Fear and anxiety disorders are potentially crippling conditions that often stem from past experience of trauma and chronic stress. One clear feature of these disorders is the failure to use proximate spatial and contextual information presented in the environment to regulate reflexive physiological threat responses. The central nervous system networks that govern spatial navigation and contextual learning and memory are a series of complex circuitries in which the hippocampus is integrally involved. Deficits in hippocampal function have been linked to severe anterograde and mild retrograde amnesia of semantic and episodic memory, and specific deficits in contextual processing. These deficits manifest as failure to distinguish between the details of contexts that help predict for danger or safety and can thus lead to the overexpression of threat responses that compose the behavioral symptoms of fear disorders. The dentate gyrus (DG) is a subdivision of the hippocampus that serves as the first filter of excitatory flow through the hippocampus. The DG is hypothesized to function in “pattern separation” or the dissociation of similar inputs into dissimilar outputs. Failure in this domain leads to generalization between contexts, a common feature of pathology. Pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor are associated with multiple behavioral disorders such as post-traumatic stress disorder, schizophrenia, and bipolar disorder. Mutations in the PAC1 receptor gene are associated with hypervigilance, and modified amygdalar and hippocampal activity. These results are mirrored by rodent studies where central PACAP infusion causes anxiety-like behavior, pain hypersensitivity, anorexia, and reinstatement of drug-seeking. PAC1 receptor transcript is found in high abundance in granule cells of the dentate gyrus and potentiation of DG synapses is impaired in PAC1 knockout mice. PACAP is known to have effects of long-duration, such as those in injury repair, growth, and development, but it also can affect ion channel physiology to control neuronal excitability through several parallel intracellular signaling cascades including those dependent on adenylyl cyclase, phospholipase C, and extracellular signal regulated kinase. Accumulated evidence suggests that recruitment of extracellular signal regulated kinase can be through either adenylyl cyclase-, phospholipase C-, or a receptor endocytosis-dependent mechanism. The experiments described in this dissertation address the role of PACAP in the DG in regulating expression of fear behavior, the effects of PACAP on the excitability of DG granule cells, and the signaling pathways and ion channels responsible for these effects. We found that PACAP infused into the DG amplifies expression of fear to a context but does not affect fear acquisition. Electrophysiology studies demonstrate that treating DG neurons with PACAP increases their excitability, and that parallel signaling mechanisms recruit extracellular signal regulated kinase to drive this excitability. Furthermore, these effects on excitability are attenuated by blocking a persistent inward sodium current. This work represents novel regulation of the DG and its impacts on behavior and identifies a current that likely participates in modulating granule cell excitability in multiple domains. In aggregate, this research traces the path from ligand, to receptor and intracellular signaling, to neurophysiology in order to propose a comprehensive description of behavioral regulation by these processes.

The incidence of neurodegenerative disorders like Parkinson’s disease (PD) and Alzheimer’s disease (AD) are increasing as the population ages. Slowing the rate of neurological decline can have a huge impact on health care costs and quality of life for both the patients and those caring for them. Pituitary adenylate cyclase activating peptide (PACAP) is a Secretin family peptide that activates the PAC1, VPAC1 and VPAC2 receptors and is associated with neuroprotection and neuronal differentiation. PACAP administration protects neurons against toxic, hypoxic, traumatic or inflammatory insults. The receptors of the Secretin family are unique due to the large extracellular domain (ECD) necessary to bind the endogenous ligand prior to receptor activation. The Secretin family ligands are all peptides, this family of receptors being responsible for regulating and maintaining homeostasis within the organism. PACAP