Free
Meeting Abstracts  |   February 2005
Action of Isoflurane on the Substantia Gelatinosa Neurons of the Adult Rat Spinal Cord
Author Affiliations & Notes
  • Ayako Wakai, M.D.
    *
  • Tatsuro Kohno, M.D., Ph.D.
  • Tomohiro Yamakura, M.D., Ph.D.
  • Manabu Okamoto, M.D., Ph.D.
  • Toyofumi Ataka, M.D., Ph.D.
  • Hiroshi Baba, M.D., Ph.D.
    §
  • * Graduate Student, † Assistant Professor, ‡ Associate Professor, § Professor and Chairman.
Article Information
Meeting Abstracts   |   February 2005
Action of Isoflurane on the Substantia Gelatinosa Neurons of the Adult Rat Spinal Cord
Anesthesiology 2 2005, Vol.102, 379-386. doi:
Anesthesiology 2 2005, Vol.102, 379-386. doi:
MOST general anesthetics can induce unconsciousness, block the motor response to noxious stimulation, and suppress autonomic responsiveness. Recent studies have revealed that the minimum alveolar concentration (MAC) is spinally mediated.1–4 The depression of the motor response to noxious stimulation may be caused by immobilization and antinociceptive effects at the spinal cord level.5,6 Cheng and Kendig7 showed that enflurane directly depresses glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and N  -methyl-d-aspartate (NMDA) currents in mouse spinal cord motor neurons, suggestive of the immobilization effect of inhaled anesthetics and its possible mechanism in the spinal cord. However, the antinociceptive effect of inhaled anesthetics may not be fully accepted because they cannot prevent the increase of blood pressure or heart rate caused by noxious stimuli at clinically relevant concentrations. Recently, Yamauchi et al.  8 reported that halothane depressed the extracellular activity of single spinal dorsal horn wide-dynamic-range neurons to noxious stimuli, suggesting that halothane has an antinociceptive effect at the spinal cord level. Furthermore, they reported that the inhibitory effect of halothane was blocked by bicuculline (γ-aminobutyric acid type A [GABAA] receptor antagonist), which suggests that the antinociceptive effect of volatile anesthetics may be related to γ-aminobutyric acid–mediated (GABAergic) transmission in the spinal dorsal horn. In fact, inhaled anesthetics enhance GABAAcurrents in cultured neurons,9 human embryonic kidney cells expressing GABAAreceptors,10,11 and brain slices12,13 at clinically relevant concentrations. Inhaled anesthetics have many effect sites other than GABAAreceptors, such as K+channels,14–17 Ca2+channels,18–21 nicotinic acetylcholine receptors,22–24 glycine receptors,25 AMPA receptors,7,26 and NMDA receptors.7,27–30 Collectively, it is considered that multiple potential anesthetic targets exist.31–33 Therefore, also in the spinal dorsal horn, the antinociceptive action of isoflurane may be mediated by the GABAergic inhibitory system and some or all of the many other mechanisms listed above.
To clarify whether isoflurane has an antinociceptive effect in the dorsal horn and whether the GABAergic system is its major anesthetic (analgesic) target, we investigated the action of isoflurane on the excitatory and inhibitory synaptic responses in lamina II (substantia gelatinosa [SG]), where the GABAergic system plays a major role in controlling nociceptive information.34,35 
Materials and Methods
Spinal Cord Slice Preparation and Electrophysiologic Recording
This study was approved by the Animal Research Committee of Niigata University Graduate School of Medical and Dental Sciences in Niigata, Japan. Thick (600- to 650-μm) spinal cord slices containing the L4 dorsal root (10–20 mm) were prepared from adult rats (aged 7–10 weeks) as described previously.36,37 After preparation, slices were perfused with oxygenated Krebs solution (10 ml/min; 36°± 1°C; composition: 117 mm NaCl, 3.6 mm KCl, 2.5 mm CaCl2, 1.2 mm MgCl2, 1.2 mm NaH2PO4, 25 mm NaHCO3, and 11 mm D-glucose) in the recording chamber for at least 30 min before recording. Blind whole cell patch clamp recordings were made from neurons located in the SG. After the whole cell configuration was established, voltage clamped neurons were held at −70 mV for recording non–NMDA receptor-mediated excitatory postsynaptic currents (EPSCs), at +40 mV for NMDA receptor–mediated EPSCs and at 0 mV for inhibitory postsynaptic currents (IPSCs).38,39 The dorsal root was stimulated using a suction electrode at 100 μA (0.05 ms) for Aδ fibers and 1,000 μA (0.5 ms) for C fibers.34–39 Aδ fiber–evoked EPSCs were judged to be monosynaptic on the basis of both their short and constant latencies and the absence of failures with repetitive stimulation at 20 Hz.40,41 Identification of C fiber–evoked monosynaptic EPSCs was based on an absence of failures with low-frequency (1 Hz) repetitive stimulation.42,43 In contrast, polysynaptic EPSCs had variable latencies and showed failures with such stimulation protocols. The monopolar silver-wire electrode (diameter, 50 μm) was used for focal stimulation, insulated except for the tip, and located within 150 μm of the recorded neurons. Whole cell patch pipettes were constructed from borosilicate glass capillaries (1.5 mm OD; World Precision Instruments, Sarasota, FL). The resistance of a typical patch pipette was 5–10 MΩ when filled with internal solution. Two pipette solutions were used: (1) Cs sulfate–based solution for voltage clamp recording, containing 110 mm Cs2SO4, 0.5 mm CaCl2, 2 mm MgCl2, 5 mm TEA-Cl, 5 mm ATP-Mg salt, 5 mm EGTA, and 5 mm HEPES, which used Cs and TEA as K+channel blockers; and (2) potassium gluconate–based solution for current clamp recording, containing 135 mm K-gluconate, 5 mm KCl, 0.5 mm CaCl2, 2 mm MgCl2, 5 mm EGTA, and 5 mm HEPES. Membrane currents were amplified with an Axopatch 200A (Axon Instruments, Foster City, CA). Signals were filtered at 2 kHz and digitized at 5 kHz. Data were collected and analyzed using pClamp6.3 (Axon Instruments).
Application of Drugs
Drugs were dissolved in Krebs solution, and the solution was applied by perfusion without an alteration in the perfusion rate and temperature. Isoflurane was applied using a carrier gas (95% O2, 5% CO2) and calibrated via  a specific vaporizer, and the concentration of isoflurane in the Krebs solution was measured by gas chromatography. We ascertained that the concentration saturated with 1.5% isoflurane was approximately 0.37 mm, nearly equal to 1 rat MAC. The drugs used in this work were strychnine, 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX), dl-2-amino-5 phosphonovaleric acid (Sigma, St. Louis, MO), tetrodotoxin (Wako, Osaka, Japan), muscimol (Sigma), NMDA (Sigma), and isoflurane (Abbott Japan, Tokyo, Japan).
Data Analysis
Numerical data are expressed as mean ± SD. Statistical differences were assessed using the paired t  test and the Kolmogorov–Smirnov test. P  < 0.05 was considered significant.
Results
Isoflurane Diminishes Dorsal Root–evoked Polysynaptic EPSCs
The dorsal root stimulation at Aδ- or C-fiber intensity evoked monosynaptic EPSCs, polysynaptic EPSCs, or both in all SG neurons tested. In some SG neurons, solely polysynaptic EPSCs were recorded at a holding potential of −70 mV, and the effects of isoflurane on these polysynaptic EPSCs were evaluated in the presence of strychnine (glycine receptor antagonist; 2 μm). Bath application of 1 MAC isoflurane reversibly suppressed Aδ- and C-fiber intensity stimulation–evoked polysynaptic EPSCs in all recorded neurons (figs. 1A and B). The integrated area of these evoked polysynaptic EPSCs was used to evaluate the isoflurane effect. Isoflurane significantly reduced the integrated area of Aδ-fiber intensity stimulation–evoked polysynaptic EPSCs from 1,328 ± 1,132 pA · ms to 756 ± 328 pA · ms (68 ± 22% of control, n = 6, P  < 0.05; fig. 1C) and C-fiber intensity stimulation–evoked polysynaptic EPSCs from 3,833 ± 2,053 pA · ms to 2,772 ± 1,701 pA · ms (70 ± 9% of control, n = 7, P  < 0.05; fig. 1C).
