Ljiljana Nikolic

Paola Nobili

Weida Shen

Etienne Audinat
email: etienne.audinat@igf.cnrs.fr

Role of astrocyte purinergic signalling in epilepsy Running title: Astrocyte purinergic signalling in epilepsy

Keywords: calcium signalling, cytokine, microglia, gliotransmission, inflammation, Main points

Epilepsy is characterized by unpredictable recurrent seizures resulting from hypersynchronous discharges from neuron assemblies. Increasing evidence indicates that aberrant astrocyte signalling to neurons plays an important role in driving the network hyperexcitability. Purinergic signalling is central in neuron-glia and glia-glia interactions and dysfunctions in communication pathways involving purinergic receptors have been reported in various CNS pathologies, such as Alzheimer disease, stroke, major depression, schizophrenia, and epilepsy. In the present review we will first discuss the mechanisms by which astrocytes influence neuronal activity. We will then review in more details recent evidence indicating that dysregulation of astrocyte purinergic signalling actively contributes to the appearance of abnormal neuronal activity in epilepsy.

Introduction

Our knowledge of astrocyte functions evolved tremendously during the last decades, adding to the classical view of supportive cells for neurons the concept that astrocytes integrate neuronal and synaptic activities and, in turn, directly influence these activities through the release of gliotransmitters. In physiological conditions, these "metabolic" and "information processing" functions usually operate on a time scale ranging from milliseconds to hours or even days, when considering brain development and hormonal cycles, and are fundamental for brain functions, including cognitive functions [START_REF] Araque | Gliotransmitters travel in time and space[END_REF][START_REF] Attwell | Glial and neuronal control of brain blood flow[END_REF][START_REF] Dallérac | Astrocytes as new targets to improve cognitive functions[END_REF][START_REF] Khakh | Diversity of astrocyte functions and phenotypes in neural circuits[END_REF][START_REF] Macdonald | Astrocytes in neuroendocrine systems: An overview[END_REF][START_REF] Pellerin | Activity-dependent regulation of energy metabolism by astrocytes: An update[END_REF][START_REF] Rusakov | Diversity of astroglial functions alludes to subcellular specialisation[END_REF][START_REF] Santello | Astrocyte function from information processing to cognition and cognitive impairment[END_REF][START_REF] Theodosis | Activity-Dependent Structural and Functional Plasticity of Astrocyte-Neuron Interactions[END_REF]. The continuous and progressive discovery of key roles played by astrocytes in regulating brain functions has also advanced our understanding of the roles of these cells in the pathogenesis of neurological and psychiatric diseases, including Alzheimer's disease, cerebral ischemia, multiple sclerosis, Huntington's disease, major depressive disorder and epilepsy [START_REF] Cresto | Do Astrocytes Play a Role in Intellectual Disabilities?[END_REF][START_REF] Halassa | The tripartite synapse: roles for gliotransmission in health and disease[END_REF][START_REF] Patel | Neuron-glia interactions in the pathophysiology of epilepsy[END_REF][START_REF] Rossi | Astrocytic dysfunction: Insights on the role in neurodegeneration[END_REF][START_REF] Steinhäuser | Crucial role of astrocytes in temporal lobe epilepsy[END_REF]Verkhratsky & Parpura, 2016).

Epilepsy is a pathological condition affecting more than 1% of the population worldwide [START_REF] Beghi | Addressing the burden of epilepsy: Many unmet needs[END_REF][START_REF] Hesdorffer | Estimating risk for developing epilepsy: A population-based study in Rochester, Minnesota[END_REF]. It is characterized by repeated and recurrent seizures altering physiological brain functioning and being responsible for the development of comorbidities such as neurological deficits. According to the International League Against Epilepsy, epilepsies should be classified as focal, generalized or combined focal and generalized, with six, non-exclusive, subgroups of etiology (structural, genetic, infectious, metabolic, immune or unknown), selected because of their potential therapeutic consequences [START_REF] Scheffer | ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology[END_REF]. Many of these epilepsies result from an acute insult (e.g. stroke, trauma, infection) that triggers progressive modifications of the brain that lead to the occurrence of spontaneous recurrent seizures. Epileptogenesis corresponds to the gradual remodelling of a formerly healthy brain into a spontaneously seizing one. The latent phase separating the initial insult from the appearance of the first spontaneous seizures can last months or even years. It is followed by the chronic phase during which recurrent spontaneous seizures occur. For a long time, it has been thought that alteration in the excitation / inhibition balance, which is at the basis of seizure generation, was the only mechanism involved in the pathogenesis of epilepsy. Consequently, anti-epileptic drugs (AEDs) developed in the last decades mostly aimed at correcting this perturbed balance between excitatory and inhibitory neurotransmission. These pharmacological interventions can, in the best scenario, control seizures but they cannot cure the disease. Furthermore, about one third of the patients are refractory to pharmacological treatments and this ratio reaches 2/3 of the patients when considering mesial temporal lobe epilepsy (MTLE), the most frequent and severe form of focal epilepsy in adults [START_REF] Asadi-Pooya | Prevalence and Incidence of Drug-Resistant Mesial Temporal Lobe Epilepsy in the United States[END_REF]. There is therefore an obvious and urgent need for the development of new AEDs. In this context, the increasing evidence that brain functions and dysfunctions rely on an integrated interplay between neurons and glial cells, and in particular astrocytes, opens new routes for the identification of promising non-neuronal targets that could lead to the development of new AEDs. Dysfunction of several astrocyte signalling pathways can contribute to hyperexcitation of neuronal networks and progression of seizures [START_REF] Steinhauser | Special Issue: Astrocytes and Epilepsy[END_REF][START_REF] Devinsky | Glia and epilepsy: excitability and inflammation[END_REF][START_REF] Patel | Neuron-glia interactions in the pathophysiology of epilepsy[END_REF]. In particular, purinergic signalling is a key signalling pathway in glia-glia and neuron-glia interactions and recent evidence indicates that this pathway contributes to the deregulation of neuronal activity in epilepsy [START_REF] Engel | ATPergic signalling during seizures and epilepsy[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF].

In a first section, we will briefly review the physiological functions of astrocytes that can impact on neuronal and synaptic activities, highlighting astrocyte dysfunctions that have been identified in pathological conditions and in particular in epilepsy. In the following sections, we will discuss more extensively how deregulation of ATP signalling in astrocytes can contribute to epileptogenesis and ictogenesis (i.e. the acute process generating seizures).

1.

Astrocytes: function and dysfunction in health and disease

Astrogliosis: an adaptive reactive phenotype

The role of astrocytes in pathologies of the central nervous system (CNS) is widely associated with reactive phenotype developed by astrocytes in response to a rupture in CNS homeostasis. This process called astrogliosis is particularly evident in epileptic syndromes associated with hippocampal sclerosis (HS) that is characterized by a glial scar [START_REF] Thom | Review: Hippocampal sclerosis in epilepsy: a neuropathology review[END_REF]. Astrogliosis involves changes at the molecular, cellular and functional levels (Dossi, Vasile, & Rouach, 2018). The degree of alterations observed in reactive astrocytes varies according to the type of insult inducing the reaction and to the possibility of reversion of the damage [START_REF] Anderson | Heterogeneity of reactive astrocytes[END_REF][START_REF] Sofroniew | Molecular dissection of reactive astrogliosis and glial scar formation[END_REF].

Transcriptomic analysis of reactive astrocytes has disclosed the occurrence of two broad phenotypes: a pro-inflammatory phenotype A1 having detrimental effects [START_REF] Lian | NFκB-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer's disease[END_REF]Liddelow et al., 2017;[START_REF] Zamanian | Genomic Analysis of Reactive Astrogliosis[END_REF] and an immune-suppressive phenotype A2 with protective functions [START_REF] Anderson | Astrocyte scar formation aids central nervous system axon regeneration[END_REF]Liddelow & Barres, 2017).

Although the specific phenotype of astrocytes in different forms of epilepsy has not yet been firmly established, different studies point rather toward a deleterious A1 phenotype.

For example, in epilepsy models, the release of Interleukin-1beta (IL-1) and High Mobility Group Box1 by astrocytes induces the activation of IL1R/TLR signalling pathways that favours the induction and maintenance of seizure activity [START_REF] Maroso | Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures[END_REF][START_REF] Vezzani | The role of inflammation in epilepsy[END_REF]. However, it should be pointed out that in most cases the population of astrocytes has been considered as a whole without taking into consideration a possible diversity of the astrocyte reactivity (e.g. lesion vs peri-lesional area). One important and still debated question is whether astrocyte reactivity is a cause or a consequence of seizures. In favour of a causative role, astrocyte-specific deletion of genes (tuberous sclerosis complex 1 or Beta 1 integrin) or mutation of an astrocyte-specific gene (GFAP) induce astrogliosis and the occurrence of spontaneous seizures [START_REF] Messing | Alexander Disease[END_REF][START_REF] Ortinski | Selective induction of astrocytic gliosis generates deficits in neuronal inhibition[END_REF][START_REF] Robel | Reactive Astrogliosis Causes the Development of Spontaneous Seizures[END_REF].

