Even if Dante's work involves a fair amount of autobiographical material and occasionally fashions itself as a record of personal experiences, his writings reveal surprisingly few facts about his life. With one exception though : Dante's banishment from Florence, which first occurred in January 1302 and soon escalated into permanent exile. As the first excerpt from [T1] shows, it is a testament to the callous expediency with which Dante's enemies took him out of the Florentine political equation that the whole proceeding (criminal conviction and first proscription) was carried out while Dante was on a diplomatic mission to Rome, precisely where his fate -and the fate of other prominent White Guelfs, his political associates within the local pro-papal faction -had been decided ahead of his embassy. As the second excerpt from [T1] also shows, exile marked something of a turning point in Dante's life, a defining moment which he came to embrace both as a man and as a literate. First and foremost, it meant leaving behind everything he held dear, living thereafter in bitter need : blame, hardship and humiliation being then and always the outcast's lot. More to the point, the death warrant, confiscation of property and loss of citizenship which were repeatedly forced upon him (and, eventually, when they came of age, upon his sons) not only crushed whatever hope he had to set foot in Florence again, but ultimately shaped his mature views on the civil unrest and periodical bloodshed that had plagued Italy's recent history. How to redeem the country from the evils partisan politics had brought upon the Italian peoples became Dante's first concern and constant preoccupation. While the solution he advocated was by no means original and -politically speaking -as much a thing of the past as the Sicilian vernacular that has risen to prominence with Frederic II and Manfred, his answers to Italy's past and current predicaments were rooted in a line of argument whose philosophical breadth, formal rigour and overall coherence were, if not unparalleled, at least second to none and -philosophically speaking -more compelling than most. Excerpts from [T2] present us with Dante's plea for peace, the cornerstone of his philosophy of man. The path that leads to peace as well as the means to maintain and eventually restore it are the keys to Dante's understanding not only of the purpose of authority throughout history, but also -and more fundamentally -of man's true nature and destination as a rational being. According to [T2], peace is the ultimate goal of human society and -by the same tokenthe very reason why men associate in the first place. Should they live together as one family instead of seeking after goods that benefit some and harm others, then humanity, free from conflict and oppression, would achieve its full potential, which Dante identified with its intellectual progress, intelligence being what sets man apart from every other worldly creature.

For the core tenets of Dante's thought revolve around the idea that, in order to reach perfection, humanity must be at peace and, in order to be at peace, humanity must be ruled by a single unified authority, it does not come as a surprise that Dante rejected everything that stood in the way of a universal monarchy, be it the greed of clergymen who turned the Church of Christ into an harlot and let the French kings seize her and hold her captive, the short-sightedness of his fellow citizens eager to fight each other as soon as they ran out of outside enemies, the cruelty of the warlords who held sway in the North of the peninsula, or the pettiness of the German princelings undeserving of sitting on the imperial throne and unable to fulfil their imperial mission. As a result, one would be hard-pressed to find a stauncher supporter of the separation between secular power and temporal government, on one side, and pastoral care and religious authority, on the other. It would be equally hard to find a more sturdy advocate of the supremacy of the Emperor over and against all kinds of local, regional or national jurisdictions. Excerpts from [T3] address both issues, most eloquently.

On the other hand, it might come as a surprise that of all the worthy men whose example prompted Dante to expose his contemporaries' failures and shortcomings, the one who definitely stands out is Manente degli Uberti, a damned soul Dante meets in his journey through the Underworld. Though it is no accident that Farinata burns in Hell, his demeanour and everything he says in [T4] speak volumes about Dante's political anthropology. As an Epicurean heretic, Farinata may very well feel nothing but contempt for divine justice and retribution ; likewise, as a Ghibelline chieftain, he may very well display an unyielding attachment to his party and lineage. That being said, his character possesses -along with honour, courtesy and valour -one redeeming quality, namely an uncompromising love for his homeland which he refused to identify with himself, his power base or his legacy. Whatever Dante deemed Farinata's ultimate destiny ought to be, he depicted him as a man who -when it mattered most -raised himself above partisan passions and saved the City he had contributed to tear apart from its destruction at the hands of his allies who were more than willing to rip their enemies' cause out, root and stem. Even though -in the aftermath of the victory at Montaperti (1260) -strength and right were on his side, Farinata refused to deal a mortal blow to those who had cast him out and chose to share his homeland with them rather than make certain that it will never be theirs again. As the dialogue between Dante and Farinata unfolds, it becomes clear that there's no love lost between the two. It also becomes clear that what drives both -the very thing Dante cherished above all and Farinata lost but managed, almost single-handedly, to pass on to the next generation -is a dream of peace, the only hope men ever had to make a broken community whole again.

Dante : Texts [T1 Dante] Excerpt 1

Dante Alighieri, Divina commedia. Paradiso, G. Petrocchi (ed), Firenze, Le Lettere, 1994, XVII, 46-69 : « Qual si partio Ipolito d'Atene per la spietata e perfida noverca, tal di Fiorenza partir ti convene.

Questo si vuole e questo già si cerca, e tosto verrà fatto a chi ciò pensa là dove Cristo tutto dì si merca.

La colpa seguirà la parte offensa in grido, come suol ; ma la vendetta fia testimonio al ver che la dispensa.

Tu lascerai ogne cosa diletta più caramente ; e questo è quello strale che l'arco de lo essilio pria saetta.

Tu proverai sì come sa di sale lo pane altrui, e come è duro calle lo scendere e 'l salir per l'altrui scale.

E quel che più ti graverà le spalle, sarà la compagnia malvagia e scempia con la qual tu cadrai in questa valle ; che tutta ingrata, tutta matta ed empia si farà contr' a te; ma, poco appresso, ella, non tu, n'avrà rossa la tempia.

Di sua bestialitate il suo processo farà la prova; sì ch'a te fia bello averti fatta parte per te stesso ».

J. and R. Hollander, Dante Alighieri. Paradiso, New York, Doubleday, 2007 : « As Hippolytus was forced to flee from Athens, because of his stepmother, treacherous and fierce, so shall you be forced to flee from Florence. This is the plan, already set in motion, that soon will bring success to him who plots it where Christ is bought and sold all day.

The populace shall blame the injured party, as it always does, but vengeance shall bear witness to the Truth that metes it out.

You shall leave behind all you most dearly love, and that shall be the arrow first loosed from exile's bow.

You shall learn how salt is the taste of another man's bread and how hard is the way, going down and then up another man's stairs.

But the heaviest burden your shoulders must bear shall be the companions, wicked and witless, among whom you shall fall in your descent.

They, utterly ungrateful, mad, and faithless, shall turn against you. But soon enough they, not you, shall feel their faces blushing past their brows.

Of their brutish state the results shall offer proof. And it shall bring you honor to have made a single party of yourself alone ».

[T1 Dante] Excerpt 2 Dante Alighieri, Convivio, F. Brambilla Ageno (ed.), Firenze, Le Lettere, 1995, I, III, 3-5: « Ahi, piaciuto fosse al dispensatore dell'universo che la cagione della mia scusa mai non fosse stata! ché né altri contra me avria fallato, né io sofferto avria pena ingiustamente, pena, dico, d'essilio e di povertate. [4] Poi che fu piacere delli cittadini della bellissima e famosissima figlia di Roma, Fiorenza, di gittarmi fuori del suo dolce seno -nel quale nato e nutrito fui in fino al colmo della vita mia, e nel quale, con buona pace di quella, desidero con tutto lo core di riposare l'animo stancato e terminare lo tempo che m'è dato -, per le parti quasi tutte alle quali questa lingua si stende, peregrino, quasi mendicando, sono andato, mostrando contra mia voglia la piaga della fortuna, che suole ingiustamente al piagato molte volte essere imputata. [5] Veramente io sono stato legno sanza vela e sanza governo, portato a diversi porti e foci e liti dal vento secco che vapora la dolorosa povertade; e sono apparito a li occhi a molti che forse che per alcuna fama in altra forma m'aveano immaginato, nel conspetto de' quali non solamente mia persona invilio, ma di minor pregio si fece ogni opera, sì già fatta come quella che fosse a fare ». R.H. Lansing, Dante's Il Convivio, New York, Garland, 1990 : « Ah, if only it had pleased the Maker of the Universe that the cause of my apology had never existed, for then neither would others have sinned against me, nor would I have suffered punishment unjustly -the punishment, I mean, of exile and poverty. [4] Since it was the pleasure of the citizens of the most beautiful and famous daughter of Rome, Florence, to cast me out of her sweet bosom--where I was born and bred up to the pinnacle of my life, and where, with her good will, I desire with all my heart to rest my weary mind and to complete the span of time that is given to me -I have travelled like a stranger, almost like a beggar, through virtually all the regions to which this tongue of ours extends, displaying against my will the wound of fortune for which the wounded one is often unjustly accustomed to be held accountable. [5] Truly I have been a ship without sail or rudder, brought to different ports, inlets, and shores by the dry wind that painful poverty blows. And I have appeared before the eyes of many who perhaps because of some report had imagined me in another form. In their sight not only was my person held cheap, but each of my works was less valued, those already completed as much as those yet to come] ».

