The awake behaving worm: Simultaneous imaging of neuronal activity and behavior in intact animals at millimeter scale

Genetically encoded optical probes of neuronal activity offer the prospect of simultaneous recordings of neuronal activity and behavior in intact animals. A central problem in simultaneous imaging is that the field of view of the high-power objective required for imaging the neuron is often too small to allow the experimenter to assess the overall behavioral state of the animal. Here we present a method that solves this problem using a microscope with two objectives focused on the preparation: a high-power lens dedicated to imaging the neuron and low-power lens dedicated to imaging the behavior. Images of activity and behavior are acquired simultaneously but separately using different wavelengths of light. The new approach was tested using the cameleon calcium sensor expressed in Caenorhabditis elegans sensory neurons. We show that simultaneous recordings of neuronal activity and behavior are practical in C. elegans and, moreover, that such recordings can reveal subtle, transient correlations between calcium levels and behavior that may be missed in nonsimultaneous recordings. The new method is likely to be useful whenever it would be desirable to record simultaneously at two different spatial resolutions from a single location, or from two different locations in space.