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Neurons that regulate mouse torpor

Abstract

The advent of endothermy, which is achieved through the continuous homeostatic regulation of body temperature and metabolism1,2, is a defining feature of mammalian and avian evolution. However, when challenged by food deprivation or harsh environmental conditions, many mammalian species initiate adaptive energy-conserving survival strategies—including torpor and hibernation—during which their body temperature decreases far below its homeostatic set-point3,4,5. How homeothermic mammals initiate and regulate these hypothermic states remains largely unknown. Here we show that entry into mouse torpor, a fasting-induced state with a greatly decreased metabolic rate and a body temperature as low as 20 °C6, is regulated by neurons in the medial and lateral preoptic area of the hypothalamus. We show that restimulation of neurons that were activated during a previous bout of torpor is sufficient to initiate the key features of torpor, even in mice that are not calorically restricted. Among these neurons we identify a population of glutamatergic Adcyap1-positive cells, the activity of which accurately determines when mice naturally initiate and exit torpor, and the inhibition of which disrupts the natural process of torpor entry, maintenance and arousal. Taken together, our results reveal a specific neuronal population in the mouse hypothalamus that serves as a core regulator of torpor. This work forms a basis for the future exploration of mechanisms and circuitry that regulate extreme hypothermic and hypometabolic states, and enables genetic access to monitor, initiate, manipulate and study these ancient adaptations of homeotherm biology.

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Fig. 1: Neuronal activity induces key features of torpor.
Fig. 2: Identification of brain regions that regulate torpor.
Fig. 3: Molecular characterization of torpor-associated avMLPA neurons.
Fig. 4: Sufficiency, necessity and natural activity of avMLPA neuronal subpopulations during torpor.

Data availability

RNA-sequencing data have been deposited in the Gene Expression Omnibus with accession number GSE149344. Additional data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

Custom code used in this study is available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank B. Sabatini, M. Andermann, B. Lowell, C. Saper and S. R. Datta for feedback on this work; members of the Sabatini and W. Regehr laboratories for reagents; members of the Greenberg, Sabatini, Andermann, Lowell and Datta laboratories for discussions; and A. Mina for technical assistance. This project relied on the Beth Israel Deaconess Medical Center Metabolic Core for experiments in metabolic cages, Boston Children’s Hospital Viral Core for AAV packaging, the Harvard NeuroDiscovery Center Enhanced Neuroimaging Core and the Neurobiology Imaging Facility (NINDS P30 Core Center grant NS072030) for imaging, the Data Analysis Core at Harvard Medical School for help with image analysis, and the Research Instrumentation Core Facility for engineering support. S.S. acknowledges support from a Herchel Smith Fellowship. This work was supported by National Institutes of Health grants R01 NS028829 and BRAIN Initiative grant R01 MH114081 to M.E.G., R01 DK107717 to A.S.B. and the Warren Alpert Distinguished Scholar Award to S.H.

Author information

Authors and Affiliations

Authors

Contributions

S.H. conceived the study and designed, performed and analysed experiments. S.S. designed, performed and analysed experiments. O.F.W., H.Y., A.J.L.-P., E.G.A. and M.E.P. performed and analysed experiments. S.A. advised on and analysed fibre photometry experiments. M.C. wrote the code to register and analyse images of brain sections. A.S.B., E.C.G. and M.E.G. advised on the study. S.H., E.C.G. and M.E.G. obtained funding for the research. S.H., S.S., E.C.G. and M.E.G. wrote the manuscript.

Corresponding authors

Correspondence to Sinisa Hrvatin or Michael E. Greenberg.

Ethics declarations

Competing interests

S.A. is the founder and CEO of Neurophotometrics Ltd., which manufactures fibre photometry systems. All other authors declare no competing interests.

