Introduction

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General anesthetics, like most other neuroactive drugs, modulate synaptic transmission in the central nervous system. The synaptic actions of general anesthetics are agent-specific, but considerable evidence indicates that they depress fast excitatory synaptic transmission mediated by glutamate and/or enhance fast inhibitory synaptic transmission mediated by GABA and glycine (MacIver, 1997). The cellular (e.g., presynaptic versus postsynaptic) and molecular (e.g., ion channel, receptor, signal transduction pathway, and fusion machinery) mechanisms of these synaptic effects remain to be elucidated. Moreover, the relative importance of the effects of various anesthetics on excitatory versus inhibitory synaptic transmission remains unclear (Perouansky and Hemmings, 2003).

Electrophysiological evidence supports both presynaptic (effects on neurotransmitter release) and postsynaptic (receptor interactions) mechanisms for the synaptic actions of general anesthetics. Prolongation of synaptic inhibition by positive modulation of postsynaptic GABA_A receptor function at GABAergic synapses is an important component of the depressant effects of volatile anesthetics and the primary action of several chemically distinct intravenous anesthetics at clinical concentrations (Hales and Lambert, 1991; Franks and Lieb, 1994; MacIver, 1997; Wakasugi et al., 1999; Buggy et al., 2000). Depression of excitatory transmission is also observed at clinical concentrations of many general anesthetics, particularly volatile anesthetics (Perouansky et al., 1995; MacIver et al., 1996; Ouanounou et al., 1998; Wakasugi et al., 1999). The mechanisms of these effects are unclear. Intravenous and volatile anesthetics decrease excitatory postsynaptic potentials in hippocampal neurons, which has been indirectly attributed to a presynaptic mechanism (Perouansky et al., 1995; MacIver et al., 1996).

Presynaptic effects of general anesthetics on glutamate release have been demonstrated directly at the neurochemical level using isolated nerve terminals (synaptosomes). Synaptosomes provide a superior experimental system for investigating presynaptic effects of drugs on synaptic transmission in isolation of indirect effects present in intact neural networks, such as brain slices. Chemical depolarization by superfusion with K⁺ channel blockers or elevated K⁺ stimulates neurotransmitter release from synaptosomes with comparable pharmacological properties to intact nerve terminals (Tibbs et al., 1989), while minimizing the effects of released transmitter (Garcia-Sanz et al., 2001). Volatile and intravenous anesthetics inhibit depolarization-induced glutamate release from isolated nerve terminals (Miao et al., 1995; Schlame and Hemmings, 1995; Ratnakumari and Hemmings, 1998). Considerable evidence indicates that this effect is due to suppression of presynaptic voltage-gated Na⁺ channels coupled to glutamate release (Ratnakumari and Hemmings, 2000).

Elucidation of the effects of general anesthetics on release of the major excitatory transmitter glutamate and major inhibitory transmitter GABA is essential to understanding the neurophysiological outcomes of presynaptic anesthetic actions. Effects on GABA release are of particular interest given the postsynaptic facilitation of GABAergic transmission by most anesthetics (Franks and Lieb, 1994). The apparent conservation in the fundamental machinery involved in mediating transmitter release among various nerve terminals (Scheller, 1995) suggests that the release of many neurotransmitters, in addition to glutamate, should also be inhibited by general anesthetics. Previous studies have failed to clarify the effects of general anesthetics on GABA release. We therefore compared the effects of a prototypical volatile (isoflurane) and intravenous (propofol) anesthetic on the release of glutamate and GABA from isolated rat cerebrocortical nerve terminals using a dual radiolabel superfusion technique.

	Materials and Methods

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Materials. Isoflurane was obtained from Abbott Laboratories (North Chicago, IL) and propofol (2,6-diisopropylphenol) from Sigma-Aldrich (St. Louis, MO). Tetrodotoxin, riluzole, and 4-aminopyridine (4AP) were from Sigma-Aldrich, L-[³H]glutamate (42 Ci/mmol) from Amersham Biosciences UK, Ltd. (Buckinghamshire, UK), and [¹⁴C]GABA (0.24 Ci/mmol) from PerkinElmer Life Sciences (Boston, MA).

