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NEUROPHARMACOLOGY

Volatile Anesthetic Effects on Glutamate versus GABA Release from Isolated Rat Cortical Nerve Terminals: 4-Aminopyridine-Evoked Release

Departments of Anesthesiology and Pharmacology, Weill Medical College of Cornell University, New York, New York

Received for publication June 7, 2005
Accepted September 15, 2005.

	Abstract

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Inhibition of glutamatergic excitatory neurotransmission and potentiation of GABA-mediated inhibitory transmission are possible mechanisms involved in general anesthesia. We compared the effects of three volatile anesthetics (isoflurane, enflurane, or halothane) on 4-aminopyridine (4AP)-evoked release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes). Synaptosomes were prelabeled with L-[³H]glutamate and [¹⁴C]GABA, and release was evoked by superfusion with pulses of 1 mM 4AP in the absence or presence of 1.9 mM free Ca²⁺. All three volatile anesthetics inhibited Ca²⁺-dependent glutamate and GABA release; IC₅₀ values for glutamate were comparable to clinical concentrations (1–1.6x MAC), whereas IC₅₀ values for GABA release exceeded clinical concentrations (>2.2x MAC). All three volatile anesthetics inhibited both Ca²⁺-independent and Ca²⁺-dependent 4AP-evoked glutamate release equipotently, whereas inhibition of Ca²⁺-dependent 4AP-evoked GABA release was less potent than inhibition of Ca²⁺-independent GABA release. Inhibition of Ca²⁺-independent 4AP-evoked glutamate release was more potent than that of GABA release for isoflurane and enflurane but equipotent for halothane. Tetrodotoxin inhibited both Ca²⁺-independent and Ca²⁺-dependent 4AP-evoked glutamate and GABA release equipotently, consistent with Na⁺ channel involvement. In contrast to tetrodotoxin, volatile anesthetics exhibited selective effects on 4AP-evoked glutamate versus GABA release, consistent with distinct mechanisms of action. Preferential inhibition of Ca²⁺-dependent 4AP-evoked glutamate release versus GABA release supports the hypothesis that reduced excitatory neurotransmission relative to inhibitory neurotransmission contributes to volatile anesthetic actions.

Action potential-evoked neurotransmitter release involves nerve-terminal depolarization that triggers Ca²⁺ entry through voltage-gated Ca²⁺ channels coupled to synaptic vesicle exocytosis (Südhof, 2004). Basal neurotransmitter release includes spontaneous vesicular fusion and nonvesicular release via reverse transporter operation independent of Ca²⁺ entry. These pathways of release are mechanistically distinct and are therefore differentially sensitive to presynaptic neuromodulators (Engelman and MacDermott, 2004). In the accompanying report (Westphalen and Hemmings, 2005), we show that volatile anesthetics produce significant transmitter-selective and agent-specific effects on basal glutamate and GABA release that results in a consistent net increase in inhibitory relative to excitatory basal transmitter release.

Neurotransmitter release from isolated nerve terminals can be evoked pharmacologically by 4AP, a K⁺ channel blocker that induces release by mimicking action potential-evoked depolarizations (Tibbs et al., 1989). Inhibition of 4AP-evoked glutamate release by volatile anesthetics seems to involve suppression of presynaptic voltage-gated Na⁺ channels coupled to transmitter release (Schlame and Hemmings, 1995; Ratnakumari et al., 2000; Westphalen and Hemmings, 2003a). However, Na⁺ channel block does not affect spontaneous transmitter release (Engelman and MacDermott, 2004), and anesthetics modulate basal release independent of their Na⁺ channel antagonism. Here we compare the effects of three widely used volatile anesthetics, the ethers isoflurane and enflurane and the alkane halothane, and the selective voltage-gated Na⁺ channel blocker tetrodotoxin on 4AP-evoked release of glutamate and GABA while compensating for their agent-specific effects on basal release (Westphalen and Hemmings, 2005).

	Materials and Methods

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Materials. Sources of all compounds are listed in the accompanying article (Westphalen and Hemmings, 2005).

