GPCR Intracellular Loop Regulation of Beta-Arrestin-Mediated Endosomal Signaling Dynamics.

G protein-coupled receptors (GPCRs) are currently appreciated to be routed to diverse cellular platforms to generate both G protein-dependent and -independent signals. The latter has been best studied with respect to ß-arrestin-associated receptor internalization and trafficking to signaling endosomes for extracellular signal-regulated kinase (ERK) activation. However, how GPCR structural and conformational variants regulate endosomal ERK signaling dynamics, which can be central in neural development, plasticity, and disease processes, is not well understood. Among class B GPCRs, the PACAP-selective PAC1 receptor is unique in the expression of variants that can contain intracellular loop 3 (ICL3) cassette inserts. The nervous system expresses preferentially the PAC1Null (no insert) and PAC1Hop (28-amino acid Hop insert) receptor variants. Our molecular modeling and signaling studies revealed that the PAC1Null and PAC1Hop receptor variants can associate with ß-arrestin differentially, resulting in enhanced receptor internalization and ERK activation for the PAC1Hop variant. The study amplifies our understandings of GPCR intracellular loop structure/function relationships with the first example of how the duration of endosomal ERK activation can be guided by ICL3. The results provide a framework for how changes in GPCR variant expression can impact developmental and homeostatic processes and may be contributory to maladaptive neuroplasticity underlying chronic pain and stress-related disorders.

Activation of Lateral Parabrachial Nucleus (LPBn) PACAP-Expressing Projection Neurons to the Bed Nucleus of the Stria Terminalis (BNST) Enhances Anxiety-like Behavior.

Anxiety disorders are among the most common psychiatric disorders, and understanding the underlying neurocircuitry of anxiety- and stress-related behaviors may be important for treatment. The bed nucleus of the stria terminalis (BNST) has been studied for its role in many stress-related pathologies, such as anxiety, pain, depression, and addiction. Our prior work has demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) receptor activation in the BNST mediates many of the behavioral consequences of chronic stress. While the BNST contains local PACAP-expressing neurons, a major source of afferent PACAP is the lateral parabrachial nucleus (LPBn), and excitotoxic lesions of the LPBn substantially decreasess PACAP immunostaining in the BNST. Here, we first assessed Cre-dependent reporter expression by injecting AAV2-hSyn-DIO-mCherry into the LPBn of PACAP-IRES-Cre mice for circuit mapping studies and identified PACAP projections to the BNST, lateral capsular central nucleus of the amygdala (CeLC), and ventromedial hypothalamus (VMH). In a second study, we assessed the effects of chemogenetically activating LPBn PACAP afferents in the BNST by injecting AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn of PACAP-IRES-Cre mice for Cre-dependent expression of excitatory designer receptors exclusively activated by designer drugs (DREADDs). Before behavioral testing, clozapine-N-oxide (CNO), the selective agonist of our DREADD, was infused directly into the BNST. We found that after specific activation of LPBn PACAP afferents in the BNST, mice had increased anxiety-like behavior compared with controls, while total locomotor activity was unaffected. These results indicate that activation of PACAPergic LPBn projections to the BNST may play an important role in producing anxiety-like behavior.

Chemoarchitecture of the bed nucleus of the stria terminalis: Neurophenotypic diversity and function.