Synaptosome Preparation. Experiments were done in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals as approved by the Weill Medical College of Cornell University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (250-350 g) were anesthetized with 80% CO₂/20% O₂ and sacrificed by cervical dislocation. The brain was rapidly removed and placed on ice. The cerebral cortex was removed and homogenized in 10 volumes of ice-cold 0.32 M sucrose with a motor-driven (500 rpm) Teflon-glass homogenizer for 10 strokes, and the homogenate was centrifuged for 2 min at 4,000g. Crude rat cerebrocortical synaptosomes (supernatant) were demyelinated by centrifugation through 0.8 M sucrose (10 ml of supernatant layered onto 10 ml of 0.8 M sucrose) for 30 min at 36,000g (Dodd et al., 1981). The pellet containing isolated nerve terminals was resuspended in ice-cold 0.32 M sucrose for use within 2 h.

Glutamate and GABA Release. Demyelinated rat cerebrocortical synaptosomes were loaded with 8 nM L-[³H]glutamate and 440 nM [¹⁴C]GABA for 15 min at 30°C in Krebs-HEPES buffer (composition: 140 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl₂, 1.2 mM Na₂HPO₄, 5 mM NaHCO₃, 0.1 mM EGTA, and 10 mM D-glucose, pH 7.4 with NaOH), pelleted by centrifugation (10 min at 20,000g at 4°C), and resuspended in ice-cold 0.32 M sucrose. For concentration-effect experiments, the prelabeling procedure was followed by a 5-min incubation with 0.2 mM D-aspartate to reduce cytoplasmic L-[³H]glutamate and, thus, Ca²⁺-independent L-[³H]glutamate release via reverse transport (Lester et al., 1994). Synaptosomes were confined by Whatman GF/B glass fiber filter disks (Maidstone, UK) and superfused at 0.5 ml/min with Krebs-HEPES buffer (initially bubbled with 95% O₂/5% CO₂ for 10 min) at 36°C using a customized (Diagram 1) Brandel SF12 superfusion apparatus (Gaithersburg, MD) set to collect 1-min fractions. Release was induced by pulses of either 30 mM K⁺ (with KCl replacing equivalent NaCl) for 3 min or 1 mM 4AP for 2 min. All pulses were induced in the presence (addition of 2 mM CaCl₂ yielding final free [Ca²⁺] of 1.9 mM; MaxChelator v2.10; http://www.stanford.edu/~cpatton/maxc.html) or absence of Ca²⁺. At the end of each experiment, synaptosomes were lysed with 0.2 M perchloric acid, and radioactivity in the synaptosomes and each fraction was quantified by liquid scintillation spectrometry with dual isotope quench correction (Beckman Coulter LS 6000IC; Beckman Coulter, Inc., Fullerton, CA) using BioSafe II scintillation cocktail (RPI, Mt. Prospect, IL). The accuracy of the dual radiolabel assay has been verified previously in detecting changes in the release of each amino acid independently of the other (Westphalen and Hemmings, 2003).

View larger version (38K): [in this window] [in a new window]   Diagram 1.   Modified superfusion system integrating a "closed" design for use with volatile anesthetics. Isoflurane or isoflurane with secretagogue are contained in separate valved glass syringes such that the superfusate can be switched between three solutions.

Application of Anesthetics. Isoflurane was added to glass syringes by dilution of saturated solutions (10-12 mM) in Krebs-HEPES buffer prepared 12 to 24 h before use. Propofol was added from concentrated solutions prepared in dimethyl sulfoxide and diluted in Krebs-HEPES buffer in glass tubes before addition to assay [maximum final [dimethyl sulfoxide] < 0.5% (v/v)]. The aqueous isoflurane concentration for initial experiments (1 mM) was approximately 3 times minimum alveolar concentration, the EC₅₀ for suppression of movement in response to a painful stimulus (Taheri et al., 1991). The propofol concentration (>12 µM) was approximately 5 times the EC₅₀ for loss of righting reflex (Tonner et al., 1992). Isoflurane or propofol were introduced through Teflon tubing from glass syringes (closed system) or glass tubes (open system), respectively. Anesthetics were added 3 min before, during, and 1 min after stimulation to insure the presence of anesthetic throughout the concurrent release pulse. In parallel experiments, isoflurane and propofol concentrations exiting the synaptosome chamber in the superfusate were determined by gas chromatography (Ratnakumari and Hemmings, 1998) or high-performance liquid chromatography (Lingamaneni et al., 2001), respectively.

