Measurement of synaptic vesicle exocytosis in aortic baroreceptor neurons.
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 microM 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 microM 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 omega-conotoxin GVIA (omega-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 microM nifedipine and only partially inhibited by bath application of 2.0 microM omega-CgTx. However, depolarization-induced exocytosis was significantly inhibited by bath application of 200 microM 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.
Dalton Cardiovascular Research Center, Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA.
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D000818:Animals; D001011:Aorta / Q000294:innervation*; D019256:Cadmium Chloride / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D004558:Electric Stimulation; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D009474:Neurons / Q000502:physiology*; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011311:Pressoreceptors / Q000502:physiology*; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000187:drug effects / Q000502:physiology*; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; C073804:FM1 43; D005456:Fluorescent Dyes; D010455:Peptides; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011189:Potassium Chloride; D020866:omega-Conotoxin GVIA; D009543:Nifedipine; D019256:Cadmium Chloride