Role of TRPM8 Channels in Altered Cold Sensitivity of Corneal Primary Sensory Neurons Induced by Axonal Damage

Abstract

The cornea is extensively innervated by trigeminal ganglion cold thermoreceptor neurons expressing TRPM8 (transient receptor potential cation channel subfamily M member 8). These neurons respond to cooling, hyperosmolarity and wetness of the corneal surface. Surgical injury of corneal nerve fibers alters tear production and often causes dry eye sensation. The contribution of TRPM8-expressing corneal cold-sensitive neurons (CCSNs) to these symptoms is unclear. Using extracellular recording of CCSNs nerve terminals combined with in vivo confocal tracking of reinnervation, Ca 2ϩ imaging and patch-clamp recordings of fluorescent retrogradely labeled corneal neurons in culture, we analyzed the functional modifications of CCSNs induced by peripheral axonal damage in male mice. After injury, the percentage of CCSNs, the cold-and menthol-evoked intracellular [Ca 2ϩ ] rises and the TRPM8 current density in CCSNs were larger than in sham animals, with no differences in the brake K ϩ current I KD. Active and passive membrane properties of CCSNs from both groups were alike and corresponded mainly to those of canonical low-and high-threshold cold thermoreceptor neurons. Ongoing firing activity and menthol sensitivity were higher in CCSN terminals of injured mice, an observation accounted for by mathematical modeling. These functional changes developed in parallel with a partial reinnervation of the cornea by TRPM8(ϩ) fibers and with an increase in basal tearing in injured animals compared with sham mice. Our results unveil key TRPM8-dependent functional changes in CCSNs in response to injury, suggesting that increased tearing rate and ocular dryness sensation derived from deep surgical ablation of corneal nerves are due to enhanced functional expression of TRPM8 channels in these injured trigeminal primary sensory neurons.

Negative Modulation of TRPM8 Channel Function by Protein Kinase C in Trigeminal Cold Thermoreceptor Neurons

“…To quantify the CTN population that would be subjected to the modulation of this proinflammatory mediator, we evaluated the functional response to cold and bradykinin from cultured adult TG neurons obtained from intact mice ( Figure 5 ). In agreement with previous studies [ 48 , 57 ], neurons that respond to cooling ramps and 100 µM menthol with [Ca 2+ ] i increases represent about 10% (167/1628) of the entire population of trigeminal primary sensory neurons ( Figure 5 A,B). The percentage of bradykinin-sensitive neurons (i.e., showing an increase in [Ca 2+ ] i during bradykinin application due to Ca 2+ -release from InsP 3 -sensitive intracellular deposits) was ~16% (264/1628).…”

“…The cold stimulus causes a dramatic increase in the firing rate, from the basal activity of around 5 spikes/s, and more bursting events (seen in the ISI plot as ISI< 100 ms). When the temperature is brought back to 33.5 °C, there is a transient period of no activity that mimics the experimental recordings [ 5 , 48 ]. When the maximal conductance of TRPM8 (akin to channel density) is diminished to 60%, there is a decrease in the basal firing rate to an average of 2–3 spikes/s.…”

“…We used our conductance-based mathematical model of corneal CTNs that includes the TRPM8 channel [ 48 , 58 , 59 ], to emulate the PKC-dependent TRPM8 functional variation and further explore its effect on basal firing and cold-evoked responses. Figure 7 A shows the result of simulating the response of our model to a cold pulse, with a normal (left, 100%) or decreased (right, 60%) maximal conductance (g max ) of the TRPM8 channel, a change similar to the reduction we observed in patch-clamp experiments.…”

“…E i is the reversal potential of the current and a i is the activation variable or open channel probability, usually voltage-dependent, except for (for other equations and details see [ 58 , 59 ]). The I KD current [ 77 , 80 ] is contained in the rest of the parameters considered in the model, and therefore it is invariant in all simulations [ 48 ].…”

“…TRPM8 has a key role in excitability disturbances of corneal fibers induced by peripheral nerve damage. In corneal physiology, local inflammation caused by surgical procedures (photorefractive or cataract surgery), tear film pathologies (dry eye disease) or long-term wearing contact lenses, among others, affect the peripheral axons and nerve terminals of corneal sensory neurons, finally altering ocular surface homeostasis [ 9 , 47 , 48 , 49 , 50 ]. Since tissue damage and inflammation can trigger signaling pathways that lead to PKC activation (reviewed by [ 51 ]), PKC-dependent regulation of TRPM8 function in corneal nerve endings is an exciting opportunity to explore its role in a physiologically relevant context.…”