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 p75NTR), 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.


| INTRODUCTION
Adolescence is an evolutionarily conserved developmental period of neurotransmitter system refinement that parallels the transition of the immature brain to the more efficient adult brain. 1 The basal forebrain cholinergic system is essential for cognitive functioning 2,3 through its acetylcholine inputs to the cortex and hippocampus. 4 Alcohol binge drinking is common during adolescence 5,6 and is associated with lasting consequences that persist into adulthood, including increased risk for diagnosis with alcohol use disorder, 7 higher rates of comorbid mental disorders, 8 and neuropathological changes in the basal forebrain. 9 9,10 ). This is accompanied by somal shrinkage of the remaining ChAT+ neurons as well as persistent reductions of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor p75 NTR , which are highly expressed on basal forebrain cholinergic neurons 10 and critical for cholinergic neuron function. 11 The AIEinduced loss of adult ChAT+IR neurons is associated with reversal learning deficits and persistent neuroimmune activation. Treatment with lipopolysaccharide induces forebrain neuroimmune genes and mimics the AIEinduced loss of basal forebrain cholinergic neurons. 9,10 Exposure to voluntary wheel running or the anti-inflammatory drug indomethacin during AIE treatment prevents ChAT+IR loss and neuroimmune gene induction. 10 These findings are consistent with AIE neuroimmune gene induction contributing to adult ChAT+IR neuron degeneration.
Forebrain ChAT+ neurons are known to be dependent upon neurotrophic (eg, NGF) inputs from the cortex and hippocampus. Previous studies find fimbria-fornix lesion-induced loss of basal forebrain cholinergic neurons (ie, ChAT+ and p75 NTR +) and cholinergic neuron shrinkage. 12,13 Recent studies suggest that AIE induces long-lasting changes in neuroimmune genes as well as Bdnf, Arc, and other trophic-neuroplasticity genes that are regulated through epigenetic mechanisms. 14 For example, AIE exposure causes a long-lasting inhibition of adult hippocampal neurogenesis that is reversed by exercise and anti-inflammatory drugs 15 as well as the histone deacetylase inhibitor TSA. 16 Fimbria-fornix lesioninduced loss of basal forebrain cholinergic neurons (ie, ChAT+ and p75 NTR +) and cholinergic neuron shrinkage can be recovered by intraventricular infusions of NGF. 12,13 Similarly, we previously reported that exercise exposure during AIE prevented the AIE-induced loss of cholinergic neuron markers (ie, ChAT+, TrkA+, and p75 NTR +) and cholinergic neuron shrinkage in the adult basal forebrain. 10 These studies suggest that the persistent AIE-induced loss of ChAT+ neurons may be because of the loss of the cholinergic phenotype, but the mechanism remains to be identified. Emerging studies reveal that ethanol elicits chromatin remodeling in brain through epigenetic modifications leading to changes in gene expression that appear to contribute to alcohol-induced neuropathology. 14,17 Epigenetic modifications involve histone acetylation and histone or DNA methylation, which can cause activation or repression of gene transcription without changing the underlying DNA sequence resulting in a specific phenotype. [18][19][20] Interestingly, AIE treatment has been shown to induce long-lasting epigenetic modifications in the amygdala and hippocampus of adult rats, and these effects can be prevented by administration of histone deacetylase inhibitors. 16,21 Acetylation and methylation of histone 3 lysine 9 (H3K9) is known to activate and repress gene transcription. 22 Recently, it was reported that AIE increased histone 3 acetyl 9 dimethylation (H3K9me2) of Bdnf gene in the amygdala during adulthood. 23 Interestingly, aerobic exercise has been shown to modulate epigenetic processes. 19,24 However, it is unknown if epigenetic processes contribute to the persistent loss of basal forebrain cholinergic neurons. We therefore tested the hypothesis that exercise exposure post-AIE treatment (ie, P56-P95) would restore cholinergic neuropathology. We report here for the first time that voluntary exercise exposure initiated 24 hours following AIE restored cholinergic neuron marker expression and blocked phosphorylation of proinflammatory NF-κB p65 in the adult basal forebrain. We did not observe formation of new basal forebrain neurons following restorative exercise exposure consistent with loss of the cholinergic phenotype. Further, we report that AIE increased H3K9me2 and DNA methylation on promoter regions of the Chat gene and H3K9me2 on the Trka gene in the adult basal forebrain, which was prevented by wheel running exercise. In addition, wheel running restored the AIE-induced reversal learning deficits on the Morris water maze. Together, these data implicate a novel neurobiological process involving neuroimmune and epigenetic mechanisms resulting in the phenotypic loss of basal forebrain cholinergic neurons following AIE.

