Opioid use disorder (OUD) manifests itself via drug tolerance, withdrawal and craving during abstinence, and relapse. Negative affect, or emotional distress, is a psychological component of withdrawal that has increasingly attracted attention from the research community, but OUD treatments for negative affect have not innovated beyond opioid agonists. Newer studies ascribe a role for the ventral tegmental area (VTA) during aversive experiences, but few studies have tested synaptic plasticity mechanisms during opioid withdrawal. Meanwhile, females have been systematically overlooked in both in human and rodent studies. In simple terms, this dissertation aims to make progress by evaluating three aspects of opioid withdrawal: synaptic plasticity in the midbrain, therapeutic targets for negative affect, and sex as a biological variable throughout. The first hypothesis tested in this work is that repeated morphine administration alters VTA inhibitory γ-aminobutyric acid (GABA) synapses in an activity-dependent way. To this end, intrinsic electrophysiological characteristics, baseline GABA release, and GABA plasticity were tested in male and female mice during morphine withdrawal. Male and female mice had vastly different physiological signatures in midbrain synapses during morphine withdrawal highlighted by preserved GABA long-term potentiation in females only. The second hypothesis tested in this work is that signaling pathways known to be modified by opioids (namely, nitric oxide signaling) can be targeted to reverse negative affect during withdrawal. Opioid withdrawal behavior was modeled in mice, and a compound was tested as a novel putative treatment for psychological opioid withdrawal behaviors. Withdrawal behaviors were similar in males and females for some tests and diverged in others. Remarkably, a nitric oxide inhibitor reversed negative affect in all conditions of negative affect regardless of sex. Taken together, males and females exhibit differences in neurochemistry and behavior during opioid withdrawal, but the nitric oxide system might be a unifying target for OUD treatment. Future work can apply these findings to identify precise sources of sex differences in the brain and test therapeutics that capitalize on individual differences in opioid neuropharmacology.