Supplementation of serotonin restored the neuronal-to-intestinal UPR ^mt activation in tmbim-2 mutants. (A) Representative photomicrographs of dve-1 reporter in animals expressing neuronal Q40 and neurotoxin TeTx in ADF neuron (ythEx768[srh-142p::TeTx::sl2::mcherry]). Scale bar, 250 μm. (B) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in A. n ≥ 15 worms. (C) Representative photomicrographs of the ADF neurotransmitter exocytosis reporter (ythEX766[srh-142p-SYP-Phluorin+unc-119(+)]) in control or neuronal cox-5B knockdown animals in WT or tmbim-2(yth130) background. The imaging used Z-stacks. Scale bar, 2 μm. (D) Quantification of srh-142p::snb-1::pHluorin fluorescence in the neurons of animals as depicted in C: WT (orange); tmbim-2(yth130) (yellow); unc-119p::cox-5B HP (ultramarine); unc-119p::cox-5B; tmbim-2(yth130) (wathet). n ≥ 12 worms. (E) Representative photomicrographs of dve-1 reporter expression in WT (a); neuronal cox-5B knockdown (b); neuronal cox-5B knockdown; tmbim-2(c); neuronal cox-5B knockdown; or ADF neuron overexpressing TeTx(ythEX768[srh-142p::TeTx::sl2::mCherry+unc-119(+)];unc-119(ed-3)III) (d) animals treated with vehicle control or 50 mM serotonin (5-HT). (F) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in E, yellow for control and blue for feeding 5-HT. n ≥ 12 worms. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test (**P < 0.01; ***P < 0.001). (G) Representative photomicrographs of dve-1 reporter in control or ADF neuron overexpressing TMBIM-2 animals in WT or tph-1(n4622) background. (H) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in G. n ≥ 15 worms. ***P < 0.001; **P < 0.01 via unpaired two-tailed Student’s t test. Error bars, SEM; Scale bar, 250 μm.