| AIE paradigm
Male Wistar rats were used in this study. See Figure 1 for description of AIE treatment paradigm. Subjects were housed in a temperature-(20°C) and humidity-controlled vivarium on a 12-hour/12-hour light/dark cycle (light onset at 0700 h) and provided ad libitum access to food and water. Experimental procedures were approved by the IACUC of the University of North Carolina at Chapel Hill and conducted in accordance with NIH regulations for the care and use of animals in research.

| Morris water maze
Spatial and reversal learning was assessed (n = 8-10 subjects per group) as previously described. 27 Briefly, habituation training was conducted one trial per day for two consecutive days beginning on P80. Twenty-four hours later, spatial learning assessment was conducted over three trials per day for five consecutive days with an intertrial interval (ITI) of approximately 80 minutes per trail each day.
The escape platform was situated in the middle of the northern quadrant 2 cm below the water line. Subjects were released into the water facing the wall at one of three pseudo-randomized locations (ie, south, southwest, and southeast). Reversal learning assessment, which started 24 hours after the last spatial trial, was conducted over three trials per day for five consecutive days with an ITI of approximately 80 minutes. For the reversal learning trials, the escape platform was situated in the middle of the southern quadrant and placed 2 cm below the water line. Subjects were released into the water tank facing the wall at one of three pseudo-randomized locations (ie, north, northwest, and northeast). A ceiling-mounted automated tracking system (Ethovision XT 8.0, Noldus Ethovision; Leesburg, VA) was used to track the subject's movement.

| Immunohistochemistry
At the conclusion of experimentation, subjects were anesthetized (n = 8 subjects per group), and tissue collected as previously described. 10 Free-floating basal forebrain samples (every sixth section; approximately Bregma: 1.60-0.20 mm based on the atlas of Paxinos and Watson 28 ) were processed as previously described. 10

| Microscopic quantification and image analysis
Across experiments, BioQuant Nova Advanced Image Analysis software (R&M Biometric, Nashville, TN) was used for image capture and quantification of immunohistochemistry as previously described. 10 Briefly, a modified unbiased stereological quantification method was used to quantify immunopositive cells in the rat basal forebrain. The outlined regions of interest were determined, and data expressed as cells per square millimeter. Somal size was determined using BioQuant Nova Advanced Image Analysis software (R&M Biometric).

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  ; 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

| Fluorescent immunohistochemistry and microscopy
Free-floating basal forebrain sections were processed as previously described. 29,30 Briefly, for assessment of cholinergic neuron marker colocalization, sections were incubated for 48 hours at 4°C in a primary antibody cocktail of goat anti-ChAT (Millipore), TrkA (Millipore), and p75 NTR (Millipore). To assess cholinergic neuron marker colocalization with BrdU, tissue was similarly incubated in a primary antibody cocktail of goat anti-ChAT (Millipore), rabbit anti-NeuN (Millipore, Cat. #MABN140), and mouse anti-BrdU (Millipore). Sections were then incubated for 2 hours at room temperature in the secondary antibody cocktail (rabbit Alexa Fluor 594, mouse Alexa Fluor 488, and goat Alexa Fluor 350; Invitrogen, Carlsbad, CA). Immunofluorescent images were obtained using a DS-RiZ scope (Nikon Inc., Melville, NY) and colocalization quantified using NIS Elements AR46 (Nikon Inc.).

| Chromatin immunoprecipitation
On P95, exercising and nonexercising CON-and AIE-treated subjects (n = 8-10 subjects per group) were anesthetized, and basal forebrain tissue dissected according to the atlas of Paxinos and Watson, 28 rapidly frozen in liquid nitrogen, and stored at −80°C for ChIP assessment. The procedure is similar to the methods reported previously. 23 Briefly, basal forebrain tissue samples were homogenized, fixed in Both immunoprecipitated DNA and input DNA were eluted in 10% (w/v) Chelex by boiling at 95°C for 10 minutes followed by centrifugation. The resulting DNA was quantified using qPCR with SSOAdvanced Universal SYBR Green Supermix (Bio-Rad, Berkeley, CA) using primers targeted against the Trka and Chat promoters (see Table 1). The ΔΔCt method was used to determine fold change relative to control and was normalized to the Input DNA fraction.

