Eléonore Bertin

Thomas Deluc

Kjara S Pilch

Audrey Martinez

Johan-Till Pougnet

Evelyne Doudnikoff

Anne-Emilie Allain

Philine Bergmann

Marion Russeau

Estelle Toulmé

Erwan Bezard

Friedrich Koch-Nolte

Philippe Séguéla

Sabine Lévi

Bruno Bontempi

François Georges

Sandrine S Bertrand

Olivier Nicole

Eric Boué-Grabot
email: eric.boue-grabot@u-bordeaux.fr

Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice

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.

expressed in the central nervous system (CNS) with varying distributions in neurons as well as in glia [START_REF] Rodrigues | Dual presynaptic control by ATP of glutamate release via 667 facilitatory P2X1, P2X2/3, and P2X3 and inhibitory P2Y1, P2Y2, 668 and/or P2Y4 receptors in the rat hippocampus[END_REF][START_REF] Rubio | Distinct localization of P2X receptors at exci-671 tatory postsynaptic specializations[END_REF][START_REF] Kaczmarek-Hajek | Re-evaluation of neuronal P2X7 expression using 674 novel mouse models and a P2X7-specific nanobody[END_REF]. P2X

receptors are ATP-gated cation channels and their activation by ATP, co-released by neurons with other neurotransmitters [START_REF] Jo | Synaptic corelease of ATP and GABA in 677 cultured spinal neurons[END_REF][START_REF] Mori | Fast synaptic transmission mediated by P2X receptors in CA3 pyramidal cells of rat hippocampal slice cultures[END_REF] or released as a gliotransmitter by astrocytes [START_REF] Gordon | Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy[END_REF][START_REF] Pougnet | ATP P2X receptors downregulate AMPA receptor trafficking and postsynaptic efficacy in hippocampal neurons[END_REF][START_REF] Lalo | Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex[END_REF], has profound modulatory actions at synapses [START_REF] Khakh | Neuromodulation by extracellular ATP and 664 P2X receptors in the CNS[END_REF][START_REF] Boué-Grabot | Modulation of central synapses by astrocyte-released ATP and postsynaptic P2X receptors[END_REF][START_REF] Illes | Purinergic neurone-glia signalling in cognitive-related pathologies[END_REF]. Among the seven P2X subunits, P2X4 displays high calcium permeability [START_REF] Kawate | Crystal structure of the ATP-gated P2X(4) ion channel in the closed state[END_REF][START_REF] Egan | Contribution of calcium ions to P2X channel responses[END_REF] and a widespread distribution in CNS neurons and glial cells as well as in peripheral tissues [START_REF] Suurväli | P2X4: a fast and sensitive purinergic receptor[END_REF][START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF][START_REF] Yeung | Enhanced expression of the P2X4 receptor in Duchenne muscular dystrophy correlates with macrophage invasion[END_REF]. Nevertheless P2X4 expression in the brain is sparse [START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF] and its contribution to the synaptic modulation in normal conditions remains debated [START_REF] Sim | Altered hippocampal synaptic potentiation in P2X4 knock-out mice[END_REF][START_REF] Pankratov | Verkhratsky A P2X receptors and synaptic plasticity[END_REF].

A growing body of evidence suggests that upregulated P2X4 expression plays important roles in various CNS disorders including chronic pain and neurodegenerative diseases such as Alzheimer's disease (AD) or amyotrophic lateral sclerosis (ALS) [START_REF] Cavaliere | Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists[END_REF][START_REF] Franke | Involvement of P2 receptors in the growth and survival of neurons in the CNS[END_REF][START_REF] Burnstock | Purinergic signalling and disorders of the central nervous system[END_REF][START_REF] Apolloni | Membrane compartments and purinergic signalling: P2X receptors in neurodegenerative and neuroinflammatory events[END_REF][START_REF] Volonte | Purinergic contribution to amyotrophic lateral sclerosis[END_REF][START_REF] Beggs | P2X4R+ microglia drive neuropathic pain[END_REF][START_REF] Varma | Amyloid-beta induces a caspase-mediated cleavage of P2X4 to promote purinotoxicity[END_REF][START_REF] Casanovas | Strong P2X4 purinergic receptor-like immunoreactivity is selectively associated with degenerating neurons in transgenic rodent models of amyotrophic lateral sclerosis[END_REF]. In the healthy organism, P2X4 is constitutively internalized by the interaction between the adapter protein 2 (AP2) and a noncanonical endocytosis motif in the C-tail of P2X4 subunit [START_REF] Bobanovic | P2X receptor trafficking in neurons is subunit specific[END_REF]28]. As a result, P2X4 is found preferentially in intracellular compartments ensuring a low surface expression not only in neurons, but also in microglia and macrophages [29][30][31][32].

Intracellular P2X4 may promote vesicle fusion of endosomes or lysosomes [32,33]. Importantly, intracellular P2X4 pools can be mobilized and trafficked to the cell surface [31,[START_REF] Robinson | The trafficking and targeting 765 of P2X receptors[END_REF], consistent with the critical role of increased P2X4 surface expression in pathological states [START_REF] Cavaliere | Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists[END_REF][START_REF] Burnstock | Purinergic signalling and disorders of the central nervous system[END_REF][START_REF] Beggs | P2X4R+ microglia drive neuropathic pain[END_REF][START_REF] Varma | Amyloid-beta induces a caspase-mediated cleavage of P2X4 to promote purinotoxicity[END_REF][START_REF] Casanovas | Strong P2X4 purinergic receptor-like immunoreactivity is selectively associated with degenerating neurons in transgenic rodent models of amyotrophic lateral sclerosis[END_REF][START_REF] Andries | 767 Ivermectin inhibits AMPA receptor-mediated excitotoxicity in 768 cultured motor neurons and extends the life span of a transgenic 769 mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Khoja | 772 Reduced expression of purinergic P2X4 receptors increases 773 voluntary ethanol intake in C57BL/6J mice[END_REF][START_REF] Wyatt | Contribution of P2X4 receptors to ethanol intake in male 777 C57BL/6 mice[END_REF]. The specific increase in P2X4 expression and surface trafficking in spinal microglia is critical for the pathogenesis of chronic pain [START_REF] Beggs | P2X4R+ microglia drive neuropathic pain[END_REF][START_REF] Tsuda | P2X4 receptors induced in spinal 780 microglia gate tactile allodynia after nerve injury[END_REF][START_REF] Ulmann | Up-regulation of P2X4 receptors in spinal 784 microglia after peripheral nerve injury mediates BDNF release and 785 neuropathic pain[END_REF][START_REF] Coull | BDNF from microglia causes the shift in neuronal anion 788 gradient underlying neuropathic pain[END_REF][START_REF] Ulmann | P2X4 receptors mediate 790 PGE2 release by tissue-resident macrophages and initiate 791 inflammatory pain[END_REF]. Increased P2X4 expression and surface density in neurons have been observed in the hippocampus or in the spinal cord of AD or ALS mouse models, respectively, suggesting that upregulated P2X4 may contribute to synaptic dysfunction and/or cell death in AD or ALS [START_REF] Volonte | Purinergic contribution to amyotrophic lateral sclerosis[END_REF][START_REF] Varma | Amyloid-beta induces a caspase-mediated cleavage of P2X4 to promote purinotoxicity[END_REF][START_REF] Casanovas | Strong P2X4 purinergic receptor-like immunoreactivity is selectively associated with degenerating neurons in transgenic rodent models of amyotrophic lateral sclerosis[END_REF][START_REF] Andries | 767 Ivermectin inhibits AMPA receptor-mediated excitotoxicity in 768 cultured motor neurons and extends the life span of a transgenic 769 mouse model of amyotrophic lateral sclerosis[END_REF]. However, the extent of the upregulated surface P2X4 state and the cellspecific functions of P2X4 in the pathological context remain elusive.

