Spinal cord slice patch-clamp recording

The methods used for obtaining a mouse spinal cord slice that retained an attached dorsal root and for patch-clamp recordings from spinal lamina I neurons have been described elsewhere³⁰. In brief, mice were anaesthetized with urethane (1.2 g/kg, i.p.), and then lumbosacral laminectomy was performed at 24 h after CFA inflammation, at which remarkable mechanical and thermal hyperalgesia was stably produced. Transverse 350–450-μm-thick spinal cord slices with dorsal roots attached were obtained. The slices were stored in an incubation solution at room temperature (in mM: NaCl, 95; KCl, 1.8; KH₂PO₄, 1.2; CaCl₂, 0.5; MgSO₄, 7; NaHCO₃, 26; glucose, 15; sucrose, 50; oxygenated with 95% O₂, 5% CO₂; pH 7.4). A slice was then transferred into a recording chamber and superfused with oxygenated recording solution at 3 ml min⁻¹ at room temperature. The recording solution was identical to the incubation solution except for (in mM): NaCl 127, CaCl₂ 2.4, MgSO₄ 1.3 and sucrose 0.

Spinal lamina I neurons were visualized with a 40X water-immersion objective using a microscope (BX51WI; Olympus) equipped with infrared differential interference contrast optics. Spinal lamina I neurons were found to lie within a distance of maximally 20 μm from the dorsal white/grey matter border. Standard whole-cell patch clamp recordings were performed with glass pipettes having a resistance of 4–6 MΩ in lamina I of spinal dorsal horn. The pipette solution consisted of (in mM): K-gluconate, 135; KCl, 5; CaCl₂, 0.5; MgCl₂, 2; EGTA, 5; HEPES, 5 and Mg-ATP, 5, pH 7.4 with KOH, measured osmolarity 300 mOsm. QX-314 (5 mM) was added to the pipette solution to prevent discharge of action potentials (APs). To measure excitatory postsynaptic currents (EPSCs) from neurons in lamina I, dorsal root was stimulated through a suction electrode with an isolated current stimulator and membrane potential was held at −70 mV. Test pulses of 0.1 ms with intensity of 3 mA were given at 30-sec intervals. Aδ-fiber or C-fiber evoked EPSCs (eEPSCs) were distinguished on the basis of the conduction velocity (CV) of afferent fibers (Aδ: 2–13 m/s; C: <0.8 m/s; calculated from the latency of EPSC from a stimulus artifact and the length of dorsal root), as described previously⁴⁷. Aδ-fiber or C-fiber responses, respectively, were considered as monosynaptic in origin when the latency remained constant and there was no failure during stimulation at 20 Hz for 1 s, or when failures did not occur during repetitive stimulation at 2 Hz for 10 s⁴⁷. Synaptic strength was quantified by the peak amplitudes of EPSCs.

In a subset of experiments, paired-pulse stimuli with an inter-stimulus interval of 110 ms (0.1 ms pulse duration, 3 mA intensity, every 30 s) were applied to dorsal root. Paired-pulse ratio (facilitation or depression) of C-fiber-evoked EPSC was calculated as the amplitude of the second C-eEPSC divided by that of the first C-eEPSC in a pair. In a subset of experiments, miniature EPSCs (mEPSCs) were recorded at membrane potential of −70 mV in the presence of gabazine (10 μM), strychnine (1 μM), AP-5 (50 μM) together with TTX (0.5 μM) to block inhibitory synaptic transmission and NMDAR-mediated excitatory synaptic transmission.

For membrane properties analysis, membrane potential was held at −70 mV under current-clamp mode. Depolarizing current steps (500 ms in duration and 2 pA increments) were used to detect the AP. The AP threshold was determined by differentiating the AP waveform and setting a rising rate of 10 mV/ms as the AP inflection point. The AP amplitude was measured from the equipotential point of the threshold to the spike peak. The AP duration was measured at the half-width of the spike. Rise and decay slopes were detected from the AP threshold to the peak.

Drugs were applied by superfusion with a change in solutions in the recording chamber, being complete within 3 min. All drugs were first dissolved at 1000 times the concentration to be used, and then diluted to the desired concentration in ACSF solution immediately before use.

Signals were gained using an Axopatch 700B amplifier (Axon Instruments, Foster City, CA, USA), low-pass-filtered at 5 kHz, and sampled at 10 kHz. Data were stored and analysed with a personal computer using the pCLAMP9.0 and Clampfit10.0 acquisition programmes (Axon Instruments).