Protocol for identifying head direction cells in mice using in vivo extracellular recordings and juxtacellular labeling

Head direction (HD) cells are required for spatial orientation. Protocols that combine functional identification with anatomical recovery of single HD neurons remain technically demanding. We present a protocol for recording HD cells in awake, head-fixed mice using in vivo extracellular single-neuron recordings during passive rotation. We describe steps for craniotomy, habituation, glass-electrode recordings, and online identification of HD cells. We then detail procedures for juxtacellular labeling with neurobiotin, high-density probe recordings, and post hoc histological recovery. For complete details on the use and execution of this protocol, refer to Hijazi et al. 1 and Jiang et al. 2