Q1 ÀQ2 ÀQ3 ÀQ4

Here we report the development of conditional knock-in (P2X4mCherryIN) mice mimicking a pathological increase of surface P2X4. We show that the increase of P2X4 at the surface of excitatory neurons decreases anxiety, impairs memory processing, and alters activity-dependent synaptic plasticity phenomena in the hippocampus suggesting that upregulation of neuronal P2X4 observed in AD [START_REF] Varma | Amyloid-beta induces a caspase-mediated cleavage of P2X4 to promote purinotoxicity[END_REF] may have key roles in AD pathogenesis. Overall, we provide an innovative knock-in P2X4 model to study the functional contributions of upregulated P2X4 in specific cells of the nervous system but also in peripheral tissues throughout the body. to visualize surface P2X4 on living cells using anti-HA antibodies. Total HA-P2X4 was revealed after cell permeabilization using anti-P2X4 antibodies while the endogenous fluorescence of mCherry fused to P2X4 allowed for direct visualization. HA-P2X4WT is weakly expressed at the surface and is found mainly in intracellular puncta restricted to the soma and proximal dendrites of neurons [START_REF] Bobanovic | P2X receptor trafficking in neurons is subunit specific[END_REF]. In contrast, HA-P2X4mCherryIN was ~10 times more expressed at the surface of neurons. Surface P2X4mCher-ryIN puncta are uniformly distributed at the surface including distal dendrites. Co-staining with the postsynaptic marker Homer-1c-GFP revealed that surface P2X4mCher-ryIN are equally distributed in small extrasynaptic clusters and in larger clusters juxtaposed to synapses (Fig. 1g, h and Supplementary Fig. S1d). Finally, we confirmed the predominant intracellular localization of untagged P2X4WT in mouse transfected hippocampal neurons using Nodu-246, a rat monoclonal antibody recognizing the native extracellular domain of mouse P2X4 [START_REF] Bergmann | Generation and characterization of specific monoclonal antibodies and Nanobodies directed against the ATPgated channel P2X4[END_REF], whereas P2X4mCherryIN is highly and uniformly distributed at the cell surface (Fig. 1i).

Generation of knock-in mice with noninternalized

P2X4mCherryIN and expression in the brain

We generated the conditional Floxed P2X4mCherryIN knock-in mice (Floxed) by homologous recombination using a targeting vector (see layer of the olfactory bulb [START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF][START_REF] Buell | An antagonist-insensitive P2X receptor expressed in epithelia and brain[END_REF], as well as a moderate presence in cerebellar Purkinje cells [START_REF] Rubio | Distinct localization of P2X receptors at exci-671 tatory postsynaptic specializations[END_REF][START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF] of CMV mice but not of CaMK2 mice, as expected. These results showed that P2X4mCherryIN distribution in CMV mice is similar to that of P2X4 in WT mice [START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF] and the restricted detection of E.M. of anti-RFP labeling in hippocampal CA1 region or cortex revealed that P2X4mCherryIN was present at postsynaptic sites of excitatory synapses, preferentially at the edge of the postsynaptic density (Fig. 2f). At some asymmetric excitatory synapses, P2X4mCherryIN was located at both pre-and post-synaptic sites (Fig. 2f) and also found intracellularly in association with the endoplasmic reticulum membranes or occasionally with mitochondria (Supplementary Fig. S3c). This suggests that replacement of P2X4WT by P2X4mCherryIN does not alter its subcellular distribution [START_REF] Rubio | Distinct localization of P2X receptors at exci-671 tatory postsynaptic specializations[END_REF]. The identification of symmetric synapses on morphological criteria being difficult, the presence of P2X4 at GABAergic synapses was not confirmed in CMV mice. In contrast, E.M. analysis revealed the presence of P2X4mCherryIN in astrocytes of CMV mice in the astrocyte end-feet encircling endothelial cells (Supplementary Fig. S3d).

Endogenous P2X4mCherryIN fluorescence is directly observable in macrophages or after LPS-induced de novo expression in microglia of CMV mice

In mice, de novo P2X4 expression was observed in spinal microglia after nerve injury or in the brain after intracerebral lipopolysaccharide (LPS) injection in tdTomato P2xr4

reporter mice [START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF][START_REF] Tsuda | P2X4 receptors induced in spinal 780 microglia gate tactile allodynia after nerve injury[END_REF][START_REF] Ulmann | Up-regulation of P2X4 receptors in spinal 784 microglia after peripheral nerve injury mediates BDNF release and 785 neuropathic pain[END_REF][START_REF] Ulmann | P2X4 receptors mediate 790 PGE2 release by tissue-resident macrophages and initiate 791 inflammatory pain[END_REF][START_REF] Ulmann | Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus[END_REF]. We performed in vivo LPS microinjections into the hippocampus of CMV, CaMK2, Floxed, and P2X4KO mice and examined

P2X4mCherryIN fluorescence (Fig. 3 and Supplementary Fig. S4). In control saline-injected mice, no endogenous mCherry fluorescence was visible in the hippocampus of all mouse genotypes and only a faint staining was revealed for the microglial marker Iba1 (Fig. 3a). In contrast, a strong increase in Iba1 staining was detected in all LPS-injected mouse lines attesting the microglial activation (Fig. 3a,b and Supplementary Fig. S4). Remarkably, endogenous P2X4mCherryIN on the surface of these hippocampal cells 324 (Fig. 4a). In cells from P2X4KO mice, no P2X4 was 325 detected on the cell surface or intracellular (Fig. 4a). Anti-326 MAP2 antibodies revealed the specific expression of 327 P2X4mCherryIN in neurons in both CMV and CaMK2 328 transgenic lines (Fig. 4b and Supplementary Fig. S5a).

329

Using antibodies against astrocytic GFAP and microglial

Iba1 markers combined with Nodu-246 and anti-RFP, revealed the presence of surface P2X4mCherryIN in microglia and astrocytes from CMV, but not CaMK2 mice (Fig. 4c, d and Supplementary Fig. S5b). Consistently, western blotting of proteins extracted from hippocampal cultures of the different knock-in lines allowed the identification of P2X4mCherryIN proteins in the CMV and CaMK2 exclusively, whereas P2X4WT was detected in Floxed and CaMK2 hippocampal cells (Fig. 4e). Next, we examined surface and total P2X4 levels using either anti-P2X4 or anti-RFP antibodies by biotinylation assays and western blotting of hippocampal cells from the different transgenic mice. Quantification of immunoblots (Fig. 4f)

clearly indicates that P2X4WT in Floxed and CaMK2 mice is mainly intracellular (Fig. 4g), while the amount of P2X4mCherryIN on the cell surface is significantly higher in both CMV and CaMK2 cells (Fig. 4a-d). Altogether, these results confirm that substitution of P2X4WT by

P2X4mCherryIN is restricted to excitatory neurons of CaMK2 mice while in CMV mice, the substitution occurs in all cells endogenously expressing P2X4 in CMV mice. In addition, our results demonstrate that P2X4mCherryIN expression leads to a stronger surface localization of P2X4mCherryIN in both CMV and CaMK2 mice (Fig. 4g).

Increased surface density of P2X4 alters LTP and LTD at CA1 hippocampal synapses

We next examined the impact of noninternalized P2X4mCherryIN in hippocampal synaptic plasticity. Field excitatory postsynaptic potential (fEPSP) were recorded in the hippocampal CA1 region after stimulation of presynaptic Schaffer collateral axons in acute brain slices from control Floxed and both CaMK2 or CMV mice (Fig. 5a-f).

The input/output curves obtained by plotting fEPSP slopes against the amplitude of stimulation (Fig. 5b) showed no significant differences between slices from CMV or CaMK2 mice compared with control Floxed mice indicating that increased P2X4 surface receptors do not disrupt basal excitatory transmission at CA1 synapses. LTP and LTD were then evaluated. Robust LTP was induced in slices from Floxed mice with persistent potentiation (Fig. 5c,d). indicating that increased surface P2X4 does not modulate basal locomotor activity (Fig. 5g, h and Supplementary Fig. S6b). Interestingly, CaMK2 mice spent significantly more times in the central zone (red area) compared with the other groups of mice (Fig. 5i), suggesting that increased surface P2X4 density may reduce anxiety-like behavior.

Neophobia-related anxiety was additionally examined by placing a novel object in the center of the arena (Fig. 5j-l; [START_REF] Dulawa | Dopamine D4 receptor-knock-out mice exhibit reduced exploration of novel stimuli[END_REF]). The latency to first exploring the novel object was slightly lower for CaMK2 than for CMV and Floxed mice but this difference failed to reach significance (Fig. 5k). Floxed mice. Thus, mice from all genotypes were capable of processing visuo-spatial information and forming shortterm spatial recognition memory.

We next asked whether impairments in learning and memory underlie the observed synaptic plasticity deficits.

