Stress is one of the leading predisposing factors for the onset of anxiety and depression. The mechanisms underlying stress vulnerability remain not fully understood and this gap significantly delays the advancement of the biomedical field. Pituitary adenylate cyclase-activating polypeptide (PACAP), a 38-amino acid peptide, has been proposed to regulate the stress response by acting at multiple levels. The central hypothesis of this work was that the PACAP system of the extended amygdala, a basal forebrain structure that includes the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST), plays a critical role in the physiological and pathological behavioral response to stress. I found that central (intracerebroventricular) administration of PACAP in rats is able to produce a depressive-like endophenotype, as measured by increased current threshold for intracranial self-stimulation (ICSS), reduced preference for a sweet solution, and reduced time spent interacting with a novel animal in a social interaction test. I then went on investigating the brain structures and mechanisms contributing to PACAP-induced behavioral effects. I found that microinfusion of PACAP, but not VIP, into the CeA and BNST caused a dose-dependent increase in acoustic startle response (ASR), a rapid defensive reflex that is an index of stress. In addition, PACAP(6-38) infusions into either of these structures was instead able to prevent the sensitization of ASR induced by footshock stress, in line with the observation that the acute exposure to footshock stress induced a significant increase in PACAP, but not VIP, levels in both the CeA and the BNST. Finally, I found that the continuous recruitment of the PACAP system of the CeA was essential to the emergence of the negative outcomes of chronic stress. Indeed, chronic social defeat stress significantly increased PACAP levels in the CeA, but not the BNST; furthermore, viral vector-mediated knockdown of the PACAP receptor PAC1R in the CeA significantly attenuated decreased body weight gain, decreased saccharin consumption, and heightened anxiety-like behavior induced by chronic social defeat and also prevented the increase in CeA corticotropin-releasing factor (CRF) levels. The results obtained provide novel insights into the neurobiological mechanisms underlying the psychopathological consequences of stress. / 2020-06-12T00:00:00Z

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.

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