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.

Behavioral model of itch, alloknesis, pain and allodynia in the lower hindlimb and correlative responses of lumbar dorsal horn neurons in the mouse.

We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the pruritogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and chloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30-min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injection of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-threshold mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2h) time course. In single-unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine-responsive neurons. This was not observed in histamine-insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLIGRL-NH2 to account for alloknesis. The presently described "calf" model appears to distinguish between itch- and pain-related behavioral responses, and provides a basis to investigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia.

Scratching inhibits serotonin-evoked responses of rat dorsal horn neurons in a site- and state-dependent manner.

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300 mN, 2 Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4-30 mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

Cross-sensitization of histamine-independent itch in mouse primary sensory neurons.

Overexpression of pruritogens and their precursors may contribute to the sensitization of histamine-dependent and -independent itch-signaling pathways in chronic itch. We presently investigated self- and cross-sensitization of scratching behavior elicited by various pruritogens, and their effects on primary sensory neurons. The MrgprC11 agonist BAM8-22 exhibited self- and reciprocal cross-sensitization of scratching evoked by the protease-activated receptor-2 (PAR-2) agonist SLIGRL. The MrgprA3 agonist chloroquine unidirectionally cross-sensitized BAM8-22-evoked scratching. Histamine unidirectionally cross-sensitized scratching evoked by chloroquine and BAM8-22. SLIGRL unidirectionally cross-sensitized scratching evoked by chloroquine. Dorsal root ganglion (DRG) cells responded to various combinations of pruritogens and algogens. Neither chloroquine, BAM8-22 nor histamine had any effect on responses of DRG cell responses to subsequently applied pruritogens, implying that their behavioral self- and cross-sensitization effects are mediated indirectly. SLIGRL unilaterally cross-sensitized responses of DRG cells to chloroquine and BAM8-22, consistent with the behavioral data. These results indicate that unidirectional cross-sensitization of histamine-independent itch-signaling pathways might occur at a peripheral site through PAR-2. PAR-2 expressed in pruriceptive nerve endings is a potential target to reduce sensitization associated with chronic itch.

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.

Activation of brain stem neurons by irritant chemical stimulation of the throat assessed by c-fos immunohistochemistry.

The method of c-fos immunohistochemistry was used to identify the brain stem distribution of neurons activated following irritant chemical stimulation of the laryngopharyngeal mucosa. In pentobarbital-anesthetized rats, either water (control), nicotine (600 mM, 1 ml) or capsaicin (330 microM, 1 ml) was applied to the pharynx via a cannula placed posterior to the soft palate. Following nicotine and capsaicin, there was a significant increase in fos-like immunoreactivity (FLI) compared with controls in the following areas: nucleus of the solitary tract from the level of the pyramidal decussation caudally to the level of the area postrema rostrally; dorsomedial aspect of trigeminal subnucleus caudalis (Vc); and paratrigeminal islands interspersed in the spinal trigeminal tract. There was significantly more FLI in Vc and paratrigeminal nuclei following capsaicin than following nicotine, while the reverse was true for NTS. In addition, there was a significant increase in FLI in area postrema and the ventrolateral medullary region dorsal to the lateral reticular nucleus following nicotine but not capsaicin. The distributions of FLI in NTS, area postrema, Vc, and paratrigeminal nuclei are consistent with prior anatomical tract-tracing studies and suggest roles for these brain stem regions in mediating sensory and reflex responses to irritant chemical stimulation of the upper respiratory mucosa.

Analgesia induced by chronic nicotine infusion in rats: differences by gender and pain test.

RATIONALE: Acute administration of nicotine induces analgesia with subsequent development of tolerance. In human studies, females are less sensitive to the analgesic effects of nicotine than males. Few previous animal studies have investigated analgesic effects of chronic nicotine administration or addressed gender differences. OBJECTIVES: To investigate whether chronic administration of nicotine induces analgesia in male and female rats as assessed by a battery of standard pain assays, if tolerance develops, and if hyperalgesia occurs following cessation of nicotine. METHODS: Nicotine (free base; 6 mg/kg/day i.v.) or saline was administered for 2 weeks via implanted osmotic pumps. Pain behavior was assessed before, during, and for 3 weeks after nicotine infusion by measuring tail flick latency, hot-plate latency, and thermal paw withdrawal latency. The paw-withdrawal threshold to non-noxious mechanical stimuli was also measured. Effects of nicotine infusion, gender, and time were assessed by three-way analyses of variance. RESULTS: Both male and female rats exhibited a comparable degree of analgesia in the hot-plate test with development of tolerance during the 2-week infusion period. Males, but not females, showed analgesia in the tail flick test. Analgesia was not observed for thermally evoked paw withdrawal in either males or females, nor did nicotine affect non-noxious mechanically evoked paw withdrawals. Males and females showed cessation of weight gain during the first week of nicotine infusion. CONCLUSIONS: Chronic nicotine-induced analgesia was confirmed in both male and female rats as assessed using the hot-plate test which reflects integrated pain behavior. Males, but not females, exhibited analgesia in a nociceptive withdrawal reflex test (tail flick), indicating that nicotine-induced analgesia may depend on both the type of pain test and gender. The lack of nicotine-induced analgesia assessed by the tail flick reflex test in female rats is consistent with recent human studies showing that nicotine reduces pain elicited by brief noxious cutaneous stimulation in male but not female subjects.

