General anesthetic actions<i>in vivo</i>strongly attenuated by a point mutation in the GABA<sub>A</sub>receptor β3 subunit

Jurd, Rachel; Arras, Margarete; Lambert, Sachar; Drexler, Berthold; Siegwart, Roland; Crestani, Florence; Zaugg, Michael; Vogt, Kaspar E.; Ledermann, Birgit; Antkowiak, Bernd; Rudolph, Uwe

doi:10.1096/fj.02-0611fje

Cited by 533 publications

(540 citation statements)

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“…Potentiation of GABA A receptors represents the major action of most i.v. anesthetics (40,41), but this mechanism alone does not account for the lesser target selectivity of volatile anesthetics compared with i.v. anesthetics (42,43).…”

Section: Discussionmentioning

confidence: 99%

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Baumgart

Zhou

Hara

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

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Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca 2+ influx without significantly altering the Ca 2+ sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca 2+ ] i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca 2+ ([Ca 2+ ] e ). Lowering external Ca 2+ to match the isoflurane-induced reduction in Ca 2+ entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca 2+ entry without significant direct effects on Ca 2+ -exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca 2+ influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. GCaMP3 | pHlourin | mechanisms of anesthesia | live cell imaging | presynaptic T he molecular and cellular mechanisms of anesthetic-induced amnesia, unconsciousness and immobilization are incompletely understood, particularly for the modern halogenated ether derivatives like isoflurane. General anesthetics, which are essential to both medical practice and experimental neuroscience, have potent and selective effects on neurotransmission (1), including both presynaptic actions (reduced neurotransmitter release) and postsynaptic actions (modulation of receptor function). These effects contribute to anesthetic-induced reductions in neuronal interactions, which are critical to information processing and consciousness (2-4). Knowledge of the fundamental synaptic effects of anesthetics is therefore essential to a molecular and physiological understanding of anesthetic mechanisms, and to development of more selective and safer anesthetics.Although postsynaptic electrophysiological effects of anesthetics can be assessed directly using whole cell recordings and heterologous expression of putative molecular targets, their presynaptic actions have been difficult to resolve by conventional approaches that do not clearly discriminate between presynaptic and postsynaptic contributions. Direct evidence for presynaptic effects of volatile anesthetics includes selective inhibition of glutamate release from isolated nerve terminals (5, 6) and of synaptic vesicle (SV) exocytosis in intact hippocampal neurons (7). However, it remains controversial whether these effects involve direct inhibition of SV exocytosis itself or of upstrea...

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Section: Discussionmentioning

confidence: 99%

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Baumgart

Zhou

Hara

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

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“…The resulting 'knock-in' mice show specific behavioral insensitivities in response to diazepam 13 , implying that important clinical properties of diazepam such as sedation 44,45 , amnesia 44 and anxiolysis 46 are attributable to GABARs containing particular α subunits. A similar strategy involving mice carrying a point mutation in β3 shows that this subunit is necessary for the anesthetic actions of etomidate 47 . The naturally occurring α6-R100Q mutation (polymorphism) in rats allowed us to examine the roles of α6 subunits and of granule cells in ethanol-induced impairment of cerebellar function.…”

