Subjects in all experiments were naïve, adult (6−12 weeks old) mice of both sexes [36
]. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME), or bred in-house from breeders so obtained. Upon weaning (at 18−21 d) or immediately after arrival, mice were housed in standard shoebox cages of 2−4 with same-sex littermates in a temperature-controlled (20 ±1°C) environment (14 h:10 h light/dark cycle; lights on at 07:00 h), and with ad lib
access to food (Harlan Teklad 8604) and tap water. Purchased mice were habituated to the laboratory for at least one week before any behavioral testing commenced.
2.2. Formalin test
Mice were habituated for 30 min in individual transparent Plexiglas cylinders prior to a subcutaneous injection of 5% formalin into the plantar right hindpaw (20 μl volume) and were digitally videotaped for 60 min. Video files were later sampled for 5 sec at 1-min intervals, and the presence or absence of right hindpaw licking/biting in that 5-sec period was scored using Observer software (Noldus, Leesburg, VA). The early (acute) phase of the formalin test was defined as 0−5 min post-injection, and the late (tonic) phase as 10−60 min post-injection. We chose to define the early phase conservatively (0−5 min, instead of the more common 0−10 min) to ensure that we were measuring only early phase behavior and not interphase or beginning late phase behavior. Across the strains shown in , the correlation between 0−5 min licking/biting and 0−10 min licking/biting (not shown) is r
<0.005), and haplotype analysis performed using 0−10 min strain means yields essentially the same list as that shown in . Data are presented in most cases as the percentage of samples in which licking/biting was detected [1
]. In some experiments, the total time spent licking in the early phase was assessed by continuous scoring of video files instead of sampling, although data were similar using either method. Hindpaw edema was quantified as done previously [56
Figure 1 Haplotype mapping implicates Atp1b3 as a candidate gene underlying variable sensitivity to formalin pain. A, Sensitivity of 16 inbred mouse strains in the early phase of the formalin test (5%, 20 μl). Bars represent mean ± SEM percentage (more ...)
2.3. Computational haplotype-based genetic mapping
SNPs were organized into haplotype blocks; only a limited number of haplotypes—typically 2, 3 or 4—are present within a haplotype block. The haplotype-based computational analysis identifies haplotype blocks in which the haplotypic strain grouping within the block correlates with the distribution of phenotypic data among the inbred strains analyzed. To do this, a p
-value that assesses the likelihood that genetic variation within each block could underlie the observed distribution of phenotypes among the inbred strains was calculated. The haplotype blocks were then ranked based upon the calculated p
-value. When this computational analysis was performed, the haplotype map had 5,694 haplotype blocks generated from 215,155 SNPs characterized across 19 inbred strains covering 2,609 genes (see http://mouseSNP.roche.com
). HapMapper computation analysis of the early phase response phenotypes proceeded as described previously [28
2.4. qPCR experiments
We quantified basal expression of Atp1b1, Atp1b2, and Atp1b3 in A/J and C57BL/6J mice in the dorsal root ganglion (DRG). All experiments were conducted in triplicate. Lumbar DRGs (L4-L6; n=6−12/mouse) were harvested and total RNA was isolated and reverse transcribed using standard protocols. Gene expression was quantified using the 7700 Sequence Detector (TaqMan) and the SYBR Green PCR Core Reagent Kit, as described in the manufacturer's manual (Applied Biosystems, Foster City, CA). In a first PCR reaction the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) content of each cDNA sample was quantified to control for variations in cDNA amounts and diluted accordingly. The values were averaged in each direction and normalized by referring mean values of the target gene to mean values of Gapdh. The specificity of the PCR products was verified by gel electrophoresis. TaqMan-PCR reactions were performed in 25-μl volumes with a final primer concentration of 900 nM at 95°C for 30 s and 60°C for 60 s for 40 cycles. Primer and probe sets for Atp1b1 (Mm00437614_m1), Atp1b2 (Mm01337151_m1), Gapdh, and Atp1b3 forward 5′- tctgagccacagacttacaaaaagt -3′ and Atp1b3 reverse 5′- cttgtgaggttcttctgttcttcca -3′ with probe 5′-6FAM- tttcctaaagccatattctg -TAMRA-3′ were obtained from Applied Biosystems.
