Project summary Opioid use disorder (OUD) is a chronic relapsing disorder that has cost the U.S. more than $1 trillion in 2017 alone. While initially driven by brain reward circuits, opioid consumption increasingly engages stress-related neural circuits that drive maladaptive emotional states. During the abstinent weeks, months, and years following opioid use, the risk of relapse is increased by emotional symptoms, such as social avoidance, depression, and opioid cravings. Similarly, tendencies to self-isolate during periods of protracted opioid withdrawal increase the risk of lethal overdose. The rapid escalation of opioid overdoses in the U.S. reflects the need for more data on neural mechanisms underlying protracted opioid withdrawal. The neuropeptide dynorphin and kappa opioid receptors (KOR) contribute to stress, aversion, and behavioral consequences of withdrawal. While evidence suggests that KOR effects may be due to modulation of dopamine and serotonin release in the nucleus accumbens (NAc), the brain regions and neural mechanisms mediating KOR control of opioid withdrawal remain a critical gap in knowledge. In this K99/R00 pathway to independence award, I aim to identify mechanisms by which KORs mediate changes in behavior during withdrawal. Using a mouse model of chronic, non-contingent morphine exposure, I have collected preliminary data indicating that 1) KOR activity in the NAc controls social avoidance and anhedonia produced during protracted withdrawal and 2) a population of dynorphin-producing neurons in the dorsal raphe nucleus, but not dynorphin neurons in the NAc itself, mediate the same effects. During the mentored phase, in Aim 1, I will learn to perform in vivo optogenetic procedures and fiber photometry recordings to characterize the role of dorsal raphe dynorphin neuron projections to the NAc in withdrawal behaviors. In Aim 2, I will expand on the model of non-contingent injections and establish a new model of remifentanil self-administration and examine relapse behavior during protracted withdrawal. I will also test changes in social and depression-like behaviors after self-administration. In the independent phase, in Aim 3, I will use my training from the K99 phase to combine optogenetics and fiber photometry recordings of dorsal raphe dynorphin inputs to the NAc with remifentanil self-administration and relapse testing. In Aim 4, I will expand on additional preliminary data collected with non-contingent injections showing that KOR expression in serotonin and dopamine neurons is critical for social avoidance and depression-like behaviors, respectively. I will use fiber photometry and fluorescent sensors of dopamine and serotonin to record release in the NAc during protracted withdrawal testing. In addition, I will examine dopamine and serotonin dynamics in the self-administration model developed in Aim 2 by testing mice with the same cell-type specific KOR knockouts and performing photometry recordings of release in the ...