[T2 Dante] Excerpt 1 Dante Alighieri, Monarchia, P.G. Ricci (ed), Firenze, Società Dantesca Italiana, 1965, I, I, 5 -III, 7 : « [I, 5] Cumque, inter alias veritates occultas et utiles, temporalis Monarchie notitia utilissima sit et maxime latens et, propter non se habere inmediate ad lucrum, ab omnibus intemptata, in proposito est hanc de suis enucleare latibulis, tum ut utiliter mundo pervigilem, tum etiam ut palmam tanti bravii primus in meam gloriam adipiscar. […]. [II, 1] Primum quidem igitur videndum quid est quod "temporalis Monarchia" dicitur, typo ut dicam et secundum intentionem. [II,2] Est ergo temporalis Monarchia, quam dicunt "Imperium", unicus principatus et super omnes in tempore vel in hiis et super hiis que tempore mensurantur. [II, 3] Maxime autem de hac tria dubitata queruntur: primo nanque dubitatur et queritur an ad bene esse mundi necessaria sit; […]. [II,4] Verum, quia omnis veritas que non est principium ex veritate alicuius principii fit manifesta, necesse est in qualibet inquisitione habere notitiam de principio, in quod analetice recurratur pro certitudine omnium propositionum que inferius assummuntur. Et quia presens tractatus est inquisitio quedam, ante omnia de principio scruptandum esse videtur in cuius virtute inferiora consistant. [II,5] Est ergo sciendum quod quedam sunt que, nostre potestati minime subiacentia, speculari tantummodo possumus, operari autem non; velut mathematica, physica et divina; quedam vero sunt que, nostre potestati subiacentia, non solum speculari sed etiam operari possumus: et in hiis non operatio propter speculationem, sed hec propter illam assummitur, quoniam in talibus operatio finis. [II,6] Cum ergo materia presens politica sit, ymo fons atque principium rectarum politiarum, et omne politicum nostre potestati subiaceat, manifestum est quod materia presens non ad speculationem per prius, sed ad operationem ordinatur. [II,7] Rursus, cum in operabilibus principium et causa omnium sit ultimus finis -movet enim primo agentem -, consequens est ut omnis ratio eorum que sunt ad finem ab ipso fine summatur. […]. [II,8] Illud igitur, siquid est, quod sit finis universalis civilitatis humani generis, erit hic principium per quo omnia que inferius probanda sunt erunt manifesta sufficienter: esse autem finem huius civilitatis et illius, et non esse unum omnium finem arbitrari stultum est. [III, 1] Nunc autem videndum est quid sit finis totius humane civilitatis : quo viso, plus quam dimidium laboris erit transactum, iuxta Phylosophum ad Nicomacum. [III,2] Et ad evidentiam eius quod queritur advertendum quod quemadmodum est finis aliquis ad quem natura producit pollicem, et alius ab hoc ad quem manum totam, et rursus alius ab utroque ad quem brachium, aliusque ab omnibus ad quem totum hominem; sic alius est finis ad quem singularem hominem, alius ad quem ordinat domesticam comunitatem, alius ad quem viciniam, et alius ad quem civitatem, et a!ius ad quem regnum, et denique optimus ad quem universaliter genus humanum Deus ecternus arte sua, que natura est, in esse producit. Et hoc queritur hic tanquam principium inquisitionis directivum. [III,3] Propter quod sciendum primo, quod Deus et natura nil otiosum facit, sed quicquid prodit in esse est ad aliquam operationem. Non enim essentia ulla creata ultimus finis est in intentione creantis, in quantum creans, sed propria essentie operatio: unde est quod non operatio propria propter essentiam, sed hec propter illam habet ut sit. [III,4] Est ergo aliqua propria operatio humane universitatis, ad quam ipsa universitas hominum in tanta multitudine ordinatur; ad quam quidem operationem nec homo unus, nec domus una, nec una vicinia, nec una civitas, nec regnum particulare pertingere potest. Que autem sit illa, manifestum fiet si ultimum de potentia totius humanitatis appareat. [III,5] Dico ergo quod nulla vis a pluribus spetie diversis participata ultimum est de potentia alicuius illorum; quia, cum illud quod est ultimum tale sit constitutivum spetiei, sequeretur quod una essentia pluribus spetiebus esset specificata: quod est impossibile. [III,6] Non est ergo vis ultima in homine ipsum esse simpliciter sumptum, quia etiam sic sumptum ab elementis participatur; nec esse complexionatum, quia hoc reperitur in mineralibus; nec esse animatum, quia sic etiam in plantis; nec esse apprehensivum, quia sic etiam participatur a brutis; sed esse apprehensivum per intellectum possibilem: quod quidem esse nulli ab homine alii competit vel supra vel infra. [III,7] Nam, etsi alie sunt essentie intellectum participantes, non tamen intellectus earum est possibilis ut hominis, quia essentie tales speties quedam sunt intellectuales et non aliud, et earum esse nichil est aliud quam intelligere quod est quod sunt, quod est sine interpolatione, aliter sempiterne non essent. Patet igitur quod ultimum de potentia ipsius humanitatis est potentia sive virtus intellectiva ». P. Shaw, Dante. Monarchy, Oxford, Oxford University Press, 1996 : « [I, 5] Now since among other truths which are hidden and useful, a knowledge of temporal monarchy is both extremely useful and most inaccessible, and since no one has attempted to elucidate it (on account of its not leading directly to material gain), I propose to draw it forth from where it lies hidden, so that my wakeful nights may be of benefit to the world, and so that I may be the first to win for my own glory the honour of so great a prize. […]. [II, 1] Firstly therefore we must see what is meant by "temporal monarchy", in broad terms and as it is generally understood. [II, 2] Temporal monarchy, then, which men call "empire", is a single sovereign authority set over all others in time, that is to say over all authorities which operate in those things and over those things which are measured by time. [II, 3] Now there are three main points of inquiry which have given rise to perplexity on this subject : first, is it is necessary to the wellbeing of the world ? […]. [II,4] Now since every truth which is not itself a first principle must be demonstrated with reference to the truth of some first principle, it is necessary in any inquiry to know the first principle to which we refer back in the course of strict deductive argument in order to ascertain the truth of all the propositions which are advanced later. And since this present treatise is a kind of inquiry, we must at the outset investigate the principle whose truth provides a firm foundation for later propositions. [II,5] For it must be noted that there are certain things (such as mathematics, the sciences and divinity) which are outside human control, and about which we can only theorise, but which we cannot affect by our actions; and then there are certain things which are within our control, where we can not only theorise but also act, and in these action is not for the sake of theory, but theorising is for the sake of taking action, since in these the objective is to take action. [II,6] Now since our present subject is political, indeed is the source and starting-point of just forms of government, and everything in the political sphere comes under human control, it is clear that the present subject is not directed primarily towards theoretical understanding but towards action. [II,7] Again, since in actions it is the final objective which sets in motion and causes everything -for that is what first moves a person who acts -it follows that the whole basis of the means for attaining an end is derived from the end itself. […]. [II,8] Therefore whatever constitutes the purpose of the whole of human society (if there is such a purpose) will be here the first principle, in terms of which all subsequent propositions to be proved will be demonstrated with sufficient rigour; for it would be foolish to suppose that there is one purpose for this society and another for that, and not a common purpose for all of them. [III, 1] We must therefore now see what is the purpose of human society as a whole; when we have seen this, more than half our work will be done, as Aristotle says in the Ethics. [III,2] And to throw light on the matter we are inquiring into, it should be borne in mind that, just as there is a particular purpose for which nature produces the thumb, and a different one for which she produces the whole hand, and again a purpose different from both of these for which she produces the arm, and a purpose different from all of these for which she produces the whole person; in the same way there is one purpose for which the individual person is designed, another for the household, another for the small community, yet another for the city, and another for the kingdom; and finally the best purpose of all is the one for which God Everlasting with his art, which is nature, brings into being the whole of mankind. And it is this purpose we are seeking here as the guiding principle in our inquiry. [III,3] Consequently the first point to bear in mind is that God and nature do nothing in vain; on the contrary whatever they bring into being is designed for a purpose. For in the intention of its creator qua creator the essential nature of any created being is not an ultimate end in itself; the end is rather the activity which is proper to that nature; and so it is that the activity does not exist for the sake of the essential nature, but the essential nature for the sake of that activity. [III,4] There is therefore some activity specific to humanity as a whole, for which the whole human race in all its vast number of individual human beings is designed; and no single person, or household, or small community, or city, or individual kingdom can fully achieve it. Now what this activity is will become clear when once we clarify what is the highest potentiality of the whole of mankind. [III, 5] I say therefore that no faculty shared by many different species is the highest potentiality of any one of them; because, since it is precisely that highest potentiality which is the defining characteristic of the species, it would follow that one and the same essential nature was specific to several species; and this is impossible. [III,6] So the highest faculty in a human being is not simply to exist, because the elements too share in the simple fact of existence; nor is it to exist in compound form, for that is found in minerals; nor is it to exist as a living thing, for plants too share in that; nor is it to exist as a creature with sense perception, for that is also shared by the lower animals; but it is to exist as a creature who apprehends by means of the potential intellect: this mode of existence belongs to no creature (whether higher or lower) other than human beings. [III,7] For while there are indeed other beings who like us are endowed with intellect, nonetheless their intellect is not "potential" in the way that man's is, since such beings exist only as intelligences and nothing else, and their very being is simply the act of understanding that their own nature exists; and they are engaged in this ceaselessly, otherwise they would not be eternal. It is thus clear that the highest potentiality of mankind is his intellectual potentiality or faculty ».

[T2 Dante] Excerpt 2 Dante Alighieri, Monarchia, I, IV, 1-2 and 5 : « [IV, 1] Now it has been sufficiently explained that the activity proper to mankind considered as a whole is constantly to actualise the full intellectual potential of humanity, primarily through thought and secondarily through action (as a function and extension of thought). [IV,2] And since what holds true for the part is true for the whole, and an individual human being "grows perfect in judgment and wisdom when he sits at rest", it is apparent that mankind most freely and readily attends to this activity -[…] -in the calm or tranquillity of peace. Hence it is clear that universal peace is the best of those things which are ordained for our human happiness. […]. [IV,5] From the arguments developed so far, it is clear what is the better, indeed the best, way of enabling mankind to engage in the activity proper to humanity; and consequently we see the most direct means of achieving the goal to which all our human actions are directed as to their final end. That means is universal peace, which is to be taken as the first principle for the arguments which follow ».

