Details for most of the procedures have been described previously (Martin & Buño, 2003). Briefly, transverse slices (400 μm) of the dorsal hippocampus from Wistar rats (13–17 days old) were prepared using conventional methods, and incubated (≈1 h at 20–22°C) in gassed (95% O2, 5% CO2 mixture) artificial cerebrospinal fluid (aCSF). The aCSF contained (in mm): NaCl 124, KCl 2.69, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and glucose 10, with a pH of 7.4. Slices were transferred to an immersion recording chamber, and infused (1.5 ml/min) with equilibrated aCSF. Recordings from CA1 hippocampal pyramidal neurons were made using the whole-cell configuration of the “blind” patch-clamp technique, as described previously (Martin & Buño, 2003). Patch electrodes had a resistance of 4–6 MΩ when filled with the internal solution that contained (in mm): K-gluconate 97.5, KCl 32.5, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 5, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 10, MgCl2 1, and ATP 4, at a pH of 7.2–7.3, and osmolarities between 280 and 290 mOsm/L. Whole-cell recordings in the current- or voltage-clamp modes were performed with a 2400 patch amplifier (A-M Systems Inc., Carlsborg, WA, U.S.A.). Fast and slow capacitances were neutralized, and series resistance was always compensated (about 70%). Cells were used only when the series resistance (6–14 MΩ) did not change >20% throughout the experiment. The membrane potential (Vm) was held at −70 mV in voltage-clamp experiments. Data were filtered at 2 KHz and transferred to the hard disk of a Pentium-based computer using a DigiData 1440A interface and the pCLAMP 10.0 software (Molecular Devices, Union City, CA, U.S.A.). Synaptic responses were evoked by bipolar Schaffer collaterals stimulation, through a pair of Elgiloy electrodes (SSM33A05, WPI, Hertfordshire, United Kingdom) placed in the stratum radiatum near the border of CA1 pyramidal layer. Stimuli were single or paired pulses (50–100 ms delay) delivered at 0.033 Hz via a 2100 isolated pulse stimulator (A-M Systems); adjusted in intensity to evoke excitatory postsynaptic currents (EPSCs) in voltage-clamp experiments, or EPSPs in current-clamp experiments. Stimulation intensity was adjusted to evoke EPSCs or EPSP amplitudes that were the maximal responses. Extracellular population spikes were recorded with a glass microelectrode (impedances 2–3 MΩ; filled with 1 m NaCl) positioned in pyramidal layer area CA1. Evoked population spikes were elicited by Schaffer collaterals stimulation as described previously for the EPSC recording. The amplitude of the population spike was measured as follows: (1) a line was drawn at the base of the population spike connecting the first and the second peaks of the field response, (2) a second line was drawn at the peak of the downward deflection of the population spike, and (3) at the peak of the spike, a line was drawn vertically between these two lines, thus, giving the amplitude of the population spike. To analyze the effects of FFA on inhibitory synaptic transmission, pairs of stimuli with an interstimulus interval ranging from 15 to 200 ms were applied to the Schaffer collaterals, and evoked population responses were recorded in the CA1 cell layer (see Fig. 3A). Data were compared using the Student’s t-test and values are presented as the mean ± standard error of the mean (SEM). p-values less than 0.05 were considered to be statistically significant.
Drugs were stored as frozen concentrated stock solutions and dissolved in oxygenated aCSF at the desired concentration immediately before use. Bicuculline methochloride, saclofen, flurbiprofen, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), and D (-)-2-amino-5-phosphonopentanoic acid (AP5) were purchased from Tocris Cookson (Bristol, United Kingdom). All other drugs were purchased from Sigma (St. Louis, MO, U.S.A.). FFA was prepared as a stock solution in dimethyl sulfoxide (DMSO) each day and diluted to the required concentrations in the extracellular solution. Epileptiform activity was induced with 100 μm 4-aminopyridine (4-AP) added to a modified Mg2+-free aCSF solution. The effectiveness of FFA in inhibiting epileptiform activity was estimated by constructing a dose–response relationship. The percentage of slices showing complete seizure cessation after the onset of the perfusion of FFA at different concentrations was averaged and plotted as a function of drug concentration (0.1–1mm). The plot was fitted to the formula:
derived from the Hill equation, where c is the half maximal inhibitory concentration (IC50) and b is the Hill coefficient.