Altered Ethanol Sensitivity Research Articles

Estrogen Receptor α Regulates Ethanol Excitation of Ventral Tegmental Area Neurons and Binge Drinking in Female Mice.

Elevations in estrogen (17β-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the VTA and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that, during high E2 states, VTA neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of ER subtypes (ERα and ERβ) in regulating the ethanol sensitivity of VTA neurons in female mice and found that ERα promotes the enhanced ethanol response of VTA neurons. We also demonstrated that enhancement of ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ERα at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus-expressing short hairpin RNAs targeting either ERα or ERβ into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ERα in the VTA had a more dramatic effect on binge-like drinking than reducing ERβ, consistent with the ability of ERα to alter ethanol sensitivity of VTA neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.SIGNIFICANCE STATEMENT Estrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances, such as alcohol. We investigated the mechanisms by which estrogen increases the response of neurons in the VTA to ethanol. We found that activation of the ERα increased the ethanol-induced excitation of VTA neurons. 17β-Estradiol-mediated enhancement of ethanol-induced excitation required the metabotropic glutamate receptor mGluR1. We also demonstrated that ERs in the VTA regulate binge-like alcohol drinking by female, but not male, mice. The influence of ERs on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC.

Adolescents primarily consume alcohol in binges, which can be particularly harmful to the developing frontal cortex and increase risk for an adult alcohol use disorder. We conducted a study investigating immediate and long lasting changes to the prefrontal cortex (PFC) transcriptome to determine the molecular mechanisms underlying adult ethanol behavioral sensitivity following binge ethanol in adolescence. DBA/2J mice were orally dosed with 4 g/kg ethanol intermittently from day 29 to 42. Adolescent mice were tested for anxiety-like behavior and ethanol sensitivity using the loss of righting reflex task. As adults, mice were tested for cognitive changes using the novel object recognition task, ethanol-induced anxiolysis and ethanol sensitivity. Adolescent binge ethanol altered ethanol sensitivity in young mice and led to lasting memory deficits in the object recognition test and greater ethanol sensitivity in adulthood. Using genomic profiling of transcripts in the PFC, we found that binge ethanol reduced myelin-related gene expression and altered chromatin modifying genes involved in histone demethylation at H3K9 and H3K36. We hypothesize that ethanol’s actions on histone methylation may be a switch for future transcriptional changes that underlie the behavioral changes lasting into adulthood.

Intersubunit interactions at putative sites of ethanol action in the M3 and M4 domains of the NMDA receptor GluN1 and GluN2B subunits.

The NMDA receptor is an important target of alcohol action in the brain. Recent studies in this laboratory have demonstrated that alcohol-sensitive positions in the intersubunit interfaces of the M3 and M4 domains of GluN1 and GluN2A subunits interact with respect to ethanol sensitivity and receptor kinetics and that alcohol-sensitive positions in the M domains of GluN2A and GluN2B subunits differ. In this study, we tested for interactions among alcohol-sensitive positions at the M domain intersubunit interfaces in GluN1/GluN2B NMDA receptors. We used whole-cell patch-clamp recording in tsA201 cells expressing tryptophan substitution mutants at ethanol-sensitive positions in the GluN1 and GluN2B NMDA receptor subunits to test for interactions among positions. Six pairs of positions in GluN1/GluN2B significantly interacted to regulate ethanol inhibition: Gly(638) /Met(824) , Gly(638) /Leu(825) , Phe(639) /Leu(825) , Phe(639) /Gly(826) , Met(818) /Phe(637) and Val(820) /Phe(637) . Tryptophan substitution at Met(824) or Leu(825) in GluN2B did not alter ethanol sensitivity but interacted with positions in the GluN1 M3 domain to regulate ethanol action, whereas tryptophan substitution at Gly(638) , which is the cognate of an ethanol-sensitive position in GluN2A, did not alter ethanol sensitivity or interact with positions in GluN1. Two and three pairs of positions interacted to regulate glutamate steady-state and peak current EC50 , respectively, and one pair interacted with respect to macroscopic desensitization. Despite highly-conserved M domain sequences and similar ethanol sensitivity in the GluN2A and GluN2B subunits, the manner in which these subunits interact with the GluN1 subunit to regulate ethanol sensitivity and receptor kinetics differs.

Region-specific disruption of synapsin phosphorylation following ethanol administration in brain-injured mice.

