PURPOSE. Stimulation to the cornea via noxious chemical and mechanical means evokes tearing, blinking, and pain. In contrast, mild cooling of the ocular surface has been reported to increase lacrimation via activation of corneal cool primary afferent neurons. The purpose of our study was to determine whether menthol induces corneal cool cell activity and lacrimation via the transient receptor potential melastatin-8 (TRPM8) channel without evoking nociceptive responses.METHODS. Tear measurements were made using a cotton thread in TRPM8 wild type and knockout mice after application of menthol (0.05-50 mM) to the cornea. In additional studies, nocifensive responses (eye swiping and lid closure) were quantified following cornea menthol application. Trigeminal ganglion electrophysiologic single unit recordings were performed in rats to determine the effect of low and high concentrations of menthol on corneal cool cells. RESULTS.At low concentrations, menthol increased tear production in TRPM8 wild type and heterozygous animals, but had no effect in TRPM8 knockout mice, while nocifensive responses remained unaffected. At the highest concentration, menthol (50 mM) increased tearing and nocifensive responses in TRPM8 wild type and knockout animals. A low concentration of menthol (0.1 mM) increased cool cell activity, yet a high concentration of menthol (50 mM) had no effect.CONCLUSIONS. These studies indicated that low concentrations of menthol can increase lacrimation via TRPM8 channels without evoking nocifensive behaviors. At high concentrations, menthol can induce lacrimation and nocifensive behaviors in a TRPM8 independent mechanism. The increase in lacrimation is likely due to an increase in cool cell activity. (Invest Ophthalmol Vis Sci. 2012;53:7034-7042)

The cornea is one of several orofacial structures requiring glandular secretion for proper lubrication. Glandular secretion is regulated through a neural reflex initiated by trigeminal primary afferent neurons innervating the corneal epithelium. Corneal sensory afferents must respond to irritating and potentially damaging stimuli, as well as drying that occurs with evaporation of the tear film, and the physiological properties of corneal afferents are consistent with these requirements. Polymodal neurons are sensitive to noxious mechanical, thermal and chemical stimuli, mechanoreceptive neurons are selectively activated by mechanical stimuli, and cool cells respond to innocuous cooling. The central terminations of corneal primary afferents are located within two regions of the spinal trigeminal nucleus. The more rostral region, located at the transition between the trigeminal subnucleus caudalis and interpolaris, represents a critical relay for the regulation of the lacrimation reflex. From this region, major control of lacrimation is carried through projections to preganglionic parasympathetic neurons located in or around the superior salivatory nucleus. Dry eye syndrome may be caused by a dysfunction in the tear secreting glands themselves or in the neuronal circuit regulating these glands. Furthermore, the dry eye condition itself may modify the properties of corneal afferents and affect their ability to regulate secretion, a possibility just now being explored.

Kurose M, Meng ID. Dry eye modifies the thermal and menthol responses in rat corneal primary afferent cool cells. J Neurophysiol 110: 495-504, 2013. First published May 1, 2013 doi:10.1152/jn.00222.2013.-Dry eye syndrome is a painful condition caused by inadequate or altered tear film on the ocular surface. Primary afferent cool cells innervating the cornea regulate the ocular fluid status by increasing reflex tearing in response to evaporative cooling and hyperosmicity. It has been proposed that activation of corneal cool cells via a transient receptor potential melastatin 8 (TRPM8) channel agonist may represent a potential therapeutic intervention to treat dry eye. This study examined the effect of dry eye on the response properties of corneal cool cells and the ability of the TRPM8 agonist menthol to modify these properties. A unilateral dry eye condition was created in rats by removing the left lacrimal gland. Lacrimal gland removal reduced tears in the dry eye to 35% compared with the contralateral eye and increased the number of spontaneous blinks in the dry eye by over 300%. Extracellular single-unit recordings were performed 8 -10 wk following surgery in the trigeminal ganglion of dry eye animals and age-matched controls. Responses of corneal cool cells to cooling were examined after the application of menthol (10 M-1.0 mM) to the ocular surface. The peak frequency of discharge to cooling was higher and the cooling threshold was warmer in dry eye animals compared with controls. The dry condition also altered the neuronal sensitivity to menthol, causing desensitization to cold-evoked responses at concentrations that produced facilitation in control animals. The mentholinduced desensitization of corneal cool cells would likely result in reduced tearing, a deleterious effect in individuals with dry eye.

These results indicate that aqueous tear deficiency produces hypersensitivity in the rat cornea. In addition, the increase in spontaneous blinks and their reduction by morphine and topical anesthesia indicate the presence of persistent irritation elicited by the activation of corneal nociceptors.

