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AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, a major subtype of ionotropic glutamate receptors, mediate the majority of fast excitatory synaptic transmission in the mammalian brain. The constitutive and regulated trafficking of AMPA receptors into and out of excitatory synapses ensures rapid responses to synaptically released glutamate and provides a mechanism for synaptic plasticity. To permit the direct, quantitative, real-time measurement of native AMPA receptor trafficking in live neurons, we designed and utilized a membrane-impermeable, photoreactive AMPA receptor antagonist to rapidly and irreversibly inactivate surface receptors with ultraviolet (UV) light. The photoreactive antagonist, 6-azido-7-nitro-1,4-dihydroquinoxaline-2,3-dione (ANQX), is an aryl azide that, when irradiated with UV light, becomes a highly reactive nitrene that can covalently cross-link to and thus irreversibly antagonize bound AMPA receptors. Thus, ANQX provides a means of rapidly silencing surface-exposed AMPA receptors. Combined with a functional AMPA receptor assay, such as continuous recording of AMPA receptor–mediated ionic currents, ANQX provides a means of directly monitoring native AMPA receptor trafficking in real time.