Introduction:Civilians and military personnel develop a range of physical and psychosocial impairments following traumatic brain injury (TBI), including alcohol abuse. As a consequence, increased rates of alcohol misuse magnify TBI-induced pathologies and impede rehabilitation efforts. Therefore, a developed understanding of the mechanisms that foster susceptibility of the injured brain to alcohol sensitivity and the response of the injured brain to alcohol is imperative for the treatment of TBI patients. Alcohol sensitivity has been demonstrated to be increased following experimental TBI and, in additional studies, regulated by presynaptic vesicle release mechanisms, including synapsin phosphorylation.Materials and Methods:Mice were exposed to controlled midline impact of the intact skull and assessed for cortical, hippocampal, and striatal expression of phosphorylated synapsin I and II in response to high-dose ethanol exposure administered 14 days following injury, a time point at which injured mice demonstrate increased sedation after ethanol exposure.Results and Discussion:Immunoblot quantitation revealed that TBI alone, compared to sham controls, significantly increased phosphorylated synapsin I and II protein expression in the striatum. In sham controls, ethanol administration significantly increased phosphorylated synapsin I and II protein expression compared to saline-treated sham controls; however, no significant increase in ethanol-induced phosphorylated synapsin I and II protein expression was observed in the striatum of injured mice compared to saline-treated TBI controls. A similar expression pattern was observed in the cortex although restricted to increases in phosphorylated synapsin II.Conclusion:These data show that increased phosphorylated synapsin expression in the injured striatum may reflect a compensatory neuroplastic response to TBI which is proposed to occur as a result of a compromised presynaptic response of the injured brain to high-dose ethanol. These results offer a mechanistic basis for the altered ethanol sensitivity observed following experimental TBI and contribute to our understanding of alcohol action in the injured brain.

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-d-aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.

The genetic relationships between ethanol preference, acute ethanol sensitivity, and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.

Arouser Reveals a Role for Synapse Number in the Regulation of Ethanol Sensitivity

A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.

Happyhour, a Ste20 Family Kinase, Implicates EGFR Signaling in Ethanol-Induced Behaviors

The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.

Molecular Weight and Volume at Position 46 in Transmembrane Domain 1 (TM1) are Important Determinants for Ethanol Sensitivity in P2X4 Receptors

Purinergic P2X receptors (P2XRs) are a family of fast acting, cation-permeable ligand-gated ion channels, which are gated by synaptically released adenosine 5′-triphosphate (ATP). Building evidence supports the notion that P2XRs play a role in mediating and/or modulating behavioral effects of ethanol. However, this work is in the early stages and the sites and mechanisms of ethanol action in P2XRs are poorly understood. Recently, we identified an amino acid located in TM1 that was conserved across P2X3 and P2X4Rs that appeared to represent an important site for the action of ethanol (i.e., Trp41 and Trp46, respectively). The current study tests the hypothesis that physical-chemical properties of the residue at position 46 play a significant role in determining ethanol sensitivity in P2X4Rs. We expressed wildtype and mutated receptors in Xenopus oocytes and determined changes in ethanol sensitivity (200mM) using an 8-channel two-electrode voltage clamp system (OpusXpress 6000; -70mV). Exchanging Trp46 residue with other aromatic residues did not significantly alter ethanol sensitivity whereas replacing Trp46 with aliphatic residues significantly reduced the action of ethanol. Correlation analysis determined that molecular weight and volume of the residue at position 46 were significantly correlated with ethanol sensitivity whereas polarity or hydrophobicity was not. The findings suggest that the size and aromaticity of the residue at position 46 in TM1 play an important role in determining ethanol sensitivity of P2X4Rs. Identifying physical-chemical properties of residues that are important for ethanol sensitivity will increase our knowledge regarding structural requirements that are necessary for ethanol to cause changes in modulation and/or transduction of agonist action in P2X4Rs. Support: NIAAA/NIH F31 AA017029, KO1 AA017243-01A1, AA013922, AA03972 and USC School of Pharmacy.

