We have discovered a highly specialized innervation of the forebrain by pituitary adenylate cyclase-activating polypeptide (PACAP) immunohistochemistry originating from the brain stem that uses glutamate, acetylcholine, and PACAP, and other peptides as neurotransmitters. The parent neurons of the axons are in the Kölliker-Fuse nucleus, and their terminals form calyx-like multirelease-site synapses in the rodent forebrain extended amygdala similar to the calyx of Held in the auditory brain stem. The latter is a giant, excitatory, cup-like axo-somatic high-fidelity synapse. The PACAP-positive terminals also form enveloping axo-somatic specialization with mixed glutamatergic and cholinergic molecular identities, co-expressing vesicular glutamate transporter 1 (VGluT1), VGluT2, vesicular acetylcholine transport (VAChT), and the neuropeptides PACAP, calcitonin gene-related peptide, and neurotensin, together with calretinin in the presynaptic compartment. We identified a distinct neuronal subpopulation in the pontine Kölliker-Fuse region of the parabrachial complex that gives rise to these calyceal terminals, which engulf Protein kinase C delta (PKCδ+) / Glutamate delta receptor 1 (GluD1+) somata in the capsular central amygdala and oval bed nucleus of the stria terminalis. Strikingly, GluD1 immunolabeling is concentrated at axo-somatic contact zones apposed to VAChT+ presynaptic vesicle cluster zones but is absent from postsynaptic densities of conventional type I synapses within the same terminals. The results demonstrate a previously unrecognized multimodal calyx-like synapse in the forebrain, with parallel fast ionotropic and modulatory peptidergic neurotransmission mechanisms, which is a substrate for high-fidelity signal transmission within viscerosensory-emotional circuits.

Immediate-early gene (IEG) induction guides elucidation of signaling pathways mediating neuronal plasticity underlying compulsive use of psychostimulants. IEG induction after psychostimulant administration has been attributed to both PKA- and RapGEF2-dependent signaling pathways initiated by D1 receptor stimulation by dopamine. However, it is not clear how each pathway contributes individually to IEG induction, dopaminoceptive neuronal activity, and neuronal plasticity. We used Cre-LoxP technology and a novel Cre-amplifier transgene to delete RapGEF2 only in D1-MSNs, and investigate its role in cocaine-induced IEG and behavioral responses. D1-MSN-specific RapGEF2 deletion blocked cocaine-induced ERK phosphorylation and Egr-1 induction, without affecting cocaine self-administration or c-Fos induction by cocaine. Deletion of Rap1 in D1-MSNs blocked cocaine-induced p-ERK and Egr-1 expression, but not the induction of c-Fos. Like RapGEF2 deletion, Rap1 deletion from D1-MSNs had no effect on final maintenance of stable cocaine self-administration, although the rate of acquisition was significantly impaired. These results suggest that D1-dependent activation of Egr1 is not ultimately required for cocaine self-administration, although it may affect the behavioral dynamics of this process. Suppressing cAMP elevation in D1-MSNs by D1-specific expression of PDE4D3-cat greatly reduced induction of both Egr-1 and c-Fos in NAc after cocaine administration, demonstrating that induction of both IEGs requires cAMP elevation in D1-MSNs. Specific inhibition of PKA activity via PKI-alpha expression in D1-MSNs also blocked both c-Fos and Egr-1 induction. Thus, acute or chronic cocaine administration activates at least two separate cAMP effectors in D1-MSNs. PKA activation leads to c-Fos induction, likely through CREB, and to Egr1 activation via Rap1, likely through a previously reported dependence on RasGRP2. RapGEF2 activation leads exclusively to Egr1 induction. The finding that PKA activates the ERK-Egr-1 signaling pathway by convergence on Rap1, and concomitantly activates c-Fos independently of Rap1, may underlie selective effects of RapGEF2 and PKA inhibition on psychostimulant-dependent behaviors in mice.

