Dual‐Channel Photostimulation for Independent Excitation of Two Populations

Manipulation of defined neurons using excitatory opsins, including channelrhodopsin, enables studies of connectivity and the functional role of these circuit components in the brain. These techniques are vital in the neocortex, where diverse neurons are intermingled, and stimulation of specific cell types is difficult without the spatial, temporal, and genetic control available with optogenetic approaches. Channelrhodopsins are effective for mapping excitatory connectivity from one input type to its target. Attempts to use multiple opsins to simultaneously map multiple inputs face the challenge of partially overlapping light spectra for different opsins. This protocol describes one strategy to independently stimulate two comingled inputs in the same brain area to assess convergence and interaction of pathways in neural circuits. This is highly relevant in the neocortex, where pyramidal neurons integrate excitatory inputs from multiple local cell types and long‐range corticocortical and thalamocortical projections. © 2018 by John Wiley & Sons, Inc.