Data Analysis. Release in each fraction was expressed as a fraction of synaptosomal content of labeled transmitter before each fraction collected (fractional release). The magnitude of release pulses was determined by subtracting baseline release (average of basal release before and after pulse) from cumulative fractional release values of the release pulse (sum FR; Garcia-Sanz et al., 2001). For analysis, sum FR data from each experiment were normalized to mean control release in the presence or absence of Ca²⁺. Concentration-effect data are expressed as a percentage of the within experiment control in the presence of Ca²⁺. Ca²⁺-dependent release is defined as release in the presence of Ca²⁺ minus release in the absence of Ca²⁺.

	Results

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4-Aminopyridine-Evoked Release. Stimulating preloaded rat cerebrocortical synaptosomes with 1 mM 4AP (Tibbs et al., 1989) induced Ca²⁺-independent release of L-[³H]glutamate and [¹⁴C]GABA. In the presence of 1.9 mM free Ca²⁺, total release increased 2.4-fold (P < 0.001) and 3.7-fold (P < 0.001) over Ca²⁺-independent release, respectively (Fig. 1A). In the absence of Ca²⁺, the release of glutamate and GABA occurs primarily by carrier-mediated reverse transport of cytoplasmic transmitter (Lester et al., 1994); the addition of Ca²⁺ triggers vesicular release, significantly increasing total release (Südhof, 1995). In the presence of Ca²⁺, 4AP evoked significantly (P < 0.001) more [¹⁴C]GABA than L-[³H]glutamate.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Release of L-[3H]glutamate and [14C]GABA from rat cerebrocortical synaptosomes preloaded with L-[3H]glutamate and [14C]GABA in the presence (filled symbols) or absence (open symbols) of 1.9 mM free Ca2+. A, 2-min pulse of 1 mM 4AP (n = 12-18). B, 3-min pulse of 30 mM K+ (n = 20-26). C, 2-min pulse of 1 mM 4AP with (n = 7) or without (n = 7) isoflurane in the presence of 1.9 mM free Ca2+. Concentrations of isoflurane were measured from the synaptosome chamber. Application bars indicate secretagogue or anesthetic entry into synaptosome chamber; lag in measurements represents chamber dead space. Anesthetic concentrations reported under Results are the average of values at 28 and 29 min. Data are shown as the mean ± S.E.M.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of isoflurane, propofol, and tetrodotoxin on 4AP-evoked (A), K+-evoked (B), and basal (C) release of L-[3H]glutamate and [14C]GABA. Isoflurane (1.07 ± 0.04, 1.08 ± 0.04, 1.03 ± 0.13 mM, respectively), propofol (12.6 ± 3.0, 17.0 ± 0.5, 13.5 ± 1.2 µM, respectively), or tetrodotoxin (2 µM) were present during stimulation of release by 1 mM 4AP or 30 mM K+. Data are shown as the mean ± S.E.M. Statistical comparisons between experimental groups (, P < 0.05; , P < 0.01; , P < 0.001) or between amino acids (, P <0.05; , P <0.01; , P <0.001) employed unpaired t tests (see Results for defined P values).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent effects of isoflurane (A), propofol (B), tetrodotoxin (C), and riluzole (D) on 1 mM 4AP-evoked release of L-[3H]glutamate and [14C]GABA. Data are shown as the percentage of control in the presence of Ca2+. Ca2+-independent controls are shown as the mean ± S.E.M. (n = 3-5). Parameters of fitted curves are presented in Table 1.

K⁺-Evoked Release. In the absence of Ca²⁺, depolarization of preloaded synaptosomes by 30 mM K⁺ evoked the release of L-[³H]glutamate and [¹⁴C]GABA. In the presence of 1.9 mM free Ca²⁺, release increased by 2.0-fold (P < 0.001) and 2.2-fold (P < 0.001) over Ca²⁺-independent release, respectively (Fig. 1B). Neither isoflurane (1.08 ± 0.04 mM) nor propofol (17.0 ± 1.5 µM) affected K⁺-evoked release in the absence or presence of Ca²⁺ (Fig. 2B). Tetrodotoxin partially inhibited Ca²⁺-independent K⁺-evoked release of L-[³H]glutamate (P = 0.010) and [¹⁴C]GABA (P = 0.033) and [¹⁴C]GABA release (P = 0.012) in the presence of Ca²⁺ (Fig. 2B).

