Anesth Analg 2004;98:647-652
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000103259.72635.72
ANESTHETIC PHARMACOLOGY
Attenuation of Gap-Junction-Mediated Signaling Facilitated Anesthetic Effect of Sevoflurane in the Central Nervous System of Rats
Eiji Masaki, MD PhD*,
Masahito Kawamura, MD
, and
Fusao Kato, PhD
Section Editor
Departments of *Anesthesiology,
Pharmacology, and
Neuroscience, Jikei University School of Medicine, Tokyo, Japan
Address correspondence and reprint requests to Eiji Masaki, Department of Anesthesiology, Jikei University School of Medicine, 3–25–8, Nishishinbashi Minato-ku, Tokyo 105–8461, Japan. Address email to ejmasaki{at}jikei.ac.jp
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Abstract
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Accumulating evidence suggests that reduction of intrinsic excitability or synaptic excitation and/or an enhancement of synaptic inhibition underlie the general anesthetic condition. Besides chemical synapse, neurons could communicate with each other by electrical coupling via gap-junctions. We hypothesized that inhibition of cell-to-cell signaling through gap-junction in the central nervous system (CNS) is involved in the anesthetic mechanism of volatile anesthetics. The minimum alveolar concentration (MAC) of sevoflurane was measured after the intracerebroventricular (ICV) or intrathecal (IT) administration of carbenoxolone (CBX), a gap-junction inhibitor, in vivo. The spontaneous oscillation in membrane currents of locus coeruleus neurons that results from electrical coupling between neurons was also recorded from young rat pontine slices by the patch clamp method, and the effect of sevoflurane on this oscillation was examined in vitro. The ICV administration of CBX (125 and 250 µg/rat) significantly reduced the MAC of sevoflurane dose-dependently, whereas IT injection failed to inhibit the MAC. Sevoflurane at clinically relevant concentrations (0.1–0.5 mM) suppressed the spontaneous oscillation in membrane current concentration-dependently. These suppressions were significant at 0.5 mM with both amplitude and frequency. We suggest that suppression of gap-junction-mediated signaling in the CNS is involved in the anesthetic-induced immobilization by sevoflurane.
IMPLICATIONS: The intracerebroventricular administration of the gap-junction inhibitor, carbenoxolone, reduced the MAC of sevoflurane, and sevoflurane suppressed the signaling through gap-junctions in the central nervous system. The inhibition of gap-junctions may be one of the mechanisms and the site of action of sevoflurane.
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Introduction
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It is now acknowledged that anesthetics exert their anesthetic effects largely through affecting the neuron-to-neuron signaling in the central nervous system (CNS) (1). Of these effects, the most well-described are those on the molecules underlying chemical synaptic transmission, such as the ionotropic glutamate, 
-aminobutyric acid (GABA), and adenosine triphosphate receptor channels expressed on postsynaptic membranes (2–4).
Another type of the cell-to-cell signaling operational in the CNS is that mediated by electrical synapses. Increasing evidence indicates that cell-to-cell communication through electrical synapses plays an important role in neuronal signaling in the CNS (5). The structural basis for the electrical synapses are now identified to be gap-junctions at which connexons expressed on the plasma membrane of each cell make direct contact to form gap-junction channels (6). Ionic currents through gap-junctions give rise to the strong electrical coupling of the neurons and glial cells in the CNS, resulting in tight and synchronized activation of multiple cell networks (7). In addition, opening and closing of the gap-junction channels are regulated by various factors, such as transjunctional voltage, intracellular pH, and other intracellular messengers including Ca2+, suggesting that modulation of gap-junction conductance by these factors allows functional regulation of the coupling (6,8).
The objective of the present study was to examine a hypothesis that the anesthetic effects of volatile anesthetics are partly mediated by their inhibitory effect on the gap-junction-mediated signaling in the CNS. This hypothesis stems from the fact that lipophilic agents such as heptanal and octanol, as well as a volatile anesthetic (halothane), reduce gap-junction channel conductance (6). Volatile anesthetics also reduce the gap-junction permeability in the nervous system and myocardial cells (9,10).
