Extended Data Fig. 8: Long-term activation of VMPO^LepR by chemogenetic (Gq-DREADD) or optogenetic (ChR2) conditioning is sufficient to induce heat tolerance in the heat endurance assay.

a, Viral injection of Cre-dependent Gq-DREADD into the rostral POA of LepR-Cre mice. VMPO^LepR neurons were chemogenetically activated (conditioned) by daily injection of CNO (0.3 mg/kg i.p.) for 1, 5 or 10 consecutive days. CNO injections were terminated 24 hours prior to heat endurance assay. b, Chemogenetic conditioning of VMPO^LepR cells via Gq-DREADD animals produced significant hypothermia that is protracted for up to 10 hours after CNO injection and could be repeated over multiple consecutive days. Traces represent group average (mean ± s.e.m.) for each day of CNO injection. N = 4 animals. c, All animals that were chemogenetically conditioned for 10 days passed the heat endurance assay. Animals that reached the cut-off temperature, demarcated by the dashed red line, were discontinued from the assay. d, Boxplots (median and IQR) showing endurance times (tE) before reaching 41.5 °C, corresponding to (c). The maximum tE was 9 hours (540 min, red dashed line). All chemogenetically conditioned animals for 10 days reached this maximum. Kruskal-Wallis test (H = 12.67, d.f = 3, P = 0.0006) with Sidak’s multiple comparison test, *P = 0.0312 **P = 0.0034. N = 5 for Control (animals that received saline injections for 10 days), N = 4 for ‘1 day’ (a single CNO injection), N = 4 for ‘5 day’ (5 days of CNO injections) and N = 5 for ‘10 day’ (10 days of CNO injections). e, Left: Representative traces of AP firing patterns of two VMPO^LepR neurons recorded 24 hours after chemogenetic conditioning for 5 or 10 days. Right: average AP firing frequency (mean ± s.e.m.) of VMPO^LepR neurons from non-stimulated control LepR-Cre;HTB animals and from animals chemogenetically conditioned for 5 or 10 days. Kruskal-Wallis test (H = 17.56, d.f = 2, P < 0.0001) with Dunn’s pairwise comparisons and Bonferroni corrections, ***P < 0.0001 (Control: 10d cond.). n = 42/4 cells per group. Neuronal recordings were performed without synaptic blockade, using ‘low-K⁺ aCSF’ and at 33 °C. f, Average body-, brown adipose- (BAT-) and tail temperature of LepR-Cre mice (N = 3) expressing Cre-dependent ChR2 and stimulated with blue light at a low frequency of 1 Hz at room ambient temperature (23 °C). g, Optogenetic control experiment: in the absence of ChR2, light stimulation of the POA/VMPO of up to 20 Hz continuously for 4 hours did not affect body temperature in freely moving mice (N = 4). h, Average body temperature (mean ± s.e.m.) during heat endurance of LepR-Cre mice expressing ChR2 in VMPO^LepR neurons that were either not optogenetically conditioned (control, light blue trace), conditioned for 1 day (1d opto, blue trace) or 3 days (3d opto, dark blue trace). All mice were optically stimulated with light pulses at 1 Hz during the heat endurance assay. N = 4 animals each.