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Sample records for intact adult mouse

  1. Radioimmunoassay for intact Gross mouse leukemia virus.

    PubMed Central

    Yalow, R S; Gross, L

    1976-01-01

    A radioimmunoassay for intact Gross leukemia virus has been developed using 125I-labeled Gross virus grown in tissue culture and guinea pig antisera to Gross virus grown either in tissue culture or harvested from leukemic C3H(f) mice. Separation of bound from free labeled virus was effected using the double antibody method. The assay can detect fewer than 10(8) virus particles and has been used to measure the viral content of individual organs from inoculated leukemic C3H(f) mice and from Ak mice with spontaneous leukemia. Organs from noninoculated healthy C3H(f) mice crossreacted poorly in the system, virus generally being detectable only in the thymus and spleen and at low concentration. In some of the inoculated C3H(f) leukemic mice the viral content of as little as 0.5 mul of plasma is measurable. That this assay is for intact virus and not for soluble antigens of the viral envelope was proven by the observation that the immunoreactive material of plasma and extracts from thymus and liver of leukemic mice has a buoyant denisty in sucrose of 1.17-1.18 g/ml, corresponding to that of intact virus grown in tissue culture. With this sensitivity it may now be possible to quantitate viral concentrations in tissue and body fluids from the time of inoculation through the development of obvious pathology. PMID:1066697

  2. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium.

    PubMed

    Nickell, William T; Kleene, Nancy K; Kleene, Steven J

    2007-09-15

    When olfactory receptor neurons respond to odours, a depolarizing Cl(-) efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl(-) against an electrochemical gradient. In isolated olfactory receptor neurons, the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is essential for Cl(-) accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl(-) efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl(-) accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl(-) transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl(-) transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl(-) transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl(-)/HCO(3)(-) exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na(+)-Cl(-) cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl(-)/HCO(3)(-) exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl(-) component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl(-) accumulation in olfactory receptor neurons.

  3. Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium

    PubMed Central

    Nickell, William T; Kleene, Nancy K; Kleene, Steven J

    2007-01-01

    When olfactory receptor neurons respond to odours, a depolarizing Cl− efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl− against an electrochemical gradient. In isolated olfactory receptor neurons, the Na+–K+–2Cl− cotransporter NKCC1 is essential for Cl− accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl− efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl− accumulation. To identify the remaining transport proteins, we first screened by RT-PCR for 21 Cl− transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl− transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl− transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl−/HCO3− exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild-type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na+–Cl− cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl−/HCO3− exchangers, reduced the EOG in epithelia from both wild-type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl− component of the response. These results support the involvement of both NKCC1 and one or more DIDS-sensitive transporters in Cl− accumulation in olfactory receptor neurons. PMID:17656441

  4. Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains

    PubMed Central

    Schwarz, Martin K.; Scherbarth, Annemarie; Sprengel, Rolf; Engelhardt, Johann; Theer, Patrick; Giese, Guenter

    2015-01-01

    In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain. PMID:25993380

  5. Imaging of the intact mouse cochlea by spectral domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Gao, Simon S.; Yuan, Tao; Xia, Anping; Raphael, Patrick; Shelton, Ryan L.; Applegate, Brian E.; Oghalai, John S.

    2011-03-01

    Current medical imaging modalities, such as MRI and CT, do not provide high enough resolution to detect many changes within the cochlea that cause hearing loss. We sought to develop the technique of optical coherence tomography (OCT) to image the cochlea noninvasively and within its native environment. We used spectral domain OCT with 950 nm as the center wavelength and a bandwidth of ~100 nm to image freshly excised normal mouse cochlea at different developmental ages. The OCT system has an axial resolution of ~4 μm (in air) and a lateral resolution of ~10 μm. When we imaged normal adult mouse cochleae through the round window membrane, Reissner's membrane, the basilar membrane, the tectorial membrane, the spiral ligament, the spiral limbus, and the modiolus could be clearly identified. When we imaged intact adult cochleae, we were able to image through ~130 μm of bone and tissue to see up to a depth of ~600 μm, and all of the previously identified structures were still visible. Imaging of early postnatal mice during the timeline of cochlear development permitted visualization of the expected structural differences from adult cochleae. Therefore, we conclude that spectral domain OCT is an effective technique for noninvasive imaging of the murine cochlea.

  6. Label-free structural photoacoustic tomography of intact mouse brain

    NASA Astrophysics Data System (ADS)

    Li, Lei; Xia, Jun; Li, Guo; Garcia-Uribe, Alejandro; Wang, Lihong V.

    2015-03-01

    Capitalizing on endogenous hemoglobin contrast, photoacoustic computed tomography (PACT), a deep-tissue highresolution imaging modality, has drawn increasing interest in neuro-imaging. However, most existing studies are limited to functional imaging on the cortical surface, and the deep-brain structural imaging capability of PACT has never been demonstrated. Here, we explicitly studied the limiting factors of deep-brain PACT imaging. We found that the skull distorted the acoustic signal and blood suppressed the structural contrast from other chromophores. When the two effects are mitigated, PACT can provide high-resolution label-free structural imaging through the entire mouse brain. With 100 μm in-plane resolution, we can clearly identify major structures of the brain, and the image quality is comparable to that of magnetic resonance microscopy. Spectral PACT studies indicate that structural contrasts mainly originate from cytochrome and lipid. The feasibility of imaging the structure of the brain in vivo has also been discussed. Our results demonstrate that PACT is a promising modality for both structural and functional brain imaging.

