Extracellular field recordings

For field recordings, after 2 h of recovery at 30 °C in an incubation chamber containing oxygenated ACSF, individual slices were transferred to the interface slice-recording chamber (BSC1, Scientific System Design Inc.) where they were maintained at 30–32 °C and constantly superfused with oxygenated ACSF at the rate of 1.5 ml/min. Solutions were applied to the slices using a peristaltic pump (Bio-Rad). Slices were visualized with a Wild M3B (Heerbrugg, Switzerland). Experiments were performed from 1 to 7 h after slicing. At the beginning of each recording, a concentric bipolar stimulating electrode (SNE-100X 50-mm-long Elektronik—Harvard Apparatus GmbH) was placed in the hippocampus CA1 stratum radiatum (SR) for stimulation of the Schaffer collateral pathway projections to the CA1. A glass micropipette (0.5–1 MΩ) filled with ACSF was placed in the CA1 hippocampal region, at 200–600 µm from the stimulating electrode, to record orthodromically evoked field extracellular post-synaptic potentials (fEPSP). fEPSPs were recorded and filtered (low pass at 1 kHz) with an Axopatch 200A amplifier (Axon Instruments, CA) and digitized at 10 kHz with an A/D converter (Digidata 1322A, Axon Instruments). Stimuli consisted of 100 μs constant current pulses of variable intensity, applied at 0.05 Hz. In each experiment, stimulus intensity was adjusted to evoke ~ 50% of the maximal fEPSP amplitude without appreciable population spike contamination. Evoked responses were monitored online and stable baseline responses (variation in the amplitude values under 10%) were recorded for at least 10 min. Only slices showing stable fEPSP amplitudes were included in the experiments. LTP was induced by high-frequency stimulation (HFS, 2 trains of stimuli at 100 Hz of 1 s duration each, 3 s inter-train interval). To analyze the time course of fEPSP amplitude, the recorded fEPSP was routinely averaged over 1 min (n = 3 traces). fEPSP amplitude changes following the LTP induction protocol were calculated with respect to the baseline (35 min after versus 1 min before LTP induction). The paired-pulse ratio (PPR) was measured from responses to two synaptic stimuli at 50 ms inter-stimulus interval. The PPR was calculated at baseline as the ratio between the fEPSP amplitude evoked by the second stimulus (A2) over the first (A1; A2/A1).

Data were stored on a computer using pClamp 10 software (Axon Instruments) and analyzed offline with Clampfit 10 program (Axon Instruments).