Astrocytic metabolic control of orexinergic activity in the lateral hypothalamus regulates sleep and wake architecture – Nature.com

Animals, housing, genotyping

All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Tufts University. Protocol number: B2022-120.

Male and female mice were bred and housed on a 12/12 light/dark cycle, given standard chow and water ad libitum. Zeitgeber time (ZT) scale, that sets the origin of the 24h period (ZT0) to the onset of the light-phase, has been used to allow comparison among studies independently of the actual clock-time settings of animal facilities. Ambient temperature was maintained at 231C, and humidity was kept within the range of 40% to 60%.

Homozygous MCT4floxed/floxed, MCT2floxed/floxed and MCT1floxed/floxed mice originated from Dr. L. Pellerins lab (Department of Physiology, University of Lausanne, Lausanne, Switzerland). Upon receival, mice were cleared from quarantine, backcrossed with C57BL/6 mice, and bred in our facility.

MCT4floxed/floxed mice present exons 3,4 and 5 of the Slc16a3 gene flanked by LoxP sites. The floxed allele showed a 516bp band, while the endogenous Slc16a3 allele showed a 388bp band (Supplementary Fig.2a). Allele from KD mice showed a 368bp band, while intact floxed allele showed a 446bp band (Supplementary Fig.2a).

MCT1floxed/floxed mice present exon 5 of the Slc16a1 gene flanked by LoxP sites. The floxed allele showed a 420-base pair (bp) band, while the endogenous Slc16a1 allele showed a 280bp band, while allele from KD mice showed a 437bp band, while intact floxed allele showed a 227bp band (Supplementary Fig.2f).

MCT2floxed/floxed mice present exons 4 and 5 of the Slc16a7 gene flanked by LoxP sites. The floxed allele showed a 436bp band, while the endogenous Slc16a7 allele showed a 308bp band, while allele from KD mice showed a 227bp band, while intact floxed allele showed a 437bp band (Supplementary Fig.5a).

Orexin-IRES-Cre mice were generated, validated, and kindly provided by Dr. Dong Kong and crossed with MCT2floxed/floxed mice.

To test the specificity of our viral promoter GFAP (0.7), Ai14 mice (#007909, Jackson Laboratory) have been kindly donated from Dr. Yongjie Yang.

Eight weeks old male and female MCT1f/f, MCT4f/f and Orexin-IRES-Cre x MCT2f/f (oMCT2 KD) mice were anesthetized with isoflurane, injected with buprenorphine (0.5mg/kg) and placed into a stereotaxic frame. Two small holes were bilaterally drilled in the skull (LH coordinates: AP=1.6, ML=0.9mm). A 2L Neuro 7002 syringe (Hamilton) was filled with AAV (1109 virus genomes per L) and the needle lowered into the holes to reach the LH (LH coordinates: DV=5.35mm). 1L of vector per hemisphere was injected at a speed of 100nL/min using a Microsyringe Pump Controller Micro4 (Harvard Apparatus). MCT4f/f or MCT1f/f mice received either AAV-PhP.eB-GFAP (0.7)-EGFP-T2A-iCre virus (aMCT4 KD or aMCT1 KD mice) or AAV-PhP.eB-GFAP (0.7)-EGFP virus (control mice) (#VB1131 Vector Biolabs) or vehicle only (control mice) (ACSF, #3525 Tocris). oMCT2 KD received AAV-PhP.eB-CAG2-DIO-mSlc16a7-P2A-GFP virus (AAV-272101, Vector Biolabs) to re-express MCT2 in these mice. To visualize orexin-expressing neurons for electrophysiological recordings oMCT2 KD and control mice received AAV9-CAG-DIO-mCherry virus (VB1326, Vector Biolabs). 7 weeks old female and male Ai14 mice have been bilaterally injected with AAV-PhP.eB-GFAP(0.7)-EGFP-T2A-iCre to test for viral transduction specificity.

