Whole nerve chorda tympani responses to sweeteners in C57BL/6ByJ and 129P3/J mice

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.     

Gustatory neural responses to umami taste stimuli in C57BL/6ByJ and 129P3/J mice

In long-term two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more monosodium L-glutamate (MSG) and inosine-5'-monophosphate (IMP) compared with mice from the 129P3/J (129) strain. The goal of this study was to assess the role of afferent gustatory input in these strain differences. We measured integrated responses of the mouse chorda tympani and glossopharyngeal nerves to lingual application of compounds that evoke umami taste in humans: MSG, monoammonium L-glutamate (NH(4) glutamate), IMP and guanosine-5'-monophosphate (GMP) and also to other taste stimuli. Chorda tympani responses to MSG and NH(4) glutamate were similar in B6 and 129 mice. Chorda tympani responses to IMP and GMP were lower in B6 than in 129 mice. Responses to umami stimuli in the glossopharyngeal nerve did not differ between the B6 and 129 strains. Responses to MSG, IMP and GMP were not affected by sodium present in these compounds because B6 and 129 mice had similar neural taste responses to NaCl. This study has demonstrated that the increased ingestive responses to the umami stimuli in B6 mice are accompanied by either unchanged or decreased neural responses to these stimuli. Lack of support for the role of the chorda tympani or glossopharyngeal nerves in the enhanced consumption of MSG and IMP by B6 mice suggests that it is due to some other factors. Although results of our previous study suggest that postingestive effects of MSG can affect its intake, contribution of other gustatory components (e.g. greater superficial petrosal nerve or central gustatory processing) to the strain differences in consumption of umami compounds also cannot be excluded. Strain differences in gustatory neural responses to nucleotides but not glutamate suggest that these compounds may activate distinct taste transduction mechanisms.     

Differences in postingestive metabolism of glutamate and glycine between C57BL/6ByJ and 129P3/J mice.

Amino acids are essential nutrients for living organisms. There are genetic differences in voluntary consumption of amino acids among mouse strains. In two-bottle preference tests, C57BL/6ByJ (B6) mice consume more glutamate (Glu) and glycine (Gly) solutions than do 129P3/J (129) mice. To examine the role of postingestive metabolism of these amino acids in regulation of their intake, we compared metabolism of orally administered Glu and Gly in B6 and 129 mice. After administration of Glu, there were increases in circulating glucose and insulin in B6 mice, whereas 129 mice had elevated blood alanine and body temperature. After ingestion of Gly, B6 mice had increases in blood glucose, whereas there was an elevation of body temperature in 129 mice. These data suggest that B6 mice preferentially convert ingested Glu and Gly to glucose in contrast to 129 mice, which preferentially use them for thermogenesis. This study strongly supports the hypothesis that the metabolic fate of a nutrient plays an important regulatory role in control of its intake. This is the first detailed study of mouse strain differences in amino acid metabolism.     

Quantitative trait loci for individual adipose depot weights in C57BL/6ByJ x 129P3/J F2 mice

To understand how genotype influences fat patterning and obesity, we conducted an autosomal genome scan using male and female F₂ hybrids between the C57BL/6ByJ and 129P3/J parental mouse strains. Mice were studied in middle-adulthood and were fed a low-energy, low-fat diet during their lifetime. We measured the weight of the retroperitoneal adipose depot (near the kidney) and the gonadal adipose depot (near the epididymis in males and ovaries in females). An important feature of the analysis was the comparison of linkage results for absolute adipose depot weight and depot weight adjusted for body size, i.e., relative weight. We detected 67 suggestive linkages for six phenotypes, which fell into one of three categories: those specific to absolute but not relative depot weight (Chr 5, 11, and 14), those specific to relative but not absolute depot weight (Chr 9, 15, and 16), and those involving both (Chr 2 and 7). Some quantitative trait loci (QTLs) affected one adipose depot more than another: Retroperitoneal depot weight was linked to Chr 8, 11, 12, and 17, but the linkage effects for the gonadal depot were stronger for Chr 5, 7, and 9. Several linkages were specific to sex; for instance, the absolute weight of gonadal fat was linked to Chromosome 7 in male (LOD = 3.4) but not female mice (LOD = 0.2). Refining obesity as a phenotype may uncover clues about gene function that will assist in positional cloning efforts.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Long-Term Artificial Sweetener Acesulfame Potassium Treatment Alters Neurometabolic Functions in C57BL/6J Mice

