Amnesic concentrations of the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, 2N) and isoflurane alter hippocampal theta oscillations in vivo

BACKGROUND: Drug-induced temporary amnesia is one of the principal goals of general anesthesia. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, also termed 2N) impairs hippocampus-dependent learning at relative, i.e., lipophilicity-corrected, concentrations similar to isoflurane. Hippocampal theta oscillations facilitate mnemonic processes in vivo and synaptic plasticity (a cellular model of memory) in vitro and are thought to represent a circuit level phenomenon that supports memory encoding. Therefore, the authors investigated the effects of F6 and isoflurane on theta oscillations (4-12 Hz). METHODS: Thirteen adult rats were implanted with multichannel depth electrodes to measure the microelectroencephalogram and were exposed to a range of concentrations of isoflurane and F6 spanning the concentrations that produce amnesia. Five of these animals also underwent control experiments without drug injection. The authors recorded the behavioral state and hippocampal field potentials. They confirmed the electrode location postmortem by histology. RESULTS: The tested concentrations for isoflurane and F6 ranged from 0.035% to 0.77% and from 0.5% to 3.6%, respectively. Isoflurane increased the fraction of time that the animals remained immobile, consistent with sedation, whereas F6 had the opposite effect. Electroencephalographic power in the theta band was less when the animals were immobile than when they explored their environment. F6 suppressed the power of oscillations in the theta band. Isoflurane slowed theta oscillations without reducing total power in the theta band. CONCLUSIONS: Drug-induced changes in theta oscillations may be a common basis for amnesia produced by F6 and isoflurane. The different patterns suggest that these drugs alter network activity by acting on different molecular and/or cellular targets.     

Isoflurane causes anterograde but not retrograde amnesia for pavlovian fear conditioning

BACKGROUND: Production of retrograde amnesia by anesthetics would indicate that these drugs can disrupt mechanisms that stabilize memory. Such disruption would allow suppression of memory of previous untoward events. The authors examined whether isoflurane provides retrograde amnesia for classic (Pavlovian) fear conditioning. METHODS: Rats were trained to fear tone by applying three (three-trial) or one (one-trial) tone-shock pairs while breathing various constant concentrations of isoflurane. Immediately after training, isoflurane administration was either discontinued, maintained unchanged, or rapidly increased to 1.0 minimum alveolar concentration for 1 h longer. Groups of rats were similarly trained to fear context while breathing isoflurane by applying shocks (without tones) in a distinctive environment. The next day, memory for the conditioned stimuli was determined by presenting the tone or context (without shock) and measuring the proportion of time each rat froze (appeared immobile). For each conditioning procedure, the effects of the three posttraining isoflurane treatments were compared. RESULTS: Rapid increases in posttraining isoflurane administration did not suppress conditioned fear for any of the training procedures. In contrast, isoflurane administration during conditioning dose-dependently suppressed conditioning (P < 0.05). Training to tone was more resistant to the effects of isoflurane than training to context (P < 0.05), and the three-trial learning procedure was more was more resistant than the one-trial procedure (P < 0.05). CONCLUSIONS: Isoflurane provided intense dose-dependent anterograde but not retrograde amnesia for classic fear conditioning. Isoflurane appears to disrupt memory processes that occur at or within a few minutes of the conditioning procedure.     

Isoflurane Hyperalgesia Is Modulated by Nicotinic Inhibition

Background: The inhaled anesthetic isoflurane inhibits neuronal nicotinic acetylcholine receptors (nAChRs) at concentrations lower than those used for anesthesia. Isoflurane produces biphasic nociceptive responses, with both hyperalgesia and analgesia within this concentration range. Because nicotinic agonists act as analgesics, the authors hypothesized that inhibition of nicotinic transmission by isoflurane causes hyperalgesia.

Methods: The authors studied female mice at 6-8 weeks of age. They measured hind paw withdrawal latency at isoflurane concentrations from 0 to 0.98 vol% after the animals had received a nicotinic agonist (nicotine), a nicotinic antagonist (mecamylamine or chlorisondamine), or saline intraperitoneally. In addition, the authors tested the interactions between mecamylamine and isoflurane and nicotine and isoflurane in heterologously expressed [alpha]4[beta]2 nAChRs.

