PATTERNING SUBSTRATES IN THE PRESENCE OF LIVING CELLS TO PRODUCE NEURAL CIRCUITS

描述（由申请人提供）：我们设计了一种新的基底图案化方法，其克服了与预图案化基底上的随机细胞结合相关的限制。 我们的方法提供了创建模式的能力，这些模式在细胞接种后限制、包含、允许或指导生长。 在铺板后图案化基底的能力允许选择特定的细胞亚群（即，转染的或敲除的细胞）和响应生长变化的模式改变。 当与包含多于一种材料的预图案化基底组合时，可以诱导神经元的极化并定向相互作用。 使用这种方法，我们能够创建具有不同复杂性和已知连接性的神经元回路。 我们的图案化技术类似于无掩模光刻，并使用高能量脉冲IR激光直接解开衬底材料。 蛋白质被去除的区域变得不允许神经元生长。 我们将使用基板图案化，微流体和电生理学的组合来创建和测试海马神经元的功能神经元回路。 一旦我们验证了功能并优化了方法，我们将专注于解决特定的生物学问题。 这将包括研究神经活动对树突棘出现，发育和动态的影响和调节。 我们还将研究应用外源性因子对脊柱形成和成熟的影响，以及在没有肌球蛋白IIB的情况下使用肌球蛋白IIB基因敲除小鼠细胞的脊柱动力学的改变。 最后，我们将研究Rab5在简单回路中连接的海马神经元突触前功能中的作用。 公共卫生相关性我们已经开发出一种在活细胞存在下图案化基底的新方法，该方法允许在细胞培养物中创建具有已知连接性的神经元回路。 这将促进我们对神经细胞之间通信的理解，并促进大脑回路的建模。 这对于了解正常的脑功能以及从脑或脊髓损伤中恢复的潜力非常重要。

DESCRIPTION (provided by applicant): We have devised a new substrate patterning method that overcomes the limitations associated with random cell binding on pre-patterned substrates. Our approach provides the ability to create patterns that restrict, contain, allow or direct growth after cells have been plated. The ability to pattern substrates after plating allows selection of specific subsets of cells (i.e., transfected or knockout cells) and modification of patterns in response to changes in outgrowth. When combined with pre-patterned substrates comprising more than one material, polarization of neurons can be induced and interactions oriented. Using this approach we are able to create neuronal circuits with varying complexity and known connectivity. Our patterning technique is similar to maskless photolithography and uses a high-energy pulsed IR laser to directly unbind substrate materials. The areas where protein is removed become non-permissive for neuronal growth. We will use the combination of substrate patterning, microfluidics and electrophysiology to create and test functional neuronal circuits of hippocampal neurons. Once we have verified function and optimized the approach, we will focus on addressing specific biological questions. This will include studying the influence and regulation of neural activity on dendritic spine emergence, development and dynamics. We will also study the influence of an applied exogenous factor on spine formation and maturation, as well as alterations in spine dynamics in the absence of myosin IIB using cells from myosin IIB knockout mice. Finally, we will study the role of Rab5 in presynaptic function of hippocampal neurons connected in simple circuits. PUBLIC HEALTH RELEVANCE We have developed a new method of patterning substrates in the presence of living cells that allows creation of neuronal circuits with known connectivity in cell culture. This will advance our understanding of communication between nerve cells and facilitate modeling of brain circuits. This is important for understanding normal brain function and the potential for recovery from brain or spinal cord injury.