Distinct input-output relationships between basal forebrain subnuclei underlie heterogeneous cholinergic modulation of the mouse cortex

The basal forebrain (BF) supports many behavioral functions via its brain-wide projections, which can be recruited independently and task specifically. However, the anatomical underpinnings for this functional heterogeneity are poorly understood. Here, we compared two mouse BF subnuclei, the horizontal diagonal band (HDB) and the substantial innominata (SI), in terms of their anatomical connectivity and functional recruitment. Using anterograde and retrograde tracing, we show that they target largely segregated forebrain regions, but their input comes from a partially overlapping subset of regions. However, many neurons within each region project to just one of the subnuclei, suggesting they can be recruited independently. To test this, we combined optogenetics and widefield Ca2+ imaging and showed that stimulating prefrontal cortical projections to the HDB or SI results in distinct large-scale patterns of cortical activity via a cholinergic mechanism. Our results provide an anatomical basis for the flexible, area-specific cholinergic modulation of brain function.