We examined spatial memory in a more cognitively challenging situation by submitting CMV, CaMK2, and Floxed

Discussion

To address pathologically increased surface P2X4 func- interacts also dynamically with various other ligand-gated ion channels such as GABA A [START_REF] Boué-Grabot | Modulation of central synapses by astrocyte-released ATP and postsynaptic P2X receptors[END_REF][START_REF] Jo | Cross-talk between P2X4 and gamma-aminobutyric acid, type 794 A receptors determines synaptic efficacy at a central synapse[END_REF][START_REF] Jo | Interplay between ionotropic receptors modulates inhibitory synaptic strength[END_REF] and may lead to NMDAR inhibition by similar crosstalk.

Behavioral phenotyping of the CMV mice did not reveal any overt alterations. In contrast, CaMK2 mice exhibited a significant decrease in anxiety-like behaviors and impaired spatial learning and memory functions. No change in locomotor, anxiety-like or cognitive functions were previously observed in P2X4KO mice [START_REF] Wyatt | Sociocommunicative and sensorimotor impairments in male P2X4-deficient mice[END_REF]. Together, these results suggest that, in contrast to the basal state, increased surface P2X4 density observed in neurons in pathological situations such as in AD [START_REF] Varma | Amyloid-beta induces a caspase-mediated cleavage of P2X4 to promote purinotoxicity[END_REF]

  extracellular action of ATP are a wide-29 spread mechanism for cell-to-cell communication in living 30 organisms through activation of P2X and P2Y receptors 31 expressed at the cell surface of most tissues, including the 32 nervous system [1]. Several P2X receptors (P2X) are These authors contributed equally: Eléonore Bertin, Thomas Deluc, Kjara S

  xxGL) was previously shown to increase the surface 128 trafficking of P2X4 without altering its functional properties 129[28,[START_REF] Toulme | Functional prop-797 erties of internalization-deficient P2X4 receptors reveal a novel 798 mechanism of ligand-gated channel facilitation by ivermectin[END_REF]. We generated a P2X4mCherryIN construct in 130 which the endocytosis motif of mouse wild-type (WT) 131 P2X4 was suppressed by swapping the last 11 amino acids 132 in the C-tail of P2X4 with the sequence coding for the red fluorescent protein mCherry (Fig.1a). ATP-evoked currents recorded from P2X4WT or P2X4mCherryIN expressing Xenopus oocytes (Fig.1b) showed that P2X4mCherryIN response amplitudes were significantly larger than those recorded from P2X4WT (Fig.1b). We next examined the surface level of P2X4WT and P2X4mCherryIN by biotinylation assays and western blotting from oocytes (Fig.1c, d). Anti-P2X4 antibodies allowed the detection of P2X4WT (70 kDa) but not P2X4mCherryIN (the anti-P2X4 epitope (amino acids 370-388) is deleted in P2X4mCherryIN constructs). P2X4mCherryIN was detected using anti-RFP antibodies showing a band at 100 kDa in agreement with the fusion of mCherry (30 kDa) to P2X4 (Fig.1c). Surface/total Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in. . . that P2X4mCherryIN is more effec-147 tively translocated to the surface than P2X4WT in Xenopus 148 oocytes (Fig. 1d, p < 0.05). 149 We next transfected hippocampal neurons and Cos cells 150 (Fig. 1e-h and Supplementary Fig. S1a-d) with extra-151 cellularly tagged HA-P2X4WT and HA-P2X4mCherryIN 152

  Fig. 2a) designed to flox the last four exons (exons 9 to 12) of P2rx4 allele (P2X4 internalization motif being located on exons 11 and 12) and followed by the insertion of a DNA fragment corresponding to the fusion of exons 9, 10, and partially 11 with mCherry cDNA. We next generated CMVCre + -P2X4mCherryIN F/F (namely CMV) mice and CaMK2Cre + -P2X4mCherryIN F/F (CaMK2) mice by breeding Floxed mice with mice either expressing the Cre recombinase under the cytomegalovirus (CMV-Cre) or the calmodulin kinase 2 (CaMK2-Cre) promoter (Supplementary Fig. S2

  displayed no obvious physical or behavioral 204 abnormalities (see Supplementary information). 205 Reverse transcription (RT)-PCR in different brain 206 regions from the different mice showed that Floxed mice Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in. . . mRNA while CMV mice only 208 expressed P2X4mCherryIN mRNA (Fig. 2c). As expected, 209 CaMK2 mice expressed both WT and knock-in mRNAs 210 since only a subset of P2X4 expressing cells (forebrain 211 excitatory neurons) replace P2X4WT by P2X4mCherryIN. 212 Next, we tested P2X4mCherryIN expression in the brain on 213 a protein level (Fig. 2d-f). The sparse expression of P2X4 214 throughout the brain observed in previous works [3, 53] was 215 recently confirmed using tdTomato p2xr4 reporter mice in 216 which the detection of cytosolic tdTomato-expressing cells 217 required the use of anti-tdTomato antibodies [15]. Not 218 surprisingly, endogenous fluorescence of mCherry fused to 219 P2X4 was not directly visible on brain slices of the different 220 knock-in mice but could be visualized using anti-RFP 221 antibodies. P2X4mCherryIN was detected in the pyramidal 222 neuron layer of the hippocampus and in the soma of neu-223 rons throughout the cortex [3, 15] in both CMV and 224 CaMK2, but not in Floxed or P2X4 knockout (P2X4KO) 225 mice (Fig. 2d). In addition, we confirmed the abundant 226 expression of P2X4mCherryIN in the epithelial glomerular 227

  P2X4mCherryIN in forebrain neurons in CaMK2 mice confirmed the specificity of Cre-dependent excision. (Supplementary Fig.S3a-b). The detection of a 100 kDa band with anti-RFP antibodies in western blots of total proteins from different brain regions confirmed the substitution of P2X4WT by P2X4mCherryIN in CMV mice (Fig.2e). The absence of detection of P2X4mCherryIN in CaMK2 mice indicated that the replacement of P2X4WT by P2X4mCherryIN is more widespread in the CMV than in CaMK2 mice, as expected. Moreover, the absence of detection of P2X4WT in Floxed or CaMK2 mice with anti-P2X4 confirmed the sparse P2X4 expression in the brain tissues (Fig.2e).

Fig. 3 Fig. 4 )

 34 Fig.2Generation of conditional P2X4mCherryIN knock-in mice and P2X4 expression in CMV-Cre or CaMK2-Cre P2X4mCher-ryIN knock-in mice. a Schematic diagram of the mouse P2xr4 gene and targeting vector strategy used to generate Floxed P2X4mCherryIN knock-in mice by homologous recombination (Floxed). By breeding Floxed P2X4mCherryIN with CMV-Cre mice or CaMK2-Cre mice, we generated constitutive CMVCre-P2X4mCherryIN (CMV) and CaMK2Cre-P2X4mCherryIN (CaMK2) to obtain a gain-of-function of P2X4 in all cells or in excitatory forebrain neurons natively expressing P2X4, respectively. b Southern blot of genomic DNA of WT and targeted allele (Floxed) using external probe (P) after digestion by MfeI or SspIenzyme (E) indicated in a. c Expression of P2X4WT and P2X4mCherryIN mRNAs by RT-PCR in cortex (Cx), cerebellum (Cb), and hippocampus (Hip) brain regions from Floxed P2X4mCherryIN, CMVCre + /Floxed P2X4mCherryIN (CMV) and CaMK2Cre + /Floxed P2X4mCherryIN (CaMK2) mice. d-f Protein expression of P2X4mCherryIN in the mouse brain revealed using anti-RFP antibodies recognizing mCherry protein. d Brain sections immunostained with anti-RFP antibodies show P2X4mCherryIN expression in the hippocampus and cortex of CMVCre-P2X4mCherryIN and CaMK2Cre-P2X4mCherryIN while no signal is visible in Floxed P2X4mCherryIN and P2X4 Knockout (KO) mice (see also Supplementary Fig.S3). Scale bar, 20 µm. e Western blotting of total proteins extracted from distinct CNS regions isolated from CMV-P2X4mCherryIN (CMV), CaMK2-P2X4mCherryIN (CaMK2), Floxed P2X4mCherryIN, and P2X4KO mice revealed with anti-RFP or anti-P2X4 antibodies. Anti-tubulin antibody was used as a loading control. OB olfactory bulb, Hip hippocampus, Cx cortex, Cb cerebellum, Hypo hypothalamus, SC spinal cord. f Electron microscopy images showing preembedding anti-RFP immunostaining in CA1 region of the hippocampus or cortex from CMV-P2X4mCherryIN and CaMK2-P2X4mCherryIN mice (see also Supplementary Fig.S3). Staining is close to the plasma membrane of pre-and post-synaptic specializations of excitatory asymmetric synapses (top). Examples of spines where P2X4mCherryIN is detected only at the postsynapse, mainly located at the edge of the postsynaptic density. t presynaptic, sp spine. Scale bar, 0.5 µm.

