Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.
Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.
复制标题
胃泌素释放肽在背根神经节和脊髓中表达的严格评估。
DOI：
10.1177/1744806916643724
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发表时间：
2016
期刊：
影响因子：
3.3
通讯作者：
Chen ZF
中科院分区：
文献类型：
--
作者：
Barry DM;Li H;Liu XY;Shen KF;Liu XT;Wu ZY;Munanairi A;Chen XJ;Yin J;Sun YG;Li YQ;Chen ZF
关于感觉神经元中胃泌素释放肽（GRP）的表达以及 GRP 抗体是否与 P 物质（SP）发生交叉反应，存在很大分歧。这些问题需要使用其他方法对 GRP 表达进行严格的重新评估。在这里，我们证明了一种广泛使用的 GRP 抗体特异性识别 GRP 但不识别 SP。在缺乏 SP (Tac1 KO) 的小鼠脊髓中，不仅 GRP 的表达显着减少，其他肽，特别是神经肽 Y (NPY) 的表达也显着减少。我们使用逆转录聚合酶链反应、原位杂交和 RNA-seq 检测了背根神经节中的 Grp mRNA。我们证明，慢性瘙痒小鼠的背根神经节中的 Grp mRNA 和蛋白质上调，但脊髓中没有上调。背神经根切断术后的脊髓切片中检测到很少的 GRP+ 免疫染色信号，并且在分离的背角神经元中未检测到 GRP+ 细胞体。超微结构分析进一步表明，与 SPergic 纤维相比，更多的 GRPergic 纤维与胃泌素释放肽受体阳性 (GRPR+) 神经元形成突触接触。我们的综合研究表明，大多数 GRPergic 纤维都是初级传入起源的。许多因素导致了这一争议，例如 Grp 转录本的拷贝数低、表达 Grp 的细胞比例小以及使用 eGFP GENSAT 转基因作为 GRP 蛋白的替代品。实验程序的优化有助于特异性检测背根神经节神经元中 GRP 的表达。
There are substantial disagreements about the expression of gastrin-releasing peptide (GRP) in sensory neurons and whether GRP antibody cross-reacts with substance P (SP). These concerns necessitate a critical revaluation of GRP expression using additional approaches. Here, we show that a widely used GRP antibody specifically recognizes GRP but not SP. In the spinal cord of mice lacking SP (Tac1 KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detected Grp mRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated that Grp mRNA and protein are upregulated in dorsal root ganglias, but not in the spinal cord, of mice with chronic itch. Few GRP+ immunostaining signals were detected in spinal sections following dorsal rhizotomy and GRP+ cell bodies were not detected in dissociated dorsal horn neurons. Ultrastructural analysis further shows that substantially more GRPergic fibers form synaptic contacts with gastrin releasing peptide receptor-positive (GRPR+) neurons than SPergic fibers. Our comprehensive study demonstrates that a majority of GRPergic fibers are of primary afferent origin. A number of factors such as low copy number of Grp transcripts, small percentage of cells expressing Grp, and the use of an eGFP GENSAT transgenic as a surrogate for GRP protein have contributed to the controversy. Optimization of experimental procedures facilitates the specific detection of GRP expression in dorsal root ganglia neurons.