Results: Female mice had significant hyperalgesia from isoflurane. Nicotine administration prevented isoflurane-induced hyperalgesia without altering the antinociception produced by higher isoflurane concentrations. Mecamylamine treatment caused a biphasic nociceptive response similar to that caused by isoflurane. Mecamylamine and isoflurane had an additive effect, both at heterologously expressed [alpha]4[beta]2 nAChRs and on the production of hyperalgesia in vivo. Mecamylamine thus potentiated hyperalgesia but did not affect analgesia.     

Isoflurane Modulation of Neuronal Nicotinic Acetylcholine Receptors Expressed in Human Embryonic Kidney Cells

Background: It is well established that neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to inhalational anesthetics. The authors previously reported that halothane potently blocked [alpha]4[beta]2-type nAChRs of rat cortical neurons. However, the effect of isoflurane, which is widely used clinically, on nAChRs largely remains to be seen. The authors studied the effects of isoflurane as compared with sevoflurane and halothane on the human [alpha]4[beta]2 nAChRs expressed in human embryonic kidney cells.

Methods: The whole-cell and single-channel patch clamp techniques were used to record currents induced by acetylcholine.

Results: Isoflurane, sevoflurane, and halothane suppressed the acetylcholine-induced currents in a concentration-dependent manner with 50% inhibitory concentrations of 67.1, 183.3, and 39.8 [mu]m, respectively, which correspond to 0.5 minimum alveolar concentration or less. When anesthetics were coapplied with acetylcholine, isoflurane and sevoflurane decreased the apparent affinity of receptor for acetylcholine, but halothane, in addition, decreased the maximum acetylcholine current. When isoflurane was preapplied and coapplied, its inhibitory action was independent of acetylcholine concentration. Isoflurane blocked the nAChR in both resting and activated states. Single-channel analyses revealed that isoflurane at 84 [mu]m decreased the mean open time and burst duration without inducing "flickering" during channel openings. Isoflurane increased the mean closed time. As a result, the open probability of single channels was greatly reduced by isoflurane.     

Isoflurane Action in the Spinal Cord Blunts Electroencephalographic and Thalamic-Reticular Formation Responses to Noxious Stimulation in Goats

Background: Isoflurane depresses the electroencephalographic (EEG) activity and exerts part of its anesthetic effect in the spinal cord. The authors hypothesized that isoflurane would indirectly depress the EEG and subcortical response to noxious stimulation in part by a spinal cord action.

Methods: Depth electrodes were inserted into the midbrain reticular formation (MRF) and thalamus of six of seven isoflurane-anesthetized goats, and needle-electrodes were placed into the skull periosteum. In five of seven goats, an MRF microelectrode recorded single-unit activity. The jugular veins and carotid arteries were isolated to permit cranial bypass and differential isoflurane delivery. A noxious mechanical stimulus (1 min) was applied to a forelimb dewclaw at each of two cranial-torso isoflurane combinations: 1.1 +/- 0.3%-1.2 +/- 0.3% and 1.1 +/- 0.3%-0.3 +/- 0.1% (mean +/- SD).

Results: When cranial-torso isoflurane was 1.1-1.2%, the noxious stimulus did not alter the EEG. When torso isoflurane was decreased to 0.3%, the noxious stimulus activated the MRF, thalamic, and bifrontal-hemispheric regions (decreased high-amplitude, low-frequency power). For all channels combined, total (-33 +/- 15%), [delta] (-51 +/- 22%), [theta] (-33 +/- 19%), and [alpha] (-26 +/- 16%) power decreased after the noxious stimulus (P < 0.05); [beta] power was unchanged. The MRF unit responses to the noxious stimulus were significantly higher when the spinal cord isoflurane concentration was 0.3% (1,286 +/- 1,317 impulses/min) as compared with 1.2% (489 +/- 437 impulses/min, P < 0.05).     

Volatile Anesthetics Enhance Flash-induced γ Oscillations in Rat Visual Cortex

Background: The authors sought to understand neural correlates of anesthetic-induced unconsciousness. Cortical [gamma] oscillations have been associated with neural processes supporting conscious perception, but the effect of general anesthesia on these oscillations is controversial. In this study, the authors examined three volatile anesthetics, halothane, isoflurane, and desflurane, and compared their effects on flash-induced [gamma] oscillations in terms of equivalent concentrations producing the loss of righting reflex (1 minimum alveolar concentration for the loss of righting [MACLR]).

