miRNA-regulated transcription associated with mouse strains predisposed to hypnotic effects of ethanol.

INTRODUCTION: Studying innate sensitivity to ethanol can be an important first step toward understanding alcohol use disorders. In brain, we investigated transcripts, with evidence of miRNA modulation related to a predisposition to the hypnotic effect of ethanol, as measured by loss of righting reflex (LORR). METHODS: Expression of miRNAs (12 samples) and expression of mRNAs (353 samples) in brain were independently analyzed for an association with LORR in mice from the LXS recombinant inbred panel gathered across several small studies. These results were then integrated via a meta-analysis of miRNA-mRNA target pairs identified in miRNA-target interaction databases. RESULTS: We found 112 significant miRNA-mRNA pairs where a large majority of miRNAs and mRNAs were highly interconnected. Most pairs indicated a pattern of increased levels of miRNAs and reduced levels of mRNAs being associated with more alcohol-sensitive strains. For example, CaMKIIn1 was targeted by multiple miRNAs associated with LORR. CAMK2N1 is an inhibitor of CAMK2, which among other functions, phosphorylates, or binds to GABAA and NMDA receptors. CONCLUSIONS: Our results suggest a novel role of miRNA-mediated regulation of an inhibitor of CAMK2 and its downstream targets including the GABAA and NMDA receptors, which have been previously implicated to have a role in ethanol-induced sedation and sensitivity.

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.

A major QTL for acute ethanol sensitivity in the alcohol tolerant and non-tolerant selected rat lines.

The Alcohol Tolerant and Alcohol Non-Tolerant rats (AT, ANT) were selectively bred for ethanol-induced ataxia as measured on the inclined plane. Here we report on a quantitative trait locus (QTL) study in an F(2) intercross population derived from inbred AT and ANT (IAT, IANT) and a follow-up study of congenics that were bred to examine one of the mapped QTLs. Over 1200 F(2) offspring were tested for inclined plane sensitivity, acute tolerance on the inclined plane, duration of the loss of righting reflex (LORR) and blood ethanol at regain of the righting reflex (BECRR). F(2) rats that were in the upper and lower 20% for inclined plane sensitivity were genotyped with 78 SSLP markers. Significant QTLs for inclined plane sensitivity were mapped on chromosomes 8 and 20; suggestive QTLs were mapped on chromosomes 1, 2 and 3. Highly significant QTLs for LORR duration (LOD = 12.4) and BECRR (LOD = 5.7) were mapped to the same locus on chromosome 1. Breeding and testing of reciprocal congenic lines confirmed the chromosome 1 LORR/BECRR QTL. A series of recombinant congenic sub-lines were bred to fine-map this QTL. Current results have narrowed the QTL to an interval of between 5 and 20 Mb. We expect to be able to narrow the interval to less than 5 Mb with additional genotyping and continued breeding of recombinant sub-congenic lines.

Quantitative genetics and mouse behavior.

Quantitative differences are observed for most complex behavioral and pharmacological traits within any population. Both environmental and genetic influences regulate such individual differences. The mouse has proven to be a superb model in which to investigate the genetic basis for quantitative differences in complex behaviors. Genetically defined populations of mice, including inbred strains, heterogeneous stocks, and selected lines, have been used effectively to document these genetic differences. Recently, quantitative trait loci methods have been applied to map the chromosomal regions that regulate variation with the goal of eventually identifying the gene polymorphisms that reside in these regions.

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

Confirmation of correlations and common quantitative trait loci between neurotensin receptor density and hypnotic sensitivity to ethanol.