4 or 10 weeks after viral injections MCT1f/f or MCT4f/f mice, depending on the experimental setting, and 7 weeks old oMCT2 KD and related controls mice were anesthetized with isoflurane, injected with buprenorphine (0.5mg/kg) and placed into a stereotaxic frame. EEG/EMG implantation surgeries have been performed as previously described21. Briefly, prefabricated EEG/EMG headmounts (8201, Pinnacle Technology, Lawrence, KS) were secured to the skull with four stainless steel EEG screws (8209, Pinnacle Technology, Lawrence, KS) into the pilot holes. Silver epoxy was applied to ensure electrical connectivity between the electrodes and the headmount. EMG leads were inserted bilaterally into the nucal muscles. The headmounts were then secured to the skull with dental acrylic. 10 days after surgery, mice were moved into an insulated sound-proof chamber and placed into individual Plexiglas circle boxes (Pinnacle Technology, Lawrence, KS) containing water and food ad libitum and headmounts were plugged to a lightweight EEG preamplifier (Pinnacle Technology) that allowed freely moving. Mice were left 3 days for habituation prior to data collection and maintained on a 12:12 light/dark cycle. EEG signals were acquired at a frequency sampling with 400Hz using Sirenia software (Pinnacle Technology, Lawrence, KS). Sleep stages were scored visually at 4s epochs by a trained experimenter using SleepSign for Animal software (Kissei Comtec). Wakefulness (W) was defined as low-amplitude, high frequency EEG and high EMG activity; REM sleep was characterized by low amplitude, desynchronized EEG with low EMG activity; and non-rapid eye movement (NREM) sleep consisted of high-amplitude, low frequency EEG with little EMG modulation. The dark phase was divided into ZT12-18 and ZT18-24 based on prior observations21,62,63 and studies showing differential activity in the dark phase64,65,66.

For in vivo lactate biosensor experiments, 10 weeks after viral injections MCT4f/f mice and 7 weeks old oMCT2 KD and related control mice were anesthetized with isoflurane, injected with buprenorphine (0.5mg/kg) and placed into a stereotaxic frame. Three small holes were drilled, one into the frontal area and two into the parietal area, and EEG screws with wire leads (8403, Pinnacle Technology, Lawrence, KS) were inserted and manually rotated into the pilot holes. One additional hole was drilled in the skull (LH coordinates: AP=1.6, ML=0.9mm) and the guide cannula was lowered into the hole to reach the LH upon insertion of the biosensor (LH coordinate DV=3.35mm). The cannula and screws were secured to the skull with dental acrylic. EEG screw wire leads were subsequently soldered to prefabricated EEG/EMG/Bio headmounts (8402, Pinnacle Technology, Lawrence, KS); EMG leads were inserted bilaterally into the nucal muscles, and headmounts were also secured with dental acrylic. 10 days after surgery, mice were moved into an insulated sound-proof chamber and let habituated as described above. 78h before the dark phase (ZT12) lactate biosensors were in vitro precalibrated with known concentration of L-lactate and the interferent Ascorbic Acid, as per manufacturers instruction, and inserted into the guide cannula. EEG and EMG signals were acquired as described above. Lactate data were also acquired through Sirenia software. EEG/EMG/biosensor recordings continued for ~18h, followed by in vitro biosensor postcalibration with known concentration of L-lactate and the interferent Ascorbic Acid, as per manufacturers instruction. Biosensors that were sensitive to the interferent Ascorbic Acid during pre or postcalibration were excluded from the analysis.

For lactate measurements analysis: The current trace (nA) recorded using the Pinnacles lactate biosensor was corrected as described in theSupplementary Materials section. The resulting trace represented the lactate concentration changes (mM) compared to the baseline (set at 1mM). The sleep (S) and wake (W) events were identified during a 6-h period (from ZT12 to ZT18) using the EEG/EMG signals, as previously described, and used to define the corresponding values of lactates change in each state. For a given state (either S or W) and for each genotype, the values of lactates change were combined and plotted as histograms (bin width: 0.01mM). To compare the CTRL vs KD conditions, the values for each group for a given state were subsequently used to compute the Kernel probability distribution (pd) using the fitdist.mat function in MATLAB; the resulting Kernel pd was used to reconstruct a smaller dataset (considering the sensitivity of the KolmogorovSmirnov test to the sample size of the analyzed vectors; n=400) using the random.mat function in MATLAB and the pd as an input. The reduced datasets were compared with a two-sample KolmogorovSmirnov test (kstest2.mat function in MATLAB) testing one of the two alternative hypotheses (using the Tail option in the kstest2.mat function): (1) H1: values in CTRL distribution greater than values in the KD distribution or (2) H1: values in CTRL distribution smaller than values in the KD distribution. H0: values in CTRL KD belongs to the same distribution. Stereotypical traces were visually identified as traces reaching stable values of lactates change during the W state for 10min and preceded by 2min of stable lactates change (during S state). The traces were then aligned, with 0min as the S-W transition and averaged for each genotype. The full trace correction description can be found here: (https://osf.io/fwru5/?view_only=48ee97f56e424b219f66ef3d9d7ed20d).