Fig. 1. Isoflurane inhibits dorsal root–evoked polysynaptic excitatory postsynaptic currents. (  A  and  B  ) Averaged traces of several consecutive Aδ fiber (  A  ) and C fiber (  B  ) intensity stimulation–evoked polysynaptic excitatory postsynaptic currents before and during the application of isoflurane (3 min) and after washout (5 min). (  C  ) Integrated area of Aδ- and C-fiber intensity stimulation evoked polysynaptic excitatory postsynaptic currents under the action of isoflurane, relative to those in the control (n = 6 and 7, respectively).  Vertical bars  represent SDs. *  P  < 0.05. The excitatory postsynaptic currents were recorded in the presence of strychnine (2 μm) at −70 mV. 
Fig. 1. Isoflurane inhibits dorsal root–evoked polysynaptic excitatory postsynaptic currents. (  A  and  B  ) Averaged traces of several consecutive Aδ fiber (  A  ) and C fiber (  B  ) intensity stimulation–evoked polysynaptic excitatory postsynaptic currents before and during the application of isoflurane (3 min) and after washout (5 min). (  C  ) Integrated area of Aδ- and C-fiber intensity stimulation evoked polysynaptic excitatory postsynaptic currents under the action of isoflurane, relative to those in the control (n = 6 and 7, respectively).  Vertical bars  represent SDs. *  P  < 0.05. The excitatory postsynaptic currents were recorded in the presence of strychnine (2 μm) at −70 mV. 
Fig. 1. Isoflurane inhibits dorsal root–evoked polysynaptic excitatory postsynaptic currents. (  A  and  B  ) Averaged traces of several consecutive Aδ fiber (  A  ) and C fiber (  B  ) intensity stimulation–evoked polysynaptic excitatory postsynaptic currents before and during the application of isoflurane (3 min) and after washout (5 min). (  C  ) Integrated area of Aδ- and C-fiber intensity stimulation evoked polysynaptic excitatory postsynaptic currents under the action of isoflurane, relative to those in the control (n = 6 and 7, respectively).  Vertical bars  represent SDs. *  P  < 0.05. The excitatory postsynaptic currents were recorded in the presence of strychnine (2 μm) at −70 mV. 
×
Stimulation of a dorsal root with Aδ-fiber intensity, C-fiber intensity, or both evoked a monosynaptic EPSC in a population of SG neurons (figs. 2A and B). These dorsal root–evoked monosynaptic EPSCs were completely blocked by 20 μm CNQX, indicating an activation of non-NMDA receptors.38 In clear contrast to polysynaptic EPSCs, isoflurane had no effect on Aδ- or C-fiber–evoked monosynaptic EPSCs in all SG neurons tested (figs. 2A and B). The peak amplitude was not significantly affected by isoflurane (Aδ fiber: 102 ± 8 of control, n = 9, P  = 0.531; C fiber: 95 ± 10% of control, n = 6, P  = 0.493; fig. 2C), suggesting that isoflurane does not affect glutamate release from primary afferent terminals and the postsynaptic non-NMDA glutamatergic receptors.
Fig. 2. Isoflurane does not affect dorsal root–evoked monosynaptic excitatory postsynaptic currents.  (A  and  B  ) Averaged traces of monosynaptic excitatory postsynaptic currents evoked by Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation before and during the application of isoflurane (3 min). These were recorded in two different cells. (  C  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on Aδ- and C-fiber–evoked monosynaptic excitatory postsynaptic currents (n = 9 and 6, respectively). 
Fig. 2. Isoflurane does not affect dorsal root–evoked monosynaptic excitatory postsynaptic currents. 
	(A  and  B  ) Averaged traces of monosynaptic excitatory postsynaptic currents evoked by Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation before and during the application of isoflurane (3 min). These were recorded in two different cells. (  C  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on Aδ- and C-fiber–evoked monosynaptic excitatory postsynaptic currents (n = 9 and 6, respectively). 
Fig. 2. Isoflurane does not affect dorsal root–evoked monosynaptic excitatory postsynaptic currents.  (A  and  B  ) Averaged traces of monosynaptic excitatory postsynaptic currents evoked by Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation before and during the application of isoflurane (3 min). These were recorded in two different cells. (  C  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on Aδ- and C-fiber–evoked monosynaptic excitatory postsynaptic currents (n = 9 and 6, respectively). 
×
Next, we tested whether isoflurane affects the activity of the NMDA receptors. In the presence of CNQX (20 μm), bicuculline (20 μm), and strychnine (2 μm), we recorded dorsal root–evoked monosynaptic NMDA receptor–mediated EPSCs at a holding potential of +40 mV (fig. 3A). The peak amplitude of these evoked NMDA currents was not affected by isoflurane (102 ± 9% of control, n = 6, P  = 0.66; fig. 3B). In addition, we investigated the effect of isoflurane on the NMDA-induced current in SG neurons. Superfusing NMDA (50 μm) elicited an inward current at −50 mV. The amplitude of the NMDA-induced current was not affected, nor was that of evoked EPSCs (104 ± 5% of control, n = 9, P  = 0.09; figs. 3C and D), suggesting that isoflurane has no effect on activation of NMDA receptors in SG neurons.
Fig. 3. Isoflurane does not affect the  N  -methyl-d-aspartate (NMDA) current. (  A  ) Monosynaptic NMDA currents evoked by dorsal root stimulation were recorded at +40 mV, in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), bicuculline (20 μm), and strychnine (2 μm). Averaged traces of several consecutive monosynaptic NMDA currents before and during the application of isoflurane (3 min). (  B  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on NMDA currents (n = 6). (  C  ) The effect of isoflurane on the current evoked by bath application of NMDA (50 μm). NMDA-induced currents before (  left  ) and during (  right  ) application of isoflurane at −50 mV. (  D  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on the NMDA-induced current (n = 9). 
Fig. 3. Isoflurane does not affect the  N  -methyl-d-aspartate (NMDA) current. (  A  ) Monosynaptic NMDA currents evoked by dorsal root stimulation were recorded at +40 mV, in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), bicuculline (20 μm), and strychnine (2 μm). Averaged traces of several consecutive monosynaptic NMDA currents before and during the application of isoflurane (3 min). (  B  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on NMDA currents (n = 6). (  C  ) The effect of isoflurane on the current evoked by bath application of NMDA (50 μm). NMDA-induced currents before (  left  ) and during (  right  ) application of isoflurane at −50 mV. (  D  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on the NMDA-induced current (n = 9). 
Fig. 3. Isoflurane does not affect the  N  -methyl-d-aspartate (NMDA) current. (  A  ) Monosynaptic NMDA currents evoked by dorsal root stimulation were recorded at +40 mV, in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), bicuculline (20 μm), and strychnine (2 μm). Averaged traces of several consecutive monosynaptic NMDA currents before and during the application of isoflurane (3 min). (  B  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on NMDA currents (n = 6). (  C  ) The effect of isoflurane on the current evoked by bath application of NMDA (50 μm). NMDA-induced currents before (  left  ) and during (  right  ) application of isoflurane at −50 mV. (  D  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on the NMDA-induced current (n = 9). 
×
We further tested the effect of isoflurane on the frequency and amplitude of miniature EPSCs (mEPSCs), indicators of action at presynaptic terminals and postsynaptic responsiveness to glutamate, respectively (fig. 4).36 In the presence of tetrodotoxin (0.5 μm), SG neurons exhibited mEPSCs with a frequency of 2.3–50.3 Hz and a mean amplitude of 8.1–24.0 pA. Neither the frequency (n = 8, P  = 0.225) nor the mean amplitude (n = 8, P  = 0.373) of mEPSCs was affected by 1 MAC isoflurane (fig. 4C). In all cells tested (n = 8), interevent interval and amplitude distributions were not changed by isoflurane (fig. 4B). These data indicate that isoflurane affects glutamate release from presynaptic terminals of neither primary afferents nor excitatory interneurons. Therefore, a clinically relevant concentration of isoflurane does not affect monosynaptic glutamatergic excitatory transmission, but instead it may suppress nociceptive transmission by acting somewhere in the polysynaptic pathways of the dorsal horn.