The PACAP receptor PAC1 is a Gs-coupled family B1 GPCR for which the highest-affinity endogenous peptide ligands are the pituitary adenylate cyclase-activating peptides PACAP38 and PACAP27, and whose most abundant endogenous ligand is PACAP38. PACAP action at PAC1 is implicated in neuropsychiatric disorders, atherosclerosis, pain chronification, and protection from neurodegeneration and ischemia. As PACAP also interacts with two related receptors, VPAC1 and VPAC2, highly selective ligands, both agonists and antagonists, for PAC1 have been sought. To date, the peptide PACAP(6-38) and polypeptide M65, which is related to maxadilan, a sandfly vasodilator peptide, have been identified as selective for PAC1. Several non-peptide small molecule compounds (SMOLs) have been reported to be specific antagonists at PAC1, albeit there is only limited literature detailing their pharmacology across different systems and within different laboratories. Here, we present a platform of cellular assays for the screening of biologically relevant antagonists at PAC1 and show that some currently proposed SMOL antagonists do not have activity in this cell reporter assay, while we confirm that PACAP(6-38) and M65 are competitive antagonists. We have used this assay system to explore other peptide antagonists at PAC1, guided by molecular dynamics analysis of the PACAP-PAC1 interaction based on cryo-EM structural models of PAC1 complexed with a number of biologically active ligands. The affinity-trap model for the PAC1-ligand interaction successfully predicts the engagement behavior of PACAP27 and PACAP38 peptide-based PAC1 inhibitors. In particular, C-terminal deletants of PACAP(6-38) that maintain equipotency to PACAP(6-38) allow the shorter sequence to function as a scaffold for further peptide-based antagonist exploration.

Immediate-early gene (IEG) induction after administration of amphetamine or cocaine has been used to trace the signaling pathways that mediate neuronal plasticity required for the short- and long-term behavioral effects of these psychostimulants. We recently reported that a novel cyclic AMP (cAMP)-dependent Rap guanine nucleotide exchange factor-2 (RapGEF2)-ERK signaling pathway is required for Egr-1 induction in D1 medium-spiny neurons (MSNs) of the nucleus accumbens (NAc) after cocaine treatment, and that its deletion from the NAc neurons attenuates cocaine-induced locomotor sensitization and conditioned place preference (CPP). However, the cell type-specific neuronal mechanisms underlying this effect remain unclear. In this study, we used Cre-LoxP technology and a novel Cre-amplifier transgene to generate conditional RapGEF2 knockout mice targeting D1-MSNs and investigated the functional role of RapGEF2 in cocaine reward. Deletion of RapGEF2 in D1-MSNs blocked cocaine-induced ERK phosphorylation (p-ERK) and Egr-1 induction. D1-MSN-specific RapGEF2 deletion did not affect intravenous cocaine self-administration, nor did it affect Fos induction by cocaine, prompting us to examine more closely the role of metabotropic (cAMP-dependent) signaling to IEGs after cocaine administration. We used a battery of D1-MSN-specific genetic interventions targeting cAMP signaling, including Drd1-Cre::Rap1A/Bfl/fl mice, and AAV injection of a Cre-dependent catalytically active phosphodiesterase (PDE4D3-cat) or a Cre-dependent protein kinase A-inhibitor (PKI) in the NAc of Drd1-Cre mice, to explore further the underlying cAMP dependence of IEG induction by acute and chronic cocaine administration, and the cAMP sensors required. Rap1 is reported as a necessary component for both RapGEF2- and PKA-dependent ERK activation, but a requirement for Rap in Fos induction by cocaine has not been examined. Deletion of Rap1A/B in D1-MSNs blocked cocaine-induced p-ERK and Egr-1 expression, but not c-Fos, supporting the idea that the RapGEF2-Rap1-ERK pathway specifically regulates Egr-1, not c-Fos, expression. D1-specific expression of the PDE4D3-cat ablated up-regulation of both Egr-1 and Fos in NAc after cocaine administration, demonstrating that induction of both IEGs requires cAMP elevation in D1-MSNs. Notably, inhibition of PKA activity via AAV-mediated expression of PKI-alpha in D1-MSNs blocked both c-Fos and Egr-1 induction. Thus, acute or chronic cocaine administration activates at least two cAMP-dependent signaling pathways in D1-MSNs: a PKA-Fos pathway and a RapGEF2-ERK-Egr-1 pathway. The finding that PKA also activates the ERK-Egr-1 signaling pathway by convergence on Rap1, and concomitantly activates c-Fos independently of Rap1, may underlie selective effects of metabotropic activation of RapGEF2 and PKA activation by cAMP on cocaine-dependent behaviors in mice.

