Adult male C57BL/6, GluA1 serine-831 and serine-845 phosphorylation site mutant (s831A and s845A) mice were used. Animals were randomly housed under a 12-h light–dark cycle (9 a.m. to 9 p.m. light), with food and water freely available, at least one week before carrying out experiments. All procedures involving animals were under the guidelines of the Fourth Military Medical University, Xi’an Jiaotong University, Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, University of Toronto, National Institute for Physiological Sciences and Johns Hopkins University.
Nerve injury model
A model of neuropathic pain was induced by the ligation of the common peroneal nerve (CPN) as described previously [21, 22]. Briefly, mice were anesthetized by an intraperitoneal injection of a mixture saline of ketamine (0.16 mg/kg) and xylazine (0.01 mg/kg). The CPN was visible between the anterior and posterior groups of muscles, running almost transversely. The left CPN was slowly ligated with chromic gut suture 5–0 until contraction of the dorsiflexor of the foot was visible as twitching of the digits. The skin was then sutured and cleaned. Sham surgery was conducted in the same manner, but the nerve was not ligated. The mice were used for behavior and/or electrophysiological studies on postsurgical days 7.
The procedure for retrograde tracer injection into the spinal cord (in the same time with CPN ligation or sham surgery) or ventral striatum (4 days after CPN ligation or sham surgery) was according to our previous works [67, 68]. The anesthetic mice were fixed on a stereotaxic frame. For the spinal cord injection, the skin between scapulas was incised and paravertebral muscles were cut off and vertebral plate of the fourth cervical vertebra was exposed. The vertebral plate was removed and the intumescentia cervialis was exposed. Then 4% FG (For FG immunostaining), 0.25% DiI or 10% Alexa-488 Dextran (for whole cell patch recording) distilled in saline solution was unilaterally pressure-injected (0.1 μl) into the C4-5 spinal cord with a Hamilton microsyringe attached with a glass micropipette (tip outer diameters ranged from 10–20 μm). Those mice were allowed to survive for one week before continuous immunostaining or whole cell patch experimental procedures. For ventral striatum (VS) injection, the skull was exposed, and a hole was drilled through the skull over the VS (0.38 mm anterior to bregma, 2.0 mm lateral to the midline and 4.5 mm ventral to the surface of the skull for the VS). 4% FG, 0.25% DiI or 10% Alexa-488 Dextran was unilaterally and iontophoretically injected (3-5 μA pulsed, 7 sec on/off) for 25 min. Those mice were allowed for three days survive before immunostaining or whole cell patch experimental procedures.
Anterograde labeling for Pha-L
The procedures for anterograde tracer phaseolus vulgaris leucoagglutinin (Pha-L; Vector Laboratories, Burlingame, CA) injection were essentially the same as described by our group previously . Briefly, The anesthetic mouse was fixed on a stereotaxic frame and Pha-L was iontophoretically injected into unilateral deep layers of the ACC according to the atlas of the mouse brain (0.98 mm anterior to Bregma, 0.35 mm lateral to the midline and 1.8 mm deep from cerebral surface). Pha-L was dissolved in a mixture of 0.05 M Tris–HCl buffer and 0.5 M KCl (pH 7.6) to a final concentration of 2.5% (W/V). The driving current (positive, 3–5 μA, 7 s on/off) was delivered for 25 min. After injection, the surgical wounds were carefully sutured. Mice were allowed to survive for approximately 2 weeks before perfusion. To examine the Pha-L injection site and the distribution of anterogradely Pha-L-labeled fibers and terminals in the spinal cord, coronal sections containing ACC and sagittal sections containing spinal cord of cervical enlargement were incubated overnight with primary antibody goat-anti-Pha-L (1:500, Vector Laboratories, Burlingame, CA) in the 5 mM sodium phosphate (pH 7.4)-buffered 0.9% saline (PBS) containing 0.3% Triton X-100, 0.12% lambda-carrageenan, 0.02% sodium azide and 1% donkey serum. On the following day, the sections were incubated in the same dilution solution containing biotinylated anti-goat IgG (Vector Laboratories, 1:200) for 4 hours. They were then incubated in an ABC complex (Vector Laboratories, 1:200) for another 90 min. Subsequently, the sections were treated with 50 mM Tris–HCl buffer (pH 7.5) solution that containing 0.02% diaminobenzidine (DAB), 0.015% H2O2 and 0.04% NiCl2 to intensify DAB-based reaction for 5–15 min. After the reaction, the sections were mounted onto gelatin-coated glass slides, dehydrated and coverslipped. To better reveal the site of ACC, sections containing injection site were further counterstained with Nissl staining. Sections were observed under a light microscope (AH-3; Olympus, Tokyo, Japan).
