Graduate School of Biomedical Sciences, Program in Neuroscience
Synaptic Transmission; Presynaptic Terminals; Neurosecretory Systems; Neuropeptides; Calcium Channels; Academic Dissertations
Life Sciences | Medicine and Health Sciences
A clear definition of the mechanisms involved in synaptic transmission is of paramount importance for the understanding of the processes governing synaptic efficacy. Despite decades of intense study, these mechanisms remain poorly understood. The work contained in this thesis examines several such mechanisms using the hypothalamic-neurohypophysial system (HNS), a classical preparation for the study of Ca2+-dependent neuropeptide release.
The first portion of this thesis is comprised of my efforts to define the cellular machinery essential for the exocytosis of secretory granules isolated from peptidergic neurohypophysial terminals of the HNS. Here, using the planar lipid bilayer model system, I have been able to show that syntaxin alone in the target membrane is sufficient to elicit fusion of modified neurohypophysial secretory granules. Surprisingly, SNAP-25 does not appear to be necessary for this process. This suggests that syntaxin may be able to substitute for SNAP-25 to form functional non-cognate fusion complexes. Additionally, the coupling of amperometric detection with the planar lipid bilayer system has allowed me to confirm these results using native, unmodified secretory granules, and also provides some insight into the kinetics of release in this reconstituted system. This model system should provide a convenient means for the study of additional regulatory factors believed to be involved in secretory vesicle exocytosis.
The second and third sections of this thesis involve my examination of the role of presynaptic Ca2+ stores in neuropeptide secretion from isolated peptidergic neurohypophysial terminals (NHT). I initially examined the source of recently discovered ryanodine-sensitive Ca2+ stores in this system. Using Immuno-electron microscopy I have found that ryanodine receptor (RyR) labeling appears to co-localize with large dense core granules. Additionally, I have shown that a large conductance cation channel, with similarities to the RyR, found in the membrane of these granules has the same characteristic response to pharmacological agents specific for the RyR. Further, application of RyR agonists modulates basal neuropeptide release from NHT. These results suggest that the large dense core granules of NHT serve as the source of a functional ryanodine-sensitive Ca2+ store.
Recent work has revealed that spark-like Ca2+ transients, termed syntillas, can be observed in NHT. These syntillas arise from ryanodine-sensitive intracellular stores. In other neuronal preparations, similar Ca2+ transients have been suggested to affect spontaneous transmitter release. However, such a role for syntillas had yet to be examined. To assess if syntillas could directly trigger spontaneous release from NHT, I used simultaneous Ca2+ imaging along with amperometric detection of release. Amperometry was adapted to this system via a novel method of false-transmitter loading. Using this approach I have found no apparent correlation between these two events, indicating that syntillas are unable to directly elicit spontaneous transmitter release.
As this finding did not rule out an indirect modulatory role of syntillas on release, I additionally present some preliminary studies examining the ability of ryanodine-sensitive Ca2+ release to modulate vesicular priming. Using immunocytochemistry, I have shown that RyR agonist treatment shifts the distribution of neuropeptides toward the plasma membrane in oxytocinergic NHT, but not in vasopressinergic NHT. RyR antagonists have the opposite affect, again only in oxytocinergic NHT. Further, I have found that application of RyR agonists result in a facilitation of elicited release in NHT using membrane capacitance recording. This facilitation appears to be due primarily to an increase in recruitment of vesicles to the readily-releasable pool. These findings suggest that ryanodine-sensitive Ca2+ stores may be involved in vesicular priming in NHTs.
Taken together, the work presented in this thesis provides some new and interesting insights into the underlying mechanisms and modulation of transmitter release in both the HNS and other CNS terminals.
McNally, James M., "Molecular Mechanisms of Neuropeptide Secretion from Neurohypophysial Terminals: a Dissertation" (2008). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 383.