| Methylated DNA immunoprecipitation
Frozen basal forebrain tissue (n = 8-10 subjects per group) was processed using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) to obtain DNA. The resulting DNA was fragmented to 200 to 500 BP, and DNA cleaned using a QIAquick PCR Purification Kit (Qiagen). DNA (1.0 μg) was then used for meDIP using the Methylated-DNA IP Kit (Zymo, Irvine, CA, Cat. #D5101) following manufacturer's instructions. Following elution, meDIP and input DNA were quantified using qPCR with SSOAdvanced Universal. SYBR Green Supermix using primers targeted against the Trka and Chat promoters (see Table 1). The ΔΔCt method was used to determine fold change relative to control and was normalized to the input DNA fraction.

| Statistical analysis
Statistical analysis was performed using SPSS (Chicago, IL). One-way analysis of variance (ANOVA) was used to assess BECs and the NeuN immunohistochemistry data. The data on body weight and Morris water maze behavior were assessed using repeated measure ANOVAs. The immunohistochemical, ChIP, and meDIP data were analyzed using 2 × 2 ANOVAs. Post hoc analyses were performed using Tukey's HSD where appropriate. All values are reported as mean ± SEM, and significance was defined as P ≤ 0.05.

| Voluntary wheel running restores AIE-induced cholinergic neuropathology in the adult basal forebrain
Adolescent intermittent ethanol treatment has previously been shown to reduce ChAT+IR neurons in the basal forebrain 9,10 that we replicated, and extended finding ChAT+IR neuron loss occurs just after the completion of AIE (ie, P56) that persists into adulthood (ie, P220) (see Figure 2). In a previous study, we reported that exercise initiated at the onset of AIE and continuing throughout experimentation (ie, P24-P80) prevented the AIE-induced loss of cholinergic neuron markers in the adult (ie, P80) basal forebrain (see Vetreno and Crews 10 ).
Previously, we reported that wheel running from the onset of AIE to the conclusion of experimentation (ie, P24-P80) prevented the AIEinduced loss of TrkA-and p75 NTR -immunoreactive cells in the adult (ie, P80) basal forebrain further establishing reproducibility across experiments (see Vetreno and Crews 10 ). In the present study, wheel running from P56 to P95 did not affect TrkA+IR or p75 NTR +IR cells in CONs, but did restore the AIE-induced loss of cells expressing TrkA and p75 NTR in the adult basal forebrain (see Figure 4). Thus, AIE reduces adult ChAT+IR, TrkA+IR, and p75 NTR +IR cholinergic neuron numbers  (±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 that persist from adolescence into adulthood that is restored to constitutive levels by wheel running.
Accumulating evidence reveals that AIE treatment causes a persistent upregulation of neuroimmune signaling molecules resulting in adult neuropathology, including loss of ChAT+IR cells. 9,10,31,32 NF-κB is a transcription factor known to induce multiple neuroimmune genes, 33 and phosphorylated NF-κB p65 has been used to follow neuroimmune activation in brain. 10,15,34 In a previous study, we  of AIE-treated animals (one-way ANOVA: F [1,14] = 8.5, P < 0.05) relative to CON, which was restored to CON levels following exercise exposure (data not shown). We next investigated colocalization of ChAT with BrdU and the neuronal marker NeuN in the adult basal forebrain (see Figure 6A). We did not observe colocalization of BrdU with either ChAT or NeuN in the adult basal forebrain following running wheel exposure. In addition, AIE treatment did not affect NeuN +IR neuron counts suggesting that populations of neurons are not reduced in the adult basal forebrain (see Figure 6B). Together, these data suggest that AIE and exercise do not alter the number of basal forebrain neurons, but do change the number of ChAT+, TrkA+, and p75NTR+ basal forebrain neurons consistent with a change in neuronal phenotype (ie, loss of the cholinergic neuron phenotype).

| Adolescent intermittent ethanol-induced epigenetic modification of the Chat and Trka gene promoters in the adult basal forebrain
To determine if Chat and Trka gene expression were altered by an epigenetic mechanism, we assessed histone acetylation and histone methylation within these genes. We found that levels of H3K9me2  Running wheel exposure from P56 to P95 did not affect levels of H3K9me2 in CONs, but did resolve the AIEinduced increase of H3K9me2 at the promoter of the Chat gene, relative to no exercise AIE subjects. B, Methylated DNA immunoprecipitation (5methyl 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 CONs (Tukey's HSD: P < 0.01). Wheel running alone did not affect Chat DNA methylation in the CONs, but did prevent the AIE-induced increase of DNA methylation at the Chat promoter CpG island (Tukey's HSD: P < 0.01; see Figure 7B). We did not observe an effect of AIE or wheel running on H3K9 acetylation associated with the Chat gene (see Supporting Information Figure Figure 8C). Thus, AIE treatment led to long-lasting increases of H3K9me2 associated with both Chat and Trka promoters that were restored by wheel running exposure.

