MBD1 Contributes to the Genesis of Acute Pain and Neuropathic Pain by Epigenetic Silencing of Oprm1 and Kcna2 Genes in Primary Sensory Neurons

Animals.

Mbd1^−/− mice, provided by Dr. Xinyu Zhao (Department of Neuroscience, University of Wisconsin-Madison, Madison, WI), have been bred onto C57B/L6 background for at least 6 generations (X. Zhao et al., 2003) and were bred by crossing the heterozygotes in our facility. Both male and female Mbd1^−/− mice are viable and fertile with normal appearance and locomotor activity (X. Zhao et al., 2003). Male Mbd1^−/− mice and the WT control littermates mice weighing 25–30 g (2–3-months of age) were used for the experiments. All animals were kept in a standard 12 h light/dark cycle, with water and food pellets available ad libitum. All procedures used were approved by the Animal Care and Use Committee at Rutgers State University of New Jersey, and consistent with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. All efforts were made to minimize animal suffering and to reduce the number of animals used. During behavioral testing, all experimenters were blind to viral treatment and/or mouse genotypes.

Animal models.

L4 spinal nerve ligation (SNL) model of neuropathic pain in mice was performed as described previously (Rigaud et al., 2008; Sun et al., 2017; J. Y. Zhao et al., 2017). Briefly, the mice were anesthetized with isoflurane and placed in a prone position. A dorsolateral skin incision was made on the lower back. The L4 transverse process was identified and freed of its muscle attachments and then removed. The underlying L4 nerve root was isolated, ligated with a 7–0 silk suture, and transected just distal to the ligature. Sham animals received an identical surgery of the SNL group, but without the ligature of the L4 spinal nerve. The capsaicin-induced acute pain model in mice was performed as described previously (Cui et al., 2016). After 10 μl of capsaicin (0.1 μg/μl in normal saline/5% ethanol, Sigma-Aldrich, M2028) solution was subcutaneously injected into the plantar side of one hindpaw, the number of shakes and flinches and the duration of lifting of ipsilateral hindpaw were recorded for the first 10 min after injection. Paw withdrawal responses to mechanical and thermal stimuli, as described below, were measured 30 min after capsaicin injection. The CFA-induced inflammatory pain model in mice was performed as described previously (Chu et al., 2005; Cui et al., 2016). A 10 μl of 50% emulsified CFA (Sigma-Aldrich, F5881; dissolved in normal saline) solution was injected subcutaneously into the plantar side of one hindpaw. A 10 μl normal saline was used as a control. The formalin-induced pain model in mice was performed as described previously (Cui et al., 2016). The mice received the injection of 0.6% formalin 10 μl (normal saline was used for control) into the plantar side of one hindpaw. The number of paw flinches and shakes were immediately counted every 5 min for 1 h. The observational session was divided into two phases: the first (0–10 min) and the second (10–60 min) phase. The model of morphine-induced analgesia and tolerance in mice was performed as described previously (Liaw et al., 2008; Liang et al., 2016b). Briefly, for morphine-induced analgesic test, paw withdrawal latency (PWL), as described below, was measured 20 min after subcutaneous injection of 1 mg/kg morphine. For the morphine-induced tolerance, the mice received subcutaneous injection of 10 mg/kg morphine twice daily for 8 d. Tail flick latency, as described below, was measured before morphine injection and 20 min after morphine injection on the mornings of day 1, 3, 5, 7, and 8.

Behavioral analysis.

Mechanical, cold, thermal, tail flick tests and locomotor performance were performed before viral microinjection or surgery and at a different time points after surgery. Each behavioral test was performed at 1 h intervals.

Paw withdrawal frequencies (PWFs) in response to mechanical stimuli (calibrated von Frey filaments) were first measured as described previously (Liaw et al., 2008; Li et al., 2017). Briefly, the animals were placed in an individual Plexiglas chamber on a raised mesh screen. Two calibrated von Frey filaments (0.07 and 0.4 g; Stoelting) were used to stimulate the hindpaw for ∼1 s, and each stimulation was repeated 10 times to both hindpaws with 5 min intervals. Paw withdrawal response in each of these 10 applications was represented as a percentage response frequency [(number of paw withdrawals/10 trials) × 100 = % response frequencies], and this percentage was obtained as an indication of the amount of paw withdrawal.

