Article Figures & Data
Figures
- Figure 1.
C. elegans generates an aversive response to mild osmotic upshifts. A, A sample image of the droplet assay (Materials and Methods). Each 6 µl droplet of buffer contains one worm, and the arrow denotes a large body bend. B, WT animals that are cultivated under the standard osmotic condition (∼150 mOsm) display a stable turning rate in a glycerol solution of 150 mOsm but gradually and significantly increase the turning rate in a hyperosmotic glycerol solution of 400 mOsm. n = 55 each. C, WT animals that are cultivated under the standard osmotic conditions (∼150 mOsm) display a significantly lower thrashing speed in a hyperosmotic glycerol solutions of 400 mOsm than in a glycerol solution of 150 mOsm. n = 55 each. D, E, WT animals that are cultivated under the standard osmotic condition (∼150 mOsm) also gradually and significantly increase the turning rate in the hyperosmotic glycerol solutions of 600 mOsm (D; n = 27 each) or 800 mOsm (E; n = 12 each). Note that in E the turning rate starts to drop after 3 min in the 800 mOsm solution, due to decreased mobility of the animals possibly caused by server dehydration. Therefore, the statistical test is performed on the turning rates in the 4th minute in E. F, G, WT animals cultivated under the standard osmotic condition (∼150 mOsm) display an increased turning rate in a 400 mOsm hyperosmotic solution that is balanced by either NaCl (F; n = 24 each) or sorbitol (G; n = 24 each) in comparison with the turning rate in the 150 mOsm solution. For B–G, the turning rates in solutions of different osmolarities in the 5th minute (in the 4th minute in E) are compared using the Student’s t test. H, WT animals that are cultivated under the standard osmotic condition (∼150 mOsm) display a stable turning rate on a NGM plate of 150 mOsm but gradually and significantly increase the turning rate on a NGM plate of 400 mOsm. The osmolarity of the plates is balanced by sorbitol. The turning rates on plates of different osmolarity are compared using the Student’s t test. n = 7 animals each. For all, ***p < 0.001, **p < 0.01. Values are reported as the mean ± SEM. WT, wild type.
- Figure 2.
The increase in the turning rate is part of the physiologic response to hyperosmotic stresses. A, Wild-type animals cultivated under the standard NGM plates of 150 mOsm display a higher turning rate in a hyperosmotic solution of 400 mOsm (NGM_400 mOsm, n = 32) than in a 200 mOsm solution (NGM_200 mOsm, n = 28); but animals that are cultivated on the plates of 400 mOsm display a turning rate in the solution of 400 mOsm (adapted_400 mOsm, n = 32) that is similar to that in the solution of 200 mOsm (adapted_200 mOsm, n = 32). Significant interaction between cultivating conditions and osmolarity is tested by two-way ANOVA. B, The osr-1(rm1) mutant animals do not display an increased turning rate in the hyperosmotic solution of 400 mOsm (n = 29) compared with the turning rate in 150 mOsm (n = 28). Both the mutant and WT animals are cultivated under the standard osmotic condition of 150 mOsm. n = 29 and 31 for wild-type animals tested in solutions of 150 and 400 mOsm, respectively. C, The dpy-2(e8) mutant animals do not display an increased turning rate in the hyperosmotic solution of 400 mOsm (n = 16) compared with the turning rate in 150 mOsm (n = 16). Both the mutant and wild-type animals are cultivated under the standard osmotic condition of 150 mOsm. n = 16 for WT animals tested in each condition. For B and C, significant interaction between genotype and osmolarity is tested by two-way ANOVA. For all, ***p < 0.001. Values are reported as the mean ± SEM. WT, wild type.
- Figure 3.
