Electrophysiological Recordings

Whole-cell patch-clamp recordings were made from Purkinje neurons. NMDA receptor-mediated evoked excitatory postsynaptic currents (eEPSCs) were recorded at +40 mV from these neurons visually identified by infrared-differential interference contrast (IR-DIC) microscopy using a 40× water immersion objective. Perfusion solution contained GYKI53655 (30 μM), to block AMPA receptors, and bicuculline (10 μM), to block GABA_A receptors. In experiments involving AMPA receptor-mediated currents, performed at −70 mV, no GYKI53655 was used, but D-AP5 (50 μM) was included to block NMDA receptors. To evoke eEPSCs, electrical pulses were delivered to granule cells axons (parallel fibers) using a monopolar electrode placed in the molecular layer at a frequency of 0.2 Hz. Patch electrodes were made from borosilicate glass and had a resistance of 4–7 MΩ when filled with (mM): 120 CsCl, 8 NaCl, 1 MgCl₂, 0.2 CaCl₂, 10 HEPES, 2 EGTA and 20 QX-314 (pH 7.2, 290 mOsm). A 40 ms paired-pulse stimulation protocol was used for pair pulse ratio (PPR) analysis. Neurons were voltage clamped, using a Multiclamp 700B amplifier (Molecular Devices, Foster City, CA, USA). Access resistance was regularly monitored during recordings, and cells were rejected if it changed >15% during the experiment. Data were filtered at 2 kHz, digitized at 10 kHz, and stored on a computer using pClamp software (Molecular Devices). Synaptic failures were identified as the lack of synaptic responses after presynaptic stimulation with the amplitude of these responses being no different from basal noise amplitude.