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Anesth Analg 2005;100:175-182 © 2005 International Anesthesia Research Society doi: 10.1213/01.ANE.0000139307.91617.6D
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Abstract |
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Lidocaine causes both inhibition and excitation in the central nervous system, including the respiratory pattern. The excitation induced by an excessive dose of local anesthetic is thought to be the result of an initial blockade of an inhibitory pathway in the cerebral cortex. To clarify the effect of lidocaine on the pre- and postsynaptic neurons of an inhibitory synapse, a cultured soma-soma respiratory pattern generator model consisting of two neurons from the snail Lymnaea stagnalis were reconstructed in vitro. First we investigated the effects of lidocaine on single presynaptic (RPeD1) or postsynaptic (VD4) neurons. While RPeD1 and VD4 were simultaneously recorded, the number of action potentials, the membrane potential, and the wavelength of the action potential were compared before and after lidocaine (0.01, 0.1, and 1 mM) administration. Lidocaine increased the number of action potentials and the wavelength of a single action potential, and it depolarized the resting membrane potential in both RPeD1 and VD4 neurons in a dose-dependent manner. Furthermore, lidocaine decreased outward potassium currents. In soma-soma pairs, RPeD1 excitation and VD4 suppression occurred in 0.01 mM lidocaine, whereas both RPeD1 and VD4 neurons were excited by 0.1 and 1 mM lidocaine. In conclusion, lidocaine causes a reduction in synaptic transmission and general neuronal excitation in both presynaptic and postsynaptic neurons.
IMPLICATIONS: Lidocaine reduces inhibitory synaptic transmission. However, lidocaine induces a decrease in the outward voltage-gated potassium current, which leads to depolarization and general excitation of both presynaptic and postsynaptic neurons. Lidocaine’s side effects, such as convulsion, seizure, and hyperventilation, may result from such changes in general neuronal excitability.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Local anesthetics are widely used in regional anesthesia for relief of pain. However, accidental intravascular injection or the administration of an excessive dose can induce excitation in the central nervous system (CNS) and respiratory rhythm (1–3). This excitation is thought to be the result of an initial blockade of inhibitory pathways in the cerebral cortex. The blockade of inhibitory pathways results in an increase in excitatory activity leading to an increase or a decrease in the respiratory rhythm (4,5). Thus, both sedative and excitatory symptoms were shown in clinical studies during increasing doses of local anesthetics. Seo et al. (6) and Kasaba et al. (7) reported that cortical and hippocampal electroencephalograms showed four different excitatory phases during increasing doses of lidocaine in cats; however, they did not find selective depression of inhibitory synapses, showing only excitation by local anesthetics. In mammals, it is difficult to investigate the specific neurons, synapses, and respiratory pattern generators because of the numerous neurons and glial cells. In contrast, in Lymnaea spp., the main respiratory pattern generator consists of only three neurons. These can be individually isolated, and the respiratory pattern can be consistently reconstructed in vitro, making it possible to exclude synaptic effects from other neurons so that electrophysiological properties can be observed from the neurons in the pattern generator only. In this study, we used a model system (8–10) in which specific inhibitory synapses can produce respiratory rhythm between individually identifiable pre- and postsynaptic neurons from the snail Lymnaea stagnalis. These soma-soma respiratory pattern generator models provided us with an excellent opportunity to test the pre- and postsynaptic effects of lidocaine.
Excitation of neurons results from a depolarization of the membrane, which opens voltage-dependent calcium channels (VDCCs). Activation of VDCCs in neurons leads to an inward calcium current and transmitter release. Outward voltage-gated potassium currents (IK+) play an important role in controlling membrane potential (11). Also, suppression of outward IK+ can lead to broadening the wavelength of the action potential and reducing the resting membrane potentials (12,13).
The purpose of this study was to determine the effect of lidocaine on pre- and postsynaptic neurons and synaptic transmission and to determine whether lidocaine selectively inhibits only the presynaptic inhibitory neuron, as suggested in previous literature (4,5).
