Robert I. Westphalen

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

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M5 is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M5 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 M5. Interaction with AGAP1 and activity of AP‐3 were required for the endocytic recycling of M5 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–M5 interaction in vivo decreased the magnitude of presynaptic M5‐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.

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 &ggr;-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 Ca2+-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 via effects on their presynaptic neuronal transporters.

Reduced inhibition of cortical glutamate and GABA release by halothane in mice lacking the K+ channel, TREK-1

Deletion of TREK‐1, a two‐pore domain K+ channel (K2P) activated by volatile anaesthetics, reduces volatile anaesthetic potency in mice, consistent with a role for TREK‐1 as an anaesthetic target. We used TREK‐1 knockout mice to examine the presynaptic function of TREK‐1 in transmitter release and its role in the selective inhibition of glutamate vs GABA release by volatile anaesthetics.

The anxiety-like phenotype of 5-HT1A receptor null mice is associated with genetic background-specific perturbations in the prefrontal cortex GABA–glutamate system

A deficit in the serotonin 5‐HT1A receptor has been found in panic and post‐traumatic stress disorders, and genetic inactivation of the receptor results in an anxiety‐like phenotype in mice on both the C57Bl6 and Swiss–Webster genetic backgrounds. Anxiety is associated with increased neuronal activity in the prefrontal cortex and here we describe changes in glutamate and GABA uptake of C57Bl6 receptor null mice. Although these alterations were not present in Swiss–Webster null mice, we have previously reported reductions in GABAA receptor expression in these but not in C57Bl6 null mice. This demonstrates that inactivation of the 5‐HT1A receptor elicits different and genetic background‐dependent perturbations in the prefrontal cortex GABA/glutamate system. These perturbations can result in a change in the balance between excitation and inhibition, and indeed both C57Bl6 and Swiss–Webster null mice show signs of increased neuronal excitability. Because neuronal activity in the prefrontal cortex controls the extent of response to anxiogenic stimuli, the genetic background‐specific perturbations in glutamate and GABA neurotransmission in C57Bl6 and Swiss–Webster 5‐HT1A receptor null mice may contribute to their shared anxiety phenotype. Our study shows that multiple strains of genetically altered mice could help us to understand the common and individual features of anxiety.

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.

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

J. Neurochem. (2010) 113, 1611–1620.

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°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 &mgr;m) stimulated Ca2+-dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5–50 &mgr;m), pentobarbital (50 and 100 &mgr;m), thiopental (20 &mgr;m), etomidate (20 &mgr;m), ketamine (20 &mgr;m), isoflurane (0.6–0.8 mm), or halothane (0.6–0.8 mm). 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.

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

J. Neurochem. (2010) 113, 1611–1620.