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

Figure 5. Multiple sensory neurons detect diacetyl and isoamyl alcohol.

(A – C) Mean ASK (A), AWC (B), and ASE (C) responses to 10 s pulses of buffer (0) or 11.5 nM or 1.15 µM diacetyl. ASK: n = 82–115; AWC: n = 52–60; ASE: n = 42–54. Shading indicates ± SEM. (D) Mean ASE responses to 1.15 µM diacetyl in WT versus unc-18 animals (synaptic transmission mutants) and odr-10 animals (AWA diacetyl receptor mutants). ASE responses in unc-18 animals are greatly diminished. Shading indicates ± SEM. (E – G) Mean ASK (E), AWC (F), and AWA (G) responses to 10 s pulses of buffer (0), 0.9 µM, 9 µM, or 90 µM isoamyl alcohol. ASK: n = 60; AWC: n = 42; AWA: n = 78–80. Shading indicates ± SEM. (H) Summary of sensory neuron responses to various stimuli. Upward arrows indicate activation; downward arrows indicate inhibition. Arrow thickness reflects response magnitude. (I – K) Cumulative response time profiles of AIA responses to 0.9 µM (I), 9 µM (J), or 90 µM (K) isoamyl alcohol in WT versus odr-7 animals (AWA cell fate mutants), ceh-36 animals (AWC and ASE cell fate mutants), and odr-7 ceh-36 animals. (L) Cumulative response time profiles of AIA responses to 1.15 µM diacetyl, 90 µM isoamyl alcohol, and E. coli OP50 bacteria-conditioned medium. Asterisks refer to Kolmogorov-Smirnov test significance versus buffer over full 10 s stimulus pulse. ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001. See Supplementary file 2 for sample sizes and test details.

Figure 5—source data 1. Source data for Figure 5 and Figure 5—figure supplements 1–3.

Figure 5.

Figure 5—figure supplement 1. Controls for diacetyl activation of sensory neurons.

Figure 5—figure supplement 1.

(A – C) Magnitude of individual ASK (A), AWC (B), or ASE (C) responses to buffer (0), 11.5 nM or 1.15 µM diacetyl shown in Figure 5A–C. Boxes show median and interquartile range. (D) Magnitude of individual ASH responses to buffer (0), 1.15 µM diacetyl, or 100 mM NaCl in WT animals. Boxes show median and interquartile range. (E – G) Cumulative response time profiles of responses from (A), (B), and (C). Only the first 5 s are shown. (H) Mean ASH responses to pulses of buffer (0), 1.15 µM diacetyl, or 100 mM NaCl in WT animals shown in (D). Shading indicates ± SEM. (I – K) Magnitude of individual ASK (I), AWC (J), or ASE (K) responses to pulses of 1.15 µM diacetyl in WT versus unc-18 and odr-10 animals. Boxes show median and interquartile range. (L and M) Mean ASK (L) and AWC (M) response to pulses of 1.15 µM diacetyl in WT versus unc-18 and odr-10 animals shown in (I) and (J). Shading indicates ± SEM. (N) Mean ASK, AWC, and ASE responses to pulses of AWA::Chrimson stimulation in WT animals. Shading indicates ± SEM. (O) Magnitude of individual responses in (N). Boxes show median and interquartile range. For (A), (B), (C), (D), (I), (J), and (K), asterisks refer to statistical significance of a one-way ANOVA with Dunnett’s multiple comparisons test versus buffer (A–D and I–K) or versus WT (I–K). ns: not significant; **: p<0.01; ***: p<0.001. For (O), ns refers to lack of statistical significance in paired t-tests comparing pre-light to within-light periods in same neurons. See Supplementary file 3 for sample sizes and test details. For (E), (F), and (G), asterisks refer to Kolmogorov-Smirnov test significance versus buffer over full 10 s stimulus pulse. ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001. See Supplementary file 2 for sample sizes and test details.
Figure 5—figure supplement 2. Simultaneous recording of multiple neurons.

Figure 5—figure supplement 2.

(A and B) Cytoplasmic AIA GCaMP5A and nuclear AWA GCaMP6s and ASK GCaMP6s responses to 10 s pulses of 1.15 µM (A) or 11.5 nM (B) diacetyl, with all three neurons recorded simultaneously in the same animal. AWA and ASK fluorescence were measured in the nucleus, AIA fluorescence was measured in the neurite. Two pulses at 11.5 nM did not elicit AIA or ASK responses. 1.15 µM diacetyl videos came from two animals; 11.5 nM diacetyl videos came from two animals. Note spontaneous ongoing activity in ASK sensory neurons, which can contribute to spontaneous AIA activity (López-Cruz et al., 2019), and may also contribute to AIA responses to weak stimuli. (C) AIA GCaMP5A and AWA GCaMP6s responses to 10 s pulses of 1.15 µM diacetyl, ordered by AWA-to-AIA lag. Right: same responses, zoomed in and re-scaled to capture response initiations. n = 12 pulses from six animals. (D) Delay between AWA and AIA neuron response initiations to 1.15 µM diacetyl pulses, measured by eye for 12 pulses shown in (A) and the 12 pulses shown in (C). Bar indicates mean. In all cases, AWA response initiated before the AIA response.
Figure 5—figure supplement 2—source data 1. Source data for Figure 5—figure supplement 2.
Figure 5—figure supplement 3. Controls and heat maps for isoamyl alcohol stimulation.

Figure 5—figure supplement 3.

(A – C) Magnitude of individual AWC (A), AWA (B), and ASK (C) responses to buffer (0), 0.9 µM, 9 µM, and 9 µM isoamyl alcohol shown in Figure 5E–G. Boxes show median and interquartile range. (D – F) Cumulative response time profiles of responses from (A), (B), and (C). Only the first 5 s are shown. (G – J) Cumulative response time profiles of AWC, AWA, AIA, and ASK (for J only) responses to 0.9 µM (G), 9 µM (H), or 90 µM (I) isoamyl alcohol, or 1.15 µM diacetyl (J) in WT animals. (K) Heat maps of AIA responses to 0.9, 9, or 90 µM isoamyl alcohol in WT versus odr-7 animals (AWA cell fate mutants), ceh-36 animals (AWC and ASE cell fate mutants), and odr-7 ceh-36 animals, shown in Figure 5I–K. (L) Mean AIA responses to 1.15 µM diacetyl, 90 µM isoamyl alcohol, AWA::Chrimson, and E. coli OP50 bacteria-conditioned medium from Figure 5L that initiated within 5 s of stimulus, aligned to the beginning of AIA activation rather than stimulus time. Shading indicates ± SEM. 1.15 µM diacetyl: n = 390; 90 µM isoamyl alcohol: n = 76; AWA::Chrimson: n = 260; OP50: n = 37. (M) Rise time of responses from (M). Bars indicate ± SEM. For (A), (B), (C), (L), and (M) asterisks refer to statistical significance of a one-way ANOVA with Dunnett’s multiple comparisons test versus buffer magnitude (A–C), versus 1.15 µM diacetyl magnitude (L), or versus 1.15 µM diacetyl rise time (M). ns: not significant; *: p<0.05; ***: p<0.001. See Supplementary file 3 (A–C and L) or Supplementary file 4 (M) for sample sizes and test details. For (D–J), asterisks refer to statistical significance of a Kolmogorov-Smirnov test versus buffer (D–F), between AWA and AIA (G–I), or between ASK and AIA (J), over full 10 s stimulus pulse. ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001. See Supplementary file 2 for sample sizes and test details.