« [IV,
[T2 Dante] Excerpt 3 Dante Alighieri, Monarchia, I, V, 1-10 ; X, 1 -XI, 2 ; XI, 8-12 and 20 : « [V, 1] Resumens igitur quod a principio dicebatur, tria maxime dubitantur et dubitata queruntur circa Monarchiam temporalem, que comuniori vocabulo nuncupatur "Imperium" ; et de hiis, ut predictum est, propositum est sub assignato principio inquisitionem facere secundum iam tactum ordinem. [V, 2] Itaque prima questio sit: utrum ad bene esse mundi Monarchia temporalis necessaria sit. Hoc equidem, nulla vi rationis vel auctoritatis obstante, potissimis et patentissimis argumentis ostendi potest, quorum primum ab autoritate Phylosophi assummatur de suis Politicis. [V, 3] Asserit enim ibi venerabilis eius autoritas quod, quando aliqua plura ordinantur ad unum, oportet unum eorum regulare seu regere, alia vero regulari seu regi; quod quidem non solum gloriosum nomen autoris facit esse credendum, sed ratio inductiva. [V, 4] Si enim consideremus unum hominem, hoc in eo contingere videbimus, quia, cum omnes vires eius ordinentur ad felicitatem, vis ipsa intellectualis est regulatrix et rectrix omnium aliarum: aliter ad felicitatem pervenire non potest. [V, 5] Si consideremus unam domum, cuius finis est domesticos ad bene vivere preparare, unum oportet esse qui regulet et regat, quem dicunt patremfamilias, vel eius locum tenentem, iuxta dicentem Phylosophum: "Omnis domus regitur a senissimo"; et huius, ut ait Homerus, est regulare omnes et leges imponere aliis. Propter quod proverbialiter dicitur illa maledictio: "Parem habeas in domo". [V, 6] Si consideremus vicum unum, cuius finis est commoda tam personarum quam rerum auxiliatio, unum oportet esse aliorum regulatorem, vel datum ab alio vel ex ipsis preheminentem consentientibus aliis; aliter ad illam mutuam sufficientiam non solum non pertingitur, sed aliquando, pluribus preheminere volentibus, vicinia tota destruitur. [V, 7] Si vero unam civitatem, cuius finis est bene sufficienterque vivere, unum oportet esse regimen, et hoc non solum in recta politia, sed etiam in obliqua; quod si aliter fiat, non solum finis vite civilis amictitur, sed etiam civitas desinit esse quod erat. [V, 8] Si denique unum regnum particulare, cuius finis est is qui civitatis cum maiori fiducia sue tranquillitatis, oportet esse regem unum qui regat atque gubernet; aliter non modo existentes in regno finem non assecuntur, sed etiam regnum in interitum labitur, iuxta illud infallibilis Veritatis: "Omne regnum in se divisum desolabitur". [V, 9] Si ergo sic se habet in hiis et in singulis que ad unum aliquod ordinantur, verum est quod assummitur supra; nunc constat quod totum humanum genus ordinatur ad unum, ut iam preostensum fuit; ergo unum oportet esse regulans sive regens, et hoc "Monarcha" sive "Imperator" dici debet. [V, 10] « [V, 1] Returning then to the point made at the beginning, there are three main points of inquiry concerning temporal monarchy (or empire as it is more commonly called) which have given rise to and continue to give rise to perplexity; and as we have already said, it is our intention to investigate these questions in the order in which we set them out and taking the principle we have just established as our starting-point. [V, 2] So the first question is this: is temporal monarchy necessary for the well-being of the world? That it is necessary can be shown with powerful and persuasive arguments, and neither reason nor authority provides any strong counter-argument. The first of these arguments may be taken from the authority of Aristotle in his Politics. [V, 3] Now this revered authority states in that work that when a number of things are ordered to a single end, one of them must guide or direct, and the others be guided or directed; and it is not only the authors illustrious name which requires us to believe this, but inductive reasoning as well. [V, 4] For if we consider a single person, we shall see that what happens in the individual is this: while all the faculties are directed towards happiness, it is the intellectual faculty which guides and directs all the others; otherwise happiness is unattainable. [V, 5] If we consider a household, whose purpose is to prepare its members to live the good life, there must be one person who guides and directs, who is called the "pater familias" or his representative, in line with Aristotle's observation that "Every household is governed by the eldest" ; and his role, as Homer says, is to guide everyone and impose rules on the others. Hence the proverbial curse: "May you have an equal in your house". [V, 6] If we consider a small community, whose purpose is neighbourly support in relation both to people and to goods, there must be one person who guides the others, either appointed by someone from outside or emerging as leader from among their number with the agreement of the others; otherwise not only will they fail to achieve that neighbourly collaboration, but sometimes, if a number of people contest the leadership, the whole community is destroyed. [V, 7] If we consider a city, whose purpose is to be selfsufficient in living the good life, there must be one ruling body, and this is so not only in just government, but in perverted forms of government as well; if this should not be the case, not only is the purpose of social life thwarted, but the city itself ceases to be what it was. [V, 8] Lastly, if we consider an individual kingdom -and the purpose of a kingdom is the same as that of a city, but with greater confidence that peace can be maintained -there must be one king who rules and governs; otherwise not only do those who live in the kingdom not achieve that purpose, but the kingdom itself falls to ruin, in accordance with those words of the infallible Truth: "Every kingdom divided against itself shall be laid waste". [V, 9] If this holds true in these cases and in individuals who are ordered to one particular goal, then the proposition advanced above is true; now it is agreed that the whole of mankind is ordered to one goal, as has already been demonstrated: there must therefore be one person who directs and rules mankind, and he is properly called "Monarch" or "Emperor". [V, 10] And thus it is apparent that the well-being of the world requires that there be a monarchy or empire.

[…]. [X, 1] Now wherever there can be conflict there must be judgment to resolve it, otherwise there would be an imperfection without its proper corrective; and this is impossible, since God and nature never fail in their provision of what is necessary. [X, 2] There is always the possibility of conflict between two rulers where one is not subject to the other's control; such conflict may come about either through their own fault or the fault of their subjects (the point is selfevident); therefore there must be judgment between them. [X, 3] And since neither can judge the other (since neither is under the other's control, and an equal has no power over an equal) there must be a third party of wider jurisdiction who rules over both of them by right. [X, 4] And this person will either be the monarch or not. If he is, then our point is proved; if he is not, he in his turn will have an equal who is outside the sphere of his jurisdiction, and then it will once again be necessary to have recourse to a third party. [X, 5] And so either this procedure will continue ad infinitum, which is not possible, or else we must come to a first and supreme judge, whose judgment resolves all disputes either directly or indirectly; and this man will be the monarch or emperor. Thus monarchy is necessary to the world. [X, 6] And Aristotle saw the force of this argument when he said: "Things do not wish to be badly ordered; a plurality of reigns is bad; therefore let there be one ruler". [XI, 1] Furthermore, the world is ordered in the best possible way when justice is at its strongest in it. […]. [XI, 2] Justice is at its strongest only under a monarch; therefore for the best ordering of the world there must be a monarchy or empire. […]. [XI,8] Building on this exposition we can argue as follows: justice is at its strongest in the world when it resides in a subject who has in the highest degree possible the will and the power to act; only the monarch is such a subject; therefore justice is at its strongest in the world when it is located in the monarch alone. [XI,9] This prosyllogism is of the second figure with intrinsic negation, and it takes this form: all B is A; only C is A; therefore only C is B. That is: all B is A; nothing except C is A; therefore nothing except C is B. [XI,10] And the first proposition is established by the preceding exposition; the second is shown as follows, firstly in relation to volition, and then in relation to power. [XI,11] To clarify the first of these it must be noted that the thing most contrary to justice is greed, as Aristotle states in the fifth book of the Ethics. When greed is entirely eliminated, nothing remains which is opposed to justice; hence Aristotle's opinion that those things which can be resolved by law should in no way be left to the judge's discretion. And it is fear of greed which makes this necessary, for greed easily leads men's minds astray. But where there is nothing which can be coveted, it is impossible for greed to exist, for emotions cannot exist where their objects have been destroyed. [XI,12] But there is nothing the monarch could covet, for his jurisdiction is bounded only by the ocean; whereas this is not the case with other rulers, whose sovereignty extends only as far as the neighbouring kingdom, as is the case, for instance, with the kings of Castille and of Aragon. From this it follows that of all men the monarch can be the purest embodiment of justice. […]. [XI,20] The minor premiss of the main syllogism has been sufficiently proved and the conclusion is certain, namely that the best ordering of the world requires the existence of a monarchy ».

[T3 Dante] Excerpt 1 Dante Alighieri, Divina commedia. Inferno, G. Petrocchi (ed), Firenze, Le Lettere, 1994, XIX, 1-6 and 112-117 e ora in te non stanno sanza guerra li vivi tuoi, e l'un l'altro si rode di quei ch'un muro e una fossa serra.

Cerca, misera, intorno da le prode le tue marine, e poi ti guarda in seno, s'alcuna parte in te di pace gode.

Che val perché ti racconciasse il freno Iustinïano, se la sella è vòta? Sanz' esso fora la vergogna meno.

Ahi gente che dovresti esser devota, e lasciar seder Cesare in la sella, se bene intendi ciò che Dio ti nota, guarda come esta fiera è fatta fella per non esser corretta da li sproni, poi che ponesti mano a la predella ». Soleva Roma, che 'l buon mondo feo, due soli aver, che l'una e l'altra strada facean vedere, e del mondo e di Deo.

L'un l'altro ha spento; ed è giunta la spade col pasturale, e l'un con l'altro insieme per viva forza mal convien che vada; però che, giunti, l'un l'altro non teme; […] Dì oggimai che la Chiesa di Roma, per confondere in sé due reggimenti, cade nel fango, e sé brutta e la soma ».

J. and R. Hollander, Dante Alighieri. Purgatorio : « Rome, which formed the world for good, once held two suns that lit the one road and the other, the world's and that to God.

The one has snuffed the other out, the sword is fastened to the crook, and these two, forced to be together, must perforce go ill, since, joined, the one fears not the other.

