A, Schematic of cre-silenced (“cre-OFF”) DNA construct containing loxP-flanked ChR2-eYFP coding region. B, Transduction of primary cultured rat neurons with cre-OFF ChR2-eYFP viral vector produces eYFP fluorescence (left) that is abolished in cells co-transduced with a vector expressing cre recombinase (right); DAPI nuclear staining (blue) is unaffected. Scale bar = 100 μm. C, Transgenic mice co-expressing cre and TdTomato in serotonergic neurons (ePet-cre; ROSA26fsTdTomato, n=4) were injected with cre-OFF ChR2-eYFP into the DRN. Inset depicts whole DRN tissue (scale bar = 200 μm) and detail demonstrating segregation of TdTomato and eYFP fluorescence into separate populations of cells. D, Serotonergic (red) and non-serotonergic (green) axons are visible in the VTA, identifiable by tyrosine hydroxylase immunoreactivity (TH, blue). Scale bar = 200 μm. E,F Quantitation of (E) eYFP and (F) TdTomato fluorescence intensity in brain regions with conspicuous eYFP expression. Abbreviations: ventral tegmental area (VTA), substantia nigra pars compacta (SNc), intermediate portion of the lateral septum (LSi), bed nucleus of the stria terminalis (BNST), red nucleus (RN), interpeduncular nucleus (IPN), nucleus accumbens (NAc), dorsal portion of the lateral septum (LSd), substantia nigra reticulata (SNr), prefrontal cortex (PFC).