Evoked Glutamate and GABA Release ± Anesthetics. 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. Synaptosomes were prepared from rat cerebral cortex and prelabeled with L-[³H]glutamate and [¹⁴C]GABA, whereupon simultaneous L-[³H]glutamate and [¹⁴C]GABA release was evoked by pulses of 1 mM 4AP as described in the accompanying article (Westphalen and Hemmings, 2005). Using [³H]H₂O as a tracer, a 2-min pulse resulted in a peak of 70 ± 4% of the applied concentration to the perfusion chamber, suggesting a maximum concentration of 0.7 mM 4AP exposure to synaptosomes (data not shown). 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, 2003b).

Anesthetics at 0.1 to 8x MAC (minimum alveolar concentration) were applied for 6 min (3 min before, during, and 1 min after stimulation with a 2-min pulse of 4AP) using a closed superfusion system (Westphalen and Hemming, 2005). This procedure insures the exposure to anesthetic throughout the 4AP-evoked release pulse (Westphalen and Hemmings, 2003a). After each experiment, anesthetic solutions were sampled and quantified by gas chromatography as described previously (Westphalen and Hemmings, 2005). In separate experiments, anesthetics were continuously applied 12 min prior to stimulation by 4AP and remained present throughout the perfusion, with comparable effects on release.

Data Analysis. Release in each fraction was expressed as a fraction of synaptosomal content of labeled transmitter prior to each fraction collected (fractional release [FR]). The magnitude of the release pulse was determined by subtracting baseline release (average of basal release before and after pulse) from cumulative fractional release values for the release pulse (sum FR; Garcia-Sanz et al., 2001). Sum FR data for each experiment were normalized by the ratio of the individual assay control to the mean of all assay controls for release in the absence or presence of Ca²⁺ for each anesthetic used prior to curve fitting.

Concentration-effect data for anesthetic effects on basal L-[³H]glutamate and [¹⁴C]GABA release were taken from Westphalen and Hemmings (2005). The basal effect on sum FR for each anesthetic concentration was determined from the fitted equation and was subtracted from the sum FR values determined for 4AP-evoked release. Likewise, the derived Ca²⁺-independent release values were subtracted from values obtained in the presence of Ca²⁺ at equivalent anesthetic concentrations to determine Ca²⁺-dependent release. This subtraction method is standard procedure when baseline measurements of x-axis data cannot be accurately duplicated (Prism, version 4.0; GraphPad Software Inc., San Diego, CA).

Data for anesthetic (0.1–8x MAC) and tetrodotoxin (0.3 nM-10 µM) inhibition of 4AP-evoked release (with basal effect subtracted) were fitted to concentration-effect curves by least-squares analysis to estimate I_max and IC₅₀ with means ± S.E. (Prism, version 4.0). Each curve fit was concurrently tested for the bottom of the curve (I_max) differing significantly from 0. If the null hypothesis (I_max = 0) was not rejected, curve fit analyses were performed with I_max constrained to 0. Sum FR values for all single concentration experimental groups followed Gaussian distributions, with some significantly differing in variance, as determined by a modification of the Kolmogorov and Smirnov test and Bartlett's test, respectively. Significant differences between mean sum FR values and between concentration-effect curve parameters were determined by unpaired Student's t test with Welch correction for variances that were not assumed to be equal (Prism, version 4.0). Sum FR values obtained for 4AP-evoked release in the presence of continuously applied anesthetics were compared with the corresponding values read from inhibition curves with the basal effect subtracted by paired Student's t test.

	Results

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4-Aminopyridine-Evoked Release. Stimulation of rat cerebrocortical synaptosomes with 1 mM 4AP in the presence of 1.9 mM free external Ca²⁺ evoked both L-[³H]glutamate and [¹⁴C]GABA release (Figs. 1, 2, 3, 4). Total release (sum FR) was greater for [¹⁴C]GABA release compared with L-[³H]glutamate, as reported previously (Westphalen and Hemmings, 2003a).

