WO2013017770A1 - Device for culturing nerve cells, and uses thereof - Google Patents

Abstract

The present invention relates to a device for culturing nerve cells on substrates, consisting of micrometric islands covered with neural cell adhesion molecules, on an evenly cytophobic base. Said system is coupled with a method for the qualitative and/or quantitative analysis of neurite growth and the synaptogenesis of said nerve cells. The invention further relates to a method for screening for compounds involved in said processes.

Description

 DEVICE FOR CULTURING NEURONAL CELLS AND

 USES

DESCRIPTION

Technical area

 The present invention relates to a support (1) for culturing neuronal cells, to the use of this support for cell culture, in screening methods and in qualitative and / or quantitative methods of analyzing growth. neuronal and synaptogenesis. The present invention also relates to a method of manufacturing said support.

 The present invention finds application, particularly in the field of research and pharmacy.

 In the description below, references in brackets ([]) refer to the list of references at the end of the text.

State of the art

 Methods for culturing living cells on substrates with micro-motifs are not new. By precise geometric control of the adhesive zones determining the possibilities of cell extension, several studies have revealed that the shape of cells in culture considerably affects cellular proliferation, differentiation and polarity (Folkman, J. & Moscona, A. Rôle of cell shape in growth control Nature 273, 345-9 (1978) [1] Watt, FM,

Jordan, P.W. & O'Neill, C.H. Cell shape controls terminal differentiation of human epidermal keratinocytes. Proc Natl Acad Sci U S A 85, 5576-80 (1988). [2] Thery, M., Racine, V., Pepin, A., Piel, M., Chen, Y., Sibarita, J. B. & Bornens, M. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol 7, 947-53 (2005). [3] Thery, M., Racine, V., Piel,

M., Pepin, A., Dimitrov, A., Chen, Y., Sibarita, JB & Bornens, M. Anisotropy of cell adhesive microenvironment governs the internai organization and orientation of polarity. Proc Natl Acad Sci USA 103, 19771 -6 (2006) [4]).

The general approach is to delineate adhesive areas in an environment covered with a release agent (cytotropic), on which the cell can not adhere, thus confining cell extension. One of the first methods described relates to a device comprising circular adhesive islands 400 to 2000 μιτι ² in diameter made by evaporation of palladium through a mask on an underlying non-adhesive surface (Ireland, GW, Dopping-Hepenstal, PJ, Jordan, PW &O'Neill, CH Limitation of Substrate Size Cytoskeletal Organisms and Behavior of Swiss 3T3 Fibroblasts Cell Biol Int Rep 13, 781-90 (1989) [5]). Current simpler techniques, such as inkjet printing methods, include depositing microscopic droplets of polylysine-type adhesive onto a non-adhesive substrate such as a polyethylene glycol coating, but the resolution is low, ie of the order of 100-200 μιτι (Sanjana, NE & Fuller, SB A fast flexible ink-jet printing method for neurons in culture dissociated patterning J Neurosci Methods 136, 151 -63 (2004). [6] Yamagata, M., Weiner, JA, Dulac, C, Roth, KA & Sanes, JR Labeled lines in the retinotectal System: markers for retinorecipient sublaminae and the retinal ganglion cell subsets that innervate them Mol Cell Neurosci 33, 296-310 (2006) [7]).

To define smaller microstructures, microcontact printing of self-assembled alkanethiolate monolayers on a metal substrate was used (Chen, CS, Mrksich, M., Huang, S., Whitesides, GM & Ingber). , DE Micropatterned surfaces for control of cell shape, position, and function, Biotechnol Prog 14, 356-63 (1998) [8]). Other micro-lithographic solutions allowing the printing of adhesion proteins on silanized glass coverslips have been developed, using micro-manufactured elastomeric pads from a chrome mask (Rozkiewicz, DI, Kraan, Y., Werten, MW, Wolf, FA, Subramaniam, V., Ravoo, BJ & Reinhoudt, DN Covalent microcontact printing of proteins for cell patterning Chemistry 12, 6290-7 (2006). [9], Shi, P., Shen, K. & Kam, LC Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro Dev Neurobiol 67, 1765-76 (2007) [10], Ladoux, B., Anon, E., Lambert, M., Rabodzey, A., Hersen, P., Buguin, A., Silberzan, P. & Mege, RM Strength dependence of cadherin-mediated adhesions Biophys J 98, 534-42 (2010). [1 1]), or a direct printing of the glass substrate or PDMS by deep UV rays going to through a chromium mask (Azioune, A., Carpi, N., Tseng, Q., Thery, M. & Piel, M. Protein micropatterns: A direct printing protocol using deep UVs., Cell Biol 97, 133-46 ( 2010). [12], Azioune, A., Carpi, N., Fink, J ., Chehimi, MM, Cuvelier, D. & Piel, M. Robust method for high-throughput surface patterning of deformable substrates. Langmuir 27, 7349-52 (2010). [13]). Another recently implemented technique consists in carrying out a covalent coupling of adhesion proteins directly on a surface via a UV-activated crosslinking chemical agent (Fink, J., Thery, M., Azioune, A., Dupont , R., Chatelain, F., Bornens, M. & Piel, M. Comparative study and improvement of current cell micro-patterning techniques, Lab Chip 7, 672-80 (2007) [14]). A very recent approach called micro-photo-printing, using a computer-controlled bi-photon laser to perform micrometric photo-ablation of a poly-vinyl-alcohol release film, was considered ( Doyle, AD, Wang, FW, Matsumoto, K. & Yamada, KM One-dimensional topography underlies three-dimensional fibrillar cell migration J Cell Biol 184, 481-90 (2009). [15]) to achieve many geometries different. However, these methods are difficult to implement, and involve expertise that often limits their use to a small number of basic research laboratories.

In the field of cellular neurobiology, fairly large adhesive islands (50-100 μιτι) were used to individually isolate primary neurons in culture and to generate synapses formed by the neuron on itself (autapses) (Allen , TG Preparation and maintenance of single-cell micro-island cultures of basal forebrain neurons Nat Protoc, 2543-50 (2006). [16], Wilson, NR, Ty, MT, Ingber, DE, On, M. & Liu , G. Synaptic Reorganization in Scaled Networks of Controlled J Neurosci 27, 13581-9 (2007) [17]). Micro-structured systems comprising parallel adhesive lines separated by non-adhesive grooves have also been used to promote directional growth of axons (Yamagata, M., Weiner, JA, Dulac, C., Roth, KA & Sanes, JR Labeled). in the retinotectal system: markers for retinorecipient sublaminae and the retinal ganglion cell subsets that innervate them Mol Cell Neurosci 33, 296-310 (2006). [7], Wilson, NR, Ty, MT, Ingber, DE, On, M. & Liu, G. Synaptic Reorganization in Scaled Networks of Controlled J Neurosci 27, 13581 -9 (2007). [17], Ruardij, TG, Goedbloed, MH & Rutten, WL Adhesion and Patterning of Cortical Neurons on Polyethylenimine and fluorocarbon-coated surfaces IEEE Trans Biomed Eng 47, 1593-9 (2000) [18]). A recent article has used a micro-contact printing technique based on an atomic force microscope to create adhesive zones of 7-10 μιτι spaced 10-20 m apart and to deposit proteins from the extracellular matrix (fibronectin or laminin) for differentially controlling the growth of neurons (Fereol, S., Fodil, R., Barnat, M., Georget, V., Milbreta, U. & Nothias, F. Micropatterned ECM substrates reveal complementary contribution of low and high affinity ligands to neurite outgrowth Cytoskeleton (Hoboken) (201 1) [19]). Another group used the micro-contact printing method to make rectangular islands of 2- 5 μηη separated by 5-10 μητι, and deposited ephrin neuronal proteins involved in axon guidance and reorientation of the growth cone (von Philipsborn, A. C, Lang, S., Bernard, A., Loeschinger, J. , David, C., Lehnert, D., Bastmeyer, M. & Bonhoeffer, F. Microcontact printing of axon guidance molecules for generation of graded patterns Nat Protoc 1, 1322-8 (2006). [20], von Philipsborn, A C, Lang, S., Loeschinger, J., Bernard, A., David, C., Lehnert, D., Bonhoeffer, F. & Bastmeyer, M. Growth cone navigation in substrate-bound ephrin gradients, Development 133, 2487 -95 (2006) [21]).

 Other approaches have also been used, micro fluidic devices associated with a culture substrate for separating two populations of different neurons, directing axonal growth along pre-determined lines and establishing synaptic contacts between the two types. of neurons (Taylor, AM, Blurton-Jones, M., Rhee, SW, Cribbs, DH, Cotman, CW & Jeon, NL A microfluidic culture platform for CNS axonal injury, regeneration and transport, Nat Methods 2, 599-605 ( 2005). [22], Paul, D., Saias, L., Pedinotti, J. C, Chabert, M., Magnifico, S., Pallandre, A., De Lambert, B., Houdayer, C, Brugg, B., Peyrin, JM & Viovy, JL A "dry and wet hybrid" lithography technique for multilevel replication templates: Biomicrofluidics 5, 24102 (201 1) [23]). This system is marketed by the company Millipore (AXIS device, ref AX15010). An improvement of this initial system with a central perpendicular microfluidic channel allows stimulations with chemical agents capable of generating a physiological response of the neurons (Taylor, AM & Jeon, NL Micro-scale and microfluidic devices for neurobiology.) Curr Opin Neurobiol 20, 640-7 (2010). [24]).

However, these methods are difficult to implement and do not make it possible to create localized adhesive contacts induced by specifically neuronal adhesion proteins, for example immunoglobulin-like adhesion molecules (IgCAM) and N-cadherin, which plays a critical role in growth cone migration and axon guidance (Bard, L, Boscher, C, Lambert, M., Mege, RM, Choquet, D. & Thoumine, O. A molecular clutch between the actin flow and N-cadherin adhesion drives growth cone migration.J Neurosci 28, 5879-90 (2008). [25], Falk, J., Thoumine, O., Dequidt, C, Choquet, D. & Faivre- Sarrailh, C. NrCAM coupling to the cytoskeleton is dependent on multiple protein domains and partitioning into lipid rafts, Mol Biol Cell 15, 4695-709 (2004) [26]). This is a limitation all the stronger as regards the study of the formation of specialized contacts allowing the communication between neurons, the "synapses", which constitutes a fundamental question of neurobiology. Synaptogenesis is a complex and multi-stage process at the level of axon / dendrite contacts, initiated by adhesion proteins and followed by recruitment of scaffold molecules and functional channel receptors (Bresler, T., Ramati, Y. , Zamorano, PL, Zhai, R., Garner, CC & Ziv, NE The Dynamics of SAP90 / PSD-95 Recruitment to New Synaptic Junctions Mol Cell Neurosci 18, 149-67 (2001). [27], Bresler, T. ., Shapira, M., Boeckers, T., Dresbach, T., Futter, M., Garner, C., Rosenblum, K., Gundelfinger, ED & Ziv, NE Postsynaptic density assembly is fundamentally different from presynaptic active zone J Neurosci 24, 1507-20 (2004). [28], Friedman, HV, Bresler, T., Garner, CC & Ziv, NE Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57-69 (2000) [29], Zito, K., Scheuss, V., Knott, G., Hill, T. & Svoboda, K. Rapid functional maturation of nascent dendritic spines Neuron 61, 247 -58 (2009) [30]). Recent studies have shown that the neurexin / neuroligin adhesion complex and the LRRTM proteins play a role in synaptic assembly (Gerrow, K., Romorini, S., Nabi, SM, Colicos, MA, Sala, C & El-Husseini, A. Neuron 49, 547-62 (2006). [31], Graf, A preformed complex of postsynaptic proteins is involved in excitatory synapse development. ER, Kang, Y., Hauner, AM & Craig, AM Functional and structural analysis of the synaptogenic activity of the neurexin-1 beta LNS domain. J Neurosci 26, 4256-65 (2006). [32], Graf, ER, Zhang, X., Jin, SX, Linhoff, MW & Craig, AM Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 19, 1013-26 (2004). [33], Heine, M., Thoumine, O., Mondin, M., Tessier, B., Giannone, G. & Choquet, D. Activity-independent and subunit-specific recruitment of functional AMPA receptors at neurexin / neuroligin contacts . Proc Natl Acad Sci USA 105, 20947-52 (2008). [34], Scheiffele, P., Fan, J., Choih, J., Fetter, R. & Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 101, 657-69 (2000). [35], Sudhof, TC Neuroligins and Neural Link Synaptic Function to Cognitive Disease. Nature 455, 903-1 (2008). [36], Ko, J., Zhang, C, Arac, D., Boucard, AA, Brunger, AT & Sudhof, TC Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation. Embo J 28, 3244-55 (2009). [37]) Linhoff, MW, Lauren, J., Cassidy, RM, Dobie, FA, Takahashi, H., Nygaard, HB, Airaksinen, MS, Strittmatter, SM & Craig, AM An unbiased expression screen for synaptogenic proteins identified LRRTM protein family as synaptic organizers. Neuron 61, 734-49 (2009). [38], Ko, J., Fuccillo, MV, Malenka, RC & Sudhof, TC LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Training. Neuron 64, 791-798 (2009). [39], Wit, J., Sylwestrak, E., O'Sullivan, M., Otto, S., Tiglio, K., Savas, JN, Yates, JR, Comoletti, D., Taylor, P. & Ghosh, A. LRRTM2 Interacts with Neurexin 1 and Regulatory Excitatory Synapse

Training. Neuron 64, 799-806 (2009). [40]).

