PURPOSE. To define the firing properties of sensory nerve terminals innervating the adult mouse cornea in response to external stimuli of differing modality.METHODS. Extracellular electrical activity of single corneal sensory nerve terminals was recorded in excised eyes of C57BL/6J mice. Eyes were placed in a recording chamber and were continuously superfused with warm saline solution. Nerve terminal impulse (NTI) activity was recorded by means of a glass pipette (tip~50 lm), applied on the corneal surface. Nerve terminal impulse discharges were stored in a computer for offline analysis.RESULTS. Three functionally distinct populations of nerve terminals were identified in the mouse cornea. Pure mechanonociceptor terminals (9.5%) responded phasically and only to mechanical stimuli. Polymodal nociceptor terminals (41.1%) were tonically activated by heat and hyperosmolal solutions (850 mOsmÁkg À1 ), mechanical force, and/or TRPV1 and TRPA1 agonists (capsaicin and allyl isothiocyanate [AITC], respectively). Cold-sensitive terminals (49.4%) responded to cooling. Approximately two-thirds of them fired continuously at 348C and responded vigorously to small temperature reductions, being classified as high-background activity, low-threshold (HB-LT) cold thermoreceptor terminals. The remaining one-third exhibited very low ongoing activity at 348C and responded weakly to intense cooling, being named low-background activity, high-threshold (LB-HT) cold thermoreceptor terminals.CONCLUSIONS. The mouse cornea is innervated by trigeminal ganglion (TG) neurons that respond to the same stimulus modalities as corneal receptors of other mammalian species. Mechano-and polymodal endings underlie detection of mechanical and chemical noxious stimuli while HB-LT and LB-HT cold thermoreceptors appear to be responsible for basal and irritation-evoked tearing and blinking, respectively.

TRPM8 is a polymodal, nonselective cation channel activated by cold temperature and cooling agents that plays a critical role in the detection of environmental cold. We found that TRPM8 is a pharmacological target of tacrolimus (FK506), a macrolide immunosuppressant with several clinical uses, including the treatment of organ rejection following transplants, treatment of atopic dermatitis, and dry eye disease. Tacrolimus is an inhibitor of the phosphatase calcineurin, an action shared with cyclosporine. Tacrolimus activates TRPM8 channels in different species, including humans, and sensitizes their response to cold temperature by inducing a leftward shift in the voltage-dependent activation curve. The effects of tacrolimus on purified TRPM8 in lipid bilayers demonstrates conclusively that it has a direct gating effect. Moreover, the lack of effect of cyclosporine rules out the canonical signaling pathway involving the phosphatase calcineurin. Menthol (TRPM8-Y745H)-and icilin (TRPM8-N799A)-insensitive mutants were also activated by tacrolimus, suggesting a different binding site. In cultured mouse DRG neurons, tacrolimus evokes an increase in intracellular calcium almost exclusively in cold-sensitive neurons, and these responses were drastically blunted in Trpm8 KO mice or after the application of TRPM8 antagonists. Cutaneous and corneal cold thermoreceptor endings are also activated by tacrolimus, and tacrolimus solutions trigger blinking and cold-evoked behaviors. Together, our results identify TRPM8 channels in sensory neurons as molecular targets of the immunosuppressant tacrolimus. The actions of tacrolimus on TRPM8 resemble those of menthol but likely involve interactions with other channel residues.