10 weeks old MCT4f/f mice or 7 weeks old C57BL/6 were anesthetized with isoflurane, injected with buprenorphine (0.5mg/kg) and placed into a stereotaxic frame. Surgeries were performed as previously described. Briefly, two small holes were bilaterally drilled in the skull (LH coordinates: AP=1.6, ML=0.9mm). Bilateral brain cannula was stereotaxically implanted into the LH (Guide cannula #8IC235G18XXC with 4mm pedestal length, internal cannula #8IC235ISPCXC 1.25mm length below the pedestal, P1 Technologies). Mice were then implanted with EEG/EMG electrodes, as described earlier. After 10 days of post-operative recovery, mice were placed into individual Plexiglas circle boxes and their microconnectors plugged to EEG preamplifier, as described above. The following drugs have been used: sodium L-lactate (5mM, equicaloric to 2.5mM glucose, Sigma, #L6022),1,4-Dideoxy-1,4-imino-D-arabinitol hydrochloride (DAB, 10mM, Sigma, #D1542), or Alpha-cyano-4-hydroxycinnamic acid (4-CIN, 1,5mM, Sigma, #C2020).To allow for continuous and controlled drug delivery, osmotic mini-pumps (model 1002; Alzet; flow rate, 0.25L/h; 2-wk duration) were secured with the flow moderator, and primed overnight in 0.9% saline solution at 37C before to be connected to the brain cannula by flexible catheter tubing (#51158, Stoelting). Osmotic mini-pumps were externalized as previously described21. Briefly, cannulas were placed in a sealed Eppendorf filled with 0.9% saline solution and maintained at 37C with an iBlock Mini Dry Bath (Midsci) placed on top of the cage. The catheter tubing was then filled with aCSF (Harvard Apparatus, 59-7316), L-lactate, DAB or 4-CIN, depending on the experimental setting, and a small air bubble was inserted into the catheter tubing during its connection with the pump to monitor the flow rate. The flexible catheter tubing was carefully aligned and attached to the EEG cable. Mice were left 3 days for habituation prior to drug delivery, recordings and data collection, and maintained on a 12:12 light/dark cycle. Each drug was delivered for 4 consecutive days, EEG/EMG recordings were acquired between day 3 and 4 of drug delivery and data analyzed as described above.

Brain slice experiments were performed on male and female adult mice (26 months depending on the experimental setting). Briefly, mice were anaesthetized with isoflurane, and after decapitation, the brain was rapidly removed and put in ice-cold (2 to 4C) carbogen-bubbled (O2 95%/CO2 5%) artificial cerebrospinal fluid (ACSF) containing the following (in mM): 120 NaCl, 3.2 KCl, 1 NaH2PO4, 26 NaHCO3, 1 MgCl2, 2 CaCl2, 2.5 glucose (osmolarity adjusted to 300mOsm with sucrose, pH 7.4). After removal of the cerebellum, the brain was glued and coronal hypothalamic slices (350m) containing the LH were cut using a vibratome (VT1200S; Leica). Before recording, slices were incubated at 33C for a recovery period of 45min. After recovery, slices were placed in a submerged recording chamber (Warner Instruments) and continuously perfused (2mL/min) with oxygenated ACSF. The glucose concentration used for recording was 2.5mM unless otherwise noted. Slices were used for maximally 4h after dissection. Experiments were performed at room temperature 21 to 24C.

Orexin neurons were visualized with an 16x objective and 2x magnification in an upright Nikon Eclipse FN1 microscope, equipped with an arc-discharge mercury lamp (Nikon intensilight C-HGFI), by using infrared differential interference contrast (IR-DIC). For patch-clamp recordings, oMCT2 KO and control mice were used at 8 weeks old (2 months). aMCT4KO and control mice were used 12 weeks post viral injection (5 months old).

In oMCT2 KD and control slices, orexin neurons were selected based on mCherry expression.

In aMCT4 KD and control slices, viral transduction was confirmed based on GFP expression brain slices showing no infection were discarded. Orexin neurons were discriminated from melanin-concentrating hormone neurons (MCH) residing in the same region for their electrophysiological fingerprints21.

