The molecular mechanisms of synaptic function and development have been studied extensively, but little is known about the energy requirements of synapses, or the mechanisms that coordinate their energy production with their metabolic demands. These are oversights, as synapses with high energy demands are more susceptible to degeneration and degrade in the early stages of diseases such as amyotrophic lateral sclerosis, spinal muscle atrophy and Parkinson’s disease. Here, in a structure-function study at Drosophila motor neuron terminals, a neurophysiological model was generated to investigate how power (ATP/s) supply is integrated to satisfy the power demand of presynaptic terminals. Power demands were estimated from six nerve terminals through direct measurements of neurotransmitter release and Ca2+ entry, as well as theoretical estimation of Na+ entry and power demands at rest (cost of housekeeping). The data was leveraged with a computational model that simulated the power demands of the terminals during their physiological activity, revealing high volatility in which power demands can increase 15-fold within milliseconds as neurons transition from rest to activity. Another computational model was generated that simulated ATP production scenarios regarding feedback to the power supply machinery (Oxphos and glycolysis) through changes in nucleotide concentrations, showing that feedback from nucleotides alone fail to stimulate power supply to match the power demands of each terminal. Failure of feedback models invokes the need for feed forward mechanisms (such as Ca2+) to stimulate power supply machinery to match power demands. We also quantified mitochondrial volume, density, number and size in each nerve terminal, revealing all four features positively correlate with the terminals power demands. This suggests the terminals enhance their oxidative capacity by increasing mitochondrial content to satisfy their power demands. And lastly, we demonstrate that abolishing an ATP buffering system (the phosphagen system) does not impair neurotransmission in the nerve terminals, suggesting motor nerve terminals are capable of satisfying their power demands without the ATP buffering system. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 84-93). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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.

A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites. At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis. Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active players in the development and integrity of the nervous system. The formation of functional neuronal circuits requires the precise addition of new synapses. Mounting evidence implicates glial function in synapse remodeling and formation. However, the precise molecular mechanisms governing these functions are poorly understood. My thesis work begins to define the molecular mechanisms by which glia communicate with neurons at the Drosophila neuromuscular junction (NMJ). During development glia play a critical role in remodeling neuronal circuits in the CNS. In order to understand how glia remodel synapses, I manipulated a key component of the glial engulfment machinery, Draper. I found that during normal NMJ growth presynaptic boutons constantly shed membranes or debris. However, a loss of Draper resulted in an accumulation of debris and ghost boutons, which inhibited synaptic growth. I found that glia use the Draper pathway to engulf these excess membranes to sculpt synapses. Surprisingly, I found that muscle cells function as phagocytic cells as well by eliminating immature synaptic ghost boutons. This demonstrates that the combined efforts of glia and muscle are required for the addition of synapses and proper growth. My work establishes that glia play a crucial role in synapse development at the NMJ and suggests that there are other glial-derived molecules that regulate synapse function. I identified one glial derived molecule critical for the development of the NMJ, a TGF-β ligand called Maverick. Presynaptically, Maverick regulates the activation of BMP pathway confirmed by reducing the transcription of the known target gene Trio. Postsynaptically, it regulates the transcription of Glass bottom boat (Gbb) in the muscle suggesting that glia modulate the function of Gbb and consequently the activation of the BMP retrograde pathway at NMJ. Surprisingly, I also found that glial Maverick regulates the transcription of Shaker potassium channel, suggesting that glia potentially could regulate muscle excitability and consequently modulate synaptic transmission. Future work will elucidate such hypothesis. My work has demonstrated two novel roles for glia at the NMJ. First is that glia engulfing activity is important for proper synaptic growth. Second is that the secretion of glial-derived molecules are required to orchestrate synaptic development. This further supports that glia are critical active players in maintaining a functional nervous system.

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