Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.

Although previously considered entirely reversible, general anaesthesia is now being viewed as a potentially significant risk to cognitive performance at both extremes of age. A large body of preclinical as well as some retrospective clinical evidence suggest that exposure to general anaesthesia could be detrimental to cognitive development in young subjects, and might also contribute to accelerated cognitive decline in the elderly. A group of experts in anaesthetic neuropharmacology and neurotoxicity convened in Salzburg, Austria for the BJA Salzburg Seminar on Anaesthetic Neurotoxicity and Neuroplasticity. This focused workshop was sponsored by the British Journal of Anaesthesia to review and critically assess currently available evidence from animal and human studies, and to consider the direction of future research. It was concluded that mounting evidence from preclinical studies reveals general anaesthetics to be powerful modulators of neuronal development and function, which could contribute to detrimental behavioural outcomes. However, definitive clinical data remain elusive. Since general anaesthesia often cannot be avoided regardless of patient age, it is important to understand the complex mechanisms and effects involved in anaesthesia-induced neurotoxicity, and to develop strategies for avoiding or limiting potential brain injury through evidence-based approaches.

Potentiation of GABAA receptor activity by volatile anaesthetics is reduced by α5GABAA receptor-preferring inverse agonists.

BACKGROUND: Animal studies have shown that memory deficits in the early post-anaesthetic period can be prevented by pre-treatment with an inverse agonist that preferentially inhibits α5 subunit-containing γ-aminobutyric acid type A (α5GABA(A)) receptors. The goal of this in vitro study was to determine whether inverse agonists that inhibit α5GABA(A) receptors reduce anaesthetic potentiation of GABAA receptor activity. METHODS: Cultures of hippocampal neurones were prepared from Swiss white mice, wild-type mice (genetic background C57BL/6J and Sv129Ev) and α5GABA(A)receptor null mutant (Gabra5-/-) mice. Whole-cell voltage clamp techniques were used to study the effects of the α5GABA(A) receptor-preferring inverse agonists L-655,708 and MRK-016 on anaesthetic potentiation of GABA-evoked currents. RESULTS: L-655,708 (50 nM) reduced sevoflurane potentiation of GABA-evoked current in wild-type neurones but not Gabra5-/- neurones, and produced a rightward shift in the sevoflurane concentration-response plot [sevoflurane EC50: 1.9 (0.1) mM; sevoflurane+L-655,708 EC(50): 2.4 (0.2) mM, P<0.05]. Similarly, L-655,708 (50 nM) reduced isoflurane potentiation of GABA-evoked current [isoflurane: 4.0 (0.6) pA pF(-1); isoflurane+L-655,708: 3.1 (0.5) pA pF(-1), P<0.01]. MRK-016 also reduced sevoflurane and isoflurane enhancement of GABA-evoked current [sevoflurane: 1.5 (0.1) pA pF(-1); sevoflurane+MRK-016 (10 nM): 1.2 (0.1) pA pF(-1), P<0.05; isoflurane: 3.5 (0.3) pA pF(-1); isoflurane+MRK-016 (1 nM): 2.9 (0.2) pA pF(-1), P<0.05]. CONCLUSIONS: L-655,708 and MRK-016 reduced the potentiation by inhaled anaesthetics of GABAA receptor activated by a low concentration of GABA. Future studies are required to determine whether this effect contributes to the memory preserving properties of inverse agonists after anaesthesia.

Modulation of NMDA receptors by pituitary adenylate cyclase activating peptide in CA1 neurons requires G alpha q, protein kinase C, and activation of Src.

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Galphas-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Galphaq subunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nM) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Galphaq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Galphaq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C beta1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase beta [CAKbeta or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.

Modulation of AMPA receptors by a novel organic nitrate.

Positive modulators of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) channels reduce desensitization and alter their gating kinetics. We have discovered a novel compound nitric oxide-mimetic that similarly modulates the AMPA receptor by reducing desensitization. This, designated GT-005, belongs to the organic nitrate family that includes the nitrovasodilator nitroglycerine. In acutely isolated hippocampal neurons, GT-005 enhanced kainate (100 microM)-evoked currents with an EC50 of 1.7+/-0.2 mM and a 176+/-10% maximal increase in the steady-state current response. Similar results were found in cultured hippocampal neurons (EC50 of 1.3+/-0.2 mM and a maximal 83+/-14% increase in the steady-state current response). GT-005 reduced the desensitization of glutamate-evoked currents and slowed the onset of desensitization. This compound also increased the rate of recovery from the desensitized state. With respect to alteration of the excitatory synaptic transmission, GT-005 delayed the decay and increased the frequency of spontaneous miniature excitatory postsynaptic currents (mepsc) recorded in cultured hippocampal neurons.

