A radioimmunoassay to monitor synaptic activity in hippocampal neurons in vitro.
Exocytosis of synaptic vesicles (SV) results in the surface exposure of lumenal epitopes of SV proteins. We have recently described the use of antibodies directed against the lumenal N-terminus of synaptotagmin I (Sytlum-Abs) to morphologically monitor exo-endocytic recycling of SVs. We report here that a radioimmunoassay based on these antibodies can be used to quantify levels of synaptic activity in primary neuronal cultures. High density cultures of hippocampal neurons grown in the absence of glia were used for these studies. A significant cell surface pool of synaptotagmin I immunoreactivity was detectable by Sytlum-Abs at steady state. The increase in the amount of Sytlum-Abs which became cell bound during a 3 min incubation at 37 degrees C over the Ab bound to this cell surface pool, was substantially higher in depolarizing media containing extracellular Ca2+ than in Ca(2+)-free media. Incubation of the cultures with Sytlum-Abs for longer time periods indicated a sustained increase in the rate of SV exocytosis in depolarizing media which lasted for at least 1 h. This increase was completely abolished by pretreating the neurons with tetanus toxin and this block correlated with a disappearance of synaptobrevin immunoreactivity. This radioimmunoassay therefore offers a new way to monitor SV exocytosis of neuronal populations in vitro irrespective of the type of neurotransmitter secreted and of postsynaptic effects.
Mundigl
O
O
Department of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06510, USA.
Verderio
C
C
Krazewski
K
K
De Camilli
P
P
Matteoli
M
M
D000818:Animals; D001692:Biological Transport; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D050683:R-SNARE Proteins; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D050863:Synaptotagmin I; D050857:Synaptotagmins; D013744:Tetanus Toxin / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D013744:Tetanus Toxin; D050857:Synaptotagmins