The effects of a co-application of menthol and capsaicin on nociceptive behaviors of the rat on the operant orofacial pain assessment device

Abstract

Background: Transient receptor potential (TRP) cation channels are involved in the perception of hot and cold pain and are targets for pain relief in humans. We hypothesized that agonists of TRPV1 and TRPM8/TRPA1, capsaicin and menthol, would alter nociceptive behaviors in the rat, but their opposite effects on temperature detection would attenuate one another if combined.

Methods: Rats were tested on the Orofacial Pain Assessment Device (OPAD, Stoelting Co.) at three temperatures within a 17 min behavioral session (33°C, 21°C, 45°C).

Results: The lick/face ratio (L/F: reward licking events divided by the number of stimulus contacts. Each time there is a licking event a contact is being made.) is a measure of nociception on the OPAD and this was equally reduced at 45°C and 21°C suggesting they are both nociceptive and/or aversive to rats. However, rats consumed (licks) equal amounts at 33°C and 21°C but less at 45°C suggesting that heat is more nociceptive than cold at these temperatures in the orofacial pain model. When menthol and capsaicin were applied alone they both induced nociceptive behaviors like lower L/F ratios and licks. When applied together though, the licks at 21°C were equal to those at 33°C and both were significantly higher than at 45°C.

Conclusions: This suggests that the cool temperature is less nociceptive when TRPM8/TRPA1 and TRPV1 are co-activated. These results suggest that co-activation of TRP channels can reduce certain nociceptive behaviors. These data demonstrate that the motivational aspects of nociception can be influenced selectively by TRP channel modulation and that certain aspects of pain can be dissociated and therefore targeted selectively in the clinic.

Figure 1. The effects of hot and cool temperatures on operant nociceptive behavior.

A single, representative rat's responses to different stimuli under baseline conditions during a (A) temperature ramping protocol for (B) licking behavior, (C) contact behavior, and (D) reward intake. (E) The Lick/Face ratio was dependent on temperature. 45°C and 21°C temperatures caused lower Lick/Face ratios (post-hoc tests, p<0.001 and p<0.01). (F) Licking events were altered by temperature. Rats licked less at 45°C compared to 33°C and 21°C (p<0.001 and p<0.001). (G) Contact behavior was altered by temperature. More contacts were made at 21°C than at 33°C (p<0.001). An ** and *** represent p-values less than 0.01 and 0.001 respectively for a Bonferroni's post-hoc test when comparing behavior to 33°C after a One-Way ANOVA. A ### represents a p-value less than 0.001 when comparing 21°C to 45°C with Bonferroni's. N = 16 for graphs E-G.

Figure 2. The effects of menthol on operant behavior.

A single, representative rat's response after a menthol application during a (A) temperature ramping protocol for (B) licking behavior, (C) contact behavior, and (D) reward intake. (E) Lick/Face values were still altered by temperature. 45°C and 21°C still lowered this value (post-hoc tests, p<0.001 and p<0.01). (F) Licking behavior was altered by temperature differently than at baseline, both 45°C and 21°C were now lower than 33°C (p<0.001 and p<0.01). (G) No differences were observed for contacts with the menthol application. An ** and *** represent p-values less than 0.01 and 0.001 respectively for a Bonferroni's post-hoc test when comparing behavior to 33°C after a One-Way ANOVA. N = 16 for graphs E-G.

Figure 3. The effects of capsaicin on operant behavior.

A single, representative rat's response to capsaicin during a (A) temperature ramping protocol for (B) licking behavior, (C) contact behavior, and (D) reward intake. (E) Temperature still had a significant effect on behavior with capsaicin application. The Lick/Face ratio was still lower at 45°C and 21°C than at 33°C (post-hoc tests, p<0.001 and p<0.001). (F) The number of licks was altered by temperature differently than at baseline. Licks at 33°C were higher than the other two temperatures (both p<0.001). (G) Contact behavior was no longer dependent on temperature when capsaicin was applied. An *** represents a p-value less than 0.001 for a Bonferroni's post-hoc test when comparing behavior to 33°C after a One-Way ANOVA. N = 16 for graphs E–G.

Figure 4. The effects of a co-application of menthol and capsaicin on operant behavior.

A single, representative rat's response to menthol and capsaicin during a (A) temperature ramping protocol for (B) licking behavior, (C) contact behavior, and (D) reward intake. (E) Temperature still had a significant effect on the Lick/Face ratio when both capsaicin and menthol were applied although no post-hoc tests were significant. (F) Licking behavior was still temperature-dependent but the combination of menthol and capsaicin restored the original relationship for licking as now licking was equal between the 33°C and 21°C periods again and 45°C was again lower than both 33°C and 21°C (p<0.001 and p<0.05). (G) The number of contacts was again not dependent on temperature. An *** represent p-values less than 0.001 for a Bonferroni's post-hoc test when comparing behavior to 33°C after a One-Way ANOVA. A # represents a p-value less than 0.05 when comparing 21°C to 45°C with Bonferroni's. N = 16 for graphs E–G.

Figure 5. The effects of menthol on a cold 7°C temperature.

(A) The Lick/Face ratio was significantly reduced by menthol application 30 mins before testing. (B) Licking behavior was also significantly reduced by menthol. (C) No effect was observed for contact behavior. A * represents a p-value of less than 0.05 with a paired t-test. N = 13 for all graphs.

Figure 6. The effects of capsaicin on behavior at 45°C at 3.5 min instead of 10.5 min.

(A) The Lick/Face ratio was significantly reduced by capsaicin application 30 mins before testing. (B) Licking behavior was also significantly reduced. (C) No effect was observed for contact behavior. A ** and *** represent p-values of less than 0.01 and 0.001 respectively, with a paired t-test. N = 14 for all graphs.