Spencer KA , Belgacem YH , Visina O , Shim S , Genus H , Borodinsky LN .

R01 NS073055 NINDS NIH HHS

	Figure 1. The Temperature of the Environment during Development Influences Early Swimming Behavior in Xenopus laevis Larvae (A) Types of behavioral responses to gentle touch; modified from [26, 27]. (B–G) Stage 37/38 (B) and 40 (C-G) larvae grown in cold (blue, 14.5°C–16°C) or warm (green, 22.5°C) temperature were subjected to 20 trials each of gentle touch at cold (14.5°C) or warm (22.5°C) temperature. Responses were video recorded at 30 Hz, n = 6 larvae per condition. (B) Average percentage of incidence of each response. (C) Example of full time course of swim speed after gentle touch (0 s) from stage 40 larva grown in and tested at warm temperature. (D) Time course of changes in speed during initial phase (first 200 ms) of the swim response. (E) Swim duration. (F) Maximum speed during initial phase of the swim response. (G) Maximum tangential acceleration during initial phase of the swim response. In (D)–(G), data are mean ± SEM. In (B) and (E)–(G), the letters on top of datasets indicate significant (different letters) or not significant (same letters) differences, p < 0.05, 2-way ANOVA, Tukey post hoc test.
	Figure 2. The Number of Spinal Motor Neurons in Developing Larvae Is Temperature Dependent (A) Images show cross-sections of immunostained spinal cord (outlined) from stage 40 larvae grown in cold (14.5°C) or warm (22.5 or 26.5°C) temperatures. (B) Graph shows number of HB9-immunopositive cells per 100 μm of spinal cord; mean ± SEM from at least 220 μm length of spinal cord per larva, n ≥ 4 larvae per condition, ∗∗∗p < 0.001, 1-way ANOVA, Tukey post hoc test. (C) Images show maximum intensity projections of whole-mount spinal cord from stage 40 larvae grown in different temperatures followed by retrograde labeling with Alexa 488-dextran conjugate from ventral axial musculature. (D) Graph shows numbers of retrogradely labeled motor neurons; mean ± SEM for each experimental group, n ≥ 10 larvae per condition, ∗∗∗∗p < 0.0001; ns, not significant’ Kruskal-Wallis test’ Dunn’s multiple comparisons test. (E) Images show maximum intensity projections of whole-mount immunostained axonal bundle #5 (with 1 being the most anterior axonal bundle innervating the axial musculature) with the axonal marker 3A10 (neurofilament associated protein), from stage 40 larvae grown in the indicated temperatures. (F) Graph shows area labeled per axonal bundles 4 through 7; mean ± SEM for each experimental group, n ≥ 10 larvae per condition, ∗p < 0.05, 1-way ANOVA, post hoc Tukey test. (G–I) Larvae grown in cold temperature exhibit fewer apoptotic spinal cord cells and motor neurons. (G) Images show cross-sections of immunostained spinal cord (outlined) from stage 40 larvae grown in different temperatures. Scale bars in (A), (C), (E), and (G), 20 μm. (H and I) Graphs show percentage of TUNEL+ (H, per total DAPI-labeled nuclei) and HB9+/TUNEL+ cells (I, per total HB9+ cells) per 100 μm of spinal cord; mean ± SEM from at least 220 μm length of spinal cord per larva, n ≥ 4 larvae per condition, ∗∗∗p < 0.001, two-tailed t test.
	Figure 3. Cold Temperature Acutely Increases Ca2+ Spike Frequency of Embryonic Ventral Spinal Neurons Ventral view of spinal cords from stage 24 embryos were Ca2+ imaged for 30-min intervals at 14.5°C, 22.5°C, and 26.5°C ± 0.5°C, in a randomized order. (A) Traces illustrate the same representative ventral spinal neuron imaged at the indicated temperatures. (B) Graph shows individual cell and mean (black squares) Ca2+ spike frequency (30 min-1) from N = 4 ventral spinal cords (n of neurons analyzed: 14.5°C, 102; 22.5°C, 104; 26.5°C, 86), ∗∗∗∗p < 0.0001; ns, not significant; repeated-measures mixed effect analysis; post hoc Tukey test. (C) Graph shows ratio of Ca2+ spikes at each temperature compared to the sum of spikes at the 3 temperatures per cell, n = 58. Teal lines represent neurons with higher spike frequency at 14.5°C, magenta lines represent neurons with higher spike frequency at 22.5°C or 26.5°C, and black lines represent neurons with no change in spike frequency across temperatures; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant; repeated-measures 2-way ANOVA, post hoc Tukey test.