Kisspeptinergic signaling is well-established as crucial for the regulation of reproduction, but its potential broader role in brain function is less understood. This study investigates the distribution and chemotyping of kisspeptin-expressing neurons within the mouse brain. RNAscope single, dual, and multiplex in situ hybridization methods were used to assess kisspeptin mRNA (Kiss1) expression and its co-expression with other neuropeptides, excitatory and inhibitory neurotransmitter markers, and sex steroid receptors in wild-type intact and gonadectomized young adult mice. Seven distinct kisspeptin neuronal chemotypes were characterized, including two novel kisspeptin-expressing groups described for the first time, that is, the Kiss1 population in the ventral premammillary nucleus and the nucleus of the solitary tract. Kiss1 mRNA was also observed to localize in both somatic and dendritic compartments of hypothalamic neurons. High androgen receptor expression and changes in medial amygdala and septo-hypothalamic Kiss1 expression following GDX in males, but not in females, suggest a role for androgen receptors in regulating kisspeptin signaling. This study provides a detailed chemoanatomical map of kisspeptin-expressing neurons, highlighting their potential functional diversity. The discovery of a new kisspeptin-expressing group and gonadectomy-induced changes in Kiss1 expression patterns suggest broader roles for kisspeptin in brain functions beyond those of reproduction.

Kisspeptin (KP) signaling in the brain is defined by the anatomical distribution of KP-producing neurons, their fibers, receptors, and connectivity. Technological advances have prompted a re-evaluation of these chemoanatomical aspects, originally studied in the early years after the discovery of KP and its receptor Kiss1r. Previously, we characterized (Hernández et al. bioRxiv 2024) seven KP neuronal populations in the mouse brain at the mRNA level, including two novel populations, and examined their response to gonadectomy. In this study, we mapped KP fiber distribution in rats and mice using immunohistochemistry under intact as well as short- and long-term post-gonadectomy conditions. Kiss1r mRNA expression was examined via RNAscope, in relation to vesicular GABA transporter (Slc32a1) in whole mouse brain, and to KP and vesicular glutamate transporter 2 (Slc17a6), Kiss1, and Slc32a1 in hypothalamic RP3V and arcuate regions. We identified KP fibers in 118 brain regions, primarily in extra-hypothalamic areas associated with sensorial processing and behavioral state control. KP-immunoreactive fiber density and distribution were largely unchanged by gonadectomy. Kiss1r was expressed prominently in sensorial and state control regions such as the septal nuclei, the suprachiasmatic nucleus, locus coeruleus, hippocampal layers, thalamic nuclei, and cerebellar structures. Co-expression of Kiss1r and Kiss1 was observed in hypothalamic neurons, suggesting both autocrine and paracrine KP signaling mechanisms. These findings enhance our understanding of KP signaling beyond reproductive functions, particularly in sensorial processing and behavioral state regulation. This study opens new avenues for investigating KP's role in controlling complex physiological processes, including those unrelated to reproduction.