Section: Gabra6 100q Allele and Behavioral Sensitivity To Ethanolmentioning

confidence: 99%

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

et al. 2005

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Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABA A receptor (GABAR), α6β3δ, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in α6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of α6β3δ GABARs. Behavioral and electrophysiological comparisons of Gabra6 100R/100R and Gabra6 100Q/100Q rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.Humans have been consuming alcohol for thousands of years, and the use of alcoholic beverages pervades human culture and society and can have substantial health effects 1 . Different mechanisms by which ethanol might depress brain function have been proposed based on ethanol's ability to modulate a wide variety of ion channels 2-4 , neurotransmitter receptors 5-10 and transporters 11 . Among these diverse targets, however, GABARs are arguably the most attractive candidates. This is in part because other classes of known GABAR modulators such as benzodiazepines, barbiturates and certain anesthetics lead to behavioral effects that closely resemble ethanol intoxication. Yet despite strong evidence implicating GABARs in ethanol's action, critical details remain unclear. For instance, although it is known that native GABARs are heteropentamers assembled from 19 possible subunits 12,13 , it has not been possible to link the activity of particular GABAR subunits to changes in behavioral sensitivity to ethanol.Recent studies suggest that specific combinations of GABAR subunits (those containing α4β3δ and α6β3δ) are uniquely sensitive to ethanol, showing dose dependencies that mirror blood alcohol levels associated with intoxication in humans 9,10 . GABARs containing α4 and δ subunits are expressed in many brain regions 14,15 , but α6 is found in only two types of neurons (cerebellar granule cells and granule cells in the cochlear nucleus) and is expressed together with δ only in cerebellar granule cells 14,16,17 . In granule cells α6 and δ combine with β subunits Correspondence should be addressed to M.W. (mwallner@mednet.ucla.edu) or T.S.O. (otist@ucla.edu). 3 These authors contributed equally to this work. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. We set out to examine whether such extrasynaptic GABARs containing α6 and δ subunits account for behavioral effects of ethanol at moderately intoxicating doses. To link these particular GABARs to behavioral sensitivity, we first characterized a naturally occurring single-nucleotide polymo...

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“…Two recent reports using 'knock-in' mouse lines have demonstrated that the clinical effects of the anaesthetic agents etomidate and propofol can be attributed to either the b2 (sedativehypnotic effect) or the b3 (anaesthetic effect) subunit (Jurd et al, 2003;Reynolds et al, 2003). In the absence of b1 knockout mice, it was hoped that having identified a b1-selective compound we would be able to perform in vivo experiments in order to understand the role of b1-containing GABA A receptors.…”

Section: Experiments To Probe the Binding Site And Role Of B1 Subunitsmentioning

confidence: 99%

Salicylidene salicylhydrazide, a selective inhibitor ofβ1‐containing GABA_Areceptors

Thompson

Wheat

Brown

et al. 2004

British J Pharmacology

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1 A high-throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of a2b1g1y GABA A receptors with a maximum inhibition of 5675% and an IC 50 of 32 (23, 45) nM. 2 Evaluation of this compound using patch-clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the b1 subunit (e.g. maximum inhibition of 68.172.7% and IC 50 value of 5.3 (4.4, 6.5) nM on a2b1g1 receptors). The presence of a b1 subunit was paramount for the inhibition with changes between a1 and a2, g1 and g2, and the presence of a y subunit having little effect. 3 On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at a1b1g1y receptors (74.371.4%). SCS displayed no use or voltage dependence, suggesting that it does not bind within the channel region. Concentration -response curves to GABA in the presence of SCS revealed a reduction in the maximum response with no change in the EC 50 or Hill coefficient. In addition, SCS inhibited pentobarbitone-induced currents. 4 Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS. 5 SCS did not compete with the known allosteric modulators, picrotoxin, pregnenolone sulphate, dehydroepiandrosterone 3-sulphate, bicuculline, loreclezole or mefenamic acid. Neither was the inhibition by SCS influenced by the benzodiazepine site antagonist flumazenil. 6 In conclusion, SCS is unique in selectively inhibiting GABA A receptors containing the b1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the b1 subunit and the lack of interaction with a range of GABA A receptor modulators suggests that SCS is interacting at a previously unidentified site.

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General anesthetic actionsin vivostrongly attenuated by a point mutation in the GABA_Areceptor β3 subunit

Cited by 533 publications

References 50 publications

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

Salicylidene salicylhydrazide, a selective inhibitor ofβ1‐containing GABA_Areceptors

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General anesthetic actionsin vivostrongly attenuated by a point mutation in the GABAAreceptor β3 subunit

Cited by 533 publications

References 50 publications

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca 2+ influx, not Ca 2+ -exocytosis coupling

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca 2+ influx, not Ca 2+ -exocytosis coupling

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

Salicylidene salicylhydrazide, a selective inhibitor ofβ1‐containing GABAAreceptors

Contact Info

Product

Resources

About

General anesthetic actionsin vivostrongly attenuated by a point mutation in the GABA_Areceptor β3 subunit

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Salicylidene salicylhydrazide, a selective inhibitor ofβ1‐containing GABA_Areceptors