Methods: Light flashes were presented every 5 s for 5 min, and event-related potentials were recorded from primary visual cortex of 15 rats with a chronically implanted bipolar electrode at increasing anesthetic concentrations (0-2.4 MACLR). Early cortical response was obtained by averaging poststimulus (0-100 ms) potentials filtered at 20-60 Hz across 60 trials. Late (100-1,000 ms) [gamma] power was calculated using multitaper power spectral technique. Wavelet decomposition was used to determine spectral and temporal distributions of [gamma] power.

Results: The authors found that (1) halothane, isoflurane, and desflurane enhanced the flash-evoked early cortical response in a concentration-dependent manner; (2) the effective concentration for this enhancement was the lowest for isoflurane, intermediate for halothane, and the highest for desflurane when compared at equal fractions of the concentration that led to a loss of righting; (3) the power of flash-induced late (> 100 ms) [gamma] oscillations was augmented at intermediate concentrations of all three anesthetic agents; and (4) flash-induced [gamma] power was not reduced below waking baseline even in deep anesthesia.     

Effects on Synaptic Inhibition in the Hippocampus Do Not Underlie the Amnestic and Convulsive Properties of the Nonimmobilizer 1,2-Dichlorohexafluorocyclobutane

Background: Although it does not suppress movement in response to noxious stimuli, the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, also known as 2N) does cause amnesia and seizures. These occur at 0.48 and 1.3 times, respectively, the concentrations that are predicted from its lipid solubility to cause immobility. The molecular and cellular basis of these effects is not known. The ionotropic [gamma]-aminobutyric acid type A (GABAA) receptor is modulated strongly by anesthetics, and it plays an important role in many seizure models. Also, the hippocampus is a structure central to the formation of memory and is susceptible to seizure generation. The authors therefore investigated the effect of F6 on GABAA receptor- mediated inhibition in hippocampal neurons.

Methods: Transverse hippocampal slices were prepared from young (12- to 21-day-old) Sprague-Dawley rats. Inhibitory postsynaptic currents were recorded from hippocampal CA1 pyramidal cells in the presence of ionotropic glutamate receptor antagonists. F6 was applied with the bath solution. The concentration of F6 achieved during the experiment at the location of synaptic inhibition was derived using a diffusion model.

Results: At tissue concentrations of up to 75 [mu]m (approximately 5 x predicted minimal alveolar concentration), F6 had no discernible effect on either the amplitude or the kinetics of GABA-mediated synaptic currents. Isoflurane, by contrast, prolonged the decay time constant of these currents at 100 [mu]m (approximately 0.3 x minimal alveolar concentration).     

Isoflurane Pretreatment Inhibits Lipopolysaccharide-induced Inflammation in Rats

Background: Previous studies have indicated that volatile anesthetic pretreatment protects cells from inflammation in vitro; therefore, the authors hypothesized that pretreatment with isoflurane may attenuate the hemodynamic and pathologic changes to the vasculature that are associated with inflammation in vivo.

Methods: Rats received intravenous lipopolysaccharide or saline placebo with and without pretreatment with isoflurane (1.4% for 30 min immediately before lipopolysaccharide). Mean arterial pressure (MAP) and response to endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasodilators were assessed hourly for 6 h. Tumor necrosis factor-[alpha] concentrations, arterial blood gases, and vascular histology were also determined.

Results: Lipopolysaccharide decreased MAP and vasodilation to acetylcholine and sodium nitroprusside. Lipopolysaccharide also caused acidosis, endothelial swelling, and endothelial detachment from the smooth muscle. Isoflurane pretreatment prevented the decrease in MAP for 5 h and attenuated the decrease at 6 h. Pretreatment increased the vasodilation to acetylcholine in lipopolysaccharide rats to control concentrations but had no effect on sodium nitroprusside. In control rats, isoflurane pretreatment increased the response to acetylcholine and sodium nitroprusside but had no effect on MAP. Isoflurane pretreatment prevented the acidosis and endothelial damage to mesenteric and aortic vessels, and attenuated the increase in tumor necrosis factor-[alpha] associated with lipopolysaccharide-induced inflammation.     

