Hippocampal slice preparation and electrophysiological recordings

Brains were removed and immersed into ice-cold (2–4°C) artificial cerebrospinal fluid (ACSF) with the following composition (in mM): 126 NaCl, 3.5 KCl, 2 CaCl₂, 1.3 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃ and 11 glucose, pH 7.4 equilibrated with 95% O₂ and 5% CO₂. Hippocampal slices (400 μm thick) were cut with a vibrating microtome (Leica VT 1000s, Germany) in ice cold oxygenated choline-replaced ACSF and were allowed to recover at least 90 min in ACSF at room (25°C) temperature. Slices were then transferred to a submerged recording chamber perfused with oxygenated (95% O₂ and 5% CO₂) ACSF (3 ml/min) at 34°C.

Whole-cell patch clamp recordings were performed from P15-P20 CA3 pyramidal neurons in voltage-clamp mode using an Axopatch 200B (Axon Instrument, USA). To record the spontaneous synaptic activity, the glass recording electrodes (4–7 MΩ) were filled with a solution containing (in mM): 100 KGluconate, 13 KCl, 10 HEPES, 1.1 EGTA, 0.1 CaCl₂, 4 MgATP and 0.3 NaGTP. The pH of the intracellular solution was adjusted to 7.2 and the osmolality to 280 mOsmol l⁻¹. The access resistance ranged between 15 to 30 MΩ. With this solution, the GABA_A receptor-mediated postsynaptic current (GABAA-PSCs) reversed at −70mV. GABA-PSCs and glutamate mediated synaptic current (Glut-PSCs) were recorded at a holding potential of −45mV. At this potential, GABA-PSCs are outwards and Glut-PSCs are inwards. All recordings were performed using Axoscope software version 8.1 (Axon Instruments) and analyzed offline with Mini Analysis Program version 6.0 (Synaptosoft). For the acute bumetanide treatment, before recording, slices were incubated during 3h in ACSF containing 10μM of bumetanide.

Single GABA_A and N-methyl-D-aspartate (NMDA) channel recordings were performed from P7 to P30 visually identified hippocampal CA3 pyramidal cells in cell-attached configuration using Axopatch-200A amplifier and pCLAMP acquisition software (Axon Instruments, Union City, CA). Data were low-pass filtered at 2 kHz and acquired at 10 kHz. The glass recording electrodes (4–7 MΩ) were filled with a solution containing (in mM) : (1) for recordings of single GABA_A channels: GABA 0.01, NaCl 120, KCl 5, TEA-Cl 20, 4-aminopyridine 5, CaCl₂ 0.1, MgCl₂ 10, glucose 10, Hepes-NaOH 10 (9, 34); (2) for recordings of single NMDA channels: nominally Mg²⁺ free ACSF with NMDA (10 μM) and glycine (1 μM) (34). The pH of pipette solutions was adjusted to 7.2 and the osmolality to 280 mOsmol l⁻¹. Both single GABA_A and single NMDA channel currents were recorded in voltage clamp mode at different membrane potentials (from −80mV to 80mV for GABA_A and from −120 to 40 mV for NMDA) in order to visualize outwardly and inwardly directed single channel currents. Analysis of currents trough single channels and I-V curves were performed using Clampfit 9.2 (Axon Instruments) as described by (34).

Sample size for each group reported in electrophysiological experiments was at least 5 neurons from at least 3 independent experiments.

Extracellular recording of the local field potentials (LFPs) were performed from P5-P20 CA3 region of hippocampus. Extracellular 50μm tungsten electrodes (California Fine Wire) were placed in the pyramidal cell layer to record the Multi Unit Activity (MUA). The signals were amplified using a DAM80i amplifier, digitized with an Axon Digidata 1550B, recorded with Axoscope software version 8.1 (Axon instruments) and analyzed offline with Mini Analysis Program version 6.0 (Synaptosoft). The frequency of MUA was analyzed before, during and after 2 min of application of isoguvacine (10μM). In order to collate the effects across experiments, we counted the proportion of slices that showed more that 20% MUA increase from the pre-drug period as excitatory, more than 20% decrease as inhibitory. The changes within range of −20% to + 20% were considered to be ‘no response’.