INTRODUCTION The presynaptic nerve terminal is a key compartment for communication within the nervous system. In fact, interneuronal communication is achieved primarily via the exocytotic release of neurotransmitters from synaptic vesicles present in nerve terminals. Physiologically, neurotransmitter release is elicited by the arrival of an action potential at a nerve terminal. This results in membrane depolarisation and a subsequent rise in the sub-plasmalemmal concentration of Ca 2+ , which is sufficient to stimulate release (Katz and Miledi, 1965; Ceccarelli and Hurlbut, 1980; Valtorta and Benfenati, 1995). It has long been known that the process of neurotransmitter release is a highly plastic phenomenon, which is sensitive to the previous history of the synapse, and can be adjusted in an extremely prompt way to changes in the intra- and extracellular environment (Jessell and Kandel, 1993). Although this feature is of paramount importance for information processing in the central nervous system and for adjusting the level of efficiency of a synapse to the physiological needs of a neurone, the bases for the high modulability of release are not completely understood. Variations in the amount of the neurotransmitter released reflect variations in the number of synaptic vesicles which undergo exocytosis following an action potential, and are caused by changes in either the number of synaptic vesicles which are immediately available for fusion or the probability of each vesicle to fuse with the plasma membrane following Ca 2+ entry (Boyd and Martin, 1956). Modulation of neurotransmitter release is thought to be achieved through fluctuations in the intra-terminal levels of second messengers such as Ca 2+ or cAMP, which in turn activate the phosphorylation of specific protein substrates by protein kinases (Llinas et al., 1981; Smith and Augustine, 1988; Zucker, 1989; Weiner, 1979). Thus far the study of signal transduction in nerve terminals has been hampered by the tiny 3573 Journal of Cell Science 113, 3573-3582 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1781 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. Key words: Synapsin, Phosphorylation, Cyclic AMP, Ca 2+ , Synaptic vesicle SUMMARY Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals Andrea Menegon 1, *, David D. Dunlap 1, *, Francesca Castano 1 , Fabio Benfenati 2 , Andrew J. Czernik 3 , Paul Greengard 3 and Flavia Valtorta 1,‡ 1 Dept Neuroscience, San Raffaele Scientific Institute, Milan, Italy 2 Dept Experimental Medicine, Section of Physiology, University of Genoa, Genoa and Dept Neuroscience, University of Rome Tor Vergata, Rome, Italy 3 Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, USA *These two authors contributed equally to the work ‡ Author for correspondence (e-mail: valtorta.flavia@hsr.it) Accepted 22 August; published on WWW 4 October 2000