is a pleiotropic peptide acting both centrally and peripherally. Exogenously administered peptide is rapidly metabolized. For neuroprotective effects, PACAP must cross the blood brain barrier (BBB). Enhancing the transport across the BBB has been accomplished through peptide glycosylation. Here we design and synthesize a series of glycosylated PACAP agonists and antagonists to evaluate them for receptor activity and ability to cross the BBB. A homology model was constructed of the full length PAC1R based on the transmembrane portion of both the mu opioid receptor and the corticotropin releasing factor-1 receptor combined with the NMR derived solution structure of the PAC1R ECD bound with the receptor antagonist, PACAP6-38. Using this model to guide us, the decision was made to place the glycosylated residue at the C-terminus of the peptide. A series of PACAP based glycopeptide agonists and antagonists were prepared using solid phase peptide synthesis (SPPS). Synthesis of PACAP analogs is complicated by the inclusion of two sites of aspartimide formation, the D3-G4 and D8-S9 sequences. Initial SPPS trials resulted in very little desired peptide formation. Reagent adjustments and using an amino-group protection strategy improved peptide yield. Methionine sulfoxide formation occurs in PACAP analogs. Substitution of methionine with leucine avoids this oxidation issue. An initial screen of PACAP and two glycosylated analogs using PC12 cells for PAC1R activation indicated that all three promoted neurite-like process outgrowths indicating PAC1R activation. The diluent treated cells did not exhibit this morphological change. Quantification of cells for assessing antiproliferative effects was not performed. More PC12 experiments should be performed to assess antiproliferative action and to screen additional glycosylated PACAP analogs for PAC1R activation. One of the glycosylated PACAP analogs was detected in CSF after i.p. administration in a mouse. Microdialysis samples obtained in vivo were analyzed by a newly developed LC/MS² technique and found to contain the administered glycosylated PACAP still intact, demonstrating that the glycopeptide crosses the BBB. Additional experiments using other glycosylated PACAP analogs are planned.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Alcohol use disorder (AUD) is a chronic, relapsing condition with a complex etiology and heritable susceptibility factors interact with environmental factors to produce and maintain the disease. One goal of current neuroscience research is to identify the neuroadaptations mediating the propensity to consume high amounts of alcohol, of either innate or environmental origin. Dysfunctional neuronal communication between prefrontal cortical regions and the nucleus accumbens (NAcc) have been implicated in excessive alcohol drinking and proposed to play a critical role in AUD. However, the exact mechanism by which altered prefrontostriatal transmission may perpetuate excessive drinking is poorly understood. In addition, the exact role of dopamine receptor 1 (D1R) or dopamine receptor 2 (D2R)-expressing medium spiny neurons in the NAcc is unclear and adds another layer of complexity to this framework. This thesis concerns pituitary adenylate cyclase-activating polypeptide (PACAP), a highly conserved 38 amino acid neuropeptide, and its receptor PAC1R. Studies in rodents and humans have implicated PACAP and PAC1R in the actions of drugs of abuse, including more recently, alcohol. Notably, the PACAP/PAC1R system has also been shown to increase glutamatergic neurotransmission in several circuits. The overall hypothesis of this project was that the PACAP/PAC1 system in the prefrontal cortex-NAcc pathway regulates excessive drinking and the long-lasting neuroplastic changes observed in alcohol addiction, via the modulation of the glutamatergic system. Using alcohol-preferring rats, a hereditary model of AUD, we found that intracerebroventricular administration of a PAC1R antagonist blocked excessive alcohol drinking, motivation to drink, and alcohol seeking behavior selectively in this line and not in outbred rats. Alcohol-preferring rats displayed a higher number of PAC1R positive cells in the NAcc Core. Blockade of PAC1R in the NAcc Core, via pharmacology or gene knockdown, resulted in reduced alcohol drinking. Conversely, we found that knockdown of the PAC1R in the NAcc Shell led to increased alcohol drinking and motivation