Gre ´gory Ghe ´zali

Glenn Dalle ´rac

Nathalie Rouach

Perisynaptic astroglial processes: dynamic processors of neuronal information

Keywords: Astrocytes, Neurons, Neuroglial interactions, Synapses, Processes, Neurotransmission

Neuroglial interactions are now recognized as essential to brain functions. Extensive research has sought to understand the modalities of such dialog by focusing on astrocytes, the most abundant glial cell type of the central nervous system. Neuron-astrocyte exchanges occur at multiple levels, at different cellular locations. With regard to information processing, regulations occurring around synapses are of particular interest as synaptic networks are thought to underlie higher brain functions. Astrocytes morphology is tremendously complex in that their processes exceedingly branch out to eventually form multitudinous fine leaflets. The latter extremities have been shown to surround many synapses, forming perisynaptic astrocytic processes, which although recognized as essential to synaptic functioning, are poorly defined elements due to their tiny size. The current review sums up the current knowledge on their molecular and structural properties as well as the functional characteristics making them good candidates for information processing units.

Introduction

Cellular composition of the brain is of two main kinds: neurons and glia. Neurons have for long been regarded as the exclusive basic structural element undertaking noble functions of the brain, allowing for subtle sensory inputs processing as well as higher abilities such as memory making and reflection. In contrast glial cells, which outnumber neurons, have been considered as a mere ''glue'' filling the space between neurons and supporting their activity by providing a metabolic bridge with blood capillaries. Although glia does play such roles, in the last decades the picture has become incredibly more complex. Astroglia, the most abundant cell type of the central nervous system, has indeed been found to regulate ionic homeostasis and neurotransmitter levels in the extracellular space and substantial evidence now shows that they are capable of releasing neuroactive molecules known as gliotransmitters, e.g., glutamate, D- serine, ATP [START_REF] Parpura | Glutamate-mediated astrocyte-neuron signalling[END_REF][START_REF] Mothet | Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine[END_REF][START_REF] Pangrsic | Exocytotic release of ATP from cultured astrocytes[END_REF]. Such finding provoked a fundamental conceptual change for neurosciences as synapses should now be considered as a party of three: the preand postsynaptic neurons as well as the astrocytes. Morphologically, the latter are characterized by a small soma from which expand manifold ramifications so that the portion of non-somatic membranes largely predominates. Classical astrocytic markers such as GFAP or SR101 actually enable to visualize only a very restricted portion of these ramifications (Fig. 1a, c-e). The radiance of astrocytes is in fact extremely complex and exceedingly branches out to eventually cover most confined intercellular spaces. The thinnest portions of these ramifications form sheet-like structures that are crucial for modeling fine brain organization during G. Dalle ´rac and N. Rouach contributed equally to this work.

& Nathalie Rouach nathalie.rouach@college-de-france.fr development as well as in adulthood. One hallmark of such function is the insulation provided by oligodendrocyte myelin sheaths allowing for accelerated action potential propagation. Similarly, astroglial processes contacting nodes of Ranvier also influence axonal spreading of electrical activity [START_REF] Butt | Astrocyte associations with nodes of Ranvier: ultrastructural analysis of HRP-filled astrocytes in the mouse optic nerve[END_REF]. The socalled perisynaptic astrocytic processes (PAPs) play a particularly prominent role at synapses where they regulate their formation, by providing an astroglial ''cradle'' and positive molecular factors [START_REF] Nedergaard | Artifact versus reality-how astrocytes contribute to synaptic events[END_REF][START_REF] Allen | Role of glia in developmental synapse formation[END_REF], as well as elimination by direct engulfing and release of negative regulators [START_REF] Allen | Role of glia in developmental synapse formation[END_REF][START_REF] Chung | Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways[END_REF]. The size and shape of PAPs enable them to come in direct apposition with the synaptic cleft (Fig. 2), where they are ideally placed to sense neuronal activity, notably through transport of potassium and glutamate [START_REF] Dalle ´rac | How do astrocytes shape synaptic transmission? Insights from electrophysiology[END_REF]).

Although it is generally well-accepted that they respond by regulating neurotransmission, whether this is mediated by gliotransmission (i.e., release of neuroactive substances from astrocytes to modulate neuronal activity) or indirectly as a result of homeostatic regulations has been and remains subject of intense debates. Such dissensions are difficult to solve in that PAP properties are not well defined given the nanometric scale at which direct optical or physiological measures would have to be performed. Here, we review the current knowledge on the molecular and functional aspects of PAPs with an integrated point of view regarding the tripartite synapse during development as well as adulthood in health and diseases.

PAP as the third synaptic compartment

In the adult brain, protoplasmic astrocytes of the cortical gray matter exhibit a complex ''spongiform'' shape made of tridimensional ramifications of processes, generally organized such that on one side an end-foot process contacts a blood vessel and on the other side numerous fine processes nestle close to active synapses. As such cells do not display the clear polarity usually provided by a basolateral organization, it has recently been proposed to define astrocytic edges according to the cellular compartment their membrane domains contact as well as their unique protein expression patterns [START_REF] Derouiche | Beyond polarity: functional membrane domains in astrocytes and Mu ¨ller cells[END_REF]. In this respect, the most distal and finest leaflets of the astrocytic tree have been shown in brain slices to preferentially localize at the vicinity of glutamatergic synapses [START_REF] Spacek | Three-dimensional analysis of dendritic spines. III. Glial sheath[END_REF]. These PAPs recruit a specific repertoire of proteins, display remarkable dynamic characteristics and form specialized signaling compartments. The current section describes these properties and further discusses how information arriving at the PAP may be conveyed to the astrocytic soma.

Specific molecular composition of PAPs

PAPs express a unique set of proteins that are particularly associated with glutamate metabolism and signaling, such as the catabolizing enzyme glutamine synthetase (GS, [START_REF] Derouiche | Astroglial processes around identified glutamatergic synapses contain glutamine synthetase: evidence for transmitter degradation[END_REF], the high-affinity glutamate transporters (GluT) GLT-1 and GLAST (Rothstein et al. 1994), the vesicular glutamate transporters VGLUT1 and 3 (Ormel et al. 2012a, b), the metabotropic glutamate receptors (mGluRs) 3 and 5 [START_REF] Lavialle | Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors[END_REF]) (Fig. 2). Astroglial processes tend to make closer contacts with the postsynaptic rather than presynaptic element [START_REF] Lehre | Asymmetry of glia near central synapses favors presynaptically directed glutamate escape[END_REF] and, in this regard, are further enriched with a repertoire of transmembrane and membrane-anchored proteins supporting bidirectional neuroglial signaling. For instance, dendritic spines express Eph receptor tyrosine kinases, while PAPs contain ephrin cell-surface ligands [START_REF] Murai | Eph receptors and ephrins in neuronastrocyte communication at synapses[END_REF], together with c-protocadherin [START_REF] Garrett | Control of CNS synapse development by {gamma}-protocadherin-mediated astrocyte-neuron contact[END_REF] and most likely integrin receptors [START_REF] Hama | PKC signaling mediates global enhancement of excitatory synaptogenesis in neurons triggered by local contact with astrocytes[END_REF][START_REF] Herrera-Molina | Astrocytic aVb3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1[END_REF]) (Fig. 2). Based on their small size (hundreds of nanometers thick), PAPs are generally assumed to not contain organelles, although they may enclose some mitochondria [START_REF] Lovatt | The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex[END_REF], which have been found in fine astroglial processes near synapses [START_REF] Genoud | Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex[END_REF][START_REF] Lavialle | Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors[END_REF]). Yet, these domains do contain cytoskeletal elements that differ from their larger parent process; namely, PAPs are filled with dense cortical actin fibers (Fig. 2) and occasionally microtubules at their base, and appear devoid of intermediate filament, including the widely used molecular marker glial fibrillary acidic protein (GFAP, Fig. 1a,c) [START_REF] Derouiche | Anatomical aspects of glia-synapse interaction: the perisynaptic glial sheath consists of a specialized astrocyte compartment[END_REF][START_REF] Haseleu | Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs)[END_REF]. Most interestingly, spatial segregation of such proteins in the astrocytic tree appears to arise from compartmentalization of messenger RNAs. Indeed, whereas mRNAs encoding for GFAP have been found in situ in retinal glial cells (Mu ¨ller cells) to be confined in the cytoplasm, actin mRNAs are mainly localized in the cell processes [START_REF] Sarthy | Subcellular localization of an intermediate filament protein and its mRNA in glial cells[END_REF]). Further, it has recently been shown that astrocyte protrusions are enriched with additional mRNAs, such as Ras-related proteins, Plakophilin-4, Ankyrin Repeat Domain 25 and inositol polyphosphate-1phosphatase [START_REF] Thomsen | A Boyden chamber-based method for characterization of astrocyte protrusion localized RNA and protein[END_REF]. To recruit molecules at PAPs, astrocytes may require scaffolding proteins, as seen in perivascular endfeet, where dystrophinassociated complex proteins undertake the anchoring of Kir4.1 potassium channels and aquaporin-4 water channels [START_REF] Enger | Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Mu ¨ller cells and brain astrocytes in mouse[END_REF]. Accordingly, actin-associated proteins of the ezrin radixin moesin (ERM) family have been described to be specifically enriched in PAPs [START_REF] Derouiche | Peripheral astrocyte processes: monitoring by selective immunostaining for the actin-binding ERM proteins[END_REF], where they dynamically regulate membrane cytoskeleton linkage. However, most scaffolding proteins in PAPs are still unknown and, therefore, represent an exciting target for future studies. Moreover, compartmentalization of the various cytoskeletal elements along the astrocytic tree supposes serial mRNA and protein transport systems. For instance, the APC tumor-suppressor protein, known to be implicated in mRNA anchoring at the leading edge of migrating fibroblasts [START_REF] Mili | Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions[END_REF] The well-known astrocytic transport of glutamate is crucial for maintaining the tonic levels of glutamate at extremely low concentrations at the synaptic cleft (see [START_REF] Moussawi | Extracellular glutamate: functional compartments operate in different concentration ranges[END_REF]. Once in the PAP, glutamate is either recycled via transformation into glutamine and vice versa, or incorporated into astrocytic vesicle through the vesicular transporters Vglut1 and 3. Direct release of glutamate mediated by opening of hemichannels is also a possible fate of PAP glutamate repertoire of proteins. However, given that mechanisms of cargo transports in astrocyte processes are still poorly understood, these hypotheses have yet to be thoroughly investigated in situ. This might be achieved using a recently described variant of the Boyden chambers assay, in which a membrane with small pores separates cultured cells from the bottom compartment, allows for the isolation of cell protrusions and thus enables genome-wide screenings of their components [START_REF] Thomsen | A Boyden chamber-based method for characterization of astrocyte protrusion localized RNA and protein[END_REF].

