Mesoscopic analysis of GABAergic marker expression in acetylcholine neurons in the whole mouse brain

In the central nervous system, acetylcholine (ACh) neurons coordinate neural network activity required for higher brain functions, such as attention, learning, and memory, as well as locomotion. Disturbances in cholinergic signaling have been described in many diseases of the developing and mature brain. Interestingly, ACh neurons can co-transmit GABA to support essential roles in brain function. However, the contributions of ACh/GABA co-transmission to brain function remain unclear. This underscores the need to better understand the heterogeneity of ACh neurons, particularly the sub-population of ACh neurons co-expressing GABAergic markers. We used various combinations of transgenic mouse lines to systematically label ACh neuron populations positive for different GABAergic markers in the brain. We developed a workflow combining tissue clearing, light-sheet fluorescence microscopy, and machine learning to image entire mouse brain hemispheres followed by quantification of ACh neurons throughout the brain. With this approach, we assessed the role of GABA co-transmission in ACh neuron function and quantified ACh and ACh/GABA neuron sub-populations in the brain. Our results suggest that GABA co-transmission from ACh neurons is not required to maintain the regular ACh neuron count in the brain. Furthermore, we report that a large subset of ACh neurons can potentially synthesize GABA by co-expressing the marker Gad2. However, most of these ACh neurons do not express vGAT, which would enable these neurons to release GABA. Taken together, we conclude that GABA co-transmission likely occurs only from a small population of ACh neurons restricted to few brain nuclei.