Neuroimmune and epigenetic involvement in adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons: Restoration with voluntary exercise

Abstract

Binge drinking and alcohol abuse are common during adolescence and cause lasting pathology. Preclinical rodent studies using the adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P]25 to P55) model of human adolescent binge drinking report decreased basal forebrain cholinergic (ie, ChAT+) neurons that persist into adulthood (ie, P56-P220). Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying molecular mechanisms contributing to the persistent loss of basal forebrain ChAT+ neurons are unknown. We report here that the AIE-induced loss of cholinergic neuron markers (ie, ChAT, TrkA, and p75^NTR ), cholinergic neuron shrinkage, and increased expression of the neuroimmune marker pNF-κB p65 are restored by exercise exposure from P56 to P95 after AIE. Our data reveal that persistently reduced expression of cholinergic neuron markers following AIE is because of the loss of the cholinergic neuron phenotype most likely through an epigenetic mechanism involving DNA methylation and histone 3 lysine 9 dimethylation (H3K9me2). Adolescent intermittent ethanol caused a persistent increase in adult H3K9me2 and DNA methylation at promoter regions of Chat and H3K9me2 of Trka, which was restored by wheel running. Exercise also restored the AIE-induced reversal learning deficits on the Morris water maze. Together, these data suggest that AIE-induced adult neuroimmune signaling and cognitive deficits are linked to suppression of Chat and Trka gene expression through epigenetic mechanisms that can be restored by exercise. Exercise restoration of the persistent AIE-induced phenotypic loss of cholinergic neurons via epigenetic modifications is novel mechanism of neuroplasticity.

Keywords: adolescence; alcohol; binge drinking; choline acetyltransferase; methylation; reversal learning.

Figure 1

Graphical representation of the adolescent intermittent ethanol (AIE) paradigm and experimental design. On postnatal day (P)21, male Wistar rats were randomly assigned to either (1) water control (CON) or (2) AIE conditions. From P25 to P55, AIE subjects received a single daily intragastric (i.g.) administration of ethanol (5.0 g/kg, 20% ethanol, w/v [black tick marks represent a single ethanol binge]) in the AM on a 2‐day on/2‐day off schedule, and CON subjects received comparable volumes of water on an identical schedule. Tail blood was collected 1 hour after treatment to assess blood ethanol concentrations (BECs) on P38 (AIE/no exercise: 162 mg/dL [±13], AIE/exercise: 176 mg/dL [±54], one‐way ANOVA: P > 0.8) and P54 (AIE/no exercise: 137 mg/dL [±21], AIE/exercise: 159 mg/dL [±13], one‐way ANOVA: P > 0.4) using a GM7 analyzer (Analox, London, UK). Both CON‐ and AIE‐treated subjects were pair‐housed on P56 (24 h post‐AIE) in their respective housing conditions 24 hours per day in either standard cages (no exercise) or cages containing running wheels (exercise). Subjects ran a cumulative average of 242 km for the duration of experimentation (CON: 236 km [±14 km]; AIE: 249 km [±47 km]; one‐way ANOVA: P > 0.8). All subjects evidenced dramatic body weight increases across age (main effect of age: P < 0.01). We did not observe an effect of treatment (P > 0.9) or exercise (P > 0.05) on body weight although exercising subjects overall evidenced an approximately 10% reduction of body weight relative to the nonexercising subjects. 5′‐Bromo‐2′‐deoxyuridine (BrdU) was administered during restorative exercise exposure (75 mg/kg, i.p., every 4 days from P56 to P79) to determine generation of new cholinergic neurons in the adult basal forebrain. Spatial and reversal learning was assessed on the Morris water maze (MWM) from P80 to P91. At the conclusion of the study (P95), subjects were sacrificed and tissue collected for analysis

