Extended Data Figure 5: Representative cholinergic DBB-innervating projection sites in the brain.

a, b, Schematic (a) and representative image (b) showing injection site in the HDBs of a Chat-cre^+/− mouse with AAV-FLEX-Syn::EGFP. Scale bar, 1 mm. c–e, Chat-cre^+/− mouse injected bilaterally into the HDB with AAV-FLEX-Syn::EGFP and stained with an anti-ChAT antibody. Staining shows high co-localization of ChAT-positive neurons with the Cre-dependent AAV, demonstrating the efficacy of the Chat-cre mouse line. Of the neurons infected with virus, only a small fraction do not express ChAT. Scale bars, 100 μm (c–e) and 50 μm (c′–e′). f–h, Cholinergic projections from the DBB can be validated by known projection sites including the olfactory bulb. Scale bar, 200 μm; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer. i–k, Hippocampus. Scale bar, 200 μm. l–n, Amygdala (basolateral amygdala shown here). Scale bar, 100 μm. o–u, Also shown are areas of the ventral hypothalamus including, Median eminence (o–q), and arcuate nucleus (r–u). Scale bars, 100 μm. Schematic in a was adapted from an image from the Allen Brain Institute Reference Atlas³⁰.