Marie Gleizes
email: marie.gleizes@inserm.fr

Caroline Fonta
email: caroline.fonta@cnrs.fr

Lionel G Nowak
email: lionel.nowak@cnrs.fr

Inhibitors of ectonucleotidases have paradoxical effects on synaptic transmission in the mouse cortex

Keywords: CD73, CD39, MLS-0038949, ARL 67156, MSCA-1, ALPL

published or not. The documents may come

INTRODUCTION

Extracellular adenosine plays a prominent role in the regulation of several functions of the central nervous system. Adenosine shows hypnogenic [START_REF] Haulică | Preliminary data on the possible hypnogenic role of adenosine[END_REF][START_REF] Porkka-Heiskanen | Brain site-specificity of extracellular adenosine concentration changes during sleep deprivation and spontaneous sleep: an in vivo microdialysis study[END_REF][START_REF] Fredholm | Actions of adenosine at its receptors in the CNS: insights from knockouts and drugs[END_REF] and antinociceptive [START_REF] Post | Antinociceptive effects in mice after intrathecal injection of 5'-N-ethylcarboxamide adenosine[END_REF][START_REF] Johansson | Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor[END_REF][START_REF] Zylka | Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine[END_REF] actions. In pathological conditions such as ischemia and epilepsy, extracellular adenosine displays neuroprotective [START_REF] Dunwiddie | The role and regulation of adenosine in the central nervous system[END_REF][START_REF] Johansson | Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor[END_REF][START_REF] Gomes | Adenosine receptors and brain diseases: neuroprotection and neurodegeneration[END_REF]) and anticonvulsive actions [START_REF] Maitre | Protective effect of adenosine and nicotinamide against audiogenic seizure[END_REF][START_REF] Dragunow | Is adenosine an endogenous anticonvulsant?[END_REF][START_REF] Gouder | Seizure suppression by adenosine A1 receptor activation in a mouse model of pharmacoresistant epilepsy[END_REF]. Correspondingly, studies have shown that the extracellular concentration of adenosine increases with physiological stimuli (e.g., [START_REF] Pull | Adenine derivatives as neurohumoral agents in the brain. The quantities liberated on excitation of superfused cerebral tissues[END_REF][START_REF] Lloyd | Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion[END_REF]Cunha et al. 1996a;[START_REF] Sperlágh | Homo-and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system: ATP-evoked purine release in the hippocampus[END_REF][START_REF] Wall | Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus[END_REF], and even larger increases have been reported in pathological conditions such as ischemia and epilepsia [START_REF] Schrader | Increase of adenosine content in cerebral cortex of the cat during bicuculline-induced seizure[END_REF][START_REF] Phillis | Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex[END_REF][START_REF] Phillis | Adenosine 5'-triphosphate release from the normoxic and hypoxic in vivo rat cerebral cortex[END_REF][START_REF] During | Adenosine: a potential mediator of seizure arrest and postictal refractoriness[END_REF][START_REF] Lloyd | Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion[END_REF][START_REF] Berman | Evidence for increased dorsal hippocampal adenosine release and metabolism during pharmacologically induced seizures in rats[END_REF][START_REF] Parkinson | Differences between rat primary cortical neurons and astrocytes in purine release evoked by ischemic conditions[END_REF][START_REF] Lopatář | Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists[END_REF]. Four adenosine receptors have been identified, the low affinity A3 and A2 B receptors and the high affinity A1 and A2 A receptors [START_REF] Fredholm | Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells[END_REF]. A2 A and A2 B receptors stimulate adenylate cyclase activity whereas A1 and A3 receptors mostly act by reducing adenylate cyclase activity [START_REF] Yaar | Animal models for the study of adenosine receptor function[END_REF].

Two mechanisms have been proposed to account for the presence of adenosine in the extracellular milieu. The first corresponds to the production of adenosine within cells through various metabolic pathways. Depending on the concentration gradient, intracellular adenosine could then be released in the extracellular milieu, primarily through bidirectional equilibrative nucleosides transporters (ENTs) [START_REF] King | Nucleoside transporters: from scavengers to novel therapeutic targets[END_REF]). However, in physiological conditions, adenosine mainly flows from the extracellular to the intracellular space, owing to the low K m of adenosine kinase [START_REF] Arch | Activities and some properties of 5′-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine[END_REF], the intracellular enzyme responsible for adenosine phosphorylation.

Adenosine can also be produced by the degradation of extracellular ATP. In the brain, ATP can be released by neurons, astrocytes, oligodendrocytes and microglial cells [START_REF] Fields | Purinergic signalling in neuron-glia interactions[END_REF][START_REF] Abbracchio | Purinergic signaling in the nervous system: an overview[END_REF][START_REF] Butt | ATP: a ubiquitous gliotransmitter integrating neuron-glial networks[END_REF]. As for adenosine, ATP is present in the extracellular milieu in resting condition [START_REF] Phillis | Adenosine 5'-triphosphate release from the normoxic and hypoxic in vivo rat cerebral cortex[END_REF]Cunha et al. 1996a;[START_REF] Sperlágh | Homo-and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system: ATP-evoked purine release in the hippocampus[END_REF][START_REF] Melani | Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum[END_REF]) and its extracellular concentration also rises under a variety of physiological and pathological conditions [START_REF] Wu | Distribution and release of adenosine triphosphate in rat brain[END_REF][START_REF] Wieraszko | Stimulation-dependent release of adenosine triphosphate from hippocampal slices[END_REF]Cunha et al. 1996a;[START_REF] Parkinson | Differences between rat primary cortical neurons and astrocytes in purine release evoked by ischemic conditions[END_REF][START_REF] Sperlágh | Homo-and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system: ATP-evoked purine release in the hippocampus[END_REF][START_REF] Frenguelli | Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus[END_REF]. Once released, extracellular ATP is rapidly degraded down to adenosine [START_REF] Dunwiddie | Adenine nucleotides undergo rapid, quantitative conversion to adenosine in the extracellular space in rat hippocampus[END_REF]. This degradation involves several types of enzymes collectively grouped under the name of ectonucleotidases [START_REF] Stefan | NPP-type ectophosphodiesterases: unity in diversity[END_REF][START_REF] Zimmermann | Cellular function and molecular structure of ecto-nucleotidases[END_REF][START_REF] Yegutkin | Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities[END_REF].

A first group of ectonucleotidases corresponds to ecto-nucleotide triphosphate diphosphohydrolases (NTPDases), which dephosphorylate ATP and ADP into AMP. Enzymes of a second group, ectonucleotide pyrophosphatases/phosphodiesterases (NPPs), produce AMP and inorganic pyrophosphate directly from ATP. In the brain parenchyma of adult rodents, these two enzyme families are mainly represented by NTPDase 1 (CD39), NTPDase 3 and NPP 1 [START_REF] Braun | Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain[END_REF][START_REF] Belcher | Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: Implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors[END_REF][START_REF] Langer | Distribution of ectonucleotidases in the rodent brain revisited[END_REF][START_REF] Homola | Expression of ectonucleotidases in the prosencephalon of melatonin-proficient C3H and melatonin-deficient C57Bl mice: spatial distribution and time-dependent changes[END_REF].

AMP produced by the first two groups of ectonucleotidases can be further degraded in adenosine by a third type of ectonucleotidases, ecto-5'-nucleotidase (eN or CD73), whose presence in the brain parenchyma is well established [START_REF] Kreutzberg | Cytochemical localization of 5′-nucleotidase in glial plasma membranes[END_REF][START_REF] Langer | Distribution of ectonucleotidases in the rodent brain revisited[END_REF]. Another enzyme, prostatic acid phosphatase (PAP), is involved in converting AMP to adenosine in the spinal cord [START_REF] Zylka | Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine[END_REF][START_REF] Street | Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord[END_REF]; in the brain however, PAP has been localized in the endosomes of inhibitory interneurons [START_REF] Nousiainen | Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations[END_REF], suggesting that PAP is not involved in the hydrolysis of extracellular AMP at excitatory synapses. phosphatases, tissue non-specific alkaline phosphatase (TNAP), has been located in the brain [START_REF] Goldstein | Mammalian brain alkaline phosphatase: expression of liver/bone/kidney locus comparison of fetal and adult activities[END_REF][START_REF] Brun-Heath | Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues[END_REF]). In the brain parenchyma of rodents and primates, alkaline phosphatase activity has been detected on both neurons and astrocytes [START_REF] Shimizu | Histochemical studies on the phosphatase of the nervous system[END_REF][START_REF] Friede | A quantitative mapping of alkaline phosphatase in the brain of the rhesus monkey[END_REF]Mori and Nagano 1985a,b;[START_REF] Fonta | Areal and subcellular localization of the ubiquitous alkaline phosphatase in the primate cerebral cortex: Evidence for a role in neurotransmission[END_REF][START_REF] Langer | Distribution of ectonucleotidases in the rodent brain revisited[END_REF][START_REF] Négyessy | Layer-specific activity of tissue non-specific alkaline phosphatase in the human neocortex[END_REF][START_REF] Fonta | Rediscovering TNAP in the brain: A major role in regulating the function and development of the cerebral cortex[END_REF]. Biochemical studies using TNAP purified from brain tissue [START_REF] Dorai | Purification and properties of brain alkaline phosphatase[END_REF], studies using immortalized neuronal cell lines [START_REF] Ohkubo | Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response[END_REF] and in vitro assays [START_REF] Street | Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord[END_REF] showed that TNAP can hydrolyze ATP, ADP and AMP to produce adenosine in the central nervous system.