Figure 2

Adolescent intermittent ethanol (AIE) exposure reduces choline acetyltransferase immunoreactive (ChAT+IR) neuron populations in the late adolescent basal forebrain that persists into adulthood. Modified unbiased stereological assessment of ChAT+IR neurons in the basal forebrain revealed an AIE‐induced reduction of 24% (±8%) on P56 (24 h post‐AIE), 31% (±4%) on P80 (25 days post‐AIE), 28% (±5%) on P95 (40 days post‐AIE), and 19% (±2%) on P220 (165 days post‐AIE), relative to CONs. Data are presented as % change relative to age‐matched CONs (n = 8/group). *P < 0.05, **P < 0.01, relative to CONs

Figure 3

Wheel running restores the adolescent intermittent ethanol (AIE)‐induced loss and somal shrinkage of choline acetyltransferase immunoreactive (ChAT+IR) cholinergic neurons in the adult basal forebrain. A, Modified unbiased stereological assessment revealed a 28% (±5%) reduction of ChAT+IR neurons in the adult (P95) basal forebrain of AIE‐treated subjects, relative to CONs. Running wheel exposure from P56 to P95 did not affect ChAT expression in CONs, but did restore the AIE‐induced loss of ChAT+IR neurons, relative to no exercise AIE subjects. B, Analysis of ChAT+IR neuron somal size revealed a 24% (±4%) reduction in the adult basal forebrain of AIE‐treated subjects, relative to CONs. Wheel running did not affect ChAT neuron somal size in CONs, but did restore the AIE‐induced ChAT+IR neuron somal shrinkage in the adult basal forebrain, relative to the no exercise AIE subjects. C, Representative photomicrographs of ChAT+IR neurons in the adult basal forebrain from CON‐ and AIE‐treated subjects across exercise conditions. Scale bar = 50 μm. Data are presented as mean ± SEM (n = 7‐8/group). *P < 0.05, **P < 0.01

Figure 4

Voluntary exercise exposure following adolescent intermittent ethanol (AIE) restores the loss of tropomyosin receptor kinase A (TrkA)‐ and p75^NTR‐immunoreactive cells in the adult basal forebrain. A, Modified unbiased stereological quantification of the high‐affinity nerve growth factor (NGF) receptor TrkA in the adult (P95) basal forebrain revealed a 27% (±6%) reduction in AIE‐treated subjects, relative to CONs. Running wheel exposure from P56 to P95 did not affect TrkA expression in CONs, but did restore the AIE‐induced loss of TrkA+IR neurons, relative to no exercise AIE subjects. Representative photomicrographs of TrkA+IR neurons in the adult basal forebrain from CON‐ and AIE‐treated subjects across exercise conditions. Scale bar = 50 μm. B, Modified unbiased stereological quantification of the low‐affinity NGF receptor p75^NTR in the adult (P95) basal forebrain revealed a significant 31% (±6%) reduction in AIE‐treated animals, relative to CONs. Wheel running alone did not affect p75^NTR expression in CONs, but did restore the AIE‐induced loss of p75^NTR+IR neurons, relative to no exercise AIE subjects. Representative photomicrographs of p75^NTR+IR neurons in the adult basal forebrain from CON‐ and AIE‐treated subjects across exercise conditions. Scale bar = 50 μm. C, Immunofluorescent colabeling revealed a high degree of TrkA (red) and p75^NTR (green) colocalization with ChAT+IR neurons (blue) in the adult (P95) basal forebrain. Data are presented as mean ± SEM (n = 8/group). *P < 0.05, **P < 0.01

Figure 5

Exercise exposure following adolescent intermittent ethanol (AIE) treatment reverses the increased expression of phosphorylated nuclear factor kappa‐light‐chain‐enhancer of activated B cells p65 (pNF‐κB p65) in the adult basal forebrain. Modified unbiased stereological quantification of pNF‐κB p65+IR cells revealed a 22% (±6%) increase in the adult (P95) basal forebrain of AIE‐treated animals, relative to CONs. Wheel running alone did not affect pNF‐κB p65+IR in CONs, but did block the AIE‐induced increase of pNF‐κB p65+IR cells, relative to no exercise AIE subjects. Scale bar = 50 μm. Data are presented as mean ± SEM (n = 7‐8/group). *P < 0.05, **P < 0.01