With the prevalence of obesity, artificial, non-nutritive sweeteners have been widely used as dietary supplements that provide sweet taste without excessive caloric load. In order to better understand the overall actions of artificial sweeteners, especially when they are chronically used, we investigated the peripheral and central nervous system effects of protracted exposure to a widely used artificial sweetener, acesulfame K (ACK). We found that extended ACK exposure (40 weeks) in normal C57BL/6J mice demonstrated a moderate and limited influence on metabolic homeostasis, including altering fasting insulin and leptin levels, pancreatic islet size and lipid levels, without affecting insulin sensitivity and bodyweight. Interestingly, impaired cognitive memory functions (evaluated by Morris Water Maze and Novel Objective Preference tests) were found in ACK-treated C57BL/6J mice, while no differences in motor function and anxiety levels were detected. The generation of an ACK-induced neurological phenotype was associated with metabolic dysregulation (glycolysis inhibition and functional ATP depletion) and neurosynaptic abnormalities (dysregulation of TrkB-mediated BDNF and Akt/Erk-mediated cell growth/survival pathway) in hippocampal neurons. Our data suggest that chronic use of ACK could affect cognitive functions, potentially via altering neuro-metabolic functions in male C57BL/6J mice.     

Aging and glucose homeostasis in C57BL/6J male mice

Age-dependent changes in glucose homeostasis were assessed in specific pathogen-free C57BL/6J male mice. Increased islet size and pancreatic insulin content in old (21-25-month-old) mice were associated with lower nonfasting plasma glucose levels and improved clearance of either an oral or an i.p. administered glucose load in comparison with young, mature (4-5-month-old) males. The almost twofold increase in islet size correlated with a twofold increase of glucose-stimulated insulin secretion from perifused islets from 25-month-old males compared with 5-month-old males. These aging male mice did not become obese, and there were no fibrotic changes associated with the hyperplastic islets observed in the old males. Thus, the findings that glucose tolerance did not deteriorate with age, coupled with the lack of evidence for impaired beta cell responsiveness to glucose in old males, suggest that deterioration in glucose homeostasis is not an inevitable consequence of aging in the mouse.     

Standardized experiments in mutant mice reveal behavioural similarity on 129S5 and C57BL/6J backgrounds

Behavioural analysis of mice carrying engineered mutations is widely used to identify roles of specific genes in components of the mammalian behavioural repertoire. The reproducibility and robustness of phenotypic measures has become a concern that undermines the use of mouse genetic models for translational studies. Contributing factors include low individual study power, non‐standardized behavioural testing, failure to address confounds and differences in genetic background of mutant mice. We have examined the importance of these factors using a statistically robust approach applied to behavioural data obtained from three mouse mutations on 129S5 and C57BL/6J backgrounds generated in a standardized battery of five behavioural assays. The largest confounding effect was sampling variation, which partially masked the genetic background effect. Our observations suggest that strong interaction of mutation with genetic background in mice in innate and learned behaviours is not necessarily to be expected. We found composite measures of innate and learned behaviour were similarly impacted by mutations across backgrounds. We determined that, for frequently used group sizes, a single retest of a significant result conforming to the commonly used P < 0.05 threshold results in a reproducibility of 60% between identical experiments. Reproducibility was reduced in the presence of strain differences. We also identified a P‐value threshold that maximized reproducibility of mutant phenotypes across strains. This study illustrates the value of standardized approaches for quantitative assessment of behavioural phenotypes and highlights approaches that may improve the translational value of mouse behavioural studies.     

