ABSTRACT The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for ERK activation in hippocampus are still largely undetermined. RapGEF2 has emerged as a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether RapGEF2 might also be required for cAMP-dependent ERK activation leading to synaptic potentiation, and how this involvement might be penetrant to hippocampus-dependent learned behavior. We demonstrate that conditional knockout of Rapgef2 in forebrain neurons, specifically in dentate gyrus and CA1 of the hippocampus, leads to an attenuation of context-dependent fear conditioning, but not of cue-dependent fear conditioning, in mice. RapGEF2 knockout is associated with a reduction in cAMP-dependent synaptic potentiation at two central hippocampal synapses-the entorhinal cortex-granule cell synapse and the CA3-CA1 synapse. Furthermore, cAMP-induced postsynaptic potentiation requires both RapGEF2 and activation of ERK. Induction of Egr-1/Zif268 (and pERK), but not of c-Fos, immediately following fear conditioning, was abolished in CA1 and detate gyrus, in the absence of RapGEF2 expression in these hippocampal regions, thus revealing a link between learning (conditioning) and molecular pathways activated during conditioned fear memory formation. Hence, we suggest that contextual fear conditioning is mediated via RapGEF2-dependent ERK activation and downstream induction of Egr-1, via an underlying mechanism of cAMP-dependent long-term potentiation at hippocampal synapses. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. This study provides a functional link between one of these cAMP-dependent GEFs, RapGEF2, and cognitive processes involved in associative learning.

peptide – ligand G-protein-coupled receptor (GPCR) reproductive hormones behaviors; neuropeptide regulation of rhythmic behavior; vasopressin, oxytocin (OT) and the neurosecretory paradigm; peptides in brain circuits specifying behavior; OT neurotherapeutics; pleiotropic actions of chromogranin-derived peptides; angiotensin II in RAS system and brain; sex hormones, neuropeptides and reproductive behavior; neuropeptides and stress; pain-related peptide signaling; peptides in appe-tite, anxiety, and motivational circuits; neuroeptides in cognitive integration; and peptide regulation of neuroglial interactions and inflammation.

Axon initial segments (AIS) of dentate granule cells in the hippocampus exhibit prominent spines (AISS) during early development that are associated with microglial contacts. In the present study, we investigated whether developmental changes in AISS could be modified by early-life stress (ELS), specifically neonatal maternal separation (MS), through stress hormones and microglial activation and examined the potential behavioural consequences. We examined AISS at postnatal day (PND)5, 15 and 50, using Golgi-Cox staining and anatomical analysis. Neurone-microglial interaction was assessed using antibodies against ankyrin-G, PSD-95 and Iba1, for AIS, AISS and microglia visualisation, respectively, in normally reared and neonatal maternally separated male and female rats. We observed a higher density of AISS in ELS rats at both PND15 and PND50 compared to controls. Effects were more pronounced in females than males. AIS-associated microglia in ELS rats showed a hyper-ramified morphology and less co-localisation with PSD-95 compared to controls at PND15. ELS-associated alteration in microglial morphology and synaptic pruning was mimicked by treatment of acute hippocampal slices of normally reared rats with vasopressin. ELS rats exhibited increased freezing behaviour during auditory fear memory testing, which was more pronounced in female subjects and corresponded with increased Fos expression in dorsal and ventral dentate granule cells. Thus, microglial synaptic pruning in dentate AIS of hippocampus is influenced by ELS, with demonstrable sex bias regarding its anatomical characteristics and subsequent fear-induced defensive behaviours.

