Neurophysiological studies were conducted in rats 7 days post-injury as described50 (link). Briefly, brains were rapidly dissected in ice-cold artificial cerebrospinal fluid (ACSF; 75 mM sucrose, 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 7 mM MgCl2, 0.5 mM CaCl2 and 10 mM glucose). Transverse hippocampal slices (350 μ) were sectioned using a vibratome (Leica Microsystems Inc., VT1000S) in NMDG cutting/recovery solution (N-methyl D-glucamine (100 mM), KCl (2.5 mM), NaH2PO4 (1.2 mM), NaHCO3 (30 mM), HEPES (20 mM), MgS04 (1 0 mM), CaCl2 (0.5 mM), and glucose (25 mM) at 30 °C (pH 7.3–7.4). After 2 min, slices were transferred to HEPES holding solution NaCl (92 mM), KCl (2.5 mM), NaH2CO3 (30 mM), NaH2PO4 (1 mM), HEPES (20 mM), D-Glucose (25 mM), MgCl2 (1 mM), CaCl2 (1 mM) for 1 h at 30 °C. Slices were allowed to incubate for 30 min in recording solution of oxygenated Kreb’s (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 1.1 mM MgCl2, 2 mM CaCl2 and 25 mM glucose) prior to recording.
Recordings were performed using a Multiclamp 700A amplifier with a Digidata 1322 and pClamp 10 software (Axon, Molecular devices, LLC). Field excitatory postsynaptic potentials (fEPSP) from CA1 were evoked by square current pulses (0.1 ms) at 0.033 Hz with a bipolar stimulation electrode (FHC, Bowdoinham, ME). Stimulus intensity was defined using a stimulus intensity required to induce 50% of the maximum EPSP slope using the input–output curves. The sample intensity was used for PPR recordings across different intervals. A stable baseline was recorded for at least 10 min prior to high frequency stimulation (HFS, 4 trains, 100 Hz, 1 s duration, separated by 20 s). Post-tetanic potentiation (PTP) was analyzed by taking the average of the slopes from the traces recorded during the first 2 min after HFS. LTP was assessed for at least 45–50 min following HFS. The PPR values were calculated by dividing the second fEPSP slope by the first fEPSP slope (fEPSP2/fEPSP1). All recordings were performed in the absence of any drug treatment and only 1 or 2 slices were recorded from each individual rat. Data were analyzed with Clampfit 10 software (Axon, Molecular devices, LLC).
Recordings were performed using a Multiclamp 700A amplifier with a Digidata 1322 and pClamp 10 software (Axon, Molecular devices, LLC). Field excitatory postsynaptic potentials (fEPSP) from CA1 were evoked by square current pulses (0.1 ms) at 0.033 Hz with a bipolar stimulation electrode (FHC, Bowdoinham, ME). Stimulus intensity was defined using a stimulus intensity required to induce 50% of the maximum EPSP slope using the input–output curves. The sample intensity was used for PPR recordings across different intervals. A stable baseline was recorded for at least 10 min prior to high frequency stimulation (HFS, 4 trains, 100 Hz, 1 s duration, separated by 20 s). Post-tetanic potentiation (PTP) was analyzed by taking the average of the slopes from the traces recorded during the first 2 min after HFS. LTP was assessed for at least 45–50 min following HFS. The PPR values were calculated by dividing the second fEPSP slope by the first fEPSP slope (fEPSP2/fEPSP1). All recordings were performed in the absence of any drug treatment and only 1 or 2 slices were recorded from each individual rat. Data were analyzed with Clampfit 10 software (Axon, Molecular devices, LLC).
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