Table 1.
List of primers for rat qPCR.
Fig 1.
Adolescent intermittent ethanol (AIE) decreases choline acetyltransferase (ChAT)+IR cholinergic neurons in the adult male and female basal forebrain throughout adolescence.
Modified unbiased stereological quantification of ChAT+IR neurons in the adult (P80) basal forebrain following AIE treatment revealed (A) a significant 27% (±5%) reduction in male subjects and (B) a significant 30% (±6%) reduction in female subjects, relative to CONs. Further, ChAT expression did not differ between male and female subjects. Given that we did not observe an effect of sex on ChAT+ expression, the remaining studies were conducted using male subjects. (C) Modified unbiased stereological quantification of ChAT+IR neurons following intermittent ethanol exposure during early adolescence (i.e. P25—P39) and late adolescence (i.e., P40 –P55) revealed a significant 27% (±6%) and 24% (±7%) reduction, respectively, in the adult male basal forebrain similar to the 29% (±4%) reduction observed following AIE treatment from P25 to P55. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, relative to CONs.
Fig 2.
Adolescent intermittent ethanol (AIE) decreases expression of multiple cholinergic neuron markers in the adult male basal forebrain.
(A) Modified unbiased stereological quantification of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) in the adult (P80) basal forebrain revealed a significant 32% (±8%) reduction in AIE-treated animals, relative to CONs. Representative photomicrographs of TrkA+IR cells in the adult basal forebrain of CON- and AIE-treated animals. (B) Modified unbiased stereological quantification of the low-affinity NGF receptor p75NTR in the adult (P80) basal forebrain revealed a significant 14% (±2%) reduction in AIE-treated animals, relative to CONs. Representative photomicrographs of p75NTR+IR cells in the adult basal forebrain of CON- and AIE-treated animals. (C) Immunofluorescent co-labeling revealed a high degree of TrkA (red) and p75NTR (green) colocalization with ChAT+IR neurons (blue) in the adult (P80) basal forebrain. (D) Quantitative PCR assessment of cholinergic neuron genes revealed a significant AIE-induced reduction of ChAT (34% [±9%]), TrkA (42% [±5%]), and p75NTR (46% [±5%]) mRNA in adult (P80) basal forebrain tissue samples, relative to CONs. qPCR analyses were run in triplicate. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, relative to CONs.
Fig 3.
Increased expression of phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells p65 (pNF-κB p65) in the basal forebrain of adolescent intermittent ethanol (AIE)-treated adult male rats.
Modified unbiased stereological quantification of pNF-κB p65+IR cells revealed a 48% (±11%) increase in the adult (P80) basal forebrain of AIE-treated animals, relative to CONs. Representative photomicrographs of pNF-κB p65+IR cells in the adult basal forebrain of CON- and AIE-treated animals. Data are presented as mean ± SEM. ** p < 0.01, relative to CONs.
Fig 4.
Wheel running prevents the adolescent intermittent ethanol (AIE)-induced loss and somal shrinkage of choline acetyltransferase (ChAT)+IR cholinergic neurons in the basal forebrain of adult male rats.
(A) Modified unbiased stereological assessment revealed a 24% (±4%) reduction of ChAT+IR neurons in the adult (P80) basal forebrain of AIE-treated subjects, relative to CONs. Running wheel exposure from P24 to P80 did not affect ChAT expression in CONs, but did prevent the AIE-induced loss of ChAT+IR neurons, relative to the non-exercising AIE animals. (B) Analysis of ChAT+IR neuron somal size revealed a significant 19% (±3%) 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 prevent the AIE-induced ChAT+IR neuron somal shrinkage in the adult basal forebrain, relative to non-exercising AIE animals. Representative photomicrographs of ChAT+IR neurons in the adult basal forebrain from CON- and AIE-treated animals across exercise conditions. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.
Fig 5.
Voluntary exercise prevents the adolescent intermittent ethanol (AIE)-induced loss of tropomyosin receptor kinase A (TrkA)- and p75NTR-immunoreactive cells as well as the increased expression of phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells p65 (pNF-κB p65) in the adult male basal forebrain.
(A) Modified unbiased stereological quantification of the high-affinity nerve growth factor (NGF) receptor TrkA in the adult (P80) basal forebrain revealed a significant 32% (±8%) reduction in AIE-treated animals, relative to CONs. Running wheel exposure from P24 to P80 did not affect TrkA expression in CONs, but did prevent the AIE-induced loss of TrkA+IR neurons, relative to the non-exercising AIE animals. (B) Modified unbiased stereological quantification of the low-affinity NGF receptor p75NTR in the adult (P80) basal forebrain revealed a significant 14% (±2%) reduction in AIE-treated animals, relative to CONs. Wheel running alone did not affect p75NTR expression in CONs, but did prevent the AIE-induced loss of p75NTR+IR neurons, relative to the non-exercising AIE animals. (C) Modified unbiased stereological quantification of pNF-κB p65+IR cells revealed a 62% (±14%) increase in the adult (P80) basal forebrain of AIE-treated animals, relative to CONs. While voluntary wheel running alone did not affect pNF-κB p65, it did prevent the AIE-induced increase of pNF-κB p65+IR cells, relative to the non-exercising AIE subjects. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.
