Whole-cell patch clamp of neurons in acute hippocampal brain slices
Whole-cell somatic recordings were performed using patch electrodes filled with a solution containing (in mM): 134 K gluconate, 10 HEPES, 10 phosphocreatine, 2 Na2ATP, 0.2 Na2GTP, and 0.2% w/v biocytin.
CA1 pyramidal cells and interneurons in SR and stratum lacunnosum moleculare were selected for recording based on the shape of their soma using video microscopy under near infrared differential interference contrast illumination. Cells were further characterised by their electrophysiological properties obtained from injecting a series of 500 ms depolarising and hyperpolarising current pulses and identified post-recording anatomically, as described previously in detail (Khan et al., 2018).
Spontaneous postsynaptic potentials were recorded from passive membrane responses and mixed spontaneous excitatory postsynaptic potentials (sEPSPs) and spontaneous inhibitory postsynaptic potentials (sIPSPs) were collected in 60 second frame samples, repeated at 0.33 Hz. Recordings were carried out under the current clamp mode of operation (NPI SEC 05LX amplifier; NPI electronics, Germany), low pass filtered at 2 KHz and digitised at 5 KHz using a CED 1401 interface (Cambridge Electronic Design, UK). Input resistance was monitored throughout experiments by means of a hyperpolarising current step (-10 pA, 10 ms). Signal (Cambridge Electronic Design, UK) was used to acquire recordings and generate current steps. The average amplitudes of spontaneous events and their frequency was measured manually from single sweep data sets of 60 second recordings, including a total sweep range of 30-50 frames (i.e., 30 – 50 minutes of recording); values below the baseline level of 0.1 mV were considered as noise, see (Ali et al., 2006) .
Paired whole-cell somatic recordings were obtained between CA1 CR interneurons in SR (for inhibitory connections). Unitary inhibitory postsynaptic potentials (IPSPs) were elicited by a depolarising current step into the presynaptic neuron (+0.05 nA, 5–10 ms) repeated at 0.33 Hz. The peak IPSP amplitudes, and width at half-amplitude measurements were obtained from averages including 100-200 unitary synaptic events.
Drugs for in vitro pharmacological studies on brain slices, zolpidem (Sigma, Aldrich, UK, 0.4 µM, dissolved first in ethanol to a final bath ethanol dilution of 1:20,000); α5-SOP002 (1-1.5 µM); diazepam (RBI, Poole UK; 1-2 µM, dissolved in ethanol to a final bath ethanol dilution of 1:5000) were bath-applied. The α5-SOP002 concentration used (1-1.5 µM) was within the range at which it is reported to act as an inverse agonist with efficacy selective for α5 containing GABAARs (Dawson et al., 2006). The concentration of zolpidem used produces near maximal effects on α1-containing receptors but submaximal effects on α2/3-containing receptors (K d 0.2 µM for α1−containing receptors; 1.5 µM for α3 containing receptors (Munakata et al., 1998).