To examine this hypothesis, we first sought to evaluate the effect of the gap-junction blockade on the general anesthetic effect of a commonly used volatile anesthetic, sevoflurane. We then evaluated the effect of sevoflurane on the gap-junction-mediated electrical activity in the CNS. For this purpose, we used acute brainstem slice preparations to measure membrane current oscillation of the neurons in the locus coeruleus (LC) as an indicator of the gap-junction-mediated signaling in the native brain tissue. The LC network was chosen because 1) the neurons in the LC, a structure rich in the connexin (subunits forming connexon hemichannels) (11), presents strongly synchronized spontaneous activities through gap-junction connections between neurons and astrocytes within the LC (7,12), and 2) LC neurons send noradrenergic projections widely to many CNS regions, including the cortex, hippocampus, thalamus, and the spinal cord. All these structures are important targets for general anesthesia, inhibition of which results in motor, mental, and sensory blockade (13). Indeed, electrical (14) and pharmacological (15) destruction of the LC produces anesthetic effects. We provide evidence that an inhibitory effect of volatile anesthetics on the gap-junction-mediated signaling in the CNS may be a part of mechanisms underlying their anesthetic effects.
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Methods
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This study was approved by the Institutional Animal Use and Care Committee and conformed to the Guiding Principles in the Care and Use of Animals by the Japanese Physiological Society and the Japanese Pharmacological Society.
In Vivo
Forty-eight hours preceding the minimum alveolar concentration (MAC) measurement, male Sprague-Dawley (SD) rats (7 wk, 150–200 g) were anesthetized with intraperitoneal injection of thiopental (50 mg/kg). A fine, L-shaped stainless tube was implanted into the right lateral ventricle as described previously (16). A silicone tube of 0.3-mm inner diameter was connected to one end of the stainless tube, tunneled through the cervical subcutaneous region, and fixed with tie ligation. After the measurement of MAC, lissamine green dye was injected through the stainless tube to confirm that the tip of the L-shaped tube was located in the ventricular space.
Intrathecal (IT) catheters (polyethylene tubing 10) were inserted in SD rats (10 wk, 250–300 g) during isoflurane anesthesia by the method described by Yaksh and Rudy (17). The IT catheter was advanced 7 cm caudal through an incision in the atlanto-occipital membrane and secured to the musculature at the incision site. Animals with obvious neurologic damage were excluded from the study. All experiments were performed 7 days after IT catheter implantation.
The MAC of sevoflurane (Dinabot Japan, Osaka, Japan) was measured according to the methods of Eger et al. (18). Briefly, each rat was placed in an individual plastic chamber (30 cm long, 5 cm in diameter) and given sevoflurane mixed with 100% oxygen (4 L/min total gas flow) to attain the final partial pressure of 1%–5%. Expired gas was continuously sampled at the outflow of the chamber to monitor the concentration of sevoflurane with an infrared analyzer (Datex Ultima, Helsinki, Finland). Rectal temperature was measured and maintained between 36.5°C–37.5°C with an electronically controlled heating mat. After initial equilibration (2.5% sevoflurane for 40 min), a tail clamp was applied for 1 min using a long hemostat, and movement in response to the stimulation was observed. The concentration of sevoflurane was increased or decreased by 0.2%–0.3% steps and each change was followed by a 20-min equilibration time. The response to the tail clamp was tested at each concentration. The mean of the smallest concentration without stimulus-evoked movement and the largest concentration with movement was taken as MAC. After measurement of baseline MAC (before the intracerebroventricular [ICV] or IT administration), carbenoxolone (CBX) disodium (Sigma Chemical, St. Louis, MO) was administered ICV or IT using a volume of 25 µL for ICV or 10 µL for IT. CBX was dissolved in normal saline at a concentration of 0, 1.25, 2.5, 5, and 10 mg/mL to give doses of 0, 31.3, 62.5, 125, and 250 µg/rat for ICV administration. The IT injections of CBX at concentration of 0, 2.5, 5, and 10 mg/mL were performed to administer doses of 0, 25, 50, and 100 µg/rat. For the control, only normal saline was injected using a volume of 25 µL for ICV or 10 µL for IT, respectively. ICV and IT injections were followed by 10-µL flush with normal saline. The MAC of sevoflurane in the presence of CBX was measured 30 min after the ICV or IT injection of CBX.
In Vitro
SD rats (7–21 days, 30–120 g) of either sex were anesthetized by intraperitoneal injection of ketamine (100–150 mg/kg) and decapitated. Two to three transverse slices of brain of 350–450 µm thickness including the LC were made in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM) CaCl2 0.1 and MgCl2 5 together with NaCl 125, KCl 2.5, NaH2PO4 1.25, D-glucose 12.5, L-ascorbic acid 0.4, and NaHCO3 25 saturated with 95% O2 + 5% CO2 (pH = 7.4) with a vibrating blade slicer (DTK-1000, Dosaka; Ted Pella, Redding, CA). The slices were incubated in aCSF in which the concentrations of CaCl2 and MgCl2 were 2 mM and 1.3 mM, respectively, for 30–40 min at 37°C, then kept at room temperature until the recording.