P7C3 Attenuates the Scopolamine-Induced Memory Impairments in C57BL/6J Mice

Memory impairment is the most common symptom in patients with Alzheimer’s disease. The purpose of this study is to evaluate the memory enhancing effects of P7C3, a recently identified compound with robust proneurogenic and neuroprotective effects, on the cognitive impairment induced by scopolamine, a muscarinic acetylcholine receptor antagonist. Different behavior tests including the Y-maze, Morris water maze, and passive avoidance tests were performed to measure cognitive functions. Scopolamine significantly decreased the spontaneous alternation and step-through latency of C57BL/6J mice in Y-maze test and passive avoidance test, whereas increased the time of mice spent to find the hidden platform in Morris water maze test. Importantly, intraperitoneal administration of P7C3 effectively reversed those Scopolamine-induced cognitive impairments in C57BL/6J mice. Furthermore, P7C3 treatment significantly enhanced the level of brain-derived neurotrophic factor (BDNF) signaling pathway in the cortex and hippocampus, and the usage of selective BDNF signaling inhibitor fully blocked the anti-amnesic effects of P7C3. Therefore, these findings suggest that P7C3 could improve the scopolamine-induced learning and memory impairment possibly through activation of BDNF signaling pathway, thereby exhibiting a cognition-enhancing potential.     

Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice

BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.     

Sugar and fat conditioned flavor preferences in C57BL/6J and 129 mice: oral and postoral interactions

C57BL/6J (B6) mice consume more sugar and fat solutions than do 129 mice in 24-h preference tests. Previous studies have attributed this observation to strain differences in taste responsiveness to these nutrients. We tested the hypothesis that differences in postingestive responsiveness contribute to the strain differences. In experiment 1, B6 and 129 mice were trained to associate consumption of a flavored solution (CS+) with intragastric (IG) infusions of 16% sucrose and a different flavored solution (CS-) with IG water infusions (22 h/day). They were then retrained with new flavors paired with IG infusions of 5.6% soybean oil and water. Although both strains developed preferences for the nutrient-paired CS+ solutions, the B6 mice displayed significantly stronger preferences. The B6 mice consumed more CS+ during training, which may have contributed to their enhanced preference. In a second experiment, training intakes were equated by giving B6 and 129 mice "isosweet" CS solutions prepared with different amounts of sucrose and saccharin. The B6 and 129 mice consumed more of the sugar- or fat-paired CS+ than the water-paired CS- during training. The two strains also displayed equally strong preferences for the CS+ over CS- in choice tests, indicating that they had similar postingestive responsiveness to the sucrose and soybean oil. We propose that B6 mice consume more sugar and fat than 129 mice because their stronger orosensory response stimulates greater intake, which leads to greater stimulation of postingestive nutrient detectors and further enhancement of consumption.     

Adrenal function and the effect of a high-fat diet on C57BL/6J and C57BL/6J-ob/ob mice.

In C57BL/6J mice and the ob/+ and ob/ob mutants total plasma corticosterone levels were found to be statistically different. In C57BL/6J mice the level was 1.9 +/- 0.2 mug/100 ml plasma, in ob/+ mice 8.6 +/- 1.6 mug/100 ml and in ob/ob mice 13.7 +/- 1.5 mug/100 ml. The percentage of protein-bound corticosterone as well as the free endogenous corticosterone levels were also different. Feeding a high-fat diet to young C57BL/6J and C57BL/6J-ob/ob mice for a period of 4 weeks had no effect upon blood glucose, plasma insulin and plasma corticosterone levels. The significantly higher increase in body weight of the high-fat diet groups of both lines of mice was mainly due to fat cell hypertrophy.     

High-efficiency CAG-FLPe deleter mice in C57BL/6J background.

The increasing popularity of conditional knockout (KO) technology has resulted in the demand for efficient FLP deleter mice. In addition, FLP deleters are needed in genetic backgrounds that are suited to behavioral studies. We generated CAG-FLPe transgenic (Tg) mice with the C57BL/6J genetic background, which is one of the most commonly-used strains in behavioral studies. We assessed the recombination efficiency of the CAG-FLPe-Tg lines by crossing them with a mouse line carrying a FRT-PGK-neo-FRT cassette. Four of five independent CAG-FLPe lines induced recombination in most (91%-100%) of their progenies, although a small fraction (0%-30%, depending on the line) showed mosaic recombination patterns. These animals are highly potent as deleters of FRT cassettes and are useful for behavioral studies involving conditional KO mice.     