BACKGROUND: In previous studies, genetic correlations were observed between hypnotic sensitivity to ethanol and high-affinity neurotensin receptor (NTS1) binding. Provisional quantitative trait loci (QTLs) were identified for these traits, and some of these QTLs were found on common chromosomal regions. In continued efforts to examine the relationship between NTS1 binding capacity and hypnotic sensitivity to ethanol, studies were designed to confirm correlations between NTS1 densities in the brain, duration of ethanol-induced loss of righting reflex (LORR), and blood ethanol concentrations at regain of righting reflex (BECRR). Another purpose of the study was to confirm QTLs for these traits. METHODS: ILS X ISS F2 mice and HAS X LAS F2 rats as well as the progenitors were tested for LORR, BECRR, and NTS1 densities. Phenotypic correlations were calculated between LORR and BECRR and between these measures and NTS1 densities in striatum from both mice and rats. The F2 mice were genotyped by using polymorphic markers for five previously reported QTLs for LORR to confirm QTLs for BECRR and NTS1 densities in striatum, ventral midbrain, and frontal cortex. RESULTS: Phenotypic correlations were found between LORR and BECRR (r = -0.66 to -0.74, p < 10(-9)) and between these measures and NTS1 densities in striatum (r = 0.28-0.38, p < 10(-2)) from both mice and rats. QTLs for LORR and BECRR (lod score = 2-6) were found in common regions of chromosomes 1, 2, and 15. By using the combined results from a previous LSXSS RI study and the current results, a suggestive QTL (lod score = 3.1) for striatal NTS1 receptor densities was found on chromosome 15 at approximately 60 cM, in the same region as the chromosome 15 LORR/BECRR QTL. CONCLUSIONS: The results are in agreement with previously reported correlations and QTLs for NTS1 receptor densities and measures of hypnotic sensitivity to ethanol in mice and extend those correlations to another species, the rat. These findings support a role for NTS1 in genetically mediated differences in hypnotic sensitivity to ethanol.

Confirmation of contextual fear conditioning QTLs by short-term selection.

A short-term selection study for contextual fear conditioning was conducted as a confirmational strategy to analyze the chromosomal locations of five previously mapped contextual fear conditioning quantitative trait loci (QTLs). The founding population was a C57BL/6 (B6) x DBA/2 (D2) F2 intercross. High and low lines were selected for three generations based on contextual fear conditioning scores. Fear conditioning was quantified as the percentage of time spent in a "frozen" posture when placed back into the chamber, where a mild footshock and a tone had been paired with exposure to the context 24 h earlier. Allele frequencies of at least three SSLP DNA markers linked to each of the five QTLs were determined in each generation. As the selection progressed, the frequency of D2 alleles decreased in the low line and increased in the high line for chromosomes 1 and 16, while the opposite was observed in chromosomes 2, 3, and 10. The direction of divergence for alleles on these five chromosomes is consistent with the original QTL mapping study. Differences between the lines in D2 allele frequencies were found to be significant for markers on chromosomes 2, 3, and 16 but did not reach significance on chromosomes 1 or 10. In general, the results are in good agreement with our original fear conditioning QTL mapping study and provide further evidence that these QTLs regulate variation in contextual fear conditioning in crosses of B6 and D2 mice.

Mapping of quantitative trait loci for hypnotic sensitivity to ethanol in crosses derived from the C57BL/6 and DBA/2 mouse strains.

BACKGROUND: Acute ethanol sensitivity is thought to be a predisposing factor toward the development of alcoholism. Accumulated evidence suggests that this characteristic may be at least partly heritable. A widely accepted approach for identifying genes thought to contribute to alcoholism is to map quantitative trait loci (QTLs) for various ethanol-related behaviors in rodent models. METHODS: Ethanol sensitivity QTLs were interval-mapped in a C57BL/6 (B6) X DBA/2 (D2) F2 intercross that contained 391 mice. Sensitivity was measured as the duration of loss of righting reflex (LORR) after 4.1 g/kg ip. LORR also was evaluated in a chromosome 1 marker-assisted congenic strain that had an approximately 30 centiMorgan (cM) portion of D2 DNA from the distal end of chromosome 1 introgressed onto a B6 background. RESULTS: A suggestive QTL was mapped on chromosome 1 (LOD = 3.3; approximately 80 cM) and a provisional QTL on chromosome 5 (LOD = 2.3; approximately 26 cM). The provisional chromosome 5 QTL was found to be sex-specific (LOD = 2.5 for males; LOD < 1 for females) with the D2 allele increasing LORR. The chromosome 1 D2 allele decreased LORR. Consistent with the F2 QTL mapping, congenic mice heterozygous for the chromosome 1 interval (B6/D2) had a significantly different mean (+/- SEM) LORR of 74.0 +/- 4.9 min (n = 36) compared with 90.8 +/- 6.2 min (n = 33) for their homozygous (B6/B6) littermates (p = 0.02). Blood ethanol concentration at regain of righting reflex was 377 +/- 10 mg% for the B6/D2 and 368 +/- 10 mg% (p = NS) for the B6/B6. CONCLUSIONS: LORR results in the chromosome 1 congenic mice were consistent with and very similar to what was predicted from the QTL analysis in the B6 X D2 F2 population. These results support a suggestive LORR QTL on the distal end of mouse chromosome 1. The results also indicate that there is a provisional sex-specific LORR QTL on chromosome 5.

