Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt chemotaxis in Caenorhabditis elegans

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1 Supplementary Information for: Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt chemotaxis in Caenorhabditis elegans Hirofumi Kunitomo, Hirofumi Sato, Ryo Iwata, Yohsuke Satoh, Hayao Ohno, Koji Yamada, and Yuichi Iino Supplementary Figures S1-S11 Supplementary Tables S1 and S2-1 -

Concentrations of chloride ion were measured by a chloride electrode and results from four independent

2 Supplementary Figure S1 NaCl gradient of the test plates for salt chemotaxis assay. (a) Generating a salt gradient on a test plate. Concentrations of chloride ion were measured by a chloride electrode and results from four independent preparations were averaged for each data point in the right panel. (b) Salt gradient at the surface of the test plates calculated by simulation of three-dimensional NaCl diffusion. See Methods for details

3 Chemotaxis index OP50 NA22 Cultivation 25 mm 50 mm 100 mm Supplementary Figure S2 Difference in bacterial strains as food source does not affect cultivation salt concentration-dependent chemotaxis. Chemotaxis of wild-type animals cultivated by OP50 (left) or NA22 (right). Mean ± s.e.m.; n = 8 assays

(b) Chemotaxis of wild-type animals cultivated at various NaCl concentrations for 6 hours with or without food. Mean ± s.e.m.; n = 5 assays.

4 Supplementary Figure S3 C. elegans avoids the salt-concentration at which it has experienced starvation. (a) Representative trajectories of wild-type animals cultivated at different NaCl concentrations for 6 hours under starvation conditions. (b) Chemotaxis of wild-type animals cultivated at various NaCl concentrations for 6 hours with or without food. Mean ± s.e.m.; n = 5 assays. (c) Shift of preferred salt concentrations under starvation. Wild-type animals were cultivated at 50 mm NaCl and transferred to bacteria-free plates with either 25 mm, 50 mm or 100 mm NaCl. Chemotaxis assays were performed at the indicated time points. Mean ± s.e.m.; n = 5 assays. (d) Migration bias in klinokinesis (left panel) and klinotaxis (right panel) of fed and starved wild-type animals. Mean ± s.e.m.; n = 16 tests; (**) p < 0.001; comparison between the same cultivation-salt concentrations; two-tailed t-test

5 Chemotaxis index Chemotaxis index a None NaCl Glycerol Sorbitol b Wild type osm-9 * * * osm-10 Cultivation 25 mm 50 mm 100 mm Supplementary Figure S4 Chemotaxis to lower salt concentrations is distinct from osmotic avoidance. (a) Chemotaxis of wild-type animals cultivated at 0 mm NaCl (None), 50 mm NaCl (NaCl), 100 mm glycerol/0 mm NaCl (Glycerol) or 100 mm sorbitol/0 mm NaCl (Sorbitol). Mean ± s.e.m.; n 4 assays. (b) Chemotaxis of osmotic avoidance mutants. Mean ± s.e.m.; n = 6 assays; (*) p < 0.01, different from wild type cultivated at the same condition; Dunnett test

6 Chemotaxis index ** n.s. n.s. (p = 0.055) n.s. NaCl Dia Bz Non Cultivation 0 mm 50 mm Supplementary Figure S5 Chemotaxis to NaCl but not to odorants is affected by NaCl concentrations during cultivation. Chemotaxis of wild-type animals to NaCl and odorants. Animals were cultivated at 0 mm or 50 mm of NaCl. Mean ± s.e.m.; n 4 assays; (**) p < 0.001, (n.s.) not significant (p > 0.05); two-tailed t-test. Dia, diacetyl; Bz, benzaldehyde; Non, 2-nonanone

Fed (a) or starved (b) wild type and fed ASER-ablated animals (c). Mean ± s.e.m.; n 9 assays; (+) p < 0.05, (*) p < 0.

7 Supplementary Figure S6 Quantitative analyses of animals' locomotion. (a-c) Migration bias of klinokinesis represented by probability of pirouette (left panels) and the bias of klinotaxis represented by curving rate (right panels). Fed (a) or starved (b) wild type and fed ASER-ablated animals (c). Mean ± s.e.m.; n 9 assays; (+) p < 0.05, (*) p < 0.01; Dunnett test compared to the basal pirouette index (dc/dt = 0) or the basal weathervane index (dc/dn = 0; n represents the coordinated perpendicular to the direction of locomotion). (d) Averaged speed of fed wild-type animals calculated according to the NaCl concentrations by 10 mm bins. Mean ± s.e.m.; n = 29 assays

Averaged fluorescence change during 5 s (ASER) or 25 s (AIB) after stimulation are shown in bar graphs. The number of tested animals for each condition is indicated in parentheses.

