Action of dexmedetomidine on the substantia gelatinosa neurons of the rat spinal cord

Abstract

Dexmedetomidine is a highly specific, potent and selective alpha(2)-adrenoceptor agonist. Although intrathecal and epidural administration of dexmedetomidine has been found to produce analgesia, whether this analgesia results from an effect on spinal cord substantia gelatinosa (SG) neurons remains unclear. Here, we investigated the effects of dexmedetomidine on postsynaptic transmission in SG neurons of rat spinal cord slices using the whole-cell patch-clamp technique. In 92% of the SG neurons examined (n = 84), bath-applied dexmedetomidine induced outward currents at -70 mV in a concentration-dependent manner, with the value of effective concentration producing a half-maximal response (0.62 microM). The outward currents induced by dexmedetomidine were suppressed by the alpha(2)-adrenoceptor antagonist yohimbine, but not by prazosin, an alpha(1)-, alpha(2B)- and alpha(2C)-adrenoceptor antagonist. Moreover, the dexmedetomidine-induced currents were partially suppressed by the alpha(2C)-adrenoceptor antagonist JP-1302, while simultaneous application of JP-1302 and the alpha(2A)-adrenoceptor antagonist BRL44408 abolished the current completely. The action of dexmedetomidine was mimicked by the alpha(2A)-adrenoceptor agonist oxymetazoline. Plots of the current-voltage relationship revealed a reversal potential at around -86 mV. Dexmedetomidine-induced currents were blocked by the addition of GDP-beta-S [guanosine-5'-O-(2-thiodiphosphate)] or Cs+ to the pipette solution. These findings suggest that dexmedetomidine hyperpolarizes the membrane potentials of SG neurons by G-protein-mediated activation of K+ channels through alpha(2A)- and alpha(2C)-adrenoceptors. This action of dexmedetomidine might contribute, at least in part, to its antinociceptive action in the spinal cord.

Fig. 1

Dexmedetomidine induces an outward current in a concentration-dependent manner in SG neurons. (A) Outward currents induced by dexmedetomidine (0.1, 1 and 10 μm). Duration of drug superfusion is shown by horizontal bars above chart recordings. Holding potential = −70 mV. (B) Relative peak amplitude was calculated as an amplitude of the dexmedetomidine (0.1–30 μm)-induced current divided by that of the current produced by dexmedetomidine (30 μm). The continuous curve was drawn according to the Hill plot with an EC₅₀ value of 0.62 μm (95% confidence interval, 0.51–0.77 μm) and a Hill coefficient of 1.34. The number next to each point denotes the number of neurons examined. Holding potential = −70 mV. (C) Outward currents elicited by dexmedetomidine (0.03 μm) in the absence and presence of TTX (1 μm). These currents were obtained from the same neuron (n= 5). Holding potential = −40 mV. (D) Relative peak amplitude was calculated as an amplitude of dexmedetomidine-induced current in the presence of TTX (5.3 ± 1.3 pA) divided by that of currents produced by dexmedetomidine (5.6 ± 1.4 pA) without TTX.

Fig. 2

Dexmedetomidine-induced currents are mediated by α₂- (α_2A- and α_2C-) but not by α₁-adrenoceptors. (A) Prazosin (2 μm), an α₁-adrenoceptor antagonist, did not affect the amplitude of the outward current induced by dexmedetomidine (0.03 μm), whereas the currents were suppressed by application of yohimbine (4 μm), an α₂-adrenoceptor antagonist. These currents were obtained from the same neuron (n= 6). Holding potential = −40 mV. (B) Relative peak amplitude was calculated as an amplitude of dexmedetomidine-induced current in the presence of prazosin (8.7 ± 2.2 pA) or yohimbine (0.1 ± 0.1 pA) divided by that of currents produced by dexmedetomidine (9.3 ± 2.6 pA) alone. *P< 0.05, control vs. yohimbine. Holding potential = −40 mV. (C) Dexmedetomidine (3 μm) produced an outward current of long duration. A persistent outward current after washout of dexmedetomidine was completely reduced in amplitude by yohimbine (4 μm). Holding potential = −40 mV. (D) The dexmedetomidine-induced outward currents in the presence of yohimbine were significantly smaller than control (*P< 0.05, n= 10). (E) Application of JP-1302 (5 μm) partially blocked the dexmedetomidine (0.03 μm)-induced current, and simultaneous application of JP-1302 (5 μm) and BRL44408 (5 μm) abolished the current completely. These currents were obtained from the same neuron (n= 6). Holding potential = −40 mV. (F) Relative peak amplitude was calculated as an amplitude of dexmedetomidine-induced current in the presence of JP-1302 (3.3 ± 0.3 pA) or JP-1302 plus BRL44408 (0.1 ± 0.1 pA) divided by that of currents produced by dexmedetomidine (6.9 ± 0.6 pA) alone. *P< 0.05, control vs. JP-1302. ^#P< 0.05, control vs. JP-1302 plus BRL44408. (G) Dexmedetomidine-induced outward current was mimicked by the α_2A-adrenoceptor agonist oxymetazoline (10 μm) on SG neurons. Holding potential = −40 mV.

Fig. 3

(A) Dexmedetomidine-induced currents show voltage dependency. (B) Relationship between holding potential and current amplitude. The regression equation was y= 0.901x + 77.1 [R² = 0.92, P= 0.002, y= amplitude of current (pA), x= holding potential (mV)].

Fig. 4

Inhibition of dexmedetomidine-induced current by GDP-β-S or Cs⁺/TEA. (A) The dexmedetomidine (3 μm)-induced outward current was examined with K-gluconate pipette solution containing GDP-β-S (2 mm). Dexmedetomidine produced an outward current just after establishing the whole-cell configuration, but this was markedly abolished when dexmedetomidine was again applied 15 min later (*P< 0.05, n= 5). Holding potential = −40 mV. (B) Dexmedetomidine (3 μm) induced an outward current just after establishing whole-cell recording with Cs⁺ and TEA-containing pipette. When dexmedetomidine was again applied 5 min later, the current was completely abolished (n= 7). Holding potential = −40 mV.

Fig. 5

Dexmedetomidine dose not affect the frequency or amplitude of spontaneous EPSCs in SG neurons. (A) Continuous chart recording of sEPSCs before and during the application of dexmedetomidine (3 μm) (upper trace). Four consecutive traces of sEPSCs for a period indicated by a short bar below the chart recording are shown on an expanded time scale (lower traces). Holding potential = −70 mV. (B) Cumulative distributions of the amplitude (left) and the inter-event interval (right) of sEPSCs before (dashed line) and during the application of dexmedetomidine (continuous line). Dexmedetomidine had no significant effect on the amplitude (P> 0.05; Kolmogorov–Smirnov test) or the inter-event interval distribution (P> 0.05).