Direct evidence for a cause-effect link between ethanol potentiation of GABA(A) receptor function and intoxication from hyperbaric studies in C57, LS, and SS mice

BACKGROUND: This article uses a direct ethanol antagonist, increased atmospheric pressure, to further test the causative link between ethanol potentiation of gamma-aminobutyric acid (GABA) type A receptor function and ethanol's behavioral effects. This was done by determining whether initial biochemical findings in long-sleep (LS) mice extended to other genotypes and whether the previously reported insensitivity of short-sleep (SS) mice to pressure antagonism of ethanol-induced loss of righting reflex extended to a nonselected ethanol-induced behavior. METHODS: The effects of 12 times normal atmospheric pressure of helium-oxygen gas (heliox) versus ethanol (25-200 mM) potentiation of GABA-activated Cl- uptake in brain membranes (microsacs) from C57, LS, and SS mice were tested by using a 36Cl- flux assay. The effects of pressure versus ethanol's (2 g/kg) anticonvulsant effect in SS mice were tested by using time to onset of isoniazid-induced myoclonic seizures. RESULTS: Exposure to 12 times normal atmospheric pressure heliox antagonized ethanol potentiation of GABA-activated Cl- uptake in all three genotypes across a range of ethanol concentrations that cause ethanol's behavioral and anesthetic effects. Pressure did not affect baseline receptor function. The threshold for initiating ethanol potentiation differed between genotypes in accordance with their behavioral sensitivities to ethanol (C57 and LS, < or =25 mM; SS, >50 mM). Pressure antagonized ethanol's anticonvulsant effect in SS mice. CONCLUSIONS: The results add important direct evidence supporting the hypothesis that ethanol potentiation of GABA(A) receptor function is an initial action of ethanol causing its behavioral effects. These findings also provide insight into possible effects of selective breeding on GABA(A) receptor function.     

Ethanol-Induced Changes in Chloride Flux are Mediated by Both GABAA and GABAB Receptors

Low concentrations of ethanol (10-30 mM) in the presence of a GABAB receptor agonist, baclofen, promoted 36Cl- uptake into membrane vesicles (microsacs) prepared from mouse cortex. Neither ethanol nor baclofen alone altered chloride influx. The GABAB antagonists, phaclofen and 2-hydroxy-saclofen, completely blocked the increase in chloride flux produced by ethanol in the presence of either baclofen or GABA. Ethanol increased the chloride conductance produced by the GABAA agonists muscimol, isoguvacine, imidazolacetic acid and amino-propane sulfonic acid and this action of ethanol was blocked by phaclofen. The specific GABAA antagonist, bicuculline, blocked ethanol-induced increase in chloride flux in the presence of either baclofen or GABA. GABA-activated chloride channels were also studied in Xenopus oocytes expressing mouse brain mRNA. In this preparation, GABA action was enhanced by ethanol, pentobarbital, and diazepam, and 2-hydroxy-saclofen partially antagonized the action of ethanol without altering the effects of pentobarbital or diazepam. These results suggest that ethanol enhancement of GABAA receptor-chloride channel function also requires activation of GABAB receptors.     

Brain Region-Dependent Sensitivity of GABAA Receptor-Mediated Responses to Modulation by Ethanol

Simultaneous extracellular and intracellular electrophysiological recordings were made from the CA1 region of rat hippocampal brain slices during superfusion with ethanol. Ethanol (80 mM) had a biphasic effect on the extracellularly recorded population spike, with an initial increase followed by a significant reduction (38%) in this response, which was maximal 10 to 15 min after the start of ethanol application. Concurrent intracellular recordings in the CA1 showed a small (0.7 mV) hyperpolarization of the resting membrane potential, with no significant change in the input impedance, EPSP, GABAA and GABAB IPSPs, or after hyperpolarization (AHP) following depolarizing current injection. Ethanol reduced the amplitude and duration of depolarizing responses to brief, localized pressure-ejection of N-methyl-D-aspartate (NMDA) onto pyramidal neuron dendrites, but did not affect the GABAA receptor-mediated depolarizing responses to the dendritic application of GABA. In parallel studies, the effect of ethanol on GABA-stimulated 36Cl- flux was measured in microsac preparations from rat hippocampus, cerebellum, and cerebral cortex. Ethanol application caused substantial enhancement of the chloride uptake from cerebellar and cerebral cortical microsacs, but had no effect on 36Cl- influx in hippocampal microsacs. These results suggest that there are important brain region-dependent differences in the sensitivity of the GABAA receptor/chloride channel to modulation by ethanol.     