Another freshly described method consists in an acute cell dissociation procedure based on cytospin centrifugation preserving the astrocytes fine morphology, which enables the precise in vitro localization of antigen in intact PAPs [START_REF] Haseleu | Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs)[END_REF]. In all, despite intense investigations of the third synaptic partner in the last decades, molecular functioning of PAPs remains poorly described; the aforementioned methodological advances may, therefore, become instrumental in unraveling their unique molecular features.

PAPs display particular dynamic morphological properties

Pioneering work performed by the group of Steven Smith in the early 90s demonstrated that cultured astrocytes respond to glutamate exposure by rapidly inducing sprouting of filopodial processes. Remarkably, similar reorganizations of the membrane surface can be induced by settling neurons on top of an astrocytic culture, suggesting that chemotropism toward neuronal diffusing signals might be the mechanism driving astrocytic plasma membrane motility [START_REF] Finkbeiner | Glutamate induces calcium waves in cultured astrocytes: longrange glial signaling[END_REF]. Further studies in brain slices have demonstrated that whereas PAPs surrounding synapses display spontaneous remodeling within the time range of a minute, this motility is not observed at the level of astrocytic somata and endfeet [START_REF] Hirrlinger | Astroglial processes show spontaneous motility at active synaptic terminals in situ[END_REF][START_REF] Haber | Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses[END_REF]). However, given the tremendous complexity of differentiated astrocyte morphology, elucidating in situ the general principle of this structural plasticity remains challenging. Thus, several in vitro assays have been developed to characterize the compartmentalized signaling pathways underpinning the asymmetric rearrangement of cellular elements in response to stimuli (Etienne-Manneville 2008). In particular, establishment of stellation in primary cultured astrocytes has been used to model the dynamic behavior of PAPs, as it fully relies on subcellular redistribution of cytoskeletal proteins [START_REF] Safavi-Abbasi | Rapid morphological changes in astrocytes are accompanied by redistribution but not by quantitative changes of cytoskeletal proteins[END_REF], and induces the rapid protrusion of filopodia at the cell surface, which are devoid of GFAP whilst expressing ezrin (Fig. 1a,b) and a-actin [START_REF] Racchetti | Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes[END_REF]). Using stellation model, filopodia extension and retraction have been shown to be controlled by the small GTPases Rac1 and Rho, respectively. Thus, activated Rac1 would relocalize under the plasma membrane at the extremity of astrocyte protrusions [START_REF] Racchetti | Astrocyte stellation, a process dependent on Rac1 is sustained by the regulated exocytosis of enlargeosomes[END_REF],

where it has been suggested to promote the polymerization of actin filaments as well as the recruitment of ezrin, which links actin to the plasma membrane when phosphorylated at T567 [START_REF] Auvinen | Regulation of ezrin localization by Rac1 and PIPK in human epithelial cells[END_REF]). In brain slices, T567-activated ezrin has been shown to be restricted to perisynaptic glial sheaths, providing a hypothetical mechanism for the stabilization of PAP remodeling after mGluRs activation [START_REF] Lavialle | Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors[END_REF]). On the other hand, L-glutamate exposure in vitro has also been found to activate the small GTPase Rho, leading to suppression of astrocyte stellation via an as yet unknown mechanism involving GluTs [START_REF] Chen | L-glutamate activates RhoA GTPase leading to suppression of astrocyte stellation[END_REF]. Therefore, the opposite effect of Rho and Rac1 activation on astrocyte morphology is consistent with the role they play in axonal growth cone and dendritic spine extension and retraction, respectively [START_REF] Tashiro | Regulation of dendritic spine morphology by the rho family of small GTPases: antagonistic roles of Rac and Rho[END_REF][START_REF] Woo | Rac1 and RhoA promote neurite outgrowth through formation and stabilization of growth cone point contacts[END_REF]. Together, these GTPases-mediated antagonistic functions of glutamate suggest that the link between synaptic activity and morphological plasticity of PAPs may not be as trivial as a chemotropism toward glutamate and likely depends on the repertoire of membrane proteins expressed at a given process. An alternative mechanism for glutamate-induced PAP structural plasticity could rely on intracellular calcium (Ca 2? ) rise upon mGluR-induced Gq GPCR activation setting off the canonical phospholipase C/inositol1,4,5-triphosphate (IP3) pathway. Recent data have indeed shown a reduced astrocytic coverage of hippocampal synapses in mice in which IP3R-induced Ca 2? release was downregulated by expressing a recombinant glutathione-S-transferase-IP3 buffer (''IP3 sponge'') specifically in astrocytes and in a temporally controlled manner [START_REF] Tanaka | Astrocytic Ca2 ? signals are required for the functional integrity of tripartite synapses[END_REF]). Moreover, neuroglial contact-mediated ephrin signaling may regulate the morphology of the tripartite synapse via the recruitment of guanine exchange factors (GEF) such as Ephexin [START_REF] Shamah | EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin[END_REF]), Tiam1 [START_REF] Tanaka | Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2[END_REF]) and the Rap1/R-Ras exchange factor SHEP1 [START_REF] Dodelet | A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A[END_REF], thereby controlling small G protein activity. Strikingly, using more integrated approaches, it has recently been shown in hippocampal acute slices that inducing long-term synaptic plasticity in CA1 pyramidal neurons, through Schaffer collaterals high-frequency stimulation, leads to a fast G protein-mediated motility of astrocytes processes, which actually move away from the dendritic shaft [START_REF] Perez-Alvarez | Structural and functional plasticity of astrocyte processes and dendritic spine interactions[END_REF]. In this regard, we have recently shown that an unexpected key determinant of synaptic astroglial coverage in the hippocampus is the gap-junction protein connexin 30 (Cx30). Indeed, in the absence of the protein, astroglial processes literally penetrate synaptic clefts and get abnormally close to postsynaptic densities (Fig. 4), thereby reducing synaptic transmission as synaptic glutamate is removed directly from the synaptic cleft by GluTs. Such finding indicates that Cx30 regulates the distance of the PAP from the synapse [START_REF] Pannasch | Connexin 30 sets synaptic strength by controlling astroglial synapse invasion[END_REF]. Further, using a mutated form (Cx30T5M) that only impairs the channel function of the protein, we showed that such regulation involves channelindependent mechanisms. Insofar as Cx30 has recently been shown to interact with the cytoskeleton [START_REF] Qu | The role of the cytoskeleton in the formation of gap junctions by Connexin 30[END_REF]), it appears that such interaction is essential to confine PAPs to perisynaptic spaces, thereby limiting the sink effect of GluT accumulation away from releasing sites. Given that Cx30 only begins to be expressed around P10 in mice, it most certainly plays a prominent role in shaping synaptic networks during postnatal development. Recent studies reminiscent of our work assessed the impact of increasing GluT expression on synaptic and neuronal function, and found impairments in presynaptic short-term plasticity, long-term plasticity as well as information processing and memory formation [START_REF] Carmona | Glial ephrin-A3 regulates hippocampal dendritic spine morphology and glutamate transport[END_REF][START_REF] Filosa | Neuron-glia communication via EphA4/ephrin-A3 modulates LTP through glial glutamate transport[END_REF]). Further research is, however, needed to unravel the molecular pathway(s) enabling Cx30 to regulate the distance of the PAP to the synaptic cleft.

