Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons

Abstract

Background: Lidocaine, which blocks voltage-gated sodium channels, is widely used in surgical anesthesia and pain management. Recently, it has been proposed that the hyperpolarization-activated cyclic nucleotide (HCN) channel is one of the other novel targets of lidocaine. Substantia gelatinosa in the spinal dorsal horn, which plays key roles in modulating nociceptive information from primary afferents, comprises heterogeneous interneurons that can be electrophysiologically categorized by firing pattern. Our previous study demonstrated that a substantial proportion of substantia gelatinosa neurons reveal the presence of HCN current (Ih); however, the roles of lidocaine and HCN channel expression in different types of substantia gelatinosa neurons remain unclear.

Methods: By using the whole-cell patch-clamp technique, we investigated the effect of lidocaine on Ih in rat substantia gelatinosa neurons of acute dissociated spinal cord slices.

Results: We found that lidocaine rapidly decreased the peak Ih amplitude with an IC50 of 80 μM. The inhibition rate on Ih was not significantly different with a second application of lidocaine in the same neuron. Tetrodotoxin, a sodium channel blocker, did not affect lidocaine's effect on Ih. In addition, lidocaine shifted the half-activation potential of Ih from -109.7 to -114.9 mV and slowed activation. Moreover, the reversal potential of Ih was shifted by -7.5 mV by lidocaine. In the current clamp, lidocaine decreased the resting membrane potential, increased membrane resistance, delayed rebound depolarization latency, and reduced the rebound spike frequency. We further found that approximately 58% of substantia gelatinosa neurons examined expressed Ih, in which most of them were tonically firing.

Conclusions: Our studies demonstrate that lidocaine strongly inhibits Ih in a reversible and concentration-dependent manner in substantia gelatinosa neurons, independent of tetrodotoxin-sensitive sodium channels. Thus, our study provides new insight into the mechanism underlying the central analgesic effect of the systemic administration of lidocaine.

Figure 1.

Lidocaine inhibits I_h in substantia gelatinosa neurons. A, Representative current responses to hyperpolarization voltage steps in the absence (control) and presence of lidocaine (100 μM), washout (recovery), and the following application of ZD7288 (10 μM) in a same neuron (upper). Lower panel shows the I_h evoking voltage protocol. Open circles in the lowest I_h trace indicate the instantaneous (I_inst) and steady state (I_ss) of I_h at −130 mV. B, Sample traces under control condition, the first perfusion of lidocaine (100 μM), washout, and the second perfusion of lidocaine. C, Superimposed traces of I_h (at −130 mV) in (B). D, Time course of the inhibition of lidocaine on I_h amplitude recorded from the same neuron in (B). Gray bars represent the periods of bath application of lidocaine. E, Summary data showing the percentage change in the peak I_h amplitude after application of lidocaine. In this and the following figures, *P < 0.05, **P < 0.01, ***P < 0.001. n.s. = no significant difference.

Figure 2.

Lidocaine-induced I_h inhibition is not affected by tetrodotoxin (TTX). A and B, In the presence of TTX (0.5 μM) in artificial cerebrospinal fluid solution, lidocaine (100 μM) still decreased the peak amplitude of I_h. C, Superimposed traces of I_h (at −130 mV) recorded in (A and B). D and E, Averaged percentage of I_h inhibition by lidocaine in the presence and absence of TTX, respectively. F, Differences of I_h amplitude under the treatment of lidocaine in the presence and absence of TTX. In this and the other figures, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Lidocaine blocks I_h in substantia gelatinosa neurons in a concentration-dependent manner. A, Example traces before and after bath application of increasing concentrations of lidocaine (in micromolar): 10, 100, and 600, which are from the same neuron. B, Superimposed traces of I_h (at −130 mV) recorded in (A). C, Dose-response curve for I_h amplitude under the effect of lidocaine. The values in parentheses indicate the number of cells examined.

Figure 4.

Lidocaine shifts I_h activation to more hyperpolarized potentials in substantia gelatinosa neurons. A, Control activation of I_h current (top) responses to the evoking protocol by hyperpolarizing voltage steps over the range from −60 to −130 mV in 10-mV increments for 1 s from the holding potential of −50 mV, which were then subjected to a voltage jump to −130 mV to obtain full activation (bottom). B, Enlargement of the tail currents in the rectangle shown in (A). Tail currents (marked as the arrow) were normalized to the maximum values [(I_{tail, max} − I_tail)/I_{tail, max}] to plot the activation curve shown in (D). C, Lidocaine markedly decreased the amplitude of tail currents (same neuron in A). D, Lidocaine clearly shifted V_0.5 to more negative values. E, Plot of I_h current density against the membrane potentials. Lidocaine reduced the current density from −70 to −130 mV. F, Time constant (τ) of I_h activation against the membrane potentials. Lidocaine significantly increased τ from −60 to −130 mV. In this and the other figures, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Lidocaine shifts the reversal potential of I_h current to more negative values in substantia gelatinosa neurons. A, Sample control traces of the deactivation of I_h current (top) and the evoking protocol (bottom: the membrane potential was stepped to −130 mV for 1 s from the holding potential of −50 mV to fully activate I_h, followed by a series of depolarization test potentials from −120 to −50 mV in 10-mV increments). B, The expanded traces of the tail currents in the rectangle shown in (A). Tail currents (marked as arrow) were measured at the onset of the test potentials. C, Lidocaine greatly reduced the amplitude of the tail currents (the same neuron in A); D, I-V curves constructed from the tail currents in the absence (black) and presence of lidocaine (gray) to each test potential. Lidocaine shifts the reversal potential to more negative values.

Figure 6.

The effect of lidocaine on firing properties in substantia gelatinosa neurons. A–C (left), Voltage responses to the current commands shown at the bottom during control (black), and the administration of different concentrations of lidocaine (red): 100, 600, and 1000 μM, respectively. Right, Enlargement of rectangular areas shown in (A–C), with a trace of recovery after washout (blue). Lidocaine reduced the frequency of sodium-dependent action potentials and the rebound firings and increased the latency of the rebound firings. Bottom, Voltage responses were recorded under a 1-s depolarization current pulse from 0 to 150 pA, followed by a 1-s hyperpolarization current pulse from 0 to −150 pA. D–G, Grouped data show the percentage change in rebound depolarization latency, frequency, resting membrane potential (RMP) changes, and R_in after application of different concentrations of lidocaine. In this and the other figures, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

I_h expression in different firing patterns of substantia gelatinosa (SG) neurons. A, Representative firing patterns in SG neurons: tonic-firing (a), delayed-firing (b), single-spike (c), initial-burst (d), phasic-bursting (e), gap-firing (f), and reluctant-firing (g) neurons evoked by the protocol in (h). B, Summary bar graph showing numbers of neurons expressing I_h with respect to cell electrophysiologic classification. C, Histogram figure showing τ values (at −130 mV) in the subtypes of SG neurons.