Electrophysiology

Five C57BL/6 N male wild‐type mice (25 g) between 8–16 weeks of age were used for in vitro electrophysiology. Mice were decapitated; their brains rapidly removed and submerged in an icy slurry of artificial CSF (ACSF) optimized for slice preparation (slice solution), containing (in mM) 118 NaCl, 3 KCl, 1.3 MgSO₄, 1.4 NaH₂PO₄, 10 glucose, 26 NaHCO₃ and 2.5 CaCl₂, which was bubbled continuously with carbogen (95% O₂, 5% CO₂). Kynurenic acid (1 mM) was also added to the slice solution to reduce glutamate‐mediated excitotoxicity. Mid‐ventral, transverse sections of the hippocampus (300 μm) were cut using a vibrating slicer (Slicer HR2; Sigmann Elektronik). Slices were then transferred to a carbogenated ACSF (bath) solution, which contained the following (in mM): 124 NaCl, 3 KCl, 1.3 MgSO₄, 1.4 NaH₂PO₄, 10 glucose, 26 NaHCO₃ and 2.5 CaCl₂ (300–305 mOsm·L⁻¹). Slices were stored at room temperature (22°C) in the bath solution for at least 30 min following slicing; the bath solution was also used to perfuse slices for all experiments. Slices were placed into a recording chamber attached to a fixed stage of a moveable upright microscope (Axioskop FS2; Carl Zeiss, Vienna, Austria) and held submerged by a platinum and polyester fibre ‘harp’. Slices were continuously perfused with a warmed (34 ± 0.5°C), carbogenated bath ACSF at a rate of 2–3 mL·min⁻¹ for at least 20 min prior to recording.

Pipettes were pulled from thin‐walled borosilicate glass (TW150F; WPI, Sarasota, FL, USA) with a two‐stage puller (PP‐83; Narishige, Amityville, NY) to a tip resistance of 5–7 MΩ when backfilled with an internal solution containing (in mM) 126 K‐gluconate, 10 HEPES, 4 KCl, 5 MgATP, 0.3 NaGTP, 1 EGTA and 0.3 CaCl_2, to which 0.05–0.1% neurobiotin was added, and the pH was adjusted to 7.27–7.30 with KOH, (275–285 mOsm·L⁻¹). Recordings were made using a Multiclamp 700B amplifier data and were acquired using a pCLAMP 10.3 via a Digidata 1322 interface (Molecular Devices, Sunnyvale, CA).

CA3 and CA1 pyramidal neurons were visually identified using infrared differential interference contrast optics and selected for recordings based on characteristic pyramidal morphology and the presence of a large apical dendrite. After obtaining the whole cell patch clamp configuration, neurons were held in a voltage clamp at −60 mV for 5–10 min before beginning experiments and between experimental measurements. Only neurons that showed stable resting membrane potential (RMP), holding a current (in a voltage clamp) in a series of control measurements, and which showed stable access resistance throughout the entire experiment, were selected for analysis.

A stimulation isolation unit (Iso‐Flex, AMPI, Jerusalem) was used to elicit synaptic responses onto recorded neurons. Stimulating electrodes (bath ACSF‐filled patch pipettes) were placed in either the mossy fibre layer for CA3 recordings or the Shaffer collaterals for CA1 recordings, exciting axons from the dentate gyrus and CA3 respectively. Stimulation intensities were adjusted to evoke responses of approximately half‐maximal amplitude. Synaptic responses that were evoked as neurons were held at −40 mV, which allowed inhibitory (likely GABAergic) and excitatory responses to be differentiated as outward and inward currents respectively. At −40 mV, most neurons showed both evoked EPSCs and inhibitory postsynaptic current (IPSCs). Holding neurons at −60 mV eliminated the IPSC component of evoked responses, suggesting the IPSC was largely GABA_A receptor‐mediated. Spontaneous IPSCs and EPSCs were also measured during 2 min continuous voltage clamp recordings at −40 mV. The RMP of neurons was measured by averaging the potential over a 30 s period of passive current clamp recording. Drug effects on postsynaptic conductances were measured using a series of hyperpolarizing voltage steps conducted from a holding potential of −40 mV. Eight successive hyperpolarizing steps were used; the initial step hyperpolarized the membrane 10 mV, and each successive step increased by a −10 mV increment such that the final step moved the membrane from −40 to −120 mV. Each successive hyperpolarizing step was shortened by Xms to limit voltage‐mediated damage to the cell membrane. Several (>2) sets of recordings of neuronal and synaptic properties (evoked synaptic responses, etc.) were taken at 5 min intervals before drug application.

6′‐GNTI, dissolved in 10 mL ACSF to a final concentration of 1 μM, was applied via bath perfusion over a period of 2–3 min and then was washed out with ACSF. Recordings were taken immediately prior to drug application, during application, 1 min after application and successively every 5 min until drug effects washed out. The electrophysiology was performed unblinded due to practical reasons.