[…]

Spread the word, then, that the Church of Rome, confounding in herself two governments, stumbles in the mud, befouling herself and her burden ». To each and every one of the Kings of Italy, and to the Senators of the Holy City, and also to Italy's Dukes, Marquises, and Counts, and to her people, a humble Italian, Dante Alighieri, a Florentine undeservedly in exile, prays for peace. […]. [11] Give up the savage ways you have adopted, you descendants of the Lombards: and if anything should remain of the seed of the Trojans and the Romans, yield to it, so that, when the heavenly eagle comes, descending like lightning, he does not see his own chicks cast out and the rightful place of hi s offspring occupied by ravens. [12] Come now, you Scandinavian race, and make yourselves eager to receive, as is your duty, the one whose arrival you justifiably fear. [13] Do not let yourselves be seduced by the ploys of cupidity, which, like the Sirens, uses its charm to overcome the vigilance of reason. [14] Hasten before him and declare your obedience to him: rejoice in singing a psalm of penitence to him, bearing in mind that "anyone who resists authority is rebelling against God's decision", while anyone who rebel s against God's decisions is kicking out against an omnipotent will, and "it is hard to kick against the goad" » [T4 Dante] Excerpt 1 Dante Alighieri, Divina commedia. Inferno, VI, 49-87 :

« "La tua città, ch'è piena d'invidia sì che già trabocca il sacco, seco mi tenne in la vita serena.

Voi cittadini mi chiamaste Ciacco: per la dannosa colpa de la gola, come tu vedi, a la pioggia mi fiacco.

E io anima trista non son sola, ché tutte queste a simil pena stanno per simil colpa." E più non fé parola.

Io li rispuosi: "Ciacco, il tuo affanno mi pesa sì, ch'a lagrimar mi 'nvita; ma dimmi, se tu sai, a che verranno li cittadin de la città partita; s'alcun v'è giusto; e dimmi la cagione per che l'ha tanta discordia assalita." E quelli a me: "Dopo lunga tencione verranno al sangue, e la parte selvaggia caccerà l'altra con molta offensione.

Poi appresso convien che questa caggia infra tre soli, e che l'altra sormonti con la forza di tal che testé piaggia.

Alte terrà lungo tempo le fronti, tenendo l'altra sotto gravi pesi, come che di ciò pianga o che n'aonti.

Giusti son due, e non vi sono intesi; superbia, invidia e avarizia sono le tre faville c'hanno i cuori accesi."

Qui puose fine al lagrimabil suono. E io a lui: "Ancor vo' che mi 'nsegni e che di più parlar mi facci dono.

Farinata e 'l Tegghiaio, che fuor sì degni, Iacopo Rusticucci, Arrigo e 'l Mosca e li altri ch'a ben far puoser li 'ngegni, dimmi ove sono e fa ch'io li conosca; ché gran disio mi stringe di savere se 'l ciel li addolcia o lo 'nferno li attosca." E quelli: "Ei son tra l'anime più nere; diverse colpe giù li grava al fondo: se tanto scendi, là i potrai vedere ». J. and R. Hollander, Dante Alighieri. Inferno : « "Your city, so full of envy that now the sack spills over, held me in its confines in the sunlit life.

You and my townsmen called me Ciacco. For the pernicious fault of gluttony, as you can see, I'm prostrate in this rain.

And in my misery I am not alone.

All those here share a single penalty for the same fault". He said no more. I answered him: 'Ciacco, your distress so weighs on me it bids me weep. But tell me, if you can, what shall be the fate of the citizens within the riven city? Are any in it just? And tell me why such discord has assailed it".

And he to me: "After long feuding they shall come to blood. The rustic faction, having done great harm, shall drive the others out.

But it in turn must fall to them, within three years, by power of him who now just bides his time.

These in their arrogance will long subject the other faction to their heavy yoke, despite its weeping and its shame.

Two men are just and are not heeded there. Pride, envy, and avarice are the sparks that have set the hearts of all on fire".

With that he ended his distressing words. And I to him: "I wish you would instruct me more, granting me the gift of further speech.

Farinata and Tegghiaio, who were so worthy, Jacopo Rusticucci, Arrigo, and Mosca, and the rest whose minds were bent on doing good, tell me where they are and how they fare. For great desire presses me to learn whether Heaven sweetens or Hell embitters them".

And he: "They are among the blacker souls. Different vices weigh them toward the bottom, as you shall see if you descend that far" ».

[T4 Dante] Excerpt 2 Dante Alighieri, Divina commedia. Inferno, X, 1-51 and 73-108 : « Ora sen va per un secreto calle, tra 'l muro de la terra e li martìri, lo mio maestro, e io dopo le spalle.

"O virtù somma, che per li empi giri mi volvi," cominciai, "com' a te piace, parlami, e sodisfammi a' miei disiri.

La gente che per li sepolcri giace potrebbesi veder? già son levati tutt' i coperchi, e nessun guardia face". E quelli a me: "Tutti saran serrati quando di Iosafàt qui torneranno coi corpi che là sù hanno lasciati.

Suo cimitero da questa parte hanno con Epicuro tutti suoi seguaci, che l'anima col corpo morta fanno.

Però a la dimanda che mi faci quinc' entro satisfatto sarà tosto, e al disio ancor che tu mi taci". E io: "Buon duca, non tegno riposto a te mio cuor se non per dicer poco, e tu m'hai non pur mo a ciò disposto".

  1] Satis igitur declaratum est quod proprium opus humani generis totaliter accepti est actuare semper totam potentiam intellectus possibilis, per prius ad speculandum et secundario propter hoc ad operandum per suam extensionem. [IV, 2] Et quia quemadmodum est in parte sic est in toto, et in homine particulari contingit quod sedendo et quiescendo prudentia et sapientia ipse perficitur, patet quod genus humanum in quiete sive tranquillitate pacis ad proprium suum opus, […], liberrime atque facillime se habet. Unde manifestum est quod pax universalis est optimum eorum que ad nostram beatitudinem ordinantur. […]. [IV, 5] Ex hiis ergo que declarata sunt patet per quod melius, ymo per quod optime genus humanum pertingit ad opus proprium. et per consequens visum est propinquissimum medium per quod itur in illud ad quod, velut in ultimum finem, omnia nostra opera ordinantur, quia est pax universalis, que pro principio rationum subsequentium supponatur ».

P. Shaw, Dante. Monarchy :

  Et sic patet quod ad bene esse mundi necesse est Monarchiam esse sive Imperium.[…]. [X, 1] Et ubicunque potest esse litigium, ibi debet esse iudicium; aliter esset inperfectum sine proprio perfectivo: quod est inpossibile, cum Deus et natura in necessariis non deficiat. [X, 2] Inter omnes duos principes, quorum alter alteri minime subiectus est, potest esse litigium vel culpa ipsorum vel etiam subditorum -quod de se patet -: ergo inter tales oportet esse iudicium. [X, 3] Et cum alter de altero cognoscere non possit ex quo alter alteri non subditur -nam par in parem non habet imperium -oportet esse tertium iurisdictionis amplioris qui ambitu sui iuris ambobus principetur.[X, 4] Et hic aut erit Monarcha aut non. Si sic, habetur propositum; si non, iterum habebit sibi coequalem extra ambitum sue iurisdictionis: tunc iterum necessarius erit tertius alius. [X, 5] Et sic aut erit processus in infinitum, quod esse non potest, aut oportebit devenire ad iudicem primum et summum, de cuius iudicio cuncta litigia dirimantur sive mediate sive inmediate: et hic erit Monarcha sive Imperator. Est igitur Monarchia necessaria mundo. [X, 6] Et hanc rationem videbat Phylosophus cum dicebat: "Entia nolunt male disponi; malum autem pluralitas principatuum: unus ergo princeps". [XI, 1] Preterea, mundus optime dispositus est cum iustitia in eo potissima est. […]. [XI, 2] Iustitia potissima est solum sub Monarcha: ergo ad optimam mundi dispositionem requiritur esse Monarchiam sive Imperium. […]. [XI, 8] Ex hac itaque declaratione sic arguatur: iustitia potissima est in mundo quando volentissimo et potentissimo subiecto inest; huiusmodi solus Monarcha est: ergo soli Monarche insistens iustitia in mundo potissima est. [XI, 9] Iste prosillogismus currit per secundam figuram cum negatione intrinseca, et est similis huic: omne B est A; solum C est A: ergo solum C est B. Quod est: omne B est A; nullum preter C est A: ergo nullum preter C est B. [XI, 10] Et prima propositio declaratione precedente apparet; alia sic ostenditur, et primo quantum ad velle, deinde quantum ad posse. [XI, 11] Ad evidentiam primi notandum quod iustitie maxime contrariatur cupiditas, ut innuit Aristoteles in quinto ad Nicomacum. Remota cupiditate omnino, nichil iustitie restat adversum; unde sententia Phylosophi est ut que lege determinari possunt nullo modo iudici relinquantur. Et hoc metu cupiditatis fieri oportet, de facili mentes hominum detorquentis. Ubi ergo non est quod possit optari, inpossibile est ibi cupiditatem esse: destructis enim obiectis, passiones esse non possunt. [XI, 12] Sed Monarcha non habet quod possit optare: sua nanque iurisdictio terminatur Occeano solum: quod non contingit principibus aliis, quorum principatus ad alios terminantur, ut puta regis Castelle ad illum qui regis Aragonum. Ex quo sequitur quod Monarcha sincerissimum inter mortales iustitie possit esse subiectum. […]. [XI, 20] Satis igitur declarata subassumpta principalis, quia conclusio certa est: scilicet quod ad optimam dispositionem mundi necesse est Monarchiam esse ».

