PACAP-PAC1R modulates fear extinction via the ventromedial hypothalamus.

Exposure to traumatic stress can lead to fear dysregulation, which has been associated with posttraumatic stress disorder (PTSD). Previous work showed that a polymorphism in the PACAP-PAC1R (pituitary adenylate cyclase-activating polypeptide) system is associated with PTSD risk in women, and PACAP (ADCYAP1)-PAC1R (ADCYAP1R1) are highly expressed in the hypothalamus. Here, we show that female mice subjected to acute stress immobilization (IMO) have fear extinction impairments related to Adcyap1 and Adcyap1r1 mRNA upregulation in the hypothalamus, PACAP-c-Fos downregulation in the Medial Amygdala (MeA), and PACAP-FosB/ΔFosB upregulation in the Ventromedial Hypothalamus dorsomedial part (VMHdm). DREADD-mediated inhibition of MeA neurons projecting to the VMHdm during IMO rescues both PACAP upregulation in VMHdm and the fear extinction impairment. We also found that women with the risk genotype of ADCYAP1R1 rs2267735 polymorphism have impaired fear extinction.

Pituitary adenylate cyclase-activating polypeptide receptor activation in the hypothalamus recruits unique signaling pathways involved in energy homeostasis.

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts pleiotropic effects on ventromedial nuclei (VMN) of the hypothalamus and its control of feeding and energy expenditure through the type I PAC1 receptor (PAC1R). However, the endogenous role of PAC1Rs in the VMN and the downstream signaling responsible for PACAP's effects on energy balance are unknown. Numerous studies have revealed that PAC1Rs are coupled to both Gαs/adenylyl cyclase/protein kinase A (Gαs/AC/PKA) and Gαq/phospholipase C/protein kinase C (Gαq/PLC/PKC), while also undergoing trafficking following stimulation. To determine the endogenous role of PAC1Rs and downstream signaling that may explain PACAP's pleiotropic effects, we used RNA interference to knockdown VMN PAC1Rs and pharmacologically inhibited PKA, PKC, and PAC1R trafficking. Knocking down PAC1Rs increased meal sizes, reduced total number of meals, and induced body weight gain. Inhibition of either PKA or PKC alone in awake male Sprague-Dawley rats, attenuated PACAP's hypophagic and anorectic effects during the dark phase. However, PKA or PKC inhibition potentiated PACAP's thermogenic effects during the light phase. Analysis of locomotor activity revealed that PKA inhibition augmented PACAP's locomotor effects, whereas PKC inhibition had no effect. Finally, PACAP administration in the VMN induces surface PAC1R trafficking into the cytosol which was blocked by endocytosis inhibitors. Subsequently, inhibition of PAC1R trafficking into the cytosol attenuated PACAP-induced hypophagia. These results revealed that endogenous PAC1Rs uniquely engage PKA, PKC, and receptor trafficking to mediate PACAP's pleiotropic effects in VMN control of feeding and metabolism.NEW & NOTEWORTHY Endogenous PAC1 receptors, integral to VMN management of feeding behavior and body weight regulation, uniquely engage PKA, PKC, and receptor trafficking to mediate the hypothalamic ventromedial nuclei control of feeding and metabolism. PACAP appears to use different signaling mechanisms to regulate feeding behavior from its effects on metabolism.

PAC1 Receptor Mediates Electroacupuncture-Induced Neuro and Immune Protection During Cisplatin Chemotherapy.

Platinum-based chemotherapy is an effective treatment used in multiple tumor treatments, but produces severe side effects including neurotoxicity, anemia, and immunosuppression, which limits its anti-tumor efficacy and increases the risk of infections. Electroacupuncture (EA) is often used to ameliorate these side effects, but its mechanism is unknown. Here, we report that EA on ST36 and SP6 prevents cisplatin-induced neurotoxicity and immunosuppression. EA induces neuroprotection, prevents pain-related neurotoxicity, preserves bone marrow (BM) hematopoiesis, and peripheral levels of leukocytes. EA activates sympathetic BM terminals to release pituitary adenylate cyclase activating polypeptide (PACAP). PACAP-receptor PAC1-antagonists abrogate the effects of EA, whereas PAC1-agonists mimic EA, prevent neurotoxicity, immunosuppression, and preserve BM hematopoiesis during cisplatin chemotherapy. Our results indicate that PAC1-agonists may provide therapeutic advantages during chemotherapy to treat patients with advanced neurotoxicity or neuropathies limiting EA efficacy.