The cGMP-gated channel subunit TAX-2 acts in the AQR, PQR, and URX neurons to regulate the aversive response to osmotic upshifts. A, The mutant animals osm-9(ky10) cultivated under the standard condition display an increased turning rate in the hyperosmotic solution of 400 mOsm that is similar to that of WT animals. n = 31 and 29 WT animals tested in 150 and 400 mOsm, respectively; n = 32 and 31 osm-9 animals tested in 150 and 400 mOsm, respectively. B, C, Two mutation alleles of tax-2, p691 and p671, are severely defective in increasing the turning rate in response to the osmotic upshift from 150 to 400 mOsm, compared with WT animals. For B, n = 24 WT animals were tested in either 150 or 400 mOsm and n = 20 and 24 p691 animals were tested in 150 and 400 mOsm, respectively; for C, n = 23 and 24 WT animals were tested in 150 and 400 mOsm, respectively, and n = 21 and 27 p671 animals were tested in 150 and 400 mOsm, respectively. D, E, Expressing the genomic DNA fragment containing the coding region and regulatory sequences of tax-2 (Ptax-2::tax-2) rescues the defects in both tax-2 alleles. For D, n = 32 and 31 WT animals tested in 150 and 400 mOsm, respectively; n = 29 and 31 transgenic animals expressing Ptax-2::tax-2 tested in 150 and 400 mOsm, respectively; and n = 28 and 26 p691 mutant sibling animals tested in 150 and 400 mOsm, respectively; for E, n = 31 and 32 WT animals were tested in 150 and 400 mOsm, respectively; n = 31 and 32 transgenic animals expressing Ptax-2::tax-2 were tested in 150 and 400 mOsm, respectively; and n = 30 p671 mutant sibling animals were tested in either 150 or 400 mOsm. F, Expressing the wild-type tax-2 cDNA in the body cavity sensory neurons AQR, PQR, URX (Pgcy-36::tax-2) rescues the defects in the tax-2(p691) mutants in generating an increased turning rate in response to the osmotic upshift from 150 to 400 mOsm. n = 24 and 22 WT animals tested in 150 and 400 mOsm, respectively, n = 22 and 24 transgenic animals expressing Pgcy-36::tax-2 tested in 150 and 400 mOsm, respectively, n = 23 and 24 p691 mutant sibling animals tested in 150 and 400 mOsm, respectively. G, Expressing cell death-promoting molecule EGL-1 in the body cavity sensory neurons AQR, PQR, and URX (Pgcy-36::egl-1) abolished the increased turning rate in response to the osmotic upshift from 200 to 400 mOsm. n = 36 and 44 WT animals were tested in 200 and 400 mOsm, respectively; n = 40 and 44 transgenic animals expressing Pgcy-36::eg-1 were tested in 200 and 400 mOsm, respectively. H, The heat map of the calcium signals in the URX neuron in individual animals that were exposed to 150 mOsm and switched to 400 mOsm and in individual animals that were exposed to 150 mOsm and switched to 150 mOsm. The calcium signal was presented as the percentage change in GCaMP6 signal using the average GCaMP6 signal in the first 60 s as the baseline [(F − Fbase)/Fbase * 100%; see Materials and Methods]. I, The average change in the GCaMP6 signal in URX is significantly higher when animals are switched from 150 to 400 mOsm (n = 15) than when animals are switched from 150 to 150 mOsm (n = 13). Box plot shows the first and third quartile, median, and the maximum and minimum. The Student’s t test was used for comparison. For A–C and G, significant interaction between genotype (wild-type vs mutant animals) and osmolarity is tested with two-way ANOVA; for D–F, significant interaction between genotype (transgenic vs nontransgenic siblings) and osmolarity is tested with two-way ANOVA. For all, animals are cultivated under the standard NGM plates with an osmotic condition of 150 mOsm. ***p < 0.001, **p < 0.01, *p < 0.05. n.s., Not significant. Values are reported as the mean ± SEM. WT, wild type.
- Figure 4.
