Supplementary Figure 1: Generation of Vglut3-ires-Cre (VT3^Cre) knock-in mice and characterization of VT3^Cre-tdTomato neurons

(a) To generate VT3^Cre knock-in mice, a cassette containing the Cre recombinase gene preceded by an internal ribosomal entry sequence (“ires”) was targeted just distal to the stop codon of the endogenous Vglut3 (Slc17a8) allele. The “neo” cassette flanked with the FRT sites was removed by crossing with Flpe deletor mouse line [Rodriguez, C.I. et al. Nat Genet. 25, 139-140 (2000)]. (b) Double staining of tdTomato with two markers (“green”), detected by in situ hybridization (ISH) for GAD1 mRNA (b, top, representative images of 9 sections from 3 mice), a marker for GABAergic inhibitory neurons, and GlyT2 mRNA (b, bottom, representative images of 9 sections from 3 mice), a marker for glycinergic neurons, on spinal sections from adult VT3^Cre-tdTomato mice. Right panels in b-d represent higher magnification of the boxed areas in left panels. Note virtually no co-localization, with only ~1% of tdTomato⁺ neurons showing detectable GAD1 mRNA (7/709) or GlyT2 mRNA (10/720). (c) Spinal sections from adult mice showing tdTomato with SOM mRNA. Arrows indicate co-localization. The table shows quantitative colocalization in different laminae. (d) Colocalization of VT3^Cre-tdTomato⁺ neurons with the Calb2/Calretinin protein. Images are representatives of 12 sections from 3 mice. Arrows indicate co-localization. 45% of adult Calb2⁺ neurons were labeled by our knock-in VT3^Cre, which is different from only 8% of adult Calb2⁺ neurons labeled by the transgenic VT3::Cre used by Peirs et al. [Peirs, C. et al. Neuron 87, 797-812 (2015)]. Additionally, transgenic VT3::Cre failed to mark neurons exhibiting delayed firing [Peirs, C. et al. Neuron 87, 797-812 (2015)], whereas a majority of type 2 neurons labeled by our knock-in VT3^Cre displayed the delayed firing pattern (see Supplementary Fig. 6f). This difference is probably due to the absence of full regulatory elements in the BAC genomic fragment used to drive their transgenic VT3::Cre. Scale bars represent 50 μm in the left panels and 20 μm in the right panels, respectively.