Animals
All procedures were carried out in accordance with the US National Institute of Health (NIH) guidelines for the care and use of laboratory animals, and approved by the Animal Care and Use Committees of Columbia University. Both male and female adult mice (820 weeks old) were used for all experiments. The following mouse lines were used in the current study: C57BL/6J (JAX 000664), VGlut2-IRES-Cre (JAX 028863), Gad2-IRES-Cre (JAX 0101802), Ai9 (JAX 007909). Mice were housed in 12-hour light-dark cycles (lights on at 07:00 a.m. and off at 07:00p.m., temperatures of 6575F with 4060% humidity) with free access to food and water.
AAV1-EF1-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA, AAV1-Ef1-DIO-EYFP, AAV1-Syn-FLEX-GCaMP6s, AAV1-Syn-FLEX-GCaMP6f, AAV8-hSyn-DIO-hM3D(Gq)-mCherry, AAV9-hSyn-DIO-hM4D(Gi)-mCherry, AAVrg-hSyn-Cre-WPRE-hGH, AAV9-CamKIIa-ChR2-eYFP, AAV1-CAG-FLEX-tdTomato, AAV1-mDlx-NLS-mRuby2 were obtained from Addgene. AAV1-EF1a-FLEX-TVA-mCherry from UNC vector core, AAV1-FLEX-2A-G(N2C)-mKate and RABV-G -GFP-EnvA, a gift from Charles Zuker.
Mice were anaesthetized with a mixture of ketamine and Xylazine (100 and 10mgkg1, intraperitoneally), then placed on a stereotaxic frame with a closed-loop heating system to maintain body temperature. After asepsis, the skin was incised to expose the skull and a small craniotomy (~0.5mm in diameter) was made on the skull above the regions of interest. A solution containing 50200nl viral construct was loaded into a pulled glass capillary and injected into the target region using a Nanoinjector (WPI). Optical fibers (0.2mm diameter, 0.39 NA, Thorlabs) were implanted into the target region with the tip 0.4mm above the virus injection site for optogenetic manipulation. For EEG and EMG recordings, a reference screw was inserted into the skull on top of the cerebellum. EEG recordings were made from two screws on top of the cortex 1mm from midline, 1.5mm anterior to the bregma and 1.5mm posterior to the bregma, respectively. Two EMG electrodes were bilaterally inserted into the neck musculature. EEG screws and EMG electrodes were connected to a PCB board which was soldered with a 5-position pin connector. All the implants were secured onto the skull with dental cement (Lang Dental Manufacturing). After surgery, the animals were returned to home-cage to recover for at least two weeks before any experiment.
For retrograde tracing, 150200nl AAVrg-hSyn.Cre.WPRE.hGH was unilaterally or bilaterally injected into the ventrolateral preoptic area (VLPO, Bregma 0.1mm, lateral 0.9mm, ventral 5.4mm) of Ai9 mice. For rabies tracing, 200nl mix of AAV-FLEX-G(N2C)-mKate and AAV-FLEX-TVA-mCherry (1:1) was unilaterally injected into the VLPO of Gad2-Cre mice. Two weeks after AAV injection, 200nl RABV-G-GFP-EnvA was unilaterally injected into the same VLPO. For anterograde tracing, 50nl AAV1-CAG-FLEX-tdTomato was unilaterally injected into the ventrolateral medulla (VLM, Bregma 6.9mm, lateral 1.1mm, ventral 5.6mm) of Vglut2-Cre mice. The ventral coordinates listed above are relative to the pial surface.
For chemogenetic inhibition, 200nl AAV9-hSyn-DIO-hM4D(Gi)-mCherry was bilaterally injected in VLM, 200nl AAVrg-hSyn.Cre.WPRE.hGH was bilaterally injected into the VLPO of C57BL/6J mice. For chemogenetic activation, 200nl AAV8-hSyn-DIO-hM3D(Gq)-mCherry was unilaterally injected into the VLM, 200nl AAVrg-hSyn.Cre.WPRE.hGH was bilaterally injected into the VLPO of C57BL/6J mice.
For optogenetic activation experiments, 200nl AAV1-EF1-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA was unilaterally injected into the VLM, an optical fiber implanted 0.4mm on top of the viral injection site in Vglut2-cre mice. For terminal stimulation, 200nl AAV1-EF1-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA was unilaterally injected into the VLM of Vglut2-Cre mice, and an optical fiber was implanted in the POA. In in vivo pharmacological inhibition experiments, Vglut2-Cre mice were unilaterally injected with AAV1-DIO-ChR2-EYFP and implanted with anoptical fiber in the VLM, then implanted with a double guide cannula (26 gauge, P1 Technologies) bilaterally above the POA. A metal head-post was attached during surgery for head fixation during the intracranial infusion.