PWLs in response to noxious thermal stimuli were examined as described previously (Liaw et al., 2008; Li et al., 2017). In brief, the mice were placed in a Plexiglas chamber on a glass plate. A radiant heat from model 336 Analgesic Meter (IITC Life Science) was applied by aiming a beam of light through a hole in the light box through the glass plate to the middle of the plantar surface of each hindpaw. When the animal lifted its foot, the light beam was automatically turned off. The length of time between the start of the light beam and the foot lift was defined as the PWL. For each side, 5 trials with 5 min interval were performed, and setting 20 s cutoff time was used to avoid tissue damage to the hindpaw.

PWLs to noxious cold (0°C) were measured with a cold aluminum plate on ice as previously described (Li et al., 2017). Each animal was placed in a Plexiglas chamber on the cold plate, the temperature of which was monitored continuingly by a thermometer, which was set at 0°C. The duration of time between the placement of the hindpaw on the plate and the animal jumping, with or without paw licking and flinching, was defined as the PWL. Each trial was repeated three times at 10 min intervals for the paw on the ipsilateral side. A cutoff time of 20 s was set to avoid tissue damage.

The tail flick test was performed on an apparatus (model 33B Tail Flick Analgesic Meter, IITC Life Science) with a radiant heat source connected to an automatic timer as described previously (Liaw et al., 2008). A cutoff time latency of 10 s was used to avoid tissue damage to the tail. Tail flick latencies were measured as the time required to induce a tail flick after applying radiant heat to the skin of the tail. The antinociceptive effects were expressed as the percentage of maximal possible analgesic effect (% MPAE): % MPAE = [(response latency − baseline latency)/(cutoff latency − baseline latency)] ×100%.

Locomotor functional activities were examined through three reflex tests as previously described (Li et al., 2017). (1) Placing reflex: The mice were held with the hind limbs slightly lower than the forelimbs, and the dorsal surfaces of the hindpaws were brought into contact with the edge of a table. The experimenters observed whether the hindpaws were placed on the table surface reflexively. (2) Grasping reflex: The mice were placed on a wire grid, and the experimenters noted whether the hindpaws grasped the wire on contact. (3) Righting reflex: The mice were placed on their back on an even surface, and the experimenters recorded whether they immediately assumed the normal upright position. Scores for placing, grasping, and righting reflexes were based on counts of each normal reflex exhibited in five trials.

Conditioned place preference test was performed as our previous described with minor modification (Li et al., 2017). Briefly, an apparatus with two Plexiglas chambers connected with an internal door (Med Associates) was used. One of the chambers was made of rough floor and walls with black and white horizontal stripes on, and another one was composed of smooth floor and walls with black and white vertical stripes on. Movement of the mice and time spent in each chamber were monitored by photobeam detectors installed along the chamber walls and automatically recorded in MED-PC IV conditioned place preference software. Mice were first preconditioned for 30 min to habituate the environment with full access to two chambers. At the end of preconditioning, basal duration time spent in each chamber was recorded within 15 min to check whether mice had a preexisting chamber bias. Animals spending >80% or <20% of the total time in any chamber were excluded from further testing. The conditioning protocol was performed for the following 3 d when the internal door was closed. The mice first received intrathecal injection of saline (5 μl) specifically paired with one conditioning chamber in the morning. At 6 h later, lidocaine (0.8% in 5 μl saline) was given intrathecally paired with another opposite conditioning chamber in the afternoon. Injection order of saline and lidocaine every day was switched. On the test day (at least 20 h after pairing), mice were placed in the chamber with door open so animals can have free access to all chambers. Movement and duration of each mouse spent in each chamber were recorded for 15 min for analysis of chamber preference. Difference scores were calculated as (test time − preconditioning time) spent in the drug chamber.

DRG microinjection.