Isoflurane Causes Anterograde but Not Retrograde Amnesia for Pavlovian Fear Conditioning

Background: Production of retrograde amnesia by anesthetics would indicate that these drugs can disrupt mechanisms that stabilize memory. Such disruption would allow suppression of memory of previous untoward events. The authors examined whether isoflurane provides retrograde amnesia for classic (Pavlovian) fear conditioning.

Methods: Rats were trained to fear tone by applying three (three-trial) or one (one-trial) tone-shock pairs while breathing various constant concentrations of isoflurane. Immediately after training, isoflurane administration was either discontinued, maintained unchanged, or rapidly increased to 1.0 minimum alveolar concentration for 1 h longer. Groups of rats were similarly trained to fear context while breathing isoflurane by applying shocks (without tones) in a distinctive environ- ment. The next day, memory for the conditioned stimuli was determined by presenting the tone or context (without shock) and measuring the proportion of time each rat froze (appeared immobile). For each conditioning procedure, the effects of the three posttraining isoflurane treatments were compared.

Results: Rapid increases in posttraining isoflurane administration did not suppress conditioned fear for any of the training procedures. In contrast, isoflurane administration during conditioning dose-dependently suppressed conditioning (P < 0.05). Training to tone was more resistant to the effects of isoflurane than training to context (P < 0.05), and the three-trial learning procedure was more was more resistant than the one-trial procedure (P < 0.05).     

Opposing Actions of Etomidate on Cortical Theta Oscillations Are Mediated by Different γ-Aminobutyric Acid Type A Receptor Subtypes

Background: Cortical networks generate diverse patterns of rhythmic activity. Theta oscillations (4-12 Hz) are commonly observed during spatial learning and working memory tasks. The authors ask how etomidate, acting predominantly via [gamma]-aminobutyric acid type A (GABAA) receptors containing [beta]2 or [beta]3 subunits, affects theta activity in vitro.

Methods: To characterize the effects of etomidate, the authors recorded action potential firing together with local field potentials in slice cultures prepared from the neocortex of the [beta]3(N265M) knock-in mutant and wild type mice. Actions of etomidate were studied at 0.2 [mu]m, which is approximately 15% of the concentration causing immobility (~1.5 [mu]m).

Results: In preparations derived from wild type and [beta]3(N265M) mutant mice, episodes of ongoing activity spontaneously occurred at a frequency of approximately 0.1 Hz and persisted for several seconds. Towards the end of these periods, synchronized oscillations in the theta band developed. These oscillations were significantly depressed in slices from [beta]3(N265M) mutant mice (P < 0.05). In this preparation etomidate acts almost exclusively via [beta]2 subunit containing GABAA receptors. In contrast, no depression was observed in slices from wild type mice, where etomidate potentiates both [beta]2- and [beta]3-containing GABAA receptors.     

Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes Predict Efficacy of Halogenated Compounds That Disobey the Meyer-Overton Rule

Background: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs.

Methods: [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chloro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique.

Results: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34[mu]M) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18[mu]M) did not alter the currents gated in either [alpha]3[beta]4 or [alpha]4[beta]2 receptors, but caused a small potentiation of [alpha]2[beta]4 hnAChRs without a concentration-response relation.     

Protein Kinase C Translocation and Src Protein Tyrosine Kinase Activation Mediate Isoflurane-induced Preconditioning In Vivo: Potential Downstream Targets of Mitochondrial Adenosine Triphosphate-sensitive Potassium Channels and Reactive Oxygen Species

Background: The authors tested the hypotheses that protein kinase C (PKC)-specific isoform translocation and Src protein tyrosine kinase (PTK) activation play important roles in isoflurane-induced preconditioning in vivo.

Methods: Rats (n = 125) instrumented for measurement of hemodynamics underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion and received 0.9% saline (control); PKC inhibitors chelerythrine (5 mg/kg), rottlerin (0.3 mg/kg), or PKC-[epsilon]V1-2 peptide (1 mg/kg); PTK inhibitors lavendustin A (1 mg/kg) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; 1 mg/kg); mitochondrial adenosine triphosphate-sensitive potassium channel antagonist 5-hydroxydecanote (10 mg/kg); or reactive oxygen species scavenger N-acetylcysteine (150 mg/kg) in the absence and presence of a 30-min exposure to isoflurane (1.0 minimum alveolar concentration) in separate groups. Isoflurane was discontinued 15 min before coronary occlusion (memory period). Infarct size was determined using triphenyltetrazolium staining. Immunohistochemistry and confocal microscopic imaging were performed to examine PKC translocation in separate groups of rats.