subunit of the Na+
-ATPase was labeled in isolated L4-L5 DRG tissue using rabbit polyclonal antibody raised against an oligopeptide corresponding to the N-terminal sequence of the rat β3
isoform plus a terminal cysteine residue [2
] (a generous gift of Dr. Kathleen J. Sweadner of Massachusetts General Hospital, Charlestown, MA). Staining for the β3
-ATPase in paraffin sections was conducted as described previously [10
]. Isolated L4-L5 DRGs with short segments of attached spinal nerve and root were rinsed in phosphate-buffered saline (PBS; pH = 7.4) and immediately immersed in fixative (4% formaldehyde in PBS) for 4−7 days at 4 °C. After fixation, tissue samples were dehydrated in graded alcohol solutions, cleared in xylene, and embedded in paraffin for cross sectioning. Five-μm thick sections were cut, and the largest sections from the middle third of the ganglia were collected and mounted on slides. Slides with sections of DRGs from both strains of mice were further processed in parallel under equivalent conditions (immunohistochemical procedures and light intensity) during image capturing.
Staining for β3
-ATPase in paraffin sections was conducted as described previously [10
]. Briefly, tissue sections were deparaffinized with xylene and rehydrated with graded alcohols and water. After high-temperature antigen retrieval in citrate buffer (pH 6), endogenous peroxidase and non-specific antibody binding sites were blocked by incubating the sections with hydrogen peroxide (3% in methanol for 10 min at room temperature) and heat-inactivated 10% donkey serum (30 min at 37 °C), respectively. Following overnight incubation with primary antibody (dilution 1:1000 in PBS containing 1% donkey serum, 4 °C), sections were incubated with secondary biotinylated antibody (donkey anti-rabbit antibodies; 1:600 in PBS/10% mouse Fab fragment; Jackson ImmunoResearch Laboratories, West Grove, PA), peroxidase-conjugated streptavidin label (1:400 in PBS), and finally with 3,3′-diaminobenzidine chromogen solution (Vector Laboratories, Burlingame, CA). Development of the staining was monitored under a microscope and stopped by washing with PBS. In negative control experiments, the incubations with primary antibody were substituted with an equivalent time of incubation in 1% donkey serum in PBS. Stained sections, dehydrated through 95% and 100% ethanol, cleared with xylene and coverslipped with Permount medium (Fisher Scientific, Fair Lawn, NJ).
Images were analyzed using Image-Pro 5.1 (Media Cybernetics, Silver Spring, MA) and custom “Clock-scan” protocol software [11
]. Cell diameters were determined in cells in which the nucleus was readily determined by calculating the average of major and minor axes of an ellipse automatically best fit to the cell outline [46
]. Average intensities of cytoplasmic and background pixels were subtracted yielding the value of cytoplasmic labelling with minimal (if any) dependence on within and between sections variations in staining or section thickness and illumination intensities [11
2.6. siRNA synthesis and validation
Five Dicer-substrate siRNA duplexes (siRNAs) [19
] were designed to be specific for the mouse Atp1b3
gene (NM_007502) using established criteria [45
]. RNA duplexes were obtained from Integrated DNA Technologies (IDT, Coralville, IA). All RNA duplexes were HPLC-purified, verified for identity by electrospray mass spectrometry, and were verified at >90% purity as assessed by analytical HPLC. Duplexes were validated for functional potency in NIH3T3 cells in vitro
prior to use in vivo
. NIH3T3 cells were obtained from ATCC and maintained under standard culture conditions in high glucose DMEM containing 10% newborn calf serum and 1% penicillin/streptomycin. Cells were plated at 3.5× 104
/well in 24-well plates the day before transfection in 0.5 ml complete media without antibiotics. Transfections were done in triplicate for each sample using 0.5 μl per well of siLentFect cationic lipid (Bio-Rad, Hercules, CA) and 5 pmol or 0.05 pmol of siRNA duplex to give a final concentration of either 10 nM or 0.1 nM. As a control, the TriFECTa DS Scrambled Negative DsiRNA duplex (Integrated DNA Technologies) was transfected in parallel at 10 nM. Cells were incubated with the transfection mixture for 24 h. Total RNA was isolated using the Promega SV96 Total RNA Isolation System (Promega, Madison, WI) as per the manufacturer's protocol. cDNA was synthesized using 150 ng total RNA the 30U SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA).
