Functional peptidomics: Combining discovery-based mass spectrometry and neurophysiology to explore communication of phase-resetting cues in the rat suprachiasmatic nucleus.

摘要

Intercellular signaling is vital to communication within neuronal circuits. The suprachiasmatic nucleus (SCN), the master circadian clock of mammals, contains a dense collection of neurons that align their intrinsic rhythmicity with environmental stimulus and physiological state. While peptide physiology has been demonstrated as a contributor to SCN signal processing, the tools to explore and directly implicate peptides in SCN cell-to-cell signaling have not been utilized. By combining mass spectrometry and physiology, this thesis used novel peptidomics methodologies to discover new peptides and evaluate their functions in circadian clock phasing of the rat SCN.;Coupling various peptide sampling techniques to MS analytical approaches enables detection with high spatial resolution. Electrical optic nerve (ON) stimulation in a horizontal SCN slice model is effective in shifting SCN clock phase. Effective stimulation parameters are time-of-day-specific; effective late night parameters are ineffective in generating early night phase shifts and vice-versa. Moreover, these phase shifting stimuli elicit peptide release from the SCN. Suites of released peptides correspond with time-of-day- and stimulus-specific phase shifting. Peptide release is not evoked by ON stimulation parameters that are ineffective in phase resetting.;One previously unreported SCN peptide, little SAAS, was selected for further characterization. Sterological analysis confirms that little SAAS-positive cells comprise the third most abundant subpopulation of peptidergic neurons in the rat SCN. Little SAAS immunohistochemistry reveals localization to the retinorecipient ventrolateral SCN core. Up-regulation of immediate-early gene product c-Fos occurs in approximately 50% of little SAAS neurons following in vivo exposure to a light pulse at early night, implicating these neurons in light-responsiveness. Exogenous little SAAS is capable of shifting the phase of rhythmic firing rates of SCN brain slices in a dose- and time-of-day-dependent manner. Glutamate-induced phase shifting is blocked by exogenous treatment of the SCN slice with little SAAS antiserum, while little SAAS peptide-induced phase shifting is not blocked by NMDA receptor antagonist APV. Gastrin-releasing peptide (GRP) and vasoactive intestinal polypeptide (VIP) are involved in relay and processing of light signal information in the SCN. Little SAAS phase shifting persists in the presence of selective GRP and VIP receptor antagonists. These studies position little SAAS downstream of NMDA receptor activation and parallel to the known intra-SCN communication signals, GRP and VIP.;This thesis justifies functional peptidomics as a useful approach to understanding intercellular signaling mechanisms regulating SCN clock phase. Directly detecting dynamic release properties of peptides from the SCN brain slice provides new insights on a complex phenomenon phenomenon. The identification and characterization of little SAAS serves as an example of the facets of SCN physiology left to be discovered. Utilization of peptidomics strategies will significantly advance understanding of timing homeostatic mechanisms.