to drink in alcohol-preferring rats, suggesting that the PACAP/PAC1R system may play an opposite role in these two NAcc subregions. Using a mouse exposure model of excessive drinking, a glutamatergic projection from PACAP-expressing cells in the prelimbic portion of the prefrontal cortex (PrL) to the NAcc Core circuit was found to be recruited by alcohol exposure. Inhibition of these neurons, as well as PACAP neuron ablation or PACAP deletion, led to decreased alcohol intake that was specific to male mice. Systemic PAC1R antagonism, and specific knockdown of PAC1R in the NAcc Core, also decreased alcohol intake in male mice. Using slice electrophysiology and channelrhodopsin assisted circuit mapping, we found that this pathway is biased to D1R-expressing neurons in the NAcc Core following alcohol exposure in males, and that PACAP application increases post-synaptic measures of glutamatergic transmission in this circuit. Overall, these data describe a key role for the corticostriatal PACAP/PAC1R system in aberrant alcohol drinking in both hereditary- and exposure-based models of AUD and give novel insights into the underlying mechanisms of alcohol addiction. / 2025-01-23T00:00:00Z

Le neuroblastome (NB) est un cancer pédiatrique dérivé de la crête neurale. Les NB à haut risque sont des tumeurs peu différenciées présentant une amplification de MYCN et les plus agressives possèdent en plus une mutation d'ALK. Pour améliorer le traitement de ces tumeurs, les nouvelles thérapies cherchent à induire la différenciation cellulaire, l'inhibition de MYCN et la réduction de la signalisation d'ALK. Les résultats obtenus indiquent que le VIP induit une neuritogenèse dans les cellules de NB à haut risque SK-N-DZ et Kelly, et réduit en plus l'expression de MYCN dans les cellules Kelly, comme précédemment observé pour les cellules IMR-32. En parallèle, le VIP diminue l'invasion des cellules Kelly et IMR-32 et réduit également l'activité d'AKT qui est impliquée dans la signalisation d'ALK, dans les cellules Kelly qui présentent la mutation ALK F1174L. Certains effets du VIP sont dépendants de la PKA. Des analogues du PACAP, un peptide apparenté au VIP, présentent une efficacité supérieure à celle du VIP dans les cellules Kelly. Les effets du VIP sur la neuritogenèse et l'expression de MYCN dans ces cellules sont médiés par le récepteur VPAC2 qui peut avoir une localisation nucléaire dans les lignées cellulaires et les cellules de patients atteints de NB. Une délocalisation de ce récepteur nucléaire par ses propres ligands est observée. De plus, des cellules souches mésenchymateuses humaines dérivées du tissu adipeux induisent la différenciation des cellules de NB via les peptides VIP et/ou PACAP. L'ensemble de ces résultats indiquent que le VIP et des analogues du PACAP agissent sur des processus moléculaires et cellulaires qui pourraient réduire l'agressivité des NB à haut risque, et pourraient donc présenter un intérêt pour une nouvelle thérapie. / Neuroblastoma (NB) is a pediatric cancer derived from neural crest. High-risk NB are poorly differentiated tumors with MYCN amplification and the most aggressive forms in addition have an ALK mutation. To improve the treatment of these tumors, the new therapies aim to induce cell differentiation, inhibition of MYCN and reduction of ALK signaling. The obtained results indicate that the neuropeptide VIP induces neuritogenesis in high-risk SK-N-DZ and Kelly NB cells, and in addition reduces the expression of MYCN in Kelly cells, as previously observed in IMR-32 cells. In parallel, VIP decreases Kelly and IMR-32 cell invasion and also reduces the activity of AKT that is involved in the signaling of ALK, in Kelly cells harboring the mutation ALK F1174L. Most of these VIP effects are PKA-dependent. Analogs of PACAP, a VIP-related peptide, exhibit a higher efficiency than VIP in Kelly cells. VIP effects on neuritogenesis and MYCN expression in these cells are mediated by the VPAC2 receptor which can have a nuclear localization in the NB cell lines and in NB from patients. A relocation of this nuclear receptor by its own ligand is observed. Moreover, human mesenchymal stem cells derived from adipose tissue induce NB cells differentiation via VIP and/or PACAP peptides. Taken together, these results indicate that VIP and PACAP analogs act on molecular and cellular processes that might reduce aggressiveness of high-risk NB, and thus could be of interest for new therapy.