Basal Release. Addition of Ca²⁺ did not affect basal (unstimulated) L-[³H]glutamate release (P = 0.29) but increased basal [¹⁴C]GABA release (P = 0.017; Fig. 2C). Isoflurane (1.03 ± 0.13 mM) inhibited basal L-[³H]glutamate release in the absence (P = 0.020) or presence (P = 0.14) of Ca²⁺ but significantly enhanced basal [¹⁴C]GABA release in the absence (P < 0.001) or presence (P = 0.017) of Ca²⁺ (Fig. 2C). Propofol enhanced basal L-[³H]glutamate release (P = 0.031) but not basal [¹⁴C]GABA release (P = 0.71) in the presence of Ca²⁺ (Fig. 2C). Tetrodotoxin inhibited basal L-[³H]glutamate release in the absence (P = 0.16) or presence (P = 0.18) of Ca²⁺ and significantly inhibited basal [¹⁴C]GABA release in the absence (P = 0.010) or presence (P = 0.006) of Ca²⁺ (Fig. 2C).

Equating Secretagogue Intensities. The sum FR of [¹⁴C]GABA released by 1 mM 4AP in the presence of Ca²⁺ (0.11) was greater (P < 0.001) than that of L-[³H]glutamate (0.055; Fig. 2A). To test the possibility that the lower sensitivity of [¹⁴C]GABA release to isoflurane was due to the greater stimulus intensity, the magnitude of [¹⁴C]GABA release was reduced to that approximating L-[³H]glutamate release by using 0.1 mM 4AP (Fig. 4). The profile of inhibition of 0.1 mM 4AP-evoked [¹⁴C]GABA release by isoflurane was similar to that observed with 1 mM 4AP (Fig. 5B; Table 1); maximum inhibition remained significantly lower (P < 0.001) than for 1 mM 4AP-evoked L-[³H]glutamate release (Fig. 3A), with no significant change in IC₅₀.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effects of 4AP and K+ on L-[3H]glutamate and [14C]GABA release in the presence of Ca2+. Data are shown as the mean ± S.E.M. (n = 4).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A, effect of isoflurane (1.15 ± 0.04 mM) on the release of L-[3H]glutamate and [14C]GABA evoked by a reduced K+ stimulus (15 mM) in the absence or presence of Ca2+. Data are shown as the mean ± S.E.M. Statistical comparisons between experimental groups used unpaired t tests (, P < 0.01; see Results for defined P values). B, concentration-effect curves for inhibition by isoflurane of the release of L-[3H]glutamate and [14C]GABA evoked by a reduced 4AP stimulus (0.1 mM) compared with data for 1 mM 4AP (from Fig. 3A).

Isoflurane-Evoked Release. Isoflurane (1.03 ± 0.13 mM) alone inhibited basal release of L-[³H]glutamate but enhanced basal [¹⁴C]GABA release in the absence or presence of Ca²⁺ (Fig. 2C). At concentrations above 0.8 mM, isoflurane evoked [¹⁴C]GABA release (Fig. 6). Above a threshold concentration of about 2 mM, isoflurane evoked L-[³H]glutamate release (Fig. 6). Statistical comparisons in the absence (2.09 ± 0.1 mM isoflurane; n = 6; Fig. 6A) or presence (2.07 ± 0.09 mM isoflurane; n = 6; Fig. 6B) of Ca²⁺ showed that isoflurane-evoked release of both L-[³H]glutamate (P = 0.079) and [¹⁴C]GABA (P = 0.68) release was Ca²⁺-independent. Isoflurane (2 mM) preferentially released [¹⁴C]GABA over L-[³H]glutamate in the absence (P = 0.0013) or presence (P = 0.015) of Ca²⁺. These effects of isoflurane were nonsaturable up to the highest concentration tested (2.4 mM).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent effects of isoflurane on basal release of L-[3H]glutamate and [14C]GABA in the absence (A) or presence (B) of 1.9 mM free Ca2+. Values for release evoked by 2 mM isoflurane are the mean ± S.E.M. (n = 6). Statistical comparisons between Ca2+ and +Ca2+ (no significant differences) and between L-[3H]glutamate and [14C]GABA (, P < 0.05; , P <0.01), determined by unpaired t tests, are shown. See Results for defined P values.