Differences in Ethanol Sensitivity of Brain NMDA Receptors of Long-Sleep and Short-Sleep Mice

Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred mice that differ in the duration of anesthesia produced by an acute dose of ethanol, were used to determine the possible association of differing ethanol sensitivity of brain NMDA receptors with differing sensitivity to the anesthetic effects of ethanol in vivo. NMDA receptor-mediated responses were determined by measurement of l -glutamate-stimulated increases in free intracellular calcium concentration (Ca_l) using the fluorescent indicator for Ca₁, Indo 1, in microsacs (a cell-free brain membrane vesicle preparation) isolated from hippocampi or cerebral cortices of the two mouse lines. In the absence of added drugs, NMDA responses did not differ between the two lines in hippocampal or cerebrocortical microsacs. However, a high concentration of ethanol (200 m_m) inhibited NMDA responses in hippocampal microsacs from LS mice. In contrast, a moderate concentration of ethanol (50 m_m) stimulated NMDA responses in hippocampal microsacs isolated from SS mice. In cerebrocortical microsacs, ethanol inhibited NMDA responses in the two lines to an equivalent degree. MK-801, a noncompetitive blocker of NMDA receptors, blocked NMDA responses at lower concentrations in hippocampal microsacs from LS mice than in SS mice, but produced a similar degree of inhibition of NMDA responses in cerebrocortical microsacs from the two lines. A high concentration of ethanol (200 mm ) increased resting Ca₁ in hippocampal microsacs from LS mice but not in hippocampal microsacs from SS mice, and increased resting Ca_l in cerebrocortical microsacs isolated from both lines of mice equally. The small change in resting Ca, produced by MK-801 in cerebrocortical microsacs did not differ between the two lines. These results show that hippocampal NMDA receptors of LS and SS mice differ in their sensitivity to ethanol, possibly because of differences in allosteric modulation at the MK-801 site or some other site that interacts with the MK-801 site of the NMDA receptor.     

Effects of Ethanol and Temperature on NMDA Receptor Function in Different Mouse Genotypes

The present study investigated whether temperature-related changes in NMDA receptor sensitivity to ethanol might play a role in mediating the effects of body temperature on behavioral sensitivity to ethanol or in determining genotypic differences in sensitivity to ethanol. We accomplished this by determining the effects of ethanol on three different mouse genotypes (C57, LS, and SS) on two types of NMDA receptor-mediated responses at 30° and 35°C (i) extracellularly recorded synaptic potentials elicited in the CA1 region of the in vitro hippocampal slice preparation by stimulation of the Schaffer-commisural pathway in the presence of the cu-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker, 6,7-dinitroqui-noxaline-2,3-dione, and low magnesium concentration; and (ii) increase in [³H]MK-801 binding elicited by glutamate in telencephalic membrane preparations. Ethanol significantly decreased NMDA receptor-mediated excitatory postsynaptic potential (EPSP) amplitude and area in the three genotypes. In C57, the effect of ethanol on NMDA receptor-mediated EPSP amplitude and area was more pronounced at 30°C, compared with that at 35°C. In most cases, there was a good correlation between the effects of ethanol on EPSP amplitude and area. The order of sensitivity between the three genotypes was C57 = LS > SS at 35°C and C57 > LS = SS at 30°C. Similarly, ethanol significantly decreased glutamate-stimulated [³H]MK-801 binding in membrane fractions. The effect of ethanol was temperature-dependent, because ethanol produced more inhibition at 30°C than at 35°C in all genotypes. The effect of ethanol on MK-801 binding was concentration-dependent, and the sensitivity to 100 mM ethanol of the genotypes at 35°C was LS > SS = C57, whereas it was SS > LS = C57 at 30°C. Collectively, the results demonstrate that temperature is an important variable that can influence NMDA receptor sensitivity to ethanol measured via electro-physiological and binding techniques, and that temperature can influence relative sensitivity of NMDA receptors to ethanol between mouse genotypes. Furthermore, the findings indicate that temperature-induced changes in sensitivity of NMDA receptors to ethanol may play a role in mediating the effects of body temperature on behavioral sensitivity to ethanol in LS, but not C57 and SS mice.     