For slice recording, 250nl AAV1-mDlx-mRuby2 was unilaterally injected in the VLPO, 250nl AAV1-DIO-ChR2-eYFP was contralaterally injected in the VLM of Vglut2-Cre mice.
For fiber photometry in GAD2-Cre mice, 200nl AAV9-CaMKIIa-ChR2-eYFP was unilaterally injected in the VLM, 200nl AAV1-FLEX-GCaMP6s was contralaterally injected in the VLPO. An optical fiber was implanted 0.2mm above the VLPO injection site.
For microendoscopic calcium imaging, 200nl AAV1-FLEX-GCaMP6f was unilaterally injected in the VLM of Vglut2-Cre mice. A GRIN lens (0.5 or 0.6mm diameter, Inscopix) was implanted 0.2mm above the injection site. After >3 weeks, the cover was removed to expose the GRIN lens and a miniaturized, single-photon, fluorescence microscope (Inscopix) was lowered over the implanted GRIN lens until the GCaMP6f fluorescence was visible under illumination with the microscopes LED. The microscopes baseplate was then secured to the skull with dental cement darkened with carbon powder for subsequent attachment of the microscope to the head. After recovery from surgery, we did not observe any gross behavioral abnormality, and these mice exhibited normal sleepwake cycles.
Mouse sleep behavior was monitored using EEG and EMG recording along with an infrared video camera at 30 frames per second. Recordings were performed for 2448h (light on at 7:00a.m. and off at 7:00p.m.) in a behavioral chamber inside a sound-attenuating cubicle (Med Associated Inc.). Animals were habituated in the chamber for at least 4h before recording. EEG and EMG signals were recorded, bandpass filtered at 0.5500Hz, and digitized at 1017Hz with 32-channel amplifiers (TDT, PZ5 and RZ5D or Neuralynx Digital Lynx 4S). Spectral analysis was carried out using fast Fourier transform (FFT) over a 5s sliding window, sequentially shifted by 2s increments (bins). Brain states were semi-automatically classified into wake, NREM sleep, and REM sleep states using a custom-written Matlab (version 2021, MathWorks) program43 (wake: desynchronized EEG and high EMG activity; NREM: synchronized EEG with high-amplitude, delta frequency (0.54Hz) activity and low EMG activity; REM: high power at theta frequencies (69Hz) and low EMG activity). Semi-auto classification was validated manually by trained experimenters. Relative delta power was calculated by dividing the delta power in the 2-s bins by the total EEG power averaged across the recording session.
Fiber photometry recordings were performed essentially as previously described44. In brief, Ca2+-dependent GCaMP fluorescence were excited by sinusoidal modulated LED light (465nm, 220Hz; 405nm, 350Hz, Doric lenses) and detected by a femtowatt silicon photoreceiver (New Port, 2151). Photometric signals and EEG/EMG signals were simultaneously acquired by a real-time processor (RZ5D, TDT) and synchronized with behavioral video recording. A motorized commutator (ACO32, TDT) was used to route electric wires and optical fiber. The collected data were analyzed by custom MATLAB scripts. They were first extracted and subject to a low-pass filter at 2Hz. A least-squares linear fit was then applied to produce a fitted 405nm signal. The DF/F was calculated as: (F-F0)/F0, where F0 was the fitted 405nm signals. Data were smoothed using a moving average method and downsampled to 5Hz. In Supplementary Fig.6, photometric data were further normalized across animals using Z-score calculation.
All optogenetic stimulation were conducted unilaterally. Mice were habituated in the behavioral chamber for at least 4hours before the experiment. Light pulses (20Hz, 10ms) with different durations (30s, 1min, 2min) from a 473nm laser diode (Shanghai laser & Optics Century Co., Ltd) were controlled by a microcontroller board (Arduino Mega 2560, Arduino). Laser power is set to 46mW for somatic stimulation in the VLM and 1015mW for terminal stimulation in the VLPO. We used two methods to trigger laser stimulation: (1) wake-trigger stimulation: An IR-camera (30 fps) was placed on the ceiling to videotape animal behavior. A custom Matlab program45 was used to real-time process video frames to detect animals location by subtracting each frame from the pre-acquired background image (without the mouse). Laser stimulation was automatically triggered when animal movement continued for a period (5min). A minimal interval of 30min was set between trials. In no-light control experiments, the same trigger method was applied except the laser power was off. (2) fixed interval stimulation: inter-stimulation interval for optogenetic stimulation is fixed to 45min. To compare optogenetic-induced and natural NREM sleep (Fig.7e), we used the first NREM sleep episode (if existed) following laser stimulation in 15-min time windows (5min before laser, 10min after laser) as optogenetic-induced sleep, used all NREM sleep episodes from 15-min time windows between laser stimulation (at least 15min away from previous or next stimulation) in the same recording sessions as natural sleep, and calculated bout durations and relative delta power of NREM sleep episodes.
