Isolating AMPAR-mediated mEPSCs in Neurons

AMPAR-mediated mEPSCs were isolated by voltage-clamping neurons at −70 mV and by supplementing the ACSF with TTX citrate (1 μM, Abcam), the GABA (A) receptor antagonist picrotoxin (50 μM, Sigma-aldrich), and the NMDA receptor antagonist D, L-2-amino-5 phosphonopentanoic acid (100 μM, AP5, Abcam)^{62 (link)}. The internal solution contained: 100 mM CsCH₃SO₃, 15 mM CsCl, 2.5 mM MgCl₂, 5 mM QX-314-Cl, 5 mM tetra-Cs-BAPTA, 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na-GTP, and 0.5% (w/v) neurobiotin with pH adjusted to 7.25 with 1 M CsOH and osmolarity adjusted to ~295 mOsm with sucrose.

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Nagendran T., Larsen R.S., Bigler R.L., Frost S.B., Philpot B.D., Nudo R.J, & Taylor A.M. (2017). Distal axotomy enhances retrograde presynaptic excitability onto injured pyramidal neurons via trans-synaptic signaling. Nature Communications, 8, 625.

Publication 2017

TTX citrate picrotoxin

Amino acid Bapta Citrate Cscl Gaba a receptor antagonist Hepes Mgcl2 Neurobiotin Neurons Nmda receptor Osmolarity Picrotoxin Qx 314 Sucrose Tetra

Corresponding Organization : UNC/NCSU Joint Department of Biomedical Engineering

Other organizations : Allen Institute, Allen Institute for Brain Science, University of Kansas Medical Center

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Variable analysis

independent variables

Voltage-clamping neurons at -70 mV
Supplementing the ACSF with TTX citrate (1 μM, Abcam)
Supplementing the ACSF with the GABA (A) receptor antagonist picrotoxin (50 μM, Sigma-aldrich)
Supplementing the ACSF with the NMDA receptor antagonist D, L-2-amino-5 phosphonopentanoic acid (100 μM, AP5, Abcam)

dependent variables

AMPAR-mediated mEPSCs

control variables

Internal solution containing: 100 mM CsCH3SO3, 15 mM CsCl, 2.5 mM MgCl2, 5 mM QX-314-Cl, 5 mM tetra-Cs-BAPTA, 10 mM HEPES, 4 mM Mg-ATP, 0.3 mM Na-GTP, and 0.5% (w/v) neurobiotin with pH adjusted to 7.25 with 1 M CsOH and osmolarity adjusted to ~295 mOsm with sucrose

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