Behavioural changes produced by transgenic overexpression of gamma2L and gamma2S subunits of the GABAA receptor.

Transgenic mice overexpressing either the mouse gamma2L or gamma2S subunit of the GABAA receptor were generated in a C57BL/6 J x DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam- and ethanol-enhanced 36Cl- flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]-flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the gamma2L or gamma2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor gamma2 subunit available (regardless of whether it is gamma2L or gamma2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Acute functional tolerance to ethanol and fear conditioning are genetically correlated in mice.

It has been speculated that tolerance to alcohol involves some form of neuronal plasticity that is similar to or the same as that mediating learning and memory. To investigate this possibility further, we tested the hypothesis that acute functional tolerance (AFT) to alcohol is genetically correlated to a Pavlovian learning task: fear conditioning. Mice selectively bred for differences in ability to acquire AFT were tested for fear conditioning. Subjects received a mild footshock paired to a broadband clicker and were tested 24 hr later for their freezing response to the conditioning chamber (context), to an altered chamber, and to the clicker. Both the original and replicate lines selected for high AFT (HAFT) were found to freeze significantly more than those selected for low AFT (LAFT) in response to the context and to the clicker. In a second experiment, an F2 population derived from the C57BL/6 (B6) and DBA/2 (D2) mouse strains were tested first for fear conditioning, followed 3 weeks later by AFT testing. AFT was defined as the difference between blood alcohol levels determined at the time of regain balance on a dowel rod first after 1.75 g/kg of ethanol and again after a subsequent dose of 2.0 g/kg. Consistent with results from HAFT and LAFT, freezing to context was found to be significantly positively correlated to AFT (r = 0.38, p = 0.04) in the F2 mice. The results suggest that co-variation in fear conditioning and AFT may be mediated by one or more of the same or at least tightly linked genes. Further dissection of this correlation may reveal neuronal mechanisms common to both AFT and fear conditioning.

Genetic relationship between central beta-endorphin and novelty-induced locomotor activity.

The goal of research presented in this report was to investigate the possibility that differences in beta-ED levels could account for some portion of genetically mediated variation in locomotor activity. Subjects representing six of the LSXSS RI mouse strains were activity tested in a novel environment. Significant effects of strain and time as well as a significant strain by time interaction were detected. Beta-ED levels were estimated from several brain regions and the pituitary of naive and activity-tested mice. Significant strain effects were detected in all brain regions but not in the pituitary. There was no overall effect of exposure to novelty, but some strains showed either a decrease or an increase in beta-ED levels in the septum, amygdala, and midbrain. A significant genetic correlation between adaptation to the novel environment (locomotor activity at 30 min subtracted from locomotor activity at 5 min) and beta-ED levels in the septum was observed. Estimates of hypothalamic mRNA for the beta-ED precursor POMC revealed no effect of strain. Finally, locomotor activity was tested following doses of the mu-opioid antagonist naltrexone. Out of six strains tested (naive to the apparatus), naltrexone dose dependently attenuated locomotor activity in only the strain that showed the highest control level of activity. The results suggest that beta-ED levels influence novelty-induced locomotor activity, but that other important genotype-dependent factors are involved.

Discovery of a new Pomc1 allele in the LS x SS recombinant inbred strains: relationship to locomotor behavior.

Various lines of evidence suggest that a polymorphism in the gene for proopiomelanocortin, Pomc1, might account for some portion of the genetic variance for open-field activity in the LS x SS RI strains. To test this hypothesis, approximately 1600 bp of Pomc1 was sequenced from genomic DNA of seven of the LS x SS strains. Two distinct alleles containing a total of five single-base pair differences were detected. A substitution was found in the coding region causing a Pro-to-Ser conversion, two substitutions occurred in the 3' untranslated region of the mRNA, and a substitution and a deletion were detected in the 3' untranscribed flanking region. The fragment containing the coding region substitution was sequenced in an additional 15 of the LS x SS strains. A total of 12 strains contained one form of the allele, while 10 had the other. The strain distribution pattern of open-field activity scores between the two allels suggests that these alleles do not contribute to the genetic variation of open-field activity in the LS x SS RI strains.

