Generation of floxed Nav1.3 mouse
An RCPI-22 129S6/SvEvTac mouse BAC library was screened using a probe representing exons 4–12. DNA from positive BAC clones was prepared and subcloned into pBluescript (BS-SKII-). Three subclones were obtained that covered Nav
1.3 sequences from exon 2–8. The construct was prepared in three steps. Firstly, the 3' arm, Neomycin cassette and one LoxP site were cloned together as follows; two oligonucleotides were cloned in BS-SKII- to form new cloning sites. One LoxP site was cloned as an EcoRI-PstI fragment from PLneo vector [26
]. The 3' arm was cloned as ClaI-BamHI 7 Kb fragment. Neomycin cassette was cloned as BamHI-SalL fragment from Py6.0 plasmid [26
]. Secondly, the 5' arm, one LoxP and the floxed exons were cloned together as follows; 7 kb fragment ClaI-SacI was cloned in BS-SKII-. A LoxP site was inserted into the BsmBI site of the genomic fragment through blunt ligation. Finally, the two halves above were cloned into a vector containing the Thymidine-Kinase (TK) cassette as follows; the 3' side was prepared as Not-ClaI fragment, the 5' side was prepared as ClaI-SacII fragment and the TK containing vector was prepared as Not-SacII fragment. All were ligated together in the same tube. Positive clones were confirmed by restriction digests and sequencing. Blastocyst injection was performed by Monica Mendelson (Colombia University). Chimeras were crossed to C57BL/6 and germ line transmission tested using Southern blotting of tail DNA derived from F1 offspring. F1 heterozygotes were crossed to FLPe deletor animals [34
] to excise the positive selection marker.
All tests were approved by the United Kingdom Home Office Animals (Scientific Procedures) Act 1986. They were performed in a Home Office designated room at 22 ± 2°C. Tests were carried out as previously descried [28
]. Formalin test: 20 μl of 5% formalin (37% formaldehyde (BDH) diluted with 0.9% saline (Sigma)) was injected subcutaneously into the plantar aspect of a hind-paw using a Hamilton syringe and 27 g needle and animals were placed in a 20 × 20 × 20 cm Plexiform chamber. The amount of time spent licking or biting the injected paw was then recorded in 5 minute periods over the next hour. Animals were habituated to the test environment for at least 1 hr before experiments were started. Activity during the first phase, from 0–10 mins, and the second phase, from 10–60 mins, was then calculated for each animal.
Induction of Neuropathic pain
Neuropathic pain was induced according to the Chung Model (ligation of spinal nerve L5). Baseline mechanical thresholds for one paw were measured from mice using von Frey hairs (using the up-down method) respectively. Animals were anaesthetised using Halothane. A midline incision was made in the skin of the back at the L2-S2 levels and the left paraspinal muscles separated from the spinal processes, facet joints and transverse processes at the L4-S1 levels. The L5 transverse process was removed and the L5 spinal nerve tightly ligated using 8-0 silk thread.
PCR on Genomic DNA
DNA was precipitated from DRG and brain after overnight incubation in lysis-buffer (100 mM Tric-Cl, 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 0.1 mg/ml Proteinase K). DNA pellet was resuspended in 200 μl TE and 1 μl was used for each 25 μl PCR reaction. Nav1.3 Primers used are (GAGAGAAAGACACTTAAATGCAGACATC), (GCTTTTTGTTCAAGTCTATCATATTCAAAG) and (AAG GAT GGC ATC ACC CAC AAG).
Deletion of exons 4 and 5 was confirmed by reverse transcription PCR (RT-PCR). RNA isolation was performed using TRIzol Reagent (Invitrogen, Paisley, UK), according to manufacturer's protocol. The reverse transcription reaction was also performed according to an Invitrogen protocol, using random primers. Primers used were (CACTAAACCCCGTTAGGAAAATTGCT) and (TCCAAGTGCTCCCTCTGTCTCCTC). The same primers were used for sequencing the RT-PCR bands.
