An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons.
An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons.
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DOI：
10.1016/j.jneumeth.2010.04.002
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发表时间：
2010-06-15
影响因子：
3
通讯作者：
MacDermott, Amy B.
中科院分区：
文献类型：
--
作者：
Joseph, Donald J.;Choudhury, Papiya;MacDermott, Amy B.
关键词：
背根神经节（DRG）神经元与脊髓背角神经元之间的突触是痛觉传递的第一个位点。我们对这些突触的分子组织和发育的了解很少，部分原因是缺乏一个可靠的模型系统来重建这两个神经元群体之间的突触发生。为了解决这个问题，我们已经建立了一个体外检测系统，包括单独纯化的DRG神经元和背角神经元的星形胶质细胞微岛。使用免疫细胞化学，我们已经发现，97%，93%，98%，96%和94%的DRG神经元在这些微岛表达标记物往往与伤害性神经元，包括P物质，TRPV1，降钙素基因相关肽（CGRP），TrKA，和peripherin，分别。三重标记与这些伤害感受样标记，突触囊泡标记VAP2和使用MAP 2作为树突状标记揭示了伤害感受样标记在突触末端的存在。使用这种免疫细胞化学方法，我们计数接触点重叠MAP2/VAP2斑点，并显示它们随着培养时间的增加。单和双膜片钳记录显示，在培养几天后观察到的重叠VAP2/MAP 2斑点可能是DRG和背角神经元之间的功能性突触在我们的体外测定系统。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表达突触前标记物并与其背角伙伴形成功能性突触的伤害感受样DRG神经元组成。因此，该模型系统可以直接应用于伤害性DRG/背角突触发育调控因子的研究。
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 micro-islands. 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.
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