Focal stimulation of single GABAergic presynaptic boutons on the rat hippocampal neuron

Abstract

Evoked inhibitory postsynaptic currents (eIPSCs) generated from a single GABAergic bouton were recorded and the functional properties were investigated. Native single boutons attached to mechanically dissociated rat hippocampal CA1 neurons, namely "synaptic bouton" preparation, were visualized with FM 1-43 dye and selectively stimulated by a glass pipette directed to a single bouton by focal stimulation. The GABAergic eIPSCs were elicited in like all-or-none fashion regarding both stimulus strength and pipette location, thus indicating that the eIPSCs result from the activation of a single bouton. The GABA release from the boutons was action potential dependent since eIPSCs were blocked in the presence of either voltage-dependent Na(+) or Ca(2+)channel blocker. Even in the presence of tetrodotoxin (TTX), eIPSCs could be elicited by additional application of a voltage-dependent K(+) channel blocker, 4-AP. The GABA release depended on external Ca(2+) concentration. Amplitude histogram of eIPSCs did not follow Poisson distribution or show discrete peaks. As a result, this new experimental approach using both focal stimulation and a synaptic bouton preparation allows for a detailed study of the native synaptic machinery in nerve terminals measuring smaller than 1 microm in size in the CNS.