Synthesis of extracellular adenosine in the brain is usually attributed to the ensemble NTPDases + NPPs + eN [START_REF] Lovatt | Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity[END_REF][START_REF] Zhang | Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73[END_REF][START_REF] Augusto | Ecto-5'nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A 2A receptor functions[END_REF][START_REF] Kulesskaya | CD73 is a major regulator of adenosinergic signalling in mouse brain[END_REF][START_REF] Wall | Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus[END_REF]). Yet a recent analysis of the metabolome suggested that TNAP is prominently involved in adenosine synthesis in the brain of 7-day-old mice [START_REF] Cruz | Identification of altered brain metabolites associated with TNAP activity in a mouse model of hypophosphatasia using untargeted NMR-based metabolomics analysis[END_REF]). In the present study, we aimed at determining, in a living brain preparation, the relative contribution of these different ectonucleotidases in adenosine synthesis at a functional level. For this purpose, we used an electrophysiological approach on adult mouse brain slices maintained in vitro. This approach rested on the action of adenosine on synaptic transmission, here examined in the piriform cortex. Adenosine indeed possesses a robust depressant effect on synaptic transmission in numerous brain regions including olfactory cortex [START_REF] Kuroda | Concomitant changes in cyclic AMP level and postsynaptic potentials of olfactory cortex slices induced by adenosine derivatives[END_REF][START_REF] Scholfield | Depression of evoked potentials in brain slices by adenosine compounds[END_REF], hippocampus [START_REF] Schubert | Analysis and quantitative evaluation of the depressive effect of adenosine on evoked potentials in hippocampal slices[END_REF], spinal cord [START_REF] Li | Adenosine inhibition of synaptic transmission in the substantia gelatinosa[END_REF] and neocortex [START_REF] Murakoshi | Multiple G-protein-coupled receptors mediate presynaptic inhibition at single excitatory synapses in the rat visual cortex[END_REF][START_REF] Fontanez | Adenosine A1 receptors decrease thalamic excitation of inhibitory and excitatory neurons in the barrel cortex[END_REF]. Examination of short-term plasticity or neurotransmitter release probability demonstrated that adenosine exerts this depressant effect mostly through presynaptic inhibition [START_REF] Hamilton | Autoreceptor-mediated purinergic and cholinergic inhibition of motor nerve terminal calcium currents in the rat[END_REF][START_REF] Prince | Adenosine decreases neurotransmitter release at central synapses[END_REF][START_REF] Debanne | Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release[END_REF][START_REF] Kerr | Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength[END_REF][START_REF] Perrier | Effect of adenosine on short-term synaptic plasticity in mouse piriform cortex in vitro: Adenosine acts as a high-pass filter[END_REF], by reducing calcium influx in synaptic terminals [START_REF] Wu | Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus[END_REF][START_REF] Gundlfinger | Adenosine modulates transmission at the hippocampal mossy fibre synapse via direct inhibition of presynaptic calcium channels: Adenosine at the hippocampal mossy fibre synapse[END_REF]). Adenosine-mediated presynaptic inhibition is produced by adenosine A1 receptors (A1Rs) [START_REF] Reddington | An A1-adenosine receptor, characterized by [ 3 H]cyclohexyladenosine binding, mediates the depression of evoked potentials in a rat hippocampal slice preparation[END_REF][START_REF] Collins | Adenosine A1 receptors mediate the inhibitory effects of exogenous adenosine in the rat olfactory cortex slice[END_REF][START_REF] Mccabe | Adenosine-induced depression of synaptic transmission in the isolated olfactory cortex: receptor identification[END_REF][START_REF] Masino | Modulation of Hippocampal Glutamatergic Transmission by ATP Is Dependent on Adenosine A1 Receptors[END_REF]. When applied alone, antagonists of A1Rs reduce presynaptic inhibition, implicating the presence of an endogenous adenosine tone [START_REF] Dunwiddie | Extracellular adenosine concentrations in hippocampal brain slices and the tonic inhibitory modulation of evoked excitatory responses[END_REF][START_REF] Pananceau | Short-term facilitation evoked during brief afferent tetani is not altered by long-term potentiation in the guinea-pig hippocampal CA1 region[END_REF][START_REF] Yang | Presynaptic adenosine A1 receptors modulate excitatory synaptic transmission in the posterior piriform cortex in rats[END_REF][START_REF] Kerr | Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength[END_REF]. Application of AMP, ADP and ATP mimics the action of adenosine on synaptic transmission [START_REF] Phillis | A potent depressant action of adenine derivatives on cerebral cortical neurones[END_REF][START_REF] Kuroda | Concomitant changes in cyclic AMP level and postsynaptic potentials of olfactory cortex slices induced by adenosine derivatives[END_REF][START_REF] Scholfield | Depression of evoked potentials in brain slices by adenosine compounds[END_REF][START_REF] Dunwiddie | Adenine nucleotides and synaptic transmission in the in vitro rat hippocampus[END_REF][START_REF] Lee | Effect of adenosine versus adenine nucleotides on evoked potentials in a rat hippocampal slice preparation[END_REF][START_REF] Hamilton | Autoreceptor-mediated purinergic and cholinergic inhibition of motor nerve terminal calcium currents in the rat[END_REF][START_REF] Wall | Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus[END_REF]. Furthermore, synaptic inhibition induced by adenine nucleotides is generally suppressed by A1R antagonists (Cunha et al. 1996b[START_REF] Cunha | Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors[END_REF][START_REF] Masino | Modulation of Hippocampal Glutamatergic Transmission by ATP Is Dependent on Adenosine A1 Receptors[END_REF][START_REF] Zhang | Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73[END_REF][START_REF] Choi | Enzymatic conversion of ATP to adenosine contributes to ATPinduced inhibition of glutamate release in rat medullary dorsal horn neurons[END_REF], implicating that bath-applied adenine nucleotides are degraded down to adenosine.

We first confirmed the presence of endogenous extracellular adenosine in resting conditions. We also confirmed that bath-applied ATP and AMP induce an increase in presynaptic inhibition, which was mediated through presynaptic A1Rs. We next used pharmacological means to inhibit the activity of ectonucleotidases, hypothesizing that it should reduce adenosine production, hence decrease presynaptic inhibition. Yet, contrary to this expectation, we found that presynaptic inhibition actually increased in most combinations of ectonucleotidase inhibitors and substrates. Monitoring neuronal activity thus revealed unexpected consequences of ectonucleotidase inhibition in an intact brain environment.

MATERIALS and METHODS

Brain slice preparation

Two-to four-month-old C57BL/6 wild-type mice were used. Due to space and husbandry constraints in our animal facility, we used female mice, which can be housed up to 5 per cage. They had access to food and water ad libidum. The method used for brain slice preparation has been previously described F o r P e e r R e v i e w [START_REF] Gleizes | Prominent facilitation at beta and gamma frequency range revealed with physiological calcium concentration in adult mouse piriform cortex in vitro[END_REF]. In brief, mice were deeply anesthetized with isoflurane and then rapidly decapitated. The brain was removed and prepared for slicing in ice-cold, oxygenated (95% O 2 / 5% CO 2 ) modified artificial cerebrospinal fluid (mACSF) in which Ca ++ is omitted and Mg ++ concentration is increased. The composition of mACSF is (mM): NaCl 124, NaHCO 3 26, KCl 3.2, NaH 2 PO 4 0.5, MgSO 4 1, MgCl 2 9, Glucose 10. Four hundred-micrometer-thick slices were then cut in the coronal plane on a vibratome in the presence of cold, oxygenated mACSF. Slices were allowed to recover for at least one hour at room temperature in a holding chamber filled with in vivo-like artificial cerebrospinal fluid (ACSF) whose composition was based on ionic concentration measurements in vivo (for references see [START_REF] Gleizes | Prominent facilitation at beta and gamma frequency range revealed with physiological calcium concentration in adult mouse piriform cortex in vitro[END_REF]): NaCl 124, NaHCO 3 26, KCl 3.2, NaH 2 PO 4 0.5, MgSO 4 1, CaCl 2 1.1, and glucose 10. Note that we have taken care to use a phosphate concentration similar to that in vivo [START_REF] Mulroney | Central control of renal sodium-phosphate (NaPi-2) transporters[END_REF]) as an excess of extracellular phosphate has been shown to inhibit alkaline phosphatase activity (e.g., [START_REF] Van Belle | Kinetics and inhibition of alkaline phosphatases from canine tissues[END_REF][START_REF] Dorai | Purification and properties of brain alkaline phosphatase[END_REF][START_REF] Coburn | Alkaline phosphatase (EC 3.1.3.1) in serum is inhibited by physiological concentrations of inorganic phosphate[END_REF]. This ACSF was continuously bubbled with a 95% O 2 / 5% CO 2 mixture (pH 7.4).

All procedures were conducted in accordance with the guidelines from the European Community (directive 2010/63/UE) and from the French Ministry of Agriculture, Agri-food and Forestry (décret 2013-118) and was approved by the local ethical committee (comité d'éthique Midi-Pyrénées pour l'expérimentation animale, N° MP/06/79/11/12) and the ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation, N° 15226-2018052417151228). The study was not pre-registered. No randomization was performed to allocate subjects in the study. No blinding was performed.

Reagents 8-cyclopentyl-1,3-dimethylxanthine (CPT) was used as a selective competitive A1R antagonist [START_REF] Bruns | Characterization of the A2 adenosine receptor labeled by [ 3 H]NECA in rat striatal membranes[END_REF]) and ZM 24185 as a selective adenosine A2 A receptor antagonist [START_REF] Poucher | The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosine receptor antagonist[END_REF]. Cibacron Blue 3G-A (CB) was used as a broad-spectrum P2 receptor antagonist; CB is an isomer of Reactive Blue 2 (RB2) and has been reported to have similar actions [START_REF] Von Kügelgen | Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex[END_REF][START_REF] Tuluc | P2-receptor antagonists: IV. Blockade of P2receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2[END_REF][START_REF] Glänzel | Constitutional isomers of Reactive Blue 2 -selective P2Yreceptor antagonists?[END_REF]. Dipyridamole (DP) was used as a non-selective ENT blocker [START_REF] Shank | Adenosine transport by rat and guinea pig synaptosomes: basis for differential sensitivity to transport inhibitors[END_REF][START_REF] Kiss | Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain[END_REF] and S-(4-nitrobenzyl)-6-thioinosine (NBTI) as a selective ENT1 blocker [START_REF] Shank | Adenosine transport by rat and guinea pig synaptosomes: basis for differential sensitivity to transport inhibitors[END_REF][START_REF] Kiss | Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain[END_REF].

We used ARL 67156 to inhibit NTPDase 1, NTPDase 3 and NPP 1 [START_REF] Crack | Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase[END_REF][START_REF] Westfall | Enhancement of sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens by the novel ecto-ATPase inhibitor ARL 67156[END_REF][START_REF] Iqbal | A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction[END_REF][START_REF] Lévesque | Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases[END_REF]. ARL 67156 is a competitive inhibitor with a relatively weak efficacy. Numerous biochemical assays showed that ATP hydrolysis by NTPDases and NPPs is reduced by ARL 67156 [START_REF] Crack | Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase[END_REF][START_REF] Newman | Glial cell inhibition of neurons by release of ATP[END_REF][START_REF] Iqbal | A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction[END_REF][START_REF] Lévesque | Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases[END_REF][START_REF] Lovatt | Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity[END_REF][START_REF] Heinrich | K + depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study[END_REF][START_REF] Bessueille | Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase[END_REF], although complete suppression of ATP hydrolysis requires low substrate and/or high inhibitor concentrations [START_REF] Iqbal | A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction[END_REF][START_REF] Lévesque | Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases[END_REF]. When applied in the absence of exogenous substrates, we used ARL 67156 at a concentration of 10-20 µM, as endogenous ATP concentration is likely to be less than 1 µM in physiological conditions (Cf. [START_REF] Phillis | Adenosine 5'-triphosphate release from the normoxic and hypoxic in vivo rat cerebral cortex[END_REF][START_REF] Melani | Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum[END_REF][START_REF] Schulz | Purinergic P2X, P2Y and adenosine receptors differentially modulate hippocampal gamma oscillations[END_REF][START_REF] Lalo | Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex[END_REF]. We used a higher concentration (100 µM) to inhibit the degradation of exogenous ATP (100 µM).

We used α,β-methylene-ADP (AOPCP) to inhibit eN activity [START_REF] Burger | 5'-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition of nucleotides[END_REF][START_REF] Fabiani | Characteristics of membrane-bound 5'-nucleotidase on human prostasomes[END_REF][START_REF] Picher | Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways[END_REF][START_REF] Sowa | Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A1 receptor activation[END_REF]. AOPCP is a competitive inhibitor of eN with a relatively weak efficacy [START_REF] Picher | Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways[END_REF][START_REF] Bhattarai | α,β-methylene-ADP (AOPCP) derivatives and analogues: Development of potent and selective ecto-5'-nucleotidase (CD73) inhibitors[END_REF]. AOPCP was used at 10 µM in the absence of exogenous substrate, as endogenous AMP concentration is probably much less than 1 µM as well. A higher concentration (100 µM) of AOPCP was used to inhibit degradation of exogenous AMP (100 µM).

For inhibiting TNAP activity, we used MLS-0038949 [START_REF] Dahl | Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)[END_REF]) rather than levamisole, which has been used in numerous studies (e.g., [START_REF] Van Belle | Kinetics and inhibition of alkaline phosphatases from canine tissues[END_REF][START_REF] Picher | Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways[END_REF]. However, when used in concentrations that completely inhibit TNAP (of the order of 1-2 mM), levamisole has some unspecific effects, including a block of action potential propagation that makes it impossible to use in preparations using living brain cells (Nowak et al. 2015). More recently developed, MLS-0038949 has been shown to specifically inhibit TNAP [START_REF] Dahl | Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)[END_REF]). The IC 50 for MLS-0038949 is below 0.2 µM [START_REF] Dahl | Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)[END_REF][START_REF] Pinkerton | Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor[END_REF]) and it should completely block TNAP activity in concentration ≥ 10 µM [START_REF] Dahl | Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)[END_REF][START_REF] Kiffer-Moreira | Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification[END_REF]. Biochemical assays using cell cultures and histochemical examination in the spinal cord showed that MLS-0038949 is highly effective at inhibiting ATP and AMP hydrolysis by TNAP [START_REF] Ciancaglini | Kinetic analysis of substrate utilization by native and TNAP-, NPP1-, or PHOSPHO1-deficient matrix vesicles[END_REF][START_REF] Street | Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord[END_REF][START_REF] Bessueille | Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase[END_REF]). In the present study we used MLS-0038949 at a concentration of 50 µM.

