KCNQ channels in nociceptive cold-sensing trigeminal ganglion neurons as therapeutic targets for treating orofacial cold hyperalgesia

Abstract

Background: Hyperexcitability of nociceptive afferent fibers is an underlying mechanism of neuropathic pain and ion channels involved in neuronal excitability are potentially therapeutic targets. KCNQ channels, a subfamily of voltage-gated K(+) channels mediating M-currents, play a key role in neuronal excitability. It is unknown whether KCNQ channels are involved in the excitability of nociceptive cold-sensing trigeminal afferent fibers and if so, whether they are therapeutic targets for orofacial cold hyperalgesia, an intractable trigeminal neuropathic pain.

Methods: Patch-clamp recording technique was used to study M-currents and neuronal excitability of cold-sensing trigeminal ganglion neurons. Orofacial operant behavioral assessment was performed in animals with trigeminal neuropathic pain induced by oxaliplatin or by infraorbital nerve chronic constrictive injury.

Results: We showed that KCNQ channels were expressed on and mediated M-currents in rat nociceptive cold-sensing trigeminal ganglion (TG) neurons. The channels were involved in setting both resting membrane potentials and rheobase for firing action potentials in these cold-sensing TG neurons. Inhibition of KCNQ channels by linopirdine significantly decreased resting membrane potentials and the rheobase of these TG neurons. Linopirdine directly induced orofacial cold hyperalgesia when the KCNQ inhibitor was subcutaneously injected into rat orofacial regions. On the other hand, retigabine, a KCNQ channel potentiator, suppressed the excitability of nociceptive cold-sensing TG neurons. We further determined whether KCNQ channel could be a therapeutic target for orofacial cold hyperalgesia. Orofacial cold hyperalgesia was induced in rats either by the administration of oxaliplatin or by infraorbital nerve chronic constrictive injury. Using the orofacial operant test, we showed that retigabine dose-dependently alleviated orofacial cold hyperalgesia in both animal models.

Conclusion: Taken together, these findings indicate that KCNQ channel plays a significant role in controlling cold sensitivity and is a therapeutic target for alleviating trigeminal neuropathic pain that manifests orofacial cold hyperalgesia.

Fig. 1

KCNQ channels in nociceptive cold-sensing trigeminal neurons. a Image shows KCNQ2 immunoreactivity (KCNQ2-ir) in a trigeminal ganglion section. b Sample traces of M-currents in the absence (black) and presence (red) of 20 µM linopirdine. The voltage step used to reveal M-currents was shown under the recording traces. The box indicates the M-currents, measured by the tail currents after the voltage step. c I–V curve of tail currents in the absence (black) and presence (red) of 20 µM linopirdine. d Ca²⁺-imaging shows an example of pre-identification of cold-sensing TG neurons with menthol (100 µM). e An example of a pre-identified cold-sensing TG neuron that responded to a cooling temperature ramp by firing action potentials. The cell was under the whole-cell current-clamp recording mode. The cooling ramp from 24 to 8°C is indicated under the recording trace. f Traces on the top panel show a nociceptive cold-sensing neuron that responds to 10 current-steps at the increment of 30 pA and the cell fires a slow action potential (Rheobase = 300 pA) with broad width and a hump in the repolarization phase. A total of 15 cold-sensing TG neurons belong to this category. Traces on the bottom panel show a non-nociceptive cold-sensing neuron that responds to 10 current-steps at the increment of 30 pA and the cell fires a fast action potential (Rheobase = 30 pA) without any hump in the repolarization phase. A total of 21 cold-sensing TG neurons belong to this category. g Traces show a nociceptive cold-sensing neuron that responds to voltage steps from −80 to −20 mV at 10 mV increment. The cell was held at −60 mV. h Summary result of M-currents recorded from nociceptive cold-sensing neurons in the absence (control) and presence of 20 µM linopirdine. The M-currents were measured by the deactivating tail currents following the voltage step of −20 mV. Data represent Mean ± SEM, **P < 0.01.

Fig. 2

Increases of the excitability of nociceptive cold-sensing trigeminal neurons by inhibiting KCNQ channels. a An example shows that blocking KCNQ channels by linopirdine (20 µM) enhances excitability of nociceptive cold-sensing TG neurons. b Summary of the changes of resting membrane potentials (RMP) following the application of 20 µM linopirdine (n = 8). c Summary of the changes of rheobase for action potential firing following the application of 20 µM linopirdine (n = 7). d Behavioral cold hypersensitivity induced by linopirdine. Orofacial operant tests were performed at 12°C following the subcutaneous injection of saline or 0.39 mg linopirdine in oral facial regions. The linopirdine-injected animals (n = 4) show a significant reduction of total contact time in comparison with saline controls (n = 4). Data represent Mean ± SEM, *P < 0.05, **P < 0.01.

Fig. 3

Suppression by retigabine of cold-induced action potential firing in nociceptive cold-sensing trigeminal neurons. a Sample traces show an example of cold-evoked action potential firing in a nociceptive cold-sensing TG neuron in the absence (control) and presence of 10 µM retigabine. Recordings were made under the whole-cell current-clamp mode. Cooling temperature ramp was applied from 24 to 8°C as indicated under the recording traces. b Summary data shows significant reduction of cold-evoked action potential firing in nociceptive cold-sensitive TG neurons (n = 8). Action potential numbers are normalized to control value. Data represent Mean ± SEM, **P < 0.01.

Fig. 4

Orofacial operant assessment of oxaliplatin-induced orofacial cold allodynia/hyperalgesia. a Images show the postures commonly seen during orofacial operant tests at 17°C for control rats (left) and rats after oxaliplatin injections (right). The control rat rested its face on the cooling module while drinking milk. The oxaliplatin-injected (18 days after the injections) rat did no contact the cooling thermal module but tried to bite it off. b Recordings of contact numbers and duration of each contact in orofacial operant tests at 17°C for the control rat (top) and the oxaliplatin-injected rat (bottom). The duration of each orofacial operant test session was 10 min. c Change of total contact time over days after the injections of oxaliplatin (n = 5–8). d Left total contact time at 17°C for the rats 18 day after injection of saline (open bar) or oxaliplatin (oxa, closed bar). Right similar to left panel except the orofacial operant tests were conducted at 12°C. Data represent Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

Alleviation of oxaliplatin-induced orofacial cold hyperalgesia by retigabine. Bar graph shows total contact time of orofacial operant tests for oxaliplatin-injected rats under the following conditions: 24, 12, 12°C with vehicle injection, 12°C with retigabine treatment at the dose of 0.19, 0.56, 1.67, and 15 mg/kg. Retigabine was administered intraperitoneally to the animals. Data represent Mean ± SEM, ***P < 0.01.

Fig. 6

Alleviation by retigabine of orofacial cold hyperalgesia in infraorbital nerve chronic constrictive injury model. Bar graph shows total contact time of orofacial operant tests for infraorbital nerve chronic constrictive injury (ION-CCI) animals under the following conditions: 24, 12, 12°C with vehicle injection, 12°C with retigabine treatment at the doses of 0.19, 0.56, and 1.67 mg/kg. Retigabine was administrated intraperitoneal to the animals. Data represent Mean ± SEM, *P < 0.05; **P < 0.01.