Imaging sessions took place during the light cycle in a behavioral chamber placed within a sound-attenuated cubicle (Med. Associates). Calcium activity was acquired using the nVista 3.0 hardware and IDPS software (Inscopix) with 475nm LED illumination (10Hz, 0.41.2mW/mm2). EEG and EMG were acquired using Neuralynx Digital Lynx 4S controlled by a custom-built MATLAB program via Neuralynx API. A TTL signal delivered from the Inscopix system to the Neuralynx system throughout the recording session was used to synchronize the timing between the imaging and EEG/EMG recordings. Each recording session lasted 60120min, and for each mouse the data from a single recording session was included. Imaging data were processed in IDPS (Inscopix, version 1.6.0.3225) and MATLAB46. First, the acquired images were spatially down-sampled by a factor of 4. To correct for lateral motion of the brain relative to the GRIN lens, we used the motion correction function in IDPS, as in previous studies47,48. Regions of interest (ROIs) were then manually identified. The pixel intensities within each ROI were averaged to create a fluorescence time series. For individual neurons, the DF/F was calculated as the difference between the calcium activity at each bin and the averaged calcium activity of the whole recording time, divided by the average. To quantify the calcium activity, we used the OASIS fast deconvolution algorithm49. The identified events were reviewed along with calcium imaging video by an experimenter and motion-induced artifacts were excluded for further analysis. To compare the activity in different brains states, we used the integrated area under curve (AUC) of detected events and normalized it to the durations (in minute) of each state, which yields relative activity per minute. The N-W or R-W selectivity index was calculated as:
$${{{{{rm{Index}}}}}}=({{{{{{rm{AUC}}}}}}}_{{{{{{rm{a}}}}}}}-{{{{{{rm{AUC}}}}}}}_{{{{{{rm{b}}}}}}})/({{{{{{rm{AUC}}}}}}}_{{{{{{rm{a}}}}}}}+{{{{{{rm{AUC}}}}}}}_{{{{{{rm{b}}}}}}})$$
where AUCa and AUCb refer to AUC activity in brain state a (e.g., NERM sleep) and b (e.g., wake) respectively. Index ranges from 1 to 1, with 0 indicating no selectivity between two states.
For the analysis of calcium activity during the transitions (Fig.2d), we calculated AUC activity in the NREM active cells before and after wake-to-NREM or NREM-to-wake transitions. A 30-s window in each brain state (e.g. 30-s wake, 30-s NREM for W-N transitions) was used for quantification. Transitions with less than 30s-episodes in either state were excluded for analysis.
After habituation for 12h in the testing chamber, C57BL/6J mice expressing hM4Di or hM3Dq in the VLM were injected with saline (day 1) and CNO (day 2, 1mg/kg body weight) intraperitoneally (i.p.) at the same time of the days. Injections were performed in light cycles (10:00a.m.) for chemogenetic inhibition and in dark cycles (9:3010:00 PM) for chemogenetic activation. In control experiments, wild type mice without viral injection were treated with CNO and saline either in light cycles or dark cycles. Sleep recording started at least 1h before saline injection and lasted 24h after CNO injection. EEG and EMG in the time window (012h after CNO or saline injection) were used for data analysis.
On the day of the experiment, mice were habituated and tested with photostimulation in a behavioral chamber for overnight (pre-test). Following the pre-test, the selective AMPA receptor antagonist NBQX (5mg/ml in 0.9% NaCl, 0.25l, Tocris Bioscience) was bilaterally infused into the POA using two 1l microsyringes (Hamilton) and two internal cannulas (P1 Technologies) inserted into the double guide cannula that implanted above the POA. The infusion rate was approximately 0.04l/min controlled by a syringe pump (Harvard Apparatus). As a control, saline (0.9% NaCl) was similarly applied. After the intracranial infusion, mice were moved back to the chamber for further optogenetic experiments. The first laser stimulation started 30min after drug infusion. A total of 68 trials in the first 4h was used for data analysis.
