Mice Of Different Genotypes Research Articles

Cooperation of Wnt/β-catenin and Dll1-mediated Notch pathway in Lgr5-positive intestinal stem cells regulates the mucosal injury and repair in DSS-induced colitis mice model.

Lgr5-positive cells located in the basal layer of crypts have self-regenerative and proliferative differentiation potentials of intestinal stem cells (ISCs), maintaining a balance of regeneration-repair in mucosal epithelium. However, the mechanisms of mucosal repair that are regulated by ISCs in ulcerative colitis (UC) remain unclear. Colon tissues from patients with UC were collected to test β-catenin and Notch1 expression by using Western blot and quantitative real-time polymerase chain reaction (PCR). β-cateninfl/fl mice, β-cateninTg mice, and Dll1tm1 Gos mice were used to cross with Lgr5-EGFP-IRES-creERT2 mice to generate mice of different genotypes, altering the activation of Wnt/β-catenin and Dll1-mediated Notch signaling in ISCs in vivo. Dextran sulfate sodium (DSS) was used to induce a colitis mice model. Intestinal organoids were isolated and cultured to observe the proliferation and differentiation levels of ISCs. β-catenin and Notch1 expression were significantly increased in the inflamed colon tissues from patients with UC. Wnt/β-catenin activation and Dll1-mediated Notch pathway inhibition in Lgr5-positive stem cells promoted the expressions of E-cadherin, CK20, and CHGA in colonic organoids and epithelium, implying the promotion of colonic epithelial integrity. Activation of Wnt/β-catenin and suppression of Dll1-mediated Notch pathway in Lgr5-positive ISCs alleviated the DSS-induced intestinal mucosal inflammation in mice. Lgr5-positive ISCs are characterized by self-renewal and high dividend potential, which play an important role in the injury and repair of intestinal mucosa. More importantly, the Wnt/β-catenin signaling pathway cooperates with the Notch signaling pathway to maintain the function of the Lgr5-positive ISCs.

GDF15 mediates renal cell plasticity in response to potassium depletion in mice.

To understand the mechanisms involved in the response to a low-K+ diet (LK), we investigated the role of the growth factor GDF15 and the ion pump H,K-ATPase type 2 (HKA2) in this process. Male mice of different genotypes (WT, GDF15-KO, and HKA2-KO) were fed an LK diet for different periods of time. We analyzed GDF15 levels, metabolic and physiological parameters, and the cellular composition of collecting ducts. Mice fed an LK diet showed a 2-4-fold increase in plasma and urine GDF15 levels. Compared to WT mice, GDF15-KO mice rapidly developed hypokalemia due to impaired renal adaptation. This is related to their 1/ inability to increase the number of type A intercalated cells (AIC) and 2/ absence of upregulation of H,K-ATPase type 2 (HKA2), the two processes responsible for K+ retention. Interestingly, we showed that the GDF15-mediated proliferative effect on AIC was dependent on the ErbB2 receptor and required the presence of HKA2. Finally, renal leakage of K+ induced a reduction in muscle mass in GDF15-KO mice fed LK diet. In this study, we showed that GDF15 and HKA2 are linked and play a central role in the response to K+ restriction by orchestrating the modification of the cellular composition of the collecting duct.

Bayesian semiparametric Markov renewal mixed models for vocalization syntax.

Speech and language play an important role in human vocal communication. Studies have shown that vocal disorders can result from genetic factors. In the absence of high-quality data on humans, mouse vocalization experiments in laboratory settings have been proven useful in providing valuable insights into mammalian vocal development, including especially the impact of certain genetic mutations. Such data sets usually consist of categorical syllable sequences along with continuous intersyllable interval (ISI) times for mice of different genotypes vocalizing under different contexts. ISIs are of particular importance as increased ISIs can be an indication of possible vocal impairment. Statistical methods for properly analyzing ISIs along with the transition probabilities have however been lacking. In this article, we propose a class of novel Markov renewal mixed models that capture the stochastic dynamics of both state transitions and ISI lengths. Specifically, we model the transition dynamics and the ISIs using Dirichlet and gamma mixtures, respectively, allowing the mixture probabilities in both cases to vary flexibly with fixed covariate effects as well as random individual-specific effects. We apply our model to analyze the impact of a mutation in the Foxp2 gene on mouse vocal behavior. We find that genotypes and social contexts significantly affect the length of ISIs but, compared to previous analyses, the influences of genotype and social context on the syllable transition dynamics are weaker.