Concentration of bath-applied AMP and ATP was 100 µM. The effective concentration is very likely to be much less due to ectonucleotidase activity and uptake mechanisms in the living brain slice: using ATP biosensors, [START_REF] Schulz | Purinergic P2X, P2Y and adenosine receptors differentially modulate hippocampal gamma oscillations[END_REF] found that the concentration of bath-applied ATP is decreased by a factor of ≈300 within 400 µM thick slices. ATP and AMP dilution in in vivo-like ACSF were prepared daily for each experiment. All other compounds were stored at -20°C as stock solutions (concentrated ×100-1000). CB, AOPCP and ARL 67156 were dissolved in in vivo-like ACSF or in distilled water. CPT, ZM 24185, DP, NBTI and MLS-003849 were first dissolved in dimethyl sulfoxide (DMSO) then diluted in ACSF. The final DMSO concentrations in ACSF were 0.1% or 0.01%. We controlled that DMSO at 0.1% did not affect electrophysiological response patterns and amplitudes. ATP, AMP, CPT, CB, DP, and NBTI were purchased from Sigma. AOPCP was purchased either from Sigma or from Tocris. ZM 24185 and ARL 67156 were purchased from Tocris. MLS-0038949 was purchased from Merck.

Stimulation and recording

For recording, an individual slice was transferred in a submersion type chamber that was continuously gravity-fed with oxygenated (95% O 2 / 5% CO 2 ) in vivo-like ACSF at a flow rate of 3-3.5 ml/min. All recordings were performed at 34-35°C in the anterior piriform cortex, a brain region with a high level of TNAP activity [START_REF] Langer | Distribution of ectonucleotidases in the rodent brain revisited[END_REF][START_REF] Brun-Heath | Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues[END_REF]; and Figure 1). Local field potentials (LFPs) were recorded in layer 1a of the anterior piriform cortex (Figure 1) through single or pairs of tungsten-in-epoxylite microelectrodes (FHC, 0.2-0.3 MΩ). The signal was amplified (×1000) and filtered (0.1 Hz-10 kHz) with a NeuroLog system (Digitimer, UK). Fifty Hz noise was eliminated with a Humbug system (Quest Scientific, Canada). All signals were digitized with a digitization rate of 20 or 50 kHz (1401plus or power1401, CED, UK). Real-time display of the signals was achieved with an oscilloscope and Spike2 software (CED, UK). Layer 1a receives excitatory inputs from the olfactory bulb via a bundle of myelinated fibers, the lateral olfactory tract (LOT). Extracellular electrical stimulation was applied in the LOT through tungsten-in-epoxylite microelectrodes (FHC, 0.2-0.3 MΩ) and consisted in monopolar cathodal square current pulses (6-35 µA, 200 µs duration) delivered by an isolated stimulator (A365 stimulus isolator, WPI). We used low-intensity electrical stimulation to restrict current spread [START_REF] Nowak | Spread of stimulating current in the cortical grey matter of rat visual cortex studied on a new in vitro slice preparation[END_REF] and to prevent polysynaptic activation.

We used three different stimulation protocols. For a minority of experiments (20/97 mice, 89 manipulations), the protocol consisted of delivering single stimuli at a frequency of 0.5 Hz throughout the recording duration (protocol 1). For most experiments (77/97 mice), we used stimulation protocols that allowed examining the paired-pulse ratio (PPR, see below). For some of these experiments, the stimulation protocol (protocol 2, 148 manipulations) was divided into two parts. First, single pulses were applied at 0.5 Hz during control, treatment and recovery epochs. Next, once responses amplitude had reached a steady-state, we applied a stimulation protocol consisting of trains of 5 pulses delivered at 6 frequencies ranging from 3 to 100 Hz [START_REF] Gleizes | Prominent facilitation at beta and gamma frequency range revealed with physiological calcium concentration in adult mouse piriform cortex in vitro[END_REF][START_REF] Perrier | Effect of adenosine on short-term synaptic plasticity in mouse piriform cortex in vitro: Adenosine acts as a high-pass filter[END_REF]. This series of 'high' frequency stimuli was repeated 10 times for averaging. This protocol was initially designed to study short-term synaptic plasticity [START_REF] Gleizes | Prominent facilitation at beta and gamma frequency range revealed with physiological calcium concentration in adult mouse piriform cortex in vitro[END_REF][START_REF] Perrier | Effect of adenosine on short-term synaptic plasticity in mouse piriform cortex in vitro: Adenosine acts as a high-pass filter[END_REF] but, for the present study, only the first two responses of the 25 Hz stimulation trains have been exploited. In the other experiments (165 manipulations), we used a stimulation protocol that consisted of pairs of stimuli, with an interpulse interval of 40 ms (25 Hz), that were delivered at 0.5 Hz throughout the recording duration (protocol 3). The choice of the 40 ms interval was motivated by the observation that it is the one that yielded the strongest short-term facilitation in our experimental conditions [START_REF] Gleizes | Prominent facilitation at beta and gamma frequency range revealed with physiological calcium concentration in adult mouse piriform cortex in vitro[END_REF]. We verified that neither our base rate at 0.5 Hz nor our short-term plasticity protocol induced lasting changes in response amplitude.

Electrophysiological data analysis

Signal processing was carried out with custom scripts within Spike2 software. The LFP recorded in layer Ia after stimulation of the LOT is composed of an early and narrow component, the fiber volley (FV), which corresponds to the summation of synchronous action potentials traveling in axons (see Figure 2A). The FV is followed by a slow negative wave, the N-wave [START_REF] Yamamoto | Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro[END_REF], which reflects the summation of monosynaptic excitatory postsynaptic potentials generated in the vicinity of the recording electrode. The time course of the effect of bath-applied reagents on field potential amplitude was monitored until a steady N-wave amplitude was reached. The control condition (before treatment) lasted 15-20 min. The recovery condition (after treatments or between treatments) consisted of a 15-30 min-long washout period. In between, the reagents for the different experimental manipulations were bath applied for 12-20 minutes.

The time course of compound actions was examined by averaging the signal over each consecutive min (thirty responses) of recording. For quantification, we used the signal averaged over the last 1-5 minutes of the 12-30 min-long series of stimuli -once compound actions have reached a plateau or once recovery was completed (stimulation protocol 1 and 3) or the average of the responses to the two first stimulus of the 5 pulse train at 25 Hz (stimulation protocol 2). Synaptic response amplitude was measured at the peak of the N-wave relative to the prestimulus baseline. The FV amplitude was measured either from baseline or from the peak of the repolarization of the stimulation artifact when this later did not rise back to the prestimulus baseline. In some cases, the FV merged either in the stimulus artifact or in the N-wave and measurements could not be considered. When pairs of stimuli were used, quantification of compounds effects on the amplitude of the FV and the N-wave was based on the amplitudes obtained in response to the first stimulus of the pairs. FV and N-wave amplitudes obtained in a given condition were expressed as percentages of the value obtained in the control condition. Data were excluded from further analysis if amplitudes in control and recovery differed by more than ±15% -except for experiments with DP, which induced irreversible changes in N-wave amplitude. Unless otherwise mentioned, population data in text and figures are displayed as the means±95% confidence interval (95% CI) of the normalized amplitudes. The dashed lines at 100% in Figures 2345indicate the control amplitude level.

Stimulation protocols 2 and 3 were used to examine the PPR. The PPR was calculated as the N-wave amplitude to the second stimulus divided by that to the first stimulus. Changes in PPR allowed determining if the changes of N-wave amplitude induced by various manipulations resulted from changes in presynaptic or postsynaptic mechanisms (for review see [START_REF] Zucker | Short-term synaptic plasticity[END_REF]. The PPR does not change when postsynaptic mechanisms are involved, but varies in a way that reflects changes in neurotransmitter release probability when presynaptic mechanisms are affected. With the 5 pulse stimulation protocol (protocol 2), the PPR was calculated from the first two responses at 25 Hz after averaging amplitudes across the 10 repeats of the stimulus train. Changes in PPR are presented as percentages relative to the PPR value in the control condition. Population data in text and figures are Whenever a compound was applied several times in a single experiment, we averaged the amplitude and PPR values obtained within that experiment before computing population data. This mainly concerned AMP and ATP applications (Figures 4 and5). For example, ATP may have been superfused up to 4 times in a single experiment (before ATP + CPT, before ATP + ARL 67156, etc). To avoid biasing population data toward experiments with multiple applications, we used the mean of the effects of these several applications in the population data. On the other hand, paired comparisons between conditions were made using the original values (before pooling).

TNAP histochemistry

To examine the efficacy of MLS-0038949 (50 µM), we performed histochemical labeling of alkaline phosphatase activity in the presence or absence of the inhibitor. Alkaline phosphatase activity was revealed as previously described [START_REF] Fonta | Vascularization in the primate visual cortex during development[END_REF]. Brains from five female mouse (2-to 4month-old) were used. Frontal and parasagittal sections (90 µm thick) were cut on a vibratome from fresh brain tissue. The sections were fixed for 40 minutes in a cold, 4% paraformaldehyde (PFA) solution, then incubated in a 0.1 M tris(hydroxymethyl)aminomethane (Tris) solution containing the TNAP substrate 5-bromo-4-chloro-3-indolyl-phosphate-4-toluidine salt (BCIP, Sigma) (0.38 mM) and the chromogen 4-nitro blue tetrazolium chloride (NBT, Sigma) (0.53 mM). The enzymatic reaction product is visualized as a dark blue deposit. Incubations were made at room temperature and were performed either with a pH close to that of the ACSF used for the electrophysiological experiments (pH 7.5) or with an alkaline pH (9.5), as is traditionally done for alkaline phosphatase histochemistry. Thirty four sections (9 NBT controls, 15 NBT/BCIP without inhibitor and 10 NBT/BCIP + MLS-0038949) have been incubated at pH 9.5 and 17 (4 NBT controls, 5 NBT/BCIP without inhibitor and 8 NBT/BCIP + MLS-0038949) at pH 7.5. Incubations lasted 30 minutes at pH 7.5 and 10 minutes at pH 9.5. The reaction was stopped in 10 mM Tris pH 7.5 solution containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM levamisole (Sigma). Sections were then rinsed in Tris, mounted, dehydrated in graded ethanol and embedded in dibutyl phthalate xylene (DPX).

As illustrated in Figure 1, blood vessels appeared strongly labeled at both pH 7.5 and 9.5 (Figures 1A,1C,1E). A high level of TNAP activity is also visible in the neuropil (Figures 1A,1C,1E), in particular in layer 1 of the anterior piriform cortex (Figure 1C,1E). MLS-0038949 at 50 µM abrogated the staining in the whole section at both pH (Figures 1B,1D), demonstrating its efficiency at inhibiting TNAP activity in our experimental conditions.

Statistical analyses

No sample calculation was performed. The sample size for each manipulation is comparable to that of recent studies combining electrophysiological and pharmacological approaches on in vitro brain slices (e.g., [START_REF] Choi | Enzymatic conversion of ATP to adenosine contributes to ATPinduced inhibition of glutamate release in rat medullary dorsal horn neurons[END_REF][START_REF] Kerr | Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength[END_REF][START_REF] Lalo | Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex[END_REF][START_REF] Lopatář | Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists[END_REF][START_REF] Lovatt | Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity[END_REF][START_REF] Muzzi | Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia[END_REF][START_REF] Street | Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord[END_REF][START_REF] Wall | Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus[END_REF][START_REF] Zhang | Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73[END_REF]. No test for outliers was conducted.

