Sleep pressure accumulates in a voltage-gated lipid peroxidation memory

Drosophila strains and culture

Flies were reared on media of cornmeal (62.5 g l⁻¹), inactive yeast powder (25 g l⁻¹), agar (6.75 g l⁻¹), molasses (37.5 ml l⁻¹), propionic acid (4.2 ml l⁻¹), tegosept (1.4 g l⁻¹) and ethanol (7 ml l⁻¹) on a 12 h light:12 h dark cycle at 25 °C. All electrophysiological and lipidomic analyses (with the exception of studies of the effects of the sni¹ mutation) were performed on randomly selected female flies aged 2–6 days post eclosion. Experimental flies were heterozygous for all transgenes and homozygous for either a wild-type or mutant (Hk¹) Hyperkinetic allele^51,52, as stated. The R23E10-GAL4 driver^16,53 controlled the expression of the fluorescent label mCD8::GFP in dFBNs, along with an N-myristoylated covalent hexamer (myr-MS6T2) of the singlet oxygen generator miniSOG⁵⁴ or catalytically defective (Hk(K289M)) or functional versions of Hyperkinetic⁴⁴, as indicated. The Dh31-GAL4 line⁵⁵ targeted mCD8::GFP to neurons of the pars intercerebralis.

Because sni is X-linked³⁸, it was most expedient to investigate its function in males. In behavioural experiments or 4-ONE analyses, hemizygous carriers of the sni¹ allele coexpressed UAS-sni³⁸, UAS-AOX⁵⁶ or UAS-Hk^RNAi (47805GD)⁵⁷ transgenes, either pan-neuronally⁵⁸ under the control of nSyb-GAL4 or in dFBNs^16,53 under the control of R23E10-GAL4, as noted. For electrophysiological recordings, dFBNs of hemizygous sni¹ mutants and wild-type males were marked with R23E10-GAL4-driven mCD8::GFP.

A Hyperkinetic allele encoding an in-frame fusion to an N-terminal Flag epitope (Hk^Flag) was created through homology-dependent repair of a CRISPR–Cas9-generated double-strand break (WellGenetics). The Flag tag was inserted immediately after the initiating methionine of isoforms Hk-PK, Hk-PE, Hk-PL, and Hk-PM and connected to the remainder of the protein via a flexible linker (4× Gly-Gly-Ser).

Sleep measurements and sleep deprivation

Females or hemizygous sni¹ mutant males³⁸ aged 2–5 days were individually inserted into 65-mm glass tubes, loaded into Drosophila Activity Monitors (Trikinetics), and housed under 12 h light:12 h dark conditions. Flies were allowed to adapt to the monitors for a day, and the activity counts during the following two 24-h periods were averaged. Inactivity periods of >5 min were classified as sleep^59,60 (Sleep and Circadian Analysis MATLAB program⁶¹). Immobile flies (<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.

SMALDI mass spectrometry imaging

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.

HPLC MS² fragmentation

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.

Electrophysiology

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 CaCl_2, 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 I_A 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 increments^9,24. Depolarizations from –110 mV produced the sum total of the cell’s potassium currents (I_total, Extended Data Fig. 2a), whereas currents evoked by voltage steps from a holding potential of –10 mV lacked the I_A (A-type or fast outward) component because voltage-gated potassium channels such as Shaker inactivated (Extended Data Fig. 2b). I_A was calculated by subtracting this non-A-type component from I_total (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 (I_A) were normalized to the maximum current amplitude (I_max) 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 \({I}_{{\rm{A}}}/{I}_{\max }=1/\left(1+{{\rm{e}}}^{\frac{V-{V}_{0.5}}{k}}\right)\) to determine the half-maximal activation and inactivation voltages (V_0.5) 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 K_V1.4 and a bicistronic mouse K_Vβ2–IRES2–EGFP cassette. A carbonyl-reactive residue⁷¹ (Cys-13) in the N-terminal inactivation peptide of K_V1.4 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).

Confocal imaging

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.

Statistics and reproducibility

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 >2 genotypes in Fig. 3a,b and Extended Data Fig. 1a and were followed by planned pairwise analyses with Holm–Šídák’s multiple comparisons test. Where the assumption of normality was violated (as indicated by D’Agostino–Pearson test), group means were compared by Mann–Whitney test, Wilcoxon test, Kruskal–Wallis ANOVA or Friedman test, followed by Dunn’s multiple comparisons test to evaluate planned pairwise differences. Test statistics, degrees of freedom, and exact P values are given in Supplementary Tables 1 and 2.

Reporting summary

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