On the other hand, there were no sign of astrogliosis in non-lesional epileptogenic cortical areas of patients with focal cortical dysplasia [START_REF] Rossini | Seizure activity per se does not induce tissue damage markers in human neocortical focal epilepsy[END_REF]. Moreover, in a guinea pig model of temporal lobe epilepsy with hippocampal sclerosis (HS-MTLE; unilateral, intra-hippocampal kainate), astrogliosis and cell damage were observed only in the injected, and not in the contralateral, hippocampus despite the occurrence of epileptiform activities in both hippocampi [START_REF] Noè | Epileptiform activity contralateral to unilateral hippocampal sclerosis does not cause the expression of brain damage markers[END_REF]. The latter two examples suggest dissociation between the occurrence of seizure and astrogliosis, as seen with the classical marker GFAP. However, they do not exclude the contribution of astrocytes to the functional remodelling that occurs during epileptogenesis and to the generation of seizures, especially in view of the many functions of astrocytes by which they influence the excitability of neuronal networks.

Another important aspect of astrocyte function that is affected by gliosis is their close interaction with brain vasculature that they contact with their endfeet. Astrocytes in physiological conditions contribute to the maintenance of blood-brain barrier (BBB) structure by increasing the expression of tight junction proteins occludin and claudin-5 and by inhibiting the expression of chemokines and leukocyte adhesion molecules, which would favour BBB disruption [START_REF] Andreone | Neuronal and Vascular Interactions[END_REF][START_REF] Zhao | Establishment and Dysfunction of the Blood-Brain Barrier[END_REF]. Modifications of astrocyte phenotype during astrogliosis could contribute to BBB impairment that occurs in epilepsy through disruption of tight-junction coupling, increased expression of adhesion molecules on endothelial cells determining an increase of pro-inflammatory cytokines and the extravasation of leukocytes in brain parenchyma [START_REF] Marchi | Inflammatory pathways of seizure disorders[END_REF][START_REF] Van Vliet | Blood-brain barrier dysfunction, seizures and epilepsy[END_REF]. Furthermore, BBB disruption in epilepsy leads to extravasation of serum albumin, which is taken up preferentially by astrocytes and activates them through transforming growth factor beta [START_REF] Heinemann | Blood-brain barrier dysfunction, TGFbeta signaling, and astrocyte dysfunction in epilepsy[END_REF]. We will now review how dysfunction of several astrocyte properties can contribute to seizure and epilepsy.

Homeostasis: keeping an eye on K + and water

One of the most important physiological functions of astrocytes is the maintenance of ion homeostasis and, in particular, the regulation of extracellular K + [K + ] 0 . The accumulation of K + in the extracellular space can lead to sustained neuronal depolarization and hyperexcitability [START_REF] Heinemann | Ceiling of stimulus induced rises in extracellular potassium concentration in the cerebral cortex of cat[END_REF][START_REF] Walz | Role of astrocytes in the clearance of excess extracellular potassium[END_REF]. Astrocytes control [K + ] 0 by two mechanisms: K + uptake and spatial buffering. The K + uptake is mediated through Na + /K + pumps, Na + /K + /Cl -cotransporters and the large expression of inward-rectifier potassium (Kir) 4.1 channels and relies also on the very negative resting potential of astrocytes [START_REF] D'ambrosio | Differential role of KIR channel and Na(+)/K(+)-pump in the regulation of extracellular K(+) in rat hippocampus[END_REF][START_REF] Kofuji | Potassium buffering in the central nervous system[END_REF][START_REF] Ransom | Activity-dependent extracellular K+ accumulation in rat optic nerve: the role of glial and axonal Na+ pumps[END_REF][START_REF] Seifert | Analysis of astroglial K+ channel expression in the developing hippocampus reveals a predominant role of the Kir4.1 subunit[END_REF]. Remarkably, mice lacking Kir4.1 channels on astrocytes were impaired for K + and glutamate uptake and were more susceptible to seizures [START_REF] Chever | Implication of Kir4.1 channel in excess potassium clearance: an in vivo study on anesthetized glial-conditional Kir4.1 knock-out mice[END_REF][START_REF] Djukic | Conditional Knock-Out of Kir4.1 Leads to Glial Membrane Depolarization, Inhibition of Potassium and Glutamate Uptake, and Enhanced Short-Term Synaptic Potentiation[END_REF]. Altered expression of astrocyte Kir4.1 channels has been reported for Alzheimer's disease [START_REF] Wilcock | Vascular amyloid alters astrocytic water and potassium channels in mouse models and humans with Alzheimer's disease[END_REF], major depressive disorder (MDD; [START_REF] Medina | Evidence for alterations of the glial syncytial function in major depressive disorder[END_REF] and for Huntington's disease [START_REF] Tong | Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice[END_REF].

Different studies, both in humans and animal models, have highlighted a clear association between uncontrolled extracellular K + increase and epilepsy [START_REF] Steinhäuser | Crucial role of astrocytes in temporal lobe epilepsy[END_REF]. In particular, measurements of extracellular K + , patch-clamp recordings of Kir currents in astrocytes, single-cell RT-PCR and immunostaining of Kir proteins revealed a reduction in Kir-mediated current and a reduction of Kir4.1 expression in surgical specimen from patients suffering from MTLE and in rodent models of MTLE [START_REF] Bordey | Properties of human glial cells associated with epileptic seizure foci[END_REF][START_REF] Heinemann | Alterations of glial cell function in temporal lobe epilepsy[END_REF][START_REF] Heuser | Loss of Perivascular Kir4.1 Potassium Channels in the Sclerotic Hippocampus of Patients With Mesial Temporal Lobe Epilepsy[END_REF][START_REF] Hinterkeuser | Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances[END_REF][START_REF] Jauch | Effects of barium, furosemide, ouabaine and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on ionophoretically-induced changes in extracellular potassium concentration in hippocampal slices from rats and from patients with epilepsy[END_REF][START_REF] Kivi | Effects of barium on stimulus-induced rises of [K+]o in human epileptic non-sclerotic and sclerotic hippocampal area CA1[END_REF][START_REF] Schröder | Functional and molecular properties of human astrocytes in acute hippocampal slices obtained from patients with temporal lobe epilepsy[END_REF].

Control of ion homeostasis by astrocytes relies also on spatial buffering [START_REF] Orkand | Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia[END_REF], which is mediated by gap junctions (GJ), channels composed of connexin (Cx) 43) and Cx30 [START_REF] Nagy | Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS[END_REF]. These channels allow the formation of a functional network between astrocytes (syncytium) and the diffusion of K + following the electrochemical gradient: .K + is taken up by astrocytes where neuronal activity is high, diffuses through the syncytium and is released at remote sites with lower [K + ] 0 . In line with the spatial buffering concept, the physiological functioning of astroglial networks should have an anti-epileptic role, and indeed, a reduced threshold for generating epileptic activity was found in mice with coupling-deficient astrocytes [START_REF] Pannasch | Astroglial networks scale synaptic activity and plasticity[END_REF][START_REF] Wallraff | The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus[END_REF]. Furthermore, evidence suggests that alterations in astrocyte coupling could be involved in the genesis and progression of epilepsy. Using a mouse model of HS-MTLE (unilateral intracortical kainate), it has been demonstrated that astrocyte coupling is impaired as early as 4 hours after the induction of status epilepticus (SE), a mechanism probably related to the release of pro-inflammatory cytokines and thus suggesting that impaired coupling represents a key event in epileptogenesis [START_REF] Bedner | Astrocyte uncoupling as a cause of human temporal lobe epilepsy[END_REF].

Moreover, the authors showed a complete lack of GJ coupling in sclerotic hippocampi of HS-MTLE patients.