To identify neurons projecting to the POA, we unilaterally injected ~200nl AAVrg-hSyn.Cre.WPRE.hGH in the VLPO of Ai9 reporter mice. Six weeks after viral injection, mice were perfused with phospho-buffered saline (PBS) containing 10 U/ml heparin, followed by 4% paraformaldehyde. Brains were then harvested and post-fixed in 4% paraformaldehyde for 3h at room temperature. The whole brains were cleared by the CUBIC method as previously described39,50. Briefly, mouse brains were washed 3 times in PBS before immersion in CUBIC reagent-1 (diluted 1:2 in water) overnight, incubated in reagent-1 for 710 days. Then, brains were washed with PBS, degassed in PBS overnight, and immersed in reagent-2 (diluted 1:2 in PBS) for 624h before incubated in reagent-2 containing TO-PRO-3 (1:5000, Thermo Fisher Scientific) for additional 710 days. Reagent 1 contained 25wt% urea (Sigma- Aldrich), 25wt% N,N,N,N-tetrakis (2-hydroxypropyl) ethylenediamine (Sigma- Aldrich) and 15wt% Triton X-100 (Nacalai Tesque). Reagent 2 contained 50wt% sucrose (Sigma-Aldrich), 25wt% urea, 10wt% triethanolamine (Sigma-Aldrich) and 0.1% (v/v) Triton X-100. All clearing procedures were performed at room temperature with gentle shake to prevent sample deformation caused by temperature fluctuation and fluorescence loss. Reagent 1 and Reagent 2 were refreshed every 3 days. Samples were imaged in an oil mix (mineral oil and silicone oil 1:1) horizontally from ventral to dorsal by Light-sheet fluorescence microscopy (UltraMicroscope, LaVision BioTec) as previously described39. The images were acquired with a z-step size of 5 m. Exposure time was 50ms per channel per z step. Data was processed in ImageJ (version 1.52i). The gamma value of the images was set to 0.5 for display purposes. The whole-brain data was registered to a reference atlas (Allen Brain Institute, 25-m resolution volumetric data with annotation map, http://www.brain-map.org) using elastix (version 5.0)51,52. The voxel size of both sample data and reference template were scaled to 6.5 m. The 3D reconstruction, cell tracing, and structure labeling were performed in Imaris (version 9.6, Bitplane).
In sleep deprivation experiments, mice were allocated into three groups: (1) control, (2) sleep deprivation (SD), (3) recovery sleep (RS) after deprivation. Each group was placed in a behavioral chamber, which was further contained in a sound-attenuating cubicle (Med Associates Inc). Mice were habituated for 48h before Fos induction. For sleep deprivation, a stepper motor controlled by an Arduino board was used to move a rod to sweep the floor from one side to the other at a speed of 2cm/s. As a motor control, Mice in the first group were subject to sweeping movement for 2h before perfusion. SD mice were subject to 30-s sweeping movement followed by a 90-s interval for 6h, and then sacrificed for perfusion. RS mice were perfused 2h after 6-h SD. All sleep manipulation was carried out in the light cycle, and animals were sacrificed between 4:00p.m. and 6:00p.m. Mouse brains were sectioned coronally at 100 m and processed for immunostaining as previously described53. Tissue sections were incubated with guinea pig c-Fos antibody (1:1000, Synaptic Systems, cat#226308) for 24h at 4C. Fluorescently tagged secondary antibodies (Alexa-488 donkey anti-guinea pig, 1:500, Jackson ImmunoResearch, cat#706-545-148) were used to visualize Fos expression. All sections were imaged using a Zeiss 810 confocal microscope. Cell counting (brain sections from AP-6.5mm to AP-7.2mm) was performed manually in ImageJ.
In optogenetic stimulation experiments (Supplementary Fig.14), Vglut2-Cre mice were injected with AAV-DIO-ChR2-eYFP unilaterally in the VLM. To maximize the c-Fos signals, a 30-min laser stimulation (20Hz, 45mW) was applied. Mice were kept in the recording chamber for additional 40minutes after stimulation, then perfused for c-Fos staining as described above. The injection site and other sleep/wake related brain regions were examined for c-Fos expression.
Mice were deeply anaesthetized, and fresh frozen brains were sectioned at 20 m thickness using a cryostat. FISH was performed using RNAscope Multiplex Fluorescent Assay V2 (Advanced Cell Diagnostics). Reagents: Fos in situ hybridization probe: cat# 316921, Slc17a6 in situ hybridization probe: cat# 319171, Slc32a1 in situ hybridization probe: cat# 319191. Tyrosine hydroxylase (TH) in situ hybridization probe: cat# 317621 (Advanced Cell Diagnostics). Images were acquired using a Zeiss 810 confocal microscope. Cell counting (brain sections from AP-6.5mm to AP-7.2mm) was performed manually in ImageJ.
