Animals and models
Adult male C57Bl/6 mice at 7–9 weeks of age were purchased from the Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. Before experimental manipulations, the mice were acclimated to the controlled conditions (22° ± 1 °C, a 12:12 light–dark cycle, 8 animals per cage, and food and water available ad libitum) for 1 week. Animal procedures performed in the present study were evaluated and approved by the Animal Care and Use Committee of Fudan University and were conducted strictly in accordance with the guidelines of the International Association for the Study of Pain. All efforts were made to minimize the number and suffering of animals. For each experiment, the animals were randomized to either the control or experimental group. We determined the sample size for each experiment based on our previous work.
The partial transection of the infraorbital nerve
Mice were placed in the supine position on a surgical pad under anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneally (i.p.). The oral cavity of the mouse was opened and the pT-ION was performed via an intraoral approach. A 5-mm long incision in the left palatal-buccal mucosa beginning from the first molar was made. The tissue was separated with a pair of forceps, and the left ION was gently dissociated with a tiny glass rod with a hooked tip. The deep branches of the ION were ligated with a catgut (4.0, BD171001, Boda Co., Ltd., Shandong, China) and distally cut off using a pair of surgery scissors, and approximately 2 mm of the nerve fibers were excised to prevent regeneration. The catgut was taken out from the bag immediately before use and immersed in 75% alcohol (80,176,961, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) to keep it sterile. The incision was closed using tissue glue, and the animals were allowed to recover from the anesthesia after the surgery in a warm chamber. The sham-operated animals underwent unilateral nerve exposure without ligation and transection. The animals were treated with daily intramuscular injection of penicillin potassium (14,005, Keda Co., Ltd., Jiangxi, China) at a dose of 50,000 units/kg (dissolved in sterilized normal saline) for 2 consecutive days after surgery. All of the surgical procedures were performed under aseptic conditions, and no severe infections or post-surgical complications were observed. No treatment was given in the naive group, and animals with emaciation or abnormal movement or mental state were excluded from subsequent experiments. According to our previous work [12], the pT-ION-induced mechanical and cold hypersensitivity is stable for at least 28 days (the longest time period that we observed) and does not spontaneously resolve over time within 28 days after surgery.
Behavioral tests
Behavioral tests were conducted by experimenters blinded to the group allocation. Pain-related behavior in mice was tested before surgery (day 0) and at 7, 14, and 21 days after surgery, and anxiety-like behaviors were tested 2 h after the pain-related behavior test at 7, 14, and 21 days after surgery. For pain-related behavior, the acetone test was performed 30 min after the von Frey test, and for anxiety-like behaviors the elevated plus maze (EPM) test was conducted 10 min after the open field test (OFT).
Von Frey test for mechanical allodynia
We performed a less stressful and more precise method of mechanical allodynia testing. All experiments were carried out in a quiet room under a soft light between 9 a.m. and 6 p.m., and the room temperature was kept at 25 °C throughout the behavioral testings. The ipsilateral hairs in the V2 and V3 measured regions of the mice were removed using a hair clipper (HC1066, Philips, Netherlands). The mice were then habituated to a box (8 cm × 8 cm × 10 cm) made of black wire mesh for 30 min per day for 3 days. A series of von Frey filaments (0.07 g, 0.16 g, 0.4 g, 0.6 g, 1.0 g, 1.4 g, and 2.0 g; Stoelting, USA) were applied to the skin in the V2 or V3 area. Each von Frey filament was applied five times at intervals of a few seconds. A period of up to 6 s was set as the cut-off time to avoid tissue damage. A quick withdrawal of the head after the filament became bent was defined as a response, and the size of the filament at which three positive responses were seen out of five stimulations was defined as the pain threshold.
Acetone test for cold allodynia
A 50 μl drop of 90% acetone (diluted in distilled water) was applied to the ipsilateral V3 skin through a 25-gauge needle attached to a 1 mL microsyringe (Hamilton, Reno, NV). Special care was taken to avoid touching the skin and to avoid acetone leakage. The animals were habituated for 3 consecutive days and then baseline values were tested. The total orofacial wiping time was obtained within a cutoff time of 2 min. Cold allodynia was considered present if the wiping time after exposure to acetone was increased compared to basal values or to sham-operated animals. Because of the difficulty in avoiding acetone being sprayed into the eyes, we did not administer acetone to the V2 area, i.e. only the secondary cold allodynia but not the primary cold allodynia was tested.
