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Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.

ABSTRACT: 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 (Tac1KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detectedGrpmRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated thatGrpmRNA 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 ofGrptranscripts, small percentage of cells expressingGrp, 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.

SUBMITTER: Barry DM

PROVIDER: S-EPMC4972254 | biostudies-literature | 2016 Jan-Dec

REPOSITORIES: biostudies-literature

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Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.

Barry Devin M DM Li Hui H Liu Xian-Yu XY Shen Kai-Feng KF Liu Xue-Ting XT Wu Zhen-Yu ZY Munanairi Admire A Chen Xiao-Jun XJ Yin Jun J Sun Yan-Gang YG Li Yun-Qing YQ Chen Zhou-Feng ZF

Molecular pain 20160101

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 (Tac1KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is signific ...[more]

PMID: 27068287

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Project description:Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from aged-matched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFβ1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middle-aged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and T-cell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain.

Dataset Information

Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.

Publications

Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.

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