Figure 3

Genetic targeting of Renshaw cells by the intersection of Calb1-dgCre and Pvalb-Flpo. (A) Schematic of the Calb1-dgCre :: Pvalb-Flpo :: R26-RCE-dual-EGFP genetic system. Genetic targeting occurs in cells that excise both stop codons in the R26-RCE:dual-EGFP reporter by expressing calbindin at the time of TMP administration and parvalbumin any time prior to age of analysis. (B) Confocal image stacks (50 µm thick sections) comparing lack of spontaneous recombination labeling (top) or labeling after two TMP injections at P15 and analysis at P60 (bottom) (Calb1/Pvalb-EGFP, green; CB-IR, blue). Most RCs (higher mag panels) and a few dorsal horn cells express EGFP and CB-IR after TMP administration. (C) Quantification of Calb1/Pvalb -EGFP inside and outside the RCA (top and bottom bars, respectively) after TMP or vehicle injections at different time points from P5 to P60 or no injections. The largest percentage of RC labeling occurs after 2 doses of TMP at P10 or P15. The number of non-RCs targeted diminishes after P15 injections compared to P10. In this intersectional Calb1/Pvalb model, there is negligible spontaneous labeling at P60, either in the RCA or outside the RCA. (Error bars = S.D.; asterisks in all graphs indicate significance level after One-Way ANOVAs across all conditions. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, n.s. non-significant. For pair-wise comparisons and details of statistics and analyses, see Supplemental Tables 7–9 and Supplemental Fig. S4).