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

FIGURE 3.

FIGURE 3

Adolescent galantamine treatment prevented the adolescent intermittent ethanol (AIE)-induced increase of proinflammatory neuroimmune signaling molecules in the adult basal forebrain. (A) Modified unbiased stereological assessment revealed a 48% (±7%) increase of high-mobility group box 1 immunoreactive (HMGB1 + IR) cells in the adult (P70) basal forebrain of AIE-treated subjects, relative to CONs. Adolescent galantamine treatment from P25 to P54 did not affect HMGB1 + IR in CONs, but blunted the AIE-induced increase of HMGB1 + IR cells in adulthood, relative to vehicle-treated AIE subjects. Representative photomicrographs of HMGB1 + IR cells in the adult basal forebrain of CON- and AIE-treated subjects. Scale bar = 50 μm. (B) Modified unbiased stereological assessment revealed a 14% (±2%) increase of Toll-like receptor 4 immunoreactive (TLR4 + IR) cells in the adult basal forebrain of AIE-treated subjects, relative to CONs. Adolescent galantamine treatment alone from P25 to P54 decreased constitutive expression of TLR4 + IR cells in CONs and prevented the AIE-induced increase of TLR4 + IR cells in adulthood, relative to vehicle-treated AIE subjects. (C) Modified unbiased stereological assessment revealed a 50% (±9%) increase of receptor for advanced glycation end-products immunoreactive (RAGE + IR) cells in the adult basal forebrain of AIE-treated subjects, relative to CONs. Adolescent galantamine treatment from P25 to P54 did not affect RAGE + IR in CONs, but prevented the AIE-induced increase of RAGE + IR cells in adulthood, relative to vehicle-treated AIE subjects. (D) Modified unbiased stereological assessment revealed an 18% (±2%) increase of phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells p65 immunoreactive (pNF-κB p65 + IR) cells in the adult basal forebrain of AIE-treated subjects, relative to CONs. Adolescent galantamine treatment alone from P25 to P54 decreased constitutive expression of pNF-κB p65 + IR cells in CONs and prevented the AIE-induced increase of pNF-κB p65 + IR cells in adulthood, relative to vehicle-treated AIE subjects. (E) Immunofluorescent co-labeling analysis revealed a 95% (±17%) increase of choline acetyltransferase immunoreactive (ChAT + IR) neurons that co-expressed pNF-κB p65 in the adult basal forebrain of AIE-treated subjects, relative to CONs. Galantamine treatment alone did not affect ChAT colocalization with pNF-κB p65 in CONs, but prevented the AIE-induced increase of ChAT colocalization with pNF-κB p65 in the adult basal forebrain, relative to vehicle-treated AIE subjects. Representative fluorescent photomicrographs of ChAT (green) and pNF-κB p65 (red) colocalization in the adult basal forebrain of CON- and AIE-treated subjects. Closed arrowheads = ChAT + IR neuron colocalization with pNF-κB p65 + (yellow); open arrowheads = ChAT + IR neurons that did not co-express pNF-κB p65. Scale bar = 50 μm. Data are presented as mean ± SEM (n = 8/group). ∗p < 0.05, ∗∗p < 0.01.