.2018 Jul 23;28(14):2263-2273.e4.

doi: 10.1016/j.cub.2018.05.054. Epub 2018 Jul 12.

A Neuronal Hub Binding Sleep Initiation and Body Cooling in Response to a Warm External Stimulus

Edward C Harding¹, Xiao Yu¹, Andawei Miao², Nathanael Andrews¹, Ying Ma¹, Zhiwen Ye¹, Leda Lignos¹, Giulia Miracca¹, Wei Ba¹, Raquel Yustos¹, Alexei L Vyssotski³, William Wisden⁴, Nicholas P Franks⁵

Affiliations

¹ Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK.
² Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK; UK Dementia Research Institute at Imperial College London, London SW7 2AZ, UK.
³ Institute of Neuroinformatics, University of Zürich/ETH Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
⁴ Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK; Centre for Neurotechnology, Imperial College London, London SW7 2AZ, UK; UK Dementia Research Institute at Imperial College London, London SW7 2AZ, UK. Electronic address: w.wisden@imperial.ac.uk.
⁵ Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK; Centre for Neurotechnology, Imperial College London, London SW7 2AZ, UK; UK Dementia Research Institute at Imperial College London, London SW7 2AZ, UK. Electronic address: n.franks@imperial.ac.uk.

PMID:30017485
PMCID: PMC6078908
DOI: 10.1016/j.cub.2018.05.054

A Neuronal Hub Binding Sleep Initiation and Body Cooling in Response to a Warm External Stimulus

Edward C Harding et al. Curr Biol.2018.

.2018 Jul 23;28(14):2263-2273.e4.

doi: 10.1016/j.cub.2018.05.054. Epub 2018 Jul 12.

Authors

Affiliations

¹ Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK.
² Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK; UK Dementia Research Institute at Imperial College London, London SW7 2AZ, UK.
³ Institute of Neuroinformatics, University of Zürich/ETH Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
⁴ Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK; Centre for Neurotechnology, Imperial College London, London SW7 2AZ, UK; UK Dementia Research Institute at Imperial College London, London SW7 2AZ, UK. Electronic address: w.wisden@imperial.ac.uk.
⁵ Department of Life Sciences, Imperial College London, South Kensington SW7 2AZ, UK; Centre for Neurotechnology, Imperial College London, London SW7 2AZ, UK; UK Dementia Research Institute at Imperial College London, London SW7 2AZ, UK. Electronic address: n.franks@imperial.ac.uk.

PMID:30017485
PMCID: PMC6078908
DOI: 10.1016/j.cub.2018.05.054

Abstract

Mammals, including humans, prepare for sleep by nesting and/or curling up, creating microclimates of skin warmth. To address whether external warmth induces sleep through defined circuitry, we used c-Fos-dependent activity tagging, which captures populations of activated cells and allows them to be reactivated to test their physiological role. External warming tagged two principal groups of neurons in the median preoptic (MnPO)/medial preoptic (MPO) hypothalamic area. GABA neurons located mainly in MPO produced non-rapid eye movement (NREM) sleep but no body temperature decrease. Nitrergic-glutamatergic neurons in MnPO-MPO induced both body cooling and NREM sleep. This circuitry explains how skin warming induces sleep and why the maximal rate of core body cooling positively correlates with sleep onset. Thus, the pathways that promote NREM sleep, reduced energy expenditure, and body cooling are inextricably linked, commanded by the same neurons. This implies that one function of NREM sleep is to lower brain temperature and/or conserve energy.

Keywords: NOS1; TetTagging; anesthesia; energy balance; hypothermia; sedation; sleep; thermoregulation; torpor; warm stimulus.

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Figures

**Figure 1**
Temperature Preference Test: Mice Prefer to Locate to a 32°C Zone (A) Protocol for testing temperature preference. A mouse was allowed to habituate in a home cage with nesting material (cyan) at ambient temperature (22°C ± 1°C) for one week. At the end of the habituation period, and two hours before “lights on,” temperature-controlled plates were turned on such that one end of the cage was set at 32°C ± 1°C while the other end remained at the ambient temperature (left schematic). Two hours after lights on, the final position of the mouse and the distribution of the nesting material were recorded (right). (B) (Left) A record of the average positions of the mice during the two hours after lights on and (right) the final position of each mouse (n = 8; green circles) and the positions of the two largest fragments of nesting material (black crosses).

