2000 — 2003 |
Wu, Ling-Gang |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Regulation of the Kinetics of Vesicle Endocytosis in a Central Synapse @ Washington University School of Medicine
0076091 Wu, Ling-Gang Abstract for "Regulation of the kinetics of vesicle endocytosis in a central synapse"
Neurons talk to each other via synapses, where the nerve impulse triggers fusion of synaptic vesicles to the presynaptic plasma membrane and release their transmitter content, which in turn acts on the postsynaptic target. To maintain synaptic transmission, fused vesicles must be retrieved from the plasma membrane (endocytosis) for re-use. The rate of endocytosis is critical because it determines whether the synapse can continuously release transmitters. Endocytosis can be either slow or fast. The mechanisms that regulate the rate of endocytosis remain unclear. The investigators hypothesize that the rate of endocytosis is regulated by both the intensity of nerve stimulation and the intracellular calcium level. This hypothesis will be tested at a calyceal synapse, located in the medial nucleus of the trapezoid body (MNTB) in the rat auditory brain stem. At this synapse, the investigators have recently made it possible to measure endocytosis with a time-resolved capacitance measurement technique. This is the first time such a technique has been applied to the fast synapse in the mammalian central nervous system. With this technique, the investigators will determine whether the intensity of nerve stimulation regulates endocytosis by comparing the rate of endocytosis following various nerve stimuli that differ in the number and frequency of nerve impulses. The investigators will also determine whether the intracellular calcium level regulates endocytosis by comparing the rate of endocytosis at various intracellular calcium levels. The proposed work will reveal the mechanisms that regulate the rate of endocytosis at mammalian central nervous system, and improve our understanding on how synapses maintain their activity.
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0.915 |
2001 |
Wu, Ling-Gang |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sustained Transmitter Release During Auditory Processing
DESCRIPTION (provide by applicant) Auditory nerves may fire at high frequency for a long time upon sound stimulation. This high frequency firing is relayed with precise timing via synapses up to the auditory brainstem nuclei for sound information processing. To achieve this task, synapses must maintain transmitter release throughout the train of firing. The mechanism underlying sustained transmitter release during repetitive stimulation is not well understood at auditory synapses. I propose a new model to account for sustained transmitter release at auditory synapses. This model is composed of three hypotheses. First, sustained release during repetitive stimulation is due to both a rapid replenishment of a pool of vesicles immediately available for release (releasable pool) and a decrease in the fraction (F) of this pool being released by each impulse. The decrease in F may allow the synapse to sustain transmitter release for a longer time because it slows the rate of depletion of the releasable pool during repetitive stimulation. Secondly, the decrease in F is caused by a decrease in the affinity of the release machinery to Ca2+ Thirdly, replenishment is rapid, but independent of the stimulus intensity and Ca2+ We will test these three hypotheses with three aims, respectively, at a calyx-type synapse in the medial nucleus of the trapezoid body in the rat auditory brainstem. We will monitor membrane capacitance at the nerve terminal, which allows for more direct measurements of the releasable pool size and F. We will determine whether sustained release and relay of action potentials during high frequency firing rely on both a decrease in F and rapid replenishment of the releasable pool (Aim 1). We will increase the Ca2+ concentration by photolysis of the caged Ca2+ compound and determine whether the affinity of the release machinery to Ca2+ is decreased during repetitive stimulation (Aim 2). Finally, we will determine whether the rate of replenishment is regulated by the frequency and duration of stimulation, by the Ca2+ buffer EGTA, or by an increase in the basal Ca2+ concentration induced by photolysis of the caged Ca2+ compound (Aim 3). These studies will improve our understanding of hearing mechanisms by revealing mechanisms underlying sustained transmitter release durng repetitive firing, which is critical for conveying sound information
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0.904 |
2002 — 2003 |
Wu, Ling-Gang |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Synaptic Inhibition by Volatile Ansethetics