Elevated plus maze test
The EPM test was performed according to a previous study [46]. The maze consisted of four arms (5 cm × 30 cm), including two closed arms having 20-cm high walls and two arms left open (open arms). The maze was elevated 40 cm above the floor. Light intensities in the central area and the opened and closed arms were set to 15 lx, 15 lx, and 5 lx, respectively, and the temperature was controlled at 22° ± 1 °C. Mice were placed in the center of the maze facing an open arm and allowed free access to the four arms for 5 min. The central platform was 6 cm × 6 cm, the two open arms were 30 cm × 6 cm, and the two closed arms were 30 cm × 6 cm × 20 cm. A video tracking system and software (Shanghai Mobile Datum Information Technology Company, Shanghai, China) was used to calculate the percentage of open-arm distance ([open distance]/[total distance] × 100), open-arm entries ([open entries]/[total entries] × 100), and open-arm time ([time in open arms]/[time in total arms] × 100).
Open field test
The OFT was administrated as described previously [46]. In brief, the mice were individually placed into the center of an open box apparatus (50 cm × 50 cm × 40 cm) with a black floor. The size of the center zone was set at 25 cm × 25 cm. After freely exploring for 5 min, the time spent in the center square and the total and central distances traveled were recorded and analyzed. Results were expressed as the time spent in the center square, the distance traveled in the center square, and the percentage of the center distance ([center distance]/[total distance] × 100). The experiment was performed under controlled conditions (22° ± 1 °C, dim light (15 lx)).
Virus injection and chemicogenetic inhibition of LHb neurons
For overexpression of Tacr3 in the LHb, mice were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally (i.p.)) and placed into a stereotaxic apparatus. The full-length coding sequence of mouse Tacr3 (NM_021382) in the AAV2/9 vector (pAAV-CMV-EGFP-2A-Tacr3-3FLAG, H12378) and the CaMKII-expressing neuron-targeting virus (pAAV overexpressing vector (AOV)-CaMKII-HM4D(Gi)-EGFP-3FLAG, H14324) were designed and purchased from OBIO Technology Co., Ltd. (Shanghai, China). A total volume of 150 nl virus (pAAV-CMV-EGFP-2A-Tacr3-3FLAG; ~ 1012 infectious units per ml) was injected into the unilateral or bilateral LHb at bregma (− 1.62 mm), midline (±0.45 mm), and the skull surface (− 2.75 mm). For chemicogenetic inhibition of CaMKII-expressing neurons in the LHb, mice were unilaterally or bilaterally injected in the LHb with 150 nl virus (pAOV-CaMKII-HM4D(Gi)-EGFP-3FLAG; ~ 1012 infectious units per ml). Mice were allowed 3 weeks for recovery and transgene expression. Mice were then injected with saline or clozapine-N-oxide (CNO, 2.5 mg/kg, Sigma, i.p.), and behavior was tested 45–55 min post injection. Chemicogenetics uses viral vector–mediated expression of the inhibitory muscarinic M4 receptor–based Gi-coupled DREADD (designer receptor exclusively activated by designer drug) hM4D(Gi) under the control of a certain promoter [43]. Gi signaling activates inward rectifying potassium channels, resulting in hyperpolarization and inhibition of neurons, and DREADD can be activated by the pharmacologically inert molecule CNO [43]. CaMKII acts as the promoter of excitatory neurons. Therefore, the injection of virus (pAOV-CaMKII-HM4D(Gi)-EGFP-3FLAG) into the LHb followed by i.p. injection of CNO at certain time points results in silencing of excitatory neurons (glutamate neurons) in the LHb.
Immunofluorescence
Immunofluorescence was used to verify viral expression in the unilateral and bilateral LHb. Mice were first anesthetized with sodium pentobarbital (100 mg/kg, intraperitoneally (i.p.)) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS) (pH 7.4) followed by ice-cold 4% paraformaldehyde in 0.1 M PBS. Mouse brains were dissected out and post-fixed at 4 °C overnight. The brains were transferred to a 20% sucrose solution for 24 h followed by a 30% sucrose solution for 48 h at 4 °C. A freezing microtome (Leica 2000, Germany) was used to cut the brain into 30 μm thickness. The slices were then washed three times using 0.3% Triton X-100 and cover-slipped. The cell nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount-G® (0100–20, Southern Biotech, Birmingham, AL, USA). Because EGFP is spontaneously fluorescent, the slices did not need to be stained with primary and secondary antibodies. A multiphoton laser point scanning confocal microscopy system (FV1000, Olympus, Tokyo, Japan) was used to obtain the images.
