Thermoresponsive polymer coatings to control the direction of synaptic transmission in artificial neuronal networks

人工神经元网络的分析提高了人们对神经元信息处理的理解和建立体外神经药理学测试系统的期望。为了在体外从分离的神经元细胞中再现神经组织的高度组织化的结构，不仅必须精确控制这种网络中单个细胞之间突触传递的位置，而且还必须精确控制突触传递的方向。然而，尽管世界各地的许多工作组作出了巨大努力，但这方面的工作尚未得到令人满意的管理。本项目的目的是调查，微结构的表面涂层的温敏聚合物（TRP）是否可以用于创建具有定义的连接模式的神经网络。TRP涂层以温度依赖性的方式经历从细胞排斥性到细胞友好状态的相变，这可以允许神经突生长的限定途径的优雅激活或失活。通过微加工技术，我们将开发一种TRP涂层的细胞培养基质，该基质具有集成的微型加热元件，使我们能够在空间上控制微米范围内的表面温度。动态和空间控制基底细胞粘附特性的可能性将形成3步细胞测定的基础，用于（1）控制基底上的细胞位置，（2）通过控制长出神经突的方向来控制细胞极化，以及（3）在此之后，允许相邻极化细胞彼此接触并形成功能性突触。为了评估和优化我们的TRP涂层和细胞培养方案的神经相容性，我们将通过单细胞力显微镜（SCFM）研究神经元细胞在涂有TRP和其他粘附促进剂的表面上的初始粘附，并进行细胞生物学，电生理学以及免疫细胞化学分析。定义的连接模式的神经元网络的创建将使重要的神经生物学问题的新的方法论方法，例如在基础科学或阿尔茨海默病或帕金森病的药物开发中的长时程增强（LTP）的背景下。

The analysis of artificial neuronal networks is raising great expectations with regard to the understanding of neuronal information processing and for the establishment of in vitro neuro-pharmacological test systems. In order to reproduce the highly organized architecture of neuronal tissue from dissociated neuronal cells in vitro, not only the position but also the direction of synaptic transmission between the individual cells in such networks has to be precisely controlled. However, despite strong efforts of numerous work groups worldwide this has not yet been satisfactorily managed. Aim of the present project is to investigate, whether microstructured surface coatings of thermoresponsive polymers (TRP) can be used for creating neuronal networks with defined connectivity patterns. TRP coatings undergo a phase transition from a cell-repellent into a cell-friendly state in a temperature-dependent manner, which could allow for the elegant activation or deactivation of defined pathways for neurite outgrowth. With microfabrication techniques we will develop a TRP-coated cell cultivation substrate with integrated micro heating elements that will allow us to spatially control the surface temperature in the µm-range. The possibility to dynamically and spatially control the substrate`s cell adhesive properties will form the basis for a 3-step cell assay for (1) controlling the cell position on the substrate, (2) controlling cell polarization by controlling the direction for outgrowing neurites and (3) after this, allowing neighboring polarized cells to contact each other and form functional synapses. For evaluation and optimization of the neurocompatibility of our TRP coatings and cell cultivation protocols we will investigate the initial adhesion of neuronal cells on surfaces coated with TRP and additional adhesion promoters via Single Cell Force Microscopy (SCFM) and conduct cell biological, electrophysiological as well as immunocytochemical analyses. The creation of neuronal networks of defined connectivity pattern will enable new methodological approaches to important neurobiological questions for example in the context of long-term potentiation (LTP) in basic science or pharmaceutical drug development for Alzheimer`s or Parkinson`s disease.