Short-sleep Mice Research Articles

Systems genetics analysis of the LXS recombinant inbred mouse strains:Genetic and molecular insights into acute ethanol tolerance.

We have been using the Inbred Long- and Short-Sleep mouse strains (ILS, ISS) and a recombinant inbred panel derived from them, the LXS, to investigate the genetic underpinnings of acute ethanol tolerance which is considered to be a risk factor for alcohol use disorders (AUDs). Here, we have used RNA-seq to examine the transcriptome of whole brain in 40 of the LXS strains 8 hours after a saline or ethanol "pretreatment" as in previous behavioral studies. Approximately 1/3 of the 14,184 expressed genes were significantly heritable and many were unique to the pretreatment. Several thousand cis- and trans-eQTLs were mapped; a portion of these also were unique to pretreatment. Ethanol pretreatment caused differential expression (DE) of 1,230 genes. Gene Ontology (GO) enrichment analysis suggested involvement in numerous biological processes including astrocyte differentiation, histone acetylation, mRNA splicing, and neuron projection development. Genetic correlation analysis identified hundreds of genes that were correlated to the behaviors. GO analysis indicated that these genes are involved in gene expression, chromosome organization, and protein transport, among others. The expression profiles of the DE genes and genes correlated to AFT in the ethanol pretreatment group (AFT-Et) were found to be similar to profiles of HDAC inhibitors. Hdac1, a cis-regulated gene that is located at the peak of a previously mapped QTL for AFT-Et, was correlated to 437 genes, most of which were also correlated to AFT-Et. GO analysis of these genes identified several enriched biological process terms including neuron-neuron synaptic transmission and potassium transport. In summary, the results suggest widespread genetic effects on gene expression, including effects that are pretreatment-specific. A number of candidate genes and biological functions were identified that could be mediating the behavioral responses. The most prominent of these was Hdac1 which may be regulating genes associated with glutamatergic signaling and potassium conductance.

Genome characterization of the selected long- and short-sleep mouse lines.

The Inbred Long- and Short-Sleep (ILS, ISS) mouse lines were selected for differences in acute ethanol sensitivity using the loss of righting response (LORR) as the selection trait. The lines show an over tenfold difference in LORR and, along with a recombinant inbred panel derived from them (the LXS), have been widely used to dissect the genetic underpinnings of acute ethanol sensitivity. Here we have sequenced the genomes of the ILS and ISS to investigate the DNA variants that contribute to their sensitivity difference. We identified ~2.7 million high-confidence SNPs and small indels and ~7000 structural variants between the lines; variants were found to occur in 6382 annotated genes. Using a hidden Markov model, we were able to reconstruct the genome-wide ancestry patterns of the eight inbred progenitor strains from which the ILS and ISS were derived, and found that quantitative trait loci that have been mapped for LORR were slightly enriched for DNA variants. Finally, by mapping and quantifying RNA-seq reads from the ILS and ISS to their strain-specific genomes rather than to the reference genome, we found a substantial improvement in a differential expression analysis between the lines. This work will help in identifying and characterizing the DNA sequence variants that contribute to the difference in ethanol sensitivity between the ILS and ISS and will also aid in accurate quantification of RNA-seq data generated from the LXS RIs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-016-9663-6) contains supplementary material, which is available to authorized users.

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1×244k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.

Transcriptome analysis of Inbred Long Sleep and Inbred Short Sleep mice

Many studies have utilized the Inbred Long Sleep and Inbred Short Sleep mouse strains to model the genetic influence on initial sensitivity to ethanol. The mechanisms underlying this divergent phenotype are still not completely understood. In this study, we attempt to identify genes that are differentially expressed between these two strains and to identify baseline networks of co-expressed genes, which may provide insight regarding their phenotypic differences. We examined the whole brain and striatal transcriptomes of both strains, using next generation RNA sequencing techniques. Many genes were differentially expressed between strains, including several in chromosomal regions previously shown to influence initial sensitivity to ethanol. These results are in concordance with a similar sample of striatal transcriptomes measured using microarrays. In addition to the higher dynamic range, RNA-Seq is not hindered by high background noise or polymorphisms in probesets as with microarray technology, and we are able to analyze exome sequence of abundant genes. Furthermore, utilizing Weighted Gene Co-expression Network Analysis, we identified several modules of co-expressed genes corresponding to strain differences. Several candidate genes were identified, including protein phosphatase 1 regulatory unit 1b (Ppp1r1b), prodynorphin (Pdyn), proenkephalin (Penk), ras association (RalGDS/AF-6) domain family member 2 (Rassf2), myosin 1d (Myo1d) and transthyretin (Ttr). In addition, we propose a role for potassium channel activity as well as map kinase signaling in the observed phenotypic differences between the two strains.