Littermate-Controlled Experiments Reveal Eosinophils Are Not Essential for Maintaining Steady-State IgA and Demonstrate the Influence of Rearing Conditions on Antibody Phenotypes in Eosinophil-Deficient Mice.

Conflicting data has emerged regarding a role for eosinophils in IgA production, with some reports that eosinophils support both secretory and circulating IgA levels during homeostasis. Previous studies have compared antibody levels between wildtype and eosinophil-deficient mice, but these mice were obtained from different commercial vendors and/or were not littermates. Thus, the possibility remains that extrinsic environmental factors, rather than an intrinsic lack of eosinophils, are responsible for the reports of reduced IgA in eosinophil-deficient mice. Here we used wild-type and eosinophil-deficient (ΔdblGATA) mice that were purchased from a single vendor, subsequently bred in-house and either co-housed as adults, co-reared from birth or raised as littermates. We found no differences in the levels of secretory IgA or in the numbers of small intestinal IgA-producing plasma cells between wild-type and ΔdblGATA mice, demonstrating that under controlled steady-state conditions eosinophils are not essential for the maintenance of secretory IgA in the intestinal tract. While we found that levels of IgM and IgE were significantly elevated in the serum of ΔdblGATA mice compared to co-reared or co-housed wild-type mice, no significant differences in these or other circulating antibody isotypes were identified between genotypes in littermate-controlled experiments. Our results demonstrate that eosinophils are not required to maintain secretory or circulating IgA production and the absence of eosinophils does not impact circulating IgG1, IgG2b, IgM, or IgE levels during homeostasis. These findings emphasize the importance of optimally controlling rearing and housing conditions throughout life between mice of different genotypes.

The Promotive Effects of PTHrP on the Union of Femoral Shaft Fracture in a Mouse Model

In order to explore the effects of PTH-related peptide on fracture healing in mice, the intervention effects and mechanism of action of PTH-related peptide on femoral shaft fracture models in mice of different genotypes were analysed to obtain a new approach for promoting the healing fractures clinically. A total of 32 8-week old PTH-related peptide wild-type and PTH-related peptide gene knockout heterozygous (PTH-related peptide +/-) mice were selected as the test system and standard femoral shaft fracture mouse models were constructed by intramedullary wire fixation. After model construction, all mice were randomly divided into the wild type control, the PTH-related peptide +/- control, the wild type treated and the PTH-related peptide +/- treated groups, with 8 mice in each group. Mice in both treated groups were administered with 80 μg/kg/day of PTH-related peptide (1-34) subcutaneously and the control groups received subcutaneous injections of saline. After 2 weeks, all mice were sacrificed and the fractured right femur of each mouse in each group was taken for X-ray examination and micro-CT scan to observe the union of the fracture. Next, the formed callus tissues were paraffin-embedded and stained to analyse and compare the bone formation changes of osteoblasts in the fracture sites of mice. Compared to the wild-type group, the saline-treated mice in the PTH-related peptide +/- group had clear fracture lines with callus volume and bone mineral density significantly reduced; after PTH-related peptide treatment, the fractures in the PTH-related peptide +/- group were tightly connected and a large number of osseous calluses were formed; in addition, the density of the callus was higher, which was significantly different from those in the control group (p<0.05). Compared to the wild-type group, the area of cartilage callus of the saline-treated PTH-related peptide +/- group increased by 97 % (p<0.05); after PTH-related peptide administration, the total callus area and osseous callus area increased significantly, while the area of cartilage osseous significantly decreased compared to the control group and after PTH-related peptide administration, the number of osteoblasts and the percent positive area of collagen I in mice were significantly increased compared to the control group. In conclusion, endogenous PTH-related peptide deficiency could lead to delayed healing of fracture, which could be compensated by PTH-related peptide administration, thereby accelerating the formation of calluses and promoting fracture healing in mice.

