All procedures were conducted in accordance with the guidelines of the Uekusa Gakuen University Laboratory Animal Care and Use Committee. Data were obtained from 54 neonatal Wistar rats (2–3 days old). The isolation of brainstem-spinal cord preparations has been described in detail previously . In brief, rats were deeply anesthetized with diethyl ether and the brainstem was isolated in a dissecting chamber at room temperature. The chamber was filled with mock CSF equilibrated with a gas mixture (5% CO2 in O2; pH 7.4). The composition of the mock CSF was as follows (in mM): NaCl, 126; KCl, 5; CaCl2, 2; MgSO4, 2; NaH2PO4, 1.25; NaHCO3, 26 and glucose, 30. The cerebrum was quickly removed by transection at the upper border of the inferior colliculus. Each trunk of the bilateral trigeminal nerves that run through the craniobasal bone was isolated to a length of 1 mm, enabling it to be pulled into a suction electrode. Subsequently, the trigeminal nerve-attached brainstem-spinal cord was cut caudally at the level of the C3 roots (Figure 1a). Furthermore, the isolated medulla was sectioned sagittally at 1–1.5 mm lateral from the midsagittal plane with a handmade slicer (Figure 1b). The trigeminal nerve-attached brainstem sagittal slice was placed in a recording chamber (volume 1.0 mL) with the medial side up and continuously superfused (flow 4–6 mL/min) at 26°C with oxygenated mock CSF.
Voltage-sensitive dye imaging
The voltage-sensitive dye imaging technique has been described in detail previously . In brief, for staining, preparations were kept for 30 min in mock CSF containing the voltage-sensitive dye Di-4-ANEPPS (7.5 mg/mL in 0.1% DMSO, Molecular Probes, Eugene, OR, USA), before being kept for at least 30 min in normal mock CSF. After staining, excess dye was removed by superfusion of the preparation with dye-free solution. After 30 min of washing, optical imaging and data analysis were performed using a MiCAM02 hardware and software package (BrainVision, Tokyo, Japan). For optical imaging, we used a fixed-stage upright fluorescence microscope (Measurescope UM-2, Nikon, Tokyo, Japan) with a low magnification objective lens (XL Fluor 4×/340, Olympus, Tokyo, Japan) and a high-resolution MiCAM02 camera.
To record the voltage-sensitive dye signals, we used light from a 150 W halogen lamp controlled by an electromagnetic shutter (Oriel Instruments, Stratford, USA). Changes in fluorescence of the dye were detected by the camera through a 510–560 nm excitation filter, a dichroic mirror, and a 590 nm absorption filter (MBE1405, Nikon). The camera captured images of 88 × 60 pixels, and the size of the area was 5.4 × 3.7 mm. Optical signals from 3 × 3 pixels (approximately 0.03 mm2) were averaged and are showed in the images.
Total frame acquisition was set to 511. Sampling time was 2.2 ms/frame; therefore, the total recording time was 1,124.2 ms. Neuronal activity was evoked by square pulse electrical stimuli (1.0 ms, 0.5–1.0 mA) delivered to the trigeminal nerve rootlet via a glass suction electrode. Acquisition was triggered by the electrical stimulus. The trigger signal was activated after one-quarter of the total recording time, corresponding to 284 ms after starting acquisition when we set the total acquisition time to 1,124.2 ms. Signal amplitude was normalized using the dF/F method, where F is the total fluorescent signal and dF corresponds to the change in fluorescence observed following evoked modification of the membrane potential. To improve the signal-to-noise ratio, we averaged signals detected in 10 consecutive trials at 0.3 Hz. To analyze the intensity of the signals, we measured the peak amplitude (at 30–40 ms after stimulation) and amplitudes at 40% (at 165 ms after stimulation) and at 60% (at 385 ms after stimulation) of the total recording time (1,124.2 ms). Measuring points at 165 and 385 ms after stimulation were selected arbitrarily.
Carbamazepine (Sigma Aldrich, Saint Louis, MO, USA) was added to perfused mock CSF at 10, 100 and 1,000 μM. Gabapentin (Sigma Aldrich) was added to perfused mock CSF at 1.0, 10 and 100 μM. NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5, Sigma Aldrich) was added to perfused mock CSF at 30 μM. These concentrations were determined from preliminary experiments and previous studies [16, 22]. Optical records using electrical stimulation were taken 20 min after the start of superfusion with control mock CSF and were taken 20 min after switching to drug-containing mock CSF. To induce activation of the NMDA receptor, we used low Mg2+ concentration solution (in mM): NaCl, 126; KCl, 5; CaCl2, 2.6; MgSO4, 0.8; NaH2PO4, 1.25; NaHCO3, 26 and glucose, 30. In these series of experiments, optical records using electrical stimulation were taken 20 min after the start of superfusion with control mock CSF and were taken 20 min after switching to low Mg2+ concentration solution, and then, taken 20 min after switching to low Mg2+ solution containing 30 μM AP5, 1,000 μM carbamazepine or 100 μM gabapentin.
Optical signal amplitudes obtained before superfusion with mock CSF containing drugs was defined as the control value. The level of statistical significance for the difference between the mean value of each variable obtained during application of different concentrations of drug was conducted by ANOVA followed by pair-wise comparisons using the Tukey–Kramer method for multiple comparisons as indicated. All statistical analyses were conducted using Statcel (OMS publisher, Japan). All values were reported as mean ± SD, and all P values < 0.05 were considered significant.