Patch electrodes were filled with an intracellular solution containing (in mM) potassium gluconate 120, NaCl 6, CaCl2 5, MgCl2 2, MgATP 2, NaGTP 0.3, EGTA 10, and HEPES 10 (adjusted to pH 7.2 with KOH). The outer wall of the electrode tip was coated with Sigmacoat (Sigma Chemical CO, St. Louis, MO). The tip resistance of the electrode was 4–6 M
. The slices were secured in a recording chamber (
0.5 mL volume) and continuously perfused with aCSF at a flow rate of 2–3 mL/min. The neurons in the LC were visually identified under infrared differential interference contrast videomicroscopy. The holding potential was kept at -80 mV in all recordings. The series resistance (8 to 20 M) and whole-cell capacitance were compensated and checked before and after pharmacological manipulations. The membrane current was recorded with the continuous voltage-clamp mode of an AxoClamp 2B (Axon Instruments) or with a CEZ-2400 (Nihon-Koden). All signals were recorded on digital audiotapes (RD-120TE, TEAC; Tokyo, Japan). After the experiments, the signal were filtered (200 or 500 Hz) and sampled at 1 kHz. For the estimation of powerspectral density functions, the membrane current signals were digitally re-filtered at 20 Hz and re-sampled at 40 Hz. The original traces in the figures and powerspectral density estimation were made with the Igor Pro graphic program (Version. 4.07; WaveMetrics, Lake Oswego, OR). The amplitude of the oscillation was measured by putting two cursors on the peak and trough of the oscillation and measuring the difference in the ordinate. The frequency of the oscillation was measured by counting the peak during 60 s. CBX was dissolved in aCSF and applied via the perfusion line. Sevoflurane was vaporized (Sevotec 3; Ohmeda, West Yorkshire, UK) with carbogen and continuously bubbled into aCSF. The actual aqueous concentration of sevoflurane was analyzed by gas chromatography (GC-14B; Shimadzu, Tokyo, Japan). The recordings were made at room temperature (24°C–26°C). The data were analyzed by analysis of variance with subsequent intragroup comparisons using the Scheffé F-test. A value of P < 0.05 was considered significant.
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Results
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In Vivo
ICV administration of CBX (125 and 250 µg/rat) significantly reduced the MAC of sevoflurane without exerting any detectable changes in the general behavior of the rat (Fig. 1A). In contrast, IT injection of CBX did not significantly affect the MAC of sevoflurane (Fig. 1B). There was no difference in the baseline MAC of sevoflurane measured before CBX administration between animals subjected to ICV and IT administration. To confirm the absence of hypoxia, hypercapnia, and acidosis, blood gas analysis was performed by sampling of arterial blood from the abdominal aorta after the second measurement of MAC. No rats exhibited hypoxia, hypercapnia, or acidosis.
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Figure 1. Effect of the intracerebroventricular (ICV) and intrathecal (IT) administration of carbenoxolone on the minimum alveolar anesthetic concentration (MAC) of sevoflurane. A, ICV (n = 10); B, IT (n = 7). After measuring the baseline MAC, the ICV and IT administration were conducted with the indicated dose of carbenoxolone in a volume of 25 and 10 µL, respectively. Data are expressed as mean ± SE. *P < 0.05 versus 0 µg.
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In Vitro
The LC neurons showed spontaneous membrane current oscillation at a holding potential of -80 mV (amplitude, 59.5 ± 6.3 pA; frequency, 0.49 ± 0.07 Hz; mean ± SE; n = 14). This oscillation was not dependent on the holding potential in the range of -100 to -50 mV (data not shown), was resistant to tetrodotoxin (1 µM; n = 3), and was blocked by CBX (Fig. 2; 1–3), showing that this oscillation is not attributable to chemical synaptic transmission but rather is generated through electrical coupling between LC neurons. Sevoflurane at clinically relevant concentration (0.5 mM) suppressed this oscillation in a manner similar to CBX (Fig. 2; 3–5). Sevoflurane also produced a slowly developing sustained inward current in contrast to the persistent outward current produced by CBX (Fig. 2, top traces), the mechanisms of which were not analyzed in detail in the present study. It is likely that the inward current by sevoflurane is mostly carried by chloride ions (expressed as an inward current at the present recording conditions where the reversal potential of Cl- was -50.3 mV) as already reported in acutely dissociated neurons of the rat hippocampus (19). The inhibition of the spontaneous oscillation by sevoflurane was accompanied by a reduction of both amplitude and frequency of the oscillation (Fig. 3A, left and right). The reduction of oscillation frequency and amplitude by sevoflurane was dependent on the concentration (Fig. 3B) and was significant at 0.5 mM concentration.