Results: Isoflurane significantly (P < 0.05) reduced infarct size (40 +/- 3% [n = 13]) as compared with control experiments (58 +/- 2% [n = 12]). Chelerythrine, rottlerin, PKC-[epsilon]V1-2 peptide, lavendustin A, PP1, 5-hydroxydecanote, and N-acetylcysteine abolished the anti-ischemic actions of isoflurane (58 +/- 2% [n = 8], 50 +/- 3% [n = 9], 53 +/- 2% [n = 9], 59 +/- 3% [n = 6], 57 +/- 3% [n = 7], 60 +/- 3% [n = 7], and 53 +/- 3% [n = 6], respectively). Isoflurane stimulated translocation of the [delta] and [epsilon] isoforms of PKC to sarcolemmal and mitochondrial membranes, respectively.     

The Effect of Hypothermia on Isoflurane MAC in Children

Background: Hypothermia has been shown to decrease the requirement for inhaled anesthetics in animals, but information in humans is limited.

Methods: Thirty-three unpremedicated children with congenital left-to-right shunt heart diseases undergoing open heart surgeries were assigned to one of three groups, with nasopharyngeal temperatures at the time of skin incision of 37, 34, or 31[degrees]C. Anesthesia was induced and maintained with isoflurane in oxygen. End-tidal isoflurane concentration and nasopharyngeal temperature were kept at stable levels for at least 15 min before the skin incision. Isoflurane minimum alveolar concentration was determined by using the Dixon up-and-down approach.

Results: Isoflurane minimum alveolar concentration values were 1.69 +/- 0.14%, 1.47 +/- 0.10%, and 1.22 +/- 0.15% (mean +/- SD) at 37, 34, and 31[degrees]C, respectively.     

Isoflurane Differentially Modulates Inhibitory and Excitatory Synaptic Transmission to the Solitary Tract Nucleus

Background: Isoflurane anesthesia produces cardiovascular and respiratory depression, although the specific mechanisms are not fully understood. Cranial visceral afferents, which innervate the heart and lungs, synapse centrally onto neurons within the medial portion of the nucleus tractus solitarius (NTS). Isoflurane modulation of afferent to NTS synaptic communication may underlie compromised cardiorespiratory reflex function.

Methods: Adult rat hindbrain slice preparations containing the solitary tract (ST) and NTS were used. Shocks to ST afferents evoked excitatory postsynaptic currents with low-variability (SEM <200 [mu]s) latencies identifying neurons as second order. ST-evoked and miniature excitatory postsynaptic currents as well as miniature inhibitory postsynaptic currents were measured during isoflurane exposure. Perfusion bath samples were taken in each experiment to measure isoflurane concentrations by gas chromatography-mass spectrometry.

Results: Isoflurane dose-dependently increased the decay-time constant of miniature inhibitory postsynaptic currents. At greater than 300 [mu]m isoflurane, the amplitude of miniature inhibitory postsynaptic currents was decreased, but the frequency of events remained unaffected, whereas at equivalent isoflurane concentrations, the frequency of miniature excitatory postsynaptic currents was decreased. ST-evoked excitatory postsynaptic current amplitudes decreased without altering event kinetics. Isoflurane at greater than 300 [mu]m increased the latency to onset and rate of synaptic failures of ST-evoked excitatory postsynaptic currents.     

Isoflurane Preconditioning Reduces the Rat NR8383 Macrophage Injury Induced by Lipopolysaccharide and Interferon [gamma]

Background: Isoflurane exposure before an insult can reduce the insult-induced injury in various organs. This phenomenon is called isoflurane preconditioning. The authors hypothesize that isoflurane can precondition macrophages, cells that travel to all tissues and are important in the host defense and inflammation responses.

Methods: Rat NR8383 macrophages were pretreated with or without 1-3% isoflurane for 1 h at 30 min before they were incubated with or without 100 ng/ml lipopolysaccharide plus 50 U/ml interferon [gamma] for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was performed after cells were stained with annexin V and propidium iodide. Inducible nitric oxide synthase protein expression in macrophages was quantified by Western blotting.