Evaluation of coronary artery hemodynamics and its relationship with atherosclerosis in west diet fed LDL-R knockout mice by ultrasound bio-microscopy

Objective To evaluate the relationship between atherosclerosis and hemodynamic of coronary artery in mice detecting by ultrasound bio-microscopy flow imaging. Methods Double 14 20-week-old LDL-R-/- and C57BL/6 male mice were selected, and randomly divided into two groups in each genotype according to weight. Each two groups were fed to 28 weeks or 36 weeks age respectively with west diet. Coronary artery hemodynamics in these mice were assessed in vivo by Vevo®2100 ultrasound imaging system, then the intima-media thickness(IMT) of aorta in histopathology were analyzed. The differences of coronary artery hemodynamic parameters such as maximum velocity (Vmax), mean velocity (Vmean) and velocity time integral (VTI) were compared between mice of different genotypes of the same week and mice of different weeks of the same genotype. And the relationship between coronary artery hemodynamic in ultrasound and aortic IMT in histopathology were analyzed. Results ①All coronary hemodynamic parameters in LDL-R-/- mice were significantly lower than those of wild-type mice except the Vmax between two 28-week-old genotypes group at the same weeks of age of different genotypes (all P 0.05). ②The histopathological measurements of aortic IMT in LDL-R-/- mice were significantly higher than those of wild type mice(all P<0.05), and those of 36-week-old mice were significantly higher than those of 28-week-old mice(all P<0.05). ③All coronary hemodynamic parameters such as Vmax, Vmean and VTI were negatively correlated with pathological measurements of aortic IMT (r=-0.532, -0.423, -0.524; all P<0.05). Conclusions The parameters of coronary artery hemodynamics obtained by ultrasound bio-microscopy are well correlated with the pathological results of atherosclerosis. Ultrasound bio-microscopic flow imaging can be used as a new method to evaluate the degree of atherosclerosis in mice by detecting the hemodynamic parameters of coronary artery. Key words: Ultrasound bio-microscopy; Mice; Coronary artery; Hemodynamics; Atherosclerosis

Corrigendum] Alterations in gene expression during sexual differentiation in androgen receptor knockout mice induced by environmental endocrine disruptors

Following the publication of the above article, an interested reader drew to our attention a number of issues and concerns related to the descriptions of the genes and mouse models in the above paper. After having consulted with the authors, they have acknowledged that their paper did indeed contain a number of errors, and these are listed below in this Corrigendum. First, the authors realized that an incorrect figure was included as Fig.1 in the paper. The correct (and entirely different) version of Fig.1, together with its figure legend, is shown opposite. The following textual errors in the paper should also be noted: i) In the Abstract, lines3‑4 in the left‑hand column (LHC): "androgen receptor (AR)‑/‑, AR+/‑ and AR+/+ male mice" should have been written as "androgen receptor (AR) conditional knockout mice"; line5 in the LHC: "flox‑AR" should have been written as "ARflox/+"; line6 in the LHC: "AR‑Cre" should have been written as "Amhr2‑Cre"; line22 in the LHC: "AR+/‑" should have been written as "ARflox/Y"; line1 in the right‑hand column (RHC): "heterozygous" should have been written as "ARflox/Y"; and line2 in the RHC, "heterozygous" should have been written as "ARflox/Y Cre+". ii) In the Introduction, paragraph 3, lines6‑7 in the LHC: "or female ARKO mice" should have been deleted; paragraph4, line5 in the LHC: "AR‑/‑", "AR+/‑" and "AR+/+" should have been deleted; paragraph4, line11 in the LHC: "AR‑/‑", and "AR+/‑" should have been written as "AR‑/Y", whereas "AR+/+" should have been written as "AR+/Y". iii) In the Materials and methods, paragraph3, the last sentence in the RHC {"The identification of the plasmid containing leydig cells expressing Cre recombinant enzyme and the specificity of the genetically modified (gm) sequence [Amhr2‑Cre (7.1)] are shown in Fig.1} should have been written as "The positive clones were identified by digestion and sequencing." iv) In the Results, the first subheading in the RHC ("...transgenic mouse model (Amhr2‑Cre)" should have been written as "...AR conditional knockout targeting vector"; paragraph1, lines1‑3 in the RHC: The sentence "The AR gene condition knockout plasmid was identified and confirmed by enzyme digestion with BamHI" should have been written as "The AR gene conditional knockout strategy is shown in Fig.1. The gene targeting vector was identified and confirmed by enzyme digestion with BamHI"; paragraph1, line4 in the RHC: "2.7kb" should have been written as "7.8 kb"; Table III, in the title and in the Table heading, "AR+/‑" should have been written as "AR+/Y", and "AR‑/‑" should have been written as "AR‑/Y"; in the subheading "Comparison between AR‑/‑ male mice and normal mice", "AR‑/‑" should have been written as "AR‑/Y", and "normal" should have been written as "AR+/Y"; and paragraph4, lines 3‑5 in the LHC: "AR+/‑" should have been written as "AR+/Y", and "AR‑/‑" should have been written as "AR‑/Y". All the authors thank the interested reader for drawing these matters to their attention, and agree to the contents of this Corrigendum. The authors also stress that these errors did not significantly influence either the results or the conclusions of the paper. Furthermore, the authors apologize to the Editor of International Journal of Molecular Medicine and to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 35: 399‑404, 2015; DOI: 10.3892/ijmm.2014.2015].

