2.2. Hippocampal slice electrophysiological studies

As previously described (Vedder et al., 2014; Widman and McMahon, 2018), adult, 16- to 18-month old male HR and LR rats were anesthetized with isoflurane and transcardially perfused with cold, high-sucrose, low-Na+ artificial cerebrospinal fluid (aCSF) containing (in mM): 85 NaCl, 2.5 KCl, 4 MgCl, 0.5 CaCl₂, 1.25 NaH₂PO₄, 25 NaHCO₃, 25 glucose, 75 sucrose. Brains were removed and coronal slices were prepared from dorsal hippocampus in ice-cold, high-sucrose, low-Na+ aCSF. Slices were allowed to recover for at least one hour at room temperature in a submersion chamber with standard aCSF containing (in mM): 119 NaCl, 5 KCl, 1.3 MgCl, 2.5 CaCl₂, 1.0 NaH₂PO₄, 26 NaHCO₃, and 11 glucose, prior to beginning recordings. To enhance slice health for experiments requiring whole-cell recordings, 2 mM kynueric acid was added to the aCSF to limit excitotoxcity It is important to note that at 16–18 months, HR and LR rats displayed similar phenotypes as previously reported in younger animals (Flagel et al., 2014; Stead et al., 2006). HR rats displayed greater locomotor response to novelty than LR rats (traveling 2677 ± 200 cm during the 5 min test compared to LRs that traveled 1595 ± 96 cm; t=4.877, df = 17.36; p=0.001). HR rats showed significantly less anxiety-like behavior than LR rats, spending more time in the center of the Open Field (30 ± 6 sec) compared to LRs (16 ± 3 sec; t=2.148, df = 16.19; p=0.04).

Slices for extracellular recordings were placed in a submersion chamber and continuously perfused (3–4 ml/min) with standard aCSF equilibrated with 95% O₂ and 5% CO₂ and maintained at 26–28⁰ C. Extracellular field excitatory postsynaptic potentials (fEPSPs) at CA3-CA1 synapses were recorded by stimulating Schaffer collaterals with pairs of pulses (0.1Hz, 100 μs duration at 50 ms interval) in stratum radiatum with a stainless steel bipolar electrode and recorded with a glass pipet filled with aCSF placed nearby in stratum radiatum. Stimulus-response curves were generated by increasing the stimulus intensity in 20 μA intervals beginning with an intensity of 20 μA, until a maximum fEPSP was achieved. 5 fEPSPs were recorded at each stimulus strength and averaged to obtain a single value. For paired-pulse ratio (PPR) experiments, pairs of stimulation were delivered at 10, 20, 30, 50, 100, 150, 200, and 400 ms inter-stimulus intervals. PPR was calculated by dividing the initial slope of the second event by the initial slope of the first event. Plasticity experiments were performed in the presence of 100 μM picrotoxin to block GABAergic transmission. After a 10 min stable baseline, long-term potentiation (LTP) was induced by theta burst stimulation (TBS), which consisted of a train of 10 bursts at 5 Hz. Each burst contained 5 pulses at 100 Hz, and the train was repeated 4 times with a 20 s interval between trains. Following TBS, stimulation returned to baseline frequency (0.1Hz) and the fEPSPs were recorded for at least 40 min. Steady state depolarization was measured during each burst from an average trace of all 4 TBS trains. Long-term depression (LTD) was induced by 1 Hz electrical stimulation for 15 min after a 10 min stable baseline. After LTD induction, stimulation returned to baseline frequency (0.1Hz) and fEPSPs were recorded for at least 40 min. The magnitude of LTP or LTD was measured by comparing the average fEPSP slope between 35–40 min to the average baseline fEPSP slope.

In whole-cell patch recordings, slices were placed in a submersion chamber and continuously perfused (2–3 ml/min) with standard aCSF (24–26⁰C). For the excitation/inhibition (E/I) ratio, NMDA receptor (NMDAR)/AMPA receptor (AMPAR) ratio, and the ratio of GluN2B subunit to number of NMDARs, CA1 pyramidal cells were blind patched (2–5 MΩ pipet resistance) with a pipet solution containing (in mM): 120 Cesium gluconate, 0.6 EGTA, 5 MgCl₂, 2 ATP, 0.3 GTP, 20 HEPES, 5 Lidocaine Bromide, pH 7.2 with CsOH. The E/I ratio was measured at −35mV to −30mV, a membrane potential between the reversal potential for excitatory and inhibitory synaptic currents. The stimulation electrode was placed more than 500μm away from the recording electrode to limit contamination by monosynaptic inhibitory input, and electrical stimulation was set to obtain a 150pA excitatory current. The E/I ratio was measured as the peak amplitude of the average excitatory current divided by the average inhibitory current. For NMDAR/AMPAR ratio and GluN2B/NMDAR ratio, slices were continuously perfused with standard aCSF containing 100 μM picrotoxin to block GABA_AR transmission and 10 μM nifedipine to block L-type Ca2+ channels. After acquisition of baseline, 1 μM Ro 25–6981, an antagonist for GluN2B-containing NMDARs, was bath applied. Once a level response was recorded, 100 μM APV was added to block the remaining NMDARs. To ensure the remaining current was mediated by AMPARs, the AMPAR antagonist, 10 μM DNQX, was bath applied at the end of each experiment. To obtain the NMDAR/AMPAR ratio, the peak amplitude of the average NMDAR current was divided by the peak amplitude of the average AMPAR current. The GluN2B/NMDAR ratio was acquired by dividing the peak amplitude of Ro 25–6981 sensitive current by the peak amplitude of total NMDAR current.

To assess differences in intrinsic excitability, direct current injection experiments were performed in CA1 pyramidal cells using a pipet solution containing (in mM): 120 Potassium gluconate, 0.6 EGTA, 5 MgCl₂, 2 ATP, 0.3 GTP, 20 HEPES, pH 7.2 with KOH. Experiments were obtained in the presence of glutamatergic antagonists, APV and DNQX, and picrotoxin, to block synaptic transmission. 10 μM nifedipine and 1 μM Ro 25–6981 were also present in the perfusion solution. In each cell, a series of increasing 100pA current steps starting at −400pA was executed three times with 5 min between each series. Resting membrane potential was obtained from the initial starting potential where no current was injected. From the hyperpolarizing current steps, input resistance was calculated and sag potential was measured. Action potential (AP) number was measured from the depolarizing current steps, and AP threshold was measured from the first AP. All electrophysiological data were acquired using Clampex 10.3 (pClamp software, Molecular Devices) and analyzed offline in Clampfit 10.3 (pClamp). For extracellular experiments, the n number represents animal number, and for whole-cell experiments, the n number represents cell number with a minimum of 4 animals used per phenotype.