Light-gated ion channels and pumps have made it possible to probe intact neural circuits by manipulating the activity of groups of genetically similar neurons. What is needed now is a method for precisely aiming the stimulating light at single neuronal processes, neurons or groups of neurons. We developed a method that combines generalized phase contrast with temporal focusing (TF-GPC) to shape two-photon excitation for this purpose. The illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups of cells. The TF-GPC two-photon excitation patterns generated large photocurrents in Channelrhodopsin-2-expressing cultured cells and neurons and in mouse acute cortical slices. The amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and, thereby, be used to trigger single action potentials or trains of action potentials.
Correlating patterned neuronal activity to defined animal behaviors is a core goal in neuroscience. Optogenetics is one large step toward achieving this goal, yet optical methods to control neural activity in behaving rodents have so far been limited to perturbing all light-sensitive neurons in a large volume. Here we demonstrate an all-optical method for precise spatial control and recording of neuronal activity in anesthetized and awake freely behaving mice. Photoactivation patterns targeted to multiple neuronal somata, produced with computer-generated holography, were transmitted to the mouse brain using a micro-objective-coupled fiber bundle. Fluorescence imaging through the same device, via epifluorescence, structured illumination, or scanless multipoint confocal microscopy, allowed imaging of neurons and recording of neuronal activity. The fiberscope was tested in mice coexpressing ChR2-tdTomato and GCaMP5-G in cerebellar interneurons, delivering near-cellular resolution photoactivation in freely behaving mice.
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