PAPs as specialized signaling compartments

PAPs account for *80 % of the astrocytic plasma membrane area [START_REF] Chao | The synapse-astrocyte boundary: an anatomical basis for an integrative role of glia in synaptic transmission[END_REF] and carry specific pools of ions channels, neurotransmitter receptors and transporters [START_REF] Wolff | Non-neuronal cells of the nervous system: function and dysfunction[END_REF]). Moreover, proteins in PAPs are expected to be concentrated in domains facing the synapse, where they would be adequately positioned to integrate synaptic events. In fact, this has recently been demonstrated for mGluRs [START_REF] Panatier | Astrocytes are endogenous regulators of basal transmission at central synapses[END_REF]) (Fig. 3b) and AMPA as well as NMDA receptors that are not directly aligned to neurotransmitter releasing sites have been shown to display a low opening probability due to their low affinity for glutamate [START_REF] Raghavachari | Properties of quantal transmission at CA1 synapses[END_REF][START_REF] Santucci | The effects of NR2 subunitdependent NMDA receptor kinetics on synaptic transmission and CaMKII activation[END_REF]. Thus, given that PAPs contact the majority of synapses in the neuropil [START_REF] Bushong | Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains[END_REF], they appear perfectly suited for locally sensing synaptic spillover. In this respect, astrocytes have been shown to respond to synaptic activation by increasing Ca 2? concentration in specific peripheral process subdomains [START_REF] Grosche | Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells[END_REF]. Strikingly, the spatial extent of these Ca 2? rises seems directly correlated to the strength of neuronal activity, such that signaling events associated to basal synaptic functioning are restricted to the tip of the astrocytic tree [START_REF] Panatier | Astrocytes are endogenous regulators of basal transmission at central synapses[END_REF] and do not involve somatic activation, which would rather be recruited during intense firing activity. Such compartmentalized Ca 2? transients most likely rely on the activation of metabotropic receptors and pathways downstream of Gq GPCR [START_REF] Volterra | Astrocytes, from brain glue to communication elements: the revolution continues[END_REF][START_REF] Agulhon | What is the role of astrocyte calcium in neurophysiology?[END_REF]. A report from Di Castro and collaborators has recently suggested that astrocytic processes may receive spatially confined synaptic inputs activating separate GPCR clusters, as pharmacological blocking of downstream GPCRs abolishes Ca 2? signals in all compartments spanning astrocytic processes, whilst independently inhibiting different types of GPCR only suppresses Ca 2? signal at specific locations. Hence, the idea that PAPs integrate input-specific signals is now emerging and may for instance explain why CA1 hippocampal astrocytes do respond to activation of Schaffer collaterals (CA3-CA1), but remain silent in response to recurrent pathway (CA1-CA1) stimulation [START_REF] Perea | Properties of synaptically evoked astrocyte calcium signal reveal synaptic information processing by astrocytes[END_REF]. Mechanistically, the functional compartmentalization of PAPs is likely to be mediated by the localized trafficking and positioning of their receptors, as is known to occur in postsynaptic elements [START_REF] Henley | Routes, destinations and delays: recent advances in AMPA receptor trafficking[END_REF]. In particular, activation of PAP mGluRs, which requires a minimal time of 50 ms glutamate exposure [START_REF] Attwell | Neuroenergetics and the kinetic design of excitatory synapses[END_REF], is predicted to be modulated by their distance from GluTs with which they compete for glutamate binding [START_REF] Tzingounis | Glutamate transporters: confining runaway excitation by shaping synaptic transmission[END_REF]. Therefore, akin to neurons, glutamate-induced Ca 2? signaling in PAPs may not only depend on the repertoire of proteins they express, but would also be modulated by the spatial arrangement of these proteins. Noteworthy, protein trafficking and PAP morphogenesis are most likely synergistically regulated, as they both rely on activity-dependent cytoskeletal remodeling [START_REF] Zhou | Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity[END_REF]) and share the same purpose of placing the receptor at the optimal location with regard to glutamate releasing sites. Thence, understanding structure/function relationship in PAPs will most certainly solve conflicting results showing on the one hand that astrocyte-specific activation of Ca 2?dependent release of glutamate does not influence excitatory synaptic activity [START_REF] Fiacco | Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity[END_REF][START_REF] Agulhon | Hippocampal shortand long-term plasticity are not modulated by astrocyte Ca 2? signaling[END_REF], whereas on the other hand other reports show that glutamate gliotransmission directly modulates neurotransmission [START_REF] Halassa | Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss[END_REF][START_REF] Fellin | Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo[END_REF]). Yet, insofar as PAPs are one order of magnitude smaller than light microscopy resolution, physiologically relevant Ca 2? ''hot spot'' in PAPs has never been observed and will require state-of-the art super resolution techniques [START_REF] Henneberger | Synaptic plasticity and Ca 2? signalling in astrocytes[END_REF].

How is information integrated from PAPs to soma? Since the early hypothesis of Smith in 1992, placing astrocytes as important contributors to information processing, a wealth of investigations have confirmed such role, in particular through the description of Ca 2? signaling occurring in astrocytes as a result of neuronal activation. In acute brain slices, bulk loading of membrane permeable Ca 2? dyes has allowed demonstrating that intercellular Ca 2? oscillations can occur independently of neuronal stimulation [START_REF] Nett | Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity[END_REF]. The latter has nevertheless been shown to regulate synchronization of spontaneous Ca 2? transients [START_REF] Aguado | Neuronal activity regulates correlated network properties of spontaneous calcium transients in astrocytes in situ[END_REF]. At the single cell level, dialysis of membrane-impermeable Ca 2? dyes via a patch pipette indicates that astrocytes can detect both basal and evoked synaptic activity in microdomains running along their processes by means of, respectively, ''focal'' (±4.2 lm) and ''expanded'' (±12.9 lm) Ca 2? elevations [START_REF] Panatier | Astrocytes are endogenous regulators of basal transmission at central synapses[END_REF][START_REF] Castro | Local Ca 2? detection and modulation of synaptic release by astrocytes[END_REF]. This body of work therefore puts forward the idea that spontaneous Ca 2? events may be spatially compartmentalized as long as they are not synchronized by activity (Fig. 3a). The occurrence of action potentials in the vicinity of PAPs may thus coordinate signaling events occurring in neighboring microcompartments and eventually lead to spreading of Ca 2? rises from PAPs to soma. This idea is further supported by in vivo data demonstrating that, after sensorimotor stimulation, Ca 2? signals in processes occur earlier than those recorded in the soma [START_REF] Wang | Astrocytic Ca 2? signaling evoked by sensory stimulation in vivo[END_REF][START_REF] Dombeck | Imaging largescale neural activity with cellular resolution in awake, mobile mice[END_REF][START_REF] Bekar | Locus coeruleus alphaadrenergic-mediated activation of cortical astrocytes in vivo[END_REF]. Further, activating different Gq GPCRs in astrocytic processes has been shown both in vitro and ex vivo [START_REF] Yagodin | Nonlinear propagation of agonist-induced cytoplasmic calcium waves in Brain Struct Funct 123 single astrocytes[END_REF][START_REF] Fiacco | Selective stimulation of astrocyte calcium in situ does not affect neuronal excitatory synaptic activity[END_REF] to induce comparable spatiotemporal patterns of Ca 2? spread, moving centripetally along the astrocytic tree. Such intracellular stream of Ca 2? may rely on local release from endoplasmic reticulum (ER) through the IP3 pathway, as ER membranes are known to express clusters of the type 2 IP3 receptors [START_REF] Sheppard | Comparison of type 2 inositol 1,4,5-trisphosphate receptor distribution and subcellular Ca 2? release sites that support Ca 2? waves in cultured astrocytes[END_REF]). However, limitations of the currently available Ca 2? imaging techniques, including the poor intracellular volume sampled by Ca 2? dyes after bulk loading [START_REF] Reeves | Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps[END_REF]) and the wash out of cytoplasmic elements induced by dialysis inherent to whole cell patch clamp, have fostered much skepticism, particularly in regard of the sites where signals are monitored, as they are not the actual loci of Ca 2? signal induction (presumably PAPs). Such information will most probably be instrumental in our understanding of astroglial integration of synaptic inputs, as it has recently been shown that local Ca 2? events within PAPs regulate both synaptic coverage and spine stability [START_REF] Bernardinelli | Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability[END_REF]. A recent study has demonstrated that genetically encoded Ca 2? indicators anchored to the membrane would be a more precise alternative, and was thus able, for the first time, to precisely monitor signaling activities occurring throughout the entire astrocytic volume [START_REF] Shigetomi | Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses[END_REF]) (Fig. 3c,d). Remarkably, these experiments revealed that spontaneous Ca 2? events in acute brain slices are independently localized in space and equivalently sized, thus delimiting functional signaling ''patches'' distributed within the astrocytic territory [START_REF] Shigetomi | Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses[END_REF]) (Fig. 3c,d). Although such investigation opens up new perspectives in astrocyte physiology, the challenge of ascertaining Ca 2? activation in PAPs still represents a major caveat that will need to be filled. Astroglial coverage of spines is heterogenous. Electron microscopy investigations indeed show that spine coverage varies from no contact within olfactory bulb glomeruli [START_REF] Chao | The synapse-astrocyte boundary: an anatomical basis for an integrative role of glia in synaptic transmission[END_REF] to ensheathment of almost all spines (*90 %) in hippocampal mushroom/perforated synapses [START_REF] Witcher | Plasticity of perisynaptic astroglia during synaptogenesis in the mature rat hippocampus[END_REF]). Such ultrastructural studies, however, provide ''snapshot'' overviews at a given timepoint, and do not appreciate the actually dynamic nature of gliasynapse interactions. Morphological relationships between the three synaptic partners have indeed been shown to change during development [START_REF] Nixdorf-Bergweiler | Developmental changes in the number, size, and orientation of GFAPpositive cells in the CA1 region of rat hippocampus[END_REF][START_REF] Bushong | Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development[END_REF]), as well as in response to neuronal activity (Gu ¨ldner [START_REF] Gu ¨ldner | Complex synaptic arrangements in the rat suprachiasmatic nucleus: a possible basis for the ''Zeitgeber'' and non-synaptic synchronization of neuronal activity[END_REF][START_REF] Oliet | Control of glutamate clearance and synaptic efficacy by glial coverage of neurons[END_REF]) and plasticity [START_REF] Wenzel | The influence of long-term potentiation on the spatial relationship between astrocyte processes and potentiated synapses in the dentate gyrus neuropil of rat brain[END_REF][START_REF] Lushnikova | Synaptic potentiation induces increased glial coverage of excitatory Brain Struct Funct 123 synapses in CA1 hippocampus[END_REF]. The following section presents the physiological properties of PAPs in these dynamic contexts and further explains how alterations of the function and three-dimensional distribution of astrocytic processes may contribute to synaptic activity.

