Characterization of basal forebrain glutamate neurons suggests a role in control of arousal and avoidance behavior

Abstract

The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three major BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamate neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed cholinergic, GABAergic and vGluT2+ neurons represent separate BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2- enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal and regions responding to aversive or rewarding stimuli such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2 neurons in a place preference paradigm elicited a striking avoidance of the area where stimulation was given. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.