In addition, these and other studies using co-culture systems between primary neurons and heterologous cells expressing neuronal adhesion proteins as well as over-expression or under-expression of these proteins in neurons , have shown that neuroexin / neuroligin heterophilic adhesion and neurexin / LRRTMs, as well as homophilic adhesion complexes between synCAMs played an important role in synaptic assembly (Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X. , Kavalali, ET, Sudhof, TC SynCAM, synaptic adhesion molecule that drives synapse assembly, Science 297 1525-31 (2002) [68]; Sara, Y., Biederer, T., Atasoy, D., Chubykin, A. , Mozhayeva, MG, Sudhof, T. C, Kavalali, and Selective Capability of SynCAM and neuroligin for functional synapse assembly, J Neuroscience 25 260-270 (2005) [69].

 However, if they make it possible to identify the important synaptogenic molecules, these co-culture systems do not allow a precise spatial control of the formation of synapses.

 Furthermore, a technique using non-neuronal neuroligin-1-expressing cells isolated in micro-wells was used to stimulate pre-synaptic differentiation in contact-forming axons (Shi, P., Scott MA, Ghosh B., Wan D., Wissner-Gross Z., Mazitschek R., Haggarty SJ, Fatih M. Synapse microarray identification of small molecules that enhance synaptogenesis Nat Nat 2, 510 (201 1) [70]), but these micro -wells have diameters of 30 μιτι, far from the physiological size of real synapses, of the order of 1 μιτι, and the hemi-synapses are always randomly formed inside these wells.

Because spontaneous neuronal contacts are formed in a very unpredictable way in time and space, and involve many adhesion molecules in unknown stoichiometric quantities, the study of synaptogenesis is a difficult task. These limitations require the development of new systems that allow robust, targeted and selective induction of synapse formation, accompanied by significant statistical data production. The culturing of neurons on micro-structured substrates covered with neuronal adhesion molecules could answer all these questions. However, one difficulty is that neurons, strongly polarized cells by nature, can have very different reactions to polarization patterns, depending on the geometry of the zones of adhesion and non-adhesion as well as depending on the molecules used. for adhesion and non-adhesion. For example, neurons may not differentiate properly in response to strong geometric limitations imposed by the adhesive microenvironment, not being able to extend their arborization of axons and dendrites. Hence the need to find the optimal geometry and coating conditions to grow primary neurons on these substrates, trying to stay as close as possible to their physiological environment.

 There is therefore a real need to find a device, a method to overcome these defects, disadvantages and obstacles of the prior art, in particular a device for culturing neuronal cells while retaining all of their properties, a device for For example, neuronal cells can be cultured to analyze the physiological properties of neuronal cells, including their axonal and dendritic development as well as the formation of synapses, and to test the effect of compounds on these processes. In addition, there is a real need to find a simple device that can be easily implemented and thus reducing costs and improve the culture of neuronal cells and their uses. Description of the invention

The present invention is specifically intended to meet these needs by providing a support (1) for culturing neuronal cells (c) comprising a substrate (3) on a surface (5) of which islet islands (7) of adhesion of said neuronal cells, said islands (7) having a diameter of 100 nm to 3 microns and being spaced between them from 1 to 10 microns. The present invention also relates to the use of the support (1) for culturing neuronal cells, in a method of quantitative and / or qualitative analysis of neuronal cells and in a screening method.

 In the present invention, neuronal cells (c) are understood to mean any cells originating from the nervous system of a mammal, bird, batrachian, or mollusc, and / or any cell derived from a neuronal cell line. It may be, for example, neuronal cells isolated from the nervous system, for example from the brain or spinal cord, rodents, for example from rats or mice, chickens, amphibians, for example Xenopus, molluscs such as Aplysia, embryonic or newborn stages, which may be located in the hippocampus, cortex, striatum, dorsal root ganglia (DRG). They may also be cells selected from the SH-SY5Y neuroblastoma line, the PC12 line, the cortical neuronal cell line (HCN-1), or the B104 neuroblastoma line.

 According to the invention, the support (1) can be of any form known to those skilled in the art, it can be for example square, rectangular or circular.

 According to the invention, the substrate (3) can be any solid substrate known to those skilled in the art. It may be for example a transparent or opaque substrate. It may be a substrate made of glass, quartz, silicon or a material of the polymer type, for example plastics, for example polycarbonate, polystyrene, polyethylene or PTFE, covered or not with a thin layer of another polymeric metal material, for example gold or metal oxides such as S102, ΤΊΟ2, NTO "indium tin oxide", or crosslinked gels, for example polydimethylsiloxane (PDMS) or polyacrylamide.

According to the invention, the thickness of the substrate (3) may be from 0.1 mm to 3 mm, from 0.1 mm to 0.290 mm, from 0.120 mm to 0.250 mm, from 0.150 mm to 0.170 mm. Advantageously, when the thickness of the substrate is between 150 μιτι and 170 μιτι, it can be used directly in an optical reading device, for example an epifluorescence microscope using a high numerical aperture oil immersion objective, allowing the illumination in total internal reflection (evanescent waves) and thus the visualization of the adhesive contact with a depth of field of a hundred nanometers.

 According to the invention the surface (5) can be any surface known to those skilled in the art on which molecules, for example chemical substances and / or proteins can be arranged. This may be for example a flat surface, a rough surface, a surface comprising depressions, for example wells or channels.

 According to the invention, the islands (7) can be arranged on the surface in such a way that they form a network, for example a hexagonal or square network. According to the invention, the islands can be located equidistant from each other or in variable intervals.

 According to the invention, the surface between the islands (7) can be a cytophobic surface (9). According to the invention, the surface can be made cytophobic because of the presence of a compound chosen from polyethylene glycol, polyethylene oxide, polyvinyl acetate, poly (2-hydroxyethyl methacrylate, polyacrylamide, poly (N-vinyl-2-pyrrolidone), poly (N-isopropyl acrylamide), silicones, for example polydimethylsiloxane (PDMS), silanes, for example perfluorinated silanes, anionic polymers, phosphoryl choline polymers, albumin, casein, hyaluronic acid, liposaccharides, glycoproteins, phospholipids or a combination thereof.

According to the invention, the application of the cytophobic compounds can be carried out by any method known to those skilled in the art, for example by liquid phase deposition, spin coating, dipping, spraying, or chemical vapor deposition, for example by plasma or controlled atmosphere or a combination of both. For example, it may be The method described in the following document Bhushan, Bharat Hansford, Derek Lee, Kang Kug "Surface modification of silicon and polydimethylsiloxane surfaces with vapor-phase-deposited ultrathin fluorosilane films for biomedical nanodevices", Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films, Jul 2006, 24 (4), 1 197 - 1202 [63].

 According to the invention, "adhesion proteins" means an adhesion protein derived from a neuronal cell membrane or a physiological membrane supporting a neuronal cell derived from non-neuronal cells, for example non-neuronal cells. neurons selected from glial cells, astrocytes, oligodendrocytes, ependymocytes, satellite cells, Schwann cells, microglial cells, or embryonic stem cells of said cells. It may be, for example, a membrane adhesion protein of neuronal cells, for example immunoglobulin-like adhesion molecules (IgCAM), for example L1 molecules, neural-type adhesion molecules (NrCAM). or transaxonal glycoprotein 1 (TAG-1), cadherins, for example N-cadherin, and proto-cadherins, neurexins, for example neurexins 1, 2 or 3, neurexins of the α or β forms, neurexins with or without alternative splicing sites in position 4, neuroligins, for example neuroligins 1, 2, 3, or 4, with or without an alternative splicing site at the A or B positions, and the leucine-rich domain transmembrane proteins ( "Leucine-Rich Repeat Transmembrane Protein") eg LRRTM 1, 2, 3 or 4.

 The adhesion proteins may also be, for example, Synaptic Cell Adhesion Molecule (SynCAM) adhesion molecules 1, 2, 3, or 4.

It may be a recombinant protein, for example a protein consisting of an N-terminal extracellular domain of adhesion proteins Membranes of neuronal cells, for example the aforementioned proteins, fused, for example, in its C-terminal part to a label.

 According to the invention, when the adhesion protein is a recombinant protein comprising a C-terminal label, the label may be chosen from the following labels: the green fluorescent protein ("green fluorescent protein" (EGFP)), the protein red fluorescent protein (RFP), polyhistidine sequences (6-10 His), haemagglutinin label (HA), myc tag, Glutathione S-transferase (GST), or a constant fragment of antibody (Fc).

 The recombinant proteins can be obtained by any method known to those skilled in the art. It may be, for example, the methods described in the document Isabelle Collin cvc, Genetic engineering, "transgenic animals", edition Essentials milans, 1999 [64], the Handbook of Biochemistry and Molecular Biology, Fourth Edition 201 1 - Editor (s): Roger L. Lundblad, Lundblad Biotechnology, Chapel Hill, North Carolina, USA; Fiona Macdonald, CRC Press, Boca Raton, Florida, USA [65], or the Practical Handbook of Biochemistry and Molecular Biology Gerald D. Fasman CRC Press, 1989 - 601 pages [66]; or the Recombinant DNA Principles and Methodologies, Editor (s): James Greene, Catholic University, Washington DC, USA CRC Press, 1998 [67].

 Preferably, the adhesion protein is chosen from neurexins, neuroligins, IgCAMs, N-cadherin, synCAMs, Leucine-rich domain transmembrane proteins ("Leucine-Rich Repeat Transmembrane" (LRRTM)) or a mixture of these. Preferably, the adhesion protein is selected from neurexins, neuroligins, N-cadherin and / or a mixture thereof.

 Advantageously, according to the invention, the adhesion protein used in the device of the invention induces growth of axons and / or dendrites and / or synaptogenesis.

According to the invention, the adhesion protein can be attached directly to the surface (5) or via an intermediate binder attached to said surface (5). According to the invention, the direct attachment of the adhesion protein on the surface (5) can be carried out by any method known to those skilled in the art, for example by adsorption, by chemical, electrochemical or thermal grafting.

 According to the invention, the intermediate binder may be chosen from a reactive chemical species which makes it possible to establish a covalent bond, for example benzophenone, an acidic, amine or thiol function, silanes, for example trichlorosilane alkyls, and / or non-covalent bond, for example nickel-nitrile triacetic acid (NTA), a protein, for example biotin, streptavidin, glutathione, an antibody for example anti-Fc, anti-GFP, anti-RFP, anti-HA, anti -myc, anti-His, anti-GST, a charged species for example poly-D-lysine or poly-ornithine, a polysaccharide.

 According to the invention, the fixing of the binder on the surface (5) can be carried out by any method known to those skilled in the art, for example by soaking the surface in a solution comprising said binder. For example, when the binder is an antibody, the fixing method may be for example a method comprising a step of soaking the surface (5) in an antibody solution and rinsing in a liquid medium.

 According to the invention, the application of the cytophobic compounds can be carried out by any method known to those skilled in the art, for example by deposition in the liquid phase, for example by spinning, dipping, spraying, or chemical vapor deposition, by example by plasma or controlled atmosphere) or a combination of both.

 Advantageously, when the binder is polylysine, it allows optimal attachment of neurons and extension of neurites.