Blockade of AMPA receptors and volatile anesthetics: reduced anesthetic requirements in GluR2 null mutant mice for loss of the righting reflex and antinociception but not minimum alveolar concentration.

BACKGROUND: The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor mediates fast excitatory neurotransmission in the central nervous system. Many general anesthetics inhibit AMPA receptors in vitro; however, it is not certain if this inhibition contributes to the behavioral properties of these drugs. AMPA receptors lacking the GluR2 subunit are resistant to blockade by barbiturates in vitro. Paradoxically, GluR2 null mutant (-/-) mice are more sensitive to barbiturate-induced loss of the righting reflex (LORR) compared with wild-type (+/+) littermates. To determine if interactions between anesthetics and AMPA receptors account for the increased sensitivity of (-/-) mice, the effects of volatile anesthetics that do not directly inhibit AMPA receptors were examined. METHODS: Isoflurane, halothane, desflurane, or sevoflurane were administered to (-/-) and (+/+) littermate controls. Anesthetic requirements for LORR, movement to tail clamp (minimum alveolar concentration [MAC]), and hind-paw withdrawal latency (HPWL) were determined. Electrophysiologic methods examined the inhibition of AMPA receptors by isoflurane and halothane. RESULTS: Anesthetic requirements for LORR and HPWL were decreased, whereas MAC values were unchanged in (-/-) mice. Isoflurane and halothane caused minimal inhibition of AMPA receptors at clinically relevant concentrations. CONCLUSIONS: Direct blockade of AMPA receptors did not account for the increased sensitivity to volatile anesthetics in GluR2 null mutant mice for HPWL or LORR. Thus, the deficiency of GluR2-containing AMPA receptors increases the sensitivity of neuronal circuitry mediating these end points, but not MAC. GluR2-containing receptors do not contribute appreciably to MAC in this mouse model. These results illustrate the difficulties in attributing behavioral responses to drug-receptor interactions in genetically engineered animals.

Positive allosteric modulators of AMPA receptors reduce proton-induced receptor desensitization in rat hippocampal neurons.

Whole-cell or outside-out patch recordings were used to investigate the effects of protons and positive modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on the desensitization of glutamate-evoked AMPA receptor currents in isolated hippocampal CA1 neurons. Protons inhibited glutamate-evoked currents (IC(50) of 6.2 pH units) but also enhanced the apparent rate and extent of AMPA receptor desensitization. The proton-induced enhancement of desensitization could not be attributed to a reduction in the rate of recovery from desensitization or to a change in the kinetics of deactivation. Non-stationary variance analysis indicated that protons reduced maximum open probability without changing the conductance of AMPA channels. The positive modulators of AMPA receptor desensitization, cyclothiazide and GT-21-005 (an organic nitrate), reduced the proton sensitivity of AMPA receptor desensitization, which suggests that they interact with protons to diminish desensitization. In contrast, the effects of wheat germ agglutinin and aniracetam on AMPA receptor desensitization were independent of pH. These results demonstrate that a reduction in the proton sensitivity of receptor desensitization contributes to the mechanism of action of some positive modulators of AMPA receptors.

The influence of glutamate receptor 2 expression on excitotoxicity in Glur2 null mutant mice.

AMPA receptor (AMPAR)-mediated ionic currents that govern gene expression, synaptic strength, and plasticity also can trigger excitotoxicity. However, native AMPARs exhibit heterogeneous pharmacological, biochemical, and ionic permeability characteristics, which are governed partly by receptor subunit composition. Consequently, the mechanisms governing AMPAR-mediated excitotoxicity have been difficult to elucidate. The GluR2 subunit is of particular interest because it influences AMPAR pharmacology, Ca(2+) permeability, and AMPAR interactions with intracellular proteins. In this paper we used mutant mice lacking the AMPAR subunit GluR2 to study AMPAR-mediated excitotoxicity in cultured cortical neurons and in hippocampal neurons in vivo. We examined the hypothesis that in these mice the level of GluR2 expression governs the vulnerability of neurons to excitotoxicity and further examined the ionic mechanisms that are involved. In cortical neuronal cultures AMPAR-mediated neurotoxicity paralleled the magnitude of kainate-evoked AMPAR-mediated currents, which were increased in neurons lacking GluR2. Ca(2+) permeability, although elevated in GluR2-deficient neurons, did not correlate with excitotoxicity. However, toxicity was reduced by removal of extracellular Na(+), the main charge carrier of AMPAR-mediated currents. In vivo, the vulnerability of CA1 hippocampal neurons to stereotactic kainate injections and of CA3 neurons to intraperitoneal kainate administration was independent of GluR2 level. Neurons lacking the GluR2 subunit did not demonstrate compensatory changes in the distribution, expression, or function of AMPARs or of Ca(2+)-buffering proteins. Thus GluR2 level may influence excitotoxicity by effects additional to those on Ca(2+) permeability, such as effects on agonist potency, ionic currents, and synaptic reorganization.

Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons.

gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABA(A) receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive GABA(A) receptor antagonists, bicuculline and picrotoxin, blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but not the tonic current were inhibited by gabazine (SR-95531). Coapplication experiments and computer simulations revealed that gabazine bound to the receptors responsible for the tonic current but did not prevent channel activation. However, gabazine competitively inhibited bicuculline blockade. The unitary conductance of the GABA(A) receptors underlying the tonic current (approximately 6 pS) was less than the main conductance of channels activated during quantal synaptic transmission (approximately 15--30 pS). Furthermore, compounds that potentiate GABA(A) receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinically-relevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs.

Medication errors in anesthetic practice: a survey of 687 practitioners.

PURPOSE: The objectives of this study were to determine: 1) if anesthesiologists had experienced a medication error and 2) to identify causal factors. The perceived value of a Canadian reporting agency for medication errors and improved standards for labels on drug ampoules was also investigated. METHODS: A self-reporting survey was mailed to members of the Canadian Anesthesiologists' Society (n = 2,266). Respondents provided free-text descriptions of medication errors and answered fixed response questions. RESULTS: Surveys from 687 anesthesiologists (30% response rate) revealed that 85% of the participants had experienced at least one drug error or "near miss". Although most errors (1,038) were of minor consequence (98%), four deaths were reported. The commonest error involved the administration of muscle relaxants instead of a reversal agent. "Syringe swaps" (70.4%) and the misidentification of the label (46.8%) were common contributing factors. Anesthesiologists (97.9%) reported that they read the ampoule label "most of the time" although the label colour was an important secondary cue. Approximately half of the participants would report the error if a reporting program existed and 84% agreed that improved standards for drug labels would reduce the incidence of error. CONCLUSIONS: Most anesthesiologists experienced at least one drug error. The commonest error was a "syringe swap" that involved a muscle relaxant. Most errors were of minor consequence, however, serious morbidity and mortality resulted from clearly preventable events. These results support the development of improved standards for drug labels and the establishment of a Canadian reporting program for medication errors.

In CA1 pyramidal neurons of the hippocampus protein kinase C regulates calcium-dependent inactivation of NMDA receptors.

The NMDA subtype of the glutamate-gated channel exhibits a high permeability to Ca(2+). The influx of Ca(2+) through NMDA channels is limited by a rapid and Ca(2+)/calmodulin (CaM)-dependent inactivation that results from a competitive displacement of cytoskeleton-binding proteins from the NR1 subunit of the receptor by Ca(2+)/CaM (Zhang et al., 1998; Krupp et al., 1999). The C terminal of this subunit can be phosphorylated by protein kinase C (PKC) (Tingley et al., 1993). The present study sought to investigate whether PKC regulates Ca(2+)-dependent inactivation of the NMDA channel in hippocampal neurons. Activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate enhanced peak (I(p)) and depressed steady-state (I(ss)) NMDA-evoked currents, resulting in a reduction in the ratio of these currents (I(ss)/I(p)). We demonstrated previously that PKC activity enhances I(P) via a sequential activation of the focal adhesion kinase cell adhesion kinase beta/proline-rich tyrosine kinase 2 (CAKbeta/Pyk2) and the nonreceptor tyrosine kinase Src (Huang et al., 1999; Lu et al., 1999). Here, we report that the PKC-induced depression of I(ss) is unrelated to the PKC/CAKbeta/Src-signaling pathway but depends on the concentration of extracellular Ca(2+). Intracellular applications of CaM reduced I(ss)/I(p) and occluded the Ca(2+)-dependent effect of phorbol esters on I(ss.) Moreover, increasing the concentration of intracellular Ca(2+) buffer or intracellular application of the inhibitory CaM-binding peptide (KY9) greatly reduced the phorbol ester-induced depression of I(ss). Taken together, these results suggest that PKC enhances Ca(2+)/CaM-dependent inactivation of the NMDA channel, most likely because of a phosphorylation-dependent regulation of interactions between receptor subunits, CaM, and other postsynaptic density proteins.

