PROJECT SUMMARY Postoperative pain is a major morbidity, and persistent opioid use after surgery has contributed to an epidemic. An improved mechanistic understanding of how pain is regulated within the brain can lead to novel non-opioid analgesic development. The long-term goal of this proposal is to understand the central regulation of postoperative pain. The objective of the current application is to test how disruptions of input circuits to the prelimbic cortex (PL) and anterior cingulate cortex (ACC), two key components of the prefrontal cortex in rodents, contribute to symptoms of chronic postoperative pain. The PL is homologous to human dorsolateral prefrontal cortex that is known to undergo synaptic changes with chronic pain, and the ACC is a well-described region for processing affective component of pain across species. Our central hypothesis is that disruptions in input circuits to the prefrontal system present a key mechanism for chronic pain and thus form important therapeutic targets. Our hypothesis is supported by the current literature showing that the PL has a pain-inhibitory role, whereas the ACC enhances pain aversion, and that chronic pain causes increased excitability in the ACC and hypo- excitability in the PL. It is also supported by our recent results demonstrating that ACC receives nociceptive inputs from the primary somatosensory cortex (S1) and the insular cortex (IC), whereas the paraventricular nuclei (PVN) of the hypothalamus projects to the PL promotes top-down pain regulation. In Aim 1, we will test the hypothesis that chronic postoperative pain strengthens the S1-ACC and IC-ACC connections and weakens the PVN-PL connection. We will combine optogenetics with in vivo Ca2+ imaging in freely moving rats to label presynaptic neurons and record ACC neuronal activity in response to nociceptive stimuli in PI and SNI models, followed by graph theoretic analysis. We will test that i) ACC pyramidal neurons receive inputs from S1 and anterior IC; ii) as pain becomes chronic after SNI (in contrast to PI which is self-resolving), there is a progressive increase in the number of ACC neurons that receive S1/IC inputs; and iii) neurons receiving S1/IC inputs are more likely to be pain-responsive to overdrive ensemble nociceptive response in the ACC. Similarly, we will show that neuronal function in the PL is enhanced by PVN inputs, but chronic pain after SNI decreases this connection to impair PL function. In Aim 2, we will test the hypothesis that altered S1-ACC, IC-ACC and PVN-PL circuits contribute to postoperative pain. We will use closed-loop neuromodulation to pair detected neural activity of S1- or IC- responsive neurons in the ACC with optogenetic inhibition, and pain relief after SNI will indicate a causal link between circuit disruption and pain behavior. We will do similar experiments by activating PL neurons. Next, we will test if normalization of S1-ACC and IC-ACC circuits with ketamine or the PVN-PL circuit with oxyt...