	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-blocking MO 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 per group (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ∗∗∗∗p < 0.001, Kolmogorov-Smirnov, two-tailed test.
	Figure 5. Electrical Activity Regulates the Number of Motor Neurons in the Spinal Cord and hb9 Transcription (A) Images show cross-sections of immunostained spinal cord (outlined) from stage 35 larvae in which activity was enhanced (veratridine [verat], Nav2a overexpression) or suppressed (voltage-gated Na+ and Ca2+ channel blockers [VGC blockers]: calcicludine, ω-conotoxin-GVIA, flunarizine, and tetrodotoxin). Scale bar, 20 μm (B) Graph shows number of HB9-immunopositive cells per 100 μm spinal cord; mean ± SEM from at least 220 μm length of spinal cord per larva, n ≥ 4 larvae per condition, ∗p < 0.05, 1-way ANOVA. (C) Schematic of luciferase reporters used to assess regulation of hb9 transcription. Gray box represents a 270-bp fragment from the M250/Region B in the hb9 5′ regulatory region. (D) Spinal cord from wild-type or Nav2a-overexpressing embryos injected with wild-type or mutant forms of the hb9 transcription reporter were incubated for 8 h with 1 μM veratridine or vehicle only and processed for luciferase activity measurements. Graph shows percentage of normalized luciferase intensity compared with control (wild-type embryo incubated with vehicle only), mean ± SEM, n > 5 spinal cords per group, ∗p < 0.05, 1-way ANOVA.
	Figure 6. TRPM8 Is Necessary for the Increase in Motor Neuron Number and the Adaptation of the Sensorimotor Response of Animals Grown in Cold Temperatures (A) Images show cross-sections of immunostained spinal cord (outlined) for HB9 from Control-MO- or TRPM8-sbMO-containing stage 40 larvae grown in cold temperature (16°C). Scale bar, 20 μm. (B) Graph shows number of HB9-immunopositive cells per 100 μm of spinal cord; mean ± SEM from at least 220 μm-length spinal cord per larva, n = 5 larvae per condition, ∗∗p < 0.005, two-tailed t test. (C–G) TRPM8-sbMO or Control-MO stage 37/38 (C) and 40 (D–G) larvae grown in cold temperature (16°C) were subjected to 20 trials each of gentle touch at cold (14.5°C) or warm (22.5°C) temperature. Responses were video recorded at 30 Hz, n = 6 larvae per condition. (C) Average percentage of incidence of each response. (D) Time course of changes in speed during initial phase (first 200 ms) of the swim response. (E) Swim duration. (F) Maximum speed during initial phase of the swim response. (G) Maximum tangential acceleration during initial phase of the swim response. In (D)–(G), data are mean ± SEM. In (C) and (E)–(G), the letters on top of datasets indicate significant (different letters) or not significant (same letters) differences, p < 0.05, 2-way ANOVA, Tukey post hoc test.
	Figure 7. Model of Mechanism of Cold-Temperature Adaptation TRPM8 activation at cold temperature results in an increase in Ca2+ spike frequency in developing spinal cord neurons. This enhanced spike frequency increases HB9-dependent motor neuron differentiation and survival. The low-temperature mediated increase in motor neuron number facilitates faster escape swimming at cold temperature compared to animals grown in warm temperature, for efficiently evading predators and increasing rate of survivability.
	Immunostained transverse section of NF stage 25 Xenopus spinal cord (outlined) using Trpm8 Ab1 (Alomone Labs Cat# ACC-049, RRID:AB_2040254). D, dorsal; V, ventral; scale bar, 20 μm. Arrows indicate TRPM8 clusters in ventral neuron domains.

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