Fig. 4. Effect of isoflurane on miniature excitatory postsynaptic currents (mEPSCs). mEPSCs were recorded at −70 mV, in the presence of tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of mEPSCs before (  left  ) and during (  right  ) application of isoflurane (Iso; 3 min). (  B  ) Cumulative distributions of interevent intervals and amplitudes of mEPSC, before (  straight line  ) and under (  dotted line  ) the action of isoflurane. Data were constructed from continuous recording for 30 s each. Isoflurane did not affect the distributions of interevent intervals and amplitudes. (  C  ) The frequency and mean amplitude under the action of isoflurane, relative to those in the control. Isoflurane affected neither the frequency nor mean amplitude of mEPSCs (n = 8). 
Fig. 4. Effect of isoflurane on miniature excitatory postsynaptic currents (mEPSCs). mEPSCs were recorded at −70 mV, in the presence of tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of mEPSCs before (  left  ) and during (  right  ) application of isoflurane (Iso; 3 min). (  B  ) Cumulative distributions of interevent intervals and amplitudes of mEPSC, before (  straight line  ) and under (  dotted line  ) the action of isoflurane. Data were constructed from continuous recording for 30 s each. Isoflurane did not affect the distributions of interevent intervals and amplitudes. (  C  ) The frequency and mean amplitude under the action of isoflurane, relative to those in the control. Isoflurane affected neither the frequency nor mean amplitude of mEPSCs (n = 8). 
Fig. 4. Effect of isoflurane on miniature excitatory postsynaptic currents (mEPSCs). mEPSCs were recorded at −70 mV, in the presence of tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of mEPSCs before (  left  ) and during (  right  ) application of isoflurane (Iso; 3 min). (  B  ) Cumulative distributions of interevent intervals and amplitudes of mEPSC, before (  straight line  ) and under (  dotted line  ) the action of isoflurane. Data were constructed from continuous recording for 30 s each. Isoflurane did not affect the distributions of interevent intervals and amplitudes. (  C  ) The frequency and mean amplitude under the action of isoflurane, relative to those in the control. Isoflurane affected neither the frequency nor mean amplitude of mEPSCs (n = 8). 
×
Isoflurane Enhances GABAergic IPSCs
As mentioned above, the most likely target of isoflurane action in the polysynaptic pathway is the GABAAreceptor. Therefore, we next examined the effect of isoflurane on inhibitory GABAergic transmission. Monosynaptic GABAergic IPSCs were evoked by focal stimulation after a blockade of glutamatergic and glycinergic transmission by CNQX (20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm), respectively (fig. 5A). Isoflurane had little effect on the amplitude of monosynaptic GABAergic IPSCs (91 ± 23%, n = 6, P  = 0.886). However, the decay time constant was significantly prolonged from 13.3 ± 6.3 to 24.4 ± 14.6 ms (177 ± 52% of control, P  < 0.05). Moreover, isoflurane significantly augmented the integrated area from 1,409 ± 588 to 1,847 ± 578 pA · ms (138 ± 23% of control, P  < 0.05; fig. 5B). These results provide evidence that isoflurane potentiates GABAergic transmission in the spinal dorsal horn neurons. In addition to these effects on the IPSC duration, isoflurane significantly delayed the time to peak of IPSC in all SG neurons tested (Δt = 1.1 ± 0.5 ms, n = 6, P  < 0.05).
Fig. 5. Isoflurane augments γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents. (  A  ) Averaged traces of focal stimulation evoked γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm) before and during the application of isoflurane (3 min). Isoflurane slightly delayed the time to peak of inhibitory postsynaptic currents. (  B  ) Amplitude, decay time constant, and integrated area of under the action of isoflurane, relative to those in the control. The amplitude was not affected by isoflurane (n = 6); however, decay time constants and integrated area were significantly increased by isoflurane. *  P  < 0.05. 
Fig. 5. Isoflurane augments γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents. (  A  ) Averaged traces of focal stimulation evoked γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm) before and during the application of isoflurane (3 min). Isoflurane slightly delayed the time to peak of inhibitory postsynaptic currents. (  B  ) Amplitude, decay time constant, and integrated area of under the action of isoflurane, relative to those in the control. The amplitude was not affected by isoflurane (n = 6); however, decay time constants and integrated area were significantly increased by isoflurane. *  P  < 0.05. 
Fig. 5. Isoflurane augments γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents. (  A  ) Averaged traces of focal stimulation evoked γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm) before and during the application of isoflurane (3 min). Isoflurane slightly delayed the time to peak of inhibitory postsynaptic currents. (  B  ) Amplitude, decay time constant, and integrated area of under the action of isoflurane, relative to those in the control. The amplitude was not affected by isoflurane (n = 6); however, decay time constants and integrated area were significantly increased by isoflurane. *  P  < 0.05. 
×
We next investigated how isoflurane enhanced GABAergic transmission, i.e.  , via  presynaptic or postsynaptic mechanisms. GABAergic miniature IPSCs (mIPSCs) were isolated by adding tetrodotoxin (0.5 μm) and strychnine (2 μm) (fig. 6A). Neither the amplitude (from 20.2 ± 6.2 pA to 19.3 ± 5.2 pA, 97 ± 12% of control, n = 8, P  = 0.470) nor the frequency (from 6.4 ± 3.2 to 6.8 ± 2.7 Hz, 113 ± 29% of control, P  = 0.245) of mIPSCs was affected by isoflurane. However, the decay time constant of mIPSCs was significantly augmented by isoflurane, as was that of evoked IPSCs (from 19.4 ± 3.8 to 26.1 ± 3.6 ms, 137 ± 22% of control, P  < 0.05; figs. 6B and C). We did not measure the time to peak of mIPSC because this amplitude was very small and hence it was difficult to measure the time to peak exactly. These results suggest that isoflurane does not alter GABA release from presynaptic terminals, but instead enhances the response of GABAAreceptor on the postsynaptic membrane.
Fig. 6. The effect of isoflurane on γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents. γ-Aminobutyric acid receptor–mediated miniature inhibitory postsynaptic currents were recorded at 0 mV in the presence of strychnine (2 μm) and tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of γ-aminobutyric acid–mediated inhibitory postsynaptic currents before (  upper  ) and during (  lower  ) the application of isoflurane (3 min). (  B  ) Averaged traces of several γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents before and after the addition of isoflurane, respectively; these are superimposed for comparison. (  C  ) The amplitude, frequency, and decay time constants under the action of isoflurane, relative to those in the control. Isoflurane affected neither the amplitude nor the frequency (n = 8). However, the decay time constants were significantly prolonged. *  P  < 0.05. 
Fig. 6. The effect of isoflurane on γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents. γ-Aminobutyric acid receptor–mediated miniature inhibitory postsynaptic currents were recorded at 0 mV in the presence of strychnine (2 μm) and tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of γ-aminobutyric acid–mediated inhibitory postsynaptic currents before (  upper  ) and during (  lower  ) the application of isoflurane (3 min). (  B  ) Averaged traces of several γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents before and after the addition of isoflurane, respectively; these are superimposed for comparison. (  C  ) The amplitude, frequency, and decay time constants under the action of isoflurane, relative to those in the control. Isoflurane affected neither the amplitude nor the frequency (n = 8). However, the decay time constants were significantly prolonged. *  P  < 0.05. 
Fig. 6. The effect of isoflurane on γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents. γ-Aminobutyric acid receptor–mediated miniature inhibitory postsynaptic currents were recorded at 0 mV in the presence of strychnine (2 μm) and tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of γ-aminobutyric acid–mediated inhibitory postsynaptic currents before (  upper  ) and during (  lower  ) the application of isoflurane (3 min). (  B  ) Averaged traces of several γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents before and after the addition of isoflurane, respectively; these are superimposed for comparison. (  C  ) The amplitude, frequency, and decay time constants under the action of isoflurane, relative to those in the control. Isoflurane affected neither the amplitude nor the frequency (n = 8). However, the decay time constants were significantly prolonged. *  P  < 0.05. 
×
Superfusing muscimol (5 μm) elicited an outward current at 0 mV in SG neurons. When we applied isoflurane before muscimol, the muscimol-induced current was potentiated markedly (fig. 7A), and the peak amplitude was augmented significantly (161 ± 57% of control, n = 8, P  < 0.05; fig. 7B). This result reinforces the notion that isoflurane changes the responsiveness of the postsynaptic GABAAreceptor and augments GABAergic transmission in the dorsal horn neurons.
