John K. Belknap

Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies

Abstract Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a manner which could severely compromise the interpretation of the mutant phenotype. The present overview summarizes the literature on a wide variety of behavioral traits for the 129, C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field activity, learning and memory tasks, aggression, sexual and parental behaviors, acoustic startle and prepulse inhibition, and the behavioral actions of ethanol, nicotine, cocaine, opiates, antipsychotics, and anxiolytics. Using the referenced information, molecular geneticists can choose optimal parental strains of mice, and perhaps develop new embryonic stem cell progenitors, for new knockouts and transgenics to investigate gene function, and to serve as animal models in the development of novel therapeutics for human genetic diseases.

Voluntary consumption of ethanol in 15 inbred mouse strains

To determine genetic differences in ethanol consumption, 15 commonly used inbred strains of mice were given ad libitum two-bottle choice between ethanol, 0.2% saccharin, or ethanol plus saccharin in one bottle versus tap water in the other bottle. Three different concentrations of ethanol were used: 3%, 6% and 10% (v/v). Of the 15 strains, the C57BL/6J, C57BR/cdJ and C57L/J strains showed the most consistent higher intake of ethanol either with or without 0.2% saccharin. In marked contrast, the DBA/1J and DBA/2J strains consistently showed the lowest intake. Consumption of 3% ethanol without saccharin was highly genetically correlated with saccharin consumption (r=0.77), suggesting that low concentrations of ethanol may have a sweet taste that affects voluntary consumption. Most strains showed very different patterns of response to ethanol with or without saccharin. Three patterns of strain responses were identified. Some strains avoided higher concentrations of ethanol whether in water or saccharin; some appeared to be sensitive to the ability of saccharin to mask the odor of ethanol; and some may have reduced consumption only when ethanol concentrations were high enough to produce aversive postingestional effects. Whereas earlier studies generally attempted to explain strain differences in consumption by invoking a single mechanism, our results demonstrate that more than one mechanism is necessary to explain the preferential ethanol intake of all strains studied.

Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice

Because of intrinsic differences between humans and mice, no single mouse model can represent all features of a complex human trait such as alcoholism. It is therefore necessary to develop partial models. One important feature is drinking to the point where blood ethanol concentration (BEC) reaches levels that have measurable affects on physiology and/or behavior (>1.0 mg ethanol/ml blood). Most models currently in use examine relative oral self-administration from a bottle containing alcohol versus one containing water (two-bottle preference drinking), or oral operant self-administration. In these procedures, it is not clear when or if the animals drink to pharmacologically significant levels because the drinking is episodic and often occurs over a 24-h period. The aim of this study was to identify the optimal parameters and evaluate the reliability of a very simple procedure, taking advantage of a mouse genotype (C57BL/6J) that is known to drink large quantities of ethanol. We exchanged for the water bottle a solution containing ethanol in tap water for a limited period, early in the dark cycle, in the home cage. Mice regularly drank sufficient ethanol to achieve BEC>1.0 mg ethanol/ml blood. The concentration of ethanol offered (10%, 20% or 30%) did not affect consumption in g ethanol/kg body weight. The highest average BEC ( approximately 1.6 mg/ml) occurred when the water-to-ethanol switch occurred 3 h into the dark cycle, and when the ethanol was offered for 4 rather than 2 h. Ethanol consumption was consistent within individual mice, and reliably predicted BEC after the period of ethanol access. C57BL/6J mice from three sources provided equivalent data, while DBA/2J mice drank much less than C57BL/6J in this test. We discuss advantages of the model for high-throughput screening assays where the goal is to find other genotypes of mice that drink excessively, or to screen drugs for their efficacy in blocking excessive drinking.

Orphanin FQ is a functional anti-opioid peptide

The heptadecapeptide orphanin FQ has recently been shown to be the endogenous agonist for the orphan opioid-like receptor, LC132. The molecular evidence that LC132 and orphanin FQ are evolutionarily related to other opioid receptors and their ligands suggests that these proteins may also play a role in modulating opiate actions. We now report that orphanin FQ (0.5-10 nmol), injected intracerebroventricularly in mice, does not produce hyperalgesia as suggested previously but rather reverses opioid-mediated (i.e. naloxone-sensitive) stress-induced antinociception in three different algesiometric assays. In addition to its antagonism of endogenous opioid antinociception, orphanin FQ dose-dependently (2.5-25 nmol) reverses systemic morphine antinociception (5 mg/kg, s.c.). Based on these data, we propose that orphanin FQ is a functional anti-opioid peptide.

