How Do the Etching Mode and Thermomechanical Loading Influence the Marginal Integrity of Universal Adhesives?

OBJECTIVE: This study evaluated the effect of etching mode and thermomechanical loading on universal adhesives. METHODS AND MATERIALS: Two universal adhesives, Peak Universal and Adhese Universal, were used in two etching modes as the experimental groups: Peak Universal etch-and-rinse (PER), Peak Universal self-etch (PSE), Adhese Universal etch-and-rinse (AER), and Adhese Universal self-etch (ASE). Two adhesives considered gold standards were used as control groups: OptiBond FL (OER) was used as a control group for the etch-and-rinse (ER) mode, and Clearfil SE Bond (CSE) was used as a control group for the self-etch (SE) mode. Standardized class V cavities were created on the buccal and lingual surface in 30 extracted caries-free human third molars. Each adhesive and resin composite was applied according to the manufacturer's instructions. The specimens were subjected to thermomechanical loading (TML) immediately after the fillings were placed. Before and after TML, replicas and photographs of the fillings were performed and evaluated quantitatively and qualitatively. The Mann-Whitney U-test or Kruskal-Wallis test was used for quantitative analyses, and Fisher exact test was used for qualitative analysis. RESULTS: Adhese Universal achieved a significantly higher percentage of continuous margin in the enamel than Peak Universal for the two types of etching both before and after TML (except for the SE group after TML). In dentin, the greatest percentage of continuous margin was achieved for Adhese Universal in the ER group (100%) before TML and for both universal adhesives in the SE groups (61%) after TML. For both etching modes and both time points, Adhese Universal had a greater percentage of continuous margin than Peak Universal for the whole margin. For the ER approach, significant differences were observed both before and after TML, and for the SE approach, significant differences were observed before TML. TML did not cause a significant decrease in the percentage of continuous margin in the enamel, but the results were the opposite in dentin. A qualitative assessment using World Dental Federation criteria did not show statistically significant differences between the groups. CONCLUSIONS: Scanning electron microscope assessment of marginal integrity showed that the evaluated factors such as etching mode and TML significantly influenced the marginal integrity of the universal adhesives. The replica method shows that laboratory and clinical assessment methods complement each other and give a broader view of marginal integrity.

A Simple Instrument Suite for Characterizing Habitability and Weathering: The Modern Aqueous Habitat Reconnaissance Suite (MAHRS).

The shallow subsurface of Mars is extremely interesting as a possible microbial habitat because it becomes temporarily wet, it is shielded from radiation, and mixing by aeolian processes could provide the sources of energy and nutrients necessary for sustaining microbial life in it. The Modern Aqueous Habitat Reconnaissance Suite (MAHRS) was developed primarily to search for potentially habitable environments in the shallow subsurface of Mars and to study weathering, but it can also be used to search for potentially habitable environments in the shallow subsurface of other planetary bodies such as the Icy Worlds. MAHRS includes an instrument developed to measure regolith wetness and search for brine in the shallow subsurface of Mars, where it is most likely to be found. The detection of brine can aid in our understanding not only of habitability but also of geochemistry and aqueous weathering processes. Besides the regolith wetness sensor, MAHRS includes an electric field sensor, an optical microscope, and a radiometer developed to characterize the near-surface environment and study mixing by aeolian processes. MAHRS was designed to aid in the selection of optimum areas for sample collection for return to Earth.

Newborn drug testing practices in Iowa birthing hospitals.

OBJECTIVES: Federal law mandates states to have policies and procedures to identify newborns exposed to maternal substance use during pregnancy. National guidelines for newborn drug testing are lacking; therefore, procedures are variable and determined by state law and local practices. In Iowa, maternal substance use during pregnancy is considered child abuse and must be reported.The objective of this study was to identify newborn drug testing policies and procedures among birthing hospitals in Iowa. METHODS: This was a cross sectional survey of all birthing hospitals in Iowa identified via the Statewide Perinatal Care Program. An electronic survey was sent to the representative at each affiliated hospital. RESULTS: Sixty-nine of 76 hospitals completed the survey for a 90.8% response rate. Newborn drug testing is ordered in 97.1% of responding hospitals with most testing 25% or less of newborns annually. The majority utilized a risk assessment tool (89.6%), although many (62.7%) also allowed for provider discretion. No hospital performed universal testing of all newborns. 86.6% of hospitals reported all positive newborn drug test results including illicit and/or prescription drugs to child protective services. 35.0% of hospitals notified mothers of the report and 45.5% offered substance abuse services and/or treatment to the mothers. CONCLUSIONS: Most Iowa birthing hospitals perform newborn drug testing and report all positive test results to child protective services. The majority use risk assessment tools. Maternal notification practices and referral for substance use disorder treatment are suboptimal and represent an area for future improvement.

