Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals.
We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.
Menegon
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Dept Neuroscience, San Raffaele Scientific Institute, Milan, Italy. valtorta.flavia@hsr.it
Dunlap
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DD
Castano
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Benfenati
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Czernik
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AJ
Greengard
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Valtorta
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D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems; D015398:Signal Transduction; D016704:Synapsins / Q000276:immunology* / Q000378:metabolism*
D016704:Synapsins; D005576:Colforsin; D003994:Bucladesine; D000242:Cyclic AMP; D002118:Calcium