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Meeting Abstracts  |   February 2001
Agent-selective Effects of Volatile Anesthetics on GABAAReceptor–mediated Synaptic Inhibition in Hippocampal Interneurons
Author Affiliations & Notes
  • Koichi Nishikawa, M.D., Ph.D.
    *
  • M. Bruce MacIver, M.Sc., Ph.D.
  • *Postdoctoral Fellow, †Associate Professor.
  • Received from the Neuropharmacology Laboratory, Department of Anesthesia, Stanford University School of Medicine, Stanford, California.
Article Information
Meeting Abstracts   |   February 2001
Agent-selective Effects of Volatile Anesthetics on GABAAReceptor–mediated Synaptic Inhibition in Hippocampal Interneurons
Anesthesiology 2 2001, Vol.94, 340-347. doi:
Anesthesiology 2 2001, Vol.94, 340-347. doi:
γ-AMINOBUTYRIC acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous systems, and GABA-mediated (GABAergic) neurons are widely distributed throughout the brain. GABA mediates neural inhibition by binding to GABAAreceptors, which are ligand-gated Clchannels and are allosterically modulated by compounds such as benzodiazepines, barbiturates, and anesthetics. 1,2 Electrophysiologic studies have demonstrated that volatile anesthetics enhance the duration of GABA-induced chloride currents in cultured hippocampal neurons 3 and that these anesthetic agents prolong the decay phase of inhibitory postsynaptic currents (IPSCs) in pyramidal neurons in brain slices. 4,5 These data support the hypothesis that general anesthesia is produced, at least in part, by enhancing neuronal inhibition mediated by GABAAreceptors.
In the hippocampus, one of the best characterized cortical circuits in the brain, GABA and its synthesizing enzyme are found in local circuit neurons (interneurons), and these neurons release GABA, providing inhibitory control of excitatory synaptic circuits and pyramidal cell discharge. 6,7 These GABAergic interneurons comprised less than 10% of the total neuron population; however, a very small number of interneurons appear to control the excitability of thousands of pyramidal cells through divergent inhibitory connections. Thus, GABAergic interneurons play an important role in regulating the excitability and synchronization of neural activity in hippocampus and other cortical regions. However, the integration of the various cellular and molecular actions of anesthetics in local brain circuitry mediated by inhibitory interneurons, and in more global brain functions, are poorly understood. Although previous reports have shown that halothane inhibited excitatory synaptic transmission recorded from interneurons located in the CA1 area, 8,9 very little is known about how volatile anesthetics modify inhibitory synaptic responses between or among hippocampal interneurons. The present study focused on volatile anesthetic modulation of synaptic inhibition between GABAergic hippocampal interneurons. The aim of the present study was to compare the actions produced by halothane, enflurane, isoflurane, and sevoflurane on IPSCs recorded from GABAergic interneurons in rat hippocampal slices.
Methods
Brain Slice Preparation
Experiments were conducted on brain slices isolated from young male Sprague-Dawley rats (55–90 g). Protocols were approved by the Institutional Animal Care Committee at Stanford University and adhered to guidelines of the National Institutes of Health and Society for Neuroscience. Rats were deeply anesthetized with diethyl ether, killed by decapitation, and brains were quickly removed and placed in cold (1–2°C) oxygenated artificial cerebrospinal fluid (ACSF; ionic composition: 151.25 mm Na+, 3.5 mm K+, 2.0 mm Ca2+, 2.0 mm Mg2+, 131.5 mm Cl, 26.0 mm HCO3−, 2.0 mm SO4−, 1.25 mm H4−, and 10.0 mm glucose). Brains were sectioned in the coronal plane into 500-μm-thick slices using a vibratome (Vibratome Series 1000, Boston, MA). Slices were then hemisected and placed on filter papers (Millipore Corp., Bedford, MA) at the interface of a humidified carbogen (95% O2–5% CO2) gas phase and ACSF liquid phase. Slices were allowed at least 1 h for recovery at room temperature (21–24°C) before submersion in ACSF in a recording chamber.
Electrophysiology
Whole cell patch-clamp recordings were made from visualized interneurons located at the stratum lacunosum–moleculare (SL-M)–stratum radiatum (SR) border in the CA1 area of rat hippocampal slices using infrared differential interference contrast microscopy. 9,10 Slices were constantly perfused with room temperature (21–24°C) ACSF at a rate of 2–2.5 ml/min in a recording chamber mounted on the stage of an upright Axioskop microscope (Zeiss, Jena, Germany). Near-infrared light illuminated the brain slice through the glass bottom of the recording chamber and was collected by a water immersion objective (40×) above the slice. The magnified image was collected by an intensified CCD camera (COHU Inc., San Diego, CA) with contrast enhancement. The image of interneurons was displayed on a video monitor, and glass patch pipettes were visually advanced using a micromanipulator (MP-285; Sutter Instrument Co., Novato, CA). Patch pipettes were made from borosilicate glass (KG33; 1.5-mm OD, 1.0-mm ID; Garner Glass Company, Claremont, CA) using an electrode puller (PP-830; Narishige, Tokyo, Japan). The resistance of the pipette was 4–6 MΩ when filled with internal solutions. Two kinds of internal solutions were used: a potassium chloride–based solution (100 mm KCl, 10 mm EGTA, 40 mm HEPES, 5 mm MgCl2, 2 mm adenosine triphosphate, and 1.5 mm guanosine triphosphate, with pH adjusted to 7.25 with KOH) and a potassium gluconate–based solution (100 mm K-gluconate, 10 mm EGTA, 40 mm HEPES, 5 mm MgCl2, 2 mm adenosine triphosphate, and 0.3 mm guanosine triphosphate, pH 7.25). In voltage-clamp recordings, N  -(2,6-Dimethylphenylcarbamoylmethyl)-triethylammonium bromide (QX-314, 1 mm) was included in the pipette solution to prevent voltage-dependent Na+currents. 11 The osmolarity of the solutions was 300 ± 5 mOsm. The series resistance was typically 10–50 MΩ immediately after obtaining the whole cell recordings and was compensated by more than 80%. Membrane potentials and currents were monitored with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), acquired through an A/D converter onto a Pentium-based computer. Data acquisition and analysis were made using pCLAMP version 8.0 (Axon Instruments) for evoked synaptic responses and DataWave version 5.1 (DataWave Technologies, Longmont, CO) for spontaneous responses. The current and voltage traces were filtered at 1 kHz and digitized at least 5 kHz. The amplitude of each spontaneous synaptic current was measured from the initial inflection point (not from the baseline) to the peak, to avoid the effects of summation on amplitude distributions.
