Amylin mediates brainstem control of heart rate in the diving reflex

Abstract

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.