The neural circuit underlying navigation regulates the aversive response to osmotic upshifts. A, Inhibiting the activity of the interneurons AIB and AIY by expressing twk-18(gf) produces significant defects in generating an increased turning rate in response to the osmotic upshift from 150 to 400 mOsm. n = 40 and 38 transgenic animals expressing twk-18(gf) in AIB and AIY were tested in 150 and 400 mOsm, respectively; and n = 40 and 38 WT sibling animals were tested in 150 and 400 mOsm, respectively. B, Inhibiting AIB, AIY, and AIZ by expressing twk-18(gf) produces significant defects in generating an increased turning rate in response to the osmotic upshift from 150 to 400 mOsm. n = 40 and 33 transgenic animals expressing twk-18(gf) in AIB, AIY, and AIZ were tested in 150 and 400 mOsm, respectively; n = 40 and 39 WT sibling animals were tested in 150 and 400 mOsm, respectively. C, Blocking the neural transmission of several command interneurons and motor neurons by expressing the tetanus toxin with the glr-1 promoter produces significant defects in generating an increased turning rate in response to the osmotic upshift from 150 to 400 mOsm. n = 24 animals in all conditions. D, Blocking the neural transmission of several motor neurons by expressing the tetanus toxin with the lad-2 promoter produces significant defects in generating an increased turning rate in response to the osmotic upshift from 150 to 400 mOsm. n = 23 transgenic animals expressing the tetanus toxin with the lad-2 promoter were tested in either 150 or 400 mOsm; and n = 22 and 23 WT sibling animals were tested in 150 and 400 mOsm, respectively. For all, animals are cultivated on the standard NGM plates with an osmotic condition of 150 mOsm, and the significant interaction between genotype (transgenic vs nontransgenic siblings) and osmolarity is tested using two-way ANOVA, ***p < 0.001, **p < 0.01. Values are reported as the mean ± SEM. WT, wild type.
Tables
Preparation of testing solutions
Balancing solute Calculated osmolarity (mOsm) Measured osmolarity (mOsm) Glycerol 150 144 400 415 600 621 800 838 NaCl 150 144 400 369 Sorbitol 150 141 400 373 Mutated gene Gene family Number of turns/min for the 5th minute p value Wild-type control Mutant 150 mOsm 400 mOsm 150 mOsm 400 mOsm aqp-1 Aquaporin 9.50 ± 1.20 (16) 27.50 ± 1.79 (16) 13.56 ± 2.48 (16) 33.80 ± 3.09 (15) 0.617 aqp-2 Aquaporin 10.88 ± 2.67 (8) 16.63 ± 3.68 (8) 8.00 ± 2.03 (8) 20.14 ± 4.98 (7) 0.357 aqp-3 Aquaporin 5.93 ± 0.91 (15) 21.87 ± 1.67 (15) 6.00 ± 1.04 (14) 16.87 ± 2.25 (15) 0.114 aqp-4 Aquaporin 8.44 ± 0.93 (16) 21.94 ± 1.87 (16) 9.94 ± 1.40 (16) 29.38 ± 3.15 (16) 0.146 aqp-6 Aquaporin 10.25 ± 1.80 (8) 28.38 ± 2.04 (8) 7.50 ± 1.38 (8) 28.13 ± 3.70 (8) 0.606 aqp-7 Aquaporin 10.33 ± 1.54 (12) 29.00 ± 1.62 (16) 9.19 ± 1.68 (16) 23.06 ± 1.68 (16) 0.156 aqp-8 Aquaporin 9.31 ± 1.76 (16) 24.63 ± 2.40 (16) 12.17 ± 1.40 (12) 27.44 ± 2.93 (16) 0.993 aqp-9 Aquaporin 11.5 ± 1.24 (32) 33.16 ± 1.44 (32) 11.78 ± 1.31 (32) 27.41 ± 1.35 (32) 0.026 aqp-10 Aquaporin 9.50 ± 1.20 (16) 27.50 ± 1.79 (16) 6.63 ± 0.80 (16) 25.67 ± 1.67 (15) 0.714 aqp-11 Aquaporin 8.44 ± 0.93 (16) 21.94 ± 1.87 (16) 7.88 ± 0.74 (16) 25.56 ± 2.22 (16) 0.187 asic-1 Epithelial sodium channel (ENaC) 13.25 ± 0.95 (36) 29.49 ± 1.47 (37) 14.74 ± 1.12 (35) 32.05 ± 1.29 (39) 0.664 mec-4 Epithelial sodium channel (ENaC) 16.59 ± 1.48 (32) 31.29 ± 1.58 (28) 20.11 ± 2.05 (28) 35.72 ± 1.57 (32) 0.784 trp-4 Transient receptor potential (TRP) channel 11.17 ± 1.02 (15) 32.29 ± 2.06 (15) 14.54 ± 1.93 (15) 35.42 ± 1.91 (15) 0.944 tmc-1 Transmembrane channel-like protein 14.75 ± 1.09 (24) 27.83 ± 1.89 (24) 12.29 ± 1.20 (24) 29.92 ± 1.39 (24) 0.115 tmc-2 Transmembrane channel-like protein 12.75 ± 1.29 (20) 28.32 ± 2.15 (22) 10.63 ± 0.98 (24) 26.09 ± 1.96 (23) 0.975 tax-4 Cyclic nucleotide-gated ion channel 10.25 ± 1.15 (28) 22.32 ± 1.35 (28) 11.22 ± 1.13 (27) 20.86 ± 1.12 (28) 0.309 Values are reported as the mean ± SEM (n animals tested). Mutants with mutations in several channel-encoding genes are examined. The number of turns per minute is shown for the 5th minute for each genotype and osmolarity treatment. The interaction of genotype and treatment is tested with two-way ANOVA on the number of turns per minute in the 5th minute. The number of animals tested under each condition is indicated in the parenthesis next to mean ± SEM.
