TRPA1 is functionally expressed primarily by IB4-binding, non-peptidergic mouse and rat sensory neurons

PLoS One. 2012;7(10):e47988. doi: 10.1371/journal.pone.0047988. Epub 2012 Oct 25.

Abstract

Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1-6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2-4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrolein / analogs & derivatives
  • Acrolein / pharmacology
  • Animals
  • Exons
  • Female
  • Fura-2 / pharmacology
  • Ganglia, Spinal / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation*
  • Green Fluorescent Proteins / metabolism
  • Isothiocyanates / pharmacology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Neurons / metabolism*
  • Plant Lectins / pharmacology
  • Protein Binding
  • Rats
  • TRPA1 Cation Channel
  • TRPC Cation Channels / biosynthesis*
  • TRPC Cation Channels / physiology*
  • Transient Receptor Potential Channels / biosynthesis*
  • Transient Receptor Potential Channels / physiology*

Substances

  • Griffonia simplicifolia lectins
  • Isothiocyanates
  • Plant Lectins
  • TRPA1 Cation Channel
  • TRPC Cation Channels
  • Transient Receptor Potential Channels
  • Trpa1 protein, mouse
  • Trpa1 protein, rat
  • Green Fluorescent Proteins
  • 2,3,4-tri-O-acetylarabinopyranosyl isothiocyanate
  • Acrolein
  • cinnamaldehyde
  • Fura-2