The metabotropic glutamate receptor subtype 5 mediates sensitivity to the sedative properties of ethanol

Inbred long-sleep and short-sleep mice (ILS and ISS) were selectively bred for differential sensitivity to the sedative effects of ethanol. Lines of mice derived from these progenitors have been used to identify several quantitative trait loci (QTLs) mediating loss of the righting reflex due to ethanol (LORE). This study investigated the metabotropic glutamate receptor subtype 5 (mGluR5) as a candidate gene underlying Lore7, a QTL mediating differential LORE sensitivity. We used knockout mice, a quantitative complementation test, pharmacological antagonism of mGluR5, real-time quantitative PCR, radioligand binding, DNA sequencing, and bioinformatics to examine the role of mGluR5 in ethanol-induced sedation. mGluR5 knockout mice had a significantly longer LORE duration than wildtype controls. Administration of the mGluR5 antagonist 2-methyl-6-(phenylethyl)-pyridine (MPEP) had differential effects on LORE in ILS and ISS mice. A quantitative complementation test also supported mGluR5 mediating LORE. Two intronic single-nucleotide polymorphisms in mGluR5 were highly correlated with LORE in recombinant inbred mice derived from a cross between ILS and ISS (LXS RIs). Differences in mGluR5 mRNA level and receptor density were observed between ILS and ISS in distinct brain regions. Finally, data from WebQTL showed that mGluR5 expression was highly correlated with several LORE phenotypes in the LXS RIs. Altogether, this data provides convincing evidence that mGluR5 mediates differential sensitivity to the sedative effects of ethanol. Studies from the human literature have also identified mGluR5 as a potential candidate gene for ethanol sensitivity.

Restraint stress and exogenous corticosterone differentially alter sensitivity to the sedative-hypnotic effects of ethanol in inbred long-sleep and inbred short-sleep mice

Decreased sensitivity to ethanol is a genetically mediated trait implicated in susceptibility to developing alcoholism. Here, we explore genotype by environment differences in ethanol sensitivity. The relationship between acute- and repeated-restraint stress, corticosterone (CORT) levels, and sensitivity to sedative-hypnotic properties of ethanol was explored using inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. In ILS mice, acute restraint decreased ethanol sensitivity at a 4.1 g/kg dose, as measured by a decrease in the duration of loss of the righting reflex (LORE) and an increase in blood ethanol concentration at regain of the righting response (BECRR). Repeated restraint also decreased LORE duration, but had no effect on BECRR. In the ISS mice, there was no effect of acute restraint on either LORE duration or BECRR. However, repeated restraint increased ethanol sensitivity at a 4.1 g/kg dose; with an increase in LORE duration, but a decrease in BECRR. Differences in hypothalamic-pituitary-adrenal (HPA) axis responsiveness to restraint stress (as measured by plasma CORT) were also examined between genotypes. ILS mice displayed habituation to repeated restraint, whereas ISS mice did not. Lastly, the effect of enhanced CORT levels independent of psychological stress was examined for its effects on the sedative-hypnotic effects of ethanol. There were no effects of CORT pretreatment on LORE duration or BECRR in ILS mice compared to saline- or noninjected littermates. In contrast, ISS mice injected with CORT showed a decreased duration of LORE, but no effects on BECRR. These findings suggest that in addition to genetic susceptibility, environmental factors (e.g., restraint stress, exogenous CORT administration) also influence sensitivity to the sedative effects of ethanol through alteration of central nervous system sensitivity and pharmacokinetic parameters, and do so in a genotype-dependent manner.

