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The Effects of a Co-Application of Menthol and Capsaicin on Nociceptive Behaviors of the Rat on the Operant Orofacial Pain Assessment Device Ethan M. Anderson1,2*, Alan C. Jenkins3, Robert M. Caudle1,2, John K. Neubert3 1 Department of Oral and Maxillofacial Surgery, University of Florida College of Dentistry, Gainesville, Florida, United States of America, 2 Department of Neuroscience, University of Florida College of Medicine, McKnight Brain Institute, Gainesville, Florida, United States of America, 3 Department of Orthodontics, University of Florida, Gainesville, Florida, United States of America 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 (33uC, 21uC, 45uC). 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 45uC and 21uC suggesting they are both nociceptive and/or aversive to rats. However, rats consumed (licks) equal amounts at 33uC and 21uC but less at 45uC 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 21uC were equal to those at 33uC and both were significantly higher than at 45uC. 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. Citation: Anderson EM, Jenkins AC, Caudle RM, Neubert JK (2014) The Effects of a Co-Application of Menthol and Capsaicin on Nociceptive Behaviors of the Rat on the Operant Orofacial Pain Assessment Device. PLoS ONE 9(2): e89137. doi:10.1371/journal.pone.0089137 Editor: Manabu Sakakibara, Tokai University, Japan Received June 13, 2013; Accepted January 20, 2014; Published February 18, 2014 Copyright: ß 2014 Anderson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by the NIH grant 5R44DA026220-03. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction TRPV1 and TRPM8/TRPA1 can modulate each other’s effects on heat and cold pain. Menthol enhances cold pain and The transient receptor potential (TRP) family of cation channels reduces heat pain [11]. It also has a ‘‘cooling’’ effect on warm are involved in the perception of hot and cold temperatures and temperatures [12]. Capsaicin evokes ‘‘burning’’ sensations in pain [1,2]. The transient receptor potential vanilloid receptor 1 humans which can alter cold perception [13]. When applied (TRPV1) is activated by heat (.43uC), low pH, and capsaicin [3] alone, capsaicin reduces heat pain thresholds but a combination of while the transient receptor potential melastatin 8 receptor menthol and capsaicin increases cool detection thresholds [13,14]. (TRPM8) is responsible for detecting cool temperatures (,23uC) Also, menthol increases cold pain thresholds alone, but this is and can be activated by menthol [4,5]. The transient receptor attenuated when capsaicin is added [13]. So it appears that some potential subfamily A1 receptor (TRPA1) can also be activated by interaction is occurring between these two types of receptors and/ menthol and is responsible for the detection of cooler temperatures or the neurons they are on.’’ [6]. Expression changes in these proteins occur in humans with In order to investigate the effects of TRPV1 and TRPM8/ pain syndromes like diabetic neuropathy [7] as well as animal TRPA1 co-activation on the behavior of awake, functioning rats models of neuropathic pain like spared nerve injury (SNI) [8] and we used an orofacial operant assay which is sensitive to heat, cold, chronic constriction injury (CCI) [9,10] suggesting that studying and mechanical nociception [15–17]. We previously demonstrated their modulation could be beneficial to treating chronic pain in the that orofacial pain assays can detect the modulation of thermal future. nociception with TRP channel agonists like capsaicin, resinifer- PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89137 TRP Channel Agonists and Operant Pain Behavior atoxin, menthol, and icillin [17–20]. We hypothesized that considered significant. GraphPad Prism 4 software was used for all menthol would reduce heat-induced nociceptive behaviors and analyses (La Jolla, CA). capsaicin would reduce cold nociceptive behaviors. Two additional experiments were performed to further examine some of the results described below. The first was on the effects of Methods menthol at a colder temperature of 7uC. The same protocols were used as stated above except that the schedule now dropped to 7uC All facilities are accredited by the Association for Assessment for 3 min instead of 21uC, this required the ramping times to be and Accreditation of Laboratory Animal Care and all procedures increased from 30 s to 1 min and therefore the total session time were approved by the University of Florida’s Institutional Animal was increased from 17 min to 18 min. A baseline session and a Care and Use Committee. menthol treated session were both performed and the data are Sixteen male, hairless Sprague-Dawley rats (Charles River, presented below. Grubbs’ test was used to remove any outliers Raleigh, NC) weighing 400–600 g were used for all procedures. before paired t-tests were performed to assess significance. The Rats were housed in pairs in 22uC temperature and 31% humidity second experiment involved placing the 45uC segment earlier in controlled rooms with a 12-hour light/dark cycle (Lights were on the protocol. For this protocol, the 3 min 33uC session occurred, during the hours of 6am-6pm and behavioral sessions were followed by a 30 s ramp, then the 45uC ensued. Rats were run as performed between 8am and 12pm.). Rats had free access to food described above for a baseline session and a capsaicin treated and water except when fasted for behavioral testing. session. Grubbs’ test and paired t-tests were again used to The methods of Anderson et al. [21] were used for this study. determine significant effects. Briefly, rats were trained on the Orofacial Pain Assessment Device (OPAD, Stoelting, Co., Wood Dale, IL) to press their faces into Results peltier devices whose temperature can be heated or cooled to aversive temperatures. If they performed this task they received Representative behavior for a single animal during baseline access to a reward bottle filled with diluted sweetened condensed readings is displayed in Figure 1B–D. Reward licking events milk (1:2, milk:water). Food-fasted rats (15+/21 hrs) were trained (Licks) are denoted as red bars as illustrated in Figure 1B. For this at a neutral temperature (33uC) until they would make at least animal, licking events were frequent at all times other than 45uC. 2,000 licks during a 20 min session as a standard. If an animal did Figure 1C illustrates the stimulus contact behavior. Note that not meet this criterion, it was excluded from the study. The each time there is a licking event a contact is being made as selection of the temperatures used was based on previous work our displayed. However, an animal can contact the stimulus but not group has done in this area. Rodents will have the largest L/F lick, indicating an unsuccessful attempt which is characteristic of a ratios and the longest bouts of reward consumption at tempera- nociceptive response [15]. For example, the increased contacts tures between 30–37uC [10,15–28] suggesting that these are the observed in the 45uC session demonstrate that the rats are making least nociceptive/aversive temperatures. Rats appear to like attempts for the reward. However, the heat is preventing them temperatures close to 33uC as they provide warm, but not hot from staying on long enough to lick and this decreases their Lick/ sensations. Next, baseline measurements were obtained for three Face values. Figure 1D displays the reward consumption during temperatures within a single session. This was accomplished by the testing session. The data for all rats are quantified below. ramping the temperature several times over the course of a 17 min During baseline conditions the Lick/Face ratio was dependent on session as illustrated in Figure 1A. temperature (Figure 1E, F(2,15) = 11.44, p = 0.0002) as they were After baseline data was collected

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