8 Supplementary Figure S7 Calcium responses of ASER and AIB are modulated by the salt concentrations of cultivation. (a,b) Calcium responses of ASER (left panels) and AIB (right panels) to a NaCl down-step from 25 mm to 0 mm (a) or that from 100 mm to 75 mm (b). Averaged fluorescence change during 5 s (ASER) or 25 s (AIB) after stimulation are shown in bar graphs. The number of tested animals for each condition is indicated in parentheses. In both panels a and b, statistics for ASER responses are: (*) p < 0.01, (n.s.) not significant; Tukey test; details are shown in Supplementary Table S1. Statistics for AIB responses are: (+) p < 0.05, (*) p < 0.01, (**) p < 0.001, (n.s.) not significant; Mann-Whitney U test compared to pre-stimulation. (c) ASER does not respond to changes in fluid flow. The direction of solution flow was switched at Time = 50 s (indicated by arrow) with NaCl concentration unchanged. (d) Responses of ASER to a stepwise increase in NaCl concentration from 25 mm to 50 mm. Procedure and time course of these experiments are the same as that used in down-step experiments (see Methods for details). Shaded region around the response curve and the error bars represents s.e.m

Averaged fluorescence change during 5 s (ASER) or 25 s (AIB) after stimulation are shown in the bar graphs. Shaded region around the response curve and the error bars represents s.e.m. The number of tested animals for each condition is indicated in parentheses.

9 Supplementary Figure S8 Plasticity of calcium responses of ASER and AIB is dependent on sensory inputs to ASER. Calcium responses of ASER (left panels) and AIB (right panels) to a NaCl down-step from 50 mm to 25 mm in dyf-11 mutants (a) and in animals whose ASER was solely rescued (b). Averaged fluorescence change during 5 s (ASER) or 25 s (AIB) after stimulation are shown in the bar graphs. Shaded region around the response curve and the error bars represents s.e.m. The number of tested animals for each condition is indicated in parentheses. Statistics for ASER responses are shown in Supplementary Table S1. Statistics for AIB responses are: (+) p < 0.05, (**) p < 0.001, (n.s.) not significant; Mann-Whitney U test compared to pre-stimulation

10 Chemotaxis index Pirouette index Weathervane index a p < p > 0.05 p < Wild type AIB-ablated p > 0.05 Wild type p > 0.05 p > 0.05 AIB-ablated Cultivation 25 mm (Ccult < Ctest) 50 mm (Ccult = Ctest) 100 mm (Ccult > Ctest) b p > 0.05 p = p > 0.05 Wild type AIB-ablated Cultivation 25 mm 50 mm 100 mm Supplementary Figure S9 Ablation of AIB affects the property of klinokinesis but not klinotaxis. (a) Bias of klinokinesis (left) and klinotaxis (right) of AIB-ablated animals. Mean ± s.e.m.; n = 16 assays; two-tailed t-test. (b) Chemotaxis of AIB-ablated animals. Mean ± s.e.m.; n = 6 assays; two-tailed t-test

Supplementary Figure S10 Gene structure and expression patterns of plc-1. (a) Schematic representation of the plc-1 locus. (Top row) Scale indicates physical position of chromosome X.

11 Supplementary Figure S10 Gene structure and expression patterns of plc-1. (a) Schematic representation of the plc-1 locus. (Top row) Scale indicates physical position of chromosome X. (Second row) Predicted gene structure of frm-9 and plc-1 in WormBase Release WS230. (Third row) Molecular lesion of the plc-1 alleles. Asterisk indicates nucleotide substitution in pe1237. Thick bars depict deletion in pe1238 and tm753. (Bottom row) Structure of the fosmids and cdna clones and results of plc-1(pe1238) rescue experiments. See Methods for details. (b) Expression patterns of a plc-1 transcriptional fusion in wild type. A variant of yellow fluorescent protein, venus, was driven by a 5-kbp 5 upstream fragment of plc-1. (Top) An adult animal. (Bottom) The anterior part of an L1 animal. A differential interference contrast (DIC) image (left) and a fluorescence image of the same field in which venus (green) and a lipophilic dye diq (red) were combined (right). diq stains several sensory neurons that act as position markers

respond to decreases in salt concentration and trigger pirouette turning (left column).

