Fig 1.
lin-32 is required for proper optimization of probability of omega turn with proportional stimulus strength.
(A) A scheme of the avoidance behaviors [11]. After contacting a noxious stimulus (x), an animal exhibits backward behavior. Short and long reversal are subdivided by the backward length with 1~2 head swings or three head swings or greater without omega turn, respectively. An omega turn is specifically defined by reorientation angles (θ ≧135) following the backward behaviors with/without either short reversal or long reversal. (B) lite-1 mutants expressing ChR2(H134R) in ASH sensory neurons (ASH::ChR2(H134R); lite-1) exhibit short reversal (left panel, dashed line) with 25% light irradiation (x) and omega turns (right panel, dashed line) with 100% light irradiation (x). (C) ASH::ChR2(H134R); lite-1 animals with ATR can optimize their dominant behaviors from reversals to omega turns with proportional to light intensity. n = 6,7,9,8,7,11. (D) Wild type animals adjust probability of omega turns depending on the sorbitol drop concentration. n = 17,30,20,20,20. (E) ASH::ChR2(H134R); lin-32 lite-1 animals showed reduced omega turns even with ATR and 100% light intensity. n = 4,7,7,4,7,7. (F) lin-32 animals show reduced omega turns independent on the sorbitol drop concentration. n = 20,7,33,20,12. n = plate (cohort) of approximately 10–20 animals. The data are presented as the mean ± SEM.
Fig 2.
lin-32 is required for the determination of AIB interneurons, which are the key neurons for the optimization of probability of omega turn.
(A) A scheme of the first central layer interneurons in the primary and secondary circuits. ASH directly connects to AVA and AIB via chemical synapses. The fax-1 gene induces expression of nmr-1 in AVA. (B) fax-1 mutants show a partial avoidance defect to 2 M sorbitol, and lin-32 fax-1 double mutants show more reductions in omega turns and long reversals than the lin-32 and fax-1 single mutants. n = 20,19,20,21,20,17,15,17. (C) Wild type animal selectively expresses an electrical synapse gene, inx-1, in its AIB neurons (arrowhead), but a typical lin-32 mutant fails. fax-1 mutant normally expresses inx-1 (arrowhead). (D) The extrachromosomal expression of lin-32 cDNA driven by both its own promoter Plin-32 and AIB lineage-selective promoter Punc-130 fully rescues avoidance behaviors in response to 2 M sorbitol. n = 21,21,13,16. n = plate (cohort) of approximately 10–20 animals. The data are presented as the mean ± SEM.
Fig 3.
Strong stimulus-dependent OFF calcium increases that are present in AIB and AVA interneurons are absent in lin-32 mutants.
(A) Average calcium responses of AIB to 30-sec 2 M sorbitol stimuli under microfluidic chips. AIB OFF calcium increases occur in wild type animals but not in lin-32 mutants. Wild type, n = 19; lin-32 mutants, n = 22. (B) A schematic drawing of how AIB connects to three downstream neurons through chemical and/or electrical synapses. (C, C') Weak ON calcium increases occur in AVA in both animals (see an enlarged view at Fig 3C' shown by a dashed line), whereas strong OFF calcium increases occur only in wild type animals but not in lin-32 mutants to 30-sec 2 M sorbitol. Wild type, n = 28; lin-32 mutants, n = 35. (D-E) ON calcium increases in RIM (D) and RIV (E) occur in both animal types to 30-sec 2 M sorbitol. Wild type, n = 15 and 28, respectively; lin-32 mutants, n = 17 and 33, respectively. (F) Both animals show normal biphasic intracellular calcium increase upon the introduction and removal of the 30-sec 2 M sorbitol stimulus (ON and OFF calcium increases). Wild type, n = 10; lin-32 mutants, n = 16. (G) Both animals show short peak duration of intracellular calcium increase upon the 30-sec 1 M sorbitol stimulus. n = 20. (H) AIB does not respond to 30-sec 1M stimuli in either type of animal. Wild type, n = 22; lin-32 mutants, n = 20, respectively. (I, I') OFF calcium increases do not occur in AVA in either type of animal upon the 30-sec 1 M sorbitol. An enlarged view at Fig 3I' shown by a dashed line. n = 21, 22. n means the number of individuals (animals). The data are presented as the mean ± SEM.
Fig 4.
AIB calcium increases probability of omega turns.
(A) AIB expressing HisCl1 (AIB::HisCl) inhibits omega turns in the presence of histamine. n = 4,6,16,7,4,5,16,5. n = plate (cohort) of approximately 10–20 animals. The data are presented as the mean ± SEM. (B) ChR2-induced excitation of AIB (AIB::ChR2(H134R)) increases omega turns during and after the 2-sec stimulation. n = 49, 57. n means the number of individuals (animals). (C) AVA expressing HisCl1 (AVA::HisCl) inhibits all avoidance behaviors in the presence of histamine. n = 6,6,7,7,6,6. n = plate (cohort) of approximately 10–20 animals. The data are presented as the mean ± SEM.
Fig 5.
AIB electrical synapses are required for the optimization of probability of omega turn.
(A) inx-1 mutants show impaired avoidance behaviors independent on the sorbitol concentration. n = 33,10,35,10,10. (B) The frequency of avoidance behaviors toward 2 M sorbitol. inx-1 mutants show reduced omega turns compared to the wild type animals. The inx-1 phenotypes were not different from those of lin-32 mutants and lin-32 inx-1 double mutants. n = 4,4,4,12. (C) Expression of inx-1 cDNA driven by its own promoter (inx-1; Pinx-1::INX-1) and the AIB lineage-selectively promoter (inx-1; Punc-130::INX-1) fully rescues osmotic avoidance defects in response to 2 M sorbitol. n = 21,21,22,13. (D) Expression of Punc-130::INX-1 cDNA fully rescues osmotic avoidance defects in lin-32 mutants in response to 2 M sorbitol. n = 21,21,13. (E) AIB::gfp::TeTx transgenic animals can adjust probability of omega turns depending on the sorbitol drop concentration. n = 8,8,9,9,9. n = plate (cohort) of approximately 10–20 animals. The data are presented as the mean ± SEM.
Fig 6.
Omega turns are correlated with strong and broad calcium mobilization, and lin-32 and inx-1 mutants show impaired calcium mobilization.
(A) Heat maps of calcium imaging of the neck muscles in vivo. Arrows indicate the area with high calcium. (B) Scatter plot of the maximum calcium fluorescence changes in individuals. The values are higher for omega turns than for reversals. **p < 0.01, ANOVA followed by the Tukey's post hoc tests. n = 24,18,17. (C) Scatter plot of the areas of the calcium increase in individuals. Areas are broader for omega turns than for reversals. ***p < 0.001, ANOVA followed by the Tukey's post hoc tests. n = 23,20,21. (D) Random samples of lin-32 and inx-1 mutants show tendency of impaired calcium fluorescence changes. *p < 0.05, ANOVA followed by the Tukey's post hoc tests. n = 20 in each strain. (E) Random samples of lin-32 and inx-1 mutants showed tendency of decreased calcium-induced areas. *p < 0.05, ANOVA followed by the Tukey's post hoc tests. n = 20 in each strain. n means the number of individuals (animals). Plots represent the mean ± SD.
Fig 7.
A hypothetical model for the optimization of avoidance behaviors.
A schematic model illustrating the neural circuit for the optimization of omega turns and reversals. The dotted arrows indicate the developmental step promoted by lin-32.