Effects of normal alcohols and isoflurane on lipid headgroup dynamics in nicotinic acetylcholine receptor-rich lipid vesicles.

The trend of evidence suggests that general anesthetics act directly on proteins in the neural membrane. However, the fact that the functions of nicotinic acetylcholine receptor (sodium permeability, desensitization rate) are modulated by the composition of the membrane in which it is reconstituted has been thought to be a result of the variation of interactions between acetylcholine receptor and membrane. In this study, protein-lipid interaction at the level of the lipid headgroup was investigated using electron paramagnetic resonance (EPR) and headgroup spin label. Lipid headgroup mobility was evaluated with rotational correlation time from the EPR spectrum. Protein-lipid interaction at headgroup depth was demonstrated from the motionally restricted component of the spectrum. Rotational correlation time increased to 13 ns from 7 ns due to protein-lipid interaction. The effect of anesthetic (ethanol, 1-hexanol, and isoflurane) on protein-lipid interaction was investigated, and the correlation time was 13 ns. It is concluded that the anesthetics used in this study did not alter protein-lipid interaction at the level of the lipid headgroup, so far as observed by rotational correlation time, without excluding the possibility that anesthetics that perturb protein-lipid interactions modulate receptor functions via this mechanism.

Lipid-dependent differential effects of stereoisomers of anesthetic alcohols.

The cis- and trans-alkenols are equally potent general anesthetics but, respectively, lower and raise the gel-to-liquid crystalline phase transition temperature of saturated phosphatidylcholines (Pringle, M.J. and Miller, K.W. (1978) Biochem. Biophys. Res. Commun. 85, 1191-1198). Here we show that although this differential effect is somewhat reduced when a double bond is introduced into the sn-2 position of phosphatidylcholine, it is abolished when the ethanolamine head group is substituted for the choline head group in dimyristoyl lipids at neutral pH. At high pH, however, dimyristoylphosphatidylethanolamine assumes a negative charge, and its phase transition temperature drops to a value close to that for the corresponding phosphatidylcholine. Under these conditions the differential effect of the alkenol isomers is restored; the cis-alkenol lowers, while the trans-alkenol raises, the phase transition temperature of deprotonated dimyristoylphosphatidylethanolamine. Thus, the differential effects of cis- and trans-alkenols on the gel-to-liquid crystalline phase transition are dependent on the physical chemical characteristics of the polar region of the perturbed lipid species, but only weakly on that of the acyl region.

Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo.

The acetylcholine receptor (AChR)-containing electroplax membranes from Torpedo californica have a relatively high cholesterol content. Reconstitution studies suggest that this cholesterol may be important in preserving or modulating the function of the acetylcholine receptor-channel complex. We have manipulated cholesterol levels in intact Torpedo AChR-rich membrane fragments using small, unilamellar phosphatidylcholine liposomes. Conditions have been established that allow further subfractionation of sucrose gradient purified Torpedo electroplax membranes into AChR-rich and ATPase-rich populations and that, at the same time, achieve cholesterol depletion without phospholipid back exchange or fusion. The incubation of membranes with excess liposomes could only achieve about a 50% reduction in the molar ratio of cholesterol to phospholipid. In no case was the number of cholesterol molecules per AChR oligomer reduced below 36. The remaining cholesterol could not be depleted either by longer incubations or by multiple, sequential depletions. Cholesterol depletion was accompanied by a significant increase in bulk membrane fluidity as measured by electron spin resonance spectroscopy, but the equilibrium binding parameters of acetylcholine to its receptor were unaltered. This suggests strongly that there exist two pools of cholesterol in the AChR-rich Torpedo electroplax membrane: an easily depleted fraction that influences bulk fluidity, and a tightly-bound fraction perhaps surrounding the AChR oligomer.

Immediate effects of lung transplantation on right ventricular morphology and function in patients with variable degrees of pulmonary hypertension.