Developmental changes in the hypothalamic mRNA expression levels of PACAP and its receptor PAC1 and their sensitivity to fasting in male and female rats.

The actions and responses of hypothalamic appetite regulatory and factors change markedly during the neonatal to pre-pubertal period. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to play pivotal roles in the regulation of metabolic and nutritional status through its specific receptor PAC1. PACAP/PAC1 have anorectic roles, and their functions are regulated by leptin in adulthood. In the present study, we showed that hypothalamic PACAP mRNA expression decreases during the neonatal to pre-pubertal period (from postnatal day 10-30) in both male and female rats. During this period, hypothalamic PACAP mRNA expression was not affected by 24h fasting in either sex, while the serum leptin levels (leptin is a positive regulator of hypothalamic PACAP expression in adulthood) of both sexes were decreased by fasting. On the other hand, hypothalamic PAC1 mRNA expression did not change during the neonatal to pre-pubertal period in either sex; however, its levels were consistently higher in males than in females. Hypothalamic PAC1 mRNA expression was decreased by 24h fasting in males, but no such changes were observed in females. These results indicate while hypothalamic PACAP expression is sensitive to a negative energy state and the serum leptin level in adulthood, no such relationships are seen in the pre-pubertal period. In addition, we speculate that differences in the gonadal steroidal milieu might induce sexual dimorphism in the basal hypothalamic PAC1 mRNA level and its response to fasting. The mechanisms responsible for and the physiological effects of such changes in hypothalamic PACAP and PAC1 expression during the developmental period remain to be clarified.

Neuroprotective effect of endogenous pituitary adenylate cyclase-activating polypeptide on spinal cord injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. To date, changes in the expression and effect of endogenous PACAP have not been clarified with respect to spinal cord injury (SCI). The aim of this study was to elucidate the expression pattern and function of endogenous PACAP on the contusion model of SCI using heterozygous PACAP knockout (PACAP(+/-)) and wild-type mice. Real-time polymerase chain reaction methods revealed that the level of PACAP mRNA increased gradually for 14 days after SCI and that PAC1R mRNA levels also increased for 7 days compared with intact control mice. PACAP and PAC1R immunoreactivities colabeled with a neuronal marker in the intact spinal cord. Seven days after SCI, PAC1R immunoreactivity was additionally co-expressed with an astrocyte marker. Wild-type mice gradually recovered motor function after 14 days, but PACAP(+/-) mice showed significantly impaired recovery from 3 days compared with wild-type mice. The injury volume at day 7 in PACAP(+/-) mice, and the number of single-stranded DNA-immunopositive cells as a marker of neuronal cell death at day 3 were significantly higher than values measured in wild-type mice. These data suggest that endogenous PACAP is upregulated by SCI and has a neuroprotective effect on the damaged spinal cord.

Changes in PACAP immunoreactivity in human milk and presence of PAC1 receptor in mammary gland during lactation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence in the nervous system and peripheral organs, including the mammary gland. Previously, we have shown that PACAP38 is present in the human milk at higher levels than in respective blood samples. However, it is not known how PACAP levels and the expression of PAC1 receptor change during lactation. Therefore, the aim of our study was to investigate PACAP38-like immunoreactivity (PACAP38-LI) in human colostrums and transitional and mature milk during lactation and to compare the expression of PAC1 receptors in lactating and non-lactating mammary glands. We found that PACAP38-LI was significantly higher in human colostrum samples than in the transitional and mature milk. PACAP38-LI did not show any significant changes within the first 10-month period of lactation, but a significant increase was observed thereafter, up to the examined 17th month. Weak expression of PAC1 receptors was detected in non-lactating sheep and human mammary glands, but a significant increase was observed in the lactating sheep samples. In summary, the present study is the first to show changes of PACAP levels in human milk during lactation. The presence of PACAP in the milk suggests a potential role in the development of newborn, while the increased expressions of PAC1 receptors on lactating breast may indicate a PACAP38/PAC1 interaction in the mammary gland during lactation.

Distribution and functional characterization of pituitary adenylate cyclase-activating polypeptide receptors in the brain of non-human primates.

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [(125)I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.

Pituitary adenylate cyclase-activating polypeptide inhibits food intake in mice through activation of the hypothalamic melanocortin system.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), exert anorexigenic activities. While alpha-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuron-expressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.

Novel stable PACAP analogs with potent activity towards the PAC1 receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.

Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia.