Fig. 7. Isoflurane augments the muscimol-induced γ-aminobutyric acid current. (  A  ) Muscimol (5 μm)-induced outward currents before (  left  ) and during (  right  ) application of isoflurane. The membrane potential was clamped at 0 mV. (  B  ) Amplitude of the current under the action of isoflurane relative to that in the control (n = 8). *  P  < 0.05. 
Fig. 7. Isoflurane augments the muscimol-induced γ-aminobutyric acid current. (  A  ) Muscimol (5 μm)-induced outward currents before (  left  ) and during (  right  ) application of isoflurane. The membrane potential was clamped at 0 mV. (  B  ) Amplitude of the current under the action of isoflurane relative to that in the control (n = 8). *  P  < 0.05. 
Fig. 7. Isoflurane augments the muscimol-induced γ-aminobutyric acid current. (  A  ) Muscimol (5 μm)-induced outward currents before (  left  ) and during (  right  ) application of isoflurane. The membrane potential was clamped at 0 mV. (  B  ) Amplitude of the current under the action of isoflurane relative to that in the control (n = 8). *  P  < 0.05. 
×
Isoflurane Does Not Affect Polysynaptic EPSCs in the Presence of Bicuculline
We tested whether the inhibitory effect of isoflurane on polysynaptic EPSCs is due to augmentation of GABAergic transmission. In the presence of bicuculline (20 μm) and strychnine (2 μm), dorsal root stimulation–evoked polysynaptic EPSCs were recorded. Under these conditions, Aδ- or C-fiber intensity stimulation usually evokes repetitive, long-lasting polysynaptic EPSCs that follow the initial fast monosynaptic or polysynaptic EPSCs.34 Isoflurane affected neither the initial fast polysynaptic nor the long-lasting polysynaptic EPSCs under the blockade of GABAergic transmission in all recorded neurons (Aδ fiber: n = 9, P  = 0.053; C fiber: n = 9, P  = 0.105; figs. 8A and B).
Fig. 8. Isoflurane has no inhibitory action in the presence of bicuculline. (  A  and  B  ) The dorsal root stimulation–evoked polysynaptic excitatory postsynaptic currents (EPSCs) were recorded in the presence of bicuculline (20 μm) and strychnine (2 μm). The integrated area of the Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation–evoked polysynaptic EPSCs were not affected by isoflurane (n = 9 and 9, respectively). 
Fig. 8. Isoflurane has no inhibitory action in the presence of bicuculline. (  A  and  B  ) The dorsal root stimulation–evoked polysynaptic excitatory postsynaptic currents (EPSCs) were recorded in the presence of bicuculline (20 μm) and strychnine (2 μm). The integrated area of the Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation–evoked polysynaptic EPSCs were not affected by isoflurane (n = 9 and 9, respectively). 
Fig. 8. Isoflurane has no inhibitory action in the presence of bicuculline. (  A  and  B  ) The dorsal root stimulation–evoked polysynaptic excitatory postsynaptic currents (EPSCs) were recorded in the presence of bicuculline (20 μm) and strychnine (2 μm). The integrated area of the Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation–evoked polysynaptic EPSCs were not affected by isoflurane (n = 9 and 9, respectively). 
×
Isoflurane Had No Effect on Action Potential Discharge Activity in SG Neurons
Finally, another possibility is that isoflurane would affect ionotropic channels, such as the K+and Ca2+channels, and, as a result, would alter the intrinsic membrane properties of SG neurons. We studied the action potential discharge activity in SG neurons in current clamp mode using patch pipettes filled with potassium gluconate instead of Cs sulfate. The resting membrane potential was not changed by isoflurane. In response to a depolarizing current injection (100 pA, 400 ms), SG neurons exhibited a train of action potentials (fig. 9A). Isoflurane had little effect on the number (from 7.2 ± 2.9 to 6.4 ± 3.2, n = 5, P  = 0.10; fig. 9B) and the threshold of action potential discharges (from −45.5 ± 1.8 mV to −45.0 ± 1.8 mV), suggesting that the effect of isoflurane on action potential–generating activity is minimal.
Fig. 9. Effect of isoflurane on action potential discharge activity. Recordings were made in whole cell current clamp. (  A  ) Representative examples of isoflurane effects on action potential discharge activity. (  B  ) The effect of isoflurane on the number of action potentials induced by current injection (100 pA, 400 ms). The number was not significantly changed by isoflurane (n = 5). (  C  ) The effect of isoflurane on the threshold of action potential discharges. Isoflurane had little effect on the threshold. 
Fig. 9. Effect of isoflurane on action potential discharge activity. Recordings were made in whole cell current clamp. (  A  ) Representative examples of isoflurane effects on action potential discharge activity. (  B  ) The effect of isoflurane on the number of action potentials induced by current injection (100 pA, 400 ms). The number was not significantly changed by isoflurane (n = 5). (  C  ) The effect of isoflurane on the threshold of action potential discharges. Isoflurane had little effect on the threshold. 
Fig. 9. Effect of isoflurane on action potential discharge activity. Recordings were made in whole cell current clamp. (  A  ) Representative examples of isoflurane effects on action potential discharge activity. (  B  ) The effect of isoflurane on the number of action potentials induced by current injection (100 pA, 400 ms). The number was not significantly changed by isoflurane (n = 5). (  C  ) The effect of isoflurane on the threshold of action potential discharges. Isoflurane had little effect on the threshold. 
×
Discussion
We have shown that 1 MAC isoflurane inhibits glutamatergic polysynaptic EPSCs in the SG neurons, leaving monosynaptic EPSCs unchanged. Conversely, isoflurane augments GABAAreceptor–mediated inhibitory transmission. Under conditions in which GABAergic and glycinergic inhibitory transmission were eliminated, the inhibitory action of isoflurane on polysynaptic EPSCs almost disappeared.
Implication of Inhibition of Polysynaptic EPSCs
Dorsal root–evoked excitatory synaptic responses in the SG consist of monosynaptic and polysynaptic EPSCs or solely polysynaptic EPSCs. SG neurons with solely monosynaptic EPSCs are relatively rare.41 The exact contribution of polysynaptic EPSCs to the excitability of SG neurons cannot be determined. However, one can reasonably speculate that the inhibition of polysynaptic EPSCs by isoflurane has considerable effects on nociceptive transmission in the superficial dorsal horn, given that more than 80% of SG neurons exhibit polysynaptic EPSCs,41 and the amplitude and duration of polysynaptic EPSCs are almost identical to those of monosynaptic EPSCs.44 
Minimal Action of Isoflurane on Glutamatergic Transmission in the SG
We have shown that isoflurane does not affect glutamate release from presynaptic terminals of primary afferents and excitatory interneurons. In addition, the responses of postsynaptic non-NMDA and NMDA receptors are also unaffected by isoflurane. These data indicate that glutamatergic transmission in the SG is not a primary target for isoflurane.
Volatile anesthetics have been reported to depress glutamate transmission via  presynaptic45 and postsynaptic actions.7 MacIver et al.  45 have reported that clinically relevant concentrations of isoflurane and halothane reduced glutamate release in rat hippocampal brain slices. Haseneder et al.  46 reported that isoflurane reduced glutamatergic transmission in SG neurons of immature rat spinal cord in presynaptic manner. An article provided evidence that enflurane directly depresses AMPA currents in mouse spinal cord motor neurons.7 Several reports have shown that isoflurane diminishes the NMDA current in cultured cortical neurons9 and Xenopus  oocytes.29 Cheng and Kendig7 showed that enflurane reduced the NMDA current in mouse spinal cord motor neurons. However, our results do not support the data listed above, at least in the superficial dorsal horn of adult rat. The exact reason for the discrepancies is unknown, but they might be caused by differences in organs and maturity of animals used. It was reported that changes in subtypes of NMDA receptors can occur during growth.47,48 Alternatively, differences of the type or concentration of volatile anesthetics used may also contribute.
Augmentation of GABAergic Transmission by Isoflurane
Classically, GABAAreceptors were thought to be located on primary afferent terminals and involved in presynaptic inhibition via  primary afferent depolarization.49 In this study, however, we did not observe inhibition of dorsal root–evoked monosynaptic EPSCs by isoflurane (fig. 2). Previously, we demonstrated that muscimol (a GABAAreceptor agonist) affected neither the amplitude of dorsal root–evoked monosynaptic EPSCs nor the frequency of mEPSCs.34 Taken together, these results indicate that facilitation of presynaptic GABAergic inhibition by isoflurane (via  primary afferent depolarization) is not prominent, at least in the fine afferent fibers in the superficial dorsal horn. Instead, postsynaptic GABAAreceptors located on somatodendritic sites of excitatory interneurons are the most likely sites of action for isoflurane.
