2 µg of siRNA were complexed with in vivo-jetPEI and injected intradermally into rats hindpaw to target neural fibers of the dorsal root ganglion. Analysis was performed 3 days later by western blot of the skin tissue, immunohistochemistry of skin and nerve and functional tests.

Ongoing/spontaneous pain behavior is associated with ongoing/spontaneous firing (SF) in adult DRG C-fiber nociceptors (Djouhri et al., 2006). Causes of this SF are not understood. We show here that conducting (sometimes called uninjured) C-nociceptors in neuropathic pain models with more hyperpolarized resting membrane potentials (Ems) have lower SF rates. Understanding the control of their Ems may therefore be important for limiting pathological pain. We report that TREK2, a leak K(+) channel, is selectively expressed in IB4 binding rat C-nociceptors. These IB4(+) C-neurons are approximately 10 mV more hyperpolarized than IB4(-) C-neurons in vivo (Fang et al., 2006). TREK2 knockdown by siRNA in these neurons in culture depolarized them by approximately 10 mV, suggesting that TREK2 is responsible for this approximately 10 mV difference. In vivo, more hyperpolarized C-nociceptor Ems were associated with higher cytoplasmic edge-TREK2 expression (edge-TREK2). Edge-TREK2 decreased in C-neurons 7 d after axotomy, and their Ems depolarized by approximately 10 mV. This again supports a contribution of TREK2 to their Ems. These relationships between (1) Em and TREK2, (2) SF rate and Em, and (3) spontaneous pain behavior and C-nociceptor SF rate suggested that TREK2 knockdown might increase spontaneous pain. After CFA-induced inflammation, spontaneous foot lifting (a measure of spontaneous pain) was (1) greater in rats with naturally lower TREK2 in ipsilateral small DRG neurons and (2) increased by siRNA-induced TREK2 knockdown in vivo. We conclude that TREK2 hyperpolarizes IB4 binding C-nociceptors and limits pathological spontaneous pain. Similar TREK2 distributions in small DRG neurons of several species suggest that these role(s) of TREK2 may be widespread.