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Figure 2. Effect of sevoflurane and carbenoxolone on the spontaneous oscillation in the membrane current of a locus coeruleus neuron. Top, a continuously recorded trace of the transmembrane whole-cell current (Im) recorded at a holding potential of -80 mV. Carbenoxolone (100 µM) and sevoflurane (5%) were dissolved in the artificial cerebrospinal fluid (aCSF) and perfused as shown in the horizontal bars. The time required from the onset of application and the establishment of the plateau concentration in the bath was <20 s. The numbers below the trace correspond to the time-extended versions (1–5) below. Note the suppression of spontaneous oscillation by both carbenoxolone (1 and 3) and sevoflurane (4 and 5).
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Figure 3. Concentration-dependent inhibition of spontaneous oscillation by sevoflurane. A, effect of increasing sevoflurane concentration on spontaneous oscillation in the locus coeruleus (LC) neuron. Left, original traces recorded in the presence of 0.1–0.5 mM sevoflurane in the perfusate. A washout for approximately 60 min, but not one for 30 min, almost completely recovered the spontaneous oscillation. Right, power spectral density functions of the transmembrane current. Note that sevoflurane decreased the height of the peaks and shifted the peak to the left (lower frequency) in a concentration-dependent manner, indicating that sevoflurane not only reduced the amplitude but also lowered the frequency of the spontaneous oscillation. B, concentration-response curves of sevoflurane on the amplitude (filled circles) and frequency (open circles) of the spontaneous oscillation in LC neurons. Data are expressed as mean ± SE *P < 0.05 versus 0%.
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Discussion
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The major findings of this study are 1) administration of a gap-junction inhibitor in the brain, but not in the spinal cord, reduced the MAC of sevoflurane and 2) sevoflurane suppressed the spontaneous oscillatory activity mediated by CBX-sensitive gap-junction-dependent mechanisms in the LC. These findings might indicate that gap-junction-mediated neuronal activities such as those observed in the LC are parts of the targets of volatile anesthetics. Suppression of gap-junction-mediated signals in the CNS by volatile anesthetics might result in anesthetic-induced immobilization.
It is generally considered that MAC principally reflects the action of volatile anesthetics in the spinal cord (20). Nevertheless, in the present study IT injection of CBX did not significantly reduce MAC even at a dose causing a significant reduction of MAC when CBX was ICV injected. Two lines of interpretations are possible. First, the reduction of gap-junction-mediated activity in the spinal cord does not affect MAC of sevoflurane; therefore it is not important in anesthetic-induced immobilization. Second, MAC of sevoflurane is affected if the gap-junction-mediated activity in the supraspinal regions controlling spinal mechanisms is modified. In the spinal cord, the presence of gap-junction-like structures has been morphologically identified (21); however, it is not known whether it is operational in neuronal signaling in the spinal cord, which argues against the first possibility. By contrast, in the brain, it has been demonstrated that gap-junction-mediated signaling underlies many important functions, such as spontaneous oscillation in the LC, which regulates norepinephrine tone in the brain and spinal cord. Therefore, it is rather more likely that ICV-injected CBX affected the gap-junction-mediated activity in the supraspinal regions, which controls the neuronal excitability of the spinal cord network. Indeed, modulation of descending modulatory systems influences anesthetic action in the cord (22). Destruction of the LC decreases the MAC of halothane and cyclopropane (14). ICV administration of GABA antagonist increases the MAC of isoflurane (23). All these findings suggest that modulation of supraspinal activity affects MAC.