Ethanol sensitivity: a central role for CREB transcription regulation in the cerebellum

BackgroundLowered sensitivity to the effects of ethanol increases the risk of developing alcoholism. Inbred mouse strains have been useful for the study of the genetic basis of various drug addiction-related phenotypes. Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice differentially express a number of genes thought to be implicated in sensitivity to the effects of ethanol. Concomitantly, there is evidence for a mediating role of cAMP/PKA/CREB signalling in aspects of alcoholism modelled in animals. In this report, the extent to which CREB signalling impacts the differential expression of genes in ILS and ISS mouse cerebella is examined.ResultsA training dataset for Machine Learning (ML) and Exploratory Data Analyses (EDA) was generated from promoter region sequences of a set of genes known to be targets of CREB transcription regulation and a set of genes whose transcription regulations are potentially CREB-independent. For each promoter sequence, a vector of size 132, with elements characterizing nucleotide composition features was generated. Genes whose expressions have been previously determined to be increased in ILS or ISS cerebella were identified, and their CREB regulation status predicted using the ML scheme C4.5. The C4.5 learning scheme was used because, of four ML schemes evaluated, it had the lowest predicted error rate. On an independent evaluation set of 21 genes of known CREB regulation status, C4.5 correctly classified 81% of instances with F-measures of 0.87 and 0.67 respectively for the CREB-regulated and CREB-independent classes. Additionally, six out of eight genes previously determined by two independent microarray platforms to be up-regulated in the ILS or ISS cerebellum were predicted by C4.5 to be transcriptionally regulated by CREB. Furthermore, 64% and 52% of a cross-section of other up-regulated cerebellar genes in ILS and ISS mice, respectively, were deemed to be CREB-regulated.ConclusionThese observations collectively suggest that ethanol sensitivity, as it relates to the cerebellum, may be associated with CREB transcription activity.

Expression profiling identifies novel candidate genes for ethanol sensitivity QTLs.

The Inbred Long Sleep (ILS) and Inbred Short Sleep (ISS) mouse strains have a 16-fold difference in duration of loss of the righting response (LORR) following administration of a sedative dose of ethanol. Four quantitative trait loci (QTLs) have been mapped in these strains for this trait. Underlying each of these QTLs must be one or more genetic differences (polymorphisms in either gene coding or regulatory regions) influencing ethanol sensitivity. Because prior studies have tended to focus on differences in coding regions, genome-wide expression profiling in cerebellum was used here to identify candidate genes for regulatory region differences in these two strains. Fifteen differentially expressed genes were found that map to the QTL regions and polymorphisms were identified in the promoter regions of four of these genes by direct sequencing of ILS and ISS genomic DNA. Polymorphisms in the promoters of three of these genes, Slc22a4, Rassf2, and Tax1bp3, disrupt putative transcription factor binding sites. Slc22a4 and another candidate, Xrcc5, have human orthologs that map to genomic regions associated with human ethanol sensitivity in genetic linkage studies. These genes represent novel candidates for the LORR phenotype and provide new targets for future studies into the neuronal processes underlying ethanol sensitivity.

Norepinephrine Transporter: A Candidate Gene for Initial Ethanol Sensitivity in Inbred Long-Sleep and Short-Sleep Mice

Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.

Genetic Dissociation Between Ethanol Sensitivity and Rapid Tolerance in Mouse and Rat Strains Selectively Bred for Differential Ethanol Sensitivity

The Inbred Long- and Short-Sleep mice (ILS and ISS) and the Inbred High- and Low-Alcohol-Sensitive rats (IHAS and ILAS) were selectively bred for differential alcohol sensitivity with use of the duration of loss-of-righting-reflex test (LORR), with the IHAS and ILS animals being much more sensitive than the ILAS and ISS animals, respectively. The current study was undertaken to determine whether acute sensitivity in these strains is genetically correlated to a rapid tolerance to alcohol, a form of tolerance that is evident 24 hr after a single alcohol dose. Separate groups of animals were administered a single pretreatment dose of alcohol (0-6 g/kg for the mice; 0-4 g/kg for the rats). Alcohol sensitivity was tested 24 hr later with the LORR test, and blood ethanol concentration was tested at regain of righting (BECRR). Alcohol-induced hypothermia also was determined in the mice. Independently derived replicate rat strains were used for all experiments (IHAS1, ILAS1; IHAS2, ILAS2); no such replicates exist for the ILS and ISS strains. Alcohol pretreatment caused a dose-dependent decrease in LORR duration accompanied by an increase in BECRR in the ILS strain, but LORR increased in the ISS strain with no effect on BECRR. Both strains became hypothermic during the LORR test on day two, but the only significant effect of alcohol pretreatment was in the ISS strain, in which alcohol-induced hypothermia was enhanced. Alcohol pretreatment caused a significant dose-dependent decrease in LORR duration accompanied by an increase in BECRR in the IHAS1 but not in the IHAS2 strain. In contrast, ILAS1 and ILAS2 strains both showed a significant increase in LORR duration and also a significant increase in BECRR. Alcohol pretreatment caused a dose-dependent decrease in LORR duration and an increase in BECRR in the IHAS1 and ILS strain, suggesting the development of functional rapid tolerance. In contrast, LORR duration increased in the ILAS1, ILAS2, and ISS groups, but BECRR either increased (ILAS1, ILAS2) or did not change (ISS). These observations suggest that central nervous system sensitivity was decreased in the ILAS1 and ILAS2 groups (i.e., rapid functional tolerance) or unchanged in the ISS strain, but that some pharmacokinetic property also was altered in these strains. Overall, the results do not support a genetic relation between alcohol sensitivity and the development of rapid tolerance.