Additional information

Peer review information Nature thanks Trygve E. Bakken, Shaun F. Morrison and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Torpor metabolic rate, brain-wide search for torpor-regulating cells and chemogenetic reactivation of FosTRAP-Gq mice.

a, Mean metabolic rate (VO2), body temperature (Tb) and gross motor activity (Act) of mice in torpor compared to mice that are fed or fasted yet not in torpor (n = 7, P values indicated on the graph). b, Schematic for the whole-brain reconstruction of FOS staining. c, Example brain slice showing Fos staining in a fasted torpid mouse (representative of n = 3 mice). d, 3D-reconstructed Fos-stained brain slices from a fasted torpid mouse. e, Average density of FOS+ cells (number of cells divided by the volume of the region, n = 3 mice, see Methods) across 179 brain regions that had on average at least 100 FOS+ cells. Paraventricular hypothalamus (PVH), a subregion of the preoptic area (POA), arcuate nucleus (ARC), dorsomedial hypothalamus (DMH) and paraventricular thalamus (PVT) are indicated. f, FOS staining of the PVH, xiphoid nucleus (Xi), POA, ARC, DMH and PVT of fasted torpid mice (n = 3 mice). g, Mean core body temperature (Tb) over 4 h after CNO administration is significantly lower in torpor-TRAP (n = 14 mice) compared to non-TRAP (n = 6 mice, P = 5.2 × 10−5) and fed-TRAP (n = 9 mice, P = 2.9 × 10−5) mice and compared to torpor-TRAP mice injected with PBS (n = 8 mice, P = 2.5 × 10−5). h, Mean activity over 4 h after CNO administration is significantly lower in torpor-TRAP (n = 14 mice) compared to non-TRAP (n = 6 mice, P = 5.2 × 10−5) and fed-TRAP (n = 9 mice, P = 9.8 × 10−3) mice and compared to torpor-TRAP mice injected with PBS (n = 8 mice, P = 2.5 × 10−5). i, Coronal brain sections from FosTRAP, LSL-Gq-DREADD-HA mice TRAPed during fasting-induced torpor (fast-TRAP, n = 2 mice) or in a fed state (fed-TRAP, n = 4 mice) and immunostained for HA. Staining in selected brain areas (PVH, POA, ARC, DMH and PVT) is shown. j, Volume-normalized signal intensity of HA staining across different hypothalamic nuclei in four fed-TRAP and two fast-TRAP mice. k, Brain-wide quantification of HA staining from four fed-TRAP and two fast-TRAP mice. Numerous (190/316) brain regions, including 32 hypothalamic areas, show increased Gq-DREADD-HA expression (>2-fold) in fast-TRAP mice compared to fed-TRAP mice. The solid line indicates unity, dashed lines indicate twofold differences. l, Correlation across brain regions between the number of FOS+ cells in torpid mice and the levels of Gq-DREADD expression in fast-TRAP mice (R = 0.83, P = 2.2 × 10−16, Pearson correlation test, n = 316 regions). All box plots indicate mean ± s.e.m. All P values are calculated using two-tailed Mann–Whitney U-tests, **P < 0.01, ***P < 0.001.

Extended Data Fig. 2 Chemogenetic reactivation of torpor-TRAPed neurons in different hypothalamic regions and anterograde projections of torpor-TRAPed avMLPA neurons.