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Methods |
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All animal experiments were approved by the Animal Care Committee of Miyazaki Medical College. Laboratory-raised stocks of the freshwater snail L. stagnalis were maintained at room temperature and fed lettuce. For cell culture studies, snails 3–6 mo old and shell lengths of 10–25 mm were selected.
Normal Lymnaea saline (NS) was prepared, consisting of NaCl 51.3 mM, KCl 1.7 mM, CaCl2 4.1 mM, MgCl2 1.5 mM, and HEPES 5.0 mM, and pH was adjusted to 7.9 with 1 M NaOH. Gentamicin (G-3632; Sigma, St. Louis, MO) was added to a concentration of 150 µg/mL. Defined medium (DM) consisted of serum-free 50% Liebowitz L-15 medium (GIBCO) with added inorganic salts and 20 µg/mL gentamycin. Inorganic salts in the medium were as in NS except that NaCl was decreased to 40 mM and pH adjusted to 7.9 (8,9).
The presynaptic (RPeD1) and postsynaptic (VD4) neurons were removed from snails. After deshelling, the snails were transferred to antibiotic NS in a sterile dissection dish. The central ganglionic rings were isolated by using standard dissection procedures and were pinned to the silicone rubber base of a tissue culture plate. The outer connective tissue of the ganglia was removed before enzyme treatment. The period of enzyme treatment was 25 min in DM with trypsin (Type III; Sigma) 2 mg/mL. Subsequently, ganglia were treated with soybean trypsin inhibitor 2 mg/mL (Sigma) for 15 min, also in DM. Before removal of identified neurons, the inner connective tissue sheath was dissected with fine forceps from the ganglia. Neurons were removed by gentle suction with a siliconized, microforge fine-polished pipette with an outside diameter of 1.5 mm (IB-150 F; WPI), and the ganglia were maintained in a high-osmolarity DM that contained 30 mM glucose. After removal, neurons were transferred to poly-L-lysine-coated culture dishes (3001; Falcon) containing 3 mL of DM. Neurons were juxtaposed in a soma-soma inhibitory configuration as reported previously (8,9). After 16–24 hours of cell pairing, the evidence for synaptic transmission between neurons was obtained via simultaneous intracellular recordings, as follows.
Neuronal activity was monitored by using conventional intracellular recording techniques. Glass microelectrodes with an outside diameter of 1.0 mm (GC100-10; CEI, UK) were filled with a saturated solution of K2SO4, yielding a tip resistance of 20–60 M
. Neurons were observed under an inverted microscope (TE-300; Nikon, Japan) and impaled by manipulators (MM 202 and M 204; Narishige, Japan). Electrical signals were amplified (IR-283; Neurodata), displayed on an oscilloscope (VC-11; Nihon Kohden, Japan), and recorded on a thermal alley recorder (RTA1200; Nihon Kohden).
The neurons were perfused with NS as control for 2 min, and then the NS was replaced by lidocaine-containing saline for 4 min. Lidocaine-containing saline had the same osmotic pressure and pH as NS.
Whole-cell voltage-clamp recordings of RPeD1 and VD4 neurons (14,15) were made with a Multiclamp 700A amplifier (Axon Instruments, Foster City, CA). Patch electrodes (the tip diameter was adjusted to 1 µm, and the resistance was 1–3 M) were pulled from glass tubing with an outside diameter of 1.5 mm with filament (MTW150F-6; WPI) on a vertical pipette puller (PA-81; Narishige). For data acquisition, analog/ digital and digital/analog converters (Powerlab; ADInstruments) were connected to a personal computer (G4; Apple). The current was filtered at 1 kHz by using a 4-pole Bessel filter and digitized at a sampling frequency of 20 kHz. To study calcium currents (ICa2+), pipettes were filled with filtered (0.22-µm filter) cesium pipette solution consisting of (mM) 29 CsCl, 11 EGTA, 5 MgCl2, 10 HEPES, 2 adenosine triphosphate-Mg, and 0.1 guanosine triphosphate-Tris, and the pH was adjusted to 7.4 with CsOH. The bath solution consisted of 47.5 mM tetrathylammonium (TEA)-Cl, 1.0 mM MgCl2, 4.0 mM CaCl2, 2.0 mM 4-aminopyridine, and 10 mM HEPES (pH 7.9 with TEA-OH). ICa2+ was measured in response to 200-ms voltage steps from a holding potential of –80 mV for 1s and 10-mV incremental steps to potentials from –80 to +100 mV. To study IK+, pipettes were filled with a filtered pipette solution contain-ing (pH 7.4) 29 mM KCl, 1/11 mM Ca2+/2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid buffer, and 10 mM HEPES, supplemented with 2 mM adenosine triphosphate-Mg and 0.1 mM guanosine triphosphate-Tris. Standard Lymnaea saline (51.3 mM NaCl, 1.7 mM KCl, 1.5 mM MgCl2, and 4.1 mM CaCl2) was used for the bath solution. For IK+ measurements, depolarizing ramps (200 mV/s) from –100 to +10 mV were used. All experiments were performed at room temperature (20°C–22°C).