Algorithms utilizing peripheral blood hematopoietic progenitor cell counts in lieu of some CD34+ cell counts predict successful peripheral blood stem cell collections with substantial time and cost savings.

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cell (HPC) counts from Sysmex hematology analyzers have been shown to correlate with peripheral blood (PB) CD34+ cell counts by flow cytometry. Algorithms utilizing HPC counts to guide stem cell collections have been proposed but rarely tested. This study describes the development and validation of algorithms utilizing HPC and PB CD34+cell counts to predict adequate peripheral blood stem cell (PBSC) collections for chemomobilized and cytokine-mobilized individuals. MATERIALS AND METHODS: Utilizing a test set of 83 PB samples from chemomobilized or cytokine-mobilized PBSC collection patients, PB CD34+ counts were correlated with HPC counts and a receiver operating characteristic curve was constructed. Cut-offs of ≤0.5 HPC/µl and ≥7 HPC/µl were established to maximize sensitivity and specificity for using HPC to predict PB CD34+ ≥ 10 cells/µl. These cut-offs were subsequently validated using a separate prospective validation set of 88 HPC/CD34+ cell sample pairs. RESULTS: Using the algorithms, all patients in the prospective validation data set achieved adequate collections of ≥1 × 106 CD34+ cells/kg, and a 67% reduction in the number of CD34+ cell counts performed was achieved. This lead to a direct cost savings of at least $18,700 USD over a 21-month period (88% reduction in direct costs). CONCLUSION: Use of the algorithms provides significant time and cost savings for the laboratory while accurately predicting (i) timing of PBSC collections to obtain adequate CD34+ product yields for chemomobilized patients and (ii) when to administer plerixafor to cytokine-mobilized patients to improve the likelihood of achieving adequate collections.

A miniature microcontroller curve tracing circuit for space flight testing transistors.

This paper describes a novel miniature microcontroller based curve tracing circuit, which was designed to monitor the environmental effects on Silicon Carbide Junction Field Effect Transistor (SiC JFET) device performance, while exposed to the low earth orbit environment onboard the International Space Station (ISS) as a resident experiment on the 7th Materials on the International Space Station Experiment (MISSE7). Specifically, the microcontroller circuit was designed to operate autonomously and was flown on the external structure of the ISS for over a year. This curve tracing circuit is capable of measuring current vs. voltage (I-V) characteristics of transistors and diodes. The circuit is current limited for low current devices and is specifically designed to test high temperature, high drain-to-source resistance SiC JFETs. The results of each I-V data set are transmitted serially to an external telemetered communication interface. This paper discusses the circuit architecture, its design, and presents example results.

Cross-reactivity studies and predictive modeling of "Bath Salts" and other amphetamine-type stimulants with amphetamine screening immunoassays.

