Lorenzo Louis-Etienne, Godin Antoine G, Wang Feng, St-Louis Manon, Carbonetto Salvatore, Wiseman Paul W, Ribeiro-da-Silva Alfredo, De Koninck Yves
Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Québec, Québec G1J 2G3, Canada, Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada, Département de Psychiatrie et Neurosciences, Université Laval, Québec, Québec G1K 7P4, Canada, Alan Edwards Center for Research of Pain, McGill University, Montréal, Québec H3A 0G1, Canada.
Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Québec, Québec G1J 2G3, Canada, Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada.
J Neurosci. 2014 Jun 11;34(24):8300-17. doi: 10.1523/JNEUROSCI.0159-14.2014.
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 α1-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 α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and β3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R β3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn 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.
虽然γ-氨基丁酸A型受体（GABA(A)Rs）和甘氨酸受体（GlyRs）在控制背角神经元兴奋性方面均发挥作用，但它们对抑制小直径初级传入终末的相对贡献仍存在争议。为解决这一问题，我们设计了一种方法，用于定量分析GABA(A)R亚基、GlyR α1亚基及其锚定蛋白桥连蛋白在大鼠脊髓感觉传入终末上的分布，这些终末通过降钙素基因相关肽（CGRP）鉴定为肽能终末，通过异凝集素B4（IB4）鉴定为非肽能终末。该方法是为光学显微镜设计的，这与免疫检测这些抗原中的几种所需的温和固定条件兼容。设计了一种算法来识别尺寸与显微镜分辨率相似的结构。为避免检测到假共定位，只有当像素重叠程度超过给定超几何分布的随机重叠像素预期程度时，才认为后者具有显著性。我们发现，CGRP(+)和IB4(+)终末均缺乏GlyR α1亚基和桥连蛋白。这些终末也不存在α1 GABA(A)R。相比之下，GABA(A)R α2/α3/α5和β3亚基在两种终末类型中均有显著表达，其他与GABA(A)R相关的蛋白（α- dystroglycan/Neuroligin-2/Collybistin-2）也是如此。超微结构免疫细胞化学证实了小传入终末中存在GABA(A)R β3亚基。实时定量PCR（qRT-PCR）证实了光学显微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与突触后部位（伤害性传入终末与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连蛋白簇表明，GABA(A)介导的突触前传递模式比突触后部位更为弥散。
