在玉米粉（62.5 g l -1），无活性酵母粉（25 g l -1），琼脂（6.75 g l -1），糖蜜（37.5 ml l -1），丙酸（4.2 mL L -1），tegosept（1.4 g l -1）和乙醇（7 ml l -l -l -l -l -l firacs）上饲养果蝇。所有电生理学和脂肪组分析（除了研究SNI1突变的作用外）均对Eclosion后2-6天的随机选择的雌性蝇进行。如前所述，实验蝇是所有转基因的杂合子，对于野生型或突变体（hk1）高运动等位基因51,52，纯合子均为纯​​合子。R23E10-GAL4驱动器16,53控制DFBN中荧光标签MCD8 :: GFP的表达，以及n-myristoymet的共价六聚体（MyR-MS6T2）单粒氧气生成器Minisog54或催化性缺陷型（k289m）或功能versinions and insiog54 singlet oxyge oxygen oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge oxyge。DH31-GAL4 LINE55将MCD8 :: GFP靶向于脑脑互动的神经元。

　　由于SNI是X-linked38，因此研究其在男性中的功能是最合意的。在行为实验或4个分析中，SNI1等位基因的半细胞载体在NSYB-GAL4或DFBNS16,53下，在NSSYB-GAL4的控制下，pan-neuronally 58在pan-neuronally 58中共表达了UAS-SNI38，UAS-SNI38，UAS-AOX56或UAS-HKRNAI（47805GD）57个转基因。对于电生理记录，用R23E10-GAL4驱动的MCD8 :: GFP标记了半合子SNI1突变体和野生型男性的DFBN。

　　编码与N末端标志表位（HKFLAG）编码框架融合的高温等位基因是通过对CRISCR-CASPR-CAS9生成的双链破裂（WellGenetics）的同源性修复来创建的。在同工型HK-PK，HK-PE，HK-PL和HK-PM的蛋氨酸引发后立即插入标志标签，并通过柔性接头（4×Gly-Gly-ser）连接到蛋白质的其余部分。

　　将2-5天的女性或半合子SNI1突变体男性分别插入65毫米玻璃管中，加载到果蝇活性监测器（Trikinetics）中，并在12小时的光线下放置：12小时的黑暗条件。允许苍蝇适应监测器一天，并且在接下来的两个24小时期间的活动计数平均。> 5分钟的不活动周期被归类为睡眠59,60（睡眠和昼夜节律分析MATLAB程序61）。固定苍蝇（<2 beam breaks per 24 h) were manually excluded.

　　To deprive flies of sleep, a spring-loaded platform stacked with Trikinetics monitors was slowly tilted by an electric motor, released, and allowed to snap back to its original position62. The mechanical cycles lasted 10 s and were repeated continuously for 12 h, beginning at zeitgeber time 12.

　　Dissected brains of rested and sleep-deprived flies were placed on PTFE-printed glass slides (Electron Microscopy Sciences), covered with ~3–5 µl gelatin (5% w/v in water), and snap frozen for shipping. For sectioning, dissected brains were thawed, suspended in 20 µl 5% gelatin, and transferred to a gelatin plateau created by removing the top half of a frozen block of 5% gelatin in a cryostat (Microm HM 525, ThermoFisher). After allowing the samples to refreeze during 10 min in the cryostat chamber, 10-µm sections were cut and thaw-mounted onto glass slides. The sections were imaged in fluorescence (BX41, Olympus) and reflected light mode (VHX 5000, Keyence) and stored at −80 °C until further use.

　　For SMALDI-MSI63, the brain sections were thawed in a desiccator and spray-coated with 80 µl of a freshly prepared solution of 2,5-dihydroxybenzoic acid (DHB, Merck) using a SMALDIPrep ultrafine pneumatic spraying system (TransMIT GmbH). The DHB solution contained 60 mg of DHB in 999 µl acetone, 999 µl water, and 2 µl pure trifluoroacetic acid (TFA, Merck). In samples destined for 4-ONE analysis, a chemical derivatization step with Girard’s reagent T (GirT, TCI Chemicals) preceded the application of the DHB matrix64. The samples were spray-coated with 35 µl of a freshly prepared solution of 15 mg ml−1 GirT in a 7:3 mixture of methanol and water containing 0.2% (v/v) TFA and incubated in a desiccator at room temperature for 2 h. Standards were prepared by applying 5-µl droplets of a tenfold dilution series of 4-ONE (Cayman Chemical) in methyl acetate, from 100 µM to 10 nM, onto blank glass slides or slides containing brain sections of rested flies. Standards underwent the same GirT-derivatization and matrix application steps as analytical samples.

