Spinal dorsal horn neurons in laminae I to II receive sensory information from myelinated Aδ and unmyelinated C-fibers encoding afferent impulses from receptors responsible for pain and thermoception.1 During spinal and epidural anesthesia, these cells are exposed to high concentrations of local anesthetics that diffuse directly into the spinal cord.2 Because the spinal cord contributes considerably to the effects of systemic anesthetics,3 spinal dorsal horn neurons are important targets for local anesthetics administered IV used for neuropathic pain therapy. After systemic application, however, these neurons are exposed to considerably lower concentrations of local anesthetics4,5 than from intrathecal application.
Based on their firing patterns as a response to a long depolarizing pulse, spinal dorsal horn neurons can be divided into 3 major physiological groups6: (1) tonic firing neurons (TFN) are characterized by tonic firing in response to depolarizing current pulses and receive sensory information about pain and thermoception.6–8 (2) Adapting firing neurons (AFN) generate an unsustained burst of spikes at the beginning of the depolarizing pulse. These neurons are nociceptive specific.6 (3) Single spike neurons (SSN) generate 1 or rarely 2 spikes at the beginning of the pulse. These neurons act as coincidence or novelty detectors.8
It is generally accepted that local anesthetics block pain transmission by blocking voltage-gated sodium channels although an increasing number of studies describe the action of local anesthetics on different types of voltage-gated potassium channels.9–12 The relevance of an inhibition of potassium channels by lidocaine for the generation of single action potentials and series of action potentials remains unclear, especially in situations when the blockade of Na+ channels is incomplete (complete blockade of voltage-gated sodium or potassium channels would result in a complete suppression of neuronal excitability).13,14
We describe the effects of low concentrations of lidocaine on the excitability of different types of spinal dorsal horn neurons in which voltage-gated sodium channels are only partially blocked (to approximately 50%). We mimicked the Na+ and K+ channel-blocking features of lidocaine by applying selective Na+ and K+ channel blockers. The results led to a better understanding of the analgesic mechanisms of local anesthetics and the role of K+ channels in different types of spinal dorsal horn neurons.
Experiments were performed using the patch-clamp technique15 on 200 μm slices cut from lumbar enlargements (L3–L6) of the spinal cords of rats aged 3 to 11 days.16,17 All animals were killed by rapid decapitation according to the standards set down by the German and Austrian guidelines. The experimental procedure was approved by the local Animal Care Committee (RP Giessen). The spinal cords was carefully excised in ice-cold preparation solution bubbled with O2 and CO2 (95%–5%). After removal of the pial membrane with fine forceps, the spinal cord was embedded in a preparation solution containing 2% agar cooled to 39°C. To accelerate solidification of the agar, the beaker with the preparation was placed in ice-cold water. The agar block containing the lumbar enlargement of the spinal cord was cut out and glued to a glass stage fixed in the chamber of the tissue slicer. The spinal cord was sectioned (200 μm) in ice-cold preparation solution under continuous bubbling. Thereafter, the slices were incubated for 45 minutes at 32°C.
Preparation solution contained (in mM) NaCl 115, KCl 5.6, CaCl2 2, MgCl2 1, glucose 11, NaH2PO4 1, and NaHCO3 25 (pH 7.4 when bubbled with 95% O2–5% CO2). In the experimental chamber, the slices were superfused with low Ca2+ solution to reduce spontaneous synaptic activity and to prevent activation of Ca2+ currents and Ca2+-dependent K+ currents. This solution was obtained from the preparation solution by setting the concentration of Ca2+ to 0.1 mM and increasing the concentration of Mg2+ to 5 mM (in the following referred to as Ringer’s solution).
Lidocaine, tetrodotoxin (TTX), and tetraethylammonium (TEA) were added directly to Ringer’s solution. The experimental chamber, with a volume of 0.4 mL, was continuously perfused with external solution at a flow rate of 2 to 3 mL/min.
The pipette solution used for current recordings from intact neurons contained (in mM) NaCl 5, KCl 144.4, MgCl2 1, EGTA 3, and HEPES 10 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). pH 7.3 was adjusted with 1 M KOH. KCl was then added to yield a final K+ concentration of 155 mM.
Pipettes pulled from borosilicate glass tubes (GC 150; Clark Electromedical Instruments, Pangbourne, United Kingdom) were fire-polished to give a final resistance of 3 to 7 MΩ. The patch-clamp amplifier was an Axopatch 200B (Axon Instruments, Foster City, CA). The effective corner frequency of the low pass filter was 5 kHz. The frequency of digitization was twice that of the filter frequency. The data were stored and analyzed using commercially available software (pCLAMP, Axon Instruments). Transients and leakage currents were digitally subtracted in all experiments using records with hyperpolarizing pulses that activated no currents. Offset potentials were nulled directly before formation of the seal. Errors in the clamped potential evoked by the series resistance of the electrode were not corrected. All experiments were performed at room temperature of 21°C to 23°C.
