Discovery and refinement of muscle weight QTLs in B6 × D2 advanced intercross mice.

The genes underlying variation in skeletal muscle mass are poorly understood. Although many quantitative trait loci (QTLs) have been mapped in crosses of mouse strains, the limited resolution inherent in these conventional studies has made it difficult to reliably pinpoint the causal genetic variants. The accumulated recombination events in an advanced intercross line (AIL), in which mice from two inbred strains are mated at random for several generations, can improve mapping resolution. We demonstrate these advancements in mapping QTLs for hindlimb muscle weights in an AIL (n = 832) of the C57BL/6J (B6) and DBA/2J (D2) strains, generations F8-F13. We mapped muscle weight QTLs using the high-density MegaMUGA SNP panel. The QTLs highlight the shared genetic architecture of four hindlimb muscles and suggest that the genetic contributions to muscle variation are substantially different in males and females, at least in the B6D2 lineage. Out of the 15 muscle weight QTLs identified in the AIL, nine overlapped the genomic regions discovered in an earlier B6D2 F2 intercross. Mapping resolution, however, was substantially improved in our study to a median QTL interval of 12.5 Mb. Subsequent sequence analysis of the QTL regions revealed 20 genes with nonsense or potentially damaging missense mutations. Further refinement of the muscle weight QTLs using additional functional information, such as gene expression differences between alleles, will be important for discerning the causal genes.

Fine-mapping of muscle weight QTL in LG/J and SM/J intercrosses.

Genetic variation plays a substantial role in variation in strength, but the underlying mechanisms remain poorly understood. The objective of the present study was to examine the mechanisms underlying variation in muscle mass, a predictor of strength, between LG/J and SM/J strains, which are the inbred progeny of mice selected, respectively, for high and low body weight. We measured weight of five hindlimb muscles in LG/J and SM/J males and females, in F(1) and F(2) intercrosses, and in an advanced intercross (AI), F(34), between the two. F(2) mice were genotyped using 162 SNPs throughout the genome; F(34) mice were genotyped at 3,015 SNPs. A twofold difference in muscle mass between the LG/J and SM/J mouse strains was observed. Integrated genome-wide association analysis in the combined population of F(2) and AI identified 22 quantitative trait loci (QTL; genome-wide P < 0.05) affecting muscle weight on Chr 2 (2 QTL), 4, 5, 6 (7 QTL), 7 (4 QTL), 8 (4 QTL), and 11 (3 QTL). The LG/J allele conferred greater muscle weight in all cases. The 1.5-LOD QTL support intervals ranged between 0.3 and 13.4 Mb (median 3.7 Mb) restricting the list of candidates to between 5 and 97 genes. Selection for body weight segregated the alleles affecting skeletal muscle, the most abundant tissue in the body. Combination of analyses in an F(2) and AI was an effective strategy to detect and refine the QTL in a genome-wide manner. The achieved resolution facilitates further elucidation of the underlying genetic mechanisms affecting muscle mass.

Genetic architecture for hole-board behaviors across substantial time intervals in young, middle-aged and old mice.

A quantitative trait locus (QTL) analysis of behaviors across the life span was conducted in F(2) mice from a C57BL/6J x DBA/2J cross and 22 BXD recombinant inbred (RI) strains. Mice of three age groups were tested in a hole-board apparatus for 3 min on three occasions approximately 1 month apart (average age at test 150, 450 and 750 days, approximately 400 mice per group, divided equally by sex). Quantitative trait loci with small effect size were found on 11 chromosomes for hole-board activity (Hbact) and hole-board rearing (Hbrear). Analysis of 22 RI strains tested at 150 and 450 days of age found only suggestive linkage, with four QTL for Hbact overlapping with those from the F(2) analysis. There was a significant phenotypic correlation between Hbact and Hbrear (approximately 0.55-0.69) and substantial commonality among QTL for the two behaviors. QTL analyses of head-pokes (HP) and fecal boli (FB) only identified QTL at the suggestive level of significance. Age accounted for approximately 15% of the phenotypic variance (sex approximately 3%), and there were genotype by age interactions at approximately 25% of the Hbact and Hbrear QTL. Quantitative trait loci for Hbrear were relatively stable across the three measurement occasions (those for Hbact somewhat less so), although mean levels of each index declined markedly comparing the first to subsequent trials. Considered as a whole, the polygenic system influencing exploratory behaviors accounts for approximately the same amount of phenotypic variance as age (within the range studied), is stable across substantial periods of time, and acts, for the most part, independently of age and sex.

Genetic determinants of weight of fast- and slow-twitch skeletal muscle in 500-day-old mice of the C57BL/6J and DBA/2J lineage.

