title: Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn.
abstract: BACKGROUND
  Gastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter.
  
  
  RESULTS
  GRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP+ cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase Cγ, which is expressed by a subset of the excitatory interneurons in this region.
  
  
  CONCLUSIONS
  These results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A.
authors.forename: Maria
authors.initials: M
authors.lastname: Gutierrez-Mecinas
authors.forename: Masahiko
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Andrew.Todd@glasgow.ac.uk.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Todd
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism; D005260:Female; D019886:Gastrin-Releasing Peptide / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007395:Interneurons / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015288:Neurokinin A / Q000378:metabolism; D009475:Neurons, Afferent / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013004:Somatostatin / Q000378:metabolism; D066148:Spinal Cord Dorsal Horn / Q000166:cytology*; D013373:Substance P / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins; D013373:Substance P; D013004:Somatostatin; D019886:Gastrin-Releasing Peptide; D015288:Neurokinin A