To investigate the effect of lidocaine on single RPeD1 or VD4 neurons, we recorded the number of action potentials, the membrane potential, and the action potential wavelength before and after lidocaine administration. The wavelength of the action potential (action potential duration) was defined and measured as the duration of the spike at a voltage halfway between the peak and the most hyperpolarized potential of the wave form (Fig. 1B).
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The action potentials of each neuron were measured from near the resting membrane potential (–60 mV), where action potentials occurred at approximately 10/min. The effects of the increasing dose of lidocaine—0.01, 0.1, and 1 mM—were evaluated. The number of action potentials were averaged 2 min before lidocaine application and compared with the average number of action potentials 3 min after each successive dose of lidocaine. Membrane potentials and action potential wavelengths were compared 1 min before and 3 min after each dose of lidocaine.
The control values were measured 2 min before lidocaine perfusion. Currents were measured 8 min after each concentration of lidocaine perfusion. Saline containing 0.1 and 1 mM lidocaine was perfused over the preparation.
First the inhibitory synapse between RPeD1 and VD4 was confirmed by stimulation of RPeD1. Then the number of action potentials, the membrane potential, and the action potential wavelength of the two neurons were simultaneously recorded at the resting membrane potential (–60 mV), and data before and after lidocaine administration were compared. The lidocaine concentrations of 0.01, 0.1, and 1 mM were evaluated. The number of action potentials was measured and averaged every 2 min. After a 2-min control period, 0.01 mM lidocaine was applied, and the lidocaine concentration was increased every 4 min to a final concentration of 1 mM lidocaine. Membrane potential and action potential wavelength were compared 2 and 4 min after each dose of lidocaine with those of the control period (1 min before the first dose of lidocaine).
To investigate the effect of lidocaine on the inhibitory synapse, RPeD1was stimulated at each lidocaine concentration. The inhibitory postsynaptic potentials (IPSPs) of VD4 to one action potential of RPeD1 were measured at each concentration of lidocaine. The IPSP of VD4 was measured at –60 mV.
Results are presented as mean ± SD. Changes in the number of action potentials, membrane potential, action potential wavelength, ICa2+, and IK+ were analyzed by repeated-measures one-way analysis of variance, followed by the Scheffé test. StatView (Abacus Concepts, Berkeley, CA) was used for statistical analyses. P < 0.05 was considered statistically significant.
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Results |
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Figure 1 displays tracings of intracellular recordings before and after lidocaine administration. Immediately after lidocaine perfusion, the membrane potential depolarized, the action potential frequency increased (Fig. 1A), and the wavelength of the action potential widened (Fig. 1B).
