Starvation transforms signal encoding in C. elegans thermoresponsive neurons and suppresses heat avoidance via bidirectional glutamatergic and peptidergic signaling
- Department of Biology, University of Fribourg, Switzerland
- Department of Biology, KU Leuven, Belgium
Figures
Figure 1
Starvation-dependent thermonociceptive plasticity in C. elegans.
(A) Schematic of the experimental procedure to quantify the impact of food deprivation on thermonociception in C. elegans adult hermaphrodites. Spontaneous and heat-evoked reversals were quantified in fed animals on food (Fed) or off food after 1, 2, 3, and 6 hr of food deprivation. Spontaneous reversals were measured as baseline reversal rates prior to any stimuli (0 W) and heat-evoked reversals were measured during a series of 4 s heat pulses at 100 W (+2°C), 200 W (+4°C), 300 W (+6°C), and 400 W (+8°C), respectively, delivered with an interstimulus interval of 20 s. (B, C) Impact of food deprivation on thermonociceptive response, showing progressive attenuation of the heat-evoked reversal response in the course of a 6-hr experiment. Results as average fractions of reversing animals (%) quantified in N ≥ 6 assays, each scoring at least 50 worms. Error bars: SEM. The same dataset is presented as heat dose–response curves (B) and as time course of the heat-evoked response decrease at each heating level (C). Spontaneous reversal rate corresponds to the baseline reversal response in the absence of heating stimuli (0 W, thermal increase = 0°C). (D) Effect of prolonged starvation (off-food for 6 hr) in the presence or absence of food odor during the food-deprivation period, indicating that external food odor cues cannot prevent the response reduction. **p < 0.01 versus the early food-deprivation condition (off-food 1 hr), by Bonferroni post hoc tests.
Figure 2 with 1 supplement
AWC mediates heat-evoked reversals upon early food deprivation and ASI mediates thermonociceptive plasticity upon starvation.
Impact of the genetic ablation of AWC, ASI, and AFD sensory neurons on thermonociceptive response and starvation-dependent plasticity. (A–D) Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) and prolonged starvation (off-food 6 hr) in wild type and in transgenic animals with caspase-mediated ablation of indicated neurons. Results are presented as average ± SEM. *p < 0.05, and **p < 0.01 versus corresponding heat level in the early food-deprivation condition, by Bonferroni post hoc tests. (E) Comparison for the highest heating level (thermal increase = 8°C) across the four genotypes presented in panels A–D. Bars as average, dots as individual assay scores, and error bars as SEM. ##p < 0.01 between early food deprivation and prolonged starvation for each genotype; **p < 0.01 versus wild type (N2) at the corresponding timepoint, by Bonferroni post hoc tests. The total number of assays (n) analyzed per condition, each scoring at least 50 worms, are indicated in panel E.
Figure 2—figure supplement 1
Heat-evoked reversal upon early food deprivation and starvation-dependent plasticity are largely intact in AFD and FLP-ablated animals.
Impact of the genetic ablation of candidate thermoresponsive neurons on thermonociceptive response and starvation-dependent plasticity. (A–C) Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) and starvation (off-food 6 hr) in wild type and in transgenic animals with caspase-mediated ablation of indicated neurons. Results are presented as average ± SEM. *p < 0.05, and **p < 0.01 versus corresponding heat level in the early food-deprivation condition, by Bonferroni post hoc tests. (D) Comparison for the highest heating level (thermal increase = 8°C) across the three genotypes presented in panels A–C. Bars as average, dots as individual assay scores, and error bars as SEM. ##p < 0.01 between early food deprivation and prolonged starvation for each genotype; **p < 0.01 versus wild type (N2) at the corresponding timepoint, by Bonferroni post hoc tests. The total number of assays (n) analyzed per condition, each scoring at least 50 worms, are indicated in panel D. The wild type (N2) dataset is the same as in Figure 2D. Data reported in the two figures were acquired in parallel, and multiple comparison corrections were made in a single analysis.
Figure 3 with 1 supplement
Differential engagement of glutamate and FLP-6 neuropeptides by AWCON and AWCOFF in the control of spontaneous and heat-evoked reversal at various heat levels.
