In Vivo Calcium Imaging with a Miniaturized Microscope in the Hypothalamus for Understanding Social Behaviors in Mice

Social behavior in vertebrates is tightly regulated through hypothalamic circuits. Molecularly defined hypothalamic neuron types were recently identified to encode social need in a homeostatic manner. Here, we describe a step-by-step protocol for performing in vivo calcium imaging in the hypothalamic medial preoptic nucleus (MPN) using a head-mounted miniaturized microscope (miniscope) in freely behaving mice. This method permits longitudinal recordings of neuronal population dynamics during social isolation and reunion, thus enabling mechanistic studies of social need and social satiety at single-neuron resolution. Specifically, adult mice receive stereotaxic injections of an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator into the MPN, followed by implantation of a gradient-index (GRIN) lens and a miniscope baseplate. After recovery, mice are habituated to the miniscope imaging setup and are subjected to scheduled social isolation and reunion, during which neuronal activities are imaged. Calcium signals are synchronized to animal behaviors for further analysis. This protocol provides comprehensive guidance for in vivo calcium imaging with miniscope in the mouse hypothalamus during social behaviors. It can be readily adapted for imaging in other deep brain regions and across different behavioral contexts. This approach bridges cellular-level neural activity and social behavior, advancing our understanding of the neural basis of complex behavior in freely moving animals.