Mutated gene/transgene Affected neurons Number of turns/min for the 5th minute p value Wild-type control Mutant/transgenic 200# mOsm 400 mOsm 200# mOsm 400 mOsm ttx-1 AFD 10.40 ± 0.79 (30) 22.20 ± 2.35 (20) 9.86 ± 0.80 (28) 21.15 ± 1.86 (27) 0.863 che-1 ASE 14.36 ± 1.21 (28) 30.31 ± 1.72 (32) 18.19 ± 1.62 (32) 28.28 ± 1.41(32) 0.057 Psrh-142::twk-18 ADF 6.75 ± 0.73 (8) 26.00 ± 2.41 (8) 8.57 ± 1.65 (7) 24.25 ± 1.47 (8) 0.298 Values are reported as the mean ± SEM (n animals tested). Mutants defective in the development and/or function of several sensory neurons are examined for osmotic upshifts. ttx-1 and che-1, as well as the wild-type controls for these two mutants, are tested at 200 and 400 mOsm for osmotic upshifts. The transgenic animals that express Psrh-142::twk-18(gf) and the nontransgenic siblings are tested at 150 and 400 mOsm for osmotic upshifts. The number of turns per minute is shown for the 5th minute for each genotype and osmolarity treatment. The interaction of genotype and treatment is tested with two-way ANOVA on the number of turns per minute in the 5th minute. The number of animals tested under each condition is indicated in the parenthesis next to mean ± SEM.
↵# ttx-1 and che-1, as well as the wild-type controls for these two mutants, were tested at 200 and 400 mOsm for osmotic upshifts. The transgenic animals that express Psrh-142::twk-18(gf) and the nontransgenic siblings are tested at 150 and 400 mOsm for osmotic upshifts.
Genetic background Transgene Expressing neurons Number of turns/min for the 5th minute
Mean ± SEM (n)p value Nontransgenic control Transgenic animals 150 mOsm 400 mOsm 150 mOsm 400 mOsm tax-2(p691) Podr-1::tax-2 AWB, AWC 5.66 ± 0.69 (29) 9.33 ± 0.89 (30) 7.63 ± 1.15 (27) 9.86 ± 1.11 (28) 0.560 tax-2(p691) Pstr-3::tax-2 ASI 6.84 ± 0.61 (31) 12.31 ± 1.25 (32) 8.55 ± 0.83 (31) 17.14 ± 2.04 (29) 0.565 Values are reported as the mean ± SEM (n animals tested). Transgenic animals that express a wild-type tax-2 cDNA in the major olfactory sensory neurons AWB and AWC (Podr-1::tax-2) or in the sensory neuron ASI (Pstr-3::tax-2) in the tax-2(p691) mutant background are examined for osmotic upshifts. The number of turns per minute is shown for the 5th minute for each genotype (transgenic vs nontransgenic siblings) and osmolarity treatment. The interaction of genotype and treatment is tested with two-way ANOVA on the number of turns per minute in the 5th minute. The number of animals tested under each condition is indicated in the parenthesis next to mean ± SEM.