  7. Isolation of intact astrocytes from the optic nerve head of adult mice

    PubMed Central

    Choi, Hee Joo; Sun, Daniel; Jakobs, Tatjana C.

    2015-01-01

    The astrocytes of the optic nerve head are a specialized subtype of white matter astrocytes that form the direct cellular environment of the unmyelinated ganglion cell axons. Due to their potential involvement in glaucoma, these astrocytes have become a target of research. Due to the heterogeneity of the optic nerve tissue, which also contains other cell types, in some cases it may be desirable to conduct gene expression studies on small numbers of well-characterized astrocytes or even individual cells. Here, we describe a simple method to isolate individual astrocytes. This method permits obtaining astrocytes with intact morphology from the adult mouse optic nerve and reduces contamination of the isolated astrocytes by other cell types. Individual astrocytes can be recognized by their morphology and collected under microscopic control. The whole procedure can be completed in 2-3 hours. We also discuss downstream applications like multiplex single-cell PCR and quantitative PCR (qPCR). PMID:26093274

  8. Imaging the Intact Mouse Cornea Using Coherent Anti-Stokes Raman scattering (CARS)

    PubMed Central

    Ammar, David A.; Lei, Tim C.; Kahook, Malik Y.; Masihzadeh, Omid

    2013-01-01

    Purpose. The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon–hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents. Methods. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed. Results. Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers. Conclusions. The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques. PMID:23821187

  9. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M. )

    1990-07-01

    During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker (Denny-Jaffee reagent) covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a ZP3-binding protein that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.

  10. Measurement of Basilar Membrane, Reticular Lamina, and Tectorial Membrane Vibrations in the Intact Mouse Cochlea

    NASA Astrophysics Data System (ADS)

    Ren, Tianying; He, Wenxuan

    2011-11-01

    A scanning low-coherence heterodyne interferometer (SLHI) was developed for measuring the microstructural vibration inside the cochlear partition of the intact living cochlea of mice. The sensitivity, frequency response, and dynamic range of the SLHI are comparable with those of a sensitive laser interferometer but the SLHI has a higher spatial resolution along the optical axis. The magnitude and phase of sound-induced vibrations were measured as a function of the focal position along the optical axis. Our data show that the SLHI has sufficient sensitivity, dynamic range, and temporal and spatial resolution to measure sub-nanometer vibrations of the basilar membrane, reticular lamina, and tectorial membrane in the intact living mouse cochlea. High spatial and temporal resolution, compact heterodyne design, and scanning capability make this interferometer an ideal tool to study molecular mechanisms of hearing in normal and genetically-modified mice.

  11. Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins.

    PubMed

    Bendahmane, Malika; Tulsiani, Daulat R P

    2003-07-15

    Capacitated acrosome-intact mouse spermatozoa bind to the egg's extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor-ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars (N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94-101]. To further elucidate the specificity of sperm-ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm-bead assay was carried out at 37 degrees C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm-bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor

  12. Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells.

    PubMed

    Calabro, Anthony R; Konsoula, Roula; Barile, Frank A

    2008-08-01

    Mouse embryonic stem (mES) cells were induced to form intact monolayers in cell culture inserts, using combinations of extracellular matrix (ECM) components and growth factors (GFs). Progressive formation of intact monolayers was monitored using transepithelial electrical resistance (TEER) and passage of paracellular permeability (PP) markers. The mES cells were initially inoculated on inactivated mouse embryonic fibroblasts (MEFs) plus leukemia inhibitory factor (LIF). At 75% confluence, cells were passaged in the absence of MEF and LIF to stimulate formation of rounded multicellular aggregates (MA). After 4 days, cultures containing MA were transferred to culture inserts coated with ECM components only, and grown in the presence of selected individual GFs. An additional 10-14 days revealed confluent monolayers with TEER values of 500-700 ohms cm2 (Omega cm2). Monolayers grown on inserts coated with ECM components, such as fibronectin or collagen-IV, in the presence of epidermal growth factor or keratinocyte growth factor in the medium, yielded the highest TEER measurements when compared to cultures grown without GFs or ECM. Acute cytotoxicity (AC) studies with confluent monolayers of mES cells in 96-well plates indicated that there is a high correlation (R2=0.91) between cell viability and TEER for 24-h exposure time. Also, decrease in TEER is inversely proportional with increase in PP of markers. In comparison to standardized Registry of Cytotoxicity (RC) data and TEER measurements, MTT IC50 values for mES cells are lower. Thus, at equivalent concentrations for the same chemicals, cell viability decreases before the integrity of the monolayer is compromised. This system represents a novel approach for the manipulation of mES cells toward specific intact monolayers, as an in vitro model for biological monolayer formation, and most importantly, for applications to cytotoxicity testing.