Figure 6

Wheel running after adolescent intermittent ethanol does not induce generation of new basal forebrain cholinergic neurons. A, Immunofluorescent assessment revealed that BrdU (green) did not colocalize with either choline acetyltransferase (ChAT; blue)‐ or NeuN (red)‐immunopositive neurons in the adult basal forebrain. Further, BrdU+IR in the basal forebrain was unaffected by AIE treatment or wheel running exposure. B, Modified unbiased stereological assessment of the neuron marker NeuN in the adult basal forebrain revealed that AIE treatment did not affect NeuN+IR neuron counts, relative to CONs. Data are presented as mean ± SEM (n = 6/group)

Figure 7

Wheel running recovers the adolescent intermittent ethanol (AIE)‐induced choline acetyltransferase (Chat) gene histone and DNA methylation in the adult basal forebrain. A, Chromatin immunoprecipitation assessment revealed that occupancy of histone 3 lysine 9 dimethylation (H3K9me2) at the promoter of the Chat gene was increased by approximately 2.2‐fold in the basal forebrain of adult (P95) AIE‐treated animals, relative to CONs. Running wheel exposure from P56 to P95 did not affect levels of H3K9me2 in CONs, but did resolve the AIE‐induced increase of H3K9me2 at the promoter of the Chat gene, relative to no exercise AIE subjects. B, Methylated DNA immunoprecipitation (5‐methyl cytosine) assessment revealed that DNA methylation at the CpG island in the Chat promoter was increased by 2.5‐fold in the adult (P95) basal forebrain of AIE‐treated animals, relative to CONs. Running wheel exposure from P56 to P95 did not affect Chat DNA methylation in CONs, but did resolve the AIE‐induced increase of DNA methylation at the CpG island in the Chat promoter, relative to no exercise AIE subjects. Data are presented as mean ± SEM (n = 8‐10/group). ***P < 0.001. TSS, transcription start site

Figure 8

Voluntary exercise exposure recovers the adolescent intermittent ethanol (AIE)‐induced tropomyosin receptor kinase A (Trka) gene histone methylation in the adult basal forebrain. A, Chromatin immunoprecipitation (ChIP) assessment revealed that occupancy of histone 3 lysine 9 dimethylation (H3K9me2) of the Trka gene at the distal promoter region was increased by approximately 1.7‐fold in the basal forebrain of adult (P95) AIE‐treated animals, relative to CONs. Running wheel exposure from P56 to P95 did not affect levels of H3K9me2 in CONs, but did resolve the AIE‐induced increase of H3K9me2 at the distal promoter of the Trka gene, relative to no exercise AIE subjects. B, ChIP assessment revealed that levels of H3K9me2 at the CpG island in the Trka promoter were increased by approximately 1.7‐fold in the basal forebrain of adult AIE‐treated animals, relative to CONs. Running wheel exposure from P56 to P95 did not affect levels of H3K9me2 in CONs, but did resolve the AIE‐induced increase of H3K9me2 at the CpG island in the Trka promoter, relative to no exercise AIE subjects. C, ChIP assessment revealed that wheel running reduced H3K9me2 at the proximal Trka promoter region of adult AIE‐treated animals, relative to no exercise AIE subjects. Data are presented as mean ± SEM (n = 8‐10/group). *P < 0.05, **P < 0.01. TSS, transcription start site

