Fig. 6.

KCl but not light evokes responses in melanopsin-expressing TG neurons. Two-month-old cultured TG neurons harvested from Opn4^cre::Ai14 mice were transfected with AAV-hSvn-floxed EFGP virus (4 μl; 6 × 10¹² titer). The premise of this method was that if cells were positive for EGFP, this increased the likelihood that melanopsin was being actively expressed. Two to three weeks after viral infection, cells on the coverslips were loaded with Fura-2 AM and 20% pluronic acid by incubating for 1 h at RT (see Materials and methods). Coverslips were individually placed in the perfusion chamber for imaging. Fluorescence in three types of ROIs was monitored. One class of ROIs was drawn on a nonfluorscent region of background. Other ROIs encircle Fura-2 loaded cells that did not show any EGFP signal. The third type of ROI encircled those cells showing both EGFP and Fura-2 signal. We first compared responses to elevated potassium (50 mM). Both the Fura-2 loaded and EGFP plus Fura-2 loaded cells responded with a KCl-induced robust calcium elevation. We next stimulated the cells with light. No light responses were found. In our survey of three coverslips containing a total of more than 30 tdTomato and EGFP cells loaded with Fura-2 AM, we detected no light-induced calcium increases.