Ethanol Interactions With Calcium‐Dependent Potassium Channels

In most neurons and other excitable cells, calcium-activated potassium channels of small (SK) and large conductance (BK; MaxiK) control excitability and neurotransmitter release. The spontaneous activity of dopamine neurons of the ventral tegmental area is increased by ethanol. This ethanol excitation is potentiated by selective blockade of SK, indicating that SK channels modulate ethanol stimulation of neurons that are critical in reward and reinforcement. On the other hand, ethanol directly modulates BK channel activity in a variety of systems, including rat neurohypophysial nerve endings, primary sensory dorsal root ganglia, nucleus accumbens neurons, Caenorhabditis elegans type-IV dopaminergic CEP neurons, and nonneuronal preparations, such as rat pituitary cells, cerebrovascular myocytes and human umbilical vein endothelial cells. Ethanol action on BK channels can modify neuropeptide and growth hormone release, nociception, cerebrovascular tone, and endothelial proliferation. Ethanol modulates BK channels even when the drug is evaluated using recombinant BK channel-forming alpha (slo) subunits or channel reconstitution in artificial, binary lipid bilayers, indicating that the slo subunit and its immediate lipid microenvironment are the essential targets of ethanol. Consistent with this, single amino acid slo channel mutants display altered ethanol sensitivity. Furthermore, C. elegans slo1 null mutants are resistant to ethanol-induced motor incoordination. On the other hand, Drosophila melanogaster slo null mutants fail to acquire acute tolerance to ethanol sedation. Ethanol action on slo channels, however, may be tuned by a variety of factors, including posttranslational modification of slo subunits, coexpression of channel accessory subunits, and the lipid microenvironment, resulting in increase, refractoriness, or even decrease in channel activity. In brief, both SK and BK channels are important targets of ethanol throughout the body, and interference with ethanol effects on these channels could form the basis for novel pharmacotherapies to ameliorate the actions or consequences of alcohol abuse.

Ethanol Tolerance Caused by slowpoke Induction in Drosophila

The large-conductance calcium-activated potassium channel encoded by the slowpoke gene has recently been implicated in the ethanol response. Caenorhabditis elegans carrying mutations in this gene have altered ethanol sensitivity and Drosophila mutant for this gene are unable to acquire rapid tolerance to ethanol or anesthetics. In Drosophila, induction of slowpoke expression has been linked to anesthetic resistance. We used Drosophila as a model system to examine the relationship between slowpoke expression and ethanol tolerance. Real-time PCR and a reporter transgene were used to measure slowpoke induction after ethanol sedation. An inducible slowpoke transgene was used to manipulate slowpoke levels in the absence of ethanol sedation. Ethanol sedation increased transcription from the slowpoke neural promoters but not from the slowpoke muscle/tracheal cell promoters. This neural-specific change was concomitant with the appearance of ethanol tolerance, leading us to suspect linkage between the two. Moreover, induction of slowpoke expression from a transgene produced a phenotype that mimics ethanol tolerance. In Drosophila, ethanol sedation induces slowpoke expression in the nervous system and results in ethanol tolerance. The induction of slowpoke expression alone is sufficient to produce a phenotype that is indistinguishable from true ethanol tolerance. Therefore, the regulation of the slowpoke BK-type channel gene must play an integral role in the Drosophila ethanol response.

Alpha5-gaba-ars are not required for memory impairment by ethanol in mice

INTRODUCTION: Ethanol has been used for centuries as an analgesic and anesthetic. The receptors and molecular mechanisms underlying the behavioral effects of ethanol remain elusive. a-aminobutyric acid type A receptors (GABA-ARs) mediate the majority of synaptic and tonic inhibitory conductances in the brain[1]. GABA-ARs contain an ethanol binding site and thus, are attractive candidates for mediating the intoxicating effects of ethanol. Previous reports showed that a tonic inhibitory conductance generated by GABA-ARs that contain the a; subunit in dentate gyrus granule cells was highly sensitive to low concentrations of ethanol (30 mM)[2]. A tonic conductance is also generated by GABA-ARs containing the a;5 subunit (a;5GABA-ARs) in CA1 hippocampal pyramidal neurons [3]. These a;5GABA-ARs have been implicated in the amnestic properties of anesthetics [4] and the rewarding properties of ethanol [5]. We used complementary electrophysiological and behavioral strategies to test the hypothesis that ethanol increases a tonic conductance in hippocampal pyramidal neurons via activation of a;5GABA-ARs and this action contributes to amnestic effect of ethanol. METHODS: The animal care committee at the University of Toronto approved the experimental protocols. To determine whether low concentrations of ethanol increased the tonic conductance in hippocampal pyramidal neurons, whole-cell currents were recorded from cultured hippocampal neurons from Swiss White mice. In other experiments, contextual and delay fear conditioning was used to study ethanol effects on memory performance in wild-type (WT) and a;5 null mutant mice (a;5-/-). Results are presented as mean ± s.e.m. and groups were compared using analysis of variance (P<0.05). RESULTS: Ethanol applied at high ( 300 mM) but not low concentrations potentiated a tonic current recorded in hippocampal neurons. Ethanol administered at low or medium doses of ethanol (0.5 or 1 g/kg) failed to impair memory performance in both genotypes whereas a higher dose (1.5 g/kg) caused a similar impairment in WT and a;5-/mice. DISCUSSION: In vitro results showed that a;5GABA-ARs are not sensitive targets for low concentrations of ethanol. The in vivo behavioral studies showed that a;5GABAARs are not necessary for memory impairment by ethanol. Deletion of the a;5 subunit gene was not associated with altered ethanol sensitivity for memory impairment. Together the results suggest that a;5GABA-ARs do not contribute to amnestic properties of ethanol.