View larger version (39K): [in this window] [in a new window]   Fig. 1. Isoflurane effects on evoked release. Isoflurane effects on L-[3H]glutamate (A) and [14C]GABA release (B) evoked by 4AP from rat cortical synaptosomes were fitted to sigmoidal concentration-effect curves. Control data in the presence of Ca2+ (glutamate, n = 12; GABA, n = 13) or absence of Ca2+ (glutamate, n = 9; GABA, n = 11) are presented as mean ± S.D. Basal effects (taken from Westphalen and Hemmings, 2005) were subtracted from evoked release data and fitted to sigmoidal concentration-effect curves (C and D). Shaded area highlights the clinical concentration range (0.5–2x MAC). Symbols in gray present sum FR data (mean ± S.D.) for 2-min pulses of 1 mM 4AP in the presence of Ca2+ plus 0.5x MAC (0.19 ± 0.003 mM, n = 3), 1x MAC (0.34 ± 0.03 mM, n = 4), or 2x MAC (0.76 ± 0.04 mM, n = 7) concentrations of isoflurane applied continuously, which do not differ from data obtained using the standard pulse application with basal effect subtracted (glutamate: P = 0.35, GABA: P = 0.17). Release in the absence of anesthetic presents mean ± S.D. of all assay controls.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Enflurane effects on evoked release. Enflurane effects on L-[3H]glutamate (A) and [14C]GABA (B) evoked by 4AP from rat cortical synaptosomes were fitted to sigmoidal concentration-effect curves. Control data in the presence of Ca2+ (glutamate, n = 20; GABA, n = 18) or absence of Ca2+ (glutamate, n = 8; GABA, n = 8) are presented as mean ± S.D. Basal effects (taken from Westphalen and Hemmings, 2005) were subtracted from evoked release data, and the difference was fitted to sigmoidal concentration-effect curves (C and D). Shaded area highlights the clinical concentration range (0.5–2x MAC). Symbols in gray present sum FR data (mean ± S.D.) for 2-min pulses of 1 mM 4AP in the presence of Ca2+ plus 0.5x MAC (0.39 mM ± 0.007, n = 3), 1x MAC (0.74 ± 0.03 mM, n = 5), or 2x MAC (1.58 ± 0.02 mM, n = 5) concentrations of enflurane applied continuously, which do not differ from data obtained using the standard pulse application with basal effect subtracted (glutamate, P = 0.09; GABA, P = 0.26). Release in the absence of anesthetic presents mean ± S.D. of all assay controls.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Halothane effects on evoked release. Halothane effects on L-[3H]glutamate (A) and [14C]GABA (B) evoked by 4AP from rat cortical synaptosomes were fitted to sigmoidal concentration-effect curves. Control data in the presence of Ca2+ (glutamate, n = 16; GABA, n = 16) or absence of Ca2+ (glutamate, n = 6; GABA, n = 6) are presented as mean ± S.D. Basal effects (taken from Westphalen and Hemmings, 2005) were subtracted from evoked release data and the difference fitted to sigmoidal concentration-effect curves (C and D). Shaded area highlights the clinical concentration range (0.5–2x MAC). Symbols in gray present sum FR data (mean ± S.D.) for 2-min pulses of 1 mM 4AP in the presence of Ca2+ plus 0.5x MAC (0.14 ± 0.005 mM, n = 3), 1x MAC (0.33 ± 0.02 mM, n = 5), or 2x MAC (0.70 ± 0.01 mM, n = 4) concentrations of halothane applied continuously, which do not differ from data obtained using the standard pulse application with basal effect subtracted (glutamate, P = 0.06; GABA, P = 0.27). Release in the absence of anesthetic presents mean ± S.D. of all assay controls.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Tetrodotoxin effects on evoked release. Tetrodotoxin effects on L-[3H]glutamate (A) and [14C]GABA (B) evoked by 4AP from rat cortical synaptosomes were fitted to sigmoidal concentration-effect curves. Control data in the presence of Ca2+ (glutamate, n = 7; GABA, n = 7) or absence of Ca2+ (glutamate, n = 6; GABA, n = 6) are presented as mean ± S.D. Basal effects (taken from Westphalen and Hemmings, 2005) were subtracted from evoked release data and the difference fitted to sigmoidal concentration-effect curves (C and D). Tetrodotoxin did not completely inhibit L-[3H]glutamate or GABA release in the presence of Ca2+, or significantly inhibit Ca2+-independent L-[3H]glutamate or GABA release. Symbols in gray present sum FR data (mean ± S.D.) for 2-min pulses of 1 mM 4AP in the presence of Ca2+ and 10 nM (n = 4), 100 nM (n = 4), or 1 µM (n = 6) tetrodotoxin applied continuously, which do not differ from data obtained using the standard pulse application with basal effect subtracted (glutamate, P = 0.16; GABA, P = 0.10). Release in the absence of anesthetic presents mean ± of all assay controls.