In the brain, high immunoreactivity to connexins is observed in the LC, substantia nigra pars compacta, and antero-ventral nucleus of the thalamus (11). Inhibition of gap-junctional signaling in these regions could underlie a descending modulation of the spinal cord function by CBX. Indeed, the LC sends a large number of descending noradrenergic projections to various subregions of the spinal cord (24). As neuron-neuron signaling via gap-junctions is present in many brain structures (5,11), it is also possible that CBX affected the excitability of such networks in addition to the LC, and consequently helped sevoflurane to exert its general anesthetic effect, leading to direct modification of the conscious level of the animals, the central pain sensation, or reflexogenic movement. In addition, the possibility that the decrease in MAC of sevoflurane with ICV-injected CBX resulted from an undescribed and unknown effect of CBX cannot be fully excluded. If CBX affects the neuronal pathway involved in MAC measurement, but not involved in sevoflurane action, such as muscle relaxation and endogenous opioid system activation, MAC of sevoflurane would be decreased, which is an inherent limitation of MAC measurement. However, as there has been no study reporting the effects of CBX on these systems and as CBX alone did not apparently affect the behavior of animals in this study (data not shown), it is more likely that CBX decreased MAC by influencing the effect of sevoflurane.
Among the brain regions mentioned above, the most likely target of CBX in the enhancement of the anesthetic effect of sevoflurane is the LC. The LC is the largest noradrenergic nucleus in the CNS and its neurons send their axons to widespread regions throughout the neuraxis. For example, the principle origin of norepinephrine release in the cortex, thalamus, and hippocampus, all of which play important roles in the general anesthetic effect, is the LC. Lines of evidence argue for an involvement of the LC in the maintenance of level of consciousness and general anesthetic effects. An inhibition of LC activity induces anesthetic effects (14,15), and anesthetics inhibit the spontaneous activity of LC (25). First, an electrolytic lesion of LC nucleus, which decreases cortical noradrenaline level, decreases the MAC of halothane and cyclopropane (14). Second, pharmacological destruction of the LC nucleus (15) with a 6-hydroxydopamine treatment enhances the anesthetic effect of thiopentone. Third, halothane, besides exerting general anesthetic effects, increases the norepinephrine content in several nuclei including the LC (25), suggesting a decrease in norepinephrine release in the regions receiving adrenergic projections from the terminals arising from adrenergic nuclei such as LC. All these observations strongly suggest an involvement of the change in LC activity underlies the general anesthetic effects.
It has been demonstrated that various types of gap-junction blockers, including CBX and intracellular acidification, disrupt the oscillation in the LC (12), suggesting that gap-junction-mediated signaling is a requisite for the spontaneous oscillation in the LC. In this context, the most plausible interpretation of the present result is that the strong suppression of the oscillation by sevoflurane resulted from blockade of gap-junctions. A number of studies have demonstrated that volatile anesthetics inhibit gap-junction-mediated neural activities (6). For example, junctional resistance between crayfish septate axons are increased by halothane and isoflurane (9), and lucifer yellow permeation through gap-junctions between cultured astrocytes is suppressed by halothane, enflurane, and isoflurane in a concentration-dependent manner (26). In the present study, we demonstrated concentration-dependent inhibition of gap-junction-mediated activity by a volatile anesthetic in the LC and further, an anesthetic-facilitatory effect of gap-junction blockers in vivo. These results strongly argue for the notion that volatile anesthetics exert their anesthetic effect partly through modulating the gap-junction-mediated activity in the CNS. However, Alvares et al. (27) recently demonstrated that in addition to the electrical coupling between cells through gap-junctions, the firing frequency of neurons in the LC also affects the frequency and amplitude of oscillation. It is therefore possible that decreasing of excitability of LC neurons by sevoflurane, together with the reduction of gap-junction-mediated signaling, might have helped to depress the spontaneous oscillation.
In summary, CBX, an inhibitor of the gap-junction, reduced the MAC of sevoflurane and sevoflurane suppressed the signaling via gap-junction in the CNS. We propose that the inhibition of gap-junction-dependent activities in the CNS partly underlies the anesthetic-induced immobilization of sevoflurane and the gap-junction-mediated interneuronal communication could be one of the sites of action of volatile anesthetics.
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Acknowledgments
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Supported, in part, by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan (No. 11671525 to EM and No. 13680902 to FK), and grants from the Japan Human Science Foundation.
The authors thank Dr. Salim Hayek (Cleveland Clinic Foundation, Cleveland, Ohio) for his help with manuscript preparation.
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Footnotes
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Presented, in part, at the annual meeting of the American Society of Anesthesiologists, Dallas, Texas, October 12, 1999.
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Accepted for publication October 1, 2003.
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Abstract
Introduction
