Experimental animals

All surgical and experimental procedures were approved by the Animal Care and Use Committee of the University of Science and Technology of China. In this study, wild-type 6–10 weeks old male C57BL/6J mice (purchased from Beijing Vital River Laboratory Animal Technology) were used. Animals were maintained under a 12-h light-dark cycle (lights on from 7:00 to 19:00) with standard lab mouse pellet food and water available ad libitum. Littermate mice were split into random groups before all behavioral experiments. After experiments, animals were euthanized by an extra dose of pentobarbital (2.5%; i.p) or isoflurane (5%; inhaling). All efforts were made to minimize the number and suffering of experimental animals.

Drug preparation and administration

Chloroquine and histamine were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in sterile saline solution. The clozapine-N-oxide (CNO) was purchased from APExBIO Technology LLC (Houston, Texas) and dissolved in saline after gently mixing with a vortex. Other detailed information for time and doses for their use was indicated in results or figure legends.

Animal modelsAcetone-ether-water model (AEW)

The acetone-ether-water model is commonly used as a non-histamine-dependent pruritus model to study chronic pruritus. We treated the mice with AEW model as previously reported [36, 47]. The hair of mice was shaved over the rostral part of the back until the skin was completely exposed at least 3 days before the experiment. Cotton (2 × 2 cm) soaked with a mixture of acetone and diethylether (1:1) was applied to the shaved area for 15 s, followed immediately after AE treatment, cotton soaked with distilled water was laid upon the same area for 30 s (AEW group). For the control group, only cotton soaked in water was used for 45 s instead. The animals were treated twice daily (9:00 and 17:00). Mice with chronic dermatitis show spontaneous scratching all day long. Thus, we observed scratching of the mouse at least 14 h after the treatment for barrier disruption on the previous day.

Neck models of acute itch

Neck models of acute itch behavioral tests were recorded and analyzed as previous studies [22, 48]. In brief, mice were shaved on the scruff of the neck before testing. Four mice were put individually into an acrylic box composed of four cells the chamber and allowed to habituate for 30 min. Then, the mice were briefly removed from the chamber and intradermally injected with the desired pruritic agent. Scratching was recorded with a digital video camera for 30 min. The video was then played back for blindly manual analysis and quantified the number of scratching bouts. A scratching bout represents a lifting of hindquarter to rub the injected site of the body and then placing it on the floor.

Stereotaxic surgery

All stereotaxic injections were performed using a stereotaxic frame (RWD, Shenzhen, China) which mice were deeply anesthetized with with 3% pentobarbital sodium (30 mg/kg, i.p.) were mounted on. A heating pad was used to maintain the core body temperature of the animals at 36 °C. Ophthalmic ointment was applied to maintain eye lubrication throughout the surgery. We used the dental drill to carefully remove the skull above the LHb region. A volume of 100 nl viruses (depending on the expression strength and viral titer) were injected at a rate of 50 nl/min using a micro-infusion pump (micro, WPI) by connecting to calibrated glass microelectrodes (tip diameter of 10–20 μm). After the injection, the glass pipettes were left in place for 5–10 min before withdrawal to allow for diffusion. The animals were allowed to recover from anesthesia on a heating blanket before returning to their home cage. Dorso-ventral (DV) from the brain surface, anterior-posterior (AP) from bregma and medio-lateral (ML) from the midline (in mm) were the coordinates.

Viral injection and optical fibers implantation

For chemogenetic inhibition of LHb glutamatergic neurons, mice were bilaterally microinjected with 120 nl rAAV2/9-CaMKIIα-hM4Di-mCherry-WPREs (titer: 5.91E + 12 vg/ml, BrainVTA, PT-0050), and rAAV2/9-CaMKIIα-mCherry-WPRE-pA (titer: 5.14E + 12 vg/ml, BrainVTA, PT-0108) as a control into the LHb. And then mice were injected with CNO (2 mg/kg, i.p.) to manipulate the activity of the LHb. To retrograde LHb-projecting neurons, the unilateral LHb of C57BL/6J mice received microinjection of 120 nl AAV2/2-retro-hSyn-EGFP-WPRE-pA (titer: 5.18E + 12 vg/ml, BrainVTA, PT-1990).