Advantageously, when the adhesion protein is a recombinant protein, it can interact with the surface (5) and / or with a binder fixed on the surface (5) thus making it possible to increase the amount of adhesion proteins fixed . For example, when the binder is an antibody, it may be specific for the adhesion protein. For example, when the protein is a recombinant protein as defined above, the antibody may be specific for the label of said recombinant protein.

 Advantageously, when the binder is an antibody, its site of interaction with the adhesive protein may be such as to allow the ectodomain of the adhesive protein to be oriented in such a way that the reactive part is available for cell adhesion. .

 According to the invention, the islands (7) can be deposited on the surface (5) by a method chosen from photolithography, dip-pen nanolithography, beam lithography, ink jet printing, printing. by microswitch.

 According to the invention, the islands (7) have a diameter of 0.2 to 2.5 μιτι, of 0.2 to 2 μηη, of 0.3 to 150 μηη.

 According to the invention, the spacing between the islands is 0.5 to 10 μιτι, 1 to 9 μιτι, 3 to 7 μιτι.

 Advantageously, the diameter of the islets makes it possible to fix the neuronal cells and makes it possible to form points of attachment between, for example, the cell body, the axons or the dendrites and the immobilized adhesion protein on the islets.

 The subject of the present invention is also a process for producing a neuronal cell culture support (1) comprising a step of depositing on a surface (5) an islet substrate (7) of neuronal cell adhesion. , said islands (7) having a diameter of 0.2 to 2.5 μιτι, 0.2 to 2 μιτι, of 0.3 to 150 μιτι and being spaced between them from 0.5 to 10 μιτι, 1 to 9 μιτι , 3 to 7 μιτι.

 The neuronal cells are as defined above.

The substrate used in the process of the invention corresponds to the substrate defined above.

The adhesion proteins used in the method of the invention correspond to the proteins defined above. According to the invention, the method may further comprise a step of coating a cytophobic compound.

 According to the invention, the step of coating the cytophobic compound can be carried out prior to the deposition step of the adhesion islands.

 According to the invention, the cytophobic compound used in the process is as defined above.

 According to the invention, the method may comprise a step of coating the surface (5) with a binder. The step of coating the surface (5) with a binder may be carried out prior to the step of depositing the adhesion islands.

 According to the invention, the binder used in the process is as defined above.

 The present invention also relates to the use of the support (1) for the culture of neuronal cells.

 The present invention also relates to the use of the support

(1) in a method of quantitative and / or qualitative analysis of neuronal cells.

 In particular, the support of the present invention can be advantageously used to analyze the effects of chemical molecules on the growth of neurites, for example on the number, the size, the geometry, the connections.

 The carrier of the present invention can be advantageously used to study the effects of chemical molecules on the formation, structure, development and migration of the axonal growth cone.

 In particular, the carrier of the present invention may be advantageously used to analyze the effects of chemical molecules on, for example, the formation, number, structure and / or function of pre-synaptic or postsynaptic hemispynapses.

The subject of the present invention is also the use of the support (1) in a method of quantitative and / or qualitative analysis of neuronal cell synaptogenesis. In particular, the carrier of the present invention may be advantageously used to study, for example, the formation, number, structure and / or function of pre-synaptic or postsynaptic hemispynapses.

 In addition, the carrier of the present invention can be advantageously used to analyze and quantify the effects of chemical molecules on, for example, synaptogenesis of neuronal cells.

 The present invention also relates to the use of the support (1) in screening methods.

 The device of the invention advantageously allows to cultivate neuronal cells and maintain these cells in culture for a time sufficient to allow their development. For example, the device of the invention allows the development of synapses. In addition, during the entire culture time, the adhesion proteins attached to the islets advantageously retain their adhesive functions, allowing, for example and advantageously the expression of endogenous counter-receptors in neurons, for example N-cadherin endogenous in axons and dendrites, endogenous neuroligins in dendrites, and endogenous neurexins in axons.

 Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the appended figures, given for illustrative purposes.

Brief description of the figures

 - Figure 1 shows a culture medium (1) comprising a substrate (3), a cytophobic surface (5), and adhesive islands (7).

Figure 2A shows a culture medium comprising a hexagonal array of micro-islets coated with neurexin-1-Fc on a surface elsewhere cytophobe. The grafting of neurexin-1β-Fc on the islets initially coated with polylysine was provided by an anti-Fc antibody. Neurons expressing neuroligin-1 develop post-synaptic compartments specifically on these islets. The sectional diagram of an individual post-synapse formed on an island of neurexin-covered is shown on the right. FIG. 2B shows a culture support comprising a hexagonal network of micro-islets covered with neurexin-1β-Fc on a surface elsewhere cytophobe. Figure 2C is a two-fold magnified diagram of a portion of the culture medium showing dendrites of neurons expressing neuroligin-1 developing post-synaptic compartments specifically on these islands. Figure 2D is a roughly 10-fold enlarged sectional diagram of an individual post-synapse formed on a neurexin-coated island.

 Figure 3 shows fluorescence images of dissociated hippocampal neurons from embryonic rats in culture on micro-islet substrates coated with neurexin-1 β-Fc. The neurons were deposited on these substrates, transfected after 4 days with neuroligin-1-HA or EGFP, and cultured in total for 8 days. (A) Image of a neuron expressing neuroligin-1-HA immuno-labeled with an anti-HA antibody. (b) Image of a neuron transfected with EGFP. The islets were coated with an anti-huFc antibody conjugated to the fluorophore Cy5 (aHuFc-Cy5), for a display of the islets (corresponding images on the right). The images clearly show a regular geometric tree structure of neurons expressing neuroligin-1 and neuroligin-1 accumulation on neurexin-1 β-Fc-coated islets, and the absence of these reactions in EGFP-expressing neurons. (c) Higher magnification images of dendrite arrays from Neuroligin-1 transfected neurons, showing the development of filopodial structures indicated by white arrows resembling dendritic spines, the tip of which forms specific contacts with the islets covered with neurexin-1 β-Fc (right).

FIG. 4 represents fluorescence images of axons and dendrites as a function of the support used. Figure 4 (a) shows photographs of a neuron expressing neuroligin-1-HA, developing on the substrate coated with neurexin-1 β-Fc. The transfected neuron was visualized by immunostaining of the HA epitope with primary anti-HA antibodies and secondary antibodies conjugated to Alexa488 fluorophore. In particular, this photograph represents the structured morphology of rat hippocampal neurons (DIV 8) grown on micro-islet substrates. Figure 4 (b) shows photographs of a neuron expressing EGFP, developing on a substrate coated with neurexin-1 β-Fc. The right panels of Figures 4a and 4b include enlargements taken in the axonal and dendritic areas highlighting the morphology of the neurites in each state. Figure 4 (c) is a bar graph showing ordinarily the percentage of islets occupied by axons or dendrites overexpressing neuroligin-1-HA, developing on substrates coated with neurexin-1 β-Fc ( Nrx1 β-Fc + Nlgn-1, 9 cells, Dendrite: n = 595 islets, axon: n = 187 islets) and for neurons expressing EGFP, developing on substrates covered with ΝΓχΙ β-Fc (control; cells, dendrites: n = 202 islets, axon: n = 148 islets).

FIG. 5 represents fluorescence images of dendrites and explains the method for quantifying the enrichment factors of neuroligin-1 and PSD-95 synaptic proteins at the micro-pattern or islet level. The image in (a) represents a dendritic region of a neuron expressing neuroligin-1-HA and PSD-95: EGFP grown on a substrate coated with ΝΓχΙ β-Fc. The corresponding image in (b) represents the islands previously coated with ahuFc-Cy5 fluorescent antibodies. The image in (c) represents the detected islets (dashed circles), which are then transferred to the PSD-95: EGFP image. Islets that are not covered with dendrites (solid circles) have been eliminated from quantification. The image in (d) represents the outlines of the remaining islets (dashed circles), transferred to the PSD-95: EGFP image, where the neurite contour is determined by a threshold function (in gray). The enrichment index is calculated by measuring the fluorescence intensity in each island (circles), and divided by the average intensity measured from dendritic zones located outside the micro-patterns (gray).

 - Figure 6 shows the results of the quantification of synaptic protein enrichment factors at the level of micro-patterns. Panel (a) shows photographs of cells showing the recruitment of Neuroligin-1 (high) and PSD-95: EGFP (medium) at micro-patterns covered with neurexin-1 β-Fc (low). Panels (b) and (c) are graphs depicting enrichment of postsynaptic proteins (endogenous neuroligins-1-HA; PSD-95: EGFP or PSD-95). The abscissa corresponds to the enrichment index which is a dimensionless number normalized to 1, dividing by the level of fluorescence at the level of the islets by a reference level on control regions. Enrichment was quantitatively determined on the islets using 3 different cultures of hippocampal neurons following the procedure described in Figure 5. The cumulative distribution of the enrichment index of neuroligins-1-HA (in b) or PSD-95: EGFP and endogenous PSD-95 (in c) is shown on the graphs. The enrichment index was calculated for each protein under the following conditions: substrates coated with neurexin-1 β-Fc and neurons expressing neuroligin-1-HA (Nrx-Fc + Nlgn-1 (solid line curve); substrates covered with human Fc and neurons expressing neuroligin-1-HA (huFc + Nlgn-1 (curve spaced lines) or substrates covered with neurexin-1 β-Fc and over-expressing neurons EGFP (Nrx-Fc + EGFP (curve in dotted line).

An enrichment index value of 1 indicates that there is no particular recruitment of the protein under study.

Figure 7 shows photographs taken over time of neuronal cells expressing neuroligin-1 and PSD-95: EGFP, in culture on a substrate whose islets are covered with neurexin-1 β-Fc. In particular, this figure represents the changes of dendritic filopodia, visualized by the fluorescence signal of PSD-95: EGFP. These figures show the contacts between the peripheral part of the dendritic filopodia enriched in PSD-95 (arrows), with islets covered with neurexin-1 β-Fc (dashed circles), precursor events of the formation of hemi-post-synapses .

 Figure 8 shows photographs of transient calcium fluxes induced by glutamate decapping. Hippocampal rat neurons (DIV 7) developing on the substrate coated with neurexin-1 β-Fc (a) and expressing neuroligin-1-HA and PSD-95: mCherry (b) were labeled with the indicator Calcium Fluo-4, to observe intracellular calcium changes (c) and were incubated in a solution containing caged glutamate (4-methoxy-7-nitroindolinyl-caged 1-glutamate). The bi-photon laser beam for decapping is focused in the vicinity of an island (arrow). It is noted that the increase of the Fluo-4 signal from the location of the stripping propagates through the dendrites over time (Panel c). Panel (d) represents a graph showing different calcium responses, normalized to a reference level (ordinate) versus time in seconds (abscissa). These responses were measured at the island closest to the location of glutamate decapping (black curve), or on neighboring islands (dashed lines), as shown in PSD-95: mCherry ( islands 1, 2 and 3 respectively).

 Figure 9 shows fluorescence images of dissociated hippocampal neurons from embryonic rats in culture on micro-islet substrates coated with N-cadherin-Fc (top panels: N-cadherin-Fc substrate) or the molecule witness Fc (bottom panels: Substrate Hu-Fc). The neurons were deposited on these substrates and cultured for 8 days. The left panels show images of neurons labeled with the phalloidin molecule.

Bodipy, for coloring the actin filaments. The images corresponding to right (anti-human Fc Cy5) show the islets coated with anti-huFc antibody conjugated to fluorophore Cy5 (aHuFc-Cy5). In other words, the corresponding images on the right show the islets coated with an anti-human Fc antibody conjugated to the Cy5 fluorophore (anti-Human Fc Cy5). These images clearly show that the growth of neurites and their orientation follow the lines defined by islets covered with N-cadherin-Fc, which is not the case for neurons grown on Fc-coated micro-patterns, whose Neurites grow badly and in random directions. Figure 9c shows the higher magnification image of a colored growth cone for actin filaments with the phalloidin-Bodipy molecule, which shows the actin accumulation at the adhesions with N-coated islets. cadherin-Fc (arrows and circles). The right panel represents the corresponding image of islets of N-cadherin-Fc. 9 d represents a culture support comprising a hexagonal network of micro-islets covered with N-cadherin-Fc on a surface elsewhere cytophobe. The grafting of the N-cadherin-Fc molecule on the islets initially coated with polylysine was provided by an anti-Fc antibody. The central diagram represents a 2-fold diagram of a portion of the culture medium showing axons and dendrites of neurons developing on the support and forming specific contacts at the islet level. The diagram on the right represents a roughly 10-fold enlarged section of an individual adhesive contact formed on an island of N-cadherin coated. Note the difference between the horizontal and vertical scales. These observations show that in this system, N-cadherin promotes selective and patterned growth, that is to say according to a particular pattern, axons, but is unable to induce pre- or post-synaptic compartments.