Another important function in which astrocytes are involved is the regulation of extracellular space (ECS) volume and fluid osmolarity through aquaporin 4 (AQP4), a channel mediating transmembrane water movements according to osmotic gradients and that is expressed by astrocytes mostly at perisynaptic processes and perivascular end-feet [START_REF] Schwartzkroin | Osmolarity, ionic flux, and changes in brain excitability[END_REF]. Regulating water flux near synapses and blood vessels may be linked to K + homeostasis [START_REF] Papadopoulos | Aquaporin water channels in the nervous system[END_REF]. Mice lacking AQP4 have a mild ECS volume expansion, an elevated threshold but prolonged duration for electrographic seizures induced in vivo by hippocampal electrical stimulation [START_REF] Binder | Functional changes in astroglial cells in epilepsy[END_REF]Binder et al., 2006). Subsequent analysis of these mice, however, indicated that they also displayed an enhanced gap junctional coupling and an improved spatial [K + ] 0 buffering [START_REF] Strohschein | Impact of aquaporin-4 channels on K+ buffering and gap junction coupling in the hippocampus[END_REF]. In human and in animal models of MTLE, there is a loss of astrocytic perivascular AQP4, which preceeds the appearance of chronic seizures [START_REF] Alvestad | Mislocalization of AQP4 precedes chronic seizures in the kainate model of temporal lobe epilepsy[END_REF][START_REF] Eid | Loss of perivascular aquaporin 4 may underlie deficient water and K+ homeostasis in the human epileptogenic hippocampus[END_REF]. This suggests that perturbed polarized expression of AQP4 during epileptogenesis, together with consequences on [K + ] 0 homeostasis, may be of pathophysiological relevance.

Neurotransmitter buffering: maintaining the equilibrium for glutamate, GABA and purines

A crucial role for astrocytes that can also impact on neuronal excitability is neurotransmitter buffering. Indeed, the removal of neurotransmitters, such as glutamate and GABA, from the synaptic cleft is crucial for maintaining a correct functioning of synapses and an appropriate balance between excitation and inhibition. Alterations in these buffering mechanisms could lead to neuronal hyper-excitability and excitotoxicity [START_REF] Choi | Glutamate neurotoxicity and diseases of the nervous system[END_REF][START_REF] Meldrum | The role of glutamate in epilepsy and other CNS disorders[END_REF]. The astrocytic buffering is mediated by neurotransmitter uptake through transporters and their metabolism through the glutamineglutamate -GABA cycle (Tore [START_REF] Eid | Roles of Glutamine Synthetase Inhibition in Epilepsy[END_REF]. Glutamate and GABA reuptake relies on specific transporters expressed by astrocytes: EAAT1 and EAAT2 (also known as GLAST and GLT1) for glutamate, and GAT1 and GAT3 for GABA. Once internalized in astrocytes, glutamate is converted into glutamine by glutamine synthetase (GS), while GABA enters the TCA cycle which produces alpha ketoglutarate, glutamate and glutamine. Glutamine produced in astrocytes is then released, up taken by neurons and used as a precursor for both glutamate and GABA synthesis.

Alterations in neurotransmitter transporters are reported in different neurological diseases, including epilepsy [START_REF] Coulter | Astrocytic regulation of glutamate homeostasis in epilepsy[END_REF]. The involvement of alterations in neurotransmitter homeostasis in epilepsy is demonstrated by an increase in extracellular glutamate levels in the sclerotic hippocampus of MTLE patients [START_REF] Cavus | Extracellular metabolites in the cortex and hippocampus of epileptic patients[END_REF][START_REF] Cavus | Decreased hippocampal volume on MRI is associated with increased extracellular glutamate in epilepsy patients[END_REF]. In experimental epilepsy, deletion of GLT1 in astrocytes determined an exaggerated response to subconvulsive doses of pentylenetetrazol [START_REF] Tanaka | Epilepsy and Exacerbation of Brain Injury in Mice Lacking the Glutamate Transporter GLT-1[END_REF].

Alterations in GABA extracellular levels can also contribute to hyper-excitability and epilepsy. Depending on the epileptic syndrome, both decreased extracellular GABA levels and increased GABA receptor mediated activity can be detrimental for the maintenance of excitation / inhibition balance. For example, in TLE an increase in the expression levels of GAT3 astrocytic GABA transporter, leading to a reduction of extracellular GABA levels, has been reported just before seizure onset [START_REF] During | Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain[END_REF][START_REF] Lee | GAT1 and GAT3 expression are differently localized in the human epileptogenic hippocampus[END_REF].

On the other hand, in absence epilepsy a dysfunction of the astrocytic GABA transporter GAT1 was responsible for the generation of non-convulsive seizures by determining an increase in GABA-A receptor mediated inhibition in thalamocortical neurons [START_REF] Cope | Enhanced tonic GABAA inhibition in typical absence epilepsy[END_REF][START_REF] Pirttimaki | Astrocytic GABA transporter GAT-1 dysfunction in experimental absence seizures[END_REF].

Reduction in GS function is involved in the pathogenesis of epilepsy by inducing extracellular glutamate accumulation, as reported in TLE patients [START_REF] Eid | Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy[END_REF][START_REF] Eid | The Glutamate-Glutamine Cycle in Epilepsy[END_REF][START_REF] Van Der Hel | Reduced glutamine synthetase in hippocampal areas with neuron loss in temporal lobe epilepsy[END_REF]. Evidence for GS involvement in the pathogenesis of epilepsy is reported also in animal models in which blocking of GS function with the inhibitor methionine sulfoximine was able to induce recurrent seizures, neuronal loss and hippocampal atrophy [START_REF] Eid | Recurrent seizures and brain pathology after inhibition of glutamine synthetase in the hippocampus in rats[END_REF][START_REF] Wang | The development of recurrent seizures after continuous intrahippocampal infusion of methionine sulfoximine in rats: a video-intracranial electroencephalographic study[END_REF].

Considering that glutamine is fundamental also for GABA synthesis in interneurons, GS dysfunction could be related also to impaired inhibitory transmission, as shown in an experimental model of TLE in which the downregulation of GS in astrocytes was responsible for a compromised synaptic inhibitory transmission [START_REF] Ortinski | Selective induction of astrocytic gliosis generates deficits in neuronal inhibition[END_REF].

Another important astrocytic enzyme involved in the balance between excitation and inhibition is adenosine kinase (ADK). By phosphorylating adenosine to adenosinemonophosphate (AMP) it exerts a key role in regulating extracellular levels of adenosine [START_REF] Etherington | Astrocytic adenosine kinase regulates basal synaptic adenosine levels and seizure activity but not activity-dependent adenosine release in the hippocampus[END_REF][START_REF] Studer | Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme[END_REF]. Since adenosine is known to dampen neuronal activity, its release exerts anti-convulsant effects [START_REF] Cunha | How does adenosine control neuronal dysfunction and neurodegeneration[END_REF][START_REF] Lado | How do seizures stop?[END_REF]. Astrogliosis occurring in epilepsy is known to be associated with increased levels of ADK both in humans and in experimental models [START_REF] Boison | Adenosinergic signaling in epilepsy[END_REF]. The increase in ADK and the consequent reduction of extracellular adenosine levels are responsible for reducing seizure threshold [START_REF] Boison | Adenosinergic signaling in epilepsy[END_REF].

Altogether these data clearly highlight how crucial is neurotransmitter buffering in order to maintain a functional synaptic activity and avoid the generation of a seizure-prone context.

Gliotransmission: sensing and modulating the surrounding environment

The concept of the "tripartite synapse" emerged from experiments demonstrating that astrocytes are able to sense neurotransmitters released by presynaptic neurons into the synaptic cleft but also can release gliotransmitters that regulate neuronal and synaptic activities [START_REF] Auld | Glial cells and neurotransmission: an inclusive view of synaptic function[END_REF][START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF].

