Ohnishi K et al. (2020), OSM-9 and OCR-2 TRPV channels are accessorial w...

XB-ART-57493

Sci Rep 2020 Oct 29;101:18566. doi: 10.1038/s41598-020-75302-3.

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OSM-9 and OCR-2 TRPV channels are accessorial warm receptors in Caenorhabditis elegans temperature acclimatisation.

Ohnishi K , Saito S , Miura T , Ohta A , Tominaga M , Sokabe T , Kuhara A .

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Caenorhabditis elegans (C. elegans) exhibits cold tolerance and temperature acclimatisation regulated by a small number of head sensory neurons, such as the ADL temperature-sensing neurons that express three transient receptor potential vanilloid (TRPV) channel subunits, OSM-9, OCR-2, and OCR-1. Here, we show that an OSM-9/OCR-2 regulates temperature acclimatisation and acts as an accessorial warmth-sensing receptor in ADL neurons. Caenorhabditis elegans TRPV channel mutants showed abnormal temperature acclimatisation. Ectopic expression of OSM-9 and OCR-2 in non-warming-responsive gustatory neurons in C. elegans and Xenopus oocytes revealed that OSM-9 and OCR-2 cooperatively responded to warming; however, neither TRPV subunit alone was responsive to warming. A warming-induced OSM-9/OCR-2-mediated current was detectable in Xenopus oocytes, yet ADL in osm-9 ocr-2 double mutant responds to warming; therefore, an OSM-9/OCR-2 TRPV channel and as yet unidentified temperature receptor might coordinate transmission of temperature signalling in ADL temperature-sensing neurons. This study demonstrates direct sensation of warming by TRPV channels in C. elegans.

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Genes referenced: dtl gprc6a
GO keywords: response to temperature stimulus