Ethanol Sensitivity of Brain NMDA Receptors in Mice Selectively Bred for Differences in Response to the Low-Dose Locomotor Stimulant Effects of Ethanol

Brain NMDA receptor responses and their sensitivity to ethanol in vitro were determined in replicate lines of FAST and SLOW mice, selectively bred for differences in sensitivity to the locomotor stimulant effects of a low dose of ethanol. L-Glutamate-stimulated increases in the intracellular free calcium concentration (Ca₁) were determined in microsacs, a cell-free brain membrane preparation, isolated from hippocampus or cerebral cortex. Previous work showed that l -glutamate-stimulated increases in Ca₁ in microsacs are mediated by activation of NMDA receptors. The concentration response for l -glutamate-stimulated increases in Ca, did not differ between the lines in either hippocampal or cerebrocortical microsacs. Ethanol produced a concentration-dependent decrease in l- glutamate-stimulated increase in Ca₁ in hippocampal and cerebrocortical microsacs from SLOW mice, but this effect of ethanol was reduced or absent in microsacs isolated from FAST mice. Resting Ca₁ and the ability of a high ethanol concentration to increase resting Ca₁ did not differ between the lines. These results suggest that differences in the sensitivity of brain NMDA receptors to the effects of ethanol determine, at least in part, differences in the locomotor stimulant effects of low doses of ethanol in FAST and SLOW mice. These differences are not due to ethanol effects on resting Ca₁.     

Effects of 6-Hydroxydopamine on Brain Catecholamines and on Acute Actions of Ethanol in LS/Ibg and SS/Ibg Mice

LS/Ibg (LS) and SS/Ibg (SS) mice differ in ethanol-induced duration of loss of righting response or sleep time, hypothermia, hyperglycemia, and blood ethanol concentrations at regaining righting response. These differences in response to ethanol are a result of differences in central nervous system sensitivity and are mediated by polygenic systems. Studies have indicated that catecholaminergic systems may be involved in the differential effects of ethanol in LS and SS lines of mice (Masserano JM, Weiner N: Investigations into the neurochemical mechanisms mediating differences in ethanol sensitivity in two lines of mice. J Pharmacol Exp Ther 221:404-408, 1982). In this study the neurotoxin, 6-hydroxydopamine (6-OHDA), intracerebroventricular, was used to test this hypothesis. Administration of 6-OHDA markedly altered thermoregulation in LS mice but produced little effect in SS mice, and ethanol-induced hyperglycemia was attenuated in both LS and SS mice by 6-OHDA. Ethanol-induced sleep time was increased in SS mice pretreated with 100 micrograms of 6-OHDA, intracerebroventricular, whereas this response in LS mice was unaffected by 6-OHDA administration. Changes in sleep time were not related to changes in blood ethanol concentrations, indicating that 6-OHDA alters ethanol-induced sleep time by mechanisms other than brain sensitivity. Levels of norepinephrine and dopamine were determined in three brain regions, and the altered capacities for thermoregulation and glucoregulation were associated with changes in hypothalamic catecholamine levels.     

Effect of Chronic Ethanol Treatment and Selective Breeding for Hypnotic Sensitivity to Ethanol on Intracellular Ionized Calcium Concentrations in Synaptosomes

The effect of chronic ethanol treatment and of selective breeding for hypnotic sensitivity to ethanol on intracellular ionized calcium concentrations (Cai) were examined in mouse whole brain synaptosomes. Following treatment with a liquid diet for 7 days, resting Cai and KCl-stimulated increases in Cai were measured in synaptosomes isolated from chronic ethanol-treated and pair-fed animals. Ethanol (350-700 mM) increased resting Cai and reduced KCl-stimulated increases in Cai in synaptosomes isolated from pair-fed animals. Ethanol-induced changes in Cai were reduced in synaptosomes isolated from chronic ethanol-treated animals. The effect of ethanol on synaptosomal Cai in long-sleep (LS) and short-sleep (SS) mice, selectively bred for differential sensitivity to the hypnotic actions of acute ethanol, was also investigated. In the absence of ethanol, resting values of Cai and KCl-stimulated increases in Cai did not differ between the two lines of mice. Ethanol (200-600 mM) increased resting Cai and reduced depolarization-stimulated increases in Cai in both long-sleep and short-sleep mice to the same degree. Similarly, KCl-stimulated increases in Ca uptake did not differ in synaptosomes isolated from whole brains and cortices of LS and SS mice, in the absence of presence of ethanol. These findings demonstrate that tolerance develops to the effect of ethanol on neuronal Cai following chronic treatment. However, sensitivity to the hypnotic action of ethanol is not related to changes in neuronal Cai in LS and SS mice.     