Quantitative real-time PCR was done using a 7900HT Real Time PCR System (Applied Biosystems, Foster City, CA) in 10 μl volume in 384-well format. Reaction mixes employed Immolase DNA Polymerase (Bioline, Randolph, MA), 200 nM primers and probe, and 10 ng cDNA. Reactions were cycled using the following program: 95 °C 10 min followed by 95 °C for 15 s, 60°C for 60 s for 40 cycles. All reactions were performed in triplicate. Primers employed were: Atp1b3 forward 5′-cagtttccagtctccttgcttg -3′ and Atp1b3 reverse 5′-gatatttgtggatatccgtctggg-3′ with probe 5′-6FAM-tccaaaggacagccttgcatccttgtga -IBFQ-3′. The fluorophore was 6-carboxyfluorescein (6FAM) and the quencher was Iowa Black FQ (IBFQ). Expression data were normalized to an internal control run in parallel for the Mus musculus ribosomal protein L23 gene, Rpl23 (NM_022891) using the ΔΔCt method. Primers and probes were Rpl23 forward: 5′- ctgtgaagggaatcaaggga -3′, Rpl23 reverse 5′- tgtcgaattaccactgctgg -3′, and probe 5′-6FAM-ctgaacagacttcctgctgctggtg -IBFQ-3′. Final expression data were normalized using the negative control DsiRNA transfections as 100%.
All five of the Atp1b3
siRNAs tested reduced the target gene expression by >90% at 10 nM. At low dose, potency differences were detectable between the reagents and duplexes 3.1 and 3.5 were most potent, suppressing target expression by 82% and 63% respectively at 0.1 nM (see Supplementary Figure 1
2.7. siRNA studies- in vivo knockdown
delivery of siRNA molecules was performed similar to a previously published protocol [30
]. The siRNA (2 μg/μl) was combined with i-Fect transfection reagent (Neuromics, Edina, MN) at a ratio of 1:5 (weight/volume) and the combination was allowed to complex for 5 min at room temperature. The siRNA/i-Fect complex was then further diluted in sterile physiological saline to give a final dose of 0.5 μg of siRNA per injection. A total of 5 μl of this solution was delivered via i.t. injection to the lumbar region using a 20-μl Hamilton microsyringe with a 30-gauge needle (Hamilton Company, Reno NV). This injection paradigm was repeated approximately 24 h and 48 h later (total of three injections).
2.8. Patch clamp electrophysiology
Under deep halothane/oxygen anesthesia, the spinal column was hemisected and dorsal root ganglia (DRG) from the lumbar region of either A/J or C57BL/6J mice were extracted and placed into Ca2+- and Mg2+-free DMEM (Invitrogen, Carlsbad, CA), then transferred at 37 °C and Ca2+- and Mg2+-free DMEM containing 1 mg/ml papain and 2 mg/ml collagenase (Sigma Aldrich, Saint Louis, MO). After enzymatic digestion, the DRGs were rinsed three times in DMEM containing 10% certified fetal bovine serum (FBS) and 2 mM L-glutamine. Mechanical trituration with a fire-polished Pasteur pipette was used to dissociate the DRG neurons, which were then rinsed three times in culturing media. Prior to plating, the DRG neurons were passed through a 100 μm nylon cell strainer (BD Biosciences, San Jose, CA). The DRG neurons were then plated onto 35-mm dishes coated with laminin (BD Biosciences) and poly-D-lysine (Sigma Aldrich), and incubated at 37 °C in 5% CO2.
