVA, ***p<0.0001) that was on average less than controls (33% average reduction) with 11 genes having a p<0.05. (C) Adolescent binge ethanol treatment potentiates the developmental reduction in gene expression, causing significant reductions in 59 genes (p<0.05, t-test of normalized ΔCt values vs P38 control) with 31 genes maintaining statistical significance following Bonferroni adjusted post-tests. (D) Adult binge ethanol resulted in only two genes showing reductions from P98 to P148 that reached a p<0.05, with zero changes following Bonferroni adjusted post-tests. Thick lines and corresponding labels illustrate which treatment was given prior to testing.

Effects of adolescent binge ethanol treatment ondopamine D4 receptor (D4DR) gene and protein expression. Adolescent mice received either water or binge ethanol (5g/kg once daily, i.g.) for ten days (P28-37). Mice were sacrificed 24 hours (P38) or 50 days (P88) after the last treatment for RT-PCR and immunohistochemistry. (A) The adolescent ethanol binge treatment caused a 71% (*p<0.05 vs P38 controls) reduction in D4DR gene expression in whole brain 24 hours after treatment at P38 that remained reduced at P88. Control mice underwent a 55% developmental reduction in D4DR gene expression (P38 to P88). (B) Quantification of punctate D4DR immunoreactivity (D4DR+IR) in the orbitofrontal cortex in adolescent mice (P38). Adolescent mice that received binge ethanol had 44% less D4DR+IR in the orbitofrontal cortex than controls (Controls: 26800 ± 5500 D4DR+IR puncta/mm2; Ethanol: 11790 ± 2900 D4DR+IR puncta/mm2, *p<0.05, t-test; N=3 Control, N=4 Ethanol). Although gene expression is whole brain and D4DR+IR protein levels are orbital frontal cortex, both show significant decreases following adolescent ethanol binge treatment. (C and D) Images of orbitofrontal cortex from adolescent (C: P38) and adult (D: P88) mice. C: Images of punctate D4DR+IR staining in P38 control and P38 binge ethanol (Etoh) treated mice (magnification 120×). D4DR+IR puncta are identified by the arrow heads (Δ). Adult D4DR+IR are both punctate and somatic. Somatic staining is identified by arrows. D: Images of orbitofrontal cortex from adult (P88) mice. Note D4DR+IR staining in P88 adult is both punctate and somatic. Quantification of P88 D4DR+IR was 6800 ± 4500 D4DR+IR puncta/mm2 for controls and 4790 ± 2900 D4DR+IR puncta/mm2 for ethanol treated animals (N=3 Control, N=4 Ethanol). (t-test; vs P38 controls, Scale bar: 20 micron).

Adolescent alcohol binge differentially alters developmental trajectory of neurotransmitter receptor and regulator gene expression. RT-PCR was performed using an RT2 Profiler PCR Array for neurotransmitter receptor and regulator genes. mRNA levels are presented as percent of P38 control to demonstrate the effects across development Patterns of adolescent ethanol binge (AE binge) treated animal developmental trajectory for Cholecystokininβ (CCKβ) and Catechol-O-methyltransferase (COMT) are shown. (Left) Binge ethanol treatment reduces P38 CCKβ expression by 64% of P38 control. Both control and ethanol groups show a similar developmental decline during the fifty days after the adolescent ethanol binge. In controls, CCKβ receptor gene expression decreases 55% across development from P38 (100%) to P88 (45% of P38 control, *p<0.05). In ethanol binge animals CCKβ mRNA levels were reduced 82% (**p<0.01 vs P38 control). However, the developmental decline from P38 AE binge (55% of P38 control) to P88 AE binge (18% of P38 control) is comparable to the control developmental decline. Adult P88 AE binge animals express 39% of P88 control CCKβ mRNA expression (†p<0.05, vs P88 control) similar to the 45% P38 AE binge animals suggesting the developmental decline continued a similar course following the initial AE binge insult. (Right) Catechol-O-methyltransferase (COMT) shows a different pattern. Binge ethanol treatment does not change P38 COMT gene expression. The 50 day developmental decline in COMT brain gene expression in controls was not statistically significant (29% reduction, p=0.24). However, AE binge treated animals showed a 64% developmental reduction in COMT gene expression (p<0.01 vs P38 controls). AE binge treatment appears to alter the developmental trajectory of COMT gene expression.