A possible involvement of immune- and vasoregulatory PACAP signaling at the PAC1 receptor in atherogenesis and plaque-associated vascular inflammation has been suggested. Therefore, we tested the PAC1 receptor agonist Maxadilan and the PAC1 selective antagonist M65 on plaque development and lumen stenosis in the ApoE−/− atherosclerosis model for possible effects on atherogenesis. Adult male ApoE−/− mice were fed a cholesterol-enriched diet (CED) or standard chow (SC) treated with Maxadilan, M65 or Sham. Effects of treatment on atherosclerotic plaques, lumen stenosis, apoptosis and pro-inflammatory signatures were analyzed in the brachiocephalic trunk (BT). The percentage of Maxadilan treated mice exhibiting plaques under SC and CED was lower than that of Sham or M65 treatment indicating opposite effects of Maxadilan and M65. Maxadilan application inhibited lumen stenosis in SC and CED mice compared to the Sham mice. In spite of increased cholesterol levels, lumen stenosis of Maxadilan-treated mice was similar under CED and SC. In contrast, M65 under SC or CED did not reveal a significant influence on lumen stenosis. Maxadilan significantly reduced the TNF-α-immunoreactive (TNF-α+) area in the plaques under CED, but not under SC. In contrast, the IL-1β+ area was reduced after Maxadilan treatment in SC mice but remained unchanged in CED mice compared to Sham mice. Maxadilan reduced caspase-3 immunoreactive (caspase-3+) in the tunica media under both, SC and CED without affecting lipid content in plaques. Despite persistent hypercholesterolemia, Maxadilan reduces lumen stenosis, apoptosis and TNF-α driven inflammation. Our data suggest that Maxadilan provides atheroprotection by acting downstream of hypercholesterolemia-induced vascular inflammation. This implicates the potential of PAC1-specific agonist drugs against atherosclerosis even beyond statins and PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors.

Abstract ID 130936Poster Board 493The receptor for pituitary adenylate cyclase-activating polypeptide (PACAP) is a Gs-coupled GPCR, designated PAC1. Antagonists of this receptor are being pursued for their potential to ameliorate atherosclerogenesis, depression, post-traumatic stress disorder, and migraine; discovery of agonists is also of interest for counteracting the neurodegenerative consequences of stroke and ischemia. However, obtaining small-molecule ligands (SMOLs) for this receptor has been challenging. Peptide-based antagonists include an N-terminally truncated version of PACAP38 (P38) itself, PACAP6-38 (P6-38). The activity of P6-38 as an antagonist is consistent with models of PACAP binding to PAC1 in which the N-terminus of PACAP is required for receptor activation leading to signaling through the Gs protein, while the C-terminus is involved in initial binding (affinity-trapping) to PAC1. P6-38 has been shown repeatedly to block the action of P38 as well as the naturally occurring ligand PACAP1-27 (P27) both in vivo and in cells expressing endogenous PAC1. Several SMOLs reported to be PAC1 antagonists in the literature were tested for antagonist activity in HEK293 cells expressing the human PAC1 receptor and a cAMP biosensor (CBS)-based cAMP detection system; however not only these but also P6 -38 were without inhibitory effect in this assay system. Accordingly, we re-tested the inhibitory activity of P6-38 against both P27 and P38 in cells expressing CBS but with native expression of PAC1: the rat NS-1 pheochromocytoma cell line, and the human SH-SY5Y neuroblastoma cell line. Both CBS-based luminescent read-out, and generation of cAMP provoked by exposure to EC50 concentrations of P27 or P38 (0.2 nM) were blocked by 1 mM P6-38 which also blocked downstream signaling effects (neuritogenesis) of P27 and P38.Recently, cryo-EM structures of a number of family B (secretin family) GPCR-ligand structures have been obtained, allowing detailed molecular dynamics analysis of ligand-receptor binding, and prediction of residues contributing most strongly to ligand engagement. Molecular dynamics simulations predicted initial receptor engagement with residues 6-30, but not 31-38, of P38. Accordingly, we synthesized a series of deletants of PACAP6-38 and tested their relative inhibitory potency in SH-SY5Y and NS-1 cells. PACAP6-30 (IC50 ∼33 nM) was nearly as potent as PACAP6-38 (IC50 ∼21 nM) as an inhibitor of cyclic AMP elevation by 0.2 nM P38 (the approximate EC50 for both the CBS-based and the direct cAMP assay, both carried out in the presence of phosphodiesterase inhibition by IBMX). PACAP6-27 was considerably less potent (IC50 >10 mM). Thus, the affinity-trap model for PAC1-ligand interaction successfully predicts the behavior of peptide-based inhibitors of P27 and P38 engagement with an endogenously expressed PAC1 receptor. We are currently engaged in peptide modification of P6-30 in order to increase the potency of this PAC1 antagonist.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

BACKGROUND:Kisspeptinergic signaling is well-established as crucial for regulation of reproduction, but its potential broader role in brain function is less understood. This study investigates the distribution and chemotyping of kisspeptin-expressing neurons within the mouse brain. METHODS:RNAscope singleplex, duplex and multiplex in situ hybridization methods were used to assess kisspeptin mRNA (Kiss1) expression and its co-expression with other neuropeptides, excitatory and inhibitory neurotransmitter markers, and sex steroid receptors in intact and gonadectomized young adult mice. RESULTS:Seven distinct kisspeptin neuronal chemotypes were characterized, including within two novel Kiss1-expressing groups described here for the first time: the ventral premammillary nucleus, and the nucleus of the solitary tract. Kiss1 mRNA was also localized in the soma, and within the dendritic compartment, of hypothalamic neurons. Altered Kiss1 expression following gonadectomy suggests a previously unappreciated role for androgen receptors in regulating kisspeptin signaling. CONCLUSION:This study provides a detailed chemoanatomical map of kisspeptin-expressing neurons in the brain, highlighting their potential functional diversity. The discovery of new kisspeptin-expressing neuronal populations, and gonadectomy-induced changes in Kiss1 expression patterns, provide a basis for further exploration of non-endocrine roles for kisspeptin in brain function.