P. Shaw, Dante. Monarchy :

  J. and R. Hollander, Dante Alighieri.Purgatorio, New York, Doubleday, 2003 :

	and let Caesar occupy the saddle,
	if you but heeded what God writes for you,
	see how vicious is the beast not goaded
	and corrected by the spurs,
	ever since you took the bridle in your hands »
	[T3 Dante] Excerpt 3
	Dante Alighieri, Divina commedia. Purgatorio, XVI, 106-112 and 127-129 :
	« Ah, Italy enslaved, abode of misery,
	pilotless ship in a fierce tempest tossed,
	no mistress over provinces but a harlot!
	How eager was that noble soul,
	only at the sweet name of his city,
	to welcome there his fellow citizen!
	Now your inhabitants are never free from war,
	and those enclosed within a single wall and moat
	are gnawing on each other.
	Search, miserable one, around your shores,
	then look into your heart,
	if any part of you rejoice in peace.
	If there is no one in your saddle, what good
	was it Justinian repaired your harness?
	Your shame would be less great had he not done so.
	Ah, you who should be firm in your devotion

  Universis et singulis Ytalie Regibus et Senatoribus alme Urbis nec non Ducibus Marchionibus Comitibus atque Populis, humilis ytalus Dantes Alagherii fiorentinus et exul inmeritus orat pacem. […]. [11] Pone, sanguis Longobardorum, coadductam barbariem; et si quid de Troyanorum Latinorumque semine superest, illi cede, ne cum sublimis aquila fulguris instar descendens adfuerit, abiectos videat pullos eius, et prolis proprie locum corvulis occupatum.[12] Eya, facite, Scandinavie soboles, ut cuius merito trepidatis adventum, quod ex vobis est presentiam sitiatis. [13] Nec seducat alludens cupiditas, more Sirenum nescio qua dulcedine vigiliam rationis mortificans. [14] Preoccupetis faciem eius in confessione subiectionis, et in psalterio penitentie iubiletis, considerantes quia "potestati resistens Dei ordinationi resistit"; et qui divine ordinationi repugnat, voluntati omnipotentie coequali recalcitrat; et "durum est contra stimulum calcitrare" ».

	[T3 Dante] Excerpt 4
	Dante Alighieri, Episolae, E. Pistelli (ed), Firenze, Bemporad, 1960, V, 1 and 11-14
	« [1] C.E. Honess, Dante Alighieri. Four Political Letters, London, Modern Humanities Research Association, 2007 :
	« [1]

le cose", disse, "che ne son lontano; cotanto ancor ne splende il sommo duce.

Quando s'appressano o son, tutto è vano nostro intelletto; e s'altri non ci apporta, nulla sapem di vostro stato umano.

Però comprender puoi che tutta morta fia nostra conoscenza da quel punto che del futuro fia chiusa la porta" ».

J. and R. Hollander, Dante Alighieri. Inferno : « Now my master takes a hidden path between the city's ramparts and the torments, and I come close behind him.

"O lofty virtue", I began, "who lead me as you will around these impious circles, speak to me and satisfy my wishes.

'The souls that lie within the sepulchres, may they be seen? For all the lids are raised and there is no one standing guard.'

And he to me: 'All will be shut and sealed when the souls return from Jehosaphat with the bodies they have left above.

'Here Epicurus and all his followers, who hold the soul dies with the body, have their burial place.

'But soon your need to have an answer will be satisfied right here, as will the wish you hide from me.' And I: 'Good leader, from you I do not keep my heart concealed except to speak few wordsas you've from time to time advised.'

'O Tuscan, passing through the city of fire, alive, and with such courtesy of speech, if it would please you, stay your steps awhile.

'Your way of speaking makes it clear that you are native to that noble city to which I was perhaps too cruel.'

This voice came suddenly from one sarcophagus, so that, startled, I drew closer to my leader.

And he to me: 'Turn back! What are you doing? Look, there Farinata stands erectyou can see all of him from the waist up.' Already I had fixed my gaze on his. And he was rising, lifting chest and brow as though he held all Hell in utter scorn.

At which my leader: 'Choose your words with care,' and his hands, ready, encouraging, thrust me toward him among the tombs.

When I stood at the foot of his tomb he looked at me a moment. Then he asked, almost in disdain: 'Who were your ancestors?' And I, eager, held nothing back, but told him who they were, at which he barely raised his eyebrows and said: 'They were most bitter enemies to me, my forebears, and my partynot once, but twice, I had to drive them out.' 'If they were banished,' I responded, 'they returned from every quarter both the first time and the second, a skill that Yours have failed to learn as well.

[…].

But the other, that great soul at whose wish I had stopped, did not change countenance, nor bend his neck, nor move his chest.

And he, continuing from where he'd paused: 'That they have badly learned this skill torments me more than does this bed.

'But the face of the lady reigning here will be rekindled not fifty times before you too shall know how difficult a skill that is to learn.

'And, so may you return to the sweet world, tell me, why are your people, in every edict, so pitiless against my kin?'

Then I to him: 'The havoc and great slaughter that dyed the Arbia red caused them to raise such prayers in our temple.'

He sighed and shook his head, then spoke: 'I was not alone, nor surely without cause would I have acted with the rest.

'But it was I alone, when all agreed to make an end of Florence, I alone who dared speak out in her defense.' 'So may Your seed sometime find peace, pray untie for me this knot,' I begged him, 'which has entangled and confused my judgment.

'From what I hear, it seems you see beforehand that which time will bring, but cannot know what happens in the present.' 'We see, like those with faulty vision, things at a distance,' he replied. 'That much, for us, the mighty Ruler's light still shines.

'When things draw near or happen now, our minds are useless. Without the words of others we can know nothing of your human state.

'Thus it follows that all our knowledge will perish at the very moment the portals of the future close" ».


domain: spi spi.nano sdv

text:
Anna Susloparova

Sophie Halliez

Séverine Begard

Morvane Colin

Luc Buée

Sébastien Pecqueur

Fabien Alibart

Vincent Thomy

Steve Arscott

Emiliano Pallecchi

Yannick Coffinier
email: yannick.coffinier@univ-lille1.fr

Low impedance and highly transparent microelectrode arrays (MEA) for in vitro neuron electrical activity probing

Keywords: microelectrode array, transparent electrodes, PEDOT:PSS, low impedance, neuronal network activity

est destinée au dépôt

Low impedance and highly transparent microelectrode arrays (MEA) for in vitro neuron electrical activity probing

Introduction

Microelectrode arrays (MEA) are recognized as standard tools allowing recording in vitro biological activity of neuronal cells [START_REF] Koutsouras | PEDOT : PSS microelectrode arrays for hippocampal cell culture electrophysiological recordings[END_REF][START_REF] Nam | [END_REF], cardiac cells [START_REF] Kshirsagar | Transparent Graphene / PEDOT : PSS Microelectrodes for Electro-and Optophysiology[END_REF][START_REF] Gerwig | PEDOT -CNT composite microelectrodes for recording and electrostimulation applications : fabrication[END_REF] and from brain slices [START_REF] Sessolo | Easy-to-Fabricate Conducting Polymer Microelectrode Arrays[END_REF][START_REF] Steidl | The adult rat hippocampal slice revisited with multi-electrode arrays[END_REF].

Recently, integrated tools combining both electro-and optophysiology (calcium or voltage sensitive imaging) have gained great interest for a better understanding of physiological phenomena within neural networks. Further development of transparent microelectrodes is required since they enable simultaneous electrophysiology and optical imaging (i.e. highresolution fluorescence imaging in tandem with transmission microscopy). Biocompatible, transparent microelectrodes have been demonstrated using a number of means. Thin titanium nitride (TiN) electrodes (30 µm in diameter) were fabricated by sputtering with an electrode surface impedance below 175 MΩ µm 2 [START_REF] Www | multichannelsystems.com, Microsoft, Microelectrode Array ( MEA ) Manual[END_REF] and a transmission at visible wavelengths of 50% [START_REF] Solovan | Electrical and Optical Properties of TiN Thin Films[END_REF]--or by atomic layer deposition (ALD) with an electrode surface impedance from 360 to 420 MΩ µm 2 and a transmission at visible wavelengths of 19-45% [START_REF] Ryynänen | Microelectrode Array With Transparent ALD TiN Electrodes[END_REF]. Due to the trade-off between metal transparency and metal conductance affecting optical transmission and electrode impedance (i.e. decreasing the metal thickness to increase transparency leads to an increase in electrical impedance due to an increase of resistance), development of both transparent and conductive materials is needed [START_REF] Lee | Transparent, conformable, active multielectrode array using organic electrochemical transistors[END_REF]. In previous publications, transparent MEAs based on indium tin oxide (ITO) with transmission at visible wavelengths of 80% [START_REF] Kwon | Opto-Array : A Hybrid Neural Interface With Transparent Electrode Array and Integrated LEDs for Optogenetics[END_REF] and graphene with transmission at visible wavelengths of 90% [START_REF] Du | Graphene microelectrode arrays for neural activity detection[END_REF][START_REF] Kuzum | Transparent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging[END_REF][START_REF] Park | applications[END_REF] were proposed. However, the impedance values of transparent ITO and graphene electrodes cannot compete with standard nontransparent MEA devices with TiN electrodes. As a comparison, the surface impedance at 1 kHz of 30 μm diameter TiN electrodes was below 70 MΩ µm 2 [START_REF] Www | multichannelsystems.com, Microsoft, Microelectrode Array ( MEA ) Manual[END_REF], whilst it was 7500 MΩ µm 2 for 200 μm diameter graphene electrodes [START_REF] Park | applications[END_REF] and 1250 MΩ µm 2 for 80 µm diameter ITO electrodes [START_REF] Ryynänen | Microelectrode array for noninvasive analysis of cardiomyocytes at the single-cell level[END_REF]. In addition, miniaturization of microelectrodes to match single neuronal cells dimensions (a few tens of µm) also affects electrodes impedance [START_REF] Kshirsagar | Semitransparent carbon microelectrodes for opto-and electrophysiology[END_REF], due to a decrease in the interfacial capacitance which is directly correlated with the microelectrode area [START_REF] Sessolo | Easy-to-Fabricate Conducting Polymer Microelectrode Arrays[END_REF]. Therefore, manufacturing small electrodes with high transparency whilst keeping the impedance as low as possible cannot be solved solely by geometrical optimization of the electrodes-and other material solutions are required.

