Regional differences in the effects of isoflurane on neurotransmitter release.

Stimulus evoked neurotransmitter release requires that Na(+) channel-dependent nerve terminal depolarization be transduced into synaptic vesicle exocytosis. Inhaled anesthetics block presynaptic Na(+) channels and selectively inhibit glutamate over GABA release from isolated nerve terminals, indicating mechanistic differences between excitatory and inhibitory transmitter release. We compared the effects of isoflurane on depolarization-evoked [(3)H]glutamate and [(14)C]GABA release from isolated nerve terminals prepared from four regions of rat CNS evoked by 4-aminopyridine (4AP), veratridine (VTD), or elevated K(+). These mechanistically distinct secretegogues distinguished between Na(+) channel- and/or Ca(2+) channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC(50) = 0.42 ± 0.03 mM) more potently than GABA release (IC(50) = 0.56 ± 0.02 mM) from cerebral cortex (1.3-fold greater potency), hippocampus and striatum, but inhibited glutamate and GABA release from spinal cord terminals equipotently. Na(+) channel-specific VTD-evoked glutamate release from cortex was also significantly more sensitive to inhibition by isoflurane than was GABA release. Na(+) channel-independent K(+)-evoked release was insensitive to isoflurane at clinical concentrations in all four regions, consistent with a target upstream of Ca(2+) entry. Isoflurane inhibited Na(+) channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC(50) = 0.30 ± 0.03 mM) more potently than GABA release (IC(50) = 0.67 ± 0.04 mM) from cortex (2.2-fold greater potency). The magnitude of inhibition of Na(+) channel-mediated 4AP-evoked release by a single clinical concentration of isoflurane (0.35 mM) varied by region and transmitter: Inhibition of glutamate release from spinal cord was greater than from the three brain regions and greater than GABA release for each CNS region. These findings indicate that isoflurane selectively inhibits glutamate release compared to GABA release via Na(+) channel-mediated transduction in the four CNS regions tested, and that differences in presynaptic Na(+) channel involvement determine differences in anesthetic pharmacology.

AGAP1/AP-3-dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release.

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M(5) is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M(5) and the AP-3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP-3 to M(5). Interaction with AGAP1 and activity of AP-3 were required for the endocytic recycling of M(5) in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP-3 or abrogation of AGAP1-M(5) interaction in vivo decreased the magnitude of presynaptic M(5)-mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor-recycling pathway that may underlie mechanisms of G-protein-coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.

Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel-dependent glutamate and GABA release in rat CNS.

We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Na(v)) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Na(v) coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [(3)H]glutamate and [(14)C]GABA release to the specific Na(v) blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Na(v) subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Na(v) subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Na(v) and Na(v)1.2. Nerve terminal-specific differences in the expression of specific Na(v) subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.

Volatile anesthetic effects on glutamate versus GABA release from isolated rat cortical nerve terminals: basal release.

The effects of three volatile anesthetics (isoflurane, enflurane, and halothane) on basal release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes) were compared using a dual isotope superfusion method. Concentration-dependent effects on basal release differed between anesthetics and transmitters. Over a range of clinical concentrations (0.5-2x minimum alveolar concentration), basal glutamate release was inhibited by all three anesthetics, whereas basal GABA release was enhanced (isoflurane) or unaffected (enflurane and halothane). These effects may represent a balance of stimulatory and inhibitory mechanisms between transmitters and anesthetics. There were no significant differences between anesthetic effects on basal release in the absence or presence of external Ca(2+), whereas intracellular Ca(2+) buffering limited volatile anesthetic inhibition of basal glutamate release. Although these results demonstrate fundamental differences in anesthetic effects on basal release between glutamatergic and GABAergic nerve terminals, all three volatile anesthetics at clinical concentrations consistently reduced the ratio of basal glutamate to GABA release. These actions may contribute to the net depression of glutamatergic excitation and potentiation of GABAergic inhibition characteristic of general anesthesia.

Volatile anesthetic effects on glutamate versus GABA release from isolated rat cortical nerve terminals: 4-aminopyridine-evoked release.

