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 rate prior to any stimuli (0 W) and heat-evoked reversals were measured during a series of 4-s heat pulses at 100, 200, 300 and 400 W, respectively, delivered with an interstimulus interval of 20 s. B-C. Impact of food deprivation on thermonociceptive response, showing progressive attenuation of heat-evoked reversal response in the course of a 6-hr experiment. Results as average fraction of reversing animals (%) quantified in N ≥ 6 assays, each scoring at least 50 worms. Error bars: S.E.M. 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). 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>.01 versus the early food deprivation condition (off-food 1hr), by Holm-Bonferroni post-hoc tests.

AWC mediates heat-evoked reversals upon early food deprivation and ASI mediates thermonociceptive plasticity upon starvation.

Impact of the genetic ablation of AWC and ASI sensory neurons on thermonociceptive response and starvation-dependent plasticity. A-C Comparison of heat-evoked reversals after early food deprivation (off-food 1hr) 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 +/- S.E.M. *, p<.05, and **, p<.01 versus corresponding heat level in the early food deprivation condition, by Bonferroni post-hoc tests. D Comparison for the 400 W heating level across the three genotypes presented in panel A to C. Bars as average, dots as individual assay scores, and error bars as S.E.M. ##, p<.01 between early food deprivation and prolonged starvation for each genotype; **, p<.01 versus wild type (N2) at the corresponding time point, by Holm Bonferroni post-hoc tests. The total number of assays analyzed per condition, each scoring at least 50 worms, are indicated in panel D.

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-D Comparison of spontaneous (0 W) and heat-evoked reversals (100 to 400 W) after early food deprivation (off-food 1hr) in wild type, eat-4(ky5), flp-6(ok3056) and in transgenic animals with AWC subtype-specific rescue of eat-4 and flp-6, respectively. Results are presented as average +/- S.E.M. **, p<.01 versus wild type (A and C) and versus non-transgenic mutants (B and D) at respective heat levels, by Holm-Bonferroni post-hoc tests. The number of assays (N), each scoring at least 50 worms, were: wild type, N=15; eat-4, N=6; flp-6, N=14; eat-4+[AWCOFF::eat-4], N=6; eat-4+[AWCON::eat-4], N=9; flp-6+[AWCOFF::flp-6], N=7; flp-6+[AWCOFF::flp-6], N=8. E Visual model illustrating the specific contribution of AWCOFF and AWCON to the regulation of spontaneous reversal, low heat-evoked reversal and high heat-evoked reversal, via the distributed action of glutamate and FLP-6 neuropeptide.

Starvation reconfigures AWC heat-evoked response from a mostly deterministic to a stochastic activity mode.

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 1hr) and prolonged starved (off-food 6hr) conditions. C-D From the same dataset as in A-B, average traces showing the dynamics of calcium up or calcium down response types with similar amplitude and globally comparable shapes.

ASI ablation prevents AWC activity pattern reconfiguration upon starvation.

(A-B) 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 1hr) and prolonged starved (off-food 6hr) conditions. The starvation impact seen in animals with intact ASI (Figure 4) is absent when ASI is ablated.

Bidirectional glutamate signaling actions modulate heat-evoked reversals following starvation.

A-C Comparison of heat-evoked reversals after early food deprivation (off-food 1hr) and prolonged starvation (off-food 6hr) in wild type, eat-4(ky5), flp-6(ok3056). D Analysis of transgenic animals with AWC subtype-specific rescue of eat-4 after prolonged starvation (off-food 6hr). Results are presented as average +/- S.E.M. **, p<.01 between the two food-deprivation time points (A-C) and versus non-transgenic mutants (D) at respective heat levels, by Holm-Bonferroni post-hoc tests. The number of assays (N), each scoring at least 50 worms, were: wild type, N=15; eat-4, N=6; flp-6, N=14; eat-4+[AWCOFF::eat-4], N=6; eat-4+[AWCON::eat-4], N=9. E Visual model illustrating the bidirectional effect of glutamatergic signaling from AWCOFF and from unidentified non-AWC neurons (other). Red: reversal-supressing pathway; Green: reversal-promoting pathway.