In the developing hypothalamus, a variety of neurons are generated adjacent to each other in a highly coordinated, but poorly understood process. A critical question that remains unanswered is how coordinated development of multiple neuronal types is achieved in this relatively narrow anatomical region. We focus on dopaminergic (DA) and oxytocinergic (OT) neurons as a paradigm for development of two prominent hypothalamic cell types. We report that the development of DA and OT-like neurons in the zebrafish is orchestrated by two novel pathways that regulate the expression of the homeodomain-containing protein Orthopedia (Otp), a key determinant of hypothalamic neural differentiation. Genetic analysis showed that the G-protein-coupled receptor PAC1 and the zinc finger-containing transcription factor Fezl act upstream to Otp. In vivo and in vitro experiments demonstrated that Fezl and PAC1 regulate Otp at the transcriptional and the post-transcriptional levels, respectively. Our data reveal a new genetic network controlling the specification of hypothalamic neurons in vertebrates, and places Otp as a critical determinant underlying Fezl- and PAC1-mediated differentiation.

Pituitary adenylate cyclase-activating polypeptide stimulates corticotropin-releasing factor, vasopressin and interleukin-6 gene transcription in hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic-pituitary-adrenal axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN) in response to stress, is secreted into the pituitary portal circulation, resulting in the release of adrenocorticotropic hormone from the anterior pituitary. AVP is synthesized in the PVN and supraoptic nucleus by various stressors. Hypothalamic 4B cells coexpress CRF and AVP. In 4B cells transfected with either a CRF or an AVP promoter-luciferase construct, forskolin increased the transcriptional activity of CRF or AVP. In the present study, we tried to determine whether pituitary adenylate cyclase-activating polypeptide (PACAP) regulates both CRF and AVP genes in the hypothalamic cells, because receptors for PACAP were expressed in the hypothalamic cells. PACAP stimulated activity of both CRF and AVP promoter via protein kinase A pathway. PACAP stimulated interleukin (IL)-6 promoter activity and the levels of IL-6 mRNA and protein. IL-6 stimulated activity of both CRF and AVP promoter in a dose-dependent manner. Finally, we found that the stimulatory effects of PACAP on both activities were significantly inhibited by treatment with anti-IL-6 monoclonal antibody. These data suggest that PACAP is involved in regulating the synthesis of IL-6 mRNA and IL-6 protein, and that the increase in endogenous IL-6 also contributes to stimulate the expression of both CRF and AVP genes. Taken together, these findings indicate that PACAP stimulates the transcription of CRF, AVP, and IL-6 genes in hypothalamic 4B cells.

Excitatory actions of vasoactive intestinal peptide on mouse thalamocortical neurons are mediated by VPAC2 receptors.

Thalamic nuclei can generate intrathalamic rhythms similar to those observed at various arousal levels and pathophysiological conditions such as absence epilepsy. These rhythmic activities can be altered by a variety of neuromodulators that arise from brain stem regions as well as those that are intrinsic to the thalamic circuitry. Vasoactive intestinal peptide (VIP) is a neuropeptide localized within the thalamus and strongly attenuates intrathalamic rhythms via an unidentified receptor subtype. We have used transgenic mice lacking a specific VIP receptor, VPAC(2), to identify its role in VIP-mediated actions in the thalamus. VIP strongly attenuated both the slow, 2-4 Hz and spindle-like 5-8 Hz rhythmic activities in slices from wild-type mice (VPAC(2)(+/+)) but not in slices from VPAC(2) receptor knock-out mice (VPAC(2)(-/-)), which suggests a major role of VPAC(2) receptors in the antioscillatory actions of VIP. Intracellular recordings revealed that VIP depolarized all relay neurons tested from VPAC(2)(+/+) mice. In VPAC(2)(-/-) mice, however, VIP produced no membrane depolarization in 80% of neurons tested. In relay neurons from VPAC(2)+/+ mice, VIP enhanced the hyperpolarization-activated mixed cation current, I(h), via cyclic AMP activity, but VIP did not alter I(h) in VPAC(2)-/- mice. In VPAC(2)-/- mice, pituitary adenylate cyclase activating-polypeptide (PACAP) depolarized the majority of relay neurons via I(h) enhancement presumably via PAC(1) receptor activation. Our findings suggest that VIP-mediated actions are predominantly mediated by VPAC(2) receptors, but PAC(1) receptors may play a minor role. The excitatory actions of VIP and PACAP suggest these peptides may not only regulate intrathalamic rhythmic activities, but also may influence information transfer through thalamocortical circuits.