Zaelzer Cristian, Gizowski Claire, Salmon Christopher K, Murai Keith K, Bourque Charles W
Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre , Montréal, Québec , Canada.
J Neurophysiol. 2018 Sep 1;120(3):1386-1396. doi: 10.1152/jn.00467.2017. Epub 2018 Jul 5.
Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq, thus VP binding to this receptor causes an increase in intracellular [Ca] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.
通过能够动态检测活组织中肽释放的方法，我们对神经网络中神经肽功能的理解将会得到提升。我们研究了嗅探细胞作为生物传感器检测大鼠下丘脑切片和离体神经垂体中内源性血管加压素（VP）释放的效用。将人胚胎肾细胞进行转染，使其表达人V1a血管加压素受体（V1aR）和基因编码的钙指示剂GCaMP6m。V1aR与Gq偶联，因此VP与该受体结合会导致细胞内[Ca]增加，这可通过GCaMP6荧光增强来检测。剂量反应分析表明，VP嗅探细胞可报告环境中VP水平>10 pM（EC = 2.6 nM），且这种效应可被V1aR拮抗剂SR 49059抑制。当置于涂有嗅探细胞的盖玻片上时，对神经垂体进行电刺激，使用低至3 Hz的60个脉冲即可引发VP释放的可逆、可重复且剂量依赖性增加。轻轻铺在切片上的悬浮嗅探细胞会附着在制剂上，并可实现离散区域中VP释放的可视化。对视交叉上核中VP神经元进行电刺激会导致显著的局部释放以及远处靶位点的VP分泌。最后，视上核中单个大细胞神经分泌细胞诱发的动作电位会引发该神经元体树突区显著的VP释放。这些结果表明，嗅探细胞可用于研究活组织中神经元不同区域的VP分泌。新发现与值得注意之处由于缺乏能够在活组织中实时检测神经肽的方法，人们对神经肽在神经网络中的具体功能作用了解甚少。在此，我们表明可通过工程改造培养的“嗅探细胞”来检测血管加压素的内源性释放，表现为荧光增强。