Evoked synaptic currents were elicited using bipolar tungsten electrodes (Frederick Haer & Co., Bowdoinham, ME). These electrodes were placed near (200–300 μm) the patched interneuron located at the border of SL-M and SR for stimulation of synaptic inputs (fig. 1A). Monosynaptic GABAAreceptor–mediated IPSCs were isolated by bath application of (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) to block N  -methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor–mediated synaptic currents. The GABAergic nature of the synaptic currents was verified by determining the reversal potential and, in some cases, by applying the GABAA-receptor antagonist, bicuculline (10 μm).
Fig. 1. Identification of interneurons located near the border between stratum lacunosum–moleculare (SL-M) and stratum radiatum (SR). (A  ) Diagram of the area CA1 of a rat hippocampal slice showing the placement of bipolar stimulating tungsten electrodes (Stim) and recording patch electrodes (Record). The Stim was placed near the SL-M–SR border to evoke inhibitory postsynaptic currents in interneurons. Under infrared differential interference contrast microscopy, visually identified interneurons located at the SL-M–SR border were recorded in the whole cell voltage–current-clamp mode. (B  ) Spontaneous action potentials (APs) with a rapid rate of spike repolarization (spike-width at half maximum < 1.5 ms using K-gluconate internal solutions) and with a large slow after-hyperpolarization (sAHP) were observed in many interneurons (> 80%), although the frequency was variable. (C  ) Voltage responses to depolarizing (300 ms, +120 pA) and hyperpolarizing (300 ms, −320 pA) current injection were recorded in the current-clamp mode. As was typical for SL-M–SR interneurons, this neuron showed little spike-frequency adaptation (accommodation) and a time-dependent inward rectification (hyperpolarizing sag). scc = Schaffer-collateral-commissural.
Fig. 1. Identification of interneurons located near the border between stratum lacunosum–moleculare (SL-M) and stratum radiatum (SR). (A 
	) Diagram of the area CA1 of a rat hippocampal slice showing the placement of bipolar stimulating tungsten electrodes (Stim) and recording patch electrodes (Record). The Stim was placed near the SL-M–SR border to evoke inhibitory postsynaptic currents in interneurons. Under infrared differential interference contrast microscopy, visually identified interneurons located at the SL-M–SR border were recorded in the whole cell voltage–current-clamp mode. (B 
	) Spontaneous action potentials (APs) with a rapid rate of spike repolarization (spike-width at half maximum < 1.5 ms using K-gluconate internal solutions) and with a large slow after-hyperpolarization (sAHP) were observed in many interneurons (> 80%), although the frequency was variable. (C 
	) Voltage responses to depolarizing (300 ms, +120 pA) and hyperpolarizing (300 ms, −320 pA) current injection were recorded in the current-clamp mode. As was typical for SL-M–SR interneurons, this neuron showed little spike-frequency adaptation (accommodation) and a time-dependent inward rectification (hyperpolarizing sag). scc = Schaffer-collateral-commissural.
Fig. 1. Identification of interneurons located near the border between stratum lacunosum–moleculare (SL-M) and stratum radiatum (SR). (A  ) Diagram of the area CA1 of a rat hippocampal slice showing the placement of bipolar stimulating tungsten electrodes (Stim) and recording patch electrodes (Record). The Stim was placed near the SL-M–SR border to evoke inhibitory postsynaptic currents in interneurons. Under infrared differential interference contrast microscopy, visually identified interneurons located at the SL-M–SR border were recorded in the whole cell voltage–current-clamp mode. (B  ) Spontaneous action potentials (APs) with a rapid rate of spike repolarization (spike-width at half maximum < 1.5 ms using K-gluconate internal solutions) and with a large slow after-hyperpolarization (sAHP) were observed in many interneurons (> 80%), although the frequency was variable. (C  ) Voltage responses to depolarizing (300 ms, +120 pA) and hyperpolarizing (300 ms, −320 pA) current injection were recorded in the current-clamp mode. As was typical for SL-M–SR interneurons, this neuron showed little spike-frequency adaptation (accommodation) and a time-dependent inward rectification (hyperpolarizing sag). scc = Schaffer-collateral-commissural.
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Application of Volatile Anesthetics
The inhalation anesthetics were applied using a carrier gas (95% O2–5% CO2) and calibrated commercial vaporizer (Fluotec 3; Fraser Harlake, Orchard Park, NY, for halothane; Foregger Enfluromatic DRV3; New York, NY, for enflurane; and Fortec; Fraser Harlake, for isoflurane and sevoflurane). During experiments, the gas-phase anesthetic concentration in the ACSF reservoir (100 ml) was continuously monitored using a Datex 254 agent monitor (Datex, Helsinki, Finland). To determine the extent of loss of volatile anesthetic agent in the recording chamber, aqueous phase concentrations of anesthetics were previously determined using an electrochemical method. 12 Clinically relevant and equipotent concentrations of volatile anesthetics (1 minimal alveolar concentration [MAC], 1 rat MAC), halothane (1.2 vol%≈ 0.35 mm), enflurane (2.2 vol%≈ 0.60 mm), isoflurane (1.4 vol%≈ 0.50 mm), sevoflurane (2.7 vol%≈ 0.40 mm) were used throughout the study. To confirm the anesthetic concentration in the recording chamber, the effects of these anesthetics on population spikes in CA1 area were compared using extracellular field recordings. The concentrations of anesthetics used in this study depressed the average population spike amplitude to 15% of control in halothane (n = 3), 22% in enflurane (n = 3), 13% in isoflurane (n = 4), and 23% in sevoflurane (n = 4), indicating that volatile anesthetics (1 rat MAC) greatly depressed population spike amplitudes to less than 25% of control. These observations are consistent with our previous findings. 13 Solutions containing anesthetics were changed rapidly and accurately using a computerized perfusion system (ValveBank8; AutoMate Scientific, Oakland, CA). High-quality polytetrafluorethylene was used for reservoirs, valves, and tubing to minimize volatile anesthetic loss and drug binding.
Animals and Chemicals
All rats were obtained from Simonsen Laboratories (Gilroy, CA) and housed on a 12-h light–dark cycle. They had free access to food and water. Chemicals for the ACSF were reagent grade or better and obtained from J.T. Baker (Philadelphia, PA) or Sigma (St. Louis, MO).