C57BL/6J (B6) and DBA/2J (D2) strains and two derivative populations, BXD recombinant inbred strains (BXD RIs) and B6D2F2, were used to explore genetic basis for variation in muscle weight at 500 days of age. In parallel with findings in 200-day-old mice (Lionikas A, Blizard DA, Vandenbergh DJ, Glover MG, Stout JT, Vogler GP, McClearn GE, and Larsson L. Physiol Genomics 16: 141-152, 2003), weight of slow-twitch soleus, mixed gastrocnemius, and fast-twitch tibialis anterior (TA) and extensor digitorum longus (EDL) muscles was 13-22% greater (P < 0.001) in B6 than in D2. Distribution of BXD RI strain means indicated that genetic influence on muscle weight (strain effect P < 0.001, all muscles) was of polygenic origin, and effect of genetic factors differed between males and females (strain-by-sex interaction: P < 0.01 for soleus, EDL; P < 0.05 for TA, gastrocnemius). Linkage analyses in B6D2F2 population identified QTL affecting muscle weight on Chr 1, 2, 6, and 9. Pleiotropic influences were observed for QTL on Chr 1 (soleus, TA), 2 (TA, EDL, gastrocnemius), and 9 (soleus, TA, EDL) and were not related to muscle type (fast/slow-twitch) or function (flexor/extensor). Effect of QTL on Chr 9 on soleus muscle was male specific. QTL on Chr 2 and 6 were previously observed at 200 days of age, whereas QTL on Chr 1 and 9 are novel muscle weight QTL. In summary, muscle weight in B6/D2 lineage is affected by a polygenic system that has variable influences at different ages, between males and females, and across muscles in a manner independent of muscle type.

Genetic architecture of fast- and slow-twitch skeletal muscle weight in 200-day-old mice of the C57BL/6J and DBA/2J lineage.

The aim of the study was to explore the genetic architecture influencing weight of fast- and slow-twitch skeletal muscles. The weights of the slow-twitch soleus, the mixed gastrocnemius, the fast-twitch tibialis anterior (TA), and extensor digitorum longus (EDL) muscles were 11-34% greater (P < 0.001) in 200-day-old C57BL/6J (B6) than in DBA/2J (D2) mice. Male muscles were 13-28% larger than female (P < 1 x 10(-5), no strain by sex interaction). The sex-related difference in muscle weight, however, varied significantly among the 23 derivative BXD recombinant inbred (RI) strains (strain by sex interaction for soleus, P < 0.01; TA, P < 1 x 10(-4); EDL, not significant; and gastrocnemius, P < 0.001). Quantitative trait loci (QTL) affecting muscle weight were mapped in an F2 intercross of B6 and D2 mice (B6D2F2) and BXD RIs. A total of 10 autosomal, muscle-specific, but not muscle-type-specific, QTL, explaining a total of 5.4, 7.7, 22.9, and 8.6% of phenotypic variance for soleus, TA, EDL, and gastrocnemius muscles, respectively, were found across chromosomes 1 (Chr 1), 2, 3 (female-specific), 5 (two), 6, 7, 8, and 9 in B6D2F2 mice. The QTL on Chr 8 for EDL and the female-specific QTL on Chr 3 for gastrocnemius muscles were statistically significant, but the remaining QTL were at the suggestive level of statistical significance. Ten QTL on Chr 1, 2, 4, 5, 7, 8, 14, 17 (two), and 19 were identified in BXD RIs. Half of the QTL in BXD RIs had pleiotropic effects and were at the suggestive level of significance (except for the significant QTL for gastrocnemius muscle on Chr 17). The B6D2F2 nominated QTL on Chr 8 for EDL weight was validated in BXD RIs (P < 0.03). Support intervals for the QTL on Chr 1 and 5 overlapped between B6D2F2 and BXD RIs. An epistatic interaction between markers on Chr 1 and 17 affected gastrocnemius weight in BXD RIs. The interaction was not, however, validated in the B6D2F2 population. Our results indicate that the differences in muscle weight in the B6 and D2 segregating populations were the outcome of a polygenic system, with each factor contributing a small amount to the phenotypic variance and the genetic architecture affecting muscle weight was muscle specific, but not muscle-type specific, and in some instances sex specific.

Association between ethanol and sucrose intake in the laboratory mouse: exploration via congenic strains and conditioned taste aversion.