INTRODUCTION: The increasing abuse of amphetamine-like compounds presents a challenge for clinicians and clinical laboratories. Although these compounds may be identified by mass spectrometry-based assays, most clinical laboratories use amphetamine immunoassays that have unknown cross-reactivity with novel amphetamine-like drugs. To date, there has been a little systematic study of amphetamine immunoassay cross-reactivity with structurally diverse amphetamine-like drugs or of computational tools to predict cross-reactivity. METHODS: Cross-reactivities of 42 amphetamines and amphetamine-like drugs with three amphetamines screening immunoassays (AxSYM(®) Amphetamine/Methamphetamine II, CEDIA(®) amphetamine/Ecstasy, and EMIT(®) II Plus Amphetamines) were determined. Two- and three-dimensional molecular similarity and modeling approaches were evaluated for the ability to predict cross-reactivity using receiver-operator characteristic curve analysis. RESULTS: Overall, 34%-46% of the drugs tested positive on the immunoassay screens using a concentration of 20,000 ng/mL. The three immunoassays showed differential detection of the various classes of amphetamine-like drugs. Only the CEDIA assay detected piperazines well, while only the EMIT assay cross-reacted with the 2C class. All three immunoassays detected 4-substituted amphetamines. For the AxSYM and EMIT assays, two-dimensional molecular similarity methods that combined similarity to amphetamine/methamphetamine and 3,4-methylenedioxymethampetamine most accurately predicted cross-reactivity. For the CEDIA assay, three-dimensional pharmacophore methods performed best in predicting cross-reactivity. Using the best performing models, cross-reactivities of an additional 261 amphetamine-like compounds were predicted. CONCLUSIONS: Existing amphetamines immunoassays unevenly detect amphetamine-like drugs, particularly in the 2C, piperazine, and ß-keto classes. Computational similarity methods perform well in predicting cross-reactivity and can help prioritize testing of additional compounds in the future.

Springtime resumption of photosynthesis in balsam fir (Abies balsamea).

Photosynthesis in balsam fir (Abies balsamea (L.) Mill.) was measured in the field at two locations in New Brunswick, Canada from late winter to late spring in 2004 and 2005. No photosynthesis was detectable while the soil remained below 0 degrees C throughout the rooting zone. In both years, photosynthesis began once soil temperature rose to 0 degrees C. In potted seedlings in growth chambers, there was no photosynthesis at an air temperature of 10 degrees C if the pots were frozen. These findings suggest that, once air temperatures permit photosynthesis, it is the availability of unfrozen soil water that triggers the onset of photosynthesis. In the field, full recovery of photosynthetic capacity following the onset of soil thaw was dependent on air temperature and took 5 weeks in 2005, but 10 weeks in 2004. There were two substantial frost events during the recovery period in 2004 that may explain the extended recovery period. In 2005, recovery was complete after the accumulation of 200 growing degree days above 0 degrees C after the start of soil thaw.

Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics.

gamma-Aminobutyric acid type A (GABA(A)) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA(A) receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA(A) receptor beta(2) subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the beta(2) subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the beta(2)(M286W) mutation resulted in a decrease in the 'cut-off' volume for propofol analog molecules to enhance GABA responses at GABA(A) alpha(1)beta(2)gamma(2s) receptors. This suggests that mutation of M286 in the GABA(A) beta(2) subunit alters the dimensions of a 'binding pocket' for propofol and related alkylphenol general anesthetics.

Anesthetic properties of 4-iodopropofol: implications for mechanisms of anesthesia.

BACKGROUND: Positive modulation of gamma-aminobutyric acid type A (GABAA) receptor function is recognized as an important component of the central nervous system depressant effects of many general anesthetics, including propofol. The role for GABAA receptors as an essential site in the anesthetic actions of propofol was recently challenged by a report that the propofol analog 4-iodopropofol (4-iodo-2,6-diisopropylphenol) potentiated and directly activated GABAA receptors, yet was devoid of sedative-anesthetic effects in rats after intraperitoneal injection. Given the important implications of these findings for theories of anesthesia, the authors compared the effects of 4-iodopropofol with those of propofol using established in vivo and in vitro assays of both GABAA receptor-dependent and -independent anesthetic actions. METHODS: The effects of propofol and 4-iodopropofol were analyzed on heterologously expressed recombinant human GABAA alpha1beta2gamma2 receptors, evoked population spike amplitudes in rat hippocampal slices, and glutamate release from rat cerebrocortical synaptosomes in vitro. Anesthetic potency was determined by loss of righting reflex in Xenopus laevis tadpoles, in mice after intraperitoneal injection, and in rats after intravenous injection. RESULTS: Like propofol, 4-iodopropofol enhanced GABA-induced currents in recombinant GABAA receptors, inhibited synaptic transmission in rat hippocampal slices, and inhibited sodium channel-mediated glutamate release from synaptosomes, but with reduced potency. After intraperitoneal injection, 4-iodopropofol did not produce anesthesia in mice, but it was not detected in serum or brain. However, 4-iodopropofol did produce anesthesia in tadpoles (EC50 = 2.5 +/- 0.5 microM) and in rats after intravenous injection (ED50 = 49 +/- 6.2 mg/kg). CONCLUSIONS: Propofol and 4-iodopropofol produced similar actions on several previously identified cellular and molecular targets of general anesthetic action, and both compounds induced anesthesia in tadpoles and rats. The failure of 4-iodopropofol to induce anesthesia in rodents after intraperitoneal injection is attributed to a pharmacokinetic difference from propofol rather than to major pharmacodynamic differences.