| Voluntary exercise restores the reversal learning deficits and increased perseveration on the Morris water maze in AIE-treated adult rats
Adolescent intermittent ethanol treatment of mice and rats has previously been found to impair behavioral flexibility inducing reversal, but not spatial learning deficits on the Morris water maze and Barnes maze, respectively. 35,36 To determine if exercise exposure might recover the reversal learning deficits in adults, AIE-treated rats were assessed on the Morris water maze. In the CON subjects, performance on the Morris water maze did not differ as a function of exercise 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 exposure (ie, CON-no exercise vs. CON-exercise; all Ps > 0.1), so CON groups were combined to gain statistical power for behavioral assessment. Spatial learning was assessed long after AIE treatment (ie, from P82 to P86), and all subjects learned to locate and escape onto the submerged platform to criterion levels by P85 (CON: 28 ± 4 s; AIE/no exercise: 24 ± 5 s; AIE/exercise: 22 ± 4 s). While AIE treatment did not affect the latency to escape or distance traveled (both Ps > 0. 3) during the spatial learning component, all subjects reduced their escape latency across testing days (main effect of day: F [4, 144] = 60.6, P < 0.01) indicating that subjects learned the spatial component. Following the completion of spatial learning, reversal learning was assessed 24 hours later beginning on P87. Latency to escape onto the submerged platform during reversal learning was increased by 53% during first day (CON: 35 ± 4 s; AIE: 52 ± 8 s; Tukey's HSD: P < 0.05) and 74% during the second day (CON: 23 ± 3 s; AIE: 39 ± 7 s; Tukey's HSD: P < 0.01) in the AIE-treated animals, relative to CONs. Importantly, voluntary wheel running blunted the AIEinduced increase in latency to escape onto the submerged platform during the first (Tukey's HSD: P = 0.08) and second (Tukey's HSD: P < 0.01) day of reversal learning (see Figure 9A). Similarly, 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 (one-way ANOVA: F [1,27] = 7.2, P < 0.05) and that wheel running blunted the AIE-induced increase in perseverative behavior during the second trial of reversal learning (one-way ANOVA: F [1,17] = 11.7, P < 0.01; see Figure 9B). These studies reveal that AIE-induced cognitive deficits in reversal learning on the Morris water maze are restored by wheel running similar to restoration of the decreases in ChAT+, TrkA+, and p75 NTR + cells in the adult basal forebrain.

| DISCUSSION
In the present study, we discovered that the AIE-  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 increases pNF-κB p65+IR and neuroimmune genes mimicking the AIE- death, but rather the loss of cholinergic phenotype. In previous studies, we found long-lasting cognitive deficits in decisionmaking, particularly reversal learning deficits. 38 We extend these studies here to voluntary wheel running restoration of long-term reversal learning deficits in adult AIE-treated subjects assessed on the Morris water maze. Together, these data suggest that AIE induces a novel neuroplastic process involving neuroimmune signaling and epigenetic gene silencing that results in the loss of the cholinergic neuron phenotype and cognitive deficits that can be restored by exercise exposure (see Figure 10).
Since AIE decreases basal forebrain ChAT+IR neurons from late adolescence (P56) to adulthood, the observed recovery of cholinergic neurons suggests that AIE does not cause cholinergic neuron death,   40 In some studies, exercise has been reported to induce trophic factors. We found that wheel running initiated during AIE treatment prevented the decrease of hippocampal NGF and concomitant loss of basal forebrain cholinergic neuron markers. 10  Emerging studies suggest that epigenetic modifications contribute to long-lasting gene expression changes in brain induced by AIE. 44 We report here that AIE treatment produced long-lasting increases of H3K9me2 at promoter regions of the Chat and Trka genes as well as DNA methylation on the CpG island located in the promoter of the  15 Together, these two studies fit with neuroimmune-trophic gene silencing contributing to persistent hippocampal pathology that can be reversed. Epigenetic processes have also been implicated in the regulation of cholinergic genes. In unmanipulated NG108-15 neuron-like cell culture, treatment with TSA increased ChAT protein expression. 46 We report here that exer- Basal forebrain cholinergic neurons innervate the cortex, hippocampus, and other brain regions critical for cognitive function, 4,48 and the AIE-induced loss of cholinergic neurons may contribute to the neurocognitive deficits observed in adulthood. 29,49 In the present study, AIE treatment did not affect spatial learning, but did impair reversal learning and increase perseveration in adult rats consistent with previously published studies. 36 Importantly, we found that exer-