	Discussion

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The general anesthetics isoflurane and propofol significantly inhibited 4AP-evoked release of glutamate and GABA. Isoflurane preferentially inhibited 4AP-evoked glutamate release compared with GABA release over a range of clinical concentrations. This was due to greater efficacy rather than potency. The Na⁺ channel blockers tetrodotoxin and riluzole also produced selective inhibition of 4AP-evoked release of glutamate versus GABA. This suggests that isoflurane, which inhibits neuronal voltage-gated Na⁺ channel currents (Rehberg et al., 1996; Rehberg and Duch, 1999; Lingameneni et al., 2000), selectively inhibits glutamate release via Na⁺ channel blockade (Tibbs et al., 1989). This conclusion is consistent with the recent finding that riluzole preferentially inhibits transmitter release from excitatory versus inhibitory neurons in hippocampal microcultures (Prakriya and Mennerick, 2000). The physiological difference(s) between glutamatergic and GABAergic nerve terminals that underlies this pharmacological selectivity remains to be determined.

In contrast to the secretagogue 4AP, neither isoflurane nor propofol affected glutamate or GABA release evoked by elevated K⁺. Previous studies support the conclusion that clinically relevant concentrations of volatile anesthetics do not affect K⁺-evoked excitatory amino acid release (Minchin, 1981; Kendall and Minchin, 1982; Lingamaneni et al., 2001) or GABA release (Minchin, 1981; Kendall and Minchin, 1982; Lecharney et al., 1995; Salford et al., 1997; Lingamaneni et al., 2001). Inhibition of K⁺-evoked release of glutamate (Miao et al., 1995; Larsen and Langmoen, 1998; Liachenko et al., 1999) or GABA (Larsen et al., 1998; Liachenko et al., 1999) by isoflurane has been reported. Interpretation of results obtained in brain slices (Liachenko et al., 1999), however, is confounded by indirect anesthetic effects mediated by postsynaptic GABA_A receptors (Buggy et al., 2000). Failure to compare anesthetic effects on release evoked by additional secretagogues (Lingamaneni et al., 2001) makes it difficult to interpret results obtained only with elevated KCl. Intravenous anesthetics, including propofol, do not affect K⁺-evoked glutamate (Lingamaneni et al., 2001) or GABA (Mantz et al., 1995; Lingamaneni et al., 2001) release from synaptosomes at clinical concentrations, a finding reproduced in this study. Greater sensitivity of 4AP-evoked versus K⁺-evoked glutamate release to volatile anesthetics and propofol has been reported in isolated nerve terminals prepared from several brain regions and species using a fluorometric assay of endogenous glutamate (Schlame and Hemmings, 1995; Lingamaneni et al., 2001). This differential sensitivity between secretagogues was not due to differences in stimulus intensity. When [K⁺]_o was reduced to 15 mM, which evoked glutamate and GABA release comparable to that evoked by 1 mM 4AP, isoflurane remained an ineffective inhibitor of K⁺-induced glutamate or GABA release.

Selective inhibition by isoflurane and propofol of 4AP-evoked over K⁺-evoked release implicates preferential blockade of voltage-gated Na⁺ channels over Ca²⁺ channels in their presynaptic actions. Repetitive depolarization due to blockade of A-type K⁺ channels by 4AP is amplified by activation of voltage-gated Na⁺ channels leading to the opening of voltage-gated Ca²⁺ channels coupled to transmitter release. Elevated K⁺ leads to synchronous activation of voltage-gated Ca²⁺ channels followed by a plateau of residual Ca²⁺ channel conductance (Tibbs et al., 1989). Blockade of Na⁺ channels is predicted to inhibit 4AP-evoked, but not K⁺-stimulated, transmitter release, as demonstrated in this study. In the presence of Ca²⁺, the Na⁺ channel blocker tetrodotoxin, like isoflurane, completely inhibited 4AP-evoked, but not K⁺-evoked, release. A role for Na⁺ channel blockade in the inhibition of glutamate release has been suggested previously for volatile anesthetics (Ratnakumari and Hemmings, 1998; Lingamaneni et al., 2001; Asai et al., 2002) and propofol (Ratnakumari and Hemmings, 1997).

Blockade of presynaptic voltage-gated Ca²⁺ channels effectively inhibits evoked transmitter release by limiting Ca²⁺ influx (Wu and Saggau, 1997), which is essential for vesicular release (Südhof, 1995). Multiple Ca²⁺ channel subtypes can coexist in presynaptic terminals to regulate neurotransmitter release (Turner et al., 1993). Electrophysiological evidence indicates that certain voltage-gated Ca²⁺ channel subtypes are blocked by general anesthetics (Topf et al., 2003). In this study, anesthetics did not affect K⁺-evoked amino acid release, a process that involves Ca²⁺ influx primarily via voltage-gated Ca²⁺ channel opening. This may be explained by a high safety factor for inhibition of transmitter release by Ca²⁺ channel blockade or by reduced anesthetic sensitivity of the presynaptic Ca²⁺ channel subtypes coupled to transmitter release, possibly due to nerve terminal-specific modulation (Turner et al., 1993).

