Transgenic Mice

Homozygous Ai32 mice (C57BL/6 background; strain no. 024109, The Jackson Laboratory, Connecticut) and homozygous VGLUT2-Cre mice (strain no. 28863, Jackson Laboratory, Connecticut) were crossbred. Ai32 mice carry the channelrhodopsin2 gene “ChR2(H134R)-EYFP” in the Gt (ROSA) 26Sor locus, which is preceded by a LoxP-flanked STOP cassette to prevent its expression. By crossing Ai32 mice with VGLUT2-Cre mice, the Cre-expressing cell population has the STOP cassette trimmed, resulting in expression of ChR2-EYFP in glutamatergic neurons expressing VGLUT2, which make up the vast majority of sensory neurons innervating the distal colon and rectum (colorectum) (29). Offspring of both sexes aged 8–14 wk heterozygous for VGLUT2-Cre and ChR2-EYFP genes (i.e., VGLUT2/ChR2) were used for the study.

To further confirm the expression efficiency of VGLUT2-Cre mice, we crossed VGLUT2-Cre mice with another reporter line, i.e., the tdTomato mice (Ai14, strain no. 7914, The Jackson Laboratory, Connecticut) as described previously (24). TdTomato filled the cytoplasmic space to allow easy quantification of DRG neurons. Offspring with heterozygous tdTomato (tdT) and VGLUT2-Cre genes (i.e., VGLUT2/tdT) were used for the study.