**Figure 2**
Neurons in the Hypothalamus Can Be Activity Tagged Using a c-*fos* Promotor (A) Compared with the expression after exposure of the mice to an ambient temperature of 22°C ± 1°C (left image), a warm stimulus of 32°C ± 1°C induces robust c-Fos expression throughout MnPO (right image) and MPO (Figures S1A and S1B). The graph on the right shows that a warm stimulus induces c-Fos expression in a significantly higher number of neurons compared to ambient temperatures (p = 1.57 × 10⁻⁴; df = 6; n = 4 mice per group). Ac, anterior commissure. The scale bar represents 200 μm.^∗∗∗p < 0.001. (B) The AAV transgenes used to activity tag warm-sensitive neurons. The hM3D_q-mCherry fusion protein is expressed, driven by theP_TRE-tight promoter, but only after neuronal activity has driven expression (by thec-fos promotor) of the tetracycline transactivator protein (tTA) and only in the absence of doxycycline (DOX). AAVs containing the transgenes were injected on the midline into the median and medial preoptic (MnPO and MPO) hypothalamus to generatePan-MnPO/MPO-ActivityTag-hM3D_q mice. ITR, inverted terminal repeats; pA, polyadenylation signal; WPRE, woodchuck-posttranscriptional-regulatory element. (C) Protocol and timeline for the activity-tagging experiments. Two days after doxycycline (200 mg/kg) was removed from the diet, thePan-MnPO/MPO-ActivityTag-hM₃D_q mice were placed in a box at a warm temperature of 32°C ± 1°C or at the ambient temperature of 22°C ± 1°C for 2 hr. (D) During this time (left and center), their average core body temperature increased by 0.8°C ± 0.09°C (n = 23) and 0.4°C ± 0.2°C (n = 10). During the warm stimulus, the temperature of the skin on the head increased by 4.5°C ± 0.3°C (n = 13) to 35.2°C ± 0.2°C, and the skin temperature measured on the tail increased by 12.5°C ± 0.8°C (n = 9) to 35.6°C ± 0.4°C. Under both warm and ambient temperatures, the Fourier transform power spectra (right) showed a waking EEG, with maxima at theta frequencies. The error bars in (A) and (D) represent the SEM, as do the error envelopes in (D).^∗p < 0.05;^∗∗∗∗p < 0.0001. See also Figure S1.

**Figure 3**
Reactivation of Neurons of Warm-Stimulus-Tagged Neurons in the MnPO-MPO Region of the Hypothalamus Induces Both a Drop in Body Temperature and Sleep (A) After exposure of mice to a warm stimulus (32°C ± 1°C for 2 hr) there was stronghM3D_q-mCherry expression (red) throughout MnPO and MPO compared to baseline levels of expression seen at ambient temperatures (22°C ± 1°C). DAPI staining is in blue. The scale bar represents 200 μm. (B) When later injected with CNO (5 mg/kg), the warm-stimulated animals (red) exhibited marked reduction in body temperature (n = 14), in contrast to animals (gray) injected with CNO after being exposed to ambient temperatures (n = 13). (C) CNO rapidly induced a state of NREM in animals (n = 9) previously exposed to a warm stimulus (red) compared to animals injected with CNO following exposure to ambient temperatures (gray; n = 30). (D) Animals (n = 9) exposed to CNO following an earlier warm stimulus had minimal waking (red) compared to animals injected with CNO following exposure to ambient temperatures (gray; n = 30). (E) The times in each vigilance state, NREM, WAKE, and REM, are shown after CNO injection after exposure to ambient temperatures (gray) and after CNO injection following exposure to a warm stimulus (red). CNO caused a significant decrease in WAKE times (two-way ANOVA; p = 5.4 × 10⁻¹²;df = 37) and a significant increase in NREM times (two-way ANOVA; p = 5.2 × 10⁻⁷;df = 37) but no change in REM times (two-way ANOVA; p = 0.66;df = 37).^∗∗∗∗p < 0.0001. (F) Representative example of EEG (electroencephalogram), EMG (electromyogram), and sleep-stage scoring after a CNO injection following ambient temperature exposure inPan-MnPO/MPO-ActivityTag-hM3D_q mice. During the 3 hr following the CNO injection, the body temperature did not change, and this individual mouse was scored as being in the waking state 43.6% of the time (dark blue), in NREM sleep 56.4% of the time (green), and REM sleep 0.0% of the time. (G) As in (C) but following exposure to a warm stimulus. During the 3 hr following the CNO injection, the body temperature dropped to a minimum of 28°C, and this individual mouse was scored as being in the waking state 25.2% of the time, in NREM sleep 74.7% of the time, and REM sleep 0.2% of the time. The error envelopes in (B)–(D) represent the SEM, as do the error bars in (E). See also Figures S2, S3, S4, and S5.

