PROJECT SUMMARY/ABSTRACT Exposure to stress can cause psychiatric illnesses including anxiety disorders. The mechanisms by which stress induces these illnesses, which tend to be both persistent and resistant to treatment, are not understood. Recent work shows that stress activates and modifies PACAP (pituitary adenylate cyclase-activating polypeptide) systems in the rat brain. Mimicking stress-induced increases in PACAP function with a single PACAP treatment causes persistent (lasting more than 1 week) increases in acoustic startle, a measure often used in both preclinical and clinical studies of anxiety. In contrast, a single CRF treatment causes increases in startle that normalize within 24 hr. PACAP's ability to produce long-lasting increases in an anxiety-related behavior in rats differentiates it from CRF and makes it an important new target for stress research. Indeed, new evidence suggests that PACAP is involved in the development of severe and debilitating forms of anxiety in humans, including post-traumatic stress disorder (PTSD), a key sign of which is persistent increases in startle (hyperarousal). This proposal examines the neurobiology of PACAP signaling in stress- and anxiety- related behaviors in rats. Considering the urgent need for new treatments for stress-related disorders, Aim 1 will focus on identifying new classes of agents that can block the acute and/or long-lasting behavioral effects of PACAP. We will examine PACAP antagonists, which are highly selective for PACAP (PAC1) receptors but not previously tested in stress studies, and kappa-opioid receptor (KOR) antagonists, which have been shown to block the long-term effects of stress. These studies may hasten medication development while providing new directions for mechanistic research. Aim 2 will examine the mechanisms by which PACAP produces persistent effects. Initial studies will focus on the bed nucleus of the stria terminalis (BNST) because (i) the BNST is a major target of PACAP innervation, (ii) stress increases PACAP expression in the BNST, and (iii) infusion of PACAP directly into the BNST is sufficient to produce long-lasting hyperarousal. One set of studies will examine how enhancing or disrupting the function of CREB, a downstream target of adenylate cyclase, affects baseline and PACAP-enhanced startle. Parallel studies will extend our new data showing that PACAP but not CRF causes marked downregulation of microRNA134 (miR134), a non-coding RNA that negatively regulates spine density and volume, by examining how enhancing or disrupting miR134 function affects baseline and PACAP-enhanced startle. These studies may identify intracellular processes that can be targeted for medication development. Aim 3 will determine if PACAP produces other core symptoms of PTSD, including persistent signs of anhedonia, social withdrawal, deficits in concentration, and impairments in extinction of fear. These studies may establish that PACAP administration provides an approach that more comprehensively models the myriad symptoms of PTSD, a finding that would also facilitate medication development. Overall, the proposed work may yield knowledge useful for the development of anti-stress agents.