Enflurane. Enflurane inhibited 4AP-evoked release of L-[³H]glutamate and [¹⁴C]GABA in a concentration-dependent manner (Fig. 2, A and B). Maximum inhibition of L-[³H]glutamate release was indeterminable from these data, which trended negative at concentrations >0.8 mM, apparently as a consequence of the inhibitory effect of enflurane on basal glutamate release (Westphalen and Hemmings, 2005). With basal release subtracted, enflurane inhibited both L-[³H]glutamate and [¹⁴C]GABA release by 100% (I_max = 0, P = 0.98 and P = 0.25, respectively; Fig. 2, C and D). The potency of enflurane for inhibition of 4AP-evoked L-[³H]glutamate release was greater than that for [¹⁴C]GABA release (P < 0.0001; Table 1). In the absence of Ca²⁺, enflurane completely inhibited 4AP-evoked release of L-[³H]glutamate and [¹⁴C]GABA (I_max = 0, P = 0.57 and P = 0.12, respectively; Table 1; Fig. 2, C and D). Enflurane inhibited Ca²⁺-independent [¹⁴C]GABA release with greater potency than Ca²⁺-dependent release (P < 0.0001; Table 1). Enflurane inhibited Ca²⁺-dependent and Ca²⁺-independent 4AP-evoked L-[³H]glutamate release at clinically relevant concentrations (IC₅₀ = 0.72 mM; 1.0x MAC) with the same potency (P = 0.66) but with greater potency (P = 0.0002) than Ca²⁺-dependent 4AP-evoked [¹⁴C]GABA release (IC₅₀ = 2.34 mM; 3.1x MAC; Table 1). Enflurane inhibited Ca²⁺-independent 4AP-evoked [¹⁴C]GABA release with greater potency than Ca²⁺-dependent release (P < 0.0001; Table 1).

Halothane. Halothane inhibited 4AP-evoked release of L-[³H]glutamate and [¹⁴C]GABA in a concentration-dependent manner (Fig. 3, A and B). With basal release subtracted, halothane inhibited both L-[³H]glutamate and [¹⁴C]GABA release by 100% (I_max = 0, P = 0.84 and P = 0.78, respectively; Fig. 3, C and D). Halothane inhibited 4AP-evoked L-[³H]glutamate release with greater potency than [¹⁴C]GABA release (P < 0.001; Table 1). In the absence of Ca²⁺, halothane inhibited 4AP-evoked release of L-[³H]glutamate and [¹⁴C]GABA with similar potencies (P = 0.93) and 100% efficacy (I_max = 0, P = 0.88 and P = 0.08, respectively; Table 1; Fig. 3, C and D). Halothane inhibited Ca²⁺-dependent L-[³H]glutamate release at clinically relevant concentrations (IC₅₀ = 0.56 mM; 1.6x MAC) with the same potency as Ca²⁺-independent 4AP-evoked L-[³H]glutamate release (P = 0.085; Table 1). Halothane inhibited Ca²⁺-dependent [¹⁴C]GABA release (IC₅₀ = 0.76 mM; 2.2x MAC) with less potency than Ca²⁺-independent release (P = 0.008; Table 1).

