PRECISELY CONTROLLED GRADIENTS FOR AXON GUIDANCE

描述（来自申请人摘要的逐字）：随着大脑的发育， 轴突通常长出很长的距离来寻找合适的目标。一些 它们用来完成这一壮举的最重要的引导线索是集中注意力 向化性分子的梯度。这些梯度是由生长 视锥，一种位于发育中的轴突顶端的复杂而敏感的结构。 了解生长锥如何检测梯度对于理解 大脑如何正常发育，正常线路的缺陷如何发生， 轴突如何在受伤后适当再生。不过有 目前关于生长锥如何响应的定量信息很少 梯度。这主要是由于缺乏实验技术 为了在体外建立可扩散轴突引导分子的梯度， 这是一个很大的问题，也是一个很大的问题。 实验者没有这种技术，就不可能系统地改变 梯度的参数，因此不可能定量确定 这些参数是如何调节轴突导向的这个项目的目标 是开发和实施一种新的技术来进行这些测量 可能 我们将建立梯度“打印”化学分子到 长而薄的胶原凝胶块的表面，轴突在其中生长，以及 使分子扩散到胶原蛋白中。精确控制 大量的趋化因子可以在精确控制的条件下沉积， 使用类似于喷墨打印机中所采用的方法的位置。因为 通过扩散的分子运动在短距离内快，但在长距离内慢 距离，浓度的初始局部不均匀性在几个小时内逐渐消失。 小时，留下一个梯度，保持平稳和稳定的几个小时， 周因此，我们可以完全控制这个梯度的形状， 控制沉积在表面上的分子图案。通过观察 轴突生长和转向行为在这些梯度，我们将获得定量 控制轴突引导的参数的空前精确的数据， 扩散梯度我们最初的模型系统将是大鼠的指导 背根神经节轴突的神经生长因子的梯度。但我们 我希望这项技术也能广泛应用于其他模型系统， 轴突引导，更一般地涉及细胞运动性和趋化性的研究。

DESCRIPTION (Verbatim from the Applicant's Abstract): As the brain develops, axons often grow over long distances to find appropriate targets. Some of the most important guidance cues they use to achieve this feat are concentration gradients of chemotropic molecules. These gradients are detected by the growth cone, a complex and sensitive structure at the tip of the developing axon. Understanding how growth cones detect gradients is crucial for understanding how the brain normally develops, how defects in normal wiring can occur, and how axons can appropriately regenerate after injury. However, there is currently very little quantitative information about how growth cones respond to gradients. This is primarily due to the absence of an experimental technique for establishing gradients of diffusible axon guidance molecules in vitro, such that the shape and steepness of the gradient is under the control of the experimenter. Without such a technique it is impossible to systematically vary the parameters of the gradient, and thus impossible to determine quantitatively how each of these parameters regulates axon guidance. The goal of this project is to develop and implement a novel technique to make these measurements possible. We will establish gradients by "printing" chemotropic molecules onto the surface of a long, thin block of collagen gel in which axons are growing, and allowing the molecules to diffuse into the collagen. Precisely controlled volumes of chemotropic factor can be deposited at precisely controlled locations using a method similar to that employed in inkjet printers. Because molecular movement by diffusion is fast over short distances but slow over long distances, initial local unevenness in the concentration smoothes out in a few hours, leaving behind a gradient that remains smooth and stable for several weeks. We thus have complete control over the shape of this gradient via control over the pattern of molecules deposited on the surface. By observing axon growth and turning behavior in these gradients we will obtain quantitative data of unprecedented precision on the parameters controlling axon guidance by diffusible gradients. Our initial model system will be the guidance of rat dorsal root ganglion axons by gradients of Nerve Growth Factor. However, we expect this technology to be also widely applicable to other model systems for axon guidance, and more generally to studies of cell motility and chemotaxis.