Figure 9

Voluntary exercise exposure following adolescent intermittent ethanol (AIE) restores reversal learning deficits on the Morris water maze. Spatial and reversal learning were assessed in adult subjects using the Morris water maze. Spatial learning was assessed from P82 to P86, and all subjects learned to locate and escape onto the submerged platform to criterion levels by P85. In the CON subjects, performance on the Morris water maze did not differ as a function of exercise exposure, and CON groups were combined to gain statistical power for behavioral assessment. While AIE treatment did not affect the latency to escape or distance traveled during the spatial learning component, all subjects reduced their escape latency across testing days. A, Latency to escape onto the submerged platform during reversal learning (ie, P87 to P91) was increased by 53% during first day and 74% during the second day in the AIE‐treated animals, relative to CONs. Voluntary wheel running blunted the AIE‐induced increase in latency to escape onto the submerged platform during the first and second days of reversal learning, relative to no exercise AIE‐treated animals. B, Assessment of perseveration, defined as time spent in the previous spatial goal quadrant during reversal learning, revealed that AIE treatment increased time spent in the previous spatial goal quadrant by 93% during the second trial of reversal learning, relative to CONs. Wheel running blunted the AIE‐induced increase in perseverative behavior during the second trial of reversal learning, relative to no exercise AIE‐treated animals. Data are presented as mean ± SEM (n = 8‐10/group). *P < 0.05, **P < 0.01

Figure 10

Simplified schematic depicting the proposed mechanism underlying the persistent adolescent intermittent ethanol (AIE)‐induced loss of basal forebrain cholinergic neurons. A, In naïve basal forebrain, cholinergic neurons express choline acetyltransferase (ChAT), the high‐affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA), and the low affinity NGF receptor p75^NTR. (Top) ChAT+IR neurons in the adult (P95) basal forebrain. (Bottom) Schematic depicting ChAT+IR basal forebrain cholinergic neurons in orange. Note that “n” in the nucleus represents NeuN immunoreactivity. B, AIE causes loss of ChAT+IR neurons in the adolescent (P56) basal forebrain that persists into adulthood (P220). (Top) AIE‐induced loss of ChAT+IR neurons and shrinkage of remaining cholinergic neurons in the adult (P95) basal forebrain. (Bottom) AIE‐induced loss of ChAT‐, TrkA‐, and p75^NTR‐immunopositive basal forebrain neurons (dashed neuron lacking ChAT [orange]) as well as shrinkage of remaining cholinergic neurons. Note that n in the nucleus represents NeuN+IR, which was unchanged by AIE consistent with loss of the cholinergic phenotype and not cell death. AIE increased dimethylation of lysine 9 of histone 3 (H3K9me2) associated with promoter regions on the Chat and Trka gene in the adult basal forebrain. AIE increased phosphorylation of the proinflammatory transcription factor NF‐κB p65 in the adult basal forebrain, and neuroimmune signaling might alter gene expression in part through epigenetic mechanisms.32 Loss of basal forebrain cholinergic neurons might contribute to the neurocognitive deficits observed in adult AIE‐treated subjects and AIE treatment caused long‐term impairments in reversal learning on the Morris water maze. C, Voluntary wheel running from P56 to P95 restored the AIE‐induced loss of basal forebrain cholinergic neurons. (Top) Restoration of ChAT+IR neurons and shrinkage of remaining cholinergic neurons in the adult (P95) basal forebrain of AIE‐treated subjects. (Bottom) Restoration of ChAT‐, TrkA‐, and p75^NTR‐immunopositive neurons and reversal of cholinergic neuron shrinkage in adult (P95) basal forebrain of AIE‐treated subjects. Importantly, the restorative effects of exercise do not appear to be because of the generation of new cholinergic neurons as ChAT did not colocalize with BrdU in the adult basal forebrain consistent an AIE‐induced loss of the basal forebrain cholinergic neuron phenotype. Restorative exercise restored to CON levels the AIE‐induced increase of H3K9me2 in promoter regions of both the Chat and Trka genes as well as the increased expression of pNF‐κB p65 in the adult basal forebrain. This suggests that restorative exercise is a potential treatment modality for maladaptive changes in neural architecture in the basal forebrain that contributes to deficits in learning and cognitive function associated with adolescent binge drinking