The nature and identification of quantitative trait loci: a community’s view

This white paper by eighty members of the Complex Trait Consortium presents a communitys view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

Identifying genes for alcohol and drug sensitivity: recent progress and future directions

New methods for identifying chromosomal regions containing genes that affect murine responses to alcohol and drugs have been used to identify many provisional quantitative trait loci (QTLs) since 1991. By 1998, 24 QTLs had been definitively mapped (P<5x10(-5)) to specific murine chromosomes, which indicates the presence of a relevant gene or genes at each location. The syntenic (homologous) region of the human genome for these genes is often known. For many mapped QTLs, candidate genes with relevant neurobiological function lie within the mapped region. Data that implicate candidate genes for specific responses include studies of knockout animals. Current strategies for gene identification include the use of congenic strains containing QTL regions introduced from another strain. There is increasing emphasis on gene-gene and gene-environment interactions in such studies.

Quantitative Trait Loci Affecting Peak Bone Mineral Density in Mice

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations, and effects of the individual genes contributing to natural variation in this trait are all unknown. Experimental animal models provide a means to circumvent complicating environmental factors, and the development of dense genetic maps based on molecular markers now provides opportunities to resolve quantitative genetic variation into individual regions of the genome influencing a given trait (quantitative trait loci, QTL). To begin to identify the heritable determinants of BMD, we have examined genetically distinct laboratory mouse strains raised under strict environmental control. Mouse whole‐body bone mineral content by dual‐energy X‐ray absorptiometry (DXA) correlated strongly with skeletal calcium content by ashing, and peak whole‐body BMD by DXA in female mice occurred at ∼80–90 days of age. We therefore determined mean body weight and peak whole body BMD values in 12‐week‐old female mice from a panel of 24 recombinant inbred (RI) BXD strains, derived from a cross between C57BL/6 and DBA/2 progenitors. The distribution of body weight and BMD values among the strains clearly indicated the presence of strong genetic influences on both of these traits, with an estimated narrow sense heritability of 60% and 35%, respectively. The patterns of differences in body weight and peak whole body BMD in the BXD strains were then integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL. After correction for redundancy among the significant correlations, QTL analysis of the BXD RI strain series provisionally identified 10 chromosomal sites linked to peak bone mass development in the female. Several of the identified sites map near genes encoding hormones, structural proteins, and cell surface receptors that are intricately involved in skeletal homeostasis. Four QTL for body weight were also identified. One of these loci was also strongly linked to inherited variation in BMD. This finding suggests that body weight and peak BMD may be influenced by linked genes or perhaps by common genes with pleiotropic effects. Our phenotyping in the RI BXD strains has allowed us to map a number of specific genetic loci strongly related to the acquisition of peak BMD. Confirmation of these findings will likely result in the understanding of which genes control skeletal health.

Functional antagonism of μ-, δ- and κ-opioid antinociception by orphanin FQ☆

Orphanin FQ (OFQ) is the recently isolated endogenous ligand for the orphan opioid-like receptor, LC132. Initial reports suggested that OFQ increased pain sensitivity when injected intracerebroventricularly (i.c.v.) in mice. However, we have recently demonstrated that OFQ is instead an anti-opioid peptide that reverses morphine- and opioid-mediated stress-induced antinociception. Morphine binds to multiple opioid receptor types (mu, delta, and kappa). The present study was designed to examine specific interactions of OFQ with antinociception mediated by each receptor type. To this end, mice were administered i.c.v. cocktails containing either vehicle or OFQ (10 nmol) and a mu-specific ([D-Ala2, N-Me-Phe4-Gly-ol]enkephalin; DAMGO; 0-0.1 nmol), delta-specific ([D-Pen2, D-Pen5]enkephalin; DPDPE; 0-50 nmol), or kappa-specific (U-50,488H; 0-1000 nmol) agonist. As we have shown previously, OFQ alone had no effect on nociceptive sensitivity. OFQ was, however, able to completely block supraspinal antinociception produced by all three receptor type-selective agonists. We conclude, therefore, that OFQ functionally antagonizes mu (and (opioid receptors, and may play a general role in opioid modulation.