ac, AAV-DIO-Gq–mCherry was injected into different hypothalamic regions of FosTRAP mice (n = 54 mice). After TRAPing during torpor, we administered CNO and measured the effect of the reactivation of torpor-TRAPed neurons within the virally injected region on core body temperature. All mice were euthanized, and the expression of the virally derived Gq-DREADD–mCherry was evaluated in each mouse across 277 hypothalamic nuclei. a, b, Each circle represents one of the 277 hypothalamic nuclei, and the y axis represents the −log10 FDR-corrected q value of the Pearson correlation (across 54 mice, q values displayed in Supplementary Table 3) between the viral expression in that nucleus and the decrease in Tb that was observed after CNO stimulation. Next, for each nucleus, 54 mice were grouped into those in which the nucleus was hit (a) and those in which it was missed (b). For each of the two groups of mice, the minimum body temperature after CNO administration was averaged and plotted. c, For each nucleus and the corresponding two groups of mice, the minimum body temperature after CNO administration was plotted (hit group, y axis; missed group, x axis). Arrows indicate anterior MPA and LPO regions. When these regions were hit with the virus and the TRAPed neurons were chemogenetically reactivated, the body temperature of the mouse decreased, whereas when these regions were missed the body temperature did not decrease. d, Mean activity over 4 h after CNO administration is significantly lower in avMLPA-hit (n = 15 mice) compared with avMLPA-missed (n = 11 mice, P = 1.4 × 10−3) mice, and compared with avMLPA-hit mice injected with PBS (n = 15 mice, P = 4.8 × 10−6). e, Mean metabolic rate (VO2) over 4 h after CNO administration is significantly lower in avMLPA-hit (n = 7 mice) compared with non-injected (n = 6 mice, P = 2.3 × 10−3) mice or avMLPA-hit mice injected with PBS (n = 6 mice, P = 1.2 × 10−3). f, Mean core body temperature (Tb) over 4 h after CNO administration is significantly lower in avMLPA-hit (n = 15 mice) compared to avMLPA-missed (n = 11 mice, P = 2.6 × 10−7) mice or avMLPA-hit mice injected with PBS (n = 15 mice, P = 9.0 × 10−8). g, Minimum metabolic rate (VO2) over 4 h after CNO administration is significantly lower in avMLPA-hit (n = 7 mice) compared with non-injected (n = 6 mice, P = 2.3 × 10−3) mice or avMLPA-hit mice injected with PBS (n = 6 mice, P = 1.2 × 10−3). h, Schematic showing projections of TRAPed avMLPAtorpor neurons. i, j, Gq-DREADD–mCherry fusion protein expression was used to visualize the projection of TRAPed avMLPAtorpor neurons across the brain (n = 4 mice). i, Expression of mCherry near the injection site (avMLPA). j, Representative images of projections to the medial habenula (MHb), PVT, DMH, periaqueductal grey (PAG), ARC and raphe pallidus (RPa). Scale bars, 50 μm. For the box plots, the centre line and box boundaries indicate mean ± s.e.m. All P values were calculated using a two-tailed Mann–Whitney U-test, **P < 0.01, ***P < 0.001.

Extended Data Fig. 3 snRNA-seq metrics.

a, b, UMAP plot of 39,562 nuclei from the avMLPA of five mice, in which the colours denote cells derived from each mouse (a) or the number of unique transcripts (UMI) per nucleus (b). c, Relative contribution of each sample (n = 5 mice) towards the total cell population making up each main cell class. d, Violin plot of the distribution of UMIs per cell for each main cell class (glutamatergic neurons, n = 11,275 cells; GABAergic neurons, n = 16,307 cells; cholinergic neurons, n = 521 cells; astrocytes, n = 3,479 cells; endothelial cells, n = 421 cells; microglia, n = 1,247 cells; oligodendrocytes, n = 4,718 cells; and OPCs, n = 1,594 cells). e, Violin plot of the distribution of genes per cell for each main cell class. f, Number of neuronal clusters formed when different fractions (25%, 50%, 75% and 90%) of total neurons (n = 7,025, 14,051, 21,077 and 25,292, respectively) are used for clustering. For each fraction a random subset of neurons was used and the analysis was repeated ten times. For the box plots, the centre line and box boundaries indicate mean ± s.e.m, and the violin plot shows the distribution from the lowest to the largest value.

Extended Data Fig. 4 Marker gene expression across neuronal cell types.

The colour denotes mean expression across all nuclei normalized to the highest mean across cell types, and the size represents the fraction of nuclei in which the marker gene was detected. Cell types are organized on the basis of hierarchical clustering across all variable genes. The five most unique makers are identified and plotted for each cell type unless a marker was identified across multiple cell types, in which case it was plotted only once.

Extended Data Fig. 5 Strategy for identifying TRAPed torpor-regulating neurons via snRNA-seq and gene expression of marker genes in the avMLPA.

