The concept of a specific circuit for the sensation of itch received a strong impetus from two papers in 2007 and 2009 [1, 2]. These studies described how gastrin releasing peptide (GRP) can specifically cause scratching behavior which is lost upon genetic or pharmacological elimination of the GRP-receptor or GRP-receptor bearing second order neurons in the spinal cord. The behavior evoked by GRP was claimed to be due to the release of this peptide from primary afferents which in turn was proposed to cause the activation of GRP-receptor-bearing neurons in the spinal cord dorsal horn [1, 2]. Recently, however the expression of GRP in DRG has been disputed [3, 4] and a role for another neuropeptide, natriuretic precursor peptide B (NPPB), in provoking scratching behavior was delineated in mice . Additionally, the capacity for a third neuropeptide, neuromedin B (NMB) to evoke scratching behaviors has been suggested .
In the present report, we address the question of expression of Grp, Nppb, and Nmb and their receptors in DRG and spinal cord through application of RNA-Seq. This method has several notable advantages over other techniques such as gene-array, RT-PCR and in situ hybridization (ISH). First, RNA-Seq uses next-generation DNA sequencing to determine every mRNA transcript in a particular sample. Since each sequenced cDNA fragment can be counted, the resulting data are quantitative. Second, because the association of transcript fragments to a particular gene is based on exact sequence alignment, rather than hybridization, gene identification is exquisitely precise. Third, the presence of rare transcripts can be captured by adjusting the depth of sequencing, thereby providing a rigorous measurement for all, or nearly all, expressed genes in a sample. These properties allowed us to accurately assess the expression level of Nppb, Grp, and Nmb and their cognate receptors.
For sensory ganglia, we examined expression in mouse and rat DRG, and mouse, rat and human trigeminal ganglia. We also determined expression of these genes in mouse, rat and human spinal cord. For mouse DRG, a genetic labeling strategy was used to isolate neurons of the transient receptor potential family V, member 1 (TRPV1) lineage and the non-TRPV1 lineage . The latter DRG preparation consists of neurons remaining following ablation of neurons in the TRPV1-lineage. The neuropeptides GRP, NPPB and NMB co-localize with TRPV1-expressing cells or the TRPV1-lineage of nociceptive neurons . Furthermore, it has been shown that the TRPV1-lineage neurons are absolutely required for pruritic responses . These two preparations were extensively sequenced (~172 million paired end reads) to reveal transcripts expressed even at very low levels. Thus, the depth of sequencing combined with our genetic enrichment strategy enhances the chances of identifying rare transcripts in mouse DRG, including those for Grp. Importantly, for comparison we also provide data on a class of mouse sensory neurons not required for itch-behavior (the non-TRPV1 transcriptome). In all other cases, we sequenced the entire DRG or trigeminal ganglion to a lower extent (~40 million paired end reads) which is sufficient to detect low expressed genes.
Our results demonstrate that mouse, rat, and human sensory neurons express natriuretic peptides capable of activating NPR1, while a role for GRP as a primary afferent itch neurotransmitter is not supported.