Cerebellar Gene Expression Profiling and eQTL Analysis in Inbred Mouse Strains Selected for Ethanol Sensitivity

Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice exhibit striking differences in a number of alcohol and drug related behaviors. This study examined the expression levels of more than 39,000 transcripts in these strains in the cerebellum, a major target of ethanol's actions in the CNS, to find differentially expressed (DE) candidate genes for these phenotypes. Genes that were differentially expressed between the strains were identified using oligonucleotide arrays as well as complimentary DNA arrays. Sequence alignment was used to locate DE genes in the mouse genome assembly. In silico expression QTL (eQTL) mapping was used to identify chromosomal regions likely to control the transcription level of DE genes, and the EASE program identified overrepresented functional themes. The genomic region immediately upstream of the cyclase associated protein homolog 1 (Cap1) gene was directly sequenced from PCR products. Nearly 300 genes were identified as differentially expressed between the cerebella of ILS and ISS. These genes and their corresponding eQTLs map to genomic regions linked to several phenotypes that differ between the ILS and ISS strains, including ethanol preference and cocaine-induced locomotor activation on Chromosomes 4 and 7 respectively. Eight genes were cross-platform validated, four of which are more highly expressed in ILS cerebellum. Three SNPs, one of which disrupts a predicted Sp1 binding site, were found in the upstream region of Cap1, a strong candidate for influencing ethanol phenotypes. Many of these DE genes are candidates to influence ethanol and drug regulated phenotypes because they either map to ethanol related QTLs in the genome or are linked to them through eQTL mapping. Genes involved in calcium ion binding and transcriptional regulation are overrepresented and therefore these gene classes may influence ethanol behaviors in mice and humans.

Formation of ethyl nitrite in vivo after ethanol administration

The purpose of the current study was to ascertain whether ethyl nitrite could be detected in vitro from the reaction of ethanol with peroxynitrite, as well as after administration of ethanol to mice. Ethyl nitrite analyte was determined by using gas chromatography–mass spectrometry with headspace analysis with the use of a solid-phase microextraction device. Peroxynitrite was allowed to react with ethanol under a variety of conditions in vitro. Ethyl nitrite was generated when peroxynitrite was allowed to react with ethanol. Male, inbred short-sleep mice were injected intraperitoneally with either ethanol [5.2 g/kg; 15.0% (weight/volume) ethanol in saline] or a 50:50 mixture of deuterium-labeled ethanol (D 5-ethanol) and ethanol. Blood samples, as well as whole brain and liver sections, were obtained from mice 30 min later for determination of ethanol, D 5-ethanol, ethyl nitrite, and deuterium-labeled ethyl nitrite (D 5-ethyl nitrite). Time courses for the appearance of ethyl nitrite in blood samples, as well as in whole brain and liver sections, obtained from mice were carried out. After ethanol administration, ethyl nitrite was detected and quantitated in mouse blood, brain, and liver. A small fraction of ethyl nitrite was present. When a 50:50 mixture of ethanol and D 5-ethanol was given to animals, both ethyl nitrite and D 5-ethyl nitrite were found in blood and brain in approximately the same ratio as that of ethanol and D 5-ethanol. The level of D 5-ethyl nitrite in liver was more than twice that of ethyl nitrite, indicating a possible isotope effect in the metabolism of ethyl nitrite. Ethyl nitrite is a new metabolite of ethanol in vivo. The mechanism of ethyl nitrite formation is most likely the reaction of ethanol with peroxynitrite generated in vivo from nitric oxide.