Recently, there has been a resurgence in regulatory peptide science as a result of three converging trends. The first is the increasing population of the drug pipeline with peptide‐based therapeutics, mainly in, but not restricted to, incretin‐like molecules for treatment of metabolic disorders such as diabetes. The second is the development of genetic and optogenetic tools enabling new insights into how peptides actually function within brain and peripheral circuits to accomplish homeostatic and allostatic regulation. The third is the explosion in defined structures of the G‐protein coupled receptors to which most regulatory peptides bind and exert their actions. These trends have closely wedded basic systems biology to drug discovery and development, creating a “two‐way street” on which translational advances travel from basic research to the clinic, and, equally importantly, “reverse‐translational” information is gathered, about the molecular, cellular and circuit‐level mechanisms of action of regulatory peptides, comprising information required for the fine‐tuning of drug development through testing in animal models. This review focuses on a small group of ‘influential’ peptides, including oxytocin, vasopressin, pituitary adenylate cyclase‐activating polypeptide, ghrelin, relaxin‐3 and glucagon‐like peptide‐1, and how basic discoveries and their application to therapeutics have intertwined over the past decade.

Defensive behaviors, including fight‐flight and freezing, are critical for animal survival. There has been significant progress in elucidating the brain regions that control decision making and locomotion, which have implicated an extended network of interconnected brain regions, local circuit computations, and cell types that interact to generate emotion‐driven behaviors. However, the roles of neuropeptides in those circuits for functional connectivity are largely unclear. We describe here a direct PACAP‐glutamatergic projection from the hindbrain parabrachial complex (PBN) to the some nuclei of pallidum, i. e. central amygdala and the bed nucleus of stria terminalis (BNST), selectively targeting GABAergic somatostatin expressing neurons. This projection establishes perisomatic glutamatergic “Calyx of Held” like synapses, in addition to axon‐dendritic synapses onto the targeting CeC and ovBNST neurons – a “high‐fidelity” (HiFi) arrangement for insuring a high response‐to‐stimulus ratio. The targeted neurons were found to project to the lateral and medial preoptical hypothalamus where the locomotor initiator and controller centers are located in rodent. To assess the behavioral implication of this pathway, we devised an experiment assessing the effect of predator odor (cat urine) exposure on defensive behavior of wild‐type compared to PACAP knock‐out C57Bl6/N mice. Mice were placed in a glass‐lidded box containing a sample of cat urine within a contained whose lid could be opened remotely. Video recording was made during a 10 min period of urine odor exposure, after which animals were euthanized by cervical dislocation and brains rapidly removed and processed for double in situ hybridization histochemistry using theRNAscope 2.5 HD Duplex Assay. The neuronal activation in the olfactory bulb (OB), piriform cortex (Pir), medial and central amygdala (MeA and CeA), bed nucleus of stria terminalis (BNST), medial and lateral preoptic hypothalamus, parabrachial nucleus (PBN) and hippocampus were assessed for fos expression within PACAP, PAC1, VGluT2 and VGAT‐positive cells. Cat urine exposure triggered purposeful movement such as object exploration/retreat and freezing behaviors in both wild‐type and knock‐out mice, with the PACAP‐deficient (knock‐out) group showing significantly less of both behaviors than the wild‐type group (p < 0.05 and p < 0.01 respectively). Fos mRNA expression was significantly less in the PACAP‐deficient compared to the wild‐type group in PBN, CeC, BNST, MPO and LPO (p < 0.001 in all cases), with similar expression in both groups in the dorsal and ventral DG and in LS. These data suggest that PACAP‐Pac1 projections to CeC are required for the expression of defensive responses elicited by predator threats and offer an initial step towards identifying the PACAP‐PAC1 signaling in sensory‐emotional circuits with locomotor consequences.Support or Funding InformationGrants CONACYT‐CB‐238744, and IN216918 to L.Z. and L.E.E. acknowledges the support of the NIMH Intramural Research Program through MH002386.

