Taylor A M, Menon S, Gupton S L
UNC/NCSU Joint Department of Biomedical Engineering, UNC-Chapel Hill, Campus Box 7575, Chapel Hill NC 27599-7575, USA.
Lab Chip. 2015 Jul 7;15(13):2781-9. doi: 10.1039/c5lc00503e. Epub 2015 May 22.
Understanding how axons are guided to target locations within the brain is of fundamental importance for neuroscience, and is a widely studied area of research. Biologists have an unmet need for reliable and easily accessible methods that generate stable, soluble molecular gradients for the investigation of axon guidance. Here we developed a microfluidic device with contiguous media-filled compartments that uses gravity-driven flow to generate a stable and highly reproducible gradient within a viewing compartment only accessible to axons. This device uses high-resistance microgrooves to both direct the growth of axons into an isolated region and to generate a stable gradient within the fluidically isolated axon viewing compartment for over 22 h. Establishing a stable gradient relies on a simple and quick pipetting procedure with no external pump or tubing. Since the axons extend into the axonal compartment through aligned microgrooves, the analysis of turning is simplified. Further, the multiple microgrooves in parallel alignment serve to increase sample sizes, improving statistical analyses. We used this method to examine growth cone turning in response to the secreted axon guidance cue netrin-1. We report the novel finding that growth cones of embryonic mouse cortical axons exhibited attractive turning in the lower concentrations of netrin-1, but were repulsed when exposed to higher concentrations. We also performed immunocytochemistry in growth cones exposed to a netrin-1 gradient within the axon viewing compartment and show that netrin receptors associated with both attraction and repulsion, DCC and UNC5H, localized to these growth cones. Together, we developed an accessible gradient chamber for higher throughput axon guidance studies and demonstrated its capabilities.
了解轴突如何被引导至脑内的目标位置对神经科学至关重要，并且是一个被广泛研究的领域。生物学家迫切需要可靠且易于获取的方法来生成稳定、可溶的分子梯度，以用于轴突导向研究。在此，我们开发了一种具有连续填充介质隔室的微流控装置，该装置利用重力驱动流在仅轴突可进入的观察隔室内产生稳定且高度可重复的梯度。此装置使用高阻微槽来引导轴突生长至一个孤立区域，并在流体隔离的轴突观察隔室内产生超过22小时的稳定梯度。建立稳定梯度依赖于一个简单快速的移液操作，无需外部泵或管道。由于轴突通过对齐的微槽延伸到轴突隔室，转弯分析得以简化。此外，多个平行排列的微槽有助于增加样本量，改善统计分析。我们使用这种方法来检测生长锥对分泌的轴突导向信号分子netrin-1的反应。我们报告了一个新发现，即胚胎小鼠皮质轴突的生长锥在较低浓度的netrin-1中表现出吸引性转弯，但在暴露于较高浓度时则被排斥。我们还在轴突观察隔室内暴露于netrin-1梯度的生长锥中进行了免疫细胞化学实验，结果表明与吸引和排斥相关的netrin受体DCC和UNC5H定位于这些生长锥。我们共同开发了一个便于使用的梯度室，用于更高通量的轴突导向研究，并展示了其功能。