Whole-cell patch-clamp recordings were performed in current-clamp mode by using a Multiclamp 700B amplifier (Molecular Devices). Data were filtered at 1kHz and sampled at 5kHz with Digidata 1322A interface and Clampex 9.2 and 10.6 from pClamp software (Molecular Devices).

Pipettes (from borosilicate capillaries; World Precision Instruments) had resistance of 67M when filled with an internal solution containing the following (in mM): 123 K-gluconate, 2 MgCl2, 8 KCl, 0.2 EGTA, 4 Na2-ATP, 0.3 Na-GTP, and 10 HEPES, pH 7.3 with KOH.

Cell-attached patch-clamp recordings were performed in neurons in voltage-clamp mode to record the spontaneous firing activity avoiding dilution of the intracellular compartments. All recordings were analyzed with Clampfit 9.2 and 10.6 from pClamp software (Molecular Devices). Mean firing rate were calculated from 2min of stable recordings.

All drugs were applied to the perfusing system (bath application) to obtain the final concentrations indicated unless otherwise stated. Alpha-cyano-4-hydroxycinnamic acid (4-CIN), sodium L-lactate and tolbutamide were obtained from Sigma.

Neurobiotin Tracer (1mg/mL; #SP-1120, Vector Laboratories) was also added to the internal solution during whole-cell recordings for post hoc immunohistochemical phenotyping. Briefly, slices were fixed overnight with 4% paraformaldehyde (PFA), then rinsed several times with a PBS solution and permeabilized and immunoblocked with 0.5% Triton X-100 and 5% NGS (Normal Goat Serum) in PBS. For immunostaining of orexin neurons, slices were incubated with rabbit anti-Orexin-A (1:500; #AB6214, Abcam) for three days at 4C. The primary antibody was visualized with anti-rabbit Alexa Fluor 633 (1:500; #A21071, Invitrogen). Sections were also incubated with streptavidin 546 (1:500; #S11225, Invitrogen) to label neurons infused with Neurobiotin, then mounted with Vectashield antifade mounting medium with DAPI (Vector Laboratories) and visualized using a confocal laser scanning microscope (Nikon A1) with a 40x oil immersion lens (NA1.0).

Due to the confined size of the lateral hypothalamus, we quantified protein downregulation from the entire isolated area, thus including all cell types within the tissue. To reduce the number of mice used, proteins were collected from 30 m sections from perfused mice (see section below). LH were isolated after confirmation of viral expression based on GFP visualization. Total protein was then extracted by resuspending the isolated tissue in radioimmunoprecipitation assay (RIPA) buffer (10mM Tris HCl, 0.1M, pH 7.2; 1% sodium deoxycholate; 1% Triton X-100; 3% sodium dodecyl sulfate [SDS]; 150mM NaCl, 1.5M; 1mM EDTA, pH 8.0, 0.5M (#AM9260G, Thermo Fisher Scientific); 1mM phenylmethanesulfonyl fluoride (#93482, Sigma); Complete Protease Inhibitor cocktail (#539131, Millipore Roche); Halt Phosphatase Inhibitor (#1862495, Thermo Fisher Scientific)) and stored at 20C until usage. Samples were then heated for 20min at 100C and then let sit on ice for additionally 20min prior to sonication. Samples were then subjected to sonication (Soniprep 150, MSE) and spun at 21,000g for 10min, supernatants were then collected for protein quantification. Protein quantification was performed using a Pierce BCA Protein Assay kit (#23227, Thermo Scientific). 10g of total protein were mixed with NuPAGE LDS Sample Buffer (#NP007, Life Technologies), NuPAGE Sample Reducing Agent (#NP0009, Life Technologies), and distilled water prior to being heated at 95C for 5min. Proteins were separated by SDS-PAGE in a 4%12% Bis-Tris gel (#NP0336, Thermofisher) using a Novex Bolt Mini Gel system and NuPAGE MOPS SDS Running Buffer (NP0001, Thermofisher) before being transferred onto Immobilon-P polyvinylidene fluoride membranes (#IPVH00010, 0.45mm pore size; Millipore) in NuPAGE Transfer Buffer with methanol 20% (NP0006, Thermofisher) for 1h 40min at 4C using a Novex Bolt Mini Blot Module. SeeBlue Plus2 standard (#LC5925, Life Technologies) was used to estimate protein sizes, and transfer was confirmed by Ponceau S (#BP103-10, Fisher Biotech) staining. Immunoblot was obtained by first blocking membranes for 1h at room temperature with a solution of 0.1% Tween 20 and 5% Bovine Serum Albumin (BSA) (#5217, Tocris) in 1 PBS (pH 7.4) and then incubated with primary antibodies overnight at 4C: rabbit anti-MCT1 (1:10.000, #20139, Proteintech) and rabbit anti-MCT4 (1:1000, #NBP1-81251, Novus Biologicals). After washing with 1 PBS containing 0.1% Tween 20, the membranes were incubated with the species-appropriate horseradish peroxidase-conjugated secondary antibodies for 1h. at room temperature at a 1:15,000 dilution in blocking solution. Immunoreactivity was revealed using SuperSignal West Pico PLUS Chemiluminescent Substrate (#34577, Thermofisher) and imaged using a Fujifilm LAS 4000 Gel Imager system with ImageQuant LAS 4000 software (Fujifilm). If needed, antibodies were stripped from membranes prior to incubation with another primary antibody or prior to incubation with mouse anti--actin (1:1000, Sigma, #A1978) in a stripping solution with 0.7% -mercaptoehtnaol (#63689, Sigma). Densitometry measurements were performed using Fiji, with each protein band being normalized to their respective -actin. Full membrane images can be found in the following link: https://osf.io/fwru5/?view_only=48ee97f56e424b219f66ef3d9d7ed20d.