Detection and Classification of Novel Renal Histologic Phenotypes Using Deep Neural Networks

With the advent and increased accessibility of deep neural networks (DNNs), complex properties of histologic images can be rigorously and reproducibly quantified. We used DNN-based transfer learning to analyze histologic images of periodic acid-Schiff-stained renal sections from a cohort of mice with different genotypes. We demonstrate that DNN-based machine learning has strong generalization performance on multiple histologic image processing tasks. The neural network extracted quantitative image features and used them as classifiers to look for differences between mice of different genotypes. Excellent performance was observed at segmenting glomeruli from non-glomerular structure and subsequently predicting the genotype of the animal on the basis of glomerular quantitative image features. The DNN-based genotype classifications highly correlate with mesangial matrix expansion scored by a pathologist (R.E.C.), which differed in these animals. In addition, by analyzing non-glomeruli images, the neural network identified novel histologic features that differed by genotype, including the presence of vacuoles, nuclear count, and proximal tubule brush border integrity, which was validated with immunohistologic staining. These features were not identified in systematic pathologic examination. Our study demonstrates the power of DNNs to extract biologically relevant phenotypes and serve as a platform for discovering novel phenotypes. These results highlight the synergistic possibilities for pathologists and DNNs to radically scale up our ability to generate novel mechanistic hypotheses in disease.

An Accumulation-of-Evidence Task Using Visual Pulses for Mice Navigating in Virtual Reality.

The gradual accumulation of sensory evidence is a crucial component of perceptual decision making, but its neural mechanisms are still poorly understood. Given the wide availability of genetic and optical tools for mice, they can be useful model organisms for the study of these phenomena; however, behavioral tools are largely lacking. Here, we describe a new evidence-accumulation task for head-fixed mice navigating in a virtual reality (VR) environment. As they navigate down the stem of a virtual T-maze, they see brief pulses of visual evidence on either side, and retrieve a reward on the arm with the highest number of pulses. The pulses occur randomly with Poisson statistics, yielding a diverse yet well-controlled stimulus set, making the data conducive to a variety of computational approaches. A large number of mice of different genotypes were able to learn and consistently perform the task, at levels similar to rats in analogous tasks. They are sensitive to side differences of a single pulse, and their memory of the cues is stable over time. Moreover, using non-parametric as well as modeling approaches, we show that the mice indeed accumulate evidence: they use multiple pulses of evidence from throughout the cue region of the maze to make their decision, albeit with a small overweighting of earlier cues, and their performance is affected by the magnitude but not the duration of evidence. Additionally, analysis of the mice's running patterns revealed that trajectories are fairly stereotyped yet modulated by the amount of sensory evidence, suggesting that the navigational component of this task may provide a continuous readout correlated to the underlying cognitive variables. Our task, which can be readily integrated with state-of-the-art techniques, is thus a valuable tool to study the circuit mechanisms and dynamics underlying perceptual decision making, particularly under more complex behavioral contexts.

