Brains were dissected and sliced at 4°C in cutting solution consisting of the following (in mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 0.1 CaCl2, 3 MgCl2, 25 glucose, 3 myo-inositol, 2 Na-pyruvate, 0.4 ascorbic acid, continuously bubbled with 95% O2/5% CO2 (pH 7.4). Slices were incubated at 32°C for at least 30 min in a bicarbonate-buffered solution composed of the following (in mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 glucose, 3 myo-inositol, 2 Na-pyruvate, 0.4 ascorbic acid, continuously bubbled with 95% O2/5% CO2 (pH 7.4). Slices were transferred to a

recording chamber at room temperature (21–24°C) in an upright click here microscope (Olympus, Center Valley, PA) equipped with a 60×, 0.9 N.A. objective. During recordings, the standard perfusion solution consisted of the bicarbonate-buffered solution (see above) with 1 μM strychnine and 25 μM bicuculline to block inhibitory synaptic transmission. Slices were superfused at 1–3 ml/min with this external

solution. Whole-cell postsynaptic patch-clamp recordings were made from visually identified cells in the MNTB region using glass pipettes of 2–3 MΩ resistance, filled with an internal recording solution of the following (in mM): 20 CsCl, 140 Cs-gluconate, 20 TEA-Cl, 10 HEPES, 5 EGTA, 5 Na2-phophocreatine, 4 ATP-Mg, 0.3 GTP-Na, pH: 7.3, 315–320 mOsm. Series resistance (Rs) was compensated by up to 70% and the membrane potential was held at −70 mV. Excitatory postsynaptic potentials (EPSCs) were evoked by stimulating presynaptic axons with a bipolar stimulating electrode (custom-made or from FHC, Bowdoin, enough ME) placed midway between the medial border of the KU-55933 price MNTB and the midline of the brainstem. Multiclamp 700A and 700B (Axon Instruments/Molecular Devices, Union City, CA) amplifiers were used. Recordings were digitized at

20 KHz with an ITC-18 A/D converter (Instrutech, Port Washington, NY) using custom macros (written by M.A. Xu-Friedman) in Igor Pro (Wavemetrics, Lake Oswego, OR) and filtered at 8 kHz. The protocol for inducing PTP was as follows: an estimate of baseline synaptic strength was obtained through low-frequency stimulation at 0.2 Hz for 25 s. PTP was induced with a 4 s stimulus train at 100 Hz, followed by low-frequency stimulation to test for PTP. Changes in miniature EPSCs (mEPSCs) were measured by delivering the same PTP-inducing train, but without the low-frequency stimulation. For phorbol ester experiments, basal synaptic strength was evaluated by paired (50 ms interval) stimuli, repeated every 20 s. During the intertrial intervals, 10 s stretches of postsynaptic current were recorded to assess the frequency and amplitude of mEPSCs. For all recordings, the access resistance and leak current were monitored, and experiments were rejected if either of these parameters changed significantly. Alexa 594 dextran and Calcium Green-1 dextran (10 kDa, Invitrogen, Carlsbad, CA) were loaded into presynaptic boutons as described previously (Beierlein et al.