Establishment of PAP-spine contacts during development

Onset of astrocytic filopodial process sprouting is concomitant to the expression of astrocytic mGluRs and GluTs at postnatal week one (Schools and Kimelberg 1999; [START_REF] Regan | Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS[END_REF]) and is hence thought to occur as a result of neuronal excitatory transmission. Over the two following weeks, astroglial processes fully infiltrate the neuropil and actively participate in the activity of synaptic circuits [START_REF] Freeman | Specification and morphogenesis of astrocytes[END_REF]. Formation of differentiated yet postsynaptically silent synapses is then subtended by the astrocytic matricellular proteins thrombospondins, especially TSP1 and 2, of which the peak of secretion is reached during early postnatal synaptogenesis [START_REF] Christopherson | Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis[END_REF]). TSPs interact with various neuronal receptors, such as integrins [START_REF] Defreitas | Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth[END_REF], neuroligins [START_REF] Xu | Thrombospondin 1 accelerates synaptogenesis in hippocampal neurons through neuroligin 1[END_REF]) and voltage-gated Ca 2? channels [START_REF] Eroglu | Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis[END_REF], thereby promoting the recruitment of adhesive as well as scaffolding proteins to nascent synapses. Thereafter, the maintenance of such connections is undertaken by trans-synaptic clustering of adhesion molecules, namely neuroligins and neurexins (Clarke and Barres 2013), mediated by the matricellular hevin-SPARC couple (Kucukdereli et al. 2011), whose expression is kept high in astrocytes during adulthood. Finally, functional maturation of synapses is regulated by the insertion and clustering of AMPA receptors in the postsynaptic density, which is directly supervised by astrocyte-secreted glypican proteins [START_REF] Allen | Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors[END_REF]. Remarkably, injection into adult cat visual cortex of immature astrocytes, which can regulate ECM composition, has been shown to be sufficient to reactivate the critical period of ocular dominance plasticity (Mu ¨ller and Best 1989). During the third and fourth postnatal weeks, glial coverage of maturing synapses and ramification of the finest leaflet of the astrocytic tree establish proper astrocytic territories by limiting processes overlap [START_REF] Bushong | Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development[END_REF]). Mechanisms behind these morphological changes involve release of glutamate from axon terminals, acting as a signal for both dendritic spines and PAPs [START_REF] Hirrlinger | Astroglial processes show spontaneous motility at active synaptic terminals in situ[END_REF][START_REF] Haber | Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses[END_REF]). Interestingly, dendritic remodeling mediated by glutamate spillover occurring in the hippocampus [START_REF] Verbich | Glial glutamate transport modulates dendritic spine head protrusions in the hippocampus[END_REF]) is associated with an adjustment of astrocytic process morphology, weakening PAP association with spines, and hence dynamically reducing GluT activity. PAP-spine association is also regulated through activation of dendritic EphA4 receptors with the astrocytic ligand ephrinA3, which results in spine retraction and reduces the lifetime of newly generated dendrites [START_REF] Murai | Control of hippocampal dendritic spine morphology through ephrin-A3/EphA4 signaling[END_REF][START_REF] Nishida | Direct astrocytic contacts regulate local maturation of dendritic spines[END_REF]. Lastly, although the formation of dendritic spine does not require the presence of astrocytes, the ability to receive synapses may not be an intrinsic neuronal property and has been shown in retinal ganglion cells to require contacts with astrocytes [START_REF] Hama | PKC signaling mediates global enhancement of excitatory synaptogenesis in neurons triggered by local contact with astrocytes[END_REF][START_REF] Barker | Developmental control of synaptic receptivity[END_REF]. In regard of such findings, the idea that astrocyte processes function as bona fide ''synaptic cradle'' nursing synaptic development has now emerged (Nedergaard and Verkhratsky 2012).

PAPs control synaptic transmission

Through shielding of chemical synapses by means of their fine distal processes, astrocytes ensure efficient spine-tospine transmission via insulation of neurotransmitter releasing sites. In particular, astroglial synaptic enwrapping is thought to prevent neurotransmitters spillover and activation of extrasynaptic receptors, thereby preserving release probability [START_REF] Oliet | Control of glutamate clearance and synaptic efficacy by glial coverage of neurons[END_REF]. In this respect, PAPs are thought to rule for synaptic versus volume mode of neurotransmission by shaping extracellular neurotransmitter diffusion and uptake [START_REF] Piet | Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk[END_REF][START_REF] Sykova | Diffusion in brain extracellular space[END_REF]. Volume transmission implies a diffusionmediated intersynaptic crosstalk that occurs when concomitant vesicular releases from distinct presynaptic elements lead to neurotransmitter spillover and activation of receptors from neighboring synapses. Interestingly, the role of volume transmission as a purely modulatory pathway has recently been revisited, as glutamate spillover (Semyanov and Kullmann 2001) and glutamate gliotransmission [START_REF] Liu | Astrocyte activation of presynaptic metabotropic glutamate receptors modulates hippocampal inhibitory synaptic transmission[END_REF]) can induce action potential firing, suggesting that PAPs allow to bypass synaptic connections. Finally, coverage of excitatory synapses has been proposed to bring astrocytic vesicles competent for glutamate release [START_REF] Parpura | Gliotransmission: exocytotic release from astrocytes[END_REF] at sufficient proximity to the synaptic cleft to activate both pre-and postsynaptic GluRs.

In the same way, astrocytic release of glutamate co-agonists has recently been shown to control synaptic transmission by unlocking the gate of NMDARs [START_REF] Papouin | Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists[END_REF]. Crucially, these mechanisms of PAP-mediated tuning of neurotransmission are nevertheless inconsistent with the functional competition brought by astrocytic glutamate transporters at the extrasynaptic level [START_REF] Huang | Astrocyte glutamate transporters regulate metabotropic glutamate receptor-mediated excitation of hippocampal interneurons[END_REF]). Indeed, GLT-1 and GLAST affinity for glutamate is comparable to NMDARs or mGluRs and their glutamate binding velocity is similar to AMPARs [START_REF] Huang | Astrocyte glutamate transporters regulate metabotropic glutamate receptor-mediated excitation of hippocampal interneurons[END_REF]). Thus, given the high expression levels of GluTs at astrocyte membranes [START_REF] Lehre | The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain[END_REF], glutamate released by PAPs would have to run away from thousands of glial GluTs to stand a chance of activating neuronal GluRs. Therefore, to solve this discrepancy, PAP glutamatergic gliotransmission must imply the spatiotemporal segregation of glutamate uptake and release events. Interestingly, such patterned membrane expression of transporters and transmitter releasing sites at PAPs has been proposed to functionally result in compartmentalization of extracellular glutamate concentrations [START_REF] Moussawi | Extracellular glutamate: functional compartments operate in different concentration ranges[END_REF], an hypothesis supported by in vitro data suggesting that, conversely, extracellular fluctuations of glutamate control membrane distribution of glial transporters [START_REF] Poitry-Yamate | Neuronal-induced and glutamate-dependent activation of glial glutamate transporter function[END_REF][START_REF] Nakagawa | Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial-neuronal cultures[END_REF]. However, most of the arguments brought forward to explain how astrocytes set the mode of synaptic transmission remain inaccurate in that the role of PAPs is often omitted. Indeed, PAPs are difficult to study in living tissue, as they are not directly accessible to traditional electrophysiological recordings, cannot be readily isolated for biochemistry, and are smaller than the optical resolution of regular light microscopy [START_REF] Reichenbach | Morphology and dynamics of perisynaptic glia[END_REF]. Therefore, despite interesting emergent hypotheses, the current lack of understanding of the PAP functional architecture is a major issue that will need to be addressed to decipher their actual role in neurotransmission.