Tetrodotoxin. Tetrodotoxin inhibited 4AP-evoked release of L-[³H]glutamate and [¹⁴C]GABA in a concentration-dependent manner (Fig. 4, A and B). With basal effects subtracted, tetrodotoxin showed incomplete efficacy for inhibition of both 4AP-evoked L-[³H]glutamate release (66% inhibition; I_max = 0, P = 0.0024) and [¹⁴C]GABA release (46% inhibition; I_max = 0, P = 0.0022) in the presence of Ca²⁺ (Fig. 4, C and D). Tetrodotoxin inhibited 4AP-evoked L-[³H]glutamate and [¹⁴C]GABA release with the same potency (P = 0.78; Table 1). The effects of tetrodotoxin on Ca²⁺-independent 4AP-evoked L-[³H]glutamate and [¹⁴C]GABA release were not statistically significant with basal effects subtracted (Fig. 4, C and D). Thus, curve parameters for Ca²⁺-dependent inhibition of evoked release of glutamate and GABA were the same (Table 1). However, inhibition of Ca²⁺-dependent glutamate release was complete (I_max = 0, P = 0.86) but was incomplete for inhibition of Ca²⁺-dependent GABA release (I_max = 0, P = 0.026). This difference in maximal inhibition of Ca²⁺-dependent release by tetrodotoxin between glutamate and GABA was also statistically significant by a comparative curve fit (P = 0.044).

Tetrodotoxin-Insensitive 4-Aminopyridine-Evoked Release. In the presence of Ca²⁺ and 1 µM tetrodotoxin (a concentration that maximally inhibited 4AP-evoked L-[³H]glutamate and [¹⁴C]GABA release; Fig. 4, C and D), the tetrodotoxin-insensitive 4AP-evoked [¹⁴C]GABA release was significantly inhibited by 0.7 mM isoflurane (P < 0.001), 1.8 mM enflurane (P < 0.001), or 0.7 mM halothane (P < 0.001) (Fig. 5). Inhibition of the residual tetrodotoxin-insensitive 4AP-evoked L-[³H]glutamate release was not significant for any anesthetic.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Additivity experiments for the effects of volatile anesthetics plus tetrodotoxin on release. Effects of 2x MAC isoflurane (0.73 ± 0.02 mM, n = 4), enflurane (1.79 ± 0.05 mM, n = 4), or halothane (0.69 ± 0.12 mM, n = 4) on tetrodotoxin (TTX)-insensitive 4AP-evoked release of L-[3H]glutamate and [14C]GABA in the presence of Ca2+ are shown as mean ± S.E.M. TTX (1 µM, n = 8) significantly inhibited 4AP-evoked L-[3H]glutamate and [14C]GABA release versus control (, P < 0.001, n = 8). In the presence of 1 µM TTX, anesthetics significantly inhibited [14C]GABA release (**, P < 0.01), with no significant additive effect on L-[3H]glutamate release.

	Discussion

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Different volatile anesthetic potencies for inhibition of glutamate versus GABA release by volatile anesthetics suggest underlying physiological difference(s) between glutamatergic and GABAergic nerve terminals resulting in distinct transmitter-selective pharmacologies. Voltage-gated Na⁺ channels are important in the generation of neuronal action potentials that trigger exocytosis of synaptic vesicles (Tibbs et al., 1989; Südhof, 2004). Mammalian Na⁺ channels show selective densities within and between neurons (Goldin, 2001; Novakovic et al., 2001), can modulate transmitter release (Engelman and MacDermott, 2004), and are inhibited by volatile anesthetics (see below). Thus, inhibition by volatile anesthetics of evoked neurotransmitter release might occur via Na⁺ channel block. This hypothesis is supported by the present demonstration that 4AP-evoked release is also inhibited by the selective Na⁺ channel blocker tetrodotoxin. Moreover, volatile anesthetics inhibit 4AP-evoked (action potential-evoked) transmitter release with greater potency than release evoked by elevated K⁺ (Schlame and Hemmings, 1995; Ratnakumari et al., 2000; Westphalen and Hemmings, 2003a; Hemmings et al., 2005b), which implicates an anesthetic target upstream of Ca²⁺ entry. Additional support for volatile anesthetic inhibition of mammalian neuronal Na⁺ channels includes anesthetic inhibition of synaptosomal influx of ²²Na⁺ and [³H]batrachotoxinin-A binding to nerve-terminal Na⁺ channels (Ratnakumari and Hemmings, 1998), inhibition of heterogeneously expressed brain Na⁺ channels (Rehberg et al., 1996; Shiraishi and Harris, 2004), and inhibition of Na⁺ currents in isolated rat dorsal root ganglion neurons (Ratnakumari et al., 2000) and isolated neurohypophysial nerve terminals (Ouyang et al., 2003; Ouyang and Hemmings, 2005).

