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.

Neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30) are 23- and 30-amino acid peptides recently isolated from the porcine hypothalamus. Immunohistochemical studies using a rabbit polyclonal antiserum against the rat NPW23 peptide revealed a limited distribution in the rat brain. NPW23-immunoreactive (irNPW) cells were detected in the paraventricular nucleus (PVH), mainly in the parvocellular division, supraoptic nucleus (SO), accessory neurosecretory nuclei, dorsal and lateral hypothalamic areas, perifornical nucleus, arcuate nucleus, and anterior and posterior pituitary; whereas, irNPW fibers were noted in the PVH and SO, retrochiasmatic nucleus, dorsal and lateral hypothalamic areas, median eminence, amygdala, and posterior pituitary. The pattern of distribution of irNPW in the hypothalamus corroborates a possible role of NPW on prolactin release and feeding behavior reported by others.

Η προκλητή δοκιμασία με GHRH + GHRP-6 είναι ένα από τα πιο ισχυρά ερεθίσματα για την έκκριση της GH. Προκειμένου να εκτιμηθεί η διαγνωστική της ικανότητα σε παιδιά με κλασσική ανεπάρκεια GH (Growth Hormone Deficiency, GHD) αλλά και με νευροεκκριτική δυσλειτουργία της GH (GH Neurosecretory Dysfunction, GHND), 35 παιδιά με μέγιστη απάντηση της GH < 10 μg/L μετά από πρόκληση με levo-dopa/κλονιδίνη (GHD), 15 με φυσιολογική απάντηση στις προκλητές εξετάσεις αλλά παθολογική αυτόνομη 24ωρη έκκριση της GH (GHND) και 20 φυσιολογικοί μάρτυρες έλαβαν 1 μg/kg GHRH και GHRP-6 i.v. και η GH μετρήθηκε στο χρόνο -15΄, 0΄, 5΄, 10΄, 15΄, 30΄, 45΄, 60΄. Έξι ασθενείς δεν απάντησαν στην συνδυασμένη προκλητή εξέταση με GHRH και GHRP-6 (μη αποκριτές), με σημαντικά χαμηλότερη μέγιστη τιμή GH: 20.7 μg/L (7.8-31.8) από τους μάρτυρες και τους υπόλοιπους ασθενείς (αποκριτές). Η απάντηση της GH (μg/L) ήταν παρόμοια μεταξύ των προεφηβικών μαρτύρων: 167±88, των προεφηβικών παιδιών με κλασσική ανεπάρκεια: 202±110 και των προεφηβικών παιδιών με νευροεκκριτική δυσλειτουργία της GH: 155±83. Οι εφηβικοί μάρτυρες είχαν υψηλότερη απάντηση: 328±149 από τους εφηβικούς ασθενείς με GHD: 203±105 και GHND: 186±105. Ενώ οι εφηβικοί μάρτυρες είχαν υψηλότερη απάντηση GH από τους προεφηβικούς, οι εφηβικοί και προεφηβικοί ασθενείς και των δύο ομάδων (GHD και GHND) είχαν παρόμοια μέγιστη απάντηση GH. Tα δεδομένα της μελέτης επιβεβαιώνουν ότι ο συνδυασμός GHRH και GHRP-6 είναι ένα ισχυρό ερέθισμα για την έκκριση της GH που μπορεί να κινητοποιήσει τα υποφυσιακά αποθέματα αυξητικής ορμόνης σε παιδιά που παρουσιάζουν τόσο κλινικά, όσο και βιοχημικά χαρακτηριστικά ανεπάρκειας αυξητικής ορμόνης. Πρόκειται για μία ασφαλή και σύντομη δοκιμασία χωρίς ανεπιθύμητες ενέργειες για τα παιδιά, η οποία μπορεί να διακρίνει τους ασθενείς με σημαντική έκπτωση των υποφυσιακών αποθεμάτων αλλά και εκκριτικής ικανότητας της GH, που παρουσιάζουν και την πιο σημαντική ανεπάρκεια στην αύξηση. Είναι πιθανό οι ασθενείς «αποκριτές» να παρουσιάζουν υποθαλαμική δυσλειτουργία στη νευρορύθμιση της έκκρισης της GH και να μπορούν να απαντήσουν θεραπευτικά σε συνθετικά εκλυταγωγά της GH.Κατά συνέπεια, η εξέταση με GHRH+GHRP-6 μπορεί να χρησιμεύσει στην επιλογή ασθενών όχι μόνο με ανεπάρκεια GH αλλά και με άλλες διαταραχές της αύξησης με θεραπευτική ένδειξη τη χορήγηση GH, οι οποίοι θα μπορούσαν να απαντήσουν στη χορήγηση συνθετικών εκλυταγωγών της GH. Περαιτέρω μελέτες χρειάζονται για να απαντήσουν στα πολύ σημαντικά αυτά κλινικά ερωτήματα. / The combined growth hormone-releasing hormone and growth hormone-releasing peptide-6 (GHRH + GHRP-6) test is most potent in evaluating GH secretion. The aim of this research was to assess its capability in children with GH deficiency and low spontaneous GH secretion (GH neurosecretory dysfunction). Thirty-five children with GH <10 ng/ml after levo-dopa/clonidine (GHD), 15 with normal provocative tests but abnormal 24-hour spontaneous GH secretion (GHND), and 20 controls (C) were given 1 μg/kg of GHRH and GHRP-6 i.v. and GH (ng/ml) was measured at -15, 0, 5, 10, 15, 30, 45 and 60 min. Six were non-responders to the combined test, with significantly lower peak GH 20.7 (7.8-31.8) than C and the rest of the patients (responders). Peak GH was similar between prepubertal (PP) controls 167 +/- 88, GHD 202 +/- 110 and GHND 155 +/- 83. Pubertal (P) controls had higher peak GH 328 +/- 149 than P-GHD 203 +/- 105 and P-GHND 186 +/- 105. While P-C had higher peak GH than PP-C, PP and P children had similar responses within the GHD and GHND groups. The GHRH + GHRP-6 test detects children with severe GH insufficiency. Patients with GHD respond similarly to those with GHND, indicating a possible hypothalamic GH neuroregulatory dysfunction in GHD. Responders to the combined test may be eligible for treatment with a synthetic GH secretagogue.