Anterograde labeling for lentivirus-assistant rabies virus
To assist the rabies virus–mediated specific labeling of ACC neurons, a lentivirus plasmid expressing TVA and mkate2 was constructed by sub-clone the fusion fragment TVA:2A:mkate2 into the plasmid FUGW (Addgene 14883). This plasmids Lenti-TVA-mKate was packaged in 293-T cells by co-transfection with pMDL g/p RRE and pMD2.G. At 48 and 72 hours post transfection, the supernatant was collected and concentrated into 1000-fold through high speed concentration . The final titer of the Lenti-TVA-mKate is 3×107 infecting unit per milliliter.
The rabies virus (RV) and the cell lines for rabies propagation and tittering were kindly supplied by Callaway, E. M and prepared in our laboratory as previously described . Briefly, RV-G pseudo typed SAD19-ΔG-mcherry was propagated in B7GG cells, and the supernatant was harvested with a titer of 105 infecting units/ml. To produce the EnvA-pseudotyped rabies, a Bhk-Enva cell was infected with filtered (0.45 μm, Millipore) RV-G-SAD19-ΔG-mcherry (EnvA-RV-mcherry). At six hours post infection, the Bhk-Enva cell were digested with 0.25% trypsin(Hyclone)to eliminate the contamination of RV-G pseudo typed rabies. During harvest of EnvA-RV-mcherry, the filtered supernatant was 2000–3000 fold concentrated through two cycles of high speed concentration as previous described . The concentrated aliquots were tittered in 293 t-tva800 cell line. The final titer of EnvA-RV-mcherry was 2x108 infecting units per milliliter. All aliquots were stored at -80°C.
Virus anterograde tracing
Virus tracing works were performed in a BSL II animal facility. To label the ACC neurons, we first micro-injected 200 nl of the VSV-G pseudotyped Lenti-TVA-mKate into unilateral deep layers of the ACC as same as the Pha-L injection site. Four days post the infection of lentivirus, 400 nl of the EnvA-RV-mcherry was microinjected into the ACC. One week after rabies infection, mice were deeply anesthetized and transcardially perfused. Coronal brain slices containing the ACC and sagittal spinal slices containing cervical enlargement were cut with a thickness of 40 μm and collected serially. For immunohistochemistry of the spinal sections, the free-floating sections were washed in 0.1 M phosphate buffered saline (PBS) solution for 3 × 5 min, followed by an incubation with 10% normal goat serum in PBS solution for 1–1.5 hour. Sections were then incubated overnight in a rabbit polyclonal anti-RFP (Abcam, ab62341, 1:500) followed with an FITC conjugated anti-rabbit serum (1:200) for 4 hour at 4°C.
For fluorescent imaging of the brain sections labeled by rabies virus or immunostained spinal sections, the sections were washed with PBS, and wet mounted directly on Vecta-Shield mounting medium (brain sections were counterstained with DAPI), sealed with nail polish, imaged with an upright fluorescence confocal microscopy (Leica TCS SP8).
Immunohistochemistry for Fos and FG
Seven days after making the nerve injury model, mice were anaesthetized and perfused with 0.1 mol/L PBS (pH 7.2–7.4) via the ascending aorta followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4). The spinal cord and brain were then removed, and cryoprotected in 0.1 M PB containing 30% sucrose overnight at 4°C. Transverse sections (30 μm thickness) of spinal cord and brain samples were cut on a freezing microtome and collected serially and seperated as three sets of sections. Sections containing cervical spinal cord and ventral striatum were collected for injection sites imaging.
