Understanding Evolution Research Group, Naturalis Biodiversity Center, Leiden, The Netherlands.
Institute for Biology Leiden (IBL), Leiden University, Leiden, The Netherlands.
J Comp Neurol. 2022 Oct;530(14):2474-2485. doi: 10.1002/cne.25343. Epub 2022 May 21.
Spider brain and central nervous system (CNS) have remained unexplored, due in part to the difficulty of observing these organs, usually only possible through histological preparations. Recently, internal anatomy studies have been supplemented by the inclusion of X- ray micro-CT. Unmineralized tissue such as the body of invertebrates requires a staining process to enhance tissue X-ray absorption and improve contrast during observation. Many current protocols are based on iodine staining requiring critical point drying (CPD) of the sample for optimal contrast. This process induces shrinking of the soft tissue generating artifacts in the morphology, volume, and even position of internal structures. Phosphotungstic acid (PTA) is an alternative staining agent recently used in marine invertebrate and plant studies. Here, we used several specimens of the common spider Araneus diadematus to visualize the spider brain and compare both contrast-enhancing ethanol-based solutions. We assessed a gradient of staining times, observed and tested the repercussions of CPD, and examined the use of vacuum to accelerate PTA diffusion. We show that PTA provides the best contrast on micro-CT scans in ethanol eliminating the need for CPD, and offering more realistic in situ visualizations of the internal organs. In combination with different scanning settings, PTA allowed observation of internal organs like the CNS, digestive system, muscles, and finer structures like the retina, visual nerves, and optic neuropiles. This fast and less invasive method could facilitate the proper documentation of the internal anatomy in the context of evolutionary, developmental and functional studies.
蜘蛛的脑部和中枢神经系统(CNS)一直未被探索,部分原因是观察这些器官存在困难,通常只能通过组织学准备来实现。最近,通过包括 X 射线微计算机断层扫描(micro-CT)在内的方法,对内部解剖结构的研究得到了补充。无矿化组织(如无脊椎动物的身体)需要进行染色处理,以增强组织对 X 射线的吸收,并在观察过程中提高对比度。许多现有的方案都基于碘染色,需要对样本进行临界点干燥(CPD)以获得最佳对比度。这一过程会导致软组织收缩,从而在形态、体积甚至内部结构的位置上产生伪影。磷钨酸(PTA)是一种最近在海洋无脊椎动物和植物研究中使用的替代染色剂。在这里,我们使用了几种常见蜘蛛 Araneus diadematus 的标本来可视化蜘蛛的大脑,并比较了两种增强对比度的基于乙醇的溶液。我们评估了一系列染色时间,观察并测试了 CPD 的影响,并检查了使用真空来加速 PTA 扩散的效果。结果表明,PTA 在乙醇中的微 CT 扫描中提供了最佳的对比度,无需 CPD,并且为内部器官提供了更逼真的原位可视化效果。结合不同的扫描设置,PTA 可以观察到 CNS、消化系统、肌肉等内部器官,以及视网膜、视觉神经和视神经节等更精细的结构。这种快速、侵入性较小的方法可以促进在进化、发育和功能研究背景下对内部解剖结构进行适当的记录。