Mice that received ethanol binge during adolescence had a deficit in reversal learning as adults. Following acquisition of reversal learning, mice were tested in a 60 second probe trial in the absence of the hidden platform. (A) Control mice spent a nearly equal amount time in the initial learning quadrant (Quadrant 1, 22.4%±3.02) and the reversal learning quadrant (Quadrant 3, 25.5%±2.5). Adult mice were treated with ethanol during adolescence spent more than twice as much time in the quadrant where the platform was during initial learning (Quadrant 1, 35.6%±5.5) than where the platform was during reversal learning (Quadrant 3, 15.5%±7.7) (**p < 0.01, t-test). Also, control mice spent significantly more time in the reversal quadrant (Quadrant 3, 25.5%±2.5) than the mice that received alcohol during adolescence (15.5%±7.7) († p<0.05, t test). Data is presented as mean ± SEM. N = 7 Control, 7 Ethanol. (B) Representative tracings of the reversal probe trial from one animal in each treatment group, depicting the increased time spent by control mice in the reversal learning quadrant (Quadrant 3) compared to adolescent ethanol treated mice, which spent more time in the initial learning quadrant (Quadrant 1).

Representative automatic segmentation of structural MRI. (A) Representative structural MRI of a control mouse. Mean diffusivity images were computed from reconstructed diffusion tensor data. (B) Brain regions were segmented in an automated fashion in order to identify candidate regions for further manual segmentation. Key: pink-olfactory bulb, blue-neocortex, brown-basal forebrain, yellow-anterior commissure, bright green-corpus callosum, red-hippocampus, purple-thalamus, turquoise-hypothalamus, dark gray-rest of midbrain, light gray- central gray, orange-superior colliculi, dark green-cerebellum, yellow green-brain stem.

Adolescent ethanol binge causes a reduction in the volume of the olfactory bulb in adults. Adolescent mice received either water or ethanol (5g/kg) once a day for ten days (P28-37). Postmortem MRI was performed on adults (P79). Following automatic segmentation, manual segmentation of the olfactory was performed by blinded investigators. (A) 3D representation of the brain showing large relative size of the olfactory bulb in the rodent brain (darkened). (B) Overlay of 3D renderings of representative control and ethanol treated adult olfactory bulbs. Arrowheads highlight volume differences between control (black) and ethanol treated (white) groups (C) Adult olfactory bulb volume quantification following manual correction. Adult mice that received binge ethanol treatment during adolescence showed a 7.8% reduction in the olfactory bulb volume (Control: 23.37 ± 0.42 mm3; Ethanol: 21.54 ± 0.33 mm3; **p<0.005; N=10 Control, 8 Ethanol).

Adolescent ethanol binge causes a reduction in the volume of the basal forebrain/medial septum brain region in adults. Adolescent mice received either water or ethanol (5g/kg) once a day for ten days (P28-37). Postmortem MRI was performed on adults (P79), the basal forebrain region was manually segmented by blinded investigators, and its volume was measured. (A) 3D schematic of the location of the basal forebrain/medial septum region from the top, sagittal, and front/coronal perspectives. (B) Basal forebrain volumes from manual segmentation. Adult mice that previously received ethanol showed a 4.6% reduction in the volume of the basal forebrain/medial septum region (Control: 11.95 ± 0.12 mm3; Ethanol: 11.40 ± 0.12 mm3, **p<0.01; N=10 Control, 8 Ethanol). (C) A regional analysis of the basal forebrain volume shows that the majority of the volume reduction (11% reduced from controls) was to the posterior segment of the basal forebrain (bregma + 0.13 to −0.46), ***p<0.001. (D) sagittal 3D rendering of two representative cases showing volume reduction in adult mice that received adolescent binge ethanol.

Basal forebrain nuclei area reduction in adults following adolescent ethanol treatment. In order to confirm MRI volume reductions, the area of the basal forebrain region nuclei was measured microscopically on sections immunolabeled for parvalbumin using bioquant software, in three different regions defined by their distances from bregma (1.34 to 1.1 mm, 0.86 to 0.14 mm, and 0.02 to −0.22 mm). Area measures were taken on up to three different immunolabeled sections per mouse, N=4-7 mice for each region. (A) The basal forebrain area was significantly reduced in the posterior bregma of the basal forebrain: 20% difference, 0.02 to −0.22 mm, *p<0.05; N=5 Control and 5 Ethanol mice. Basal forebrain areas measured at bregma 0.14mm (B) and −0.22mm (C) are shown from representative sections. The anterior commissure (open arrow) and internal capsule (closed arrow) were used as landmarks and are labeled on the atlas and image to help with orientation. Key: VP-ventral pallidum, SI-substantia innominata, HDB-horizontal diagonal band, MCPO-magnocellular preoptic nucleus, LPO-lateral preoptic area, IPAC-interstitial nucleus of the posterior limb of the anterior commissure, BST-bed nucleus of stria terminalis, AAD-anterior amygdaloid area dorsal, aca-anterior commissure anterior.