Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to broadly regulate the cellular stress response. In contrast, it is unclear if the PACAP/PAC1 receptor pathway has a role in human psychological stress responses, such as posttraumatic stress disorder (PTSD). In heavily traumatized subjects, we find a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females (N=64, replication N=74, p<0.005). Using a tag-SNP genetic approach (44 single nucleotide polymorphisms, SNPs) spanning the PACAP (ADCYAP1) and PAC1 (ADCYAP1R1) genes, we find a sex-specific association with PTSD. rs2267735, a SNP in a putative estrogen response element within ADCYAP1R1, predicts PTSD diagnosis and symptoms in females only (combined initial and replication samples: N=1237; p<2x10−5). This SNP also associates with fear discrimination and with ADCYAP1R1 mRNA expression. Methylation of ADCYAP1R1 is also associated with PTSD (p < 0.001). Complementing these human data, ADCYAP1R1 mRNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP/PAC1 pathway are involved in abnormal stress responses underlying PTSD. These sex-specific effects may occur via estrogen regulation of ADCYAP1R1. PACAP levels and ADCYAP1R1 SNPs may serve as useful biomarkers to further our mechanistic understanding of PTSD.

Exposure to chronic stress has been argued to produce maladaptive anxiety-like behavioral states, and many of the brain regions associated with stressor responding also mediate anxiety-like behavior. Pituitary adenylate cyclase activating polypeptide (PACAP) and its specific G protein-coupled PAC 1 receptor have been associated with many of these stress-and anxiety-associated brain regions, and signaling via this peptidergic system may facilitate the neuroplasticity associated with pathological affective states. Here we investigated whether chronic stress increased transcript expression for PACAP, PAC 1 receptor, brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB) in several nuclei. In rats exposed to a 7 day chronic variate stress paradigm, chronic stress enhanced baseline startle responding induced by handling and exposure to bright lights. Following chronic stress, quantitative transcript assessments of brain regions demonstrated dramatic increases in PACAP and PAC 1 receptor, BDNF, and TrkB receptor mRNA expression selectively in the dorsal aspect of the anterolateral bed nucleus of the stria terminalis (dBNST). Related vasoactive intestinal peptide (VIP) and VPAC receptor, and other stress peptide transcript levels were not altered compared to controls. Moreover, acute PACAP38 infusion into the dBNST resulted in a robust dose-dependent anxiogenic response on baseline startle responding that persisted for 7 days. PACAP/PAC 1 receptor signaling has established trophic functions and its coordinate effects with chronic stress-induced dBNST BDNF and TrkB transcript expression may underlie the maladaptive BNST remodeling and plasticity associated with anxiety-like behavior.

Half of the data points were inadvertently omitted from the published version of Fig. 4a; the statistical analyses in the text and figure legend, however, do refer to the complete data set. The corrected figure is shown here and has been corrected in the online versions of the paper.In addition, we present additional information to clarify two results reported in the Article regarding plasma pituitary adenylate cyclaseactivating polypeptide (PACAP) levels and post-traumatic stress disorder (PTSD) symptom associations. In the Article, we reported replication of the association between PACAP levels and hyperarousal subscale, because this was the most robust association in the initial cohort. We now present the analyses separately for initial, replication and combined cohorts in Table 1. All associations but one are significant in the replication cohort. The second issue concerns potential medical confounds that could underlie the reported association. Although we do not have medical chart data on most patients, we do have responses from a health questionnaire administered during collection of trauma history and other data. We have now reanalysed the associations for the PTSD symptom scale (PSS) hyperarousal and total symptoms using subjective reports of health condition from the questionnaires as covariates. These data are presented in Table 2 and do not show any effect of health-and illness-related questions on the relationship between PACAP and PTSD symptoms. None of these additions affect the results and conclusions of the original Article.

Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

Pituitary adenylate cyclase-activating polypeptides (PACAP) have potent regulatory and neurotrophic activities on superior cervical ganglion (SCG) sympathetic neurons with pharmacological profiles consistent for the PACAP-selective PAC 1 receptor. Multiple PAC 1 receptor isoforms are suggested to determine differential peptide potency and receptor coupling to multiple intracellular signaling pathways. The current studies examined rat SCG PAC 1 receptor splice variant expression and coupling to intracellular signaling pathways mediating PACAP-stimulated peptide release. PAC 1 receptor mRNA was localized in over 90% of SCG neurons, which correlated with the cells expressing receptor protein.The neurons expressed the PAC 1 (short)HOP1 receptor but not VIP/PACAP-nonselective VPAC 1 receptors; low VPAC 2 receptor mRNA levels were restricted to ganglionic nonneuronal cells. PACAP27 and PACAP38 potently and efficaciously stimulated both cAMP and inositol phosphate production; inhibition of phospholipase C augmented PACAP-stimulated cAMP production, but inhibition of adenylyl cyclase did not alter stimulated inositol phosphate production. Phospholipase C inhibition blunted neuron peptide release, suggesting that the phosphatidylinositol pathway was a prominent component of the secretory response. These studies demonstrate preferential sympathetic neuron expression of PACAP-selective receptor variants contributing to regulation of autonomic function.The identification of pituitary adenylate cyclase-activating polypeptide (PACAP) 1 and vasoactive intestinal peptide (VIP)/ PACAP receptors has broadened our understanding of the mechanisms underlying the regulatory and neurotrophic roles of this family of peptides. The PACAP precursor molecule is tissue-specifically posttranslationally processed to two biologically active ␣-amidated products, PACAP38 (pro-PACAP-(131-168)) and PACAP27 (pro-PACAP-(131-157)) (1-5), which share amino acid homology with VIP. In the nervous system, PACAP induces neuronal calcium flux, facilitates membrane depolarization, and increases spike frequency (6 -8, 10).2 PACAP peptides also enhance neuroblast survival, proliferation, differentiation, and neurite outgrowth and prevent neuronal apoptosis upon growth factor or stimulus withdrawal and ischemic insult (11-16).The cloning of cDNAs for three putative seven-transmembrane G-protein-coupled receptors for VIP and PACAP demonstrated receptor subtype diversity and functional heterogeneity (17-25). The PACAP-selective PAC 1 receptor demonstrates high affinity for only PACAP38 and PACAP27 and is coupled to multiple intracellular signaling cascades. The VPAC 1 and VPAC 2 receptors, in contrast, exhibit approximate equal high affinity for the PACAP38, PACAP27, and VIP peptides and are coupled to adenylyl cyclase. Multiple PAC 1 receptor isoforms result from the alternative splicing of two exons in the aminoterminal extracellular domain and/or two (HIP and HOP) exons in the third cytoplasmic loop (19,23,24,26). Cell-specific expression of PAC 1 receptor sp...

Pituitary adenylate cyclase activating polypeptide (PACAP) peptides are expressed and regulated in sensory afferents of the micturition pathway. Although these studies have implicated PACAP in bladder control, the physiological significance of these observations has not been firmly established. To clarify these issues, the roles of PACAP and PACAP signaling in micturition and cystitis were examined in receptor characterization and physiological assays. PACAP receptors were identified in various tissues of the micturition pathway, including bladder detrusor smooth muscle and urothelium. Bladder smooth muscle expressed heterogeneously PAC 1null, PAC1HOP1, and VPAC2 receptors; the urothelium was more restricted in expressing preferentially the PAC 1 receptor subtype only. Immunocytochemical studies for PAC 1 receptors were consistent with these tissue distributions. Furthermore, the addition of 50 -100 nM PACAP27 or PACAP38 to isolated bladder strips elicited transient contractions and sustained increases in the amplitude of spontaneous phasic contractions. Treatment of the bladder strips with tetrodotoxin (1 M) did not alter the spontaneous phasic contractions suggesting direct PACAP effects on bladder smooth muscle. PACAP also increased the amplitude of nerve-evoked contractions. By contrast, vasoactive intestinal polypeptide had no direct effects on bladder smooth muscle. In a rat cyclophosphamide (CYP)-induced cystitis paradigm, intrathecal or intravesical administration of PAC 1 receptor antagonist, PACAP6 -38, reduced cystitis-induced bladder overactivity. In summary, these studies support roles for PACAP in micturition and suggest that inflammation-induced plasticity in PACAP expression in peripheral and central micturition pathways contribute to bladder dysfunction with cystitis.neuropeptides; urinary bladder; bladder overactivity; dorsal root ganglia; spinal cord; inflammation THE STORAGE AND PERIODIC ELIMINATION of urine requires a complex neural control system that coordinates the activities of the smooth muscle of the urinary bladder and the smooth and striated muscle of the urethral sphincters (13,32,33). Coordination between these organs is mediated by a complex neural control system located in the brain, spinal cord, and peripheral ganglia (12). Experiments with a chemically (cyclophosphamide, CYP)-induced bladder inflammation (11, 34,38) rodent model have demonstrated alterations in neurochemical (52, 53, 66, 69), electrophysiological (29, 72), organizational (65, 68), and functional properties of the micturition reflex (24, 38, 39), suggesting dramatic reorganization of the micturition reflex pathways. Alterations in peripheral bladder afferent (sensory)/ efferent (autonomic and motor) and central interneuronal pathway functions may underlie detrusor overactivity that accompany CYP-induced cystitis.Pituitary adenylate cyclase activating polypeptide (PACAP) peptides have diverse functions in the endocrine, nervous, gastrointestinal, and cardiovascular systems (1, 6) through PAC 1 , VPAC 1 and VPAC ...

Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxietylike emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala (CeA) as critical structures mediating fear-and anxiety-like behavior in both humans and animals. These areas organize coordinated fear-and anxiety-like behavioral responses as well as peripheral stress responding to threats via direct and indirect projections to the paraventricular nucleus of the hypothalamus (PVN) and brainstem regions (Walker et al., 2003;Ulrich-Lai and Herman, 2009;Walker et al., 2009). In particular, the BNST has been argued to mediate these central and peripheral responses when the perceived threat is of long duration (Waddell et al., 2006) and/or when the anxiety-like response is sustained (Walker and Davis, 2008); hence, the BNST may mediate pathological anxiety-like states that result from exposure to chronic stress. Indeed, chronic stress paradigms result in enhanced BNST neuroplasticity that has been associated with pathological anxiety-like states (Vyas et al., 2003;Pego et al., 2008). Here we review evidence that suggests that pituitary adenylate cyclase activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH) work together to modulate BNST function and increase anxiety-like behavior. Moreover, we have shown that BNST PACAP as well as its cognate PAC1 receptor are substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al., 1997;Hammack et al., 2009). We describe how interactions between PACAP and CRH in the BNST may mediate stress-associated behaviors, including anorexia and anxiety-like behavior. These studies have the potential to define specific mechanisms underlying anxiety disorders, and may provide important therapeutic strategies for stress-and anxiety-management.

Summary Chronic or repeated stressor exposure can induce a number of maladaptive behavioral and physiological consequences and among limbic structures, the bed nucleus of the stria terminalis (BNST) has been implicated in the integration and interpretation of stress responses. Previous work has demonstrated that chronic variate stress (CVS) exposure in rodents increases BNST pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) and PAC1 receptor (Adcyap1r1) transcript expression, and that acute BNST PACAP injections can stimulate anxiety-like behavior. Here we show that chronic stress increases PACAP expression selectively in the oval nucleus of the dorsolateral BNST in patterns distinct from those for corticotropin releasing hormone (CRH). Among receptor subtypes, BNST PACAP signaling through PAC1 receptors not only heightened anxiety responses as measured by different behavioral parameters but also induced anorexic-like behavior to mimic the consequences of stress. Conversely, chronic inhibition of BNST PACAP signaling by continuous infusion with the PAC1 receptor antagonist PACAP(6-38) during the week of CVS attenuated these stress-induced behavioral responses and changes in weight gain. BNST PACAP signaling stimulated the hypothalamic-pituitary-adrenal (HPA) axis and heightened corticosterone release; further, BNST PACAP(6-38) administration blocked corticosterone release in a sensitized stress model. In aggregate with recent associations of PACAP/PAC1 receptor dysregulation with altered stress responses including post-traumatic stress disorder, these data suggest that BNST PACAP/PAC1 receptor signaling mechanisms may coordinate the behavioral and endocrine consequences of stress.