Mice were anesthetized with isoflurane and transcardially perfused with saline (sodium chloride 0.9%), followed by cold 4% PFA in PBS pH 7.4. The brains were post-fixed in the same solution for 12h at 4C and then cryoprotected for at least 24h in 30% sucrose (Sigma) in 1 PBS at 4C. Coronal sections containing the LH were cut serially on a freezing microtome (SM2000R; Leica) at a thickness of 30m and then stored in an antifreeze solution at 20C until immunostaining.

For evaluation of cell-specificity upon AAV injection: 4 serial sections 120m apart were used and Cre-induced Td-Tomato expression was evaluated in Ai14 mice. Free-floating sections were washed twice in 1x PBS for 10min and then incubated with 10% normal goat serum (NGS, #5425, Cell Signaling) blocking solution (1x PBS, 0.3% Triton) for 1h. at room temperature, followed by incubation with primary antibodies (chicken anti-GFAP 1:500, Abcam, #AB4674; or rabbit anti-Orexin-A 1:500, Millipore, #AB3704) overnight at 4C on a shaking platform. Sections were rinsed three times with 1x PBS solution and then incubated with secondary antibodies (633 goat anti-rabbit, Invitrogen, #A21071; or 633 goat anti-chicken, Ivitrogen, #A21103) for 2h. at room temperature. All sections were counterstained with DAPI, mounted on slides with Vectashield antifade mounting medium (#H-1800, Vector Laboratories). Fluorescent images were acquired with a confocal laser scanning microscope (Nikon A1) using a 20x objective. Image analysis and counting of cells expressing Td-tomato with Orexin-A or with GFAP were performed within the LH by using Cell-Counter plug-in in Fiji software. Data are expressed as percentage of double positive cells over the total number of Td-Tomato positive cells. More than 50 cells per animal were counted.

For cannula placement verification: 5 serial sections 120m apart were used for assessing cannula placement (as well as biosensor placement). Free-floating sections were washed twice in 1x PBS for 10min and then incubated with 10% normal goat serum (NGS) blocking solution (1x PBS, 0.3% Triton) for 1h at room temperature, followed by incubation with primary antibodies (chicken anti-GFP 1:500, Abcam, AB13970; or rabbit anti-Orexin-A 1:500, Abcam, #AB6214) overnight at 4C on a shaking platform. Sections were rinsed three times with 1x PBS solution and then incubated with secondary antibodies (546 goat anti-rabbit, Invitrogen, #A11035; and/or 488 anti-chicken, Invitrogen, #A11039) for 2h. at room temperature. All sections were counterstained with DAPI, mounted on slides with Vectashield antifade mounting medium and checked at an epifluorescence microscope Nikon E800. Bilateral cannula placement representative images were acquired using an epifluorescence microscope (Keyence BZ-X700) with a 10x objective and stitched with Keyence software. Biosensor placement representative images were acquired with a confocal laser scanning microscope (Nikon A1) using a 20x objective. For beautification purpose, brightness of representative images has been changed to increase contrast using Fiji software. (https://osf.io/fwru5/?view_only=48ee97f56e424b219f66ef3d9d7ed20d).

