Figure 4. TRPM8 Is Necessary for the Cold-Temperature-Mediated Increase in Ca²⁺ Spike Frequency in the Ventral Spinal Cord.

(A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA.

(B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord.

(C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control.

(D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining.

(E) Stage 24 ventral spinal cord from wild-type embryos was Ca²⁺ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials.

(F) Stage 24 ventral spinal cord from wild-type embryos was Ca²⁺ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca²⁺ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test.

(G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc: ornithine decarboxylase (101 bp) as positive control.

(H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca²⁺-imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca²⁺ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test.

See also Figures S2 and S3.