DRG microinjection was performed as described with minor modification (Liang et al., 2016a; Li et al., 2017). Briefly, a midline incision was made in the lower lumbar back region, and the L₃ and/or L₄ articular processes were exposed and then removed with small forceps. After the DRG was exposed, viral solution (0.5 μl each site) was injected into two sites in the L₃ and L₄ DRGs with a glass micropipette connected to a Hamilton syringe. The pipette was retained for 10 min. After injection, the surgical field was irrigated with sterile saline and the skin incision closed with wound clips. The injected mice displayed no sign of paresis or other abnormalities.

For siRNA injection, Mbd1 siRNA (catalog #sc-35864) and its negative control (NC) siRNA (catalog #sc-44230) were purchased from Santa Cruz Biotechnology. To improve delivery and prevent degeneration of siRNA, TurboFect in vivo transfection reagent (Thermo Fisher Scientific) was used as a delivery vehicle as described previously (Li et al., 2017).

Plasmid constructs and virus production.

Mouse Mbd1 cDNA was synthesized and amplified from the total RNA of mouse DRG using the SuperScript III One-Step RT-PCR System with the Platinum Taq High Fidelity Kit (Invitrogen) and the primers (Table 1). A second PCR step was performed using the Platinum Pfx DNA Polymerase (Invitrogen) and the primers (Table 1). Fragments harboring full-length Mbd1 was ligated into the proviral plasmids (University of North Carolina, Chapel Hill, NC) using the BspEI and NotI restriction sites (New England Biolabs). The resulting vectors expressed the genes under the control of the cytomegalovirus promotor. Recombinant adeno-associated virus Type 5 (AAV5) viral particles carrying the Mbd1 cDNAs were produced at the University of North Carolina Vector Core. AAV5 viral particles carrying enhanced GFP was purchased from University of North Carolina Vector Core. Herpes simplex virus (HSV)-GFP and HSV-Dnmt3a were provided by Dr. Eric J Nestler.

RNA extraction and RT-PCR.

RNA extraction and reverse transcription RT-PCR were performed according to our previously published protocols (Liang et al., 2016a; Li et al., 2017). Total RNA from tissues or culture cells were extracted using miRNeasy Mini Kit (QIAGEN) and reverse-transcribed using the Thermo Script reverse transcriptase (Invitrogen) follow the manufacturer's instructions with either the Oligo(dT) primers or specific reverse primers (Table 1). cDNA was amplified in a Bio-Rad CFX96 real-time PCR system using the primers listed in the Table 1 (Integrated DNA Technologies). GAPDH was used as an internal control. Each sample was run in triplicate in a 20 μl reaction with 250 nm forward and reverse primers, 10 μl of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), and 20 ng of cDNA. The cycle parameters were set as follows: an initial 3 min incubation at 95°C, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Relative changes of mRNA levels were calculated by using the ΔCt method (2^−ΔΔCt).

For single-cell real-time RT-PCR, freshly dissociated DRG neurons from adult mice were first prepared as described previously (Liang et al., 2016a; Li et al., 2017). Briefly, 4 h after plating, a single living small DRG neuron was collected under an inverted microscope fit with a micromanipulator and microinjector and placed in a PCR tube with 5–10 μl of cell lysis buffer (Signosis). After centrifugation, the supernatants were collected. The remaining real-time RT-PCR procedure was performed following the manufacturer's instructions with the single-cell real-time RT-PCR assay kit (Signosis). All primers used are listed in Table 1.

DRG neuronal culture and transfection.

