PRECISE CLOSED LOOP TWO-PHOTON ALL-OPTICAL MANIPULATION OF HIPPOCAMPAL CIRCUITS IN UNRESTRAINED BEHAVING MICE

Hippocampal pyramidal neurons involved in spatial navigation, known as place cells, are located mainly in the CA1 region. The spatial region where a place cell is active is referred to as its place field. Behavioral timescale synaptic plasticity (BTSP) is a hippocampal learning mechanism capable of generating new place fields during navigation (Bittner KC., et al 2017). To study the BTSP during the place-cell formation and within hippocampal neuronal ensembles, it is necessary to measure and manipulate neuronal activity with high spatial and temporal resolution across neuronal populations. All-optical methods combining two-photon (2P) calcium imaging with 2P optogenetic photostimulation enable in vivo circuit interrogation; however these methods have been limited to head-fixed animals (Emiliani et al., 2022). We previously developed a flexible 2P holographic microendoscope (2P FENDO) for all-optical circuit interrogation in freely behaving mice, implemented with either a GRIN lens (Accanto et al., 2023) or a mini-objective (Blot et al., 2025). We demonstrated fast 2P functional imaging and 2P holographic photostimulation at single-cell resolution in cortical circuits during free behavior. Here, we further extend the capabilities of 2P FENDO to access deep regions such as the hippocampus. By integrating behavioral tracking with closed-loop optogenetics, we demonstrate calcium imaging at up to 80 Hz and real-time manipulation of spatially tuned neurons in freely behaving mice with single-cell resolution. This platform supports scalable, causal links between neuronal activity and behavior.