  13. ERK phosphorylation in intact, adult brain by alpha(2)-adrenergic transactivation of EGF receptors.

    PubMed

    Du, Ting; Li, Baoman; Liu, Shufang; Zang, Peizhuo; Prevot, Vincent; Hertz, Leif; Peng, Liang

    2009-12-01

    Our previous work demonstrated dexmedetomidine-activated phosphorylation of extracellular regulated kinases 1 and 2 (ERK(1/2)) in primary cultures of mouse astrocytes and showed that it is evoked by alpha(2)-adrenoceptor-mediated transactivation of epidermal growth factor (EGF) receptors, a known response to activation of G(i/o)- or G(q)-coupled receptors [Li, B., Du, T., Li, H., Gu, L., Zhang, H., Huang, J., Hertz, L., Peng, L., 2008a. Signaling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons. Br. J. Pharmacol. 154, 191-203]. Like most studies of transactivation, that study used cultured cells, raising the question whether a similar effect can be demonstrated in intact brain tissue and the brain in vivo. In the present study we have shown that (i) dexmedetomidine-mediated ERK(1/2) phosphorylation occurs in mouse brain slices with a similar concentration dependence as in cultured astrocytes (near-maximum effect at 50nM); (ii) intraperitoneal injection of dexmedetomidine (3microg/kg) in adult mice causes rapid phosphorylation of the EGF receptor (at Y845 and Y992) and of ERK(1/2) in the brain; (iii) both EGF receptor and ERK(1/2) phosphorylation are inhibited by intraventricular administration of (a) AG 1478, a specific inhibitor of the receptor-tyrosine kinase of the EGF receptor; (b) GM 6001, an inhibitor of metalloproteinase(s) required for release of EGF receptor agonists from membrane-bound precursors; or (c) heparin, neutralizing heparin-binding EGF (HB-EGF). Thus, in intact brain HB-EGF, known to be expressed in brain, may be the major EGF agonist released in response to stimulation of alpha(2)-adrenoceptors, the released agonist(s) activate(s) EGF receptors, and ERK(1/2) is phosphorylated as a conventional response to EGF receptor activation. Our previous paper (see above) showed that dexmedetomidine evokes no ERK(1/2) phosphorylation in cultured neurons, but neurons

  14. Histaminergic modulation of the intact respiratory network of adult mice.

    PubMed

    Dutschmann, M; Bischoff, A M; Büsselberg, D; Richter, D W

    2003-02-01

    Histaminergic modulation of neuronal activity in the respiratory network was investigated under normoxic and hypoxic conditions in the working heart-brainstem preparation of adult mice. Systemic application of histamine, as well as the H-1 and H-3 receptor agonists 6-[2-(4-imidazolyl)ethylamino]- N-(4-trifluoromethylphenyl) heptanecarboxamide (HTMT) and imetit, 0.5-10 micro M, significantly increased the frequency of respiratory burst discharges. Dimaprit, an H-2 receptor agonist, had no effect on respiratory activity. To test for ongoing histaminergic modulation we applied the histamine receptor antagonists pyrilamine (H-1); cimetidine (H-2) and thioperamide (H-3), each 0.5-10 micro M. Only the H-1 receptor antagonist had significant effects, viz. reduction of respiratory frequency and depression of burst amplitude. Underlying effects of histamine receptor activation were identified at the cellular level. Intracellular recordings showed that histamine mediated an increase in synaptic drive potentials in inspiratory neurones while augmentation of inhibitory and excitatory synaptic activity was observed in expiratory neurones. The augmented synaptic depolarisation of inspiratory neurones was blocked by the H-1 receptor antagonist. Histaminergic modulation is also involved in the hypoxic response of the respiratory network. Blockade of H-1 receptors significantly attenuated secondary depression of the biphasic hypoxic responses, while hypoxic augmentation was not affected. We conclude that histamine is a functional neuromodulator, which is tonically active in the respiratory network and is activated further during hypoxia. The data indicate that histaminergic neuromodulation acts predominantly via H-1 receptors.

  15. Differential stress reactivity in intact and ovariectomized prepubertal and adult female rats.

    PubMed

    Romeo, Russell D; Lee, Susan J; McEwen, Bruce S

    2004-01-01

    The pubertal development of the hypothalamic-pituitary-adrenal (HPA) axis has received relatively little experimental attention. As puberty is marked by an increase in the susceptibility to various psychiatric disorders that may be related to HPA dysfunction, it is imperative to elucidate the pubertal development of this neuroendocrine axis. To date, the limited research in this area has been conducted primarily on males. Presently, we investigated stress responsiveness, as measured by both stress hormones (e.g., corticotropin (ACTH) and corticosterone) and gonadal steroids, in intact and ovariectomized prepubertal and adult female rats before and after a 30-min session of restraint stress. We report here that intact prepubertal females exhibit an extended corticosterone stress response (30-45 min longer) compared to intact adults. Moreover, ovariectomized prepubertal females continue to exhibit a prolonged stress-induced corticosterone and progesterone response compared to ovariectomized adults, indicating these protracted responses prior to puberty are independent of ovarian hormones. ACTH levels were not significantly different between intact and ovariectomized prepubertal and adult animals at all the post-stress time points measured, suggesting that the prolonged corticosterone response in prepubertal females is due to an enhanced sensitivity to ACTH at the level of the adrenal cortex. Taken together, these data indicate that stress reactivity changes dramatically during puberty in females. Furthermore, these data demonstrate additional development of the HPA axis during pubertal maturation, resulting in a more quickly terminated stress response in adulthood.