Figures 10a and b show fluorescence images of dissociated hippocampal neurons from embryonic rats in culture on micro-islet substrates coated with N-cadherin-Fc (N-cadherin-Fc substrate). The neurons were deposited on these substrates, transfected after 4 days with red fluorescent protein (RFP) (a) or N-cadherin-RFP (b), and cultured in total for 8 days. The corresponding images of the islets coated with an anti-huFc antibody conjugated to the fluorophore Cy5 (αHuFc-Cy5), are presented in the right panels. These images clearly show the enrichment of N-cadherin-RFP, which is the membrane receptor of the N-cadherin-Fc ligand, but not of the RFP, on N-cadherin-Fc coated islands (arrows). Figure 10 (c) is a bar graph depicting on the ordinate the enrichment index of RFP and N-cadherin-RFP on N-cadherin-Fc coated islets.

 Figure 11a shows a culture medium comprising a hexagonal array of micro-islets coated with 6His-neuroligin-1 on a surface elsewhere cytophobe. Figure 11b shows a two-fold magnification diagram of a portion of the culture medium showing axons and dendrites of neurons developing on the support and forming specific island-level contacts. Figure 11 represents a roughly 10-fold enlarged sectional diagram of an individual pre-synapse formed on an island of neuroligin-coated graft onto islands initially coated with polylysine via anti-Histidine antibodies. In this figure, synaptic vesicles are also represented and the term CASK stands for calcium / calmodulin-dependent serine protein kinase. Figure 11 represents a fluorescence image of a culture support on which the islets are coated with an anti-6His antibody conjugated to the fluorophore Cy5 and on which 6His-neuroligin-1 was fixed. The

Figure 1 1 e represents a fluorescence image of the axon of a neuron transfected with neurexin-1 β-GFP. In this image, the clear spots correspond to the accumulation of neurexin-1 β-GFP at the level of the islets covered with 6His-neuroligin-1 and show the growth of the axons.

FIG. 12a shows a culture support comprising a hexagonal network of micro-islets coated with adhesion molecules synaptic 1 fused to the Fc fragment (SynCAM-1-Fc) and grafted onto the islets initially coated with polylysine via an anti-Fc antibody on a surface elsewhere cytophobe. Figure 12b is a two-fold magnification diagram of a portion of the culture medium showing axons and dendrites of neurons developing on the support and forming specific island-level contacts, in particular, they form pre-synaptic compartments. specifically on these islets. Figure 12c shows a roughly 10-fold enlarged sectional diagram of a presynapse formed on a SynCAM-1-Fc coated island. Figure 12d is a fluorescence photograph of a culture medium showing the islets coated with a Cy5 fluorophore-conjugated anti-Fc antibody to which SynCAM-1-Fc proteins (clear dots in the photograph) have been attached. Figure 12e shows a fluorescent image of the immunofluorescence staining of the endogenous SynCAMI receptor after deposition and culture for 8 days of dissociated hippocampal neurons from embryonic, untransfected rats on the culture device. Figure 12f shows a fluorescence image of the immunofluorescence staining of presynaptic vesicles (synapsin) after culture on the dissociated hippocampal neuron device, from embryonic rats.

EXAMPLES

Example 1 Manufacture of the Cell Culture Support

Islet substrates

The matrices of hydrophilic islands of 0.5 to 2 μιτι and separated from 3 to 6 μιτι in a cytophobic environment were obtained from CYTOO on glass slides of 2x2cm ² and 170μηη thick (custom products, CYTOOchips Custom, catalog number 10-950-00). The substrates were then treated with Poly (L-lysine) PLL (40 g / ml, Sigma P2636- 1G) which was adsorbed on the hydrophilic points. The substrates were then dried and stored at 4 ° C.

Production and purification of neurexin-1 β-Fc

The production protocol for neurexin-1β-Fc has been published previously (Heine, M., Thoumine, O., Mondin, M., Tessier, B., Giannone, G. & Choquet, D. Activity-independent and subunit-specific recruitment of functional AMPA receptors at neurexin / neuroligin contacts Proc Natl Acad Sci USA 105, 20947-52 (2008) [34]). The pcDNA neomycin plasmid containing the gene encoding the neurexin-1 β protein without the alternative S4 splicing site, fused to the C-terminal with the sequence coding for the constant fragment of human IgG (Fc) (Scheiffele, P., Fan , J., Choih, J., Fetter, R. & Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons Cell 101, 657-69 (2000) [35]) provided by P. Scheiffele (Biozentrum, Basel, CH). This plasmid was subcloned between the HindIII / XhoI sites in the pcDNAhygro (+) vector. HEK293 cells (5 × 10 ⁶ cells) were electroporated (Biorad Pulser Xcell) with 30 to 50 g of this plasmid and then cultured in DMEM High glucose medium (DMEMNA0500, Midimed) containing 10% fetal calf serum (SVFBRE500, Midimed), 1 mM MEM Sodium pyruvate (1 1360039, Fisher Scientific), 1% GlutaMAX (Trade Mark) supplement I (35050038, Fisher Scientific) without antibiotic for 36 h, then supplemented with 0.5 mg / mL. hygromycin B (10687-010, Fisher Scientific) to select a stable clone expressing neurexin-1β-Fc (10 days). The cells were cultured on supports coated with Poly-L-lysine hydrobromide (1 mg / ml, P2636-1G, Sigma-Aldrich) for 3 months in Medium AIM V (registered trademark) (31035-025, Fisher Scientific) supplemented with 0.5 mg / mL of hygromycin B. 60 mL of conditioned medium was taken twice a week and then frozen at -20 ° C. All the media collected were mixed, filtered (0.2 μιτι), treated with 10 mM DTT (DL-dithiothreitol, D9779-10G, Sigma) and the pH was adjusted to 7. Using a peristatic pump, this mixture was passed on a column of protein G (HiTrap Protein G HP, 17-0407-03, GE Healthcare) previously balanced with 20 mM sodium phosphate pH = 7. Elution of the protein was performed with Glycine (Sigma G-8898) 0.1 M pH = 2.7, and the eluate was collected in 1 ml fractions in tubes containing Tris buffer (Trizma Base, Sigma T6066) 1 M, pH = 9 adjusted with HCl. Quantification of the protein concentration in these fractions is performed by a BCA assay (W9981 L, Fisher Scientific). The protein is stored at -80 ° C in 20-50 μl aliquots.

Production and purification of SvnCAM-1-Fc svnaptic adhesion protein

 The plasmid encoding the SynCAM-1-Fc protein has been generously provided by T. Biederer (Yale University, USA) and is described in the reference (Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D. ., Liu,

X., Kavalali, E.T., Sudhof, T.C. SynCAM, synaptic adhesion molecule that drives synapse assembly, Science 297 1525-31 (2002) [68]). The SynCAM-1-Fc molecule was produced from this gene according to the method described above for neurexin-1-Fc, according to the protocol described in Breillat, C., Thoumine O., Choquet, D. Characterization of SynCAM surface SynCAM derived ligand with high homophilic binding affinity, Biochem Biophys Res Commun, 359, 655-659 (2007) [71]. Alternatively, the recombinant adhesion proteins were purchased from the manufacturer R & D Systems: N-cadherin-Fc (6626-NC-050), neurexin1-Fc (4485-NX-050), neurexin-1β-Fc (5268-NX-050), Neuroligin-1-6His (4340-NL-050), Neuroligin-2-6His (5645-NL-050), and LRRTM-1-6His (4897-LR-050), LRRTM- 3-6His (4898-LR-050), LRRTM-4-6His (5377-LR-050), SynCAM1-Fc (3519-S4), SynCAM2-Fc (4290-S4),

SynCAM3-Fc (3678-S4), SynCAM4-Fc (4164-S4). Recouyrement substrates including islets

 One day before depositing the cells, the substrates were treated with goat anti-human Fc antibodies (Jackson ImmunoResearch, ref 109-005-098) conjugated to the Cy5 fluorophore through a coupling kit (PA25001 GE Healthcare) and diluted in borate buffer (Merck boric acid, 1001650500, 0.2 M, pH = 8.5), to a final concentration of 0.023 mg / ml. Prior to incubation, the diluted antibodies were centrifuged for 10 min at 1400 rpm at 4 ° C. Each coverslip obtained was inverted over 200 μl of this antibody solution on parafilm (Dutscher, 090998) previously sterilized for 1 h with a UV lamp (365 nm) under a laminar flow hood (TELSTAR, AV-100). and incubated for 4 to 5 hours at 23 ° C to ensure uniform antibody distribution on the micro-patterns.

 The example was also carried out using anti-6His mouse antibodies (Santa Cruz, ref SC-8036) conjugated to the Cy5 fluorophore by means of a coupling kit (PA25001 GE Healthcare) and diluted in borate buffer (Merck boric acid). 1001650500, 0.2 M, pH = 8.5) at a final concentration of 0.023 mg / ml.

 Subsequently, the substrates were thoroughly washed with borate buffer, and then incubated at 4 ° C overnight with 100 μl of purified neurexin-i-Fc, purified N-cadherin-Fc (R & D Systems, 1388-NC-050) or human Fc (Jackson Immunoresearch, P / N 009-000-008) used as a control, all the proteins being diluted to 0.04 mg / ml in the borate buffer. These protein solutions were previously centrifuged for 10 minutes at 14,000 rpm at 4 ° C.

 In another embodiment, the substrates were thoroughly washed with borate buffer, and then incubated at 4 ° C overnight, ie 16 hours with 100 μl of SynCAM-1 solutions.

Fc or 6His-neuroligin-1 purified and diluted to 0.04 mg / ml in buffer borate. These protein solutions were previously centrifuged for 10 minutes at 14,000 rpm at 4 ° C.

 The next day, the substrates were placed in a 6-well plate, washed 3 times with 1 ml of borate buffer and once with 1 ml of minimal essential medium (MEM) (21430-020, Gibco / Invitrogen) supplemented with % horse serum (16050-122, Gibco / Invitrogen) and left in 2.5 ml MEM supplemented with 10% horse serum for 1 to 2 hours in an incubator at 37 ° C and 5% CO2 (Heraeus , HERACell 150).

EXAMPLE 2 Culture of Neuronal Cells with the Support of the Invention and Study of the Development of Cultured Cells

 In the following examples, the methods and materials used are those described below:

 Cell culture and transfection

 Neurons dissociated from the hippocampus from E18 rat embryos were spread on substrates (100,000 cells / substrate). After 4-5 hours, the substrates were thoroughly washed with Neurobasal (NB) medium (Gibco, 12348-017) supplemented with B27 (Gibco, 17504-044) and L-Glutamine (Glu200100, MidiMed) at 37 ° C. to remove unbound cells, then incubated in 2.5 ml of this same medium for 8 to 10 days. After 3 days the neurons were transfected with the plasmids encoding Neuroligin-1-HA (provided by P. Scheiffele), PSD-95: EGFP (provided by S. Okabe) or EGFP vectors (Qiagen), using the Effectene kit (Qiagen, 301427), according to the manufacturer's instructions, using a total of 2 μg of DNA per well. For co-transfections of Neuroligin-1-HA and EGFP or Neuroligin-1-HA and PSD-95: EGFP, the plasmids were used in a ratio of 2: 1.

In another embodiment, the neurons were transfected with plasmids encoding Neurexin-i-GFP (provided by M. Missler), N-cadherin-RFP (provided by RM Mege) or vectors for red fluorescent protein (RFP) (Qiagen).