Relationship of brain ethanol metabolism to the hypnotic effect of ethanol. II: Studies in selectively bred rats and mice

BACKGROUND: To clarify the role of brain acetaldehyde in the hypnotic effect of ethanol, we compared the ethanol-oxidizing capacity (rate of acetaldehyde accumulation) and catalase and aldehyde dehydrogenase activity in the brains of animals genetically selected for different sensitivities to the hypnotic effect of ethanol. METHODS: We used high, low, or control alcohol-sensitive rats (HAS, LAS, and CAS) and short- and long-sleep mice (SS and LS), as well as SS x LS recombinant inbred mice with known strain differences in mean duration of ethanol-induced sleep. We studied the rate of accumulation of acetaldehyde from ethanol in brain homogenates of these animals and correlated those values with their hypnotic sensitivity to ethanol. RESULTS: Acetaldehyde accumulation from ethanol was significantly higher in the brain homogenates from HAS rats and LS mice with high sensitivity to the hypnotic effect of ethanol in vivo, compared with LAS rats and SS mice with low sensitivity to ethanol. A correlation was found between the duration of ethanol-induced sleep and the in vitro rate of accumulation of ethanol-derived acetaldehyde in the brains of recombinant SS x LS mice strains. There was no correlation of sleep time with brain catalase levels. There were no line differences in brain catalase or aldehyde dehydrogenase or in alcohol or aldehyde dehydrogenase activity in livers of LAS, CAS, and HAS rats or in SS and LS mice. CONCLUSIONS: A correlation between the brain acetaldehyde accumulation, but not catalase levels, and the central effect of ethanol was demonstrated in animals genetically differing in initial sensitivity to the hypnotic effect of ethanol.     

Influence of Thyrotropin-Releasing Hormone and Catecholaminergic Interactions on CNS Ethanol Sensitivity

The role of catecholamine neuronal systems in mediating the analeptic and thermogenic effects of thyrotropin-releasing hormone (TRH) was examined in long-sleep (LS) and short-sleep (SS) mice. TRH [0.1 to 40 μg, intracerebroventricularly (icv)] was associated with a reduction in the sleep times of LS mice, but no dose of TRH had any effect on sleep times of SS mice. However, TRH (20 μg, icv) produced a 1.0° to 1.5°C attenuation of the ethanol-induced hypothermia in both LS and SS mice. TRH did not change the rate of ethanol elimination in either line of mice, suggesting that the reduction in LS sleep times and attenuation of LS and SS hypothermia were due to decreased CNS ethanol sensitivity rather than an increase in the rate of ethanol metabolism. TRH (20 μg, icv) given alone produced an activation of central and peripheral catecholamine systems in LS, but not SS mice, as reflected by an increase in the invivo tyrosine hydroxylase (TH) activity in the brain and adrenal gland. TRH, given with ethanol, prevented or attenuated ethanol-induced decreases in the brain and adrenal gland in vivo TH activity in LS mice but not SS mice. Thus, there was an association between the ability of TRH to produce an activation of catecholamine neuronal systems (increased rate of catecholamine biosynthesis) and the analeptic action of TRH to reduce the CNS depressant effects of ethanol (decreased sleep times). TRH was able to attenuate the hypothermic effect of ethanol in both LS and SS mice, despite a lack of effect on TH activity, suggesting that catecholamine neurons may not have a major role in the thermogenic action of TRH in LS and SS mice.     