In the past decades, conducting polymers have attracted great interest and is currently a buoyant field of research due to their lower microelectrode impedance compare to inorganic materials [START_REF] Ganji | Development and Translation of PEDOT : PSS Microelectrodes for Intraoperative Monitoring[END_REF]. Of particular interest for the development of transparent and low impedance MEA electrodesis Poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) which is considered as a very promising candidate. In addition to chemical stability [START_REF] Groenendaal | Poly(3,4ethylenedioxythiophene) and its derivatives: past, present, and future[END_REF] and biocompatibility [START_REF] Kim | Organic electrochemical transistor based immunosensor for prostate specific antigen (PSA) detection using gold nanoparticles for signal amplification[END_REF], PEDOT:PSS displays good optical transmittance (> 90%) and high conductivity (>10 3 S cm -1 ) [START_REF] Kim | High-performance, polymer-based direct cellular interfaces for electrical stimulation and recording[END_REF]. Also, a combination of both electronic and ionic conductivity in the bulk of the material provides an additional degree of freedom to be able to optimize its capacitance (i.e. volumetric capacitance rather than surface capacitance).

PEDOT:PSS has been already used to produce low impedance microelectrodes by coating such materials as gold [START_REF] Pas | Neurospheres on Patterned PEDOT : PSS Microelectrode Arrays Enhance Electrophysiology Recordings[END_REF][START_REF] Koutsouras | PEDOT : PSS microelectrode arrays for hippocampal cell culture electrophysiological recordings[END_REF][START_REF] Sessolo | Easy-to-Fabricate Conducting Polymer Microelectrode Arrays[END_REF][START_REF] Gerwig | PEDOT -CNT composite microelectrodes for recording and electrostimulation applications : fabrication[END_REF][START_REF] Khodagholy | Highly Conformable Conducting Polymer Electrodes for In Vivo Recordings[END_REF] or platinum [START_REF] Ganji | Development and Translation of PEDOT : PSS Microelectrodes for Intraoperative Monitoring[END_REF]-as well as to reduce the impedance of graphene microelectrodes [START_REF] Kshirsagar | Transparent Graphene / PEDOT : PSS Microelectrodes for Electro-and Optophysiology[END_REF]. Whereas in previous publications, PEDOT:PSS was used to improve the recording performance of the opaque as well as transparent microelectrodes, in this work, PEDOT:PSS will be used as the sole conductive element of the electrodes. Although fabrication of flexible PEDOT:PSS based MEAs by means of inkjet-printing [START_REF] Garma | [END_REF] with surface impedance of 1380 MΩ µm 2 at 1 kHz has been demonstrated, the main issues of this process were linked to the electrode dimension (diameter of 300 μm) as well as the resolution and reproducibility of the patterns [START_REF] Garma | [END_REF].

The goal of this work is to develop small, biocompatible, transparent electrodes with low impedance and high transparency. The approach used in this work solves the main challenges of dimension/impedance and transparency/impedance by microfabrication of the microelectrodes from PEDOT:PSS material. For the first time, the fabrication of transparent electrodes made of PEDOT:PSS was demonstrated. A PEDOT:PSS solution is mixed with ethylene glycol, dodecyl benzene sulfonic acid (DBSA) and 1wt.% of (3-Glycidyloxypropyl)trimethoxysilane (GOPS). Ethylene glycol is added to enhance the conductivity of PEDOT:PSS, DBSA helps to adjust the surface tension and GOPS enhances the stability of PEDOT:PSS in aqueous environments [START_REF] Sessolo | Easy-to-Fabricate Conducting Polymer Microelectrode Arrays[END_REF]. From now on, this mixture will be denoted as PEDOT:PSS for simplicity since it is not deposited on either metallic electrodes or other conductive material such as ITO or even don't mixed with carbon nanotubes or graphene.

The electrical characteristics of the electrodes and their robustness/stability were assessed over time by long term electrical impedance measurements. The biocompatibility of the different materials of the MEA device was also validated. Neural cells culture activity was demonstrated with electrodes composed of pure PEDOT:PSS. The work shows that microelectrodes made of PEDOT:PSS are very promising for both electrophysiology and optical characterization (fluorescence)/stimulation (optogenetics) experiments on neurons.

Material and methods

MEA fabrication process

A schematic representation of the microfabrication process of the MEA devices with PEDOT:PSS electrodes is shown in Fig. 1a-f. Briefly, the process involves the patterning of gold contact pads and tracks as well as the patterning of the PEDOT:PSS microelectrodes onto a glass substrate (1 mm thick). Parylene C is used as an insulating layer on the circuit where required. The specific details of the fabrication process are presented in supplementary material.

Results and discussion

The MEA device consists of 59 round recording electrodes and one large reference electrode

Electrical characterization of the MEA devices

MEA require low impedance to achieve a high signal-to-noise ratio. The MEA devices with PEDOT:PSS electrodes were characterized using impedance spectroscopy (KEYSIGHT E4990A Impedance Analyzer). The measurements were performed in presence of phosphate buffer saline (PBS) solution by applying a 100 mV sine wave with the frequency varied from 20 Hz to 1 MHz.

Influence of electrode diameter

Firstly, the influence of the electrode size on the surface impedance was investigated. Fig. 2a compares the impedance spectra measured on the same MEA device with the same thickness of PEDOT:PSS electrodes (200 nm) but with different diameters (10 and 30 µm). By comparing the values of surface impedance at 1 kHz, the typical frequency used for neuronal signals recording, it was observed that electrodes having a larger diameter have a lower surface impedance value--due to expected higher interfacial capacitance [START_REF] Sessolo | Easy-to-Fabricate Conducting Polymer Microelectrode Arrays[END_REF]. The surface impedance values (mean ± standard deviation) at 1 kHz were 35.4 ± 8MΩ µm 2 for electrodes with a diameter of 10 µm and 33 ± 2.6 MΩ µm 2 for electrodes with a diameter of 30 µm, respectively. electrodes of 30 µm in diameter [START_REF] Www | multichannelsystems.com, Microsoft, Microelectrode Array ( MEA ) Manual[END_REF]. The crossing effect of the impedance spectra at around 4 kHz could be mostly attributed to post-measurement normalization of the impedance with area that induces additional error bars (i.e. electrode size accuracy).

Influence of PEDOT:PSS thickness

The influence of the thickness of the PEDOT:PSS electrodes on the surface impedance was investigated. Fig. 2b shows the impedance spectra of PEDOT:PSS electrodes having the same sizebut with different thicknesses. A significant surface impedance decrease for the thicker PEDOT:PSS electrodes was observed due to a corresponding capacitance increase [START_REF] Rivnay | High-performance transistors for bioelectronics through tuning of channel thickness[END_REF] [START_REF] Rivnay | High-performance transistors for bioelectronics through tuning of channel thickness[END_REF]. For example, the surface impedance value at 1 kHz for electrodes having a diameter of 30 µm and a PEDOT:PSS thickness of 100 nm is 95.2 ± 3.7 MΩ µm 2 , whilst the surface impedance value for the PEDOT:PSS layer having a thickness of 200 nm is 33 ± 2.6 MΩ µm 2 , respectively (Fig. 2b). This effect can be explained by the dependence of the capacitance on the thickness. Indeed, an increase of the PEDOT:PSS thickness is correlated with the increase of PEDOT:PSS volume and corresponds to the increase of capacitance, which, as a consequence, leads to a decrease of the surface impedance [START_REF] Rivnay | High-performance transistors for bioelectronics through tuning of channel thickness[END_REF]. A similar dependence of surface impedance to the thickness of the electrode material was already shown by Kshirsagar et al. [START_REF] Kshirsagar | Transparent Graphene / PEDOT : PSS Microelectrodes for Electro-and Optophysiology[END_REF] who demonstrated that increasing the PEDOT:PSS layer thickness (electrodeposited by different time durations) led to a decrease of the impedance value. As a comparison, the impedance of a PEDOT:PSS layer electrodeposited during 0.2 sec is around 700 kΩ whilst those of PEDOT:PSS layers electrodeposited during 10 sec is around 50 kΩ-the thickness of the PEDOT:PSS layer is proportional to the deposition time [START_REF] Kshirsagar | Transparent Graphene / PEDOT : PSS Microelectrodes for Electro-and Optophysiology[END_REF].

The low frequency part of the curves is mostly related to the electrode impedance contribution whereas the high frequency part is mostly related to the electrolyte impedance. Since the measured electrode impedance was normalized to the surface, there is small impedance differences at low frequencies. In the case of thickness, there is no normalization of the thickness-so the impedance differences are very pronounced at low frequencies. Due to the different distances between electrodes and the reference electrode, the electrolyte impedance can vary from electrode to electrode, which leads to the impedance differences at high frequencies. Averaged impedance spectra measured during 3 weeks of all electrodes with a diameter of 10 and 30 µm on the same MEA device with PEDOT:PSS thickness of 200 nm. (d) Surface impedance values at 1 kHz over 22 days for all electrodes (10 and 30 µm of diameter).

Electrode to electrode impedance variability

Variation àof the surface impedance value from electrode to electrode on the same device is crucial to assess to detect the similar extracellular signals in neurophysiology.

