Kung Frank, Wang Jianfeng, Perez-Castillejos Raquel, Townes-Anderson Ellen
Rutgers Biomedical Health and Sciences, Graduate School of Biomedical Sciences, New Jersey Institute of Technology, Joint Program in Biomedical Engineering, 185 South Orange Ave, Newark, NJ 07103, USA.
Integr Biol (Camb). 2015 Mar;7(3):313-23. doi: 10.1039/c4ib00213j. Epub 2015 Jan 23.
In retinal degeneration, death of photoreceptors causes blindness. Repair of the retina by transplanting photoreceptors has resulted in limited functional connectivity between transplanted and host neurons. We hypothesize that absence of appropriate biological cues, specifically positional (retinotopographic) cues, reduces synaptogenesis. Here we use micropatterning to test whether regional origin affects the early synaptic development of photoreceptors. Right and left retinas from salamanders were first labelled with dextran tetramethyl-rhodamine and fluorescein, respectively, bisected into nasal (N)/temporal (T) or dorsal (D)/ventral (V) halves, individually dissociated, mixed, and cultured for 1 week. Origin of cells was identified by the fluorescent label. Interactions between photoreceptors and neighboring (target) cells were assessed by the number of neuritic contacts with a presynaptic swelling (varicosity). Randomly-plated photoreceptors showed no preference for cellular origin, likely due to multiple potential interactions available to each cell. To reduce cell-cell interactions, culture substrate was patterned using a microfluidic device with 10 μm-wide channels separated by 200 μm, thus allowing only 1-2 targets per photoreceptor. In patterned cultures, 36.89% of N rod cells contacted T targets but only 27.42% of N rod cells contacted N targets; similarly 35.05% of T rod cells contacted N cells but only 17.08% contacted T cells. Thus, opposite regions were more permissive of contact. However, neither cone nor D/V rod cells showed preferences for positional origin of targets. In conclusion, micropatterning demonstrated that neuritic differentiation by rod cells depends on retinotopographic cues along the nasal/temporal plane, suggesting that transplanting rod cells of known positional origin will increase transplant success.
在视网膜变性中，光感受器死亡会导致失明。通过移植光感受器来修复视网膜，在移植的神经元与宿主神经元之间产生的功能连接有限。我们推测，缺乏适当的生物学信号，特别是位置（视网膜拓扑）信号，会减少突触形成。在这里，我们使用微图案化技术来测试区域来源是否会影响光感受器的早期突触发育。首先分别用葡聚糖四甲基罗丹明和荧光素标记蝾螈的右视网膜和左视网膜，将其切成鼻侧（N）/颞侧（T）或背侧（D）/腹侧（V）两半，分别解离、混合并培养1周。通过荧光标记确定细胞的来源。通过具有突触前肿胀（曲张体）的神经突接触数量来评估光感受器与相邻（靶）细胞之间的相互作用。随机接种的光感受器对细胞来源没有偏好，这可能是因为每个细胞都有多种潜在的相互作用。为了减少细胞间相互作用，使用微流控装置对培养底物进行图案化，该装置具有宽度为10μm、间隔为200μm的通道，因此每个光感受器仅允许1 - 2个靶细胞。在图案化培养中，36.89%的鼻侧视杆细胞与颞侧靶细胞接触，但只有27.42%的鼻侧视杆细胞与鼻侧靶细胞接触；同样，35.05%的颞侧视杆细胞与鼻侧细胞接触，但只有17.08%与颞侧细胞接触。因此，相对的区域更易于形成接触。然而，视锥细胞和背侧/腹侧视杆细胞对靶细胞的位置来源均无偏好。总之，微图案化技术表明视杆细胞的神经突分化取决于鼻侧/颞侧平面的视网膜拓扑信号，这表明移植已知位置来源的视杆细胞将提高移植成功率。