Activity-dependent plasticity of PAP-spine functional interactions

Subtle changes in the efficacy of synaptic transmission are believed to be at the core of memory formation and can be experimentally assessed through the induction of long-term potentiation (LTP) and long-term depression, respectively corresponding to activity-dependent increase and decrease in synaptic strength [START_REF] Bliss | Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path[END_REF][START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF][START_REF] Cooke | Plasticity in the human central nervous system[END_REF]. The so-called Hebbian synapse increases its efficacy when pre-and postsynaptic activity concurs, and, conversely, decreases its strength as a result of disparate activity. NMDAR-dependent LTP and LTD require coincident presynaptic glutamate release and depolarization of the postsynaptic membrane, enabling synchronous Mg 2? block relief and NMDAR activation (Lu ¨scher and Malenka 2012). Strikingly, astrocytes have recently been proposed to provide additional sources of activity underlying atypical form of Hebbian plasticity. NMDAR-independent LTP of presynaptic neurotransmitter release could indeed be induced by gliotransmission of glutamate coupled to postsynaptic depolarization [START_REF] Perea | Astrocytes potentiate transmitter release at single hippocampal synapses[END_REF], thereby implying that astrocytes would contribute to the long-term storage of synaptic information. Although this hypothesis has recently been validated in vivo for both LTP and LTD [START_REF] Min | Astrocyte signaling controls spike timingdependent depression at neocortical synapses[END_REF][START_REF] Navarrete | Astrocyte calcium signal and gliotransmission in human brain tissue[END_REF], we are still far from understanding the exact mechanisms underlying these astrocyte-dependent forms of plasticity. For instance, signaling dynamics at the PAPs may be several orders of magnitude slower than neurotransmission events, which would imply that the pairing of pre-and postsynaptic activities is likely achieved long before gliotransmission has occurred. In this respect, a recent study proposed that for certain patterns of correlated activity, astrocytes may act as a ''thresholding unit'', meaning that PAPs-to-neuron signaling would be triggered in response to local build-up of neurotransmitters accumulated as a result of repeated stimulations [START_REF] Min | Astrocyte signaling controls spike timingdependent depression at neocortical synapses[END_REF]. However, further studies are still required to fully decipher how PAPs manage to sense differences in extracellular neurotransmitters concentration. In a more integrated perspective, astrocytes may control metaplasticity, i.e., the ability of synapses to exhibit long-term modifications. Indeed both LTP (Henneberger et al. 2010) and LTD [START_REF] Zhang | Bell-shaped D-serine actions on hippocampal long-term depression and spatial memory retrieval[END_REF] are in part gated by the astrocyte-secreted NMDAR coagonist D-serine, known to be enriched in glial processes [START_REF] Schell | mGluR3 and mGluR5 are the predominant metabotropic glutamate receptor mRNAs expressed in hippocampal astrocytes acutely isolated from young rats[END_REF]. Thus, PAPs have been proposed to set the ''sliding threshold'' for long-term synaptic plasticity by controlling the number of NMDARs that may be activated during synaptic transmission [START_REF] Panatier | Glia-derived Dserine controls NMDA receptor activity and synaptic memory[END_REF]. This hypothesis has, however, recently been challenged by reports showing that serine racemase (SR), the enzyme undertaking the synthesis of D-serine, is predominantly expressed in glutamatergic neurons and that genetic suppression of neuronal, but not astroglial, SR leads to alteration in synaptic functions and plasticity [START_REF] Rosenberg | Neuronal release of D-serine: a physiological pathway controlling extracellular D-serine concentration[END_REF][START_REF] Benneyworth | Cell selective conditional null mutations of serine racemase demonstrate a predominate localization in cortical glutamatergic neurons[END_REF]. Moreover, given that PAPs enwrap *60 % of hippocampal excitatory synapses and support activity-dependent spreading of Ca 2? signaling, such structures are ideally positioned to coordinate plasticity at neighboring inactivated synapses along dendritic shafts (known as heterosynaptic plasticity). It has, thus, been proposed that gliotransmitter-mediated homeostatic plasticity may serve as an efficient way to regulate the overall neuronal outputs, via coordinated scaling of synaptic weights [START_REF] Beattie | Control of synaptic strength by glial TNFalpha[END_REF][START_REF] Gordon | Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy[END_REF]. In addition to the functional strengthening or weakening of pre-existing synapses, activity-dependent long-term plasticity is known to induce the de novo formation and elimination of synapses.

Strikingly, it has also recently been described that LTP induces the structural remodeling of PAPs. Indeed, LTP was found to induce a transient increase in PAP motility [START_REF] Bernardinelli | Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability[END_REF][START_REF] Perez-Alvarez | Structural and functional plasticity of astrocyte processes and dendritic spine interactions[END_REF], leading to a decreased astrocytic coverage of dendritic spines [START_REF] Perez-Alvarez | Structural and functional plasticity of astrocyte processes and dendritic spine interactions[END_REF]) although an increased coverage preferentially occurring on enlarged spines was also reported [START_REF] Bernardinelli | Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability[END_REF]. Thus, LTP regulates, both in vitro and in vivo, synaptic coverage and, in doing so, influences synaptic fate. Remarkably, a decrease in astroglial synapse coverage in the amygdala was also recently evidenced during consolidation of contextual memory [START_REF] Ostroff | Synapses lacking astrocyte appear in the amygdala during consolidation of Pavlovian threat conditioning[END_REF]. These latter evidence suggest that astrocytic withdrawal may promote synaptic efficacy.

Finally, as astrocyte territories contact several individual neurons, PAPs most probably connect synaptic ensembles belonging to separate neuronal circuits. It would, therefore, be interesting to ascertain in future investigations whether astrocytes may compute information stored from dispersed synaptic networks.

Implication of PAP-spine interactions in synaptopathies?

Homeostatic fluctuation of extracellular glutamate levels is a key modulator of synaptic function. The amount of neurotransmitters flowing in the extracellular space is set by the dynamic balance between neuroglial uptake and release events, as well as the coordinated synaptic recycling of neurotransmitter precursor and metabolites [START_REF] Parpura | The astrocyte excitability brief: from receptors to gliotransmission[END_REF]. Alterations in the core elements making up this system results in a vast repertoire of neurodegenerative and neuropsychiatric disorders. For instance, excessive or prolonged activation of excitatory amino acid receptors results in the massive influx of ions, and eventually leads to activation of Ca 2? -dependent inflammatory pathways and cell death cascades [START_REF] Puyal | Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: a challenge for neuroprotection[END_REF]. Such glutamatergic excitotoxic signaling is heavily involved in the etiology of neurodegenerative disorders such as Alzheimer's or Huntington's disease [START_REF] Hynd | Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease[END_REF][START_REF] Mehta | Excitotoxicity: bridge to various triggers in neurodegenerative disorders[END_REF], multiple sclerosis [START_REF] Kostic | Multiple sclerosis and glutamate excitotoxicity[END_REF]) and neuropathic pain [START_REF] Osikowicz | The glutamatergic system as a target for neuropathic pain relief[END_REF]. Excessive glutamatergic gliotransmission [START_REF] Ding | Enhanced astrocytic Ca 2? signals contribute to neuronal excitotoxicity after status epilepticus[END_REF]), reversion of astrocytic glutamate transporters activity [START_REF] Grewer | Glutamate forward and reverse transport: from molecular mechanism to transportermediated release after ischemia[END_REF], connexin (and likely pannexin) hemichannels [START_REF] Bennett | Connexin and pannexin hemichannels in inflammatory responses of glia and neurons[END_REF]) and glutamate-permeable ion channels opening [START_REF] Woo | TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation[END_REF], may represent a significant source of glutamate release leading to excitotoxicity. Supporting this notion, Papouin et al. have recently demonstrated that PAP-secreted D-serine gates the glutamate-induced Ca 2? overload that mediates neuronal death in hippocampal slices, hence suggesting that perisynaptic neuroglial interactions play a crucial role in neurodegeneration [START_REF] Papouin | Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists[END_REF]. Other mechanisms subtending neurological disorders rely on long-lasting adaptation of glutamate homeostasis regulators such as the glial glutamate transporter EAAT2/GLT-1 and the glutamate-catabolizing enzyme GS as a result of prolonged exposure to inadequate ECS glutamate concentrations [START_REF] Javitt | Translating glutamate: from pathophysiology to treatment[END_REF]. These persistent changes have indeed been shown to mediate symptoms of mental disorders with known environmental causes, such as substance abuse [START_REF] Kalivas | The glutamate homeostasis hypothesis of addiction[END_REF]) and mood disorders [START_REF] Mathews | Targeting the glutamatergic system to treat major depressive disorder: rationale and progress to date[END_REF]. Moreover, failure of the system to maintain the overall excitation/inhibition balance of synaptic inputs is thought to result in a pathophysiological pathway shared by most psychiatric conditions [START_REF] Yizhar | Neocortical excitation/ inhibition balance in information processing and social dysfunction[END_REF]. In this respect, ''glutamate hypotheses'' accounting for neuropsychiatric disorders such as schizophrenia [START_REF] Moghaddam | From revolution to evolution: the glutamate hypothesis of schizophrenia and its implication for treatment[END_REF]), Huntington's chorea [START_REF] Cummings | Alterations in cortical excitation and inhibition in genetic mouse models of Huntington's disease[END_REF]) and Down's syndrome [START_REF] Baroncelli | Brain plasticity and disease: a matter of inhibition[END_REF]) have emerged. Interestingly, this framework is currently being reconsidered in of the tripartite nature of the synapse. For instance, the function of astrocytic cysteine/glutamate exchangers and glutamate transporters has been shown to be strongly altered after prolonged exposure to drugs of abuse, thereby mediating symptoms of drug-seeking patients [START_REF] Reissner | Using glutamate homeostasis as a target for treating addictive disorders[END_REF]. In the same way, given that astrocytes processes could control the synchronized activity of neuronal networks dispersed across their territory of influence [START_REF] Halassa | Synaptic islands defined by the territory of a single astrocyte[END_REF][START_REF] Pannasch | Emerging role for astroglial networks in information processing: from synapse to behavior[END_REF], astroglial processes have been proposed to participate in the spatiotemporal coordination of excitation/ inhibition balance via the release of gliotransmitters [START_REF] Fellin | Astrocytic glutamate is not necessary for the generation of epileptiform neuronal activity in hippocampal slices[END_REF]. Therefore, in all, implication of the third synaptic compartment in our understanding of synaptopathies has yet to be considered and is likely to bring forth new pharmacological targets and therapeutic strategies.

Conclusions and perspectives

The PAP has recently been recognized as the third active element of the chemical synapse [START_REF] Araque | Tripartite glia, the unacknowledged partner[END_REF], thereby challenging the previous neurocentric dogma of synaptic transmission. The unique molecular composition as well as structural plasticity and signaling properties of perisynaptic glial sheaths endow them with the ability to interfere dynamically with neuronal information processing. Although the PAP is now viewed as the initiation locus of astrocyte excitability, most studies have, however, yielded unreliable monitoring of these synaptically evoked glial events due to spatial resolution limitations of conventional approaches at the nanometric scale. Thus, force is to conclude that our understanding of the mechanisms used by PAP for sensing and responding to ongoing neurotransmission is still lagging behind in experimental evidences and many questions consequently linger. Do all PAPs cover synapses the same way? Does this depend on brain areas? On neuronal populations? On the type of synapses? What are the signaling cues regulating glia-synapse contacts? Are all PAPs expressing the same pool of proteins or mRNAs? Are all PAPs equipped for gliotransmission? Addressing these questions of fine brain organization will most probably require the synergistic combination of cutting-edge optical and electrophysiological methodologies.