Lidocaine increased the number of action potentials to 36 ± 5 in RPeD1 (n = 10) and to 30 ± 14 in VD4 at a concentration of 0.01 mM (n = 9); to 42 ± 6 in RPeD1 and to 45 ± 4 in VD4 at a concentration of 0.1 mM; and to 52 ± 19 in RPeD1 and to 50 ± 17 in VD4 at a concentration of 1 mM (Fig. 2). Lidocaine also significantly increased membrane potentials (RPeD1: from –59 ± 4 mV to –56 ± 4 mV at 0.01 mM, to –51 ± 4 mV at 0.1 mM, and to –27 ± 7 at 1 mM; VD4: from –56 ± 4 mV to –53 ± 4 mV at 0.01 mM, to –47 ± 4 mV at 0.1 mM, and to –25 ± 3 at 1 mM, respectively) and action potential wavelengths (RPeD1: from 0.5 ± 0.1 ms to 0.8 ± 0.1 ms at 0.01 mM, to 1.5 ± 0.4 ms at 0.1 mM, and to 5.1 ± 0.5 ms at 1 mM; VD4: from 0.6 ± 0.1 ms to 0.8 ± 0.1 ms at 0.01 mM, to 1.7 ± 0.4 ms at 0.1 mM, and to 6.2 ± 0.5 ms at 1 mM) (Fig. 2). There were no differences between RPeD1 and VD4 neurons (Fig. 2).
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Lidocaine decreased the maximum current response (RPeD1: to –3.6 ± 0.4 nA at 0.1 mM and to –2.3 ± 0.3 nA at 1 mM at 0 mV; VD4: to –3.2 ± 0.3 nA at 0.1 mM and to –2.0 ± 0.3 nA at 1 mM at +10 mV) in a dose-dependent manner (Fig. 3). The reversal potential of the IK+ was –65 mV (Fig. 4). At levels above the reversal potential, lidocaine decreased outward IK+ in a dose-dependent manner in both VD4 and RPeD1.
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= 0.1 mM lidocaine perfusion (n = 6); 
= 1 mM lidocaine perfusion (n = 6). Results are presented as mean ± SD. Vm = membrane potential.An inhibitory synapse between RPeD1 and VD4 was reestablished in the soma-soma configuration (Fig. 5A). The change in action potential frequency in RPeD1 and VD4 synaptic pairs before and after lidocaine perfusion is represented in Figure 5B. Increasing the concentration of lidocaine displays two different types of electrophysiological patterns between the two neurons of this inhibitory synapse. Lidocaine 0.01 mM caused RPeD1 excitation and VD4 suppression, likely as a result of RPeD1 excitation and subsequent synaptic inhibition of VD4. However, lidocaine 0.1 and 1.0 mM significantly increased the number of action potentials in both RPeD1 and VD4. Changes in the number of action potentials, the membrane potential, and the action potential wavelength between RPeD1 and VD4 synaptic pairs at each concentration of lidocaine are shown in Figure 6. Lidocaine significantly increased membrane potential (RPeD1: from –59 ± 4 mV to –56 ± 4 mV at 0.01 mM, to –51 ± 4 mV at 0.1 mM, and to –27 ± 7 at 1 mM; VD4: from –56 ± 4 mV to –53 ± 4 mV at 0.01 mM, to –47 ± 4 mV at 0.1 mM, and to –25 ± 3 at 1 mM) (n = 10) and the wavelength of action potentials (RPeD1: from 0.5 ± 0.1 ms to 0.7 ± 0.1 ms at 0.01 mM, to 1.4 ± 0.3 ms at 0.1 mM, and to 5.3 ± 0.5 ms at 1 mM; VD4: from 0.6 ± 0.1 ms to 0.8 ± 0.1 ms at 0.01 mM, to 1.7 ± 0.4 ms at 0.1 mM, and to 6.1 ± 0.6 ms at 1 mM). The number of action potentials after 0.01 mM lidocaine perfusion was significantly decreased in VD4 compared with RPeD1, as shown by the black arrows at 2–4 min in the time scale of Figure 6. There was no difference in membrane potentials or wavelengths of action potentials between RPeD1 and VD4 before and after lidocaine perfusion.
