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 (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.

Molecular and neural basis of contagious itch behavior in mice

“…This prompted us to examine the expression of GRP, a well-established neuropeptide marker in the SCN that has been implicated in photic modulation of circadian phase (10, 12, 13), as well as in dorsal root ganglion neurons that are required for transmission of itch information to GRPR in the spinal cord (14). Consistent with our recent study (15), we confirmed the specificity of the GRP antibody using Grp knockout (KO) mice (fig. S2).…”

“…2, F to H). Because immunohistochemistry largely revealed GRP-expressing fibers that arborize densely in the core and shell of the SCN where Grpr expression is detected (16), we used in situ hybridization to investigate whether Grp + cells are located in the core area (15). Consistent with previous studies (10), Grp + cell bodies were detected in the core, but not the shell, of the SCN (Fig.…”

Comparative Anatomy of Gastrin-releasing Peptide Pathways in the Trigeminal Sensory System of Mouse and the Asian House Musk Shrew <i>Suncus murinus</i>

“…GRP-immunoreactivity was located mainly in smaller DRG neurons in mice [2, 8, 11, 12, 26, 34] and rats [28], and also smaller TG neurons in rats [28]. We found a similar distribution of GRP-ir neurons in suncus and mouse TG neurons.…”

“…We found a similar distribution of GRP-ir neurons in suncus and mouse TG neurons. GRP-expressing neurons account for 2%–9% of mouse DRG neurons [2, 8, 11–13, 26, 34], 6% of rat DRG neurons [28], and 12% of rat TG neurons [28]. In this study, we also found that ~9% of TG neurons in suncus and ~7% of TG neurons in mice expressed GRP.…”

“…The expression levels of GRP in the DRG and GRPR in the spinal dorsal horn were both increased in allergic contact dermatitis and in dry skin mouse models [34]. Similar results were also reported in monkeys and mice with chronic pruritus [2, 15, 24]. …”