Western blotting
Radio immunoprecipitation assay lysis buffer was used to homogenize the LHb sample on ice, and then the samples were centrifuged at 12,000 × rpm for 20 min. The Pierce bicinchoninic acid (BCA) kit (Thermo Scientific, Rockford, IL) was used to measure the protein concentration in the supernatant. The samples were boiled with protein loading buffer, and 30 μg total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk in TBST (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature. Membranes were incubated with primary antibodies against p-CaMKII (Thr286, 1:1000 dilution, SC-12886-R, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (1:10,000 dilution, 51,332, Proteintech, Manchester, UK) at 4 °C overnight. The PVDF membranes were washed using TBST and incubated with the HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:10,000 dilution, SA00001–2, Proteintech) for 1 h at room temperature. An ImageQuant LAS4000 mini image analyzer (GE Healthcare, UK) was used to capture the images, and the Quantity One Analysis Software (Version 4.6.2, Bio-Rad Laboratories, Hercules, USA) was used to quantify the band intensities.
Microarray analysis and real-time PCR (RT-PCR)
Tissue collection
For gene expression profiling (n = 15 mice per group) and RT-PCR validation (n = 8 adult mice per group), mice were anesthetized by i.p. injection of sodium pentobarbital (50 mg/kg) on day 21 after pT-ION surgery. The LHbs were removed and soaked in RNAlater (R0901, Sigma, USA) and then snap-frozen in liquid nitrogen and kept at − 80 °C. Fifteen Hbs were pooled for tissue sampling, whereas for RT-PCR validation two Hbs were used for the final tissue sample.
Microarray analysis
Trizol reagent (Invitrogen) and the mirVana micro RNA (miRNA) Isolation Kit (Ambion, Austin, TX, USA) were used to extract total RNA from the mouse LHbs in both the sham and pT-ION groups according to the manufacturer’s protocol. A NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was used to determine the RNA yield, and agarose gel electrophoresis stained with ethidium bromide was used to evaluate the RNA integrity. The Agilent Mouse Gene Expression Kit (8*60 K, Design ID:014850) was used in this experiment, and data analysis was performed on the six samples (three samples for each group, with 15 Hbs in each sample). Total RNA was transcribed to double-stranded cDNA, synthesized into cRNA, labeled with Cyanine-3-CTP, and then hybridized onto the microarray. After washing, the Agilent Scanner G2505C (Agilent Technologies) was used to scan the arrays, and the array images were analyzed using the Feature Extraction software (version 10.7.1.1, Agilent Technologies). The raw data were analyzed by Genespring, normalized with the quantile algorithm, and flagged as “Detected”. DEGs were defined as genes with a fold change ≥2.0 and a p-value < 0.05.
Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses
The Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/home.jsp) was used to analyze the GO and KEGG pathways. These data were Log2 transformed and median centered using the Adjust Data function of the R package gplots. Hierarchical clustering using the R package average linkage was then performed, and DAVID assigned these genes into relevant GO biological pathways and KEGG molecular pathways. Related and significant GO biological pathways were identified by EASE score p-values < 0.01, and significant KEGG molecular pathways were identified by EASE score p-values < 0.05. Higher enrichment and gene counts indicated more important pathways. Finally, tree visualization was performed using Java Treeview (Stanford University School of Medicine, Stanford, CA, USA).
Correlation and coexpression analysis
The Search Tool for the Retrieval of Interacting Genes (STRING) database and Pearson’s correlation coefficient were used in the gene-gene interaction and coexpression analysis. In the present study, the interaction of the DEGs was screened using the STRING 10.5 online tool (http://string-db.org). The DEGS with a combined score ≥ 0.2 were selected, and Cytoscape 3.6.1 was used to construct and visualize the gene-gene interaction network.
RT-qPCR validation of regulated genes
We performed RT-qPCR to validate that the Tacr3 expression was changed only in the LHb and not in other negative emotion-related brain areas including the ACC, PFC, and hippocampus. Total RNA from mouse LHb, ACC, PFC, and hippocampus was isolated using Trizol (Sigma), and the Prime Script RT Kit (Takara) was used for reverse transcription. The Takara SYBR Green reagents were used for RT-qPCR with the Applied Biosystems 7300 plus Detection System according to the manufacturer’s instructions. The sequences of the primers used for the RT-qPCR were as follows: Tacr3 Forward: TTC ATT CTC ACT GCG ATC TAC CA, Reverse: GCC TGC ACG AAA TCT TTT GTT CA; Mylk3 Forward: ACC ATG TAC TGA CTA CAG GAG G, Reverse: CCA CTG TTC GCA CAG GTA TGT; Itpka Forward: ACT GGC AGA AGA TCC GTA CCA, Reverse: CCG GCA GCT TTG AAA CTC C.
