Immunostaining for Homer reveals the majority of excitatory synapses in laminae I-III of the mouse spinal dorsal horn.
Immunostaining for Homer reveals the majority of excitatory synapses in laminae I-III of the mouse spinal dorsal horn.
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DOI：
10.1016/j.neuroscience.2016.05.009
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发表时间：
2016-08-04
期刊：
影响因子：
3.3
通讯作者：
Todd AJ
中科院分区：
文献类型：
--
作者：
Gutierrez-Mecinas M;Kuehn ED;Abraira VE;Polgár E;Watanabe M;Todd AJ
识别突触能突触对于追踪突触回路是重要的。大多数神经元突触上的蛋白质被组织固定所掩盖。Homer可以在不需要抗原修复的情况下显示突触。脊髓背角在将躯体感觉信息传递到大脑之前处理躯体感觉信息。背角的神经元组织仍然知之甚少，尽管最近的研究已经在中间神经元中定义了几个不同的群体，这些群体占其组成神经元的大部分。所有的初级传入神经，以及在板层I-III的神经元的绝大多数是突触能的，和一个主要的因素限制了我们的理解的突触回路一直是难以识别突触能突触与光学显微镜。尽管抗体有许多潜在的靶点，但由于组织固定后的蛋白质交联，这些靶点难以用免疫细胞化学观察。虽然这可以通过抗原修复方法来克服，但这些方法导致难以检测其他抗原。本研究的目的是测试突触后蛋白Homer是否可以用来揭示背角中的突触能突触。荷马的免疫染色给点状标记时，通过共聚焦显微镜观察，这是有限的突触在超微结构水平。我们发现Homer puncta与AMPA受体GluR 2亚基共定位，但不与抑制性突触相关蛋白gephyrin共定位。我们还研究了几个种群的神经元轴突，发现大多数终扣接触至少一个荷马点。这些结果表明，Homer抗体可用于显示绝大多数的神经元突触，而无需抗原修复。这对于追踪突触回路，以及研究痛觉神经元突触的可塑性具有重要价值。
Identifying glutamatergic synapses is important for tracing synaptic circuits. Most proteins at glutamatergic synapses are masked by tissue fixation. Homer can reveal glutamatergic synapses without the need for antigen retrieval. The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I–III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states.