1. Dynamics of Presynaptic Diacylglycerol in a Sensory Neuron Encode Differences between Past and Current Stimulus Intensity 
Hayao Ohno, Naoko Sakai, Takeshi Adachi, Yuichi Iino 
Cell Reports 
Volume 20, Issue 10, Pages (September 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 2294-2303DOI: (10.1016/j.celrep.2017.08.038)
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3. Figure 1
The DAG/PKC Pathway in ASER Controls the Salt Concentration Preferences
(A) Schematic of the salt concentration preference assay. Worms conditioned with 0, 50, or 100 mM of NaCl were placed on an agar assay plate with a gradient of NaCl. An assay plate was divided into six areas, and the chemotaxis (CTX) index was calculated as shown.
(B) Worms were fed (top) or starved (bottom) at the indicated NaCl concentrations, and their distributions on assay plates were tested. n ≥ 19 plates (4–6 animals per plate).
(C) The salt concentration preference assay was performed on goa-1(n1134) conditioned with 50 mM NaCl and food. n = 9 plates (5–6 animals per plate). The CTX index is shown in (D).
(D) CTX indices of wild-type, egl-30(pe914 gf), goa-1(n1134), ttx-4(nj3), and Is[ASERp::ttx-4(gf)] worms conditioned with 50 mM NaCl and food. n ≥ 9 plates (4–6 animals per plate). ∗∗∗p < (ANOVA with Dunnett’s post-test).
(E–G) The salt concentration preference assay was performed on animals carrying Pgcy-5::Cas9 and PU6::goa-1 sgRNA (E and F) or animals carrying PU6::goa-1 sgRNA (G). n ≥ 12 plates (2–5 transgene-bearing animals per plate). An sgRNA without a guide sequence was used as a control in (F). ∗∗∗p < (two-tailed t test). n.s., not significant.
Error bars indicate SEM. See also Figure S1.
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4. Figure 2
The Abundance of Presynaptic DAG in ASER Changes in Response to Shifts in External Salt Concentrations in a Food-Dependent Manner
(A and B) Localization of Downward DAG2 (A) and TTX-4::3×Venus (B) in the distal synaptic neurite of ASER. A presynaptic marker, mCherry::RAB-3, was co-expressed. The approximate observed area (red dotted box) is shown in the schematic of the head (A). Scale bars, 5 μm.
(C and D) Fluorescence intensity ratios of Downward DAG2 to soluble mCherry (control) were quantified in the synaptic neurite of ASER of animals in which Downward DAG2 and mCherry were expressed in ASER.
(C) Worms were treated with 1 μg/ml PMA (a DAG analog), DMSO (vehicle control), or 1 μg/ml 4α-PMA (an inactive PMA control) for 3 hr with 50 mM NaCl. n ≥ 18 animals. ∗∗∗p < (ANOVA with Dunnett’s post-test).
(D) Worms were cultured on NGM containing 0, 50, or 100 mM NaCl and observed in buffer containing 0, 50, or 100 mM NaCl, respectively. n = 15 animals (ANOVA with Dunnett’s post-test).
(E and F) Averaged Downward DAG2 responses measured in the synaptic region (left) or the cell body (right) of ASER. After conditioning with 50 mM NaCl and food, external NaCl concentrations were shifted (time = 0) from 50 mM to 100 mM (E) or to 0 mM (F). n ≥ 16 animals.
(G) Averaged peak fluorescence change of Downward DAG2 after the shift in NaCl concentrations. n ≥ 16 animals. ∗∗∗p < (ANOVA with Tukey’s post-test).
(H and I) Averaged Downward DAG2 responses measured in the ASER synaptic region. After conditioning for 4 hr with 50 mM NaCl and starvation, external NaCl concentrations were shifted (time = 0) from 50 mM to 100 mM (H) or to 0 mM (I). n ≥ 16 animals.
Error bars and the shaded regions around the response curves indicate SEM. See also Figure S2.
