Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, United States.
J Neurosci Methods. 2013 Oct 30;220(1):18-23. doi: 10.1016/j.jneumeth.2013.08.007. Epub 2013 Aug 16.
To assess gap junctional intercellular communication we have developed a tracer-based methodology which is both highly sensitive and potentially adaptable for in vivo measurements. We found that injection of serotonin revealed significantly more intercellular communication than that injection of the most permeant synthetic tracer currently in use, neurobiotin. Furthermore, mechanical tracer loading steps can be replaced by transfection with human serotonin transporter and the inclusion of serotonin in the medium. Tracer and transporter are detected using immunocytochemical techniques and the presence of cells that are tracer-positive but transporter-negative indicates junctional communication. Tracer loading in vivo using transgenesis, electroporation or viral transduction to direct expression of transporter should be more easily accomplished than with mechanical loading methods.
为了评估缝隙连接细胞间通讯，我们开发了一种示踪剂基础的方法，该方法既高度敏感又可潜在适用于体内测量。我们发现，与目前最常用的通透性最强的合成示踪剂神经生物素相比，注射血清素显示出显著更多的细胞间通讯。此外，机械示踪剂加载步骤可以被转染人血清素转运体和在培养基中包含血清素所取代。示踪剂和转运体通过免疫细胞化学技术检测，并且存在示踪剂阳性但转运体阴性的细胞表明存在缝隙连接通讯。使用转基因、电穿孔或病毒转导在体内进行示踪剂加载，以指导转运体的表达，应该比机械加载方法更容易实现。
