Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca2+ Imaging.

Abstract

Calcium-permeable AMPA receptors (CP-AMPARs) and kainate receptors (CP-KARs) play crucial roles in synaptic plasticity and are implicated in various neurological processes. Current methods for identifying neurons expressing these receptors, such as electrophysiological recordings and immunostaining, have limitations in throughput or inability to distinguish functional receptors. This protocol describes a novel approach for the vital identification of neurons containing CP-AMPARs and CP-KARs using calcium imaging. The method involves loading neurons with Fura-2 AM, a calcium-sensitive fluorescent probe, KCl application to identify all neurons, and further addition of specific AMPAR agonists (e.g., 5-fluorowillardiine) in the presence of voltage-gated calcium channel blockers and NMDAR/KAR antagonists to identify CP-AMPAR-containing neurons. CP-KAR-containing neurons are identified using domoic acid applications in the presence and absence of NASPM (a CP-AMPAR antagonist). This technique offers several advantages over existing methods, including the ability to assess large neuronal populations simultaneously, distinguish between different receptor types, and provide functional information about CP-AMPAR and CP-KAR expression in living neurons, making it a valuable tool for studying synaptic plasticity and neurological disorders. Key features • The described protocol allows vital identification of neurons containing calcium-permeable AMPA (CP-AMPARs) and kainate receptors (CP-KARs). • This approach can be combined with other methods, such as electrophysiological recordings or immunostaining. • The method is fast, reproducible, and allows non-invasive simultaneous identification of numerous CP-AMPAR-/CP-KAR-containing neurons. • The described protocol can be used for pharmacological screening of different drugs, including neuroprotectors, or investigation of features of CP-AMPAR-/CP-KAR-containing neurons in health and disease.