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	Figure 1. Thermosensory neurons regulate cold tolerance and temperature acclimatisation in C. elegans. (a) Cultivation conditions for the temperature acclimatisation assay. 15 °C-cultivated N2 wild-type animals can survive at 2 °C. 25 °C-cultivated N2 wild-type animals failed to survive at 2 °C. When 15 °C-cultivated wild-type animals were transferred and conditioned at 25 °C for 3 or 5 h, they exhibited decreased survival at 2 °C. (b) Cold tolerance and temperature acclimatisation are regulated by a subset of sensory neurons. ADL sensory neurons can detect temperature, and TRPV channels in ADL are involved in temperature acclimatisation.
	Figure 2. Temperature acclimatisation assay. Animals were assayed using the 15 °C → 25 °C (0, 3, or 5 h) → 2 °C protocols. (a,b,c) TRPV mutants osm-9(ky10), ocr-2(ak47), osm-9(ky10) ocr-2(ak47) and osm-9(ky10) ocr-2(ak47); ocr-1(ak46) showed abnormally elevated survival rate (Number of assays ≥ 9, mean ± SEM). Comparisons were performed with Dunnett’s test for each condition: 15 °C → 25 °C (0, 3, 5 h) → 2 °C (p < 0.01). (d,e) The abnormally elevated cold tolerance of osm-9 and ocr-2 mutants was partially rescued by expression of osm-9 and ocr-2 cDNA in ADL sensory neurons, respectively (number of assays ≥ 9, mean ± SEM). Statistical significance was assessed using ANOVA followed by a Bonferroni multi-comparison test (n.s. p ≥ 0.05, p < 0.01, *p < 0.05).
	Figure 3. Ca2+ imaging of ADL sensory neurons in TRPV mutants. Average thermal responses in ADL of each strain cultivated at 15 °C. Line graphs indicate YFP/CFP ratio changes under warming and cooling. (a) Data for wild-type in left and middle graphs are the same, given that the experiments were conducted simultaneously. Comparisons were performed with Dunnett’s test (n.s. p ≥ 0.05, p < 0.01). The bar graph indicates the average ratio change from 230 to 235 s, a maximum point of ratio changes in wild-type (n ≥ 22, mean ± SEM). (b, c) Abnormal temperature responses of ADL in osm-9 and ocr-2 mutants were rescued by expression of osm-9 and ocr-2 cDNA in ADL, respectively. The bar graph indicates the average ratio change from 230 to 235 s (n ≥ 30, mean ± SEM). Statistical significance was assessed using ANOVA followed by a Bonferroni multi-comparison test (n.s. p ≥ 0.05, p < 0.01, *p < 0.05).
	Figure 4. Ca2+ imaging of a gustatory neuron additively expressing OSM-9 and OCR-2. osm-9 cDNA or osm-9 cDNA with ocr-2 cDNA were expressed in a non-warmth-sensing gustatory neuron, ASER of a glr-3 mutant lacking a cold receptor GLR-3. Ca2+ imaging was performed using G-CaMP8. Line graphs indicate the G-CaMP8/tagRFP ratio change under warming. The bar graph indicates the average ratio change from 181 to 200 s, a temperature maximum point (n ≥ 27, mean ± SEM). Comparisons were performed with Dunnett’s test (n.s. p ≥ 0.05, **p < 0.01).
	Figure 5. Electrophysiological analysis of TRPV OSM-9, OCR-2, and OCR-1 using Xenopus oocytes. (a) Representative traces of currents (upper) and temperature (lower) for distilled water (DW)-injected Xenopus oocytes or Xenopus oocytes expressing OSM-9, OCR-2, OCR-1, OSM-9/OCR-2, or OSM-9/OCR-2/OCR-1. The membrane potential was set at − 60 mV. (b) Comparison of normalised warming-evoked currents in DW-injected Xenopus oocytes and Xenopus oocytes expressing OSM-9, OCR-2, OCR-1, OSM-9/OCR-2, or OSM-9/OCR-2/OCR-1 (n ≥ 6 oocytes per group, mean ± SEM). Amplitudes of warming-evoked currents were calculated by subtracting the peak inward currents at basal temperature (approximately 25 °C) from the peak inward currents after temperature changes for each Xenopus oocyte. Statistical significance was assessed using ANOVA followed by a Bonferroni multi-comparison test for results detected between groups marked with “a” and “b” (p < 0.05). (c) Representative traces for cool- or warm-stimulation (upper) and temperature (lower) for DW-injected Xenopus oocytes or Xenopus oocytes expressing OSM-9, OCR-2, or OSM-9/OCR-2. The membrane potential was set at − 60 mV. (d) Comparison of normalised cool-evoked currents in DW-injected Xenopus oocytes and Xenopus oocytes expressing OSM-9, OCR-2, or OSM-9/OCR-2 (n ≥ 6 oocytes per group, mean ± SEM). Amplitudes of cool-evoked currents were calculated by subtracting the peak inward currents at basal temperature (approximately 25 °C) from the peak inward currents at approximately 15 °C for each Xenopus oocyte (left panel). Amplitudes of warming-evoked currents were calculated by subtracting the peak inward currents at approximately 15 °C from the peak inward currents at approximately 35 °C for each Xenopus oocyte (right panel). Statistical significance was assessed using ANOVA followed by a Bonferroni multi-comparison test for results detected between groups marked with “a” and “b” (p < 0.01). (e) Averaged current–voltage (I–V) relationships for DW-injected Xenopus oocytes or for Xenopus oocytes expressing OSM-9, OCR-2, or OSM-9/OCR-2 in response to warm stimuli. Ramp pulses from − 80 to + 80 mV were applied at 3-s intervals and I-V curves were obtained at indicated temperatures. The blue traces represent the I–V relationship at room temperature, while the red traces represent the warming-evoked I–V relationship (n ≥ 6 oocytes per group, mean ± SEM).
	Figure 6. Model of temperature sensation in ADL neurons for cold tolerance and temperature acclimatisation modulated by TRPV channels and unidentified temperature receptors, such as GPCRs. Temperature is sensed by both unidentified GPCRs and OSM-9/OCR-2 TRPV channels. GPCR-mediated G protein signalling regulates OSM-9/OCR-2 activity, which controls cold tolerance and temperature acclimatisation.

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