Statistical analyses were constrained by the design of compound application. We performed paired ttests on raw data when a single manipulation followed a control (e.g., control → CPT → recovery). We used repeated-measures ANOVA (mixed-effects analysis, Prism 9, GraphPad) on raw data when more than one manipulation was inserted between one control and one recovery (a "sequence" of manipulation; e.g., control → ATP → ATP + MLS-0038949 → recovery). In these cases, Tukey's test was used for post-hoc analysis and for comparing between experimental manipulations and between control and manipulations; Tukey's test also includes a correction for multiple comparisons. The repeated-measures ANOVA also includes a correction for deviation from sphericity (i.e., unequal variances of the differences between conditions), which reduces the number of degrees of freedom and increases the P value in proportion to the deviation from sphericity (method of Greenhouse and Geisser). Unless otherwise mentioned, the P values given in the text correspond to those obtained with Tukey's test. The "n" reported together with Tukey's test P values corresponds to the number of pairs of values that have been compared. Unpaired t-tests were used with normalized data to compare treatments made in different experiments or in different sequences of manipulation in a single experiment. Assessment of normality has been performed using Shapiro-Wilk test. Departure from normality has been found in 5 out of 33 manipulations (DP, ATP alone, ATP+MLS-0038949, AMP alone and AMP+AOPCP) for N-wave amplitude distribution and in 2 manipulations (DP, AMP+AOPCP+CPT) for PPR distribution. However, significance of differences in comparisons involving these manipulations remained in all cases when using non-parametric tests (Wilcoxon test for paired comparisons, Kolmogorov-Smirnov test for unpaired comparisons). Effects of experimental manipulations were considered significant when P ≤ 0.05. A full statistical report is available as supplementary material. The report includes the degrees of freedom and P values, as well as the F values for ANOVA, t values for paired and non-paired t-tests, and the q values for Tukey's tests.

RESULTS

The electrophysiological results presented in this study were obtained from 97 adult mice (105 slices). Extracellular recordings of LFPs were performed in layer 1a of the anterior piriform cortex. Responses were evoked by electrical stimulation applied in the LOT. The mean amplitude of the evoked synaptic response (N-wave) was -0.39 mV (mean of all control conditions, n=287, standard deviation = 0.20 mV). In most experiments (77/97 mice), we examined the PPR (interstimulus interval: 40 ms), which in control conditions averaged 1.26 (standard deviation: 0.20, n=246). This value witnesses the presence of short-term facilitation at the LOT-layer 1a synapse in our experimental conditions [START_REF] Gleizes | Prominent facilitation at beta and gamma frequency range revealed with physiological calcium concentration in adult mouse piriform cortex in vitro[END_REF].

Endogenous adenosine acts through A1 receptors and is generated in the extracellular

space in mouse piriform cortex In a first step, we confirmed that endogenous adenosine was present in the extracellular space. To reveal ambient adenosine action, we used CPT to block A1Rs. Blocking A1Rs should prevent the inhibition generated by endogenous adenosine, which would result in an increase in N-wave amplitude. N-wave amplitude indeed consistently increased in the presence of CPT (Figure 2A). On average (Figure 2C), N-wave amplitude in the presence of CPT (0.2-1 µM) increased by +20.8%±4.8%, relative to control response amplitude (n=44, paired t-test: P<0.0001). This increase in N-wave amplitude was accompanied by a significant decrease in PPR (Figure 2D) by -7.6%±3.3% relative to control (n=27, paired t-test: P=0.0002). These results indicate that endogenous adenosine tonically inhibits synaptic transmission by acting on A1Rs located on the presynaptic side of the synapses. Similar observations have been reported in layer 1 of the posterior piriform cortex [START_REF] Yang | Presynaptic adenosine A1 receptors modulate excitatory synaptic transmission in the posterior piriform cortex in rats[END_REF], as well as at many other synapses (e.g., [START_REF] Hamilton | Autoreceptor-mediated purinergic and cholinergic inhibition of motor nerve terminal calcium currents in the rat[END_REF][START_REF] Dunwiddie | Extracellular adenosine concentrations in hippocampal brain slices and the tonic inhibitory modulation of evoked excitatory responses[END_REF][START_REF] Calabresi | Endogenous adenosine mediates the presynaptic inhibition induced by aglycemia at corticostriatal synapses[END_REF][START_REF] Gundlfinger | Adenosine modulates transmission at the hippocampal mossy fibre synapse via direct inhibition of presynaptic calcium channels: Adenosine at the hippocampal mossy fibre synapse[END_REF][START_REF] Kerr | Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength[END_REF].

In contrast, blocking adenosine A2 A receptors with ZM 24185 (0.2 µM) had no significant effect, neither on N-wave amplitude (paired t-test: P=0.4, n=11, Figure 2C), nor on PPR (P=0.8, n=10, Figure 2D).

Studies reported that extracellular ATP acting through metabotropic nucleotide receptors (P2Y receptors) could inhibit neurotransmitter release at some synapses, hence generate presynaptic inhibition [START_REF] Cunha | Purinergic modulation of the evoked release of [ 3 H]acetylcholine from the hippocampus and cerebral cortex of the rat: role of the ectonucleotidases[END_REF][START_REF] Von Kügelgen | Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex[END_REF][START_REF] De Lorenzo | Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction[END_REF]). Yet CB (30 µM), a broad-spectrum P2 receptor antagonist, had no effect on N-wave amplitude (paired t-test, P=0.6, n=6, not illustrated), suggesting that endogenous extracellular ATP did not contribute significantly to presynaptic inhibition at the LOT-layer 1a synapse. Ambient adenosine could have two origins: adenosine could be produced inside brain cells and released in the extracellular space through ENTs. In this case, blocking ENTs should reduce the outflow of adenosine, hence should reduce the extracellular concentration of adenosine, which would be read out as an increase of N-wave amplitude and a decrease in PPR. Alternatively, adenosine could be produced outside the cells by ectonucleotidases, then reintegrated within cells through ENTs. In this case, blocking ENTs should increase the extracellular concentration of adenosine, which should manifest as a decrease of N-wave amplitude and an increase in PPR. Experiments performed with DP (20 µM), a non-selective inhibitor of ENTs, largely supported the second alternative: in the presence of DP, Nwave amplitude was reduced and facilitation was increased (Figure 2B). At the population level (Figures 2C,2D), N-wave amplitude decreased by 47.7%±6.7% in the presence of DP, (P<0.0001, n=15) and the PPR increased by 18.7%±9.2% (P=0.02, n=15). Furthermore, co-application of DP and CPT completely antagonized the action of DP (Figure 2B). N-wave amplitude and PPR did not differ from those obtained in the control condition (P=0.6 and P=0.1 respectively, n=5, Figures 2C,2D) when CPT (0.2-0.4 µM) was coapplied with DP. This result confirmed that the decrease in N-wave amplitude and the associated increase in PPR in the presence of DP were due to adenosine accumulating in the extracellular space and acting on presynaptic A1Rs.

In contrast to DP that blocks both ENT1 and ENT2, the block of ENT1 alone with the ENT1 specific blocker NBTI (10 µM) had no significant effect on N-wave amplitude and PPR (paired t-test: P=0.8 and n=8, P=0.6 and n=4 respectively, Figure 2C, 2D), although we used a concentration largely above that known to completely block ENT1 in the mouse [START_REF] Kiss | Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain[END_REF]. That NBTI fails to block adenosine flow has also been observed in the hippocampus [START_REF] Dunwiddie | Extracellular adenosine concentrations in hippocampal brain slices and the tonic inhibitory modulation of evoked excitatory responses[END_REF].

These experiments confirmed that endogenous adenosine was present in the extracellular space and supported the hypothesis that, in our experimental conditions, adenosine was essentially produced in the extracellular space, in all likelihood from hydrolysis of extracellular adenine nucleotides by ectonucleotidases.

Effects of inhibiting ectonucleotidases on presynaptic inhibition mediated by endogenous adenosine

We first examined whether TNAP was implicated in endogenous adenine nucleotides hydrolysis. For this purpose, we blocked TNAP activity with a specific TNAP inhibitor, MLS-0038949 (50 µM). If TNAP was responsible for the bulk of endogenous adenosine production, then inhibiting TNAP should increase response amplitude and decrease PPR to values similar to those obtained with CPTwhich prevents the action of endogenous adenosine on A1Rs. Contrary to this expectation (example in Figure 3A), TNAP inhibition had no significant effect on N-wave amplitude (-3.3±6.3%, P=0.14, n=14, Figure 3D) and the PPR did not differ from that measured in the control condition (+6.2±8.0%, P=0.5, n=13, Figure 3F). Furthermore, inhibition mediated by A1Rs seemed to be left intact, as the combination of CPT (0.2-1 µM) and MLS-0038949 led to an increase in N-wave amplitude relative to the amplitude measured in MLS-0038949 alone (P=0.0015, n=7) and this increase (+19.1±14.7%, Figure 3E) did not differ from that obtained with CPT alone (unpaired t-test, P=0.8).

One possible explanation for the lack of effect of TNAP inhibition on adenosine-mediated presynaptic inhibition is its relatively high K m for ATP and AMP hydrolysis. Ambient adenosine concentration in resting brain slices has been estimated to be a couple of hundred nM (Dunwiddie and Diao 1994; Lee and Venton 2018) and it is expected that the concentration of adenosine precursors is not much higher [START_REF] Melani | Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum[END_REF][START_REF] Schulz | Purinergic P2X, P2Y and adenosine receptors differentially modulate hippocampal gamma oscillations[END_REF][START_REF] Lalo | Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex[END_REF]. In contrast, the K m of TNAP for AMP or ATP hydrolysis is of several tens or hundreds of µM [START_REF] Dorai | Purification and properties of brain alkaline phosphatase[END_REF][START_REF] Pizauro | Allosteric modulation by ATP, calcium and magnesium ions of rat osseous plate alkaline phosphatase[END_REF][START_REF] Ohkubo | Ecto-alkaline phosphatase in NG108-15 cells : a key enzyme mediating P1 antagonist-sensitive ATP response[END_REF][START_REF] Picher | Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways[END_REF][START_REF] Simão | Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics[END_REF]). In the brain parenchyma, the first two steps of ATP hydrolysis can also be performed by NTPDase 1, NTPDase 3 and NPP 1. All three enzymes usually display lower K m for ATP than TNAP -of the order of 10 to few tens of µM [START_REF] Hosoda | Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1 by its association with glycosaminoglycans[END_REF][START_REF] Kukulski | Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8[END_REF][START_REF] Iqbal | A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction[END_REF][START_REF] Simão | Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics[END_REF]. Likewise, the K m of eN for AMP -usually a few µM to few tens of µM [START_REF] Burger | 5'-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition of nucleotides[END_REF][START_REF] Fabiani | Characteristics of membrane-bound 5'-nucleotidase on human prostasomes[END_REF][START_REF] Picher | Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways[END_REF][START_REF] Sowa | Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A1 receptor activation[END_REF][START_REF] Grković | Developmental increase in ecto-5'-nucleotidase activity overlaps with appearance of two immunologically distinct enzyme isoforms in rat hippocampal synaptic plasma membranes[END_REF] -is lower than that of TNAP.

To examine the contribution of NTPDase 1, NTPDase 3 and NPP 1 to ambient ATP degradation, we used the inhibitor ARL 67156. Preventing ATP degradation should reduce ambient adenosine level and consequently increase N-wave amplitude. Yet, addition of ARL 67156 (10-20 µM; see methods) led to a significant decrease of N-wave amplitude (P=0.007, n=12; example in Figure 3B). On average, N-wave amplitude was reduced by 9.9±5.1% (Figure 3D) relative to control amplitude. The PPR showed a weak (+2.9±2.3%), non significant increase (P=0.06, n=12, Figure 3F). Thus, presynaptic inhibition appeared stronger, rather than weaker, when ATP and ADP breakdown was antagonized with ARL 67156. This result suggested that a compound accumulated when NTPDase 1, NTPDase 3 and NPP 1 were inhibited, which induced stronger presynaptic inhibition.