Cyclic GMP-dependent feedback inhibition of AMPA receptors is independent of PKG.

In central neurons, the second messenger cGMP is believed to induce long-term changes in efficacy at glutamatergic synapses through activation of protein kinase G (PKG). Stimulating nitric oxide synthase, activating soluble guanylyl cyclase or elevating concentrations of intracellular cGMP depressed excitatory synaptic transmission in CA1 hippocampal neurons. Unexpectedly, intracellular cGMP depressed responses of AMPA receptors and inhibited excitatory postsynaptic currents in hippocampal neurons independently of phosphorylation. Our findings demonstrate that cGMP's modulation of excitatory transmission may involve a coupling of AMPA channel activity to levels of cGMP.

The general anesthetic propofol slows deactivation and desensitization of GABA(A) receptors.

Propofol (2,6-di-isopropylphenol) has multiple actions on GABA(A) receptor function that act in concert to potentiate GABA-evoked currents. To understand how propofol influences inhibitory IPSCs, we examined the effects of propofol on responses to brief applications of saturating concentrations of GABA (1-30 mM). GABA was applied using a fast perfusion system to nucleated patches excised from hippocampal neurons. In this preparation, propofol (10 microM) had no detectable agonist effect but slowed the decay, increased the charge transfer (62%), and enhanced the peak amplitude (8%) of currents induced by brief pulses (3 msec) of GABA. Longer pulses (500 msec) of GABA induced responses that desensitized with fast (tau(f) = 1.5-4.5 msec) and slow (tau(s) = 1-3 sec) components and, after the removal of GABA, deactivated exponentially (tau(d) = 151 msec). Propofol prolonged this deactivation (tau(d) = 255 msec) and reduced the development of both fast and slow desensitization. Recovery from fast desensitization, assessed using pairs of brief pulses of GABA, paralleled the time course of deactivation, indicating that fast desensitization traps GABA on the receptor. With repetitive applications of pulses of GABA (0.33 Hz), the charge transfer per pulse declined exponentially (tau approximately 15 sec) to a steady-state value equal to approximately 40% of the initial response. Despite the increased charge transfer per pulse with propofol, the time course of the decline was unchanged. These experimental data were interpreted using computer simulations and a kinetic model that assumed fast and slow desensitization, as well as channel opening developed in parallel from a pre-open state. Our results suggest that propofol stabilizes the doubly liganded pre-open state without affecting the isomerization rate constants to and from the open state. Also, the rate constants for agonist dissociation and entry into the fast and slow desensitization states were reduced by propofol. The recovery rate constant from fast desensitization was slowed, whereas that from slow desensitization appeared to be unchanged. Taken together, the effects of propofol on GABA(A) receptors enhance channel opening, particularly under conditions that promote desensitization.

Blockade of glutamate receptors and barbiturate anesthesia: increased sensitivity to pentobarbital-induced anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice.

BACKGROUND: Barbiturates enhance gamma-aminobutyric acid type A (GABA(A)) receptor function and also inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates. METHODS: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABA(A) receptors. RESULTS: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 microM), thiopental (IC50, 153 vs. 34 microM), and phenobarbital (IC50, 930 vs. 205 microM) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABA(A) receptors by pentobarbital. CONCLUSIONS: The GluR2 null mutant mice were more sensitive to pentobarbital anesthesia despite a reduced sensitivity of GluR2-deficient AMPA receptors to barbiturate blockade. Our results indicate that the inhibition of AMPA receptors does not correlate with the anesthetic effects of barbiturates in this animal model. We postulate that the increase in the sensitivity to anesthetics results from a global suppression of excitatory neurotransmission in GluR2-deficient mice.

Platelet-derived growth factor receptor-induced feed-forward inhibition of excitatory transmission between hippocampal pyramidal neurons.

Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity of N-methyl-D-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-gamma, and by blocking the intracellular release of Ca(2+). Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca(2+)- and PKA-dependent inhibition of their modulation by c-Src.