Variation in Galr1 expression determines susceptibility to exocitotoxin-induced cell death in mice

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death ( Sicd1 ). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp , map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1 ) and the FVB/NJ (susceptible at Sicd1 ) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6- Sicd1 congenic mice when compared with FVB/NJ or B6.FVB- Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that 'susceptible' strains showed a reduction in Galr1 expression as compared with 'resistant' strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.     

Mapping a locus for alcohol physical dependence and associated withdrawal to a 1.1 Mb interval of mouse chromosome 1 syntenic with human chromosome 1q23.2-23.3

Physiological dependence and associated withdrawal episodes are thought to constitute a motivational force perpetuating continued alcohol use/abuse. Although no animal model duplicates alcoholism, models for specific factors, like the withdrawal syndrome, are useful to identify potential determinants of liability in humans. We previously detected quantitative trait loci (QTLs) with large effects on predisposition to physical dependence and associated withdrawal following chronic or acute alcohol exposure to a large region of chromosome 1 in mice ( Alcdp1 and Alcw1 , respectively). Here, we provide the first confirmation of Alcw1 in a congenic strain, and, using interval-specific congenic strains, narrow its position to a minimal 1.1   Mb (maximal 1.7   Mb) interval syntenic with human chromosome 1q23.2-23.3. We also report the development of a small donor segment congenic that confirms capture of a gene(s) affecting physical dependence after chronic alcohol exposure within this small interval. This congenic will be invaluable for determining whether this interval harbors a gene(s) involved in additional alcohol responses for which QTLs have been detected on distal chromosome 1, including alcohol consumption, alcohol-conditioned aversion and -induced ataxia. The possibility that this QTL plays an important role in such diverse responses to alcohol makes it an important target. Moreover, human studies have identified markers on chromosome 1q associated with alcoholism, although this association is still suggestive and mapped to a large region. Thus, the fine mapping of this QTL and analyses of the genes within the QTL interval can inform developing models for genetic determinants of alcohol dependence in humans.     

A locus for circadian period of locomotor activity on mouse proximal chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (~1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.     

Acute locomotor responses to cocaine in adolescents vs. adults from four divergent inbred mouse strains

Growing evidence suggests that adolescent mice display differential sensitivity to the acute locomotor activating effects of cocaine as compared to adults, but the direction of the difference varies across studies and the reasons are not clear. Few studies have directly examined genetic contributions to age differences in locomotor stimulation from cocaine. The goal of this study was to determine the extent to which reduced stimulation in C57BL/6J adolescents as compared to adults generalizes to other strains. Therefore, we examined male and female mice from four genetically divergent inbred stains (BALB/cByJ, C57BL/6J, DBA/2J and FVB/NJ) at two ages, postnatal day 30 and postnatal day 65. Mice received either saline or cocaine (15 or 30 mg/kg), and then immediately were placed back into their home cages. Locomotor activity was recorded continuously in the home cage by video tracking. Adolescents displayed reduced stimulation as compared to adults for C57BL/6J, BALB/cByJ and female FVB/NJ mice. No age differences were observed for DBA/2J or male FVB/NJ. No main effects of sex were observed. Strain differences in pharmacokinetics, neural development or physiology could contribute to the observed differences between ages across strains. Future comparative studies could discover biological differences between strains that explain age differences in cocaine sensitivity.     

Genetic basis for the psychostimulant effects of nicotine: a quantitative trait locus analysis in AcB/BcA recombinant congenic mice

Genetic differences in sensitivity to nicotine have been reported in both animals and humans. The present study utilized a novel methodology to map genes involved in regulating both the psychostimulant and depressant effects of nicotine in the AcB/BcA recombinant congenic strains (RCS) of mice. Locomotor activity was measured in a computerized open-field apparatus following subcutaneous administration of saline (days 1 and 2) or nicotine on day 3. The phenotypic measures obtained from this experimental design included total basal locomotor activity, as well as total nicotine activity, nicotine difference scores, nicotine percent change and nicotine regression residual scores. The results indicated that the C57BL/6J (B6) were insensitive to nicotine over the entire dose-response curve (0.1, 0.2, 0.4 and 0.8 mg/kg). However, the 0.8-mg/kg dose of nicotine produced a significant decrease in the locomotor activity in the A/J strain and a wide and continuous range of both locomotor excitation and depression among the AcB/BcA RCS. Single-locus association analysis in the AcB RCS identified quantitative trait loci (QTL) for the psychostimulant effects of nicotine on chromosomes 11, 12, 13, 14 and 17 and one QTL for nicotine-induced depression on chromosome 11. In the BcA RCS, nicotine-induced locomotor activation was associated with seven putative regions on chromosomes 2, 7, 8, 13, 14, 16 and 17. There were no overlapping QTL and no genetic correlations between saline- and nicotine-related phenotypes in the AcB/BcA RCS. A number of putative candidate genes were in proximity to regions identified with nicotine sensitivity, including the alpha2 subunit of the nicotinic acetylcholine receptor and the dopamine D3 receptor.     

Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice

The ability to genetically manipulate mice has led to rapid progress in our understanding of the roles of different gene products in human disease. Transgenic mice have often been created in the FVB/NJ (FVB) strain due to its high fecundity, while gene-targeted mice have been developed in the 129/SvJ-C57Bl/6J strains due to the capacity of 129/SvJ embryonic stem cells to facilitate germline transmission. Gene-targeted mice are commonly backcrossed into the C57Bl/6J (B6) background for comparison with existing data. Genetic modifiers have been shown to modulate mammary tumor latency in mouse models of breast cancer and it is commonly known that the FVB strain is susceptible to mammary tumors while the B6 strain is more resistant. Since gene-targeted mice in the B6 background are frequently bred into the polyomavirus middle T (PyMT) mouse model of breast cancer in the FVB strain, we have sought to understand the impact of the different genetic backgrounds on the resulting phenotype. We bred mice deficient in the inducible nitric oxide synthase (iNOS) until they were congenic in the PyMT model in the FVB and B6 strains. Our results reveal that the large difference in mean tumor latencies in the two backgrounds of 53 and 92 days respectively affect the ability to discern smaller differences in latency due to the Nos2 genetic mutation. Furthermore, the longer latency in the B6 strain enables a more detailed analysis of tumor formation indicating that individual tumor development is not stoichastic, but is initiated in the #1 glands and proceeds in early and late phases. NO production affects tumors that develop early suggesting an association of iNOS-induced NO with a more aggressive tumor phenotype, consistent with human clinical data positively correlating iNOS expression with breast cancer progression. An examination of lung metastases, which are significantly reduced in PyMT/iNOS^−/− mice compared with PyMT/iNOS^+/+ mice only in the B6 background, is concordant with these findings. Our data suggest that PyMT in the B6 background provides a useful model for the study of inflammation-induced breast cancer.     

A major QTL on chromosome 11 influences psychostimulant and opioid sensitivity in mice

The identification of genes influencing sensitivity to stimulants and opioids is important for determining their mechanism of action and may provide fundamental insights into the genetics of drug abuse. We used a panel of C57BL/6J (B6; recipient)× A/J (donor) chromosome substitution strains (CSSs) to identify quantitative trait loci (QTL) for both open field activity and sensitivity to the locomotor stimulant response to methamphetamine (MA). Mice were injected with saline (days 1 and 2) and MA (day 3; 2 mg/kg i.p.). We analyzed the total distance traveled in the open field for 30 min following each injection. CSS-8, -11 and -16 showed reduced MA-induced locomotor activity relative to B6, whereas CSS-10 and -12 showed increased MA-induced locomotor activity. Further analysis focused on CSS-11 because it was robustly different from B6 following MA injection, but did not differ in activity following saline injection and because it also showed reduced locomotor activity in response to the mu-opioid receptor agonist fentanyl (0.2 mg/kg i.p.). Thus, CSS-11 captures QTLs for the response to both psychostimulants and opioids. Using a B6 × CSS-11 F₂ intercross, we identified a dominant QTL for the MA response on chromosome 11. We used haplotype association mapping of cis expression QTLs and bioinformatic resources to parse among genes within the 95% confidence interval of the chromosome 11 QTL. Identification of the genes underlying QTLs for response to psychostimulants and opioids may provide insights about genetic factors that modulate sensitivity to drugs of abuse.     

Genetic architecture of the mouse hippocampus: identification of gene loci with selective regional effects