OBJECTIVES: This study sought to determine the immediate effects of lung transplantation on right ventricular morphology and function in patients with variable degrees of pulmonary hypertension and to evaluate these features as potential markers of immediate outcome. BACKGROUND: Selected lung transplant recipients with severe preoperative pulmonary hypertension have previously been shown to have a reduction in right ventricular size and improved function at follow-up evaluation. METHODS: Thirty-two consecutive patients (mean [+/- SD] age 44 +/- 11 years) were prospectively classified into three groups according to their pretransplantation pulmonary artery systolic pressure: severe pulmonary hypertensive group > or = 75 mm Hg, intermediate pulmonary hypertensive group 40 to 74 mm Hg and non-pulmonary hypertensive group < 40 mm Hg. Hemodynamic and transesophageal echocardiographic variables were measured immediately before and after lung transplantation. RESULTS: Pulmonary artery systolic and mean pressures markedly decreased after transplantation in the severe pulmonary hypertensive group (from 115 +/- 26 to 45 +/- 19 mm Hg and from 76 +/- 14 to 31 +/- 11 mm Hg, respectively, both p < 0.05). Mean pulmonary artery pressure decreased in the intermediate group (from 34 +/- 7 to 26 +/- 7 mm Hg, p < 0.05). Right ventricular end-diastolic area, end-systolic area and eccentricity index decreased in the severe pulmonary hypertensive group after transplantation. End-diastolic area also decreased in the intermediate pulmonary hypertensive group. Right ventricular fractional area change was not significantly different between groups and did not change consistently after transplantation. Three patients with severe pulmonary hypertension who had continued depression of right ventricular function after transplantation died in the immediate postoperative period. CONCLUSIONS: Lung transplantation is associated with an immediate decrease in pulmonary artery pressures and right ventricular size and normalization of septal geometry but variable changes in right ventricular function. Continued depression of right ventricular fractional area change may be a potential marker of poor outcome.

Alcohol and anesthetic mechanisms in genetically engineered mice.

Genetically engineered animals (e.g., transgenics, gene knockouts, gene knockins) are being utilized with increasing frequency to investigate the mechanisms of action of alcohol and anesthetics. By creating and analyzing animals that harbor precise, preplanned changes in candidate genes, researchers are rapidly making progress toward uncovering how these drugs exert their effects on the central nervous system to bring about their behavioral effects. Since these sedative / hypnotic agents are likely to exert their effects by altering neurotransmission, the majority of investigations to date have focused on neurotransmitter receptors and modulators of neurotransmission such as kinases.

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.

Sensory thresholds and the antinociceptive effects of GABA receptor agonists in mice lacking the beta3 subunit of the GABA(A) receptor.

A line of mice was recently created in which the gabrb3 gene, which encodes the beta3 subunit of the GABA(A) receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA(A) receptors in the CNS enabled an investigation of the role of GABA and GABA(A) receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA(A) and GABA(B) receptor agonists. Homozygous null (beta3-/-) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (beta3+/+) and heterozygous (beta3+/-) littermates. The beta3-/- mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their beta3+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in beta3-/- mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA(A) receptors in the spinal cord. As expected, subcutaneous administration of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in beta3-/- mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, beta3+/+ and beta3+/- mice. However, the antinociceptive effect of the GABA(B) receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in beta3-/- mice compared to the progenitor strains, beta3+/+ or beta3+/- mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA(A) receptors can affect the production of antinociception by other analgesic drugs as well.

Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.

1. We have redetermined the anaesthetic potencies (EC50S) for a series of primary alkanols, to resolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use of data from different laboratories. 2. For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were plotted for over one hundred tadpoles, and the median effective concentrations determined. Aqueous concentrations present during potency assays were determined independently, and for alkanols with chain length greater than nonanol, correction was made for depletion from the aqueous phase. 3. The EC50S were found to decrease logarithmically with increasing number of carbon atoms in the hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene group was associated with an approximately four fold increase in potency. 4. The relationship between log EC50 and CN was best described by the quadratic equation, log EC50 = 0.022 (+/- 0.0038) CN2 + 0.76 (+/- 0.051) CN + 3.7 (+/- 0.14) (r2 = 0.9951). 5. A previously described correlation between the apparent changes in the free energy of binding of an additional methylene group both to luciferase and to the sites for LRR in tadpoles was not confirmed. 6. A cut-off in potency beyond dodecanol was established in experiments where tadpoles were maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR. 7. These findings indicate that the soluble enzyme firefly luciferase does not adequately model the anaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparent changes in the free energy of binding, per methylene group, of an alkanol to luciferase do not parallel that for tadpoles.

Preoperative risk models for minimally invasive coronary bypass: a preliminary study.

