Figure 1

Expression of mouse Sstr2 and human CCR5 in nociceptive neurons in C. elegans generates agonist specific avoidance behaviour. (A) Flow sorting of transgenic nematodes expressing heterologous GPCRs and gut specific GFP marker (elt-2::GFP). Graph shows a plot of forward scatter (size) against GFP fluorescence intensity. GFP-positive animals are sorted into larval stages L1, L2 and L3 according to size with 90% purity and 95% viability (sort gates R1, R2 and R3 respectively). (B) Wild type and Sstr2 transgenic animals were tested with 1–50 μM of SST-28. As controls, the responses to 1% SDS, M9 diluent, the unrelated neuropeptides, neurotensin and MIP-1α were determined. Sstr2 transgenic animals did not avoid 25 μM cyclosomatostatin. Both strains display a normal avoidance response to 1% SDS. Asterisks indicate a statistically significant difference between wild type and Sstr2 transgenic animals. (C) Avoidance responses of wild type and CCR5 transgenic animals to various concentrations of MIP-1α, M9 diluent, 1% SDS and 25 μM SST-28. Error bars denote the standard error of the mean. Asterisks indicate a statistically significant difference between wild type and CCR5 transgenic animals. (D) Animals were either directly tested for their response to M9, SST-28 or 1% SDS or pre-incubated with 25 μM cyclosomatostatin (denoted cyclosst in figure) for 3 minutes before the assays. Pre-treatment with 25 μM cyclosomatostatin abolished the avoidance response of Sstr2 transgenic animals to 25 μM SST-28. Avoidance responses can be recovered by an additional 5 minutes wash to remove cyclosomatostatin. Asterisks indicate a statistically significant difference between cyclosomatostatin treated and untreated transgenic animals. In all panels, each data point represents an average of at least five independent assays for wild-type and each respective strain. Error bars denote the standard error of the mean. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005). Avoidance index = number of worms behind barrier/total number of worms