Under 2–3% isoflurane anesthesia, the lumbar segment of the rat spinal cord was removed by laminectomy. The spinal cord tissue was immediately placed in ice-cold sucrose artificial cerebrospinal fluid (aCSF) presaturated with 95% O2 and 5% CO2. The sucrose aCSF contained (in mM) 234 sucrose, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 12.0 glucose, and 25.0 NaHCO3. Transverse spinal cord slices (400 μm) were cut in the ice-cold sucrose aCSF using a vibratome and preincubated in Krebs solution oxygenated with 95% O2 and 5% CO2 at 34°C for at least 1 hour before recording. The Krebs solution contained (in mM) 117.0 NaCl, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 11.0 glucose, and 25.0 NaHCO3.
Recordings of excitatory postsynaptic currents (EPSCs) were performed using the whole-cell voltage-clamp method as we described previously (Li et al., 2002 (link); Zhou et al., 2008a (link)). The spinal cord slice was continuously perfused with Krebs solution at 5.0 ml/min at 34°C maintained by an inline solution heater. The lamina II is identified as a distinct translucent band across the superficial dorsal horn under a microscope with transmitted illumination. Neurons in the outer zone of lamina II were studied in most of the experiments because they receive input from both TRPV1- and non-TRPV1-expressing C-fiber afferent terminals (Pan et al., 2003 (link); Pan and Pan, 2004 (link); Chen and Pan, 2006 (link)). In some experiments, large-diameter lamina I neurons were also selected for recording. Lamina I and II neurons in the slice were identified on a fixed-stage microscope using differential interference contrast/infrared illumination. EPSCs were recorded with an electrode (impedance was 5–8 MΩ) filled with the following internal solution (in mM): potassium gluconate, 135.0; KCl, 5; MgCl2, 2.0; CaCl2, 0.5; HEPES, 5.0; EGTA, 5.0; ATP-Mg, 5.0; and Na-GTP, 0.5; adjusted to pH 7.2–7.3 with 1 M of KOH (290–300 mOsm). EPSCs were evoked by electrical stimulation (0.6 mA, 0.2 ms) of the attached dorsal root, and monosynaptic EPSCs were identified on the basis of the constant latency of evoked EPSCs and the absence of conduction failure of evoked EPSCs in response to a 20-Hz electrical stimulation as we described previously (Li et al., 2002 (link); Zhou et al., 2008a (link)).
To determine the paired-pulse depression or facilitation, two EPSCs were evoked by a pair of stimuli given at 50 ms intervals. The paired-pulse ratio was expressed as the ratio of the amplitude of the second synaptic response to the amplitude of the first synaptic response. The AMPA receptor-mediated EPSCs were recorded at a holding potential of −60 mV in the presence of 10 μM bicuculline and 2 μM strychnine. The NMDA receptor-mediated EPSCs were recorded at +40 mV in the presence of 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 μM bicuculline, and 2 μM strychnine (Zhou et al., 2008b (link)). To record the miniature EPSCs (mEPSCs), 0.5 μM tetrodotoxin was added to the perfusion solution. In some experiments, to examine the presynaptic effects of the μ opioid receptor agonist (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO), we used the following internal solution (in mM): 110 Cs2SO4, 5 TEA, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, 1 guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S), and 10 lidocaine N-ethyl bromide that had been adjusted to pH 7.2–7.3 with 1 M CsOH (290–300 mΩsm). In this case, GDP-β-S and Cs2+ were used to inhibit the postsynaptic effect of the opioid receptor agonist (Zhou et al., 2008a (link)).
The postsynaptic G protein-coupled inwardly rectifying K+ channel (GIRK) currents were recorded from lamina II neurons at a holding potential of −60 mV using the pipette internal solution containing (in mM) 135.0 potassium gluconate, 5.0 KCl, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP; the solution was adjusted to pH 7.2–7.4 with 1 M KOH (290–300 mΩsm) (Zhou et al., 2008a (link)). Signals were recorded using an amplifier (MultiClamp700B; Axon Instruments Inc.), filtered at 1–2 kHz, digitized at 10 kHz, and stored into a computer. DAMGO, D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), BAPTA, GDP-β-S, CNQX, 2-amino-5-phosphonopentanoic acid (AP-5), and MK-801 were obtained from Sigma-Aldrich. Bicuculline, strychnine, and BAPTA-AM were purchased from Tocris Cookson Inc.