Sex-specific strategies in spatial orientation in C57BL/6J mice

Sex differences in spatial learning are found in many species of mammals and even in invertebrates. Results from laboratory mouse studies, however, have been inconsistent in comparison to studies of humans, laboratory rats and wild rodent species. Here we re-examined this question in C57BL/6J mice that were exposed to enriched environments using two tasks, an object recognition task and a place learning task where mice were motivated by exploratory drive, not aversive conditioning or food restriction. Using these methods, we found a female advantage for object recognition, similar to the female advantage found in humans and laboratory rats. In the place learning task, male performance was unimpaired by intra-maze cue deletion but impaired by extra-maze cue masking. Female mice, in contrast, were able to navigate accurately under both cue conditions. In summary, by utilizing testing and housing methods that were more species appropriate, we found sex-specific patterns of cue encoding and place learning in better accordance with prior results from other mammalian species. The implication of these results is that the C57BL/6J mouse is an appropriate model for the study of cognitive sex differences in mammals.     

Ventilatory behavior after hypoxia in C57BL/6J and A/J mice.

Given the environmental forcing by extremes in hypoxia-reoxygenation, there might be no genetic effect on posthypoxic short-term potentiation of ventilation. Minute ventilation (VE), respiratory frequency (f), tidal volume (VT), and the airway resistance during chemical loading were assessed in unanesthetized unrestrained C57BL/6J (B6) and A/J mice using whole body plethysmography. Static pressure-volume curves were also performed. In 12 males for each strain, after 5 min of 8% O2 exposure, B6 mice had a prominent decrease in VE on reoxygenation with either air (-11%) or 100% O2 (-20%), due to the decline of f. In contrast, A/J animals had no ventilatory undershoot or f decline. After 5 min of 3% CO2-10% O2 exposure, B6 exhibited significant decrease in VE (-28.4 vs. -38.7%, air vs. 100% O2) and f (-13.8 vs. -22.3%, air vs. 100% O2) during reoxygenation with both air and 100% O2; however, A/J mice showed significant increase in VE (+116%) and f (+62.2%) during air reoxygenation and significant increase in VE (+68.2%) during 100% O2 reoxygenation. There were no strain differences in dynamic airway resistance during gas challenges or in steady-state total respiratory compliance measured postmortem. Strain differences in ventilatory responses to reoxygenation indicate that genetic mechanisms strongly influence posthypoxic ventilatory behavior.     

Orthotopic ovarian transplantations in young and aged C57BL/6J mice

Orthotopic ovarian transplantations were done between young (6-wk-old) and aged (17-mo-old) C57BL/6J mice. The percentages of mice mating following surgery from the four possible ovarian transfer combinations were as follows: young into young, 83%; young into aged, 46%; aged into young, 83%; and aged into aged, 36%. The percentages of these mice that were pregnant 10 days following the presence of a vaginal sperm plug were as follows: young into young, 58%; young into aged, 9%; aged into young, 50%; and aged into aged, 0%. Some of the fetuses derived from matings of the above mice were dissociated and their cells prepared for chromosomal smears. No evidence of aneuploidy or mosaicism was found in fetuses derived from ovaries of young or aged mice. Aged ovaries, transferred to either young or aged recipients, were found to have fewer developing follicles and lower weight, which was most apparent in recipients that failed to mate or to get pregnant. Concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), and prolactin in plasma from each of the pregnant recipients were analyzed by radioimmunoassay. The only statistical differences found between the transfer groups occurred in FSH concentrations. Plasma FSH was markedly elevated (P less than 0.005) in young recipients with ovaries transplanted from aged donors, in comparison to young recipients with ovaries from young donors. These data indicate that the aging ovary and uterus play a secondary role in reproductive failure and that the aging hypothalamic-hypophyseal complex is primarily responsible for the loss of fecundity in older female C57BL/6J mice.     