  16. 3D perfusion mapping in the intact mouse heart after myocardial infarction using myocardial contrast echocardiography

    NASA Astrophysics Data System (ADS)

    Li, Yinbo; Yang, Zequan; French, Brent A.; Hossack, John A.

    2005-04-01

    An intact mouse model of surgically-induced myocardial infarction (MI) caused by permanent occlusion of the Left Anterior Descending (LAD) coronary artery was studied. Normal mice with no occlusion were also studied as controls. For each mouse, contrast enhanced ultrasound images of the heart were acquired in parallel cross-sections perpendicular to the sternum at millimeter increments. For accurate 3D reconstruction, ECG gating and a tri-axial adjustable micromanipulator were used for temporal and spatial registration. Ultrasound images at steady-state of blood refilling were color-coded in each slice to show relative perfusion. Myocardial perfusion defects and necrosis were also examined postmortem by staining with Phthalo blue and TTC red dyes. Good correlation (R>0.93) in perfused area size was observed between in vivo measurements and histological staining. A 3D multi-slice model and a 3D rendering of perfusion distribution were created and showed a promising match with postmortem results, lending further credence to its use as a more comprehensive and more reliable tool for in vivo assessment of myocardial perfusion than 2D tomographic analysis.

  17. High-resolution in vivo imaging of mouse brain through the intact skull

    PubMed Central

    Park, Jung-Hoon; Sun, Wei; Cui, Meng

    2015-01-01

    Multiphoton microscopy is the current method of choice for in vivo deep-tissue imaging. The long laser wavelength suffers less scattering, and the 3D-confined excitation permits the use of scattered signal light. However, the imaging depth is still limited because of the complex refractive index distribution of biological tissue, which scrambles the incident light and destroys the optical focus needed for high resolution imaging. Here, we demonstrate a wavefront-shaping scheme that allows clear imaging through extremely turbid biological tissue, such as the skull, over an extended corrected field of view (FOV). The complex wavefront correction is obtained and directly conjugated to the turbid layer in a noninvasive manner. Using this technique, we demonstrate in vivo submicron-resolution imaging of neural dendrites and microglia dynamics through the intact skulls of adult mice. This is the first observation, to our knowledge, of dynamic morphological changes of microglia through the intact skull, allowing truly noninvasive studies of microglial immune activities free from external perturbations. PMID:26170286

  18. Development of an intact hepatocyte activation system for routine use with the mouse lymphoma assay

    SciTech Connect

    Brock, K.H.; Moore, M.M.; Oglesby, L.A.

    1987-01-01

    The authors have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK/sup +/-/-3.7.2C mouse lymphoma cells. This method should provide a means of simulating more closely in-vivo metabolism compared to metabolism by liver homogenates, while still being useful for routine screening. Hepatocytes were isolated from 200-250 gm adult male Sprague-Dawley rats; 1 x 10/sup 6/ viable hepatocytes were seeded per flask. Rapid attachment of the hepatocytes (2 hr) was obtained by using fibronectin-coated 25-cm/sup 2/ tissue culture flasks. Cocultivated cultures were incubated at 37/sup 0/C on a platform rocker at 32 oscillations per minute. A 16-hr cocultivated period was selected. With this hepatocyte activation methodology, CP, DMN, DMBA, and B(a)P, genotoxins that require metabolic activation, could be detected as mutagens in L5178Y/TK/sup +/-/ cells.

  19. Positive control study for the intact immature Swiss-Webster mouse uterotrophic assay

    NASA Astrophysics Data System (ADS)

    Alisjahbana, Arlisa; Yusuf, Ayda T.

    2014-03-01

    Endocrine disruptors are chemicals in the environment that can disrupt the normal functions of hormones in the body. A majority of these endocrine disruptors are estrogenic. The most commonly used in vivo method to test the estrogenicity of substances is the uterotrophic assay. This assay has many procedural variations, but works on the principle of measuring increase of uterine weight (uterotrophic response) as a response to estrogenic chemicals in animals with low levels of endogenous estrogens. Positive control study should be performed before using this method for the first time. The Developmental Biology laboratory in ITB plans to test pollutants for estrogenicity, therefore this study is conducted. In this study, intact immature Swiss-Webster mice (Mus musculus L.) were used. Twenty four female mice were weaned at 21 days old and divided into four groups. Each group were given 0, 5, 50, or 500 μg/kg bw 17β-estradiol daily for three days. 17β-estradiol dissolved in 10% ethanol-corn oil were administered via subcutaneous injection to the dorsoscapular and lumbar region with an injection volume of 0,05 mL/10 g bw. Twenty four hours after the last injection, the mice were killed by cervical dislocation and then dissected. The uterus is removed and weighted to obtain the uterine wet weight and blotted weight, and then processed for histological observation. Uterine gland number were calculated form transverse sections of the uterus, luminal epithelium height and cell number were measured form longitudinal sections. Both wet and blotted uterine weights per body weight ratio increased in a dose dependent manner with significant differences to controls (p< 0.05). Uterine gland number and luminal epithelium cell height also increase in a dose dependent manner with significant differences to controls (p<0.05). Cell number showed no significant difference to controls. The results are consistent with the hypothesis except for cell number which did not differ

  20. Three-dimensional laser-induced photoacoustic tomography of mouse brain with the skin and skull intact

    NASA Astrophysics Data System (ADS)

    Wang, Xueding; Pang, Yongjiang; Ku, Geng; Stoica, George; Wang, Lihong V.