 Immunolabeling

To visualize the recruitment of neuroligin-1 on the surface of neurons, cultures transfected with Neuroligin-1-HA were fixed at 37 ° C in a solution of phosphate buffered saline (PBS) (Euromedex ET330) supplemented with 4% of paraformaldehyde (VWR 28794.295,) and 4% sucrose (Fluka 84097) for 10 min, and the remaining active sites were saturated with 1 ml of a 50 mM NH Cl solution in PBS for 15 min. Nonspecific binding was blocked with 1 ml of PBS containing 1% bovine serum albumin (Sigma, A3059). Then, the neurons were stained with anti-HA rat antibody diluted 1: 400 in phosphate buffered saline (PBS) (Roche, 11867423001) followed by Alexa568 conjugated goat anti-rat antibody (Invitrogen A1 1077; 2 mg / ml) diluted 1: 800 in PBS for 30 min at 23 ° C. To label endogenous PSD-95 proteins, the cells were fixed and permeabilized in 0.1% Triton X 100 (T9284, Sigma) in PBS for 5 min. Nonspecific binding was blocked with 1 ml of PBS containing 1% BSA. The neurons were labeled with anti-PSD-95 mouse antibody (Neuromab 75-028, 1: 400) followed by Alexa568 fluorophore-conjugated goat anti-mouse antibodies (2 mg / ml dilution 1: 800, Invitrogen A1 1004). To label the actin filaments, the cells were fixed and permeabilized in 0.1% Triton X 100 (T9284, Sigma) in PBS for 5 min. Nonspecific binding was blocked with 1 ml of PBS containing 1% BSA, and then the neurons were treated with phallicidin-Bodipy (trademark) FL (Molecular Probes, Invitrogen, B607) diluted 1: 100 in PBS. The slides were then mounted on glass slides (Waldemar Knittel, Braunschweig, Germany) with a drop of Mowiol (Calbiochem) and sealed with varnish (Pig). Immunostaining was also performed with anti-DsRed rabbit antibodies (Clontech 632496, 1: 800) followed by Alexa568-conjugated secondary antibodies to visualize the transfected RFP and N-cadherin-RFP, according to the method described above. above.

Immunostaining was also performed with anti-synapsin mouse antibodies (Synaptic Systems 106001, 1: 400), or anti-SynCAM-1 rabbit antibodies (Novus NB 300-186, 1: 400) followed by antibodies. Fluorescent side effects for labeling synapsin or endogenous SynCAM-1 adhesion molecules according to the method described above.

Image analysis

 Immunolabels were visualized on a Leica DM R-type right epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63x / 1, 32 NA objective and a Chroma Technology filter set (Bellows Falls, VT, USA) for EGFP: excitation: S490 / 20 nm, emission: S528 / 38 nm, dichroic: 86100bs; TRITC or RFP: excitation: S555 / 28 nm, emission: S617 / 73, dichroic: 101848; the Cy5: excitation: S635 / 20, emission: S685 / 40 nm, dichroic: 101848. The images were acquired with a CCD camera (HQ Coolsnap, Roper Scientific, Evry, France), using the software Metamorph (Universal Imaging Corp. .)

 The enrichment factors of Neuroligin-1 and PSD-95 were measured on 3 dendritic regions randomly selected on each neuron and were calculated using an automatic program written in the Metamorph (Universal Imaging Corp) software, and a procedure explained in Figure 5.

 The RFP and N-cadherin-RFP enrichment factors were measured on 3 axonal regions randomly selected on each neuron and were calculated according to the procedure explained in Figure 5.

Decay of glutamate The dissociated hippocampal neurons transfected with Neuroligin-1-HA and PSD-95: mCherry, which grew for 7 to 8 days on substrates comprising islets coated with neurexin-β-Fc, were labeled with 2.5 μl of Fluo-4-AM (Invitrogen; F23917) for 5 min in Neurobasal (NB) culture medium (Gibco, 12348-017) supplemented with B27 (Gibco, 17504-044) and L-Glutamine ( Glu200100, MidiMed) then placed in 400 μl of Tyrode (30 mM glucose, 120 mM NaCl, 5 mM KCl, 0.1 mM MgCl ₂ , 2 mM CaCl ₂ , 25 mM HEPES (Sigma, glucose: G6152 NaCl: S7653, KCI: P4504, MgCl _2: M8266, CaCl _2: C7902, HEPES: H4034) supplemented with 5 mM 4-methoxy-7-nitroindolinyl cage-L-glutamate (MNI-glu) (Tocris, 1490) , and 1 μΜ TTX (Ascent Scientific, ASC-055) to avoid the generation of potential action potentials.

 The observation chamber was placed on a confocal scanning laser microscope (TCS SP5, Leica) thermostated at 37 ° C and equipped with a bi-photon pulsed laser (Mai Tai Spectra-Physics) set at 750 nm. For fluorescence recordings, an area of 100 x 100 m was scanned at 700 Hz by an Argon laser at 488 nm and the fluorescence was collected between 500 and 530 nm by a photomultiplier, using an HCX PL APO CS oil lens. 63X / 1 .32 NA and an iris open three times the Airy disk (180 μηη).

 After the recording of a baseline, a bi-photon light pulse with a duration of 10 ms corresponding to a scan of a restricted zone of 2 μιτι in diameter, near an island, was imposed to discard caged glutamate. Fluorescence signals from Fluo-4 were then recorded for 1 minute.

To represent the calcium response obtained during MNI-glu decapping, the fluorescence intensity values of fluo-4 at a given location were normalized to the average fluorescence intensity obtained before the bi-photon light pulse. Live cell imaging

The hippocampal primary neurons (DIV7) cultured on neurexin-1 β-Fc covering the micro-motifs and expressing Nlgn-1 WT and PSD-95: EGFP were placed in an open chamber containing a 1: 1 mixture of a solution. of Tyrode and NB conditioned medium. The cells were then observed on a Leica 6000 DMI inverted microscope (Leica Microsystems, Wetzlar, Germany), equipped with a CCD camera (Coolsnap HQ2, Roper Scientific, Evry, France) and a thermostatic box (Life Imaging Services, Basel, Switzerland) providing 37 ° C and 5% CO ₂ .

 Acquisitions over periods of several hours were made using mercury lamp illumination, an HCX PL APO CS oil objective 63X1, 32 NA and interferential filters from Chroma Technology (Bellows Falls, VT). USA), for the observation of EGFP (excitation HQ 480/30 nm, dichroic 86100bs, emission S528 / 38 nm) and Cy5 (excitation HQ630 / 20 nm, dichroic 101848, emission S685 / 40 nm). Images of PSD-95: EGFP were recorded every 10 min for 4 h, while images of the Cy5 patterns were taken only twice, one before and one after the acquisition.

Results and discussion

Transmembrane adhesion proteins, neurexins, and neuroligins are key players in synapse formation (Sudhof, TC Neuroligins and neurexins link synaptic function to cognitive disease, Nature 455, 903-1 1 (2008) [36]). These molecules form a link between the pre- and postsynaptic membranes by a high affinity recognition between their ectodomains (Craig, AM & Kang, Y. Neurexineuroligin signaling in synapse development, Curr Opin Neurobiol 17, 43-52 (2007). [41]). When present on the surface of heterologous cells or microspheres, neuroligin and neurexin have the ability to generate pre- and post-synapses, respectively. functional, in contact with primary neurons (Graf, ER, Zhang, X., Jin, SX, Linhoff, MW & Craig, AM Neurexins induce differentiation of GABA and glutamate postsynaptic special izations via neuroligins Cell 1 19, 1013-26 (2004) [34], Heine, M., Thoumine, O., Mondin, M., Tessier, B., Giannone, G. & Choquet, D. Activity-independent and subunit-specific recruitment of functional AMPA receptors. neuromycin / neuroligin contacts Proc Natl Acad Sci USA 105, 20947-52 (2008) [35], Scheiffele, P., Fan, J., Choih, J., Fetter, R. & Serafini, T. Neuroligin Nonneuronal cells triggers presynaptic development in contacting axons, Cell 101, 657-69 (2000). [35], Sudhof, TC Neuroligins and neurexins link synaptic function to cognitive disease, Nature 455, 903-1 1 (2008). ], Ko, J., Zhang, C., Arac, D., Boucard, AA, Brunger, AT & Sudhof, TC Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation. [Embo] J 28, 3244- 55 (2009). [37] ]). The importance of neurexin and neuroligin in synaptic function is further underlined by the abnormal phenotypes of knockout mouse models (Chubykin, AA, Atasoy, D., Etherton, MR, Brose, N., Kavalali). , And, Gibson, JR & Sudhof, TC Activity-Dependent Validation of Excitatory versus Inhibitory Synapses by Neuroligin-1 versus Neuroligin-2 Neuron 54, 919-31 (2007).

[42] Missler, M., Zhang, W., Rohlmann, A., Kattenstroth, G., Hammer, R.E., Gottmann, K. & Sudhof, T.C. Alpha-neurexins Ca2 + couple channels to synaptic vesicle exocytosis. Nature 423, 939-48 (2003). [43] Varoqueaux, F., Aramuni, G., Rawson, RL, Mohrmann, R., Missler, M., Gottmann, K., Zhang, W., Sudhof, TC & Brose, N. Neuroligins determines synapse maturation and function. Neuron 51, 741-54 (2006). [44]).

 These elements clearly show the importance of adhesion molecules in axonal growth and synaptogenesis.

The present example via the use of the culture support of the invention has advantageously allowed to cultivate neuronal cells and a spatial control of the formation of synapses at a resolution micrometric, due to the adhesion properties of the synaptogenic neurexin / neuroligin complex (Figures 1 and 2).

 The rat hippocampal neuron culture on substrates with islets coated with purified neurexin was performed and the cells obtained are shown in Figures 3 and 4. The islet substrates have a regular network of chemically activated islets (1, 5). μιτι of diameter, separated by 5 μιτι), surrounded by a non-adhesive bottom. The islets were coated with a recombinant neurexin-1 β fused with the constant fragment of human IgG, Fc (ΝΓχΙ β-Fc), or pure Fc used as a control molecule. The coupling was done via a secondary anti-Fc antibody labeled with the Cy5 fluorophore, advantageously allowing both the good orientation of the adhesive ectodomain and the visualization of the fluorescence of the micro-islets.

 Dissociated hippocampal neurons were placed on these substrates and allowed their development for 7 to 10 days. The neurons were transfected after 4 days with neuroligin-1 (Nlgn-1), or green fluorescent protein (EGFP) as a control.

 Recognition between immobilized ΝΓχΙ β-Fc and neuroligin-1 expressed in neurons strongly affected cellular morphology and limited growth as a function of island geometry and positioning as shown in Figure 3. Surprisingly, Advantageously, the dendrites of the neurons expressing neuroligin-1 propagated preferentially along the islet lines covered with neurexin-1 β-Fc and invaded them to a much higher degree than the axons of these same cells ( Figure 4).

 This demonstrates the contact between the neurons and the device of the invention.

Study of neuronal cell growth and formation of post-svnaptic assemblages To examine the formation of post-synapses on these substrates, the accumulation of post-synaptic neuroligin-1 and PSD-95 proteins, or the EFGP control protein, on islets coated with neurexin-1 β-Fc or Fc a been examined for each condition. An enrichment index was calculated by dividing the average fluorescence intensity of the given protein into co-localization dendritic zones with protein-coated islands, by the average fluorescence intensity of the dendritic zones located between the islets ( Figure 5). Since each neuron comes into contact with hundreds of micro-islands, the values of the statistical samples allowing the comparisons quickly become very high. The quantitation shown in Figure 6 shows that both proteins were significantly recruited to Nrx1-Fc coated islands (Nmin-1: 26 cells, n = 2608 islets, NLGN-1 + PSD-95: EGFP: 17 cells, n = 993 islands). The detected recruitment was specific for the neurexin / neuroligin interaction (EGFP: 24 cells, n = 1045 islets, EGFP + PSD-95: mCherry: 7 cells, n = 446 islets) or islet coating with Fc instead of ββ-Fc (Nlgn-1: 1 1 cells, n = 608 islets, Nlgn-1 + PSD-95: EGFP: 3 cells, n = 158 islets). The endogenous PSD-95 protein was also significantly recruited at the islet level (15 cells, n = 1482 islets).

Dendritic spine-like structures have been observed showing an accumulation of neuroligin-1 in the peripheral part of the filopodia attached to the micro-pattern coated with neurexin-1β-Fc (Figure 3c). Also, in order to better understand the dynamics of synapse formation, this system was combined with video microscopy to live imaging the fluorescence of PSD-95: EGFP. One of the advantages of using the culture substrate of the invention comprising islets with respect to random observations of synapse formation is that the position where synapse formation is expected is controlled. One point that is not yet clear in the literature is whether the Synapses are formed between the axonal filopodia contacting the dendrites, or between the dendritic filopodia contacting the axons. This example clearly demonstrates that the dendritic filopodia, thanks to an aggregate of the PSD-95 scaffold protein located at the end of the filopodium, can reach micro-islets of neurexin and establish firm contact within 3 hours (Figure 7). . These preformed complexes containing Neuroligin-1 and PSD-95 are likely to serve as predetermined post-synaptic precursors for the establishment of new functional excitatory synapses.

