ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.
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We thank G. Dabee for the production of all transgenic mice at the animal facility, H. Orignac for help with Xenopus facilities and E. Normand for stereotaxic injection. We thank the Mouse Clinical Institute (Institut Clinique de la Souris, MCI/ICS) in the Genetic Engineering and Model Validation Department who established the mouse mutant floxed P2X4mCherryIN line. We are grateful to F. Rassendren (IGF, Montpellier) for providing P2X4KO mice. We also thank the biochemistry facility of Bordeaux Neurocampus. Electron microscopy was performed at the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University. This work was supported by CNRS, University of Bordeaux, a grant LabEx BRAIN ANR-10-LABX-43 to EB-G and EB, a grant from Inserm for the generation of the mouse line to EB-G, the Louise and Alan Edwards Foundation, an awarded grant from Quebec Pain Research Network (QPRN) to TD, International Ph. D program of the IdEx of Bordeaux to EB-G and PS and DFG grant SFB1328-Z02 to FK-N.
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All experimental procedures complied with official European guidelines for the care and use of laboratory animals (Directive 2010/63/UE).
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Bertin, E., Deluc, T., Pilch, K.S. et al. Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice. Mol Psychiatry 26, 629–644 (2021). https://doi.org/10.1038/s41380-019-0641-8
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