General anesthetic potencies of a series of propofol analogs correlate with potency for potentiation of gamma-aminobutyric acid (GABA) current at the GABA(A) receptor but not with lipid solubility.

A series of 27 analogs of the general anesthetic propofol (2,6-diisopropylphenol) were examined for general anesthetic activity in Xenopus laevis tadpoles and for the ability to produce enhancement of submaximal GABA responses and/or direct activation at recombinant GABA(A) receptors. Fourteen of the propofol analogs produced loss of righting reflex in the tadpoles, whereas 13 were inactive as anesthetics. The same pattern of activity was noted with the actions of the compounds at the GABA(A) alpha(1)beta(2)gamma(2s) receptor. The potencies of the analogs as general anesthetics in tadpoles correlated better with potentiation of GABA responses than direct activation at the GABA(A) alpha(1)beta(2)gamma(2s) receptor. The calculated octanol/water partition coefficients for the analogs did not explain the lack of activity exhibited by the 13 nonanesthetic analogs, although this physicochemical parameter did correlate modestly with in vivo anesthetic potency. The actions of one nonanesthetic analog, 2,6-di-tert-butylphenol, were examined in detail. 2,6-Di-tert-butylphenol was inactive at GABA(A) receptors, did not function as an anesthetic in the tadpoles, and did not antagonize any of the actions of propofol at GABA(A) receptors or in tadpoles. A key influence on the potency of propofol analogs appears to be the size and shape of the alkyl groups at positions 2 and 6 of the aromatic ring relative to the substituent at position 1. These data suggest steric constraints for the binding site for propofol on the GABA(A) receptor.

Human dopamine transporter gene: coding region conservation among normal, Tourette's disorder, alcohol dependence and attention-deficit hyperactivity disorder populations.

The dopamine transporter (DAT) provides major regulation of the synaptic levels of dopamine and is a principal target of psychostimulant drugs. Associations between DAT gene polymorphisms and human disorders with possible links to dopaminergic neurotransmission, including attention-deficit/hyperactivity disorder (ADHD) and consequences of cocaine and alcohol administration, have been reported. We now report approximately 60000 bp of genomic sequence containing the entire DAT gene. This sequence was used to amplify each of the 15 DAT gene exons and several introns and analyze these amplification products by single-stranded sequence conformation (SSCP) and/or direct sequencing. These results define silent allelic single nucleotide sequence variants in DAT gene exons 2, 6, 9 and 15. Rare conservative mutations are identified in amino acids encoded by DAT exons 2 and 8. Analyses of the common nucleotide variants and the previously reported VNTR in the non-coding region of exon 15 define the pattern of linkage disequilibrium across the DAT locus. These comprehensive analyses, however, fail to identify any common protein coding DAT sequence variant in more than 150 unrelated individuals free of neuropsychiatric disease, 109 individuals meeting City of Hope criteria for Tourette's syndrome, 64 individuals with DSM-IV diagnoses of ethanol dependence, or 15 individuals with ADHD. These data are consistent with substantial evolutionary conservation of the DAT protein sequence. They suggest that gene variants that alter levels of DAT expression provide the best current candidate mechanism for reported associations between DAT gene markers, ADHD and other more tentatively associated neuropsychiatric disorders.

Cholinesterase inhibition by potato glycoalkaloids slows mivacurium metabolism.