The Na⁺ channel blocker tetrodotoxin inhibited basal and Ca²⁺-independent K⁺-evoked glutamate and GABA release. Changes in the [Na⁺]_o/[Na⁺]_i ratio alter Ca²⁺-independent reverse transport of transmitters that are coupled to Na⁺ cotransport (Lester et al., 1994). The disparity between inhibition of Ca²⁺-independent release by tetrodotoxin and by anesthetics may stem from the different potencies or efficacies by which they inhibit Na⁺ entry (Ratnakumari and Hemmings, 1997; Ratnakumari and Hemmings, 1998) and/or from anesthetic actions at other presynaptic targets (MacIver, 1997). Recent evidence suggests, however, that isoflurane or propofol do not directly affect presynaptic neuronal transporters of glutamate or GABA (Westphalen and Hemmings, 2003).

Isoflurane, like tetrodotoxin, inhibited basal glutamate release, apparently through Na⁺ channel blockade. In contrast, isoflurane enhanced basal GABA release, particularly at higher concentrations (>1.5 mM), whereas tetrodotoxin was inhibitory. This stimulatory effect of isoflurane occurred at higher than clinical concentrations (Taheri et al., 1991), was not saturable over the concentration range studied, and was Ca²⁺-independent. General anesthetics also evoke the release of norepinephrine (Pashkov and Hemmings, 2002), whereas halothane produces an increase in mini-excitatory postsynaptic potential frequency in a rat hippocampal slice preparation (Nishikawa and MacIver, 2000). These observations suggest that volatile anesthetics may stimulate a low level of spontaneous Ca²⁺-independent vesicular release from certain terminals by a mechanism resistant to inhibition by Na⁺ channel blockade. The mechanisms underlying Ca²⁺-independent vesicular transmitter release and biochemical differences between glutamatergic and GABAergic terminals remain unclear; whether these mechanisms are directly affected by anesthetics warrants further investigation.

Significantly more GABA was released compared with glutamate for a given concentration of 4AP (Tapia and Sitges, 1982). When the level of 4AP-evoked GABA release was equalized with that of glutamate by reducing the concentration of 4AP, selective inhibition of glutamate release by isoflurane was maintained, thus eliminating a difference in secretagogue intensity as a cause. Preferential stimulation of basal GABA release by isoflurane alone could also contribute to the lower maximal inhibition of evoked GABA release compared with that of glutamate. Selective inhibition of 4AP-evoked glutamate versus GABA release, however, was also produced by other Na⁺ channel blockers that are not known to stimulate basal release.

Selective inhibition of amino acid release by general anesthetics and Na⁺ channel blockers and differential sensitivity to the secretagogue 4AP suggest fundamental physiological differences between glutamatergic and GABAergic nerve terminals. Mounting evidence supports the notion of distinct patterns of presynaptic ion channel distributions within and between neurons by demonstrating selective densities and/or function of K⁺ channels (Veh et al., 1995; Southan and Robertson, 1998) and Na⁺ channels (Stuart et al., 1997; Martina et al., 2000). Differential involvement of a presynaptic phorbol ester receptor (Munc13-1) between glutamate and GABA terminals has also been reported (Augustin et al., 1999). Our findings, and those of Prakriya and Mennerick (2000), demonstrate the pharmacological implications of such transmitter-specific presynaptic specialization.

Inhibition of excitatory amino acid transmitter release, which may be enhanced by possible blockade of postsynaptic glutamate receptors (Perouansky and Antognini, 2003), appears to be an important mechanism of neuronal depression by clinical concentrations of volatile anesthetics. Concurrently, partial inhibition of inhibitory amino acid transmitter release may be balanced by potentiation of postsynaptic GABA_A receptors. The presynaptic effects of the intravenous anesthetic propofol are less potent in relation to its clinically relevant concentrations; its marked effects on GABA_A receptors (Hales and Lambert, 1991) may play a more important role in producing anesthesia. For isoflurane, selective depression of glutamate release, stimulation of spontaneous GABA release, and potentiation of postsynaptic GABA_A receptors provide complementary actions to depress excitatory and enhance inhibitory central nervous system transmission.