Browsing by Subject "Neurotransmitter Agents"
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Item Membrane Lipids and Synaptic Vesicle Trafficking in the CNS(2009-01-14) Wasser, Catherine Rebecca; Kavalali, Ege TMost vesicles within a synapse are dormant. The rest participate in synaptic neurotransmission, with a portion of these preferentially fusing first. Moreover, all synapses experience spontaneous neurotransmitter release which may originate from the random exocytosis of vesicles prepared to fuse immediately upon calcium influx; however, spontaneously fusing vesicles may be independent because they prefer spontaneous fusion. The functional separation argues that the compositions the synaptic vesicle membranes are somehow unique between pools. The first three chapters explore the role of cholesterol in synaptic transmission. We treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed augmented spontaneous neurotransmission. In contrast, the same treatments severely impaired responses evoked by action potentials and hypertonicity. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis. Chapter five examines the role of sphingosine on neurotransmitter release. By adding sphingosine to hippocampal cultures, we found that sphingosine enhances neurotransmission in a synaptobrevin-2-dependent manner. Chapter six investigates the stability of actively recycling synaptic vesicles. We employed several approaches (fluorescent and ultrastructural imaging) to monitor not only the fate recycling vesicles, but also the origin and reuse of spontaneously fusing vesicles. We conclude that at rest, the total recycling pool remains active and resists spontaneous fusion up to at least six hours; while spontaneous fusion of spontaneously fusing vesicles is much faster. This argues that vesicles fusing spontaneously do not originate from the recycling pool. In chapter seven, we observe how modifying synaptic vesicle membranes might affect neurotransmitter release. By the uptake of horseradish peroxidase into vesicles followed by hydrogen peroxide perfusion, we induced free radical modification of vesicle membranes and found that modifying recycling pool vesicles increased spontaneous fusion and attenuated evoked release. Taken together, the results of each chapter appear to suggest that the fusion of action potential-dependent and-independent vesicles are regulated by different mechanisms, supporting the theory that some vesicles may be unique within a synapse.Item Molecular and Functional Determinants of Synaptic Vesicle Recycling In CNS Synapses(2007-05-23) Virmani, Tuhin; Kavalali, Ege T.Chemical neurotransmission is the basis for information processing in the brain, and presynaptic terminals respond to a large range of stimulation patterns including baseline rhythms of activity that coordinate neuronal ensembles, to short bursts of activity that encode information. They also release neurotransmitter spontaneously in the absence of any activity. The question then is how can a single subcellular compartment with approximately 100 synaptic vesicles coordinate these complex functions? We used a multifaceted approach to address this question. We first studied the role of synaptotagmin 7 (syt7), a highly alternatively spliced synaptic plasma membrane protein, whose short splice forms inhibit clathrin-mediated endocytosis. We found that in hippocampal synapses, the splice variants formed a bi-directional molecular switch targeting vesicles to kinetically distinct recycling pathways. Additionally, syt7 knockout synapses had less fast endocytosis, while calcium binding site mutant synapses showed increased vesicle endocytosis. We further investigated the slower recycling pathways by exploring rab5 function. A dominant negative rab5 mutation did not alter synaptic function, but constitutively active rab5 or the inhibition of vesicle budding from endosomes by the PI3-kinase inhibitor wortmannin, decreased vesicle pool size and release kinetics. This suggests that central synapses are tuned towards faster modes of recycling. The model of spontaneous neurotransmitter release from this same evoked recycling pool places additional constraints on this system. We explored this hypothesis by directly visualizing presynaptic recycling of spontaneous vesicles using FM dyes, syt1 antibodies and HRP uptake. We found that there are actually two sets of vesicle pools, one for evoked release, and one for spontaneous release that have minimal interaction with one another. Can presynaptic function be a substrate for diseases of the CNS? To test this important question, we studied a mouse model for infantile Batten disease. We found that underlying synaptic deficits in vesicle pool size and mini frequency could produce the neurological phenotypes exhibited by patients well before the onset of neurodegeneration. Taken together, these results show that synaptic vesicle recycling is a very plastic entity and that synapses have the intrinsic ability to modulate their vesicle trafficking pathways in response to the varying demands placed on them.Item Structure and Function of Proteins Involved in Regulated Secretion: Synaptotagmins and Complexin(2010-01-12T18:50:31Z) Craig, Timothy Kellog; Rizo-Rey, JosepThe release of neurotransmitter from neurons is a tightly regulated process. There are a number of proteins required for membrane fusion to occur, and then there are regulatory proteins that allow membrane fusion to proceed at incredible speed with the precise timing necessary for complex functions such as sight, motor control, and conscious thought. This study will explore the role of three such regulators through biophysical and structural methods. There are a number of proteins that are essential for membrane fusion. The SNARE proteins are the plasma membrane protein Syntaxin, the vesicle membrane protein Synaptobrevin, and the plasma membrane associated protein SNAP25. These proteins form a tight complex called the SNARE complex that is required for neurotransmitter release. This complex bridges the vesicle and plasma membranes, bringing them into close proximity. Formation of this complex is thus an important point of regulation for the neurotransmitter release process. This SNARE complex serves not only to bridge two membranes, but also to become an anchoring point for a number of regulators of neurotransmitter release such as Complexin, and Synaptotagmin as well as other required proteins such as Munc13 and Munc18. Complexin is a small soluble protein that binds to the SNARE complex with high affinity and regulates the formation of the SNARE complex. Synaptotagmin is the calcium sensor for fast release of neurotransmitter. Here I present data showing that the N-terminus of Complexin is involved in a critical interaction with the C-terminus of the SNARE complex that is responsible for the excitatory effect of complexin in neurotransmitter release. Synaptotagmins work with Complexins to trigger rapid membrane fusion in response to calcium influx. Synaptotagmin VII is an important protein for the release of glucagon from islets of langerhans. The C2B domain of this protein is nearly 50% identical to the C2B domain of SytI, but when the C2B domains of SytVII and SytI are switched, the protein does not function correctly. In this study the structure of SytVII was determined by x-ray crystallography to 1.44Å resolution in order to determine if the C2B domain of SytVII is structurally different from other C2B domains. Additionally I crystallized and solved the structure of the C2A domain of Synaptotagmin IX in an effort to compare it to the C2 domains of the other members of the synaptotagmin family. This analysis resulted in the surprising conclusion that a high degree of structural similarity does not necessarily relate to interoperability of the domains. [Keywords: Synaptotagmin; Complexin; Neurotransmitter release; membrane fusion; C2B; C2A; Synaptotagmin IX; Synaptotagmin VII; NMR; crystallography]