Miniscope Imaging of Nucleus Accumbens Neural Activity in Freely Behaving Rats: Virus Injection, Gradient Index Lens Implantation, Recording Strategies, and Analytical Methods.

In vivo calcium imaging in freely moving rats using miniscopes provides valuable information about the neural mechanisms of behavior in real time. A gradient index (GRIN) lens can be implanted in deep brain structures to relay activity from single neurons. While such procedures have been successful in mice, few reports provide detailed procedures for successful surgery and long-term imaging in rats, which are better suited for studying complex human behaviors. We present a robotic surgical protocol for same-day virus injection and GRIN lens implantation into the rat nucleus accumbens core. Our procedure utilizes a direct lens insertion without tissue aspiration and produces quality image retention for months of recording. We also describe daily recording strategies to minimize damage and promote long-term imaging. Finally, we present custom protective strategies to eliminate the need to remove miniscopes between sessions. This methodology protects rats from repeated isoflurane exposure and ensures a consistent focal plane for the entirety of the experiment. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Craniotomy Basic Protocol 2: Virus injection Basic Protocol 3: GRIN lens implantation Basic Protocol 4: Baseplate mounting and assessment of the anesthetized rat Basic Protocol 5: Assessment of the awake, behaving rat Support Protocol 1: Protective miniscope cone fabrication Support Protocol 2: Miniscope cable fabrication.