PRECISELY CONTROLLED GRADIENTS FOR AXON GUIDANCE

描述(逐字摘自申请者摘要)：随着大脑的发育， 为了找到合适的靶点，轴突通常会长出很长的距离。其中一些 他们用来实现这一壮举的最重要的指导线索是专注 趋化分子的梯度。这些梯度是通过生长检测到的 锥体，发育中轴突顶端的一种复杂而敏感的结构。 了解生长锥体如何检测梯度对于理解 大脑是如何正常发育的，正常线路的缺陷是如何发生的 轴突如何在受伤后适当再生。然而，还有 目前关于生长锥体如何反应的定量信息很少 到渐变。这主要是因为没有实验技术。 为了在体外建立可扩散轴突引导分子的梯度，例如 渐变的形状和陡度由 实验者。如果没有这样的技术，就不可能有系统地改变 梯度的参数，因此不可能定量确定 这些参数中的每一个如何调节轴突引导。这个项目的目标是 是开发和实施一种新的技术来进行这些测量 有可能。 我们将通过将趋化分子“打印”到 长而薄的胶原凝胶表面，轴突在其中生长，以及 让分子扩散到胶原蛋白中。精确控制 可精确控制趋化因子的体积 使用与喷墨打印机中使用的方法类似的方法。因为 分子扩散运动短距离快，长距离慢 距离，浓度中的初始局部不均匀在几个时间内消除 几个小时，留下了一个在几个小时内保持平滑和稳定的梯度 几周。因此，我们可以完全控制这个梯度的形状。 控制沉积在表面的分子图案。通过观察 在这些梯度中轴突的生长和转向行为我们将获得定量的 关于控制轴突引导的参数的空前精确的数据 扩散渐变。我们的初始模型系统将以大鼠为指导 神经生长因子梯度作用下的背根节轴突。然而，我们 希望这项技术也能广泛应用于其他模型系统 轴突指导，以及更广泛的细胞运动性和趋化性研究。

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.