High frequency stimulation failed

  to trigger a potentiation in CaMK2 slices, while in CMV slices a potentiation was induced, but evoked responses returned rapidly to baseline after the induction. At 40 min post induction, LTP was absent in slices from both CMV and CaMK2 mice (Fig.5c, d). Increased surface P2X4 appears to block LTD as well (Fig.5e, f). LTD was induced in slices from Floxed, CMV and CaMK2 mice but a significant persistent synaptic depression was recorded solely in Floxed mice only (Fig.5e, f). LTD from CMV and CaMK2 slices was not significantly different from baseline. Altogether, these E. Bertin et al. increased surface P2X4 blocks LTP 382 and alters LTD at hippocampal CA1 synapses. Moreover, 383 the effects are more pronounced when surface P2X4 is 384 increased exclusively in forebrain excitatory neurons. general activity were assessed during open field exploration (Fig.5g-i). Floxed, CaMK2, and CMV mice showed similar velocity and total distance traveled in the arena,

  mice spent significantly more time 406 exploring the object than Floxed or CMV mice (Fig. 5l), 407 suggesting that anxiety is lower in these mice. To corro-408 borate these findings, the three mouse lines were tested in 409 the elevated plus maze (Fig. 5m). Compared with CMV and 410 Floxed mice, CaMK2 mice spent a greater proportion of 411 time in the open arms of the maze, confirming their 412 anxiolytic phenotype. These results indicate that neuronal 413 P2X4 is involved in the regulation of anxiety-like behavior, 414 with an increased number of P2X4 on neuronal surfaces 415 resulting in reduced anxiety. 416 Spatial recognition memory was then evaluated using a 417 modified version of the Y-maze two-trial arm discrimina-418 tion task (Fig. 5n and Supplementary Fig. S6; [57]). After a 419 single encoding phase of 10 min with only two accessible 420 arms, an inter-trial interval of 4 h resulted in a robust and 421 similar preference for the unexplored, previously inacces-422 sible, arm during the test phase in CMV, CaMK2, and

  mice to spatial discrimination tests in the eight-arm radial maze. Mice were required to locate the three constantly baited arms of the maze (Fig.5o). To provide a rigorous control over the time course of neuronal events induced by spatial learning, we trained the mice over one single day. Reference memory training consisted of four equivalent blocks of six trials separated by a 1 h interval. Each trial started with all eight arms opened and terminated when the mouse entered the third baited arm and returned to the central platform of the maze. While the number of reference memory errors (i.e., entries into unbaited arms) decreased significantly over the four training blocks in all three mouse lines (Fig. 5o), CaMK2 mice made significantly more errors than CMV and Floxed mice (Fig. 5k, p = 0.0094). Poorer performance of CaMK2 was particularly apparent in training blocks 2 and 4 compared with Floxed and CMV mice.Subsequently recent and long-term memory were evaluated by submitting mice to retrieval testing 1 and 14 days after training completion (Fig.5p). As expected, Floxed mice exhibited memory decay over time with an increased number of reference memory errors at day 14. A similar decay was observed in CMV mice, indicating that forgetting was unaffected in this mouse line. In contrast, the poorer performance of CaMK2 mice achieved upon training remained stable across delays and was not further exacerbated by the passage of time. At day 14, the number of reference memory errors was elevated but similar for all three genotypes. Thus, selective expression of noninternalized P2X4mCherryIN in excitatory forebrain neurons translated into impaired spatial memory processing.

  functions indicating that increased neuronal 474 P2X4 observed in AD models may have a key role in the 475 pathogenesis of AD [25]. Importantly, analysis of P2X4mCherryIN expression showed that substitution of P2X4WT by P2X4mCherryIN occurred in the expected brain regions in both strains. In the expression pattern of P2X4mCherryIN is 480 consistent with what was described for WT P2X4 in the 481 CNS, i.e., high expression in the olfactory epithelium and a 482 sparse expression in the hippocampus, cortex, cerebellum, 483 and spinal cord [3, 15, 53]. In addition, and in line with 484 other findings [41, 58], P2X4mCherryIN was also found 485 expressed in peritoneal macrophages of CMV mice. In 486 CaMK2 mice, P2X4mCherryIN expression was mainly 487 restricted to hippocampal and cortical regions, in line with 488 previous reports of CaMK2 promoter selectivity [59]. The 489 in vitro expression of P2X4mCherryIN construct revealed 490 that the substitution of the last 11 amino acids of the C-tail 491 of mouse P2X4 by the red fluorescent protein mCherry 492 increases surface trafficking without altering P2X4 function 493 and subcellular targeting [3, 27, 28, 30, 42, 43]. Indeed, in 494 both CMV and CaMK2 knock-in mice, E.M. confirmed that 495 P2X4mCherryIN are localized at both pre-and post-496 synaptic specializations in hippocampal or cortical excita-497 tory neurons, as expected [2, 3, 15]. Inhibitory symmetric 498 synapses are difficult to identify based on morphology criteria, thus the presence of P2X4mCherryIN at theGABAergic synapse, where P2X4 was reported to be present, remains to be shown by including specific markers[START_REF] Lalo | Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex[END_REF][START_REF] Boué-Grabot | Modulation of central synapses by astrocyte-released ATP and postsynaptic P2X receptors[END_REF][START_REF] Jo | Cross-talk between P2X4 and gamma-aminobutyric acid, type 794 A receptors determines synaptic efficacy at a central synapse[END_REF]. Surface and intracellular staining on hippocampal cultures as well as biotinylation experiments from the different knock-in mice demonstrate that substitution of P2X4WT by P2X4mCherryIN induced a significant increase in surface P2X4 in both CaMK2 and CMV neurons. In addition, P2X4mCherryIN was found to be present and upregulated at the surface of hippocampal microglia and astrocytes as well as macrophages of CMV mice. P2X4 expression in astrocytes has been previously observed, but was debated[START_REF] Stokes | P2X4 receptor function in the nervous system and current breakthroughs in pharmacology[END_REF]. Notably, P2X4mCherryIN was detected in hippocampal astrocytes in vitro, but was detected on brain slices by E.M. only.In contrast to transfected cells overexpressing P2X4mCherryIN, direct fluorescence of P2X4mCherryIN was undetectable from brain slices and hippocampal cultures of both knock-in mice, preventing the direct identification and functional characterization of P2X4mCherryIN-

Fig.

  Fig.5Surface increase of neuronal P2X4 impairs LTP and LTD at CA1 hippocampal synapses and alters anxiety, spatial learning, and memory. a Schematic drawing of the experimental protocol and example of an fEPSP recorded from the hippocampal CA1 region (Erec). fEPSP were induced by the electrical stimulation (stim) of the Schaffer collaterals (Sch) in the CA3 area and recorded in the stratum radiatum layer in brain slices of Floxed, CaMK2, and CMV P2X4mCherryIN mice. DG dentate gyrus. b The relative field slope is plotted against stimulation intensity. The input/output curves for neurons in hippocampal slices from Floxed (gray circles, n = 7), CaMK2 (orange circles, n = 7) and CMV P2X4mCherryIN (red circles, n = 6) mice show similar linear regression (mean ± s.e.m., oneway ANOVA, F (2, 18) = 0.15; p = 0.85) indicating that basal synaptic transmission is not affected by the increase of surface P2X4 expression. c, d Surface P2X4 increase impairs LTP. c Plots of normalized fEPSP slopes recorded in CA1 over time before and after the induction of LTP. LTP was induced by three tetanic trains (100 Hz, 1 s) with a 20 sec interval) in control Floxed (n = 10), CaMK2 (n = 10) or CMV mice (n = 9). d Bar graph summary showing the relative change of fEPSP slope 30-40 min after the induction of LTP in Floxed (144.7 ± 11.3% of baseline, n = 10), CMV mice (113.2 ± 5.27% of baseline, n = 9) or CaMK2 (115.6 ± 3.25% of baseline, n = 10), oneway ANOVA and tuckey's post hoc test; F (3, 25) = 9,41; p = 0.002, ***p < 0.001;. Error bars: s.e.m.; the number of mice is indicated in parentheses. e, f Increased surface P2X4 impairs LTD. e Plots of normalized fEPSP slopes recorded in CA1 over the time before and after the induction of LTD with a 1 Hz, 15 min train Floxed (n = 21), CaMK2 (n = 11), and CMV mice (n = 9). f Bar graph summary showing the relative change of fEPSP slope 30-40 min after the induction of LTD in Floxed (79.09 ± 3.63% of the baseline, n = 22, p < 0.001), CMV (84.58 ± 5.97% of baseline, n = 8, p > 0.05) and CaMK2 mice (94.24 ± 3.89% of baseline, n = 9, p > 0.05). one-way ANOVA and tuckey's post hoc test; F (3, 44) = 5.44; p = 0.0028, **p < 0.01; Error bars: s.e.m.; the number of mice is indicated in parentheses. g-i Open field assessments of control (Floxed mice, n = 24) and CaMK2 (n = 13) or CMV (n = 12) P2X4mCherryIN mice. g Examples of exploration trajectories (top view) in the empty arena for each type of mice. Peripheral and central activities are indicated by yellow and red traces, respectively. h Similar total travel distance by