Several classical neurotransmitters have been also identified as gliotransmitters, among which glutamate, D-serine, ATP, adenosine and GABA have been the most studied [START_REF] Angulo | GABA, a forgotten gliotransmitter[END_REF][START_REF] Araque | Gliotransmitters travel in time and space[END_REF][START_REF] Harada | Gliotransmitter Release from Astrocytes: Functional, Developmental, and Pathological Implications in the Brain[END_REF][START_REF] Papouin | Astrocytic control of synaptic function[END_REF][START_REF] Petrelli | Novel insights into gliotransmitters[END_REF]). An increase in intracellular Ca 2+ is often crucial for the release of gliotransmitters [START_REF] Araque | Gliotransmitters travel in time and space[END_REF][START_REF] Bazargani | Astrocyte calcium signaling: the third wave[END_REF][START_REF] Khakh | Diversity of astrocyte functions and phenotypes in neural circuits[END_REF][START_REF] Rusakov | Diversity of astroglial functions alludes to subcellular specialisation[END_REF][START_REF] Volterra | Astrocyte Ca2+ signalling: an unexpected complexity[END_REF] that can be mediated through exocytotic or non-exocytotic pathways [START_REF] Hamilton | Do astrocytes really exocytose neurotransmitters?[END_REF] Following the pioneer work of Parri et al [START_REF] Parri | Spontaneous astrocytic Ca2+ oscillations in situ drive NMDAR-mediated neuronal excitation[END_REF] in the thalamus, we and others showed that astrocytes in acute slices of the hippocampus release glutamate that induces slow inward currents (SICs) in CA1 pyramidal cells due to the activation of extrasynaptic NMDA receptors [START_REF] Angulo | Glutamate released from glial cells synchronizes neuronal activity in the hippocampus[END_REF][START_REF] Fellin | Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors[END_REF][START_REF] Perea | Properties of synaptically evoked astrocyte calcium signal reveal synaptic information processing by astrocytes[END_REF]. SICs generated by a single astrocyte have the capacity to synchronize a small ensemble of neurons whose soma or dendrites are within the morphological domain of this astrocyte. It was later on proposed that this form of gliotransmission could be at the origin of hypersynchronous excitation of neurons in models of epilepsy [START_REF] Kang | Astrocytic glutamate releaseinduced transient depolarization and epileptiform discharges in hippocampal CA1 pyramidal neurons[END_REF][START_REF] Tian | An astrocytic basis of epilepsy[END_REF]. However, although Ca 2+ signalling in astrocytes and SICs frequency are enhanced during ictal events triggered in acute hippocampal slices, blocking action potentials with tetrodoxin blocks ictal events but not SICs whereas blocking NMDA receptors blocks SICs but not ictal events [START_REF] Fellin | Astrocytic glutamate is not necessary for the generation of epileptiform neuronal activity in hippocampal slices[END_REF]). Yet, astrocyte glutamate also contribute to set the extracellular concentration of ambient glutamate that tonically activate NMDA receptors of CA1 neurons [START_REF] Cavelier | Tonic release of glutamate by a DIDS-sensitive mechanism in rat hippocampal slices[END_REF][START_REF] Jabaudon | Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin[END_REF][START_REF] Le Meur | Tonic activation of NMDA receptors by ambient glutamate of non-synaptic origin in the rat hippocampus[END_REF] and regulate synaptic glutamate release through the activation of presynaptic metabotropic glutamate receptors or NMDA receptors in CA1 and in the dentate gyrus, respectively [START_REF] Jourdain | Glutamate exocytosis from astrocytes controls synaptic strength[END_REF][START_REF] Perea | Astrocytes potentiate transmitter release at single hippocampal synapses[END_REF]. In the context of epilepsy, in vitro models of seizure showed that an excitatory loop involving astrocyte Ca 2+ signalling, glutamate and ATP signalling lowers seizure threshold for neurons [START_REF] Gomez-Gonzalo | An excitatory loop with astrocytes contributes to drive neurons to seizure threshold[END_REF]. Moreover, increased astrocyte Ca 2+ signalling and glutamate release contribute to neuronal cell death in the neocortex in vivo after a pilocarpine induced SE [START_REF] Ding | Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus[END_REF].

Finally, it should be noted also that astrocyte Ca 2+ signalling is modulated by inhibitory interneurons through GABA signalling and the activation of astrocyte GABA-B receptors and GAT-3 transporters [START_REF] Mariotti | The inhibitory neurotransmitter GABA evokes long-lasting Ca2+ oscillations in cortical astrocytes[END_REF][START_REF] Matos | Astrocytes detect and upregulate transmission at inhibitory synapses of somatostatin interneurons onto pyramidal cells[END_REF]Mederos et al., 2019) but also through the release of somatostatin by specific interneurons [START_REF] Mariotti | Interneuron-specific signaling evokes distinctive somatostatinmediated responses in adult cortical astrocytes[END_REF]. This interneuron-induced astrocyte Ca 2+ signalling differentially modulates inhibitory synaptic transmission originating from different types of interneurons [START_REF] Matos | Astrocytes detect and upregulate transmission at inhibitory synapses of somatostatin interneurons onto pyramidal cells[END_REF]Mederos & Perea, 2019). Knowing the importance of neuronal inhibitory networks in different forms of epilepsy, such as temporal lobe or focal cortical epilepsies [START_REF] Trevelyan | The Role of Inhibition in Epileptic Networks[END_REF] changes in the activity or survival of inhibitory interneurons can lead to impaired astrocyte signalling that may be relevant in the context of these epilepsies.

Astrocyte release of ATP (mechanisms of release)

ATP plays an important role in physiological and pathological conditions, via activating various receptors on glia and neurons [START_REF] Illes | Purinergic neurone-glia signalling in cognitive-related pathologies[END_REF])(Fig. 1). Under physiological conditions, the concentration of ATP in extracellular space is very low.

However, it can dramatically increase under pathological conditions such as trauma, ischemia/hypoxia, inflammation or excessive neuronal activity [START_REF] Engel | ATPergic signalling during seizures and epilepsy[END_REF][START_REF] Franke | Pathophysiology of astroglial purinergic signalling[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF][START_REF] Rodrigues | ATP as a multi-target danger signal in the brain[END_REF]. The sustained increase of extracellular ATP concentration under pathological conditions indicates that mechanisms regulating ATP release exist.

As for other gliotransmitters (see section 1.4), a variety of molecular mechanisms of ATP release from astrocytes have been suggested, including calcium-dependent exocytosis and opening of large conductance channels such as hemichannels and anion channels [START_REF] Araque | Gliotransmitters travel in time and space[END_REF][START_REF] Engel | ATPergic signalling during seizures and epilepsy[END_REF][START_REF] Hamilton | Do astrocytes really exocytose neurotransmitters?[END_REF][START_REF] Harada | Gliotransmitter Release from Astrocytes: Functional, Developmental, and Pathological Implications in the Brain[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF][START_REF] Xiong | Ca2+-Dependent and Ca2+-Independent ATP Release in Astrocytes[END_REF]. Regarding vesicular release, ATP can be packaged into vesicles and co-released with other transmitters [START_REF] Zimmermann | Signalling via ATP in the nervous system[END_REF]. A vesicular nucleotide transporter (VNUT) concentrating ATP into vesicles has been identified [START_REF] Sawada | Identification of a vesicular nucleotide transporter[END_REF] and is also expressed in non-neuronal cells where it localizes with vesicles of the regulatory secretary pathway [START_REF] Lazarowski | Vesicular and conductive mechanisms of nucleotide release[END_REF]. Moreover, VNUT and fluorescent ATP co-labelled lysosomes in astrocytes have been observed and several groups have shown that astrocytes release ATP by lysosome exocytosis [START_REF] Oya | Vesicular nucleotide transporter is involved in ATP storage of secretory lysosomes in astrocytes[END_REF][START_REF] Verderio | TI-VAMP/VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes[END_REF][START_REF] Zhang | Regulated ATP release from astrocytes through lysosome exocytosis[END_REF]. Verderio and coworkers further identified that vesicle-associated membrane protein-7 (VAMP7), which is insensitive to the tetanus toxin, mediated lysosome exocytosis and contributed to ATP release from astrocytes [START_REF] Verderio | TI-VAMP/VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes[END_REF]. Different functions of astrocyte vesicular release of ATP have been proposed. In cultured astrocytes, ATP vesicular release is the predominant cause of intercellular calcium waves [START_REF] Bowser | Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy[END_REF]. In vivo, brainstem astrocytes were shown to control breathing through a pH-dependent vesicular release of ATP [START_REF] Gourine | Astrocytes control breathing through pH-dependent release of ATP[END_REF][START_REF] Kasymov | Differential Sensitivity of Brainstem versus Cortical Astrocytes to Changes in pH Reveals Functional Regional Specialization of Astroglia[END_REF]. Moreover, the use of transgenic mice expressing a dominant-negative cytoplasmic SNARE domain of the synaptobrevin-2 protein specifically in astrocytes (dnSNARE mice) provided evidence indicating that ATP vesicular release from astrocytes regulates synaptic strength and plasticity in the hippocampus [START_REF] Pascual | Astrocytic Purinergic Signaling Coordinates Synaptic Networks[END_REF], sleep homeostasis in the hypothalamus [START_REF] Halassa | Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss[END_REF], inhibition and induction of long-term potentiation in the neocortex [START_REF] Lalo | Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex[END_REF]Rasooli-Nejad, Palygin, Lalo, & Pankratov, 2014).

Although the specificity of the astrocyte expression of the transgene in these mice has been questioned [START_REF] Fujita | Neuronal Transgene Expression in Dominant-Negative SNARE Mice[END_REF], a more recent report has excluded any neuronal expression in the hippocampus [START_REF] Papouin | Septal Cholinergic Neuromodulation Tunes the Astrocyte-Dependent Gating of Hippocampal NMDA Receptors to Wakefulness[END_REF].

Overall, these studies indicate that astrocytes have the capability to release ATP from synaptic-like vesicles and that this process regulates the physiology of different neuronal networks.