　　A home-built SMALDI-MS imaging ion source based on an AP-SMALDI5 AF system (TransMIT GmbH) was coupled to an orbital trapping mass spectrometer (Q Exactive, ThermoFisher). Mass spectra were acquired at a mass resolution of 140,000 in positive-ion mode. A high voltage of 4 kV was applied to the sample holder. The standard pixel size of 5 µm × 5 µm in lipid analyses was increased to 25 µm × 25 µm for 4-ONE measurements to facilitate the detection of low-intensity signals. A single-ion-monitoring (SIM) experiment was performed first for 4-ONE, followed by a full MS scan.

　　SMALDI-MS images were created in Mirion65 (TransMIT GmbH) using a bin width of ∆(m/z) = 0.004; the images were normalized to total ion charge66. A digital mask created from a ubiquitous lipid signal was applied to the measurement area in order to exclude off-tissue pixels, and all images were stitched together in a single file to ensure uniform evaluation. An automatically generated list of all signals found in at least ten pixels in the stitched file was applied to the separate images to obtain the summed intensity of each signal. Signals were annotated in a bulk search against LIPID MAPS67, allowing for [M + H]+, [M+Na]+, and [M + K]+ adducts and selecting the most likely lipid(s) for each measured mass. All annotations with a mass deviation <5 ppm were exported for further validation in HPLC MS2 fragmentation experiments.

　　Approximately 1,300 rested and 1,300 sleep-deprived brains were collected in batches of 20–50 per session and snap frozen in plastic tubes. The frozen batches were combined in a glass Potter homogenizer, suspended in 50 µl ice-cold ammonium acetate (0.1% in water, Honeywell), manually homogenized, and transferred to a pre-cleaned Eppendorf tube. Lipids were extracted with 600 µl ice-cold methyl tert-butyl ether (MTBE, Sigma-Aldrich) and 150 µl methanol (VWR). After shaking the mixture for 1 h at 4 °C, 200 µl water (VWR) was added, the mixture was shaken for another 10 min, and the organic phase was collected after centrifugation for 5 min at 1,000g. The aqueous phase was re-extracted using an additional 400 µl MTBE, 120 µl methanol, and 100 µl water. The organic phases from both extraction steps were combined, and the solvent was evaporated under a stream of nitrogen for 30 min, leaving ~700 µg and ~800 µg of dry extract of rested and sleep-deprived samples, respectively. The extracts were stored at –80 °C until further use. An extraction blank was created by performing these steps without brain tissue.

　　Lipid extracts were thawed, dissolved in 650 µl acetonitrile, 300 µl isopropanol, and 50 µl water (all VWR) in an ultrasonic bath, and separated on a C18 column (100 mm × 2.1 mm, 2.6 µm particle size, 100 Å pore size; Phenomenex) in an UltiMate 3000 Rapid Separation System (ThermoFisher) coupled to an orbital trapping mass spectrometer (Q Exactive HF-X, ThermoFisher) using a heated electrospray ionization source (HESI II, ThermoFisher). Data-dependent acquisition and MS2 fragmentation experiments were based on the inclusion list obtained from SMALDI-MSI annotations, with [M + H] +, [M+Na] +, [M + K] + and [M + NH4]+ adducts in positive-ion mode. Since the ionization mechanisms of MALDI and electrospray MS differ, MS2 fragmentation of lipid extracts was additionally performed in negative-ion mode, considering [M–H]− and [M + CHO2]− adducts, to increase the molecular coverage of SMALDI-MSI hits. Lipids were identified using LipidMatch68. All MS2-verified lipid annotations were validated by accurate mass and the detection of all fatty acids plus the head group. only one annotation (PE 27:2) was based on accurate mass and head group alone.