Isoflurane is known to have two distinct effects on synaptic GABAAresponses: prolongation of the decay phase and reduction of the peak amplitude (blocking effect) of GABAergic IPSCs in rat hippocampal slices12 and human embryonic kidney 293 cells.11 Furthermore, Banks and Pearce12 have reported that the concentration of isoflurane revealed a dissociation between the effects on the time course and the amplitude of IPSCs (prolonging and blocking effects), suggesting that distinct mechanisms underlie the two actions. These results indicate that the blocking effect of isoflurane at 1 MAC was not remarkable, but this concentration was enough to observe the prolongation of the decay phase. Our current results showed the amplitude of GABAergic evoked and miniature IPSCs were not significantly reduced by isoflurane at 1 MAC. However, we observed a prolongation of the decay time course of both of GABAergic evoked and miniature IPSCs. We speculate that similar blocking effects of IPSCs may be observable also in the dorsal horn at higher concentrations of isoflurane.
On the other hand, the amplitude of the bath-applied muscimol-induced current was apparently augmented by isoflurane at 1 MAC (fig. 7), although isoflurane had no effect on the peak amplitude of GABAergic IPSCs (figs. 5B and 6C). There are several possibilities for the difference in the action of isoflurane between exogenous and synaptic GABA responses. First, isoflurane may positively modulate extrasynaptic but not synaptic GABAAreceptors in SG neurons. Naturally expressed GABAAreceptors are thought to be heteromeric and comprised of forms of some subunits, such as α, β, and γ. This variety of GABAAreceptor subunits and a different combination of subunits results in the formation GABAAreceptors exhibiting a distinct pharmacology, including isoflurane actions. Second, an augmentation of IPSC amplitude by isoflurane may have been occluded, owing to the release of GABA into the synaptic cleft at a concentration high enough to saturate the GABAAreceptors on the postsynaptic neurons. A single quantum of GABA is known to saturate postsynaptic GABAAreceptors.50 Hapfelmeier et al.  11 showed that isoflurane significantly increased the amplitude and rise times of currents elicited by subsaturating GABA concentrations (10−7m∼10−5m) but decreased the amplitude of currents elicited by a saturating GABA concentration (10−4m). They suggested that isoflurane would decrease the dissociation rate of GABA from GABAAreceptors. In addition to the prolonging effect, we also demonstrated that isoflurane significantly delayed the time to peak of the evoked IPSC. These results may support dissociation rate theory.
Is the Facilitation of GABAergic Transmission a Major Mechanism of Isoflurane on Polysynaptic EPSCs?
As discussed above, isoflurane inhibits polysynaptic excitatory transmission, and the augmentation of GABAergic inhibition may be a most likely mechanism for this. To reinforce this hypothesis, we tested the effect of isoflurane in the presence of bicuculline and strychnine, a condition in which both GABAergic and glycinergic inhibitions are eliminated. In this situation, the inhibitory action of isoflurane almost disappeared (figs. 8A and B), suggesting that the effect on the GABAAreceptor is a major action of isoflurane in the inhibition polysynaptic EPSCs. In the dorsal horn neurons that receive direct primary afferent input, the monosynaptic EPSC is generally followed by GABAergic IPSCs, glycinergic IPSCs, or both. Therefore, when the duration of these IPSCs is prolonged, the number of spikes should be decreased, and consequently the peak amplitude and integrated area of polysynaptic EPSCs can be reduced in the recorded neuron.
The remaining possibility was that isoflurane might directly suppress the excitability of excitatory interneurons by affecting membrane properties for generating action potentials. Many studies have reported that inhaled anesthetics affect several types of K+and Ca2+channels,14–21 which may alter action potential–generating membrane properties. However, our data have shown that isoflurane had no effect on the resting membrane potential and the threshold of action potential discharges (fig. 9). Therefore, a direct inhibitory action of isoflurane on membrane excitability is unlikely as a mechanism for the inhibition of polysynaptic EPSCs.
It is well known that inhaled anesthetics also enhance the glycine-induced Clcurrent.31 However, in this study, we did not examine the isoflurane action on the glycine receptor, because the inhibitory effect of glycinergic transmission in nociceptive transmission is much less prominent than that of GABAergic transmission in the SG.34 The glycine receptor may also play a role, at least in part, in the antinociceptive action of isoflurane in the dorsal horn.
In conclusion, we have demonstrated that 1 MAC isoflurane markedly inhibits dorsal root–evoked polysynaptic EPSCs. The augmentation of GABAergic transmission by isoflurane is the most likely mechanism for this phenomenon. Our results presented here may provide a cellular basis for the antinociceptive action of isoflurane at the spinal cord level.
The authors thank Hidemasa Furue, Ph.D. (Lecturer, Department of Integrative Physiology, Graduate School Sciences, Kyusyu University, Fukuoka, Japan), and Naoshi Fujiwara, Ph.D. (Professor and Chairman, Department of General Education, Niigata University School of Health Sciences, Niigata, Japan), for technical advice.
References
Rampil IJ, Mason P, Singh H: Anesthetic potency (MAC) is independent of forebrain structures in the rat. Anesthesiology 1993; 78:707–12Rampil, IJ Mason, P Singh, H
Antognini JF, Schwartz K: Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 1993; 79:1244–9Antognini, JF Schwartz, K
Borges M, Antognini JF: Does the brain influence somatic responses to noxious stimuli during isoflurane anesthesia? Anesthesiology 1994; 81:1511–5Borges, M Antognini, JF
Rampil IJ: Anesthetic potency is not altered after hypothermic spinal cord transection in rats. Anesthesiology 1994; 80:606–10Rampil, IJ
Savola MKT, Woodley SJ, Maze M, Kendig JJ: Isoflurane and an alpha 2-adrenoceptor agonist suppress nociceptive neurotransmission in neonatal rat spinal cord. Anesthesiology 1991; 75:489–98Savola, MKT Woodley, SJ Maze, M Kendig, JJ
Collins JG, Kendig JJ, Mason P: Anesthetic actions within the spinal cord: Contributions to the state of general anesthesia. Trends Neurosci 1995; 18:549–53Collins, JG Kendig, JJ Mason, P
Cheng G, Kendig JJ: Enflurane directly depresses glutamate AMPA and NMDA currents in mouse spinal cord motor neurons independent of actions on GABAAor glycine receptors. Anesthesiology 2000; 93:1075–84Cheng, G Kendig, JJ
Yamauchi M, Sekiyama H, Shimada SG, Collins JG: Halothane suppression of spinal sensory neuronal responses to noxious peripheral stimuli is mediated, in part, by both GABAAand glycine receptor systems. Anesthesiology 2002; 97:412–7Yamauchi, M Sekiyama, H Shimada, SG Collins, JG
Ming Z, Knapp DJ, Mueller RA, Breese GR, Criswell HE: Differential modulation of GABA- and NMDA-gated currents by ethanol and isoflurane in cultured rat cerebral cortical neurons. Brain Res 2001; 920:117–24Ming, Z Knapp, DJ Mueller, RA Breese, GR Criswell, HE
Topf N, Jenkins A, Baron N, Harrison NL: Effects of isoflurane on γ-aminobutyric acid type A receptors activated by full and partial agonists. Anesthesiology 2003; 98:306–11Topf, N Jenkins, A Baron, N Harrison, NL
Hapfelmeier G, Haseneder R, Eder M, Adelsberger H, Kochs E, Rammes G, Zieglgansberger W: Isoflurane slows inactivation kinetics of rat recombinant α1β2γ2L GABAAreceptors: Enhancement of GABAergic transmission despite an open-channel block. Neurosci Lett 2001; 307:97–100Hapfelmeier, G Haseneder, R Eder, M Adelsberger, H Kochs, E Rammes, G Zieglgansberger, W
Banks MI, Pearce RA: Dual actions of volatile anesthetics on GABAAIPSCs: Dissociation of blocking and prolonging effects. Anesthesiology 1999; 90:120–34Banks, MI Pearce, RA
Nishikawa K, MacIver MB: Agent-selective effects of volatile anesthetics on GABAAreceptor–mediated synaptic inhibition in hippocampal interneurons. Anesthesiology 2001; 94:340–7Nishikawa, K MacIver, MB
Friederich P, Benzenberg D, Trellakis S, Urban BW: Interaction of volatile anesthetics with human Kv channels in relation to clinical concentrations. Anesthesiology 2001; 95:954–8Friederich, P Benzenberg, D Trellakis, S Urban, BW