Additional mechanisms contributing to the release of ATP from astrocytes corresponds to the so-called conductive pathways allowing direct exchange between the cytoplasmic compartment and the extracellular space through large pores [START_REF] Lazarowski | Vesicular and conductive mechanisms of nucleotide release[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF]. The best documented of these pathways is ATP permeation through hemichannels, either constituted by pannexins or by connexins.

Although these two families of proteins have similar membrane topology (i.e. 4 transmembrane domains, 2 extracellular and 1 intracellular loop, along with intracellular Nand C-terminal tails), they have no amino acid sequence homology and differ in their activation mode. Several studies have shown that pannexin-1 (PANX1) can release ATP in physiological conditions in response to the activation of different pathways, including purinergic receptor activation [START_REF] Bao | Pannexin membrane channels are mechanosensitive conduits for ATP[END_REF][START_REF] Pelegrin | Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor[END_REF].

Since PANX1 is widely expressed and can be activated at membrane potentials in the physiological range, it is considered as the major cause of ATP efflux through hemichannels. However, the positive feedback loop of ATP-induced ATP release may also constitute a danger signal under pathological conditions [START_REF] Rodrigues | ATP as a multi-target danger signal in the brain[END_REF] and it should be noted that the activity of PANX1 channels is much higher in acute slices (human and mice) experiencing ictal discharges than in slices without ictal discharges [START_REF] Dossi | Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy[END_REF]. Conditions leading to ATP efflux through hemiconnexins require low extracellular calcium concentration and strong depolarization, indicating that this pathway operates mostly in pathological conditions. The results obtained from pannexin-and connexin-deficient mice have led to somewhat controversial interpretations [START_REF] Bedner | Functional redundancy and compensation among members of gap junction protein families?[END_REF][START_REF] Hanstein | Promises and pitfalls of a Pannexin1 transgenic mouse line[END_REF] but they nevertheless support the idea that hemichannels contribute to the release of ATP, particularly in pathological conditions.

Anion channels have long been suspected to mediate ATP release from astrocytes [START_REF] Kimelberg | Anion channels in astrocytes: biophysics, pharmacology, and function[END_REF]. In cultured astrocytes, ATP release through Maxi-anion channels may play a role in glutamate-induced release of ATP [START_REF] Zhao | Maxi-anion channels play a key role in glutamate-induced ATP release from mouse astrocytes in primary culture[END_REF] whereas volume-regulated anion channels (VRAC) contribute to the propagation of calcium waves [START_REF] Fujii | Astrocyte calcium waves propagate proximally by gap junction and distally by extracellular diffusion of ATP released from volumeregulated anion channels[END_REF] through an autocrine mechanism [START_REF] Akita | Ca2+ nanodomain-mediated component of swelling-induced volume-sensitive outwardly rectifying anion current triggered by autocrine action of ATP in mouse astrocytes[END_REF]. The exact contribution of astrocyte anion channels to physiological and pathological processes in vivo remains largely unknown but the recent molecular identification of several subunits constituting these channels should help resolving this issue [START_REF] Han | Tweetyhomolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC swell ) in the Brain[END_REF][START_REF] Osei-Owusu | Molecular Biology and Physiology of Volume-Regulated Anion Channel (VRAC)[END_REF]. In the context of epilepsy, additional studies are required to evaluate the relative contribution of astrocytic exocytotic and conductive pathways to a rise in ATP in the extracellular space during episodes of intense neuronal activities. Interestingly, PANX1 channel activation was shown recently to promote seizure generation through ATP signalling involving P2 receptors in human cortical brain tissue and in a mouse model of MTLE (intra-hippocampal kainate) [START_REF] Dossi | Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy[END_REF]. However, both neuronal and astroglial PANX1 channels were activated in these conditions and the specific role of PANX1dependent release of ATP from astrocytes remains to be tested.

Purinergic signalling in the onset and progression of epilepsy

Despite the long-lasting interest in the study of ATP as a signalling messenger the exact roles of purinergic signalling in pathological conditions is far from being elucidated. Once released into the extracellular space, ATP is rapidly hydrolysed to ADP and adenosine by different ectonucleotidases present in the extracellular environment (Haydon & Carmignoto, 2006). Thereby, the release of ATP generates different purines that can activate different types of receptors, including P2X and P2Y receptors as well as adenosine receptors [START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF]. The activation of these receptors may have a variety of effects on the onset and progression of epilepsy and this will depend on the extracellular concentrations of the different purines. It is commonly believed that these concentrations increase under pathological conditions, [START_REF] Franke | Pathophysiology of astroglial purinergic signalling[END_REF][START_REF] Rodrigues | ATP as a multi-target danger signal in the brain[END_REF] but in the context of epilepsy this has only been convincingly established for adenosine [START_REF] During | Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain[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]. However, a recent in vivo study reported increased ATP concentrations after spontaneous seizures in the chronic phase of the rat pilocarpine model [START_REF] Doná | Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy[END_REF]. New genetically encoded sensors based on the use of mutated P2 receptors should help clarifying this issue for ATP and ADP.

Adenosine has a well-established anticonvulsant action [START_REF] Boison | Adenosine dysfunction in epilepsy[END_REF][START_REF] Boison | Adenosinergic signaling in epilepsy[END_REF][START_REF] Cieślak | Role of the purinergic signaling in epilepsy[END_REF][START_REF] Masino | Adenosine Receptors and Epilepsy[END_REF]. This primarily relies on the activation of adenosine A1 receptors that decrease excitatory synaptic transmission and, for instance, mice lacking A1 receptors have lower threshold of seizure propagation [START_REF] Boison | Adenosine dysfunction in epilepsy[END_REF][START_REF] Fedele | Adenosine A1 receptors are crucial in keeping an epileptic focus localized[END_REF]) whereas A2 and A3 receptors have rather convulsive or deleterious effects [START_REF] Cunha | How does adenosine control neuronal dysfunction and neurodegeneration[END_REF][START_REF] Matute | Neuroglial interactions mediated by purinergic signalling in the pathophysiology of CNS disorders[END_REF]. The astrocytic enzyme adenosine kinase (ADK) appears to play a key role in regulating the anticonvulsant effect of adenosine. The expression of ADK is upregulated during epilepsy, leading to a deficiency in adenosine concentration and thus of A1 receptor activation [START_REF] Boison | Adenosinergic signaling in epilepsy[END_REF].

In contrast, the role of ATP signalling in epilepsy is less well understood. There is, however, a general agreement that ATP signalling contributes the pathology of epilepsy.

Direct application of ATP analogues in the brain evokes neuronal excitation and promotes seizure activity [START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF]. There is a profound remodelling of the expression of hippocampal P2 receptors following SE in rodents [START_REF] Avignone | Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling[END_REF][START_REF] Engel | ATPergic signalling during seizures and epilepsy[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF], suggesting that these receptors contribute to epileptogenesis. Analysis of the role of specific P2 receptors led to contrasting results depending on the identity of these receptors, their cellular expression but also on the type of epilepsy or on the experimental model. Many of these studies were directed toward the analysis of P2X7, P2X4 and P2Y12 receptors expressed by microglia and have been reviewed previously [START_REF] Engel | ATPergic signalling during seizures and epilepsy[END_REF][START_REF] Eyo | Microglia: Lifelong patrolling immune cells of the brain[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF]. Without entering in too many details, it is nevertheless worth mentioning that P2X4 and P2X7 have rather detrimental effects whereas P2Y12 have more protective effects on cell survival and disease progression in experimental SE or TLE. It should also be remembered that purinergic signalling is an important communication pathway between astrocytes and microglia. For The role of metabotropic P2Y1 receptors in epilepsy has been also studied. P2Y1 is expressed mostly in astrocytes, oligodendrocytes and in neurons, in particular hippocampal inhibitory interneurons [START_REF] Bowser | ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks[END_REF]. A recent study based on the analysis of P2Y1 deficient mice but also on the use of selective agonists and antagonists of P2Y1 indicates that these receptors have an anticonvulsant action at the onset of SE, either triggered by systemic pilocarpine or by intra-amygdala kainate, but have proconvulsant effects once SE has been initiated and favour spontaneous seizures during the chronic phase of these models [START_REF] Alves | Context-Specific Switch from Anti-to Proepileptogenic Function of the P2Y 1 Receptor in Experimental Epilepsy[END_REF]. Surprisingly, the same study reported an up-regulation of P2Y1 in microglia and in neurons, but no expression of these receptors in astrocytes after SE. Considering the many studies involving a role of P2Y1 in astrocytes, cell type specific knockout of P2Y1 receptors are now needed to help clarifying these complex effects (see also below). Overall these observations support the idea that ATP signalling intervenes in epilepsy, but the exact mechanisms involved and their influence on epileptogenesis and ictogenesis are still unclear. However, several mechanisms involving ATP signalling in astrocytes have been recently updated under pathological conditions and in particular in the context of epilepsy.