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The IPSPs in VD4 were not suppressed in the small concentration of lidocaine (0.01 mM) (Fig. 7). Presynaptic RPeD1 excitation was able to suppress the postsynaptic VD4 in the small concentration of lidocaine (<0.1 mM) (Figs. 5B and 6). However, in the large concentration of lidocaine (1 mM), the IPSPs were suppressed, and presynaptic RPeD1 could not suppress postsynaptic VD4 excitation (Figs. 5B, 6, and 7A). Lidocaine suppressed IPSP amplitude in a dose-dependent manner (n = 10 at each concentration) (Fig. 7B). In 0.01 mM lidocaine perfusion, the IPSP remained at 90% of control. In 0.1 mM lidocaine, the IPSP was reduced to 30% of control, and in 1 mM lidocaine it was reduced to 10% of control. All IPSPs were measured during current clamping of the postsynaptic VD4 at –60 mV.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the respiratory pattern generator of L. stagnalis, the excitation induced by lidocaine was due to direct simulation of each neuron. Increasing the concentration of lidocaine suppressed synaptic transmission while individually exciting both pre- and postsynaptic neurons. Our results demonstrate that lidocaine induces depolarization and excitation in both pre- and postsynaptic neurons. In addition, our results demonstrate that lidocaine increases the wavelength of the action potential (Fig. 1). This spike broadening suggests that lidocaine decreases voltage-dependent IK+, which is confirmed in Figure 4 (13). In our experiments, lidocaine depolarized the cell to potentials that generate inward calcium currents through VDCCs (Fig. 3). In previous studies, lidocaine was found to be a sodium and calcium channel blocker (16,17). Our results also confirm the reduction in calcium current, and this should reduce synaptic transmitter release. In the CNS, neuronal excitation does not necessarily result in network excitation, because chemical synapses can exhibit both excitation and inhibition. Excitation of individual neurons does not always result in the excitation of the brain, because there are many inhibitory and excitatory synapses. In this experiment, a simple inhibitory synapse model demonstrated that lidocaine can induce two patterns of activity in inhibitory synapses. One of the patterns occurs at 0.01 mM lidocaine, when synaptic pairs show presynaptic RPeD1 excitation and postsynaptic VD4 suppression (Figs. 5 and 6). When in isolation, 0.01 mM lidocaine increases the number of action potentials in both RPeD1 and VD4 (Fig. 2). Therefore, excitation of the presynaptic neuron (RPeD1) by 0.01 mM lidocaine can suppress the postsynaptic neurons’ (VD4) lidocaine-induced excitability through inhibitory chemical synaptic transmission. In the other pattern, both presynaptic RPeD1 and postsynaptic VD4 neurons are excited in 0.1 and 1 mM of lidocaine. The increase in both presynaptic and postsynaptic excitation, along with the inhibition of chemical synaptic transmission (Fig. 7), leads to uncontrolled excitation. Dopamine is the main transmitter at this inhibitory synapse (10). Suppression of dopaminergic inhibitory synaptic transmission by lidocaine (Fig. 7B) would lead to excitation of the postsynaptic neuron.
Our results indicate that the excitation induced with local anesthetics not only affected the presynaptic neuron, but also directly stimulated both pre- and postsynaptic neurons. However, in this experiment, we studied only inhibitory synaptic transmission. Inhibitory and excitatory synaptic connection may produce further complex inhibition and/or excitation phenomena in the CNS with lidocaine exposure. We need to further investigate excitation and suppression by lidocaine.
In conclusion, the mechanism of excitation induced by lidocaine was not through the inhibition of the presynaptic neuron but was through the direct stimulation of both the pre- and postsynaptic neurons. Our data suggest that one of the mechanisms for excitation by lidocaine is through the depolarization of the resting membrane potential, likely the result of a reduction in voltage-gated potassium current. Lidocaine also decreases the inward calcium current and IPSP amplitude. The reduction in inward calcium current shared reduces the probability of transmitter release; the reduction in IPSP amplitude confirms this, but measurement of change in quantal content with lidocaine will be required to conclude that lidocaine works presynaptically to reduce transmitter release. At large doses of lidocaine, the reduction in inhibitory synaptic transmission promotes the excitation of postsynaptic neurons.
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Acknowledgments |
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We thank Tyler Dunn for helping with the manuscript preparation.
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References |
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