    2003-10-01

    Three-dimensional laser-induced photoacoustic tomography, also referred to as optoacoustic tomography, is developed to image animal brain structures noninvasively with the skin and skull intact. This imaging modality combines the advantages of optical contrast and ultrasonic resolution. The distribution of optical absorption in a mouse brain is imaged successfully. The intrinsic optical contrast reveals not only blood vessels but also other detailed brain structures, such as the cerebellum, hippocampus, and ventriculi lateralis. The spatial resolution is primarily diffraction limited by the received photoacoustic waves. Imaged structures of the brain at different depths match the corresponding histological pictures well.

  1. Effective protein inhibition in intact mouse oocytes through peptide nanoparticle-mediated antibody transfection

    PubMed Central

    Li, Ruichao; Jin, Zhen; Gao, Leilei; Liu, Peng

    2016-01-01

    Female meiosis is a fundamental area of study in reproductive medicine, and the mouse oocyte model of in vitro maturation (IVM) is most widely used to study female meiosis. To investigate the probable role(s) of an unknown protein in female meiosis, the method traditionally used involves microinjecting a specific antibody into mouse oocytes. Recently, in studies on somatic cells, peptide nanoparticle-mediated antibody transfection has become a popular tool because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, untill now no researchers have tried using this technique on mouse oocytes because the zona pellucida surrounding the oocyte membrane (vitelline membrane) is usually thought or proved to be a tough barrier to macromolecules such as antibodies and proteins. Therefore, we attempted to introduce an antibody into mouse oocytes using a peptide nanoparticle. Here we show for the first time that with our optimized method, an antibody can be effectively delivered into mouse oocytes and inhibit its target protein with high specificity. We obtained significant results using small GTPase Arl2 as a test subject protein. We propose peptide nanoparticle-mediated antibody transfection to be a superior alternative to antibody microinjection for preliminary functional studies of unknown proteins in mouse oocytes. PMID:27114861

  2. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches.

    PubMed

    Giannakis, Marios; Stappenbeck, Thaddeus S; Mills, Jason C; Leip, Douglas G; Lovett, Michael; Clifton, Sandra W; Ippolito, Joseph E; Glasscock, Jarret I; Arumugam, Manimozhiyan; Brent, Michael R; Gordon, Jeffrey I

    2006-04-21

    We have sequenced 36,641 expressed sequence tags from laser capture microdissected adult mouse gastric and small intestinal epithelial progenitors, obtaining 4031 and 3324 unique transcripts, respectively. Using Gene Ontology (GO) terms, each data set was compared with cDNA libraries from intact adult stomach and small intestine. Genes in GO categories enriched in progenitors were filtered against genes in GO categories represented in hematopoietic, neural, and embryonic stem cell transcriptomes and mapped onto transcription factor networks, plus canonical signal transduction and metabolic pathways. Wnt/beta-catenin, phosphoinositide-3/Akt kinase, insulin-like growth factor-1, vascular endothelial growth factor, integrin, and gamma-aminobutyric acid receptor signaling cascades, plus glycerolipid, fatty acid, and amino acid metabolic pathways are among those prominently represented in adult gut progenitors. The results reveal shared as well as distinctive features of adult gut stem cells when compared with other stem cell populations.

  3. Malnutrition among Cognitively Intact, non-Critically Ill Older Adults in the Emergency Department

    PubMed Central

    Pereira, Greg F.; Bulik, Cynthia M.; Weaver, Mark A.; Holland, Wesley C.; Platts-Mills, Timothy F.

    2014-01-01

    Objectives We estimate the prevalence of malnutrition among older patients presenting to an emergency department (ED) in the southeastern United States and identify subgroups at increased risk. Methods We conducted a cross-sectional study with random time block sampling of cognitively intact patients aged 65 years and older. Nutrition was assessed using the Mini Nutritional Assessment Short-Form (0–14 scale) with malnutrition defined as a score of 7 or less and at-risk for malnutrition defined as a score of 8–11. The presence of depressive symptoms was defined as a Center for Epidemiological Studies Depression-10 score of 4 or more (0–10 scale). Results Among 138 older adults, 16% (95% Confidence Interval [CI], 10%–22%) were malnourished and 60% (95% CI, 52%–69%) were either malnourished or at-risk for malnutrition. Seventeen of the 22 malnourished patients (77%) denied previously being diagnosed with malnutrition. The prevalence of malnutrition was not appreciably different between males and females, across levels of patient education, or between those living in urban and rural areas. However, the prevalence of malnutrition was higher among patients with depressive symptoms 52%, those residing in assisted living 50%, those with difficulty eating 38%, and those reporting difficulty buying groceries 33%. Conclusion Among a random sample of cognitively intact older ED patients, more than half were malnourished or at-risk for malnutrition, and the majority of malnourished patients had not previously been diagnosed. Higher rates of malnutrition among those with depression, difficulty eating, and difficulty buying groceries suggest the need to explore multifaceted interventions. PMID:25129819

