Chronic pain alters sensory responses and carries a strong emotional component. Persistent pain can heighten pain experiences, resulting in hyperalgesia and allodynia. Further, patients suffering from chronic pain are more prone to experience a range of affective disorders including depression, sleep dysregulation, panic disorders, anxiety abnormalities and stress-related disorders including post-traumatic stress disorder (PTSD). Hence while pain serves a protective function to prevent additional physiological harm by driving behavioral and cognitive responses, chronic or persistent pain can lead to maladaptive nociceptive responses and exacerbate psychopathologies. Among brain regions, the amygdala is centrally situated to integrate the many descending and ascending signals to modulate the sensory and emotional components of pain. The amygdala is well studied for its role in fear and stress-related behavioral processes. The central nucleus of the amygdala (CeA), and in particular the lateral capsular subdivision of the CeA (CeLC), receives prominent ascending pain neurotransmission via the spino- parabrachioamygdaloid tract. In this pathway, peripheral nociceptive signals carried via primary sensory Aδ- and C-fibers terminate in the dorsal horn where second order neurons send projections via the spino-parabrachial pathway to the lateral parabrachial nucleus (LPBn). Thus, the LPBn collects cutaneous (mechanical and thermal), deep (muscular and articular) and visceral nociceptive signals and relays the information in a highly organized manner principally to the CeLC for nociceptive processing. In pain, the CeA and the LPBn-CeLC projections have been shown to undergo plasticity in the forms of enhanced synaptic transmission and alterations in neurotransmitter and receptor expression. Accordingly, the neurocircuit intersections in the CeA can modulate the sensory and emotional responses to pain. Yet despite these associations, the mediators and mechanisms underlying the emotional consequences of pain are poorly understood. Pituitary adenylate cyclase activating polypeptide (PACAP) is a neural and endocrine pleiotropic peptide important in the development and homeostatic regulation of many physiological systems. Recently, the expression of PACAP and its cognate PAC1 receptor has been shown to be upregulated in specific limbic regions by chronic stress. PACAP infusions into several limbic regions is anxiogenic, and altered blood PACAP levels and PAC1 receptor polymorphism have been associated with PTSD and other stress-related disorders. Here, we establish that CeLC PACAP originates from the LPBn as part of the spino-parabrachoamygdaloid pathway. Chronic pain enhanced PACAP expression along LPBn-CeLC projections, indicating it may be a component of pain- related plasticity. CeA PACAP signaling was sufficient to induce nociceptive hypersensitivity and anxiety-like behaviors. In a chronic neuropathic pain model, CeA PACAP signaling was found to contribute to heightened anxiety-like behaviors and nociceptive responses. Further, we characterized one prominent intracellular signaling mechanism through which CeA PACAP signaling influences these behaviors. In these experiments we provide evidence that CeA PACAP signaling plays an important role in the emotional components of pain and that alterations in CeA PACAP signaling are part of pain-related plasticity. This work establishes novel molecular mechanisms that underlie the emotional component of pain and may contribute to the development of chronic pain and associated affective disorders.

Cystic Fibrosis (CF) is caused by mutations in CFTR, a protein for chloride efflux in epithelial cells. VIP is a peptide that activates CFTR and improves membrane stability; VIP has 3 receptors VPAC1, VPAC2 and PAC1 that can cause CFTR phosphorylation. VIP-knockout (VIPKO) mice experience inflammation and reduced CFTR membrane localization comparable to CF phenotypes, that’s reversible after 3 weeks of VIP treatment (VIPKOT). In this thesis western blotting showed VPAC1 and VPAC2 expression increased in VIPKO and VIPKOT lung and duodenum tissues. The expression and maturation of CFTR was unchanged in both VIPKO and VIPKOT tissues. The results showed absence of VIP caused increased receptor expression in VIPKO mice, after VIP treatment VIPKO mice maintained increased receptor expression. VIP treatment reduces inflammation and restores existing CFTR membrane localization in VIPKO mice. VIP receptor expression may be important for future treatment of CF for CFTR localization and reducing tissue inflammation.

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Autonomic control of cardiac function depends on the coordinated activity generated by neurons within the intracardiac ganglia, and intrinsic feedback loops within the ganglia provide precise control of cardiac function. Both pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are important regulators of cell-to-cell signaling within the intracardiac ganglia, and PACAP and VIP action on these ganglia, mediated through associated receptors, play an important role in the regulation of coronary blood flow, cardiac contraction, relaxation, and heart rate. Results reported here using PACAP and VIP provide direct evidence of some of the complex signaling which occurs in neurons of the rat intracardiac ganglia.

Dynein is a minus end directed molecular motor required for numerous cellular processes during intracellular transport and mitosis. Pac1/LIS1 and Bik1/CLIP-170 are two proteins required for targeting dynein to cytoplasmic microtubule plus ends in budding yeast. The lab previously proposed a model whereby Pac1/LIS1 binds to the motor domain of dynein heavy chain, Dyn1/HC, forming a complex that interacts with the +TIP protein Bik1/CLIP170 at plus ends. This project focused on using Bimolecular Fluorescence Complementation (BiFC) to visualize protein-protein interactions among dynein pathway components in vivo. Budding yeast, Saccharomyces cerevisiae is an ideal system to manipulate dynein as it is a non-essential protein in this system. The BiFC assay fuses two non-fluorescent halves of Venus, a YFP-derivative, to proteins of interest. If an interaction between the proteins occur, the two halves are brought to close proximity and the fluorophore is reconstituted. Cells co-expressing Dyn1-VN with Pac1-VC or Bik1-VC exhibited fluorescent foci associated with microtubule plus ends, the cell cortex and spindle pole bodies (SPBs). Additionally, cells co-expressing Pac1-VC with Bik1-VN exhibited fluorescent foci associated with microtubule plus ends. Cells coexpressing Tub1-VC and Bik1-VN or Dyn1-VN have BiFC signal indicating that both interact with the microtubule directly. Pac-1 coexpressed with Tub1 had no signal above background. These data support that these three components associate at microtubule plus ends. Dyn1 and Pac1 interact with Bik1 at microtubule plus ends. Bik1 serves as a docking platform for the two, but dynein is still able to interact with microtubules, while Pac1 is not.