Anesth Analg 2006;102:124-128
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000184829.25310.38
ANESTHETIC PHARMACOLOGY
Actions of Norepinephrine and Isoflurane on Inhibitory Synaptic Transmission in Adult Rat Spinal Cord Substantia Gelatinosa Neurons
Stefan K. Georgiev, MD,
Ayako Wakai, MD, PhD,
Tatsuro Kohno, MD, PhD,
Tomohiro Yamakura, MD, PhD, and
Hiroshi Baba, MD, PhD
Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences
Address correspondence and reprint requests to Hiroshi Baba, MD, PhD, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachidori, 951-8510 Niigata, Japan. Address e-mail baba{at}med.niigata-u.ac.jp.
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Abstract
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Volatile inhaled anesthetics and nitrous oxide (N2O) are often used together in clinical practice to produce analgesia. Because the analgesic effect of N2O is, at least in part, mediated by norepinephrine (NE) release in the spinal cord, we examined the interaction between isoflurane (ISO) and NE in the adult rat spinal cord with respect to central nociceptive information processing. The effects of clinically relevant concentrations of ISO (1 MAC) and NE (20 µM) on spontaneous inhibitory transmission in substantia gelatinosa (SG) neurons were examined using the blind whole-cell patch-clamp method. ISO prolonged the decay time and increased the total charge transfer of spontaneous inhibitory postsynaptic currents. NE increased the frequency and mean amplitude of inhibitory postsynaptic currents and the charge transfer as well. Coapplication of both drugs led to an additive increase of the charge transfer and frequent temporal summation of inhibitory postsynaptic currents. We conclude that both ISO and NE enhance the inhibitory synaptic transmission in the rat SG neurons and their interaction is additive, suggesting that ISO may add to the analgesic action of N2O at the spinal cord dorsal horn level.
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Introduction
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Despite the more than 150-yr history of general anesthesia, the cellular mechanisms of anesthetic and analgesic actions are still not fully understood. There is ample evidence indicating that inhaled volatile anesthetics enhance 
-aminobutyric acid (GABA)-ergic and glycinergic inhibitory synaptic transmission and, more specifically, that they enhance the inhibitory postsynaptic currents (IPSC) through ligand gated chloride channels (1–4). In rat hippocampal neurons isoflurane (ISO) prolongs the half decay time (T1/2) and reduces the amplitude of IPSCs, but the total charge transfer of the IPSCs is always increased (5,6). Thus, the net action of ISO seems to be facilitation of inhibition, at least in the hippocampus.
The spinal cord is a crucial point in the modulation of nociceptive input (7–9), and it is believed that at least part of the analgesic and immobilizing effects of inhaled anesthetics are mediated by GABAA and glycine receptors in the spinal cord (10,11). The action of volatile anesthetics on the inhibitory transmissions in the spinal cord dorsal horn has not been fully examined, except in a recent study of Wakai et al. (12), who established that augmentation of GABAergic inhibitory transmission by ISO is likely to be a major mechanism of its antinociceptive action.
In clinical anesthesia, nitrous oxide (N2O) is widely used as a supplement to volatile inhaled anesthetics. It is believed that various pathways at different levels of the neural axis mediate its different pharmacological effects. Regarding the mechanism of analgesic action, N2O is believed to act on opioid and noradrenergic receptors in the brainstem, leading to activation of descending noradrenergic neurons and facilitation of descending inhibition, mediated by norepinephrine (NE) release in the spinal cord (13–15). In other words, the analgesic effect of N2O is most likely dependent on NE release in spinal cord (16). Hashimoto et al. (14) demonstrated that NE, released from descending noradrenergic fibers, activates GABAergic neurons at the dorsal horn. Furthermore, it has been reported that NE increases the frequency and amplitude of miniature IPSCs in rat substantia gelatinosa (SG) neurons (17). Thus, the analgesic effect of N2O is mediated, at least in part, by NE-evoked IPSCs in the spinal cord dorsal horn.
Little is known about ISO's action on the analgesic effect of N2O. The interaction between N2O and ISO might also occur at supraspinal centers, but in this study we explored the final stage of the laughing gas antinociceptive effects transduction cascade-NE release in the dorsal horn. When ISO is applied in combination with N2O, it could be expected that the duration of N2O-induced (i.e., NE-induced) IPSCs in the spinal cord will be increased, and the amplitude will be decreased by ISO; but the effect on total charge transfer of IPSC is difficult to predict, as discussed above. Nishikawa et al. (18) recently found that sevoflurane facilitated the NE-induced enhancement of GABAergic IPSCs in hippocampal neurons. In the present study we used the blind whole-cell patch-clamp technique to examine the effects of coapplication of ISO and NE on spontaneous IPSCs (sIPSCs) in rat spinal cord SG (lamina II), where GABAergic and glycinergic systems play a major role in controlling nociceptive information (7).
