Basal forebrain cellular composition was investigated using IHC. As mentioned above, histochemistry for GABA interneuron markers calretinin, calbindin, and parvalbumin found no marked changes after adolescent alcohol treatment. Similarly, astroglial marker glial fibrillary acidic protein was similar between groups (data not shown). Our gene expression studies found ChAT gene expression was decreased 71% at P38 following adolescent binge treatment (Figure 10). The basal forebrain is rich in cholinergic neurons, critical for learning and memory, that project to multiple brain regions to integrate cortical activity. We used ChAT+IR to identify cholinergic neurons (Figure 10). The density of ChAT+IR neurons was decreased 7.5% from 166 to 153 cells/mm2 in the Ch4 region (see Methods) of the basal forebrain (Figure 10A, *p<0.05). This change is similar in magnitude to the observed volumetric reduction in these mice, suggesting that cholinergic neuron loss contributes to the reduction in basal forebrain volume. Thus, adolescent binge drinking reduced density of ChAT+IR neurons in the adult basal forebrain.