Differential regulation of cocaine-induced locomotor activity in inbred long-sleep and short-sleep mice by dopamine and serotonin systems

Acute injection of cocaine increases locomotor activity of inbred long-sleep (ILS) mice to a greater extent than inbred short-sleep (ISS) mice. Strain differences in dopamine and/or serotonin (5-HT) neurotransmission could underlie these behavioral differences. Here, we found that dopamine D1, 5-HT 2A and 5-HT 3 receptor antagonists reduced cocaine-stimulated activity selectively in ILS mice. In contrast, 5-HT transporter (SERT) or 5-HT 1A receptor antagonists potentiated cocaine-stimulated activity in ISS, but not in ILS, mice; this potentiation in ISS mice was abolished by dopamine D1 receptor blockade. Thus, in ILS mice, cocaine-induced activation of D1, 5-HT 2A or 5-HT 3 receptors is sufficient to produce locomotor stimulation. In contrast, ISS mice require pharmacologically increased 5-HT levels, which appear to result in increased dopamine neurotransmission, for cocaine-induced activation. Our results demonstrate strain differences in dopamine/5-HT receptor subtypes and their interactions that contribute to the differential behavioral responsiveness of ILS and ISS mice to cocaine.

MK-801- and ethanol-induced activity in inbred long-sleep and short-sleep mice: dopamine and serotonin systems

Low doses of (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cyclohepten-5,10-imine (MK-801; dizocilpine) or ethanol induce less locomotor activation in inbred long-sleep (ILS) than short-sleep (ISS) mice. These differences may involve altered dopamine and/or 5-hydroxytryptamine (serotonin; 5-HT) neurotransmission. To address this possibility, the dopaminergic and serotonergic mechanisms underlying the locomotor-stimulant effects of MK-801 and ethanol in ILS and ISS mice were studied. Dopamine D1, D2 and 5-HT 2A receptor antagonists reduced MK-801-stimulated activity in ILS mice without having any effect in ISS mice. The 5-HT reuptake inhibitor fluoxetine potentiated MK-801-stimulated activity selectively in ILS mice. Strain differences in 5-HT transporters do not explain this selective effect of fluoxetine in ILS mice since [ 3H]citalopram binding and [ 3H]5-HT uptake studies found no differences in the affinity, number or function of 5-HT transporters between ILS and ISS mice. Ethanol-induced activity in ISS mice was depressed by dopamine D2 and 5-HT 2C receptor antagonists and enhanced by a 5-HT 1A receptor antagonist. These results suggest that in ILS mice the locomotor-stimulant effects of MK-801 require increased dopamine and/or 5-HT neurotransmission. Conversely, in ISS mice, the effects of MK-801 appear to be monoamine-independent. Thus, even though both MK-801 and ethanol inhibit N-methyl- d-aspartate receptors, their stimulant effects appear to involve different neuronal systems.

Locomotor Activity Induced by Noncompetitive NMDA Receptor Antagonists Versus Dopamine Transporter Inhibitors: Opposite Strain Differences in Inbred Long-Sleep and Short-Sleep Mice

Locomotor Activity Induced by Noncompetitive NMDA Receptor Antagonists Versus Dopamine Transporter Inhibitors: Opposite Strain Differences in Inbred Long-Sleep and Short-Sleep Mice

Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes.

Several studies have shown that genetic factors influence the effects of nicotine on respiration, acoustic startle, Y-maze crosses and rears, heart rate and body temperature in the mouse. Recently, we identified restriction fragment length polymorphisms (RFLPs) associated with the alpha4 (Chrna4) and alpha6 (Chrna6) nicotinic cholinergic receptor genes in the recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines. The alpha4 polymorphism has been identified as a point-mutation at position 529 (threonine to alanine) and the alpha6 polymorphism has not yet been identified. The studies described here evaluated the potential role of these polymorphisms in regulating sensitivity to nicotine by constructing dose-response curves for the effects of nicotine on six responses in the LSxSS RI strains. The results obtained suggest that both of the polymorphisms may play a role in regulating variability in sensitivity to nicotine. Those RI strains carrying the LS-like alpha4 RFLP were significantly more sensitive to the effects of nicotine on Y-maze crosses and rears, temperature and respiration and were less sensitive to the effects of nicotine on acoustic startle than those strains carrying the SS-like alpha4 RFLP. Those RI strains carrying the LS-like alpha6 RFLP were more sensitive to the effects of nicotine on respiration and acoustic startle, and less sensitive to the effects of nicotine on Y-maze crosses than those strains carrying the SS-like alpha6 RFLP. These results suggest that genetically determined differences in sensitivity to nicotine may be explained, in part, by variability associated with at least two of the neuronal nicotinic receptor genes, alpha4 and alpha6.