Statistics
Data are expressed as mean ± SD, and levels of significance of data were set at 0.05. Statistical significance of data was determined using one-way analysis of variance (ANOVA) to compare differences between groups.
Results
Identification of Interneurons Located Near the Border of the Stratum Lacunosum–Moleculare and Stratum Radiatum
To date, information regarding anesthetic modulation of hippocampal interneurons has been limited because interneurons are sparse and widely scattered, making them unlikely targets for an electrode aimed blindly at brain slices. Using infrared differential interference contrast microscopy, 28 whole cell recordings from interneurons located near the SL-M–SR border were successfully made. These SL-M–SR interneurons have been reported to receive less spontaneous synaptic input and to fire at slower rates. 14 Spontaneous solitary action potentials (APs) and voltage responses of an interneuron to depolarizing and hyperpolarizing currents injection are presented in figures 1B and C. Spontaneous APs showed a rapid rate of spike repolarization and a large after-hyperpolarization (fig. 1B). Interneurons were also characterized by their fast APs and high-frequency discharge with little frequency adaptation (fig. 1C). These findings are in accordance with the previous reports for firing patterns of CA1 interneurons. 14,15 
Volatile Anesthetics Depressed the Amplitude of Evoked Inhibitory Postsynaptic Currents and Prolonged Their Decay in an Agent-specific Manner
In interneurons voltage clamped at −60 mV using KCl-based internal solutions, monosynaptic GABAAIPSCs were evoked using bipolar tungsten electrodes. These inward currents were abolished by an application of the GABAA-receptor antagonist, bicuculline (10 μm). Evoked IPSCs had slower rise times (1.5 ± 0.5 ms; n = 20) and slower decay time constants (36 ± 2.5 ms; n = 20) than those of spontaneous IPSCs (0.8 ± 0.2 ms in the rise time and 19 ± 5 ms in the decay; n = 10). Clinically relevant and equipotent concentrations (1.0 rat MAC) of volatile anesthetics depressed the amplitude of evoked IPSCs (fig. 2A). Interestingly, the relative degree of amplitude depression produced by these anesthetics was different (fig. 2B), i.e.  , halothane (1.2 vol%≈ 0.35 mm) depressed IPSC amplitudes to 79.8 ± 9.3% of control (n = 5;P  < 0.05), enflurane (2.2 vol%≈ 0.60 mm) to 38.2 ± 8.6% (n = 6;P  < 0.001), isoflurane (1.4 vol%≈ 0.50 mm) to 52.4 ± 8.4% (n = 5;P  < 0.001), and sevoflurane (2.7 vol%≈ 0.40 mm) to 46.1 ± 16.0% (n = 8;P  < 0.001). On the other hand, these anesthetics also preferentially prolonged the decay time constant of evoked IPSCs (fig. 2C). All anesthetics significantly prolonged the decay of evoked IPSCs; 290.1 ± 33.2% of control in halothane (n = 5;P  < 0.001), 423.6 ± 47.1% in enflurane (n = 6;P  < 0.001), 277.0 ± 32.2% in isoflurane (n = 5;P  < 0.001), and 529.4 ± 48.5% in sevoflurane (n = 8;P  < 0.001). In particular, the effects of enflurane and sevoflurane on the decay were significantly greater than those of halothane and isoflurane (P  < 0.05, ANOVA). These data suggest that all volatile anesthetics have similar but agent-specific effects on the evoked IPSC decay independent of the relative degree of amplitude depression.
Fig. 2. The effects of volatile anesthetics on stimulus-evoked GABAAreceptor–mediated inhibitory postsynaptic currents (IPSCs) in interneurons. (A  ) Representative traces of evoked IPSCs in response to electrical stimulation of the stratum lacunosum–moleculare and stratum radiatum border were obtained in the presence of glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm). Interneurons were voltage clamped at −60 mV. All anesthetics depressed evoked IPSC amplitude and prolonged their decays in an agent-specific manner. These effects were completely reversed 60 min after washout of anesthetics. (B  ) All anesthetics significantly depressed evoked IPSC amplitudes (P  < 0.05 for halothane [Hal];P  < 0.001 for enflurane [Enf], isoflurane [Iso], and sevoflurane [Sevo]vs.  control). There was no significant difference among three anesthetics: enflurane, isoflurane, and sevoflurane (analysis of variance). (C  ) All anesthetics markedly prolonged the decay time constants of evoked IPSCs in an agent-specific manner (P  < 0.001 for all anesthetics; n = 5 for halothane, n = 6 for enflurane, n = 5 for isoflurane, and n = 8 for sevoflurane). The decay phase was fitted by a single exponential curve, and the time from peak amplitude to 1/e of peak amplitude was measured in averaged traces from at least 10 recordings. The effects produced by enflurane and sevoflurane were greater than those of halothane and isoflurane (P  < 0.05, analysis of variance). No significant difference was observed between enflurane and sevoflurane.
Fig. 2. The effects of volatile anesthetics on stimulus-evoked GABAAreceptor–mediated inhibitory postsynaptic currents (IPSCs) in interneurons. (A 
	) Representative traces of evoked IPSCs in response to electrical stimulation of the stratum lacunosum–moleculare and stratum radiatum border were obtained in the presence of glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm). Interneurons were voltage clamped at −60 mV. All anesthetics depressed evoked IPSC amplitude and prolonged their decays in an agent-specific manner. These effects were completely reversed 60 min after washout of anesthetics. (B 
	) All anesthetics significantly depressed evoked IPSC amplitudes (P 
	< 0.05 for halothane [Hal];P 
	< 0.001 for enflurane [Enf], isoflurane [Iso], and sevoflurane [Sevo]vs. 
	control). There was no significant difference among three anesthetics: enflurane, isoflurane, and sevoflurane (analysis of variance). (C 
	) All anesthetics markedly prolonged the decay time constants of evoked IPSCs in an agent-specific manner (P 
	< 0.001 for all anesthetics; n = 5 for halothane, n = 6 for enflurane, n = 5 for isoflurane, and n = 8 for sevoflurane). The decay phase was fitted by a single exponential curve, and the time from peak amplitude to 1/e of peak amplitude was measured in averaged traces from at least 10 recordings. The effects produced by enflurane and sevoflurane were greater than those of halothane and isoflurane (P 
	< 0.05, analysis of variance). No significant difference was observed between enflurane and sevoflurane.