Mapping of provisional quantitative trait loci influencing temporal variation in locomotor activity in the LS x SS recombinant inbred strains.

The finding that stress-induced locomotor activity exhibited a significant strain x time interaction in the LS x SS RI strains prompted examination of QTL influencing this behavior as a function of time. The degree of genetic determination for locomotor activity was 0.26 for the first 5 min and decreased to 0.16 for the last 5 min of a 30-min test but the number of genetic factors stayed relatively constant (three or four) across time. A QTL point analysis revealed a total of 15 QTL, 5-8 per 5-min time block. Few of the QTL were detected across all time points and different combinations of QTL were evident for each time period. Five of the QTL were in common with those reported by other investigators for similar behaviors. The results suggest that locomotor behavior is under a greater degree of genetic control during the initial part of the test but environmental factors become increasingly important as the test progresses. Furthermore, different genetic factors appear to be mediating genetic variation in locomotor behavior at any given time point.

Quantitative trait locus analysis of contextual fear conditioning in mice.

Family, twin and adoption studies provide evidence for a substantial genetic component underlying individual differences in general intelligence, specific cognitive abilities and susceptibility to psychopathologies related to fear-inducing events. Contextual fear conditioning, which is highly conserved across species, can serve as a model for elucidating genes that regulate individual differences in learning and emotion. In fear conditioning, an initially neutral stimulus, such as a tone or a particular environment (context), elicits a fear response after it has been paired with an aversive stimulus, such as shock. Two neural circuits have been implicated in fear conditioning. The fear component is regulated by amygdaloid pathways, while the contextual component is, at least in part, dependent on the hippocampus. C57BL/6J (B6) and DBA/2J (D2) mice differ in several types of complex learning. including contextual fear conditioning. A quantitative trait locus (QTL) analysis of contextual fear conditioning was performed in a B6/D2 F2 intercross population. Two QTLs for contextual conditioning (lod score > 4.3) were identified on chromosomes 10 and 16. QTLs for conditioning to the auditory cue (lod score > 4.3) were localized to chromosomes 1 and 10. Suggestive QTLs (lod score = 2.8-4.1) for contextual conditioning were detected on chromosomes 1, 2 and 3.

Common quantitative trait loci for alcohol-related behaviors and central nervous system neurotensin measures: locomotor activation.

We have analyzed LSXSS recumbinant inbred for ethanol-induced activity using 2.0 g/kg ethanol and a new method we call ethanol activation slope. The ethanol activation slope provides a robust dose-response measure of ethanol activation, independent of both activity after saline and the inhibitory effects of ethanol on locomotor activity. These behavioral data were used in a quantitative trait locus analysis to map chromosomal loci involved in ethanol-induced locomotor activity. We tentatively identified seven loci that mediate the low-dose stimulatory effect of ethanol and six loci involved in locomotion after 2.0 g/kg ethanol. Only one of the loci are in common between the two behaviors. We also compared the behavioral quantitative trait locus to those previously identified that are involved in regulating central nervous system neurotensin levels and neurotensin receptor densities. Six chromosomal regions were identified that regulate at least one central nervous system neurotensin measure and an ethanol-induced locomotor behavior. The identification of loci controlling both central nervous system neurotensin levels or neurotensin receptor densities and ethanol-induced locomotor activity strengthens the proposal that neurotensin regulates, in part, ethanol-induced behaviors and central nervous system sensitivity to ethanol.

Alterations in locomotor activity after microinjections of GBR-12909, selective dopamine antagonists or neurotensin into the medial prefrontal cortex.

