The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VTP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

Neuropeptide signaling play critical roles in maintaining distinct behavioral states and orchestrating transitions between them. However, elucidating the mechanisms underlying neuropeptide modulation of neural circuits in vivo remains a major challenge. The nematode Caenorhabditis elegans serves as an excellent model organism to study neuropeptide signaling mechanisms encoded in relatively simple neural circuits. We have used the C. elegans egg-laying circuit as a model to understand how neuropeptide signaling modifies circuit activity to generate opposing behavioral outcomes. C. elegans egg-laying behavior is composed of alternating cycles of two states – short bursts of egg deposition (active phases) and prolonged periods of quiescence (inactive phases). We have identified two neuropeptides (NLP-7 and FLP-11) that are locally released from a group of neurosecretory cells (uv1) and coordinate the temporal organization of egglaying by prolonging the duration of inactive phases. These neuropeptides regulate activity within the core circuit by inhibiting serotonergic transmission between its individual components (HSN motorneurons and Vm2 vulval muscles). This inhibition is achieved at least in part, by reducing synaptic vesicle abundance in the HSN synaptic regions. To identify potential downstream signaling components that mediate the actions of these neuropeptides, we have performed a forward genetic screen and have identified a strong candidate. In addition, we are trying to identify the receptor(s) of these neuropeptides by using a candidate gene approach. Together, we demonstrate that local neuropeptide signaling maintains the periodicity of distinct behavioral states by regulating serotonergic transmission in the core neural circuit.

Neuropeptide und ihre Rezeptoren regulieren im menschlichen Körper essentielle Funktionen. Fehlfunktionen können zu schwerwiegenden Krankheiten führen, weswegen die Erforschung dieser Peptid-Rezeptor-Systeme von hohem Wert ist. Die Komplexität der Signalisierung erschwert die Forschung in Säugetiermodellen, weswegen auch Modelle von Invertebraten herangezogen werden können, wo viele homologe Neuropeptide zu finden sind. In dieser Arbeit wurde das ein Homolog zum Neuropeptid Y (NPY) im Rundwurm Caenorhabditis elegans (C. elegans) identifiziert und charakterisiert. Das NPY-System besteht aus 3 Peptiden und 4 Rezeptoren, welche in Säugetieren vor allem den Energiehaushalt regulieren, aber auch bei Stress, Depression und Angstzuständen eine Rolle spielen. In C. elegans wurden 41 dem NPY ähnliche Rezeptoren (NPR) und über 30 Neuropeptide identifiziert, welche auch Funktionen in der Nahrungssuche zeigen. In dieser Arbeit wurde gezeigt, dass das humane und C. elegans System pharmakologische und funktionale Gemeinsamkeiten aufweisen. Hier wurden der NPR-1 und NPR-11 als NPY-ähnlichste Rezeptoren identifiziert und erstmals ein NPY-ähnliches Peptid beschrieben, das FLP-34-1. Des Weiteren wurde eine bekannte Methode für Zellkulturexperimente in C. elegans etabliert, der Biolumineszenz-Energietransfer (BRET), welcher die Bindung von FLP-34-1 an NPR-11 in vivo zeigte. Zudem konnte mit dieser Methode eine Peptid-induzierte Internalisierung von NPR-11 Richtung Endosomen in vivo in Echtzeit gezeigt werden. Die Ergebnisse dieser Arbeit helfen die molekularen Mechanismen der Peptid-Rezeptor-Interaktionen besser zu verstehen und unterstützen damit auch die Forschung an höheren Tieren.

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Circadian rhythms in physiology and behavior are synchronized by a central pacemaker, the suprachiasmatic nuclei (SCN) of the hypothalamus. Shift work, jet lag and sleep disorders can disrupt circadian rhythms, negatively impacting health and well-being. The SCN pacemaker resets rapidly in response to changes in the daily light cycle, however, adjustment of peripheral oscillators to changing time zones or work shifts is more gradual, leading to internal desynchrony. In addition, many diseases can impair the SCN’s ability to adjust to changes in the light cycle. My research investigated whether combined pharmacological inhibition of neuropeptide Y and serotonin could enhance resetting and attenuate transient cycles in locomotor activity following a sudden change in light exposure. I found that simultaneously blocking neuropeptide Y and serotonin receptors potentiated phase shifts during the late subjective night and significantly reduced transient cycles of locomotor activity in hamsters. Development of treatments that enhance the circadian system’s response to light may alleviate some of the negative health consequences experienced by travelers, shift workers and individuals with disease-related circadian desynchrony.

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Amylin, or islet amyloid polypeptide is a 37-amino acid member of the calcitonin peptide family. Amylin role in the brainstem and its function in regulating heart rates is unknown. The diving reflex is a powerful autonomic reflex, however no neuropeptides have been described to modulate its function. In this thesis study, amylin expression in the brainstem involving pathways between the trigeminal ganglion and the nucleus ambiguus was visualized and characterized using immunohistochemistry. Its functional role in slowing heart rate and also its involvement in the diving reflex were elucidated using stereotaxic microinjection, whole-cel patch-clamp, and a rat diving model. Immunohistochemical and tract tracing studies in rats revealed amylin expression in trigeminal ganglion cells, which also contained vesicular glutamate transporter 2 positive. With respect to the brainstem, amylin containing fibers were discovered in spinal trigeminal tracts. These fibers curved dorsally toward choline acetyltransferase immunoreactive neurons of the nucleus ambiguus, suggesting that amylin may synapse to parasympathetic preganglionic neurons in the nucleus ambiguus. Microinjection of fluorogold to the nucleus ambiguus retrogradely labeled a population of trigeminal ganglion neurons; some of which also contained amylin. In urethane-anesthetized rats, stereotaxic microinjections of amylin to the nucleus ambiguus caused a dose-dependent bradycardia that was reversibly attenuated by microinjections of the selective amylin receptor antagonist, salmon calcitonin (8-32) (sCT (8-32)) or AC187, and abolished by bilateral vagotomy. In an anesthetized rat diving model, diving bradycardia was attenuated by glutamate receptor antagonists CNQX and AP5, and was further suppressed by AC187. Whole-cel patch-clamp recordings from cardiac preganglionic vagal neurons revealed that amylin depolarizes neurons while decreasing conductance. Amylin also resulted in a reduction in whole cell currents, consistent with the decrease in conductance. Amylin is also found to increase excitability of neurons. In the presence of TTX, spontaneous currents in cardiac preganglionic vagal neurons were observed to decrease in frequency in response to amylin while amplitude remained constant, signifying that amylin reduces presynaptic activity at cardiac preganglionic vagal neurons. Finally, evoked synaptic currents revealed that amylin decreases evoked currents, further demonstrating that amylin depolarization and increase in excitability of cardiac preganglionic vagal neurons is also associated with simultaneous inhibition of presynaptic transmission. Our study has demonstrated for the first time that the bradycardia elicited by the diving reflex is mediated by amylin from trigeminal ganglion cells projecting to cardiac preganglionic neurons in the nucleus ambiguus. Additionally, amylin results in the depolarization and increased excitability of cardiac preganglionic vagal neurons while inhibiting presynaptic transmission. / Pharmacology