OBJECTIVE: Available risk assessment models are designed for standard coronary artery bypass grafting. We hypothesized that minimally invasive coronary bypass could improve on predicted outcome in extremely high-risk patients (Parsonnet score > 20%) by the current risk models. METHODS: From September 1996 to September 1997, 27 consecutive extremely high-risk patients underwent minimally invasive coronary bypass. Seventeen patients were male; age was 73 +/- 12 years, and 63% of patients were older than 75 years. Left ventricular ejection fraction was 33.7% +/- 15% and 63% had an ejection fraction of less than 35%. The predicted 30-day mortality according to the System 97 model was 25.6% +/- 11.3%. The Parsonnet risk score was 36.2% +/- 11%; the predicted length of stay in the hospital was 15.3 +/- 3 days. The predicted risk of stroke according to the Multicenter Perioperative Stroke Risk Index was 22.3% +/- 11.7%. RESULTS: Minimally invasive coronary bypass was isolated in 20 patients and integrated with angioplasty and stenting in 7 patients. The observed 30-day mortality was 0% (P < .01 vs predicted): at an average follow-up of 10.8 +/- 4.1 months, 26 patients (96.3%) are alive without angina; one patient with acquired immunodeficiency syndrome died on postoperative day 40 of acute pancreatitis. No patient had a stroke or neurologic deficit (P < .01 vs predicted). Patency of internal thoracic artery anastomosis was confirmed by angiography in all 27 patients. No patient required reoperation. Eighteen patients (67%) were extubated in the operating room. The observed length of hospital stay after minimally invasive coronary bypass was 3.8 +/- 2.6 days (P < .01 vs predicted). CONCLUSION: On the basis of our results on a relatively small series of patients, we suggest that risk models geared for standard coronary bypass grafting may not be appropriate for minimally invasive coronary bypass.

Platelet membrane fluidity individuals at risk for Alzheimer's disease: a comparison of results from fluorescence spectroscopy and electron spin resonance spectroscopy.

RATIONALE: Previous fluorescence studies employing 1,6-diphenyl-1,3,5-hexatriene (DPH) have revealed an increase in the fluidity of platelet membranes from individuals with Alzheimer's disease (AD) and their first-degree relatives. This biophysical alteration has been reported to be relatively specific for the hydrocarbon core of platelet membranes, where DPH preferentially localizes; this effect is not reflected by the fluorescent reporter triethylamino-DPH, which labels membranes at the lipid-aqueous interface. OBJECTIVE: The goal of this study was to explore the validity and reproducibility of these findings using an independent biophysical technique, electron spin resonance (ESR) spectroscopy. METHODS: Platelet membranes prepared from first-degree relatives of patients with AD were labeled with DPH, or the spin-labeled fatty acid probes 5-doxylstearate (5-DS) and 12-doxylstearate (12-DS). These spin labeled probes provide an index of structural order at the respective depths of their nitroxide moieties in the membrane. The resulting preparations were examined by fluorescence and ESR spectroscopy. RESULTS: Increased platelet membrane fluidity (PMF), as determined by the fluorescence anisotropy of DPH, was associated with only a modest reduction in the order parameter derived for 5-DS labeled membranes. In contrast, the mean order parameters derived from the paired samples labeled with 12-DS differed substantially from each other, and revealed decreased order (increased fluidity) in the hydrocarbon 12-C region where DPH preferentially localizes. CONCLUSIONS: These results provide an independent validation of the biophysical alterations of platelet membranes that are manifested by a subgroup of patients with AD and their first-degree relatives.

Inhaled nitric oxide partially reverses hypoxic pulmonary vasoconstriction in the dog.

Nitric oxide (NO) inhaled during a hypoxia-induced increase in pulmonary vasomotor tone decreases pulmonary arterial pressure (Ppa). We conducted this study to better characterize the hemodynamic effects induced by NO inhalation during hypoxic pulmonary vasoconstriction in 11 anesthetized ventilated dogs. Arterial and venous systemic and pulmonary pressures and aortic flow probe-derived cardiac output were recorded, and nitrosylhemoglobin (NO-Hb) and methemoglobin (MetHb) were measured. The effects of 5 min of NO inhalation at 0, 17, 28, 47, and 0 ppm during hyperoxia (inspiratory fraction of O2 = 0.5) and hypoxia (inspiratory fraction of O2 = 0.16) were observed. NO inhalation has no measurable effects during hyperoxia. Hypoxia induced an increase in Ppa that reached plateau levels after 5 min. Exposure to 28 and 47 ppm NO induced an immediate (< 30 s) decrease in Ppa and calculated pulmonary vascular resistance (P < 0.05 each) but did not return either to baseline hyperoxic values. Increasing the concentration of NO to 74 and 145 ppm in two dogs during hypoxia did not induce any further decreases in Ppa. Reversing hypoxia while NO remained at 47 ppm further decreased Ppa and pulmonary vascular resistance to baseline values. NO inhalation did not induce decreases in systemic arterial pressure. MetHb remained low, and NO-Hb was unmeasurable. We concluded that NO inhalation only partially reversed hypoxia-induced increases in pulmonary vasomotor tone in this canine model. These effects are immediate and selective to the pulmonary circulation.

