Functional calcium imaging of nine neuronal populations in a freely moving social task

Current capabilities of state-of-the-art in vivo imaging during freely moving behavior can record two, spectrally distinct, fluorophores. This severely limits the number of cell types identifiable in a functional imaging experiment. Here we present a pipeline that enables the distinction of nine neuronal subtypes from regions defined by behaviorally relevant cells during in vivo GCaMP imaging. These subtypes are identified utilizing unique fluorophores that are co-expressed with GCaMP, unmixed by multispectral lambda imaging on a confocal microscope, and spectral fingerprints co-registered with functional data obtained on miniaturized microscopes. Using this method, we recorded pyramidal neurons in the medial prefrontal cortex categorized by projection region during a freely moving social memory task. As neural activity in each category was concurrently imaged, we can determine which projection neurons most preferentially encode identity versus novelty/familiarity and the types of social behaviors which are most important for this distinction. This method will not only increase efficiency for calcium imaging experiments by enabling nine neuronal populations to be investigated simultaneously but also enhances the statistical power of the results by utilizing within subject comparisons.