Amanda H. Klein

Topical application of L-menthol induces heat analgesia, mechanical allodynia, and a biphasic effect on cold sensitivity in rats

Menthol is used in analgesic balms and also in foods and oral hygiene products for its fresh cooling sensation. Menthol enhances cooling by interacting with the cold-sensitive thermoTRP channel TRPM8, but its effect on pain is less well understood. We presently used behavioral methods to investigate effects of topical menthol on thermal (hot and cold) pain and innocuous cold and mechanical sensitivity in rats. Menthol dose-dependently increased the latency for noxious heat-evoked withdrawal of the treated hindpaw with a weak mirror-image effect, indicating antinociception. Menthol at the highest concentration (40%) reduced mechanical withdrawal thresholds, with no effect at lower concentrations. Menthol had a biphasic effect on cold avoidance. At high concentrations (10% and 40%) menthol reduced avoidance of colder temperatures (15 degrees C and 20 degrees C) compared to 30 degrees C, while at lower concentrations (0.01-1%) menthol enhanced cold avoidance. In a -5 degrees C cold plate test, 40% menthol significantly increased the nocifensive response latency (cold hypoalgesia) while lower concentrations were not different from vehicle controls. These results are generally consistent with neurophysiological and human psychophysical data and support TRPM8 as a potential peripheral target of pain modulation.

Facial injections of pruritogens or algogens elicit distinct behavior responses in rats and excite overlapping populations of primary sensory and trigeminal subnucleus caudalis neurons

In the present study, we investigated whether intradermal cheek injection of pruritogens or algogens differentially elicits hindlimb scratches or forelimb wipes in Sprague-Dawley rats, as recently reported in mice. We also investigated responses of primary sensory trigeminal ganglion (TG) and dorsal root ganglion (DRG) cells, as well as second-order neurons in trigeminal subnucleus caudalis (Vc), to pruritic and algesic stimuli. 5-HT was the most effective chemical to elicit dose-dependent bouts of hindlimb scratches directed to the cheek, with significantly less forelimb wiping, consistent with itch. Chloroquine also elicited significant scratching but not wiping. Allyl isothiocyanate (AITC; mustard oil) elicited dose-dependent wiping with no significant scratching. Capsaicin elicited equivalent numbers of scratch bouts and wipes, suggesting a mixed itch and pain sensation. By calcium imaging, ∼ 6% of cultured TG and DRG cells responded to 5-HT. The majority of 5-HT-sensitive cells also responded to chloroquine, AITC, and/or capsaicin, and one-third responded to histamine. Using a chemical search strategy, we identified single units in Vc that responded to intradermal cheek injection of 5-HT. Most were wide dynamic range (WDR) or nociceptive specific (NS), and a few were mechanically insensitive. The large majority additionally responded to AITC and/or capsaicin and thus were not pruritogen selective. These results suggest that primary and second-order neurons responsive to pruritogens and algogens may utilize a population coding mechanism to distinguish between itch and pain, sensations that are behaviorally manifested by distinct hindlimb scratching and forelimb wiping responses.

Three functionally distinct classes of C-fibre nociceptors in primates

In primate C-fiber polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically-sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, a MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically-insensitive C-fibers (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin responsive neurons in primate DRG. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibers likely corresponding to murine MrgprD- expressing, non-peptidergic nociceptive afferents.

Behavioral evidence of thermal hyperalgesia and mechanical allodynia induced by intradermal cinnamaldehyde in rats

TRPA1 agonists cinnamaldehyde (CA) and mustard oil (allyl isothiocyanate=AITC) induce heat hyperalgesia and mechanical allodynia in human skin, and sensitize responses of spinal and trigeminal dorsal horn neurons to noxious skin heating in rats. TRPA1 is also implicated in cold nociception. We presently used behavioral methods to investigate if CA affects sensitivity to thermal and mechanical stimuli in rats. Unilateral intraplantar injection of CA (5-20%) induced a significant, concentration-dependent reduction in latency for ipsilateral paw withdrawal from a noxious heat stimulus, peaking (61.7% of pre-injection baseline) by 30 min with partial recovery at 120 min. The highest dose of CA also significantly reduced the contralateral paw withdrawal latency. CA significantly reduced mechanical withdrawal thresholds of the injected paw that peaked sooner (3 min) and was more profound (44.4% of baseline), with no effect contralaterally. Bilateral intraplantar injections of CA resulted in a significant cold hyperalgesia (cold plate test) and a weak enhancement of innocuous cold avoidance (thermal preference test). The data are consistent with roles for TRPA1 in thermal (hot and cold) hyperalgesia and mechanical allodynia.

