Simultaneous visualization of multiple neuronal properties with single-cell resolution in the living rodent brain.

To understand the fine-scale structures and functional properties of individual neurons in vivo, we developed and validated a rapid genetic technique that enables simultaneous investigation of multiple neuronal properties with single-cell resolution in the living rodent brain. Our technique PASME (promoter-assisted sparse-neuron multiple-gene labeling using in uteroelectroporation) targets specific small subsets of sparse pyramidal neurons in layer 2/3, layer 5 of the cerebral cortex and in the hippocampus with multiple fluorescent reporter proteins such as postsynaptic PSD-95-GFP and GFP-gephyrin. The technique is also applicable for targeting independently individual neurons and their presynaptic inputs derived from surrounding neurons. Targeting sparse layer 2/3 neurons, we uncovered a novel subpopulation of layer 2/3 neurons in the mouse cerebral cortex. This technique, broadly applicable for probing and manipulating neurons with single-cell resolution in vivo, should provide a robust means to uncover the basic mechanisms employed by the brain, especially when combined with in vivo two-photon laser-scanning microscopy and/or optogenetic technologies.