Starvation reconfigures neuropeptidergic signaling by ASI.

A Comparison of heat-evoked reversals after early food deprivation (off-food 1hr) and prolonged starvation (off-food 6hr) in wild type, ins-4(ok3534), ins-6(tm2008), nlp-18(ok1557) and ins-32(tm6109). *, p<.05, and **, p<.01 between the two food deprivation time points, by Holm Bonferroni post-hoc tests. #, p<.05, and ##, p<.01 versus wild type (N2) at the corresponding time point, by Holm Bonferroni post-hoc tests. B-E 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-hocs tests were conducted on the genotype factor only. Indicated p values were corrected with Holm 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.

Visual model of the cellular and molecular signaling pathways mediating and modulating heat-evoked reversals according to feeding state.

A Situation upon early food deprivation (off-food 1hr). The two AWC neurons produce mostly deterministic 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 neuropeptide from ASI neurons, as well as NLP-18 neuropeptides from an unidentified source, also promote heat-evoked reversals. B Situation following prolonged starvation (off-food 6hr). Starvation causes several functional changes. First, AWC calcium activity pattern switches from a deterministic mode to a stochastic mode, with a mix of calcium elevations, calcium decreases and variable responses; this effect is mediated by ASI. Second, INS-32 and NLP-18 neuropeptides switch from a reversal-promoting effect to a reversal-suppressing effect; for INS-32, ASI remains a relevant peptide source, whereas for NLP-18 ASI becomes a relevant peptide source. Third, glutamatergic signaling switches from a mostly reversal-promoting effect to a reversal-suppressing effect; this switch is associated with different glutamate sources (in 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 thermosensory encoding change and complex neuromodulation using bidirectional signaling molecules that produce reversal-promoting (green pathways) or reversal-suppressing effects (red pathways) in a context-dependent manner.

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 thermo-responsive neurons on thermonociceptive response and starvation-dependent plasticity. A-C Comparison of heat-evoked reversals after early food deprivation (off-food 1hr) 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 +/- S.E.M. *, p<.05, and **, p<.01 versus corresponding heat level in the early food deprivation condition, by Bonferroni post-hoc tests. E Comparison for the 400 W heating level across the three genotypes presented in panel A to C. Bars as average, dots as individual assay scores, and error bars as S.E.M. ##, p<.01 between early food deprivation and prolonged starvation for each genotype; **, p<.01 versus wild type (N2) at the corresponding time point, by Holm Bonferroni post-hoc tests. The total number of assays analyzed per condition, each scoring at least 50 worms, are indicated in panel D.

A-B Comparison of spontaneous (0 W) and heat-evoked reversals (100 to 400 W) after early food deprivation (off-food 1hr) in wild type, nlp-5(ok1981) and ins-22(ok3616).

Results are presented as average +/- S.E.M. *, p<.05 and **, p<.01 versus wild type by Holm-Bonferroni post-hoc tests. The number of assays (N), each scoring at least 50 worms, were: wild type, N=15; nlp-5, N=6; ins-22, N=6.

AWC subtype differentiation is partly required for proper heat-evoked reversal response after 1hr off food.

A-B Comparison of spontaneous (0 W) and heat-evoked reversals (100 to 400 W) after early food deprivation (off-food 1hr) in wild type, nsy-1(ok593), in which both AWCs differentiate as AWCON and nsy-7(tm3080), in which both AWCs differentiate as AWCOFF. Results are presented as average +/- S.E.M. *, p<.05 and **, p<.01 versus wild type by Holm-Bonferroni post-hoc tests. The number of assays, each scoring at least 50 worms, were: wild type, N=15; nsy-1, N=6; nsy-7, N=6.