Recently, there has been a resurgence in regulatory peptide science as a result of three converging trends. The first is the increasing population of the drug pipeline with peptide-based therapeutics, mainly in, but not restricted to, incretin-like molecules for treatment of metabolic disorders such as diabetes. The second is the development of genetic and optogenetic tools enabling new insights into how peptides actually function within brain and peripheral circuits to accomplish homeostatic and allostatic regulation. The third is the explosion in defined structures of the G-protein coupled receptors to which most regulatory peptides bind and exert their actions. These trends have closely wedded basic systems biology to drug discovery and development, creating a "two-way street" on which translational advances travel from basic research to the clinic, and, equally importantly, "reverse-translational" information is gathered, about the molecular, cellular and circuit-level mechanisms of action of regulatory peptides, comprising information required for the fine-tuning of drug development through testing in animal models. This review focuses on a small group of 'influential' peptides, including oxytocin, vasopressin, pituitary adenylate cyclase-activating polypeptide, ghrelin, relaxin-3 and glucagon-like peptide-1, and how basic discoveries and their application to therapeutics have intertwined over the past decade.

The origin and functional significance of vasopressin (AVP)-containing fibres in limbic regions has been an ongoing subject of investigation for several years. We have previously identified AVP-magnocellular neurones of rat hypothalamus that provide glutamatergic projections to the hippocampus, amygdala, lateral habenula and locus coeruleus. However, we also reported AVP-immunopositive fibres in those regions that are thin and make Gray type II synapses, which are unlikely to be of magnocellular origin. Therefore, in the present study, we characterised AVP mRNA co-expression with expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) neuronal traits in rat and mouse brain, using high-resolution in situ hybridisation methods, including a radio-ribonucleotide and RNAscope 2.5 HD duplex assay, with Slc17a7, Slc17a6, Slc32a1 and Avp probes corresponding to mRNAs of VGLUT1, VGLUT2, VGAT and AVP, respectively. We located 18 cell groups expressing Avp and identified their molecular signatures for VGLUT and VGAT mRNA expression. Avp cell groups of hypothalamus and midbrain are mainly VGLUT mRNA-expressing, whereas those in regions derived from cerebral nuclei are mainly VGAT mRNA-expressing, suggesting a functional segregation of glutamate/GABA co-transmission with AVP. A newly identified Slc17a7 and Slc17a6 (but not Slc32a1) expressing vasopressinergic cell group was found in layer II-III neurones of the central entorhinal cortex, which projects to the hippocampus. These data support the notion of a complex role for AVP with respect to modulating multiple central circuits controlling behaviour in specific ways depending on co-transmission with glutamate or GABA, potentially giving rise to a functional classification of AVPergic neurones in the central nervous system.

Axon initial segments (AIS) of dentate granule cells (GC) in hippocampus exhibit prominent spines during early development that are associated with microglial contacts. Here, we asked if developmental changes in axon initial segment spines (AISS) could be modified by neonatal maternal separation through stress hormones and microglial activation and examined the potential behavioral consequences. We examined AISS densities at postnatal day (PND) 15 and 50, using Golgi-Cox staining and anatomical analysis. Neuron-microglial interaction was assessed using antibodies against ankyrinG, PSD95 and Iba1, for AIS, AISS and microglia, respectively, in normally reared and neonatal maternally separated (MS) male and female rats. We observed a higher density of AISS in MS groups at both PND15 and PND50 compared to control. Effects were more pronounced in female than in male rats. AIS-associated microglia showed a hyper-ramified morphology and less co-localization with PSD95 in MS compared to normally reared animals at PND 15. An MS-like alteration in microglial morphology and synaptic pruning could be produced ex vivo by vasopressin application in acute hippocampal slices from normally reared animals. MS rats exhibited increased freezing behavior during auditory fear memory testing which, like effects on AISS density, was more pronounced in females than males. Freezing behavior was associated with Fos expression in dorsal and ventral dentate GC. In summary, AIS associated microglial activity is altered by MS. Sex differences in the long-term effects of MS on AISS density are penetrant to a behavioral phenotype of increased stimulus reactivity in adult female subjects.