To record calcium fluorescence of LHb glutamatergic neurons, the unilateral LHb (AP: -1.9 mm, ML: -0.45 mm, DV: -2.65 mm) of C57BL/6J mice received microinjection of 120 nl rAAV2/9-CaMKIIα-GCaMP6s-WPRE-pA (titer: 5.31E + 12 vg/ml, BrainVTA, PT-0110) or control virus rAAV2/9-CaMKIIα-EGFP-WPRE-pA (titer: 5.82E + 12 vg/ml, BrainVTA, PT-0959). After 14 days of virus injection, an optic fiber (diameter: 200 μm; length, 4 mm; N.A., 0.37; Inper) was implanted 100 μm above the viral injection site. The optical fiber was secured with skull-penetrating screws and dental acrylic. Photometric recordings were conducted using the fiber photometry recording system after the fibers-implantation procedures to ensure adequate animal recovery.

In vitro electrophysiologySlice preparation

Animals were anesthetized using the intraperitoneal administration of pentobarbital sodium (30 mg/kg) followed by intracardially perfused with ~ 20 ml of ice-cold oxygenated Nmethyl-d-glucamine (NMDG)-based artificial cerebrospinal fluid (ACSF) composed of the following (in mM): 93 NMDG, 1.2 NaH2PO4, 2.5 KCl, 20 HEPES, 30 NaHCO3, 2 thiourea, 25 glucose, 3 Na-pyruvate, 5 Na-ascorbate, 10 MgSO4, 0.5 CaCl2, and 3 glutathione (GSH). The 300-µm-thick coronal brain slices containing the LHb were cut in ice-cold cutting solution sectioned at 0.34 mm/s using a Leica VT1000s vibratome. Slices were initially warmed in cutting solution at 33 °C for 10 min and then placed into N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) ACSF that contained (in mM) 92 NaCl, 2.5 KCl, 30 NaHCO3, 20 HEPES, 1.2 NaH2PO4, 25 glucose, 2 MgSO4, 2 CaCl2, 2 thiourea, 3 Na-pyruvate, 5 Na-ascorbate, and 3 GSH at 25 °C for at least 1 h. After incubation, the slices were transferred into an immersion recording chamber (Warner Instruments, USA) and were continuously perfused with normal ACSF that contained (in mM) 2.4 CaCl2, 3 KCl, 129 NaCl, 1.3 MgSO4, 1.2 KH2PO4, 20 NaHCO3and 10 glucose at a rate of 2.5-3 ml/min at room temperature. During preparation and recording, all solutions were continuously bubbled with 95% O2/5% CO2 to maintain stable hydrogen and continuously provide oxygenation.

In vitro electrophysiological recordings

Whole-cell patch-clamp recordings on the target LHb neurons were visually guided by IR-DIC visualization and an infrared-sensitive charge-coupled device (CCD) camera using a fluorescent Olympus BX51WI microscope. Recording pipettes (5–8 MΩ) were pulled from glass capillaries using a four-stage horizontal micropipette puller (P1000, Sutter Instruments) and backfilled with intracellular solution containing (in mM) 130 K-gluconate,10 HEPES, 5 KCl, 0.6 EGTA, 2 MgCl2, 2 Mg-ATP, and 0.3 Na-GTP. Recordings were acquired with a Multiclamp 700B amplifier (Molecular Devices). Data were sampled at low-pass filtered at 2 kHz, digitized at 10 kHz. Further analysis was performed offline by Clampfit 10.0 software (Molecular Devices). These recordings were excluded when more than 20% of changes in the series resistance occur. CNO (10 µM) was bath-applied to confirm the efficacy of hM4Di-mediated inhibition. To record firing rates of neuronal excitability, 10 to 150 pA of 500 ms-current pulses were injected with an increment of 10 pA per step, and the firing numbers were quantified for each step.

Fiber photometry recording

Calcium-dependent fluorescence signals (470 nm) were background-corrected with autofluorescence signals (405 nm) to control for movement and bleaching artifacts. The laser power was adjusted at the tip of optical fiber to approximately 20–23 µW. Baseline fluorescence (F0) was calculated by the running average of the whole individual recording. All GCaMP6s signal data were normalized to fluorescence change (ΔF/F) by calculating ΔF/F0=(F-F0)/F0. The 15 min timestamps of behavioral event associated with histamine, chloroquine or AEW model-evoked scratching behaviors, air puff was plotted and aligned with fluorescence signal in the custom-written MATLAB codes (R2017b, MathWorks) that were produced by ThinkerTech Nanjing Bioscience. Averaged traces of Ca2+ fluorescent signal changes and heatmaps were analyzed using the MATLAB program.