Stability of PEDOT:PSS electrode impedance

Next, the stability of PEDOT:PSS MEA devices for long-term experiments was studied. The electrical performance of the electrodes needs to be stable over time because typical neurons' cell cultures start exhibiting synchronized burst events around 3 weeks of DIV culture [START_REF] Koutsouras | PEDOT : PSS microelectrode arrays for hippocampal cell culture electrophysiological recordings[END_REF]. For example, commercial MEAs with TiN electrodes (Multi Channel System) are stable for longterm experiments (up to several weeks or even month) [START_REF] Rivnay | High-performance transistors for bioelectronics through tuning of channel thickness[END_REF]. In order to investigate the stability of our MEA devices, their impedances were measured during several weeks (3 -4 measurements per week) in a PBS solution. Between the measurements, the MEA devices were stored in deionized water in order to avoid an increase of impedance. Following the characterization, the following parameters (30 µm diameter and with the PEDOT:PSS thickness of 200 nm) were finally chosen and used for the fabrication of the MEA devices intended for the recording of the electrical neuronal activity. In biological applications, and notably for neuron activity measurements, data confidence is crucial, and so reliable MEA devices are mandatory. In the following section, we have tested several devices and have investigated the impedance variation from device to device.

Impedance variation from device to device

Fot this part of the study, six MEA devices with electrodes having a diameter of 30 µm and a PEDOT:PSS thickness of 200 nm have been fabricated and characterized. All devices were entirely fabricated separately from each other by using a freshly prepared PEDOT:PSS mixture for spin-coating. The values shown in Fig. 3 were obtained by averaging the impedance values at 1 kHz of all electrodes of each device. The averaging values of surface impedance at 1 kHz are in the range of 24 -35 MΩ µm 2 .

Fig. 3

The device to device reproducibility. Averaging the surface impedance values at 1 kHz of all electrodes with a diameter of 30 µm and the PEDOT:PSS thickness of 200 nm for 6 different MEAs.

The impedance values of PEDOT:PSS electrodes are comparable to the impedance values of state-of-the-art MEA devices (Table 1). Comparison of the impedance values at 1 kHz and transmission values of the electrodes from different materials.

Transmission measurement

In order to combine both electrophysiology measuements and the ablity of optical microscopy observation, we aimed to fabricate both transparent and low impedance electrodes (32.9 ± 2.6

MΩ µm 2 at 1 kHz for 30 µm diameter electrodes). The average transmission at visible wavelengths was 97% and 93% for plain PEDOT:PSS layers deposited by spin-coating on glass slides with thickness of 100 nm and 200 nm, respectively (Fig. S6 in supplementary material).

As a comparison, a transparency of 84% was obtained for graphene/PEDOT:PSS-based MEAbut with higher surface impedance value of 117.3 ± 9.2 MΩ µm 2 at 1 kHz (30 μm diameter) [START_REF] Kshirsagar | Transparent Graphene / PEDOT : PSS Microelectrodes for Electro-and Optophysiology[END_REF] while an 80% transparency was obtained using ITO-based MEAs [START_REF] Kwon | Opto -μECoG Array : A Hybrid Neural Interface with Transparent μECoG Electrode Array and Integrated LEDs for Optogenetics[END_REF] with a surface impedance value of 1250 MΩ µm 2 (80 μm diameter) [START_REF] Ryynänen | Microelectrode Array With Transparent ALD TiN Electrodes[END_REF]. Table 1 and Fig. S7 (supplementary material)

shows impedance values (MΩ µm 2 ) and optical transmissions (%) of our MEA compared to other transparent MEA described in literature and to non-transparent commercial TiN based MEA.

Biocompatibility assessment

The biocompatibility of all MEA device materials intended to be in contact with cells (PDMS, PEDOT:PSS and Parylene) was tested by performing metabolic activity assays that will directly reflect the living cell numbersand cell death assays. Rat cortical cells grown on PDMS, PEDOT:PSS and Parylene on top of PEDOT:PSS were compared to cells grown on glass after 7, 14, 22 and 28 days of culture in vitro (DIV)--results are shown in Fig. 4 and Tables S4 andS5 in supplementary material). The mean metabolic activity of cells grown on PDMS was significantly lower at 14, 22 and 28 DIV-and the cell death significantly increased at 22 and 28 DIV. This indicates a biocompatibility clearly inferior to the biocompatibility of glass.

However, in our final device, as only the walls of the well are made of PDMS, we assumed this would not have a significant impact on the results. The mean metabolic activity of cells grown on PEDOT:PSS was significantly lower at 7 and 14 DIV. It was also lower at 22 and 28 DIV but no significant difference was observed. The cell death was significantly increased only at 7

DIV. This suggests that PEDOT:PSS does not promote cell adherence when compared to glass but, once cells have adhered to it, their viability is quite similar to cells grown on glass. This would explain both the reduced number of living cells observed right after cell seeding and the similar mortality rate compared to control observed at more advanced times. Regarding the possible leaching of toxic additives mixed with the PEDOT:PSS solution (ethylene glycol, DBSA and GOPS), as we did not change the cell culture medium during the entire culture ( 28DIV), we assume the sensitivity of our assay to such leaching is quite high. In that case, even a slight leaching would be expected to impact cell viability at some point through accumulation of these additives in the medium. Yet, we did not observe lower cell viability at advanced time points ruling out any leaching-induced toxicity.

The mean metabolic activity of cells grown on parylene was mildly lower through the 4 weeks of culture but the reduction was only found to be significant at 14 DIV. Moreover, the cell death was never found to be significantly increased. This indicates that parylene biocompatibility is only slightly lower than glass biocompatibility. This slight difference is probably due to a reduced cellular adherence during seeding process rather than to an increased cellular death.

Altogether, these results indicate that our final device will allow the growth of neuronal cells although a slight reduction in the number of living cortical cells could be expected.

Fig. 4

Biocompatibility assessment of materials composing the MEA device. The biocompatibility of PDMS, PEDOT:PSS and parylene was compared to glass biocompatibility through metabolic activity assays (a) and death cell assays (the ratio of cell death on metabolic activity is presented in b). Rat primary cortical cells were seeded on the different substrates and tests were performed after 7, 14, 22 and 28 DIV. Mean values are indicated in black. n=4 biological replicates for each condition. Significant differences between results obtained for cells grown on glass and results obtained for other substrates were examined using the nonparametric Mann-Whitney and Wilcoxon tests (*:p<0.05).

Cell culture on the PEDOT:PSS electrodes

We and others have characterized extensively cortical cell cultures using MEA technology and immunofluorescence stainings [START_REF] Ito | Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks[END_REF][START_REF] Wagenaar | An extremely rich repertoire of bursting patterns during the development of cortical cultures[END_REF]. Typically, our cultures start exhibiting spontaneous globally-synchronized burst events after around 3 weeks of culture (Fig. S9 in supplementary material). Based on these observations, we chose to follow the establishment of functional neural networks from dissociated cortical cells during 4 weeks in order to provide a proof-ofconcept for the fabrication of biocompatible electrodes using PEDOT:PSS. A cortical cell culture was established on a MEA device with PEDOT:PSS electrodes . Spontaneous neuronal cell activity was recorded after 14, 21 and 28 DIV (Fig. 5, culture 1, Tables S6-S8 in supplementary material). As commercial MEAs are reusable, we investigated to see if the PEDOT:PSS MEA could also sustain a standard cleaning method in order to be reused afterwards. After culture 1, the cells and extracellular proteins were washed out from the MEA device using an anionic detergent with protease enzyme. The electrode impedance was checked out and no change was observed (data not shown). We then established a new cortical cell culture and new recordings were performed (Fig. 5, culture 2, Tables S6-S8 in supplementary material). Several parameters (number of active electrodes, spike and burst frequencies, burst duration and number of spikes per burst) were extracted from the two series of recordings and compared to data obtained with control cortical cell cultures established on commercial MEAs with electrodes from TiN (Fig. 5, commercial MEAs n = 3, Tables S6-S8 in supplementary material). After 3 weeks of culture, the majority of the PEDOT:PSS electrodes allow recording neuronal activity (Fig. S9a in supplementary material) that was found comparable to control cultures (Fig. 5). The increased firing rate and the network activity synchronization (Fig. 5e and5f) observed over time show proper maturation of the neural network as expected. However, cultures established on commercial MEAs seem to reach a high level of synchronization sooner (Fig. 5e). One can note that the baseline noise level observed during neuronal activity recording is smaller with the PEDOT:PSS electrodes (around 9 µV) when compared to TiN electrodes from commercial MEAs (around 15 µV) (Fig. S9b in supplementary material) suggesting a higher signal-to-noise ratio and therefore a better ability to detect extracellular activity. The

MEA device with PEDOT:PSS electrodes detects lower intensity signals than commercial

MEAs and this for every stage assayed (Tables S6-S8 in supplementary material). Further experiments are needed to determine if the apparent delayed in synchronization we observed (Fig. 5e) is related to a wider range of detection and therefore to improved technical performances (depending of the algorithm and method selected to quantify activity correlation) rather than to a biological cause (i.e. differences in the neural network maturation rate). Usually, synchronised burst events correspond to high intensity signals, so signals easily detectable.

Depending of the network structure and positioning of the electrodes, non-synchronised events correspond partially to high intensity signals and partially to lower intensity signals and therefore are partially easily detectable, partially less easily detectable. Lowering the detection threshold will not modify the occurrence of synchronised events per se but depending on the network structure and positioning of the electrodes, this can allow increasing the detection of non-synchronised events and therefore decreasing the proportion of synchronised events.

Alternatively, although similar numbers of cortical cells had been seeded on the different types of MEAs, we cannot rule out that a slightly lower number of cells adhered to MEA device with PEDOT:PSS electrodes as suggested by our biocompatibility study. As cell density impacts network maturation [START_REF] Wagenaar | An extremely rich repertoire of bursting patterns during the development of cortical cultures[END_REF], this could be also explained by the synchronization delay. In that case, a slight increase in cell concentration at the seeding step would solve the problem. Both explanations are not mutually exclusive.

To image live neurons on top of the PEDOT:PSS electrodes, we used a fluorescent probe that selectively labels living neurons. After one month of culture, fluorescent-positive living neurons were perfectly visible on top of the electrodes and everywhere on the recording area (Fig. 5).