**Figure 4**
Survey of Neurochemical Markers for Neurons Activity Tagged by External Warmth in the MnPO-MPO Area Double-label immunohistochemistry of warm-labeled (activity-tagged) neurons (red) in the preoptic hypothalamus ofPan-MnPO/MPO-ActivityTag-hM3D_q mice with candidate gene markers (green). (A) Choline acetyltransferase (ChAT), somatostatin (SOM), calretinin (CR), and parvalbumin (PV) all stained negative. The scale bars represent 200 μm. (B) NOS1 antibody (green) stained approximately 25% of hM3D_q-mCherry-positive neurons (red), indicating nitrergic neurons. Examples of double-labeled cells are indicated with arrows. (C) Many warm-activatedNos1-Cre neurons (red) in the MnPO and MPO areas co-stain with VGlut2 antisera (green). Examples of double-labeled cells are indicated with arrows. (D) Glutamate decarboxylase 67 (GAD67) antibody (green) stained approximately 30% of hM3D_q-mCherry-positive neurons (red), indicating GABAergic neurons. Examples of double-labeled cells are indicated with arrows. (E) The mouse MnPO and MPO hypothalamic area has a population of neurons with strongNos1 expression, as visualized byin situ hybridization with aNos1-selective probe (top three images), taken from the Allen Brain Atlas [36] and immunohistochemistry with a NOS1 antibody (lower image). In the images on the left of (B)–(D), the scale bars represent 200 μm, and for the larger images on the right of each panel the scale bars represent 100 μm. The scale bar in (E) represents 200 μm.

