title: Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.
abstract: During development of neuronal circuits, presynaptic and postsynaptic functions are adjusted in concert, to optimize interneuronal signaling. We have investigated whether activation of glutamate receptors affects presynaptic function during synapse formation, when constitutive synaptic vesicle recycling is downregulated. Using primary cultures of hippocampal neurons as a model system, we have found that chronic exposure to both NMDA and non-NMDA glutamate receptor blockers during synaptogenesis produces an increase in miniature EPSC (mEPSC) frequency, with no significant changes in mEPSC amplitude or in the number of synapses. Enhanced synaptic vesicle recycling, selectively in glutamatergic nerve terminals, was confirmed by the increased uptake of antibodies directed against the lumenal domain of synaptotagmin. No increased uptake was detected in neuronal cultures grown in the chronic presence of TTX, speaking against an indirect effect caused by decreased electrical activity. Enhanced mEPSC frequency correlated with a reduction of synaptophysin-synaptobrevin-vesicle-associated membrane protein 2 (VAMP2) complexes detectable by immunoprecipitation. Intracellular perfusion with a peptide that inhibits the binding of synaptophysin to synaptobrevin-VAMP2 induced a remarkable increase of mEPSC frequency in control but not in glutamate receptor blocker-treated neurons. These findings suggest that activation of glutamate receptors plays a role in the downregulation of the basal rate of synaptic vesicle recycling that accompanies synapse formation. They also suggest that one of the mechanisms through which this downregulation is achieved is an increased interaction of synaptophysin with synaptobrevin-VAMP2.
authors.affiliation: Consiglio Nazionale delle Ricerche, Cellular and Molecular Pharmacology and "B. Ceccarelli" Centers, Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Bacci
authors.forename: S
authors.initials: S
authors.lastname: Coco
authors.forename: E
authors.initials: E
authors.lastname: Pravettoni
authors.forename: U
authors.initials: U
authors.lastname: Schenk
authors.forename: S
authors.initials: S
authors.lastname: Armano
authors.forename: C
authors.initials: C
authors.lastname: Frassoni
authors.forename: C
authors.initials: C
authors.lastname: Verderio
authors.forename: P
authors.initials: P
authors.lastname: De Camilli
authors.forename: M
authors.initials: M
authors.lastname: Matteoli
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D006624:Hippocampus; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D050683:R-SNARE Proteins; D051381:Rats; D017470:Receptors, Glutamate / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors; D009435:Synaptic Transmission / Q000187:drug effects; D013572:Synaptic Vesicles / Q000378:metabolism; D016708:Synaptophysin / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D018691:Excitatory Amino Acid Antagonists; D046911:Macromolecular Substances; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D016708:Synaptophysin; D013779:Tetrodotoxin