a, Schematic for the identification of Cre-dependent AAV-DIO-Gq-DREADD–mCherry mRNA with or without recombination. Top, AAV-DIO-Gq-DREADD–mCherry vector map before (Cre) and after (Cre+) Cre-mediated recombination. Blue and white triangles surrounding the Gq-GREADD–mCherry indicate loxP sites. Black arrows indicate the binding site of the sequencing primer. ITR, inverted terminal repeats; WPRE, Woodchuck Hepatitis Virus post-transcriptional regulatory element; Poly-A, polyadenylation signal. Bottom, owing to the Cre-mediated inversion in the AAV-DIO-Gq-DREADD–mCherry vector, the mRNA transcript sequence 3′ of the sequencing primer is different after Cre-mediated recombination, enabling us to identify TRAPed cells during snRNA-seq as those cells in which the viral mRNA contains the recombined (Cre+) sequence. b, Quantification of the number of virally transduced cells in TRAPed (n = 4 mice) and non-TRAPed (n = 1 mouse) samples. c, Quantification of the number of TRAPed cells in TRAPed (86 ± 27 cells) and non-TRAPed (1 cell) samples. d, The percentage of transduced cells that are TRAPed in TRAPed (2.3 ± 0.7%, n = 4 mice) and non-TRAPed (0.04%, n = 1 mouse) samples based on snRNA-seq analysis. e, The percentage of TRAPed neurons in TRAPed samples (1.8 ± 0.3%, n = 4 mice) based on fluorescence in situ hybridization analysis. fh, Mean transcripts per cell across all neuronal cell types identified in snRNA-seq for Vgat (Slc32a1, marker of GABAergic neurons) (f), Vglut2 (Slc17a6, marker of glutamatergic neurons) (g) and Adcyap1 (adenylate cyclase-activating peptide 1) (h). i, snRNA-seq indicates that e2, e5, e10, e11, 16 and e30 represent Vglut2+Adcyap1+ cell types, whereas e22, e27, h33, h12 and h24 are Vglut2+Adcyap1. On the basis of this categorization, 72.4 ± 2.2% of Vglut2+ neurons are Adcyap1+ (n = 5 mice). For the box plot, the centre line and box boundaries indicate mean ± s.e.m. j, Mean transcripts per cell across all neuronal cell types identified in snRNA-seq for Lepr. Adcyap1+ clusters e5 and e10 express Lepr. Data are mean ± 2s.e.m.

Extended Data Fig. 6 In situ hybridization analysis of torpor-regulating avMLPA neurons.

a, Coronal sections showing the avMLPA of FosTRAP mice (n = 4 mice) injected with AAV-DIO-Gq-DREADD–mCherry and torpor-TRAPed. Immunofluorescent staining against mCherry indicates the location of avMLPAtorpor neurons (cyan), whereas in situ hybridization indicates the expression of the marker gene Adcyap1. b, High-magnification images of staining shown in a indicate the location of mCherry+ avMLPAtorpor neurons (cyan), whereas in situ hybridization indicates the expression of marker genes Adcyap1 and Vglut2. Example avMLPAtorpor mCherry+ cells are circled. Several mCherry+ cells express Adcyap1 and/or Vglut2. c, Quantification of the fraction of avMLPAtorpor neurons that express Adcyap1 (28.8 ± 3.5%, n = 4 mice) and Vglut2 (38.5 ± 3.8%, n = 4 mice). d, Quantification of the fraction of avMLPAAdcyap1+ (14.3 ± 0.5%, n = 4 mice) and avMLPAVglut2+ (13.0 ± 1.8%, n = 4 mice) neurons that are torpor-TRAPed. e, Coronal section showing the avMLPA of FosTRAP mice. In situ hybridization shows cells that are positive for Adcyap1 (cyan), Vgat (yellow) and Vglut2 (purple). The composite image indicates co-expression of multiple markers. f, High-magnification image with example Adcyap1+ cells circled. White circles indicate Adcyap1+ cells that are positive for Vglut2 and negative for Vgat, whereas yellow circles indicate all Adcyap1+ cells that are positive for Vgat (even if co-positive with Vglut2). g, The fraction of Adcyap1+ cells that are positive for Vglut2 or Vgat (82 ± 3% or 14 ± 1%, respectively, n = 3 mice). For the box plots, the centre line and box boundaries indicate mean ± s.e.m.

Extended Data Fig. 7 Expression pattern of Vgat, Vglut2 and Adcyap1 in the anterior POA.

Coronal sections adapted from the Allen Mouse Brain Atlas32. Anterior–posterior coordinates relative to bregma are indicated for each set of images.

