Gephyrin Clusters Are Absent from Small Diameter Primary Afferent Terminals Despite the Presence of GABAA Receptors
Gephyrin Clusters Are Absent from Small Diameter Primary Afferent Terminals Despite the Presence of GABAA Receptors
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DOI：
10.1523/jneurosci.0159-14.2014
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发表时间：
2014-06-11
影响因子：
5.3
通讯作者：
De Koninck, Yves
中科院分区：
文献类型：
--
作者：
Lorenzo, Louis-Etienne;Godin, Antoine G.;De Koninck, Yves
虽然GABA（A）受体（GABA（A）Rs）和甘氨酸受体（GlyRs）都在控制背角神经元兴奋性中起作用，但它们对抑制小直径初级传入终末的相对贡献仍有争议。为了解决这一问题，我们设计了一种方法来定量分析GABA（A）R-亚基，GlyR α 1-亚基及其锚定蛋白，gephyrin，在大鼠脊髓感觉传入终末的分布，通过降钙素基因相关肽（CGRP）的肽能终末和Isolectin-B4（IB 4）的非肽能终末鉴定。该方法是专为光学显微镜，这是兼容的几个这些抗原的免疫检测所需的温和的固定条件。设计了一种算法来识别尺寸与显微镜分辨率相似的结构。为了避免检测到错误的共定位，后者被认为是显着的，只有当像素重叠的程度超过预期的随机重叠像素给定的超几何分布。我们发现CGRP（+）和IB 4（+）末端均缺乏GlyR α 1亚单位和桥蛋白。α 1 GABA（A）R也不存在于这些终末。相反，GABA（A）R α 2/α 3/α 5和β 3亚基在两种末端类型中显著表达，其他GABA（A）R相关蛋白（α-肌营养不良聚糖/神经连接素-2/Collybistin-2）也是如此。超微结构免疫细胞化学证实了GABA（A）R β 3亚单位在小的传入终末的存在。荧光定量PCR（qRT-PCR）证实了光镜免疫化学分析的结果。这些结果表明背角抑制性突触在突触前和突触后部位（伤害性传入终末和背角神经元上的抑制性突触）遵循不同的组织规则。初级传入终末缺乏桥蛋白簇，表明GABA（A）介导的传递在突触前比在突触后更弥散。
Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR alpha 1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR alpha 1-subunit and gephyrin. The alpha 1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R alpha 2/alpha 3/alpha 5 and beta 3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (alpha-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R beta 3 subunits in small afferent terminals. Real-time quantitativePCR(qRT-PCR) confirmed the results of light microscopy immuno-chemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynapticsites(nociceptive afferentterminals vs inhibitory synapses on dorsalhorn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.