It has been postulated that increased dopamine (DA) activity in the medial prefrontal cortex (mPFC) exerts an inhibitory influence over DA release in the nucleus accumbens and, thus, also over locomotor activity. Experiments were designed to examine the role of mPFC DA and neurotensin (NT), a neuropeptide which interacts with DA, in spontaneous locomotor activity. LS/IBG mice were injected bilaterally with either GBR-12909, a selective DA uptake blocker, the DA D1 receptor antagonist R-(+)-SCH-23390, the DA D2 receptor antagonist epidepride, NT or a combination of drugs. GBR-12909 produced a U-shaped dose-response curve with a maximum inhibition of 47% of control. Postmortem tissue levels of DA, 5-hydroxytryptamine, norepinephrine and their major metabolites were determined after microinjections of GBR-12909. Tissue levels of these compounds were not significantly affected by GBR-12909. However, the ratios of homovanilic acid/DA and homovanilic acid + 3,4-dihyroxyphenylacetic acid/DA were significantly decreased, whereas the 5-hydroxyindoleacetic acid/5-hydroxytryptamine ratio was not affected by GBR-12909, suggesting a selective effect on DAergic processes. By itself, R-(+)-SCH-23390 had no effect on locomotor activity except at a very high dose which caused locomotor inhibition (49% of control). Epidepride caused a dose-dependent inhibition of locomotor activity with a maximum inhibition of 49% of control. When coinjected with an inhibitory dose of GBR-12909, both epidepride and R-(+)-SCH-23390 attenuated the GBR-12909 effect in a dose-dependent manner. A broad range of doses of NT was found to have no consistent effect on locomotor activity. However, when coinjected with an inhibitory dose of GBR-12909, NT attenuated the GBR-12909-induced inhibition in a dose-dependent manner. The results suggest that stimulation of DA receptors in the mPFC, both DA D1 and DA D2 receptors mediates locomotor inhibition. Furthermore, stimulation of NT receptors appears to antagonize the effects of DA.

Characterization of neurotensin-stimulated phosphoinositide hydrolysis in brain regions of long sleep and short sleep mice.

Previous studies have shown that long sleep (LS) and short sleep (SS) mice, which were selectively bred for differences in brain sensitivity to ethanol, differ in neurotensin (NT) receptor densities in specific brain regions. The present study was designed to determine whether these receptor differences mediate differences in the effects of NT on the phosphoinositide (PI) second messenger system in four brain regions from LS and SS mice. Baseline and NT- or carbachol-stimulated PI hydrolysis were Ca(2+)-dependent. Stimulation of PI hydrolysis by NT or carbachol was region specific; NT effect was approximately equal in ventral midbrain (VMB) and entorhinal cortex (EC) with slightly less stimulation in nucleus accumbens (NA) and no effect in the cerebellum (CE). Carbachol-enhanced PI hydrolysis was greatest in the VMB followed by EC and NA with no stimulation in the CE. There were no between line (LS vs. SS) differences in carbachol effects, but stimulation by NT was greater in EC and NA from LS than from SS mice. Ethanol enhanced NT-stimulated, but not carbachol-stimulated, PI metabolism in SS and LS NA brain slices. Results with levocabastine, an inhibitor of low-affinity NT receptor (NTL) binding, suggest that NT may stimulate PI hydrolysis via NTL, as well as high-affinity receptors.

Pharmacogenetics of cocaine: I. Locomotor activity and self-selection.

We investigated the effects of cocaine on multiple activity measures and cocaine self-selection in C57BL/6Ibg and DBA/2Ibg mice. Male mice were tested in an automated activity monitor at three doses of cocaine, 5, 15 and 30 mg kg-1. Activity measures included locomotion, rearings, stereotyped movements and wall-seeking. Testing was conducted on 2 days with saline injection, i.p. on day one and cocaine i.p. injected on day two. We also tested other mice of both strains for cocaine ingestion in a two-choice test, pairing tap water with 40 mg% cocaine HCl in tap water. Two separate groups of mice received 15 or 30 mg kg-1 of cocaine i.p., killed at 5 min and brain cocaine levels were determined by HPLC. Cocaine produced dose-related increases in locomotion in both strains, with a delay in initial activation noticed at 30 mg kg-1 in C57s but not in DBAs. In DBAs, cocaine suppressed rearings and increased stereotyped movements while having no consistent effect on either behaviour in C57s. At all doses, cocaine produced moderate increases in proximity to the wall in DBAs and 30 mg kg-1 produced pronounced wall-seeking in C57s. At 15 and 30 mg kg-1 DBAs tended to have higher levels of cocaine in whole brain than did C57s. Finally, C57s consumed significantly more cocaine than did the DBAs.