The bed nucleus of the stria terminalis (BNST) is a compact but neurophenotypically complex structure in the ventral forebrain that is structurally and functionally linked to other limbic structures, including the amygdala nuclear complex, hypothalamic nuclei, hippocampus, and related midbrain structures, to participate in a wide range of functions, especially emotion, emotional learning, stress-related responses, and sexual behaviors. From a variety of sensory inputs, the BNST acts as a node for signal integration and coordination for information relay to downstream central neuroendocrine and autonomic centers for appropriate homeostatic physiological and behavioral responses. In contrast to the role of the amygdala in fear, the BNST has gained wide interest from work suggesting that it has main roles in mediating sustained responses to diffuse, unpredictable and/or long-duration threats that are typically associated with anxiety-related responses. Further, some BNST subregions are highly sexually dimorphic which appear contributory to the differential stress and social interactive behaviors, including reproductive responses, between males and females. Notably, maladaptive BNST neuroplasticity and function have been implicated in chronic pain, depression, anxiety-related abnormalities, and other psychopathologies including posttraumatic stress disorders. The BNST circuits are predominantly GABAergic-the glutaminergic neurons represent a minor population-but the complexity of the system results from an overlay of diverse neuropeptide coexpression in these neurons. More than a dozen neuropeptides may be differentially coexpressed in BNST neurons, and from variable G protein-coupled receptor signaling, may inhibit or activate downstream circuit activities. The mechanisms and roles of these peptides in modulating intrinsic BNST neurocircuit signaling and BNST long-distance target cell projections are still not well understood. Nevertheless, an understanding of some of the principal players may allow assembly of the circuit interactions.

PACAP orchestration of stress-related responses in neural circuits.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic polypeptide that can activate G protein-coupled PAC1, VPAC1, and VPAC2 receptors, and has been implicated in stress signaling. PACAP and its receptors are widely distributed throughout the nervous system and other tissues and can have a multitude of effects. Human and animal studies suggest that PACAP plays a role responding to a variety of threats and stressors. Here we review the roles of PACAP in several regions of the central nervous system (CNS) as they relate to several behavioral functions. For example, in the bed nucleus of the stria terminalis (BNST), PACAP is upregulated following chronic stress and may drive anxiety-like behavior. PACAP can also influence both the consolidation and expression of fear memories, as demonstrated by studies in several fear-related areas, such as the amygdala, hippocampus, and prefrontal cortex. PACAP can also mediate the emotional component of pain, as PACAP in the central nucleus of the amygdala (CeA) is able to decrease pain sensitivity thresholds. Outside of the central nervous system, PACAP may drive glucocorticoid release via enhanced hypothalamic-pituitary-adrenal axis activity and may participate in infection-induced stress responses. Together, this suggests that PACAP exerts effects on many stress-related systems and may be an important driver of emotional behavior.

PAC1 Receptor Internalization and Endosomal MEK/ERK Activation Is Essential for PACAP-Mediated Neuronal Excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) can significantly increase the excitability of diverse neurons through differential mechanisms. For guinea pig cardiac neurons, the modulation of excitability can be mediated in part by PAC1 receptor plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. By contrast, PAC1 receptor-mediated excitability of hippocampal dentate gyrus granule cells appears independent of membrane-delimited AC/cAMP/PKA and PLC/PKC signaling. For both neuronal types, there is mechanistic convergence demonstrating that endosomal PAC1 receptor signaling has prominent roles. In these models, neuronal exposure to Pitstop2 to inhibit ß-arrestin/clathrin-mediated PAC1 receptor internalization eliminates PACAP modulation of excitability. ß-arrestin is a scaffold for a number of effectors especially MEK/ERK and notably, paradigms that inhibit PAC1 receptor endosome formation and ERK signaling also blunt the PACAP-induced increase in excitability. Detailed PAC1 receptor internalization and endosomal ERK signaling mechanisms have been confirmed in HEK PAC1R-EGFP cells and shown to be long lasting which appear to recapitulate the sustained electrophysiological responses. Thus, PAC1 receptor internalization/endosomal recruitment efficiently and efficaciously activates MEK/ERK signaling and appears to represent a singular and critical common denominator in regulating neuronal excitability by PACAP.

