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. 2019 Nov 13;8:e50566. doi: 10.7554/eLife.50566

Figure 4. Glutamatergic sensory neurons cooperate to inhibit AIA.

(A) Simplified diagram of connections between AWA, AIA and four glutamatergic sensory neurons, based on White et al. (1986). (B) Schematic of cell-selective glutamate knockout genetic strategy (López-Cruz et al., 2019). The eat-4 locus is excised only in the presence of flippase. ORF: open reading frame; UTR: untranslated region; FRT: flippase recombinase target. (C–H) Cumulative response time profiles of AIA responses to AWA::Chrimson stimulation in various animals lacking either glutamate release or cellular function of specific sensory neurons. For (D–G), dotted black and blue lines are control and eat-4-FRT; AWC,ASE,ASK,ASG::nFlippase, respectively, from (C). (C) Control (eat-4-FRT genetic background with no flippase expression), unc-18, and eat-4-FRT; AWC,ASE,ASK,ASG::nFlippase animals. (D) eat-4-FRT; ASK::nFlippase animals. (E) eat-4-FRT; ASG::nFlippase animals. (F) eat-4-FRT; AWC::nFlippase animals. (G) eat-4-FRT; AWC+ASE::nFlippase animals. (H) WT and che-1 animals (ASE cell fate mutants). (I) Cumulative response time profiles of AIA responses to AWA::Chrimson stimulation in WT, unc-18 animals, unc-18; AWC,ASE::unc-18(+) transgenic rescue animals (two lines), and unc-18; AWC,ASE::unc-18(e234) transgenic control animals. Asterisks refer to Kolmogorov-Smirnov significance versus eat-4-FRT controls (C–G) or WT (H, I) over full 10 s stimulus pulse. ns: not significant; **: p<0.01; ***: p<0.001. See Supplementary file 2 for sample sizes and test details. Heat maps of data from Figure 4 appear in Figure 4—figure supplement 1. Additional representations of data from Figures 1–4 appear in Figure 4—figure supplement 2.

Figure 4—source data 1. Source data for Figure 4 and figure supplements.

Figure 4.

Figure 4—figure supplement 1. Controls and heat maps for FRT-FLP recombination.

Figure 4—figure supplement 1.

(A) Heat maps of AIA responses to AWA::Chrimson in eat-4-FRT control strain, unc-18, FRT+nFlippase animals lacking glutamate release in specific sensory neurons, shown in Figure 4C–G, WT, and WT control strain expressing AWC,ASE,ASK,ASG::nFlippase. (B) Cumulative response time profiles of AIA responses to AWA::Chrimson stimulation in WT animals, animals with the genetically modified eat-4-FRT locus alone, or animals with the AWC,ASE,ASK,AWG::nFlippase transgene alone. All genotypes include the AWA::Chrimson transgene. (C) Heat maps of AIA responses to AWA::Chrimson in WT and che-1 animals (ASE cell fate mutants), shown in Figure 4H. (D) Heat maps of AIA responses to AWA::Chrimson in WT, unc-18, unc-18; AWC,ASE::unc-18(e234) transgenic control animals bearing an inactivating point mutation, and unc-18; AWC,ASE::unc-18(+) transgenic rescue animals (two lines).
Figure 4—figure supplement 2. Additional representations of AIA data from Figures 1–4.

Figure 4—figure supplement 2.

(A – C) Negative correlation between AIA GCaMP fluorescence at baseline and magnitude of responses to AWA::Chrimson (A), 11.5 nM diacetyl (B), and 1.15 µM diacetyl (C). Only responses to the first stimulus pulse are included in this analysis. AWA::Chrimson: n = 176; 11.5 nM diacetyl: n = 91; 1.15 µM diacetyl: n = 208. (D – I) Mean AIA responses to various stimuli that resulted in AIA activation within 5 s of stimulus, aligned to the beginning of AIA activation rather than stimulus time. Shading indicates ± SEM. (D) WT versus odr-7 and odr-10 to 1.15 µM diacetyl, shown in Figure 2A. (E and F) WT versus animals expressing a transgene encoding Tetanus Toxin Light Chain A (TeTx) in AWA to 1.15 µM diacetyl (E) or AWA::Chrimson (F), shown in Figure 2C and Figure 2—figure supplement 1A. (G and H) WT versus unc-7 unc-9 animals to 11.5 nM diacetyl (G) or AWA::Chrimson (H), shown in Figure 2—figure supplement 1B and D. (I) unc-7 mutants versus unc-7 unc-9 double mutants, unc-7 unc-9; AWA,AIA::unc-9(+) transgenic rescue animals, and unc-7 unc-9; AWA,AIA::unc-9(fc16) transgenic control animals with an inactivating point mutation, shown in Figure 2F. (J – L) Mean AIA responses to 11.5 nM diacetyl (J), 1.15 µM diacetyl (K), and AWA::Chrimson (L) that resulted in activation within 5 s of stimulus, aligned to the beginning of AIA activation rather than stimulus time, in WT versus unc-13, unc-18(e234), and unc-18(e81) animals, shown in Figure 3A, Figure 3—figure supplement 1A, and Figure 3C. (M) Rise times of AIA responses to AWA::Chrimson in WT, unc-13, unc-18(e234), and unc-18(e81) synaptic transmission mutants, shown in Figure 3C. (N – P) Mean AIA responses to AWA::Chrimson stimulation that resulted in activation within 5 s of stimulus, aligned to the beginning of AIA activation rather than stimulus time, in various animals lacking either glutamate release or cellular function of specific sensory neurons, shown in Figure 4. (N) eat-4-FRT, eat-4-FRT; AWC,ASE,ASK,ASG::nFlippase, eat-4-FRT; ASG::nFlippase, eat-4-FRT; AWC,ASE::nFlippase, eat-4-FRT; ASK::nFlippase, and eat-4-FRT; AWC::nFlippase. (O) eat-4-FRT, unc-18(e234), WT, and WT expressing nFlippase in AWC, ASE, ASK, and ASG. (P) WT and che-1 animals (ASE cell fate mutants). For (A), (B), and (C), asterisks refer to significance of linear regression slope differing from 0. ***: p<0.001. For (M), ns refers to a lack of statistical significance of one-way ANOVA with Dunnett’s multiple comparisons test. See Supplementary file 4 for sample sizes and test details. For (P), ns refers to lack of statistical significance of an unpaired t-test. For all others, asterisks refer to statistical significance of one-way ANOVA with Dunnett’s multiple comparisons test versus unc-7 (for I only) or versus WT (for all others). ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001. See Supplementary file 3 for sample sizes and test details.