2.3. Field Excitatory Postsynaptic Potential (fEPSP) Recordings in Hippocampal Slices

Extracellular fEPSP recordings were performed according to standard methods as previously described [30,31].

In brief, 400 µm thick coronal brain slices were cut on a vibratome (Leica VT 1000S) in ice-cold dissection buffer (mM): 127 NaCl, 1.9 KCl, 1.2 KH₂PO₄, 26 NaHCO₃, 10 D-glucose, 2 MgSO₄, and 1.1 CaCl₂, constantly saturated with 5% CO₂ and 95% O₂ (pH 7.4). Slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF, in mM: 127 NaCl, 1.9 KCl, 1.2 KH₂PO₄, 26 NaHCO₃, 10 D-glucose, 1 MgSO_4, 2.2 CaCl₂; pH 7.4) for 1 h at 30 °C and afterwards stored at room temperature. Recordings were performed in a submerged-type recording chamber (Warner Instruments). Stimulation (TM53CCINS, WPI) and recording (ACSF-filled glass pipettes, 2–3 MΩ) electrodes were positioned in the stratum radiatum (SR) to record Schaffer collateral field excitatory postsynaptic potentials (fEPSPs). Signals were amplified with an Axopatch 200B (Molecular Devices), digitized at 5 kHz with an ITC-16 (HEKA) and recorded using WinWCP from the Strathclyde Electrophysiology Suite. Stimuli (100 µs) were delivered through a stimulus isolator (WPI). For each individual slice the strength of the stimulation (typically between 30–125 µA) was chosen to evoke 40–60% of the maximal response, defined by initial fEPSP slope. Only slices that showed stable fiber volley and fEPSP were used for further recording. The same stimulus intensity was applied during baseline recording (0.067 Hz, 20–30 min) and induction of LTP using 100 stimuli during 1 s (100 Hz, 1 s). The baseline was determined by averaging fEPSP initial slopes from the period before the tetanic stimulation (at least 15 min of stable recording). The level of LTP was determined by averaging fEPSP slopes from the period between 50 and 60 min after the high-frequency stimulation. Before the tetanic stimulation, each slice was used to record input–output relationship (IOR, 25–150 µA in 25 µA steps) and paired-pulse facilitation (PPF, 10–20–50–100–200–500 ms interpulse interval at the same stimulation strength as LTP recordings). IOR changes in fEPSP slope and fiber volley amplitude were normalized within each slice (% from the maximal response at the highest stimulus strength was calculated) and averaged values for each group were plotted against the stimulus intensity. For PPF paired-pulse ratio of EPSP2/EPSP1 slope and amplitude at each interstimulus interval were defined per slice and mean values per group were plotted. EPSP1 was calculated as an average of EPSP1s from all interstimulus intervals for each single slice.

Four traces were averaged for each single data point analyzed.