Extended Data Fig. 8 Chemogenetic stimulation and silencing of avMLPAVgat, avMLPAVglut2 or avMLPAAdcyap1 neurons.

ac, Stereotaxic viral injection of AAV-DIO-Gq-DREADD and subsequent chemogenetic stimulation of avMLPAVgat (n = 6 mice), avMLPAVglut2 (n = 5 mice) or avMLPAAdcyap1 (n = 8 mice) neurons. a, Experimental schematic. b, Minimum core body temperature of avMLPAVgat mice (orange, P = 0.48), avMLPAVglut2 mice (light blue, P = 8 × 10−3) and avMLPAAdcyap1 mice (dark blue, P = 1.6 × 10−4) before and after chemogenetic stimulation with CNO. c, Mean activity of the same avMLPAVgat mice (P = 0.24), avMLPAVglut2 mice (P = 0.032) and avMLPAAdcyap1 mice (P = 1.6 × 10−4), before and after chemogenetic stimulation with CNO. d, Schematic showing the unilateral stereotaxic viral injection of AAV-DIO-Gq-DREADD and subsequent chemogenetic stimulation of avMLPAAdcyap1 neurons. e, Change in mean core body temperature after bilateral (n = 8 mice) and unilateral (n = 4 mice) chemogenetic stimulation of avMLPAAdcyap1 neurons. The dashed line indicates CNO administration. Coloured lines indicate the mean core body temperature across mice; grey shading indicates the 95% confidence interval. f, Mean core body temperature of mice before and after bilateral (n = 8 mice, P = 1.6 × 10−4) or unilateral (n = 4 mice, P = 0.03) chemogenetic stimulation of avMLPAAdcyap1 neurons. g, Schematic for the stereotaxic viral co-injection of AAV-Flex-TeLC and AAV-DIO-Gq-DREADD and subsequent chemogenetic stimulation of avMLPAVglut2 and avMLPAAdcyap1 neurons. h, Changes in mean core body temperature after chemogenetic stimulation of avMLPAVglut2 and avMLPAAdcyap1 neurons that either express the excitatory Gq-DREADD receptor (n = 6 and n = 8 mice, respectively) or co-express the Gq-DREADD receptor and TeLC, which inhibits synaptic transmission (n = 2 and n = 4 mice, respectively). The dashed line indicates CNO administration. Coloured lines indicate the mean core body temperature across mice; grey shading indicates the 95% confidence interval. i, Quantification of mean core body temperature over 4 h after chemogenetic stimulation in avMLPAVglut2 and avMLPAAdcyap1 (P = 1 × 10−6) neurons that either solely express the excitatory Gq-DREADD receptor (n = 6 and n = 8 mice, respectively) or co-express the Gq-DREADD and TeLC (n = 2 and n = 4 mice, respectively). jo, Stereotactic injection of AAV-Flex-TeLC to inhibit synaptic transmission in avMLPAVglut2 and avMLPAAdcyap1 neurons. j, k, Core body temperature of fed and fasted Vglut2-IRES-Cre mice from Fig. 4e (the number of mice in each group is indicated on the graph). j, The minimum Tb is not significantly different between control-fed and TeLC-fed (P = 0.72) mice, but is significantly lower in control-fast (P = 0.018), and pre-fast (P = 0.01) compared to TeLC-fast mice, suggesting that avMLPAVglut2 activity is necessary for torpor. k, Time needed to reach the minimum body temperature (Fig. 4e) is significantly longer in TeLC-fast compared with either pre-fast or control-fast mice (P = 9.2 × 10−3 for both sets). l, m, Body temperature of fed and fasted Adcyap1-2A-Cre mice from Fig. 4f (the number of mice in each group is indicated on the graph). l, The minimum Tb is not significantly different between control-fed and TeLC-fed (P = 0.41) mice, or in TeLC-fast compared to control-fast (P = 0.71) and pre-fast (P = 0.19) mice. m, Time needed to reach the minimum body temperature (Fig. 4f) is significantly longer in TeLC-fast compared to pre-fast (P = 2 × 10−3) and control-fast (P = 7 × 10−3) mice. n, o, Core body temperature (measured in 1-min intervals) of fed mice during the 12-h light and 12-h dark cycle in which avMLPAVglut2 (n) or avMLPAAdcyap1 (o) neurons were injected with either AAV-Flex-TeLC (TeLC), a control AAV (control), or remained un-injected (Pre). The core body temperature is significantly different between the dark and light cycle across pre-fed (n = 3 mice, n = 3,960 temperature data points, P = 2 × 10−16), control-fed (n = 2 mice, n = 2,640 temperature data points, P = 2 × 10−16) and TeLC-fed (n = 5 mice, n = 6,600 temperature data points, P = 2 × 10−16) Vglut2-IRES-Cre mice (n) as well as pre-fed (n = 4 mice, n = 5,280 temperature data points, P = 2 × 10−16), control-fed (n = 7 mice, n = 9,240 temperature data points, P = 2 × 10−16) and TeLC-fed (n = 8 mice, n = 10,560 temperature data points, P = 2 × 10−16) Adcyap-2A-Cre mice (o). In the box plots the centre line denotes the median, the box boundaries mark the interquartile range (IQR) and the whiskers extend to 1.5 × IQR and any data points outside this range. p, q, Coronal section showing the avMLPA of Adcyap1-2A-Cre mice (n = 2 mice) injected with AAV-Flex-TeLC-eYFP. Immunofluorescent staining against eYFP indicates the location of silenced TeLC+ neurons (green), whereas in situ hybridization indicates the expression of the Adcyap1 mRNA. q, High-magnification image with example Adcyap1+ cells circled. White circles indicate Adcyap1+ cells that co-express TeLC–eYFP (43 ± 5%, n = 2 mice), yellow circles indicate Adcyap1+ that do not co-express TeLC–eYFP. All P values are calculated using a two-tailed Mann–Whitney U-test. NS indicates not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001. In the box plots in bm, the centre line and box boundaries indicate mean ± s.e.m.