**Figure 5**
Cell-Type-Selective Activity Tagging Reactivating warm-stimulus-tagged nitrergic neurons in the MnPO-MPO hypothalamus induces both sleep and hypothermia, whereas reactivating warm-stimulus-tagged GABAergic neurons in the MnPO-MPO hypothalamus induces only sleep. (A) The AAV transgenes used to selectively activity tag genetically defined (Cre-positive) and warm-sensitive neurons. TheP_TRE-tight promoter (TRE) responds to thec-Fos-promoter-controlled tetracycline transactivator protein (tTA) to drivehM3D_q-mCherry transgene expression restricted to Cre-positive cells and only in the absence of doxycycline. (B) Schematic illustrating midline injection of the two AAV constructs into MnPO-MPO inVgat-ires-Cre mice andNos1-ires-Cre mice to produceNos1-MnPO/MPO-ActivityTag-hM3D_q mice andVgat-MnPO/MPO-ActivityTag-hM3D_q mice, respectively. (C) Differential distribution of warm-taggedNos1-Cre neurons and warm-taggedVgat-Cre neurons in MnPO and MPO. The taggedNos1 neurons are in both MnPO and MPO areas, whereas the taggedVgat neurons are mainly in MPO area as determined by mCherry immunohistochemistry to detect the hM3D_q-mCherry receptor. The scale bar represents 150 μm. (D) When later injected with CNO, the warm-stimulatedNos1-MnPO/MPO-ActivityTag-hM3D_q mice exhibited marked hypothermia, which lasted for several hours (n = 5) and which was indistinguishable from that seen inPan-MnPO/MPO-ActivityTag-hM3D_q mice (shown in red—data from Figure 3B). In contrast,*Vgat-MnPO/MPO-ActivityTag-hM3D*_q mice injected with CNO showed more modest and transient hypothermia (n = 6). (E) With animals previously exposed to a warm stimulus, CNO rapidly induced a state of NREM in bothNos1-MnPO/MPO-ActivityTag-hM3D_q (blue) andVgat-MnPO/MPO-ActivityTag-hM3D_q (green) mice. In both cases, cell-type-selective tagging elicited NREM sleep that was not significantly different to pan-tagging in thePan-MnPO/MPO-ActivityTag-hM3D_q mice (shown in red—data from Figure 3C). (F) With animals previously exposed to a warm stimulus, CNO rapidly induced a state of minimal waking in bothNos1-MnPO/MPO-ActivityTag-hM3D_q (blue) andVgat-MnPO/MPO-ActivityTag-hM3D_q (green) mice. In both cases, cell-type-selective tagging elicited a waking state sleep that was not significantly different to pan-tagging in thePan-MnPO/MPO-ActivityTag-hM3D_q mice (shown in red—data from Figure 3D). (G) The times in each vigilance state, NREM, WAKE, and REM, are shown after CNO injection intoNos1-MnPO/MPO-ActivityTag-hM3D_q mice (blue; n = 5) and intoVgat-MnPO/MPO-ActivityTag-hM3D_q mice (green; n = 6). The times for thePan-MnPO/MPO-ActivityTag-hM3D_q mice are shown for comparison (shown in red—data from Figure 3E). (H) Representative example of EEG, EMG, and sleep-stage scoring after a CNO injection (5 mg/kg) inNos1-MnPO/MPO-ActivityTag-hM3D_q mice following a warm stimulus. During the 3 hr following the CNO injection, the body temperature dropped to a minimum of 27.5°C, and this individual mouse was scored as being in the waking state 20.0% of the time, in NREM sleep 79.9% of the time, and REM sleep 0.1% of the time. (I) Representative example of EEG, EMG, and sleep-stage scoring after a CNO injection (5 mg/kg) inVgat-MnPO/MPO-ActivityTag-hM3D_q mice following a warm stimulus. During the 3 hr following the CNO injection, the body temperature dropped to a minimum of 34.2°C, and this individual mouse was scored as being in the waking state 29.1% of the time (dark blue), in NREM sleep 70.6% of the time (green), and REM sleep 0.3% of the time. Data are expressed as mean ± SEM.

**Figure 6**
Conceptual Circuit Model for How External Warming Induces and Links Sleep and Body Cooling Skin warming activates a pathway that runs through the spinal cord and is relayed by glutamatergic neurons in the lateral parabrachial nucleus [16, 22, 23]. This activates theNos1-positive glutamatergic neurons in MnPO-MPO whose output drives GABAergic neurons in both MPO and GABAergic neurons in the lateral preoptic (LPO) areas [25]. Others have demonstrated that activating glutamatergic cells in MnPO-MPO induces hypothermia [26, 28] and that GABAergic neurons in the MnPO-MPO area do not regulate body temperature [25, 28]. We suggest the MPO GABA neurons inhibit monoaminergic arousal pathways, such as the histaminergic tuberomammillary nucleus, resulting in sleep; by contrast, the LPO GABA neurons inhibit both heat-promoting glutamatergic and GABAergic neurons in the dorsomedial hypothalamus (DM), resulting in body cooling [25].

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Comment in

Neuroscience: A 'Skin Warming' Circuit that Promotes Sleep and Body Cooling.
Peever J.Peever J.Curr Biol. 2018 Jul 23;28(14):R800-R802. doi: 10.1016/j.cub.2018.06.043.Curr Biol. 2018.PMID:30040944

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A Neuronal Hub Binding Sleep Initiation and Body Cooling in Response to a Warm External Stimulus

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A Neuronal Hub Binding Sleep Initiation and Body Cooling in Response to a Warm External Stimulus

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