We first examined whether ATP could be this compound. Biochemical assays indeed showed that ectonucleotidase inhibition by ARL 67156 results in an increase in extracellular ATP concentration [START_REF] Newman | Glial cell inhibition of neurons by release of ATP[END_REF][START_REF] Badimon | Negative feedback control of neuronal activity by microglia[END_REF]. Extracellular ATP may exert presynaptic inhibition through P2Y receptor activation [START_REF] Cunha | Purinergic modulation of the evoked release of [ 3 H]acetylcholine from the hippocampus and cerebral cortex of the rat: role of the ectonucleotidases[END_REF][START_REF] Von Kügelgen | Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex[END_REF][START_REF] De Lorenzo | Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction[END_REF]. Therefore, we used CB (30 µM) in the presence of ARL 67156 to determine whether accumulating ATP could explain the decrease in N-wave amplitude. However, N-wave amplitude in this condition (89.8±3.7% of control, n=6, Figure 3E) did not differ from that in the presence of ARL 67156 alone (P=0.09).

Instead, the accumulating compound appeared to act through A1Rs: the application of CPT in the presence of ARL 67156 (Figure 3E) resulted in an N-wave amplitude that did not differ significantly from that obtained in the control condition (+6.3±6.3% relative to control, P=0.1, n=4) but that tended to be larger than in the presence of ARL 67156 alone (P=0.055). Altogether, the lack of action of CB and the restoration of response amplitude by CPT suggest that the strengthening of presynaptic inhibition observed in the presence of ARL 67156 was mediated by A1Rs.

We next examined the contribution of eN in degrading endogenous AMP by blocking its activity using AOPCP (10 µM; example in Figure 3C). This blocking should increase N-wave amplitude and decrease PPR to values similar to those obtained in the presence of CPT. Yet N-wave amplitude was reduced by 10.9±5.5% (P=0.001, n=15, Figure 3D) and PPR was increased by 6.3±3.8% (P=0.04, n=10, Figure 3F).

Thus inhibiting eN led to a further increase in presynaptic inhibition. Here again, this suggested that a compound accumulated during eN inhibition, which increased presynaptic inhibition. We tested whether this compound action depended on A1Rs. Blocking A1Rs with CPT (0.2-1 µM) during AOPCP application increased N-wave amplitude to values significantly higher than in the presence of AOPCP alone (+11.5%±12.4%, P=0.009, n=9, Figure 3E). Furthermore, N-wave amplitude obtained in AOPCP + CPT did not differ from that obtained in CPT alone (unpaired t-test, P=0.14). These results show that inhibiting endogenous AMP degradation by eN increased presynaptic inhibition and suggest that this increased presynaptic inhibition was, unexpectedly, mediated through A1Rs. Inhibition of ectonucleotidases increases, rather than decreases, presynaptic inhibition produced by exogenous ATP and AMP As mentioned above, one possibility for explaining our negative results with regard to TNAP inhibition and ambient adenosine synthesis is the relatively high K m of TNAP for both ATP and AMP hydrolysis, compared to their endogenous concentrations. We therefore examined the effect of blocking TNAP activity during the application of exogenous TNAP substrates in concentrations within the operating range of TNAP.

We first used ATP at 100 µM as an exogenous substrate. ATP reduced N-wave amplitude to 50.4±5.6% of the control value (P<0.0001, n=32, Figure 4C). We examined whether P2Y receptor activation could contribute to the response suppression induced by ATP using CB (30 µM). Yet CB did not counteract the inhibition induced by ATP (P=0.08, n=7, not illustrated). On the other hand, blocking A1Rs with CPT abrogated ATP-induced inhibition such that N-wave amplitude was significantly higher than in the control condition (+14.9±9.6%, P=0.002, n=22, Figure 4D) and did not differ from that measured in CPT alone (unpaired t-test, P=0.2; Figures 2C and4D). In parallel to the decrease in N-wave amplitude, ATP induced an increase in PPR (Figure 4E) by 17.6±5.8% (P<0.0001, n=27). As for N-wave amplitude, addition of CPT suppressed this change in PPR (Figure 4F), which returned to values similar to those measured in control conditions (P=0.9, n=18). These results implicate that bath-applied ATP was hydrolyzed down to adenosine by ectonucleotidases.

We next inhibited TNAP activity with MLS-0038949 during ATP infusion. If TNAP was responsible for the bulk of exogenous ATP hydrolysis, then inhibiting TNAP should lead to a response amplitude comparable to that observed in ATP + CPT -when the degradation products of ATP are prevented from acting on A1Rs. An opposite effect was observed (example in Figure 4A): N-wave amplitude remained well below control response amplitude in the presence of ATP and MLS-0038949 (-62.8±6.6%, P<0.0001, n=14) and was actually significantly lower, by about 12% on average, than in the presence of ATP alone (ATP vs. ATP + MLS-0038949: P<0.0001, Figure 4C). In addition, the coapplication of ATP and MLS-0038949 led to a significant (P<0.0001, n=14) increase in PPR (+27.8±8.8% relative to control value) in comparison to that measured in ATP alone (Figure 4E).

We next examined the consequences of inhibiting NTPDase 1, NTPDase 3 and NPP 1 with ARL 67156 (100 µM) on the response changes induced by exogenous ATP. Here again, N-wave amplitude was reduced, rather than increased, in comparison to that obtained with ATP alone (Figure 4B). Hence at the population level (Figure 4C), N-wave amplitude remained significantly lower than in the control condition (-66.1±6.6%, P<0.0001, n=14) during the coapplication of ATP and ARL 67156. Furthermore, as with MLS-0038949, N-wave amplitude during coapplication of ATP and ARL 67156 was significantly reduced (-15.7%) in comparison to that in the presence of ATP alone (P=0.01). The PPR was also larger (P=0.03, n=14) during coapplication of ATP and ARL 67156 (+25.6±7.7% relative to control, Figure 4E) than in the presence of ATP alone.

Altogether, the decrease in N-wave amplitude and the increase of the PPR implicate that, contrary to our expectations, presynaptic inhibition was stronger when ATP breakdown was inhibited with MLS-0038949 or with ARL 67156. The gain in inhibition induced by ectonucleotidase inhibitors was not correlated with the amount of inhibition induced by the nucleotides alone (not illustrated).

ATP and ADP should have accumulated in the extracellular space when TNAP or NTPDase 1, NTPDase 3 and NPP 1 were inhibited. ATP and ADP in turn might have contributed to presynaptic inhibition through P2Y receptors. Yet adding CB (30 µM) to ATP and MLS-0038949 (n=4) or to ATP and ARL 67156 (n=4) failed to counteract the additional inhibition produced by MLS-0038949 or by ARL 67156. Instead, CB tended to further decrease N-wave amplitude (not illustrated). This decrease was not significant in the ARL 67156 case (ATP + ARL 67156 vs. ATP + ARL 67156 + CB: P=0.1)

F o r P e e r R e v i e w
but was significant (-6%) in the MLS-0038949 case (ATP + MLS-0038949 vs. ATP + MLS-0038949 + CB: P=0.03). CB action as an ectonucleotidase inhibitor [START_REF] Tuluc | P2-receptor antagonists: IV. Blockade of P2receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2[END_REF]) could explain this additional increase in presynaptic inhibition.

Rather than a participation of P2Y receptors, coapplication of CPT with the inhibitors showed that presynaptic inhibition remained essentially mediated by A1Rs. Indeed, adding CPT (0.2-0.4 µM) to MLS-0038949 (50 µM) and ATP (100 µM) suppressed all the inhibitory effects of the combination of ATP + MLS-0038949. N-wave amplitude returned to a value (+10.2%±11.1% relative to control value, n=6, Figure 4D) that did not differ (P=0.6, unpaired t-test) from that measured during the coapplication of ATP and CPT (+14.9±9.6%, n=22). In the same condition, the PPR decreased back to a value comparable to that measured in ATP + CPT (P=0.9, unpaired t-test, Figure 4F). Likewise, N-wave amplitude during the coapplication of CPT (0.2-0.4 µM), ARL 67156 (100 µM) and ATP (100 µM) (+13.1%±13.8%, n=4, Figure 4D) did not differ from that measured during the coapplication of ATP and CPT (P=0.9, unpaired t-test). The PPR also did not differ from that measured in ATP + CPT (P=0.7, unpaired t-test, Figure 4F). These results suggest that inhibiting ATP degradation reinforced presynaptic inhibition and that, quite unexpectedly, this presynaptic inhibition remained mediated by adenosine A1 receptors.

We next used AMP (100 µM) as an exogenous substrate. As with ATP, the application of AMP resulted in a reduction of N-wave amplitude to 47.0%±6.7% of control amplitude (P<0.0001, n=33, Figure 5C). This reduction of response amplitude was abrogated in the presence of CPT (0.2-1 µM), such that response amplitude in the presence of AMP + CPT did not differ from that in control condition (P=0.9, n=23, Figure 5D). AMP infusion also induced an increase in PPR by 24.2±7.5% (P<0.0001, n=28, Figure 5E). CPT reversed the effect of AMP on PPR, such that PPR was decreased below that measured in the control condition (-5.9%±4.8%, P=0.03, n=20, Figure 5F). These results suggest that the AMP-induced inhibition depended on its dephosphorylation to adenosine by TNAP or eN.

We first examined the effect of inhibiting TNAP on the presynaptic inhibition induced by AMP and found that AMP-induced inhibition was not modified by MLS-0038949 (50 µM) (example in Figure 5A). N-wave amplitude in the presence of AMP and MLS-0038949 (41.3%±10.1% of control amplitude, n=12) did not differ from that measured in AMP alone (P=0.9, Figure 5C). Likewise, PPR in the presence of AMP and MLS-0038949 (+33.3%±19.1%, n=12) did not differ (P=0.4, Figure 5E) from that measured in AMP alone. In summary, there was no evidence that TNAP inhibition prevented AMP-induced presynaptic inhibition.

We next examined the contribution of eN using AOPCP (100 µM). Yet AOPCP did not suppress the presynaptic inhibition induced by AMP (100 µM); instead, AMP-induced inhibition appeared to be reinforced in the presence of AOPCP, as witnessed by a further decrease in N-wave amplitude (example in Figure 5B). On average (Figure 5C), N-wave amplitude remained much lower than in the control condition (P=0.0002), reaching a value representing 31.6±7.4% (n=9) of the control response amplitude. This value was also significantly (P=0.003) lower, by 15.4%, than that obtained in the presence of AMP alone (Figure 5C). In parallel, the PPR during AMP and AOPCP coapplication (+30.4±14.5%) appeared significantly higher (P=0.01) than that obtained with AMP alone (Figure 5E).

Altogether, these results show that inhibiting the two ectonucleotidases involved in AMP to adenosine conversion did not prevent and possibly reinforced AMP-induced presynaptic inhibition. Furthermore, this persisting inhibition appeared to be mediated through A1Rs, as it was completely counteracted by CPT (0.2-1 µM). Blockade of A1Rs with CPT in the presence of MLS-0038949 or AOPCP canceled AMP effect on N-wave amplitude (Figure 5D). In these conditions, N-wave amplitude did not differ from that obtained in the presence of AMP + CPT without inhibitors (unpaired t-test, P=0.8 for MLS-

DISCUSSION

The present study was aimed at examining the involvement of ectonucleotidases in extracellular adenosine synthesis in living brain tissue. We used an electrophysiological approach, which allowed examining the functional consequences of inhibiting ectonucleotidases at the synaptic level. We evaluated the consequence of inhibiting ectonucleotidase activity by measuring a physiological response, adenosine-mediated presynaptic inhibition.