　　Adult flies aged 2–6 days post eclosion were head-fixed to a custom mount using eicosane (Sigma). Cuticle, trachea, excess adipose tissue, and the perineural sheath were removed to create a small window, and the brain was continuously superfused with extracellular solution equilibrated with 95% O2–5% CO2 and containing (in mM) 103 NaCl, 3 KCl, 5 TES, 8 trehalose, 10 glucose, 7 sucrose, 26 NaHCO3, 1 NaH2PO4, 1.5 CaCl2, 4 MgCl2, pH 7.3, 275 mOsM. GFP-positive cells were visualized on a Zeiss Axioskop 2 FS mot microscope equipped with a 60×/1.0 NA water-immersion objective (LUMPLFLN60XW, Olympus) and a pE-300 white LED light source (CoolLED). Borosilicate glass electrodes (9–11 MΩ for dFBNs, 5–7 MΩ for neurons of the pars intercerebralis) were fabricated on a PC-10 micropipette puller (Narishige) or a DMZ Universal Electrode Puller (Zeitz) and filled with intracellular solution containing (in mM) 10 HEPES, 140 potassium aspartate, 1 KCl, 4 MgATP, 0.5 Na3GTP, 1 EGTA, pH 7.3, 265 mOsM. Where indicated, 50 µM 4-ONE or 200 µM 4-hydroxynonenal (4-HNE, Cayman Chemical) were added directly to the intracellular solution; in recordings from neurons of the pars intercerebralis, during which larger-diameter electrodes were used than in recordings from dFBNs, the 4-ONE concentration was lowered to 1 µM. Stock solutions of 4-ONE and 4-HNE were prepared in methyl acetate and ethanol, respectively; vehicle concentrations were not allowed to surpass 0.15% of the total volume after dilution. Recordings were obtained at room temperature with a MultiClamp 700B amplifier, lowpass-filtered at 10 kHz, and sampled at 20 or 50 kHz using Digidata 1440A or 1550B digitizers controlled through pCLAMP 10 or 11 (Molecular Devices). For photostimulation of miniSOG during whole-cell recordings9, a 455-nm LED (Thorlabs M455L3) with a mounted collimator lens (Thorlabs ACP2520-A) and T-Cube LED Driver (Thorlabs) delivered 3.5–5 mW cm−2 of optical power to the sample. Data were analysed using the NeuroMatic package69 (http://neuromatic.thinkrandom.com) in Igor Pro (WaveMetrics).

　　Whole-cell capacitance compensation and bridge balance were used in voltage- and current-clamp recordings, respectively. Series resistances were monitored but not compensated and allowed to rise at most 20% above baseline—but never beyond 50 MΩ—during a recording. Uncompensated mean series resistances of ~40 MΩ in dFBNs (Extended Data Fig. 3c) caused predicted voltage errors of ~16 mV at typical IA amplitudes of ~400 pA (Extended Data Figs. 3a,b, 4 and 6). Input resistances were calculated from linear fits of the steady-state voltage changes elicited by 1-s steps of hyperpolarizing current (5-pA increments) from a pre-pulse potential of –60 ± 5 mV. Membrane time constants were estimated by fitting a single exponential to the voltage deflection caused by a hyperpolarizing 5-pA current step lasting 200 ms. Voltage-spike frequency functions were determined from voltage responses to a series of depolarizing current steps from a membrane potential of –60 ± 5 mV. To account for variations in input resistance within the dFBN population, the current required to produce a 5-mV hyperpolarizing voltage deflection from a pre-pulse potential of –60 ± 5 mV was used as a cell-specific unitary current step instead of a static 5-pA increment. Spikes were detected by finding minima in the time derivative of the membrane potential trace.

　　Voltage-clamp experiments on dFBNs and neurons of the pars intercerebralis were performed in the presence of 1 µM tetrodotoxin (Tocris) and 200 µM cadmium to block sodium and calcium currents, respectively. Potassium currents were measured by stepping neurons from holding potentials of –10 or –110 mV for 400 ms to a series of test potentials spanning the range from –100 mV to +30 mV in 10-mV increments9,24. Depolarizations from –110 mV produced the sum total of the cell’s potassium currents (Itotal, Extended Data Fig. 2a), whereas currents evoked by voltage steps from a holding potential of –10 mV lacked the IA (A-type or fast outward) component because voltage-gated potassium channels such as Shaker inactivated (Extended Data Fig. 2b). IA was calculated by subtracting this non-A-type component from Itotal (Extended Data Fig. 2c). To determine the fast and slow inactivation time constants9, double-exponential functions were fit to the decaying phase of A-type currents elicited by 400-ms steps to +30 mV (Extended Data Fig. 2d). In cases where the fits of slow inactivation time constants were poorly constrained, only the fast inactivation time constants were included in the analysis. Spiking was simulated by 3-ms depolarizing pulses to +10 mV, repeated at 10 Hz for 20 min.

　　Steady-state activation parameters were determined by applying depolarizing 400-ms voltage pulses from holding potentials of –10 or –110 mV; the pulses covered the range from –60 to +60 mV in steps of 10 mV. Linear leak currents were estimated from the slope of the current-voltage relationship at hyperpolarized potentials and subtracted. Steady-state inactivation parameters were obtained with the help of a two-pulse protocol, in which a 300-ms pre-pulse (–120 to +60 mV in 10-mV increments) was followed by a 400-ms test pulse to +30 mV; non-inactivating outward currents, measured from a pre-pulse potential of +10 mV, were subtracted. Peak A-type currents (IA) were normalized to the maximum current amplitude (Imax) of the respective cell and plotted against the test or pre-pulse potentials (V). An estimated liquid junction potential70 of 16.1 mV was subtracted post hoc. Curves were fit to the Boltzmann function to determine the half-maximal activation and inactivation voltages (V0.5) and slope factors (k).