Recordings of excitatory postsynaptic currents (EPSCs) were performed using the whole-cell voltage-clamp method as we described previously (Li et al., 2002 (link); Zhou et al., 2008a (link)). The spinal cord slice was continuously perfused with Krebs solution at 5.0 ml/min at 34°C maintained by an inline solution heater. The lamina II is identified as a distinct translucent band across the superficial dorsal horn under a microscope with transmitted illumination. Neurons in the outer zone of lamina II were studied in most of the experiments because they receive input from both TRPV1- and non-TRPV1-expressing C-fiber afferent terminals (Pan et al., 2003 (link); Pan and Pan, 2004 (link); Chen and Pan, 2006 (link)). In some experiments, large-diameter lamina I neurons were also selected for recording. Lamina I and II neurons in the slice were identified on a fixed-stage microscope using differential interference contrast/infrared illumination. EPSCs were recorded with an electrode (impedance was 5–8 MΩ) filled with the following internal solution (in mM): potassium gluconate, 135.0; KCl, 5; MgCl2, 2.0; CaCl2, 0.5; HEPES, 5.0; EGTA, 5.0; ATP-Mg, 5.0; and Na-GTP, 0.5; adjusted to pH 7.2–7.3 with 1 M of KOH (290–300 mOsm). EPSCs were evoked by electrical stimulation (0.6 mA, 0.2 ms) of the attached dorsal root, and monosynaptic EPSCs were identified on the basis of the constant latency of evoked EPSCs and the absence of conduction failure of evoked EPSCs in response to a 20-Hz electrical stimulation as we described previously (Li et al., 2002 (link); Zhou et al., 2008a (link)).
To determine the paired-pulse depression or facilitation, two EPSCs were evoked by a pair of stimuli given at 50 ms intervals. The paired-pulse ratio was expressed as the ratio of the amplitude of the second synaptic response to the amplitude of the first synaptic response. The AMPA receptor-mediated EPSCs were recorded at a holding potential of −60 mV in the presence of 10 μM bicuculline and 2 μM strychnine. The NMDA receptor-mediated EPSCs were recorded at +40 mV in the presence of 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 μM bicuculline, and 2 μM strychnine (Zhou et al., 2008b (link)). To record the miniature EPSCs (mEPSCs), 0.5 μM tetrodotoxin was added to the perfusion solution. In some experiments, to examine the presynaptic effects of the μ opioid receptor agonist (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO), we used the following internal solution (in mM): 110 Cs2SO4, 5 TEA, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, 1 guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S), and 10 lidocaine N-ethyl bromide that had been adjusted to pH 7.2–7.3 with 1 M CsOH (290–300 mΩsm). In this case, GDP-β-S and Cs2+ were used to inhibit the postsynaptic effect of the opioid receptor agonist (Zhou et al., 2008a (link)).
The postsynaptic G protein-coupled inwardly rectifying K+ channel (GIRK) currents were recorded from lamina II neurons at a holding potential of −60 mV using the pipette internal solution containing (in mM) 135.0 potassium gluconate, 5.0 KCl, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP; the solution was adjusted to pH 7.2–7.4 with 1 M KOH (290–300 mΩsm) (Zhou et al., 2008a (link)). Signals were recorded using an amplifier (MultiClamp700B; Axon Instruments Inc.), filtered at 1–2 kHz, digitized at 10 kHz, and stored into a computer. DAMGO, D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), BAPTA, GDP-β-S, CNQX, 2-amino-5-phosphonopentanoic acid (AP-5), and MK-801 were obtained from Sigma-Aldrich. Bicuculline, strychnine, and BAPTA-AM were purchased from Tocris Cookson Inc.
