All animal procedures were approved by the Harvard Medical Area Standing Committee on Animals. Parasagittal cerebellar slices, 250 μm thick, were cut from the vermis of 13–19 day-old Sprague-Dawley rats as described previously (Brenowitz and Regehr, 2003
; Brenowitz and Regehr, 2005
). The extracellular ACSF contained: 125 mM NaCl, 26 mM NaHCO3
, 25 mM glucose, 2.5 mM KCl, 1.25 mM NaH2
, 1 mM MgCl2
, 2 mM CaCl2
, and was bubbled with 95% O2
. For measurements of CF mEPSCs, CaCl2
was replaced with 2.5 mM SrCl2
was increased to 4 mM to prevent CF hyperexcitability.
Drugs were bath applied. NBQX, picrotoxin, UK14304, yohimbine, DHPG, AM251, CGP55845A, DPCPX, MCPG, phenylephrine, and isoproterenol were purchased from Tocris Bioscience (Ellisville, MO). Fura-2 and fura-FF were purchased from Invitrogen (Carlsbad, CA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Statistical significance was assessed with unpaired Student’s T-tests except where noted. Data are presented as mean±SEM.
Whole-cell voltage-clamp recordings were performed in PCs at room temperature using a Multiclamp 700B (Axon Instruments/Molecular Devices, Union City, CA) and glass electrodes (1–2 MΩ) filled with an internal solution consisting of: 35 mM CsF, 100 mM CsCl, 10 mM EGTA, 10 mM HEPES. Bicuculline (20 μM) was added to the ACSF to block inhibitory currents. NBQX (250–350 nM) was included in the external solution to reduce the amplitude of the CF-EPSC and minimize voltage-clamp errors. For experiments testing the effects of GTPγS in blocking the suppression of CF synapses by DHPG, the internal solution consisted of 145 mM CsMeSO4, 15 mM HEPES, 0.2 mM EGTA, 1 mM MgCl2, 5 mM TEA-Cl, 2 mM Mg-ATP, 10 mM Phosphocreatine (tris), 2 mM QX-314, and either 0.4 mM Na-GTP or 1 mM GTPγS.
Recordings were performed at 34°C in ACSF containing picrotoxin (20 μM) to block inhibitory currents. CGP55845A (2 μM) was added to the ACSF for experiments in which high frequency stimulus trains were presented. Glass electrodes (2–3 MΩ) were filled with an internal solution containing: 120 mM KMeSO3, 5 mM NaCl, 2 mM MgCl2, 0.05 mM CaCl2, 0.1 mM EGTA, 10 mM HEPES, 2 mM Na2ATP, 0.4 mM NaGTP, 14 mM tris-creatine phosphate (pH 7.3). For calcium imaging experiments using Fura-2, EGTA was omitted. In some experiments GTP was replaced with GTPγS (1 mM). Small hyperpolarizing currents were injected to prevent spontaneous spiking and maintain the resting membrane potential at a constant level throughout each experiment. Hyperpolarization was reduced during conditioning trains to permit robust spiking in response to PF stimulation. 13 of the 25 total LTD experiments were performed with the experimenter blind to the drug treatment.
Imaging was carried out as previously described (Brenowitz and Regehr, 2003
; Brenowitz and Regehr, 2005
). The ratiometric calcium indicators Fura-2 (200 μM, ) or Fura-FF (500 μM, ) were added to the intracellular solution to measure postsynaptic calcium transients. Images were acquired at 50 Hz with 383 nm excitation, beginning 150–250 ms prior to the onset of CF stimuli or conditioning trains. Images with excitation at the isosbestic point of Fura-2 (360 nm) or Fura-FF (357 nm) were taken immediately before and after 383 nm excitation. Fluorescence ratios were converted to calcium concentrations using a value for the KD
of 131 nM for Fura-2 (Brenowitz and Regehr, 2003
; Grynkiewicz et al., 1985
) and 3.5 μM for Fura-FF (Brenowitz et al., 2006).