Effect of chronic corticosterone application on depression‐like behavior in C57BL/6N and C57BL/6J mice

Many studies using genetic mouse models are performed with animals on either one of the two closely related genetic backgrounds, C57BL/6J or C57BL/6N. These strains differ only in a few genetic loci, but have some phenotypic differences that also affect behavior. In order to determine the effects of chronic stress hormone exposure, which is relevant for the pathogenesis of psychiatric disorders, we investigated here the behavioral manifestations of long‐term increase in corticosterone levels. Thus, male mice from both sub‐strains were subcutaneously implanted with corticosterone (20 mg) or placebo pellets that released the hormone for a period of 21 days and resulted in significantly elevated plasma corticosterone levels. Corticosterone significantly increased food intake in B6N, but not in B6J mice. At various time points after pellet implantation, we performed tests relevant to activity and emotional behaviors. B6J mice displayed a generally higher activity in the home cage and the open field. Corticosterone decreased the activity. In B6N mice, corticosterone also decreased sucrose preference, worsened the coat state and increased forced swim immobility, while it had no effect in the B6J strain. Altogether, these results indicate that B6N mice are more sensitive to some of the effects of chronic corticosterone treatment than B6J mice.     

QTL Analysis of Dietary Obesity in C57BL/6byj X 129P3/J F2 Mice: Diet- and Sex-Dependent Effects

Obesity is a heritable trait caused by complex interactions between genes and environment, including diet. Gene-by-diet interactions are difficult to study in humans because the human diet is hard to control. Here, we used mice to study dietary obesity genes, by four methods. First, we bred 213 F₂ mice from strains that are susceptible [C57BL/6ByJ (B6)] or resistant [129P3/J (129)] to dietary obesity. Percent body fat was assessed after mice ate low-energy diet and again after the same mice ate high-energy diet for 8 weeks. Linkage analyses identified QTLs associated with dietary obesity. Three methods were used to filter candidate genes within the QTL regions: (a) association mapping was conducted using >40 strains; (b) differential gene expression and (c) comparison of genomic DNA sequence, using two strains closely related to the progenitor strains from Experiment 1. The QTL effects depended on whether the mice were male or female or which diet they were recently fed. After feeding a low-energy diet, percent body fat was linked to chr 7 (LOD = 3.42). After feeding a high-energy diet, percent body fat was linked to chr 9 (Obq5; LOD = 3.88), chr 12 (Obq34; LOD = 3.88), and chr 17 (LOD = 4.56). The Chr 7 and 12 QTLs were sex dependent and all QTL were diet-dependent. The combination of filtering methods highlighted seven candidate genes within the QTL locus boundaries: Crx, Dmpk, Ahr, Mrpl28, Glo1, Tubb5, and Mut. However, these filtering methods have limitations so gene identification will require alternative strategies, such as the construction of congenics with very small donor regions.     

The mapping of quantitative trait loci underlying strain differences in locomotor activity between 129S6 and C57BL/6J mice

Performance in the open field and rotarod paradigms, two common assessments of locomotor function, have been demonstrated to be strain dependent in mice. In this study, eight significant quantitative trait loci (QTL) for behavior phenotypes in either the open field or rotarod paradigms were identified between the 129S6 (129/SvEvTac) and C57BL/6J strains. These strains were chosen for comparison because of their frequent use in the generation of mutant mice from gene-targeted, embryonic stem cells. Two of the QTLs for horizontal distance traveled are located on Chromosomes (Chrs) 1 and 12 and closely replicate the findings of other groups using different strains of mice. Rotarod performance was influenced in an oppositional manner by two separate QTLs on Chr 1 and 2. Additionally, examination of several different aspects of behavior in the open field revealed significant QTLs for average speed (Chr 12), duration (Chrs 2, 16, and 18), time spent in motion (Chr 16), vertical movements (rearing) (Chrs 6 and 12), and vertical time (rearing time) (Chrs 6 and 12). Our finding of independent QTLs for these topographic components of open field activity supports the idea that they are separate and distinct from total horizontal distance traveled and should be studied independently. The QTLs described in this study, in combination with our panel of polymorphic chromosomal markers for 129S6 and C57BL/6J strains, will be useful in assessing the potential epistatic effects of parental strain background on the phenotypes of genetically modified mice.