Astrocyte ATP-mediated intercellular signalling in epilepsy

ATP is an essential mediator of communication between astrocytes and between astrocytes and other types brain cells [START_REF] Butt | ATP: A ubiquitous gliotransmitter integrating neuron-glial networks[END_REF](Fig. 1). Whether astrocyte purinemediated signalling is impaired in epilepsy is an intriguing question receiving increased attention. Genetic impairment of the vesicular ATP release from astrocytes by expressing a dominant-negative SNARE (see above and [START_REF] Pascual | Astrocytic Purinergic Signaling Coordinates Synaptic Networks[END_REF]) delays the onset of recurrent seizures, decrease epileptiform activity and reduce hippocampal damage after a pilocarpine-induced SE [START_REF] Clasadonte | Astrocyte control of synaptic NMDA receptors contributes to the progressive development of temporal lobe epilepsy[END_REF]. These effects probably involve the control of NMDA receptor expression that is regulated by adenosine A1 receptor activation following ATP release from astrocyte [START_REF] Deng | Astrocytic activation of A1 receptors regulates the surface expression of NMDA receptors through a Src kinase dependent pathway[END_REF][START_REF] Fellin | Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo[END_REF]. On the other hand, ATP release through PANX1 appears to contribute to ictal discharges in human epileptic brain tissue and in mouse models of MTLE (intra-hippocampal kainate) or SE (systemic kainate) [START_REF] Dossi | Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy[END_REF][START_REF] Santiago | Targeting Pannexin1 Improves Seizure Outcome[END_REF], although in these two studies the source of ATP was not specifically determined. Once released by astrocytes, ATP could have several effects. Selective activation of hippocampal astrocytes by optogenetics was shown to induce ATP release that led to the excitation of CCK-expressing interneurons, through P2Y1 receptor activation, and to the inhibition of pyramidal cells, through A1 receptors [START_REF] Tan | Glia-derived ATP inversely regulates excitability of pyramidal and CCK-positive neurons[END_REF]. These effects should rather have anti-convulsive effects but it should be noted that they have been observed and studied in the presence of glutamate and GABA-A receptor antagonists, making it difficult to evaluate their consequences on the whole hippocampal network. Using a similar approach, but in absence of blockers of synaptic transmission, we observed that the initial and dominant effect of optogenetic activation of hippocampal astrocytes was an excitation of pyramidal neurons [START_REF] Shen | An autocrine purinergic signaling controls astrocyte-induced neuronal excitation[END_REF]. This excitation was the consequence of an autocrine ATP signalling in astrocytes involving P2Y1 receptors and boosting Ca 2+

signalling and glutamate release from astrocytes. In CA1, this astrocyte glutamate release led to the activation of post-synaptic NMDA receptors and pre-synaptic mGluR receptors on excitatory terminals. Of note, this initial excitation of pyramidal neurons was followed by an inhibition due to A1 receptor activation [START_REF] Shen | An autocrine purinergic signaling controls astrocyte-induced neuronal excitation[END_REF], as observed by [START_REF] Tan | Glia-derived ATP inversely regulates excitability of pyramidal and CCK-positive neurons[END_REF]. It is difficult to extrapolate from these observations made with optogenetic activation of astrocytes in non-epileptic tissue what would be the net effect of astrocyte ATP signalling in the epileptic hippocampus where concentration of purines and expression of their receptors are modified (see section 3 above). However, enhanced Ca 2+ signalling in astrocytes, P2Y1 receptors and presynaptic mGluR receptors had been already associated with epilepsy. Ca 2+ signalling is enhanced in cortical astrocytes during several days after pilocarpine-induced SE and contribute to neuronal excitotoxicity [START_REF] Ding | Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus[END_REF]. Ca 2+ signals in hippocampal astrocytes facilitate the propagation of epileptiform activity induced by kainate in vivo [START_REF] Heuser | Ca2+ Signals in Astrocytes Facilitate Spread of Epileptiform Activity[END_REF] and Ca 2+ signalling is enhanced in astrocyte endfeet in vivo during the latent phase of a mouse model of MTLE (unilateral intra-cortical kainate; [START_REF] Szokol | Augmentation of Ca(2+) signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy[END_REF]). In a rat model (rapid kindling) of MTLE, activation of P2Y1 receptors is responsible for astrocyte Ca 2+ hyperactivity that induces an increase in glutamate release from astrocytes and a downstream activation of neuronal mGluR5, thereby increasing release probability at the CA3-CA1 synapses [START_REF] Álvarez-Ferradas | Enhanced astroglial Ca2+ signaling increases excitatory synaptic strength in the epileptic brain[END_REF]. Blocking astrocyte Ca 2+ signalling or inhibiting P2Y1 or mGluR5 receptors relieved this abnormal enhancement of synaptic strength. The same group reported subsequently that astrocyte PANX1 hemichannels are the route through which ATP is released before activating P2Y1 receptors and triggering the increased astroglial Ca 2+ signalling [START_REF] Wellmann | Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy[END_REF]. These observations are consistent with an enhanced autocrine ATP signalling being responsible for an increased glutamate release from astrocytes promoting neuronal excitation in the epileptic hippocampus. It is not clear yet whether this autocrine signalling pathway is involved in the purine-dependent action of astrocytes favouring ictal discharges in human epileptic brain tissue (see above and [START_REF] Dossi | Pannexin-1 channels contribute to seizure generation in human epileptic brain tissue and in a mouse model of epilepsy[END_REF]). It should be noted, however, that in a recent study using a model of SE induced by systemic injection of kainate, targeted deletion of PANX1 from astrocytes lowers the level of ATP but potentiates seizures during SE [START_REF] Scemes | Astrocyte and Neuronal Pannexin1 Contribute Distinctly to Seizures[END_REF]. This effect was found to be related to the lower level of adenosine consecutive to the increased level of ADK in astrocytes.

Altogether, the majority of the above cited observations point to the conclusion that the actions of astrocyte purinergic signalling determined by the combined effects of ATP, adenosine and glutamate can displace the equilibrium between excitation and inhibition and promote appearance of epileptiform activity. It is important to note that P2Y1 regulation of astrocyte Ca 2+ signalling described in the context of epilepsy has been also

reported in a mouse model of Alzheimer's disease [START_REF] Delekate | Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model[END_REF][START_REF] Reichenbach | P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer's disease model[END_REF] and in a model of traumatic brain injury [START_REF] Choo | Antagonism of purinergic signalling improves recovery from traumatic brain injury[END_REF]. In both cases, blocking this pathway had beneficial consequences on outcome of these models. Impaired astrocyte purinergic signalling could thus be a common signature of different CNS pathologies.

Purinergic interactions between microglia and astrocytes are likely to influence how astrocytes regulate neuronal excitability and epilepsy progression. Together with astrogliosis, reactivity of microglia is another typical feature of human and experimental epilepsies and purinergic signalling is enhanced in both cell types [START_REF] Devinsky | Glia and epilepsy: excitability and inflammation[END_REF][START_REF] Rassendren | Purinergic signaling in epilepsy[END_REF]. In the hippocampus of naïve animals, activation of microglial TLR4 receptors rapidly induces a release of ATP that activates astrocytic P2Y1 receptors and the release of glutamate by astrocytes, which in turn activates mGluR5 receptors on presynaptic glutamatergic terminals [START_REF] Pascual | Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission[END_REF]. This cascade eventually leads to an enhancement of excitability and promotes the appearance of epileptiform activities in vitro in experimental conditions where GABA receptors are blocked and external Mg 2+ is low. Since microglial cells also express purinergic receptors, the release of ATP by astrocytes consecutively to the activation of P2Y1 receptors (see above) could initiate a vicious circle by recruiting further microglia. This possibility has not been specifically tested in the context of epilepsy but purine-mediated crosstalk between these glial cells has been reported in the corpus callosum [START_REF] Davalos | ATP mediates rapid microglial response to local brain injury in vivo[END_REF][START_REF] Schipke | Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices[END_REF] and also in the dentate gyrus following pilocarpine induced SE by showing that blocking microglial P2X7 receptors prevents astroglial death [START_REF] Kim | Blockade of P2X receptor prevents astroglial death in the dentate gyrus following pilocarpine-induced status epilepticus[END_REF]. Interestingly, in a model of traumatic brain injury, reactive microglia induces a protective phenotype of astrocytes by down-regulating the expression of astrocytic P2Y1 receptors [START_REF] Shinozaki | Transformation of Astrocytes to a Neuroprotective Phenotype by Microglia via P2Y 1 Receptor Downregulation[END_REF].

