PROJECT SUMMARY Subthalamic nucleus (STN) deep brain stimulation (DBS) is a common surgical treatment for Parkinson’s disease (PD). Despite over 20 years of clinical success, the therapeutic mechanisms of STN DBS remain elusive. However, it has become clear that DBS acts at the molecular, cellular, and systems levels in complex and sometimes contradictory ways. Current techniques such as electrophysiology, electrochemistry, and functional imaging commonly used to study pieces of this puzzle are limited in either resolution or behavioral paradigms these can be applied to, and have thus not provided all the information needed to parse out the complicated relationships between stimulation and evoked effects. Here, we propose the use of fluorescence calcium microscopy in GCaMP6f-expressing rats using a head-mounted miniature single photon system as a novel tool to bridge the gap between cellular and system level understanding of DBS in awake behaving animal models of PD. To this end, we will analyze neural activity changes in motor cortex evoked by stimulation of the STN during open field, stepping, cylinder tests, and apomorphine-induced rotations, all of which are stereotypical tests that have shown predictive validity for evaluation of movement and therapeutic efficacy in parkinsonian animals. The techniques proposed here provide a unique approach for answering questions about DBS mechanisms such as whether DBS-induced activation or pharmacologic inhibition of excitatory STN glutamatergic neuronal projections to the globus pallidus internus / substantia nigra reticulata produces detectable changes in motor cortex activity associated with changes in behavioral outcomes (e.g., open field, kinematic assessment of stepping, cylinder, and apomorphine-induced rotation tests).