12 Supplementary Figure S11 A possible neuronal mechanism that regulates cultivation concentration-dependent salt chemotaxis. When salt concentration of cultivation was higher than that of current condition (C cult > C test ), ASER and AIB neurons respond to decreases in salt concentration and trigger pirouette turning (left column). But if C cult < C test, AIB does not respond and animals maintain the direction of migration toward lower salt (right column). See text for details

13 Supplementary Table S1 Summary of the calcium responses of the ASER neuron. No. Figure Genetic background of the host strain Cultivation Stimulus n Ratio change *1 Response *2 Comparison p value *3 1 0 mm ± vs 2 < mm ± vs 3 < a from 50 mm to 25 mm 3 50 mm ± vs 4 < mm ± vs 3 < mm ± vs S7a 25 mm from 25 mm to 0 mm ± vs 4 > 0.05 Wild-type 7 50 mm ± vs 6 > mm ± 0.07 n.s. 5 vs 7 > mm ± vs 7 > 0.05 S7b from 100 mm to 75 mm mm ± vs 9 < mm ± vs 10 < S7c 50 mm from 50 mm to 50 mm ± 0.06 n.s. 8 vs 11 < mm ± vs 10 > S8a dyf-11(pe554) 50 mm from 50 mm to 25 mm ± vs 11 < mm ± vs mm ± vs 14 > S8b dyf-11(pe554); Ex[gcy-5p::dyf-11(+)] 50 mm from 50 mm to 25 mm ± vs 15 > mm ± vs 15 > mm ± vs 17 < c plc-1(pe1237) 50 mm from 50 mm to 25 mm ± vs 18 < mm ± vs 18 > mm ± vs 20 < d plc-1(pe1238) 50 mm from 50 mm to 25 mm ± vs 21 < mm ± vs 21 > mm ± vs 23 < e plc-1(pe1238); Ex[gcy-5p::plc-1(+)] 50 mm from 50 mm to 25 mm ± vs 24 < mm ± vs 24 > 0.05 *1 Averaged ratio change after stimulation ± SD 25 vs 26 < 0.01 *2 Comparison of fluorescence intensity between pre- and post- stimulation by Mann-Whitney U test. (+) p < 0.01, (n.s.) p > vs 27 < 0.01 *3 Comparison between the indicated data sets by Tukey test. 26 vs 27 >

