Retrograde transgene expression in cortical pyramidal neurons projecting to different targets. A–F, Low-magnification epifluorescence images (top) and higher-magnification confocal stacks (bottom) of mCherry fluorescence in S1 after injection of rAAV2/1.mCherry into superior colliculus (A, D, SC), thalamus (B, E), and contralateral S1 (C, F, cS1). For all images, the pial surface is toward the top. mCherry-expressing layer 5 pyramidal neurons are apparent in each preparation. In the contralateral-injected animal (C, F), the brightest fluorescence is from anterograde labeling of contralateral-projecting neurons infected at the injection site, but mCherry-expressing cell bodies are apparent in layer 5 (F). G, Injection of two different Cre-dependent viruses into SC and cS1 in the same Emx1-Cre mouse labels distinct populations of pyramidal neurons. 2/1.FLEX.tdTomato (red) was injected into cS1; 2/1.FLEX.GCaMP3 into SC. Low-magnification image (top) shows GCaMP3-expressing somata and anterograde mCherry labeling in contralaterally projecting axon terminals. Retrogradely expressing somata from cS1 are less clear at this magnification and in this section, but are apparent in higher-magnification confocal stacks (bottom). Retrogradely labeled pyramidal neuron populations from SC and cS1 are nonoverlapping.