To examine cell-targeting specificity, Cre-induced mCherry expression in orexin neurons, was evaluated in 6 brain sections from 4 Orexin-cre mice injected in the LH with the AAV9-CAG-DIO-mCherry virus.

Free-floating sections were washed twice in 1x PBS for 10min and then incubated with 10% normal goat serum (NGS, #5425, Cell Signaling) blocking solution (1x PBS, 0.3% Triton) for 1h at room temperature, followed by incubation with primary antibodies (mouse anti-Orexin-A 1:200, R&D Systems, #MAB763, and rabbit anti-mCherry 1:500, Thermo Fisher, #600-401-P16) overnight at 4C on a shaking platform. Sections were rinsed three times with 1x PBS solution and then incubated with secondary antibodies (488 goat anti-mouse, Invitrogen, #A-11001; and 546 goat anti-rabbit, Invitrogen, #A11035) for 2h at room temperature. All sections were counterstained with DAPI, mounted on slides with Vectashield antifade mounting medium. Confocal stacks were acquired using a confocal laser scanning microscope (Nikon A1) using a 40x oil immersion lens (NA1.0). Cell counting was performed using Fiji by scanning through the z-planes manually, DAPI staining was used to colocalize each cells nucleus with an orexin-specific marker (Orexin-A) and the reporter (mCherry). In the results section shown in Supplementary Fig.7, we found that 94%2.1% of mCherry expressing cells (107 cells counted, from 6 brain sections) were Orexin-A+. On average 85.6%4.9% of all orexin neurons (Orexin-A+, 120 cells from 6 brain sections) were mCherry+ (expressing Cre recombinase). Data are expressed as meanSEM of brain sections analyzed.

For orexin neuron quantification: 5 serial sections 120m apart from Orexin-Cre negative or Orexin-Cre positive mice were used for quantification of orexin neurons. Sections were pre-treated with peroxidase 3% for 15min and then incubated for 1h at room temperature in the blocking solution (1 PBS, 10% NGS, 0.3% Triton X-100), followed by incubation with Rabbit anti-Orexin-A primary antibody (rabbit anti-Orexin-A 1:500, Abcam, #AB6214) in blocking solution and incubated at 4C overnight. The following day, after washing the sections with 1 PBS, sections were incubated with biotinylated secondary antibody (biotinylated goat anti-rabbit, 1:500, Abcam, #AB6720) for 1h at room temperature. Sections were then incubated with A and B components of Vectastain Elite ABC kit (PK-4000, Vector Laboratories) for 1h at room temperature, and finally the reaction was developed using 3,3-diaminobenzidine (DAB) following the suppliers instructions (SK4100 Vector Laboratories). The reaction was blocked by washing the sections with distilled water and mounted with Poly-Mount (#08381, Polysciences). Brightfield images were acquired with Nikon E800 microscope, and of Orexin-positive cells within the LH were counted by using Cell-Counter plug-in in Fiji software. Data are expressed as average of total cells per animal.

For slice electrophysiology, normal distribution was assessed using KolmogorovSmirnov test. Comparisons between two groups were conducted with the paired or unpaired Students t test or the nonparametric MannWhitney test as appropriate. Repeated-measures ANOVAs followed by Tukeys test were used when multiple measurements were made over time in the same groups, the data were analyzed by fitting a linear mixed-effects model in case of missing values. All values and related statics can be found in Source Data file.

EEG/EMG: Repeated-measures ANOVA was used when multiple measurements were made over time in the same groups followed by Tukeys post hoc multiple comparisons tests. Comparisons between two groups were conducted with the paired or unpaired Students t test. Lactate biosensor traces were analyzed by using two-sample KolmogorovSmirnov test. The level of significance was set at p<0.05. Statistical analysis was carried out using the Prism7 and Prism8 software (GraphPad Software, Inc). All values, sample size and related statistics for 24EEG analysis can be found in the Statistics Table (https://osf.io/fwru5/?view_only=48ee97f56e424b219f66ef3d9d7ed20d).

Schematic representations throughout the manuscript have been created with licensed BioRender.com.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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Astrocytic metabolic control of orexinergic activity in the lateral hypothalamus regulates sleep and wake architecture - Nature.com