Research Article: Adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons and neuroimmune activation are prevented by exercise and indomethacin

Date Published: October 8, 2018

Publisher: Public Library of Science

Author(s): Ryan P. Vetreno, Fulton T. Crews, Thomas Arendt.

http://doi.org/10.1371/journal.pone.0204500

Abstract

Basal forebrain cholinergic neurons mature in adolescence coinciding with development of adult cognitive function. Preclinical studies using the rodent model of adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-days on/2-days off from postnatal day [P]25 to P55) reveal persistent increases of brain neuroimmune genes that are associated with cognitive dysfunction. Adolescent intermittent ethanol exposure also reduces basal forebrain expression of choline acetyltransferase (ChAT), an enzyme critical for acetylcholine synthesis in cholinergic neurons similar to findings in the post-mortem human alcoholic basal forebrain. We report here that AIE decreases basal forebrain ChAT+IR neurons in both adult female and male Wistar rats following early or late adolescent ethanol exposure. In addition, we find reductions in ChAT+IR somal size as well as the expression of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor p75NTR, both of which are expressed on cholinergic neurons. The decrease in cholinergic neuron marker expression was accompanied by increased phosphorylation of NF-κB p65 (pNF-κB p65) consistent with increased neuroimmune signaling. Voluntary wheel running from P24 to P80 prevented AIE-induced cholinergic neuron shrinkage and loss of cholinergic neuron markers (i.e., ChAT, TrkA, and p75NTR) as well as the increase of pNF-κB p65 in the adult basal forebrain. Administration of the anti-inflammatory drug indomethacin (4.0 mg/kg, i.p prior to each ethanol exposure) during AIE also prevented the loss of basal forebrain cholinergic markers and the concomitant increase of pNF-κB p65. In contrast, treatment with the proinflammatory immune activator lipopolysaccharide (1.0 mg/kg, i.p. on P70) caused a loss of cholinergic neuron markers that was paralleled by increased pNF-κB p65 in the basal forebrain. These novel findings are consistent with AIE causing lasting activation of the neuroimmune system that contributes to the persistent loss of basal forebrain cholinergic neurons in adulthood.

Partial Text

Adolescence is a conserved neurodevelopmental period characterized by significant refinement of neurotransmitter systems that parallels the transition of the immature brain to the more efficient adult brain [1]. The basal forebrain cholinergic system, which is the primary source of acetylcholine innervation to the cortex and hippocampus [2], plays a crucial role in cognitive functioning [3, 4]. While cholinergic neurons are generated early in embryonic development [5–8], these neurons undergo maturational refinement during adolescence [9, 10] that is accompanied by consolidation of cholinergic projections [11–13]. In humans, adolescence is also associated with high levels of alcohol binge drinking [14, 15], which can negatively impact the maturing basal forebrain cholinergic system. Indeed, preclinical rat studies find that adolescent basal forebrain cholinergic neurons are particularly sensitive to ethanol-induced neurodegeneration [16]. Adolescent intermittent ethanol (AIE), which models human adolescent binge drinking, causes a loss of cholinergic neurons immediately following the conclusion of AIE treatment that persists well into adulthood (i.e., P220) [16–20]. Studies comparing our adolescent intermittent ethanol exposure model to an identical adult intermittent ethanol exposure model reveal that adolescents are uniquely sensitive to ChAT+ neuron loss [16] whereas adult loss of ChAT may require months of continuous ethanol exposure [21]. Loss of adult ChAT+ neurons following AIE has been shown to correlate with disruption of novel object recognition memory [20]. The heightened vulnerability of the adolescent brain, coupled with the importance of acetylcholine in cognitive functioning, underscores the importance of identifying the mechanism underlying the persistent loss of basal forebrain cholinergic neurons following adolescent binge ethanol exposure.

In the present study, we extend our research on the persistent AIE-induced reduction of ChAT+IR cholinergic neurons in the male basal forebrain with the discovery of (1) a loss of ChAT+ cholinergic neurons in AIE-treated adult female subjects, (2) a reduction of additional cholinergic neuron markers following AIE treatment, and (3) neuroimmune involvement in the AIE-induced loss of cholinergic neurons. We report here for the first time that AIE treatment, which models human adolescent binge drinking, led to a reduction of ChAT+IR neurons in the basal forebrain of adult female subjects similar to male AIE-treated rats. We also report that both early (i.e., P25 –P39) and late (i.e., P40 –P55) adolescent intermittent ethanol treatment similarly reduces ChAT+IR neurons. We also discovered that AIE treatment decreased expression of the high-affinity nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TrkA) and the low-affinity NGF receptor p75NTR, both of which are markers of cholinergic neurons, in the adult basal forebrain. Further, we observed increased expression of pNF-κB p65+IR in the adult basal forebrain, which is a neuroimmune marker consistent with previous studies finding increased neuroimmune activation following AIE treatment [30]. We report here for the first time that exposure to wheel running from P24 –P80 prevented the AIE-induced loss of cholinergic neuron markers (i.e., ChAT, TrkA, and p75NTR) as well as the increased expression of pNF-κB p65 in the adult basal forebrain. These observations are consistent with previous studies finding that exercise blunts induction of proinflammatory cytokines in the basal forebrain following middle cerebral artery occlusion [37]. Further, the anti-inflammatory drug indomethacin blocked the AIE-induced loss of cholinergic neuron markers (i.e., ChAT and TrkA) as well as the increased expression of pNF-κB p65 in the basal forebrain. Previous studies have found that indomethacin blocks adolescent ethanol-induced neuroimmune induction and neurodegeneration in the hippocampus and cortex [30, 53]. In addition, the inflammagen LPS mimicked the AIE-induced loss of cholinergic neuron markers (i.e., ChAT and TrkA) and increased pNF-κB p65+IR in the adult basal forebrain. Taken together, these data support the hypothesis that adolescent binge ethanol-induced neuroimmune signaling contributes to the persistent loss of cholinergic neuron markers in the adult basal forebrain (see Fig 8).

 

Source:

http://doi.org/10.1371/journal.pone.0204500

 

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