Ethanol Teratogenesis in Selectively Bred Long-Sleep and Short-Sleep Mice: A Comparison to Inbred C57BL/6J Mice

Sensitivity to alcohol may influence the severity of ethanol teratogenesis. To examine this hypothesis, the teratogenic effects of ethanol were compared in Long-Sleep (LS) and Short-Sleep (SS) mice, selectively bred for differences in ethanol-induced narcosis. Inbred C57BL/6J (B6) mice were included to confirm previously reported teratogenic effects using our own treatment regimen and standard assessment technique. Intragastric administration of ethanol (5.8 g/kg) on Days 9 and 10 of pregnancy resulted in growth retardation and an increase in prenatal mortality in LS litters but not in SS litters. Therefore, alcohol sensitivity plays a role in the severity of prenatal alcohol effects. B6 mice showed more ethanol teratogenicity than either LS or SS mice, even though maternal blood ethanol levels were similar across genotypes. This result suggests genetic variations other than alcohol sensitivity also influence ethanol teratogenesis.     

Differences in NMDA Receptor Antagonist-Induced Locomotor Activity and [3H]MK-801 Binding Sites in Short-Sleep and Long-Sleep Mice

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl d -aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater loco-motor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [³H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [³H]MK-801 and [³H]CPP and brain levels of [³H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [³H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.     

Effect of Exogenous GM1 on Ethanol Sensitivity in Selectively Bred Mouse Lines

Ethanol sensitive long-sleep (LS) and ethanol resistant short-sleep (SS) mice are lines that have been genetically selected for differential central nervous system sensitivities to the hypnotic effect of ethanol. Because they were genetically selected only for differences in sensitivity to ethanol hypnosis, biochemical and physiological differences between them are likely related to their differential ethanol sensitivity. The synaptosomal and whole brain concentration of GM1 ganglioside was previously shown to differ significantly between the lines. Further, GM1 alters membrane responses to ethanol, including a differential effect on LS and SS synaptosomal membrane disordering. Therefore, GM1 was administered intracerebroventricularly (icv) with micro-osmotic pumps, to partially bypass the blood-brain barrier and to test its effect on CNS sensitivity to ethanol hypnosis in LS and SS mice. In the first experiment, 3 days' infusion of GM1 (20 μg/μl, 24 μl/day), saline control and treated LS and SS mice were tested for both regaining of the righting reflex and waking brain ethanol concentration. Incorporation of ³H-GM1 into brain membranes was verified by scintillation spectroscopy. GM1 did not alter ethanol sensitivity or brain ethanol concentration at time of waking in LS mice. Conversely, SS mice treated with GM1 were significantly more sensitive to ethanol hypnosis than saline controls as measured by the time to regain the righting reflex (“sleep time”? and waking brain ethanol concentrations. In the second experiment, GM1-treated SS mice were again significantly more sensitive to ethanol hypnosis than saline controls. GM1 incorporation into the contralateral and ipsilateral cerebral hemispheres was determined by high-performance liquid chromatography.     

Genetically Determined Differences in the Antagonistic Effect of Pressure on Ethanol-Induced Loss of Righting Reflex in Mice

Hyperbaric exposure antagonizes ethanol's behavioral effects in a wide variety of species. Recent studies indicating that there are genetically determined differences in the effects of body temperature manipulation on ethanol sensitivity suggested that genotype might also influence the effects of hyperbaric exposure on ethanol intoxication. To investigate this possibility, ethanol injected long sleep (LS)/Ibg (2.7 g/kg), short sleep (SS)/Ibg (4.8 g/kg), 129/J (2.9 g/kg), and C57BL/6J (3.6 g/kg) mice were exposed to one atmosphere absolute (ATA) air or to one or 12 ATA helium-oxygen (heliox) at ambient temperatures selected to offset ethanol and helium-induced hypothermia. Hyperbaric exposure significantly reduced loss of righting reflex (LORR) duration in LS, 129, and C57 mice, but not in SS mice. A second experiment found that hyperbaric exposure significantly reduced LORR duration and increased the blood ethanol concentration (BEC) at return of righting reflex (RORR) in LS mice, but did not significantly affect either measure in SS mice. These results indicate that exposure to 12 ATA heliox antagonizes ethanol-induced LORR in LS, 129 and C57 mice, but not in SS mice. Taken with previous results, the present findings suggest that the antagonism in LS, 129, and C57 mice reflects a pressure-induced decrease in brain sensitivity to ethanol and that the lack of antagonism in SS mice cannot be explained by pressure-induced or genotypic differences in ethanol pharmacokinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface exposure of synaptosomal gangliosides from long-sleep and short-sleep mice.

