Nerve injury elevates functional Cav3.2 channels in superficial spinal dorsal horn

Abstract

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial spinal dorsal horn. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2 but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 knockout mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial spinal dorsal horn may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.

Keywords: Cav3 channels; lamina II neuron; neuropathic pain; spinal dorsal horn; whole-cell patch-clamp recording.

Figure 1.

Analgesic effects of Cav3 channel blockers on rat neuropathic pain model induced by PSNL. (a) and (b) Time courses of mechanical allodynia and thermal hyperalgesia of sham (n = 6) and PSNL (n = 6) rats over a period of 14 days after operation. (c) and (d) Time courses of mechanical allodynia and thermal hyperalgesia of rat PSNL models treated with NiCl₂ (0.5 µg/h, i.t., n = 6), TTA-A2 (0.35 µg/h, i.t., n = 7), and ascorbic acid (0.5 g/kg, i.p., n = 5). Intrathecal (n = 5) or intraperitoneal (n = 3) injection of saline was used as the control. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control. PSNL: partial sciatic nerve ligation; PWT: paw withdrawal threshold; PWL: paw withdrawal latency; i.t.: intrathecal injection; i.p.: intraperitoneal injection; TTA-A2: [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide].

Figure 2.

Expression of Cav3.1–3.3 in superficial SDH in sham and PSNL rats. (a) qRT-PCR results of Cav3 mRNA expression in superficial SDH relative to β-actin in sham (n = 6) and PSNL (n = 6) rats. (b) and (c) Representative western blot (b) and the quantitative analysis (c) of Cav3 protein obtained from the superficial SDH of sham (n = 6) and PSNL (n = 6) rats. The molecular weights of Cav3 and β-actin were ∼250 and ∼42 kDa, respectively. (d) Representative western blot of Cav3.2 from WT and KO mice. *p < 0.05, compared with sham control. PSNL: partial sciatic nerve ligation; S: sham; P: PSNL; WT: wild-type; KO: knockout.

Figure 3.

PSNL-induced mechanical allodynia and thermal hyperalgesia in WT and Cav3.2 KO mice. (a) and (b) Time courses of mechanical allodynia and thermal hyperalgesia of sham and PSNL mice over a period of 14 days after operation (WT-sham: n = 4, WT-PSNL: n = 4; KO-sham: n = 9, and KO-PSNL: n = 10). *p < 0.05, **p < 0.01, ***p < 0.001, compared with sham. ^#p < 0.05, ^##p < 0.01, compared with KO-PSNL mice. PSNL: partial sciatic nerve ligation; WT: wild-type; KO: knockout; PWT: paw withdrawal threshold; PWL: paw withdrawal latency.

Figure 4.

The proportion and electrophysiological characteristics of Cav3 currents in SG neurons of PSNL rats. (a) The proportion of neurons with or without I_T in sham (41/69, 59.4%) and PSNL rats (46/60, 76.7%). (b) Peak amplitudes of I_T (V_hold = −110 mV) from sham (n = 41) and PSNL (n = 46) rats. (c) Current density (peak current amplitude divided by cell’s capacitance) against test potential of I_T from sham (n = 41) and PSNL (n = 46) rats. (d) and (e) Representative activation and inactivation traces of I_T in SG neurons from sham and PSNL rats. Activation currents (d) were evoked by the voltage steps from −110 mV (V_hold) to test potentials from −70 mV through −40 mV in a 5-mV step. Inactivation currents (e) were evoked by test steps to −50 mV after a 0.5-s prepulse at potentials from −60 mV to −110 mV in a 5-mV step. (f) Steady-state activation and inactivation of I_T in the sham and PSNL rats. (g) Bar graphs of half-activation or half-inactivation potentials of I_T (activation: sham, n = 11, PSNL, n = 16; inactivation: sham, n = 14, PSNL, n = 22). *p < 0.05, compared with sham control. PSNL: partial sciatic nerve ligation.

Figure 5.

Cav3 currents in SG neurons of Cav3.2 KO mice. (a) The proportion of neurons with or without I_T in sham and PSNL mice. (b) Peak amplitudes of I_T (V_hold = −110 mV) from sham and PSNL KO mice. (c) Current density against test potential of I_T from sham and PSNL mice. Sham, n = 21; PSNL, n = 25 (p > 0.05). PSNL: partial sciatic nerve ligation.

Figure 6.

Blockade effects of Cav3 currents in SG neurons from sham and PSNL rats by NiCl₂. (a) and (b) The current–voltage (I–V) curves (a) and representative traces (b) of I_T from sham and PSNL rats before or after the treatment of NiCl₂ (sham, n = 11; PSNL, n = 16). (c) Inhibitory ratio of NiCl₂ on I_T amplitudes between sham and PSNL rats at −45 mV (sham, n = 11; PSNL, n = 16). (d) Steady-state activation and inactivation of I_T between sham and PSNL groups after treated with NiCl₂. (e) Effects of NiCl₂ on half-activation or half-inactivation potentials of Cav3 currents in sham and PSNL rats (activation: sham, n = 11, PSNL, n = 16; inactivation: sham, n = 14, PSNL, n = 22). *p < 0.05, **p < 0.01, ***p < 0.001, compared with control. PSNL: partial sciatic nerve ligation.