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-[(3)H]glutamate and [(14)C]GABA, and release was evoked by superfusion with pulses of 1 mM 4AP in the absence or presence of 1.9 mM free Ca(2+). All three volatile anesthetics inhibited Ca(2+)-dependent glutamate and GABA release; IC(50) values for glutamate were comparable to clinical concentrations (1-1.6x MAC), whereas IC(50) values for GABA release exceeded clinical concentrations (>2.2x MAC). All three volatile anesthetics inhibited both Ca(2+)-independent and Ca(2+)-dependent 4AP-evoked glutamate release equipotently, whereas inhibition of Ca(2+)-dependent 4AP-evoked GABA release was less potent than inhibition of Ca(2+)-independent GABA release. Inhibition of Ca(2+)-independent 4AP-evoked glutamate release was more potent than that of GABA release for isoflurane and enflurane but equipotent for halothane. Tetrodotoxin inhibited both Ca(2+)-independent and Ca(2+)-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 Ca(2+)-dependent 4AP-evoked glutamate release versus GABA release supports the hypothesis that reduced excitatory neurotransmission relative to inhibitory neurotransmission contributes to volatile anesthetic actions.

The general anesthetic isoflurane depresses synaptic vesicle exocytosis.

General anesthetics have marked effects on synaptic transmission, but the mechanisms of their presynaptic actions are unclear. We used quantitative laser-scanning fluorescence microscopy to analyze the effects of the volatile anesthetic isoflurane on synaptic vesicle cycling in cultured neonatal rat hippocampal neurons monitored using either transfection of a pH-sensitive form of green fluorescent protein fused to the luminal domain of VAMP (vesicle-associated membrane protein), (synapto-pHluorin) or vesicle loading with the fluorescent dye FM 1-43. Isoflurane reversibly inhibited action potential-evoked exocytosis over a range of concentrations, with little effect on vesicle pool size. In contrast, exocytosis evoked by depolarization in response to an elevated extracellular concentration of KCl, which is insensitive to the selective Na+ channel blocker tetrodotoxin, was relatively insensitive to isoflurane. Inhibition of exocytosis by isoflurane was resistant to bicuculline, indicating that this presynaptic effect is not caused by the well known GABA(A) receptor modulation by volatile anesthetics. Depression of exocytosis was mimicked by a reduction in stimulus frequency, suggesting a reduction in action potential initiation, conduction, or coupling to Ca2+ channel activation. There was no evidence for a direct effect on endocytosis. The effects of isoflurane on synaptic transmission are thus caused primarily by inhibition of action potential-evoked synaptic vesicle exocytosis at a site upstream of Ca2+ entry and exocytosis, possibly as a result of Na+ channel blockade and/or K+ channel activation, with the possibility of lesser contributions from Ca2+ channel blockade and/or soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated vesicle fusion.

The identification and characterization of excitotoxic nerve-endings in Alzheimer disease.

Regionally specific neuronal loss is a distinguishing feature of Alzheimer disease (AD). Excitotoxicity is a mechanism commonly invoked to explain this. We review the accumulating evidence for such a hypothesis, particularly the altered expression and pharmacology of glutamate receptors and transporters in pathologically susceptible regions of the AD brain. Loss of neurons would be expected to lead to the retrograde degeneration of their afferents, which should be reflected in a loss of presynaptic markers such as synaptophysin. We discuss the possibility that neurons may be destroyed locally, but that glutamatergic presynaptic terminals may remain, or even re-proliferate. The reduced glutamate uptake site density in AD brain may signify a loss of the transporters on otherwise intact terminals, rather than the loss of glutamatergic afferents. Neuronal death may follow if cells are exposed to excessive amounts of glutamate; the loss of transporters from functioning, but defective, glutamate terminals would mean they could continue to release glutamate to exacerbate excitotoxicity. We discuss experimental methods to quantitate synapses, which are crucial for deciding between the various possibilities.

Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals.