Animals were terminally anaesthetised with 150 mg/kg sodium pentobarbitone (Rhône Mérieux), and then intracardially perfused with ice cold PBS (0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mMKH2PO4) followed by ice cold 4% paraformaldehyde/PBS. The tissues were removed and placed in O.C.T compound. 10 μm sections were prepared and mounted on Superfrost Plus slides (BDH). Sections were fixed in 4% paraformaldehyde for 5 mins and washed 3 × 10 mins in PBS + 0.1% Triton-X. They were blocked and permeabilised in 10% goat serum (GibcoBRL)/PBS + 0.1% Triton-X for at least 3 hrs at room temperature. Sections were then incubated in the following antisera in 10% goat serum/PBS + 0.3% Triton-X overnight at 4°C: rabbit anti-beta-galactosidase (5'-3') (1:500), mouse anti-peripherin (Chemicon) (1:1000) or mouse anti-neurofilament (Sigma N-52) (1:1000). They were then washed 3 × 10 mins in PBS + 0.1% Triton-X. The primary antisera were localised by immunofluorescence with Alexa Fluor 594 (Molecular Probes) (1:1000) and Alexa Fluor 488 (Molecular Probes) (1:1000). The slides were finally washed 3 × 30 mins at 4°C and mounted using Aqueous Mounting Solution (Sigma).
Measuring Peak sodium current
Cultures of dorsal root ganglion (DRG) neurons were prepared from adult Nav1.3 null and WT littermate mice. The animals were killed by cervical dislocation, and dorsal root ganglia (DRG) were removed and enzymatically dissociated (Dispase/collagenase; Sigma, Poole, Dorset, UK). The isolated and pooled DRG neurons were plated onto poly-L-lysine coated glass coverslips and maintained in culture for 1–2 days.
Electrophysiology and solutions
Conventional voltage-clamp recordings in the whole-cell patch-clamp configuration were made from neurones of varying sizes (corresponding to small and medium sized neurons with a maximum apparent diameter of 43 μm) using an Axopatch 200B amplifier (Axon Instruments, Union City, California, USA) driven from a PC generating pulse protocols (Pclamp 9, Axon Instruments). The neuronal capacitance was estimated through the procedure of capacity transient cancellation, soon after attaining the whole-cell configuration. Electrodes were made from thin-walled glass (Harvard Apparatus, Edenbridge, Kent, UK) and had an initial resistance of between 1.5 and 2 MΩ when filled with intracellular solution. Series-resistance compensation was set near 70% with a nominal feed-back lag of 12 ms. Recordings were filtered at 5 KHz (4-pole Bessel filter) and sampled at 10 KHz. Recordings were made at room temperature.
TTX-s and TTX-r Na+ currents were recorded in relative isolation by including pharmacological blockers of both K+ and Ca2+ currents in both extracellular and intracellular media. In order to distinguish TTX-s and TTX-r currents, recordings were made before and after the addition of 250 nM TTX to the extracellular solution, where the TTX-s component was subsequently derived by off-line analysis using digital subtraction. The extracellular solution contained (in mM): NaCl 43.3, Tetraethylammonium Chloride 96.7, HEPES 10, CaCl2 2.1, MgCl2 2.12, 4-Aminopyridine 0.5, CsCl 10, KCl 7.5, CdCl2 0.1. The intracellular solution contained (in mM): CsCl 130, CsF 13, EGTA (Na) 3, Tetraethylammonium Chloride 10, HEPES 10, CaCl2 1.21, ATP(Mg) 3 mM, GTP (Li) 500 μM. External and internal solutions were buffered to pH 7.2–7.3 with the addition of CsOH. Tetrodotoxin (TTX, 250 nM) was locally applied and removed by gravity fed superfusion. The reagents, with the exception of TTX, were obtained from Sigma-Aldrich (Poole, Dorset, UK). TTX was purchased from Alomone Labs (TCS Biologicals, Botolph Claydon, Bucks, UK).
