NEURONS IN THE VENTROLATERAL PERIAQUEDUCTAL GRAY IN FENTANYL USE AND WITHDRAWAL

Fentanyl withdrawal produces persistent negative affect that promotes continued drug use, yet the neural mechanisms underlying this process remain less understood. This dissertation identifies the ventrolateral periaqueductal gray (vlPAG) as a critical brain region that mediates fentanyl withdrawal and regulates drug use. The dissertation integrates behavioral, systems, and network-level approaches. Two complementary mouse models of fentanyl dependence and withdrawal were established, including a self-administration model that resulted in escalating intake and withdrawal-associated negative affect, and a osmotic minipump model that also produced withdrawal-associated negative affect. Whole-brain Fos mapping, to mark for neuronal activity, following naloxone-precipitated fentanyl withdrawal, revealed that the periaqueductal gray as a hub region of brain activity within distributed functional networks. To determine whether withdrawal-activated neurons in the vlPAG causally contribute to behavior, neurons recruited during fentanyl withdrawal were tagged and later reactivated chemogenetically. Reactivation of these neurons promoted hyperalgesia and anxiety-like behavior, accompanied by altered activity across amygdalar and thalamic circuits and a distinct pattern of global functional connectivity compared to controls. In parallel, GABAergic vlPAG neurons exhibited increased neuronal and synaptic activity during withdrawal, and chemogenetic inhibition of these neurons prevented withdrawal-induced hyperalgesia and decreased fentanyl associated active lever presses. Together, these findings demonstrate that discrete vlPAG ensembles encode the affective component of fentanyl withdrawal and exert widespread influence over distributed brain networks. GABAergic vlPAG neurons were further shown to mediate withdrawal-associated negative affect and fentanyl use. This work establishes a neuron-to-behavior framework for understanding how vlPAG neurons govern negative affect and fentanyl use in dependence.