This, in combination with the neuronal activity recordings, provides proof-of-concept for the fabrication of a biocompatible MEA with totally transparent electrodes made from PEDOT:PSS. Live-imaging of neurons on a MEA with totally transparent electrodes from PEDOT:PSS (i, j) and neurons on a commercial MEA that display opaque electrodes (made of titanium nitride) preventing us to image cells on top of them (k). Note that for MEA made with transparent electrodes from PEDOT:PSS, the connectors are opaque as they are made of titanium/gold. Scale bars, 20 µm (j, k) and 50 µm (i).

Conclusion

Our findings clearly demonstrate the viability of MEA technology for neuronal studies based on PEDOT:PSS microelectrodes. The devices were electrically characterized and showed high optical transmission (as high as 97%) and low electrical impedance values (33 MΩ µm 2 )comparable to the impedance of commercially available opaque MEA devices). The biocompatibility of PEDOT:PSS was also investigated and successfully validated, meaning that our approach is suitable for measuring signals from neurons. In addition, we have demonstrated that our systems were able to sustain neuron networks growing with maturation and synchronization after 4 weeks-and able to perform neuronal activity recording with a lower baseline noise compared to TiN-based commercial MEA. We were also able to live-image fluorescent-labelled neurons located on top of our PEDOT:PSS electrodes. Although MEA systems enable the extracellular recording of large populations of neuronal (or any other excitable or neuron-like cells) for weeks and even months; these recording could be less informative compared to intracellular ones. As most of MEA systems are composed by nontransparent electrodes, this prevents optical characterization of cells located on top of them, thus limiting the amount of additional information that can be obtained. For example, does the absence of signal from a given electrode come from absence of neurons, of living neurons, lack of neuronal activity or no neuronal activity triggering spike generation? Using our device, we showed that we could determine how many living neurons are present on top of the electrodes.

In addition, other dyes sensitive to specific parameters (calcium concentration, reactive oxygen species, etc) can also be used to specifically assay the physiological state of the recorded cells.

Moreover, a very important issue when working on neuronal networks is their complexity and potential cellular heterogeneity: this can be intrinsic to the model (for example cortical cells encompass different types of neurons) or induced by the experimenter to model a physiological or pathological situation. The use of fluorescent reporter genes (for example, genes specific to a type of neurons or other fluorescent markers can allow visualizing such heterogeneity. Thus, performing extracellular recording combined to a well-defined cell population is crucial to conclude on the role of specific cells' status regarding the global network activity. Another advantage of our device is the possibility to perform optomodulation experiments (activation/inactivation of some functions) of our neuron network including both optogenetic and photochemical modulation (photoswitchable, photocleavable or drug release). Indeed, development of optomodulation tools in neuroscience are still growing, leading to device and probes able to perform simultaneously optical modulation and monitoring of neural activity.

One of the very promising tool is, for instance, the genetically encoded voltage indicators (GEVI) system that has shown higher performances in terms of voltage detection sensibility over classical methods.

Fig. 1g presents

  Fig.1gpresents a photograph of a fabricated MEA device complete with a cell culture chamber.

Fig. 1h show

  Fig. 1h show a photograph (plus zoom) of the PEDOT:PSS electrodes, the openings in the Parylene, and the gold contact lines.

Fig. 1

 1 Fig. 1 Schematic representation of the fabrication process of the MEA devices with electrodes from pure PEDOT:PSS (a) patterning of the gold contact pads and interconnects onto a glass wafer via Ebeam lithography and lift-off (b) spincoating of an anti-adhesive layer, deposition of 2 µm thick parylene C layer, spin-coating of the photoresist and openings of the microelectrodes by 2 nd Ebeam lithography followed by oxygen plasma etching (c) removal of the photoresist and spin-coating of the PEDOT:PSS solution (d) peeling-off the parylene C sacrificial layer (e) deposition of the second 2 µm thick parylene C layer, spin-coating of the photoresist and openings of the contact pads and electrodes by 3 rd Ebeam lithography step and oxygen plasma etching (f) removal of the photoresist (g) A photograph of a fabricated MEA device with gold contact pads and tracks, PEDOT:PSS microelectrodes in the center and the cell culture chamber made of PDMS on top. h) Microscopic image of the PEDOT:PSS microelectrodes. The thickness of spin-coated PEDOT:PSS layer is 200 nm.

(

  740 µm in diameter) (Fig S1 in supplementary material). The dimensions of the device (49 × 49 × 1 mm 3 ), the configuration of the contact pads, and of the internal reference electrode are compatible with commercial MEA-System (MEA2100-System, Headstage for 2 MEAs with 60 electrodes, Multi Channel Systems). The surface of the patterned microelectrodes was characterized by means of atomic force microscopy (AFM) as well as scanning electron microscopy (SEM) (Fig. S2 in supplementary material).

  The values were obtained by averaging the impedance spectra of 20 electrodes for each diameter. The similar dependence of the impedance on the electrode size was demonstrated for the commercial MEA devices. As a comparison, the surface impedance of TiN electrodes ranges between 21.2 -35.3 MΩ µm 2 for electrodes of 10 µm in diameter and 19.6 -31.4 MΩ µm 2 for

Fig. 2

 2 Fig. 2 (a) Influence of electrode diameter on surface impedance. The impedance spectra measured for PEDOT:PSS electrodes (200 nm thick) for different electrode sizes (10 and 30 µm). (b) Influence of PEDOT:PSS thickness on surface impedance. The impedance spectra measured for PEDOT:PSS electrodes (30 µm in diameter) for different thicknesses (100 and 200 nm). Long-term stability measurement done in PBS solution over 3 weeks. (c)

Fig. 2c shows

  Fig.2cshows the impedance spectra obtained by averaging several impedance spectra measured in a PBS solution during 3 weeks for electrodes having a diameter of 10 and 30 µm on the same device with a PEDOT:PSS thickness of 200 nm. The surface impedance values (mean ± standard deviation) at 1 kHz were 37.6 ± 6.4 MΩ µm 2 and 37.1 ± 3.3 MΩ µm 2 for electrodes with a diameter of 10 and 30 µm, respectively.

Fig. 2d presents

  Fig. 2d presents the evolution over time of the impedance values at 1 kHz for electrodes with a diameter of 10 and 30 µm. The first impedance measurement was performed immediately after fabrication of the device. The next measurement of the surface impedance was performed on the next day after storing overnight the device in deionized water. The increase of the surface impedance can be explained by a stabilization of PEDOT:PSS and by the removal of any excess

Fig. 5

 5 Fig. 5 Neuronal activity characterization and live-imaging of rat cortical cells cultured on a MEA with electrodes made from PEDOT:PSS compared to commercial MEAs with electrodes made of titanium nitride. Spontaneous neuronal activity was recorded after 14, 21 and 28 DIV. For commercial MEAs, n = 3 MEAs. The mean spike frequency (a), the mean number of bursts per minute (b), the mean burst duration in ms (c) the mean number of spikes per burst (d) and the results

  The values indicate a small variation from electrode to electrode on the same device and thus validate the good quality of the fabrication process. Moreover, the slight variation ofsurface impedance values from electrode to electrode are comparable to those from electrodes made of other materials. Aa a comparison, the surface impedance values (mean ± standard deviation) at 1 kHz of 24 electrodes with a diameter of 30 µm 37 ± 3.3 MΩ µm 2 , 14 ± 1.3 MΩ µm 2 and 11.5 ± 0.7 MΩ µm 2 , made of TiN, gold coated with PEDOT and gold coated with PEDOT-CNT, respectively[START_REF] Gerwig | PEDOT -CNT composite microelectrodes for recording and electrostimulation applications : fabrication[END_REF].

	The surface
	impedance values (mean ± standard deviation) at 1 kHz were 35.4 ± 8MΩ µm 2 for electrodes
	with a diameter of 10 µm and 33 ± 2.6 MΩ µm 2 for electrodes with a diameter of 30 µm,
	respectively.

Electrode dimension Electrode material |Z| [kΩ] at 1 kHz |Z| [MΩ µm 2 ] at 1 kHz Transmission [%]

	d = 30 µm	TiN	< 100	21.2 -35.3	Opaque	Multi Channel
						Systems
	d = 10 µm	TiN	250 -400	19.6 -31.4	Opaque	Multi Channel
						Systems
	d = 30 µm	ALD TiN	510 -590	360-390	18 -45	[9]
	d = 30 µm	Transparent TiN	< 250	177		Multi Channel
						Systems
	d = 200 µm	graphene	243 ± 6	7650 ± 3	90	[14]
	d = 30 µm	Graphene/	166 ± 13	117 ± 9	84 ± 4	[3]
		PEDOT:PSS
	d = 80 µm	ITO	250	1250	80	[15]
	d = 30 µm	PEDOT:PSS	46.5 ± 3.7	32.9 ± 2.6	93	this work
		(200 nm)
	d = 10 µm	PEDOT:PSS	433.2 ± 58.5	35.4 ± 8	93	this work
		(200 nm)
						Reference
	10 × 10 µm 2	Ti/Au	35	3.5	Opaque	[1]
		+PEDOT:PSS
		(350 nm)
	d = 50 µm	Ti/Au	20	39	Opaque	[17]
		+PEDOT:PSS
	20 × 20 µm 2	Ti/Au	23	9.2	Opaque	[5]
		+PEDOT:PSS
		(380 nm)
	d = 30 µm	Au +PEDOT	20.0 ± 1.9	14.1 ± 1.3	Opaque	[4]
		(247 ± 33 nm)
		Au +PEDOT-CNT	16.2 ± 1.1	11.4 ± 0.8
		(352 ± 27 nm)

Table 1

 1

Acknowledgements

We acknowledge the Labex DistalZ project (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease), the EU: ERC-2017-COG project IONOS (# GA 773228), the University of Lille, CNRS and PEpS project for financial support. This work was also partly supported by the French RENATECH network (French national nanofabrication platform).