Goshgarian Harry G, Buttry Janelle L
Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA.
Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA.
J Neurosci Methods. 2014 Jan 30;222:156-64. doi: 10.1016/j.jneumeth.2013.11.003. Epub 2013 Nov 12.
The first aim of the study was to determine if WGA-Alexa 488 would undergo retrograde transsynaptic transport in the phrenic motor system as we have shown with WGA-HRP in a previous study. The advantage of using WGA-Alexa 488 is that labeled neurons could be isolated and analyzed for intracellular molecular mechanisms without exposing tissue sections to chemicals for histochemical staining. The second aim of the study was to investigate the pattern and extent of labeling that occurs when WGA-Alexa 488 is applied to the cervical phrenic nerve as compared to intradiaphragmatic injection. After injecting the hemidiaphragm ipsilateral to a C2 spinal cord hemisection, WGA-Alexa 488 presumably diffused to the contralateral hemidiaphragm and labeled the phrenic nuclei bilaterally. In all animals with hemidiaphragmatic injection, the rostral ventral respiratory group (rVRG) was also labeled bilaterally in the medulla. Thus, injection of WGA-Alexa 488 into the diaphragm results in retrograde transsynaptic transport in the phrenic motor system. After applying WGA-Alexa 488 to the ipsilateral intact cervical phrenic nerve in both C2 hemisected rats and rats with a sham hemisection, only ipsilateral phrenic neurons were labeled; there was no labeling of the rVRG or any other center in the medulla. These results suggest that WGA-Alexa 488 must be applied in the vicinity of the phrenic myoneural junction where there is a high concentration of WGA receptors in order for transsynaptic transport to occur. The present study provides investigators with a new tool to study plasticity in the respiratory system after spinal cord injury.
本研究的首要目的是确定WGA-Alexa 488是否会在膈神经运动系统中进行逆行跨突触运输，正如我们在之前的一项研究中使用WGA-HRP所显示的那样。使用WGA-Alexa 488的优势在于，标记的神经元可以被分离出来，并对细胞内分子机制进行分析，而无需将组织切片暴露于用于组织化学染色的化学物质中。本研究的第二个目的是研究将WGA-Alexa 488应用于颈膈神经时与膈内注射相比所发生的标记模式和范围。在对与C2脊髓半横断同侧的半膈进行注射后，WGA-Alexa 488可能扩散到对侧半膈，并双侧标记膈神经核。在所有进行半膈注射的动物中，延髓中的双侧头端腹侧呼吸组（rVRG）也被标记。因此，将WGA-Alexa 488注射到膈肌中会导致膈神经运动系统中的逆行跨突触运输。在C2半横断大鼠和假半横断大鼠中，将WGA-Alexa 488应用于同侧完整的颈膈神经后，仅同侧膈神经元被标记；延髓中的rVRG或任何其他中枢均未被标记。这些结果表明，为了发生跨突触运输,WGA-Alexa 488必须应用于膈肌神经肌接头附近，此处存在高浓度的WGA受体。本研究为研究人员提供了一种新工具，用于研究脊髓损伤后呼吸系统的可塑性。