Fig 6.
Indomethacin treatment prevented the adolescent intermittent ethanol (AIE)-induced loss of cholinergic neuron markers and increased phosphorylation of the proinflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells p65 (phosphorylated [pNF-κB p65]) in the male basal forebrain.
(A) Modified unbiased stereological assessment of choline acetyltransferase (ChAT)+IR neurons revealed a significant 28% (±10%) reduction in the basal forebrain of AIE-treated animals, relative to CONs. While indomethacin alone did not affect ChAT expression, it did prevent the AIE-induced loss of ChAT+IR neurons, relative to vehicle-treated AIE subjects. (B) Modified unbiased stereological assessment of the low-affinity nerve growth factor (NGF) receptor p75NTR revealed a significant 18% (±6%) reduction in the AIE-treated animals, relative to CONs. Indomethacin treatment did not affect p75NTR expression in CON- or AIE-treated subjects. (C) Modified unbiased stereological quantification of the high-affinity NGF receptor tropomyosin receptor kinase A (TrkA) in the adult (P80) basal forebrain revealed a significant 31% (±6%) reduction in AIE-treated animals, relative to CONs. Indomethacin treatment alone did not affect TrkA expression, but did prevent the AIE-induced loss of TrkA+IR neurons, relative to vehicle-treated AIE subjects. (D) Modified unbiased stereological quantification of pNF-κB p65+IR cells revealed a 50% (±4%) increase in the basal forebrain of AIE-treated animals, relative to CONs. While indomethacin alone did not affect pNF-κB p65, it did prevent the AIE-induced increase of pNF-κB p65+IR cells, relative to the vehicle-treated AIE subjects. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.
Fig 7.
Lipopolysaccharide (LPS) treatment mimicked the adolescent intermittent ethanol (AIE)-induced loss of cholinergic neuron markers and increased phosphorylation of the proinflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells p65 (phosphorylated [pNF-κB p65]) in the adult male basal forebrain.
(A) Modified unbiased stereological assessment of choline acetyltransferase (ChAT)+IR neurons in the basal forebrain of adult (P80) rats revealed a significant reduction in AIE- (31% [±4]), CON+LPS- (32% [±8%]), and AIE+LPS-treated animals (28% [±5%]), relative to CONs. (B) Modified unbiased stereological assessment of p75NTR+IR neurons in the basal forebrain of adult (P80) rats revealed a significant reduction in AIE-treated animals (18% [±5]) while LPS treatment did not alter expression of p75NTR. (C) Modified unbiased stereological assessment of tropomyosin receptor kinase A (TrkA)+IR neurons in the basal forebrain of adult (P80) rats revealed a significant reduction in AIE- (22% [±5]), CON+LPS- (26% [±2]), and AIE+LPS-treated animals (28% [±5]), relative to CONs. (D) Modified unbiased stereological assessment of pNF-κB p65+IR cells in the basal forebrain of adult (P80) rats revealed a significant increase in AIE- (48% [±11%]), CON+LPS- (54% [±11%]), and AIE+LPS-treated animals (93% [±12%]), relative to CONs. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.
Fig 8.
Simplified schematic depicting the proposed mechanism underlying the adolescent intermittent ethanol (AIE)-induced loss of basal forebrain cholinergic neurons.
Adolescent binge ethanol exposure increases phosphorylation of the proinflammatory transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells p65 (phosphorylated [pNF-κB p65]). This in turn results in the induction of neuroimmune signaling molecules that activates positive loops of amplification that persist into adulthood [54]. The present study suggests that this persistent proinflammatory neuroimmune activation contributes to the loss of basal forebrain cholinergic neurons that continues into adulthood. Indeed, exposure to either voluntary wheel running, which conveys immune modulatory effects, or treatment with the anti-inflammatory drug indomethacin prevents the loss of basal forebrain cholinergic neurons by preventing the AIE-induced increase of pNF-κB p65. Moreover, treatment with the endotoxin lipopolysaccharide (LPS) mimicked the AIE-induced loss of cholinergic neurons while increasing expression of pNF-κB p65. While these data implicate the neuroimmune system in the persistent AIE-induced loss basal forebrain cholinergic neurons, the precise mechanism remains to be fully elucidated. Interestingly, LPS treatment has previously been found to decrease hippocampal expression of the neurotrophin nerve growth factor (NGF) [55, 56], which is critical for basal forebrain cholinergic maintenance and survival [10]. Thus, AIE-induced neuroimmune activation may disrupt NGF inputs to the basal forebrain culminating in the persistent loss of cholinergic neurons. Thus, these data provide support for a neuroimmune mechanism underlying the AIE-induced loss of basal forebrain cholinergic neurons.