Primary DRG neuronal cell cultures and viral transduction were performed as described previously (Liang et al., 2016a; Li et al., 2017). Briefly, all DRGs from cervical to lumbar levels were collected in cold Neurobasal medium (Invitrogen/Thermo Fisher Scientific) with 10% FBS (Invitrogen/Thermo Fisher Scientific), 2% B-27 supplement (Invitrogen/Thermo Fisher Scientific), 1% l-glutamine (Invitrogen/Thermo Fisher Scientific), 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen/Thermo Fisher Scientific) after adult WT or Mbd1^−/− mice were killed with isoflurane. The DRGs were then treated with enzyme solution containing collagenase Type I (1 mg/ml) and dispase (5 mg/ml) in Hanks' balanced salt solution (HBSS) (Invitrogen/Thermo Fisher Scientific). After trituration and centrifugation, the dissociated cells were resuspended in mixed neurobasal medium and plated at a density of 1.5 × 10⁵ to 4 × 10⁵ cells on glass coverslips (for calcium image experiment or whole-cell patch-clamp recording) or in a six-well plate coated with poly-d-lysine (50 μg/ml, Sigma-Aldrich) for viral transfection or cAMP measurement. The cells were incubated in an incubator (95% O₂/5% CO₂) at 37°C. One day later, the drugs or virus (0.5–2 μl, titer ≥1 × 10¹²/ml) was added to each well based on the experimental design. Cells were collected for detecting mRNA or protein at 48 or 72 h later.

Chromatin immunoprecipitation assay.

The chromatin immunoprecipitation assays were conducted by using the EZ ChIP Kit (Millipore) as described previously (Liang et al., 2016a; Li et al., 2017). The homogenization DRG solution from the WT and Mbd1^−/− mice was cross-linked with 1% formaldehyde at 37°C for 10 min and the reaction terminated by the addition of 0.25 m glycine. After centrifugation, the pellets were collected, washed, and suspended in lysis buffer containing 0.1% SDS, 1% sodium deoxy cholate, and 1% NP-40 in the presence of protease inhibitors. The suspension was sonicated to shear chromatin and produce 0.2–1 kb DNA fragments. After the samples were precleared with protein G agarose, they were immunoprecipitated with 5 μg of rabbit anti-MBD1 antibody (sc-10751, Santa Cruz Biotechnology) or normal rabbit serum. Input (10% of the sample for immunoprecipitation) was used as a positive control. The fragment of Oprm1 and Kcna2 gene promoters containing the predicted MBD1 binding sites was detected by PCR with the primers listed in Table 1.

Cresyl violet histochemical staining.

The WT and Mbd1^−/− mice were deeply anesthetized with isoflurane and transcardially perfused with 50–100 ml of 4% PFA in 0.1 m PBS, pH 7.4. The DRG was removed, postfixed overnight at 4°C, and cryoprotected in 30% sucrose in 0.1 m PBS overnight. Two sets of 20 μm sections were harvested from each DRG by grouping every third sections (3 or 4 sections per set per DRG). Each set (one set for the morphological observation and another for counting number of stained cells) was subjected to cresyl violet (Nissl) staining as described previously (Liaw et al., 2005) with slight modification. Briefly, after being dehydrated, the sections were stained in 0.1% cresyl violet solution at 37°C for 30 min. The sections were then rinsed in distilled water for 2 times and differentiated in 95% ethyl alcohol with 2 drops glacial acetic acid for 15 min. The morphology of the stained cells was observed under a light microscope. The number of the stained cells with the nucleus was counted randomly in a specific size of rectangular area (130 × 160 μm²) with the staining cells.

Immunohistochemistry.

The sections from the DRG and spinal cord of the WT and Mbd1^−/− mice were prepared as described above. All the sections were blocked for 1 h at 25°C in 0.01 m PBS containing 10% goat serum and 0.3% Triton X-100 before being incubated with the guinea pig anti-MOR antibody (1:1000; Millipore), rabbit anti-Kv1.2 antibody (1:200; Alomone Labs), rabbit anti-NF200 (1:100, Sigma-Aldrich), biotinylated IB4 (1:200, Sigma-Aldrich), or mouse anti-calcitonin gene-related peptide (CGRP, 1:200, Abcam) overnight at 4°C. The sections were then incubated with goat anti-rabbit, anti-guinea pig, or anti-mouse IgG conjugated with Cy3 (1:200, Jackson ImmunoResearch Laboratories) or with FITC-labeled Avidin D (1:200, Sigma-Aldrich) for 1 h at room temperature. Control experiments included omission of the primary antiserum and substitution of normal guinea pig, rabbit serum, or mouse serum for the primary antiserum. All immunofluorescence-labeled images were examined under the DMI4000 fluorescence microscope (Leica Microsystems) and captured with the DFC365FX camera (Leica Microsystems). Positive neuron counting and integrated densities after background subtraction were performed by National Institutes of Health ImageJ Software.

