A genetic approach to optically investigate P2X2 receptor activation in vivo using an activity‐dependent FRET based reporter tag

Abstract

P2X receptors are ATP‐gated cation selective ion‐channels found throughout the body, including the CNS where ATP signaling is proposed to contribute to normal brain physiology, whereas aberrant P2X receptor expression may result in phenotypes relating to nervous system dysfunction, including epilepsy. However, a precise mechanistic understanding of P2X receptors in the brain does not exist and their role in epileptiform activity remains unclear. Our goal was to generate transgenic P2X2 reporter mice and, thus investigate in vivo P2X2 receptor mediated ATP signaling and its involvement in the hyperexcitability of neural networks. We have developed a non‐invasive approach to optically track the location and activation of P2X2 receptors, using a calcium sensitive FRET based reporter, with sensitivity equal to whole‐cell patch clamp recording (P2X2‐YC; Nature Methods, 2008). Here, we describe the generation of lentiviruses expressing P2X2‐YC and the DNA construct design and bacterial artificial chromosome (BAC) based transgenic method to generate mice expressing P2X2‐YC under the influence of the CaMKIIα gene promoter. We have successfully generated six ‘founder’ mouse lines verified by genomic PCR and have tested lentiviruses in vitro. The mice and viruses will be further characterised using immunohistochemistry, fluorescence imaging and electrophysiology to assess P2X2‐YC gene expression.This research was supported by the NIH.