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.
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University.
Sivan
S
Kanner
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia.
Marta
M
Bisio
School of Electrical Engineering, Tel-Aviv University.
Gilad
G
Cohen
School of Physics and Astronomy, Tel-Aviv University.
Miri
M
Goldin
Department of Informatics, Bioengineering, Robotics and System Engineering, University of Genova.
Marieteresa
M
Tedesco
School of Electrical Engineering, Tel-Aviv University.
Yael
Y
Hanein
School of Physics and Astronomy, Tel-Aviv University.
Eshel
E
Ben-Jacob
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University.
Ari
A
Barzilai
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia.
Michela
M
Chiappalone
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel-Aviv University; School of Physics and Astronomy, Tel-Aviv University; paol.bonifazi@gmail.com.
Paolo
P
Bonifazi
D000818:Animals; D001369:Axons / Q000502:physiology; D001921:Brain / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D003712:Dendrites / Q000502:physiology; D009434:Neural Pathways / Q000502:physiology; D009474:Neurons / Q000502:physiology*