The locus coeruleus (LC)-norepinephrine (NE) system modulates a range of salient brain functions, including memory and response to stress. The LC-NE system is regulated by neurochemically diverse inputs, including a range of neuropeptides such as arginine-vasopressin (AVP). Whilst the origins of many of these LC inputs, their synaptic connectivity with LC neurons, and their contribution to LC-mediated brain functions, have been well characterized, this is not the case for the AVP-LC system. Therefore, our aims were to define the types of synapses formed by AVP+ fibers with LC neurons using immunohistochemistry together with confocal and transmission electron microscopy (TEM), the origins of such inputs, using retrograde tracers, and the plasticity of the LC AVP system in response to stress and spatial learning, using the maternal separation (MS) and Morris water maze (MWM) paradigms, respectively, in rat. Confocal microscopy revealed that AVP+ fibers contacting tyrosine hydroxylase (TH)+ LC neurons were also immunopositive for vesicular glutamate transporter 2, a marker of presynaptic glutamatergic axons. TEM confirmed that AVP+ axons formed Gray type I (asymmetric) synapses with TH+ dendrites thus confirming excitatory synaptic connections between these systems. Retrograde tracing revealed that these LC AVP+ fibers originate from hypothalamic vasopressinergic magnocellular neurosecretory neurons (AVPMNNs). MS induced a significant increase in the density of LC AVP+ fibers. Finally, AVPMNN circuit upregulation by water-deprivation improved MWM performance while increased Fos expression was found in LC and efferent regions such as hippocampus and prefrontal cortex, suggesting that AVPMMN projections to LC could integrate homeostatic responses modifying neuroplasticity.

Results from a variety of sources indicate a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in light/glutamate-induced phase resetting of the circadian clock mediated by the retinohypothalamic tract (RHT). Attempts to block or remove PACAP's contribution to clock-resetting have generated phenotypes that differ in their responses to light or glutamate. For example, previous studies of circadian behaviors found that period-maintenance and early-night phase delays are intact in PACAP-null mice, yet there is a consistent deficit in behavioral phase-resetting to light stimulation in the late night. Here we report rodent stimulus-response characteristics of PACAP release from the RHT, and map these to responses of the suprachiasmatic nucleus (SCN) in intact and PACAP-deficient mouse hypothalamus with regard to phase-resetting. SCN of PACAP-null mice exhibit normal circadian rhythms in neuronal activity, but are "blind" to glutamate stimulating phase-advance responses in late night, although not in early night, consistent with previously reported selective lack of late-night light behavioral responsiveness of these mice. Induction of CREB phosphorylation, a hallmark of the light/glutamate response of the SCN, also is absent in SCN-containing ex vivo slices from PACAP-deficient mouse hypothalamus. PACAP replacement to the SCN of PACAP-null mice restored wild-type phase-shifting of firing-rate patterns in response to glutamate applied to the SCN in late night. Likewise, ex vivo SCN of wild-type mice post-orbital enucleation are unresponsive to glutamate unless PACAP also is restored. Furthermore, we demonstrate that the period of efficacy of PACAP at SCN nerve terminals corresponds to waxing of PACAP mRNA expression in ipRGCs during the night, and waning during the day. These results validate the use of PACAP-deficient mice in defining the role and specificity of PACAP as a co-transmitter with glutamate in ipRGC-RHT projections to SCN in phase advancing the SCN circadian rhythm in late night.

EDITORIAL article Front. Endocrinol., 04 December 2019Sec. Neuroendocrine Science Volume 10 - 2019 | https://doi.org/10.3389/fendo.2019.00793

