Whole-brain mapping of basal forebrain cholinergic neurons reveals a long-range reciprocal input-output loop between distinct subtypes.

Basal forebrain cholinergic neurons (BFCNs) influence cognition and emotion through specific acetylcholine release in various brain regions, including the prefrontal cortices and basolateral amygdala (BLA). Acetylcholine release is controlled by distinct BFCN subtypes, modulated by excitatory and inhibitory inputs. However, the organization of the whole-brain input-output networks of these subtypes remains unclear. Here, we identified two distinct BFCN subtypes-BFCN(→ACA) and BFCN(→BLA)-innervating the anterior cingulate cortex (ACA) and BLA, each with unique distributions, electrophysiological properties, and projection patterns. Combining rabies-virus-assisted mapping and triple-plex RNAscope hybridization, we characterized their whole-brain input networks, identifying unique excitatory and shared inhibitory inputs for these subtypes. Moreover, our results reveal a long-range reciprocal input-output loop: BFCN(→ACA) neurons target the isocortex, their shared excitatory-input source, whereas BFCN(→BLA) neurons target shared inhibitory-input sources such as the striatum and pallidum, thus enabling dynamic interactions among these BFCN subtypes. Our study deepens understanding of cholinergic modulation in cognition and emotion and provides insights into their functional interactions.