Brain slice electrophysiology
Brain slice preparation
Mice were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and intracardially perfused with ~ 20 ml ice-cold oxygenated modified artificial cerebrospinal fluid (ACSF) that contained 93 mM N-methyl-d-glucamine (NMDG), 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2, 10 mM MgSO4, and 3 mM glutathione (GSH). The pH of the ACSF was 7.3–7.4, and the osmolarity was 300–305 mOsm·kg− 1. Coronal slices (300 μm) that contained the LHb were sectioned at 0.18 mm·s− 1 on a vibrating microtome (VT1200s, Leica). The brain slices were initially incubated in NMDG ACSF for 10–15 min at 33 °C, followed by HEPES ACSF that contained 92 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 2 mM CaCl2, 2 mM MgSO4, and 3 mM GSH (pH 7.3–7.4, osmolarity 300–305 mOsm·kg− 1) for at least 1 h at 25 °C. The brain slices were transferred to a slice chamber for electrophysiological recording and were continuously perfused with standard ACSF that contained 129 mM NaCl, 2.4 CaCl2, 3 mM KCl, 1.3 mM MgSO4, 20 mM NaHCO3, 1.2 mM KH2PO4, and 10 mM glucose (pH 7.3–7.4, osmolarity 300–305 mOsm·kg− 1) at 3–4 ml·min− 1 at 32 °C. The temperature of the ACSF was maintained by an in-line solution heater (TC-344B, Warner Instruments). The recorders were blinded to the group identity during recording and analysis.
Whole-cell patch clamp recordings
Neurons in the slices were visualized using a 40× water-immersion objective on an upright microscope (BX51WI, Olympus) equipped with infrared-differential interference contrast and an infrared camera connected to the video monitor. Whole-cell patch clamp recordings were obtained from visually identified LHb cells. Patch pipettes (3–5 MΩ) were pulled from borosilicate glass capillaries (VitalSense Scientific Instruments Co., Ltd) with an outer diameter of 1.5 mm on a four-stage horizontal puller (P1000, Sutter Instruments). The signals were acquired via a Multiclamp 700B amplifier, low-pass filtered at 2.8 kHz, digitized at 10 kHz, and analyzed with Clampfit 10.7 software (Molecular Devices). If the series resistance changed more than 20% during the recording, the experimental recording was immediately terminated.
Synaptic transmission
Neurons were held at − 70 mV using the voltage clamp mode for recording spontaneous excitatory postsynaptic currents (sEPSCs). The pipettes were filled with intracellular solution that contained 0.5 mM EGTA, 10 mM HEPES, 125 mM K-Gluconate, 15 mM KCL, 10 mM Na2-phosphocreatine, 2 mM Mg-ATP, and 0.5 mM Na-GTP. The osmolarity of the solution was adjusted to 285–290 mOsm·kg− 1 and the pH was adjusted to 7.2 with KOH. The baseline was recorded for at least 5 min with standard ACSF. Strychnine (5 μM) and bicuculline (10 μM) were added to the standard ACSF to eliminate inhibitory components, and the slices were incubated in this drug solution for at least 10 min before the experiments. The recording of sEPSCs lasted at least 5 min.
Statistical analysis
All data are shown as the mean ± SEM. For the pain-related experiments, differences between groups for the von Frey and acetone tests were determined using two-way repeated-measures ANOVA (listed in Supplementary Table S1) followed by Tukey’s multiple comparison test. For the anxiety-like behaviors, differences between groups in the EPM and OFT were determined using one-way ANOVA (listed in Supplementary Table S2) followed by Dunnett’s post hoc multiple comparison test. For the RT-PCR and western blotting results, group differences were determined using one-way ANOVA (listed in Supplementary Table S2) followed by Dunnett’s post hoc multiple comparison test. For the electrophysiological experiments and other comparisons between two groups, differences were determined using unpaired Student’s t-test (listed in Supplementary Table S3). p < 0.05 was considered as the threshold of significance in all tests. All statistical analyses were performed using GraphPad Prism 6.0 software (San Diego, CA, USA).