Fig. 2. The effects of volatile anesthetics on stimulus-evoked GABAAreceptor–mediated inhibitory postsynaptic currents (IPSCs) in interneurons. (A  ) Representative traces of evoked IPSCs in response to electrical stimulation of the stratum lacunosum–moleculare and stratum radiatum border were obtained in the presence of glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm). Interneurons were voltage clamped at −60 mV. All anesthetics depressed evoked IPSC amplitude and prolonged their decays in an agent-specific manner. These effects were completely reversed 60 min after washout of anesthetics. (B  ) All anesthetics significantly depressed evoked IPSC amplitudes (P  < 0.05 for halothane [Hal];P  < 0.001 for enflurane [Enf], isoflurane [Iso], and sevoflurane [Sevo]vs.  control). There was no significant difference among three anesthetics: enflurane, isoflurane, and sevoflurane (analysis of variance). (C  ) All anesthetics markedly prolonged the decay time constants of evoked IPSCs in an agent-specific manner (P  < 0.001 for all anesthetics; n = 5 for halothane, n = 6 for enflurane, n = 5 for isoflurane, and n = 8 for sevoflurane). The decay phase was fitted by a single exponential curve, and the time from peak amplitude to 1/e of peak amplitude was measured in averaged traces from at least 10 recordings. The effects produced by enflurane and sevoflurane were greater than those of halothane and isoflurane (P  < 0.05, analysis of variance). No significant difference was observed between enflurane and sevoflurane.
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Volatile Anesthetic Effects on Spontaneous Inhibitory Postsynaptic Currents in Stratum Lacunosum–Moleculare and Stratum Radiatum Interneurons
Spontaneous IPSCs, including AP-independent and -dependent components, were recorded from SL-M–SR interneurons in the absence of tetrodotoxin. The frequencies of spontaneous IPSCs were variable from cell to cell but ranged between 0.5 and 5 Hz in the control conditions. The effects of halothane (1.2 vol%≈ 0.35 mm) on spontaneous IPSCs are illustrated in figure 3A. In all interneurons (n = 6), the frequencies of spontaneous IPSCs were increased by halothane to twofold or threefold (fig. 3B). The amplitude distribution and cumulative probability of spontaneous IPSCs in the absence and presence of halothane were also analyzed (fig. 3C). Similar to evoked IPSCs, halothane produced a small reduction of spontaneous IPSC amplitudes.
Fig. 3. Halothane increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and prolonged their decay phases. Interneurons were voltage clamped at −60 mV using KCl-based internal solutions, and GABAAsIPSCs were recorded in the presence of 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) and (±)-2-amino-5-phosphonopentanoic acid (100 μm) and in the absence of tetrodotoxin. (A  ) Continuous recordings (2 s/record) in control (top four traces) and in the presence of halothane (0.35 mm, 20 min, bottom four traces). (B  ) Halothane-induced increase in spontaneous IPSC frequency plotted against experimental time. The frequency increased from 0.5 to approximately 2.5 Hz in control to more than 6 Hz in the presence of halothane. This effect was completely reversed after halothane washout. (C  ) Halothane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs. Halothane considerably depressed sIPSC amplitudes. Synaptic currents with amplitudes less than 15 pA and with slow rise times were excluded in this study.
Fig. 3. Halothane increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and prolonged their decay phases. Interneurons were voltage clamped at −60 mV using KCl-based internal solutions, and GABAAsIPSCs were recorded in the presence of 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) and (±)-2-amino-5-phosphonopentanoic acid (100 μm) and in the absence of tetrodotoxin. (A 
	) Continuous recordings (2 s/record) in control (top four traces) and in the presence of halothane (0.35 mm, 20 min, bottom four traces). (B 
	) Halothane-induced increase in spontaneous IPSC frequency plotted against experimental time. The frequency increased from 0.5 to approximately 2.5 Hz in control to more than 6 Hz in the presence of halothane. This effect was completely reversed after halothane washout. (C 
	) Halothane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs. Halothane considerably depressed sIPSC amplitudes. Synaptic currents with amplitudes less than 15 pA and with slow rise times were excluded in this study.
Fig. 3. Halothane increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and prolonged their decay phases. Interneurons were voltage clamped at −60 mV using KCl-based internal solutions, and GABAAsIPSCs were recorded in the presence of 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) and (±)-2-amino-5-phosphonopentanoic acid (100 μm) and in the absence of tetrodotoxin. (A  ) Continuous recordings (2 s/record) in control (top four traces) and in the presence of halothane (0.35 mm, 20 min, bottom four traces). (B  ) Halothane-induced increase in spontaneous IPSC frequency plotted against experimental time. The frequency increased from 0.5 to approximately 2.5 Hz in control to more than 6 Hz in the presence of halothane. This effect was completely reversed after halothane washout. (C  ) Halothane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs. Halothane considerably depressed sIPSC amplitudes. Synaptic currents with amplitudes less than 15 pA and with slow rise times were excluded in this study.
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The effects produced by enflurane (2.2 vol%≈ 0.60 mm) on spontaneous IPSCs are shown in figure 4A. Similar to effects observed with halothane, spontaneous IPSC frequency was increased by enflurane application (twofold to threefold of control;fig. 4B). The depressant effect produced by enflurane on spontaneous IPSC amplitudes was greater than those of halothane (fig. 4C). The effects of isoflurane (1.4 vol%≈ 0.50 mm) on spontaneous IPSCs are presented in figure 5A. In some interneurons (two of five), isoflurane caused a transient increase in spontaneous IPSC frequency (fig. 5B), which appeared to come from AP-dependent discharge of synaptically connected interneurons, because larger amplitude events contributed to this frequency increase (fig. 5C). Finally, sevoflurane effects (2.7 vol%≈ 0.40 mm) on spontaneous IPSCs were studied (figs. 6A–C). In summary, the most striking effects produced by these volatile anesthetics on spontaneous IPSCs were an increased frequency of spontaneous IPSCs (at least twofold) and a prolongation of their decay times.