  might play essential roles in the regulation of synaptic plasticity and behavior such as anxiety and learning and memory. The absence of a phenotype of the CMV mice, although in agreement with the weaker effects on the synaptic plasticity of CMV compared with CaMK2 mice, is intriguing since substitution of P2X4WT by P2X4mCherryIN in all cells natively expressing P2X4 was expected to have more pronounced effects than specific P2X4mCherryIN expression solely in forebrain excitatory neurons. However, this could be explained by temporal differences between CMV and CaMK2 promoter activation. CMV is a ubiquitous early gene promoter leading to the genetic excision of Floxed P2X4mCherryIN in the germ-line and consequently from the beginning of the embryonic development while the CaMK2 promoter is considered as an adult promoter with postnatal activity reaching its maximum around the third postnatal week [67]. Expression of P2X4mCherryIN in all cells natively expressing P2X4 during the developmental period and thereafter may have deleterious effects leading to developmental compensations. Another possibility is that the expression of P2X4mCherryIN in CMV mice in other types of neurons along with expression in glial cells may counterbalance the effect of its increased surface density specifically in excitatory neurons. Indeed, increased P2X4 expression in spinal cord microglia during neuropathic conditions triggers the release of BDNF by microglia resulting in neuronal hyperexcitability [39, 40]. In addition, microglia and BDNF have been shown to influence synaptic plasticity and learning in the brain [68]. These results suggest that increased surface P2X4 in microglia of CMV mice may promote synaptic plasticity and counterbalance the negative impact of an increase of surface P2X4 specifically in excitatory neurons of CaMK2 mice.P2X4 is present in multiple cell types in the peripheral or CNS and in various epithelial, endothelial, or immune cells.After injury and during inflammation and cell damage, high levels of ATP are released and P2X4 is strongly upregulated on cell surfaces. This upregulation seems to orchestrate key events during neurodegenerative diseases, neuropathic and inflammatory pain, ischemia-induced inflammation, alcohol inflammation in asthma, rheumatoid arthritis 626 or postsurgical liver regeneration (see for review[START_REF] Suurväli | P2X4: a fast and sensitive purinergic receptor[END_REF]). 627 These novel conditional knock-in mice defective for 628 P2X4 internalization provide a valuable tool which will 629 allow to further decipher the role of upregulated P2X4 state 630 not only in the context of neurological diseases, but also of 631 peripheral inflammation, infection as well as in lung, car-632 diac or liver functions [69-73].

  Fig. 2 Generation of conditional P2X4mCherryIN knock-in mice and P2X4 expression in CMV-Cre or CaMK2-Cre P2X4mCher-ryIN knock-in mice. a Schematic diagram of the mouse P2xr4 gene and targeting vector strategy used to generate Floxed P2X4mCherryIN knock-in mice by homologous recombination (Floxed).

	By breeding
	Floxed P2X4mCherryIN with CMV-Cre mice or CaMK2-Cre mice,
	we generated constitutive CMVCre-P2X4mCherryIN (CMV) and
	CaMK2Cre-P2X4mCherryIN (CaMK2) to obtain a gain-of-function of
	P2X4 in all cells or in excitatory forebrain neurons natively expressing
	P2X4, respectively. b Southern blot of genomic DNA of WT and
	targeted allele (Floxed) using external probe (P) after digestion by
	MfeI or SspIenzyme (E) indicated in a. c Expression of P2X4WT and
	P2X4mCherryIN mRNAs by RT-PCR in cortex (Cx), cerebellum
	(Cb), and hippocampus (Hip) brain regions from Floxed
	P2X4mCherryIN, CMVCre + /Floxed P2X4mCherryIN (CMV) and
	CaMK2Cre + /Floxed P2X4mCherryIN (CaMK2) mice. d-f Protein
	expression of P2X4mCherryIN in the mouse brain revealed using anti-
	RFP antibodies recognizing mCherry protein. d Brain sections
	immunostained with anti-RFP antibodies show P2X4mCherryIN
	expression in the hippocampus and cortex of CMVCre-
	P2X4mCherryIN and CaMK2Cre-P2X4mCherryIN while no signal
	is visible in Floxed P2X4mCherryIN and P2X4 Knockout (KO) mice
	(see also Supplementary Fig. S3). Scale bar, 20 µm. e Western blotting
	of total proteins extracted from distinct CNS regions isolated from
	CMV-P2X4mCherryIN (CMV), CaMK2-P2X4mCherryIN (CaMK2),
	Floxed P2X4mCherryIN, and P2X4KO mice revealed with anti-RFP
	or anti-P2X4 antibodies.

Anti-tubulin antibody was used as a loading control. OB olfactory bulb, Hip hippocampus, Cx cortex, Cb cerebellum, Hypo hypothalamus, SC spinal cord. f Electron microscopy images showing preembedding anti-RFP immunostaining in CA1 region of the hippocampus or cortex from CMV-P2X4mCherryIN and CaMK2-P2X4mCherryIN mice (see also Supplementary

  Surface P2X4mCherryIN expression is increased in hippocampal neurons of CaMK2 or CMV mice as well as in glial cells of CMV mice. a Representative images of surface and total P2X4WT and/or P2X4mCherryIN in Floxed, CMV, and CaMK2 primary hippocampal cell cultures reveal that surface P2X4mCherryIN is strongly increased compared with P2X4WT. Extracellular surface P2X4 and P2X4mCherryIN are visualized using Nodu-246 (green) antibodies. P2X4mCherryIN is expressed only in astrocytes and microglia of CMV mice solely. c Surface and total P2X4mCherryIN revealed using Nodu-246 and anti-RFP antibodies (red) respectively, in GFAP-positive astrocytes (green) from seven div primary hippocampal CMV cells. Nuclear marker DAPI is shown in blue. d Surface and total P2X4mCherryIN using Nodu-246 or anti-RFP antibodies (red), respectively, in microglia stained using anti-Iba1 antibodies (green) from 21 div primary hippocampal cells from CMV mice. Nuclear marker DAPI is shown in blue. e Western blotting of total proteins from primary hippocampal cells from Floxed, CMV, CaMK2, and P2X4KO mice using anti-RFP and anti-P2X4 antibodies reveal P2X4mCherryIN specifically in CMV and CaMK2 mice while P2X4WT is detected in CaMK2 and Floxed mice. f Representative immunoblots of total and biotinylated surface proteins from primary hippocampal cells isolated from Floxed, CMV, and CaMK2 mice revealed with either anti-P2X4, anti-RFP or both antibodies, respectively. As in e, total proteins of P2X4KO macrophages were used as a negative control. g Quantification of surface/total P2X4WT and P2X4mCherryIN in hippocampal cells. Bars represent mean ± s.e.m. of