Mutated gene Expressing neurons+ Number of turns/min for the 5th minute p value Wild-type control Mutant 150 mOsm 400 mOsm 150 mOsm 400 mOsm gpa-8 AQR, PQR, URX 9.58 ± 1.02 (24) 24.61 ± 1.51 (23) 7.33 ± 0.85 (21) 20.67 ± 1.89 (21) 0.538 gpa-13 PQR 7.19 ± 1.13 (16) 16.13 ± 2.91 (15) 9.63 ± 2.23 (16) 21.69 ± 1.98 (16) 0.467 gcy-1 URX 8.19 ± 1.50 (16) 29.00 ± 1.91 (16) 5.88 ± 1.01 (16) 25.31 ± 2.22 (16) 0.691 gcy-25 AQR, PQR, URX 11.07 ± 1.52 (15) 26.73 ± 2.10 (15) 8.50 ± 1.10 (16) 23.13 ± 1.26 (16) 0.734 gcy-28 Widely expressed 9.31 ± 1.76 (16) 24.63 ± 2.40 (16) 9.00 ± 1.14 (16) 32.06 ± 2.18 (16) 0.049# gcy-32 AQR, PQR, URX 11.06 ± 1.80 (16) 27.60 ± 1.77 (15) 7.71 ± 0.71 (14) 25.81 ± 1.61 (16) 0.622 gcy-33 AQR, PQR, URX 10.94 ± 2.24 (16) 22.88 ± 2.67 (16) 8.88 ± 1.13 (16) 18.87 ± 2.34 (15) 0.655 gcy-34 AQR, PQR, URX 7.33 ± 0.84 (15) 22.94 ± 2.14 (16) 7.73 ± 1.01 (15) 20.19 ± 2.37 (16) 0.377 gcy-35 AQR, PQR, URX 9.83 ± 1.39 (23) 30.08 ± 1.25 (24) 9.68 ± 1.26 (22) 28.46 ± 1.40 (24) 0.578 gcy-36 AQR, PQR, URX 9.83 ± 1.39 (23) 30.08 ± 1.25 (24) 8.42 ± 1.18 (24) 27.39 ± 1.99 (23) 0.665 gcy-37 AQR, PQR, URX 9.94 ± 1.25 (16) 26.69 ± 2.15 (16) 6.93 ± 0.73 (15) 21.87 ± 2.14 (15) 0.592 Values are reported as the mean ± SEM (n animals tested). Mutants that are mutated for genes that encode G-protein pathway components and are expressed in AQR, PQR, and URX are examined. The number of turns per minute is shown for the 5th minute for each genotype and osmolarity treatment. The interaction of genotype and treatment is tested with two-way ANOVA on the number of turns per minute in the 5th minute. The number of animals tested under each condition is indicated in the parenthesis next to mean ± SEM.
↵+ Only the expression in AQR, PQR or URX neurons is indicated.
↵# The rate of turns increases more in the mutant than in the wild-type control.
Transgene Affected neurons Number of turns/min for the 5th minute p value Nontransgenic control Transgenic worms 150 mOsm 400 mOsm 150 mOsm 400 mOsm Podr-2b(3a)::twk-18(gf) AIB, AIZ 9.38 ± 1.19 (16) 25.69 ± 1.96 (16) 5.63 ± 1.38 (16) 22.60 ± 2.24 (15) 0.848 Pinx-1::twk-18(gf) AIB 7.44 ± 1.19 (16) 29.31 ± 2.22 (16) 1.63 ± 0.51 (16) 26.56 ± 2.04 (16) 0.355 Pttx-3::twk-18(gf) AIY 7.81 ± 0.99 (16) 25.06 ± 2.26 (16) 7.56 ± 1.08 (16) 23.40 ± 1.92 (15) 0.669 Values are reported as the mean ± SEM (n animals tested). Transgenic animals that express a gain-of-function isoform of the potassium channel TWK-18 to inhibit the activity of specific interneurons and/or motor neurons are examined. The number of turns per minute is shown for the 5th minute for each genotype (transgenic vs nontransgenic siblings) and osmolarity treatment. The interaction of genotype and treatment is tested with two-way ANOVA on the number of turns per minute in the 5th minute. The number of animals tested under each condition is indicated in the parenthesis next to mean ± SEM.
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