  4. Cell proliferation and neurogenesis in adult mouse brain.

    PubMed

    Bordiuk, Olivia L; Smith, Karen; Morin, Peter J; Semënov, Mikhail V

    2014-01-01

    Neurogenesis, the formation of new neurons, can be observed in the adult brain of many mammalian species, including humans. Despite significant progress in our understanding of adult neurogenesis, we are still missing data about the extent and location of production of neural precursors in the adult mammalian brain. We used 5-ethynyl-2'-deoxyuridine (EdU) to map the location of proliferating cells throughout the entire adult mouse brain and found that neurogenesis occurs at two locations in the mouse brain. The larger one we define as the main proliferative zone (MPZ), and the smaller one corresponds to the subgranular zone of the hippocampus. The MPZ can be divided into three parts. The caudate migratory stream (CMS) occupies the middle part of the MPZ. The cable of proliferating cells emanating from the most anterior part of the CMS toward the olfactory bulbs forms the rostral migratory stream. The thin layer of proliferating cells extending posteriorly from the CMS forms the midlayer. We have not found any additional aggregations of proliferating cells in the adult mouse brain that could suggest the existence of other major neurogenic zones in the adult mouse brain.

  5. Cell proliferation and neurogenesis in adult mouse brain.

    PubMed

    Bordiuk, Olivia L; Smith, Karen; Morin, Peter J; Semënov, Mikhail V

    2014-01-01

    Neurogenesis, the formation of new neurons, can be observed in the adult brain of many mammalian species, including humans. Despite significant progress in our understanding of adult neurogenesis, we are still missing data about the extent and location of production of neural precursors in the adult mammalian brain. We used 5-ethynyl-2'-deoxyuridine (EdU) to map the location of proliferating cells throughout the entire adult mouse brain and found that neurogenesis occurs at two locations in the mouse brain. The larger one we define as the main proliferative zone (MPZ), and the smaller one corresponds to the subgranular zone of the hippocampus. The MPZ can be divided into three parts. The caudate migratory stream (CMS) occupies the middle part of the MPZ. The cable of proliferating cells emanating from the most anterior part of the CMS toward the olfactory bulbs forms the rostral migratory stream. The thin layer of proliferating cells extending posteriorly from the CMS forms the midlayer. We have not found any additional aggregations of proliferating cells in the adult mouse brain that could suggest the existence of other major neurogenic zones in the adult mouse brain. PMID:25375658

  6. Localized nuclear and perinuclear Ca2+ signals in intact mouse skeletal muscle fibers

    PubMed Central

    Georgiev, Tihomir; Svirin, Mikhail; Jaimovich, Enrique; Fink, Rainer H. A.

    2015-01-01

    Nuclear Ca2+ is important for the regulation of several nuclear processes such as gene expression. Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27 μm (NLCSs) and 2.55 ± 0.17 μm (PLCSs), S.E.M.]. Additionally, global nuclear Ca2+ signals (NGCSs) were observed. To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect. PMID:26483696

  7. Histomorphological Phenotyping of the Adult Mouse Brain.

    PubMed

    Mikhaleva, Anna; Kannan, Meghna; Wagner, Christel; Yalcin, Binnaz

    2016-01-01

    This article describes a series of standard operating procedures for morphological phenotyping of the mouse brain using basic histology. Many histological studies of the mouse brain use qualitative approaches based on what the human eye can detect. Consequently, some phenotypic information may be missed. Here we describe a quantitative approach for the assessment of brain morphology that is simple and robust. A total of 78 measurements are made throughout the brain at specific and well-defined regions, including the cortex, the hippocampus, and the cerebellum. Experimental design and timeline considerations, including strain background effects, the importance of sectioning quality, measurement variability, and efforts to correct human errors are discussed. © 2016 by John Wiley & Sons, Inc. PMID:27584555

  8. ATM localization and gene expression in the adult mouse eye

    PubMed Central

    Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stéphanie; Aoufouchi, Said; Menasche, Maurice

    2009-01-01

    Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RT–PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue

  9. A Comprehensive Atlas of the Adult Mouse Penis.

    PubMed

    Phillips, Tiffany R; Wright, David K; Gradie, Paul E; Johnston, Leigh A; Pask, Andrew J

    2015-01-01

    Mice are routinely used to study the development of the external genitalia and, in particular, the process of male urethral closure. This is because misplacement of the male penile urethra, or hypospadias, is amongst the most common birth defects reported in humans. While mice present a tractable model to study penile development, several structures differ between mice and humans, and there is a lack of consensus in the literature on their annotation and developmental origins. Defining the ontology of the mouse prepuce is especially important for the relevance and interpretation of mouse models of hypospadias to human conditions. We have developed a detailed annotation of the adult mouse penis that addresses these differences and enables an accurate comparison of murine and human hypospadias phenotypes. Through MRI data, gross morphology and section histology, we define the origin of the mouse external and internal prepuces, their relationship to the single human foreskin as well as provide a comprehensive view of the various structures of the mouse penis and their associated muscle attachments within the body. These data are combined to annotate structures in a novel 3D adult penis atlas that can be downloaded, viewed at any angle, and manipulated to examine the relationship of various structures.

  10. A Comprehensive Atlas of the Adult Mouse Penis

    PubMed Central

    Phillips, Tiffany R.; Wright, David K.; Gradie, Paul E.; Johnston, Leigh A.; Pask, Andrew J.