Role of neuronal nicotinic-acetylcholine receptors in the activation of neurons in trigeminal subnucleus caudalis by nicotine delivered to the oral mucosa.

To characterize the role of neuronal nicotinic acetylcholine receptors (nAChRs) in oral irritation and pain, we employed the method of c-fos immunohistochemistry to map the locations and numbers of brainstem neurons that express the immediate-early gene, c-fos, after application of nicotine to the tongue, either alone or after pretreatment with cholinergic antagonists. Groups of anesthetized rats received the following chemicals delivered bilaterally to the dorsal tongue: (1) 0.9% NaCl followed by nicotine (1%, 61 mM), (2) the nAChR antagonist, mecamylamine 0.1% (= 4.9 mM) followed by nicotine, (3) the muscarinic antagonist atropine (0.1% 1.46 mM) followed by nicotine, (4) atropine (1%, 14.6 mM) followed by nicotine, (5) 0.9% NaCl as a control, and (6) unstimulated controls. Two hours later, animals were perfused with phosphate-buffered saline followed by 4% paraformaldehyde through the aorta. Post-fixed brainstems were cut in 50-micron frozen sections and immunohistochemically processed for fos-like immunoreactivity (FLI). Following application of nicotine, there were significant increases in FLI compared with saline-treated controls in dorsomedial and ventrolateral aspects of the trigeminal caudalis. Pretreatment with either mecamylamine or the high (1%) concentration of atropine significantly reduced nicotine-evoked FLI in these areas, while pretreatment with the low (0.1%) atropine concentration did not significantly affect FLI. These results are consistent with the idea that nicotine activates nAChRs residing on lingual nociceptive fibers, which, in turn, excite neurons in trigeminal caudalis.

Psychophysical and neurobiological evidence that the oral sensation elicited by carbonated water is of chemogenic origin.

The sensation produced by carbonated beverages has been attributed to chemical excitation of nociceptors in the oral cavity via the conversion of CO(2) to carbonic acid in a reaction catalyzed by carbonic anhydrase. In separate studies, we tested if the carbonic anyhdrase blocker, acetazolamide, reduced either the intensity of sensation in humans or c-fos expression by trigeminal neurons in rats, evoked by application of carbonated water to the tongue. In the psychophysical experiment, one-half of the dorsal tongue was pretreated with acetazolamide (1 or 2%), after which the tongue was exposed bilaterally to carbonated water. In a two-alternative forced-choice paradigm, subjects chose which side of the tongue yielded a stronger sensation and additionally rated the magnitude of sensation on each side. Pretreatment with acetazolamide reduced the magnitude of sensation elicited by carbonated water in a concentration-dependent manner, since a significant majority of subjects chose the untreated side of the tongue as having a stronger sensation and assigned significantly higher intensity ratings to that side. Acetazolamide did not affect the irritant sensation from citric acid, while capsaicin pretreatment reduced both the sensation elicited by carbonated water and the irritation induced by citric acid application. In a separate experiment using rats, delivery of carbonated water to the tongue significantly increased the number of cells expressing c-fos-like immunoreactivity in the dorsomedial trigeminal nucleus caudalis (versus saline controls); this was significantly reduced by pretreatment with acetazolamide. Our results support the hypothesis that carbonated water activates lingual nociceptors via conversion of CO(2) to carbonic acid; the nociceptors in turn excite trigeminal neurons involved in signaling oral irritation.

Neurobiological and psychophysical mechanisms underlying the oral sensation produced by carbonated water.

Carbonated drinks elicit a sensation that is highly sought after, yet the underlying neural mechanisms are ill-defined. We hypothesize that CO(2) is converted via carbonic anhydrase into carbonic acid, which excites lingual nociceptors that project to the trigeminal nuclei. We investigated this hypothesis using three methodological approaches. Electrophysiological methods were used to record responses of single units located in superficial laminae of the dorsomedial aspect of trigeminal subnucleus caudalis (Vc) evoked by lingual application of carbonated water in anesthetized rats. After pretreatment of the tongue with the carbonic anhydrase inhibitor dorzolamide, neuronal responses to carbonated water were significantly attenuated, followed by recovery. Using c-Fos immunohistochemistry, we investigated the distribution of brainstem neurons activated by intraoral carbonated water. Fos-like immunoreactivity (FLI) was significantly higher in the superficial laminae of dorsomedial and ventrolateral Vc in animals treated with carbonated water versus controls. Dorzolamide pretreatment significantly reduced FLI in dorsomedial Vc. We also examined the sensation elicited by carbonated water in human psychophysical studies. When one side of the tongue was pretreated with dorzolamide, followed by bilateral application of carbonated water, a significant majority of subjects chose the untreated side as having a stronger sensation and assigned significantly higher intensity ratings to that side. Dorzolamide did not reduce irritation elicited by pentanoic acid. The present data support the hypothesis that carbonated water excites lingual nociceptors via a carbonic anhydrase-dependent process, in turn exciting neurons in Vc that are presumably involved in signaling oral irritant sensations.