Extended Data Fig. 2: Heat acclimation-induced upregulation of tonic warm-sensitive AP firing is most robustly detected in the VMPO^LepR population.

a, Comparison of membrane capacitances in acclimated neurons from various VMPO populations recorded in acute slices, selecting cells with similar soma sizes. b, On average, acclimated VMPO^LepR neurons had a significantly higher firing frequency compared to acclimated VMPO^Vgat neurons, acclimated VMPO^Vglut2 neurons as well as randomly sampled VMPO acclimated neurons. Non-acclimated VMPO^LepR neurons plotted for reference. One-way ANOVA, P < 0.0001; Sidak’s multiple comparison test, ***P < 0.0001 (LepR+ Accl. : LepR+ Non-accl.), ***P < 0.0001 (LepR+ Accl. : Vgat+ Accl.), *P = 0.0362 (LepR+ Accl. : Vglut2+ Accl.), ***P < 0.0001 (LepR+ Accl. : VMPO random Accl.). n = 40/7 (LepR+ Accl), n = 40/6 (LepR+ Non accl), n = 31/3 (Vgat+ Accl.), n = 39/4 (Vglut2+ Accl.) and n = 21/2 (VMPO Random Accl.) cells. c, VMPO^LepR neurons (green) and DMH^LepR neurons (orange) were recorded in whole-cell patch clamp configuration to assess acclimation-induced AP firing frequency increases in acclimated and non-acclimated animals (n=35/5 per group). Kruskal-Wallis test (H = 46.20, d.f. = 3, P < 0.0001); Dunn’s pairwise comparisons with Bonferroni corrections (p < 0.0001 for Non-Accl. : Accl.POA LepR). No change in the average AP firing frequency was observed in DMH^LepR neurons. d, AP firing rates at 36 °C in both non-acclimated and acclimated VMPO^LepR neurons in ex vivo brain slices are comparable in the presence and absence of the fast synaptic transmission blockers (CNQX 10 µM, APV 50 µM and Gabazine 5 µM). n = 12/2 (Non-accl. –Syn. block), n = 15/3 (Non-accl. +Syn. block), n = 11/2 (Accl. –Syn. block), n = 15/3 (Accl. +Syn. block) cells. e, Adding acetylcholine receptor antagonists tubocurarine (10 µM) and scopolamine (10 µM) to the solution with CNQX, APV, and Gabazine slightly (but insignificantly) reduced AP firing in non-acclimated VMPOLepR neurons and did not affect AP firing in acclimated VMPOLepR cells: n = 25/5 (Non-accl. / syn. block), n = 25/4 (Non-accl. / syn. block+tubocurarine+scopolamine), n = 25/6 (Accl. / syn. block), n = 25/4 (Accl. / syn. block+tubocurarine+scopolamine). Data recorded at 33 °C. f, Frequencies of VMPOLepR at sub-physiological and physiological temperatures: regression analysis (grey and red lines) of non-acclimated and acclimated VMPOLepR firing rates at 33 °C, 36 °C, and 39 °C (data in main Fig. 1e). The analysis predicted that firing rates would be indistinguishable at ~29.1 °C (intersection of red and grey lines), confirmed experimentally by recording at 27 °C and 30 °C. Data partially overlapping with Fig. 1e. g, Warm sensitivity (measured by the temperature coefficient T_c) of tested VMPO neuronal populations after heat acclimation. One-way ANOVA, P < 0.0001; Tukey’s multiple comparison test, ***P < 0.0001 (LepR+ : Vgat+), ***P < 0.0001 (LepR+ : Vglut2+), ***P < 0.0001 (LepR+ : Pacap+), ***P < 0.0001 (LepR+ : FosTRAP 4h), ***P < 0.0001 (LepR+ : FosTRAP 8h). n = 38/7 (LepR+), n = 31/3 (Vgat+), n = 36/4 (Vglut2+), n = 31/3 (Pacap+), n = 18/2 (FosTRAP 4h) and n = 26/3 (FosTRAP 8h) cells. h, Distribution of ex vivo recorded temperature-insensitive, cold-sensitive (CSN, temperature coefficient < −0.6 Hz/°C), warm-sensitive (WSN, temperature coefficient ≥ 0.75 Hz/°C) and silent neurons within the acclimated VMPO neuronal populations demarcated by the expression of Vgat (n = 31/3) and Vglut2 (n = 36/4) as well as ‘warm TRAPped’ neurons for either 4h (n = 18/2) or 8h (n = 26/3). Compare with Fig. 1d. i, Spontaneous activity pattern in representative non-acclimated and acclimated VMPO^LepR differs not only by frequency but also regularity of action potential firing as evidenced by the different inter spike interval coefficient of variation (ISI CoV, a measure of AP firing regularity, is the standard deviation of the interspike interval (ISI) divided the mean ISI). Analysed recordings were performed at 36 °C bath temperature. j Interspike interval coefficient of variation (ISI CoV) for the indicated neuronal populations obtained from heat acclimated mice. One-way ANOVA, P < 0.0001; Tukey’s multiple comparison test, ***P < 0.0001 (LepR+ Non-accl. : LepR+ Accl.), *P = 0.0215 (LepR+ Accl. : Vgat+), **P = 0.0011 (LepR+ Accl. : FosTRAP 4h). n = 63/7 (LepR+ Non-accl.), n = 57/7 (LepR+ Accl.) n = 22/3 (Vgat+), n = 31/4 (Vglut2+), n = 30/3 (Pacap+), n = 16/2 (FosTRAP 4h), n = 28/3 (FosTRAP 8h) and n = 15/2 (Random) cells. Neuronal activity was recoded in brain slices under fast synaptic transmission blockade and using ‘high-K+ aCSF’ except for panels (c) and (e) where ‘low-K+ aCSF’ and 33 °C bath temperature were used. Boxplots in (a), (b), (d), (e), (f), (g), and (j) represent median and interquartile range; data in (c) are shown as mean ± s.e.m.