Fig. 4. The effects of enflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of enflurane (2.2 vol%≈ 0.60 mm, bottom  ). (B  ) Enflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Enflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 4. The effects of enflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from interneurons. Recording conditions were the same as described in figure 3. (A 
	) Continuous recordings of sIPSCs (2 s/record) in control (top 
	) and in the presence of enflurane (2.2 vol%≈ 0.60 mm, bottom 
	). (B 
	) Enflurane-induced increase in sIPSC frequency plotted against experimental time. (C 
	) Enflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 4. The effects of enflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of enflurane (2.2 vol%≈ 0.60 mm, bottom  ). (B  ) Enflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Enflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
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Fig. 5. The effects of isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of isoflurane (1.4 vol%≈ 0.50 mm, bottom  ). (B  ) Isoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Isoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 5. The effects of isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were the same as described in figure 3. (A 
	) Continuous recordings of sIPSCs (2 s/record) in control (top 
	) and in the presence of isoflurane (1.4 vol%≈ 0.50 mm, bottom 
	). (B 
	) Isoflurane-induced increase in sIPSC frequency plotted against experimental time. (C 
	) Isoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 5. The effects of isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of isoflurane (1.4 vol%≈ 0.50 mm, bottom  ). (B  ) Isoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Isoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
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Fig. 6. The effects of sevoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were similar to those described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of sevoflurane (2.7 vol%≈ 0.40 mm, bottom  ). (B  ) Sevoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Sevoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 6. The effects of sevoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were similar to those described in figure 3. (A 
	) Continuous recordings of sIPSCs (2 s/record) in control (top 
	) and in the presence of sevoflurane (2.7 vol%≈ 0.40 mm, bottom 
	). (B 
	) Sevoflurane-induced increase in sIPSC frequency plotted against experimental time. (C 
	) Sevoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 6. The effects of sevoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were similar to those described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of sevoflurane (2.7 vol%≈ 0.40 mm, bottom  ). (B  ) Sevoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Sevoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
×
To examine the relative degree of increase in synaptic inhibition, we analyzed the total negative charge transfer mediated by GABAAreceptors. As observed for evoked IPSCs, all anesthetics depressed spontaneous IPSC amplitudes to some degree but greatly prolonged the time course of these synaptic currents. The total negative charge transfer of evoked IPSCs was greatly increased by all anesthetics (P  < 0.05 vs.  control;fig. 7A), because the small reduction of amplitude was more than offset by a considerable increase of the duration of inhibitory currents produced by all of the volatile anesthetics studied. Similarly, spontaneous IPSC charge transfer was also greatly increased by the anesthetics (P  < 0.05 vs.  control;fig. 7B). The large variability observed for charge transfer data mainly comes from the variability of IPSC frequency increases as previously shown in figures 3B, 4B, 5B, and 6B.
Fig. 7. The negative charge transfer mediated by GABAAreceptors in interneurons was increased by all anesthetics. (A  ) The increase in total negative charge transfer for evoked inhibitory postsynaptic currents (IPSCs) is shown. The total area under IPSCs was plotted as a percentage change of control values. All anesthetics increased the total negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics. Ten recordings (2 s/record) were measured in both control and the presence of the anesthetic. (B  ) The increase in total negative charge transfer for spontaneous IPSCs is shown. All anesthetics greatly increased the negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics.
Fig. 7. The negative charge transfer mediated by GABAAreceptors in interneurons was increased by all anesthetics. (A 
	) The increase in total negative charge transfer for evoked inhibitory postsynaptic currents (IPSCs) is shown. The total area under IPSCs was plotted as a percentage change of control values. All anesthetics increased the total negative charge transfer (P 
	< 0.05 vs. 
	control; n = 5 each), but there was no significant difference among four anesthetics. Ten recordings (2 s/record) were measured in both control and the presence of the anesthetic. (B 
	) The increase in total negative charge transfer for spontaneous IPSCs is shown. All anesthetics greatly increased the negative charge transfer (P 
	< 0.05 vs. 
	control; n = 5 each), but there was no significant difference among four anesthetics.
Fig. 7. The negative charge transfer mediated by GABAAreceptors in interneurons was increased by all anesthetics. (A  ) The increase in total negative charge transfer for evoked inhibitory postsynaptic currents (IPSCs) is shown. The total area under IPSCs was plotted as a percentage change of control values. All anesthetics increased the total negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics. Ten recordings (2 s/record) were measured in both control and the presence of the anesthetic. (B  ) The increase in total negative charge transfer for spontaneous IPSCs is shown. All anesthetics greatly increased the negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics.
×
Discussion
Major findings of our study are that all volatile anesthetics tested depressed the amplitude of evoked IPSCs recorded from hippocampal interneurons and considerably prolonged IPSC decay times in an agent-specific manner. Similar results were also apparent for spontaneous IPSCs. Although all agents depressed IPSC amplitudes to some degree, the dominant effect was an increase of inhibition by prolonging the duration of these inhibitory synaptic currents. As a result, these data suggest that volatile anesthetics depress hippocampal interneuron circuits by enhancing GABAAreceptor–medicated inhibition in an agent-specific manner. To our knowledge, this is the first study to compare effects produced by four volatile anesthetics on synaptic inhibition between connected interneurons. These results have important implications for theories of anesthetic action, as well as for the consequences of studying neural network circuit function in anesthetized animals.
Several studies have shown that the anesthetics enhance GABAAreceptor–mediated synaptic inhibition via  effects at both presynaptic and postsynaptic sites of action. 5,9 We extended these observations to include hippocampal interneurons and showed that hippocampal interneuron circuits were depressed by anesthetic-induced enhancement of GABAAreceptor–mediated synaptic inhibition. At presynaptic sites, our data show that clinically relevant concentrations of anesthetics (1 rat MAC) increase the spontaneous release of GABA from interneuron nerve terminals, evident as an increased frequency of spontaneous IPSCs recorded from interneurons (figs. 3–6). However, we did not rule out the possibility that anesthetics increase the action potential discharge activity of connected interneurons at presynaptic sites upstream from nerve terminals. 16 In fact, a minor population of hippocampal interneurons has been shown to be depolarized by halothane. 9 In addition, there can be no doubt that volatile anesthetics exert their dominant effect postsynaptically, evident in the prolongation of IPSCs. Volatile anesthetics appear to act on not only inhibitory synapses but also depress glutamate-mediated excitation of interneurons and stabilize membrane electrical properties of synaptically connected interneurons. 9 The overall effect on cortical circuit function would be to diminish the role that enhanced GABA-mediated inhibition plays during anesthesia, because the GABAergic interneurons are themselves depressed by volatile anesthetics.