Shin WJ, Winegar BD: Modulation of noninactivating K+channels in rat cerebellar granule neurons by halothane, isoflurane, and sevoflurane. Anesth Analg 2003; 96:1340–4Shin, WJ Winegar, BD
Gray AT, Winegar BD, Leonoudakis DJ, Forsayeth JR, Yost CS: TOK1 is a volatile anesthetic stimulated K+channel. Anesthesiology 1998; 88:1076–84Gray, AT Winegar, BD Leonoudakis, DJ Forsayeth, JR Yost, CS
Rajan S, Wischmeyer E, Karschin C, Preisig-Muller R, Grzeschik KH, Daut J, Karschin A, Derst C: THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+channels. J Biol Chem 2001; 276:7302–11Rajan, S Wischmeyer, E Karschin, C Preisig-Muller, R Grzeschik, KH Daut, J Karschin, A Derst, C
Study RE: Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons. Anesthesiology 1994; 81:104–16Study, RE
Kameyama K, Aono K, Kitamura K: Isoflurane inhibits neuronal Ca2+channels through enhancement of current inactivation. Br J Anaesth 1999; 82:402–11Kameyama, K Aono, K Kitamura, K
Kamatchi GL, Chan CK, Snutch T, Durieux ME, Lynch C III: Volatile anesthetic inhibition of neuronal Ca channel currents expressed in Xenopus oocytes. Brain Res 1999; 831:85–96Kamatchi, GL Chan, CK Snutch, T Durieux, ME Lynch, C
Kamatchi GL, Durieux ME, Lynch C III: Differential sensitivity of expressed L-type calcium channels and muscarinic M(1) receptors to volatile anesthetics in Xenopus oocytes. J Pharmacol Exp Ther 2001; 297:981–90Kamatchi, GL Durieux, ME Lynch, C
Matsuura T, Kamiya Y, Itoh H, Higashi T, Yamada Y, Andoh T: Inhibitory effects of isoflurane and nonimmobilizing halogenated compounds on neuronal nicotinic acetylcholine receptors. Anesthesiology 2002; 97:1541–9Matsuura, T Kamiya, Y Itoh, H Higashi, T Yamada, Y Andoh, T
Violet JM, Downie DL, Nakisa RC, Lieb WR, Franks NP: Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. Anesthesiology 1997; 86:866–74Violet, JM Downie, DL Nakisa, RC Lieb, WR Franks, NP
Flood P, Ramirez-Latorre J, Role L: Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected. Anesthesiology 1997; 86:859–65Flood, P Ramirez-Latorre, J Role, L
Mascia MP, Machu TK, Harris RA: Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics. Br J Pharmacology 1996; 119:1331–6Mascia, MP Machu, TK Harris, RA
Joo DT, Gong D, Sonner JM, Jia Z, MacDonald JF, Eger EI II, Orser BA: Blockade of AMPA receptors and volatile anesthetics: Reduced anesthetic requirements in GluR2 null mutant mice for loss of the righting reflex and antinociception but not minimum alveolar concentration. Anesthesiology 2001; 94:478–88Joo, DT Gong, D Sonner, JM Jia, Z MacDonald, JF Eger, EI Orser, BA
Ming Z, Griffith BL, Breese GR, Mueller RA, Criswell HE: Changes in the effect of isoflurane on N  -methyl-d-aspartic acid–gated currents in cultured cerebral cortical neurons with time in culture: Evidence for subunit specificity. Anesthesiology 2002; 97:856–67Ming, Z Griffith, BL Breese, GR Mueller, RA Criswell, HE
Nishikawa K, MacIver MB: Excitatory synaptic transmission mediated by NMDA receptors is more sensitive to isoflurane than are non–NMDA receptor-mediated responses. Anesthesiology 2000; 92:228–36Nishikawa, K MacIver, MB
Yamakura T, Harris RA: Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels: Comparison with isoflurane and ethanol. Anesthesiology 2000; 93:1095–101Yamakura, T Harris, RA
de Sousa SL, Dickinson R, Lieb WR, Franks NP: Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon. Anesthesiology 2000; 92:1055–66de Sousa, SL Dickinson, R Lieb, WR Franks, NP
Franks NP, Lieb WR: Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367:607–14Franks, NP Lieb, WR
Krasowski MD, Harrison NL: General anaesthetic actions on ligand-gated ion channels. Cell Mol Life Sci 1999; 55:1278–303Krasowski, MD Harrison, NL
Yamakura T, Bertaccini E, Trudell JR, Harris RA: Anesthetics and ion channels: Molecular models and sites of action. Annu Rev Pharmacol Toxicol 2001; 41:23–51Yamakura, T Bertaccini, E Trudell, JR Harris, RA
Baba H, Ji RR, Kohno T, Moore KA, Ataka T, Wakai A, Okamoto M, Woolf CJ: Removal of GABAergic inhibition facilitates polysynaptic A fiber-mediated excitatory transmission to the superficial spinal dorsal horn. Mol Cell Neurosci 2003; 24:818–30Baba, H Ji, RR Kohno, T Moore, KA Ataka, T Wakai, A Okamoto, M Woolf, CJ
Moore KA, Kohno T, Karchewski LA, Scholz J, Baba H, Woolf CJ: Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord. J Neurosci 2002; 22:6724–31Moore, KA Kohno, T Karchewski, LA Scholz, J Baba, H Woolf, CJ
Baba H, Shmoji K, Yoshimura M: Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord: I. Anesthesiology 2000; 92:473–84Baba, H Shmoji, K Yoshimura, M
Kohno T, Moore KA, Baba H, Woolf CJ: Peripheral nerve injury alters excitatory synaptic transmission in lamina II of the rat dorsal horn. J Physiol 2003; 548:131–8Kohno, T Moore, KA Baba, H Woolf, CJ
Yoshimura M, Nishi S: Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: Pharmacological properties of synaptic currents. Neuroscience 1993; 53:519–26Yoshimura, M Nishi, S
Baba H, Goldstein PA, Okamoto M, Kohno T, Ataka T, Yoshimura M, Shimoji K: Norepinephrine facilitates inhibitory transmission in substantia gelatinosa of adult rat spinal cord: II. Anesthesiology 2000; 92:485–92Baba, H Goldstein, PA Okamoto, M Kohno, T Ataka, T Yoshimura, M Shimoji, K
Kohno T, Kumamoto E, Higashi H, Shimoji K, Yoshimura M: Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult rat spinal cord. J Physiol 1999; 518:803–13Kohno, T Kumamoto, E Higashi, H Shimoji, K Yoshimura, M
Baba H, Doubell TP, Woolf CJ: Peripheral inflammation facilitates Aβ fiber-mediated synaptic input to the substantia gelatinosa of the adult rat spinal cord. J Neurosci 1999; 19:859–67Baba, H Doubell, TP Woolf, CJ
Ataka T, Kumamoto E, Shimoji K, Yoshimura M: Baclofen inhibits more effectively C-afferent than Aδ-afferent glutamatergic transmission in substantia gelatinosa neurons of adult rat spinal cord slices. Pain 2000; 86:273–82Ataka, T Kumamoto, E Shimoji, K Yoshimura, M
Nakatsuka T, Ataka T, Kumamoto E, Tamaki T, Yoshimura M: Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of the rat spinal dorsal horn during postnatal development. Neuroscience 2000; 99:549–5Nakatsuka, T Ataka, T Kumamoto, E Tamaki, T Yoshimura, M
Yoshimura M, Nishi S: Excitatory amino acid receptors involved in primary afferent-evoked polysynaptic EPSPs of substantia gelatinosa neurons in the adult rat spinal cord slice. Neurosci Lett 1992; 143:131–4Yoshimura, M Nishi, S
Maclver MB, Mikulec AA, Amagasu SM, Monroe FA: Volatile anesthetics depress glutamate transmission via  presynaptic actions. Anesthesiology 1996; 85:823–34Maclver, MB Mikulec, AA Amagasu, SM Monroe, FA
Haseneder R, Kurz J, Dodt HU, Kochs E, Zieglgansberger W, Scheller M, Rammes G, Hapfelmeier G: Isoflurane reduces glutamatergic transmission in neurons in the spinal cord superficial dorsal horn: Evidence for a presynaptic site of an analgesic action. Anesth Analg 2004; 98:1718–23Haseneder, R Kurz, J Dodt, HU Kochs, E Zieglgansberger, W Scheller, M Rammes, G Hapfelmeier, G
Kuehl-Kovarik MC, Partin KM, Magnusson KR: Acute dissociation for analyses of NMDA receptor function in cortical neurons during aging. J Neurosci Methods 2003; 129:11–7Kuehl-Kovarik, MC Partin, KM Magnusson, KR
Law AJ, Weickert CS, Webster MJ, Herman MM, Kleinman JE, Harrison PJ: Expression of NMDA receptor NR1, NR2A and NR2B subunit mRNAs during development of the human hippocampal formation. Eur J Neurosci 2003; 18:1197–205Law, AJ Weickert, CS Webster, MJ Herman, MM Kleinman, JE Harrison, PJ
MacDermott AB, Role LW, Siegelbaum SA: Presynaptic ionotropic receptors and the control of transmitter release. Annu Rev Neurosci 1999; 22:443–85MacDermott, AB Role, LW Siegelbaum, SA
Mody I, De Koninck Y, Otis TS, Soltesz I: Bridging the cleft at GABA synapses in the brain. Trends Neurosci 1994; 17:517–25Mody, I De Koninck, Y Otis, TS Soltesz, I
Fig. 1. Isoflurane inhibits dorsal root–evoked polysynaptic excitatory postsynaptic currents. (  A  and  B  ) Averaged traces of several consecutive Aδ fiber (  A  ) and C fiber (  B  ) intensity stimulation–evoked polysynaptic excitatory postsynaptic currents before and during the application of isoflurane (3 min) and after washout (5 min). (  C  ) Integrated area of Aδ- and C-fiber intensity stimulation evoked polysynaptic excitatory postsynaptic currents under the action of isoflurane, relative to those in the control (n = 6 and 7, respectively).  Vertical bars  represent SDs. *  P  < 0.05. The excitatory postsynaptic currents were recorded in the presence of strychnine (2 μm) at −70 mV. 
