Fig 1: NaV1.3 is required for acclimation-induced, tonic warm-sensitive firing and heat tolerance.a, LepR-Cre mice POA injected with Cre-dependent constructs encoding shRNAs against Scn3a or scrambled control (scram-Scn3a shRNA). b, Acclimated VMPOLepR neurons labeled with GFP encoded within the shRNA constructs. Scale bars, 250 μm. ac, anterior commissure. c, Left: average INaP in VMPOLepR neurons expressing Scn3a shRNA and scrambled control. Traces are presented as mean ± s.e.m. Right: quantification (mean ± s.e.m.) of INaP at −35 mV, showing a reduction of INaP in Scn3a shRNA expressing acclimated LepR+ neurons. Unpaired, two-tailed Student’s t-test, *P = 0.0174; n = 8/4 (Scn3a shRNA) and n = 8/3 (scram-Scn3a shRNA) cells. d, Firing frequency of acclimated VMPOLepR neurons significantly reduced by the functional shRNAs. Unpaired two-tailed Student’s t-test, **P = 0.0044; n = 30/5 (Scn3a shRNA) and n = 20/3 (scram-Scn3a shRNA) cells. e, Distribution of temperature-insensitive, WSN and silent neurons within the acclimated VMPOLepR neuron population expressing either Scn3a (n = 47/5) or scram-Scn3a (n = 19/3) shRNA. f, NaV1.3fl/fl and WT controls were injected with an AAV encoding the Cre recombinase into the POA (cKO). g, Most of the WT animals able to defend their body temperature within physiological range. In contrast, all but one of the NaV1.3 cKO animals were unable to maintain their body temperature <41.5 °C (N = 9 for each group). h, Left: range of body temperatures of animals shown in g. Right: quantification of endurance time at 36 °C acclimation temperature of mice shown in g. Cut-off time was 72 h (dashed line). Mann–Whitney U-test, *P = 0.0155 (N = 9 each). i, Left: Allen Brain Atlas annotation of human POAs. Right: human tissue block covering POAs MnPO/MPA/OVLT (LFB/H&E stain). j, LEPR coexpression in human VMPO with RNAscope ISH. Left: PACAP + LEPR-tv1 (long isoform; coexpression in yellow). Middle: vGLUT2 + LEPR-alltv (all isoforms; coexpression in yellow). Right: LEPR-alltv + LEPR-tv1 (coexpression in yellow). Electrophysiological recordings were performed with fast synaptic transmission blockade and at 36 °C. Box plots show the median and IQR (Extended Data Fig. 10 and Supplementary Figs. 4 and 5).
Fig 2: Validation and electrophysiological characterization of Nav1.3 knock-down.a, Left: in order to test the efficacy of shRNA against the NaV1.3 mRNA, Cre-dependent shRNA-carrying AAVs were co-injected together with AAV encoding the Cre recombinase into the POA of C57/BL6 mice. Following 3 weeks of virus expression, mRNA was extracted from the POA tissue. Non-injected C57BL/6 mouse POA tissue served as control. Right: boxplot (median and interquartile range) of relative NaV1.3 mRNA expression normalized to the housekeeping genes Tubb3 and Ube2l3 in mouse POA. Unpaired two-tailed t-test, *P = 0.0226. N = 3 (WT) and N = 6 (Scn3a shRNA) mice. b, Firing frequencies of non-acclimated VMPOLepR neurons expressing either scrambled shRNA (scram-Scn3a shRNA; n = 27/2) or functional shRNAs against Nav1.3 (Scn3a shRNA; n = 27/3) at 36 °C. Shown as median and interquartile range. c, Left: plot showing AP firing frequency vs regularity of firing in VMPOLepR neurons expressing either functional shRNAs against NaV1.3 / Scn3a mRNA or scrambled control. Right: quantification of the firing regularity between the two groups (plotted as mean ± s.e.m.). Unpaired two-tailed t-test, *P = 0.0226. n = 30/5 (Scn3a shRNA) and n = 17/3 (scram-Scn3a shRNA) cells. d, Firing frequencies of acclimated VMPOLepR neurons expressing either scrambled shRNA (scram-Scn3a shRNA; n = 19/3) or functional shRNAs against NaV1.3 (Scn3a shRNA; n = 47/7) at the three indicated bath temperatures. Individual cells are plotted in color; black lines represent linear regression for each group. Slope=temperature coefficient (TC) = 1.2651 for scram-Scn3a shRNA and TC = 0.0474 for Scn3a shRNA, demonstrating that NaV1.3 knock-down significantly reduced warm sensitivity of acclimated VMPOLepR. e, Example traces of spontaneous warm-sensitive activity of acclimated VMPOLepR expressing either scrambled or functional shRNAs against NaV1.3 recorded at 33 °C, 36 °C and 39 °C.