Self- and Cross-desensitization of Oral Irritation by Menthol and Cinnamaldehyde (CA) via Peripheral Interactions at Trigeminal Sensory Neurons

Menthol and cinnamaldehyde (CA) are plant-derived spices commonly used in oral hygiene products, chewing gum, and many other applications. However, little is known regarding their sensory interactions in the oral cavity. We used a human psychophysics approach to investigate the temporal dynamics of oral irritation elicited by sequential application of menthol and/or CA, and ratiometric calcium imaging methods to investigate activation of rat trigeminal ganglion (TG) cells by these agents. Irritancy decreased significantly with sequential oral application of menthol and CA (self-desensitization). Menthol cross-desensitized irritation elicited by CA, and vice versa, over a time course of at least 60 min. Seventeen and 19% of TG cells were activated by menthol and CA, respectively, with ∼50% responding to both. TG cells exhibited significant self-desensitization to menthol applied at a 5, but not 10, min interval. They also exhibited significant self-desensitization to CA at 400 but not 200 μM. Menthol cross-desensitized TG cell responses to CA. CA at a concentration of 400 but not 200 μM also cross-desensitized menthol-evoked responses. The results support the argument that the perceived reductions in oral irritancy and cross-interactions between menthol and CA and menthol observed (at least at short interstimulus intervals) can be largely accounted for by the properties of trigeminal sensory neurons innervating the tongue.

Eugenol and carvacrol induce temporally desensitizing patterns of oral irritation and enhance innocuous warmth and noxious heat sensation on the tongue

Summary Eugenol and carvacrol are derived from the oils of clove and oregano, respectively, and impart warming, numbing, and irritant sensations in the oral cavity by interacting with the heat‐sensitive ion channel TRPV3. Here we show that these agents induce oral irritation in a manner that exhibits self‐desensitization and cross‐desensitization of capsaicin‐evoked irritancy, and enhancement of warmth and heat pain, but not cold sensation. Abstract Eugenol and carvacrol, from the spices clove and oregano, respectively, are agonists of TRPV3, which is implicated in transduction of warmth and possibly heat pain. We investigated the temporal dynamics of lingual irritation elicited by these agents, and their effects on innocuous warmth and heat pain, using a half‐tongue method in human subjects. The irritant sensation elicited by both eugenol and carvacrol decreased across repeated applications at a 1‐minute interstimulus interval (self‐desensitization) which persisted for at least 10 minutes. Both agents also cross‐desensitized capsaicin‐evoked irritation. Eugenol and carvacrol significantly increased the magnitude of perceived innocuous warmth (44 °C) for >10 minutes, and briefly (<5 minutes) enhanced heat pain elicited by a 49 °C stimulus. Similar albeit weaker effects were observed when thermal stimuli were applied after the tongue had been desensitized by repeated application of eugenol or carvacrol, indicating that the effect is not due solely to summation of chemoirritant and thermal sensations. Neither chemical affected sensations of innocuous cool or cold pain. A separate group of subjects was asked to subdivide eugenol and carvacrol irritancy into subqualities, the most frequently reported being numbing and warmth, with brief burning, stinging/pricking, and tingle, confirming an earlier study. Eugenol, but not carvacrol, reduced detection of low‐threshold mechanical stimuli. Eugenol and carvacrol enhancement of innocuous warmth may involve sensitization of thermal gating of TRPV3 expressed in peripheral warm fibers. The brief heat hyperalgesia following eugenol may involve a TRPV3‐mediated enhancement of thermal gating of TRPV1 expressed in lingual polymodal nociceptors.

A tingling sanshool derivative excites primary sensory neurons and elicits nocifensive behavior in rats

Szechuan peppers contain hydroxy-α-sanshool that imparts desirable tingling, cooling, and numbing sensations. Hydroxy-α-sanshool activates a subset of sensory dorsal root ganglion (DRG) neurons by inhibiting two-pore potassium channels. We presently investigated if a tingle-evoking sanshool analog, isobutylalkenyl amide (IBA), excites rat DRG neurons and, if so, if these neurons are also activated by agonists of TRPM8, TRPA1, and/or TRPV1. Thirty-four percent of DRG neurons tested responded to IBA, with 29% of them also responding to menthol, 29% to cinnamic aldehyde, 66% to capsaicin, and subsets responding to two or more transient receptor potential (TRP) agonists. IBA-responsive cells had similar size distributions regardless of whether they responded to capsaicin or not; cells only responsive to IBA were larger. Responses to repeated application of IBA at a 5-min interstimulus interval exhibited self-desensitization (tachyphylaxis). Capsaicin did not cross-desensitize responses to IBA to any greater extent than the tachyphylaxis observed with repeated IBA applications. These findings are consistent with psychophysical observations that IBA elicits tingle sensation accompanied by pungency and cooling, with self-desensitization but little cross-desensitization by capsaicin. Intraplantar injection of IBA elicited nocifensive responses (paw licking, shaking-flinching, and guarding) in a dose-related manner similar to the effects of intraplantar capsaicin and serotonin. IBA had no effect on thermal sensitivity but enhanced mechanical sensitivity at the highest dose tested. These observations suggest that IBA elicits an unfamiliar aversive sensation that is expressed behaviorally by the limited response repertoire available to the animal.