Effect of inhaled nitric oxide on pulmonary hemodynamics after acute lung injury in dogs.

Increased pulmonary vascular resistance (PVR) and mismatch in ventilation-to-perfusion ratio characterize acute lung injury (ALI). Pulmonary arterial pressure (Ppa) decreases when nitric oxide (NO) is inhaled during hypoxic pulmonary vasoconstriction (HPV); thus NO inhalation may reduce PVR and improve gas exchange in ALI. We studied the hemodynamic and gas exchange effects of NO inhalation during HPV and then ALI in eight anesthetized open-chest mechanically ventilated dogs. Right atrial pressure, Ppa, and left ventricular and arterial pressures were measured, and cardiac output was estimated by an aortic flow probe. Shunt and dead space were also estimated. The effect of 5-min exposures to 0, 17, 28, 47, and 0 ppm inhaled NO was recorded during hyperoxia, hypoxia, and oleic acid-induced ALI. During ALI, partial beta-adrenergic blockade (propranolol, 0.15 mg/kg i.v.) was induced and 74 ppm NO was inhaled. Nitrosylhemoglobin (NO-Hb) and methemoglobin (MetHb) levels were measured. During hyperoxia, NO inhalation had no measurable effects. Hypoxia increased Ppa (from 19.8 +/- 6.1 to 28.3 +/- 8.7 mmHg, P < 0.01) and calculated PVR (from 437 +/- 139 to 720 +/- 264 dyn.s.cm-5, P < 0.01), both of which decreased with 17 ppm NO. ALI decreased arterial PO2 and increased airway pressure, shunt, and dead space ventilation. Ppa (19.8 +/- 6.1 vs. 23.4 +/- 7.7 mmHg) and PVR (437 +/- 139 vs. 695 +/- 359 dyn.s.cm-5, P < 0.05) were greater during ALI than during hyperoxia. No inhalation had no measureable effect during ALI before or after beta-adrenergic blockade. MetHb remained low, and NO-Hb was unmeasurable. Bolus infusion of nitroglycerin (15 micrograms) induced an immediate decrease in Ppa and PVR during ALI.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma colloid oncotic pressure-pulmonary artery occlusion pressure gradient. A poor predictor of pulmonary edema in surgical intensive care unit patients.

Because Starling's equation contains four factors that theoretically influence fluid movement across the pulmonary capillary bed, we prospectively examined the relationship between the plasma colloid oncotic pressure (PCOP) minus the mean pulmonary artery occlusion pressure (PAOP) (the only two presently available clinically for measurement) and pulmonary edema determined in terms of percent venous admixture (Qs/Qt) and roentgenographically in 17 consecutive patients in a surgical intensive care unit to determine whether this PCOP-PAOP gradient could accurately predict the presence of pulmonary edema. The PCOP-PAOP gradient proved to be a poor predictor of pulmonary edema determined by these means. We believe this resulted from the inherent insensitivity of the PCOP-PAOP gradient as an estimate of the net intravascular filtration pressure, and Qs/Qt and roentgenograms as measurements of lung edema, as well as the multiplicity of variables involved in the genesis of pulmonary edema in the clinical situation.

Transcutaneous oxygen and carbon dioxide monitoring. Use in adult surgical patients in an intensive care unit.

We evaluated a combined transcutaneous oxygen and carbon dioxide (tcPO2 and tcPO2) monitor in 33 adult surgical patients in an intensive care unit. Surgical procedures included cardiothoracic, general, vascular, and orthopedic operations. Ninety-three paired, transcutaneous values were compared with simultaneously determined arterial blood gas measurements. The correlation coefficient for PaO2 was .75 (P less than .01) whereas for PaCO2 it was .55 (P less than .01). Although statistically significant, individual transcutaneous values differed by as much as 50 mm Hg from levels determined from arterial samples. Such differences make it inappropriate to use this monitor to predict actual PaO2 or PaCO2. By contrast, continuous monitoring allowed observation of acute changes associated with respiratory-care procedures and cardiovascular decompensation. Such acute changes changes were appreciated on the tcPO2 tracing but not the tcPO2 tracing. We conclude that the tcPO2 monitor is a valuable trend indicator of cardiopulmonary dysfunction, but that the tcPCO2 monitor is not.

Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam.

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain gamma-aminobutyric acidA (GABAA) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABAA-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABAA-receptors would also exhibit a differential response to halothane as assayed by an in vitro 36Cl- influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 mumol/l GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC50 336 +/- 64 mumol/l (S.E.M.) vs. 605 +/- 110 mumol/l, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7 +/- 0.4 nmol.mg-1 x 3 s-1, vs. DR 4.7 +/- 0.5 nmol.mg-1 x 3 s-1). Halothane (500 mumol/l) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC50 for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC50 1.8 +/- 0.4 mumol/l vs. 14.7 +/- 0.9 mumol/l without halothane) than in the DR mice (GABA EC50 4.7 +/- 0.6 mumol/l vs. 14.7 +/- 0.9 mumol/l without halothane).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Genetic dissection of the molecular target(s) of anesthetics with the gene knockout approach in mice.

Techniques have recently been developed that enable the creation of mice that harbor specific, predetermined genetic changes. These 'gene knockout mice', which contain a single genetic modification that is determined by the investigator, can subsequently be analyzed with tests that span the molecular, cellular, and behavioral levels. Application of such a multi-level approach to mechanisms of drug action should ultimately allow general anesthetic responses to be properly attributed to a molecular site.

Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors.

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.

Mice lacking the long splice variant of the gamma 2 subunit of the GABA(A) receptor are more sensitive to benzodiazepines.

The gamma 2 subunit is required for benzodiazepine modulation of the GABA(A) receptor. Alternate splicing of precursor GABA(A) gamma 2 mRNA results in two splice variants, a short (gamma 2S) and a long (gamma 2L) variant. We investigated the roles of these splice variants in benzodiazepine pharmacology using mice lacking genes for the gamma 2L splice variant. Sleep time responses to midazolam and zolpidem were 20 and 18% greater, respectively, in null allele mice compared with wild-type mice, while responses to nonbenzodiazepine agents such as etomidate and pentobarbital were unchanged. Although the GABA(A) receptor number was not altered in null allele mice, there was a corresponding increase in affinity of brain membranes for benzodiazepine agonists (midazolam, diazepam, and zolpidem), while affinity for benzodiazepine inverse agonists (beta CCM and Ro15-4513) was decreased. These changes were not observed in inbred mice of the parental strains (C57BL/6J and 129/SvJ) used to create the genetically altered mice, indicating that differences between gamma 2L null allele and wild-type mice were unlikely to be simply due to cosegregation of linked alleles. Absence of the gamma 2L splice variant increases the affinity of receptors for benzodiazepine agonists, and is associated with a modest increase in behavioral sensitivity to benzodiazepine agonists. Lack of the gamma 2L subunits may shift the GABA(A) receptor from an inverse agonist-preferring toward an agonist-preferring configuration.

Pharmacologic and behavioral responses of inbred C57BL/6J and strain 129/SvJ mouse lines.

Gene-targeting technology is creating an explosion in the number of animals available with single gene mutations that affect the function of the central nervous system. Most gene-targeted mice are produced on a mixed genetic background of C57BL/6J and substrains of Strain 129. Understanding the behavioral characteristics and responses to various drugs of these parental strains is vital to interpreting data from gene-targeted mice. We directly compared C57BL/6J and Strain 129/SvJ mouse lines on several behavioral paradigms and in response to several hypnotic and anesthetic drugs. Compared to Strain 129/SvJ mice, C57BL/6J animals are more sensitive to the hypnotic effects of midazolam, zolpidem, and propofol, less sensitive to etomidate and ethanol, and do not differ in sensitivity to Ro15-4513 or pentobarbital. These strains do not differ in their sensitivity to the motor ataxic effects of the volatile anesthetics enflurane or halothane. However, Strain 129/SvJs are more sensitive to the immobilizing effects of halothane but not enflurane. Motor coordination differs initially, but with repeated testing strain differences are no longer apparent. Strain 129/SvJ mice are more anxious on the elevated plus maze and open-field activity assays. Thus, these mouse strains harbor polymorphisms that influence some, but not all, traits of interest to behavioral neuroscientists.