New optical approaches for monitoring PAP structure and function

Three-dimensional reconstruction of electron microscopic serial section is a suitable technique for resolving the ultrastructural architecture of the tortuous perisynaptic astroglial membranes [START_REF] Ventura | Three-dimensional relationships between hippocampal synapses and astrocytes[END_REF][START_REF] Witcher | Three-dimensional relationships between perisynaptic astroglia and human hippocampal synapses[END_REF]. Although electron microscopy provides the most accurate description and quantification of PAP morphology and synapse coverage, such approach can, however, only provide static observations on fixed tissue and is notoriously tedious.

Alternative approaches have recently been developed by tempting to overcome the diffraction resolution limit inherent to light microscopy [START_REF] Maglione | Seeing the forest tree by tree: superresolution light microscopy meets the neurosciences[END_REF], thus providing a way to study PAPs dynamics in physiological contexts. Stimulated emission depletion (STED) microscopy is the first modality of super-resolution techniques that successfully broke the diffraction limit [START_REF] Hell | Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy[END_REF] by reducing the size of the blurry excitation spot called point-spread function (PSF). This is achieved by restricting the emission light to a central spot which is slightly smaller than the diffraction barrier. Such technique has been used to resolve changes in synapse shape [START_REF] Tønnesen | Spine neck plasticity regulates compartmentalization of synapses[END_REF], neurotransmitter vesicles fusion [START_REF] Willig | STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis[END_REF] as well as single-molecule trafficking in dendritic spines [START_REF] Blom | Spatial distribution of DARPP-32 in dendritic spines[END_REF][START_REF] Westin | Nanoscopic spine localization of Norbin, an mGluR5 accessory protein[END_REF]. STED microscopy is thus a particularly promising approach to unravel the intimate structure of the tripartite synapse. However, one may keep in mind that STED microscopy induces relatively high phototoxicity, does not improve Z-axis spatial resolution and displays a lower temporal resolution than traditional scanning microscopy techniques. In addition, to work in tissues at a reasonable depth, STED microscopy should be combined to 2-photon excitation, otherwise limiting access to cultured cells or organotypic slices. More recent methodologies have further emerged, offering alternative possibilities to investigate PAPs dynamics. Photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) combine the iterative and random switching on/off of fluorophores with the computational localization of its PSF center [START_REF] Henriques | PALM and STORM: unlocking live-cell super-resolution[END_REF]. Such multicolor photo-switchable super-resolution imaging has for example recently enabled highly specific single molecules detection with less than 50 nm lateral resolution [START_REF] Bates | Multicolor superresolution imaging with photo-switchable fluorescent probes[END_REF]). However, PALM and STORM are thus far only applicable to in vitro cultured cells. Future development of these techniques may enable unraveling PAPs proteins/mRNAs compartmentalization and activation by fusing chromatically distinguishable photo-activatable probes [START_REF] Heilemann | Light at the end of the tunnel[END_REF] to neurotransmitter receptors and transporter molecules. Furthermore, novel Ca 2? indicators have recently been generated. Of particular interest, the recent use of a membrane-targeted ''GCaMP'', a well-known Ca 2? indicator expressed in situ or in vivo in astrocytes [START_REF] Shigetomi | A genetically targeted optical sensor to monitor calcium signals in astrocyte processes[END_REF][START_REF] Shigetomi | Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses[END_REF], has revealed unprecedented intracellular near-membrane Ca 2? events within astrocyte processes microdomains [START_REF] Shigetomi | A genetically targeted optical sensor to monitor calcium signals in astrocyte processes[END_REF], 2013) (Fig. 3c, d). Noteworthy, this non-invasive approach enables to assess Ca 2? signaling in fine astroglial processes. One should nevertheless not overlook the fact that Ca 2? indicators can strongly buffer intracellular Ca 2? , thereby altering important signaling pathways, while low-affinity indicators possibly operate below detection level. Finally, the use of membrane voltage reporters has not yet been exploited to study PAP excitability but should, in the near future, help to unravel the mechanisms enabling PAPs to endorse synaptic neuroglial ionic regulation.

Adapting electrode-based electrophysiological techniques to record PAP signaling

Electrophysiology is the gold standard for assessing electrical activity. Techniques such as patch clamp recordings are usually performed at cell bodies, enabling the detection of currents originating mostly from somas and proximal processes. At distal processes, currents might be detectable at the somatic level only in the context of strong and coordinated activity generated in multiple loci. However, basal activity of channels and receptors from individual PAPs is currently hardly detectable. This is partly due to restricted spatial and temporal control of astrocyte membrane potentials and currents recorded in situ with the whole cell patch clamp technique. Thus, despite the fact that the surface of the numerous tiny distal astroglial processes exceeds by far the membrane area of soma and main proximal processes, up to now, we cannot directly and reliably record their electrophysiological activity. This is particularly unfortunate, since PAPs precisely contain the receptors and channels that are functionally relevant and are most likely the ones playing a crucial role in regulating activity of adjacent synapses. There is, therefore, an urge to develop refined patch clamp approaches designed to overcome current limitations and record from fine astroglial distal processes with dimensions typically below hundreds of nanometers, as performed nowadays for dendrites [START_REF] Davie | Dendritic patch-clamp recording[END_REF]. One of the main challenges is to visualize PAPs as their dimensions, typically below hundreds of nanometers, cannot be resolved by regular light microscopy. In addition, they can only be identified topologically, based on their close association with synapses as there is, as yet, no specific known marker for PAPs. Thus, only super resolution techniques such as STED microscopy coupled to two photon excitations can provide the required resolution to visualize dynamically such structures in live tissues. To identify PAPs, one therefore needs to label both astrocytes, using for instance dye loading via a patch pipette (e.g., Alexa-488, Fig. 1e), and spines, taking for example advantage of transgenic mice such as Thy1-YFPH mice where spines are sparsely labeled. Another important challenge is to improve the signal to noise ratio of the patch clamp technique, since the presumably small electrophysiological responses of tiny astrocytic processes are likely to be below the present current threshold detection, i.e., 3-5 pA. Finally one also needs to design electrodes with tip size below 1 lm. Although this can be presently achieved using filamented glass with micropipette puller, nanotechniques such as focused ion beam microscopy [START_REF] Qiao | Fabrication of nanoelectrodes for neurophysiology: cathodic electrophoretic paint insulation and focused ion beam milling[END_REF] or carbon nanotubes [START_REF] Krapf | Experimental observation of nonlinear ionic transport at the nanometer scale[END_REF] are very promising approaches to produce finer electrodes with a better control. Remarkably, such electrodes will not only enable to patch tiny processes, but shall also contribute to a better signal to noise ratio due their high resistance, as well as minimize deformation of tissue [START_REF] Scanziani | Electrophysiology in the age of light[END_REF]. In fact, in depth study of the intimate dialog between PAPs and adjacent synapses will most likely rely in combinations of electrophysiology and Ca 2? imaging, as both approaches give access to complementary relevant information at the level of PAPs. Indeed, whilst Ca 2? imaging only provides information about one, yet crucial signaling molecule involved in numerous transduction pathways and thought to engage astroglial excitability, patch clamp recordings give access to quantitative information concerning all ionic currents induced by activation of membrane receptors and channels. Such interdisciplinary strategy should, therefore, be the key to unravel online the dynamics of neuroglial signaling between PAPs and adjacent synapses, as well as their role in processing brain information.

Fig. 1

 1 Fig.1The astrocyte arborescence viewed through different methodological approaches. a-c Immunolabeling of acutely dissociated and morphologically well-preserved mouse astrocytes showing that ezrin covers the entire astrocytic surface (a) and mainly localizes to the PAPs (b) whereas GFAP is restricted to the main processes. The ezrin IR-positive processes extend beyond the main processes by a mean distance of 6.64 lm (c). d Images of confocal Z-series projection for

Fig. 2

 2 Fig.2The unique molecular composition of the PAP supports morphology dynamics and glutamate signaling. Structure (left): current knowledge on the molecular content of PAPs indicate that they express specific structural proteins such as dense actin fibers (unlike astrocytic branches that instead typically express intermediate filaments) as well as the ezrin protein, linking actin fibers to the plasma membrane. Spatial segregation of actin and possibly ezrin appears to arise from the compartmentalization of messenger RNAs followed by local synthesis. Regulation of the PAP morphology is thought to involve Ephrin signaling, implying the ephrin-receptor (Eph-R) and guanine exchange factors (GEF), which regulate extension and retraction of actin fibers through modulation of the Rho-GTPase activity. Glutamatergic function (right): in addition to the glutamate transporters (GLT1/GLAST), PAPs express a set of glutamate receptors mainly represented by the metabotropic types (mGluRs). The well-known astrocytic transport of glutamate is crucial for maintaining the tonic levels of glutamate at extremely low concentrations at the synaptic cleft (see[START_REF] Moussawi | Extracellular glutamate: functional compartments operate in different concentration ranges[END_REF]. Once in the PAP, glutamate is either recycled via transformation into glutamine and vice versa, or incorporated into astrocytic vesicle through the vesicular transporters Vglut1 and 3. Direct release of glutamate mediated by opening of hemichannels is also a possible fate of PAP glutamate