Extended Data Fig. 9 Fibre-photometry set-up, recordings and torpor model.

a, Schematic showing the fibre-photometry set-up. Three LED lights (415 nm, 470 nm and 560 nm) were used as excitation light sources. For all recordings, 470 nm and 560 nm light sources were driven in phase, with 415 nm driven out of phase (Methods). The emitted signals were detected by a digital camera at the end of a patch cord. b, Example coronal brain slice from an Adcyap1-2A-Cre mouse co-injected with AAV-DIO-Gq-DREADD–mCherry and AAV-Flex-GCaMP6s and used for fibre photometry studies (n = 8 mice). The white dashed lines indicate the location of the optical fibre. Cells co-expressing GCaMP6s (green) and mCherry (red) appear yellow. c, Example fibre-photometry recording (from mouse shown in b) showing the core body temperature (top) followed by three different signals (470 nm, 415 nm and 560 nm). Here, the 470-nm signal represents the calcium-dependent GCaMP6s signal, the 415-nm signal represents the Ca2+-independent isosbestic GCaMP6s signal, and the 560-nm signal represents the mCherry signal. The red line indicates the scaled fit of the Ca2+-independent 415-nm signal used to normalize the Ca2+-dependent 470-nm signal for Ca2+-independent changes in signal intensity. Both the 415-nm and 560-nm channels serve as controls for heat-mediated LED decay, bleaching of GCaMP6s and movement artefacts. d, Recordings of a representative fasting session. Top panel, core body temperature of mice during each recording session (dashed line indicates the threshold body temperature below which the mouse is considered torpid); second panel, raw Ca2+-dependent 470-nm GCaMP6s signal (the red line indicates the scaled fit of the Ca2+-independent 415-nm signal used to normalize for bleaching or other Ca2+-independent changes in signal intensity); third panel, dF/F value relative to the Ca2+-independent scaled fit (blue line indicates the local baseline, which is determined as the 10th percentile of the dF/F value within a sliding three-minute interval); fourth panel, the standard deviation of the dF/F value calculated within a sliding three-minute interval; bottom panel, the dF/F values of the most prominent peaks identified (top 1% of all peaks in the session). eg, Quantification of baseline dF/F (%) (e), peak frequency (per min) (f) and standard deviation (g) for non-torpid (yellow), torpor entry (light blue), torpor (blue) and torpor arousal (teal) in 8 individual mice across all 3-min intervals (left to right: n = 251, 62, 97, 17, 321, 46, 57, 15, 203, 52, 39, 19, 269, 44, 66, 18, 141, 30, 59, 2, 250, 57, 42, 5, 43, 31, 23, 7, 80, 44, 51, 8 time intervals). In box plots, the centre line and box boundaries indicate mean ± s.e.m. P values greater than 0.05 are indicated. h, Example fibre-photometry signal (top) clustered into two states and coloured by state. State 0 corresponds to the mouse being out of torpor or exiting torpor, whereas state 1 corresponds to the mouse entering or maintaining torpor. i, Core body temperature (left) and motor activity (right) are significantly lower during state 1 compared with state 0 of the photometry-based model (n = 8 mice, P = 1.6 × 10−4). j, The time that a mouse spent in torpor (entry or maintenance) was accurately calculated by the model based on the photometry data 82.3 ± 3.2% of the time (model sensitivity). Conversely, whenever the model determined that the mouse was entering or maintaining torpor, its estimation was 88.4 ± 2.8% accurate (specificity). k, Model sensitivity and specificity were significantly lower (P = 1.6 × 10−4, n = 8 mice) when the temporal relationship between the temperature and the fibre-photometry data was removed. In box plots, the centre line and box boundaries indicate mean ± s.e.m. All P values were calculated using a two-tailed Mann–Whitney U-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Fig. 10 Fibre-photometry recordings of avMLPAAdcyap1 neurons in fed freely moving mice with CHA-induced hypothermia and changes in ambient temperature.