Isoflurane, enflurane, and halothane completely inhibited Ca²⁺-dependent 4AP-evoked glutamate and GABA release with potencies comparable to inhibition of mammalian voltage-gated Na⁺ channels (Rehberg et al., 1996; Ouyang et al., 2003). In contrast, the selective Na⁺ channel blocker tetrodotoxin completely inhibited Ca²⁺-dependent 4AP-evoked glutamate release but only partially inhibited GABA release. This is consistent with a previous study that showed tetrodotoxin-resistant Ca²⁺-dependent 4AP-evoked inhibitory postsynaptic currents in rat hippocampus (Akaike, 2002). Preferential inhibition of autaptic excitatory postsynaptic currents versus inhibitory postsynaptic currents by Na⁺ channel blockers in hippocampal neuron microcultures (Prakriya and Mennerick, 2000) suggests fundamental differences in the distribution and/or function of Na⁺ channels between glutamate and GABA nerve terminals. The potency of tetrodotoxin in inhibiting both glutamate and GABA release corresponds to the tetrodotoxin-sensitive Na⁺ channel subtypes Na_V1.1, 1.2, 1.3, 1.6, and/or 1.7 (Novakovic et al., 2001; Lai et al., 2004). Perhaps the tetrodotoxin-insensitive fraction of 4AP-evoked GABA release involves expression of tetrodotoxin-resistant Na⁺ channel subtypes (e.g., Na_V1.8 and/or 1.9; Novakovic et al., 2001; Lai et al., 2004) in a subset of GABAergic terminals. Variability in depolarization threshold and/or release probability of vesicular transmitter exocytosis between nerve terminals containing the same (Rosenmund et al., 1993; Murthy et al., 1997) and/or different transmitters (this study; Prakriya and Mennerick, 2000; Akaike, 2002) may provide the basis for preferential inhibition of evoked glutamate versus GABA release by volatile anesthetics. Because inhibition of 4AP-evoked glutamate and GABA release by tetrodotoxin was neither totally effective nor transmitter-selective in potency, volatile anesthetic effects on transmitter release that were fully efficacious and transmitter-selective can only partially be attributable to Na⁺ channel inhibition or require the distinct state-dependent Na⁺ channel-blocking effects of volatile anesthetics (Ouyang et al., 2003). The ability of clinical concentrations of volatile anesthetics to inhibit the tetrodotoxin-insensitive component of 4AP-evoked GABA release further supports the involvement of additional mechanism(s). GABAergic synaptic diversity in release probability, number of release sites, presynaptic GABA_A receptor subtypes, modulation by endogenous and exogenous factors (Cherubini and Conti, 2001), and tetrodotoxin-insensitive nicotinic-mediated increases in GABA release (Zhu and Chiappinelli, 2002) suggest possible mechanisms for transmitter-selective anesthetic effects.