One set of sections containing ACC was used for Fos and FG immunostaining according to our previous works [21, 72]. In brief, sections were sequentially incubated with the following solutions: (1) PBS solution of 3% bovine serum, 0.3% Triton X-100 (PBS-TX) containing mouse antisera against Fos (1:500, ab11959, Abcam) and rabbit antisera against FG (1:500, AB153, Millipore) for 2 days at 4°C, (2) an Alexa-594 conjugated anti-mouse (1:200, Invitrogen) and Alexa-488 conjugated anti-rabbit (1:200, Invitrogen) antibody in PBS-TX for 24 hrs at 4°C. Sections were then rinsed in PBS, mounted onto glass slides, air dried, cover-slipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine in 0.01 M PBS. The signals were visualized under confocal microscope (FV-1000; Olympus, Tokyo, Japan) under appropriate filter for Alexa-488 (excitation 495 nm; emission 519 nm) and Alexa -594 (excitation 590 nm; emission 617 nm). For obsevation of the FG/Fos neurons, a careful focusing through the thickness of all sections determined that the immunolabeling had penetrated the whole thickness of the sections and only the neuronal cell bodies with obvious light emission were counted. Since the light from some positive neurons might be too weak to detect, the numbers of Fos-ir neurons and/or FG labeled neurons in Tables 1 and 2 should be regarded as representing the minimum of the real positive neurons in the sections. In addition, to avoid possible double counting of positive neurons the sections were carefully moved across the stage and analyzed from left to right.
One set of sections containing ACC was used for FG immunostaining and Nissl counterstaining. Sections were sequentially induced with (1) rabbit antisera against FG (1:500) for 2 days at 4°C, (2) biotin conjugated goat anti-rabbit antibody (1:200, Millipore) for 24 hrs at 4°C, (3) ABC elite kit (1:100) for 2 hrs. Finally, the sections were reacted with 0.05 M Tris–HCl buffer (pH 7.6) containing 0.04% DAB (Dojin) and 0.003% H2O2 for visualizing FG-like immunoreactive neurons. Then the sections were mounted onto gelatin-coated glass slides and processed for standard Nissl staining.
Another set of sections were used for control staining. The primary antibodies were omitted or replaced with normal rabbit/mouse serum and the other procedures were the same as those for the first 2 sets of sections in all groups. No staining was observed on brain sections when the primary antibody was omitted or replaced from the protocol.
Whole-cell patch-clamp recordings
Coronal brain slices (300 μm) at the level of the ACC were prepared using standard methods1,9,11. Slices were transferred to a submerged recovery chamber containing oxygenated (95% O2 and 5% CO2) ACSF (124 mM NaCl, 4.4 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 25 mM NaHCO3, 1 mM NaH2PO4, and 10 mM glucose) at room temperature for at least 1 hr and then heated up to 32°C for recording. Evoked EPSCs were recorded from layer V neurons in randomly selected sides of the ACC, with an Axon 200B amplifier, and the stimulations were delivered by a bipolar tungsten stimulating electrode placed in layer II/III of the ACC. AMPA receptor-mediated EPSCs were induced by repetitive stimulations at 0.02 Hz, and neurons were voltage-clamped at -60 mV in the presence of AP5 (50 μM). The recording pipettes (3–5 MΩ) were filled with a solution containing (in mM) 112 Cs-Gluconate, 5 TEA-Cl, 3.7 NaCl, 0.2 EGTA, 10 HEPES, 2MgATP, 0.3 Na3GTP and 5 QX-314 (adjusted to PH 7.2 with CsOH, 290 mOsmol). 0.1 mM spermine was included into the solution when recording AMPA I-V curve. Picrotoxin (100 μM) and AP5 (50 μM) was always present to block γ-aminobutyric acid (A) (GABAA) and NMDA receptor mediated synaptic currents in all experiments. To test the miniature EPSC, tetrodotoxin (1 mM) was added into the ACSF. The initial access resistance was 15–30 MΩ, and it was monitored throughout the experiment. Data were discarded if the access resistance changed >15% during experiment. Data were filtered at 1 kHz, and digitized at 10 kHz.
For recording ACC-spinal cord or ACC-ventral striatum projecting neurons, the ACC sections were observed under FV-1000 confocal microscope under proper filters for DiI (excitation 549 nm; emission 565 nm) or Alexa-488 Dextran Amine. In some cases, Alexa-488 or Alexa-594 was introduced into the recording solution for dual-labeling of the DiI or Dextran retrograde labeled neurons, respectively.
All experiments were carried out as blind to genotype and the conditions of the experiments. Data were collected and processed randomly, and no data points were excluded. No statistical methods were used to predetermine sample sizes, but our sample sizes were similar to those reported in previous publications. Statistical comparisons were made using the unpaired, paired t-test, or two-way ANOVA (Tukey test was used for post hoc comparison). The normal distribution and the variation within each group of data was verified by using Sigmaplot 11.0 software before applying statistical comparison. Analyzed numbers (n) for each set of experiments are indicated in the corresponding figure legends or main text sections. The examples shown in each figure are representative and were reproducible at least three times for each set of experiments. All data were presented as the Mean ± S.E.M. In all cases, p < 0.05 was considered statistically significant.