Generation of transgenic mice
The dominant-negative pore mutation of rat KCNQ2 was generated by mutating the residue 279-Gly to Ser (rQ2-G279S). rQ2-G279S cDNA was subcloned into a 265-plasmid with a forebrain-specific promoter of αCaMKII and an SV40 poly A. The αCaMKII promoter is specifically expressed in the forebrain and has been widely used for genetic manipulation of region- specific gene expression [25–28]. Transgenic mice of the B6D2F1/Crl strain were generated by pronuclear microinjection of linearized rQ2-G279S DNA in fertilized eggs. Transgenic mice were selected using genomic PCR with transgene-specific primers (upstream 5'-GCT AGA GGA TCT TTG TGT AAG GAA C-3', downstream 5'-GGA AAG TCC TTG GGG TCT TCT ACC-3'). Expression of the transgene coding for mutant rQ2-G279S in the forebrain was verified using real-time RT PCR and in situ hybridization.
Female mice (both rQ2-G279S transgenic and wild type) of 8-12 weeks age were used in this study. In one experiment, ICR mice from the Laboratory Animal Center, Peking University Health Science Center were also used. Mice were housed in groups (two to five per cage) under a controlled temperature (23 ± 2°C) and humidity (50 ± 5%) environment with ad libitum access to food and water. Animals were maintained on a reverse 12 h/12 h light/dark cycle (lights on at 7:00 AM and off at 19:00 PM). The animal experimental protocols were approved by the Animal Use and Care Committee of Peking University and were consistent with the Ethical Guidelines of the International Association for the Study of Pain.
The Kv7/KCNQ channel opener retigabine or the blocker XE991 (Tocris, UK) was dissolved in a mixture of tween-80 (Sigma, St. Louis, MO, USA) and saline (0.9% NaCl, autoclaved before use) at a ratio of 1:9 (v/v). For intracerebroventricular (i.c.v) injection, drugs were dissolved in DMSO and diluted with saline at a ratio of 1:1 (v/v). Drugs were diluted to desired concentrations one day before experiments and were stored at -20°C. Drug solutions were administered to mice intraperitoneally at a volume of 10 ml per kilogram body weight. Injection of drug solutions and behavior testing were conducted on the basis of a double-blind and randomized manner, in which one experimenter carried out drug injection and randomized division of mice, and another experimenter who was blinded to drug administration and mouse groups, conducted measurement of pain response.
Patch clamp recordings of brain slices
Brain slices were transferred to a small volume (~0.5 ml) recording chamber that was mounted on a fixed-stage, upright microscope (Nikon, Japan) (IR-DIC) optics. The recording chamber was superfused with ACSF containing (mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 1.5 MgCl2, 1.0 CaCl2, 16 glucose, 1.3 Na L-ascorbate, 0.6 Na-pyruvate and saturated with 95% O2 - 5% CO2 at a flow rate of ~2-3 ml/min. In some current-clamp experiments, 10 μm DNQX, 50 μm APV and 10 μm Gabazine were added to the extracellular medium to block spontaneous synaptic transmission. Patch electrodes were fabricated from filamented, thick-walled borosilicate glass pipettes and heat-polished to a resistance of 3~4 M when filled with an internal solution consisting of the following (mM): 130 K-gluconate, 10 HEPES, 0.6 EGTA, 5 KCl, 3 Na2ATP, 0.3 Na3GTP, 4 MgCl2, 10 Na2-phosphocreatine; pH adjusted to 7.25 with KOH. The final osmolarity of the solution was ~290 mOsm. Liquid junction potential (~7 mV) was not corrected. Recordings were made using a Multiclamp 700B amplifier (Molecular Devices, Union City, CA) operating in either voltage-clamp or current-clamp mode on the soma of CA1 pyramidal neurons. All recordings were acquired at 5 or 10 kHz with a Digidata 1440A interface (Molecular Devices) in conjunction with a PC, and filtered at 1 or 2 kHz, respectively, with a low-pass Bessel filter. Stimulus generation and data acquisition were performed using pClamp10 (Molecular Devices). The series resistance was 10-40 MΩ in whole-cell current clamp recording. Cells with a stable resting membrane potential and stable AP amplitudes (> 110 mV) were used, and all recordings were performed at room temperature (22°C ± 2).
Locomotion was measured with the Animal Locomotor Video Analysis System (JLBehv-LAG-8, Shanghai Jiliang Software Technology Co. Ltd, China), which consists of eight identical black plexiglass chambers (30 ' 30 ' 65 cm) in light- and sound-controlled cubes. Each chamber was equipped with a video camera (winfast vc100) on the top. All cameras were connected to a PC computer for recording video files of movement of mice in chambers (n = 7 per genotype). The locomotor activity of each mouse was analyzed with the DigBehv analysis software (Shanghai Jiliang Software Technology Co. Ltd, China) and expressed as a total distance (cm) traveled in the period of 30 min .
