title: TRPA1 is functionally expressed primarily by IB4-binding, non-peptidergic mouse and rat sensory neurons.
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.
authors.affiliation: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Marie E
authors.initials: ME
authors.lastname: Barabas
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kossyreva
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Stucky
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D005091:Exons; D005260:Female; D016257:Fura-2 / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017879:Isothiocyanates / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism*; D037121:Plant Lectins / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D000074025:TRPA1 Cation Channel; D050052:TRPC Cation Channels / Q000096:biosynthesis* / Q000502:physiology*; D050051:Transient Receptor Potential Channels / Q000096:biosynthesis* / Q000502:physiology*
chemical_list: C045475:Griffonia simplicifolia lectins; D017879:Isothiocyanates; D037121:Plant Lectins; D000074025:TRPA1 Cation Channel; D050052:TRPC Cation Channels; D050051:Transient Receptor Potential Channels; C475685:Trpa1 protein, mouse; C480976:Trpa1 protein, rat; D049452:Green Fluorescent Proteins; C041942:2,3,4-tri-O-acetylarabinopyranosyl isothiocyanate; D000171:Acrolein; C012843:cinnamaldehyde; D016257:Fura-2