In order to isolate NMDA EPSCs, 3 μM NBQX was added and Vh = −40 mV; in some cases, D-AP5 (50 μM) was added to confirm that synaptic responses were NMDAR mediated. When measuring RI, 100 μM spermine was added to the intracellular solution in order to prevent dilution of cytoplasmic polyamines and 50–100 μM AP5 was added to the bath solution. RI was calculated as the ratio of the slope
0–40 mV and −70 to 0 mV; the average EPSC (−70 mV) was averaged with the one following the depolarization period. Two stimulating electrodes were placed in the Schaffer collateral-commissural pathway and stimulated at 0.05–0.1 Hz to record AMPAR EPSCs and at 0.03 Hz for NMDAR EPSCs. When investigating mGluR-LTD, L-689,560 (5 μM) was added Alectinib mouse to the bath solution and (S)-3,5-DHPG (100 μM) http://www.selleckchem.com/products/abt-199.html was bath applied for 5 min. Data were acquired and analyzed with WinLTP
(Anderson and Collingridge, 2007). Average amplitudes of EPSCs over a period of 5 min immediately before and 25 min after LTD were considered to determine the magnitude of LTD. Statistical analysis was performed using the Student’s t test or one-way ANOVA as appropriate, and significance was set at p < 0.05. See Supplemental Experimental Procedures for further details. We thank P. Rubin and P. Tidball for technical assistance, R. Kahn for Arf1 plasmids, and T. Bouschet for Arf6 plasmids. This work was funded by BBSRC, MRC, The Wellcome Trust, and the WCU Program (Korea). D.L.R. designed the research and performed all biochemistry experiments and some imaging experiments; M.A. designed the research and performed all electrophysiology experiments; A.A. performed Megestrol Acetate live imaging experiments; E.B.S., N.H., J.M., and N.J. performed imaging experiments; K.M. performed molecular biology; J.R.M. advised electrophysiology experiments; G.L.C. designed research and supervised electrophysiology experiments; J.G.H. designed the research, supervised the biochemistry and imaging experiments, performed imaging experiments, supervised the project, and wrote the paper. “
“The molecular architecture of synapses determines the synaptic strength
at a given steady state. Modular scaffold proteins are decisive factors for the internal organization of synapses. They provide binding sites for the transient immobilization of neurotransmitter receptors in the postsynaptic membrane, thus setting the gain on synaptic transmission. In addition, synaptic scaffold proteins bind to cytoskeletal elements and regulate downstream signaling events in the postsynaptic density (PSD). In view of this, it is essential to know the actual numbers of scaffold proteins to assess their roles for the ultrastructure, function, and plasticity of synapses in quantitative terms. Here, we have developed nanoscopic techniques based on single-molecule imaging that enable us to gain quantitative insights into the molecular organization of inhibitory synapses in spinal cord neurons.