Other potential presynaptic targets of volatile anesthetics include K⁺ and Ca²⁺ channels (Topf et al., 2003; Franks and Honoré, 2004), ligand-gated ion channels (Franks and Lieb, 1994), cell signaling machinery, such as protein kinase C (Hemmings and Adamo, 1998) and G-protein coupled receptors (Yamakura et al., 2001), and synaptic vesicle fusion machinery (Hawasli et al., 2004), all of which can modulate transmitter release (Miller, 1998; Engelman and MacDermott, 2004). Genetic inactivation of components of the presynaptic release machinery differentially affect basal and evoked release (SNAP-25; Washbourne et al., 2001) or evoked glutamate and GABA release (Munc 13-1; Augustin et al., 1999), suggesting these proteins as potential targets for the pathway and transmitter-selective effects of volatile anesthetics. Like Na⁺ channels, these putative anesthetic targets may have distinct patterns of expression within and between neurons that may contribute to transmitter-selective effects.

Preferential inhibition of Ca²⁺-independent 4AP-evoked GABA versus glutamate release by isoflurane and enflurane, but not halothane, was in contrast to preferential inhibition of Ca²⁺-dependent glutamate versus GABA release by all three anesthetics tested. This suggests more than one presynaptic site of action for these effects. A potential target for anesthetic effects on Ca²⁺-independent transmitter release is inhibition of reverse transmitter transporter function (Attwell et al., 1993). Inhibition by isoflurane of glutamate uptake into rat cerebrocortical synaptosomes occurs with similar potency (IC₅₀ = 0.7 mM) to inhibition of Ca²⁺-independent release, but inhibition of GABA uptake (IC₅₀ = 0.8 mM) is less potent than inhibition of Ca²⁺-independent release (Westphalen and Hemmings, 2003b). The maximal efficacy of isoflurane inhibition of glutamate and GABA uptake also varies (Westphalen and Hemmings, 2003b), consistent with a greater role of transporter inhibition to glutamate release than GABA release. In view of the insensitivity of GABA uptake to volatile anesthetics, the greater sensitivity of Ca²⁺-independent 4AP-evoked GABA release to isoflurane further suggests that nerve-terminal GABA transporters are not sensitive to volatile anesthetics.

In summary, three volatile anesthetics consistently inhibited Ca²⁺-dependent 4AP-evoked glutamate release with greater potency than GABA release, which supports distinct presynaptic targets between glutamatergic and GABAergic nerve terminals. Although glutamate is released from synaptic vesicles at saturating concentrations for its receptors, even moderate reductions in release probability could affect the kinetics of synaptic glutamate concentration (Cavelier et al., 2005) and the release probability of low probability nerve terminals (Prakriya and Mennerick, 2000) to depress excitatory transmission. The less potent inhibition of GABA release by volatile anesthetics is balanced by postsynaptic potentiation of GABA_A receptors, such that net inhibitory GABAergic transmission is enhanced (Stucke et al., 2002). The correlation between anesthetic potency in vivo (MAC) and potency of evoked glutamate release inhibition supports this action as a relevant volatile anesthetic target. The differing potencies for inhibition of Ca²⁺-dependent and Ca²⁺-independent 4AP-evoked GABA and glutamate release provide evidence for multiple transmitter and pathway-specific presynaptic targets. The biological difference(s) between glutamatergic and GABAergic nerve terminals that underlie this selectivity in volatile anesthetic action remains to be determined. Comparable transmitter-selective inhibition was not produced by tetrodotoxin, indicating that a mechanism(s) in addition to Na⁺ channel block must be involved. Selective depression of evoked glutamate versus GABA release depressed basal glutamate relative to GABA release, and potentiation of postsynaptic GABA_A receptors provide synergistic mechanisms by which volatile anesthetics may depress excitatory and enhance net inhibitory central nervous system transmission.

	Footnotes

 
This work was supported by National Institutes of Health Grant GM 58055 and by Department of Anesthesiology, Weill Medical College, Cornell University.

doi:10.1124/jpet.105.090662.

ABBREVIATIONS: 4AP, 4-aminopyridine; MAC, minimum alveolar concentration; FR, fractional release.

Address correspondence to: Dr. Hugh C. Hemmings, Jr., Department of Anesthesiology, LC-203, Box 50, 1300 York Avenue, New York, NY 10021. E-mail: hchemmi{at}med.cornell.edu

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