Novel strategy to selectively label excitatory and inhibitory neurons in the cerebral cortex of mice

Revealing the connections of neuronal systems is critical for understanding how they function. The vast majority of neurons in all cortical areas consist of excitatory cells whose activity is controlled by inhibitory cells. Distribution and projection patterns of inhibitory and excitatory cells are key information to understand the organization of the nervous system. To investigate axonal projections, we developed a method to uniquely distinguish excitatory axons from inhibitory ones in the cortex using transgenic mice expressing Cre recombinase in the Ca2+/calmodulin-dependent protein kinase IIα-containing neurons. These animals were injected by an adenoviral vector engineered so that it directs red fluorescent protein expression in non-Cre-expressing cells, and green fluorescent protein in Cre-positive neurons. We demonstrated in vitro and in vivo that GFP-expressing neurons are GABA-immunonegative (excitatory), while the RFP-expressing cells are either GABAergic neurons or glial cells. One week after the viral vector injection RFP and GFP signals overlapped in a subset of cells but after 1 month, the two signals showed total segregation. Six months post-inoculation, GFP-labelling was clearly visible in axons but RFP remained only in somata and proximal dendrites. This technique can thus be used to differentiate excitatory axonal projections from inhibitory ones, and represent a unique tool in neuronal circuit analysis.