BACKGROUND: The neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. METHODS: We used AAV (adeno-associated virus) neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57BL/6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. RESULTS: PACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex to hypothalamus, impairs c-fos activation and corticotropin-releasing hormone (CRH) messenger RNA elevation in the paraventricular nucleus after 2 hours of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in nonhypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala, on the other hand, attenuates ARS-induced hypophagia, along with extended amygdala fos induction, without affecting ARS-induced CRH messenger RNA elevation in the paraventricular nucleus. PACAP projections to extended amygdala terminate at protein kinase C delta type (PKCd) neurons in both the central amygdala and the oval bed nucleus of the stria terminalis. Silencing of PKCd neurons in the central amygdala, but not in the oval bed nucleus of the stria terminalis, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n >= 3 per group. CONCLUSIONS: A frontocortical descending PACAP projection controls paraventricular nucleus CRH messenger RNA production to maintain hypothalamic-pituitary-adrenal axis activation and regulate the endocrine response to stress. An ascending PACAPergic projection from the external lateral parabrachial nucleus to PKCd neurons in the central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.

Journal of NeuroendocrinologyVolume 35, Issue 11 e13345 EDITORIAL Advances in peptide modulation in systems physiology Lee E. Eiden, Lee E. Eiden orcid.org/0000-0001-7524-944X Section on Molecular Neuroscience, National Institute of Mental Health, NIH, Bethesda, Maryland, USA Contribution: Writing - original draft, Writing - review & editingSearch for more papers by this authorDavid Grattan, David Grattan orcid.org/0000-0001-5606-2559 Centre for Neuroendocrinology, Division of Health Sciences, University of Otago, Dunedin, New Zealand Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorXiao-Dong Wang, Xiao-Dong Wang orcid.org/0000-0001-7730-3710 School of Brain Science and Brain Medicine, Zhejiang University, Hangzhou, China Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorLimei Zhang, Corresponding Author Limei Zhang [email protected] orcid.org/0000-0002-7422-5136 Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico Correspondence Limei Zhang, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. Email: [email protected] Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this author Lee E. Eiden, Lee E. Eiden orcid.org/0000-0001-7524-944X Section on Molecular Neuroscience, National Institute of Mental Health, NIH, Bethesda, Maryland, USA Contribution: Writing - original draft, Writing - review & editingSearch for more papers by this authorDavid Grattan, David Grattan orcid.org/0000-0001-5606-2559 Centre for Neuroendocrinology, Division of Health Sciences, University of Otago, Dunedin, New Zealand Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorXiao-Dong Wang, Xiao-Dong Wang orcid.org/0000-0001-7730-3710 School of Brain Science and Brain Medicine, Zhejiang University, Hangzhou, China Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this authorLimei Zhang, Corresponding Author Limei Zhang [email protected] orcid.org/0000-0002-7422-5136 Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico Correspondence Limei Zhang, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. Email: [email protected] Contribution: Conceptualization, Writing - original draft, Writing - review & editingSearch for more papers by this author First published: 03 October 2023 https://doi.org/10.1111/jne.13345 Special Issue of papers from the 24th International Symposium on Regulatory Peptides (RegPep24 World Conference), 1st–5th August 2022, University of Stirling, Scotland, UK. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Open Research PEER REVIEW The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/jne.13345. DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed in this study. REFERENCES 1Hernandez-Perez OR, Hernández VS, Zetter MA, Eiden LE, Zhang L. Nucleus of the lateral olfactory tract: a hub linking the water homeostasis-associated supraoptic nucleus-arginine vasopressin circuit and neocortical regions to promote social behavior under osmotic challenge. J Neuroendocrinol. 2022;223:e13202. 2Peles G, Swaminathan A, Levkowitz G. Glucocorticoid-sensitive period of corticotroph development-implications for mechanisms of early life stress. J Neuroendocrinol. 2022;e13229. 3Sabban EL, Serova L, Nahvi RJ, Liu X. Potential benefits of intranasal neuropeptide Y include sustained extinction of fear memory. J Neuroendocrinol. 2023;223:e13279. 4Chen X, Coffman BL, Brindley RL, et al. Phospholipase C-epsilon defines a PACAP-stimulated pathway for secretion in the chromaffin cell. J Neuroendocrinol. 2023;223:e13255. 5Bakalar D, Gavrilova O, Jiang SZ, et al. Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP. J Neuroendocrinol. 2023;223:e13286. 6Kozlova EV, Bishay AE, Denys ME, et al. Gene deletion of the PACAP/VIP receptor, VPAC2R, alters glycemic responses during metabolic and psychogenic stress in adult female mice. J Neuroendocrinol. 2023;223. 7Paes-Leme B, Monteiro LRN, Gholami K, et al. Fasting increases circulating angiotensin levels and brain Agtr1a expression in male rats. J Neuroendocrinol. 2023;223:e13334. 8Chen D, Hagen SJ, Boyce M, Zhao CM. Neuroendocrine mechanism of gastric acid secretion: historical perspectives and recent developments in physiology and pharmacology. 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Background Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE −/− ) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE −/− mouse model of atherosclerosis and under the influence of a high-fat diet. Methods For this purpose, PACAP −/− /ApoE −/− , PAC1 −/− /ApoE −/− , and ApoE −/− mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1 + ], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified. Results ApoE −/− mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1 −/− /ApoE −/− mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE −/− mice. In contrast, PACAP deficiency in ApoE −/− mice did not affect the chondrogenic signature under either diet. Conclusions Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase-activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Homeostatic challenges increase the drive for social interaction. The neural activity that prompts this motivation remains poorly understood. Here, we identify direct projections from hypothalamic supraoptic nucleus (SON) to the cortico-amygdalar nucleus of the lateral olfactory tract (NLOT). Dual in situ hybridization (DISH) with probes for PACAP, and VGLUT1, VGLUT2, V1a and V1b revealed a population of vasopressin-receptive PACAPergic neurons in NLOT layer 2 (NLOT2). Water deprivation (48 hours, WD48) increased sociability compared to euhydrated subjects, assessed with the three-chamber social interaction test (3CST). Fos expression immunohistochemistry showed NLOT and its main efferent regions had further increases in rats subjected to WD48+3CST. These regions strongly expressed PAC1 mRNA. Microinjections of AVP into NLOT produced similar changes in sociability to water deprivation, and these were reduced by co-injection of V1a or V1b antagonists along with AVP. We conclude that during challenge to water homeostasis, there is a recruitment of a glutamatergic-multi-peptidergic cooperative circuit that promotes social behavior. Supported by grants UNAM-DGAPA-PAPIIT- PAPIIT-IN216918 & GI200121 & CONACYT-CB-283279 (LZ); MH002386, NIMH, NIH, USA (LEE).