Using CPT, we evidenced the presence of an endogenous adenosine-mediated presynaptic inhibition at the LOT-layer 1a synapse (Figure 2). We also showed that endogenous adenosine was most probably mainly generated in the extracellular space: by blocking both ENT1 and ENT2 with DP, we observed, as shown previously [START_REF] Motley | Endogenous adenosine inhibits excitatory transmission in the rat olfactory cortex slice[END_REF][START_REF] Sanderson | Effects of adenosine uptake blockers and adenosine on evoked potentials of guinea-pig olfactory cortex[END_REF][START_REF] Phillis | Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex[END_REF][START_REF] Dunwiddie | Extracellular adenosine concentrations in hippocampal brain slices and the tonic inhibitory modulation of evoked excitatory responses[END_REF][START_REF] Cunha | Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors[END_REF]), a substantial increase in presynaptic inhibition, suggesting an increase in extracellular endogenous adenosine concentration; this was confirmed by blocking A1Rs with CPT, which canceled the action of DP (Figure 2). These results implicate that, in our experimental conditions, endogenous adenosine was mainly generated in the extracellular space and thereafter pumped into cells through ENTs. This proposal is at variance with studies that showed that an outflow of adenosine per se largely contributes to extracellular adenosine level [START_REF] Lloyd | Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion[END_REF][START_REF] Phillis | Adenosine 5'-triphosphate release from the normoxic and hypoxic in vivo rat cerebral cortex[END_REF]Cunha et al. 1996a;[START_REF] Brager | Activity-dependent release of adenosine contributes to short-term depression at CA3-CA1 synapses in rat hippocampus[END_REF][START_REF] Lovatt | Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity[END_REF][START_REF] Wall | Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus[END_REF]. However, these studies used activation protocols such as prolonged electrical stimulation or experimentally induced epilepsy or ischemia, which are metabolically more demanding than the low intensity, low frequency electrical stimulation used in our experiments. In our conditions of minimal activation, the bulk of extracellular adenosine likely derived from extracellular ATP. This implicates that ectonucleotidases degrade ambient extracellular adenine nucleotides into adenosine.

We also confirmed that bath-applied AMP and ATP exert a depressant effect on synaptic transmission. This depressant effect was similar to that of adenosine in the same settings [START_REF] Perrier | Effect of adenosine on short-term synaptic plasticity in mouse piriform cortex in vitro: Adenosine acts as a high-pass filter[END_REF]. As shown previously (Cunha et al. 1996b[START_REF] Cunha | Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors[END_REF][START_REF] Masino | Modulation of Hippocampal Glutamatergic Transmission by ATP Is Dependent on Adenosine A1 Receptors[END_REF][START_REF] Zhang | Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73[END_REF][START_REF] Muzzi | Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia[END_REF][START_REF] Choi | Enzymatic conversion of ATP to adenosine contributes to ATPinduced inhibition of glutamate release in rat medullary dorsal horn neurons[END_REF], AMP-and ATP-induced inhibition involved A1Rs as CPT counteracted their actions (Figures 4 and5). These results obtained with bath-applied adenine nucleotides also implicates that ectonucleotidases are involved in extracellular adenosine synthesis.

Using a metabolomics approach, [START_REF] Cruz | Identification of altered brain metabolites associated with TNAP activity in a mouse model of hypophosphatasia using untargeted NMR-based metabolomics analysis[END_REF] showed that TNAP is strongly involved in adenosine synthesis in the whole brain of the developing mouse. We first examined whether TNAP contributes to adenosine-mediated presynaptic inhibition through the hydrolysis of extracellular adenine nucleotides by inhibiting its activity with MLS-0038949. We showed, for the first time in the cerebrum, that MLS-0038949 is able to suppress TNAP activity when it is assessed using a histochemical approach (Figure 1). Yet we found that inhibition of TNAP: (1) did not prevent presynaptic inhibition mediated by endogenous purines; (2) did not alleviate presynaptic inhibition induced by exogenous AMP or ATP; (3) and instead significantly reinforced, rather than reduced, presynaptic inhibition induced by exogenous ATP. Brain TNAP activity decreases during development [START_REF] Goldstein | Mammalian brain alkaline phosphatase: expression of liver/bone/kidney locus comparison of fetal and adult activities[END_REF]. This could offer an explanation for our negative results in the endogenous and in the exogenous AMP conditions; however, this does not explain the opposite-toour-expectation result we obtained with exogenous ATP.

Only few studies have examined the consequences of inhibiting TNAP with MLS-0038949 on synaptic transmission. [START_REF] Zhang | Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73[END_REF] reported that MLS-0038949 suppresses both endogenous purinergicand exogenous AMP-induced inhibition in mice knocked-down for the CD73 gene, but has no effect in wild-type mice [START_REF] Zhang | Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73[END_REF]). In the dorsal horn of the spinal cord, [START_REF] Street | Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord[END_REF] found that MLS-0038949 reduced AMP-induced inhibition in mice in which both PAP and CD73 genes were knocked-down but did not alter AMP-induced inhibition in normal mice. Yet [START_REF] Choi | Enzymatic conversion of ATP to adenosine contributes to ATPinduced inhibition of glutamate release in rat medullary dorsal horn neurons[END_REF] reported that MLS-0038949 reduced both AMP-and ATP-induced presynaptic inhibition in the medullary dorsal horn of the rat. None of these studies reported the enhancement of ATP-induced presynaptic inhibition that we observed in the presence of MLS-0038949.

These negative or unexpected results obtained with TNAP inhibition led us to examine the involvement of the other ectonucleotidases present in the brain parenchyma. Beside TNAP, AMP hydrolysis can also be carried out by eN. However, blocking eN with AOPCP produced an increase in presynaptic inhibition -rather than a decrease -, both in the endogenous condition (Figure 3) and the exogenous AMP condition (Figure 5). Similarly, an increase in inhibition, rather than a decrease, has been reported in several studies using AOPCP in endogenous condition (Cunha et al. 1996b[START_REF] Cunha | Inhibition by ATP of hippocampal synaptic transmission requires localized extracellular catabolism by ecto-nucleotidases into adenosine and channeling to adenosine A1 receptors[END_REF][START_REF] Lee | Effect of adenosine versus adenine nucleotides on evoked potentials in a rat hippocampal slice preparation[END_REF][START_REF] Muzzi | Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia[END_REF]) and a strengthening by AOPCP of exogenous AMP effect has been noticed in some studies [START_REF] Moody | Stimulation of P1-purinoceptors by ATP depends partly on its conversion to AMP and adenosine and partly on direct action[END_REF][START_REF] Muzzi | Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia[END_REF]. We moreover found that this strengthening of presynaptic inhibition was suppressed by CPT, implicating an action mediated through A1Rs (Figures 3 and5).

A possible explanation for these results is that AMP itself could act as an agonist of A1Rs. Previous studies have shown persisting AMP action in the presence of bath-applied adenosine deaminase and its suppression in the presence of bath-applied AMP deaminase [START_REF] Moody | Stimulation of P1-purinoceptors by ATP depends partly on its conversion to AMP and adenosine and partly on direct action[END_REF][START_REF] Wu | Evidence of direct activation of adenosine A1 receptor by 5'-adenosine monophosphate in isolated guinea pig atrial myocytes[END_REF][START_REF] Ross | Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors[END_REF]). More recently, [START_REF] Rittiner | AMP is an adenosine A1 receptor agonist[END_REF] demonstrated that AMP and a non-hydrolyzable AMP analog can directly activate A1Rs in embryonic neuron cultures and in transfected HEK293 cells. The strengthening of presynaptic inhibition we observed in the presence of AOPCP could then be explained by AMP accumulating in the extracellular space. Direct activation of A1Rs by AMP would compensate for the reduction of extracellular adenosine synthesis.

In addition, as only dephosphorylated compounds can be transported through ENTs, the extracellular concentration of AMP would increase, resulting in a stronger activation of A1Rs, hence stronger inhibition. Following this rationale, the role of ectonucleotidases in degrading AMP could be envisioned as a mechanism for allowing adenosine recycling within cells, rather than as a necessary step for allowing A1R activation.

We also examined the putative involvement of NTPDases and NPPs in ATP-induced presynaptic inhibition. Yet in the presence of exogenous ATP, we observed stronger -rather than weakerpresynaptic inhibition when NTPDases and NPPs activities were inhibited with ARL 67156 (Figure 4). ARL 67156 reinforced presynaptic inhibition also in endogenous condition (Figure 3), as shown previously in the hippocampus [START_REF] Pascual | Astrocytic purinergic signaling coordinates synaptic networks[END_REF][START_REF] Lopatář | Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists[END_REF]. It has been reported that ARL 67156 may have unspecific effects by altering action potential propagation in axons [START_REF] Lopatář | Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists[END_REF]. However, in our experiments, the amplitude of the FV did not seem to be modified by ARL 67156 (endogenous condition: P=0.13, n=8; with ATP: P=0.2, n=10, not illustrated), indicating that the decrease in N-wave amplitude did not result from changes in axonal excitability and that it was of synaptic origin. This synaptic origin is further supported by the increase in PPR observed with ARL 67156 in the presence of bath-applied ATP (Figure 4).

As extracellular ATP accumulates when ectonucleotidases are inhibited [START_REF] Newman | Glial cell inhibition of neurons by release of ATP[END_REF][START_REF] Heinrich | K + depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study[END_REF][START_REF] Badimon | Negative feedback control of neuronal activity by microglia[END_REF][START_REF] Bessueille | Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase[END_REF], we envisioned that this accumulating ATP might have activated P2Y receptors, which contribute to presynaptic inhibition at some synapses (noradrenergic inputs: von Kügelgen 1994; acetylcholinergic inputs: [START_REF] Cunha | Purinergic modulation of the evoked release of [ 3 H]acetylcholine from the hippocampus and cerebral cortex of the rat: role of the ectonucleotidases[END_REF][START_REF] De Lorenzo | Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction[END_REF]). Yet CB did not compensate for the supposed inhibitory action of accumulated ATP on P2Y receptors. Instead, it tended to generate an increase in presynaptic inhibition in addition to that already induced by ectonucleotidase inhibitors. This result can be interpreted by the observation that CB can also act as an inhibitor of ectonucleotidases [START_REF] Tuluc | P2-receptor antagonists: IV. Blockade of P2receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2[END_REF]) -thus reinforcing the actions of MLS-0038949 and Besides, we came to the conclusion that, despite the presence of ARL 67156 or MLS-0038949 that should have reduced extracellular adenosine level, ATP-induced presynaptic inhibition remained primarily mediated by adenosine (or AMP): the blockade of A1Rs with CPT suppressed ATP-induced inhibition in a similar fashion, irrespective of the presence or lack of ectonucleotidase inhibitors (Figure 4). Altogether our experiments show that attempts at preventing ATP-induced presynaptic inhibition by preventing adenosine (or AMP) production resulted in a more potent presynaptic inhibition, which seems to be mediated only through A1Rs. We can only speculate on the origin of this paradoxical result.

A reversal of adenosine transport direction may provide a first hypothesis. In control condition, the flow of adenosine through ENTs is from the extracellular to the intracellular space. However, inhibiting ectonucleotidases may reverse the concentration gradient for adenosine, such that adenosine would be released, rather than taken-up, by cells. Outflow of intracellular adenosine would then compensate for the depletion of extracellular adenosine, although this mere compensation does not readily account for the strengthening of presynaptic inhibition.

Another hypothesis is that the ectonucleotidase inhibitors may also function as positive allosteric modulators of A1Rs. As we did not inhibit all ectonucleotidases at the same time, TNAP activity on one hand, NTPDase 1, NTPDase 3 and NPP 1 activity or eN activity on the other hand, may have compensated for the inhibition of each other. Thus part of the extracellular nucleotides could still have been degraded down to adenosine. If, in addition, AOPCP, ARL 67156 and MLS-0038949 were able to boost A1R mediated responses, then presynaptic inhibition may remain unchanged or may even be enhanced despite the reduction of extracellular adenosine concentration.

