Single-bouton-mediated synaptic transmission: postsynaptic conductance changes in their relationship with presynaptic calcium signals.

Abstract

Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals. This paper describes a method that was designed to activate transmitter release from solitary synaptic boutons in culture. Neurons from the embryonic rat superior colliculus were grown at low density and double-loaded with a fluorescent marker of synaptic vesicles (FM1-43 or RH414) and a fluorescent Ca2+ indicator (Fura-2, Mag-fura-2, Oregon Green BAPTA-1 or Oregon Green BAPTA-5N). Action potential generation was blocked by tetrodotoxin. Appropriate synaptic boutons were selected under phase-contrast and fluorescence illumination at a magnification of 1000. They were activated by short electrical pulses via a fine-tipped glass pipette filled with bath solution. Presynaptic Ca2+ transients were measured in a region delineated by the FM1-43/RH414 fluorescence. By simultaneous presynaptic Ca2+ imaging and whole-cell recording of postsynaptic responses to single depolarizing pulses, the quantitative relationships between pre- and postsynaptic parameters of synaptic strength in a small synapse of central origin could, for the first time, be analysed. The experiments showed that the average postsynaptic currents depend strongly on the size of the presynaptic Ca2+ transients. However, at any level of presynaptic Ca2+ concentration postsynaptic responses fluctuated in amplitude.