Fig. 1. Isoflurane inhibits dorsal root–evoked polysynaptic excitatory postsynaptic currents. (  A  and  B  ) Averaged traces of several consecutive Aδ fiber (  A  ) and C fiber (  B  ) intensity stimulation–evoked polysynaptic excitatory postsynaptic currents before and during the application of isoflurane (3 min) and after washout (5 min). (  C  ) Integrated area of Aδ- and C-fiber intensity stimulation evoked polysynaptic excitatory postsynaptic currents under the action of isoflurane, relative to those in the control (n = 6 and 7, respectively).  Vertical bars  represent SDs. *  P  < 0.05. The excitatory postsynaptic currents were recorded in the presence of strychnine (2 μm) at −70 mV. 
Fig. 1. Isoflurane inhibits dorsal root–evoked polysynaptic excitatory postsynaptic currents. (  A  and  B  ) Averaged traces of several consecutive Aδ fiber (  A  ) and C fiber (  B  ) intensity stimulation–evoked polysynaptic excitatory postsynaptic currents before and during the application of isoflurane (3 min) and after washout (5 min). (  C  ) Integrated area of Aδ- and C-fiber intensity stimulation evoked polysynaptic excitatory postsynaptic currents under the action of isoflurane, relative to those in the control (n = 6 and 7, respectively).  Vertical bars  represent SDs. *  P  < 0.05. The excitatory postsynaptic currents were recorded in the presence of strychnine (2 μm) at −70 mV. 
×
Fig. 2. Isoflurane does not affect dorsal root–evoked monosynaptic excitatory postsynaptic currents.  (A  and  B  ) Averaged traces of monosynaptic excitatory postsynaptic currents evoked by Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation before and during the application of isoflurane (3 min). These were recorded in two different cells. (  C  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on Aδ- and C-fiber–evoked monosynaptic excitatory postsynaptic currents (n = 9 and 6, respectively). 
Fig. 2. Isoflurane does not affect dorsal root–evoked monosynaptic excitatory postsynaptic currents. 
	(A  and  B  ) Averaged traces of monosynaptic excitatory postsynaptic currents evoked by Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation before and during the application of isoflurane (3 min). These were recorded in two different cells. (  C  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on Aδ- and C-fiber–evoked monosynaptic excitatory postsynaptic currents (n = 9 and 6, respectively). 
Fig. 2. Isoflurane does not affect dorsal root–evoked monosynaptic excitatory postsynaptic currents.  (A  and  B  ) Averaged traces of monosynaptic excitatory postsynaptic currents evoked by Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation before and during the application of isoflurane (3 min). These were recorded in two different cells. (  C  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on Aδ- and C-fiber–evoked monosynaptic excitatory postsynaptic currents (n = 9 and 6, respectively). 
×
Fig. 3. Isoflurane does not affect the  N  -methyl-d-aspartate (NMDA) current. (  A  ) Monosynaptic NMDA currents evoked by dorsal root stimulation were recorded at +40 mV, in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), bicuculline (20 μm), and strychnine (2 μm). Averaged traces of several consecutive monosynaptic NMDA currents before and during the application of isoflurane (3 min). (  B  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on NMDA currents (n = 6). (  C  ) The effect of isoflurane on the current evoked by bath application of NMDA (50 μm). NMDA-induced currents before (  left  ) and during (  right  ) application of isoflurane at −50 mV. (  D  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on the NMDA-induced current (n = 9). 
Fig. 3. Isoflurane does not affect the  N  -methyl-d-aspartate (NMDA) current. (  A  ) Monosynaptic NMDA currents evoked by dorsal root stimulation were recorded at +40 mV, in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), bicuculline (20 μm), and strychnine (2 μm). Averaged traces of several consecutive monosynaptic NMDA currents before and during the application of isoflurane (3 min). (  B  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on NMDA currents (n = 6). (  C  ) The effect of isoflurane on the current evoked by bath application of NMDA (50 μm). NMDA-induced currents before (  left  ) and during (  right  ) application of isoflurane at −50 mV. (  D  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on the NMDA-induced current (n = 9). 
Fig. 3. Isoflurane does not affect the  N  -methyl-d-aspartate (NMDA) current. (  A  ) Monosynaptic NMDA currents evoked by dorsal root stimulation were recorded at +40 mV, in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), bicuculline (20 μm), and strychnine (2 μm). Averaged traces of several consecutive monosynaptic NMDA currents before and during the application of isoflurane (3 min). (  B  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on NMDA currents (n = 6). (  C  ) The effect of isoflurane on the current evoked by bath application of NMDA (50 μm). NMDA-induced currents before (  left  ) and during (  right  ) application of isoflurane at −50 mV. (  D  ) The amplitude under the action of isoflurane, relative to that in the control. Isoflurane had no effect on the NMDA-induced current (n = 9). 
×
Fig. 4. Effect of isoflurane on miniature excitatory postsynaptic currents (mEPSCs). mEPSCs were recorded at −70 mV, in the presence of tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of mEPSCs before (  left  ) and during (  right  ) application of isoflurane (Iso; 3 min). (  B  ) Cumulative distributions of interevent intervals and amplitudes of mEPSC, before (  straight line  ) and under (  dotted line  ) the action of isoflurane. Data were constructed from continuous recording for 30 s each. Isoflurane did not affect the distributions of interevent intervals and amplitudes. (  C  ) The frequency and mean amplitude under the action of isoflurane, relative to those in the control. Isoflurane affected neither the frequency nor mean amplitude of mEPSCs (n = 8). 
Fig. 4. Effect of isoflurane on miniature excitatory postsynaptic currents (mEPSCs). mEPSCs were recorded at −70 mV, in the presence of tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of mEPSCs before (  left  ) and during (  right  ) application of isoflurane (Iso; 3 min). (  B  ) Cumulative distributions of interevent intervals and amplitudes of mEPSC, before (  straight line  ) and under (  dotted line  ) the action of isoflurane. Data were constructed from continuous recording for 30 s each. Isoflurane did not affect the distributions of interevent intervals and amplitudes. (  C  ) The frequency and mean amplitude under the action of isoflurane, relative to those in the control. Isoflurane affected neither the frequency nor mean amplitude of mEPSCs (n = 8). 