Ion currents were recorded before action potential recordings. They were activated by voltage steps to different potentials after a 50-millisecond prepulse to –120 mV; holding potential was –80 mV in the voltage-clamp configuration.
Action potentials were recorded in current-clamp mode. To make the action potentials or trains of action potentials comparable, the membrane potential was maintained at approximately –80 mV by administering sustained depolarizing or hyperpolarizing currents through the recording electrode.
Identification of Dorsal Horn Neurons
The dorsal horn neurons were identified in spinal cord slices as multipolar cells with a soma (8–12 μm diameter) located in laminae I to II. Neurons were distinguished from glial cells in voltage-clamp mode on the basis of a procedure described previously.17 During the experiments, the cells were monitored under infrared video microscopy (Hamamatsu Photonics, Shizuoka Pref., Japan).
Based on our data from studies with the same experimental model,18 we performed a power calculation with α 0.05 and found 10 experiments per group for a power of 80%. We therefore aimed to obtain at least 10 complete sets of data for each parameter. Only neurons with 1 recording under control conditions and 1 after superfusion with the drug were included in the analysis.
The inclusion criterion for all patch-clamp experiments was the anatomical location (see identification of dorsal horn neurons). For current-clamp studies, resting membrane potential is more negative than −50 mV and an action potential in response to a 500-millisecond current pulse is generated. Because there is no visual tool to distinguish between tonically firing (TFNs), adapting firing (AFNs), and single spike (SSNs) dorsal horn neurons, all neurons were chosen blindly and retrospectively stratified into 1 of 3 groups (TFN, AFN, SSN) based on their first response. Inclusion criteria for voltage-clamp studies of potassium channels were resting membrane potential more negative than −50 mV and generation of an action potential in response to a 1-millisecond current pulse. The inclusion criterion for voltage-clamp studies of sodium channels was sodium current larger than 1 nA.17
Statistical Analysis and Fitting
Numerical values are expressed as means ± standard error of the mean (SEM); the differences between control and drug are given as median and range. The maximum number of action potentials that the neurons generated was determined as f = (N − 1)*(ΔT) − 1, where N is the number of spikes and ΔT the time interval between the first and the last spike. The trace with the maximum firing frequency was selected for further analysis. Five consecutive pulses (1 millisecond) with identical interspike intervals were used to determine the theoretical maximum firing frequency of the neurons. Five single action potentials exceeding 0 mV with the lowest possible interspike intervals were selected to define this maximum possible frequency.
Analysis of Changes in Action Potentials and Firing Behavior
For each neuron, a pair of recordings before and after drug administration was subjected to statistical analysis. The exact Wilcoxon signed rank test was used throughout. P values <0.05 are given. The test was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, http://www.graphpad.com/scientific-software/prism/).
Voltage-Gated Sodium and Potassium Currents
The concentrations of lidocaine and TTX were selected to block approximately 50% of the voltage-gated sodium current.12,14,19 Lidocaine (100 μM) and TTX (10 nM) blocked voltage-gated Na+ currents in all 3 types of neurons. The estimated peak amplitude of voltage-gated sodium current was reduced to 56% ± 3% (n = 23) and 54% ± 3% (n = 34), respectively (Fig. 1, A and B). While 100 μM lidocaine reduced the amplitude of voltage-gated potassium currents (Fig. 1A), 10 nM TTX had no effect on voltage-gated potassium currents (Fig. 1B). To mimic the effects of 100 μM lidocaine on voltage-gated sodium and potassium currents in TFNs, we combined 10 nM TTX with 10 mM TEA to block voltage-gated sodium and potassium currents to about 50% (Fig. 1C; n = 7).
Effect of Lidocaine and TTX on Single Action Potentials
The properties of single action potentials (peak potential, width at half-maximum potential, maximum positive and maximum negative slope) were investigated in current-clamp mode in all 3 types of neurons. The action potentials were elicited by 1-millisecond current pulses. Lidocaine and TTX at the concentrations chosen affected the properties of single action potentials. Table 1 has detailed the analysis of changes in different single action potential parameters.