Volatile anesthetics achieve their anesthetic effects partly by depressing excitatory glutamatergic synaptic transmission. Evidence suggests that depression of glutamatergic synaptic transmission is caused by inhibition of transmitter release. However, the cellular and molecular mechanisms underlying inhibition of transmitter release remain unclear. Based on our preliminary results, I hypothesize that volatile anesthetics depress glutamatergic synaptic transmission by reducing the presynaptic Ca2+ influx by two mechanisms: 1) inhibition of presynaptic Na+ channels, which decreases the action potential amplitude and thus the action potential-evoked Ca2+ influx, and 2) inhibition of presynaptic Ca2+ channels. We will test this hypothesis at a glutamatergic synapse in the medial nucleus of the trapezoid body in rat brainstem slices. This synapse offers a significant advantage over other synapses, because it has a large nerve terminal that allows for direct recordings of presynaptic action potentials, Na+, K+ and Ca2+ currents and fluorescence recordings of Ca2+ influx. These presynaptic recordings can be performed simultaneously with recordings of the postsynaptic excitatory current (EPSC) at the same synapse, which allows us to quantitatively evaluate the involvement of each presynaptic ion channel type in controlling action potential-evoked transmitter release. With these techniques, we will study the action of three commonly used volatile anesthetics, isoflurane, halothane and sevoflurane at clinically relevant concentrations. We will characterize the effects of these anesthetics on presynaptic Na+, K+ and Ca2+ channels and the contribution of each of these effects to depression of the EPSC. In addition, we will investigate whether these anesthetics also inhibit the EPSC by a mechanism independent of modulation of ion channels, i.e., direct inhibition of the release machinery. By revealing mechanisms underlying volatile anesthetic-induced depression of glutamate release, the proposed work will significantly contribute to our understanding of the cellular and molecular mechanisms of general anesthesia, and may ultimately help to design better general anesthetics.
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0.904 |
2002 |
Wu, Ling-Gang |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Sustained Transmitter Release During Repetitive Firing
DESCRIPTION (provide by applicant) Auditory nerves may fire at high frequency for a long time upon sound stimulation. This high frequency firing is relayed with precise timing via synapses up to the auditory brainstem nuclei for sound information processing. To achieve this task, synapses must maintain transmitter release throughout the train of firing. The mechanism underlying sustained transmitter release during repetitive stimulation is not well understood at auditory synapses. I propose a new model to account for sustained transmitter release at auditory synapses. This model is composed of three hypotheses. First, sustained release during repetitive stimulation is due to both a rapid replenishment of a pool of vesicles immediately available for release (releasable pool) and a decrease in the fraction (F) of this pool being released by each impulse. The decrease in F may allow the synapse to sustain transmitter release for a longer time because it slows the rate of depletion of the releasable pool during repetitive stimulation. Secondly, the decrease in F is caused by a decrease in the affinity of the release machinery to Ca2+ Thirdly, replenishment is rapid, but independent of the stimulus intensity and Ca2+ We will test these three hypotheses with three aims, respectively, at a calyx-type synapse in the medial nucleus of the trapezoid body in the rat auditory brainstem. We will monitor membrane capacitance at the nerve terminal, which allows for more direct measurements of the releasable pool size and F. We will determine whether sustained release and relay of action potentials during high frequency firing rely on both a decrease in F and rapid replenishment of the releasable pool (Aim 1). We will increase the Ca2+ concentration by photolysis of the caged Ca2+ compound and determine whether the affinity of the release machinery to Ca2+ is decreased during repetitive stimulation (Aim 2). Finally, we will determine whether the rate of replenishment is regulated by the frequency and duration of stimulation, by the Ca2+ buffer EGTA, or by an increase in the basal Ca2+ concentration induced by photolysis of the caged Ca2+ compound (Aim 3). These studies will improve our understanding of hearing mechanisms by revealing mechanisms underlying sustained transmitter release durng repetitive firing, which is critical for conveying sound information
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0.904 |