La forme la plus agressive des gliomes, tumeurs cérébrales primitives, est l'astrocytome de grade IV appelé aussi glioblastome (GBM). Le pronostic des patients atteints d'un GBM est très sombre du fait de la nature infiltrante de ces cancers et de la présence de cellules souches cancéreuses (CSC) peu sensibles aux thérapies actuelles. Il est donc primordial d'étudier ces deux caractéristiques des GBM. Mes travaux de thèse ont porté sur ces deux aspects.La connaissance de l'implication de la voie de signalisation Hedgehog (Hh) dans le maintien du pool de CSC permet d'envisager de nouvelles stratégies thérapeutiques visant à les éliminer. La voie Hh joue un rôle important dans le développement embryonnaire. L'activation de cette voie a été mise en évidence dans de nombreux cancers dont les GBM. La cyclopamine est une substance alcaloïde inhibitrice de la voie Hh. L'inconvénient de l'utilisation de la cyclopamine pour une thérapie anticancéreuse est que cette voie est aussi active dans les cellules souches somatiques adultes. Pour cibler la tumeur, une prodrogue glucuronylée de la cyclopamine a été synthétisée. La partie glucuronide peut être hydrolysée par la β-glucuronidase, une enzyme présente dans la zone nécrotique des GBM, conduisant à la libération de la cyclopamine active de façon ciblée, dans le microenvironnement tumoral. Des tests de viabilité indiquent que cette prodrogue ne présente pas latoxicité de la cyclopamine envers les cellules C6 de GBM de rat, leur population de cellule souches (C6-GSC) ainsi que dans des coupes de cerveau de rat adulte. En revanche, l'hydrolyse par la β-glucuronidase de cette prodrogue permet de restaurer la toxicité de la cyclopamine sur les cellules de GBM. L'analyse par RT-qPCR de l'expression d'un gène cible de la voie Hh, le facteur de transcription GLI1, permet de vérifier l'inhibition spécifique de la voie Hh par la cyclopamine dans les cellules C6 et les C6-GSC. Ainsi, la prodrogue en libérant la cyclopamine dans l'environnement tumoral devrait permettre de limiter l'étendue des effets de la drogue sur les cellules saines du cerveau.La voie Hh joue un rôle dans la régulation de la migration et de l'invasion des cellules de GBM. Mon équipe d'accueil a montré que le vasoactive intestinal peptide (VIP) régule la migration et l'invasion de deux lignées cellulaires M059K et M059J. Dans ce travail de thèse, les effets du VIP et de son analogue le pituitary adenylate cyclase-activating peptide(PACAP) sur la migration et l'invasion des cellules C6 de rat et U87 humaines ont été étudiés. Les résultats obtenus indiquent que les agonistes VIP, PACAP-38 et l'antagoniste synthétique VIP10-28 des récepteurs de ces peptides contrôlent la migration et l'invasion dans les deux lignées de GBM. Le VIP et le PACAP-38 inhibent l'invasion ex vivodes cellules C6 dans des coupes de cerveau. De plus, le VIP10-28 augmente la migration et l'invasion de ces cellules de façon PKA-, Akt- et Hh-dépendante. L'étude des voies detransduction impliquées dans l'action des neuropeptides sur la migration et l'invasion des GBM a ensuite été approfondie. Les données obtenues indiquent que le VIP et le PACAP diminuent le taux de la forme active nucléaire de la protéine GLI1 dans les cellules C6 et U87 de façon PKA-dépendante. Inversement, le VIP10-28 augmente l'expression de GLI1 dans les cellules C6 de façon Akt-dépendante. Enfin, nous avons constaté que le PACAP diminue la phosphorylation d'Akt dans les cellules C6 etaugmente celle de la protéine PTEN, en accord avec le rôle de ces kinases dans la régulation de la migration cellulaire et de l'activité GLI1. L'ensemble de ces résultats indique que le système VIP-récepteurs régule la migration et l'invasion par des interactions entre les voies PKA, Hh et Akt/PTEN dans les cellules de GBM. / The most aggressive form of glioma, primary brain tumors, is grade IV astrocytoma also called glioblastoma (GBM)...

Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in brain structures involved in emotion. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. In order to determine whether chronic stress would potentiate the behavioral and/or endocrine response to subthreshold BNST PACAP infusion, rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, stressed and control rats were bilaterally infused into the BNST with 0.5 µg PACAP. Startle response to intra-BNST PACAP infusion was assessed post-infusion in Experiment 1. In Experiments 2 and 3, blood was sampled via a tail nick 30 min following PACAP infusion to assess the corticosterone response to PACAP following CVS. We found an increase in startle amplitude and an increase in plasma corticosterone levels 30 minutes following BNST PACAP infusion only in rats that had been previously exposed to CVS. These results were likely mediated via PAC1 receptors, as equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels. These results suggest that repeated exposure to stressors sensitizes the neural circuits underlying the behavioral and endocrine responses to BNST PACAP infusion and BNST PACAP/PAC1 receptor signaling likely plays a critical role in mediating stress responses.

