Two weeks after behavioral testing, transverse hippocampal slices from one hemisphere (400-μm thickness) were cut in an ice-cold cutting solution using a Vibratome (Leica VT1000 s; Leica Microsystems, Wetzlar, Germany). The cutting solution contained (in mm) 220 sucrose, 3.0 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, and 13 d-glucose, and was oxygenated with 95% O2 and 5% CO2 (pH 7.4 was adjusted with NaOH, and osmolarity was maintained at 350–360 mOsm). Slices were allowed to recover in oxygenated extracellular solution at room temperature (approximately 23°C) for at least 1 h before transferring to a recording chamber. The extracellular solution contained (in mm) 125 NaCl, 3.0 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, 13 d-glucose (pH 7.4 was adjusted with NaOH, and osmolarity was maintained at 305–315 mOsm). Individual slices were transferred to a submerged recording chamber perfused with oxygenated extracellular solution at constant flow rate of 3 ml/min. Brain regions, neurons, and electrodes were visualized through a 4× or 40× lens using an upright microscope equipped with infrared-differential interference contrast optics (Nikon Eclipse E600FN, Nikon Instruments Inc., Melville, NY, U.S.A.). Glass electrodes were pulled in four stages on a horizontal pipette puller (Model P-87 Flaming/Brown Micropipette Puller; Sutter Instruments, Novato, CA, U.S.A.) from Wiretrol II capillary glass (Drummond Scientific, Broomall, PA, U.S.A.). Recordings of fEPSPs were made in the stratum radiatum of the hippocampal CA1 region at 30°C. Electrodes for recording and stimulation were filled with 2 m NaCl (approximately 5 MΩ) and extracellular solution (approximately 1.5 MΩ), respectively. The stimulating electrode was placed in the stratum radiatum to activate Schaffer collateral/commissural afferents. The recording and stimulating electrodes were placed approximately 600 μm away from each other. Monopolar stimulation was applied with a stimulus isolator (WPI, Sarasota, FL, U.S.A.). To obtain input–output curves, we applied different stimulation intensities ranging from 0–400 μA in steps of 50 μA (50 μs for stimulation duration, 0.05 Hz). Short-term plasticity was examined using pairs of stimuli that were delivered at varying interstimulus intervals (30–500 msec) and intensities (0–400 μA) at 0.05 Hz. The baseline stimulation intensity was adjusted to elicit a fEPSP slope of 35% of the maximal response (Kluge et al., 2008). After establishing a stable baseline for 20 min (stimulation parameters: 0.05 Hz; duration, 50 μs; intensity, approximately 100 μA in control and approximately 80 μA in irradiated rats), LTP was induced with a single train of high-frequency stimulation (100 Hz for 1 s) and LTD was induced by low-frequency stimulation (1 Hz for 900 s) in separate slices. Stimulation intensity for LTP and LTD was same as the baseline stimulation intensity. Poststimulation responses were recorded for at least 60 min, with stimuli using baseline stimulation parameters. One recording for LTP and another one for LTD were obtained from two slices of each rat. Potentials were amplified using a multiClamp 700B (Molecular Devices, Union City, CA, U.S.A.). Amplifier control and data acquisition were performed using CLAMPEX 10.1 software via 16-bit data acquisition system Digidata 1320A (Molecular Devices). Signals were digitized at 10 kHz and analyzed off-line.
The slope of fEPSP was measured off-line using the clampfit analysis program (Molecular Devices). For analyzing paired-pulse facilitation (PPF), the slope ratio of the second to the first fEPSP was calculated, and plotted for different interstimulus intervals or stimulation intensities. For analyzing LTP or LTD (the increase or decrease of fEPSP slope after stimulation, respectively), values obtained after stimulation were expressed as percentage of the baseline values; the magnitude of LTP or LTD was compared between control and irradiated rats.