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. 2021 Feb 26;15:652494. doi: 10.3389/fnbeh.2021.652494

FIGURE 4.

FIGURE 4

Adolescent galantamine treatment prevented the adolescent intermittent ethanol (AIE)-induced increase of histone methylation markers in the adult basal forebrain. (A) Modified unbiased stereological assessment revealed a 45% (± 6%) increase of histone 3 lysine 9 dimethylation immunoreactive (H3K9me2 + IR) cells in the adult (P70) basal forebrain of AIE-treated subjects, relative to CONs. Adolescent galantamine treatment alone from P25 to P54 did not affect expression of H3K9me2 + IR cells in CONs, but prevented the AIE-induced increase of H3K9me2 + IR cells in adulthood (P70), relative to vehicle-treated AIE subjects. Representative photomicrographs of H3K9me2 + IR cells in the adult basal forebrain of CON- and AIE-treated subjects. (B) Modified unbiased stereological assessment revealed a 21% (±6%) increase of histone 3 lysine 9 trimethylation immunoreactive (H3K9me3 + IR) cells in the adult basal forebrain of AIE-treated subjects, relative to CONs. Adolescent galantamine treatment alone from P25 to P54 did not affect expression of H3K9me3 + IR cells in CONs, but prevented the AIE-induced increase of H3K9me3 + IR cells in adulthood, relative to vehicle-treated AIE subjects. Representative photomicrographs of H3K9me3 + IR cells in the adult basal forebrain of CON- and AIE-treated subjects. Scale bar = 50 μm. Data are presented as mean ± SEM (n = 8/group). ∗p < 0.05, ∗∗p < 0.01. (C) Simplified schematic depicting the proposed neuroimmune and epigenetic mechanism underlying the persistent AIE-induced loss of basal forebrain cholinergic neurons (BFCNs). (Left) AIE treatment from P25 to P54 causes the loss of BFCN markers and somal shrinkage of the residual cholinergic neurons in the adult (P70) basal forebrain. (Small panel). Representative photomicrograph of ChAT + IR neurons in the adult (P70) AIE-treated basal forebrain. (Large panel) Schematic depicting AIE-induced decreased expression of BFCN markers (i.e., ChAT, TrkA, p75NTR), somal shrinkage of the residual ChAT + neurons, and increased expression of activated pNF-κB p65 within ChAT + BFCNs. In the proposed mechanism, AIE treatment increases expression of the endogenous proinflammatory TLR4/RAGE ligand HMGB1 and the HMGB1 receptors TLR4 and RAGE leading to increased BFCN expression of activated pNF-κB p65 and increased expression of the histone 3 lysine 9 (H3K9) methylation markers (i.e., dimethylation and trimethylation) resulting in loss of BFCN markers. (Right) Galantamine treatment combined with AIE prevents the AIE-induced loss of BFCN markers and somal shrinkage of the residual cholinergic neurons in the adult basal forebrain. (Small panel) Representative photomicrograph of ChAT + IR neurons in the adult AIE-treated basal forebrain following adolescent galantamine treatment. (Large panel) Adolescent galantamine treatment (i.e., P25–P54) combined with AIE blocks the induction of HMGB1-TLR4/RAGE-pNF-κB p65 signaling, increase of histone 3 methylation markers, and provides long-lasting recovery of the somal shrinkage and loss of BFCN markers in the adult basal forebrain.