PROJECT SUMMARY Adolescent alcohol misuse is a significant risk factor for alcohol use disorder in adulthood. Particularly concerning are the long-term effects on brain maturation and associated cognitive function, including the ability to appropriately process and respond to pain. A growing body of research indicates that adolescent alcohol misuse enhances pain sensitivity and anxiety in adulthood, and increased pain sensitivity and heightened anxiety may increase the risk for alcohol misuse later in life. Recently, a role for a circuit involving the basolateral amygdala (BLA), prelimbic (PrL) cortex, and ventrolateral periaqueductal gray (vlPAG) in pain processing has been described. Within this circuit, activation of the BLA by nociceptive stimuli results in increased feedforward inhibition of PrL neurons projecting to the vlPAG (PrLPAG). This feedforward inhibition is mediated by parvalbumin interneurons (PVINs) in the PrL. Within this circuit, PVINs modulate pain sensitivity and pain-related affect by regulating the activity of PrLPAG neurons. Adolescence is a critical period for development. Environmental insults occurring during adolescence, such as those caused by repeated episodes of binge-like alcohol consumption, disrupt normal development of PrL circuitry. These changes include reduced intrinsic excitability of and evoked AMPA and NMDA mediated currents onto PVINs. In addition, preliminary data we have collected indicates that following adolescent intermittent alcohol exposure the excitatory/inhibitory (E/I) balance evoked by optogenetic stimulation of BLA terminals in the PrL cortex is reduced at PrL PVINs and increased at PrLPAG neurons. Further, following alcohol exposure during adolescence, the density of perineuronal nets (PNNs) which enwrap PVINs and restrict plasticity is increased in the PrL. The overarching hypothesis of this proposal is that persistent changes in synaptic function following AIE are associated with altered activation patterns of PrL PVINs and PrLPAG neurons that are correlated with altered pain sensitivity. We further hypothesize that digestion of PNNs using chondroitinase ABC will reverse changes in the E/I balance at PrL PVINs and PrLPAG neurons. The proposed studies will use a combination of cell-type and projection specific chemogenetic manipulations to determine the effect of changes in the activity of PrL PVINs and PrLPAG neurons on nociception and pain-related affect following adolescent alcohol exposure. Changes in the activation patterns of PrL PVINs and PrLPAG neurons in response to nociceptive stimuli will also be assessed using fiber photometry. Finally, to determine whether PNNs stabilize altered synaptic function following adolescent alcohol exposure, PNNs will be digested and the E/I balance at BLA inputs onto PVINs and PrLPAG neurons will be measured using acute slice electrophysiology. These experiments will provide valuable insights into how changes in PrL PVIN and PrLPAG synaptic function r...