Chromaffin cells (CCs) of the adrenal gland and the sympathetic nervous system produce the catecholamines (epinephrine and norepinephrine; EPI and NE) needed to coordinate the bodily "fight-or-flight" response to fear, stress, exercise, or conflict. EPI and NE release from CCs is regulated both neurogenically by splanchnic nerve fibers and nonneurogenically by hormones (histamine, corticosteroids, angiotensin, and others) and paracrine messengers [EPI, NE, adenosine triphosphate, opioids, γ-aminobutyric acid (GABA), etc.]. The "stimulus-secretion" coupling of CCs is a Ca2+ -dependent process regulated by Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ pumps, and exchangers and intracellular organelles (RE and mitochondria) and diffusible buffers that provide both Ca2+ -homeostasis and Ca2+ -signaling that ultimately trigger exocytosis. CCs also express Na+ and K+ channels and ionotropic (nAChR and GABAA ) and metabotropic receptors (mACh, PACAP, β-AR, 5-HT, histamine, angiotensin, and others) that make CCs excitable and responsive to autocrine and paracrine stimuli. To maintain high rates of E/NE secretion during stressful conditions, CCs possess a large number of secretory chromaffin granules (CGs) and members of the soluble NSF-attachment receptor complex protein family that allow docking, fusion, and exocytosis of CGs at the cell membrane, and their recycling. This article attempts to provide an updated account of well-established features of the molecular processes regulating CC function, and a survey of the as-yet-unsolved but important questions relating to CC function and dysfunction that have been the subject of intense research over the past 15 years. Examples of CCs as a model system to understand the molecular mechanisms associated with neurodegenerative diseases are also provided. Published 2019. Compr Physiol 9:1443-1502, 2019.

The beta 1 and beta 2 adrenoceptors (β1 and β2 AR) are GPCRs for the catecholamines norepinephrine and epinephrine. Both of these receptors couple to Gsα. Their activation causes cAMP elevation, which in turn controls cellular signaling through its downstream effectors PKA and the cAMP guanine nucleotide exchange factors (GEFs) Epac1 and Epac2. The β2AR has also been shown to signal via engagement of β‐arrestin, which acts as a scaffold for a variety of signaling proteins, including the MAP kinase ERK. We have recently characterized a neuronal and endocrine‐specific cAMP sensor, a GEF related to Epac 1 and 2, and to the previously characterized non‐cAMP‐activated GEF PDZ‐GEF1, which we have named NCS (neuritogenic cyclic AMP sensor)‐Rapgef2 (a protein product of the Rapgef2 gene). This sensor mediates activation of ERK leading to neuritogenesis in the PC12 and NS‐1 neuroendocrine cell lines (Emery et al., Sci. Signal. 6, ra51, 2013; Emery et al., J. Biol. Chem. 289: 10125, 2014). We have created NS‐1 cell lines stably expressing either β1AR or β2AR, and examined signaling to each of the three cAMP sensors present in these cells following treatment with isoproterenol. In β1AR‐expressing cells, agonist treatment caused activation of all measurable cAMP‐dependent signaling pathways in these cells: Epac2/p38‐dependent growth arrest; PKA‐dependent CREB phosphorylation; and NCS‐Rapgef2/ERK‐dependent neuritogenesis. In contrast, agonist stimulation of β2AR‐expressing cells caused isoproterenol‐initiated Epac2/p38‐dependent growth arrest and PKA‐dependent CREB phosphorylation, but did not couple to NCS‐Rapgef2/ERK‐dependent neuritogenesis. To compare the desensitization profiles of the two receptors, a biosensor that allowed for continuous real‐time cyclic AMP measurements was co‐expressed in each cell line (Emery et al., Peptides, 79: 39, 2016). In β2AR‐expressing cells, the maximal effect of isoproterenol on cAMP was observed after 10 minutes of treatment and decreased rapidly thereafter. In contrast, isoproterenol‐dependent β1AR activation caused persistent cAMP elevation, observed at approximately maximal levels at least 40 minutes following agonist addition. Unlike the mode of ERK phosphorylation observed following β1AR activation (NCS‐Rapgef2‐dependent), ERK activation elicited by β2AR (NCS‐Rapgef2‐independent) most likely occurs in a cellular compartment restricted from transcriptional regulation, which is required for neuritogenesis in this cell type (Ravni et al., Mol. Pharmacol. 73: 1688, 2008). We conclude that there is an inverse relationship between adrenergic receptor desensitization, and engagement of NCS‐Rapgef2 of sufficient duration to support the sustained activation of ERK necessary to promote neuritogenesis in NS‐1 cells. We notice the same inverse relationship in receptors for dopamine (D1) and adenosine (A2A), as well as the neuropeptides PACAP, VIP, and GLP‐1 (PAC1, VPAC1, VPAC2, and GLP‐1R).Support or Funding InformationThis work was supported by NIMH Intramural Research Program Project ZIAMH002386 and by a 2014 NARSAD Young Investigator Grant to A.C.E. from the Brain and Behavior Research Foundation (Grant 21356).