Locomotor Activity Induced by Noncompetitive NMDA Receptor Antagonists Versus Dopamine Transporter Inhibitors: Opposite Strain Differences in Inbred Long‐Sleep and Short‐Sleep Mice

The actions of ethanol in the brain involve multiple neuroreceptor systems, including glutamatergic N-methyl-D-aspartate receptor (NMDAR) channels. In a novel environment, both ethanol and the noncompetitive NMDAR antagonist MK-801 stimulate locomotor activity to a lesser extent in inbred long-sleep (ILS) mice compared with inbred short-sleep (ISS) mice. The behaviorally activating effects of noncompetitive NMDAR antagonists are thought to involve increased monoamine neurotransmission. Thus, in this study, we sought to determine whether: (1) habituation to the behavioral environment alters the differential locomotor-stimulant effects of noncompetitive NMDAR antagonists in ILS and ISS mice and (2) the differential behavioral sensitivity of ILS and ISS mice to noncompetitive NMDAR antagonists is mediated through direct inhibition of the dopamine transporter (DAT). Open field locomotor activity was measured following acute systemic injection of saline or drug. [3H]DA uptake parameters were determined in striatal synaptosomes prepared from drug-naïve mice. Habituation to the testing environment abolished the strain differences in saline-induced locomotor activity. However, ethanol- as well as MK-801-treated ILS mice still exhibited reduced locomotor activity compared with ISS mice, suggesting that a drug-environment interaction is not the primary explanation for the strain differences. The noncompetitive NMDAR antagonists phencyclidine and ketamine also induced significantly lower locomotor activity in ILS than in ISS mice. In contrast, the DAT inhibitors cocaine and GBR 12909 and the DA releaser amphetamine induced greater locomotor activity in ILS than in ISS mice, a strain difference opposite that of the noncompetitive NMDAR antagonists. Furthermore, the differential behavioral effect found with DAT inhibitors was not mediated by differences in the affinity nor number of striatal DATs between ILS and ISS mice. Our results support the conclusion that the differential locomotor-stimulant effects of ethanol and noncompetitive NMDAR antagonists in ILS and ISS mice are not mediated through direct inhibition of DAT.

Alcohol differentially affects c-Fos expression in the supraoptic nucleus of long-sleep and short-sleep mice

Ethanol administration in long-sleep (LS) and short-sleep (SS) mice results in a large number of Fos-IR neurons in the supraoptic nucleus (SON) in LS, and almost no Fos-IR neurons in the same nucleus in SS mice. In contrast, isotonic saline, hypertonic saline, with or without ethanol, resulted in a similar pattern of Fos-IR in both strains. These data indicate a differential effect of ethanol on c-Fos signaling specifically in the SON. Since the LS and SS mice were specifically selected for differential sensitivity to the sedative/hypnotic effects of ethanol, this differential in c-Fos activity may be causally implicated in their differential sensitivity to ethanol.

Genetic analysis of low-dose ethanol-induced activation (LDA) in inbred long sleep (ILS) and inbred short sleep (ISS) mice.

Ethanol-induced activation (LDA) is a measure of alcohol action on behavior and a predictor of future human alcoholism. Previous studies have shown that the long sleep (LS) and short sleep (SS) selected lines of mice differ in LDA and that two to five genes specify this phenotypic difference. The current study examines the genetic components of LDA in the ILS and ISS mice that are inbred derivatives of LS and SS and examines the effects of inbreeding on LDA. We provide estimates of heritability and number of loci that are consistent with those found in previous studies of the LS and SS. We conclude that LDA is a polygenic trait, specified by two to six loci, which have not been affected by inbreeding or genetic drift in the ILS and ISS populations.

Direct Evidence for a Cause-Effect Link Between Ethanol Potentiation of GABAA 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 γ-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, ≤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 GABAA receptor function is an initial action of ethanol causing its behavioral effects. These findings also provide insight into possible effects of selective breeding on GABAA receptor function.