11 ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders 12 including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of 13 P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in 14 P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization 15 motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, 16 cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing 17 noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. 18 The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not 19 alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of 20 the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the 21 hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal 22 excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our 23 results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and 24 memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. 25 Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in 26 various physiological and pathological contexts. 27