Kanner Sivan, Bisio Marta, Cohen Gilad, Goldin Miri, Tedesco Marieteresa, Hanein Yael, Ben-Jacob Eshel, Barzilai Ari, Chiappalone Michela, Bonifazi Paolo
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University.
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia.
J Vis Exp. 2015 Apr 15(98):52572. doi: 10.3791/52572.
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.
大脑通过神经元组件的协同激活和动态通信来运作。一个主要的开放性问题是，构成大多数不同脑功能基础的大量动态基序，如何能从大脑回路固定的拓扑和模块化组织中产生。与存在内在实验困难的神经元回路体内研究相比，体外制备为操纵和探究实验性神经元系统的结构、动态和化学特性提供了更大的可能性。这项工作描述了一种体外实验方法，该方法允许生长由空间上不同、功能上相互连接的神经元组件组成的模块化网络。该方案允许在不同拓扑复杂度水平上控制神经元网络的二维（2D）结构。在规则的盖玻片和嵌入微电极阵列的基板上都可以实现所需的网络图案化。微加工结构被压印在硅片上，并用于创建生物相容性聚合物模板，该模板包含所需网络架构的负特征。在表面涂覆促进细胞粘附的分子层的过程中，将模板放置在培养基板上。去除模板后，接种神经元，它们会自发地重新定向到涂覆区域。通过减小隔室间距离，可以获得孤立的或相互连接的神经元回路。为了促进细胞存活，将细胞与位于培养皿周边的支持性神经元网络共培养。展示了分别使用嵌入微电极阵列的基板和钙成像获得的模块化网络活动的电生理和光学记录。虽然每个模块都显示出自发的全局同步，但模块间同步的发生受回路间连接密度的调节。
