Two-Photon Optogenetic Control of Neuronal Activity with Single Synapse Precision by Sculpted Light

Abstract

Recent advances in optogenetic techniques have provided new tools for controlling neuronal activity, opening up the way to a range of studies in neuroscience. The most widely used approach has been the optical activation of the genetically expressed light-gated ion channel Channelrhodopsin-2 (ChR2) to initiate population activity in neuronal circuits. However, single cell resolution of optogenetic activation has remained challenging. This is because neither single-photon nor conventional two-photon excitation provides the necessary combination of high spatial selectivity and the simultaneous stimulation of a sufficiently large membrane area necessary to induce fast and significant depolarizations by ChR2 in a single neuron.The presented work reports on two-photon excitation of ChR2 allowing the generation of fast and large ChR2-mediated currents in single cells with high spatial and temporal resolution by using temporally focused beams. It is demonstrated that this technique efficiently induces strong depolarization and reliable action potential firing in single ChR2-expressing neurons in rat and mouse hippocampal slices. It is further shown that subcellular compartments such as dendrites and large presynaptic terminals can be activated by the TF-2P technique. The superb spatial and temporal resolution provided by this technique allows so far unattainable precision for fine manipulation of neuronal activity to study and control the function of neuronal microcircuits in vitro and in vivo.