This work was supported by the European Project BRIANBOW (FP7- Young Explorers, The authors would like to thank Dr. Jacopo Tessadori for useful comments on the manuscript, and Silvia Chiappalone for her help in producing the graphics used in the video.

The procedure was done in accordance with the NIH standards for care and use of laboratory animals and was approved by the Tel-Aviv University Animal Care and Use Committee (permit number &#8211; L-14-019).
1. Preparation of Instruments and PDMS
<ol>
	<li>Prepare the wafer (Table of Materials, or order the wafer from a microfabrication lab), a scalpel, and a pair of tweezers &#8211; sterilization is not needed.</li>
	<li>Make poly-D-lysine (PDL) solution according to the fol...

<ol>
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The authors have nothing to disclose.

A protocol to grow 2D modular neuronal networks in vitro composed of functionally inter-connected circuits is described. The procedure is based on patterning a cellular adhesive layer. Patterning is achieved with PDMS stencils reproducing the negative feature of the desired network architecture. PDMS stencils define the areas where the cellular adhesive layer is deposited. Once cells are plated, they spontaneously assemble to the coated islands and self-organize into active inter-connected circuits. Recordings o...

Experimental and theoretical evidences support the possibility that the brain operates through coordinated activation of cell assemblies1-5, which can be regarded as dynamic functional units that transiently interact with each other, shaping and underlying different brain states. Functional modularity is also dependent on and associated with the structural modular organization of the brain circuits6,7. How function and structure of brain circuits mutually shape each other is still one of the main open questions in neuroscience. To provide a deeper understanding of this question, it is important to identify optimal experimental frameworks where it is possible to address, at least partially, those issues. Since controlled manipulation of the spatio-temporal dynamics of neuronal networks in in vivo experiments is challenging, the development of in vitro neuronal networks models is of significant interest due to their easy accessibility, monitoring, manipulation and modeling8,9. In recent years, in vitro technologies supported by advanced substrate patterning methods have allowed to induce neuronal networks to develop a range of predefined modular structures3 and to study the functional properties of networks with imposed topologies10. In particular, methods were recently used to organize networks by imposing physical constraints4,11. Indeed, to study the link between structure and function in neuronal networks and to provide a simplified but plausible representation of interacting neuronal assemblies, in vitro systems should provide inter-connected neuronal sub-populations. Widely studied 2D homogenous neuronal cultures do not impose any spatial constraints on the self-organized emergent wiring of the circuits. Therefore a possible approach to shape artificially interconnected cell assemblies is to position different neuronal populations in spatially distinct areas. The distance among these areas does not prevent the inter assemblies connections. This approach, while ensuring a considerable control over network complexity, has been shown to provide a richer repertoire of synchronization models6,7,12.
In order to facilitate a reproducible culturing of modular neuronal assemblies, a protocol to assemble the self-organization of networks into neuronal clusters linked by axons and dendrites is presented and described. The polymeric structure for the physical confinement of neuronal cultures has been created from polydimtheylsiloxane (PDMS). PDMS is an elastomer widely used for biomedical applications owing to its biocompatibility, transparency and permeability to gases13. The PDMS is prepared and excluded from the micromachined SU8 207514,15 structures by spin-coating a liquid PDMS onto a &#34;master&#34; as described previously in Jackman et al.16 The achieved patterned neuronal networks are composed of inter-connected modules of different size and they have been successfully obtained on both coverslips and Micro Electrode Arrays (MEAs)17-20. The density of connections between the modules can change the features of the network synchronization, from a fully synchronized network, typical of uniform cultures, to transient states of synchronization among modules.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PDMS, Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Vacuum Chamber</td>
			<td>Thermo</td>
			<td>5305-0609</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine PDL</td>
			<td>Sigma</td>
			<td>P7886</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell - Technologies Corporation</td>
			<td>WS-650-23</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well culture plate</td>
			<td>Sigma</td>
			<td>CLS3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8217;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA1060-Inv-BC</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC02</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Biological Industries Beit Haemek</td>
			<td>03-033-1c</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Minimum Essential Medium-Eagle</td>
			<td>Biological Industries Beit Haemek</td>
			<td>01-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Electrode Arrays 4Q</td>
			<td>Multi Channel Systems</td>
			<td>60-4QMEA1000iR-Ti-pr</td>
			<td>cleaning manual: http://www.multichannelsystems.com</td>
		</tr>
		<tr>
			<td>silicon wafer</td>
			<td>microchem</td>
			<td>SU8-2075</td>
			<td>Preparation protocol: www.microchem.com</td>
		</tr>
	</tbody>
</table>

design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

A SU8-2075 mold on a silicon wafer with a feature thickness of approximately 100 &#181;m was used to shape the PDMS. The pattern was composed of squares of several dimensions, with a side length and distance varying between 200 and 700 &#181;m (Figure 1B). The size of the square was chosen to fit the field of view of a 10X (for islands with a side length &#60;800 &#181;m) and of a 20X objective (for islands with a side length &#60;400 &#181;m). Three parameters, namely cell plating density, distance betw...

Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

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8.4K 조회수.  Tel-Aviv University.  이 원고는 공간적으로 제한, 기능적으로 상호 연결된 신경 회로로 구성된 체외 모듈 형 네트워크에서 성장하는 프로토콜을 설명합니다. 마스크는 폴리머 기판 위에 배양 세포 접착을 촉진하는 단백질 패턴 층을 사용한다. 도금 뉴런 코팅 분야 자발적인 연결을 설정 및 전기 생리 활성을 나타내는에서 성장. 