Excitability of Tonic Firing Neurons
To investigate the excitability of TFNs, sustained current (5–100 pA) was applied for 500 milliseconds (Fig. 2). Lidocaine (100 μM) reduced the number of spikes (P = 0.0016; Fig. 2A; Table 2). The ability to generate a series of action potentials was lost in all 13 neurons; the firing pattern was converted into an adapting one. TTX application reduced firing frequency (P = 0.0038; Fig. 2B), but the tonic firing pattern remained unchanged. When TEA (10 mM) was applied secondary to TTX (10 nM), the reduction in spike frequency was similar to the effects of lidocaine (Fig. 2C). The number of action potentials was reduced from 11.3 ± 1.4 to 2.3 ± 0.6 (P = 0.014), and the firing pattern changed to an adapting one.
Five consecutive current pulses (1 millisecond) with different interspike intervals were used to determine the theoretical maximum firing frequency of the neurons (up to 200 Hz), depending predominantly on the reserve of sodium channels in the neurons. The application of lidocaine (Fig. 3A) or TTX (Fig. 3B) reduced the maximum elicitable frequency in these cells, but all the cells were still able to generate the 5 consecutive action potentials with frequencies higher than those generated by the neuron itself after a sustained current application (P = 0.0020 and 0.031 for lidocaine and TTX, respectively; Table 2).
Excitability of Adapting Firing Neurons
AFNs generated an unsustained burst of spikes at the beginning of a depolarizing pulse of 500 milliseconds. Lidocaine at a concentration of 100 μM reduced the action potentials (P = 0.0038; Fig. 4A; Table 2). A similar reduction was observed after application of 10 nM TTX (P = 0.0057; Fig. 4B). Because TTX mimicked the effects of lidocaine sufficiently, no experiments with additional TEA were performed. Five consecutive pulses (1 millisecond) with different interspike intervals elicited sequences of 5 single action potentials with high frequencies. The application of lidocaine (Fig. 5A) or TTX (Fig. 5B) reduced the maximum frequency (P = 0.0050), but all the cells were still able to generate 5 consecutive action potentials with high frequencies (Table 2).
Excitability of Single Spike Neurons
SSNs generate only 1 or rarely 2 spikes at the beginning of the pulse. Neither 100 μM lidocaine (Fig. 6A) nor 10 nM TTX (Fig. 6B) was able to change the firing pattern of these cells significantly; only the shape of the single action potential was slightly altered. In addition, in the experiments applying 5 consecutive pulses with different interspike intervals, the neurons were still able to generate 5 consecutive action potentials (Fig. 7, A and B; Table 2).
The local anesthetic lidocaine is widely used in local anesthesia and is sometimes administered for systemic analgesia.20 The clinical effects of lidocaine are usually explained by a blockade of voltage-gated sodium channels of different neuronal structures. We showed that the mechanism of action of lidocaine at low concentrations (blockade of 50% of available sodium channels) depends on the type of neuron investigated.
In addition to this generally accepted view, local anesthetics such as lidocaine have multiple sites of action in the nervous system. In particular, interactions with different types of K+ channels12,21,22 and hyperpolarization-activated cation channels23 have been described. Because we intended to determine the impact of the K+ channel inhibiting properties of lidocaine in different types of spinal dorsal horn neurons, we investigated the effects of lidocaine (which blocks voltage-gated sodium and potassium currents),12,24 TTX (blocks voltage-gated sodium currents; TTX-resistant Na+ channels are absent in spinal dorsal horn neurons),14,17,19,25 and, if applicable, TEA (blocks potassium channels).13,19 The concentration of lidocaine was in the clinically relevant range4,5 and was selected to produce a moderate blockade of voltage-gated Na+ channel-mediated effects (the reserve of Na+ channels in neurons is high)14,19,26 and a pronounced blockade of voltage-gated K+ channels (the safety factor for K+ channels is rather low)19 to demonstrate the special relevance of a potassium channel blockade by lidocaine. The concentrations of TTX and TEA were selected to produce an equivalent blockade of voltage-gated Na+ and K+ channels, respectively, as we described previously for TEA on voltage-gated Na+ and K+ channels and action potentials.19
The specific simulation of lidocaine’s action by TTX and if necessary (in TFN) by TEA is problematic because TTX cannot exactly mimic the use-dependent blockade of voltage-gated sodium channels by lidocaine. The application of TTX in AFN, however, yielded comparable results, suggesting that lidocaine’s effects in these cells depend predominantly on a tonic blockade of voltage-gated sodium channels. Furthermore, we performed experiments in each cell type with 5 consecutive, separately elicited single action potentials. We demonstrated that all cells were able to generate artificial series of action potentials with high frequencies after application of lidocaine and TTX. We therefore concluded that the use-dependent blockade of voltage-gated sodium channels by lidocaine has little effect on those frequencies of stimulated action potentials that we investigated in the neurons. The effect of use-dependent blockade of lidocaine could become more pronounced in neurons generating higher action potential frequencies than those we observed. In addition, lidocaine blocks A-type potassium currents in a concentration-dependent manner,12,24 and voltage-gated delayed rectifier channel conductance is slightly increased at low concentrations and slightly decreased at higher concentrations of lidocaine.12,27 Contrary to the effects of lidocaine, TEA produces an exclusive blockade of voltage-gated delayed rectifier channels.17,19,28 We were unable to find a drug with the same potassium channel-blocking properties as lidocaine that did not block voltage-gated sodium channels. A variety of sodium channel blockers in clinical use also block potassium channels in different patterns.12,18,19,23,29–34 Applied at concentrations approximating the half-maximal inhibitory concentration (IC50) value for sodium channels, these drugs are usually able to reduce the frequency of series of action potentials in TFN in a similar manner.18,19,29,30