BACKGROUND: The duration of action for many pharmaceutical agents is dependent on their breakdown by endogenous hydrolytic enzymes. Dietary factors that interact with these enzyme systems may alter drug efficacy and time course. Cholinesterases such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze and inactivate several anesthetic drugs, including cocaine, heroin, esmolol, local ester anesthetics, and neuromuscular blocking drugs. Natural glycoalkaloid toxins produced by plants of the family Solanaceae, which includes potatoes and tomatoes, inhibit both AChE and BuChE. Here the authors assess the extent to which two solanaceous glycoalkaloids (SGAs), alpha-solanine and alpha-chaconine, can alter the effects of neuromuscular blocking drugs and cholinesterase inhibitors in vivo and in vitro. METHODS: Inhibition of purified human AChE and BuChE by SGAs, neuromuscular blocking drugs, and cholinesterase inhibitors was assessed by an in vitro colorimetric cholinesterase assay. In vivo experiments were carried out using anesthetized rabbits to test whether SGAs affect recovery from mivacurium-induced paralysis. RESULTS: SGAs inhibited human BuChE at concentrations similar to those found in serum of individuals who have eaten a standard serving of potatoes. Coapplication of SGAs (30-100 nm) with neuromuscular blocking drugs and cholinesterase inhibitors produced additive cholinesterase inhibition. SGA administration to anesthetized rabbits inhibited serum cholinesterase activity and mivacurium hydrolysis. In addition, SGA prolonged the time needed for recovery from mivacurium-induced paralysis (149 +/- 12% of control; n = 12). CONCLUSIONS: These findings support the hypothesis that inhibition of endogenous enzyme systems by dietary factors can influence anesthetic drug metabolism and duration of action. Diet may contribute to the wide variation in recovery time from neuromuscular blockade seen in normal, healthy individuals.

Differential modulatory actions of the volatile convulsant flurothyl and its anesthetic isomer at inhibitory ligand-gated ion channels.

A challenge for theories of general anesthesia is the existence of compounds predicted to be anesthetics but which, instead, do not produce anesthesia and often elicit other behavioral effects such as convulsions. This study focused on flurothyl (bis[2,2, 2-trifluoroethyl] ether), a potent volatile convulsant, and its anesthetic isomer, 'iso-flurothyl' (1,1,1,3,3, 3-hexafluoro-2-methoxypropane). The effects of flurothyl and iso-flurothyl were studied using the whole-cell patch-clamp technique on agonist-activated chloride currents in human GABA(A), glycine, and GABA(C) rho(1) receptors expressed in HEK 293 cells. GABA(A) and glycine receptors are promising molecular targets for the actions of inhaled ether general anesthetics. Flurothyl acted as a non-competitive antagonist at GABA(A) alpha(2)beta(1) and alpha(2)beta(1)gamma(2s) receptors, but had no effect at glycine alpha(1) receptors. Flurothyl had biphasic actions on GABA responses at GABA(C) rho(1) receptors. In contrast, iso-flurothyl enhanced ('potentiated') submaximal agonist responses at GABA(A) and glycine receptors, but had no effect on GABA responses at GABA(C) rho(1) receptors. Point mutations in GABA(A) and glycine receptor subunits, which have been previously shown to abolish potentiation of agonist responses by the ether anesthetics enflurane and isoflurane, also ablated potentiation of agonist responses by iso-flurothyl. These same mutations in the GABA(A) receptor had only modest effects on the inhibitory actions of flurothyl. GABA(A) receptors with mutations conferring insensitivity to antagonism by picrotoxin were still inhibited by flurothyl, suggesting that picrotoxin and flurothyl antagonize GABA responses by distinct sites or mechanisms of action. In summary, antagonism of GABA(A) receptors is likely to account for the convulsant effects of flurothyl, while the general anesthetic actions of iso-flurothyl, like those of other ether anesthetics, may be related to positive modulation of GABA(A) and/or glycine receptors.

Intravenous anesthetics differentially modulate ligand-gated ion channels.