 This example clearly demonstrates that the device of the invention makes it possible to cultivate and study the physiological mechanisms, for example the development of synapses.

Study of the formation of synapses by neuronal cells in culture on the substrate of the invention

 This step is also a study of the function of the postsynaptic assemblies on the device of the invention covered with neurexin.

 In a second step, it was tested whether the newly formed neuronal contacts on the islets of neurexin contained functional glutamate receptors, using the glutamate decapping technique and calcium imaging. Neurons were transfected with PSD-95: mCherry to visualize post-synaptic differentiations and incubated with a cell-penetrating calcium indicator (Fluo-4).

When M NI -glutamate was photo-released near neurexin islands showing PSD-95-mcherry accumulation, local and selective transient calcium fluxes were recorded (Figure 8), demonstrating the presence of functional channels. glutamate receptors. In a previous study, using beads coated with neurexin-1 β-Fc, it was shown that transient calcium fluxes are due to voltage-gated calcium channels that open through the Membrane depolarization induced by AMPA receptor activation by glutamate (Heine, M., Thoumine, O., Mondin, M., Tessier, B., Giannone, G. & Choquet, D. Activity-independent and subunit -specific recruitment of functional AMPA receptors at neurexin / neuroligin contacts Proc Natl Acad Sci USA 105, 20947-52 (2008) [34]).

Study of the neuritic growth and the formation of pre-synapses of neuronal cells on the substrate of the invention

 This example also relates to the study of the growth of neurites and the formation of pre-synapses by the neuronal cells in culture on the substrate of the invention.

 In addition the device of the invention allows via the use of different immobilized adhesion proteins at the islet level, the selective study of different synaptic systems, or proteins involved in the growth of axons and dendrites. For example, when N-cadherin-Fc was deposited, significant growth of neurites was observed in directions related to islet disposition, accompanied by adhesion and specific migration of growth cones. which was not the case for substrates coated with the control molecule Fc (FIG. 9). In addition, cells transfected with N-cadherin-RFP, but not cells transfected with the RFP control protein, showed local accumulation of N-cadherin-RFP on the N-cadherin-Fc coated islands (Figure 10). This example also relates to the study of axonal growth and the formation of pre-synapses by neuronal cells in culture using the device of the invention as well as the study of the formation of pre-synapses by the cells. neurons in contact with the substrate of the invention coated with Neuroligin-1 or SynCAM-1.

The device of the invention also makes it possible, via the use of different immobilized adhesion proteins at the level of the islets, the study selective of different synaptic systems, or proteins involved in the growth of axons and dendrites. For example, when the 6His-Neuroligin-1 molecule was deposited on the islets, significant growth of axons was observed in directions related to islet disposition, as well as accumulation of neurexin-GFP in the axons of the islets. transfected neurons (Figure 1 1). Similarly, when the SynCAM-1-Fc molecule was deposited on the islets, significant growth of the axons was observed in directions related to the islet disposition, as well as an accumulation of endogenous SynCAM-1 receptors and Presynaptic vesicles labeled with synapsin (Figure 12).

Other applications

 As demonstrated in this example, the device of the invention makes it possible to study the formation of synapses and synaptic function with a high production of statistical and reproducible data. In addition, the device of the invention can be widely used with other synaptogenic molecules, such as neuroligins, for example to induce the formation of pre-synapses and / or with newly identified proteins such as LRRTMs and TrkCs (Sudhof , TC Neuroligins and neurexins link synaptic function to cognitive disease, Nature 455, 903-1 (2008) [36], Linhoff, MW, Lauren, J., Cassidy, RM, Dobie, FA, Takahashi, H., Nygaard. , HB, Airaksinen, MS, Strittmatter, SM & Craig, Neuron 61, Neuron 61,

734-49 (2009). [38], Ko, J., Fuccillo, M.V., Malenka, R.C. & Sudhof, T.C. LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation. Neuron 64, 791-798 (2009). [39], Wit, J., Sylwestrak, E., O'Sullivan, M., Otto, S., Tiglio, K., Savas, JN, Yates, JR, Comoletti, D., Taylor, P. & Ghosh, A. LRRTM2 Interacts with Neurexin and Regulatics

Excitatory Synapse Training. Neuron 64, 799-806 (2009). [40], Takahashi, H., Arstikaitis, P., Prasad, T., Bartlett, TE, Wang, YT, Murphy, TH & Craig, AM Postsynaptic TrkC and presynaptic PTPsigma function as bidirectional excitatory synaptic organizing complex. Neuron 69, 287-303 (201 1). [45]).

 Thus, the device of the invention can be used in processes in which molecules are screened to study their effect on synaptic differentiation. It also allows the detection of new therapeutic targets for pathologies such as autism, schizophrenia, and X-linked mental retardation in humans, pathologies in which mutations of the genes encoding neuroligins and neurexins appear to be involved (Jamain, S., Quach, H., Betancur, C., Rastam, M., Colineaux, C., Gillberg, I. C., Soderstrom, H., Giros, B., Leboyer, M., Gillberg, C. & Bourgeron, T. Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism.Nat Genet 34, 27-9 (2003). [46], Talebizadeh, Z., Lam, DY, Theodoro, MF, Bittel, D.C., Lushington, GH & Butler, M.

G. Novel splice isoforms for NLGN3 and NLGN4 with possible implications in autism. J Med Genet 43, e21 (2006). [47], Rujescu, D., Ingason, A., Cichon, S., Pietilainen, OP, Barnes, MR, Toulopoulou, T., Picchioni, M., Vassos, E., Ettinger, U., Bramon, E. Murray, R., Ruggeri, M., Tosato, S.,

Bonetto, C, Steinberg, S., Sigurdsson, E., Sigmundsson, T., Petursson,

H., Gylfason, A., Olason, PI, Hardarsson, G., Jonsdottir, GA, Gustafsson, O., Fossdal, R., Giegling, I., Moller, HJ, Hartmann, AM, Hoffmann, P., Crombie , C, Fraser, G., Walker, N., Lonnqvist, J., Suvisaari, J., Tuulio-Henriksson, A., Djurovic, S., Miss, I., Andreassen, OA, Hansen, T., Werge , T., Kiemeney, LA, Franke, B., Veltman, J., Buizer-Voskamp, JE, Sabatti, C, Ophoff, RA, Rietschel, M., Nothen, MM, Stefansson, K., Peltonen, L. , St Clair, D., Stefansson, H. & Collier, DA Disruption of the neurexin 1 gene is associated with schizophrenia. Hum Mol Genet 18, 988-96 (2009). [48]). Example 3 Method for Screening Molecules on Neuronal Cell Cultures with the Support of the Invention

 Cells and culture substrates are obtained according to the method described in Examples 1 and 2 above.

 After 4 days of culture, the neuronal cells cultured on substrates coated with N-cadherin-Fc (BD Transduction Laboratories (trademark), ref. 610921) are placed in the presence of peptides competing with cadherin interactions, for example containing the HAV sequences. (1 mM) (Williams, EJ, Williams, G., Gour, B., Blaschuk, O. & Doherty, P. INP, a novel N-cadherin antagonist targeted to amino acids that flank the HAV motif.) Mol Cell Neurosci 15, 456-64 (2000) [49], Williams, E., Williams, G., Gour, BJ, Blaschuk, OW & Doherty, P. A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined. by amino acids that flank the HAV motif, J Biol Chem 275, 4007-12 (2000). [50]), adhesive protein fragments derived from cadherins, for example the EC1-EC2 domains (Perret, E., Benoliel, AM , Nassoy, P., Stones, A., Delmas, V., Thiery, JP, Bongrand, P. & Feracci, H. Fast dissociation kinetics between individual Ec adherin fragments revealed by flow chamber analysis. Embo J 21, 2537-46 (2002).

[51], Perret, E., Leung, A., Feracci, H. & Evans, E. Trans-bonded peers of E-cadherin, a remarkable hierarchy of mechanical strengths. Proc Natl Acad Sci USA 101, 16472-7 (2004). [52]) or transfected with siRNAs directed against cadherins (Paradis, S., Harrar, DB, Lin, Y., Koon, A.C, Hauser, JL, Griffith, E.C., Zhu, L., Brass , LF, Chen, C. & Greenberg, ME An RNAi-based approach identified molecules required for glutamatergic and GABAergic synapse development Neuron 53, 217-32 (2007) [53]) or catenins (Bard, L., Boscher , C, Lambert, M., Mege, RM, Choquet, D. & Thoumine, O. A molecular clutch between the actin flow and N-cadherin adhesion drives growth cone migration J Neurosci 28, 5879-90 (2008). 25]) or mutated cadherin molecules (in the extracellular domains to prevent interactions with cadherin counter-receptors, or in intracellular domains to prevent binding with intracellular partners). The effect of these compounds on the development of neurites and synapses after 8 days of culture will be examined and quantified as explained in Figures 5 and 6.

 After 4 days of culture, the neuronal cells cultured on micro-patterned substrates coated with neurexin-1-Fc are placed in the presence of recombinant neurexin proteins (Scheiffele, P., Fan, J., Choih, J., Fetter, R Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons Cell 101, 657-69 (2000) [35], Dean, C, Scholl, FG, Choih, J., DeMaria, S. , Berger, J., Isacoff, E. & Scheiffele, P. Neurexin mediates the assembly of presynaptic terminais.N Nat Neurosci 6, 708-16 (2003). [54], Levinson, JN, Chery, N., Huang, K., Wong, TP, Gerrow, K., Kang, R., Prange, O., Wang, YT & El-Husseini, A. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1 beta in Biol Chem 280, 17312-9 (2005), [55]), competing cell-permeate peptides of post-synaptic protein interactions (Sainlos, M., Tigaret , C, Poujol, C, Olivier, N.B., Bard, L., Breillat, C, Thiolon, K., Choquet, D. & Imperiali, B. Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization. Nat Chem Biol 7, 81-91 (2010). [56], Bard, L., Sainlos, M., Bouchet, D., Cousins, S., Mikasova, L., Breillat, C., Stephenson, FA, Imperiali, B., Choquet, D. & Groc, L. Dynamic and specifies interaction between synaptic NR2-NMDA receptor and PDZ proteins. Proc Natl Acad Sci U S A 107,

19561 -6 (2010). [57]) or transfected with siRNAs directed against neuroligins (Chih, B., Engelman, H. & Scheiffele, P. Control of excitatory and inhibitory synapse formation by neuroligins, Science 307, 1324-8 (2005). ]) or LRRTMs, or mutant molecules of neuroligins or LRRTMs (in their extracellular domains to prevent interactions with neurexin, or in intracellular domains for prevent linkage with postsynaptic partners). The effect of these compounds on the development of neurites, growth cones, and synapses after 8 days of culture is examined and quantified as described in Figures 5 and 6.

 After 4 days of culture, neuronal cells cultured on substrates coated with neuroligins or LRRTMs are brought into contact with neuroligin proteins or recombinant LRRTMs (Scheiffele, P., Fan, J., Choih, J., Fetter, R. & Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons Cell 101, 657-69 (2000) [35], Dean, C, Scholl, FG, Choih, J., DeMaria, S., Berger, J., Isacoff, E. & Scheiffele, P. Neurexin mediates the assembly of presynaptic terminais.N Nat Neurosci 6, 708-16 (2003). [54], Levinson, JN, Chery, N., Huang, K ., Wong, TP, Gerrow, K., Kang, R., Prange, O., Wang, YT & El-Husseini, A. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1 beta in neuroligin-induced synaptic specificity J Biol Chem 280, 17312-9 (2005).

[55]), competing cell-permeate peptides for interactions between proteins at the pre-synaptic level, or transfected with siRNAs directed against neurexins or mutated neurexin molecules (in their extracellular domains to prevent interactions with neuroligins or LRRTMs , or in the intracellular domains to prevent binding with pre-synaptic partners). The effect of these compounds on the development of neurites and synapses after 8 days of culture is examined and quantified as described in Figures 5 and 6.