	Intracellular P2X4WT is revealed by anti-P2X4 antibodies (red) in
	Floxed and P2X4KO mice while intracellular P2X4mCherryIN is
	revealed using anti-RFP antibodies (red) from CaMK2 and CMV
	mice. Staining was performed at 21 div. Scale bars, 10 µm. b High
	density of surface P2X4mCherryIN using Nodu-246 (green) is
	detected in CaMK2 and CMV neurons identified using neuronal
	MAP2 marker (magenta). Surface P2X4WT is almost absent in Floxed
	mice and not detected in P2X4KO neurons (see Supplementary
	Fig. S5). c, d the surface/total ratio of P2X4WT (gray bar) in Floxed and CaMK2
	mice and P2X4mCherryIN in CaMK2 (orange bar) or CMV mice (red
	bar) and show that P2X4 surface expression is strongly increased in
	cells expressing P2X4mCherryIN mice compared with those expres-
	sing P2X4WT. The number of independent experiments is indicated
	by circles. one-way ANOVA, and Bonferroni's post hoc test, F (3, 20)
	= 17.2; *p < 0.05, **p < 0.01, ***p < 0.001.
	tions, we created a Floxed knock-in P2X4mCherryIN
	mouse line and generated two distinct conditional
	P2X4mCherryIN	lines,	expressing	noninternalized
	P2X4mCherryIN either in all cells natively expressing
	P2X4 (CMV mice) or in excitatory forebrain neurons
	(CaMK2 mice), respectively. Both CMV and CaMK2
	P2X4mCherryIN knock-in mice were viable, reproduced

normally and displayed no manifest phenotypic issues. The key finding of this study is that the increased surface density of P2X4 in forebrain excitatory neurons is a major regulator of hippocampal synaptic plasticity, learning and memory Fig. 4

5

  Surface increase of neuronal P2X4 impairs LTP and LTD at CA1 hippocampal synapses and alters anxiety, spatial learning, and memory. a Schematic drawing of the experimental protocol and example of an fEPSP recorded from the hippocampal CA1 region (Erec). fEPSP were induced by the electrical stimulation (stim) of the Schaffer collaterals (Sch) in the CA3 area and recorded in the stratum radiatum layer in brain slices of Floxed, CaMK2, and CMV P2X4mCherryIN mice. DG dentate gyrus. b The relative field slope is plotted against stimulation intensity. Increased surface P2X4 impairs LTD. e Plots of normalized fEPSP slopes recorded in CA1 over the time before and after the induction of LTD with a 1 Hz,[START_REF] Xu | P2X4 receptor reporter mice: sparse brain expression and feedingrelated presynaptic facilitation in the arcuate nucleus[END_REF] min train Floxed (n = 21), CaMK2 (n = 11), and CMV mice (n = 9). f Bar graph summary showing the relative change of fEPSP slope 30-40 min after the induction of LTD in Floxed (79.09 ± 3.63% of the baseline, n = 22, p < 0.001), CMV (84.58 ± 5.97% of baseline, n = 8, p > 0.05) and CaMK2 mice (94.24 ± 3.89% of baseline, n = 9, p > 0.05). one-way ANOVA and tuckey's post hoc test; F (3, 44) = 5.44; p = 0.0028, **p < 0.01; Error bars: s.e.m.; the number of mice is indicated in parentheses. g-i Open field assessments of control (Floxed mice, n = 24) and CaMK2 (n = 13) or CMV (n = 12) P2X4mCherryIN mice. g Examples of exploration trajectories (top view) in the empty arena for each type of mice. Peripheral and central activities are indicated by yellow and red traces, respectively. h Similar total travel distance by each mouse line in the empty arena during 10 min was similar. i CaMK2 mice spent more time in the central arena compared with Floxed and CMV mice, one-way ANOVA and Bonferroni's post hoc test; F (2, 46) = 5.139; p = 0.0097, **p < 0.01. j-l One object exploration test in control (Floxed mice, n = 24) and CaMK2 (n = 13) or CMV (n = 12) P2X4mCherryIN mice. j Examples of exploration trajectories (top view) for each type of mice. Activities far away or at proximity of the object are indicated in yellow or red, respectively. k Latency to first interaction with the novel object is shown for each type of mouse. One-way ANOVA and Bonferroni's post hoc test, F (2, 46) = 2.868; p = 0.067. l CaMK2 mice spent more time exploring the novel object than Floxed and CMV mice. One-way ANOVA, F (2, 46) = 10.27; p = 0.0002; **p < 0.01, ***p < 0.001. m Mean percent of time spent in the open arms of the elevated plus maze for Floxed mice (n = 22), CaMK2 (n = 14), and CMV (n = 11) mice. CaMK2 spent more time in the open arms, indicating reduced anxiety compared with the other mouse lines. One-way ANOVA and Bonferroni's post hoc test, F (2, 44) = 5.186; p = 0.0095; *p < 0.05. n Spatial recognition memory evaluated in the Y-maze. Floxed (n = 24), CaMK2 (n = 13),or CMV (n = 12) mice were able to recognize the novel arm of the maze that was rendered accessible 4 h after encoding (one-way ANOVA, F (2, 46) = 0.097; p = 0.907). o, p Spatial learning and memory assessment using the eight-radial maze in Floxed mice (gray), CaMK2 (orange) and CMV (red) mice. The number of mice in each group is indicated in parentheses. o Learning occurred over one single day and consisted of four blocks of six trials separated by a 1 h interval. The location of the three constantly baited arms of the maze is indicated in red. CaMK2 mice committed more reference memory errors (visits to unbaited arms) compared with Floxed and CMV mice.

	519	expressing cells. Fluorescence of P2X4mCherryIN was
	520	directly visible solely in isolated peritoneal macrophages of
	521	CMV mice in agreement with its high basal expression in
	522	macrophages [41]. Interestingly, following LPS injection in
	523	the hippocampus to induce microglial activation and
	524	increase de novo P2X4 expression [15], endogenous
	525	The input/output curves for fluorescence of P2X4mCherryIN was revealed in Iba1-
	526	neurons in hippocampal slices from Floxed (gray circles, n = 7), positive microglia exclusively in CMV mice. These results
	527 528 529	CaMK2 (orange circles, n = 7) and CMV P2X4mCherryIN (red cir-indicate that the P2X4mCherryIN fluorescence may repre-cles, n = 6) mice show similar linear regression (mean ± s.e.m., one-sent a unique tool to directly monitor increased P2X4 way ANOVA, F (2, 18) = 0.15; p = 0.85) indicating that basal synaptic transmission is not affected by the increase of surface P2X4 expression in pathological models such as chronic pain,
	530	expression. c, d Surface P2X4 increase impairs LTP. c Plots of nor-AD, ALS, alcohol intake, inflammation, epilepsy, ischemia,
	531 532 533	malized fEPSP slopes recorded in CA1 over time before and after the or brain trauma [14, 19, 22, 23, 25, 26, 35-38, 55, 61]. induction of LTP. LTP was induced by three tetanic trains (100 Hz, LTP and LTD phenomena are widely recognized as 1 s) with a 20 sec interval) in control Floxed (n = 10), CaMK2 (n = 10) or CMV mice (n = 9). d Bar graph summary showing the relative crucial molecular mechanisms underlying cognitive func-
	534	change of fEPSP slope 30-40 min after the induction of LTP in Floxed tions such as learning and memory [62]. Although activa-
	535 536 537	(144.7 ± 11.3% of baseline, n = 10), CMV mice (113.2 ± 5.27% of tion of P2X receptors by glial ATP was recently shown to baseline, n = 9) or CaMK2 (115.6 ± 3.25% of baseline, n = 10), one-directly modulate glutamatergic synaptic strength [10], P2X way ANOVA and tuckey's post hoc test; F (3, 25) = 9,41; p = 0.002, ***p < 0.001;. Error bars: s.e.m.; the number of mice is indicated in receptors exert modulatory actions exclusively during
	538 539 540 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567	N C O R R E C T E D P R O O F activity-dependent plasticity at central synapses and do not influence basal synaptic transmission [17, 18, 63]. Previous studies have shown P2X4 modulating hippocampal LTP via using P2X4KO mice revealed that LTP in CA1 neurons was slightly reduced compared with WT mice and that poten-tiation of P2X4 by ivermectin enhanced LTP only in WT mice [17]. Results suggested that P2X4 may enhance the content of NR2B subunits in synaptic NMDARs [63]. In contrast, an other study showed that the pharmacological blockade of P2X4 facilitated the induction of NMDAR-dependent LTP indicating an inhibitory impact of P2X4 [18, 64]. Furthermore, increased P2X4 expression was reported in AD models [25] suggesting that P2X4 might also contribute to synaptic dysfunction and memory deficits. Field potential recordings show that P2X4mCherryIN expression does not change the basal excitatory transmis-sion at CA1 synapses but causes impairments in both LTP and LTD in agreement with a negative impact of P2X4 on synaptic plasticity. Surprisingly, but in full agreement with our behavioral results, these deficits were stronger in CaMK2 than in CMV mice. Impaired LTP observed in CaMK2 mice occurred immediately suggesting a deficit of LTP induction. In contrast, in CMV mice, LTP was induced but progressively returned to baseline, rather indicating a deficit in LTP maintenance. Since basal excitation mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is unchanged, and LTD or LTP in parentheses. e, f U hippocampal CA1 neurons are mainly initiated post
	568	synaptically by NMDAR [62], our results suggest that
	569	increased P2X4 in CA1 neurons might alter NMDAR