BACKGROUND: Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates gamma-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior. METHODS: A heteromeric nAChR composed of alpha4 and beta4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of alpha1beta2gamma2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 microm to 10 mm. RESULTS: Ketamine caused potent, concentration-dependent inhibition of the alpha4beta4 nAChR current with an IC50 of 0.24 microm. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the alpha1beta2gamma2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the alpha4beta4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations. CONCLUSIONS: The alpha4beta4 nAChR, which is predominantly found in the central nervous system (CNS), is differentially affected by clinically relevant concentrations of intravenous anesthetics. Ketamine, commonly known to be an inhibitor at the N-methyl-D-aspartate receptor, is also a potent inhibitor at a central nAChR. It has little effect on a common CNS GABAA receptor in a clinically relevant concentration range. Interaction between ketamine and specific subtypes of nAChRs in the CNS may result in anesthetic behaviors such as inattention to surgical stimulus and in analgesia. Thiopental causes minor inhibition at the alpha4beta4 nAChR. Modulation of the alpha4beta4 nAChR by etomidate is unlikely to be important in anesthesia practice based on the insensitivity of this receptor to clinically used concentrations.

The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations.

The actions of 13 general anaesthetics (diethyl ether, enflurane, isoflurane, methoxyflurane, sevoflurane, chloral hydrate, trifluoroethanol, tribromoethanol, tert-butanol, chloretone, brometone, trichloroethylene, and alpha-chloralose) were studied on agonist-activated Cl(-) currents at human GABA(A) alpha(2)beta(1), glycine alpha(1), and GABA(C) rho(1) receptors expressed in human embryonic kidney 293 cells. All 13 anaesthetics enhanced responses to submaximal (EC(20)) concentrations of agonist at GABA(A) and glycine receptors, except alpha-chloralose, which did not enhance responses at the glycine alpha(1) receptor. None of the anaesthetics studied potentiated GABA responses at the GABA(C) rho(1) receptor. Potentiation of submaximal agonist currents by the anaesthetics was studied at GABA(A) and glycine receptors harbouring mutations in putative transmembrane domains 2 and 3 within GABA(A) alpha(2), beta(1), or glycine alpha(1) receptor subunits: GABA(A) alpha(2)(S270I)beta(1), alpha(2)(A291W)beta(1), alpha(2)beta(1)(S265I), and alpha(2)beta(1)(M286W); glycine alpha(1)(S267I) and alpha(1)(A288W). For all anaesthetics studied except alpha-chloralose, at least one of the mutations above abolished drug potentiation of agonist responses at GABA(A) and glycine receptors. alpha-Chloralose produced efficacious direct activation of the GABA(A) alpha(2)beta(1) receptor (a 'GABA-mimetic' effect). The other 12 anaesthetics produced minimal or no direct activation of GABA(A) and glycine receptors. A non-anaesthetic isomer of alpha-chloralose, beta-chloralose, was inactive at GABA(A) and glycine receptors and did not antagonize the actions of alpha-chloralose at GABA(A) receptors. The implications of these findings for the molecular mechanisms of action of general anaesthetics at GABA(A) and glycine receptors are discussed.

General anaesthetic actions on ligand-gated ion channels.

The molecular mechanisms of general anaesthetics have remained largely obscure since their introduction into clinical practice just over 150 years ago. This review describes the actions of general anaesthetics on mammalian neurotransmitter-gated ion channels. As a result of research during the last several decades, ligand-gated ion channels have emerged as promising molecular targets for the central nervous system effects of general anaesthetics. The last 10 years have witnessed an explosion of studies of anaesthetic modulation of recombinant ligand-gated ion channels, including recent studies which utilize chimeric and mutated receptors to identify regions of ligand-gated ion channels important for the actions of general anaesthetics. Exciting future directions include structural biology and gene-targeting approaches to further the understanding of general anaesthetic molecular mechanisms.

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane.

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

A deficit of functional GABA(A) receptors in neurons of beta 3 subunit knockout mice.

Mice whose gamma-aminobutyric acid type A (GABA(A)) beta3 subunit gene is inactivated ('beta3 knockout mice') have been previously shown to have epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal mortality. In this study, we analyze whole-cell responses to GABA in neurons from beta3+/+, beta3+/- and beta3-/- mice. We demonstrate markedly decreased responses to GABA in both hippocampal and dorsal root ganglion neurons isolated from beta3-/- mice without major differences in the GABA concentration-response curves. We also utilize the subunit selective pharmacology of Zn2+ and the anticonvulsant drug loreclezole to help infer the presence of beta2 and gamma subunits in the GABA(A) receptors remaining in neurons from beta3-/- mice.