 After 4 days of culture, the neuronal cells cultured on substrates coated with SynCAMs are placed in the presence of recombinant SynCAM proteins (Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, ET, Sudhof, TC SynCAM, Synaptic adhesion molecule that drives synapse assembly, Science 297 1525-31 (2002) [72];

Sara, Y., Biederer, T., Atasoy, D., Chubykin, A., Mozhayeva, MG, Sudhof, T. C, Kavalali, and Selective Capability of SynCAM and Neuroligin for functional synapse assembly. J Neuroscience 260-270 (2005) [73]; Breillat, C., Thoumine O., Choquet, D. Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity, Biochem Biophys Res Commun, 359, 655-659 (2007) [74], of cell-cell peptides. Competitive permeations of interactions between proteins at the pre-synaptic level, or transfected with siRNAs directed against SynCAMs or mutated SynCAM molecules (in their extracellular domains to prevent homophilic interactions with SynCAM receptors, or in intracellular domains to prevent liaison with pre-synaptic partners). The effect of these compounds on the development of neurites and synapses after 8 days of culture is examined and quantified as described in Figure 1 1.

 Peptides and proteins are used at concentrations 10 times greater than the dissociation constants of the targeted interactions, ie typically 1 μΜ for cadherin interactions, 10 nM for neurexin / neuroligin interactions, and 5 μΜ for intracellular level of pre- and post-synaptic scaffolds. The siRNAs are used at concentrations of 0.5-2 g / carrier using the Lipofectamine 2000 transfection agent, as recommended by the supplier (Invitrogen).

 For synCAMs, peptides and proteins were used at concentrations 10 times higher than the dissociation constants of the targeted interactions, ie 100 nM for interactions between

SynCAMs.

 Alternatively, the substrates are seeded with neurons derived from knockout mice for certain genes, for example, neurexins (Missler, M., Zhang, W., Rohlmann, A., Kattenstroth, G., Hammer, RE, Gottmann , K. & Sudhof, TC Alpha-neurexins couple

Ca2 + channels to synaptic vesicle exocytosis. Nature 423, 939-48 (2003). [43]), neuroligins (Varoqueaux, F., Aramuni, G., Rawson, R. L, Mohrmann, R., Missler, M., Gottmann, K., Zhang, W., Sudhof, TC & Brose, N. Neuroligins determines synapse maturation and function, Neuron 51, 741-54 (2006). [44]), IgCAMs (Kamiguchi, H., Hlavin, ML & Lemmon, V. Role of L1 in neural development: what the knockouts Mol Cell Neurosci 12, 48-55 (1998) [59]) or cadherins (Suzuki, S.C., Fume, H., Koga, K., Jiang, N., Nohmi, M., Shimazaki , Y., Katoh-Fukui, Y., Yokoyama, M., Yoshimura, M. & Takeichi, M. Cadherin-8 is required for the first relay synapses to receive functional inputs from primary sensory afferents for cold sensation. , 3466-76 (2007). [60]), or mutated ("knock-in") mice expressing adhesion proteins carrying mutations involved in certain pathologies such as autism, schizophrenia, or mental retardation ( Etherton, MR, Tabuchi, K., Sharma, M., Ko, J. & Sudhof, TC An autism-associated point mutation in the cytoplasmic cytoplasmic tail selectively odd AMPA receptor-mediated synaptic transmission in hippocampus. EMBO J 30, 2908-19. [61], Tabuchi, K., Blundell, J., Etherton, MR, Hammer, RE, Liu, X., Powell, CM & Sudhof, TC A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318, 71-6 (2007). [62]), and the development of hemi-synapses is studied as described in Figures 5 and 6.

List of references

 1. Folkman, J. & Moscona, A. Role of cell shape in growth control.

 Nature 273, 345-9 (1978).

 2. Watt, F.M., Jordan, P.W. & O'Neill, C.H. Cell shape controls terminal differentiation of human epidermal keratinocytes. Proc NatI

Acad Sci U S A 85, 5576-80 (1988).

 3. Thery, M., Racine, V., Pepin, A., Piel, M., Chen, Y., Sibarita, J. B. & Bornens, M. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol 7, 947-53 (2005).

4. Thery, M., Racine, V., Piel, M., Pepin, A., Dimitrov, A., Chen, Y., Sibarita, JB & Bornens, M. Anisotropy of cell adhesive microenvironment governs cell internai organization orientation of polarity. Proc NatI Acad Sci U S A 103, 19771 -6 (2006).

 5. Ireland, G. W., Dopping-Hepenstal, P. J., Jordan, P. W. & O'Neill, C.

 H. Limitation of substrate size cytoskeletal alters organization and behavior of Swiss 3T3 fibroblasts. Cell Biol Int Rep 13, 781-90 (1989).

 6. Sanjana, N. E. & Fuller, S. B. A fast flexible ink-jet printing method for dissociated neurons in culture. J Neurosci Methods 136, 151-63 (2004).

 7. Yamagata, M., Weiner, J.A., Dulac, C., Roth, K.A. & Sanes, J.R.

 Labeled lines in the retinotectal System: markers for retinorecipient sublaminae and the retinal ganglion cell subsets that innervate them. Mol Cell Neurosci 33, 296-310 (2006).

8. Chen, C.S., Mrksich, M., Huang, S., Whitesides, G.M. & Ingber, D.

 E. Micropatterned surfaces for control of cell shape, position, and function. Biotechnol Prog 14, 356-63 (1998).

9. Rozkiewicz, D.I., Kraan, Y., Werten, M.W., Wolf, F.A., Subramaniam, V., Ravoo, B.J. & Reinhoudt, D.N.Covalent microcontact printing of proteins for cell patterning. Chemistry 12,

6290-7 (2006). 10. Shi, P., Shen, K. & Kam, LC Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro. Dev Neurobiol 67, 1765-76 (2007).

 H. Ladoux, B., Anon, E., Lambert, M., Rabodzey, A., Hersen, P., Buguin, A., Silberzan, P. & Mege, R. M. Strength dependence of cadherin-mediated adhesions. Biophys J 98, 534-42 (2010).

 12.Azioune, A., Carpi, N., Tseng, Q., Thery, M. & Piel, Protein M. micropatterns: A direct printing protocol using deep UVs. Methods CelI Biol 97, 133-46 (2010).

13.Azioune, A., Carpi, N., Fink, J., Chehimi, M. M., Cuvelier, D. & Piel, M. Robust method for high-throughput surface patterning of deformable substrates. Langmuir 27, 7349-52 (2010).

 14. Fink, J., Thery, M., Azioune, A., Dupont, R., Chatelain, F., Bornens, M. & Piel, M. Comparative study and improvement of current cell micro-patterning techniques. Lab Chip 7, 672-80 (2007).

 15. Doyle, A.D., Wang, F.W., Matsumoto, K. & Yamada, K.M. One-dimensional topography underlies three-dimensional fibrillar cell migration. J Cell Biol 184, 481-90 (2009).

 16. Allen, T. G. Preparation and maintenance of single-cell micro-island cultures of basal forebrain neurons. Nat. Protoc. 1, 2543-50 (2006).

17. Wilson, N.R., Ty, M.T., Ingber, D.E., Sur, M. & Liu, G. Synaptic reorganization in scaled networks of controlled size. J Neurosci 27, 13581-9 (2007).

 18. Ruardij, T.G., Goedbloed, M.H. & Rutten, W. L. Adhesion and patterning of cortical neurons on polyethylenimine- and fluorocarbon-coated surfaces. IEEE Trans Biomed Eng 47, 1593-9 (2000).

 19. Fereol, S., Fodil, R., Barnat, M., Georget, V., Milbreta, U. & Nothias, F. Micropatterned ECM substrates reveal complementary contribution of low and high affinity ligands to neurite outgrowth.

Cytoskeleton (Hoboken) (201 1). 20. von Philipsborn, A. C, Lang, S., Bernard, A., Loeschinger, J., David, C., Lehnert, D., Bastmeyer, M. & Bonhoeffer, F. Microcontact printing of axon guidance molecules for generation of graded patterns. Nat. Protoc, 1322-8 (2006).

Philipsborn, A. C, Lang, S., Loeschinger, J., Bernard, A., David, C., Lehnert, D., Bonhoeffer, F. & Bastmeyer, M. Growth cone navigation in substrate-bound ephrin gradients. Development 133, 2487-95 (2006).

 22. Taylor, A.M., Blurton-Jones, M., Rhee, S.W., Cribbs, D.H., Cotman, C.W. & Jeon, N.L. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2, 599-605 (2005).

 23. Paul, D., Saias, L., Pedinotti, J.C., Chabert, M., Magnifico, S., Pallandre, A., De Lambert, B., Houdayer, C., Brugg, B., Peyrin, JM & Viovy, JL A "dry and wet hybrid" lithography technique for multilevel replication templates: Applications to microfluidic neuron culture and two-phase global mixing. Biomicrofluidics 5, 24102 (201 1).

 24. Taylor, A. M. & Jeon, N. L. Micro-scale and microfluidic devices for neurobiology. Curr Opin Neurobiol 20, 640-7 (2010).

 25. Bard, L., Boscher, C., Lambert, M., Mege, R. M., Choquet, D. & Thoumine, O. A molecular clutch between the actin flow and N-cadherin adhesions drives growth cone migration. J Neurosci 28, 5879-90 (2008).

26. Falk, J., Thoumine, O., Dequidt, C., Choquet, D. & Faivre-Sarrailh, C. NrCAM coupling to the cytoskeleton depend on multiple protein domains and partitioning into lipid rafts. Mol Biol Cell 15, 4695-709 (2004).

27. Bresler, T., Ramati, Y., Zamorano, PL, Zhai, R., Garner, CC & Ziv, The Dynamics of SAP90 / PSD-95 Recruitment to New Synaptic Junctions. Mol Cell Neurosci 18, 149-67 (2001). Bresler, T., Shapira, M., Boeckers, T., Dresbach, T., Futter, M., Garner, C., Rosenblum, K., Gundelfinger, ED & Ziv, NE Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly. J Neurosci 24, 1507-20 (2004). Fhedman, HV, Bresler, T., Garner, CC & Ziv, NE Assembly of new individual excitatory synapses: time and synaptic sequence of molecule recruitment. Neuron 27, 57-69 (2000).

Zito, K., Scheuss, V., Knott, G., Hill, T. & Svoboda, K. Rapid functional maturation of nascent dendritic spines. Neuron 61, 247-58 (2009).

Gerrow, K., Romorini, S., Nabi, S.M., Colicos, M.A., Sala, C. & El-Husseini, A. Preformed complex of postsynaptic proteins is involved in excitatory synapse development. Neuron 49, 547-62 (2006).

Graf, E.R., Kang, Y., Hauner, A.M., & Craig, A.M., Functional and Splice Site Analysis of the Synaptogenic Activity of the Neurexin-1 Beta LNS domain. J Neurosci 26, 4256-65 (2006).

Graf, E.R., Zhang, X., Jin, S.X., Linhoff, M.W. & Craig, A.M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 19, 1013-26 (2004).

Heine, M., Thoumine, O., Mondin, M., Tessier, B., Giannone, G. & Choquet, D. Activity-independent and subunit-specific recruitment of functional AMPA receptors at neurexin / neuroligin contacts. Proc Natl Acad Sci U S A 'I OO, 20947-52 (2008).

Scheiffele, P., Fan, J., Choih, J., Fetter, R. & Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 101, 657-69. (2000).

Sudhof, TC Neuroligins and Neuromins. Nature 455, 903-1 (2008). Ko, J., Zhang, C, Arac, D., Boucard, AA, Brunger, AT & Sudhof, TC Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation. Embo J 28, 3244-55 (2009).

Linhoff, MW, Lauren, J., Cassidy, RM, Dobie, FA, Takahashi, H., Nygaard, HB, Airaksinen, MS, Strittmatter, SM & Craig, AM An expression expression screen for synaptogenic proteins identified the LRRTM protein family as synaptic organizers. Neuron 61, 734-49 (2009).

Ko, J., Fuccillo, M.V., Malenka, R.C. & Sudhof, T.C. LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation. Neuron 64, 791-798 (2009).

of Wit, J., Sylwestrak, E., O'Sullivan, M., Otto, S., Tiglio, K., Savas, JN, Yates, JR, Comoletti, D., Taylor, P. & Ghosh, A. LRRTM2 Interacts with Neurexin 1 and Regulatory Excitatory Synapse Formation. Neuron 64, 799-806 (2009).

Craig, A. M. & Kang, Y. Neurexin-neuroligin signaling in synapse development. Curr Opin Neurobiol M, 43-52 (2007).