Viral expression and placement of optical implants were verified at the termination of the experiments using DAPI counterstaining of 100 m coronal sections (Prolong Gold Antifade Mountant with DAPI, Invitrogen). Images were acquired using a Zeiss 810 confocal microscope. Cell numbers were counted manually in ImageJ.
Four weeks after viral injection, mice were quickly decapitated under isoflurane sedation. Brains were placed in artificial cerebral spinal fluid (aCSF). Coronal slices (300m) containing the POA were cut on a vibratome (Leica VT1200) in sucrose cutting solution containing 2.5mM KCl, 10mM MgCl2, 0.5mM CaCl2, 1.25mM NaH2PO4, 26mM NaHCO3, 234mM sucrose, 11mM glucose. Slices were then transferred to aCSF containing 126mM NaCl, 26mM NaHCO3, 2.5mM KCl, 1.25mM NaH2PO4, 2mM CaCl2, 1mM MgCl2, and 10mM d-glucose bubbled with 95% O2 and 5% CO2. Slices were incubated at 341C for 30min before resting at room temperature prior to the experiment. Regular pipette intracellular solution contained 127mM potassium gluconate, 8mM NaCl, 4mM ATP-Mg, 0.6mM EGTA, 0.3mM GTP, 10 HEPES, and 8.1mM biocytin adjusted to pH 7.37.4 with KOH. Osmolarity was adjusted to 290300mOsm.
Inhibitory GABAergic cells in the POA were located using the presence of mRuby fluorescence. Cells were targeted approximately 20100m from the slice surface. Patch-clamp Scientifica MicroStar micromanipulators were controlled using LinLab2 software (Scientifica). Recordings were performed in juxtacellular or whole-cell mode with MultiClamp 700B amplifiers (Molecular Devices) in the current clamp or voltage clamp mode at 341C bath temperature. Data acquisition was performed through an Axon Digidata 1550B (Molecular Devices) connected to a PC running pClamp 11 (Molecular Devices). Recordings were sampled at 10kHz and filtered with a 2-kHz Bessel filter. Patch pipettes were pulled with a Flaming/Brown micropipette puller P-80PC (Sutter Instruments) and had an initial resistance of 38 M. Series resistance was automatically compensated at 15M. The membrane potential values given were not corrected for the liquid junction potential which was approximately 16mV. Cells were stimulated with blue LED light through a 40 water-immersion objective using pE-300ultra (CoolLED Limited) at approximately ~18.8mW. Stimulation protocols comprised of 10 sweeps of paired pulses at 2, 5, 10, 20, and 40, and 60Hz delivered with a light on time of 1ms. 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 10M) and DL-2-Amino-5-phosphonopentanoic acid (DL-AP5; 10M) was bath applied in some recordings.
Unitary excitatory post-synaptic potentials (EPSPs) and post-synaptic currents (EPSCs) were analyzed using custom Matlab scripts. EPSPs or EPSCs were considered if the absolute value is >2 times ofthe standard deviation of the baseline. Baseline was calculated as the mean of 10ms prior to the stimulation or pre-synaptic action potential. Amplitudes were found at the maximum or minimum point within a 1.518ms time window after the stimulation onset. Lower- or upper-bounds of detection windows were adjusted when necessary. The rise time was determined as the time interval encompassing 2080% of the amplitude. Latencies were determined by calculating the onset time of the PSP or PSC and subtracting the stimulation onset. This onset time represented the intersection of the line at baseline and the line through the 20% and 80% amplitude points.
No statistical methods were used to predetermine sample size, and investigators were not blinded to group allocation. No method of randomization was used to determine how animals were allocated to experimental groups. Mice in which post hoc histological examination showed viral targeting or fiber implantation was in the wrong location were excluded from analysis. Two-sided paired t-test, unpaired t-test, and MannWhitney U-test were used and indicated in the respective Figure legends. Values are tested against normal distributions using the KolmogorovSmirnov test with statistical significance set at p<0.05. All analyses were performed in MATLAB. Data are presented as meanSEM.
Further information on research design is available in theNature Research Reporting Summary linked to this article.
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Control of non-REM sleep by ventrolateral medulla glutamatergic neurons projecting to the preoptic area - Nature.com