Extended Data Fig. 2: Expression of DREADD receptors in Qrfp^iCre neurons.

We generated Rosa26^dreaddm3 mice and crossed them with Qrfp^iCre mice (Extended Data Fig. 1) to obtain mice that express hM3Dq-mCherry exclusively in iCre-expressing cells (Qrfp^iCre;Rosa26^dreaddm3 mice). a, Generation of mice that express hM3Dq and hM4Di in Cre-expressing neurons. Targeting vectors and structures of the targeted alleles of Rosa26^dreaddm3 and Rosa26^dreaddm4 mice. We mated these mice with FLP66 mice to delete the pgk-Neo cassette. Orange boxes indicate hM3Dq-mCherry or hM4Di-mCherry. Because the CAG promoter drives expression of hM3Dq-mCherry or hM4Dq-mCherry only after Cre-mediated excision of the floxed stopper element, this allowed us to express hM3Dq or hM4Di specifically in Cre-expressing neurons. b, Horizontal and coronal sections of brain prepared from a Qrfp^iCre;Rosa26^dreaddm3 mouse, showing the distribution of mCherry-positive neurons in the hypothalamus. c, Top, strategy for chemogenetic excitation or inhibition of whole iCre-positive neuronal populations in Qrfp^iCre mice. Bottom, chemogenetic excitation of iCre-positive cells in Qrfp^iCre mice induced hypothermia. Heterozygous (Q-het) or homozygous (Q-homo) Qrfp^iCre mice with heterozygous Rosa26^dreaddm3 (M3) and/or Rosa26^dreaddm4 (M4) alleles were subjected to experiments. CNO was administered at ZT12 (start of the dark period). The ambient temperature was 23 °C. We found that excitatory manipulation of Qrfp^iCre neurons in mice resulted in severe immobility. As the posture of these mice was similar to that observed during daily torpor, we initially postulated that activation of iCre-positive cells induced a daily torpor-like state. To evaluate this hypothesis, we measured body temperature and found that the induced state of immobility was accompanied by marked, long-lasting hypothermia. T_BAT decreased beginning about 5 min after CNO administration and lasted 12 h. Mice spontaneously recovered without external warming. By contrast, inhibitory DREADD manipulation of iCre-positive neurons did not have any effect on T_BAT. Notably, hM3Dq-mediated activation of iCre-positive neurons in Qrfp^iCre;Rosa26^dreaddm3 mice induced robust hypothermia, even in homozygous Qrfp^iCre mice in which Qrfp sequences are completely replaced by iCre in both alleles. This suggests that QRFP itself does not have a role in inducing hypothermia. The degree of hypothermia was greater in QRFP-deficient mice, which indicates that endogenous QRFP itself counteracts the hypothermia. d, Excitatory manipulation of Q neurons in Qrfp^iCre;Rosa26^dreaddm3 mice in the light period (at ZT1) also induced a long-lasting hypothermic state (n = 4 mice for each condition). Line and shading in c, d denote mean and s.d. of each group. AHA, anterior hypothalamus; ARC, arcuate nucleus; LPO, lateral preoptic area; MM, medial mammillary nucleus; SON, supraoptic nucleus; TMN, tuberomammillary nucleus; VMH, ventromedial hypothalamus.