BACKGROUND: A substantial body of literature indicates that intakes of "sweet" solutions and ethanol are positively correlated across inbred strains of rats and mice but there has been speculation that the correlation is fortuitous and there is no agreement on the underlying mechanism. METHODS AND RESULTS: We assessed the correlation between intake of sucrose and ethanol in congenic mice created by backcrossing alleles favoring sucrose intake from the BXD RI-5 strain into DBA/2J. In addition, to probe more specifically the interrelationship between intake of the two solutions, we examined aversion generalization from sucrose to ethanol in C57BL/6J mice. Among the congenic mice, a statistically significant product-moment correlation of r = 0.36 (p < 0.02) was found between 6-hr intake of sucrose corrected for differences in baseline water intake and preference for 10% ethanol presented in a 96-hr 2-bottle test. Furthermore, C57BL/6J male mice conditioned to avoid a 0.2 M sucrose solution generalized their aversion to a 10% ethanol solution presented in the same 2-bottle test, drinking 42.1 +/- 9.38% (mean +/- SE) of their total fluid intake from the ethanol tube, compared with the control group mean of 69.86 +/- 8.84%. CONCLUSIONS: The positive association between intake of sucrose and ethanol in congenic mice provides strong evidence that the previously demonstrated genetic correlation between intake of these solutions is not the result of fortuitous fixation of unrelated alleles and provides suggestive evidence that, at least in the B6/D2 lineage, the genetic association between intakes of the two solutions reflects close linkage or the pleiotropic effects of the same genes. The demonstration that a conditioned taste aversion to sucrose generalized to ethanol in the C57BL/6J inbred mouse strain is an extension of similar observations in outbred rats and specifically demonstrates that intake of the two solutions is controlled by some of the same physiologic or neurological processes and thus is consistent with the pleiotropic interpretation of the genetic correlation.

Quantitative trait loci associated with short-term intake of sucrose, saccharin and quinine solutions in laboratory mice.

The goal of this study was simultaneously to map two genetic loci which, collectively, have a large effect on intake of sucrose, saccharin and quinine solutions in mice. These loci had been previously identified using long-term measurements with the traditional two-bottle test, but the present study used a short-term, one-bottle test. Intake of distilled water, 100 mM sucrose, 10 mM sodium saccharin and 1.1 mM quinine HCl over 6 h was measured on two occasions from a non-deprived group of 61 male and 72 female F2 mice derived from a cross of the C57BL/6J and DBA/2J mouse strains and used to detect quantitative trait loci (QTL). DNA from each animal was typed for polymorphisms in anonymous microsatellite markers on mouse chromosomes 4 and 6. Saccharin and sucrose relevant QTL were detected on distal chromosome 4 and a quinine relevant QTL was detected on medial/distal chromosome 6 in the region of Prp. The location of these QTL and the proportion of phenotypic variance they accounted for were similar to those arrived at following previous determinations using the two-bottle test. Measurement stability for the three gustatory phenotypes was high, product-moment correlation coefficients between first and second determinations varying between approximately 0.80 for sucrose and saccharin and 0.73 for quinine. QTL parameters assessed independently for first and second presentations of sucrose and saccharin were stable, but the location of the quinine QTL differed between presentations. The present experiment illustrates the utility of a 6 h fluid intake test in the mapping of Sac and Qui loci. The short duration of the test provides a simple means of measuring variation in gustatory processes and the discovery that these loci influence short-term as well as long-term fluid intake extends understanding of the mechanism of gene action.

Chorda tympani responses in two inbred strains of mice with different taste preferences.

Behavioral studies suggest that there are significant differences in the taste systems of the inbred mouse (Mus musculus) strains: C57BL/6J (B6) and DBA/2J (D2). In an attempt to understand the biological basis of the behavioral differences, we recorded whole-nerve chorda tympani responses to taste solutions and compared the results to intake of similar solutions in nondeprived mice. Stimuli included a test series composed of 0.1 M sodium chloride, 0.3 M sucrose, 10 mM sodium saccharin, 3 mM hydrochloric acid, and 3 mM quinine hydrochloride, as well as concentration series for the same substances. Neural activity of the chorda tympani that was evoked by sucrose, saccharin, or NaCl was greater in B6 than D2 mice; and neural threshold for sucrose was lower in B6 mice, but neural thresholds for HCl and quinine were lower in D2 mice. B6 mice drank more sucrose and saccharin but less quinine than D2 mice; thus, sucrose and saccharin preference were positively correlated, but NaCl and quinine aversiveness were negatively correlated with the chorda tympani results. Nonetheless, genes involved in the structuring of taste receptors and/or the chordae tympani, which transduce taste stimuli having diverse perceptual qualities, differ for the two mouse strains.

Ethanol consumption in rats selectively bred for differential saccharin intake.

