Ca(v)3.2 T-type Ca2+ channel-dependent activation of ERK in paraventricular thalamus modulates acid-induced chronic muscle pain

Abstract

Treatments for chronic musculoskeletal pain, such as lower back pain, fibromyalgia, and myofascial pain syndrome, remain inadequate because of our poor understanding of the mechanisms that underlie these conditions. Although T-type Ca2+ channels (T-channels) have been implicated in peripheral and central pain sensory pathways, their role in chronic musculoskeletal pain is still unclear. Here, we show that acid-induced chronic mechanical hyperalgesia develops in Ca(v)3.1-deficient and wild-type but not in Ca(v)3.2-deficient male and female mice. We also show that T-channels are required for the initiation, but not maintenance, of acid-induced chronic muscle pain. Blocking T-channels using ethosuximide prevented chronic mechanical hyperalgesia in wild-type mice when administered intraperitoneally or intracerebroventricularly, but not intramuscularly or intrathecally. Furthermore, we found an acid-induced, Ca(v)3.2 T-channel-dependent activation of ERK (extracellular signal-regulated kinase) in the anterior nucleus of paraventricular thalamus (PVA), and prevention of the ERK activation abolished the chronic mechanical hyperalgesia. Our findings suggest that Ca(v)3.2 T-channel-dependent activation of ERK in PVA is required for the development of acid-induced chronic mechanical hyperalgesia.

Figure 1.

Acid-induced chronic mechanical hyperalgesia develops in WT and Ca_v3.1^−/− but not Ca_v3.2^−/− mice. A–C, Mechanical responses of WT (A, n = 7), Ca_v3.1^−/− (B, n = 5) and Ca_v3.2^−/− mice (C, n = 8) following the injection of acid into the gastrocnemius muscle. In all panels, arrows indicate days on which mice received acid injections. Filled circles represented ipsilateral side, and open-circles represented contralateral side. Error bars represent SEM.

Figure 2.

Blocking T-channels prevents the development of acid-induced chronic mechanical hyperalgesia in WT mice in a dose- and time-dependent manner. A, Effects of different dosages (in mg/kg) of ETX administered intraperitoneally. One hundred milligrams per kilogram is the lowest dosage tested that prevented hyperalgesia. *p < 0.05, compared with saline-treated animal. B, Effects of different dosages of the L-channel blocker nicardipine. None of the dosages tested had a significant effect on acid-induced hyperalgesia (n = 4). C, Effects of ETX injection before and after the second acid injection. Treatment with ETX 15 min before but not 30 min after the second acid injection prevented acid-induced chronic hyperalgesia. *p < 0.05, compared with group treated with ETX 15 min before acid injection. D, Effects of ETX administration at different time points (0, 5, 10, 15, 20, and 25 min) after the second acid injection. ETX given at 10 min (or less) after second acid injection prevented the development of mechanical hyperalgesia. In contrast, ETX given at 15 min (or more) after second acid injection had no effect on the development of mechanical hyperalgesia. Numbers in parentheses indicate the number of animals tested. *p < 0.05, compared with 0 min group. Error bars represent SEM.

Figure 3.

Peripheral and spinal T-channels are not involved in the development of acid-induced chronic hyperalgesia. A, Intramuscular injection of ETX (0.1 and 0.2 mg) or NNC 55-0396 (NNC, 1 μg) did not prevent acid-induced chronic hyperalgesia. B, Current–voltage curves of T-currents recorded from Fluorogold-labeled muscle afferents DRG neurons. Numbers in parentheses indicate the number of cells recorded. Inset shows representative current traces evoked by a depolarizing step to −40 mV from a holding potential of −90 mV. C, Intrathecal injection of ETX (0.1 and 0.2 mg) or NNC (1 μg) did not prevent acid-induced chronic hyperalgesia. D, The induction of spinal c-Fos expression in response to repeated acid injection was similar in WT and Ca_v3.2^−/− mice. E, The number of c-Fos-positive cells in the dorsal horn in WT and Ca_v3.2^−/− mice that received pH 4.0 or pH 7.2 saline. Averaged numbers of c-Fos-positive cells from three consecutive sections were counted per animal. Three animals were used for each treatment. *p < 0.05, compared with group treated with pH 7.2 saline. Error bars represent SEM.

Figure 4.

T-channels in the brain are required for the development of acid-induced chronic muscle hyperalgesia. A, Intracerebroventricular injection of ETX (0.2 mg) or NNC 55-0396 (NNC, 1 μg), but not nicardipine (0.125 mg), prevented acid-induced chronic mechanical hyperalgesia in WT mice. *p < 0.05, compared with saline-treated animals. B, C, pERK staining in the central nucleus of the amygdala (Amyg) (B) and the piriform cortex (Pir) and paraventricular hypothalamic nucleus (PVN) (C) following acid injection was similar in WT and Ca_v3.2^−/− mice at 10 min after second acid injection. D, Acid-induced, Ca_v3.2 T-channel-dependent pERK staining was observed in the PVA (outlined by dashed line). pERK staining in the PVA was observed in pH 4.0 saline-injected WT but not Ca_v3.2^−/− mice. E, Quantification of pERK-positive cells in the PVA from WT and Ca_v3.2^−/− mice that received pH 4.0 or pH 7.2 saline. Average numbers of pERK-positive cells from three consecutive sections were counted per animal. Three animals were used for each treatment. *p < 0.05, compared with WT group. Error bars represent SEM.

Figure 5.

T-channels in the brain are required for the development of acid-induced chronic muscle hyperalgesia. A, pERK staining in the PVA following acid injection was observed in vehicle but not U0126 intra-PVA infusion in WT mice. Amygdala pERK staining was similar in both vehicle and U0126 infusion groups. B, Intra-PVA infusion of U0126 (1.5 nmol) but not vehicle before the second acid injection into muscle prevented acid-induced chronic muscle hyperalgesia in WT mice. C, pERK staining in the PVA following acid injection was observed in PDBU but not vehicle intra-PVA infusion in Ca_v3.2^−/− mice. Amygdala pERK staining was similar in both vehicle and PDBU infusion groups. D. Intra-PVA infusion of PDBU (20 pmol) but not vehicle before second acid injection into muscle enabled acid-induced chronic muscle hyperalgesia in Ca_v3.2^−/− mice. *p < 0.05, compared with vehicle-treated group. Error bars represent SEM.