Chubykin, A.A., Atasoy, D., Etherton, M.R., Brose, N., Kavalali, E.T., Gibson, J.R. & Sudhof, T.C.Activity-Dependent Validation of Excitatory versus Inhibitory Synapses by Neuroligin-1 versus Neuroligin-2. Neuron 54, 919-31 (2007).

Missler, M., Zhang, W., Rohlmann, A., Kattenstroth, G., Hammer, R.E., Gottmann, K. & Sudhof, T.C. Alpha-neurexins Ca2 + couple channels to synaptic vesicle exocytosis. Nature 423, 939-48 (2003). Varoqueaux, F., Aramuni, G., Rawson, R. L, Mohrmann, R., Missler, M., Gottmann, K., Zhang, W., Sudhof, TC & Brose, N. Neuroligins determines synapse maturation and function . Neuron 51, 741-54 (2006).

Takahashi, H., Arstikaitis, P., Prasad, T., Bartlett, TE, Wang, YT, Murphy, TH & Craig, AM Postsynaptic TrkC and presynaptic PTPsigma function as a bidirectional excitatory synaptic organizing complex. Neuron 69, 287-303 (201 1).

Jamain, S., Quach, H., Betancur, C., Rastam, M., Colineaux, C., Gillberg, I. C., Soderstrom, H., Giros, B., Leboyer, M., Gillberg, C. & Bourgeron , T. Mutations of the X-linked Gene Encoding Neuroligins NLGN3 and NLGN4 are associated with autism. Nat. Genet 34, 27-9 (2003).

Talebizadeh, Z., Lam, D.Y., Theodoro, M.F., Bittel, D.C., Lushington, G.H. & Butler, M.G. Novel splice isoforms for NLGN3 and NLGN4 with possible implications in autism. J Med Genet 43, e21 (2006).

Rujescu, D., Ingason, A., Cichon, S., Pietilainen, OP, Barnes, MR, Toulopoulou, T., Picchioni, M., Vassos, E., Ettinger, U., Bramon, E., Murray, R., Ruggeri, M., Tosato, S., Bonetto, C., Steinberg, S., Sigurdsson, E., Sigmundsson, T., Petursson, H., Gylfason, A., Olason, PI, Hardarsson, G. , Jonsdottir, GA, Gustafsson, O., Fossdal, R., Giegling, I., Moller, HJ, Hartmann, AM, Hoffmann, P., Crombie, C, Fraser, G., Walker, N., Lonnqvist, J ., Suvisaari, J., Tuulio-Henriksson, A., Djurovic, S., Melle, I., Andreassen, OA, Hansen, T., Werge, T., Kiemeney, LA, Franke, B., Veltman, J ., Buizer-Voskamp, JE, Sabatti, C, Ophoff, RA, Rietschel, M., Nothen, MM, Stefansson, K., Peltonen, L., St Clair, D., Stefansson, H. & Collier, DA Disruption of the neurexin 1 gene is associated with schizophrenia. Hum Mol Genet 18, 988-96 (2009). Williams, E.J., Williams, G., Gour, B., Blaschuk, O. & Doherty, P. INP, has a novel N-cadherin antagonist targeted to amino acids that flank the HAV motif. Mol Cell Neurosci 15, 456-64 (2000).

Williams, E., Williams, G., Gour, BJ, Blaschuk, OW & Doherty, P. A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif. J Biol Chem 275, 4007-12 (2000). 51. Perret, E., Benoliel, AM, Nassoy, P., Stones, A., Delmas, V., Thiery, JP, Bongrand, P. & Feracci, H. Fast dissociation kinetics between individual E-cadherin fragments revealed by flow chamber analysis. Embo J 2 †, 2537-46 (2002).

52. Perret, E., Leung, A., Feracci, H. & Evans, E. Trans-bonded peers of E-cadherin, a remarkable hierarchy of mechanical strengths. Proc Natl Acad Sci U S A 101, 16472-7 (2004).

 53. Paradis, S., Harrar, DB, Lin, Y., Koon, A.C, Hauser, J.L., Griffith, E.C., Zhu, L., Brass, LF, Chen, C. & Greenberg, ME An RNAi-based approach identified molecules required for glutamatergic

GABAergic synapse development. Neuron 53, 217-32 (2007).

 54. Dean, C, Scholl, F.G., Choih, J., DeMaria, S., Berger, J., Isacoff, E.

 & Scheiffele, P. Neurexin medias the assembly of presynaptic terminais. Nat Neurosci 6, 708-16. (2003).

55. Levinson, JN, Chery, N., Huang, K., Wong, TP, Gerrow, K., Kang, R., Prange, O., Wang, YT & El-Husseini, A. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1 beta in neuroligin-induced synaptic specificity. J Biol Chem 280, 17312-9 (2005).

56. Sainlos, M., Tigaret, C, Poujol, C, Olivier, NB, Bard, L., Breillat, C, Thiolon, K., Choquet, D. & Imperiali, B. Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization. Nat Chem Biol 7, 81-91 (2010).

 57. Bard, L., Sainlos, M., Bouchet, D., Cousins, S., Mikasova, L., Breillat, C., Stephenson, F. A., Imperiali, B., Choquet, D. & Groc, L.

Dynamic and specifies interaction between synaptic NR2-NMDA receptor and PDZ proteins. Proc Natl Acad Sci U S A 107, 19561 -6 (2010).

 58. Chih, B., Engelman, H. & Scheiffele, P. Control of excitatory and inhibitory synapse formation by neuroligins. Science 307, 1324-8

(2005). Kamiguchi, H., Hlavin, ML & Lemmon, V. Role of L1 in neural development: what the knockouts tell us. Mol Cell Neurosci 12, 48-55 (1998).

Suzuki, S. C, Smoke, H., Koga, K., Jiang, N., Nohmi, M., Shimazaki, Y., Katoh-Fukui, Y., Yokoyama, M., Yoshimura, M. & Takeichi, M. Cadherin-8 is required for the first relay synapses to receive functional inputs from primary sensory afferents for cold sensation. J Neurosci 27, 3466-76 (2007).

Etherton, M.R., Tabuchi, K., Sharma, M., Ko, J. & Sudhof, T.C.A. autism-associated point mutation in the cytoplasmic cytoplasmic tail selectively odd AMPA receptor-mediated synaptic transmission in hippocampus. EMBO J 30, 2908-19.

Tabuchi, K., Blundell, J., Etherton, M.R., Hammer, R.E., Liu, X., Powell, C.M. & Sudhof, T.C. Neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318, 71-6 (2007).

Bhushan, Bharat Hansford, Derek Lee, Kang Kug "Surface modification of silicon and polydimethylsiloxane surfaces with vapor-phase-deposited ultrathin fluorosilane films for biomedical nanodevices", Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films, Jul 2006, 24 (4), 1 197 - 1202

Isabelle Collin cvc, Genetic engineering, "transgenic animals", edition Les Essentiels milans, 1999,

 Handbook of Biochemistry and Molecular Biology, Fourth Edition 201 1 - Editor (s): Roger L. Lundblad, Lundblad Biotechnology, Chapel Hill, North Carolina, USA; Fiona Macdonald, CRC Press, Boca Raton, Florida, USA,

Gerald D. Fasman Practical Handbook of Biochemistry and Molecular Biology CRC Press, 1989 - 601 pages; Recombinant DNA Principles and Methodologies, Editor (s): James Greene, Catholic University, Washington, DC, USA CRC Press, 1998.

Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, ET, Sudhof, TC SynCAM, Synaptic adhesion molecule that drives synapse assembly, Science 297 1525-31 (2002) )

Sara, Y., Biederer, T., Atasoy, D., Chubykin, A., Mozhayeva, M.G., Sudhof, T.C., Kavalali, E. T. Selective capability of SynCAM and neuroligin for functional synapse assembly. J Neuroscience 25 260-270 (2005).

Shi P., Scott M.A., Ghosh B., Wan D., Wissner-Gross Z., Mazitschek R., Haggarty S.J., Fatih M. Synapse microarray identification of small molecules that enhance synaptogenesis. Nat. Commun. 2, 510 (201 1).

Breillat, C., Thoumine O., Choquet, D. Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity, Biochem Biophys Res Commun, 359, 655-659 (2007).

Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, ET, Sudhof, TC SynCAM, Synaptic adhesion molecule that drives synapse assembly, Science 297 1525-31 (2002) ).

Sara, Y., Biederer, T., Atasoy, D., Chubykin, A., Mozhayeva, M.G., Sudhof, T.C, Kavalali, E. T. Selective capability of SynCAM and neuroligin for functional synapse assembly. J Neuroscience 25 260-270 (2005).

Breillat, C., Thoumine O., Choquet, D. Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity, Biochem Biophys Res Commun, 359, 655-659 (2007).

Claims

1. Support (1) for culturing neuronal cells (c) comprising a substrate (3) on a surface (5) of which islets (7) of adhesion proteins of said neuronal cells chosen from the immunoglobulin-type adhesion molecules are arranged (IgCAM), cadherins, neurexins, neuroligins, and leucine-rich domain transmembrane proteins or a mixture thereof, said islands (7) having a diameter of 0.2 to 2.5 μιτι and being spaced apart between them from 0.500 to 10 pm. The carrier of claim 1 wherein the adhesion proteins of said neuronal cells are selected from synaptic adhesion molecules (SynCAM) or a mixture thereof. 3. Support according to claim 1 or 2, wherein the surface (5) between the islands (7) is a cytophobic surface (9). The carrier of any one of claims 1 to 3, wherein said adhesion proteins are chemically bonded either directly to the surface (5) or via an intermediate binder attached to said surface. (5), said binder being selected from a reactive chemical species, a protein, a polysaccharide, an antibody, a polymer, a charged species. The carrier of any one of claims 1 to 4, wherein the neuronal cell adhesion protein is an adhesion protein derived from a neuronal cell membrane or a physiological membrane of non-neuronal cells supporting a neuronal cell. 6. Support according to any one of claims 1 to 5 wherein the surface (5) is made cytophobic because of a compound selected from polyethylene glycol, poly (vinyl acetate), poly (2-hydroxyethyl methacrylate, polyacrylamide, poly (N-vinyl-2-pyrrolidone), poly (N-isopropyl acrylamide), anionic polymers, phosphoryl choline polymers, albumin, casein, hyaluronic acid , liposaccharides, glycoproteins, phospholipids or a mixture thereof. 7. Support according to any one of claims 1 to 6, wherein the islands (7) are deposited on the surface (5) by a method selected from photolithography, dip-pen nanolithography, beam lithography, inkjet printing, microcontact printing. 8. Support according to any one of claims 1 to 7 comprising neuronal cells on the islands (7). The carrier of claim 8 wherein said neuronal cells are selected from the group consisting of neuronal cells derived from the nervous system of a mammal, bird, batrachian, or mollusk, and / or any cell derived from a neuronal cell line. . 10. Support according to claims 8 or 9 wherein the neuronal cells are isolated from the nervous system of rodents, chickens, amphibians, molluscs. 1 1. A carrier according to any one of claims 8 to 10 wherein the neuronal cells are selected from the SH-SY5Y neuroblastoma line, the PC12 line, the cortical neuronal cell line (HCN-1), or the B104 neuroblastoma line. 12. A method of manufacturing a support according to any one of claims 1 to 1 1, comprising a step of depositing on a surface (5) of an islet substrate (7) of neuronal cell adhesion proteins. selected from immunoglobulin-like molecules (IgCAM), cadherins, neurexins, neuroligins, and leucine-rich domain transmembrane proteins or a mixture thereof, said islets (7) having a diameter of 0, 2 to 2.5 μιτι and being spaced between them from 0.500 to 10 μιτι. The method of claim 12, wherein the method further comprises a step of coating a cytophobic compound selected from polyethylene glycol, polyvinyl acetate, poly (2-hydroxyethyl methacrylate, polyacrylamide, poly (N) -vinyl-2-pyrrolidone), poly (N-isopropyl acrylamide), anionic polymers, phosphoryl choline polymers, albumin, casein, hyaluronic acid, liposaccharides, glycoproteins, phospholipids or a mixture of these, said coating step being prior to the deposition step of the islands of adhesion proteins. 14. Use of a carrier according to any one of claims 1 to 1 1 for culturing neuronal cells. 15. Use of a support according to any one of claims 1 to 1 1 in a qualitative and / or quantitative analysis method of neurite growth and synaptogenesis of said neuronal cells. 16. Use of a support according to any one of claims 1 to 1 1 in a screening method.