In conclusion, these data provide evidence that the four volatile anesthetics tested enhanced GABAergic synaptic inhibition between interneurons. Differential effects on IPSC amplitude and on the prolongation of these currents were observed for each agent, similar to effects observed for CA1 pyramidal neurons. 5 Thus, hippocampal interneuron circuits are depressed by these anesthetics in an agent-specific manner, consistent with a multisite agent-specific theory of anesthetic action. 13,17 
The authors thank Frances A. Monroe, B.A., (Research Technician, Stanford University School of Medicine, Stanford, California) for technical assistance.
References
Tanelian DL, Kosek P, Mody I, MacIver MB: The role of the GABAAreceptor/chloride channel complex in anesthesia. A nesthesiology 1993; 78: 757–76Tanelian, DL Kosek, P Mody, I MacIver, MB
MacDonald RL, Olsen RW: GABAAreceptor channels. Annu Rev Neurosci 1994; 17: 569–602MacDonald, RL Olsen, RW
Jones MV, Brooks PA, Harrison NL: Enhancement of gamma-aminobutyric acid-activated Clcurrents in cultured rat hippocampal neurones by three volatile anaesthetics. J Physiol (Lond) 1992; 449: 279–93Jones, MV Brooks, PA Harrison, NL
Lukatch HS, MacIver MB: Voltage-clamp analysis of halothane effects on GABA (A fast) and GABA (A slow) inhibitory currents. Brain Res 1997; 765: 108–12Lukatch, HS MacIver, MB
Banks MI, Pearce RA: Dual actions of volatile anesthetics on GABAAIPSCs: Dissociation of blocking and prolonging effects. A nesthesiology 1999; 90: 120–34Banks, MI Pearce, RA
Woodson W, Nitecka L, Ben-Ari Y: Organization of the GABAergic system in the rat hippocampal formation: A quantitative immunocytochemical study. J Comp Neurol 1989; 280: 254–71Woodson, W Nitecka, L Ben-Ari, Y
Freund TF, Buzsaki G: Interneurons of the hippocampus. Hippocampus 1996; 6: 347–470Freund, TF Buzsaki, G
Perouansky M, Kirson ED, Yaari Y: Halothane blocks synaptic excitation of inhibitory interneurons. A nesthesiology 1996; 85: 1431–8Perouansky, M Kirson, ED Yaari, Y
Nishikawa K, MacIver MB: Membrane and synaptic actions of halothane on rat hippocampal pyramidal neurons and inhibitory interneurons. J Neurosci 2000; 20: 5915–23Nishikawa, K MacIver, MB
Dodt HU, Zieglgansberger W: Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res 1990; 537: 333–36Dodt, HU Zieglgansberger, W
Regehr WG, Tank DW: Calcium concentration dynamics produced by synaptic activation of CA1 hippocampal pyramidal cells. J Neurosci 1992; 12: 4202–23Regehr, WG Tank, DW
Hagan CE, Pearce RA, Trudell JR, MacIver MB: Concentration measures of volatile anesthetics in the aqueous phase using calcium sensitive electrodes. J Neurosci Methods 1998; 81: 177–184Hagan, CE Pearce, RA Trudell, JR MacIver, MB
MacIver MB: General anesthetic actions on transmission at glutamate and GABA synapses, Anesthesia: Biologic Foundations. Edited by Yaksh TL, Lynch C, Zapol WM, Maze M, Biebuyck JF, Saidman LJ. Philadelphia, Lippincott-Raven, 1997, pp 277–86
Lacaille JC, Schwartzkroin PA: Stratum lacunosum-moleculare interneurons of hippocampal CA1 region. I. Intracellular response characteristics, synaptic responses, and morphology. J Neurosci 1988; 8: 1400–10Lacaille, JC Schwartzkroin, PA
Williams S, Samulack DD, Beaulieu C, LaCaille JC: Membrane properties and synaptic responses of interneurons located near the stratum lacunosum-moleculare/radiatum border of area CA1 in whole-cell recordings from rat hippocampal slices. J Neurophysiol 1994; 71: 2217–35Williams, S Samulack, DD Beaulieu, C LaCaille, JC
Murugaiah KD, Hemmings HC Jr: Effects of intravenous general anesthetics on [3H]GABA release from rat cortical synaptosomes. A nesthesiology 1998; 89: 919–28Murugaiah, KD Hemmings, HC
Harrison NL: Ion channels take center stage: Twin spotlights on two anesthetic targets. A nesthesiology 2000; 92: 936–8Harrison, NL
Fig. 1. Identification of interneurons located near the border between stratum lacunosum–moleculare (SL-M) and stratum radiatum (SR). (A  ) Diagram of the area CA1 of a rat hippocampal slice showing the placement of bipolar stimulating tungsten electrodes (Stim) and recording patch electrodes (Record). The Stim was placed near the SL-M–SR border to evoke inhibitory postsynaptic currents in interneurons. Under infrared differential interference contrast microscopy, visually identified interneurons located at the SL-M–SR border were recorded in the whole cell voltage–current-clamp mode. (B  ) Spontaneous action potentials (APs) with a rapid rate of spike repolarization (spike-width at half maximum < 1.5 ms using K-gluconate internal solutions) and with a large slow after-hyperpolarization (sAHP) were observed in many interneurons (> 80%), although the frequency was variable. (C  ) Voltage responses to depolarizing (300 ms, +120 pA) and hyperpolarizing (300 ms, −320 pA) current injection were recorded in the current-clamp mode. As was typical for SL-M–SR interneurons, this neuron showed little spike-frequency adaptation (accommodation) and a time-dependent inward rectification (hyperpolarizing sag). scc = Schaffer-collateral-commissural.
Fig. 1. Identification of interneurons located near the border between stratum lacunosum–moleculare (SL-M) and stratum radiatum (SR). (A 
	) Diagram of the area CA1 of a rat hippocampal slice showing the placement of bipolar stimulating tungsten electrodes (Stim) and recording patch electrodes (Record). The Stim was placed near the SL-M–SR border to evoke inhibitory postsynaptic currents in interneurons. Under infrared differential interference contrast microscopy, visually identified interneurons located at the SL-M–SR border were recorded in the whole cell voltage–current-clamp mode. (B 
	) Spontaneous action potentials (APs) with a rapid rate of spike repolarization (spike-width at half maximum < 1.5 ms using K-gluconate internal solutions) and with a large slow after-hyperpolarization (sAHP) were observed in many interneurons (> 80%), although the frequency was variable. (C 
	) Voltage responses to depolarizing (300 ms, +120 pA) and hyperpolarizing (300 ms, −320 pA) current injection were recorded in the current-clamp mode. As was typical for SL-M–SR interneurons, this neuron showed little spike-frequency adaptation (accommodation) and a time-dependent inward rectification (hyperpolarizing sag). scc = Schaffer-collateral-commissural.