Rat lines selectively bred for high ethanol consumption consume more saccharin solution than do their low-ethanol-consuming counterparts. The present study utilized the technique of reciprocal selection to examine the reliability of the saccharin/ethanol relationship; specifically, consumption of 1-10% ethanol solution was measured in rats selectively bred for high vs. low saccharin consumption (Occidental HiS and LoS lines). HiS rats consumed more ethanol than did LoS rats. These results support the idea that individual differences in ethanol and saccharin consumption share some common mechanism(s).

Measuring gustatory variation in mice: a short-term fluid-intake test.

A short-term fluid-intake test is described which is directed at the study of individual variation in gustation in laboratory mice. To avoid position preferences associated with two-bottle tests, single graduated cylinders are used to present the solution. Intake of water and solutions is recorded for a 6-h period beginning 3 h prior to the dark phase of the light cycle. The timing of data collection ensures a stable baseline of fluid intake because it coincides with the period in which mice begin to drink. Food and water are available ad lib. at all other times so the test avoids the water restriction that is often used in gustatory studies. We report normative data on ten commonly used inbred strains for sucrose (100 mM), saccharin (10 mM), quinine (1.1 mM), HCI (1 and 3 mM). NaCl (320 mM), and monosodium glutamate (150 mM). Strain differences were pronounced for all tastants. Concurrent measures of food and fluid intake by C57BL/6J and DBA/2J mice demonstrated that the short-term reduction of fluid intake resulting from 6-h quinine administration, which was restricted to C57BL/6J mice, was associated with a minor reduction in food intake during the 6-h test and had no statistically significant effect on food or fluid intake during the 18-h post-test period or during a 6-h period the next day. The absence of large-scale or persistent nonspecific effects supports the use of the paradigm for screening of multiple solutions on the same animals. The reliability of the test is supported by positive correlations between repeated measurements of the same solution across substantial time intervals. Its ease of use, substantial reliability, and avoidance of water restriction make the test a very useful addition to screening tools in the field of gustation research.

Genotypic selection provides experimental confirmation for an alcohol consumption quantitative trait locus in mouse.

Quantitative genetic research has produced a wealth of basic information concerning genetic influence on alcohol-related processes. Recent developments in quantitative trait locus (QTL) methodology were promptly applied to the task of individuating polygenes affecting alcohol-related attributes in animal models and a body of reliable data is gradually coming into focus as a result of replication and convergence of evidence from a variety of methods. A key issue in QTL research is the need to distinguish true positive results from the false positive results that are inherent in analytical procedures requiring large numbers of significance tests. One school of thought holds that stringent significance levels should be imposed; another suggests more modest criteria for QTL nomination, with subsequent confirmation trials with independent samples. Recombinant inbred strains and various types of intercrosses have been used in correlational designs, both for nomination and confirmation studies. Alternative experimental procedures include knockout preparations and short-term phenotypic selective breeding. We present here results from a third experimental method-that of marker-based genotypic selection--in evaluation of two nominated QTLs for alcohol acceptance in mice.

Alcohol acceptance, preference, and sensitivity in mice. II. Quantitative trait loci mapping analysis using BXD recombinant inbred strains.

Quantitative trait loci (QTL) mapping of complex phenotypes has emerged as an important feature of the recombinant inbred (RI) strain methodology. In this second study of our series on alcohol-related behaviors in mice, we examine alcohol acceptance, preference, and hypnotic dose sensitivity (HDS) to a standard dose of alcohol measured in BXD RI strains to identify candidate QTL regions responsible for their heritability. We detected highly significant marker associations for acceptance on chromosome 12 (Eif4e), for preference on chromosome 1 (D1Rti2) and chromosome 7 (D7Mit7), and for HDS on chromosome 7 (Mpmv1). These are the strongest QTL associations that we detected, but several other candidate QTL regions are reported. Given the limited number of BXD RI strains available, the large number of markers used herein, and the consequent chance of identifying false marker associations, these RI QTL mapping results must be seen as tentative, but an important first step toward identifying QTL for alcohol-related behaviors.

Alcohol acceptance, preference, and sensitivity in mice. I. Quantitative genetic analysis using BXD recombinant inbred strains.

Although the recombinant inbred strain method was designed for molecular genetic analysis of linkage, it also provides powerful quantitative genetic analyses of heritability and genetic correlations. Measures of alcohol acceptance, alcohol preference, and hypnotic dose sensitivity (HDS) were assessed in 21 strains of mice from the BXD RI series. Sex differences were found to be significant at a phenotypic level. However, heritability estimates for acceptance, preference, and HDS are similar in males and females. Heritability estimates for the three measures are approximately 0.20 for acceptance and preference, and 0.10 for HDS. Analyses of genetic correlations reveal that acceptance and preference share some degree of genetic influence, although they mostly operate under different genetically mediated mechanisms. HDS did not show a significant genetic relationship to either acceptance or preference. Strong correlations were obtained when acceptance, preference, and HDS strain means were correlated across male and female recombinant inbreds, suggesting substantial genetic similarity across sexes.