a, Fibre-photometry recording data displayed as in Extended Data Fig. 9d. The dashed line indicates the time of CHA administration. bd, Baseline dF/F (b), peak frequency (c) and standard deviation (d) measured for each mouse before and after CHA administration across all recorded three-minute intervals (left to right: n = 69, 17, 63, 23, 140, 24, 161 and 37 time intervals). e, The mean baseline decreases after CHA treatment (P = 0.03, n = 4 mice). f, Schematic showing the fibre-photometry recording of avMLPAAdcyap1 neurons when mice are exposed to different environmental temperatures with food provided in the chamber. g, Mean GCaMP6s signal (n = 6 mice) of avMLPAAdcyap1 neurons with environmental temperature changes along a programmed sequence: 25 °C → 37 °C → 25 °C → 10 °C → 25 °C. Grey shading indicates the 95% confidence interval. h, Example fibre-photometry recording showing the ambient (chamber) temperature (top) followed by three different signals (470 nm, 415 nm and 560 nm). Signals from the 415-nm and 560-nm channels are used as controls for any potential effects of temperature on the photometry signal. i, Mean neuronal responses at different ambient temperatures. avMLPAAdcyap1 neurons are not sensitive to increases in the ambient temperature to 37 °C (P = 0.59), and instead appear to be sensitive to a decrease in environmental temperature (n = 6 mice, P = 0.0021). In box plots, the centre line and box boundaries indicate mean ± s.e.m. All P values were calculated using a two-tailed Mann–Whitney U-test. NS indicates not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1: FosTRAP-Gq-HA signal and Fos+ cells across brain regions. Average HA-signal from Fast-TRAP or Fed-TRAP animals quantified across 316 brain regions and normalized to the volume of the region. Fold difference in normalized HA-signal between Fast-TRAP and Fed-TRAP animals. Number of Fos+ cells across the same brain regions in WT mice analyzed during fasting-induced torpor.

Supplementary Table

Supplementary Table 2: Quantification of viral expression across hypothalamic areas. Viral mCherry expression across 54 animals and 277 hypothalamic areas, spanning AP coordinates +0.61 to -2.53mm relative to bregma. Expression was semi-quantitatively assessed across each of the two hemispheres as either none (N), minimal (M), partial (P), total (T), or NA (data not available, “Expression key” tab).

Supplementary Table

Supplementary Table 3: Hypothalamic screen analysis. For 226 brain areas that were transduced in at least three animals, the correlation between the viral expression and the decrease in core body temperature is displayed along with the average minimum temperature observed following chemogenetic stimulation of animals in which the area was either hit or missed.

Supplementary Table

Supplementary Table 4: Markers of neuronal cell types. Differential gene expression analysis identified marker genes enriched in each of the 36 neuronal clusters. Top twenty markers ranked by Bonferroni-corrected p-value are displayed for each cell type along with the log2 fold enrichment over other cell types.

Supplementary Table

Supplementary Table 5: Markers of TRAPed Adcyap1+ neurons. Differential gene expression analysis between TRAPed and non-TRAPed Adcyap1+ neurons in the snRNA-seq dataset.

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Hrvatin, S., Sun, S., Wilcox, O.F. et al. Neurons that regulate mouse torpor. Nature 583, 115–121 (2020). https://doi.org/10.1038/s41586-020-2387-5

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