DOI: 10.5061/dryad.k3j9kd551
title: Data from: Hypothalamic circuitry underlying stress-induced insomnia and peripheral immunosuppression

description: The neural substrates of insomnia/hyperarousal induced by stress remain
 unknown. Here, we show that restraint stress leads to hyperarousal
 associated with strong activation of corticotropin-releasing hormone
 neurons in the paraventricular nucleus of hypothalamus (CRHPVN) and
 hypocretin neurons in the lateral hypothalamus (HcrtLH). CRHPVN neurons
 directly innervate HcrtLH neurons and optogenetic stimulation of
 LH-projecting CRHPVN neurons elicits hyperarousal. CRISPR-Cas9-mediated
 knockdown of the crh gene in CRHPVN neurons abolishes hyperarousal induced
 by stimulating LH-projecting CRHPVN neurons. Genetic ablation of Hcrt
 neurons or crh gene knockdown significantly counteracts restraint
 stress-induced hyperarousal. As stress is further associated with changes
 in immune function, we used single-cell mass cytometry by time of flight
 (CyTOF) to analyze peripheral blood and found extensive changes to immune
 cell distribution and functional responses during wakefulness upon
 optogenetic stimulation of CRHPVN neurons. Our findings suggest both
 central and peripheral systems are synergistically engaged in the response
 to stress via CRHPVN circuitry.
description: CRH::Cre mice were equipped
 with EEG/EMG electrodes as described previously. AAVs encoding the
 optogenetic actuator ChR2 (AAV-DJ-DIO-EF1α-ChR2-eYFP; Lot # 5227; 4.12 ×
 10<sup>12</sup> gc/ml) were infused into the paraventricular
 nucleus (unilateral; 0.4 μl; -0.90 mm AP, ±0.25 mm ML, -4.85 mm DV
 (injection), -4.6 mm DV (optic fiber)) over the course of 3 min followed
 by 10 min for viral distribution to the tissue. Then, a fiber optic
 implant (200 µm diameter, 5.5 mm length, Doric Lenses, Inc.) was slowly
 lowered and cemented in place above the PVN. Mice were allowed to recover
 for at least 3 weeks prior to starting the experiment. Following recovery, 15 ms 10 mW
 (LaserGlow, calibrated with Thorlabs light meter) 473 nm light pulses at
 10 Hz (10 sec ON, 20 sec OFF) were delivered to the PVN for 1 hour, and
 then whole blood was collected 3 hours following the start of stimulation
 using microcapillary tubes (retro-orbital bleed; approximately 350 ml
 blood). Blood was immediately stabilized using a protein stabilization
 reagent, allowed to sit for 10 minutes, and then placed at -80 ℃ in
 cryovials until further processing. Red blood cells (RBCs) were lysed
 using 1× Thaw-lyse buffer (Smart Tube Inc., San Carlos, CA) in
 ddH<sub>2</sub>O. After lysing, cells were pelleted at 600× g
 for 5 min at room temperature. RBC depleted samples were barcoded using
 palladium isotopes. In brief, cells were washed with cell staining media
 (CSM), 1× PBS, and 1× PBS containing 0.02% saponin and then placed into a
 deep well block. Unique barcoding reagents were added to each well of the
 block containing cells from each mouse and incubated for 15 minutes at
 room temperature. After incubation, cells were washed with CSM and then
 resuspended via brief vortexing. Then, all samples were pooled into a
 single fluorescence-activated cell sorting (FACS) tube for the next
 staining step. After washing and pelleting the pooled cells, 150 ml of Fc
 block was added and cells were incubated at room temperature (RT) with
 gentle shaking (600 rpm) for 10 minutes. Then, 300 ml of the surface
 antibody mix (see Table. S1) was added and cells were incubated at RT for
 30 minutes with gentle shaking. Then, cells were washed, pelleted, and the
 supernatant was collected and discarded. To permeabilize cells for
 intra-cellular staining, 700 ml of MeOH was added to the tube and cells
 were incubated at 4 ℃ for 10 minutes. Then, cells were washed with 1× PBS
 and CSM, followed by incubation with the intracellular antibody mix (see
 Table. S1) for 30 minutes at RT with gentle shaking (600 rpm). Cells were
 washed and then a DNA intercalator (DVS # Inter-1X-natir) was added in a
 mixture of 16% paraformaldehyde/PBS. This mixture was incubated at 4 °C
 overnight. On the following day, the pooled sample was
 washed and then 1× normalization “beta” beads were added and cells were
 strained into a new FACS tube. Cells were run on a Helios (Fluidigm)
 machine using the SuperSampler attachment. Following the run, the data
 were normalized to the normalization bead signal. Then, data was run
 through de-barcoder software (MATLAB; bead distance 0.2) and individual
 .FCS files were gated and analyzed manually using ImmuneAtlas and Cytobank
 platforms. tSNE plots were generated for filtered singlet/leukocyte (i.e.,
 red blood cells excluded) populations for each file and formed into a
 composite plot. In brief, .FCS files were arcsinh transformed (a = 0, b =
 1/5, c = 0) and 3000 randomly selected events were sampled for each file.
 tSNE was computed on the aggregated matrix with all events (3000 × 12 =
 36,000). The following parameters were used for tSNE in the RTsne package
 (https://CRAN.R-project.org/package = Rtsne): perplexity = 30, max_iter = 500. Then, the transformed coordinate events were mapped back to the sample they come from, as well as composite maps for each condition and the total population.
description: The files are in .fcs (flow cytometry standard) format. The files
 contain discrete events (each one is a cell or bead) detected by the cytof
 machine. Each event can be associated with any number of antibodies in the
 panel, a palladium barcode, and DNA intercalator staining.
 Control group files are labelled with CRH_Cre. Stimulated group
 files are labelled with CRH_Cre_10Hz.