Fig. 1. Identification of interneurons located near the border between stratum lacunosum–moleculare (SL-M) and stratum radiatum (SR). (A  ) Diagram of the area CA1 of a rat hippocampal slice showing the placement of bipolar stimulating tungsten electrodes (Stim) and recording patch electrodes (Record). The Stim was placed near the SL-M–SR border to evoke inhibitory postsynaptic currents in interneurons. Under infrared differential interference contrast microscopy, visually identified interneurons located at the SL-M–SR border were recorded in the whole cell voltage–current-clamp mode. (B  ) Spontaneous action potentials (APs) with a rapid rate of spike repolarization (spike-width at half maximum < 1.5 ms using K-gluconate internal solutions) and with a large slow after-hyperpolarization (sAHP) were observed in many interneurons (> 80%), although the frequency was variable. (C  ) Voltage responses to depolarizing (300 ms, +120 pA) and hyperpolarizing (300 ms, −320 pA) current injection were recorded in the current-clamp mode. As was typical for SL-M–SR interneurons, this neuron showed little spike-frequency adaptation (accommodation) and a time-dependent inward rectification (hyperpolarizing sag). scc = Schaffer-collateral-commissural.
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Fig. 2. The effects of volatile anesthetics on stimulus-evoked GABAAreceptor–mediated inhibitory postsynaptic currents (IPSCs) in interneurons. (A  ) Representative traces of evoked IPSCs in response to electrical stimulation of the stratum lacunosum–moleculare and stratum radiatum border were obtained in the presence of glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm). Interneurons were voltage clamped at −60 mV. All anesthetics depressed evoked IPSC amplitude and prolonged their decays in an agent-specific manner. These effects were completely reversed 60 min after washout of anesthetics. (B  ) All anesthetics significantly depressed evoked IPSC amplitudes (P  < 0.05 for halothane [Hal];P  < 0.001 for enflurane [Enf], isoflurane [Iso], and sevoflurane [Sevo]vs.  control). There was no significant difference among three anesthetics: enflurane, isoflurane, and sevoflurane (analysis of variance). (C  ) All anesthetics markedly prolonged the decay time constants of evoked IPSCs in an agent-specific manner (P  < 0.001 for all anesthetics; n = 5 for halothane, n = 6 for enflurane, n = 5 for isoflurane, and n = 8 for sevoflurane). The decay phase was fitted by a single exponential curve, and the time from peak amplitude to 1/e of peak amplitude was measured in averaged traces from at least 10 recordings. The effects produced by enflurane and sevoflurane were greater than those of halothane and isoflurane (P  < 0.05, analysis of variance). No significant difference was observed between enflurane and sevoflurane.
Fig. 2. The effects of volatile anesthetics on stimulus-evoked GABAAreceptor–mediated inhibitory postsynaptic currents (IPSCs) in interneurons. (A 
	) Representative traces of evoked IPSCs in response to electrical stimulation of the stratum lacunosum–moleculare and stratum radiatum border were obtained in the presence of glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm). Interneurons were voltage clamped at −60 mV. All anesthetics depressed evoked IPSC amplitude and prolonged their decays in an agent-specific manner. These effects were completely reversed 60 min after washout of anesthetics. (B 
	) All anesthetics significantly depressed evoked IPSC amplitudes (P 
	< 0.05 for halothane [Hal];P 
	< 0.001 for enflurane [Enf], isoflurane [Iso], and sevoflurane [Sevo]vs. 
	control). There was no significant difference among three anesthetics: enflurane, isoflurane, and sevoflurane (analysis of variance). (C 
	) All anesthetics markedly prolonged the decay time constants of evoked IPSCs in an agent-specific manner (P 
	< 0.001 for all anesthetics; n = 5 for halothane, n = 6 for enflurane, n = 5 for isoflurane, and n = 8 for sevoflurane). The decay phase was fitted by a single exponential curve, and the time from peak amplitude to 1/e of peak amplitude was measured in averaged traces from at least 10 recordings. The effects produced by enflurane and sevoflurane were greater than those of halothane and isoflurane (P 
	< 0.05, analysis of variance). No significant difference was observed between enflurane and sevoflurane.
Fig. 2. The effects of volatile anesthetics on stimulus-evoked GABAAreceptor–mediated inhibitory postsynaptic currents (IPSCs) in interneurons. (A  ) Representative traces of evoked IPSCs in response to electrical stimulation of the stratum lacunosum–moleculare and stratum radiatum border were obtained in the presence of glutamate receptor antagonists (±)-2-amino-5-phosphonopentanoic acid (100 μm) and 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm). Interneurons were voltage clamped at −60 mV. All anesthetics depressed evoked IPSC amplitude and prolonged their decays in an agent-specific manner. These effects were completely reversed 60 min after washout of anesthetics. (B  ) All anesthetics significantly depressed evoked IPSC amplitudes (P  < 0.05 for halothane [Hal];P  < 0.001 for enflurane [Enf], isoflurane [Iso], and sevoflurane [Sevo]vs.  control). There was no significant difference among three anesthetics: enflurane, isoflurane, and sevoflurane (analysis of variance). (C  ) All anesthetics markedly prolonged the decay time constants of evoked IPSCs in an agent-specific manner (P  < 0.001 for all anesthetics; n = 5 for halothane, n = 6 for enflurane, n = 5 for isoflurane, and n = 8 for sevoflurane). The decay phase was fitted by a single exponential curve, and the time from peak amplitude to 1/e of peak amplitude was measured in averaged traces from at least 10 recordings. The effects produced by enflurane and sevoflurane were greater than those of halothane and isoflurane (P  < 0.05, analysis of variance). No significant difference was observed between enflurane and sevoflurane.
×
Fig. 3. Halothane increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and prolonged their decay phases. Interneurons were voltage clamped at −60 mV using KCl-based internal solutions, and GABAAsIPSCs were recorded in the presence of 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) and (±)-2-amino-5-phosphonopentanoic acid (100 μm) and in the absence of tetrodotoxin. (A  ) Continuous recordings (2 s/record) in control (top four traces) and in the presence of halothane (0.35 mm, 20 min, bottom four traces). (B  ) Halothane-induced increase in spontaneous IPSC frequency plotted against experimental time. The frequency increased from 0.5 to approximately 2.5 Hz in control to more than 6 Hz in the presence of halothane. This effect was completely reversed after halothane washout. (C  ) Halothane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs. Halothane considerably depressed sIPSC amplitudes. Synaptic currents with amplitudes less than 15 pA and with slow rise times were excluded in this study.