Targeting the PAC1 Receptor for Neurological and Metabolic Disorders.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (PAC1R, ADCYAP1R1) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of G protein-coupled receptors (GPCRs). PAC1R has been shown to play crucial roles in the central and peripheral nervous systems. The activation of PAC1R initiates diverse downstream signal transduction pathways, including adenylyl cyclase, phospholipase C, MEK/ERK, and Akt pathways that regulate a number of physiological systems to maintain functional homeostasis. Accordingly, at times of tissue injury or insult, PACAP/PAC1R activation of these pathways can be trophic to blunt or delay apoptotic events and enhance cell survival. Enhancing PAC1R signaling under these conditions has the potential to mitigate cellular damages associated with cerebrovascular trauma (including stroke), neurodegeneration (such as Parkinson's and Alzheimer's disease), or peripheral organ insults. Conversely, maladaptive PACAP/PAC1R signaling has been implicated in a number of disorders, including stressrelated psychopathologies (i.e., depression, posttraumatic stress disorder, and related abnormalities), chronic pain and migraine, and metabolic diseases; abrogating PAC1R signaling under these pathological conditions represent opportunities for therapeutic intervention. Given the diverse PAC1R-mediated biological activities, the receptor has emerged as a relevant pharmaceutical target. In this review, we first describe the current knowledge regarding the molecular structure, dynamics, and function of PAC1R. Then, we discuss the roles of PACAP and PAC1R in the activation of a variety of signaling cascades related to the physiology and diseases of the nervous system. Lastly, we examine current drug design and development of peptides and small molecules targeting PAC1R based on a number of structure- activity relationship studies and key pharmacophore elements. At present, the rational design of PAC1R-selective peptide or small-molecule therapeutics is largely hindered by the lack of structural information regarding PAC1R activation mechanisms, the PACAP-PAC1R interface, and the core segments involved in receptor activation. Understanding the molecular basis governing the PACAP interactions with its different cognate receptors will undoubtedly provide a basis for the development and/or refinement of receptor-selective therapeutics.

Parabrachial Pituitary Adenylate Cyclase-Activating Polypeptide Activation of Amygdala Endosomal Extracellular Signal-Regulated Kinase Signaling Regulates the Emotional Component of Pain.

BACKGROUND: Chronic pain and stress-related psychopathologies, such as depression and anxiety-associated abnormalities, are mutually reinforcing; however, the neuronal circuits and mechanisms that underlie this reinforcement are still not well understood. Pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate PAC1 receptor (Adcyap1r1) are expressed in peripheral nociceptive pathways, participate in anxiety-related responses and have been have been linked to posttraumatic stress disorder and other mental health afflictions. METHODS: Using immunocytochemistry, pharmacological treatments and behavioral testing techniques, we have used a rodent partial sciatic nerve chronic constriction injury model (n = 5-8 per group per experiment) to evaluate PACAP plasticity and signaling in nociceptive and stress-related behaviors. RESULTS: We show that chronic neuropathic pain increases PACAP expression at multiple tiers along the spinoparabrachioamygdaloid tract. Furthermore, chronic constriction injury bilaterally augments nociceptive amygdala (in the central nucleus of the amygdala [CeA]) PACAP immunoreactivity, extracellular signal-regulated kinase phosphorylation, and c-Fos activation, in parallel with heightened anxiety-like behavior and nociceptive hypersensitivity. Acute CeA infusions with the PACAP receptor antagonist PACAP(6-38) blocked chronic constriction injury-induced behavioral responses. Additionally, pretreatments with inhibitors of mitogen-activated protein kinase enzymes or endocytosis to block endosomal PACAP receptor extracellular signal-regulated kinase signaling attenuated PACAP-induced CeA neuronal activation and nociceptive responses. CONCLUSIONS: Our data suggest that chronic pain-induced PACAP neuroplasticity and signaling in spinoparabrachioamygdaloid projections have an impact on CeA stress- and nociception-associated maladaptive responses, which can be ameliorated upon receptor antagonism even during injury progression. Thus, the PACAP pathway provides for an important mechanism underlying the intersection of stress and chronic pain pathways via the amygdala.

