Dl ap5

All recordings were performed in a submersion chamber with continuous perfusion of oxygenated standard aCSF. Whole-cell voltage clamp recordings were carried out in blind patched CA1 pyramidal neurons. Inhibitory postsynaptic currents (IPSCs) were pharmacologically isolated with bath perfusion of DNQX (10 μM; Sigma) and DL-AP5 (50 μM; Tocris). Spontaneous IPSCs (sIPSCs) were recorded using CsCl internal solution (in mM: 140.0 CsCl, 10.0 EGTA, 5.0 MgCl₂, 2.0 Na-ATP, 0.3 Na-GTP, 10.0 HEPES; E_Cl = 0 mV). All cells were dialized for 3–7 min prior to the beginning of experimental recordings. Stability of series resistance during the recording was verified posthoc through comparing the average rise and decay time of sIPSCs. Recordings were discarded if the rise or decay time changed by >20%.

Experiments were conducted at room temperature on a Nikon TI-Eclipse inverted microscope equipped with a 60×, 1.2 NA water immersion objective and Perfect Focus System. Fluorescent dyes were excited by a Nikon Intensilight C-HGFI lamp (Neutral Density Filter 16) through excitation filters centred at 482 nm and 640 nm, using dichroic longpass mirrors (cut-off wavelength 500 nm and 650 nm). The emitted light passed emission band-pass filters ranging from 500 nm–550 nm and 660 nm–730 nm, respectively (Semrock, Rochester, NY) and was projected onto a cooled EM-CCD camera (iXonEM DU-885, Andor).
Cover slips were placed into a perfusion chamber (volume  = 500 µl) containing saline (144 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 2.5 mM MgCl₂, 10 mM Glucose, 10 mM Hepes, pH 7.5). Synaptic boutons were stimulated by electric field stimulation (platinum electrodes, 10 mm spacing, 1 ms pulses of 50 mA and alternating polarity); 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris Bioscience) and 50 µM DL-2-Amino-5-phosphonopentanoic acid (DL-AP5, Tocris Bioscience) were added to prevent recurrent activity.

The following (concentrations of) drugs were delivered through the recording aCSF solution: GABA_A receptor antagonist: Picrotoxin, PTX (50 μM), AMPA receptor antagonist NBQX (1, 2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulf- onamide hydrate; 10 μM), NMDA receptor antagonist DL-AP5 (DL-2-Amino-5-phosphonopentanoic acid; 25 μM), EAAT blocker DL-TBOA (DL-threo-beta-Hydroxyaspartic acid; 50 μM), Group II mGluR antagonist LY341495 were from Tocris Bioscience (United Kingdom). Barium chloride, BaCl₂ (100 μM) and NMDA antagonist MK-801 [(5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; 10 μM] were from Sigma-Aldrich (United States). Both DL-AP5 and MK-801 completely blocks NMDA receptors under these conditions and are considered equivalent.

CRF, CGP 55845A, DL-AP5, and DNQX were obtained from Tocris (Ellisville, MO, USA). Drugs were added to the aCSF from stock solutions to obtain known concentrations in the superfusate. Stock solutions of AP-5, CGP 55845A and CRF were prepared in distilled water, while DNQX and R121919 hydrochloride (R12) were dissolved in 100% DMSO. All drugs were applied to the bath solution to achieve the final desired concentrations. The final DMSO concentration in the bath solution did not exceed 0.15%.