Fig. 3. Halothane increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and prolonged their decay phases. Interneurons were voltage clamped at −60 mV using KCl-based internal solutions, and GABAAsIPSCs were recorded in the presence of 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) and (±)-2-amino-5-phosphonopentanoic acid (100 μm) and in the absence of tetrodotoxin. (A 
	) Continuous recordings (2 s/record) in control (top four traces) and in the presence of halothane (0.35 mm, 20 min, bottom four traces). (B 
	) Halothane-induced increase in spontaneous IPSC frequency plotted against experimental time. The frequency increased from 0.5 to approximately 2.5 Hz in control to more than 6 Hz in the presence of halothane. This effect was completely reversed after halothane washout. (C 
	) Halothane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs. Halothane considerably depressed sIPSC amplitudes. Synaptic currents with amplitudes less than 15 pA and with slow rise times were excluded in this study.
Fig. 3. Halothane increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and prolonged their decay phases. Interneurons were voltage clamped at −60 mV using KCl-based internal solutions, and GABAAsIPSCs were recorded in the presence of 6-cyano-7-nitro-quinoxaline-2,3-dione (17.2 μm) and (±)-2-amino-5-phosphonopentanoic acid (100 μm) and in the absence of tetrodotoxin. (A  ) Continuous recordings (2 s/record) in control (top four traces) and in the presence of halothane (0.35 mm, 20 min, bottom four traces). (B  ) Halothane-induced increase in spontaneous IPSC frequency plotted against experimental time. The frequency increased from 0.5 to approximately 2.5 Hz in control to more than 6 Hz in the presence of halothane. This effect was completely reversed after halothane washout. (C  ) Halothane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs. Halothane considerably depressed sIPSC amplitudes. Synaptic currents with amplitudes less than 15 pA and with slow rise times were excluded in this study.
×
Fig. 4. The effects of enflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of enflurane (2.2 vol%≈ 0.60 mm, bottom  ). (B  ) Enflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Enflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 4. The effects of enflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from interneurons. Recording conditions were the same as described in figure 3. (A 
	) Continuous recordings of sIPSCs (2 s/record) in control (top 
	) and in the presence of enflurane (2.2 vol%≈ 0.60 mm, bottom 
	). (B 
	) Enflurane-induced increase in sIPSC frequency plotted against experimental time. (C 
	) Enflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 4. The effects of enflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of enflurane (2.2 vol%≈ 0.60 mm, bottom  ). (B  ) Enflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Enflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
×
Fig. 5. The effects of isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of isoflurane (1.4 vol%≈ 0.50 mm, bottom  ). (B  ) Isoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Isoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 5. The effects of isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were the same as described in figure 3. (A 
	) Continuous recordings of sIPSCs (2 s/record) in control (top 
	) and in the presence of isoflurane (1.4 vol%≈ 0.50 mm, bottom 
	). (B 
	) Isoflurane-induced increase in sIPSC frequency plotted against experimental time. (C 
	) Isoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 5. The effects of isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were the same as described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of isoflurane (1.4 vol%≈ 0.50 mm, bottom  ). (B  ) Isoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Isoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
×
Fig. 6. The effects of sevoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were similar to those described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of sevoflurane (2.7 vol%≈ 0.40 mm, bottom  ). (B  ) Sevoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Sevoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 6. The effects of sevoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were similar to those described in figure 3. (A 
	) Continuous recordings of sIPSCs (2 s/record) in control (top 
	) and in the presence of sevoflurane (2.7 vol%≈ 0.40 mm, bottom 
	). (B 
	) Sevoflurane-induced increase in sIPSC frequency plotted against experimental time. (C 
	) Sevoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
Fig. 6. The effects of sevoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in interneurons. Recording conditions were similar to those described in figure 3. (A  ) Continuous recordings of sIPSCs (2 s/record) in control (top  ) and in the presence of sevoflurane (2.7 vol%≈ 0.40 mm, bottom  ). (B  ) Sevoflurane-induced increase in sIPSC frequency plotted against experimental time. (C  ) Sevoflurane effects on the amplitude distribution of sIPSCs and on the cumulative probability of sIPSCs.
×
Fig. 7. The negative charge transfer mediated by GABAAreceptors in interneurons was increased by all anesthetics. (A  ) The increase in total negative charge transfer for evoked inhibitory postsynaptic currents (IPSCs) is shown. The total area under IPSCs was plotted as a percentage change of control values. All anesthetics increased the total negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics. Ten recordings (2 s/record) were measured in both control and the presence of the anesthetic. (B  ) The increase in total negative charge transfer for spontaneous IPSCs is shown. All anesthetics greatly increased the negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics.
Fig. 7. The negative charge transfer mediated by GABAAreceptors in interneurons was increased by all anesthetics. (A 
	) The increase in total negative charge transfer for evoked inhibitory postsynaptic currents (IPSCs) is shown. The total area under IPSCs was plotted as a percentage change of control values. All anesthetics increased the total negative charge transfer (P 
	< 0.05 vs. 
	control; n = 5 each), but there was no significant difference among four anesthetics. Ten recordings (2 s/record) were measured in both control and the presence of the anesthetic. (B 
	) The increase in total negative charge transfer for spontaneous IPSCs is shown. All anesthetics greatly increased the negative charge transfer (P 
	< 0.05 vs. 
	control; n = 5 each), but there was no significant difference among four anesthetics.
Fig. 7. The negative charge transfer mediated by GABAAreceptors in interneurons was increased by all anesthetics. (A  ) The increase in total negative charge transfer for evoked inhibitory postsynaptic currents (IPSCs) is shown. The total area under IPSCs was plotted as a percentage change of control values. All anesthetics increased the total negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics. Ten recordings (2 s/record) were measured in both control and the presence of the anesthetic. (B  ) The increase in total negative charge transfer for spontaneous IPSCs is shown. All anesthetics greatly increased the negative charge transfer (P  < 0.05 vs.  control; n = 5 each), but there was no significant difference among four anesthetics.
×