A galactose oxidase/NaB[3H]4 technique was used to examine the relative surface exposure of gangliosides from whole brain synaptosomes of long-sleep (LS) and short-sleep (SS) mice. The surface exposure of the monosialoganglioside, GM1, did not differ between the two lines. Surface exposure of the polysialogangliosides GD1a, GD1b, and GT1b, however, was significantly greater in LS synaptosomes than in SS. Hydrolysis of the polysialogangliosides by neuraminidase to the end-product, GM1, at early time periods occurred more rapidly in LS than in SS synaptosomes. Upon exposure to either 250 mM or 50 mM ethanol, LS synaptosomal ganglioside surface exposure was decreased, but that of SS was increased. Pairwise comparisons of the individual ganglioside classes indicated that the decrease in LS synaptosomal ganglioside surface exposure was attributable to decreases in the polysialogangliosides, compared with controls. The ethanol-induced increase in SS synaptosomal ganglioside surface exposure, however, was mainly due to an increased surface exposure of only GD1a. These results suggest that intrinsic differences in the surface exposure of gangliosides and/or the magnitude and direction of ethanol-induced changes in ganglioside surface distribution may reflect biophysical or modulatory mechanisms by which this class of compounds modifies membrane sensitivity to ethanol. These results suggest that further studies should be performed to determine whether gangliosides are factors in genetically determined sensitivity to ethanol.     

The Effects of Chronic Ethanol Ingestion on Ethanol Binding to Hepatic Cytochrome P-450 and on Certain Hepatic and Renal Parameters in the "Long Sleep" and "Short Sleep" Mouse

Male mice selected for genetic differences in ethanol-induced sleep time, thereby designated long sleep (LS) and short sleep (SS), were treated with the Lieber-DeCarli liquid diet for 25 days. This chronic ethanol treatment produced an increase in liver/body weight and kidney/body weight in SS mice only. In addition, chronic ethanol treatment produced significant increases in both LS and SS treated mice in in vivo ethanol elimination, hepatic cytochromes P-450 and B_s, NADPH cytochrome c reductase and hepatic and renal 7-ethoxycoumarin O-de-ethylase activity. Geno-typic differences were observed in the magnitude of response of microsomal ethanol oxidation per mg of microsomal protein (SS > LS). Further, control LS and SS mice possessed substantially different activity of renal 7-ethoxycoumarin O-de-ethylase. Both lines exhibited similar induced renal 7-ethoxycoumarin O-de-ethylase activity after chronic ethanol ingestion. Ethanol binding spectra produced when ethanol was added to hepatic microsomes were examined using double reciprocal plots. Chronic ethanol ingestion produced genotypically related (LS > SS) increases in the absorbance change maximum per mg of microsomal protein. No significant changes in the spectral dissociation constant or absorbance change maximum per nM cytochrome P-450 were observed following ethanol treatment.     

A Genetic Analysis of Ethanol, Pentobarbital, and Methyprylon Sleep-Time Response

The sleep-time responses to ethanol, pentobarbital, and methyprylon were assessed in various generations derived from crossing the long-sleep (LS) and short-sleep (SS) mouse lines in order to assess whether common or different genes regulate response to these agents. The LS and SS mice were selectively bred for differences in duration of ethanol-induced sleep time. Ethanol and pentobarbital responses segregate in a different fashion into F1 and F2 generations derived from the LS and SS lines, indicating different genie control and probably different mechanisms of action for these two agents. Ethanol and methyprylon response segregated similarly but fewer genes seem to influence methyprylon response. These results support the notion that water-soluble depressants have common mechanisms of action.     

Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes.

The effects of ethanol on Cl- uptake were studied using a cell-free subcellular preparation from brain that contains a gamma-aminobutyric acid (GABA)/barbiturate receptor-sensitive Cl- transport system. In isolated vesicles prepared from rat cerebral cortex, ethanol, at concentrations that are present during acute intoxication (20-50 mM), stimulated 36Cl- uptake in a concentration-dependent and biphasic manner. The ethanol-stimulated uptake of 36Cl- was markedly inhibited by the GABA antagonists picrotoxin and bicuculline but not by a variety of other neurotransmitter receptor antagonists. The effects of ethanol in stimulating 36Cl- uptake in isolated brain vesicles were qualitatively and quantitatively similar to that of pentobarbital. Ethanol also markedly potentiated both muscimol- and pentobarbital-stimulated 36Cl- uptake at concentrations below those that directly stimulate 36Cl- uptake. Under our incubation conditions, ethanol did not release GABA, suggesting that it interacts with the postsynaptic GABA/barbiturate receptor complex. The ability of ethanol to stimulate GABA/barbiturate receptor-mediated Cl- transport may explain many of its pharmacological properties and provides a mechanism for the common psychopharmacological actions of ethanol, barbiturates, and benzodiazepines.     

