Cheryl Reed

Gene expression differences in mice divergently selected for methamphetamine sensitivity

AbstractIn an effort to identify genes that may be important for drug-abuse liability, we mapped behavioral quantitative trait loci (bQTL) for sensitivity to the locomotor stimulant effect of methamphetamine (MA) using two mouse lines that were selectively bred for high MA-induced activity (HMACT) or low MA-induced activity (LMACT). We then examined gene expression differences between these lines in the nucleus accumbens, using 20 U74Av2 Affymetrix microarrays and quantitative polymerase chain reaction (qPCR). Expression differences were detected for several genes, including Casein Kinase 1 Epsilon (Csnkle), glutamate receptor, ionotropic, AMPA1 (GluR1), GABA B1 receptor (Gabbr1), and dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32). We used the www.WebQTL.org database to identify QTL that regulate the expression of the genes identified by the microarrays (expression QTL; eQTL). This approach identified an eQTL for Csnkle on Chromosome 15 (LOD=3.8) that comapped with a bQTL for the MA stimulation phenotype (LOD=4.5), suggesting that a single allele may cause both traits. The chromosomal region containing this QTL has previously been associated with sensitivity to the stimulant effects of cocaine. These results suggest that selection was associated with (and likely caused) altered gene expression that is partially attributable to different frequencies of gene expression polymorphisms. Combining classical genetics with analysis of whole-genome gene expression and bioinformatic resources provides a powerful method for provisionally identifying genes that influence complex traits. The identified genes provide excellent candidates for future hypothesis-driven studies, translational genetic studies, and pharmacological interventions.

A strategy for the integration of QTL, gene expression, and sequence analyses.

Although hundreds if not thousands of quantitative trait loci (QTL) have been described for a wide variety of complex traits, only a very small number of these QTLs have been reduced to quantitative trait genes (QTGs) and quantitative trait nucleotides (QTNs). A strategy, Multiple Cross Mapping (MCM), is described for detecting QTGs and QTNs that is based on leveraging the information contained within the haplotype structure of the mouse genome. As described in the current report, the strategy utilizes the six F2 intercrosses that can be formed from the C57BL/6J (B6), DBA/2J (D2), BALB/cJ (C), and LP/J (LP) inbred mouse strains. Focusing on the phenotype of basal locomotor activity, it was found that in all three B6 intercrosses, a QTL was detected on distal Chromosome (Chr) 1; no QTL was detected in the other three intercrosses, and thus, it was assumed that at the QTL, the C, D2, and LP strains had functionally identical alleles. These intercross data were used to form a simple algorithm for interrogating microsatellite, single nucleotide polymorphism (SNP), brain gene expression, and sequence databases. The results obtained point to Kcnj9 (which has a markedly lower expression in the B6 strain) as being the likely QTG. Further, it is suggested that the lower expression in the B6 strain results from a polymorphism in the 5′-UTR that disrupts the binding of at least three transcription factors. Overall, the method described should be widely applicable to the analysis of QTLs.

Quantitative-trait loci analysis of cocaine-related behaviours and neurochemistry

We recently conducted a dose-response study of the effects of cocaine on several activity measures in the panel of BxD/Ty recombinant inbred mice. Animals were tested in an automated activity chamber over 2 days with i.p. saline on day 1 and i.p. cocaine on day 2, at one of four doses, 5, 15, 30 or 45 mg kg(-1). The monitor recorded total distance traveled, nosepokes in a holeboard, repeated movements and time spent by an individual in proximity to the centre of the apparatus. Dose-response curves for locomotor activation, i.e. the difference between cocaine and saline scores, showed that for all strains tested, scores increased 5-30 mg kg(-1). With few exceptions, locomotor activity at 45 mg kg(-1) was not significantly higher than that at 30 mg kg(-1). Repeated movement scores showed patterns similar to locomotor activity and nosepokes tended to be progressively inhibited by increasing doses of cocaine. Recombinant inbred strain mean distributions for all behaviours and at all doses exhibited continuous, rather than discrete variation, thus providing evidence of multiple-gene effects on cocaine-related behaviours. Quantitative trait loci (QTL) analysis pointed to several chromosomal locations associated with variations in cocaine-related behaviours and some are either identical or close to QTL reported by others. In separate groups of animals, densities of dopamine D1, and D2 receptors and dopamine uptake transporters were measured in the medial prefrontal cortex, caudate-putamen, nucleus accumbens and ventral midbrain. In all areas, all measures showed distributions consistent with polygenic influence and were associated with QTL. Of particular interest was our finding of a large segment on chromosome 15, which is related to dopamine receptor densities and cocaine-related behaviours.