Identification of T helper (Th)1- and Th2-associated antigens of Cryptococcus neoformans in a murine model of pulmonary infection

Cryptococcosis, caused by Cryptococcus neoformans, has been demonstrated to be controlled by T helper (Th)1 cells while Th2 cells are associated with fungal growth and dissemination. Although cryptococcal immunoreactive protein antigens were previously identified, their association with Th1 or Th2 immune responses was not provided. In mice, Th1-dependent IFN-γ induces the production of IgG2a, whereas the Th2 cytokine IL-4 stimulates the expression of IgG1 rendering each isotype an indicator of the underlying Th cell response. Therefore, we performed an immunoproteomic study that distinguishes Th1- and Th2-associated antigens by their reactivity with Th1-dependent IgG2a or Th2-dependent IgG1 antibodies in sera from C. neoformans-infected wild-type mice. We additionally analysed sera from Th2-prone IL-12-deficient and Th1-prone IL-4Rα-deficient mice extending the results found in wild-type mice. In total, ten, four, and three protein antigens associated with IgG1, IgG2a, or both isotypes, respectively, were identified. Th2-associated antigens represent promising candidates for development of immunotherapy regimens, whereas Th1-associated antigens may serve as candidates for vaccine development. In conclusion, this study points to intrinsic immunomodulatory effects of fungal antigens on the process of Th cell differentiation based on the identification of cryptococcal protein antigens specifically associated with Th1 or Th2 responses throughout mice of different genotypes.

Abstract 201: Maternal HDL-Cholesterol Levels in Women From the Gambia are Directly Related to Infant Birthweight

Babies born with low birthweight are often at a health disadvantage. Previous studies have shown direct relationships between maternal plasma cholesterol and infant birthweight in resource-rich countries. As plasma cholesterol levels are often decreased in resource-poor countries, the purpose of these studies was to evaluate the relationship between plasma cholesterol and birthweight in women enrolled in the ENID trial (ISRCTN49285450) in rural Gambia, West Africa. Plasma was obtained at enrolment (13.6±3.3 wk) and at 20 and 30 weeks of gestation; samples were obtained from women with term infants that weighed &lt;2.75 kg or &gt;3.25 kg at birth. Women with lower HDL-cholesterol (HDL-C) concentrations in mid-pregnancy had lower birthweight infants compared to women with higher HDL-C concentrations. There was no significant association between LDL-C or total cholesterol concentrations and birthweight. The relationship with HDL-C and birthweight was maintained when maternal BMI was included in the model. To begin to elucidate the processes involved in the regulation of fetal growth, placental function was examined in mice with increasing maternal HDL-C concentrations based on apoA-I levels; mice were lacking apoA-I (apoA-I -/- ), were wildtype (apoA-I +/+ ), or had excess apoA-I (apoA-I tg/tg ). HDL decreased in size as plasma apoA-I levels increased, and there were no statistical differences in the proteins carried by HDL, except for apoA-I, in pregnant mice of different genotypes. However, pregnancy alone led to changes in the HDL proteome. Importantly, fetuses of mice with lower concentrations of maternal HDL-C had reduced growth rates, not due to a lack of fetal apoA-I. The murine fetal growth rates were directly related to nutrient uptake by and transport across the placenta. This work suggests that maternal HDL affects placental function leading to enhanced nutrient supply and improved growth in utero, making HDL a potential biomarker for fetal growth and putative target for intervention.

Are Changes in Composition in Response to Treatment of a Mouse Model of Osteogenesis Imperfecta Sex-dependent?

