Acetylcholine (ACh) plays important roles in memory encoding and attention in the hippocampus. However, changes in ACh signaling patterns during different neural and behavioral states remain poorly understood. Here, we used a genetically encoded ACh sensor and multi-plane, dual-color two-photon microscopy to establish the ACh signaling patterns in hippocampal CA1 of mice performing spatial behaviors. We observed spatially homogeneous signaling across volumes spanning hundreds of microns, which was positively correlated with locomotion speed. In novel environments, there was an increase in release persisting for dozens of laps while maintaining a positive speed correlation. When mice voluntarily disengaged, the magnitude of the speed-correlated release decreased, and this was accompanied by reduced place cell numbers and less precise place maps. Administration of scopolamine mimicked the effects of voluntary disengagement in terms of behavior and place cell metrics. These findings establish behaviorally correlated ACh signaling patterns in the hippocampus.

The granular retrosplenial cortex (RSG) supports memory, orientation, and fear processing. The mouse RSG contains several cell types that are remarkably distinct from those found in other cortical regions, including low rheobase neurons that dominate layer 2/3 (L2/3 LR) and similarly exclusive pyramidal cells in layer 5a (L5a RSG). While the functions of the RSG are extensively studied in both mice and rats, it remains unknown if the transcriptomically unique cell types of the mouse RSG are evolutionarily conserved in rats. Here, we show that mouse and rat RSG contain the same unique cell types, with L2/3 LR and L5a RSG cell types together representing more than 50% of all RSG neurons in each species. This preservation of cell types in male and female rats happens despite dramatic changes in key cell-type-specific marker genes, with the expression that selectively tags mouse L5a RSG neurons completely absent in rats. Important for Cre-driver line development, we identify alternative, cross-species genes that can be used to selectively target the cell types of the RSG in both mice and rats. Our results show that the unique cell types of the RSG are conserved across millions of years of evolution and emphasize stark species-specific differences in marker genes that need to be considered when making cell-type-specific knock-in lines across species. The retrosplenial cortex is important for memory, spatial orientation, fear processing, and imagining oneself in the future. Lesions to this brain region in humans lead to an inability to find one's way home. The mouse granular retrosplenial cortex (RSG) contains neuron types that are particularly distinct from those found in neighboring regions. Whether these distinct neurons are preserved across species remains unknown. Here, we show that all cell types of the mouse RSG are also found in rats, and the unique RSG cell types dominate the region in each species. These results suggest that the unique RSG neurons support evolutionarily important functions that facilitate the preservation of these neurons across millions of years of evolution.