Recording Neural Activity in Unrestrained Animals with Three-Dimensional Tracking Two-Photon Microscopy

摘要

h2 class="section-title"Summary/h2 p id="abspara0010"Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but brain motion, which often accompanies behavior, compromises these measurements. Two-photon point-scanning microscopy is especially sensitive to motion artifacts, and two-photon recording of activity has required rigid coupling between the brain and microscope. We developed a two-photon tracking microscope with extremely low-latency (360?μs) feedback implemented in hardware. This microscope can maintain continuous focus on neurons moving with velocities of 3?mm/s and accelerations of 1?m/ssup2/sup both in-plane and axially. We recorded calcium dynamics of motor neurons and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentations and behavioral outputs, and we measured light-induced depolarization of a visual interneuron in a moving animal using a genetically encoded voltage indicator. Our technique can be extended to stabilize recordings in a variety of moving substrates.