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衬底图案化方法，该方法克服了与预先图案化的衬底上的随机细胞结合相关的限制。我们的方法提供了在细胞电镀后创建限制、遏制、允许或直接生长的模式的能力。电镀后构图底物的能力允许选择特定的细胞亚群(即，转基因或敲除细胞)，并根据生长的变化修改图案。当与包含一种以上材料的预先图案化的衬底结合时，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电路。我们的图案化技术类似于无掩模光刻，使用高能脉冲红外激光直接解粘衬底材料。蛋白质被去除的区域变得不允许神经元生长。我们将使用底物构图、微流体和电生理学相结合的方法来创建和测试海马神经元的功能神经元电路。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的发生、发育和动力学的影响和调节。我们还将利用Myosin IIB基因敲除小鼠的细胞，研究外源因子对脊柱形成和成熟的影响，以及在没有Myosin IIB的情况下脊柱动力学的变化。最后，我们将研究Rab5在简单电路连接的海马神经元突触前功能中的作用。与公众健康相关我们已经开发出一种在活细胞存在的情况下对底物进行图案化的新方法，这种方法可以在细胞培养中创建具有已知连接性的神经元电路。这将促进我们对神经细胞之间通信的理解，并促进对大脑回路的建模。这对于了解正常的大脑功能和从脑或脊髓损伤中恢复的潜力很重要。

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.