A last possibility resides in the finding that application of exogenous ATP can cause the release of endogenous ATP [START_REF] Sperlágh | Homo-and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system: ATP-evoked purine release in the hippocampus[END_REF]. In particular, both astrocytes and microglial cells possess ATP receptors whose activation leads to the release of ATP [START_REF] Anderson | ATP-induced ATP release from astrocytes[END_REF][START_REF] Pascual | Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission[END_REF][START_REF] Imura | Microglia release ATP by exocytosis[END_REF][START_REF] Shinozaki | Microglia trigger astrocytemediated neuroprotection via purinergic gliotransmission[END_REF]. Thus in the presence of ectonucleotidase inhibitors, extracellular ATP concentration was likely to increase, leading to the release of larger amounts of endogenous ATP. Since ectonucleotidases were not all inhibited at the same time, part of the extracellular ATP could still have been degraded down to adenosine, this latter mediating presynaptic inhibition through A1Rs. So there might be an equilibrium point where the increase in ATP-induced ATP release compensates for the reduction of adenosine production due to partial ectonucleotidase inhibition. Beyond this equilibrium point, extracellular adenosine concentration might increase above the concentration in control conditions, which may explain the paradoxical increase in A1R-dependent presynaptic inhibition observed in the presence of ARL 67156 or MLS-0038949. This hypothesis is apparently at variance with the effect of CB in our experiments, but the action of CB as an ectonucleotidase inhibitor complicates the figure. Direct monitoring of extracellular adenosine concentration should help to solve this issue.

From a phenomenological point of view, a strengthening of presynaptic inhibition by ectonucleotidase inhibitors was almost systematically observed: altogether, we observed a 10-16% increase in presynaptic inhibition in 5 out of 7 combinations of inhibitors and conditions. These changes may be considered negligible, yet it is worth confronting these values with the dose-response relationship for adenosine obtained in the same experimental conditions [START_REF] Perrier | Effect of adenosine on short-term synaptic plasticity in mouse piriform cortex in vitro: Adenosine acts as a high-pass filter[END_REF]. Due to the nonlinearity of this relationship, a decrease of N-wave amplitude from, for example, 50% to 35% of control amplitude would actually be equivalent to a doubling of the concentration of adenosine. While biochemical studies have long shown how ectonucleotidases orchestrate nucleotide degradation, our results indicate an unpredicted degree of complexity when experiments are carried out in an intact brain environment. activity. Frontal sections from mouse brains were stained using NBT/BCIP histochemistry at pH 7.5 (A, B) or 9.5 (C, D, E) in the absence (A, C, E) or presence (B, D) of MLS-0038949 (50 µM). The square in C delimits the piriform cortex and is enlarged in E. Pir: piriform cortex; L1, L2: layer 1 and layer 2 of piriform cortex; LOT: lateral olfactory tract. ac: anterior commissure; cc: corpus callosum. Mx: motor cortex; S1: primary sensory cortex. St: striatum. Alkaline phosphatase activity was detected throughout the mouse brain. Intense staining is observed in layer 1 of the piriform cortex. The LOT is visible in the sections in C, D and E, which were made at the level of the anterior piriform cortex. The LOT is not visible in A and B, which correspond to sections made at the level of the posterior piriform cortex. B, D: Staining was suppressed by MLS-0038949 in both vessels and parenchyma. Scale bar in B represents 1 mm and applies to panels A-D. Scale bar in E represents 200 µm.

Figure 2. Endogenous adenosine generates presynaptic inhibition through adenosine A1 receptors (A1Rs) and is generated in the extracellular space in the anterior piriform cortex. A, B. Example traces show the responses evoked in layer 1a after electrical stimulation in the lateral olfactory tract. For revealing short-term plasticity, stimuli were given in pairs ("pulse 1" and "pulse 2"), spaced apart by 40 ms. The fiber volley (FV) corresponds to the narrow peak that immediately follows the stimulation artifact. The slow negativity that follows the FV corresponds to the N-wave. The amplitude of the N-wave induced by the second pulse is larger than that evoked by the first pulse, corresponding to the phenomenon of short-term facilitation, quantified with the paired-pulse ratio (PPR). The graphs below traces show the time course of compound effects on N-wave amplitude ("Amp.") for the first stimulus (upper graph) and on paired-pulse ratio ("PPR", lower graph). Note that the absolute value of the amplitude is used to illustrate time course of compound effects on N-wave amplitude. Timing of manipulations is indicated above graphs. A. N-wave amplitude increased in the presence of the A1R antagonist CPT (1 µM). This was accompanied by a decreased facilitation. Results obtained with stimulation protocol 2 (see Methods) that provided one single PPR value for each condition. Each trace is the average of 10 responses. was reduced below control value in the presence of ARL (10-20 µM) or AOPCP (10 µM). E. The addition of CPT (0.2-1 µM) to ectonucleotidase inhibitors reveals that presynaptic inhibition mediated through adenosine A1 receptors was essentially left intact in the presence of MLS-0038949, ARL 67156 and AOPCP. On the other hand, the decrease in N-wave amplitude induced by ARL 67156 was not altered by the metabotropic nucleotide receptor antagonist CB (30 µM). F. PPR was not significantly modified in the presence of MLS or ARL and it increased above control level in the presence of AOPCP. G. The increase in PPR observed in the presence of AOPCP was not maintained in the presence of CPT. Except for CPT alone (see legend of Figure 2), repeated measures ANOVAs were used to compare control conditions with inhibitor conditions and inhibitor + receptor antagonist conditions (e.g., control vs. MLS vs. MLS+CPT); number of sharps in panel E indicates significance level after post-hoc Tukey's tests between inhibitor alone and inhibitor with CPT; ##: 0.001 < P ≤ 0.01. Other conventions as in Figure 2. On the other hand, the addition of AOPCP (100 µM) produced a supplementary decrease in N-wave amplitude compared to that measured in AMP alone. D. CPT (0.2-1 µM) suppressed the reduction of N-wave amplitude observed with AMP alone, with AMP in the presence of MLS and with AMP in the presence of AOPCP. E. PPR was increased in the presence of AMP alone. In the presence of MLS, the PPR did not differ from that measured with AMP alone. On the other hand, in the presence of AOPCP, PPR was further increased compared to that measured with AMP alone. F. CPT abolished the increase in PPR observed with AMP alone, or with AMP in the presence of MLS or AOPCP. Repeated measures ANOVAs were used to compare control conditions with AMP alone and AMP + ectonucleotidase inhibitors (e.g., control vs. AMP vs. AMP+AOPCP). The number of sharps indicates significance level of post-hoc Tukey's tests between AMP alone and AMP+AOPCP; ##: 0.001 < P ≤ 0.01; #: 0.01 < P ≤ 0.05. Other conventions as in Figures 2.

Page

Graphical abstract text

Ectonucleotidases refers to several types of enzymes, which collectively play an essential role in the degradation of extracellular nucleotides to adenosine. Inhibiting their activities should reduce extracellular adenosine concentration. Using an electrophysiological approach on mouse brain slices, we examined how inhibition of ectonucleotidases affects adenosine-mediated presynaptic inhibition. Unexpectedly, we found that inhibitors of ectonucleotidases almost systematically strengthened, rather than reduced, the presynaptic inhibition mediated by adenosine A1 receptors. This result suggests that, in an intact brain environment, manipulating the activity of ectonucleotidases in order to control extracellular adenosine concentration might generate physiological outcomes opposite to those anticipated.

  means±95% CI of the normalized PPRs. The dashed lines at 100% in Figures2-5indicate the control PPR level.

  .6 for AOPCP). Likewise, the PPR was not different from that measured in AMP + CPT without inhibitor (Figure5F, P=0.1 for MLS-0038949; P=0.2 for AOPCP).

  to control ectonucleotidase activities in an integrated functional system might generate physiological outcomes opposite to those anticipated.

Figure 1 .

 1 Figure 1. Histochemical staining shows that the TNAP inhibitor MLS-0038949 suppresses alkaline phosphatase

  Figure2. Endogenous adenosine generates presynaptic inhibition through adenosine A1 receptors (A1Rs) and is generated in the extracellular space in the anterior piriform cortex. A, B. Example traces show the responses evoked in layer 1a after electrical stimulation in the lateral olfactory tract. For revealing short-term plasticity, stimuli were given in pairs ("pulse 1" and "pulse 2"), spaced apart by 40 ms. The fiber volley (FV) corresponds to the narrow peak that immediately follows the stimulation artifact. The slow negativity that follows the FV corresponds to the N-wave. The amplitude of the N-wave induced by the second pulse is larger than that evoked by the first pulse, corresponding to the phenomenon of short-term facilitation, quantified with the paired-pulse ratio (PPR). The graphs below traces show the time course of compound effects on N-wave amplitude ("Amp.") for the first stimulus (upper graph) and on paired-pulse ratio ("PPR", lower graph). Note that the absolute value of the amplitude is used to illustrate time course of compound effects on N-wave amplitude. Timing of manipulations is indicated above graphs. A. N-wave amplitude increased in the presence of the A1R antagonist CPT (1 µM). This was accompanied by a decreased facilitation. Results obtained with stimulation protocol 2 (see Methods) that provided one single PPR value for each condition. Each trace is the average of 10 responses. B. Blockade of equilibrative nucleoside transporters (ENTs) with dipyridamole (DP, 20 µM) decreased N-wave amplitude and increased facilitation. CPT (0.4 µM) canceled the action of DP on Nwave amplitude and PPR. Results obtained with stimulation protocol 3 (see Methods). Stimulus pairs were delivered every 2 s. Each trace is the average of 30 responses corresponding to the last minutes of recording in each condition. C, D. population data. C. Blocking A1Rs with CPT (0.2-1 µM) led to an increase in N-wave amplitude. In contrast, blocking adenosine A2 A receptors with ZM 24185 (0.2 µM) had no significant effect. Blocking ENT1 and ENT2 with DP (20 µM) induced a significant reduction in N-wave amplitude. Blocking A1Rs with CPT (0.2-0.4 µM) counteracted the action of DP (DP+CPT). The mean amplitude during DP+CPT does not reach the value in CPT alone probably owing to the competitive nature of CPT antagonism; adenosine accumulating during ENTs inhibition may have partially displaced CPT from A1Rs. The blockade of ENT1 with the selective ENT1 inhibitor NBTI (10 µM) had no significant effect on N-wave amplitude. D. PPR decreased with CPT and increased in the presence of DP. The other manipulations had no significant effect. The numbers in the bars correspond to the number of manipulations for each condition. Number of asterisks above bars indicates outcomes of paired t-test (CPT, ZM 24185 and NBTI experiments) and of post-hoc Tukey's test (DP, DP+CPT experiments) between controls and tests: ***: P ≤ 0.001; **: 0.001 < P ≤ 0.01; *: 0.01 < P ≤ 0.05). Sharps indicate significance level obtained with post-hoc Tukey's test between DP and DP + CPT; #: 0.01 < P ≤ 0.05.

Figure 3 .

 3 Figure 3. Presynaptic inhibition by endogenous adenosine in mouse piriform cortex is not suppressed and may be strengthened by ectonucleotidase inhibitors. A.-C.: examples. A. Synaptic response was barely affected by the specific TNAP inhibitor, MLS-0038949 (MLS, 50 µM). Stimulation protocol type 2 was used for this experiment. B. Superfusion of ARL 67156 (ARL, 10 µM), an inhibitor of NTPDase 1, NTPDase 3 and NPP 1, led to a reversible decrease in N-wave amplitude. This amplitude decrease was associated with an increase in paired-pulse ratio (PPR), implicating stronger presynaptic inhibition. C. Superfusion of AOPCP (10 µM), an eN inhibitor, produced a reversible decrease in N-wave amplitude associated with an increase in PPR. Experiments in B and C were performed using stimulation protocol 3. D.-F.:population data. D. Addition of MLS (50 µM) did not change N-wave amplitude compared to the control condition. On the other hand, N-wave amplitude

Figure 4 .