The role of presynaptic mechanisms in general anesthetic depression of excitatory glutamatergic neurotransmission and facilitation of GABA-mediated inhibitory neurotransmission is unclear. A dual isotope method allowed simultaneous comparisons of the effects of a representative volatile (isoflurane) and intravenous (propofol) anesthetic on the release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes). Synaptosomes were prelabeled with L-[(3)H]glutamate and [(14)C]GABA, and release was determined by superfusion with pulses of 30 mM K(+) or 1 mM 4-aminopyridine (4AP) in the absence or presence of 1.9 mM free Ca(2+). Isoflurane maximally inhibited Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (99 +/- 8% inhibition) to a greater extent than [(14)C]GABA release (74 +/- 6% inhibition; P = 0.023). Greater inhibition of L-[(3)H]glutamate versus [(14)C]GABA release was also observed for the Na(+) channel antagonists tetrodotoxin (99 +/- 4 versus 63 +/- 5% inhibition; P < 0.001) and riluzole (84 +/- 5 versus 52 +/- 12% inhibition; P = 0.041). Propofol did not differ in its maximum inhibition of Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (76 +/- 12% inhibition) compared with [(14)C]GABA (84 +/- 31% inhibition; P = 0.99) release. Neither isoflurane (1 mM) nor propofol (15 microM) affected K(+)-evoked release, consistent with a molecular target upstream of the synaptic vesicle exocytotic machinery or voltage-gated Ca(2+) channels coupled to transmitter release. These findings support selective presynaptic depression of excitatory versus inhibitory neurotransmission by clinical concentrations of isoflurane, probably as a result of Na(+) channel blockade.

Effects of isoflurane and propofol on glutamate and GABA transporters in isolated cortical nerve terminals.

BACKGROUND: Depression of glutamate-mediated excitatory transmission and potentiation of gamma-aminobutyric acid (GABA)-mediated inhibitory transmission appear to be primary mechanisms by which general anesthetics produce anesthesia. Since effects on transmitter transport have been implicated in anesthetic actions, the authors examined the sensitivity of presynaptic glutamate and GABA transporters to the effects of a representative volatile (isoflurane) and a representative intravenous (propofol) anesthetic. METHODS: A dual-isotope (l-[3H]glutamate and [14C]GABA) approach allowed simultaneous comparisons of anesthetic effects on three independent assays of glutamate and GABA transporters in adult rat cerebral cortex: transmitter uptake into isolated nerve terminals (synaptosomes), transmitter binding to lysed and washed synaptosomes (synaptic membranes), and carrier-mediated release (reverse transport) of transmitter from preloaded synaptosomes using a modified superfusion system. RESULTS: Isoflurane produced small but statistically significant inhibition of l-[3H]glutamate and [14C]GABA uptake, while propofol had no effect. Inhibition of uptake by isoflurane was noncompetitive, an outcome that was mimicked by indirectly affecting transporter function through synaptosomal depolarization. Neither isoflurane nor propofol affected l-[3H]glutamate or [14C]GABA binding to synaptic membranes or Ca(2+)-independent carrier-mediated l-[3H]glutamate or [14C]GABA release (reverse transport). CONCLUSIONS: These findings suggest that isoflurane and propofol at clinical concentrations do not affect excitatory glutamatergic transmission or inhibitory GABAergic transmission directly effects on their presynaptic neuronal transporters.

General anesthetics do not affect release of the neuropeptide cholecystokinin from isolated rat cortical nerve terminals.

BACKGROUND: General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized. METHODS: The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35 degrees C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration. RESULTS: Elevation of extracellular KCl concentration (to 15-30 mM) or veratridine (10-20 microm) stimulated Ca2+ -dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 microm), pentobarbital (50 and 100 microm), thiopental (20 microm), etomidate (20 microm), ketamine (20 microm), isoflurane (0.6-0.8 mM), or halothane (0.6-0.8 mMm). CONCLUSIONS: Clinically relevant concentrations of several classes of general anesthetics did not affect basal, KCl-evoked, or veratridine-evoked CCK8s release from isolated rat cortical nerve terminals. This is in contrast to the demonstrable effects of certain general anesthetics on the release of amino acid and catecholamine transmitters. These transmitter-specific presynaptic effects of general anesthetics suggest that anesthetic-sensitive presynaptic targets are not common to all transmitter classes.