Immunohistochemistry

Mice were deeply anesthetized with an 3% isoflurane and perfused with 20 ml ice-cold phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA). Brains were carefully removed and postfixed in 4% PFA for 6–8 h, and cryoprotected with 30% sucrose for 48 h. The brain was 30 μm thick sections were coronally prepared using a freezing microtome (Leica CM1950). For immunofluorescent staining, the sections were incubated with PBS containing 0.3% Triton X-100 for 1 h at room temperature and subsequently allowed to react with primary antibodies (rabbit anti-glutamate, 1:500, Sigma; rabbit anti-c-Fos, 1:1000, Synaptic Systems) at 4˚C overnight. After washing with PBS, the sections were subsequently coupled with the corresponding fluorophore-conjugated secondary antibodies for 1.5 h at room temperature. Finally, after rinsing 3 times in PBS, sections were mounted with DAPI staining. Confocal images were acquired under a 10x or 20x objective with a 1024 × 1024 resolution using the Olympus confocal microscopes (FV3000, Olympus). Double fluorescence labelled cells were manually counted using ImageJ software. The Paxinos and Franklin atlas (2013) was employed to define the regions of interest.

Itch-induced conditioned place aversion test

To directly test the role of LHb glutamatergic neurons in negative experience of itch, we used itch-related conditioned place aversion (CPA) protocols, based on previous publications [23]. A three-compartment place preference apparatus for mouse was used. One chamber has white walls, the other chamber has black walls and the middle chamber has gray walls as a neutral zone. There were two manual doors between three chambers. The procedure consists of four phases: habituation, pre-test, conditioning and preference testing (post-test). In habituation phase, animals are allowed to freely explore the apparatus to reduce the effects of novelty for 30 min. During pre-test phase, mice were placed into the middle compartment and allowed to freely explore the entire apparatus. We determined the time of one chamber for 15 min as the baseline preference. Following testing of baseline preference, the conditioning phase is performed twice per day (morning and afternoon) for three consecutive days. In the morning, each animal was injected saline (i.d.) into the nape and assigned one compartments for 30 min, which was accordingly designated the “itch-unpaired” compartment. In the afternoon, the same mice received a subcutaneous injection of chloroquine (i.d.) in the opposite compartment (itch-paired compartment) for 30 min. Animals are only allowed to access to one chamber in one conditioning trial. After six conditioning trials, animals are allowed to freely explore the whole apparatus on the preference testing day for 15 min. The time spent in the itch-paired chamber was analyzed.

In the process of the AEW model treatment (0–6 days), we used a chronic itch-CPA test procedure. Mice were placed in a 3-chamber CPA arena and given free access to entire chambers for 30 min on day one. The apparatus was re-designated as a CNO-paired chamber in which the mice were injected with CNO (i.p) and the other as saline-paired chamber. From day 3 to day 5, mice were conditioned for 3 continuous days with two pairing sessions each day. 24 h after the last pairing, mice were given free access to all chambers, and the time spent in the CNO-paired chamber was analyzed.

Assessment of motor function

A rotarod system of accelerating treadmills (IITC, Inc., Life Sciences, St. Petersburg, FL) was used to assess coordinate motor activity and general motor disability. On day before the test, mice were placed on an apparatus that accelerated 5–20 revolutions per minute (r.p.m.) for 5 min, and trained to maintain its balancing walking. Mice were gently placed on a rod that gradually accelerated from 5 r.p.m. to 40 r.p.m. over a 5 min period, and the latencies of the mice to drop were recorded.

Data analysis and statistics

Software used for data analysis and plotting the results included: MATLAB R2017a, Olympus FV10-ASW 4.0a Viewer, GraphPad Prism v.8.0.1, Adobe Illustrator 2021 and Adobe Photoshop 2021. All experiments and data analyses such as immunohistochemistry, electrophysiology and behavior were performed blindly. For the quantitation of c-Fos+ cells, we selected 3–4 continuous sections containing LHb from each animal. The average number of c-Fos+ neurons in the bilateral LHb per section was calculated as “NO. of c-Fos+ neurons”. Statistical detection methods include: student’s paired t test, student’s unpaired t test, Mann-Whitney U test, Wilcoxon signed-rank test, two-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons. Before applying the student’s t test statistics, the data were conformed to normal distribution. The data were analyzed with Mann-Whitney U test for unpaired t test and Wilcoxon signed-rank test for paired t test if normality was violated. Statistical significances were represented as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. These data were presented as the mean ± standard error of the mean (SEM).