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Methods
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This study was approved by the Animal Care and Use Committee at Niigata University School of Medicine. As previously described (19,20), adult (6-8 wk) male Wistar rats were anesthetized with urethane (1.5 g/kg intraperitoneally). The lumbosacral segment of the spinal cord was removed through a limited laminectomy and placed in a preoxygenated ice-cold Krebs solution. After removing the dura mater, all the ventral and dorsal roots, and the pia-arachnoid membrane, a 500- to 600-µm thick transverse slice was cut on a vibrating microslicer (DTK-1500, Dosaka Co. Ltd., Kyoto, Japan). The slice was placed on a nylon mesh in a recording chamber, perfused with 36°C Krebs solution (containing in mM: NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, Na2HPO4 1.2, NaHCO3 25, glucose 11), bubbled with a gas mixture of 95% O2 and 5% CO2. The recording electrode was a fire-polished glass pipette with resistance 4-8 M
. Pipette solution was standard cesium sulfate based solution (containing in mM: Cs2SO4 110, CaCl2 0.5, MgCl2 2, ethyleneglycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 5, hydroxyethylpiperazineethanesulfonic acid (HEPES) 5, tetraethyl ammonium chloride (TEA) 5, adenosine triphosphate (ATP) 5) to which 1 mM guanosine thiodiphosphate (GDP-ß-S) was added to block the postsynaptic effects of NE. Drugs were applied through the bathing solutions. NE (20 µM, Sigma, St. Louis, MO) was protected from light exposure. Isoflurane (1.5 vol%, Abbott) was delivered through a commercial vaporizer (Muraco, Tokyo, Japan) using a carrier gas (95% O2, 5% CO2) and its concentration was confirmed to approximate rat's 1 MAC using gas chromatography. Axopatch 200A (Axon Instruments Inc., Union City, CA) was used for voltage-clamp recordings. sIPSCs were recorded at a holding potential of 0 mV. To compare the effects of independent and common administration of both drugs, the neurons were divided into 2 groups. In Group I (n = 20) ISO was perfused for at least 15 min, then NE was added to the perfusing solution. In Group II (n = 20), NE was perfused first for at least 5 min, followed by coapplication of ISO and NE (ISO+NE). Each drug was applied for a sufficient period of time to ensure that its effect reached steady-state before the recording. The signal was filtered, amplified, digitalized, and stored in a PC for further analysis. IPSCs kinetics were analyzed using pClamp 8 software (Axon Instruments)—amplitude, T1/2, frequency of IPSCs were analyzed from at least a 10-s period and integrated area from 20 s. Numeric data are presented as mean ± sd, unless otherwise stated. For statistical analysis Student's t-test (GraphPad Prism 4 software; GraphPad, San Diego, CA) was used. P < 0.05 was considered significant.
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Results
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Whole-cell patch-clamp recordings were made from 40 SG neurons. The baseline sIPSC frequency was 2.4 ± 0.8 Hz, the mean amplitude – 14.8 ± 5.4 pA, and the time to half decay – 7.5 ± 2.7 ms. Isoflurane affected neither the amplitude (14.5 pA, 98% of control) nor the frequency (2.4 Hz, 99% of control) of IPSCs but prolonged the half decay time to 13.8 ± 4.5 ms, which is 182% of control (P < 0.0001, n = 20, paired Student's t-test) and increased the charge transfer (Fig. 1), expressed by an increase of the area under the curve by 18.7 pA/s or 165% of control values (P < 0.0001, n = 20, paired Student's t-test). The effect of ISO was time dependent, reaching steady-state after 10-15 min. Applying NE with the perfusion solution (Fig. 2) resulted in marked increase of IPSC frequency (17.3 ± 8.4 Hz, or 692% of control, P < 0.0001, n = 20, paired Student's t-test) and increase in mean IPSC amplitude (20.5 ± 9.1 pA, or 135% of control, P < 0.0001, n = 20, paired Student's t-test), without affecting the half decay time (7.5 ± 2.3 ms, 100% of control). The integrated area increased by 154.4 pA/s or 642% (P < 0.0001, n = 20, paired Student's t-test). The effects of NE reached steady state for 2-3 min, which is considerably faster than ISO.
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Figure 1. Isoflurane (ISO) prolongs the time to half decay and increases the total charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs) in substantia gelatinosa neurons. A: sIPSCs recorded at holding potential of 0 mV before and during application of 1.5 vol. % ISO. B: Effect of ISO on IPSC amplitude, frequency, half decay time, and area under the curve. *P < 0.05.
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Figure 2. Norepinephrine (NE) increases the frequency, amplitude and the total charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from substantia gelatinosa neurons. A: sIPSCs recorded at holding potential of 0 mV before and during application of NE (20 µM). B: Effect of NE on IPSC amplitude, frequency, half decay time, and area under the curve. *P < 0.05.