ISH histochemistry.

ISH histochemistry was performed as described previously (X. Zhao et al., 2013; Li et al., 2017). Briefly, the Mbd1 antisense RNA probe (0. 433 kb) was first prepared by in vitro transcription and labeled with digoxigenin-dUTP according to the manufacturer's instructions (Roche Diagnostic). Primers are listed in Table 1. Bilateral L3/4 DRGs from WT and Mbd1^−/− mice were harvested for the single labeling of Mbd1 mRNA and double labeling of Mbd1 mRNA with DRG marker immunohistochemistry. For the single labeling, after treatment of proteinase K and prehybridization, the sections (20 μm at the thickness) were hybridized with digoxigenin-dUTP-labeled Mbd1 antisense RNA probe (2 ng/μl) overnight at 67°C. After being washed, the sections were then incubated with alkaline-phosphatase-conjugated anti-digoxigenin antibody (1:1000) overnight. The fluorescent signals were finally developed with HNPP/Fast Red (Roche Diagnostic) in detection buffer 30 min twice. The sections were then rinsed in PBS and mounted with antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). For the double labeling, all protocols before being blocked with PBT containing 10% normal goat serum are similar to those for the single labeling. The sections were then incubated with alkaline-phosphatase-conjugated anti-digoxigenin (1:1000, Roche Diagnostic) and chicken anti-β tubulin III (1:500, EMD Millipore), rabbit anti-glutamine synthetase (GS, 1:500, Sigma-Aldrich), rabbit anti-NF200 (1:100, Sigma-Aldrich), biotinylated IB4 (1:200, Sigma-Aldrich), or mouse anti-CGRP (1:200, Abcam) at 4°C overnight in 20% goat serum blocking solution. After being washed, the sections were incubated with species-appropriate FITC or Cy2-conjugated secondary antibody (1:500) or with FITC-labeled Avidin D (1:200, Sigma-Aldrich) for 1 h at room temperature followed by HNPP/FastRed detection.

Western blotting.

The tissues or cultured neuron cells were homogenized in the lysis buffer containing 10 mm Tris, 1 mm PMSF, 5 mm MgCl₂, 5 mm EGTA, 1 mm EDTA, 1 mm DTT, 40 μm leupeptin, and 250 mm sucrose. After centrifugation at 4°C for 15 min at 1000 × g, the supernatant was collected for cytosolic proteins and the pellets were sonicated in the lysis buffer plus 1% SDS and 0.1% Triton X-100 for nuclear proteins. After protein concentration was measured, the samples were heated at 99°C for 5 min and loaded onto a 4%–15% stacking/7.5% separating SDS-polyacrylamide gel (Bio-Rad). The proteins were then electrophoretically transferred onto a PVDF membrane (Bio-Rad). According to the targeted protein molecular weights, the membranes were cut into several small strips and then blocked with 3% nonfat milk in TBS containing 0.1% Tween 20 for 1 h. The following primary antibodies were used: mouse anti-Kv1.1 (1:200, NeuroMab), mouse anti-Kv1.2 (1:200, NeuroMab), mouse anti-Kv1.4 (1:300, NeuroMab), rabbit anti-MOR (1:500, Neuromics), rabbit anti-κ opioid receptor (KOR) (1:500, Novus Biologicals), rabbit anti-GAPDH (1:1000; σ), mouse anti-MBD1 (1:500, gift from the X.Z. laboratory), rabbit anti-DNMT3a (1:500, Cell Signaling Technology), and rabbit anti-histone H3 (1:1000, Cell Signaling Technology). The proteins were detected by HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:3000 Jackson ImmunoResearch Laboratories) and visualized with peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad) using a ChemiDoc XRS System (Bio-Rad). The intensity of blots was quantified with densitometry by System with Image Lab software (Bio-Rad). The band intensities for cytosol proteins were normalized to GAPDH and those for nucleus proteins to total histone H3.