Abstract Background Fibrosis is a hallmark of cardiac remodeling and is present in a variety of cardiac diseases. In particular, in the highly prevalent heterogeneous group of cardiac dysfunctions with preserved left ventricular ejection fraction (LVEF), interstitial fibrosis has been identified as a pivotal pathophysiological factor. Current evidence suggests that comorbidities, mainly from the metabolic spectrum, promote chronic systemic low-grade inflammation with secondary affection of the heart, leading to fibrosis and thus impairing cardiac structure and function. In contrast to cardiac dysfunction with reduced LVEF, which is predominantly of ischemic origin, effective antifibrotic treatment options are very limited emphasizing the urgent need for new therapeutic targets. In this context, the recent report of an antifibrotic effect of the pleiotropic neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) in irradiation-induced myocardial damage [1] renders it an intriguing new candidate. Purpose The aim of this study is to elucidate the role of PACAP and of PAC1 receptor in cardiac fibrosis in a murine model of metabolic stress. Methods PACAP−/− and PAC1−/− C57/Bl6 J mice were crossbred with an ApoE−/− strain and metabolic stress was induced by feeding a cholesterol-enriched diet (CED) for 10 weeks. Standard chow (SD) served as control diet. Tissue samples of the ventricles were processed for FFPE histological analysis with hematoxylin/eosin, Giemsa and picrosirius red stains and subjected to morphometric quantification of cardiomyocyte cross diameters and fibrosis using a software-based image analysis approach. Results After 10 weeks of CED, a statistically significant higher extent of myocardial fibrosis was detected in PACAP−/−/ApoE−/− and PAC1−/−/ApoE−/− compared to ApoE−/− mice (Fig. 1). The pattern of fibrous deposition was consistent with that of reactive interstitial fibrosis and affected both ventricles uniformly. Morphometric analysis revealed no macroscopic or cellular hypertrophy in the PACAP−/−/ApoE−/− and PAC1−/−/ApoE−/− mice compared with ApoE−/− mice after CED. All morphological changes were exclusively present after feeding CED and were not detectable with SD feeding. Conclusions This study provides novel evidence for a significant involvement of PACAP and its receptor PAC1 in the development of metabolically induced cardiac fibrosis. The antifibrotic effect of PACAP, which can be inferred in this context, is mediated to a significant extent via the PAC1 receptor, and only becomes apparent under metabolic stress conditions. The absence of macroscopic and cellular cardiac hypertrophy in PACAP deficient or PAC1 deficient mice as demonstrated here precludes relevant hemodynamic effects of PACAP and PAC1 receptor, respectively. According to the results of our pilot study, PACAP analogues or PAC1 receptor agonists may offer a therapeutic potential to attenuate metabolically triggered cardiac fibrosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This research was partly supported by the Von Behring-Röntgen-Stiftung Schloss 1 D-35037 Marburg, Germany.

Homeostatic challenges may alter the drive for social interaction. The neural activity that prompts this motivation remains poorly understood. In the present study, we identify direct projections from the hypothalamic supraoptic nucleus to the cortico‐amygdalar nucleus of the lateral olfactory tract (NLOT). Dual in situ hybridization with probes for pituitary adenylate cyclase‐activating polypeptide (PACAP), as well as vesicular glutamate transporter (VGLUT)1, VGLUT2, V1a and V1b, revealed a population of vasopressin‐receptive PACAPergic neurons in NLOT layer 2 (NLOT2). Water deprivation (48 h, WD48) increased sociability compared to euhydrated subjects, as assessed with the three‐chamber social interaction test (3CST). Fos expression immunohistochemistry showed NLOT and its main efferent regions had further increases in rats subjected to WD48 + 3CST. These regions strongly expressed PAC1 mRNA. Microinjections of arginine vasopressin (AVP) into the NLOT produced similar changes in sociability to water deprivation, and these were reduced by co‐injection of V1a or V1b antagonists along with AVP. We conclude that, during challenge to water homeostasis, there is a recruitment of a glutamatergic‐multi‐peptidergic cooperative circuit that promotes social behavior.