We have identified in a neuroendocrine cell line, NS‐1, three separate pathways for cAMP signaling mediated through Epac2 via p38 phosphorylation (subserving growth arrest); through PKA via CREB phosphorylation (subserving neuronal survival and neuron‐specific gene expression); and through the novel cAMP sensor NCS‐Rapgef2 (Emery et al., Sci. Signal. 6,281:ra51, 2013) via ERK phosphorylation (subserving neuritogenesis) (Emery et al., J. Biol. Chem. 289:10126–39, 2014). These pathways are parcellated, exhibiting little if any signaling cross‐talk, and are activated by a variety of CNS Gs‐coupled receptors, including the PAC1 PACAP receptor (Emery & Eiden, FASEB J, 26, 3199–3211, 2011) and the D1 dopamine receptor (Eiden et al., FASEB J, 30, supplement 1, 1265.5, 2016). Thus, points within each pathway may eventually represent targets for intracellular therapeutics aimed at modulation of individual components of neuronal behavior, in the context of CNS disorders relating to addiction and anxiety. Accordingly we have embarked on a drug discovery program, using assays developed in cellula for each pathway, for specific inhibitors of NCS‐Rapgef2.The adenylyl cyclase inhibitor SQ22,536 (9‐tetrahydrofuranyladenine) (IC50 = 10 mM to inhibit forskolin‐induced Elk1 activation) has in addition inhibitory activity against NCS‐Rapgef2 (IC50 = 170 mM to inhibit 8‐Br‐cAMP‐induced Elk1 activation): this work was carried out using a battery of high‐content in cellula assays that distinguish signaling through the three intracellular cAMP sensors NCS‐Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA) (Emery et al., Mol Pharmacol 83, 95–105, 2013). We have now examined the activities of compounds with modified substituents at several positions of adenine (6‐amino‐purine) to enhance selectivity for NCS‐Rapgef2 by decreasing affinity for adenylyl cyclase (AC) without increasing affinity for PKA or Epac. The compound N6‐phenyl‐9‐tetrahydrofuranyladenine is a potent NCS‐Rapgef2 inhibitor (IC50 = 10 mM to inhibit 8‐Br‐cAMP‐dependent ERK phosphorylation) with greatly diminished AC inhibitory activity (IC50 >300 mM for cyclic AMP measurements following 40 min treatment with IBMX + forskolin), without noticeable inhibitory or stimulatory activity at either PKA or Epac. These measurements were carried out in the neuroendocrine pheochromocytoma (PC12)‐derived neuroendocrine cell line NS‐1, engineered to allow high‐content screening for activation and inhibition of AC, PKA, Epac and NCS‐Rapgef2. Exploration of further modification to create a sufficiently potent and selective NCS‐Rapgef2 inhibitor for use in vivo for blockade of D1 dopamine receptor‐dependent effects of psychomotor stimulants that require signaling through ERK are in progress.Support or Funding InformationThis work was supported by the National Institute of Mental Health Intramural Research Program, Projects MH002386 to L.E.E. and MH002592 to M.V.E.