Effects of a low dose of ethanol in an animal model of premenstrual anxiety

Low (1 mM), but not 10 mM, concentrations of ethanol selectively potentiate current gated by α 4β 2δ subunit combinations of the gamma-aminobutyric acid type A (GABA A) receptor, a subtype increased in hippocampus after withdrawal from progesterone in a rodent model of premenstrual anxiety. In the current study, we tested the hypothesis that the anxiolytic effect of ethanol would exhibit a similar dose-response effect by using the acoustic startle response (ASR) and elevated plus-maze as behavioral models. To this end, adult, female rats were tested (1) 24 h after removal of a progesterone-filled capsule implanted subcutaneously for 21 days (progesterone withdrawal) or (2) on the day of diestrus, a low hormone state. Low doses of ethanol (0.2–0.4 mg/kg) produced a significant 60%–70% decrease in the ASR only in animals undergoing progesterone withdrawal. However, higher doses of ethanol (0.8–1.2 g/kg) were ineffective in these animals, resulting in an “inverted U” ethanol dose effect similar to that observed at recombinant α 4β 2δ subunit combinations of the GABA A receptor. Consistent with these findings, significant 70% attenuation of the ASR was also achieved after progesterone withdrawal with 3 mg/kg of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a GABA A receptor partial agonist with greater potency at α 4βδ receptors than at other known isoforms. In contrast, this partial agonist was not anxiolytic in control animals. These results support the suggestion that very low doses of ethanol are anxiolytic in a model of premenstrual anxiety, whereas higher, potentially sedative, doses are without effect. The results may be relevant for altered ethanol sensitivity during premenstrual syndrome, when increased ethanol consumption has been reported.

Altered effects of ethanol in NR2A ΔC/ΔC mice expressing C-terminally truncated NR2A subunit of NMDA receptor

Phosphorylation of C-termini of receptor subunits is thought to play a significant role in modulation of N-methyl- D-aspartic acid (NMDA) receptor function. To investigate whether the C-terminus of the NR2A subunit is involved in determining the sensitivity of NMDA receptors to ethanol we compared the effects of ethanol in vitro on NMDA-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 and dentate gyrus (DG) of adult male NR2A ΔC/ΔC mice lacking the C-terminus of NR2A subunit and in their parental strain C57Bl/6. We also tested the in vivo effects of a hypnotic dose of ethanol in C57Bl/6 and NR2A ΔC/ΔC mice and their F2 offspring. Ifenprodil (10 μM) was used to distinguish between the NR2A and NR2B components of NMDA fEPSPs. Ethanol (100 mM) in the presence of ifenprodil inhibited the CA1 NR2A-mediated component of NMDA fEPSPs two times more in NR2A ΔC/ΔC than in C57Bl/6. Ethanol inhibition of the CA1 NR2B-mediated component was five to seven times lower in NR2A ΔC/ΔC than in C57Bl/6. In the DG ethanol had similar effects in the two strains. In vivo administration of ethanol (4 g/kg) induced sedation of similar duration in both strains of mice. A second administration of ethanol 7 days after the initial injection revealed an increased ethanol sensitivity of NR2A ΔC/ΔC and F2 ΔC/ΔC mice including a shortened time to loss of righting reflex and an increased sleep time. The sensitization of NR2A ΔC/ΔC mice to alcohol was not accompanied by an altered ethanol sensitivity of NMDA fEPSPs recorded in vitro. Our data are consistent with the inhibitory action of ethanol on NMDA receptors being mediated by a site other than the intracellular C-terminus of the NR2A subunit. The altered sensitivities to ethanol of both NR2A- and NR2B-mediated responses in the CA1 of NR2A ΔC/ΔC imply that NR2A- and NR2B subunit-containing NMDA receptors may be linked by a common target of ethanol.

Alcohols inhibit N-methyl- d-aspartate receptors via a site exposed to the extracellular environment

N-Methyl- d-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC 50 of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC 50 of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.

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 (i.c.v.) 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 micrograms/microliters, 24 microliters/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.