A Functional Study of Single Mammalian CNS “Synaptic Bouton”

Abstract

Single CNS neurons could be dissociated with adherent functional synaptic boutons without using any enzyme, namely when preparing a “synaptic bouton.” This allows experimenters to investigate the effects of presynaptic modulators of synaptic transmission with unprecedented case and accuracy. Moreover, a single bouton can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this communication, high voltage-dependent Ca2+ channels of nerve endings, as one of experimental examples using the “synaptic bouton” preparation, are described. Ca2+ channels belonging to different subtypes, which trigger GABA release from nerve terminals (boutons) projecting to rat hippocampal CA1 pyramidal neurons, were studied. GABA-ergic evoked inhibitory postsynaptic currents (eIPSCs) were recorded; these currents were evoked by focal stimulation of single boutons in mechanically dissociated neurons and by stimulation of a nerve bundle in slice preparations. Nilvadipine, an L-type Ca2+ channel blocker, completely inhibited eIPSCs evoked by stimulation of single boutons but exerted no effect on eIPSCs evoked by low-frequency stimulation of the nerve bundle. Nilvadipine did, however, prevent potentiation of the eIPSC amplitude following high-frequency stimulation of the nerve bundles in slice preparations. ω-Conotoxin-GVIA, an N-type Ca2+ channel blocker, and ω-Agatoxin-IVA, a P/Q-type Ca2+ channel blocker, completely inhibited the eIPSCs in 33.3 and 83.3% of the recordings from single boutons, respectively. In response to low-frequency nerve bundle stimulation in the slice preparation, both ω-Conotoxin-GVIA and ω-Agatoxin-IVA partially reduced the amplitude of eIPSC, and the residual component could be abolished by Cd2+. From these results, the following hypotheses could be drawn. (i) The distribution of P/Q- and N-type Ca2+ channels at a single bouton is nonuniform; (ii) when a focal stimulation is applied to a single bouton, L-type Ca2+ channels play a significant role in generation of action potentials, which subsequently activate P/Q- and N-type Ca2+ channels at GABA release sites; and (iii) action potentials conducted through axons in the slice preparation are sufficient to depolarize the bouton membrane, even when L-type Ca2+ channels are suppressed.