Results: Lipopolysaccharide plus interferon [gamma] decreased cell viability by approximately 50%. This decrease was dose-dependently inhibited by aminoguanidine, an inducible nitric oxide synthase inhibitor. Lipopolysaccharide plus interferon [gamma] caused inducible nitric oxide synthase expression. This expression was inhibited by pretreatment with 2% but not 1% or 3% isoflurane. Isoflurane at 2% inhibited lipopolysaccharide plus interferon [gamma]-induced accumulation of nitrite, an oxidation product of nitric oxide. Pretreatment with 2% but not 1% or 3% isoflurane improved cell viability. Lipopolysaccharide plus interferon [gamma] increased the number of propidium iodide-positive staining cells. This increase was attenuated by 2% isoflurane pretreatment. The protective effect of 2% isoflurane was abolished by chelerythrine, calphostin C, or bisindolylmaleimide IX, protein kinase C inhibitors.     

Interaction of Isoflurane with the Dopamine Transporter

Background: Isoflurane administration is known to increase extracellular dopamine (DA) concentration. Because the dopamine transporter (DAT) is a key regulator of DA, it is likely affected by isoflurane. This study investigates the hypothesis that isoflurane inhibits DA reuptake by causing DAT to be trafficked into the cell.

Methods: Rhesus monkeys were scanned with positron emission tomography (PET) using [18F]FECNT (a highly specific DAT ligand) while anesthetized with 1% isoflurane. The isoflurane was increased to 2%, and the animals were rescanned. Uptake was analyzed with the tissue reference method using the cerebellum as the reference tissue to determine the binding potential in the putamen. Immunohistochemistry and Western blot analyses were performed in vivo in rats to determine if isoflurane administration would change the total amount of DAT. Rats breathed air plus 2% isoflurane for 30 min, and then striatal DAT assays were rapidly performed. In vitro immunocytochemistry experiments were performed using human embryonic kidney (HEK) cells stably transfected with human DAT. The cells were exposed to 4% isoflurane for 1 h while the location of DAT was observed with fluorescent confocal microscopy.

Results: The [18F]FECNT binding potential in rhesus monkeys decreased by 63 +/- 6% (SEM, n = 5) when isoflurane was increased from 1 to 2% as compared with no significant change (0.7 +/- 2.5%; SEM, n = 5) when the isoflurane concentration was not changed (P < 0.001). No difference in DAT staining between isoflurane-treated and control rats was apparent from visual inspection, and quantitative Western blot analyses showed no significant change in total DAT protein. After isoflurane treatment, focal puncta of intense fluorescence was visible inside the HEK cells.     

The Inhibitory Effects of Isoflurane on Protein Tyrosine Phosphorylation-modulated Contraction of Rat Aortic Smooth Muscle

Background: Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in initiating and modulating vascular smooth muscle contraction. The aim of the current study was to examine the effects of isoflurane on sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor-induced, tyrosine phosphorylation-mediated contraction of rat aortic smooth muscle.

Methods: The Na3VO4-induced contraction of rat aortic smooth muscle and tyrosine phosphorylation of proteins including phospholipase C[gamma]-1 (PLC[gamma]-1) and p44/p42 mitogen-activated protein kinase (MAPK) were assessed in the presence of different concentrations of isoflurane, using isometric force measurement and Western blotting methods, respectively.

Results: Na3VO4 (10-4 m) induced a gradually sustained contraction and significant increase in protein tyrosine phosphorylation of a set of substrates including PLC[gamma]-1 and p42MAPK, all of which were markedly inhibited by genistein (5 x 10-5 m), a tyrosine kinase inhibitor. Isoflurane (1.2-3.5%) dose-dependently depressed the Na3VO4-induced contraction (P < 0.05-0.005; n = 8). Isoflurane also attenuated the total density of the Na3VO4-induced, tyrosine-phosphorylated substrate bands and the density of tyrosine-phosphorylated PLC[gamma]-1 band and p42MAPK band (P < 0.05-0.005; n = 4) in a concentration-dependent manner.     

Effect of Isoflurane and Other Potent Inhaled Anesthetics on Minimum Alveolar Concentration, Learning, and the Righting Reflex in Mice Engineered to Express α1 γ-Aminobutyric Acid Type A Receptors Unresponsive to Isoflurane

Background: Enhancement of the function of [gamma]-aminobutyric acid type A receptors containing the [alpha]1 subunit may underlie a portion of inhaled anesthetic action. To test this, the authors created gene knock-in mice harboring mutations that render the receptors insensitive to isoflurane while preserving sensitivity to halothane.