The Effects of Prior Stress on Anxiety-Like Responding to Intra-BNST Pituitary Adenylate Cyclase Activating Polypeptide in Male and Female Rats.

Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in anxiety-related brain structures. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In male rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. Here, we examined whether CVS would sensitize the behavioral and/or endocrine response to a subthreshold BNST PACAP infusion. Male and cycling female rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, rats were bilaterally infused into the BNST with a normally subthreshold dose of PACAP. We found an increase in startle amplitude and plasma corticosterone levels 30 min following intra-BNST PACAP infusion in male rats that had been previously exposed to CVS. CVS did not enhance the startle response in cycling females. Equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels even in previously stressed male rats. These results suggest that repeated exposure to stressors may differentially alter the neural circuits underlying the responses to intra-BNST PACAP, and may result in different anxiety-like responses in males and females.

Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain.

The intricate relationships that associate pain, stress responses and emotional behavior have been well established. Acute stressful situations can decrease nociceptive sensations and conversely, chronic pain can enhance other pain experiences and heighten the emotional and behavioral consequences of stress. Accordingly, chronic pain is comorbid with a number of behavioral disorders including depression, anxiety abnormalities and associated stress-related disorders including post traumatic stress disorder (PTSD). The central nucleus of the amygdala (CeA) represents a convergence of pathways for pain, stress and emotion, and we have identified pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in fiber elements in the lateral capsular division of the CeA (CeLC). The PACAP staining patterns colocalized in part with those for calcitonin gene related peptide (CGRP); anterograde fiber tracing and excitotoxic lesion studies demonstrated that the CeLC PACAP/CGRP immunoreactivities represented sensory fiber projections from the lateral parabrachial nucleus (LPBn) along the spino-parabrachioamygdaloid tract. The same PBn PACAP/CGRP fiber system also projected to the BNST. As in the BNST, CeA PACAP signaling increased anxiety-like behaviors accompanied by weight loss and decreased feeding. But in addition to heightened anxiety-like responses, CeA PACAP signaling also altered nociception as reflected by decreased latency and threshold responses in thermal and mechanical sensitivity tests, respectively. From PACAP expression in major pain pathways, the current observations are novel and suggest that CeA PACAP nociceptive signaling and resulting neuroplasticity via the spino-parabrachioamygdaloid tract may represent mechanisms that associate chronic pain with sensory hypersensitivity, fear memory consolidation and severe behavioral disorders.

PAC1 receptor antagonism in the bed nucleus of the stria terminalis (BNST) attenuates the endocrine and behavioral consequences of chronic stress.

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.

Pleiotropic functions of pituitary adenylyl cyclase-activating polypeptide on retinal ontogenesis: involvement of KLF4 in the control of progenitor cell proliferation.

We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.

Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

Regulation of bed nucleus of the stria terminalis PACAP expression by stress and corticosterone.

Single-nucleotide polymorphisms (SNPs) in the genes for pituitary adenylyl cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with stress-related psychiatric disorders. Although, from recent work, we have argued that stress-induced PACAP expression in the bed nucleus of the stria terminalis (BNST) may mediate stress-related psychopathology, it is unclear whether stress-induced increases in BNST PACAP expression require acute or repeated stressor exposure and whether increased BNST PACAP expression is related to stress-induced increases in circulating glucocorticoids. In the current work, we have used real-time quantitative polymerase chain reaction (qPCR) to assess transcript expression in brain punches from rats after stressor exposure paradigms or corticosterone injection. BNST PACAP and PAC1 receptor transcript expression was increased only after 7 days of repeated stressor exposure; no changes in transcript levels were observed 2 or 24 hours after a single-restraint session. Moreover, repeated corticosterone treatment for 7 days was not sufficient to reliably increase BNST PACAP transcript levels, suggesting that stress-induced elevations in corticosterone may not be the primary drivers of BNST PACAP expression. These results may help clarify the mechanisms and temporal processes that underlie BNST PACAP induction for intervention in stress-related anxiety disorders.