Osteogenesis imperfecta (OI) is a genetic disease characterized by skeletal fragility and deformity. There is extensive debate regarding treatment options in adults with OI. Antiresorptive treatment reduces the number of fractures in growing oim/oim mice, an animal model that reproducibly mimics the moderate-to-severe form of OI in humans. Effects of long-term treatments with antiresorptive agents, considered for treatment of older patients with OI with similar presentation (moderate-to-severe OI) are, to date, unknown. Fourier transform infrared (FTIR) imaging, which produces a map of the spatial variation in chemical composition in thin sections of bone, was used to address the following questions: (1) do oim/oim mice show a sex dependence in compositional properties at 6.5 months of age; (2) is there a sex-dependent response to treatment with antiresorptive agents used in the treatment of OI in humans; and (3) are any compositional parameters in oim/oim mice corrected to wild-type (WT) values after treatment? FTIR imaging data were collected from femurs from four to five mice per sex per genotype per treatment. Treatments were 24 weeks of saline, alendronate, or RANK-Fc; and 12 weeks of saline+12 weeks RANK-Fc and 12 weeks of alendronate+RANK-Fc. FTIR imaging compositional parameters measured in cortical and cancellous bones were mineral-to-matrix ratio, carbonate-to-mineral ratio, crystal size/perfection, acid phosphate substitution, collagen maturity, and their respective distributions (heterogeneities). Because of the small sample size, nonparametric statistics (Mann-Whitney U- and Kruskal-Wallis tests with Bonferroni correction) were used to compare saline-treated male and female mice of different genotypes and treatment effects by sex and genotype, respectively. Statistical significance was defined as p<0.05. At 6.5 months, saline-treated male cortical oim/oim bone had increased mineral-to-matrix ratio (p=0.016), increased acid phosphate substitution (p=0.032), and decreased carbonate-to-mineral ratio (p=0.016) relative to WT. Cancellous bone in male oim/oim also had increased mineral-to-matrix ratio (p=0.016) relative to male WT. Female oim/oim mouse bone composition for all cortical and cancellous bone parameters was comparable to WT (p>0.05). Only the female WT mice showed a response of mean compositional properties to treatment, increasing mineral-to-matrix after RANK-Fc treatment in cancellous bone (p=0.036) compared with saline-treated mice. Male oim/oim increased mineral-to-matrix cortical and cancellous bone heterogeneity in response to all long-term treatments except for saline+RANK-Fc (p<0.04); female oim/oim cortical mineral-to-matrix bone heterogeneity increased with ALN+RANK-Fc and all treatments increased cancellous female oim/oim bone acid phosphate substitution heterogeneity (p<0.04). Both oim/oim and WT mice, which demonstrate sex-dependent differences in composition with saline treatment, showed few responses to long-term treatment with antiresorptive agents. Female WT mice appeared to be more responsive; male oim/oim mice showed more changes in compositional heterogeneity. Changes in bone composition caused by these agents may contribute to improved bone quality in oim/oim mice, because the treatments are known to reduce fracture incidence. The optimal drug therapy for long-term treatment of patients with moderate-to-severe OI is unknown. Based on bone compositional changes in mice, antiresorptive treatments are useful for continued treatment in OI. There is a reported sexual dimorphism in fracture incidence in adults with OI, but to date, no one has reported differences in response to pharmaceutical intervention. This study suggests that such an investigation is warranted.

Alterations in gene expression during sexual differentiation in androgen receptor knockout mice induced by environmental endocrine disruptors.

In the present study, we aimed to explore the effect of environmental endocrine disruptors (EEDs) on sexual differentiation in androgen receptor (AR)-/-, AR+/- and AR+/+ male mice. By using a Cre-loxP conditional knockout strategy, we generated AR knockout mice. By mating flox-AR female mice with AR-Cre male mice, the offspring male mice which were produced were examined. Mice not subjected to any type of intervention were used as the controls. Furthermore, male mice of different genotypes were selected and further divided into subgroups as follows: the control group, bisphenol A (BPA) group and the dibutyl phthalate [corrected] (DBP) group. The expression of the Wilms tumor 1 (WT1), lutropin/choriogonadotropin receptor (LHR), 17-β-hydroxysteroid dehydrogenase type 3 (17βHSD3) and steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2) genes was determined by RT-qPCR and western blot analysis. There was no statistically significant difference in the weight of the mice between the control group and the knockout group (P>0.05). The results revealed that, compared with the control group, in the knockout group, anogenital distance was shortened, and testicular weight and testosterone levels were decreased; estradiol levels were elevated; the differences were statistically significant (P<0.05). In the group of AR+/- male mice exposed to 100 mg/l EEDs, hypospadias was successfully induced, suggesting that EEDs are involved in the embryonic stage of sexual development in male mice. The quantitative detection of WT1, LHR, 17βHSD3 and SRD5A2 gene expression by RT-qPCR and western blot analysis indicated that these genes were significantly downregulated in the mice in the BPA group. In conclusion, exposure to EEDs induces hypospadias in heterozygous and wild-type male mice offspring during sexual differentiation, but has no effect on homozygous offspring. Therefore, EEDs play an important role during the third stage of sexual differentiation.