(A–C, E, F) Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) among wild type, eat-4(ky5), flp-6(ok3056), eat-4;flp-6 double mutants, and transgenic animals with AWC subtype-specific rescue of eat-4 and flp-6, respectively. Results are presented as average ± S.E.M. **p < 0.01 versus wild type (A–C) and versus non-transgenic mutants (E, F) at respective thermal increase levels, by Bonferroni post hoc tests. The number of assays (n), each scoring at least 50 worms, were: wild type, n = 27; eat-4, n = 12; flp-6, n = 15; eat-4;flp-6, n = 9; eat-4+[AWCOFF::eat-4], n = 6; eat-4+[AWCON::eat-4], n = 9; flp-6+[AWCON::flp-6], n = 7; flp-6+[AWCOFF::flp-6], n = 8. (D) Epistasis analysis of eat-4 and flp-6 mutation effects on the heat-evoked reversal (thermal increase = 8°C). Bars as average, dots as individual assay scores, and error bars as SEM. A two-way ANOVA showed no significant interaction of the two mutations, and Bonferroni post hoc tests confirmed significant cumulative effects of the two mutations (##p < 0.001). (G) Visual model illustrating the specific contribution of AWCOFF and AWCON to the regulation of heat-evoked reversals via the distributed action of glutamate and FLP-6 neuropeptide. A single wt control dataset is reported across panels.
Figure 3—figure supplement 1
Alteration of heat-evoked reversals in nlp-5 and ins-22 mutants.
(A, B) Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) in wild type, nlp-5(ok1981), and ins-22(ok3616). Results are presented as average ± SEM. *p < 0.05 and **p < 0.01 versus wild type by Bonferroni post hoc tests. The number of assays (n), each scoring at least 50 worms, were wild type, n = 27; nlp-5, n = 6; ins-22, n = 6. A single wt control dataset is reported across panels in this figure and in Figure 3.
Figure 4 with 1 supplement
Starvation reconfigures AWC heat-evoked response polarity distribution from mostly excitatory to a heterogeneous mix combining excitatory and inhibitory responses.
(A, B) Calcium activity in AWCOFF (left) and AWCON (right) in response to a series of four thermal up-steps. Upper plots show the average traces, lower heat maps show individual neuron traces, in the early food-deprivation (off-food 1 hr) and prolonged starvation (off-food 6 hr) conditions. (C, D) Histograms showing the distribution of calcium peak magnitudes (all thermal increase levels pooled) and highlighting the shift from mostly excitatory responses (early food deprivation, upper plot) to an equal mix of excitatory and inhibitory responses (prolonged starvation, lower plot) taking place for both AWCON and AWCOFF. Results of Kruskal–Wallis non-parametric tests comparing the two food-deprivation timepoints are indicated to the left of each graph.
Figure 4—figure supplement 1
Similar kinetics of calcium up and calcium down responses under the early food-deprivation and the prolonged starvation condition.
(A, B) From the same dataset as in Figure 4A, B, average traces showing the dynamics of calcium up (excitatory) or calcium down (inhibitory) response types with similar amplitude and globally comparable shapes.
Figure 5
ASI ablation prevents AWC activity pattern reconfiguration upon starvation.
Calcium activity in AWCOFF (A) and AWCON (B) in response to a series of four thermal up-steps in ASI-ablated animals. Upper plots show the average traces, lower heat maps show individual neuron traces, in the early food deprivation (off-food 1 hr) and prolonged starvation (off-food 6 hr) conditions. The starvation impact seen in animals with intact ASI (Figure 4) is absent when ASI is ablated. (C, D) Histograms showing the distribution of calcium peak magnitudes (all thermal increase levels pooled) for both AWCON and AWCOFF. Results of Kruskal–Wallis non-parametric tests comparing the two food-deprivation timepoints are indicated to the left of each graph.
Figure 6 with 1 supplement
Bidirectional glutamate signaling actions modulate heat-evoked reversals following starvation.
(A–D) Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) and prolonged starvation (off-food 6 hr) in wild type, eat-4(ky5), flp-6(ok3056), and double mutant. (E) Heat-evoked reversal rate averaged over the four heating levels for the prolonged starvation condition. Same data as in panels A–D. (F) Analysis of transgenic animals with AWC subtype-specific rescue of eat-4 after prolonged starvation (off-food 6 hr). Results are presented as average ± SEM. **p < 0.01 between the two food-deprivation timepoints (A–E) and versus non-transgenic mutants (F) at respective heat levels, by Bonferroni post hoc tests. The number of assays (n), each scoring at least 50 worms, were: wild type, n = 27; eat-4, n = 15; flp-6, n = 15; eat-4+[AWCOFF::eat-4], n = 6; eat-4+[AWCON::eat-4], n = 9. (G) Visual model illustrating the bidirectional effect of glutamatergic signaling from AWCOFF and from unidentified non-AWC neurons (other).