    2016-01-01

    Mice are routinely used to study the development of the external genitalia and, in particular, the process of male urethral closure. This is because misplacement of the male penile urethra, or hypospadias, is amongst the most common birth defects reported in humans. While mice present a tractable model to study penile development, several structures differ between mice and humans, and there is a lack of consensus in the literature on their annotation and developmental origins. Defining the ontology of the mouse prepuce is especially important for the relevance and interpretation of mouse models of hypospadias to human conditions. We have developed a detailed annotation of the adult mouse penis that addresses these differences and enables an accurate comparison of murine and human hypospadias phenotypes. Through MRI data, gross morphology and section histology, we define the origin of the mouse external and internal prepuces, their relationship to the single human foreskin as well as provide a comprehensive view of the various structures of the mouse penis and their associated muscle attachments within the body. These data are combined to annotate structures in a novel 3D adult penis atlas that can be downloaded, viewed at any angle, and manipulated to examine the relationship of various structures. PMID:26112156

  11. Evaluating a Genetically Encoded Optical Sensor of Neural Activity Using Electrophysiology in Intact Adult Fruit Flies

    PubMed Central

    Jayaraman, Vivek; Laurent, Gilles

    2007-01-01

    Genetically encoded optical indicators hold the promise of enabling non-invasive monitoring of activity in identified neurons in behaving organisms. However, the interpretation of images of brain activity produced using such sensors is not straightforward. Several recent studies of sensory coding used G-CaMP 1.3—a calcium sensor—as an indicator of neural activity; some of these studies characterized the imaged neurons as having narrow tuning curves, a conclusion not always supported by parallel electrophysiological studies. To better understand the possible cause of these conflicting results, we performed simultaneous in vivo 2-photon imaging and electrophysiological recording of G-CaMP 1.3 expressing neurons in the antennal lobe (AL) of intact fruitflies. We find that G-CaMP has a relatively high threshold, that its signal often fails to capture spiking response kinetics, and that it can miss even high instantaneous rates of activity if those are not sustained. While G-CaMP can be misleading, it is clearly useful for the identification of promising neural targets: when electrical activity is well above the sensor's detection threshold, its signal is fairly well correlated with mean firing rate and G-CaMP does not appear to alter significantly the responses of neurons that express it. The methods we present should enable any genetically encoded sensor, activator, or silencer to be evaluated in an intact neural circuit in vivo in Drosophila. PMID:18946545

  12. Relaxation, [Ca2+]i and [Mg2+]i during prolonged tetanic stimulation of intact, single fibres from mouse skeletal muscle.

    PubMed Central

    Westerblad, H; Allen, D G

    1994-01-01

    1. In skeletal muscle there is generally a slowing of relaxation with increasing tetanus duration and it has been suggested that this is due to Ca2+ loading of parvalbumin (PA). To study this we have produced prolonged tetani in intact, single fibres from a mouse foot muscle which contain a high concentration of PA. We measured the rate of tension relaxation and also various aspects of Ca2+ handling. 2. During 'interrupted' tetani (15 repeated cycles of 100 ms with stimulation and 50 ms without) we observed a marked slowing of the relaxation both under control conditions and in acidosis (obtained by increasing the bath CO2 content). This slowing was not accompanied by any reduction of the initial rate of decline of the free myoplasmic Ca2+ concentration ([Ca2+]i), which was measured with indo-1. 3. The functioning of the sarcoplasmic reticulum (SR) pump after tetani of various durations was analysed by plotting d[Ca2+]i/dt vs. [Ca2+]i during the final slow decline of [Ca2+]i after tetani. This analysis showed that the rate of SR Ca2+ pumping after a 1 s tetanus is less than half of that after a 100 ms tetanus. 4. The amplitude of the tail of [Ca2+]i 250 ms into relaxation was measured after tetani of various durations. This amplitude increased with tetanus duration and could be fitted to the sum of one exponential and one linear function. The exponential component increased with a time constant of 0.17 s and probably reflects Ca2+ loading of PA. 5. Ca2+ binding to PA will displace Mg2+ and hence the free myoplasmic concentration of Mg2+ ([Mg2+]i) will increase. To study this we used the fluorescent Mg2+ indicator furaptra. The results showed an increase of [Mg2+]i during prolonged tetani which, after removing the Ca2+ component of the fluorescent signal, amounted to about 0.5 mM. 6. A model of Ca2+ movements and tension production in skeletal muscle was used. The model showed that the increase of the amplitude of [Ca2+]i tails after tetani of various durations can

  13. Exogenous androgen during development alters adult partner preference and mating behavior in gonadally intact male rats.

    PubMed

    Henley, C L; Nunez, A A; Clemens, L G

    2010-04-01

    In the rat, neonatal administration of testosterone propionate to a castrated male causes masculinization of behavior. However, if an intact male is treated neonatally with testosterone (hyper-androgen condition), male sexual behavior in adulthood is disrupted. There is a possibility that the hyper-androgen treatment is suppressing male sexual behavior by altering the male's partner preference and thereby reducing his motivation to approach the female. If so, this would suggest that exposure to supra-physiological levels of androgen during development may result in the development of male-oriented partner preference in the male. To test this idea, male rats were treated either postnatally or prenatally with testosterone, and partner preference and sexual behavior were examined in adulthood. The principal finding of this study was that increased levels of testosterone during early postnatal life, but not prenatal, decreased male sexual behavior and increased the amount of time a male spent with a stimulus male, without affecting the amount of time spent with a stimulus female during partner preference tests. Thus, the reduction in male sexual behavior produced by early exposure to high levels of testosterone is not likely due to a reduction in the male's motivation to approach a receptive female.