MK-801-induced locomotor activity in long-sleep x short-sleep recombinant inbred mouse strains: correlational analysis with low-dose ethanol and provisional quantitative trait loci

BACKGROUND: Low doses of the N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine) or ethanol increase locomotor activity to a lesser extent in long-sleep (LS), than in short-sleep (SS), mice. LS mice also have fewer brain [3H]MK-801 binding sites than SS mice. In this study, LSXSS recombinant inbred (RI) mice were used to investigate whether different NMDAR densities contribute to differential MK-801 activation and whether common genes are involved in initial sensitivity to MK-801-and ethanol-induced activation. METHODS: Locomotor activity was measured for 90 min after saline or MK-801 injection. Quantitative autoradiographic analysis of [3H]MK-801 binding was used to measure densities of NMDARs in seven brain regions. The ethanol (1-2 g/kg) activation scores from Erwin and colleagues (1997) were used for correlational analysis, as was their method for quantitative trait loci (QTL) analysis. RESULTS: Both saline and MK-801 (0.3 mg/kg, given intraperitoneally) induced a continuum of locomotor responses across the LSXSS RI strains. There was a 4-fold range of MK-801 difference scores (MK-801 score-saline baseline), with the RI 9 and RI 4 strains representing low and high responders, respectively. Dose-response experiments with these two strains confirmed that 0.3 mg/kg MK-801 produced significant activation, similar to previous results with LS and SS mice. However, unlike previous LS/SS results, lower densities of NMDARs were not observed in the RI 9 than in the RI 4 mouse brains. No significant genetic correlations were observed between MK-801-induced and ethanol-induced responses in the LSXSS RI mice. Two provisional MK-801 activation QTLs were identified (p < 0.01) on chromosomes 11 and 19, neither in common with those mapped for ethanol activation. CONCLUSIONS: Different densities of brain NMDARs are unlikely to account for the differential activation of LSXSS RI mice by MK-801. Additionally, in the RI mice either separate sets of genes regulate low dose MK-801- and ethanol-induced locomotor responses or the overlapping subset of genes controlling these two behaviors is small (< or =10%).     

Calcium Differentially Alters Behavioral and Electrophysiological Responses to Ethanol in Selectively Bred Mouse Lines

Sensitivity to the hypnotic action of ethanol has been found to increase in SS/Ibg (SS) but not in LS/Ibg (LS) mice after intracerebroventricular (icv) administration of calcium. In the present investigation, a correlation was found between calcium-induced changes in behavioral sensitivity and in the sensitivity of cerebellar Purkinje neurons to the depressant effects of locally applied ethanol. Cerebellar Purkinje neuron sensitivity was measured as the dose of ethanol pressure ejected from a multibarreled micropipette required to produce a 50% depression of spontaneous firing rate of single neurons. Administration of 0.2-0.4 mumol calcium chloride into the lateral ventricle of the brain increased the sensitivity of SS but not LS mice to the hypnotic behavioral effect of systemically administered ethanol. Similarly, Purkinje neuron sensitivity to locally applied ethanol was also enhanced in SS but not in LS mice 15 min following administration of calcium (0.25 mumol) icv. Furthermore, locally applied ethanol was more effective in depressing spontaneous Purkinje neuron discharge in SS mice when a 1 mM calcium solution was concomitantly pressure ejected with ethanol from the micropipette. Magnesium chloride did not mimic the effects of calcium on either behavioral or electrophysiological effects of ethanol, suggesting that the action of calcium is not a nonspecific effect of divalent cations. These data suggest that calcium-dependent processes may be involved in behavioral and electrophysiological effects associated with ethanol intoxication. Further research will be required to determine if the genetically selected difference in ethanol sensitivity expressed in LS and SS mice is regulated by calcium mechanisms.