In order to derive current density measurements, peak Na+ currents were recorded in response to a family of incrementing, depolarizing clamp-steps, filtered at 5 KHz (4-pole Bessel filter) and normally sampled at 20 KHz. The holding potential was -80 mV, and the clamp-steps were preceded by a 20 ms pre-pulse. These values of membrane potential, coupled with the presence of internal F-, were not optimal for recording TTX-r persistent Na+ current, and the TTX-r currents analysed always exhibited the voltage-dependent and kinetic characteristics of Nav1.8. Current density estimates were found by dividing the peak-current values by the value estimated for membrane capacitance. TTX-s current amplitudes were found by off-line digital subtraction.
Recording of spontaneously active fibres
At defined time points after L5 spinal nerve ligation mice were anaesthetised via a brief induction with isofluorane gas followed by an i.p. injection of pentobarbitone (100 mg/kg). A lumbar laminectomy was performed and the L4 and L5 dorsal roots were identified and cut close to the spinal cord. Both the ventral roots and the L4 spinal nerve were cut as far distally as possible and then the dorsal roots, ventral roots, DRGs and spinal nerves (in continuity) from L4 and L5 were carefully and quickly removed. Throughout the process of dissection the tissue was kept hydrated by regularly applying oxygenated (95% O2, 5% CO2) aCSF solution (in millimolar: NaCl 118; KCl 4.7; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25; CaCl 2.5 and glucose 11) to the preparation. The tissue was then kept in oxygenated aCSF at room temperature for 30 minutes before the start of electrophysiological recording. The peripheral nerve preparations were mounted in a two chamber recording bath with the spinal nerve and DRG on one side continuously perfused with oxygenated aCSF at a rate of 10 ml/min. This solution was kept at 35 ± 1°C by running the perfusion tube through a jacket containing water heated and circulated by a thermostatically controlled water bath. The dorsal and ventral roots were sealed in the other chamber and covered in mineral oil which had been previously oxygenated with an air tube. Oil in this chamber was periodically replaced to maintain hydration and oxygenation of the dorsal root. A leak-proof partition barrier of perspex and high-vacuum grease ensured electrical isolation from the other compartment. For the purposes of electrical stimulation the end of the spinal nerve was taken up into a suction electrode with bipolar silver/silver chloride wires. Fine filaments were teased from the dorsal root using sharpened watchmakers forceps. These strands were placed over electrodes made of sliver wire coated with teflon that had been stripped away from the tip. A crushed strand from the ventral root was placed across another silver wire electrode to act as an indifferent recording. Signals from the electrodes were passed through a head-stage which was also connected to earth. From here the signal was amplified and filtered (low pass 500 Hz, high pass 5 KHz) using Neurolog equipment (Digitimer, UK). Residual 50 Hz noise was removed with a Humbug noise eliminator (Quest Scientific, Canada). The signal was viewed on a digital oscilloscope (Tektronix, USA) and also recorded for off-line analysis (Powerlab and Chart 5 software, ADI instruments, UK).
Spontaneously active units were recorded in both the L5 and L4 dorsal roots. In strands with multiple ectopically firing units separation was achieved on the basis of differing amplitude, action potential shape and interspike interval. The number of conducting fibres in each strand was determined by incremental electrical stimulation of the spinal nerve whilst observing the recruitment of units on a storage oscilloscope (Gould, UK). Strands that were studied typically contained a total of 8 to 12 conducting units and around 50 to 60 units were sampled from each dorsal root. The proportion of spontaneously active units in each preparation was calculated as a percentage and then a mean obtained for all data in a group with n representing a single L4 or L5 root taken from one animal. A large proportion of units observed firing spontaneously did so in a bursting manner and so further analysis of these patterns was undertaken using specialised software (Spike 2, Cambridge Electronic Design, UK).
Unpaired t-test was used to compare data unless otherwise indicated.