CONTRIBUTION OF SEX AND GENETICS TO NEUROENDOCRINE ADAPTATION TO STRESS IN MICE

Male and female C57BL/6 (B6) and DBA/2 (D2) mice were subjected to either acute or 5 days of repeated restraint in ventilated, 50 ml centrifuge tubes. Control animals were not disturbed. The acute restraint animals were killed immediately following 15, 30 or 60 min of restraint and blood collected for corticosterone (CORT) analysis. The results of the acute restraint procedure revealed a strain difference in time to peak CORT in plasma with D2 animals showing an earlier peak. The males of both strains evinced similar maximum response and similar to B6 females; however, the D2 females showed a 2-fold greater CORT response than did the B6 females. Repeated restraint consisted of 5 days of 12 h in the tubes. At the end of 5 days, the animals were weighted and adrenalectomized in preparation for determination of brain corticosteroid receptors. Upon sacrifice, brains, thymus, adrenals and blood were harvested, the last for corticosteroid binding globulin (CBG). Five days of repeated restraint produced body weight loss in both strains, with B6s less affected than D2s. Repeated restraint reduced the mass of the adrenals in the B6s only. Restraint also reduced the mass of the thymus in both strains and sexes, but to a greater extent in the B6s. Plasma CBG densities were also sensitive to restraint, but only in females, showing a restraint-related decrease. Repeated restraint had no effect on hippocampal glucocorticoid or mineralocorticoid receptors; however for the latter, we observed significant strain and sex effects with D2 having higher Bmax than B6 and females having higher Bmax than males. In the pituitary, glucocorticoid receptors (GR) were reduced by repeated restraint in males, but increased in females, especially in the B6. These findings lend preliminary evidence for involvement of sex and genetics as sources of individual differences in bioadaptation to stress.

Sensitivity to psychostimulants in mice bred for high and low stimulation to methamphetamine

Methamphetamine (MA) and cocaine induce behavioral effects primarily through modulation of dopamine neurotransmission. However, the genetic regulation of sensitivity to these two drugs may be similar or disparate. Using selective breeding, lines of mice were produced with extreme sensitivity (high MA activation; HMACT) and insensitivity (low MA activation; LMACT) to the locomotor stimulant effects of acute MA treatment. Studies were performed to determine whether there is pleiotropic genetic influence on sensitivity to the locomotor stimulant effect of MA and to other MA‐ and cocaine‐related behaviors. The HMACT line exhibited more locomotor stimulation in response to several doses of MA and cocaine, compared to the LMACT line. Both lines exhibited locomotor sensitization to 2 mg/kg of MA and 10 mg/kg of cocaine; the magnitude of sensitization was similar in the two lines. However, the lines differed in the magnitude of sensitization to a 1 mg/kg dose of MA, a dose that did not produce a ceiling effect that may confound interpretation of studies using higher doses. The LMACT line consumed more MA and cocaine in a two‐bottle choice drinking paradigm; the lines consumed similar amounts of saccharin and quinine, although the HMACT line exhibited slightly elevated preference for a low concentration of saccharin. These results suggest that some genes that influence sensitivity to the acute locomotor stimulant effect of MA have a pleiotropic influence on the magnitude of behavioral sensitization to MA and sensitivity to the stimulant effects of cocaine. Further, extreme sensitivity to MA may protect against MA and cocaine self‐administration.

