Transient receptor potential channel A1 and noxious cold responses in rat cutaneous nociceptors

Abstract

The role of transient receptor potential channel A1 (TRPA1) in noxious cold sensation remains unclear. Some data support the hypothesis that TRPA1 is a transducer of noxious cold whilst other data contest it. In this study we investigated the role of TRPA1 in cold detection in cutaneous nociceptors in vivo using complementary experimental approaches. We used noxious withdrawal reflex electromyography, and single fibre recordings in vivo, to test the hypothesis that TRPA1-expressing primary afferents mediate noxious cold responses in anaesthetised rats. TRPV1 and TRPM8 agonists sensitise their cognate receptors to heat and cold stimuli respectively. Herein we show that the TRPA1 agonist cinnamaldehyde applied to the skin in anaesthetised rats did not sensitise noxious cold evoked hind limb withdrawal. In contrast, cinnamaldehyde did sensitise the C fibre-mediated noxious heat withdrawal, indicated by a significant drop in the withdrawal temperature. TRPA1 agonist thus sensitised the noxious reflex withdrawal to heat, but not cold. Thermal stimuli also sensitise transient receptor potential (TRP) channels to agonist. Activity evoked by capsaicin in teased primary afferent fibres showed a significant positive correlation with receptive field temperature, in both normal and Freund's complete adjuvant-induced cutaneous inflammation. Altering the temperature of the receptive field did not modulate TRPA1 agonist evoked-activity in cutaneous primary afferents, in either normal or inflamed skin. In addition, block of the TRPA1 channel with Ruthenium Red did not inhibit cold evoked activity in either cinnamaldehyde sensitive or insensitive cold responsive nociceptors. In cinnamaldehyde-sensitive-cold-sensitive afferents, although TRPA1 agonist-evoked activity was totally abolished by Ruthenium Red, cold evoked activity was unaffected by channel blockade. We conclude that these results do not support the hypothesis that TRPA1-expressing cutaneous afferents play an important role in noxious cold responses.

Fig. 1

Cinnamaldehyde enhances heat sensitivity but not cold sensitivity. (A) Slow heat ramps evoked withdrawal at 52 °C and this was not changed after dimethylsulphoxide (DMSO) (100%) vehicle. Topical cinnamaldehyde (10% in DMSO) caused a significant reduction in withdrawal threshold (data from three animals with up to three ramps per treatment, ** P<0.01, 10% cinn vs. DMSO). (B) Noxious cold evoked withdrawal at −10 °C and this was also not affected by DMSO (100%) treatment. Cinnamaldehyde did not cause an increased sensitivity to cold (data from three animals with up to three ramps per treatment, n.s.=not significantly different).

Fig. 2

Capsaicin, but not cinnamaldehyde, evoked activity is temperature sensitive. This relationship is not altered by complete Freund's adjuvant (CFA) induced cutaneous inflammation. (A) In naïve rat skin increasing temperature increased the activity evoked by capsaicin (data from four animals, total of 10 units, activity of each recorded at three different temperatures. Spearman's correlation r=0.52, P<0.01). (B) In contrast, increasing temperature did not affect the activity evoked by cinnamaldehyde (data from four animals, 8 units each at three different temperatures. Spearman's correlation r=0.34, P>0.05). (C) Three days after CFA induction of cutaneous inflammation, increasing temperature still increases activity evoked by capsaicin (data from four animals, total of 17 units tested at three different temperatures. Spearman's correlation r=0.34, P<0.01). (D) Three days after CFA induction of cutaneous inflammation, increasing temperature still did not affect cinnamaldehyde evoked activity (data from four animals, total of 19 units each tested at three different temperatures. Spearman's correlation r=−0.09, P>0.05).

Fig. 3

Ruthenium Red (RuR) inhibits cinnamaldehyde but not cooling evoked activity in 2A delta nociceptors. The two units are represented by either a square or circle. Cinnamaldehyde evoked activity is shown by the dotted line. Cooling evoked activity is shown by the black line. To aid comparison both cooling evoked activity and cinnamaldehyde evoked activity have been normalised to baseline (0 mM Ruthenium Red, 100%).