Fig 5. MIP-signaling mediates salt avoidance learning that shows hallmarks of long-term memory.

(A) Overview of taste associative learning assays. Synchronized young adult C. elegans are conditioned for six hours on NaCl-containing plates with or without bacterial food. Salt chemotaxis behavior of worms is then tested on NaCl concentration gradients. The CI is calculated from the number of worms that migrated toward the lower and upper parts of the gradient [8–10]. Worms chemotax to the salt concentrations at which they were fed, but avoid salt concentrations at which they were starved. (B) Mutants of mip-1 and sprr-2 chemotax to the salt concentrations at which they were fed and do not show any significant salt avoidance learning defects compared to wild-type animals when conditioned at 25 mM NaCl in the absence of food, but do show significantly impaired salt avoidance learning at 100 mM NaCl. (C) Expression of sprr-2 under its promoter sequence [sprr-2p::sprr-2] rescues the salt avoidance learning defect of sprr-2 mutants. (D) Expression of mip-1 under control of its endogenous promoter sequence [mip-1p::mip-1] rescues the salt avoidance learning defect of mip-1 mutants. (E) A mip-1; sprr-2 double mutant has no additive learning defect. (F) Blocking translation (cycloheximide) or transcription (actinomycin D) during conditioning impairs salt avoidance learning in wild-type animals. (G) Salt avoidance learning of mutants for transcription factors and regulators linked to learning and memory. (H) A sprr-2; crh-1 double mutant displays a statistically significant additive learning defect as compared to the single mutants. For (B-H), boxplots indicate 25^th (lower boundary), 50^th (line), and 75^th (upper boundary) percentiles. Whiskers show minimum and maximum values. Individual CIs, blue dots. Outliers, black dots. Statistical comparisons by one-way ANOVA and Tukey post-hoc test (n ≥ 6). *p<0.05; **p<0.005; ***p<0.001; n.s., not significant.