Bidirectional interactions between microglia and astrocytes in epilepsy will not only set the level of purinergic signalling but probably also that of microglial release of cytokines. This is particularly important since cytokines are implicated in the control of glutamate release from astrocytes (see next section). Careful examination of purinergic receptors types that contribute to the crosstalk between astrocytes and microglia is required to better characterize paracrine glial signalling in epilepsy. Clearly more targeted genetic approaches involving deletion or activation of specific type of purinergic receptor on astrocytes or microglia could clarify the actual outcome of such purinergic communication on the functional changes in the epileptic brain.

Cytokine control of astrocyte purinergic signalling in epilepsy

There is strong evidence that high levels of key pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and IL-1β, and downstream signalling that these cytokines promote, contribute to the neuronal hyper-excitability and degeneration in epilepsy (van Vliet, Aronica, [START_REF] Van Vliet | Review: Neuroinflammatory pathways as treatment targets and biomarker candidates in epilepsy: emerging evidence from preclinical and clinical studies[END_REF].These neuromodulatory actions of cytokines, distinct from their canonical function in the immune system, could induce seizures by acting on astrocytes in multiple ways. For instance, these cytokines released by microglia inhibit gap junctions in astrocytes in vitro [START_REF] Meme | Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by beta-amyloid[END_REF] and in MTLE, inhibition of gap junctions by TNFα and IL-1β leads to the impairment of astrocytic buffering of extracellular K + [START_REF] Bedner | Astrocyte uncoupling as a cause of human temporal lobe epilepsy[END_REF]. Furthermore, TNFα could induce seizures through excessive astrocyte-neuron gliotransmitter mediated signalling. Clues supporting this idea can be drawn from studies showing, on the one hand, that cytokines can boost glutamate release from astrocytes [START_REF] Vesce | Glutamate release from astrocytes in physiological conditions and in neurodegenerative disorders characterized by neuroinflammation[END_REF] and, on the other hand, that astrocytic glutamate promotes abnormal neuronal activity in epilepsy (see sections 1.4 and 4).

In the context of purinergic signalling in astrocytes, TNFα is the key cytokine that can amplify astrocytic glutamate release through coupling with P2Y1 receptors (Fig. 1). It was first shown that binding of TNFα to the astrocytic TNFR1 receptors is a powerful positive modulator of glutamate release from astrocytes [START_REF] Bezzi | CXCR4-activated astrocyte glutamate release via TNFalpha: amplification by microglia triggers neurotoxicity[END_REF]. Later on, the same group showed that TNFα controls P2Y1R-dependent glutamate release from astrocytes [START_REF] Domercq | P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins[END_REF] and that gliotransmission driven by this pathway loses its efficacy in modulating glutamatergic synaptic activity in TNFα knockout mice [START_REF] Santello | TNFalpha controls glutamatergic gliotransmission in the hippocampal dentate gyrus[END_REF]. This stimulation of astrocyte glutamate release by a cytokine was demonstrated in the dentate gyrus and leads to increase the release probability at glutamatergic synapses impinging onto granule cells through the activation of pre-synaptic NMDA receptors. It is not clear yet whether TNFα also potentiate other outcomes of astrocyte glutamate release in the hippocampus, such as the generation of SICs [START_REF] Angulo | Glutamate released from glial cells synchronizes neuronal activity in the hippocampus[END_REF][START_REF] Fellin | Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors[END_REF] or the mGluR-dependent potentiation of synaptic excitation in CA1 (Gertrudis [START_REF] Perea | Astrocytes potentiate transmitter release at single hippocampal synapses[END_REF].

Although P2Y1R-dependent signalling is one of the main pathways regulating astrocyteneuron interactions through the release of glutamate, it has not been considered as a specific cytokine target in the epileptic brain. However, TNFα signalling is rapidly upregulated in the hippocampus after SE [START_REF] Avignone | Status epilepticus induces a particular microglial activation state characterized by enhanced purinergic signaling[END_REF] and astrocytic purinergic P2Y1 signalling is highly active in models of MTLE (see above section 4). Our recent findings indicate that TNFα signalling is one pathway that increases astrocyte purinergic signalling in epilepsy. We indeed confirmed that TNFα triggers a Ca 2+ -dependent glutamate release from astrocytes that boosts excitatory synaptic activity in granule cells of the dentate gyrus through presynaptic NMDA receptors (see above). We further showed that this effect of TNFα can be mimicked by optogenetic activation of astrocyte and demonstrated that this action involves the autocrine activation of astrocyte P2Y1 receptors [START_REF] Nikolic | Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis[END_REF]. In a mouse model of MTLE (unilateral intracortical kainate), this autocrine loop seems permanently activated at the end of the latent phase that precedes the appearance of spontaneous seizures: In the ipsilateral dentate gyrus, the frequency of mEPSCs is higher than in the control conditions, exogenous TNFα does not trigger anymore the increase of excitatory synaptic transmission and blocking P2Y1 receptors, which has no effect in control conditions, reduces excitatory synaptic activity to the level of control conditions [START_REF] Nikolic | Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis[END_REF]. This signalling pathway could therefore be responsible for the lowering of seizure threshold by TNFα [START_REF] Iori | Modulation of neuronal excitability by immune mediators in epilepsy[END_REF] and thus be an important component of the functional remodelling (epileptogenesis) that will drive the hippocampus to spontaneous seizures.

Targeting the coupling of TNFα with astrocyte purinergic signalling may thus hold the potential to be a useful therapeutic strategy for reducing astrocytic glutamate release and normalizing synaptic activity in epilepsy. It should be kept in mind, however, that the precise mechanism of the cytokine-P2Y1 interaction in epilepsy is still unclear. A promising candidate to be considered is of course TNFR1 whose expression increased in animal model of seizures and has been reported to mediate the ictogenic effects of TNFα [START_REF] Iori | Modulation of neuronal excitability by immune mediators in epilepsy[END_REF]. Interestingly, TNFR1 has been shown to mediate TNFα effects on astrocyte glutamate signalling in an animal model of multiple sclerosis [START_REF] Habbas | Neuroinflammatory TNFalpha Impairs Memory via Astrocyte Signaling[END_REF].

Inhibiting the function of astrocyte TNFR1, or P2Y1, should prevent the lowering of seizure threshold. Our observation that this TNFα-P2Y1 pathway is activated during the latent phase, i.e. before the appearance of the first spontaneous seizures, suggests that targeting this pathway could be a disease-modifying strategy through which the occurrence of spontaneous seizures could be delayed or event prevented.

  instance, microglial cells sense ATP released by astrocytes[START_REF] Davalos | ATP mediates rapid microglial response to local brain injury in vivo[END_REF][START_REF] Schipke | Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices[END_REF] and ATP released by microglia enhances Ca 2+ signalling and glutamate release by astrocytes (O.[START_REF] Pascual | Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission[END_REF]. It is therefore very likely that microglia and astrocyte purinergic signalling influence each other in pathological settings known to favour the release of ATP (see above).

Figure 1 :

 1 Figure 1: Purinergic signalling between astrocytes, neurons and microglial cells in epilepsy. The mechanisms of ATP release and the involvement of specific purinergic P2X and P2Y receptors types are depicted for astrocytes and microglia. Activation of astrocytic P2Y1 receptors leads to the rise in intracellular Ca 2+ that promotes astrocyte release of glutamate, which acts on pre-and post-synaptic neuronal glutamate receptors and increases excitation. ATP released from astrocytes can be converted into adenosine and promote activation of adenosine A1 receptors which will favour inhibition. The cytokine TNF likely released by microglia, activates the P2Y1R-dependent glutamate release from astrocytes and thus neuronal excitation.

Acknowledgments

Work in Audinat Lab is supported by grants from the Fédération pour la Recherche sur le Cerveau (FRC) and the European Commission (EC-H2020 MSCA-ITN EU-GliaPhD No. 722053). Ljiljana Nikolic is supported by Ministry of Education, Science and Technological Development of Republic of Serbia (grant no. 173027).

Conflict of Interest Statement

The authors declare no conflict of interest.


Muhammad Usman
email: muhammad.usman@squ.edu.om

Muhammad Farooq
email: muhammad.farooq@uvas.edu.pk

Khalil Hanna
email: khalil.hanna@ensc-rennes.fr

Environmental side effects of the injudicious use of antimicrobials in the era of COVID-19

Moreover, a large number of people are self-medicating with antibiotics in a misguided attempt to protect themselves from the virus. This practice is especially prevalent in developing communities. CoV-2). Therefore, researchers have become increasingly concerned about the inclinic impacts of COVID-19 on overall levels of antimicrobial resistance (AMR) [START_REF] Hsu | How covid-19 is accelerating the threat of antimicrobial resistance[END_REF].