We recently mapped two quantitative trait loci that have widespread effects on hippocampal architecture in mouse: Hipp1a and Hipp5a. We also noted remarkable strain differences in the relative sizes of different hippocampal regions. Estimated heritable variation for these differences was 42% in hippocampus proper, 40% in dentate gyrus, 31% in granule cell layer and 18% in pyramidal cell layer. Region size varied at least 50% from largest to smallest measurement. Here we have utilized these differences to identify loci with effects on the dentate gyrus, granule cell layer, hippocampus proper and pyramidal cell layer. Our sample consists of C57BL/6J and DBA/2J and 32 BXD recombinant inbred strains. Volumetric data were corrected for shrinkage and for differences in brain weight. We identified significant loci on chromosomes (Chr) 6, 13 and 15, and a significant interaction locus on proximal Chr 11. A suggestive distal Chr 1 locus overlaps with Hipp1a. HipV13a (Chr 13, 42-78Mb) has an additive effect of 0.56 mm3 (12.1%) on dentate gyrus volume, while GrV6a (Chr 6, 29-65 Mb) has additive effects of 0.14 mm3 (16.0%) on the volume of the granule cell layer. HipV13a also interacts with DGVi11a, a locus on proximal Chr 11 that operates exclusively through its epistatic effect on HipV13a and has no independent main effect HipV15a (Chr 15, 0-51 Mb) has an additive effect of 1.76 mm3 (9.0%) on the volume of the hippocampus proper. We used WebOTL, a recently described web-based tool, to examine genetic correlation of gene expression with hippocampal volume. We identified a number of genes that map within the OTL intervals and have highly correlated expression patterns. Using WebQTL's extensive database of published BXD phenotypes, we also detected a strong and potentially biologically meaningful correlation between hippocampal volume and the acoustic startle response.     

Stimulus processing of glycine is dissociable from that of sucrose and glucose based on behaviorally measured taste signal detection in Sac 'taster' and 'non-taster' mice

Mouse strains have been divided into 'tasters' and 'non-tasters' based on their relatively high and low preference, respectively, for low concentrations of sucrose and saccharin. These phenotypic differences appear to be due to a polymorphism in the gene at the Sac locus encoding for the T1R3 taste receptor selectively affecting the functionality of the T1R2+3 heterodimer. To psychophysically examine whether these phenotypes are due to sensory sensitivity as opposed to hedonic responsiveness, we measured taste signal detection of sucrose, glucose, and glycine by Sac taster (C57BL/6J and SWR/J) and non-taster (129P3/J and DBA/2J) strains in an operant conditioning paradigm using a gustometer. The taster mice had lower detection thresholds for sucrose and glucose compared with the non-taster mice. The detection thresholds corresponded well with reported responsiveness to low concentrations of these sugars in two-bottle intake tests suggesting that the Sac taster phenotype has a sensory basis and is not simply a matter of strain differences in the hedonic evaluation of weak intensities of the stimuli. Taster status did not entirely account for the strain differences in detection thresholds for glycine, a 'sweet' tasting amino acid. Collapsed across strains, detection thresholds for sucrose and glucose were highly correlated with each other (r = 0.81), but only modestly correlated with those for glycine (r < or = 0.43). This suggests that stimulus processing of glycine in the perithreshold intensity domain can be dissociated from that of sucrose and glucose. The mechanism underlying this difference may be related to the ability of glycine to bind with the T1R1+3 heterodimer.     

A Susceptibility Locus on Chromosome 13 Profoundly Impacts the Stability of Genomic Imprinting in Mouse Pluripotent Stem Cells

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A Locus for Circadian Period of Locomotor Activity on Mouse Proximal Chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (?1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.     

The relative affective potency of glycine, L-serine and sucrose as assessed by a brief-access taste test in inbred strains of mice

In general, rodents prefer both sucrose and L-serine relative to water and treat both compounds as possessing a similar taste quality (e.g. 'sweetness') despite that they are believed to bind with different T1R heterodimeric receptors in taste bud cells. We assessed the affective potency of these compounds along with glycine, which is thought to bind with both T1R receptor complexes, using a brief-access taste test in a gustometer. Unconditioned licking responses of two 'taster' strains (C57BL/6J and SWR/J), which display high preference for low concentrations of sucrose, and two 'non-taster' (129P3/J and DBA/2J) strains, which display blunted preference for low concentrations of sucrose, were measured during 5 s trials of varying concentrations of a single compound when mice (n=10/strain/stimulus) were non-deprived and when access to home-cage water was restricted. In non-deprived mice, sucrose generated monotonically increasing concentration-response curves regardless of strain, whereas glycine was only marginally effective at stimulating licking and L-serine produced relatively flat functions. The profile of responsiveness across strains was more complex than expected. For example, when tested with sucrose in the non-deprived condition, the 129P3/J non-taster strain surpassed the responsiveness of taster mice at mid-range to high concentrations. Under water-restricted conditions, these mice also were significantly more responsive to high concentrations of both sucrose and glycine compared with the other strains when stimulus licking was standardized relative to water. Thus, the affective potency of the stimuli tested here seems to be related to the ability of the compounds to bind with the T1R2+3 receptor complex. However, the profile of strain responsiveness to these tastants in the brief-access test does not appear to be simply explained by the sweetener 'taster' status of the strain.