*p < 0.05, **p < 0.01. Paired two-way ANOVA and Bonferroni's post hoc test, Time effect, F (3, 255) = 205.3, p < 0.0001. Paired two-way ANOVA, genotype effect, F (2, 85) = 4927, p = 0.0094. p Retrieval testing 1 (recent) or 14 (long-term) days after initial training in the eight-arm radial maze for all three mouse lines. Performance achieved on the fourth training block (D0) is shown for comparison. The poorer performance exhibited by CaMK2 mice (higher number of errors) was not exacerbated by the passage of time. *p < 0.05, paired two-way ANOVA and Bonferroni's post hoc test, delay effect, F (1, 35) = 11.70; p = 0.0016. All data are presented as mean ± s.e.m. 541 N-methyl-D-aspartate receptor receptors (NMDAR), how-542 ever these findings remained controversial. The first work 570 function. P2X4 is highly permeable to calcium and can 571 mediate a strong calcium influx at the resting membrane potential that may induce NMDAR inactivation [64]. P2X4

Acknowledgements We thank G. Dabee for the production of all 634 transgenic mice at the animal facility, H. Orignac for help with

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S Teyssedre

M Mouaci

Saidi

Space Charge in Electron-Beam Irradiated Cross-Linked Polyethylene (XLPE): Charge Implantation and

Release from the Bulk

Space Charge in Electron-Beam Irradiated Cross-Linked Polyethylene (XLPE): Charge Implantation and Release from the Bulk 1 C. Mouchache, 2 V.Griseri, 1 N.Saidi-Amroun, 2 G. Teyssèdre, 1 S. Mouaci and 1 M. Saidi 1 Material Physics Laboratory, Physics Faculty, University of Sciences and Technology (USTHB) BP 32 El-Alia, Bab-Ezzouar, Algiers, Algeria, 2 LAPLACE, Université de Toulouse and CNRS, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France.

Abstract-. The aim of the present work is to study the dynamics of the space charge in crosslinked polyethylene (XLPE)

induced by electron beam irradiation. For this purpose three energies have been chosen so as to implant charges in a controlled way, in the volume, at different depths not too close to the electrodes. Pulsed Electro Acoustic (PEA) method was used to measure space charge in each sample, at the end of the irradiation process, in ambient conditions. The space charge measurements show the existence of two electron implantation sites after irradiation, one in the bulk at a depth depending on the irradiation energy and the other near the irradiated surface and a fixed position (25 µm). The application of the electric field shows that the charge implanted in the volume tends to be evacuated during the polarization. The charge accumulated persists during the relaxation and is stable after 1 hour. The current measurements reveal the impact of the irradiation in XLPE sample: the steady current measured on the irradiated sample is substantially higher compared to the one recorded on the non-irradiated specimens. Also a change in conduction mechanism is observed. The current recorded on XLPE irradiated weakly diverges from linearity with the electric field in the field range up to 40 kV/mm.

I. INTRODUCTION

Cross-linked polyethylene (XLPE) is one of the most used electrical polymeric insulation for DC power cables for its good electrical and thermal properties. The issues with the presence of space charge in XLPE have led to a large amount of research [START_REF] Teyssedre | Charge distribution and electroluminescence in cross-linked polyethylene under dc field[END_REF][START_REF] He | Space charge behavior in XLPE cable insulation under ac stress and its relation to thermo-electrical aging[END_REF] into the way to study the electric behavior and the way to improve material compounding. Space charge plays an important role in aging of XLPE and strongly affects its electrical characteristic [START_REF] Zhang | Evidence of strong correlation between space-charge buildup and breakdown in cable insulation[END_REF], therefore the behavior and the dynamic of apparition and accumulation is continuously investigated. When XLPE is submitted to a high DC field a space charge is developed in the bulk. However, many processes can be involved in charge accumulation and unravelling their nature is tricky.

An electron beam represents an alternative ways to build up a space charge in insulating materials, as reported using a recently built irradiation means [START_REF] Griseri | Charge build-up and transport in electron beam irradiated polymers in a new irradiation chamber[END_REF][START_REF] Banda | Polarization of electron-beam irradiated LDPE films: contribution to charge generation and transport[END_REF]. The irradiation by electrons allowed to deposit charges at specific location, and to control the quantity of deposited charge with varying the beam current or the time specimen is exposed to irradiation. This constitutes a marked difference compared to charge implantation with DC field.

In this study of space charges in XLPE, electronic charges where deposited at different depths using an electron beam. We focus on charges deposited in the bulk of XLPE materials. To probe the distribution and the dynamic of charge implanted in the bulk after the irradiations and during the polarisation under dc field, the pulsed electro-pulsed acoustic (PEA) method has been chosen. Isothermal charging current measurements on XLPE have been done using different electric field levels up to 40kV/mm at different temperatures. Experimental results were used to determine the mechanism of conduction on both irradiated and non-irradiated sample and evaluate the difference induced by the irradiation.

II. EXPERIMENTAL SET-UP

A. XLPE Samples

The specimens were taken from rolls issued from peeled HV cable produced in the frame of the European project Artemis [START_REF] Fothergill | Electrical, microstructural, physical and chemical characterization of HV XLPE cable peelings for an electrical aging diagnostic data base[END_REF][START_REF] Teyssedre | Semi-quantitative analysis of photoluminescence in thermoelectrically aged cables: II-Analysis of a population of cables[END_REF]. The cable peelings were 150 µm in thickness and 80 mm wide. Prior to peeling, the cable, with 15mm thick insulation, was submitted to an electrical and thermal aging under 225 kVac voltage with 90°C at the inner screen. More information on samples and peelings are available elsewhere [START_REF] Teyssedre | Semi-quantitative analysis of photoluminescence in thermoelectrically aged cables: II-Analysis of a population of cables[END_REF]. Electrical and structural characterizations of the aged cable were achieved in a previous work and it was considered that the induced ageing is negligible [START_REF] Griseri | Dynamic of space charge in electron-beam irradiated cross-linked polyethylene[END_REF].

The sample was irradiated with an electron beam using irradiation chamber called Matspace, which is equipped with an electron gun with energy in the range [10-100 keV] [START_REF] Griseri | Analysis of the Pulsed Electroacoustic signal treatment recorded on electron beam irradiated dielectrics[END_REF]. The irradiation was performed under vacuum of about 10 -6 mbar, we can consider that the irradiation to be homogeneous on the sample, with a flux of 1 nA/cm 2 . The irradiation time was limited to 5 minutes to avoid surface discharges.

B. Space charge measurements

Space charge measurements were performed by classic pulsed electro acoustic (PEA) [START_REF] Maeno | Measurement of spatial charge distribution in thick dielectrics using the Pulsed Electroacoustic method[END_REF][START_REF] Li | Pulsed Electroacoustic method for measurement of charge for accumulation in solid dielectrics[END_REF] method. The test bench is based on a PEA cell developed in the Laboratory. This technique is currently used to measure the distribution of the charge in solid dielectric materials.

The specimen was placed between a semiconducting material layer (carbon black doped polymer) acting as high voltage electrode and an aluminum bottom electrode to the ground. The measurements were carried out under dc field of 40 kV/mm in air at a controlled temperature of 25 °C. The polarization was applied to all samples for 10000 s with a depolarization time of 3600 s. The space charge profile was not recorded during the application ramp at 1 kV/mm.s -1 for electric field to reach the desired voltage.