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Kurose M, Meng ID. Corneal dry-responsive neurons in the spinal trigeminal nucleus respond to innocuous cooling in the rat. J Neurophysiol 109: 2517-2522, 2013. First published February 27, 2013 doi:10.1152/jn.00889.2012.-Corneal primary afferent neurons that respond to drying of the ocular surface have been previously characterized and found to respond to innocuous cooling, menthol, and hyperosmotic stimuli. The purpose of the present study was to examine the receptive field properties of second-order neurons in the trigeminal nucleus that respond to drying of the ocular surface. Single-unit electrophysiological recordings were performed in anesthetized rats, and dry-responsive corneal units were isolated in the brain stem at the transition zone between the spinal trigeminal subnucleus caudalis and subnucleus interpolaris. Corneal units were characterized according to their responses to changes in temperature (cooling and heating), hyperosmotic artificial tears, menthol, and low pH. All dry-responsive neurons (n ϭ 18) responded to cooling of the ocular surface. In addition, these neurons responded to hyperosmotic stimuli and menthol application to the cornea. One-half of the neurons were activated by low pH, and these acid-sensitive neurons were also activated by noxious heat. Furthermore, neurons that were activated by low pH had a significantly lower response to cooling and menthol. These results indicate that dry-responsive neurons recorded in the trigeminal nucleus receive input from cold, sensitive primary afferent neurons, with a subset of these neurons receiving input from corneal primary afferent neurons sensitive to acid and noxious heat. It is proposed that acid-insensitive corneal neurons represent a labeled line for lacrimation in response to evaporation of tears from the ocular surface, whereas acid-sensitive neurons are involved in tearing, elicited by damaging or potentially damaging stimuli.

This study aimed to determine whether psychophysical stress conditionings had facilitatory effects on masseter muscle nociception in the central nervous system via serotonergic mechanisms in rats. Two experiments were conducted to assess: (1) whether repeated forced swim stress for 3 days increased the number of Fos-positive neurons evoked by masseter muscle injury due to formalin injection; and (2) whether serotonin-reuptake inhibitor, fluoxetine, administered daily after each stress conditioning, had modulatory roles on Fos expression. The number of Fos-positive cells was quantified in several areas within the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord regions (Vc areas), including the ventrolateral area of the trigeminal subnucleus interpolaris/Vc transition, and the middle or caudal portion of the Vc regions, since nociceptive neural activity in the Vc region could play critical roles in deep craniofacial nociception. We found that forced swim stress conditionings increased depression-like behaviors, which was prevented by fluoxetine. Repeated forced swim stress significantly increased Fos expression in all Vc areas compared with those of non-stressed rats, while systemic administration of fluoxetine significantly decreased Fos expression in all areas, but mainly in the caudal Vc region, in stressed rats. Fluoxetine had no effect on Fos expression in non-stressed rats. These results indicate that repeated forced swim stress conditionings increase Fos expression in the Vc areas, and the contribution of serotonergic mechanisms to masseter muscle nociception could be greater in stressed rats than in sham rats. These results support the hypothesis that changes in brain function, including serotonergic mechanisms, in the Vc areas play critical roles in enhanced masseter muscle nociceptive responses under psychophysical stress conditions.

Corneal cool cells are sensitive to the ocular fluid status of the corneal surface and may be responsible for the regulation of basal tear production. Previously, we have shown that dry eye, induced by lacrimal gland excision (LGE) in rats, sensitized corneal cool cells to the transient receptor potential melastatin 8 (TRPM8) agonist menthol and to cool stimulation. In the present study, we examined the effect of dry eye on the sensitivity of cool cells to the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin. Single-unit recordings in the trigeminal ganglion were performed 7–10 days after LGE. At a concentration of 0.3 μM, capsaicin did not affect ongoing or cool-evoked activity in control animals yet facilitated ongoing activity and suppressed cool-evoked activity in LGE animals. At higher concentrations (3 μM), capsaicin continued to facilitate ongoing activity in LGE animals but suppressed ongoing activity in control animals. Higher concentrations of capsaicin also suppressed cool-evoked activity in both groups of animals, with an overall greater effect in LGE animals. In addition to altering cool-evoked activity, capsaicin enhanced the sensitivity of cool cells to heat in LGE animals. Capsaicin-induced changes were prevented by the application of the TRPV1 antagonist capsazepine. With the use of fluorescent in situ hybridization, TRPV1 and TRPM8 expression was examined in retrograde tracer-identified corneal neurons. The coexpression of TRPV1 and TRPM8 in corneal neurons was significantly greater in LGE-treated animals when compared with sham controls. These results indicate that LGE-induced dry eye increases TRPV1-mediated responses in corneal cool cells at least in part through the increased expression of TRPV1. NEW & NOTEWORTHY Corneal cool cells are known to detect drying of the ocular surface. Our study is the first to report that dry eye induced alterations in cool cell response properties, including the increased responsiveness to noxious heat and activation by capsaicin. Along with the changes in cell response properties, it is possible these neurons also function differently in dry eye, relaying information related to the perception of ocular irritation in addition to regulating tearing and blinking.