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 varia tions in the concentration of second messengers (Ca2+, cyclic AMP) in singl e presynaptic boutons by detecting the phosphorylation of the synapsins, ex cellent nerve terminal substrates for cyclic AMP- and Ca2+/calmodulin-depen dent protein kinases, For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca2+/calmo dulin-dependent protein kinase II (at site 3) or synapsins I/II phosphoryla ted by either cAMP-dependent protein kinase or Ca2+/calmodulin-dependent pr otein kinase I (at site 1), Cerebellar granular neurones in culture were do uble-labelled with a monoclonal antibody to synapsins I/II and either of th e polyclonal antibodies. Digitised images were analysed to determine the re lative 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 phosphor ylation; (ii) depolarisation in the presence of extracellular Ca2+ was foll owed by a selective and widespread increase in site 3 phosphorylation, alth ough the relative phosphorylation stoichiometry varied among individual ter minals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often oc curred in specific nerve terminal sub-populations aligned along axon branch es, In addition to shedding light on the regulation of synapsin phosphoryla tion in living nerve terminals, this approach permits the spatially-resolve d analysis of the activation of signal transduction pathways in the presyna ptic compartment, which is usually too small to be studied with other curre ntly available techniques.