Fig 3: Electrophysiological characterization of VMPOLepR neurons.a, RMP in acclimated VMPOLepR neurons (n = 19/3) depolarized compared with the RMP of non-acclimated VMPOLepR (n = 17/3) cells. Unpaired, two-tailed Student’s t-test, ***P = 0.0001. b, Membrane input resistance (Rm) comparable between non-acclimated (n = 37/9) and acclimated (n = 41/10) VMPOLepR neurons. c, Left: membrane hyperpolarization in non-acclimated (n = 17/2) and acclimated (n = 19/2) VMPOLepR neurons caused by replacement Na+ for NMDG+ in aCSF. Right: the difference in membrane potential (Δ) between Na+-based aCSF and NMDG+-based aCSF is larger in acclimated VMPOLepR neurons. Unpaired, two-tailed Student’s t-test, *P = 0.0201. d, Left: AP phase plot of non-acclimated (gray, n = 9/4) and acclimated (red, n = 10/5) VMPOLepR neurons. Right: both AP 10–90% rise time (Wilcoxon’s test, **P = 0.0046) and 90% to 10% decay time (unpaired, two-tailed Student’s t-test, ***P = 0.0006) are significantly faster in VMPOLepR neurons after acclimation. e, Left: current–voltage relationship for VMPOLepR neuron peak transient NaV currents recorded in nucleated patches. Two-way ANOVA (effect of acclimation voltage, *P < 0.0001; Tukey’s multiple-comparison test, **P = 0.0016 (−25 mV), ***P = 0.0003 (−20 mV), ***P = 0.0002 (−15 mV), ***P < 0.0001 (−10 mV), ***P = 0.0005 (−5 mV) and **P = 0.0072 (0 mV); n = 6/2 (Non-accl.) and n = 6/2 (Accl.) cells). Right: example of transient NaV current recordings from VMPOLepR neurons. Inset: voltage step protocol used. f, Left: average INaP, revealed by slow depolarizing voltage ramp, enhanced after heat acclimation (n = 12/4 (Non-accl.) and n = 10/4 (Accl.) cells). Inset: ramp protocol used to record INaP. Right: quantification of INaP at −35 mV based on data shown on the left. Unpaired, two-tailed Student’s t-test, *P = 0.0055. g, Left: INaP in acclimated VMPOLepR neurons reduced by riluzole (10 µM) and completely blocked by TTX (1 µM). Right: quantification of INaP at −35 mV based on data shown on the left. One-way ANOVA, P < 0.0001; Tukey’s multiple-comparison test, ***P < 0.0001 (Accl.:Accl. + riluzole), ***P < 0.0001 (Accl.:Accl. + TTX), *P = 0.0170 (Accl. + riluzole:Accl. + TTX) (n = 9/2 (Accl.), n = 10/2 (riluzole) and n = 7/2 (TTX) cells). h, Left: firing frequency (fAP) of acclimated VMPOLepR neurons reduced by riluzole (10 µM) and ICA121431 (200 nM). One-way ANOVA, P < 0.0001; Tukey’s multiple-comparison test, ***P < 0.0001 (Accl.:Accl. + riluzole), ***P < 0.0001 (Accl.:Accl. + ICA121431); n = 40/10 (Accl.), n = 35/4 (riluzole) and n = 39/6 (ICA121431) cells. Right: example traces of the three conditions shown. i, Left: NaV1.3 antagonist ICA121341 blocking INaP in acclimated VMPOLepR neurons to a similar extent to riluzole. Right: quantification of INaP at −35 mV based on data shown on the left. One-way ANOVA, P = 0.0002; Tukey’s multiple-comparison test, **P = 0.0029 (Accl.:Accl. + ICA121431), ***P = 0.0001 (Accl.:Accl. + ICA121431 + riluzole); n = 8/3 (Accl.), n = 12/4 (ICA121431) and n = 10/2 (ICA121431 + riluzole). Part of the Accl. INaP data shown in g was repurposed for comparisons shown here. j, Distribution of temperature-insensitive, CSN, WSN and silent neurons within acclimated VMPOLepR neuron populations recorded with either riluzole (10 µM) or ICA121431 (200 nM) in perfusion fluid (n = 33/4 for riluzole and n = 24/4 for ICA121431). k, Firing frequencies of acclimated VMPOLepR control cells (n = 30/5), acclimated VMPOLepR cells recorded with riluzole (n = 33/4) and acclimated VMPOLepR cells recorded with ICA121431 (n = 24/4). Individual cells are plotted in color; black lines represent linear regression for each group Tcore (slope or temperature coefficient) = 1.9 for Accl. control, Tcore = 0.68 for riluzole and Tcore = 0.29 for ICA121431. Acclimated control cells were randomly sampled from the acclimated VMPOLepR cells plotted in Fig. 1e. Box plots in a–c and h represent the median and IQR; elsewhere data are shown as mean ± s.e.m (Extended Data Fig. 9 and Supplementary Figs. 1 and 2). Neuronal activity and currents were recoded under fast synaptic transmission blockade and at 36 °C.
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