It is well recognized that excessive use of antimicrobials in medicine and agriculture drives AMR which is a global threat to human, animal and environmental health [START_REF] Maillard | Reducing antibiotic prescribing and addressing the global problem of antibiotic resistance by targeted hygiene in the home and everyday life settings: A Position Paper[END_REF]. According to the US Center for Disease Prevention and Control (2019), up to 50% of antibiotic use is not medically justified. This situation is particularly worse in developing communities which have unregulated over-the-counter sales and a weak water and sewage infrastructure. For example, a recent typhoid fever outbreak in Pakistan has been associated to the antibiotic resistance caused by inappropriate use of drugs as well as poor sanitation and contaminated water supplies [START_REF] Cohen | Frightening' typhoid fever outbreak spreads in Pakistan[END_REF]. Recent estimates by UN Interagency Coordination Group on Antimicrobial Resistance (2019) suggest that AMR diseases are responsible for about 700,000 annual deaths worldwide. Without concrete action, the death toll could reach up to 10 million a year by 2050.

A c c e p t e d m a n u s c r i p t

The intent of this communication is to highlight the consequences of COVID-19 management on the environment. The focus will be on the unnecessary use of antibacterial drugs and personal hygiene products in the treatment and prevention of COVID-19. A brief description of these issues is provided below.

Excessive use of therapeutic antimicrobials

Antibiotics have reasonable roles in the management of COVID-19 particularly to treat suspected or confirmed bacterial co-infections (Rawson et al., 2020a). The World Health Organization has advised that antibiotics should not be used for the purposes of treating COVID-19 except in the presence of bacterial co-infections (WHO, 2020).

However, growing evidence suggest that very high proportion of COVID-19 patients are being unnecessarily treated with antibiotics. Empirical antibiotics were prescribed for 90% of patients despite low confirmation of secondary bacterial infections (<10%) [START_REF] Lai | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges[END_REF]. Wei et al. ( 2020) also noted that antibiotics were started on admission in 59% of COVID-19 patients despite the absence of definitive evidence of bacterial coinfection in these patients. Moreover, 98% of these prescriptions were empiric.

Similarly, another very recent review of data from covid-19 cases, mostly from Asia, reported that >70% of patients received antimicrobial treatment despite only <10%

having bacterial or fungal coinfections (Rawson et al., 2020a). Moreover, the same study found that broad spectrum antibiotics, intended to kill a wide range of bacteria, are being administered. Overuse of these antibiotics can stimulate AMR in the acute care settings [START_REF] Hsu | How covid-19 is accelerating the threat of antimicrobial resistance[END_REF]Rawson et al., 2020b). Premature hype surrounding possible treatments for COVID-19 could also spike the prescription of antibiotics. This has been seen for the antibiotic azithromycin in combination with the drug hydroxychloroquine causing shortages in the US (Reardon, 2020). Though unmeasured, there is a possibility of a large number of people taking antibiotics in misguided attempts to selfmedicate to protect themselves from the virus. This has the potential to be very prevalent in developing communities where antibiotics can be easily obtained without Release of antibiotic resistant bacteria along with large quantities of antibiotics to the wastewater would spur the AMR and other unintended consequences on the environment. A recent review detailing pharmaceutical pollutants in global aquatic systems found antibiotics as the most-frequently detected compounds (Patel et al., 2019). It should be noted that wastewater is the major hotspot for AMR as it is characterized by extremely high bacterial loads coupled with subtherapeutic antibiotics [START_REF] Berendonk | Tackling antibiotic resistance: the environmental framework[END_REF].

Excessive use of non-therapeutic antimicrobials

Proper hand hygiene has been recognized as the first line of defense against this pandemic. Therefore, frequent hand-washing with plain soap and water for at least 20 seconds has been advised as an impactful way to prevent the spread of COVID-19 (WHO, 2020). Soaps contain surfactants that dissolve the lipid-bilayer, an integral part of SARS-CoV-2 envelop, leading to the deactivation of virus. These deactivated viral molecules are then encapsulated within micelles which are then washed away [START_REF] Chaudhary | Fighting the SARS CoV-2 (COVID-19) Pandemic with Soap[END_REF]. A 20-second wash provides sufficient time for the soap molecules to dissolve the lipid-bilayer of the SARS-CoV-2 virus.

All regular soaps available in the marketplace have the basic ingredients and chemistry required to disrupt the virus [START_REF] Chaudhary | Fighting the SARS CoV-2 (COVID-19) Pandemic with Soap[END_REF]. Common soaps are effective at inactivating the enveloped viruses such as the SARS-CoV-2 therefore, antibacterial soaps are not required (UNESCO, 2020). The US-Food and Drug Administration (FDA) has also advocated the same and moved to ban 19 antimicrobial chemicals from soaps acknowledging their dangerous impacts [START_REF] Mcnamara | Triclosan: an Instructive Tale[END_REF]. Despite this, use of antibacterial soaps and disinfectants has increased during this pandemic as evident from sharp increase in their sale worldwide [START_REF] Matters | Coronavirus: Supplies run low of anti-bacterial hand-soaps[END_REF]. Similar increase A c c e p t e d m a n u s c r i p t in demand for antibacterial products/soaps was noted in the era of 2009 Swine flu [START_REF] Hickman | Soap and medicine sales soar due to swine flu[END_REF]Slater et al., 2011).

Antibacterial products contain high amounts of biocides which result in a sharp increase in their presence in wastewater. As a matter of fact, antimicrobial chemicals from soaps could occur in wastewater biosolids (that are land applied as soil conditioners) at multiple orders of magnitude higher than the concentrations of antibiotics [START_REF] Mcclellan | Pharmaceuticals and personal care products in archived U.S. biosolids from the 2001 EPA national sewage sludge survey[END_REF]. This may pose severe risks to ecosystem and to the spread of AMR in the environment. In addition to the AMR, persistence of these biocides in the environment is a major environmental threat that calls for suitable remediation [START_REF] Mcnamara | Triclosan: an Instructive Tale[END_REF]. Moreover, presence of these biocides can sabotage the wastewater treatment processes that rely on microbial activity [START_REF] Mcnamara | The Impacts of Triclosan on Anaerobic Community Structures, Function, and Antimicrobial Resistance[END_REF]. These compounds may enter into soil system disrupting the functioning of native biota playing crucial roles in biogeochemical cycling of elements and environmental remediation (Patel et al., 2019).

Concluding remarks and recommendations

Antibiotic stewardship principles should continue to be applied and promoted in the times of COVID-19 [START_REF] Huttner | COVID-19: don't neglect antimicrobial stewardship principles![END_REF]. However, development of an antimicrobial policy specific for COVID-19 is urgently needed. For this, coordinated strategies at the individual, health-care and policy levels are essential (Rawson et al., 2020b).

Since current wastewater treatment techniques are unable to offer complete elimination of these compounds [START_REF] Kümmerer | A path to clean water[END_REF]Rodriguez-Mozaz et al., 2020), our daily exposure to various antibiotics, biocides and their active metabolites in environment becomes inevitable. Pollutant input prevention at the production source complemented with wastewater treatment is crucial to ensure water safety [START_REF] Kümmerer | A path to clean water[END_REF]. However, effective input prevention in case of antimicrobials and other pharmaceuticals calls for responsible use of drugs and personal care products by individuals. Intensive information campaigns should be launched to educate the masses about the ecological consequences of misusing antibacterial products. A large

  in the treatment and prevention of COVID-19, caused by novel coronavirus SARS-CoV-2, is on the rise. The increased use of antimicrobials can have serious consequences on the environment. Antibiotics have had a reasonable role in bacterial co-infections with regards to the management of COVID-19. However, recent evidences suggest that there has been injudicious prescription of antimicrobials.

  high proportion of these drugs in bioactive forms is excreted into the wastewater from where they may enter in natural systems(Slater et al., 2011).

  population is creating the habit of regular hand washing as a result of the pandemic. These practices will remain embedded even after the pandemic subsides. products during the pandemic will have the greatest opportunity to instilling change. Governments should ensure that advertising material and message of hygienic products are aligned with COVID-19 guidelines. We also urge researchers to evaluate the environmental consequences of the response measures to COVID-19 by focusing on therapeutic as well as non-therapeutic antimicrobials.

Acknowledgments Dr. M. Usman gratefully acknowledges the research grant from PEIE Research Chair for the Development of Industrial Estates and Free Zones, Sultan Qaboos University, Oman (CHAIR/DVC/MADAYN/20/02).

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