Behavioral responses and tests for visceral and cutaneous pain
Visceral pain induced by injection of capsaicin into the mouse colon
The chemical capsaicin was purchased from Sigma, and dissolved in 10% ethanol, 10% Tween 80 and 80% saline (according to 0.1% w/vol), and this solution of 10% ethanol, 10% Tween 80 and 80% saline was used as the vehicle. Mice were habituated to the glass observation chambers for 30 min before experiments. Petroleum jelly (Vaseline) was applied in the perianal area to avoid stimulation of somatic areas by contact with the irritant chemicals. 50 μl of chemical solution was administered by introducing a fine cannula with a rounded tip (external diameter 0.61 mm; 4 cm long) into the colon via the anus . Spontaneous behavior was then observed directly over a period of 20 min. Postures defined as pain-related behavior were (i) licking of the abdomen, (ii) stretching the abdomen, (iii) squashing of the lower abdomen against the floor and (iv) abdominal retractions.
Visceral pain induced by i.p. injection of acetic acid or magnesium sulfate
After habituation in the glass observation chambers for 30 min, mice were given intraperitoneal (i.p.) injections (10 ml/kg) with 0.3%, 0.6%, or 1% acetic acid (n = 7-10 per genotype per concentration), and the number of lengthwise abdominal constrictions ("writhes") were counted over 15 min . The concentration of magnesium sulfate was 12 mg/kg (n = 10 per genotype), and the number of writhes were counted over 10 min [23, 44]. To test the antinociceptive effect of retigabine on visceral pain, mice received an intraperitoneal injection of vehicle or retigabine (7.5 mg/kg) or retigabine (7.5 mg/kg) co-injected with XE991 (1 mg/kg) at volume of 10 ml/kg, 20 min prior to 1% acetic acid injection (n = 8-13, per group). The vehicle used in this study was a mixture of tween-80 (Sigma, St. Louis, MO, USA) and saline (0.9% NaCl, autoclaved before use) in a ratio of 1:9 (v/v).
While lightly restrained in a cloth holder, the distal half of mouse's tail was dipped into a bath of water thermostatically controlled at 47.0°C or 49.0°C (± 0.1°C) (n = 7-11, per genotype). The latency to respond to the heat stimulus by vigorous flexion of the tail was measured. Mice were tested for twice, and two latency determinations (separated by > 60 s) were measured and averaged .
Hot plate test
Mice were brought to testing room and allowed to acclimatize for 10 minutes before the test begins. Pain reflexes in response to a thermal stimulus were measured using a Hot Plate Analgesia Meter. The surface of the hot plate was heated to a constant temperature of 53°C (± 0.2°C). Mice were placed on the hot plate (25.4 cm × 25.4 cm), which is surrounded by a clear acrylic cage (19 cm tall, close top), and the Start/Stop button on the timer is activated. The latency to respond to hindpaw flick or lick was measured to the nearest 0.1 seconds by deactivating the timer when the response was observed. The mouse was immediately removed from the hot plate and returned to its home cage. If a mouse did not respond within 60 seconds, the test was terminated and the mouse was removed from the hot plate. Animals were tested one at a time and were not habituated to the apparatus prior to testing. Each animal was tested only once, because repeated testing can lead to systematic latency alterations [44–46].
After habituation to individual glass observation chambers, mice were given a subcutaneous injection of 5% formalin into the plantar right hindpaw (25 μl volume) and observed for 60 min after the formalin injection. The presence of right hindpaw licking/biting was recorded. The acute phase of formalin test was defined as 0-10 min after injection and the tonic phase as 10-60 min after injection. Data are presented as the time spent in each phase in which licking/biting was detected .
Intracerebroventricular (i.c.v.) injection
Intracerebroventricular (i.c.v.) injections were made directly into the lateral ventricle (1 mm lateral and 0.4 mm caudal to the bregma at a depth of 2.5 mm) according to modification of a previously described method . For i.c.v. injection, wild type mice were lightly anesthetized with ethylether. XE991, retigabine or vehicle (50% DMSO in saline) in 5 μl was injected directly into the lateral ventricle 15 min before 0.6% acetic acid (i.p.) injection. After behavioral evaluation, mice were sacrificed for examination of accuracy of the site of i.c.v injection. The dose of XE991 used was 30 nmol or 10 nmol, and the dose of retigabine was 49 nmol or 16 nmol based on our previous study .
Tissue Preparations and Nissl straining
Adult mice were deeply anesthetized and transcardially perfused with normal saline followed by 4% paraformaldehyde. Brains were removed and post-fixed overnight at 4°C following submersion in 20% and 30% sucrose in 0.1 M phosphate buffer. Coronal sections were cut on a cryostat (Leica CM1900) into a series of 30 μm thick sections and mounted on glass slides for Nissl straining. For of c-Fos immunohistochemistry staining, 20 μm thick sections were cut and stored at -20°C in a solution containing 30% (v/v) ethylene glycol, 30% (v/v) glycerol, and 0.1 M sodium phosphate buffer. For Nissl straining, brain slides were stained with Nissl substance at 37°C for 1 hour and then rinsed with water, dehydrated in alcohol series and xylene, before coversliped with resinous mounting medium .