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Coapplication of ISO and NE led to an increase of all observed variables (Fig. 3). There were slight differences in the degree of increase of IPSCs amplitude, frequency, half decay time, and integrated area depending on the drugs' perfusion order (e.g., half decay time increased more in Group 1, where ISO was applied first and frequency increased more in Group 2, where NE was applied first), but because they were not statistically significant, they were not studied further (Table 1). Mean IPSC amplitude reached 20.5 ± 9.2 pA, which is 138% of control (P < 0.0001, n = 40, paired Student's t-test). The frequency of sIPSC increased to 16.8 ± 8.4 Hz or 698% (P < 0.0001, n = 40, paired Student's t-test). Half decay time was prolonged to 13.3 ± 4.3 ms or 178% of control values (P < 0.0001, n = 40, paired Student's t-test). The under the curve area increased by 188.3 pA.s or 759% compared to control (P < 0.0001, n = 40, paired Student's t-test). All changes were reversible after 30 min washout.
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Figure 3. Coapplied norepinephrine and isoflurane (NE+ISO) increase the amplitude, frequency, half decay time, and the total charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs). A: sIPSCs recorded at holding potential of 0 mV before and during application of 1.5 vol. % ISO and NE (20 µM). Note the resulting frequent temporal summation of IPSC. B: Effect of NE+ISO on IPSC amplitude, frequency, half decay time and area under the curve. All parameters are additively increased. *P <0.05.
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Table 1. Changes of Inhibitory Postsynaptic Current Amplitude, Frequency, Half Decay Time and Area Under the Curve According to the Perfused Drugs Order
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Discussion
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We have demonstrated that 1 MAC ISO prolonged the decay phase kinetics and increased the charge transfer without affecting the sIPSC amplitude or frequency, whereas NE markedly increased IPSC frequency, amplitude, and the charge transfer without affecting the half decay time.
Volatile inhaled anesthetics increased IPSC decay time in cortical (21), hippocampal (5,6,22), trigeminal (3), spinal motor (23), and SG (12) neurons. They also decreased the IPSC amplitude at larger concentrations (5,6) and increased (3,6,22,23) or decreased (21) miniature IPSC frequency. All of these data suggest that ISO acts on both presynaptic and postsynaptic sites, thus increasing the transmitter release and modulating the GABAA receptor, which, in turn, occurs through binding to two distinct sites (6). In our study on SG neurons, 1 MAC ISO prolonged the decay phase kinetics in a similar manner and increased the charge transfer, but it did not affect either the spontaneous IPSC amplitude or frequency. The exact reason for these discrepancies is unknown, but they might have been caused by the type or concentration of volatile anesthetics used. Alternatively, differences in organ maturity of the animals used may also contribute. ISO at 1 MAC concentration did not affect the amplitude of sIPSCs, but the effect on amplitude is reported to be concentration-dependent (5,6). Therefore, it may be possible that ISO at larger concentrations depresses NE-evoked IPSCs by decreasing IPSC amplitude; however, this needs to be investigated further.
It was demonstrated that NE increased the amplitude and frequency of spontaneous IPSCs in hippocampal interneurons (24) and of miniature IPSCs in spinal cord slice (20), although the sIPSC amplitude was not increased in cerebellar stellate cells (25). In this study, we found that NE considerably increased the inhibitory transmission charge transfer in rat spinal cord dorsal horn neurons by increasing the sIPSC amplitude and frequency.
Some of the results listed above are consistent with previous findings (12,18,20). Our major concern in this study was how ISO modulates NE (i.e., N2O)-induced augmentation of sIPSCs. The integrated area was increased to 642% of control by NE alone, to 165% by ISO alone, and to 759% by coadministration of NE and ISO. This means that the interaction of both drugs is apparently additive with respect to the effect on total charge transfer of IPSCs. In addition to the effect on total charge transfer, coadministration of these drugs resulted in very frequent summation of IPSCs (Fig. 3), suggesting that postsynaptic GABAA receptors are almost continuously activated. Thus, we can speculate that the membrane potential of many SG neurons is continuously hyperpolarized (when the resting potential is more positive than the Cl–-equilibrium potential) and the membrane resistance is continuously decreased by opening Cl– channels during the coadministration, leading to a state in which the excitability of SG neurons is tonically inhibited.
In conclusion, both ISO and NE enhance the inhibitory synaptic transmission in rat SG neurons and their interaction is additive, which may be a mechanism of ISO (at least at 1 MAC concentration) that facilitates the analgesic action of N2O at the spinal cord dorsal horn level.
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Footnotes
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Supported by grant-in-aid for scientific research Nos. 13470318 and 16591529 from the Ministry of Education, Science, Sports and Culture of Japan, Tokyo, Japan.
August 17, 2005.
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Abstract
Introduction