 4 Figure 4. Effect of ectonucleotidase inhibitors on presynaptic inhibition induced by exogenous ATP. A., B.: examples. A. ATP (100 µM) induced a decrease in N-wave amplitude and an increase in paired-pulse ratio (PPR). These two effects were enhanced in the presence of the TNAP inhibitor, MLS-0038949 (MLS, 50 µM). B. The NTPDases/NPPs inhibitor, ARL 67156 (ARL, 100 µM), strengthened the decrease in N-wave amplitude and increase in PPR induced by ATP (100 µM). The examples in A and B were among those showing the strongest potentiation of presynaptic inhibition by ectonucleotidase inhibitors. C-F.: population data. C. ATP alone (100 µM) reduced N-wave amplitude by 50%. The addition of either MLS (50 µM) or ARL (100 µM)resulted in a reduction of N-wave amplitude significantly greater than that obtained with ATP alone. D. CPT (0.2-1 µM) suppressed the reduction in N-wave amplitude observed with ATP alone, or with ATP in the presence of MLS or ARL. E. Superfusion of ATP increased the mean PPR value. In the presence of either MLS or ARL, the PPR reached values significantly higher than in the presence of ATP alone. F. CPT abolished the increase in PPR observed with ATP alone, or with ATP in the presence of MLS or ARL. Repeated measures ANOVAs were used to compare control conditions with ATP and ATP + ectonucleotidase inhibitors (for example: control vs. ATP vs. ATP+MLS). Number of sharps indicate significance level of post-hoc Tukey's tests between ATP and ATP + ectonucleotidase inhibitors; ###: P ≤ 0.001; #: 0.01 < P ≤ 0.05. Other conventions as in Figures 2.

Figure 5 .

 5 Figure 5. Effects of inhibiting ectonucleotidases on presynaptic inhibition induced by exogenous AMP. A., B.: examples.A. AMP (100 µM) induced a decrease in N-wave amplitude and an increase in paired-pulse ratio (PPR). These two effects were not affected by the addition of the TNAP inhibitor, MLS-0038949 (MLS, 50 µM). B. Infusion of the eN inhibitor AOPCP (100 µM) in the presence of AMP induced a further decrease of Nwave amplitude. C.-F.: population data. C. AMP (100 µM) reduced N-wave amplitude to 47% of control amplitude. Adding MLS (50 µM) did not significantly change the N-wave amplitude compared to AMP alone. On the other hand, the addition of AOPCP (100 µM) produced a supplementary decrease in N-wave amplitude compared to that measured in AMP alone. D. CPT (0.2-1 µM) suppressed the reduction of N-wave amplitude observed with AMP alone, with AMP in the presence of MLS and with AMP in the presence of AOPCP. E. PPR was increased in the presence of AMP alone. In the presence of MLS, the PPR did not differ from that measured with AMP alone. On the other hand, in the presence of AOPCP, PPR was further increased compared to that measured with AMP alone. F. CPT abolished the increase in PPR observed with AMP alone, or with AMP in the presence of MLS or AOPCP. Repeated measures ANOVAs were used to compare control conditions with AMP alone and AMP + ectonucleotidase inhibitors (e.g., control vs. AMP vs. AMP+AOPCP). The number of sharps indicates significance level of post-hoc Tukey's tests between AMP alone and AMP+AOPCP; ##: 0.001 < P ≤ 0.01; #: 0.01 < P ≤ 0.05. Other conventions as in Figures 2.

Inhibitors of ectonucleotidases have paradoxical effects on synaptic transmission in the mouse cortex Marie Gleizes 1 , Caroline Fonta 1* and Lionel G. Nowak 1*

	A	Pulse 1			Pulse 2	B		Pulse 1	Pulse 2
			100 µV					100 µV
		2 ms					2 ms
			Control		AMP				Control	AMP
			AMP + MLS	Recovery			AMP + AOPCP	Recovery
		Ctrl	AMP	AMP+MLS	Recovery		Ctrl	AMP	AMP+AOPCP	Recovery
	Amp. (|mean|	± SEM, mV)				Amp. (|mean|	± SEM, mV)
								1,6
		PPR					PPR	1,4
				Time (min)				Time (min)
		1: CerCo, Université Toulouse 3, CNRS, Pavillon Baudot, CHU Purpan, BP 25202, 31052 Toulouse Cedex.
		marie.gleizes@inserm.fr, caroline.fonta@cnrs.fr, lionel.nowak@cnrs.fr
		*: Correspondence:
						F o r
								P e e
									r
									R
									e v i e w

lionel.nowak@cnrs.fr, caroline.fonta@cnrs.fr Supplementary material: full statistical report

  The four tables presented below report the details of the results of the statistical tests used in our study: repeated measures ANOVA (table 1), post-hoc Tukey's tests (table 2), paired t-test (table3) and unpaired t-tests (table 4). The degrees of freedom and P values are reported in each table, as well as the F values for ANOVA, t values for paired and non-paired t-tests, and the q values for Tukey's tests. We also provide the number of the Figures where the results of the comparisons are presented, when available.

Table 1 . Repeated measures ANOVAs (mixed-effects model)

 1

	Conditions compared	Variable	Figure number	Degrees of freedom (DFn, DFd)	F value	P value
	Ctrl / DP /	N-wave amplitude	2C	1.9, 17	36	<0.0001
	DP+CPT	PPR	2D	0.69, 6.2	7	0.04
	Ctrl / MLS /	N-wave amplitude	3D, 3E	1.0, 6.0	8.3	0.0281
	MLS+CPT	PPR	3F, 3G	0.91, 8.2	1.0	0.33
	Ctrl / ARL /	N-wave amplitude	3D, 3E	1.1, 8.0	11	0.010
	ARL+CPT	PPR	3F, 3G	1.1, 7.7	1.9	0.21
	Ctrl / ARL /	N-wave amplitude	3D, 3E	0.78, 6.3	12	0.0149
	ARL+CB	PPR	3F, 3G	1.0, 8.3	2.8	0.128
	Ctrl / AOPCP /	N-wave amplitude	3D, 3E	1.2, 11	10	0.0071
	AOPCP+CPT	PPR	3F, 3G	0.33, 2.1	3.8	0.14
	Ctrl / ATP /	N-wave amplitude	4C, 4D	0.005, 0.12	100	0.037
	ATP+CPT	PPR	4E, 4F	0.47, 10	34	0.0008
	Ctrl / ATP /	N-wave amplitude	NI	1.6, 19	78	<0.0001
	ATP+CB
	Ctrl / ATP / ATP+MLS	N-wave amplitude PPR	4C 4E	0.58, 18 0.46, 14	82 34	<0.0001 0.0004
	Ctrl / ATP / ATP+ARL	N-wave amplitude PPR	4C 4E	1.0, 3.0 1.5, 19	82 32	<0.0001 <0.0001
	Ctrl / ATP / ATP+MLS /	N-wave amplitude	NI	1.0, 3.1	15	0.029
	ATP+MLS+CB
	Ctrl / ATP / ATP+ARL /	N-wave amplitude	NI	1.1, 3.2	19	0.019
	ATP+ARL+CB
	Ctrl / AMP /	N-wave amplitude	5C, 5D	1.1, 23	36	<0.0001

Table 2 . Post-hoc Tukey's tests Conditions compared

 2

	Page 65 of 69		Journal of Neurochemistry
		Variable	Figure number	Degrees of	q value	P value
				freedom
	Ctrl vs DP	N-wave amplitude	2C	14	11	<0.0001
		PPR	2D	14	4.5	0.017
	Ctrl vs DP+CPT	N-wave amplitude	2C	4	1.4	0.6
		PPR	2D	4	1.3	0.14
	DP vs DP+CPT	N-wave amplitude	2C	4	5.6	0.035
		PPR	2D	4	6.7	0.023
	Ctrl vs MLS	N-wave amplitude	3D	6	3.2	0.14
		PPR	3F	12	1.5	0.54
	Ctrl vs MLS+CPT	N-wave amplitude	3E	6	2.0	0.38
		PPR	3G	6	2.1	0.38
	MLS vs MLS+CPT N-wave amplitude	3D, 3E	5	11	0.0015
		PPR	3F, 3G	5	0.58	0.91
	Ctrl vs ARL	N-wave amplitude	3D	11	5.4	0.0075
		PPR	3F	11	3.6	0.065
	Ctrl vs ARL+CB	N-wave amplitude	3E	5	8.1	0.0054
		PPR	3G	5	3.7	0.10
	ARL vs ARL+CB	N-wave amplitude	3D, 3E	5	3.9	0.086
		PPR	3F, 3G	5	3.3	0.14
	Ctrl vs ARL+CPT	N-wave amplitude	3E	3	4.2	0.11
		PPR	3G	3	2.7	0.28
	ARL vs ARL+CPT N-wave amplitude	3D, 3E	3	5.7	0.055
		PPR	3F, 3G	3	3.6	0.16
	Ctrl vs AOPCP	N-wave amplitude	3D	10	7.3	0.0012
		PPR	3F	9	4.2	0.036
	Ctrl vs AOPCP+CPT	N-wave amplitude PPR	3E 3G	8 4	5.1 4	0.018 0.098
	AOPCP vs AOPCP+CPT	N-wave amplitude PPR	3D, 3E 3F, 3G	7 3	6.1 3.7	0.0088 0.15
	Ctrl vs ATP	N-wave amplitude	4C	31	15	<0.0001
		PPR	4E	26	9.4	<0.0001
	Ctrl vs ATP+CPT	N-wave amplitude	4D	17	7.2	0.0002
		PPR	4F	17	0.22	0.99
	ATP vs ATP+CPT N-wave amplitude	4C, 4D	17	18	<0.0001
		PPR	4E, 4F	11	14	<0.0001

Table 3 . Paired t-tests

 3

	Conditions compared	Variable	Figure number	Degrees of freedom	t value	P value
	Ctrl vs CPT	N-wave amplitude	2C, 3D	43	12.56	<0.0001
		PPR	2D, 3E	26	4.411	0.0002
	Ctrl vs ZM241385	N-wave amplitude	2C	10	0.8278	0.43
		PPR	2D	9	0.3217	0.75
	Ctrl vs NBTI	N-wave amplitude	2C	7	0.2957	0.78
		PPR	2D	3	0.5872	0.60
	Ctrl vs CB	N-wave amplitude	NI	5	0.5411	0.61
		PPR	NI	4	4.773	0.0088
	Ctrl vs MLS +	N-wave amplitude	NI	10	1.724	0.11
	AOPCP	PPR	NI	7	0.05464	0.96
	AMP vs AMP +	N-wave amplitude	NI	5	0.4542	0.67

Table 4 . Unpaired t-tests

 4

	Page 67 of 69		Journal of Neurochemistry
	Conditions compared	Variable	Figure number	Degrees of	t value	P value
				freedom
	CPT vs MLS + CPT	N-wave amplitude	3D, 3E	49	0.2521	0.80
	CPT vs ARL + CPT	N-wave amplitude	3D, 3E	46	1.760	0.085
	CPT vs AOPCP + CPT N-wave amplitude	3D, 3E	51	1.526	0.14
	CPT vs ATP + CPT	N-wave amplitude	2C, 4D	64	0.2315	0.23
	ATP + CPT vs ATP + MLS + CPT	N-wave amplitude PPR	4D 4F	26 22	0.4800 0.0155	0.63 0.99
	ATP + CPT vs ATP + ARL + CPT	N-wave amplitude PPR	4D 4F	24 20	0.1464 0.3578	0.88 0.72
	AMP + CPT vs AMP + MLS + CPT	N-wave amplitude PPR	5D 5F	27 24	0.1906 1.598	0.85 0.12
	AMP + CPT vs AMP + AOPCP + CPT	N-wave amplitude PPR	5D 5F	27 24	0.5843 1.308	0.56 0.20

Journal of Neurochemistry

Funding:

This research was supported by CNRS, by University of Toulouse 3 (IDEX transversalité 2015, UFTMIP: 2016-106-CIF-D-DRDV) and by Hypophosphatasie Europe foundation (ref CNRS 194679).

Author Contributions: Conceptualization, CF and LGN; methodology, LGN; software, LGN; validation, MG, CF and LGN; formal analysis, MG, CF and LGN; investigation, MG, LGN and CF; resources, CF and LGN; data curation, LGN; writing-original draft preparation, MG and LGN; writing-review and editing, LGN and CF; visualization, MG, LGN and CF; supervision, LGN and CF; project administration, CF; funding acquisition, LGN and CF. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.