Fig. 3

 3 Fig. 3 Perisynaptic astrocyte processes have unique and compartmentalized activities. a Three-dimensional representation of a hippocampal astrocyte arborization derived from a two photon Z-series projection of Texas Red dextran signal. The astrocyte process (red) display locally confined Ca 2? activities within subdomains (multicolor). b High-magnification confocal images of hippocampal immunolabeling showing a portion of an S100b-IR-positive astrocyte process (gray), synaptotagmin-IR-positive presynaptic

Fig. 4

 4 Fig. 4 Astroglial processes can invade synaptic clefts. a Serial electron microscopy images illustrating a hippocampal asymmetric synapse [PSD, marked by arrow, axonal bouton (b), dendritic spine (S)] contacted by astroglial processes (green) in Cx30 ?/? and Cx30 -/-mice. Deficiency for astroglial Cx30 leads to penetration of synaptic clefts by glial processes. Scale bar, 0.2 lm. b Reconstructions of the entire dendritic spine (gray) with PSD (red) and the surrounding astrocytic elements (green) are illustrated for Cx30 ?/? (c) and Cx30 -/-mice (d), showing a closer association of Cx30 -/- astrocytic processes to dendritic spines and a shorter distance to PSD. Adapted from Pannasch et al. (2014)

Acknowledgments This work was supported by grants from French Research Agency (ANR, Programme Blanc), City of Paris (Programme Emergence), INSERM, CNRS and Colle `ge de France to N.R., Labex Memolife to G.D. and from the Paris 6 University doctoral school ED3C to G.G.

Conflict of interest Authors have no conflict of interest to declare.


E Pousse

P A Glaude

R Fournet

F Battin-Leclerc

Experimental study of the structure of a lean premixed indane/CH 4 /O 2 /Ar flame

In order to better understand the chemistry involved during the combustion of components of diesel fuel, the structure of a laminar lean premixed methane flame doped with indane has been investigated. The gases of this flame contains 7.1% (molar) of methane, 36.8% of oxygen and 0.90% of indane corresponding to an equivalence ratio of 0.74 and a ratio C 9 H 10 /CH 4 of 12.75%. The flame has been stabilized on a burner at a pressure of 6.7 kPa using argon as dilutant, with a gas velocity at the burner of 49.2 cm/s at 333 K. Quantified species included usual methane C 0 -C 2 combustion products, but also 11 C 3 -C 5 hydrocarbons and 3 C 1 -C 3 oxygenated compounds, as well as 17 aromatic products, namely benzene, toluene, phenylacetylene, styrene, ethylbenzene, xylenes, trimethylbenzenes, ethyltoluenes, indene methylindane, methylindene, naphthalene, phenol, benzaldehyde, benzofuran. The temperature was measured thanks to a thermocouple in PtRh (6%)-PtRh (30%) settled inside the enclosure and ranged from 800 K close to the burner up to 2000 K in the burned gases.

INTRODUCTION

If many detailed kinetic models are available for the oxidation of mixtures representative of gasolines, they are much less numerous in the case of diesel fuels because of their more complex composition. The constituents of diesel fuel contain from 10 to 20 carbon atoms and include about 30% (mass) of alkanes, the remaining part being mainly alkylcyclohexanes (24%), alkyldecalines (15%), alkylbenzenes (10%), indanes (7%) and polycyclic naphtenoaromatic compounds [START_REF] Guibet | Fuels and engines[END_REF]. If the oxidation of alkanes has been extensively studied, the abundance of models diminishes considerably when other families of components are considered and very few models exist for substituted cycloalkanes and aromatics compounds, except from toluene [START_REF] Battin-Leclerc | Detailed chemical kinetic models for the low-temperature combustion of hydrocarbons with application to gasoline and diesel fuel surrogates[END_REF]. The oxidation of alkylbenzenes with alkylic side-chains from C 2 to C 4 has been studied in a flow reactor in Princeton [START_REF] Litzinger | The oxidation of ethylbenzene near 1060 K[END_REF] [4] [START_REF] Litzinger | Gas-phase oxidation of isopropylbenzene at high temperature[END_REF] and that of n-propylbenzene in a jet-stirred reactor at Orleans [START_REF] Dagaut | Experimental and kinetic modeling study of the oxidation of n-propylbenzene[END_REF]. The autoignition of alkylbenzenes (toluene, xylenes, ethylbenzene, trimethylbenzenes, propylbenzene, ethyltoluenes and butylbenzene) has also been investigated in the rapid compression machine of Lille [START_REF] Roubaud | Oxidation and combustion of low alkylbenzenes at high pressure: comparative reactivity and auto-ignition[END_REF] [START_REF] Roubaud | High pressure auto-ignition and oxidation mechanism of o-xylene, o-ethytoluene, and n-butylbenzene between 600 and 900[END_REF]. The oxidation of indane has only been studied in a jet stirred reactor [START_REF] Dagaut | Kinetic effect of dimethoxymethane on the oxidation of indane[END_REF] at Orleans. No measurement in flame has yet been reported for this species. The purpose of the present paper is to experimentally investigate the structure of a premixed laminar methane flame containing indane. The use of a methane flame will allow us to have a reactive mixture rich in methyl radicals and to be more representative of combustion mixtures containing larger hydrocarbons than hydrogen or C 2 flames. This study will be performed using a lean flame for the chemistry to be better representative of that occurring in engines controlled via Homogenous Charge Combustion Ignition (HCCI) which are under development. These results will be use in order to develop a new mechanism for the oxidation of indane based on our experience in modelling the reactions of both alkanes [10] and light aromatic compounds (benzene [START_REF] Costa | Experimental and modelling study of the oxidation of benzene[END_REF] and toluene [START_REF] Bounaceur | Experimental and modeling study of the oxidation of toluene[END_REF]).

EXPERIMENTAL PROCEDURE

The experiments were performed using an apparatus developed in our laboratory to study temperature and stable species profiles in a laminar premixed flat flame at low pressure and recently used in the case of rich methane flames doped by light unsaturated soot precursors [START_REF] Gueniche | Rich methane premixed laminar flames doped with light unsaturated hydrocarbons. I. Allene and propyne[END_REF] [14] [START_REF] Gueniche | Rich methane premixed laminar flames doped by light unsaturated hydrocarbons. Part III: cyclopentene Combust[END_REF]. The body of the flat flame matrix burner, provided by McKenna Products, was made of stainless steel, with an outer diameter of 120 mm and a height of 60 mm (without gas/water connectors). This burner was built with a bronze disk (95% copper, 5% tin). The porous plate (60 mm diameter) for flame stabilization was water cooled (water temperature: 333 K) with a cooling coil sintered into the plate. The burner could be operated with an annular co-flow of argon to favor the stabilization of the flame. This horizontal burner was housed in a water-cooled vacuum chamber evacuated by two primary pumps and maintained at 6.7 kPa by a regulation valve. This chamber was equipped of four quartz windows for an optical access, a pressure transducer (MKS 0-100 Torr), a microprobe for samples taking and a thermocouple for temperature measurements. The burner could be vertically translated, while the housing and its equipments were kept fixed. A sighting telescope measured the position of the burner relative to the probe or the thermocouple with an accuracy of 0.01 mm. The flame was lighted on using an electrical discharge. Gas flow rates were regulated by RDM 280 Alphagaz and Bronkhorst (El-Flow) mass flow regulators. Methane (99.95 % pure) was supplied by Alphagaz -Air Liquide. Oxygen (99.5% pure) and argon (99.995% pure) were supplied by Messer. Liquid indane, supplied by Alfa Aesar (purity 99%), was contained in a glass vessel pressurized with argon. After each load of the vessel, argon bubbling and vacuum pumping were performed in order to remove oxygen traces dissolved in the liquid hydrocarbon fuel. The liquid reactant flow rate was controlled by using a liquid mass flow controller, mixed to the carrier gas and then evaporated by passing through a single pass heat exchanger, the temperature of which was set above the boiling point of the mixture. Carrier gas flow rate was controlled by a gas mass flow controller located before the mixing chamber. Temperature profiles were obtained using a PtRh (6%)-PtRh (30%) type B thermocouple (diameter 200 m). The thermocouple wire was sustained by a fork and crosses the flame horizontally to avoid conduction heat losses. The junction was located at the centre of the burner. The thermocouple was coated with an inert layer of BeO-Y 2 O 3 to prevent catalytic effects [START_REF] Kent | A noncatalytic coating for platinum-rhodium thermocouples[END_REF]. The ceramic layer was obtained by damping in a hot solution of Y 2 (CO 3 ) 3 (93% mass.) and BeO (7% mass.) followed by a drying in a Meker burner flame. This process was reiterated (about ten times) until the whole metal was covered. Radiative heat losses are corrected using the electric compensation method [START_REF] Bonne | Messungen in der reaktionszone von wasserstoffsauerstoff -und methan -sauerstoff -flammen[END_REF]. The sampling probe was in quartz with a hole of about 50 μm diameter (d i ). The probe was finished by a small cone with an angle to the vertical of about 20°. For temperature measurements in the flames perturbed by the probe, the distance between the junction of the thermocouple and the end of the probe was taken equal to two times d i , i.e. to about 100 μm. The sampling quartz probe was directly connected to a heated passivated transfer line heated at 423 K. This line was itself connected to a heated pressure transducer (MKS 0-100 Torr) and through heated valves to a turbomolecular pomp, a pyrex line and a heated stainless steel loop located inside a gas chromatograph. The pressure in the transfer line was always below 1.3 kPa so that the pressure drop between the flame and the inlet of the probe ensured reactions to be frozen. The on-line connection to a chromatograph via a heated transfer line was found necessary in order to analyse compounds above C 6 . The analyses could be then performed according to two methods:

Gas samples of compounds with a sufficient vapour pressure were directed through the pyrex line towards a volume which was previously evacuated by the turbo molecular pump down to 10 -7 kPa and which was then filled up to a pressure of 1.3 kPa (pressures in the pyrex line were measured by a MKS 0-10 Torr pressure transducer before compression and by a MKS 0-1000 Torr one after compression) and collected in a pyrex loop after compression by a factor 5 by using a column with rising mercury pressure. A chromatograph with a Carbosphere packed column and helium or argon as carrier gas was used to analyse O 2 , H 2 , CO and CO 2 by thermal conductivity detection and C 2 H 2 , C 2 H 4 , C 2 H 6 by flame ionisation detection (FID). Water was detected by TCD but not quantitatively analyzed. Calibrations were performed by analysing a range of samples containing known pressures of each pure compound to quantify and mole fractions were derived from the known total pressure in the pyrex loop. This method of sampling could allow us to analyse heavier hydrocarbons from allene to toluene using a chromatograph with a Haysep packed column with FID and nitrogen as gas carrier gas as in our previous study [START_REF] Gueniche | Rich methane premixed laminar flames doped by light unsaturated hydrocarbons. Part III: cyclopentene Combust[END_REF], but it was found more accurate and faster to use the second method. When both methods were used for these compounds, a very good agreement was obtained between both of them. Gas samples were directed towards the loop located inside the gas chromatograph and previously evacuated by the turbo molecular pump down to 10 -7 kPa. The loop was then filled up to a pressure of 1.3 kPa and its content was just after analyzed using FID and helium as gas carrier gas by using a capillary column (a HP-Plot Q or a HP-1 column). This method of sampling was used to analyze methane, all the hydrocarbons from C 3 and the oxygenated products other than carbon oxides.

RESULTS AND DISCUSSION

A laminar premixed flat flame has been stabilized on the burner at 6.7 kPa (50 Torr) with a gas flow rate of 5.44 l/min corresponding to a gas velocity at the burner of 49.2 cm/s at 340 K and with mixtures containing 7.1% (molar) of methane, 36.8% of oxygen and 0.90% of indane corresponding to an equivalence ratio of 0.74.

Figure 1 displays the experimental temperature profiles obtained with and without the probe showing that the presence of the probe induces a thermal perturbation involving a lower temperature. Without the probe, the lowest temperatures measured the closest to the burner (0.4 mm above) were around 1115 K. Due to the thinness of this lean flame and the size of the thermocouple, it was not possible to measure lower temperatures. The highest temperatures were reached from 6 mm above the burner and were around 1995 K. FIGURE 1 Figure 2 presents the profiles of both hydrocarbons reactants and shows that indane is completely consumed close to the burner, at 2 mm height, while some methane remains up to 3 mm. FIGURE 2 Figures 3 present the profiles of oxygen (fig. 3a), hydrogen (fig. 3a), water (fig. 3a) and of carbon monoxide (fig. 3b) and carbon dioxide (fig. 3b) above the burner. The mole fraction of water has been obtained from a material balance on the other major species. In this lean flame, the profiles of carbon monoxide and hydrogen display a marked maximum at 2.5 mm height and the major final products are for a large extent only carbon dioxide and water. FIGURE 3 Figure 4 present the profiles of C 2 species. Acetylene is the most abundant C 2 species (peak mole fraction of 1800 ppm) and is produced last. It reaches its maximum concentration close to the burner around 2.5 mm. Ethylene is slightly less abundant than acetylene. The profiles of ethylene and ethane peak around 2 mm. FIGURE 4 Figure 5 presents the profiles of the observed C 3 products, with propene (fig. 5a) and propane (fig. 5a) peaking first around 2 mm above the burner, while the maxima for allene (fig. 5b) and propyne (fig 5b) are around 2.2 mm. The peak mole fraction observed for propene (80 ppm) is close to those of propyne and allene but it's more than ten times larger than that of propane. FIGURE 5 Figure 6 displays the profiles of C 4 species. Butenes (fig. 6a) are produced first and reach its maximum concentration close to the burner, around 1.9 mm. The profiles of diacetylene (fig. 6b), vinylacetylene (fig. 6b) and butadienes (fig. 6b) peak around 2.2 mm. The most abundant C 4 compounds are vinylacetylene and 1,3-butadiene with peak mole fractions of 70 and 100 ppm respectively. FIGURE 6 Figure 7 presents the profiles of cyclopentadiene (CPTD) and methylcyclopentadiene (MCPTD). The profiles of methylcyclopentadiene peak further around 1.8 mm and the maxima of that of cyclopentadiene, the most abundant specie with a peak mole fraction of 70 ppm, is around 2.2 mm. FIGURE 7 Figure 8 displays the profiles of light oxygenated species: methanol, acetaldehyde and acroleïn which are intermediate products of the combustion of methane. Methanol and acetaldehyde, which are the most abundant species with peak mole fractions of 160 and 60 ppm respectively, are produced early and reach their maximum concentration close to the burner, around 1.9 mm. The profiles of acroleïn peak around 2.1 mm. FIGURE 8 Figure 9 present the profiles of monocyclic aromatic products. The maxima of the profiles of, ethylbenzene (fig. 9c) and ethyltoluene (fig. 9d) occur first around 1.8 mm above the burner, followed by that of toluene (fig. 9a), xylenes (fig. 9b) and trimethylbenzene (fig. 9d) around 2.0 mm and finally that of benzene (fig. 9a), styrene (fig. 9c) and phenylacetylene (fig. 9c) around 2.3 mm. The most abundant of these species are benzene and toluene, with peak mole fractions of 900 and 600 ppm respectively, and to a lesser extent styrene (with 250 ppm), ethylbenzene (with 200 ppm) and phenylacetylene (with 100 ppm). FIGURE 9 Figure 10 displays the profiles of bicyclic aromatic compounds. The maxima of the profiles of indene (fig. 10a) and methylindane (fig. 10c) are observed first around 1.7 mm above the burner, followed by that of methylindene (fig. 10c) and benzocyclobutane (fig. 10b) around 2.2 mm and finally that of naphthalene (fig. 10c) around 2.3 mm. The most abundant of these species is indene. Indene is the major intermediate C 2 + species detected in this flame with a peak mole fraction of 1600 ppm. FIGURE 10 Finally figure 11 presents the profiles of oxygenated aromatic species. Benzaldehyde, the most abundant of these species (peak mole fraction of 80 ppm), reach its maxima first around 2.0 mm above the burner. The profiles of phenol peak further around 2.2 mm and the maximum of that of benzofuran is the last one around 2.3 mm. FIGURE 11 With the addition of indane, the formation of large amounts of acetylene, ethylene, indene, benzene, toluene, styrene, ethylbenzene and phenylacetylene were observed. The most important ways of decomposition of indane is probably H-atom abstraction by metatheses or by bimolecular initiation with oxygen yields 1-indanyl and 2-indanyl. The formation of 1indanyl should be favored on the basis of the number of available abstraction sites (2 CH 2 groups) and the fact that this radical is stabilized by resonance. -scission C-C bond in 1indanyl followed by addition of hydrogen or methyl rdical CH 3 can yields respectivelymethylstyrene or -ethylstyrene (wich experimentally observed but not quantified). Thermal decomposition of indanyl radicals can also led to acetylene and benzyl radical. Benzyl yields toluene via hydrogen addition, whereas its oxidation yields benzaldehyde. The recombination of benzyl and methyl radicals yields ethylbenzene. Indanyl radicals also can decomposate to benzene and propargyl radical (C 3 H 3 ) or to phenylacetylene and CH 3 . Indene is formed viascission C-H bond in 1-indanyl and 2-indanyl. Its probably the most important voice decomposition of indanyl radicals by the fact that indene is the most intermediaire important specie experimentally observed. Combinations of 1-indanyl with methyl radical CH 3 yields 1methylindane. Indene oxidation yields 1-indanone whereas H atom abstraction on indene yields indenyl. Indenyl oxidation yields indenonyl that can rearrange to eliminate CO and 2-phenylvinyl that in turn decomposes to form benzene, or forms styrene by hydrogenation. Indenyl can rearrange to yield -ethynylbenzyl that,by oxidation yields benzaldehyde.

CONCLUSION

This paper presents new experimental results for a lean premixed laminar flame of methane seeded with indane which can be considered as an important component of diesel fuels. Profiles of temperature have been measured and mole fraction profiles have been obtained for 41 identified stable species from C 0 to C 10 , including 17 aromatic products and 6 oxygenated compounds other than the reactants.

FIGURE CAPTIONS

Figure 1: Temperature profiles: experimental measurements performed without and with the sampling probe. Figure 2: Profiles of the mole fractions of both hydrocarbon reactants. Figure 3: Profiles of the mole fractions of oxygen and the main oxygenated products. Figure 4: Profiles of the mole fractions of C 2 species. Figure 5: Profiles of the mole fractions of C 3 species. Figure 6: Profiles of the mole fractions of C 4 species. Figure 7: Profiles of the mole fractions of cyclopentadiene and methylcyclopentadiene. Figure 8: Profiles of the mole fractions of oxygenated C 1 -C 3 species. Figure 9: Profiles of the mole fractions of C 6 -C 10 monoaromatic species. Figure 10: Profiles of the mole fractions of biaromatic species. Figure 11: Profiles of the mole fractions of oxygenated aromatic species. Figure 12: Proposed major pathways in the oxidation of indane in flame

  Figure 3 a) b)

Figure 7 Figure 8

 78 Figure 7