Measurement of synaptic vesicle exocytosis in aortic baroreceptor neurons

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Abstract

The purpose of this study was to evaluate the use of the fluorescent membrane label FM1-43 as a measure of synaptic terminal exocytosis during stimulation of labeled aortic baroreceptor and unlabeled nodose ganglia neurons. Activation of the nerve terminals with electrical stimulation or depolarization with 90 mM KCl in the presence of 2.0 μM FM1-43 resulted in bright, punctate staining of synaptic boutons. Additional depolarization in the absence of dye resulted in destaining with a time course that was consistent and repeatable in multiple boutons within a given terminal. Destaining was dependent on calcium influx and was blocked by bath application of 100 μM CdCl2. Whole cell patch-clamp studies have reported that depolarization-induced calcium influx in aortic baroreceptor cell bodies is predominantly caused by the activation of ω-conotoxin GVIA (ω- CgTx)sensitive N-type calcium channels. In addition, these N-type channels have been shown to be inhibited by activation of metabotropic glutamate receptors. In the present study, exocytosis in aortic baroreceptor terminals was not affected by bath application of 5 μM nifedipine and only partially inhibited by bath application of 2.0 μM ω-CgTx. However, depolarization- induced exocytosis was significantly inhibited by bath application of 200 μM L-AP4, a type III metabotropic glutamate receptor agonist. Results from this study suggest that 1) FM1-43 can be used to measure synaptic vesicle exocytosis in baroreceptor neurons; 2) the N-type calcium channel may not be involved in the initial phase of vesicle exocytosis; and 3) activation of L- AP4-sensitive metabotropic glutamate receptors inhibits 90 mM KCl-induced vesicle release.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume275
Issue number2 44-2
StatePublished - 1998
Externally publishedYes

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Pressoreceptors
Synaptic Vesicles
Exocytosis
Baths
Metabotropic Glutamate Receptors
Neurons
N-Type Calcium Channels
Presynaptic Terminals
Conotoxins
Nodose Ganglion
Calcium
Excitatory Amino Acid Agonists
Cadmium Chloride
Nifedipine
Electric Stimulation
Coloring Agents
Staining and Labeling
Membranes
FM1 43

Keywords

  • Metabotropic glutamate receptors
  • Nodose neurons

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

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abstract = "The purpose of this study was to evaluate the use of the fluorescent membrane label FM1-43 as a measure of synaptic terminal exocytosis during stimulation of labeled aortic baroreceptor and unlabeled nodose ganglia neurons. Activation of the nerve terminals with electrical stimulation or depolarization with 90 mM KCl in the presence of 2.0 μM FM1-43 resulted in bright, punctate staining of synaptic boutons. Additional depolarization in the absence of dye resulted in destaining with a time course that was consistent and repeatable in multiple boutons within a given terminal. Destaining was dependent on calcium influx and was blocked by bath application of 100 μM CdCl2. Whole cell patch-clamp studies have reported that depolarization-induced calcium influx in aortic baroreceptor cell bodies is predominantly caused by the activation of ω-conotoxin GVIA (ω- CgTx)sensitive N-type calcium channels. In addition, these N-type channels have been shown to be inhibited by activation of metabotropic glutamate receptors. In the present study, exocytosis in aortic baroreceptor terminals was not affected by bath application of 5 μM nifedipine and only partially inhibited by bath application of 2.0 μM ω-CgTx. However, depolarization- induced exocytosis was significantly inhibited by bath application of 200 μM L-AP4, a type III metabotropic glutamate receptor agonist. Results from this study suggest that 1) FM1-43 can be used to measure synaptic vesicle exocytosis in baroreceptor neurons; 2) the N-type calcium channel may not be involved in the initial phase of vesicle exocytosis; and 3) activation of L- AP4-sensitive metabotropic glutamate receptors inhibits 90 mM KCl-induced vesicle release.",
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N2 - The purpose of this study was to evaluate the use of the fluorescent membrane label FM1-43 as a measure of synaptic terminal exocytosis during stimulation of labeled aortic baroreceptor and unlabeled nodose ganglia neurons. Activation of the nerve terminals with electrical stimulation or depolarization with 90 mM KCl in the presence of 2.0 μM FM1-43 resulted in bright, punctate staining of synaptic boutons. Additional depolarization in the absence of dye resulted in destaining with a time course that was consistent and repeatable in multiple boutons within a given terminal. Destaining was dependent on calcium influx and was blocked by bath application of 100 μM CdCl2. Whole cell patch-clamp studies have reported that depolarization-induced calcium influx in aortic baroreceptor cell bodies is predominantly caused by the activation of ω-conotoxin GVIA (ω- CgTx)sensitive N-type calcium channels. In addition, these N-type channels have been shown to be inhibited by activation of metabotropic glutamate receptors. In the present study, exocytosis in aortic baroreceptor terminals was not affected by bath application of 5 μM nifedipine and only partially inhibited by bath application of 2.0 μM ω-CgTx. However, depolarization- induced exocytosis was significantly inhibited by bath application of 200 μM L-AP4, a type III metabotropic glutamate receptor agonist. Results from this study suggest that 1) FM1-43 can be used to measure synaptic vesicle exocytosis in baroreceptor neurons; 2) the N-type calcium channel may not be involved in the initial phase of vesicle exocytosis; and 3) activation of L- AP4-sensitive metabotropic glutamate receptors inhibits 90 mM KCl-induced vesicle release.

AB - The purpose of this study was to evaluate the use of the fluorescent membrane label FM1-43 as a measure of synaptic terminal exocytosis during stimulation of labeled aortic baroreceptor and unlabeled nodose ganglia neurons. Activation of the nerve terminals with electrical stimulation or depolarization with 90 mM KCl in the presence of 2.0 μM FM1-43 resulted in bright, punctate staining of synaptic boutons. Additional depolarization in the absence of dye resulted in destaining with a time course that was consistent and repeatable in multiple boutons within a given terminal. Destaining was dependent on calcium influx and was blocked by bath application of 100 μM CdCl2. Whole cell patch-clamp studies have reported that depolarization-induced calcium influx in aortic baroreceptor cell bodies is predominantly caused by the activation of ω-conotoxin GVIA (ω- CgTx)sensitive N-type calcium channels. In addition, these N-type channels have been shown to be inhibited by activation of metabotropic glutamate receptors. In the present study, exocytosis in aortic baroreceptor terminals was not affected by bath application of 5 μM nifedipine and only partially inhibited by bath application of 2.0 μM ω-CgTx. However, depolarization- induced exocytosis was significantly inhibited by bath application of 200 μM L-AP4, a type III metabotropic glutamate receptor agonist. Results from this study suggest that 1) FM1-43 can be used to measure synaptic vesicle exocytosis in baroreceptor neurons; 2) the N-type calcium channel may not be involved in the initial phase of vesicle exocytosis; and 3) activation of L- AP4-sensitive metabotropic glutamate receptors inhibits 90 mM KCl-induced vesicle release.

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