We have recently reported the existence of a vasopressinergic input from the hypothalamic paraventricular nucleus (PVN) to the medial division of the lateral habenula (LHbM), reporting to a subpopulation of putative GABAergic interneurons there. Stimulation of this pathway influences escape behaviors in the rat. Here, we identify additional hunger‐related orexinergic and reward‐related midbrain aminergic (dopaminergic and serotoninergic) inputs converging on this habenular cell group, and uniquely expressing the estrogen receptor ERalpha. The origins of each of the projection types were determined by fluorogold retrograde tracing. Orexinergic, dopaminergic and serotonergic projections originate in lateral hypothalamus (LH), ventral tegmental area (VTA) and substantia nigra (SN), and dorsal raphe (DR), respectively. They share, with the vasopressinergic inputs from the PVN, sex‐steroid responsivity, i.e. they express androgen receptors (ARs) and aromatase, the latter visualizable in axon terminals within the habenula. The distinct LHbM cell population onto which these inputs converge are likely to be functionally GABAergic, as determined by in situ hybridization histochemistry identifying the expression of gad1, gad2, Slc6A1, Slc6A11, Slc32A1 within them. This cell group also expresses mRNA encoding receptors for serotonin, dopamine, vasopressin, and orexin. Of physiological importance, AR and aromatase expression in the input neurons from PVN, LH, VTA/SN and DR are dependent on both estrogen levels, in female rats, and history of sexual activity, in male rats. Castration‐induced behavioral changes in passive vs active coping strategies before a predator were also observed. We postulate that all four pathways studied here contribute to the hormonal/homeostatic control of behavioral motivation in a coordinated or convergent fashion, through the use of estrogen as a co‐transmitter impingent on LHbM neurons. These findings are relevant within a broader physiological context, reflecting the influence of hunger and thirst circuitry on contingency‐dependent motivated behavior. Support or Funding Information Partially supported by: DGAPA‐UNAM‐PAPIIT‐IN216214, CONACYT‐CB‐176919 & CB‐238744 to LZ and NIMH‐IRP‐1ZIAMH002386 to LEE. LZ is a Fulbright visiting scholar, also supported by PASPA‐DGAPA‐UNAM fellowships for her sabbatical research stay hosted by LEE of SMN‐NIMH‐NIH‐USA.

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF–small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor–Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element–binding protein–dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein—coupled receptor → Gs → adenylate cyclase → cAMP → PKA → cAMP response element–binding protein pathway mediating cell survival and the G protein—coupled receptor → Gs → adenylate cyclase → cAMP → neuritogenic cAMP sensor–Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.

Gs‐coupled GPCR signaling in the NS‐1 pheochromocytoma cell line leads to the activation of three distinct cyclic AMP sensors, Epac, PKA and NCS/Rapgef2 mediate distinct cellular outputs: p38‐dependent growth arrest; CREB‐dependent cell survival and neuron‐specific gene expression; and ERK‐dependent neuritogenesis, respectively (Emery et al., Sci. Sig. 6 : ra51, 2013; Emery et al., JBC 289 : 10126, 2014). Expression of D1 dopamine receptors from a stably‐integrated, CMV promoter‐driven expression cassette in NS‐1 cells causes cyclic AMP elevation and activation of all three of these pathways, leading to D1 receptor‐dependent activation of all three downstream pathways, as previously reported for activation of the endogenous PAC1hop receptor by the neuropeptide PACAP. To investigate which of the three pathways might mediate cAMP‐dependent immediate‐early gene (IEG) transcription relevant to neurotransmitter action in vivo, we examined genome‐wide transcriptional regulation by cyclic AMP in NS‐1 cells, using microarray analysis. Treatment of NS‐1 cells for one hour with the cell‐permeant cyclic AMP analog 8‐CPT‐cAMP (100 micromolar), an equipotent activator of Epac2, PKA and NCS/Rapgef2 (Emery et al., JBC 289 : 10126, 2014) resulted in significant up‐regulation of 174 mRNAs. Of these, ten encoded transcription factors, and six of these encoded IEGs: Ier‐3/Egr3, Zif268/Egr1, Nr4a1, Nr4a3, c‐Fos, and JunB. Cyclic AMP‐dependent up‐regulation of the Ier‐3/Egr2 and Zig268/Egr1 genes, but not the Nr1a1, Nr4a3, or JunB genes, was ERK‐dependent (blocked by the MEK inhibitor U0126, at 10 micromolar), while up‐regulation of the c‐Fos gene appeared to be partially ERK‐dependent. These results suggest that regulation of IEG expression by D1 and PAC1 receptor stimulation may involve two cAMP‐dependent pathways, one mediated by NCS/Rapgef2, and the other by either Epac or PKA. These novel signaling pathways may be relevant to IEG regulation by dopamine in the striatum in vivo (Gerfen et al., J. Neurosci. 15: 8167, 1995) and by the stress‐regulatory neuropeptide PACAP in hypothalamus and extended amygdala (Stroth and Eiden, Neuroscience 165: 1025, 2010; Emery and Eiden, FASEB J. 26: 3199, 2012). Support or Funding Information Supported by NIMH‐IRP project ZO1‐MH002386