Luciferase assay.

To construct the Oprm1 and Kcna2 gene reporter plasmids, we amplified the fragments from the Oprm1 and Kcna2 gene promoter (including the MBD1-binding motif) by PCR from genomic DNA. The PCR products were cloned into the KpnI and HindIII restriction sites of the pGL3-Basic vector (Promega). After the sequences of recombinant clones were verified, both constructs were prepared using Plasmid Midi Kit (QIAGEN) and in vitro methylated by CpG methyltransferase M.SssI (New England Biolabs). A catecholaminergic cell (CAD) line derived from central nervous neurons was cultured in DMEM/F-12 medium (catalog 11330–032; Invitrogen/Thermo Fisher Scientific), supplemented with 10% FBS (JR Scientific), 100 units/ml penicillin, and 100 μg/ml streptomycin (Quality Biological) on 12-well plates in a humidified 5% CO₂ incubator. Each well was transfected with 300 ng of the constructed overexpression plasmid and luciferase gene reporter plasmid along with 10 ng of the pRL-TK plasmid (a normalizing control; Promega) using Lipofectamine 2000 (Invitrogen). After 48 h culture, the transfected cells were lysed with passive lysis buffer and 10 μl supernatant was assayed for luciferase activity with the dual-luciferase reporter assay system (Promega). The relative reporter activity was obtained by normalization of the firefly activity to Renilla activity with a Centro XS3 LB 960 Microplate Luminometer (Berthold Technologies). Three independent transfection experiments were performed.

Coimmunoprecipitation assay.

Coimmunoprecipitation was performed as described previously (Tao et al., 2003; Park et al., 2009). The DRGs from WT and Mbd1^−/− mice were collected in the lysis buffer containing 50 mmol Tris, pH 8.0, 150 mmol NaCl, 0.1% SDS, 1.0% NP-40, protease inhibitor mixture, and 10 μm TSA and homogenized with glass tissue grinders. After the centrifugation, the supernatant was collected and then preincubated with 50 μl protein A/G mouse agarose (Santa Cruz Biotechnology). After overnight incubation, 400 μg proteins were used to incubate with rabbit anti-DNMT3a serum (Ab13888, Abcam) plus Protein A/G mouse Agarose (Santa Cruz Biotechnology) by rotation for 4 h at 4°C. After the mixture was washed four times with the lysis buffer, the proteins were eluted by boiling loading buffer before being loaded onto an SDS-polyacrylamide gel (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad) followed by Western blot analysis. MBD1 and DNMT3a were detected as described above.

Whole-cell patch-clamp recording.

The acute disassociated DRG neurons from adult WT and Mbd1^−/− mice were cultured as described above. Whole-cell voltage-clamped recording as described previously (Liang et al., 2016a; J. Y. Zhao et al., 2017) was performed 4–10 h after plating. The micropipettes' electrode resistances ranged from 3 to 5 mΩ. The neurons were voltage-clamped with an Axopatch-700B amplifier (Molecular Devices). The intracellular pipette solution consisted of the following (in mm): potassium gluconate 120, KCl 20, MgCl₂ 2, EGTA 10, HEPES 10, and Mg-ATP 4, pH 7.3 (with KOH, 310 mOsm). To diminish the Na⁺ and Ca²⁺ component in voltage-gated potassium current, an extracellular solution contained the following (in mm): choline chloride 150, KCl 5, CdCl₂ 1, CaCl₂ 2, MgCl₂ 1, HEPES 10, and glucose 10, pH 7.4 (with Tris base, 320 mOsm). Signals were filtered at 1 kHz and digitized using a DigiData 1550 with pClamp 10.4 software (Molecular Devices). Series resistance was compensated by 60%–80%. Cell membrane capacitances were acquired by reading the value for whole-cell capacitance compensation directly from the amplifier. An online P/4 leak subtraction was performed to eliminate leak current contribution. The data were stored on computer by a DigiData 1550 interface and were analyzed by the pCLAMP 10.4 software package (Molecular Devices).