Figure 6—figure supplement 1
Intact starvation-induced thermonociceptive plasticity in ins-1 mutants.
Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) and prolonged starvation (off-food 6 hr) in wild type (N2), and ins-1(nj32). *p < 0.05, and **p < .01 between the two food-deprivation timepoints, by Bonferroni post hoc tests. #p < 0.05, and ##p < 0.01 versus wild type (N2) at the corresponding timepoint, by Bonferroni post hoc tests.
Figure 7
Starvation reconfigures neuropeptidergic signaling by ASI.
(A) Comparison of heat-evoked reversals after early food deprivation (off-food 1 hr) and prolonged starvation (off-food 6 hr) in wild type, ins-4(ok3534), ins-6(tm2008), nlp-18(ok1557), and ins-32(tm6109). *p < 0.05, and **p < 0.01 between the two food-deprivation timepoints, by Bonferroni post hoc tests. #p < 0.05, and ##p < 0.01 versus wild type (N2) at the corresponding timepoint, by Bonferroni post hoc tests. Data for wild type are the same as the one depicted in Figure 6. Impact of transgenic rescue in ins-32 (B, C) and nlp-18 (D, E) background. Two-way ANOVA showing no significant heating power x genotype interaction, but a significant genotype main effect; post hoc tests were conducted on the genotype factor only. Indicated p values were corrected with Bonferroni correction. (F) Model of the bidirectional actions and the sources for NLP-18 and INS-32 neuropeptides in the modulation of heat-evoked reversal. Red: reversal-suppressing pathway; Green: reversal-promoting pathway. Additional neuropeptides (such as INS-6) may also be involved, but in the absence of direct evidence for their origin from ASI, they were not included in this scheme.
Figure 8
Visual model of the cellular and molecular signaling pathways mediating and modulating heat-evoked reversals according to food-deprivation duration.
(A) Situation upon early food deprivation (off-food 1 hr). The two AWC neurons produce mostly stimulus-locked calcium elevations in response to heat stimuli and make a major contribution to heat-evoked reversals. Glutamate signaling from AWCON and AWCOFF, as well as FLP-6 neuropeptide signaling from AWCOFF, mediates heat-evoked reversals. INS-32 and NLP-18 neuropeptide from ASI neurons, as well as potentially from additional unidentified sources (not depicted), also promote heat-evoked reversals. (B) Situation following prolonged starvation (off-food 6 hr). Starvation causes several functional changes. First, AWC calcium activity pattern shifts from a mostly excitatory response (calcium elevations) to less predictable response patterns combining calcium elevations (excitation), calcium decreases (inhibition), and variable responses; this effect is mediated by ASI (purple arrow). Second, INS-32 and NLP-18 neuropeptides switch from a reversal-promoting effect to a reversal-suppressing effect. One simple model for INS-32 and NLP-18 action would be that they mediate the ASI effect on AWC activity patterns (purple arrow), but this possibility remains hypothetical. Third, glutamatergic signaling switches from a mostly reversal-promoting effect to a reversal-suppressing effect; this switch is associated with different glutamate sources (AWC and non-AWC neurons, respectively). Of note, residual reversal response upon starvation might be mediated by FLP-6 and glutamate from AWCOFF. In summary, our findings highlight a complex reconfiguration of the thermoresponsive circuit following starvation, which includes a change in thermosensory encoding and complex neuromodulation using bidirectional signaling molecules that produce reversal-promoting (green pathways) or reversal-suppressing effects (red pathways) in a context-dependent manner.
Additional files
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Supplementary file 1
Primer, plasmid, and strain details.
- https://cdn.elifesciences.org/articles/108246/elife-108246-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108246/elife-108246-mdarchecklist1-v1.pdf
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Source data 1
Raw data and statistics.
- https://cdn.elifesciences.org/articles/108246/elife-108246-data1-v1.xlsx
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Starvation transforms signal encoding in C. elegans thermoresponsive neurons and suppresses heat avoidance via bidirectional glutamatergic and peptidergic signaling
eLife 14:RP108246.
https://doi.org/10.7554/eLife.108246.3
