Can Hippocampal Neurites and Growth Cones Climb over Obstacles?

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 µm wide, with 4 µm period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 – do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles.导向分子（guidance molecules）如Sema3A或Netrin-1，在平坦表面环境下可诱导生长锥（growth cone, GC）产生排斥或吸引反应，但目前针对障碍物存在时导向分子的作用机制，相关研究仍十分匮乏。为此，本研究结合化学与机械信号调控手段，向接种于经图案化处理的聚二甲基硅氧烷（polydimethylsiloxane, PDMS）表面的解离海马神经元的生长锥施加持续的Netrin-1流；该PDMS表面形成了宽度2 μm、周期4 μm、高度范围100~600 nm的线条结构。接种后24小时开展的生长锥转向实验结果显示，丝状伪足（filopodia）可在数分钟内爬越此类线条，且未检测到黏附标志物桩蛋白（Paxillin）的染色信号。生长锥与神经突可顺利爬越高度为100 nm的线条，但爬越高度超过300 nm的线条时，频率显著降低且耗时更长；神经突始终无法跨越600 nm高的线条。当神经元在图案化表面培养3天后，神经突也可跨越300 nm与600 nm高的线条，沿线条表面平行生长并表达桩蛋白。经SMI 312选择性染色的轴突，其跨越此类线条的能力与树突无显著差异。本研究结果表明，诸如丝状伪足这类高活动性神经元结构，可响应化学信号爬越高障碍物；而体积更大的神经元结构响应速度较慢，需要数小时乃至数天才能完成同类障碍物的跨越。