Methods: The authors recorded miniature inhibitory synaptic currents in hippocampal neurons from hippocampal slices from knock-in and wild-type mice. They also determined the minimum alveolar concentration (MAC), and the concentration at which 50% of animals lost their righting reflexes and which suppressed pavlovian fear conditioning to tone and context in both genotypes.

Results: Miniature inhibitory postsynaptic currents decayed more rapidly in interneurons and CA1 pyramidal cells from the knock-in mice compared with wild-type animals. Isoflurane (0.5-1 MAC) prolonged the decay phase of miniature inhibitory postsynaptic currents in neurons of the wild-type mice, but this effect was significantly reduced in neurons from knock-in mice. Halothane (1 MAC) slowed the decay of miniature inhibitory postsynaptic current in both genotypes. The homozygous knock-in mice were more resistant than wild-type controls to loss of righting reflexes induced by isoflurane and enflurane, but not to halothane. The MAC for isoflurane, desflurane, and halothane did not differ between knock-in and wild-type mice. The knock-in mice and wild-type mice did not differ in their sensitivity to isoflurane for fear conditioning.     

Modulation of γ-Aminobutyric Acid Type A Receptor-mediated Spontaneous Inhibitory Postsynaptic Currents in Auditory Cortex by Midazolam and Isoflurane

Background: Anesthetic agents that target [gamma]-aminobutyric acid type A (GABAA) receptors modulate cortical auditory evoked responses in vivo, but the cellular targets involved are unidentified. Also, for agents with multiple protein targets, the relative contribution of modulation of GABAA receptors to effects on cortical physiology is unclear. The authors compared effects of the GABAA receptor-specific drug midazolam with the volatile anesthetic isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal cells of auditory cortex.

Methods: Whole cell recordings were obtained in murine brain slices at 34[degrees]C. GABAA sIPSCs were isolated by blocking ionotropic glutamate receptors. Effects of midazolam and isoflurane on time course, amplitude, and frequency of sIPSCs were measured.

Results: The authors detected no effect of midazolam at 0.01 [mu]m on sIPSCs, whereas midazolam at 0.1 and 1 [mu]m prolonged the decay of sIPSCs by approximately 25 and 70%, respectively. Isoflurane at 0.1, 0.25, and 0.5 mm prolonged sIPSCs by approximately 45, 150, and 240%, respectively. No drug-specific effects were observed on rise time or frequency of sIPSCs. Isoflurane at 0.5 mm caused a significant decrease in sIPSC amplitude.     

Isoflurane Preconditioning Improves Long-term Neurologic Outcome after Hypoxic-Ischemic Brain Injury in Neonatal Rats

Background: Preconditioning the brain with relatively safe drugs seems to be a viable option to reduce ischemic brain injury. The authors and others have shown that the volatile anesthetic isoflurane can precondition the brain against ischemia. Here, the authors determine whether isoflurane preconditioning improves long-term neurologic outcome after brain ischemia.

Methods: Six-day-old rats were exposed to 1.5% isoflurane for 30 min at 24 h before the brain hypoxia-ischemia that was induced by left common carotid arterial ligation and then exposure to 8% oxygen for 2 h. The neuropathology, motor coordination, and learning and memory functions were assayed 1 month after the brain ischemia. Western analysis was performed to quantify the expression of the heat shock protein 70, Bcl-2, and survivin 24 h after isoflurane exposure.

Results: The mortality was 45% after brain hypoxia-ischemia. Isoflurane preconditioning did not affect this mortality. However, isoflurane preconditioning attenuated ischemia-induced loss of neurons and brain tissues, such as cerebral cortex and hippocampus in the survivors. Isoflurane also improved the motor coordination of rats at 1 month after ischemia. The learning and memory functions as measured by performance of Y-maze and social recognition tasks in the survivors were not affected by the brain hypoxia-ischemia or isoflurane preconditioning. The expression of Bcl-2, a well-known antiapoptotic protein, in the hippocampus is increased after isoflurane exposure. This increase was reduced by the inhibitors of inducible nitric oxide synthase. Inducible nitric oxide synthase inhibition also abolished isoflurane preconditioning-induced neuroprotection.