A tale of two methods: Identifying neuronal CB1 receptors.

A paper by Dr. G. Marsicano's group [1] in this issue of Molecular Metabolism is clarifying and further elaborating on some of their previous results on the action of cannabinoid type 1 receptor (CB1) in neuronal mitochondria [2]. This new article was written, at least in part, in response to our finding that anti-CB1 serum used in this as well as in other studies also recognizes mitochondrial stomatin-like protein 2 [3]. Although we did not specifically criticize their major findings and general approach, we did emphasize the possible technical issues that can occur when these antibodies are used. We believe that it is a constructive course of action when different laboratories publicly discuss limitations of methodologies and reliability of the data interpretation in biomedicine. Study of mitochondria in the brain is one of those topics that merit such discourse. “Hard core” mitochondrial bioenergetics has been most extensively studied in tissues with homogenous cell populations, such as the liver, muscle, and heart, that most reliably (albeit not perfectly) provide isolation of mitochondria from the cell type of interest (e.g., hepatocytes and myocytes). The brain, however, consists of multiple cell types and subclasses, and it is virtually impossible to isolate very pure mitochondrial fractions using currently available technologies. In accord, Marsciano and colleagues previously reported that the CB1 agonist, WIN 55,212-2 (WIN), at concentrations of 50 nM and 100 nM, reduced mitochondrial respiration by about 40% and 50%, respectively [2]. In the new study, they report approximately 7% and 20% reduction of mitochondrial respiration at corresponding concentrations of WIN [1]. A confounding factor may be contamination of mitochondrial fractions with the fragments of axonal cell membranes that are known to contain CB1. Hebert-Chatelain et al. [1] also used more sensitive immunoperoxidase cytochemical method with DAB–Ni as a chromogen. They now report mitochondrial labeling in mice of different genotypes, including CB1-KO mice, similar to what we reported. We agree with Dr. Marsicano that adapted experimental procedures and proper controls are essential for correct interpretations of the data. Their and our observations are not too dissimilar; nevertheless, different conclusions may be drawn. We hope that these constructive interactions will increase the drive to improve currently available experimental technologies so that fundamental issues regarding brain mitochondria can be more conclusively addressed in future studies.

Quantitative Fundus Autofluorescence in Mice: Correlation With HPLC Quantitation of RPE Lipofuscin and Measurement of Retina Outer Nuclear Layer Thickness

Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age. Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4(-/-), Abca4(+/-), and Abca4(+/+) mice (ages 2-12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed. The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2-12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4(-/-) mice than in Abca4(+/+) mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4(-/-) and wild-type mice was more pronounced (approximately 3-4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4(-/-) mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age. The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.

Enhanced sensitivity of skeletal muscle growth in offspring of mice long-term selected for high body mass in response to a maternal high-protein/low-carbohydrate diet during lactation

To investigate the effects of a high-protein/low-carbohydrate diet fed to mice of different genotypes during pregnancy and/or lactation on offspring skeletal muscle growth and metabolism. Pregnant mice from strains selected for high body mass (DU6) or endurance running performance (DUhLB) and from an unselected control strain (DUK) were fed iso-energetic diets containing 20 % (C) or 40 % protein and low carbohydrate (HP) from mating to weaning at day 21 of age. At birth, offspring were cross-fostered resulting in different exposure to maternal prenatal-preweaning diets (C-C, HP-C, C-HP, HP-HP). Rectus femoris muscle of male mice (n = 291) was examined at day 23, 44, 181 and 396 of age for cellular growth and metabolism. At day 23 of age, body and muscle growth was retarded by 30-40 % (P < 0.0001) in response to the C-HP and HP-HP, but not to the HP-C diet, due to reduced fibre size (P < 0.0001) but not fibre number. DNA was highly reduced in DU6, less in DUhLB, but not in DUK muscle (strain × diet; P < 0.0001). Despite some compensation, muscle growth was still impaired (P < 0.001) in adulthood (day 44; day 181), but at senescence only in DU6 mice (strain × diet; P < 0.05). Only at weaning, isocitrate and lactate dehydrogenase activities were increased or decreased (P < 0.0001), respectively, without influence on fibre type composition. A high-protein/low-carbohydrate diet fed to dams during lactation, but not during pregnancy, retards skeletal muscle growth in offspring with greater response of a heavy, obese compared with a physically fit and a control genotype and causes a transient shift towards oxidative versus glycolytic muscle metabolism.

Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Circadian Regulation of ATP Release in Astrocytes

Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high-level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP(3) signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP(3) signaling to express daily rhythms in ATP release.

Mutant Huntingtin in Glial Cells Exacerbates Neurological Symptoms of Huntington Disease Mice

Huntington disease (HD) is caused by an expansion of the polyglutamine (polyQ) repeat (>37Q) in huntingtin (htt), and age of onset is inversely correlated with the length of the polyQ repeat. Mutant htt with expanded polyQ is ubiquitously expressed in various types of cells, including glia, but causes selective neurodegeneration. Our recent study demonstrated that expression of the N-terminal mutant htt with a large polyQ repeat (160Q) in astrocytes is sufficient to induce neurological symptoms in mice (Bradford, J., Shin, J. Y., Roberts, M., Wang, C. E., Li, X.-J., and Li, S. H. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 22480-22485). Because glia-neuron interactions are critical for maintaining the normal function and survival of neurons in the brain and because mutant htt is more abundant in neurons than in glial cells, it is important to investigate whether glial htt can still contribute to HD pathology when mutant htt is abundantly expressed in neuronal cells. We generated transgenic mice that express mutant htt with 98Q in astrocytes. Unlike our recently generated htt-160Q transgenic mice, htt-98Q mice do not show obvious neurological phenotypes, suggesting that the length of the polyQ repeat determines the severity of glial dysfunction. However, htt-98Q mice show increased susceptibility to glutamate-induced seizure. Mice expressing mutant htt in astrocytes were mated with N171-82Q mice that express mutant htt primarily in neuronal cells. Double transgenic mice expressing mutant htt in both neuronal and glial cells display more severe neurological symptoms and earlier death than N171-82Q mice. These findings indicate a role of glial mutant htt in exacerbating HD neuropathology and underscore the importance of improving glial function in treating HD.

Somatostatin receptor subtypes 2 and 5 mediate inhibition of egg yolk-induced gall bladder emptying in mice

Somatostatin inhibits gall bladder contraction. Impaired gall bladder emptying is associated with gall bladder stone formation. The incidence of cholecystolithiasis is high in patients treated with a somatostatin agonist octreotide, which predominantly interacts with somatostatin receptor subtype 2 (SSTR2). Therefore, it is believed that SSTR2 regulates gall bladder contraction; however, evidence has not been provided. Here, we evaluate the effects of SSTR1-SSTR5-selective agonists on egg yolk-induced gall bladder contraction in mice. Homozygous deletion of SSTR2 and SSTR5 was generated by cross-mating of SSTR2(-/-) with SSTR5(-/-) mice. Mice of different genotypes were injected with SSTR1-5-selective agonists or octreotide 15 min before induction of gall bladder emptying by egg yolk. One hour later, gall bladders were removed and weighed. Egg yolk-reduced gall bladder weights in all mice, irrespective of their genotype. Octreotide was the most potent inhibitor of gall bladder emptying in wild-type mice. In contrast, agonists with high selectivity for SSTR2 or SSTR5 inhibited gall bladder emptying by approximately 50-60%, whereas SSTR1-, SSTR3- and SSTR4-selective agonists failed to influence gall bladder contraction. In SSTR2(-/-) mice, octreotide and an SSTR5-selective agonist inhibited gall bladder emptying by approximately 50%, whereas SSTR2-selective agonists were inactive. Octreotide inhibited gall bladder emptying in SSTR5(-/-) mice by approximately 50%, without any effect in SSTR2(-/-)/SSTR5(-/-) mice. Our study provides evidence for the role of SSTR2 and SSTR5 in regulating gall bladder emptying in mice.