Corticotropin-releasing factor-1 receptor involvement in behavioral neuroadaptation to ethanol: A urocortin1-independent mechanism

A common expression of neuroadaptations induced by repeated exposure to addictive drugs is a persistent sensitized behavioral response to their stimulant properties. Neuroplasticity underlying drug-induced sensitization has been proposed to explain compulsive drug pursuit and consumption characteristic of addiction. The hypothalamic-pituitary-adrenal (HPA) axis-activating neuropeptide, corticotropin-releasing factor (CRF), may be the keystone in drug-induced neuroadaptation. Corticosterone-activated glucocorticoid receptors (GRs) mediate the development of sensitization to ethanol (EtOH), implicating the HPA axis in this process. EtOH-induced increases in corticosterone require CRF activation of CRF1 receptors. We posited that CRF1 signaling pathways are crucial for EtOH-induced sensitization. We demonstrate that mice lacking CRF1 receptors do not show psychomotor sensitization to EtOH, a phenomenon that was also absent in CRF1 + 2 receptor double-knockout mice. Deletion of CRF2 receptors alone did not prevent sensitization. A blunted endocrine response to EtOH was found only in the genotypes showing no sensitization. The CRF1 receptor antagonist CP-154,526 attenuated the acquisition and prevented the expression of EtOH-induced psychomotor sensitization. Because CRF1 receptors are also activated by urocortin-1 (Ucn1), we tested Ucn1 knockout mice for EtOH sensitization and found normal sensitization in this genotype. Finally, we show that the GR antagonist mifepristone does not block the expression of EtOH sensitization. CRF and CRF1 receptors, therefore, are involved in the neurobiological adaptations that underlie the development and expression of psychomotor sensitization to EtOH. A CRF/CRF1-mediated mechanism involving the HPA axis is proposed for acquisition, whereas an extrahypothalamic CRF/CRF1 participation is suggested for expression of sensitization to EtOH.

On the Integration of Alcohol-Related Quantitative Trait Loci and Gene Expression Analyses

BACKGROUND Quantitative trait loci (QTLs) have been detected for a wide variety of ethanol-related phenotypes, including acute and chronic ethanol withdrawal, acute locomotor activation, and ethanol preference. This study was undertaken to determine whether the process of moving from QTL to quantitative trait gene (QTG) could be accelerated by the integration of functional genomics (gene expression) into the analysis strategy. METHODS Six ethanol-related QTLs, all detected in C57BL/6J and DBA/2J intercrosses were entered into the analysis. Each of the QTLs had been confirmed in independent genetic models at least once; the cumulative probabilities for QTL existence ranged from 10 to 10. Brain gene expression data for the C57BL/6 and DBA/2 strains (n = 6 per strain) and an F2 intercross sample (n = 56) derived from these strains were obtained by using the Affymetrix U74Av2 and 430A arrays; additional data with the U74Av2 array were available for the extended amygdala, dorsomedial striatum, and hippocampus. Low-level analysis was performed by using multiple methods to determine the likelihood that a transcript was truly differentially expressed. For the 430A array data, the F2 sample was used to determine which of the differentially expressed transcripts within the QTL intervals were cis-regulated and, thus, strong candidates for QTGs. RESULTS Within the 6 QTL intervals, 39 transcripts (430A array) were identified as being highly likely to be differentially expressed between the C57BL/6 and DBA/2 strains at a false discovery rate of 0.01 or better. Twenty-eight of these transcripts showed significant (logarithm of odds > or =3.6) to highly significant (logarithm of odds >7) cis-regulation. The process correctly detected Mpdz (chromosome 4) as a candidate QTG for acute withdrawal. CONCLUSIONS Although improvements are needed in the expression databases, the integration of QTL and gene expression analyses seems to have potential as a high-throughput strategy for moving from QTL to QTG.