Gender specificity in the genetic determinants of peak bone mass

Peak bone mass is a major determinant of osteoporotic fracture risk. Gender differences in peak bone mass acquisition are well recognized in humans and may account for a substantial share of the increased prevalence of fragility fractures in women compared with men. Skeletal development is regulated by both heritable and environmental factors. Experimental animal models provide a means to circumvent complicating environmental factors. In this study we examined the heritability of peak bone mineral density (BMD) in genetically distinct laboratory mouse strains raised under strict environmental control and sought to identify genetic loci that may contribute to gender differences in this skeletal phenotype. Peak whole body BMD of male and female mice from a panel of 18 recombinant inbred (RI) strains derived from a cross between C57BL/6 and DBA/2 progenitors (BXD) was measured by dual‐energy X‐ray absorptiometry (DXA). A highly significant relationship existed between body weight and BMD in the BXD RI mice (r2 = 0.25; p = 1 × 10−43). To allow for comparison between male and female RI strains, whole body BMD values were corrected for the influence of body weight. The distribution of weight‐corrected BMD (WC‐BMD) values among the strains indicated the presence of strong genetic influences in both genders, with an estimated narrow sense heritability of 45% and 22% in male and female mice, respectively. Comparison of RI strain results by two‐way analysis of variance (ANOVA) revealed a significant strain‐by‐gender interaction (F1,17,479 = 6.13; p < 0.0001). Quantitative trait locus (QTL) analysis of the BXD RI strain series provisionally identified nine chromosomal sites linked to peak bone mass development in males and seven regions in females. In two cases, the provisional chromosomal loci were shared between genders, but in most cases they were distinct (five female‐specific QTLs and six male‐specific QTLs). QTL analysis of a genetically heterogeneous F2 population derived from the B6 and D2 progenitor strains provided additional support for the gender specificity of two loci. A significant phenotype‐genotype correlation was only observed in male F2 mice at microsatellite marker D7Mit114 on chromosome 7, and a correlation at D2Mit94 on chromosome 2 was only observed in female F2 mice. The present data highlight the important role of gender in the genetic basis of peak bone mass in laboratory mice. Because the male phenotype is associated with considerable fracture risk reduction, an elucidation of the nature of that effect could provide the basis for novel diagnostic, preventative, or therapeutic approaches.

Type I and type II error rates for quantitative trait loci (QTL) mapping studies using recombinant inbred mouse strains

Effective mapping strategies for quantitative traits must allow for the detection of the more important quantitative trait loci (QTLs) while minimizing false positives. Type I (false-positive) and Type II (false-negative) error rates were estimated from a computer simulation of QTL mapping in the BXD recombinant inbred (RI) set comprising 26 strains of mice, and comparisons made with theoretical predictions. The results are generally applicable to other RI sets when corrections are made for differing strain numbers and marker densities. Regardless of the number or magnitude of simulated QTLs contributing to the trait variance, thep value necessary to provide genome-wide. 05 Type I error protection was found to be aboutp=.0001. To provide adequate protection against both Type I (α=.0001) and Type II (β=.2) errors, a QTL would have to account for more than half of the between-strain (genetic) variance if the BXD or similar set was used alone. In contrast, a two-step mapping strategy was also considered, where RI strains are used as a preliminary screen for QTLs to be specifically tested (confirmed) in an F2 (or other) population. In this case, QTLs accounting for ∼16% of the between-strain variance could be detected with an 80% probability in the BXD set when α=0.2. To balance the competing goals of minimizing Type I and II errors, an economical strategy is to adopt a more stringent α initially for the RI screen, since this requires only a limited genome search in the F2 of the RI-implicated regions (∼10% of the F2 genome whenp<.01 in the RIs). If confirmed QTLs do not account in the aggregate for a sufficient proportion of the genetic variance, then a more relaxed α value can be used in the RI screen to increase the statistical power. This flexibility in setting RI α values is appropriate only when adequate protection against Type I errors comes from the F2 (or other) confirmation test(s).