The action potential (AP) was also recorded with the use of whole-cell current clamp. The coverslips were placed in the chamber and perfused with extracellular solution consisting of the following (in mm): NaCl 140, KCl 4, CaCl₂ 2, MgCl₂ 2, HEPES 10, and glucose 5, with pH adjusted to 7.38 by NaOH. The intracellular pipette solution contained the following (in mm): KCl 135, Mg-ATP 3, Na₂ATP 0.5, CaCl₂ 1.1, EGTA 2, and glucose 5; pH was adjusted to 7.38 with KOH and osmolality adjusted to 300 mOsm with sucrose. The resting membrane potential was taken 3 min after a stable recording was first obtained. To evoke AP, we delivered depolarizing currents from 100 to 1400 pA (100 pA increment, 200 ms duration). The AP threshold was defined as the first point on the rapid rising phase of the spike at which the change in voltage exceeded 50 mV/ms. The membrane potential was held at the existing resting membrane potential during the current injection. The AP amplitude was measured between the peak and the baseline, and the AP overshoot was measured between the AP peak and 0 mV. To measure the membrane input resistance, each cell was delivered with a series of hyperpolarizing currents, 200 ms duration delivered in steps of 100 pA from 200 pA to 2000 pA, and the membrane input resistance was obtained from the slope of a steady-state I–V plot. The after-hyperpolarization amplitude was measured between the maximum hyperpolarization and the final plateau voltage. The data were stored on computer by a DigiData 1500 interface and were analyzed by the pCLAMP 10.4 software package (Molecular Devices). All experiments were performed at room temperature.

Calcium image.

Intracellular KCl-induced [Ca²⁺]i transients via voltage-dependent Ca²⁺ channels in the DRG neurons were measured with TILLvisION Imaging System version 3.3 (T.I.L.L. Photonics) as described previously (Vetter et al., 2006; Thyagarajan et al., 2009; Han et al., 2013; Lukacs et al., 2013). Acute disassociated DRG cultured neurons from WT and Mbd1^−/− mice were prepared as described above. Three days after plating, the neurons were washed to remove the serum and then loaded with 2 μm fura-2 AM (Invitrogen) for 30 min in the dark at room temperature. After washing, the neurons were placed in a recording chamber mounted on an inverted IX51 microscope (Olympus) coupled to a dual excitation Ca²⁺ imaging system (Photon Technology International). Ca²⁺ responses were induced by the application of 100 mm KCl (osmotically corrected by reducing the Na⁺ concentration) with or without 200 μm DAMGO/400 μm D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and imaged at 340 and 380 nm excitation wavelengths to detect intracellular free calcium. CTOP was given 5 min before DAMGO/KCl application. The experiments were performed in triplicate and repeated with at least 3 separate batches of culture.

cAMP assay.

cAMP was measured by using a radioimmune assay kit (GE Healthcare) as described by the manufacturer (Vetter et al., 2006; Udina et al., 2010). Acute disassociated DRG cultured neurons from WT and Mbd1^−/− mice were prepared in 6-well plates as described above. Three days after plating, the neurons were stimulated with or without DAGMO (160 nm) or coadministration with CTOP (400 nm) in the presence or absence of 50 μm forskolin (FSK, catalog #1099, Tocris Bioscience, an adenylyl cyclase activator). CTOP was given 5 min before DAGMO for 15 min. FSK was then added for another 15 min. After incubation, reactions were terminated. Intracellular cAMP levels were measured with a competitive cAMP EIA kit in triplicate as per the manufacturer's instructions. The experiments were performed in triplicate and repeated with at least 3 separate batches of culture.

Statistical analysis.

All data are presented as mean ± SEM. The data were statistically analyzed with two-tailed, unpaired Student's t test and a one-way or two-way ANOVA. When ANOVA showed significant difference, pairwise comparisons between means were tested by the post hoc Turkey method (Sigma-Aldrich, Plot 12.5). The cells were equally suspended and then randomly distributed in each tested well for the in vitro experiments. The mice were distributed into different treated groups randomly for the in vivo experiments. p < 0.05 was considered statistically significant in all analyses.