C. Current measurements

The charging current measurements were performed in air at different temperature ranging from 20 to 80 °C by step of 20 °C, with a various DC voltage varying from 5 to 40 kV/mm for each temperature. Gold electrodes of 20 mm diameter and 30 nm thickness were deposited on both faces of the samples. The sample was then placed between two brass electrodes for the measurements. The protocol used in the measurements consisted of 10000 s of polarization and a depolarization time lasting 3600 s.

III. RESULTS AND DISCUSSION

A. Charge implantation profiles

Fig. 1 shows space charge distributions obtained in air just after the electronic irradiation in XLPE, with different irradiation energies of 60, and 80 keV. Peaks detected on the electrodes (abscissa 0 and 150µm) represent the image charges. As we can see, a broad negative charge peak is detected in the bulk at different depth as function of the energy of electrons. For the irradiation with 60 keV the peak maximum is observed at 65 µm from the irradiated surface. With the increase of the energy to 70 keV the electrons penetrate deeper, the peak maximum being detected at 80 µm. After the irradiation under 80 keV the peak is observed at about 100 µm. The profile for the irradiation under 80 keV is with a shoulder near the ground electrode. It gives the impression that some charges have drifted to the ground electrode during or after thermalization. During irradiation, one of the faces of the sample is at floating potential. As a result, a high field region settles between the region of implanted charges and the ground electrode, and this space-charge induced field is higher as the charge region is close to the ground. This feature main explain the drift of negative charges for the highest electron beam energy used here.

On the other hand a negative charge is detected near to the surface where the maximum of the peak observed at 25 µm. This deposited charge its common for all irradiations used. The presence of this unexpected charge near the surface is still not clearly understood. It could correspond to drift back of implanted electrons, to artefacts in the irradiation chamber producing low energy electrons, or to an evolution of the surface of the films during their storage time.

B. Space charges under DC fields

The measurements of space charge on non-irradiated XLPE performed under polarization using DC field of 40 kV/mm for 1 hour is shown in Fig. 2. Negative charge injection appears clearly from the ground electrode. Over time the negative charge grows slowly and the image charge on the ground electrode decreases. Also the injection of a small amount of positive charges near to the anode is detected, but without evolving much with time.

The space charge profiles obtained in Volts-off for 1 h are plotted in Fig. 2. The negative charges injected during the polarization step appear spread nearly homogeneously into the all insulation. The charge decays uniformly with time. The positive induced charges on the electrodes representing the image charge are also decreasing slowly during the relaxation. In the last profile recorded after 1 hour of relaxation a negligible amount of negative charges is still detected in the volume, and we can consider that the charges are totally recombined or extracted. During polarization of the sample irradiated under 70 keV, Fig. 3, the distribution of the space charge shows much change in the 3 h charging time. Indeed, the charge previously stored starts to decrease as soon as the field is applied. The initial peak vanishes after about 1 h of polarization. At the same time an accumulation of negative charge near to the irradiated surface (at 25 µm from it) is detected. This accumulation of negative is added to previously stored negative charge at this depth.

Once the initial peak has completely disappeared, a prominent positive charge peak appears at 50 to 120 µm from the ground electrode. The peak maximum is observed at about 75 µm; the peak grows continuously during the polarization. The influence charges on both electrodes are also changing continuously during the polarization due to internal charges redistribution. A small amount of positive heterocharge is detected in the non-irradiated region.

During electron-beam irradiation primary electrons are deposited and electron-hole pairs are created due to the energy deposition. In fact the density of electron-hole pairs creation is theoretically much higher than primary electrons deposition, the energy amount to create a pair being taken as 30 eV [START_REF] Sessler | Models of charge transport in electron-beam irradiated insulators[END_REF]. Most of the pairs created presumably disappear by geminate recombination but of them lead to charge trapping with a zero net charge as long as the charges don't move. With the application of a DC field, redistribution of the charges appears and net positive charges can be revealed. In the configuration used for applying the field, electrons are drifted towards the right electrode, i.e. in the irradiated region and positive charges to the non-irradiated region. As transport is most effective in the irradiated region, negative charges tend to be extracted at the anode or trapped nearby it whereas positive charges do not move substantially. This leads to the build-up of a net positive charge.

During Volt-off, Fig. 4, it is obvious that the positive charge created when the DC field is applied decreases very slowly. The same behavior holds for the net charge peaks near cathode and anode. The distribution of space charge during relaxation is same as in the last profile observed during polarization. An electrostatic equilibrium seems to be reached between the different kinds of charges. Results for different e-beam energy, not shown here for the sake of space saving show similar trends, i.e. change of the sign of the charge from negative to positive followed by stable charge in volts-off.

C. Charging current

Charging currents were measured for about 10000s under DC field of 40 kV/mm at a temperature of 20°C. The charging current recorded is plotted on Fig. 5, for both irradiated under 70 keV and non-irradiated samples. Just after voltage application, a fast decrease in current is observed (Fig. 5) in the non-irradiated sample tending to a stable value after about 4000 s. For the irradiated specimen the current deceases continuously over the 3 h charging time, and it seems not reaching a steady state value. Because of this strong difference in transient currents, there is a crossover of the curves, the final current being higher for the irradiated material. In these experiments, currents were measured consecutively at different field steps followed by discharging (see below). The presence of slowly moving charges inside the irradiated sample certainly brings complex response when submitted to step voltages followed by discharging.

Fig. 6 shows the evolution of current density vs. electric field at a temperature of 20°C for irradiated and non-irradiated XLPE. The values of current are taken after 3 h of polarization for each sample (even though the charging current is still decreasing). Between each polarization step the specimen was short-circuited for about 1 h. A linear fitting was applied in order to the determine the dominating regime of conduction.

In non-irradiated XLPE sample we find two linear regions; the intersection of these lines defines a threshold field [START_REF] Montanari | Space charge phenomenology in polymeric insulating materials[END_REF] between two conduction regimes at low and high field. The threshold field is at 20 kV/mm. The slope at low field is about 1.4. We consider that the conduction is dominated by ohmic mechanism. At high field the slope is estimated to be equal to 3.6 which is indicative of conduction process governed by space charge limited current.

The current density vs. electric field characteristic on the irradiated XLPE is very different from that of the reference sample. A much slower field dependence of the current appears, with a slope of 0.65 and no transition of mechanism is detected. The current here is modified by the presence of internal charges generated by irradiation. The highest value of current density at 5 kV/mm compared to the one at 10 kV/mm, can be explained by the transport of charge deposited by the electron beam encompassing excess electrons deposited as well as electronic or ionic carriers created upon energy deposition.

IV. CONCLUSION

Two charge clouds have been observed in XLPE peelings during and after e-beam irradiation. One of the charge clouds appears in the bulk and corresponds to primary electron deposition. The second one is near the irradiated face; the charge appears very stable there. Several reasons may explain this feature: either positive charges are accumulated during polarisation and persist during relaxation or traps are formed near the surface during long-term storage of cable peelings or blocking conditions are at play for charge release. Current transients and current-field characteristics show that the electrical properties are substantially influenced by the irradiation process. It is obvious that application of DC field for 1h did not erase the history of the samples regarding irradiation effects. The open question is whether only electrostatic effects are at play during irradiation or should we consider an evolution of the trapping and transport sites, i.e. chemical evolution of the material. This will be investigated in the next steps.

Fig. 1 .

 1 Fig. 1. Space charge profiles recorded on a XLPE after irradiation under 60, 70 and 80 keV electron beam for 5 min with a flux of 1 nA/cm². The film surfaces are at 0 and 150µm.

Fig. 2 .

 2 Fig. 2. Space charge profile recorded on a non-irradiated XLPE, during (a) 1 h of polarisation under 40 kV/mm and (b) 1 h of relaxation in Volt-off. Arrows indicate the trend regarding charge evolution in time.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Space charge profile recorded on a XLPE, after irradiation by electrons under 70 keV for 5 min. during 3 h of polarization under 40 kV/mm. Irradiated face to the right.

Fig. 5 .

 5 Fig. 5. Charging current measured in 10000 s at 20°C and 40 kV/cm for nonirradiated and irradiated 70 keV XLPE.

Fig. 6 .

 6 Fig. 6. Current density as function of electric field in log-log scale for nonirradiated and 70 keV irradiated XLPE at 20 °C

ACKNOWLEDGMENT

The work is supported by French-Algerian Tassili research program in the frame of Partenariat Hubert Curien (PHC).