Eugenol and carvacrol excite first- and second-order trigeminal neurons and enhance their heat-evoked responses

Eugenol and carvacrol from clove and oregano, respectively, are agonists of the warmth-sensitive transient receptor potential channel TRPV3 and the irritant-sensitive transient receptor potential ankyrin (TRPA)-1. Eugenol and carvacrol induce oral irritation that rapidly desensitizes, accompanied by brief enhancement of innocuous warmth and heat pain in humans. We presently investigated if eugenol and carvacrol activate nociceptive primary afferent and higher order trigeminal neurons and enhance their heat-evoked responses, using calcium imaging of cultured trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons, and in vivo single-unit recordings in trigeminal subnucleus caudalis (Vc) of rats. Eugenol and carvacrol activated 20-30% of TG and 7-20% of DRG cells, the majority of which additionally responded to menthol, mustard oil and/or capsaicin. TG cell responses to innocuous (39°) and noxious (42 °C) heating were enhanced by eugenol and carvacrol. We identified dorsomedial Vc neurons responsive to noxious heating of the tongue in pentobarbital-anesthetized rats. Eugenol and carvacrol dose-dependently elicited desensitizing responses in 55% and 73% of heat-sensitive units, respectively. Responses to noxious heat were briefly enhanced by eugenol and carvacrol. Many eugenol- and carvacrol-responsive units also responded to menthol, cinnamaldehyde and capsaicin. These data support a peripheral site for eugenol and carvacrol to enhance warmth- and noxious heat-evoked responses of trigeminal neurons, and are consistent with the observation that these agonists briefly enhance warmth and heat pain on the human tongue.

Novel menthol-derived cooling compounds activate primary and second-order trigeminal sensory neurons and modulate lingual thermosensitivity.

We presently investigated 2 novel menthol derivatives GIV1 and GIV2, which exhibit strong cooling effects. In previous human psychophysical studies, GIV1 delivered in a toothpaste medium elicited a cooling sensation that was longer lasting compared with GIV2 and menthol carboxamide (WS-3). In the current study, we investigated the molecular and cellular effects of these cooling agents. In calcium flux studies of TRPM8 expressed in HEK cells, both GIV1 and GIV2 were approximately 40- to 200-fold more potent than menthol and WS-3. GIV1 and GIV2 also activated TRPA1 but at levels that were 400 times greater than those required for TRPM8 activation. In calcium imaging studies, subpopulations of cultured rat trigeminal ganglion and dorsal root ganglion cells responded to GIV1 and/or GIV2; the majority of these were also activated by menthol and some were additionally activated by the TRPA1 agonist cinnamaldehyde and/or the TRPV1 agonist capsaicin. We also made in vivo single-unit recordings from cold-sensitive neurons in rat trigeminal subnucleus caudalis (Vc). GIV 1 and GIV2 directly excited some Vc neurons, GIV1 significantly enhanced their responses to cooling, and both GIV1 and GIV2 reduced responses to noxious heat. These novel cooling compounds provide additional molecular tools to investigate the neural processes of cold sensation.

Topical hindpaw application of L-menthol decreases responsiveness to heat with biphasic effects on cold sensitivity of rat lumbar dorsal horn neurons.

Menthol is used in pharmaceutical applications because of its desired cooling and analgesic properties. The neural mechanism by which topical application of menthol decreases heat pain is not fully understood. We investigated the effects of topical menthol application on lumbar dorsal horn wide dynamic range and nociceptive-specific neuronal responses to noxious heat and cooling of glabrous hindpaw cutaneous receptive fields. Menthol increased thresholds for responses to noxious heat in a concentration-dependent manner. Menthol had a biphasic effect on cold-evoked responses, reducing the threshold (to warmer temperatures) at a low (1%) concentration and increasing threshold and reducing response magnitude at high (10%, 40%) concentrations. Menthol had little effect on responses to innocuous or noxious mechanical stimuli, ruling out a local anesthetic action. Application of 40% menthol to the contralateral hindpaw tended to reduce responses to cooling and noxious heat, suggesting a weak heterosegmental inhibitory effect. These results indicate that menthol has an analgesic effect on heat sensitivity of nociceptive dorsal horn neurons, as well as biphasic effects on cold sensitivity, consistent with previous behavioral observations.