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Presynaptic terminals of neurons are optimized for neurotransmitter release, which is tightly controlled by presynaptic calcium. Here, we evaluate the role of calcium influx through voltage-gated calcium channels (VGCCs) in regulating the initial vesicular release probability (p) and the number of vesicles available for release by action potentials (effective RRP) at the calyx of Held synapse in mice. Two established methods of estimating effective RRP size and p reveal that both are calcium dependent. Reducing calcium influx by blocking R-type (VGCCs) or P/Q-type VGCCs also reduces EPSC amplitude via p and effective RRP size. Furthermore, activation of gamma-aminobutryic acid class B (GABAB) receptors, which reduces presynaptic calcium by regulating VGCCs without other significant effects on release, also reduces the effective RRP size and p. These findings suggest that the calcium dependence of RRP size may influence the manner in which certain neuromodulators affect neurotransmitter release.

This thesis describes the design, synthesis and development of fluorescent probes to monitor synaptic transmission at individual presynaptic terminals in the mouse brain. Two distinct approaches to accomplish this are discussed. The first approach seeks to monitor synaptic activity by using pH-sensitive endocytic membrane probes to label active presynaptic terminals. The second approach seeks to monitor synaptic activity by loading small fluorescent molecules into presynaptic vesicles and studying their evoked release upon stimulation. The first chapter of this thesis describes currently available techniques that are used to study synaptic transmission in the brain. The use of electrochemical techniques is discussed and the use of fluorescent reporters is introduced as a means to image single synapses with high resolution. Chapter II of this thesis describes the rational design of pH-sensitive membrane probes for labeling recycling vesicles. The synthesis, photophysical properties and biological characterization of these probes are described. Although these probes proved to be too lipophilic to work well in the brain tissue and neuronal culture, their use on the cell surface is demonstrated. Furthermore, the structure activity relationship established by this library of probes can be used to direct the future development of pH-sensing endocytic dyes. Chapter III and IV of this thesis describe the development of new generations of Fluorescent False Neurotransmitters (FFNs) for imaging vesicular content release from individual presynaptic terminals in the brain. Chapter III introduces a novel imaging agent, FFN200, for monitoring and quantifying dopamine release from individual synaptic terminals in the mouse brain. Chapter IV describes the exploration and screening of small fluorescent molecules in the mouse brain for the purpose of developing FFNs at synaptic terminals other than dopamine. FFN7122 is introduced as the first FFN to be developed for terminals outside of dopamine. FFN7122 is shown to be a marker for glutamatergic terminals in the hippocampus, dorsal striatum, and motor cortex of the mouse brain. The evoked release of this probe from presynaptic vesicles is demonstrated and two hypotheses for its uptake mechanism are proposed.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 119-134). Also available in electronic version.

Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.

Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2003. / Vita. Includes bibliographical references.

Drosophila melanogaster is an excellent model system to identify genes involved in synaptic growth and function. In Drosophila, the Serrate (Ser) gene encodes a transmembrane protein that is a ligand for Notch receptor. Several previous studies implicated a role for Serrate in normal wing development and patterning. In this study, I demonstrate that Serrate is required for normal synaptic growth and function. I characterized the phenotype of a Serrate mutation (serB936) that was identified by an EMS-induced genetic screen aimed at identifying novel genes that play a role in synaptic growth and function. Co-localization studies show that Serrate protein is expressed at both the pre- and postsynaptic side of larval neuromuscular junctions (NMJs). Mutations in ser impair synaptic transmission at larval NMJs. This defect is entirely presynaptic, as nerve-evoked excitatory junction potentials (EJP) and quantal content (QC) of neurotransmitter release are significantly reduced when compared to wild-type control. Further, mutations in ser also alter the growth of the NMJ and the underlying muscle. Mutations in ser significantly reduce the size of larval body wall muscles (length and surface area) as well as the number and size of synaptic boutons, and the number of secondary axonal branches. Ubiquitous or muscle-specific expression of normal Serrate in serB936 mutants restores a normal muscle size but not a normal size and structure of the innervating NMJ. However, expression of normal Serrate in the motor axon restores a normal number of synaptic boutons and secondary branches at serB936 mutant NMJs. In addition, it restores normal neurotransmitter release. These data suggest that Serrate protein is required presynaptically for normal synaptic growth and function. Interestingly, overexpression of Serrate in a wild type background resulted in similar phenotypes than to those of loss-of-function mutants. In conclusion, these data suggest a new functional role for Serrate in synaptic growth and function.

<p>Long-term plasticity, the long-lasting, activity-dependent change in synaptic efficacy, is a fundamental property of the nervous system. Presynaptic forms of long-term plasticity are widely expressed throughout the brain, having been described in regions such as the cortex, cerebellum, hippocampus, thalamus, amygdala and striatum. Presynaptic long-term potentiation (LTP) is associated with an increase in presynaptic release probability, but further evidence of the cellular basis for the change in release probability is not known. At the molecular level, presynaptic LTP is known to require protein kinase A, the synaptic vesicle protein, Rab3A, and the active zone protein, RIM1alpha. RIM1alpha, a presynaptic scaffold protein, binds to many molecules with known functions at different stages of the neurotransmitter release process and the synaptic vesicle cycle. Understanding which interactions of RIM1alpha mediate presynaptic LTP would shed light on the molecular and cellular mechanisms for presynaptic long-term plasticity.</p><p>Here I developed a novel platform to achieve robust acute genetic</p><p>manipulation of presynaptic proteins at hippocampal mossy fiber synapses, where presynaptic LTP is expressed. With this platform, I perform structure-function analysis of RIM1alpha in presynaptic LTP. I find that RIM1alpha phosphorylation by PKA at serine 413 is not required for mossy fiber LTP, nor does RIM1alpha-Rab3A interation. These findings suggest that RIM1alpha, Rab3A and PKA signaling, instead of functioning synergistically, may represent separate requirements for presynaptic long-term plasticity. I then tested whether Munc13-1, a priming protein, is an effector for RIM1alpha in presynaptic LTP and provide the first evidence for the involvement of Munc13-1 in presynaptic long-term synaptic plasticity. I further demonstrate that the interaction between RIM1alpha and Munc13-1 is required for this plasticity. These results further our understanding of the molecular mechanisms of presynaptic plasticity and suggest that modulation of vesicle priming may provide the cellular substrate for expression of LTP at mossy fiber synapses.</p> / Dissertation

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