Dissociated DRG neurons were cultured in F-12 media supplemented with 10% certified FBS, 2 mM L-glutamine, and 100 U/ml penicillin and streptomycin (Invitrogen). The external solution (ECS) used during patch-clamp recordings contained (in mM): 145 NaCl, 5.4 KCl, 1 MgCl2, 10 HEPES, 0.2 CdCl2, 2 BaCl2, and 5.1 D-glucose. Suppression of K+ and Ca2+ currents was achieved with the addition of Cd2+ and Ba2+, and the absence of Ca2+. NaOH was used to adjust the ECS to a pH of ~7.4. The pipette solution or intracellular solution (ICS) contained (in mM): 60 Na-gluconate, 70 Cs-gluconate, 10 CsCl, 5 sodium phosphocreatine, 10 HEPES, 5 EGTA, 10 TEA-Cl, 4 Mg-ATP and 0.3 GTP. High concentrations of Na+ as well as ATP, creatine phosphate, TEA and pyruvate were used to enhance pump activation while further suppressing K+ currents. The ICS (osmolarity of ~290 mOsm) was adjusted to a pH of ~7.2.
Dorsal root ganglion neurons were cultured for 24 h prior to experiments. Currents were recorded in the whole-cell patch clamp mode using an Axopatch 200B amplifier with onboard lowpass Bessel filter at 5 kHz and digitized through a Digidata 3200A at a sampling rate of 2 kHz. Recording electrodes (borosilicate glass , P-97 puller, Sutter Instruments, Novato, CA), were fire-polished to a tip resistance of 3−6 MΩ (MP-830 microforge, Narishige, East Meadow, NY). During patch-clamp recording, cells were perfused with ECS at a rate of 1 ml/min. Fast solution exchange was achieved using a three barrel perfusion system (SF-77B; Warner Instruments, Hamden, CT). Small cells were verified to be neurons by eliciting voltage-dependent Na+ current through gradual depolarization above −60mV in whole cell configuration. Neuronal current activities or holding currents (Ih) were continuously recorded under condition of holding potential (Vh) clamped at −40 mV. Membrane capacitance (Cm) and series resistance (Rs) were measured through the peak amplitude and decay constant of transients induced by repetitive depolarizing pulses of 10 mV.
To evaluate the ability of A/J and C57BL/6 DRG neurons to sustain repetitive firing, they were stimulated by trains of depolarizing currents set at 2 times their action potential (AP) threshold at a frequency of 10 Hz for 1 second, repeated 50 times with 5 second intervals between stimulations. The AP threshold of each neuron was determined by applying 500 ms-long depolarizing current steps by increments of 10 pA. The acquisition frequency was 50 kHz to fully capture the AP spikes and the highest amplitude of response to each stimulus train was used as index of the gradual decay of cellular response over the 50 trials, with or without treatment with 10 μM ouabain.
2.9. Rotarod test
Motor ataxia was determined using a standard mouse rotarod (Ugo Basile, Verese, Italy) which provided a accelerating rotational speed from 22 rpm at the start of the test to a maximum speed of 50 rpm. Mice were baseline tested 2−4 times prior to any experiments. The latency to fall off of the rotarod was determined automatically by a timer that measured to the nearest second. A cutoff latency of 300 sec was used for all rotarod assessments.
Data were analyzed by ANOVA followed by Student's t-test or Bonferroni multiple comparison post hoc test, as appropriate. The criterionα=0.05 was used throughout.