PACAP in the BNST produces anorexia and weight loss in male and female rats.

Recent gene association studies have implicated pituitary adenylate cyclase-activating peptide (PACAP) systems in several psychiatric disorders associated with stressor exposure, and we have argued that many of the behavioral consequences of repeated stressor exposure may depend on the expression of PACAP in the bed nucleus of the stria terminalis (BNST). One behavioral consequence of the activation of stress systems can be anorexia and subsequent weight loss, and both the activation of central PACAP systems as well as neuronal activity in the BNST have also been associated with anorexic states in rodents. Hence, we investigated the regulation of food and water intake and weight loss following BNST PACAP infusion. BNST PACAP38 dose-dependently decreased body weight, as well as food and water intake in the first 24 h following infusion. Because different BNST subregions differentially regulate stress responding, we further examined the effects of PACAP38 in either the anterior or posterior BNST. Anterior BNST PACAP38 infusion did not alter weight gain, whereas posterior PACAP38 infusion resulted in weight loss. PACAP38 infused into the lateral ventricles did not alter weight, suggesting that the effects of BNST-infused PACAP were not mediated by leakage into the ventricular system. These data suggest that PACAP receptor activation in posterior BNST subregions can produce anorexia and weight loss, and corroborate growing data implicating central PACAP activation in mediating the consequences of stressor exposure.

Pituitary adenylate cyclase-activating polypeptide (PACAP) potently dilates middle meningeal arteries: implications for migraine.

Migraine is a debilitating neurological disorder characterized by mild to severe headache that is often accompanied by aura and other neurological symptoms. Among proposed mechanisms, dilation of the dural vasculature especially the middle meningeal artery (MMA) has been implicated as one component underlying this disorder. Several regulatory peptides from trigeminal sensory and sphenopalatine postganglionic parasympathetic fibers innervating these vessels have been implicated in the process including pituitary adenylate cyclase-activating polypeptide (PACAP). Although PACAP has been well described as a potent dilator in many vascular beds, the effects of PACAP on the dural vasculature are unclear. In the current study, we examined the ability of PACAP to dilate MMAs that were isolated from rats and pressurized ex vivo. PACAP38 potently dilated pressurized MMAs with an EC(50) of 1 pM. The PAC1 receptor antagonist, PACAP(6-38), abolished MMA dilation caused by picomolar concentrations of PACAP. In contrast, cerebellar arteries isolated from the brain surface were ~1,000-fold less sensitive to PACAP than MMAs. Although cerebellar arteries expressed transcripts for all three PACAP receptor subtypes (PAC1, VPAC1, and VPAC2 receptors) by RT-PCR analyses, MMA demonstrated only PAC1 and VPAC2 receptor expression. Further, multiple variants of the PAC1 receptor were identified in the MMA. The expression of PAC1 receptors and the high potency of PACAP to induce MMA vasodilation are consistent with their potential roles in the etiology of migraine.

Excitotoxic lesions of the bed nucleus of the stria terminalis (BNST) attenuate the effects of repeated stress on weight gain: evidence for the recruitment of BNST activity by repeated, but not acute, stress.

Exposure to repeated stress can lead to diverse and widespread behavioral consequences, including reduction in food and water intake and subsequent diminution in weight gain. Many reports have suggested that repeated stress substantially alters the neurochemistry, morphology and physiology of neurons within the bed nucleus of the stria terminalis (BNST). Here we investigate the role of the BNST in mediating the reduced weight gain observed during repeated stress. Rats exposed to a one-week variate stress paradigm exhibited a reduction in weight gain over the course of the 7-day paradigm. Excitotoxic lesions to a subregion of the anterolateral BNST containing the oval nucleus had no effects early in the 7-day paradigm, but significantly attenuated the effects of repeated stress on weight gain by the last day of stress. These data suggest that at least two mechanisms mediate the effects of stress on body weight gain, and that when stressor exposure becomes repeated, the BNST is recruited, worsening the symptoms of stressor exposure.

Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor.

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 post-traumatic stress disorder (PTSD). Here we find, in heavily traumatized subjects, a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females. We examined 44 single nucleotide polymorphisms (SNPs) spanning the PACAP (encoded by ADCYAP1) and PAC1 (encoded by ADCYAP1R1) genes, demonstrating a sex-specific association with PTSD. A single SNP in a putative oestrogen response element within ADCYAP1R1, rs2267735, predicts PTSD diagnosis and symptoms in females only. This SNP also associates with fear discrimination and with ADCYAP1R1 messenger RNA expression in human brain. Methylation of ADCYAP1R1 in peripheral blood is also associated with PTSD. Complementing these human data, ADCYAP1R1 mRNA is induced with fear conditioning or oestrogen 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 oestrogen regulation of ADCYAP1R1. PACAP levels and ADCYAP1R1 SNPs may serve as useful biomarkers to further our mechanistic understanding of PTSD.

PACAP/VIP and receptor characterization in micturition pathways in mice with overexpression of NGF in urothelium.

Urothelium-specific overexpression of nerve growth factor (NGF) in the urinary bladder of transgenic mice stimulates neuronal sprouting or proliferation in the urinary bladder, produces urinary bladder hyperreflexia, and results in increased referred somatic hypersensitivity. Additional NGF-mediated changes might contribute to the urinary bladder hyperreflexia and pelvic hypersensitivity observed in these transgenic mice such as upregulation of neuropeptide/receptor systems. Chronic overexpression of NGF in the urothelium was achieved through the use of a highly urothelium-specific, uroplakin II promoter. In the present study, we examined pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), and associated receptor (PAC1, VPAC1, VPAC2) transcripts or protein expression in urothelium and detrusor smooth muscle and lumbosacral dorsal root ganglia in NGF-overexpressing and littermate wildtype mice using real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical approaches. Results demonstrate upregulation of PAC1 receptor transcript and PAC1-immunoreactivity in urothelium of NGF-OE mice whereas PACAP transcript and PACAP-immunoreactivity were decreased in urothelium of NGF-OE mice. In contrast, VPAC1 receptor transcript was decreased in both urothelium and detrusor smooth muscle of NGF-OE mice. VIP transcript expression and immunostaining was not altered in urinary bladder of NGF-OE mice. Changes in PACAP, VIP, and associated receptor transcripts and protein expression in micturition pathways resemble some, but not all, changes observed after induction of urinary bladder inflammation known to involve NGF production.

Roles for pituitary adenylate cyclase-activating peptide (PACAP) expression and signaling in the bed nucleus of the stria terminalis (BNST) in mediating the behavioral consequences of chronic stress.

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 anxiety-like 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 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 and brainstem regions (Walker et al. Eur J Pharmacol 463:199-216, 2003, Prog Neuropsychopharmacol Biol Psychiatry 33(8):1291-1308, 2009; Ulrich-Lai and Herman Nat Rev Neurosci 10:397-409, 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. Behav Neurosci 120:324-336, 2006) and/or when the anxiety-like response is sustained (Walker and Davis Brain Struct Funct 213:29-42, 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. Brain Res 965:290-294, 2003; Pego et al. Eur J Neurosci 27:1503-1516, 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 is substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al. Brain Res 767:109-119, 1997; Hammack et al. Psychoneuroendocrinology 34:833-843, 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.

Pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1HOP1 receptor activation coordinates multiple neurotrophic signaling pathways: Akt activation through phosphatidylinositol 3-kinase gamma and vesicle endocytosis for neuronal survival.

MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC(1) receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC(1)HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC(1)HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC(1)HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on G alpha(q)/phosphatidylinositol 3-kinase gamma activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase gamma-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC(1)HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.