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首页> 外文期刊>Journal of Neuroscience Methods >An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons.
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An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons.

机译：用于研究伤害性背根神经节和背角神经元之间突触形成的体外测定系统。

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摘要

Synapses between nociceptive dorsal root ganglion (DRG) neurons and spinal cord dorsal horn neurons represent the first loci for transmission of painful stimuli. Our knowledge of the molecular organization and development of these synapses is sparse due, partly, to a lack of a reliable model system that reconstitutes synaptogenesis between these two neuronal populations. To address this issue, we have established an in vitro assay system consisting of separately purified DRG neurons and dorsal horn neurons on astrocyte microislands. Using immunocytochemistry, we have found that 97%, 93%, 98%, 96%, and 94% of DRG neurons on these microislands express markers often associated with nociceptive neurons including Substance P, TRPV1, calcitonin-gene related peptide (CGRP), TrKA, and peripherin, respectively. Triple labeling with these nociceptive-like markers, synaptic vesicle marker Vglut2 and using MAP2 as a dendritic marker revealed the presence of nociceptive-like markers at synaptic terminals. Using this immunocytochemical approach, we counted contact points as overlapping MAP2/Vglut2 puncta and showed that they increased with time in culture. Single and dual patch-clamp recordings showed that overlapping Vglut2/MAP2 puncta observed after a few days in culture are likely to be functional synapses between DRG and dorsal horn neurons in our in vitro assay system. Taken together, these data suggest our co-culture microisland model system consists of mostly nociceptive-like DRG neurons that express presynaptic markers and form functional synapses with their dorsal horn partners. Thus, this model system may have direct application for studies on factors regulating development of nociceptive DRG/dorsal horn synapses.
机译：伤害性背根神经节（DRG）神经元和脊髓背角神经元之间的突触代表了疼痛刺激传递的第一个位点。我们对这些突触的分子组织和发育的了解很少，部分原因是缺乏可重构这两个神经元群体之间突触形成的可靠模型系统。为了解决这个问题，我们建立了一个体外测定系统，该系统由星形胶质细胞微岛上分别纯化的DRG神经元和背角神经元组成。通过免疫细胞化学，我们发现这些微岛上的DRG神经元中有97％，93％，98％，96％和94％的DRG神经元表达的标志物通常与伤害性神经元有关，包括P物质，TRPV1，降钙素基因相关肽（CGRP）， TrKA和外周蛋白。使用这些伤害性样标记物，突触囊泡标记物Vglut2进行三次标记，并使用MAP2作为树突状标记物，表明突触末端存在伤害性样标记物。使用这种免疫细胞化学方法，我们将接触点计数为重叠的MAP2 / Vglut2点，并显示它们随着培养时间的增加而增加。单次和两次膜片钳记录表明，在我们的体外分析系统中，培养几天后观察到的重叠的Vglut2 / MAP2点很可能是DRG和背角神经元之间的功能性突触。综上所述，这些数据表明我们的共培养微岛模型系统主要由类似伤害感受的DRG神经元组成，这些神经元表达突触前标记并与其背角伴侣形成功能性突触。因此，该模型系统可直接用于调节伤害性DRG /背角突触发育的因素的研究。

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