14 Supplementary Table S2 C. elegans strains and transgenic lines used in this study. Strain Genotype Description Bristol N2 C. elegans wild isolate Wild type. JN929 egl-30(pe914) I. Putative gain-of-function (gf) allele of egl PR674 che-1(p674) I. TJ1052 age-1(hx546) II. CB1370 daf-2(e1370) III. MT3641 osm-10(n1602) III. CX10 osm-9(ky10) IV. ins-1(nr2091) IV. RB759 akt-1(ok525) V. IK130 pkc-1(nj3) V. FX02364 gcy-22(tm2364) V. JN554 dyf-11(pe554) X. JN1239 plc-1(pe1237) X. JN1240 plc-1(pe1238) X. Genetic ablation of ASE and AIB OH8585 otis4[gcy-7p::gfp]; otex3822[ceh-36p::cz-caspase3(p17) gcy-7p::caspase3(p12)-nz myo-3p::mcherry]. Genetic ablation of ASEL 4. OH8593 ntis1[gcy-5p::gfp] V; otex3830[ceh-36p::cz-caspase3(p17) gcy-5p::caspase3(p12)-nz myo-3p::mcherry]. Genetic ablation of ASER 4. Is[npr-9p::casp1 unc122p::mcherry]; Is[npr-9p::venus]. Genetic ablation of AIB. Cell-specific rescue of dyf-11 Ex[myo-3p::venus]. Control with wild-type background. dyf-11(pe554) X; Ex[myo-3p::venus]. dyf-11(pe554) control. dyf-11(pe554) X; Ex[dyf-11p::dyf-11(+) myo-3p::venus]. Rescued in ciliated neurons. dyf-11(pe554) X; Ex[gcy-5p::dyf-11(+) myo-3p::venus]. Rescued specifically in ASER. dyf-11(pe554) X; Ex[gcy-7p::dyf-11(+) myo-3p::venus]. Rescued specifically in ASEL. dyf-11(pe554) X; Ex[gcy-5p::dyf-11(+) gcy-7p::dyf-11(+) myo-3p::venus]. Rescued in both ASEL and ASER. Stimulation of ASER by TRPV1 Ex[unc-122p::mCherry]. Control. Ex[gcy-5p::trpv1 unc-122p::mcherry]. ASER-specific expression of TRPV1. Calcium imagig Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. Wild-type background 18. plc-1(pe1237) X; Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. plc-1(pe1237) background. plc-1(pe1238) X; Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. plc-1(pe1238) background. plc-1(pe1238) X; Ex[unc-122p::mCherry]; Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. plc-1(pe1238) control. plc-1(pe1238) X; Ex[gcy-5p::plc-1(+) unc-122p::mcherry]; Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. ASER-specific rescue of plc-1(pe1238). dyf-11(pe554) X; Ex[unc-122p::mCherry]; Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. dyf-11(pe554) control. dyf-11(pe554) X; Ex[gcy-5p::dyf-11(+) unc-122p::mcherry]; Ex[gcy-5p::GCaMP2 lin-44p::gfp]. Imaging of ASER. ASER-specific rescue of dyf-11(pe554). Ex[odr-2p::GCaMP1; unc-122p::gfp]. Imaging of AIB. Wild-type background 22. plc-1(pe1237) X; Ex[odr-2p::GCaMP1 unc-122p::gfp]. Imaging of AIB. plc-1(pe1237) background. plc-1(pe1238) X; Ex[odr-2p::GCaMP1 unc-122p::gfp]. Imaging of AIB. plc-1(pe1238) background. plc-1(pe1238) X; Ex[unc-122p::mCherry]; Ex[odr-2p::GCaMP1 unc-122p::gfp]. Imaging of AIB. plc-1(pe1238) control. plc-1(pe1238) X; Ex[gcy-5p::plc-1(+) unc-122p::mcherry]; Ex[odr-2p::GCaMP1 unc-122p::gfp]. Imaging of AIB. ASER-specific rescue of plc-1(pe1238). dyf-11(pe554) X; Ex[unc-122p::mCherry]; Ex[odr-2p::GCaMP1 unc-122p::gfp]. Imaging of AIB. dyf-11(pe554) control. dyf-11(pe554) X; Ex[gcy-5p::dyf-11(+) unc-122p::mcherry]; Ex[odr-2p::GCaMP1 unc-122p::gfp]. Imaging of AIB. ASER-specific rescue of dyf-11(pe554). Optogenetics lite-1(ce314)x; peis1091[gcy5p::chr2 unc122p::mcherry]. Optical stimulation of ASER. lite-1(ce314)x; peis1091[gcy5p::chr2 unc122p::mcherry]; Is[npr-9p::casp1 unc122p::mcherry]; Is[npr-9p::venus]. Optical stimulation of ASER under genetic ablation of AIB. lite-1(ce314)x; Ex[npr-9p::ChR2 unc-122p::mcherry]. Optical stimulation of AIB. Cell-specific activation or knock-down of pkc-1 Ex[unc-122p::mCherry]. Control. Ex[gcy-5p::pkc-1(gf) unc-122p::mcherry]. ASER-specific expression of pkc-1(gf) 10. Ex[gcy-5p::ccdB(hpRNAi) myo-3p::venus]. ASER-specific RNAi control. Ex[gcy-5p::pkc-1(hpRNAi) myo-3p::venus]. ASER-specific knock-down of pkc-1. Cell-specific rescue of plc-1 Ex[unc-122p::mCherry]. Control with wild-type background plc-1(pe1238) X; Ex[unc-122p::mCherry]. plc-1(pe1238) control. plc-1(pe1238) X; Ex[flp-6p::plc-1(+) unc-122p::mcherry]. Rescued in several neuron classes including ASE. plc-1(pe1238) X; Ex[gcy-5p::plc-1(+) unc-122p::mcherry]. Rescued specifically in ASER. plc-1(pe1238) X; Ex[gcy-7p::plc-1(+) unc-122p::mcherry]. Rescued specifically in ASEL. plc-1(pe1238) X; Ex[npr-9p::plc-1(+) unc-122p::mcherry]. Rescued in AIB. plc-1(pe1238) X; Ex[odr-2p::plc-1(+) unc-122p::mcherry]. Rescued in several neuron classes including AIB

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nature10643 Supplementary Table 1. Identification of hecw-1 coding polymorphisms at amino acid positions 322 and 325 in 162 strains of C. elegans. WWW.NATURE.COM/NATURE 1 Supplementary Figure