The ability to generate action potential series in TFN depends on a critical equilibrium of sodium and potassium currents.35 Because the reserve of sodium channels is rather high in these cells compared with the reserve of potassium currents,19 a blockade of voltage-gated potassium channels by lidocaine or TEA led to a significant depression in firing frequency and changes in the firing pattern (the effects of an exclusive application of TEA on series of action potentials in TFN has been described).19 However, these cells continued to generate stimulated single spikes with high frequencies that exceeded those generated by the neurons themselves. It seems that the lack of adequate repolarization by a blockade of voltage-gated potassium channels, reducing the number of available sodium channels, is a highly sensitive mechanism that reduces the excitability of tonically firing spinal dorsal horn neurons. Similar effects were observed using ketamine,30 meperidine,29 or clonidine18 to block voltage-gated sodium and potassium currents. The impact of potassium channel blockade in TFN becomes important at low concentrations of lidocaine when the remaining Na+ channels still suffice to generate action potentials. This situation might occur during the onset of spinal anesthesia, when the Na+ channel blockade is not fully developed or during the systemic administration of local anesthetics. A similar suppression of tonic firing properties by lidocaine was reported for brain neurons.23 It was explained by a blockade of the hyperpolarization-activated cation current, I(h), by lidocaine. Interestingly, I(h) currents were absent in dorsal horn neurons of Wistar rats.36 Furthermore, the reduction in the number of action potentials in TFN by lidocaine in our experiments was also seen in experiments with TEA, which was ineffective in blocking I(h) currents. We postulate that a different action mechanism of lidocaine was responsible for the reduction in the number of action potentials in TFN of Wistar rats. Presumably, a blockade of voltage-gated potassium channels by lidocaine was responsible for the reduction in the number of action potentials in spinal dorsal horn TFN. The effects of lidocaine and TTX on firing properties of AFN were similar in our study. We conclude that in these cells the reduction in the number of available sodium channels is the critical event to reduce the number of generated action potentials.37 The spike of SSN seems to be resistant to a 50% blockade of voltage-gated sodium currents. Almost no change in firing pattern was observed; however, a more elaborate analysis of single action potentials revealed some minor changes in single action potential variables. Because these cells may act as coincidence detectors,8 this phenomenon may help explain the delayed loss of touch sensibility during the onset of spinal anesthesia.
Because the spinal cord contributes to the effects of systemically administered anesthetics,38 their action can be explained in part by an interaction on different types (TFN, AFN) of spinal dorsal horn neurons. The pain relief after IV administration of lidocaine could be explained by the reduction of firing frequency in spinal dorsal horn TNF and AFN.
In conclusion, lidocaine at low concentrations suppresses the tonic firing pattern of TFN by an interaction with voltage-gated potassium channels. The effects on AFN can be explained by an interaction with voltage-gated sodium channels. The different sensitivity to a blockade of voltage-gated potassium and sodium channels in different types of neurons offers a differentiated approach in pain therapy depending on the type of neurons involved in the transduction of pain.
Name: Matthias Wolff, PD, Dr. med.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Matthias Wolff has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Rose Schnöbel-Ehehalt, Dr. med.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Rose Schnöbel-Ehehalt has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Jörg Mühling, Dr. med.
Contribution: This author helped write the manuscript.
Attestation: Jörg Mühling approved the final manuscript.
Name: Markus A. Weigand, Dr. med.
Contribution: This author helped write the manuscript.
Attestation: Markus A. Weigand has seen the original study data and approved the final manuscript.
Name: Andrea Olschewski, Dr. med.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Andrea Olschewski has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Jianren Mao, MD.
We thank Horst Olschewski for critical discussion of the manuscript and Eugenia Lamont, BA, for language editing. Excellent technical assistance by B. Agari and O. Becker is gratefully acknowledged.
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© 2014 International Anesthesia Research Society
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