Fig. 4. Effect of isoflurane on miniature excitatory postsynaptic currents (mEPSCs). mEPSCs were recorded at −70 mV, in the presence of tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of mEPSCs before (  left  ) and during (  right  ) application of isoflurane (Iso; 3 min). (  B  ) Cumulative distributions of interevent intervals and amplitudes of mEPSC, before (  straight line  ) and under (  dotted line  ) the action of isoflurane. Data were constructed from continuous recording for 30 s each. Isoflurane did not affect the distributions of interevent intervals and amplitudes. (  C  ) The frequency and mean amplitude under the action of isoflurane, relative to those in the control. Isoflurane affected neither the frequency nor mean amplitude of mEPSCs (n = 8). 
×
Fig. 5. Isoflurane augments γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents. (  A  ) Averaged traces of focal stimulation evoked γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm) before and during the application of isoflurane (3 min). Isoflurane slightly delayed the time to peak of inhibitory postsynaptic currents. (  B  ) Amplitude, decay time constant, and integrated area of under the action of isoflurane, relative to those in the control. The amplitude was not affected by isoflurane (n = 6); however, decay time constants and integrated area were significantly increased by isoflurane. *  P  < 0.05. 
Fig. 5. Isoflurane augments γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents. (  A  ) Averaged traces of focal stimulation evoked γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm) before and during the application of isoflurane (3 min). Isoflurane slightly delayed the time to peak of inhibitory postsynaptic currents. (  B  ) Amplitude, decay time constant, and integrated area of under the action of isoflurane, relative to those in the control. The amplitude was not affected by isoflurane (n = 6); however, decay time constants and integrated area were significantly increased by isoflurane. *  P  < 0.05. 
Fig. 5. Isoflurane augments γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents. (  A  ) Averaged traces of focal stimulation evoked γ-aminobutyric acid receptor–mediated monosynaptic inhibitory postsynaptic currents in the presence of 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX; 20 μm), dl-2-amino-5 phosphonovaleric acid (50 μm), and strychnine (2 μm) before and during the application of isoflurane (3 min). Isoflurane slightly delayed the time to peak of inhibitory postsynaptic currents. (  B  ) Amplitude, decay time constant, and integrated area of under the action of isoflurane, relative to those in the control. The amplitude was not affected by isoflurane (n = 6); however, decay time constants and integrated area were significantly increased by isoflurane. *  P  < 0.05. 
×
Fig. 6. The effect of isoflurane on γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents. γ-Aminobutyric acid receptor–mediated miniature inhibitory postsynaptic currents were recorded at 0 mV in the presence of strychnine (2 μm) and tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of γ-aminobutyric acid–mediated inhibitory postsynaptic currents before (  upper  ) and during (  lower  ) the application of isoflurane (3 min). (  B  ) Averaged traces of several γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents before and after the addition of isoflurane, respectively; these are superimposed for comparison. (  C  ) The amplitude, frequency, and decay time constants under the action of isoflurane, relative to those in the control. Isoflurane affected neither the amplitude nor the frequency (n = 8). However, the decay time constants were significantly prolonged. *  P  < 0.05. 
Fig. 6. The effect of isoflurane on γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents. γ-Aminobutyric acid receptor–mediated miniature inhibitory postsynaptic currents were recorded at 0 mV in the presence of strychnine (2 μm) and tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of γ-aminobutyric acid–mediated inhibitory postsynaptic currents before (  upper  ) and during (  lower  ) the application of isoflurane (3 min). (  B  ) Averaged traces of several γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents before and after the addition of isoflurane, respectively; these are superimposed for comparison. (  C  ) The amplitude, frequency, and decay time constants under the action of isoflurane, relative to those in the control. Isoflurane affected neither the amplitude nor the frequency (n = 8). However, the decay time constants were significantly prolonged. *  P  < 0.05. 
Fig. 6. The effect of isoflurane on γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents. γ-Aminobutyric acid receptor–mediated miniature inhibitory postsynaptic currents were recorded at 0 mV in the presence of strychnine (2 μm) and tetrodotoxin (0.5 μm). (  A  ) The consecutive traces of γ-aminobutyric acid–mediated inhibitory postsynaptic currents before (  upper  ) and during (  lower  ) the application of isoflurane (3 min). (  B  ) Averaged traces of several γ-aminobutyric acid–mediated miniature inhibitory postsynaptic currents before and after the addition of isoflurane, respectively; these are superimposed for comparison. (  C  ) The amplitude, frequency, and decay time constants under the action of isoflurane, relative to those in the control. Isoflurane affected neither the amplitude nor the frequency (n = 8). However, the decay time constants were significantly prolonged. *  P  < 0.05. 
×
Fig. 7. Isoflurane augments the muscimol-induced γ-aminobutyric acid current. (  A  ) Muscimol (5 μm)-induced outward currents before (  left  ) and during (  right  ) application of isoflurane. The membrane potential was clamped at 0 mV. (  B  ) Amplitude of the current under the action of isoflurane relative to that in the control (n = 8). *  P  < 0.05. 
Fig. 7. Isoflurane augments the muscimol-induced γ-aminobutyric acid current. (  A  ) Muscimol (5 μm)-induced outward currents before (  left  ) and during (  right  ) application of isoflurane. The membrane potential was clamped at 0 mV. (  B  ) Amplitude of the current under the action of isoflurane relative to that in the control (n = 8). *  P  < 0.05. 
Fig. 7. Isoflurane augments the muscimol-induced γ-aminobutyric acid current. (  A  ) Muscimol (5 μm)-induced outward currents before (  left  ) and during (  right  ) application of isoflurane. The membrane potential was clamped at 0 mV. (  B  ) Amplitude of the current under the action of isoflurane relative to that in the control (n = 8). *  P  < 0.05. 
×
Fig. 8. Isoflurane has no inhibitory action in the presence of bicuculline. (  A  and  B  ) The dorsal root stimulation–evoked polysynaptic excitatory postsynaptic currents (EPSCs) were recorded in the presence of bicuculline (20 μm) and strychnine (2 μm). The integrated area of the Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation–evoked polysynaptic EPSCs were not affected by isoflurane (n = 9 and 9, respectively). 
Fig. 8. Isoflurane has no inhibitory action in the presence of bicuculline. (  A  and  B  ) The dorsal root stimulation–evoked polysynaptic excitatory postsynaptic currents (EPSCs) were recorded in the presence of bicuculline (20 μm) and strychnine (2 μm). The integrated area of the Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation–evoked polysynaptic EPSCs were not affected by isoflurane (n = 9 and 9, respectively). 
Fig. 8. Isoflurane has no inhibitory action in the presence of bicuculline. (  A  and  B  ) The dorsal root stimulation–evoked polysynaptic excitatory postsynaptic currents (EPSCs) were recorded in the presence of bicuculline (20 μm) and strychnine (2 μm). The integrated area of the Aδ- (  A  ) and C-fiber (  B  ) intensity stimulation–evoked polysynaptic EPSCs were not affected by isoflurane (n = 9 and 9, respectively). 
×
Fig. 9. Effect of isoflurane on action potential discharge activity. Recordings were made in whole cell current clamp. (  A  ) Representative examples of isoflurane effects on action potential discharge activity. (  B  ) The effect of isoflurane on the number of action potentials induced by current injection (100 pA, 400 ms). The number was not significantly changed by isoflurane (n = 5). (  C  ) The effect of isoflurane on the threshold of action potential discharges. Isoflurane had little effect on the threshold. 
Fig. 9. Effect of isoflurane on action potential discharge activity. Recordings were made in whole cell current clamp. (  A  ) Representative examples of isoflurane effects on action potential discharge activity. (  B  ) The effect of isoflurane on the number of action potentials induced by current injection (100 pA, 400 ms). The number was not significantly changed by isoflurane (n = 5). (  C  ) The effect of isoflurane on the threshold of action potential discharges. Isoflurane had little effect on the threshold. 
Fig. 9. Effect of isoflurane on action potential discharge activity. Recordings were made in whole cell current clamp. (  A  ) Representative examples of isoflurane effects on action potential discharge activity. (  B  ) The effect of isoflurane on the number of action potentials induced by current injection (100 pA, 400 ms). The number was not significantly changed by isoflurane (n = 5). (  C  ) The effect of isoflurane on the threshold of action potential discharges. Isoflurane had little effect on the threshold. 
×