Sensory nerve terminals detect peripheral stimuli in order to discern touch, temperature and pain. These neurons make up a considerably heterogeneous population that can be classified into subgroups based on peripheral targets, functional properties and central projections
. Larger-diameter neurons (≥27 um) tend to have myelinated axons, as with Aβ and Aδ fibers in vivo
, whereas smaller-diameter neurons (<27 µm) tend to have unmyelinated, C fiber axons in vivo
. Calcitonin Gene-Related Peptide (CGRP) expression and isolectin B4 (IB4) binding are two common histochemical markers used to define subpopulations of small-diameter neurons
. CGRP is expressed by peptidergic sensory neurons
, whereas IB4 binds to α-D-galactose carbohydrate residues typically expressed on the plasma membrane of non-peptidergic neurons
. In peripheral skin targets, IB4-binding neurons terminate superficially between keratinocytes within the epidermis, whereas peptidergic neurons terminate in deeper layers of the epidermis and dermis 
. Centrally, peptidergic neurons terminate in the outer laminae (I and outer II) of the spinal dorsal horn and target spinal neurons that transmit nociceptive information to the thalamus or brainstem nuclei, regions that mediate the sensory discriminative aspects of pain
. In contrast, the non-peptidergic, IB4-binding population primarily terminates on interneurons within the inner lamina II of the spinal cord and target interneurons that express PKCγ
. Rostrally, the input from IB4-binding neurons ultimately projects to brain areas including the amygdala and hypothalamus, regions involved in affective components of pain 
. Therefore, it is reasonable to hypothesize that specific receptors that transduce environmental or endogenous stimuli would distribute preferentially between peptidergic and non-peptidergic subpopulations in order to provide selective input to these diverse neural pain pathways.
The Transient Receptor Potential Ankyrin 1 (TRPA1) channel has been the focus of intense interest for its role in inflammatory nociception
and its potential function in transduction of mechanical and cold signals
. Previous investigations of the distribution of TRPA1 among peptidergic and non-peptidergic (IB4-positive) neurons have largely employed in situ
hybridization and immunohistochemical techniques, which have produced disparate results
. Some studies indicate greater TRPA1 expression in peptidergic, IB4-negative neurons 
, while others found more TRPA1 in non-peptidergic, IB4-binding neurons
. For example, Story and colleagues 
found TRPA1 mRNA in 3.6% of dorsal root ganglia (DRG) neurons from adult rat, and a majority of these TRPA1-positive cells expressed CGRP. On the other hand, Caspani and colleagues 
reported that 28% of lumbar 3–6 DRG neurons from adult mouse contain TRPA1 mRNA but only 2–3% of these neurons were CGRP-positive. Discrepancies between studies may have resulted from the inherent limitations of in situ
hybridization and immunohistochemistry techniques. For in situ
hybridization, the presence of mRNA does not always accurately predict that the respective protein will be expressed, as RNA can have a high turnover rate and become degraded prior to translation. Immunohistochemistry can sometimes lead to false-positives due to non-specific binding of antibodies or false-negatives due to lower sensitivity of antibodies. Further, receptors might be retained in internal organelles and not functionally expressed at the cell membrane, as has been shown for TRPA1 
. The sensitivity of both mRNA and antibody staining approaches depends on the thresholds established for positive versus negative cells.
Calcium imaging combined with live cell markers is a useful approach to determine functional expression of receptors. However, inconsistent results have also been reported for the TRPA1 agonists, allyl isothiocyanate (AITC) and cinnamaldehyde (CINN). Investigators report rates as low as 3–7% 
to as high as 30% 
for response to 100 µM CINN. Reports for AITC vary in kind, with 18 to 45% of neurons responding to 50 µM AITC 
. The discrepant results may be due to the wide range of agonists concentrations and duration of application used in these studies 
, the use of different species (mouse versus rat), or the duration neurons are maintained in culture.
Therefore, we set out to resolve the discrepancies in functional TRPA1 expression in identified populations of mouse and rat DRG neurons by using ratiometric calcium imaging and internally consistent parameters. We used lumbar 1–6 DRG ganglia because somata within these DRGs project to skin areas typically probed by behavior assays, such as the plantar hind paw. For both mouse and rat, we used similar isolation protocols, culture duration and media. We did not routinely add exogenous growth factors to the media because adult DRG neurons do not require growth factors for survival 
, and growth factors, such as nerve growth factor (NGF), have been shown to increase the functional expression of TRP channels, including TRPA1 
. We identified populations of C fiber-type, small-diameter neurons in live cultures by using IB4-FITC as well as a novel CGRP-GFP mouse where GFP is expressed in CGRPα-expressing neurons. Under these conditions, functional TRPA1 is predominantly found in small-diameter neurons that are IB4-positive and CGRP-negative.