Multiple Cross Mapping (MCM) markedly improves the localization of a QTL for ethanol‐induced activation

This study examines the use of multiple cross mapping (MCM) to reduce the interval for an ethanol response QTL on mouse chromosome 1. The phenotype is the acute locomotor response to a 1.5‐g/kg i.p. dose of ethanol. The MCM panel consisted of the six unique intercrosses that can be obtained from the C57BL/6J (B6), DBA/2J (D2), BALB/cJ (C) and LP/J (LP) inbred mouse strains (N ≥ 600/cross). Ethanol response QTL were detected only with the B6xD2 and B6xC intercrosses. For both crosses, the D2 and C alleles were dominant and decreased ethanol response. The QTL information was used to develop an algorithm for sorting and editing the chromosome 1 Mit microsatellite marker set (http://www.jax.org). This process yielded a cluster of markers between 82 and 85 cM (MGI). Evidence that the QTL was localized in or near this interval was obtained by the analysis of a sample (n = 550) of advanced cross heterogenous stock animals. In addition, it was observed that one of the BXD recombinant inbred strains (BXD‐32) had a recombination in the interval of interest which produced the expected change in behavior. Overall, the data obtained suggest that the information available within existing genetic maps coupled with MCM data can be used to reduce the QTL interval. In addition, the MCM data set can be used to interrogate gene expression data to estimate which polymorphisms within the interval of interest are relevant to the QTL.

Quantitative Genetic Analysis of Ventral Midbrain and Liver Iron in BXD Recombinant Inbred Mice

Abstract Male and female mice from 15 of the BXD/Ty recombinant inbred strain panel were examined for regional brain and liver iron content. Brain regions included medial prefrontal cortex, nucleus accumbens, caudate-putamen and ventral midbrain. Our focal tissue was the ventral midbrain, containing the ventral tegmentum and substantia nigra. This area contains the perikarya of the dopamine neurons that project to nucleus accumbens and caudate-putamen. Genetic correlations between ventral midbrain and liver iron content were not statistically significant, suggesting that peripheral and central iron regulatory systems are largely independent. Correlations between ventral midbrain iron and iron in the caudate-putamen and nucleus accumbens, but not the prefrontal cortex were moderately high and significant. Ventral midbrain and liver iron contents were subjected to quantitative trait loci analysis to identify associated chromosomal locations. This analysis revealed several suggestive loci for iron content in ventral midbrain but fewer loci for liver. Genetic correlations between ventral midbrain iron and published dopamine functional indices were significant, suggesting a link between ventral midbrain iron status and central dopamine neurobiology. This work shows the value of quantitative genetic analysis in the neurobiology of iron and in showing the close association between ventral midbrain iron and nigrostriatal/mesolimbic dopamine function.

Sensitivity to rewarding or aversive effects of methamphetamine determines methamphetamine intake.

Amphetamines have rewarding and aversive effects. Relative sensitivity to these effects may be a better predictor of vulnerability to addiction than sensitivity to one of these effects alone. We tested this hypothesis in a dose–response study in a second replicate set of mouse lines selectively bred for high vs. low methamphetamine (MA) drinking (MADR). Replicate 2 high (MAHDR‐2) and low (MALDR‐2) MA drinking mice were bred based on MA consumption in a two‐bottle choice procedure and examined for novel tastant drinking. Sensitivities to the rewarding and aversive effects of several doses of MA (0.5, 2 and 4 mg/kg) were measured using a place conditioning procedure. After conditioning, mice were tested in a drug‐free and then drug‐present state for time spent in the saline‐ and MA‐paired contexts. Similar to the first set of MADR lines, by the end of selection, MAHDR‐2 mice consumed about 6 mg MA/kg/18 h, compared to nearly no MA in MALDR‐2 mice, but had similar taste preference ratios. MAHDR‐2 mice exhibited place preference in both the drug‐free and drug‐present tests, and no significant place aversion. In contrast, MALDR‐2 mice exhibited no place preference or aversion during the drug‐free test, but robust place aversion in the drug‐present test. These data extend our preliminary findings from the first set of MADR lines and support the hypothesis that the combination of greater sensitivity to the rewarding effects of MA and insensitivity to the aversive effects of MA is genetically associated with heightened risk for MA consumption.