The Maudsley rat strains as a probe to investigate noradrenergic-cholinergic interaction in cognitive function.

Central noradrenergic function in relation to cognitive performance was studied in the Maudsley rat strains. Neurochemical studies revealed a higher response to acute stress in the locus coeruleus (LC) in the Maudsley reactives (MR) than in the Maudsley non-reactives (MNRA). Autoradiographic studies showed that MNRAs had greater 125I clonidine binding to alpha 2 receptors in LC, which was accompanied by a higher behavioral sensitivity to clonidine. MRs had a deficit in working memory, but were superior to MNRAs in two reference memory tasks. MRs displayed a stronger preference for novel objects, with no strain differences in general exploratory activity. The behavioral profile of the MRs is similar to rats treated with drugs which enhance noradrenergic function. Furthermore, MNRA rats had greater availability of muscarinic receptors, which correlated with behavioral performance in the spatial working memory task. The differences in noradrenergic and cholinergic systems and their relationship to the behavioral profile make the Maudsley strains a useful tool to probe the interaction between two neurotransmitter systems in cognitive function.

The utilization of quantitative trait loci in toxicogenetics.

With the increasing number of marker loci available in mouse, the traditional method of recombinant inbred strains can be extended to searches for quantitative trait loci (QTL) influencing continuously distributed phenotypes. These loci have effect sizes too small to be easily detectable by conventional single-locus techniques. Collective, several QTL may account for a substantial proportion of the phenotypic variability. Prospective advantages of location of QTL in toxicology-relevant phenotypes include opportunities for the study of mechanism, prospects of generating animal models by genotypic selection, study of the dynamics of gene interaction, and guiding the search for homologous genes in human beings.

Recombinant-inbred strains: general methodological considerations relevant to the study of complex characters.

If appropriately determined, recombinant-inbred (RI) strain means provide an excellent method for determining genetic correlations among complex characters. However, little systematic attention has been paid to important environmental influences on strain means such as random effects due to litter membership or systematic maternal influences, which are inevitably confounded with genetic effects. It is suggested that users of RI strains would do well to control for litter effects by sampling appropriately from many litters and assessing the potential role of maternal influences by appropriate fostering procedures. Concern for these and other environmental sources of variation has caused reliability of strain means to emerge as an important issue in studies with RIs which focus on complex characters. Examples of estimating the reliability of RI strain means are provided to draw attention to the value of this kind of information in both gene-mapping studies and genetic correlational analyses. In addition, particularly in the case of behavioral tests which are susceptible to considerable day-to-day variation, repeated testing of the same animals can serve to diminish the influence of extreme deviations which are due to random variations in the manner in which the test is conducted on any given day. The advantages of RIs for gene mapping are well established. However, via the power of genetic correlational analysis, the RI methodology is emerging as a major alternative method, e.g., as distinct from lesion studies, pharmacological interventions, etc., in the bio-behavioral sciences to explore relationships between different domains of inquiry. Via its cumulative and integrative power, it is likely to make a major contribution to investigations of relationships between complex characters at various levels of and this application which should be considered separately from its application to gene mapping.

Maternal influences on cardiovascular pathophysiology.

Elevated blood pressure (BP) is of special clinical significance because of its association with pathophysiologies such as heart disease, renal failure, and stroke. We described the development of a protocol for use with hypertensive rats in which prepubertal exposure to a high salt (8% NaCl) diet results in a pathophysiological syndrome including rapid increase in BP, failure to maintain normal weight gain, renal damage, cerebrovascular lesions, and early mortality. These phenomena are described for the inbred spontaneously hypertensive rat (SHR), and for reciprocal F1 hybrids of a cross between SHR and the Dahl salt-sensitive (SS/Jr) inbred strain. The study with reciprocal F1s revealed striking effects of maternal environment on pathophysiological response to a high salt diet. F1s nurtured by SHR mothers weighed less at 35 days of age, and after exposure to the high salt diet suffered more rapid BP increases, greater incidence of stroke, body weight loss, and mortality, than F1s nurtured by SS/Jr dams. These results suggest that maternal mediation of the nutritional status of the animal may play an important role in determining susceptibility to elevated BP and subsequent pathophysiology associated with exposure to a high salt diet. The implication of these findings for human hypertension is briefly discussed.