　　HEK-293 cells (CRL-1573, American Type Culture Collection) were grown at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v) fetal bovine serum and 100 U ml−1 penicillin plus 100 µg ml−1 streptomycin (ThermoFisher). The cells were neither externally authenticated nor routinely tested for mycoplasma contamination. Cells were transfected (Lipofectamine 3000, ThermoFisher) with a 1:1 mixture of CMV promoter-driven expression vectors encoding mouse KV1.4 and a bicistronic mouse KVβ2–IRES2–EGFP cassette. A carbonyl-reactive residue71 (Cys-13) in the N-terminal inactivation peptide of KV1.4 was mutated to serine. The growth medium was replaced during whole-cell recordings with extracellular solution containing (in mM) 10 HEPES, 140 NaCl, 5 KCl, 10 glucose, 2 CaCl2, 1 MgCl2, pH 7.4. Where indicated, HEK-293 cells were pre-incubated in extracellular solution supplemented with 12 mM methylglyoxal19 for 1 h, followed by three washes with methylglyoxal-free solution, before data acquisition. GFP-positive cells were visually targeted with borosilicate glass electrodes (2–3 MΩ) filled with intracellular solution containing (in mM) 10 HEPES, 80 potassium aspartate, 60 KCl, 10 glucose, 2 MgATP, 1 MgCl2, 5 EGTA, pH 7.3. Signals were acquired at room temperature with a MultiClamp 700B amplifier, lowpass-filtered at 10 kHz, and sampled at 20 kHz using a Digidata 1440 A digitizer controlled through pCLAMP 10 (Molecular Devices). Because untransfected HEK-293 cells lack voltage-gated conductances (Extended Data Fig. 7f), no channel blockers were present. To determine the fast and slow inactivation time constants, double-exponential functions were fit to the decaying phase of A-type currents elicited by 1-s steps to +30 mV. Spiking was simulated by 3-ms depolarizing pulses to +10 mV, repeated at 10 Hz for 20 min. Data were analysed using the NeuroMatic package69 (http://neuromatic.thinkrandom.com) in Igor Pro (WaveMetrics).

　　Dissected brains were fixed for 20 min in PBS with 4% (w/v) paraformaldehyde, washed 3 times for 20 min with 0.5% (v/v) Triton X-100 in PBS (PBST), and incubated sequentially at 4 °C in blocking solution (10% goat serum in PBST) overnight, with mouse monoclonal anti-Flag M2 antibodies (1:500, Sigma) in blocking solution for 2 days, and with goat anti-Mouse IgG Alexa Fluor 633 antibodies (1:500, ThermoFisher) for one day. The samples were washed 5 times with blocking solution before and after the addition of the secondary antibody, mounted in Vectashield, and imaged on a Leica TCS SP5 confocal microscope with an HCX IRAPO L 25×/0.95 water-immersion objective.

　　With the exception of sleep measurements, no statistical methods were used to predetermine sample sizes. Flies of the indicated genotype, sex and age were selected randomly for analysis and assigned randomly to treatment groups if treatments were applied (for example, sleep deprivation). The investigators were not blinded to group allocation.

　　SMALDI-MSI signal intensities were analysed in LipidSig72 and MATLAB (The MathWorks). Global differences between normalized glycerophospholipid intensities in cryosections of rested and sleep-deprived brains were evaluated by multiple t-tests with FDR-adjusted P < 0.05, using the method of Benjamini–Hochberg. Statistical associations with sleep history of user-defined lipid features, such as the indicated double-bond equivalent ranges or phospholipid head groups, were computed by Fisher’s exact test in LipidSig72. Principal component and hierarchical cluster analyses were performed in MATLAB. The list of significantly different signals was exported and re-imported into Mirion to generate SMALDI-MS images for display. Behavioural and electrophysiological data were analysed in Prism 10 (GraphPad).

　　All null hypothesis tests were two-sided. To control type I errors, P values were adjusted to achieve a joint α of 0.05 at each level in a hypothesis hierarchy; multiplicity adjusted P values are reported in cases of multiple comparisons at one level. Group means or their time courses were compared by paired t-test, one- or two-way repeated-measures ANOVA, or mixed-effects models in cases where a variable was not measured in all cells at all time points, as indicated in figure legends. Repeated-measures ANOVA and mixed-effect models used the Geisser–Greenhouse correction in all instances except the comparisons of >图3a，b和扩展数据中的2个基因型图1a，然后进行了霍尔姆·什沙克的多重比较测试的计划成对分析。如果违反了正常性的假设（如D'Agostino -Pearson检验所示），则通过Mann -Whitney测试，Wilcoxon检验，Kruskal -Wallis Anova或Friedman测试进行比较，然后进行Dunn的多重比较测试，以评估计划的成对差异。在补充表1和2中给出了测试统计，自由度和精确的P值。

　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得。