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5. Figure 3
The TAX-4 CNG Channel Subunit and EGL-8/PLC-β Are Required for Normal DAG Responses
(A, B, and E) Averaged Downward DAG2 responses measured in the ASER synaptic region of the tax-4(p678) mutants (A), the tax-4(p678) mutants that expressed tax-4 specifically in ASER (B), and the egl-8(n488) mutants (E). After conditioning with 50 mM NaCl and food, external NaCl concentrations were shifted (time = 0) from 50 mM to 100 mM (top) or to 0 mM (bottom). n ≥ 7 animals.
(C) Averaged peak fluorescence change of Downward DAG2 in the ASER synaptic region of the wild-type, the tax-4(p678) mutants, and the tax-4(p678) mutants that expressed tax-4 specifically in ASER. n ≥ 7 animals.
(D) Fluorescence intensity ratios of Downward DAG2 to mCherry (control) were quantified in the synaptic neurite of ASER of the wild-type, dgk-1(sy428);dgk-2(gk124); dgk-3(gk110), and egl-8(n488) animals that express Downward DAG2 and mCherry in ASER from a genome-integrated array. n ≥ 17 animals. ∗∗∗p < 0.001 (ANOVA with Dunnett’s post-test).
(F and G) Averaged peak fluorescence change after the NaCl shifts of Downward DAG2 in the ASER synaptic region of the wild-type, DAG-related mutants (F), and egl-19(n582) (G). n ≥ 10 animals. ∗∗∗p < 0.001, ∗p < 0.05; ANOVA with Tukey’s post-test (F) or two-tailed t test (G) compared with the wild-type unless indicated otherwise.
Error bars indicate SEM. See also Figures S3 and S4.
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6. Figure 4
The Synaptic Insulin/PI3K/Akt Pathway Regulates the Plasticity of the DAG Dynamics
(A) The salt concentration preference assay was performed on daf-2(pe1230) mutants that had experienced 50 mM NaCl with (left) or without (right) food. n = 14 plates (5–7 animals per plate).
(B–D) The salt concentration preference assay was performed on daf-18(e1375) (B), goa-1(pe773) (C), and daf-18(e1375); goa-1(pe773) (D) mutants conditioned with 50 mM NaCl and food. n = 16 plates (5–8 animals per plate). The CTX indices are shown in (E).
(E) CTX indices of wild-type, daf-18, daf-18; goa-1, and goa-1 worms conditioned with 50 mM NaCl and food. n ≥ 12 plates (5–8 animals per plate). ∗∗∗p < 0.001, ∗∗p < 0.01 (ANOVA with Tukey’s post-test).
(F) The salt concentration preference assay was performed on goa-1 animals that expressed the goa-1 cDNA under the gcy-5 or goa-1 promoter and had been conditioned with 50 mM NaCl and food. n ≥ 16 plates (1–6 transgene-bearing animals per plate). ∗∗∗p < 0.001, ∗∗p < 0.01 (ANOVA with Dunnett’s post-test).
(G and H) Averaged Downward DAG2 responses measured in the ASER synaptic region of the akt-1(ok525) mutants. After worms had been exposed to 50 mM NaCl with (G) or without (H) food, external NaCl concentrations were shifted (time = 0) from 50 mM to 100 mM (top) or to 0 mM (bottom). n ≥ 9 animals.
(I and J) Averaged peak fluorescence change after the NaCl shifts of Downward DAG2 in the ASER synaptic region of the wild-type, daf-2(pe1230), akt-1(ok525), casy-1(tm718), and daf-18(e1375). After worms had been exposed to 50 mM NaCl with or without food, external NaCl concentrations were shifted (time = 0) from 50 mM to 100 mM (I) or to 0 mM (J). n ≥ 6 animals. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05 (ANOVA with Tukey’s post-test, compared with the wild-type unless indicated otherwise).
(K) A model for the mechanism of salt concentration memory in C. elegans.
Error bars indicate SEM. See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
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