The tendency of users to relapse severely hinders adequate treatment of addiction. Physical and psychological stressors often contribute to difficulties in maintaining behavior change, and may play a significant role in relapse. We have previously shown that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediates many consequences of chronic stressor exposure. Hence, chronic stress substantially increased BNST PACAP levels, intra-BNST PACAP infusions produced the behavioral and endocrine consequences of stressor exposure, and BNST PACAP antagonism blocked many of the consequences of chronic stress. In the present set of studies, we investigated the role of BNST PACAP in stress-induced reinstatement of cocaine seeking. Rats self-administered cocaine (3mg/ml; 0.5mg/ig/infusion, i.v.) for 1hr daily over 10 days, which was followed by extinction training in which lever pressing no longer resulted in cocaine delivery. In the first experiment we showed that intra-BNST PACAP infusion (1 μg; 0.5 μl per side) reinstated previously extinguished cocaine seeking behavior. In the second experiment intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38 (1 μg; 0.5 μl per side) blocked stress-induced reinstatement. Hence, stressor exposure (5 sec 2mA footshock) caused significant reinstatement of cocaine seeking behavior, which was blocked by intra-BNST PACAP6-38 infusion. Overall, these data suggest that BNST PACAP systems mediate stress-induced reinstatement to drug seeking. Understanding the neuropharmacology of BNST PACAP in stress-induced reinstatement and the role of PACAP systems may lead to viable targets for relapse prevention.

Migraine is one of the most prevalent contributors to the global burden of mental and neurological disorders. It is a complex episodic condition that presents as intense recurrent unilateral headaches lasting hours to days that can be accompanied by nausea, photophobia, phonophobia and other neurological symptoms. The causes of migraine appear multifactorial and are not fully understood. However, activation of the trigeminovascular system and sphenopalatine parasympathetic neurons and the resulting vasodilation of meningeal arteries have been associated with the development of migraine pain. Recently, the neurotransmitter and neurotrophic peptide pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in this migraine headache pathway. The effects of PACAP parallel those of other migraine inducing agents and notably PACAP induces vasodilation of the MMA concurrent with the genesis of migraine headache when administered to human subjects. The mechanisms by which PACAP induces dilation are presently unclear. The objective of this present work was to elucidate the signaling pathways linking PACAP to MMA dilation. To achieve this objective, we developed an ex vivo approach to study isolated MMA at physiologically relevant intravascular pressure. Using this preparation we found that PACAP dilates MMA at picomolar concentrations via PAC1 receptors. Further, in MMA, PACAP-induced dilation is mediated exclusively though activation of KATP channels. While investigating the mechanisms of PACAP-induced dilation of MMA we discovered that basal KATP channel activity influences MMA diameter. Inhibition of KATP channels with glibenclamide or PNU37883 at physiological intravascular pressure resulted in a vasoconstriction of ≈ 20 %. Also consistent with basal KATP activity, glibenclamide induced a membrane potential depolarization of ≈ 14 mV. Further, in MMA loaded with the ratiometric Ca2+ indicator, Fura-2-AM, glibenclamide-induced MMA constriction was correlated with a simultaneous increase in the ratio of 340 nm/380 nm excited fura-2 fluorescence, consistent with an increase in intracellular Ca2+. Vascular smooth muscle KATP channels can be phosphorylated and activated by PKA, resulting in membrane potential hyperpolarization. KT5720, a PKA inhibitor, induced a constriction in MMA similar to that of glibenclamide (≈ 25 %). Additional treatment with glibenclamide did not induce further constriction suggesting that PKA activity may underlie tonic KATP channel activation. Together these results suggest that tonic PKA activity underlies basal KATP channel activity and together play a key role in regulation of MMA diameter. In summary, results presented in this dissertation suggest that picomolar PACAP-induced dilation of MMA is via activation of the PAC1-Hop1 receptor splice variant and KATP channel activation. Furthermore, KATP channels are also involved in tonic regulation of MMA diameter due to basal PKA activity. These unique features of the MMA provide additional insight into potential therapeutic targets in the development of treatments for migraine.