We measured serum CORT elevation in wild-type and PACAP-deficient C57BL/6N male mice after acute (1h) or prolonged (2-3h) daily restraint stress for 7 d. The PACAP dependence of CORT elevation was compared to that of stress-induced hypophagia. Daily restraint induced unhabituated peak CORT elevation, and hypophagia/weight loss, of similar magnitude for 1, 2, and 3h of daily restraint, in wild-type mice. Peak CORT elevation, and hypophagia, were both attenuated in PACAP-deficient mice for 2 and 3h daily restraint. Hypophagia induced by 1-h daily restraint was also greatly reduced in PACAP-deficient mice, however CORT elevation, both peak and during recovery from stress, was unaffected. Thus, hypothalamic PACAPergic neurotransmission appears to affect CRH gene transcription and peptide production, but not CRH release, in response to psychogenic stress. A single exposure to restraint sufficed to trigger hypophagia over the following 24h. PACAP deficiency attenuated HPA axis response (CORT elevation) to prolonged (3h) but not acute (1h) single-exposure restraint stress, while hypophagia induced by either a single 1h or a single 3h restraint were both abolished in PACAP-deficient mice. These results suggest that PACAP's actions to promote suppression of food intake following an episode of psychogenic stress is unrelated to the release of CRH into the portal circulation to activate the pituitary-adrenal axis. Furthermore, demonstration of suppressed food intake after a single 1-h restraint stress provides a convenient assay for investigating the location of the synapses and circuits mediating the effects of PACAP on the behavioral sequelae of psychogenic stress.

PACAP-27 and PACAP-38 are the exclusive physiological ligands for the mammalian PAC1 receptor. The role of C-terminal amidation of these ligands at that receptor was examined in neuroendocrine cells expressing the PAC1 receptor endogenously and in non-neuroendocrine cells in which the human and rat PAC1 receptors were expressed from stable single-copy genes driven by the CMV promoter, providing stoichiometrically appropriate levels of this Gs-coupled GPCR in order to examine the potency and intrinsic activity of PACAP ligands and their des-amidated congeners. We found that replacement of the C-terminal glycine residues of PACAP-27 and -38 with a free acid; or extension of either peptide with the two to three amino acids normally found at these positions in PACAP processing intermediates in vivo following endoproteolytic cleavage and after exoproteolytic trimming and glycine-directed amidated, were equivalent in potency to the fully processed peptides in a variety of cell-based assays. These included real-time monitoring of cyclic AMP generation in both NS-1 neuroendocrine cells and non-neuroendocrine HEK293 cells; PKA-dependent gene activation in HEK293 cells; and neuritogenesis and cell growth arrest in NS-1 cells. The specific implications for the role of amidation in arming of secretin-related neuropeptides for biological function, and the general implications for neuropeptide-based delivery in the context of gene therapy, are discussed.