Plasmids and crosses

To construct the ZM10104 imaging strain, we created and then crossed two integrated lines, one expressing GCaMP6s and one expressing the wCherry landmark. The first of these lines, ADS700, was made by coinjecting lin-15(n765) animals with pJH4039 (ift-20 GCaMP6s::3xNLS) and a lin-15–rescuing plasmid. A stable transgenic line (hpEx3942) with consistent GCaMP expression in the chemosensory neurons was selected for integration, and transgenic animals were irradiated with ultraviolet (UV) light to integrate the transgenes into the genome. The resulting integrated line (aeaIs008) was backcrossed four times against N2 wild type. The second line, ADS701, was similarly made by coinjecting lin-15(n765) animals with pJH4040 (gpc-1 wCherry) and a lin-15–rescuing plasmid. A stable transgenic line with good wCherry expression was selected for integration, and transgenic animals were irradiated with UV light to integrate the transgenes into the genome. The resulting integrated line (hpIs728) was backcrossed four times against N2 wild type. To make ZM10104, ADS700 hermaphrodites were crossed with N2 males. Heterozygous aeaIs008/+ male progeny were then crossed with ADS701 hermaphrodites. F1 progeny were picked for wCherry expression, and F2 progeny were picked for both GCaMP6s and wCherry expression. The line was then homozygozed in the F3 generation.

The ADS707 mutant imaging line was created by crossing the ZM10104 line with EG9631, an unc-13(s69) mutant obtained from the Caenorhabditis Genetics Center (CGC) (53). EG9631 hermaphrodites were crossed with ZM10104 males. Heterozygous (aeaIs008/+; hpIs728/+; +/unc-13) F1 hermaphrodite progeny were selected by GCaMP6s and wCherry expression and wild-type locomotion (unc-13 is recessive). F2 progeny were picked for fluorescence and the unc-13 uncoordinated phenotype. The line was homozygozed for fluorescence in the F3 generation.