4.3. Slice Preparation and Electrophysiological Recordings

Electrophysiological experiments were performed 30–35 days after the injection of pi-locarpine. Rats were anesthetized with isoflurane (Laboratorios Karizoo S.A., Barcelona, Spain), decapitated, and their brains quickly removed. Brain slices containing the hippocampus and entorhinal cortex were sectioned using a Microm International HM 650 V vibratome (Microm, Walldorf, Germany) in ice-cold carbogen-aerated ACSF containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂, 24 mM NaHCO₃, and 10 mM glucose. The slices were then transferred to oxygenated ACSF and incubated at 35 °C for 1 h before electrophysiological recording. The brain slices were then transferred to a recording chamber and perfused with ACSF at a constant flow rate of 5 mL/min for 15–20 min at 30 °C. Local field potentials (LFPs) were recorded simultaneously from the CA1 stratum radiatum and deep layers of the entorhinal cortex using glass microelectrodes (0.2–1.0 MΩ). LFPs were recorded with a Model 1800 amplifier (A-M Systems, Carlsborg, WA, USA) and digitized with an ADC/DAC NI USB-6211 (National Instruments, Austin, TX, USA). Recording was performed using WinWCP v5.7.8 software (University of Strathclyde, Glasgow, UK).

Whole-cell voltage clamp recordings of EPSCs were performed on pyramidal neurons in the CA1 field of the hippocampus. Neurons were visualized using a Zeiss Axioscop 2 microscope (Zeiss; Oberkochen, Germany) equipped with differential interference contrast optics and a PointGrey Grasshopper3 GS3-U3-23S6M-C video camera (FLIR Integrated Imaging Solutions Inc., Wilsonville, OR, USA). Signals were recorded using a Multiclamp 700B (Molecular Devices, Sunnyvale, CA, USA) patch clamp amplifier and an NI USB-6343 A/D converter (National Instruments, Austin, TX, USA) with WinWCP 5 software (University of Strathclyde, Glasgow, UK). A CsMeSO₄-based pipette solution was used, containing (in mM): 127 CsMeSO₄, 10 NaCl, 5 EGTA, 10 HEPES, 6 QX314, 4 ATP-Mg, and 0.3 GTP. The pH was adjusted to 7.25 with CsOH. Patch electrodes (4–5 MΩ) were pulled from borosilicate glass capillaries using a P-1000 micropipette puller (Sutter Instrument, Novato, CA, USA). The voltage clamp holding potential was fixed at -20 mV and the response recording potential was −70 mV.

Synaptic responses at a CA1 pyramidal neuron were elicited by stimulating two different inputs using bipolar stimulating electrodes placed at the Shaffer collaterals and the temporoammonic pathway (Figure 4a). An A365 stimulus isolator (World Precision Instruments, Sarasota, FL, USA) was used for extracellular stimulation. The NMDAR channel blocker AP-5 (50 mM, Sigma-Aldrich, St. Louis, MO, USA) and the GABAa blocker bicuculline (20 mM, Sigma-Aldrich) were applied in the bath.

The independence of the inputs was tested by stimulating the first and second pathways with 50 and 200 ms delay, respectively; if the amplitude of the responses at 50 ms did not exceed the amplitude at 200 ms by more than 15%, the stimulating inputs were considered independent. Next, two stimulation protocols were used: paired-pulse (50 ms interstimulus interval) and short-train (5 pulses at 50 Hz) protocols. Stimulation of Schaffer’s collaterals and the temporoammonic pathway was applied alternately at 10 s intervals.

Passive and active membrane properties were investigated using neuronal responses to current steps (1.5 s duration every 3.5 s, amplitudes from −100 to +600 pA in 10–20 pA increments) as previously described [75]. Signals were recorded with the Heka EPC8 amplifier, digitized at 50 kHz frequency using the LIH 8+8 AD-DA converter and PatchMaster software version 2x92 (all from HEKA Elektronik GmbH, Lambrecht, Germany). The patch electrode was filled with the internal solution containing the following (in mM): 135 potassium gluconate, 5 KCl, 5 NaCl, 5 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP-Na, 0.5% biocytin, pH 7.25, adjusted with KOH.

The resting membrane potential was measured as the voltage at zero input current. Input resistance was estimated as the slope of the linear regression of the current–voltage relationship for the subthreshold steps. Membrane τ was calculated as a parameter of a single exponential function fitted to the onset of the voltage response to a hyperpolarizing current step.

APs generated at the rheobase current were selected for analysis. The rheobase current was determined as the minimum current sufficient to induce AP generation. The voltage threshold was determined as the point at which the first derivative of the voltage (dV/dt) exceeded 5 mV/ms. The time of the first spike was measured from the start of the current step to the time of the first AP threshold. The amplitude was determined as the peak AP voltage relative to its threshold. The rise time was measured from 10% to 90% of the peak amplitude relative to the AP threshold. Half-width was determined as the AP width at the voltage level of its half amplitude relative to a threshold. The fAHP peak was determined as the point at which the voltage decay slowed to less than 5 mV/ms. The mAHP peak was measured as the lowest value of the voltage after the AP peak relative to its threshold. The time to mAHP was measured between the fAHP and mAHP peaks. ADP amplitude was measured as the peak voltage on the portion of the AP between the fAHP and mAHP peaks relative to the fAHP peak voltage.

For each neuron, the maximum AP frequency and the maximum slope of the current–frequency curves were measured. The current that elicited the maximum AP frequency was determined as the minimum current sufficient to elicit AP generation at the maximum frequency. Early frequency adaptation was determined as the ratio of the second interspike interval to the first, and late adaptation was determined as the ratio of the last interspike interval to the first at the next current step after the rheobase (rheobase + 10 pA).