Free-floating sections were rinsed in 0.1 M PBS (pH7.4), incubated for 30 min in PBS containing 3% H2O2 at 37°C, and rinsed three times for 5 min each time in PBS. After 60 min incubation in 0.3% Triton X-100 in PBS containing 12.5% goat serum, these brain sections (n = 3-4) were incubated overnight at 4°C with c-Fos primary antibody (1:1000; Santa Cruz Biotechnology). Sections were rinsed for 5 min in PBS (three times) and incubated for 60 min with secondary antibody (Zhongshan Golden Bridge Biotechnology company, Beijing, China). After rinsing three times in PBS, the reaction was detected using 3, 3'-diaminobenzidine (DAB). Analysis of c-Fos staining was carried out by an observer who was blinded to the genotype and drug treatment of the mice. Cells containing a nuclear brown-black reaction product were considered to be c-Fos-positive. All c-Fos-positive cells that were distinguishable from background staining were counted in each region of interest within a defined area (0.01 mm2) .
In situ hybridization
PCR primers used for amplifying the transgene vector SV40 probe were as follows: upstream primer 5' GAGATGTGGCTTGGGCAG 3' and downstream primer 5' CATTCATCAGTTCCATAGGTT 3'; and the PCR product was 248 bp in size. Using the Gel DNA Extraction kit (Tiangen Biotech, Beijing, China), the resulting PCR product was extracted from the gel and subcloned into the pSPT18 vector with T4 ligase (Takara). The identity of the plasmid was confirmed by DNA sequencing. The plasmid was linearized with HindIII or EcoRI and used for RNA transcription with T7 or Sp6 RNA polymerase to generate antisense or sense probes, respectively, in the presence of digoxigenin-labeled rUTP (Roche Diagnostics). Deparaffinized and dehydrated paraffin-embedded tissue sections (5 μm) were incubated in 0.1 M HCl for 10 min, and treated with Proteinase K (20 μg/ml in TBS which contains 2 mM CaCl2) for 20 min at 37°C. The slides were cooled to room temperature, washed with TBS (150 mM NaCl, 50 mM Tris, pH7.5), fixed in 4% paraformaldehyde for 10 min, and hybridized overnight at 47°C with the SV40 cRNA probe. After hybridization, sections were washed in 2xSSC plus 50% formamide for 30 min and 2xSSC twice for 15 min (37°C). The samples were incubated with antidigoxigenin antibody conjugated with alkaline phosphatase (dilution 1:500; Roche) for 1h. 5-Bromo-4-chloro-3-indolyl phosphate and nitro-blue-tetrazolium (Roche) were used for the color reaction .
Real time RT-PCR
60 μg of RNA extracted from mouse brain were digested with 6 μl Turbo DNase for 1 hour. cDNA was synthesized using oligo dT and M-MLV reverse transcriptase according to the manufacturer's instructions (Invitrogen). The PCR amplification mixtures (20 μl) contained 1 μl template cDNA, 10 μl 2 × SYBR® Premix Ex TaqTM (Takara) containing SYBR Green and 5 μM forward and reverse primers. The sequences of these primers (for β-actin, upstream 5'-TGT TAC CAA CTG GGA CGA C-3', downstream 5'-GGT GTT GAA GGT CTC AAA CAT-3'; for rQ2-G279S, upstream 5'-CTC TGC CAC TGG TGA AGG-3', downstream 5'-TGT AGG TAG TTT GTC CAA TTA TGT C-3', for genomic DNA primers, upstream 5'-CCC ATA CAC CTC CTC TGA A-3', downstream 5'-CTG AAG TGT CTA CCC TTA CGG-3') were designed using Oligo software. The product of the rQ2-G279S primers used to amplify the KCNQ2 mutant gene within SV40 poly A was 160 bp. The genomic DNA primers located in the 265-plasmid after SV40 poly A were used to detect any contamination of transgenic genomic DNA with product length of 148 bp. β-actin was used as an internal reference and the product length was 163 bp. Amplification was carried out using the ABI PRISM 7500™ with cycling conditions as follows: there was an initial denaturation step at 95°C for 10 s, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s with fluorescent detection at 60°C. Melting curve analysis was performed from 60°C to 95°C in 1°C step. Results were analyzed using the 2-ΔΔCt method to compare expression of genes of interest with that of β-actin .
Statistical analysis was performed with GraphPad Prism (the 5th edition) and SPSS 13.0 for Windows. All data are presented as mean ± s.e.m. Statistical significance between multiple groups was examined by the Student's t test or one-way or two-way ANOVA with an appropriate post hoc test. A value of p < 0.05 was considered to be statistically significant.