Gs-coupled GPCRs signal through the second messenger cAMP. In the neuroendocrine NS-1 cell line, cAMP acts through three parcellated signal pathways using distinct cAMP sensors as third messenger: protein kinase A (PKA) is linked to neuroendocrine-specific gene expression, the exchange protein (EPAC) to cell proliferation arrest, and the neuritogenic cAMP sensor NCS-Rapgef2 to neuritogenesis (Xu et al., J. Neuroendocrinol. 33: e12974). NCS-RapGEF2-mediated neuritogenesis occurs via the Rap-Braf-MEK-ERK pathway, which is activated by a variety of Gs-coupled GPCRs, including the endogenously expressed PAC1 receptor, and receptors for GLP-1 (GLP1R) and VIP (VIPR1, VIPR2) when exogenously expressed in NS-1 cells, followed by exposure to exendin-4 or VIP (Xu et al., ibid) or by ADRB2, after exposure to isoproterenol. We have noted previously that other Gs-coupled GPCRs expressed in NS-1 cells mediate measurable cAMP elevation, which however is apparently below the threshold required for triggering and maintaining neuritogenesis (Xu et al., ibid). Here, we identify changes in the NS-1 cell transcriptome associated with PACAP activation of PAC1 receptor and the role of ERK phosphorylation in sustaining gene expression potentially associated with cAMP-dependent neuritogenesis. Continuous ERK phosphorylation is required for neuritogenic signaling: inhibition of ERK phosphorylation with U0126 for up to 24 hours after PACAP treatment arrests neuritogenesis, while inhibition of PKA with H89 has no effect on neuritogenesis. NS-1 cells were treated for 24 hours with PACAP in the presence and absence of either the MEK inhibitor U0126 or the PKA inhibitor H89, and subjected to transcriptomic analysis. We identified 25 genes significantly upregulated by PACAP but downregulated (P<0.001, >2 fold, n=3) by co-treatment with U0126. DCLK1 is a serine/threonine protein kinase important for neuronal development. Dclk1 mRNA is upregulated ~six-fold at 8 hr and four-fold at 24 hr after PACAP treatment, and this was reduced to 2 fold compared to no treat) and its upregulation by PACAP is also blocked by U0126. The gene encoding the orphan GPCR, GPR50, is upregulated more than 100-fold by 8 hours after PACAP, and induction of GPR50 mRNA is blocked more than 97% by U0126, with partial blockade (80%) by H89. We are currently investigating the impact of GPR50 on neuritogenesis using GPR50 knockdown with shRNA in NS-1 cells. Forty-five genes upregulated by PACAP and blocked by H89, but not U0126, include Slit2 and Gas1. Regulation of these genes by Gs-coupled GPCR signaling through PKA may mediate NS-1 cell responses separate from activation of neuritogenesis. Cyclic AMP elevation associated with Gs-coupled GPCR signaling in NS-1 cells results in upregulation of both RapGEF2/ERK-dependent and PKA-dependent gene induction. A threshold for cAMP elevation, either in amount or duration of elevation, or both, appears to be a requirement for Gs-coupled GPCR-mediated neuritogenesis. NS-1 cells provide a model for investigating the functional importance of RapGEF2- and PKA-dependent gene regulation initiated by cAMP in neuroendocrine cells.

Neuropeptides are expressed in cell-specific patterns throughout mammalian brain. Neuropeptide gene expression has been useful for clustering neurons by phenotype, based on single-cell transcriptomics, and for defining specific functional circuits throughout the brain. How neuropeptides function as first messengers in inter-neuronal communication, in cooperation with classical small-molecule amine transmitters (SMATs) is a current topic of systems neurobiology. Questions include how neuropeptides and SMATs cooperate in neurotransmission at the molecular, cellular and circuit levels; whether neuropeptides and SMATs always co-exist in neurons; where neuropeptides and SMATs are stored in the neuron, released from the neuron and acting, and at which receptors, after release; and how neuropeptides affect 'classical' transmitter function, both directly upon co-release, and indirectly, via long-term regulation of gene transcription and neuronal plasticity. Here, we review an extensive body of data about the distribution of neuropeptides and their receptors, their actions after neuronal release, and their function based on pharmacological and genetic loss- and gain-of-function experiments, that addresses these questions, fundamental to understanding brain function, and development of neuropeptide-based, and potentially combinatorial peptide/SMAT-based, neurotherapeutics.