  14. Adult mouse brain gene expression patterns bear an embryologic imprint.

    PubMed

    Zapala, Matthew A; Hovatta, Iiris; Ellison, Julie A; Wodicka, Lisa; Del Rio, Jo A; Tennant, Richard; Tynan, Wendy; Broide, Ron S; Helton, Rob; Stoveken, Barbara S; Winrow, Christopher; Lockhart, Daniel J; Reilly, John F; Young, Warren G; Bloom, Floyd E; Lockhart, David J; Barlow, Carrolee

    2005-07-19

    The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional "imprint" consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior-posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org).

  15. Intact lipid imaging of mouse brain samples: MALDI, nanoparticle-laser desorption ionization, and 40 keV argon cluster secondary ion mass spectrometry.

    PubMed

    Mohammadi, Amir Saeid; Phan, Nhu T N; Fletcher, John S; Ewing, Andrew G

    2016-09-01

    We have investigated the capability of nanoparticle-assisted laser desorption ionization mass spectrometry (NP-LDI MS), matrix-assisted laser desorption ionization (MALDI) MS, and gas cluster ion beam secondary ion mass spectrometry (GCIB SIMS) to provide maximum information available in lipid analysis and imaging of mouse brain tissue. The use of Au nanoparticles deposited as a matrix for NP-LDI MS is compared to MALDI and SIMS analysis of mouse brain tissue and allows selective detection and imaging of groups of lipid molecular ion species localizing in the white matter differently from those observed using conventional MALDI with improved imaging potential. We demonstrate that high-energy (40 keV) GCIB SIMS can act as a semi-soft ionization method to extend the useful mass range of SIMS imaging to analyze and image intact lipids in biological samples, closing the gap between conventional SIMS and MALDI techniques. The GCIB SIMS allowed the detection of more intact lipid compounds in the mouse brain compared to MALDI with regular organic matrices. The 40 keV GCIB SIMS also produced peaks observed in the NP-LDI analysis, and these peaks were strongly enhanced in intensity by exposure of the sample to trifluororacetic acid (TFA) vapor prior to analysis. These MS techniques for imaging of different types of lipids create a potential overlap and cross point that can enhance the information for imaging lipids in biological tissue sections. Graphical abstract Schematic of mass spectral imaging of a mouse brain tissue using GCIB-SIMS and MALDI techniques. PMID:27549796

  16. Intact lipid imaging of mouse brain samples: MALDI, nanoparticle-laser desorption ionization, and 40 keV argon cluster secondary ion mass spectrometry.

    PubMed

    Mohammadi, Amir Saeid; Phan, Nhu T N; Fletcher, John S; Ewing, Andrew G

    2016-09-01

    We have investigated the capability of nanoparticle-assisted laser desorption ionization mass spectrometry (NP-LDI MS), matrix-assisted laser desorption ionization (MALDI) MS, and gas cluster ion beam secondary ion mass spectrometry (GCIB SIMS) to provide maximum information available in lipid analysis and imaging of mouse brain tissue. The use of Au nanoparticles deposited as a matrix for NP-LDI MS is compared to MALDI and SIMS analysis of mouse brain tissue and allows selective detection and imaging of groups of lipid molecular ion species localizing in the white matter differently from those observed using conventional MALDI with improved imaging potential. We demonstrate that high-energy (40 keV) GCIB SIMS can act as a semi-soft ionization method to extend the useful mass range of SIMS imaging to analyze and image intact lipids in biological samples, closing the gap between conventional SIMS and MALDI techniques. The GCIB SIMS allowed the detection of more intact lipid compounds in the mouse brain compared to MALDI with regular organic matrices. The 40 keV GCIB SIMS also produced peaks observed in the NP-LDI analysis, and these peaks were strongly enhanced in intensity by exposure of the sample to trifluororacetic acid (TFA) vapor prior to analysis. These MS techniques for imaging of different types of lipids create a potential overlap and cross point that can enhance the information for imaging lipids in biological tissue sections. Graphical abstract Schematic of mass spectral imaging of a mouse brain tissue using GCIB-SIMS and MALDI techniques.

  17. Dissection of complex adult traits in a mouse synthetic population.

    PubMed

    Burke, David T; Kozloff, Kenneth M; Chen, Shu; West, Joshua L; Wilkowski, Jodi M; Goldstein, Steven A; Miller, Richard A; Galecki, Andrzej T

    2012-08-01

    Finding the causative genetic variations that underlie complex adult traits is a significant experimental challenge. The unbiased search strategy of genome-wide association (GWAS) has been used extensively in recent human population studies. These efforts, however, typically find only a minor fraction of the genetic loci that are predicted to affect variation. As an experimental model for the analysis of adult polygenic traits, we measured a mouse population for multiple phenotypes and conducted a genome-wide search for effector loci. Complex adult phenotypes, related to body size and bone structure, were measured as component phenotypes, and each subphenotype was associated with a genomic spectrum of candidate effector loci. The strategy successfully detected several loci for the phenotypes, at genome-wide significance, using a single, modest-sized population (N = 505). The effector loci each explain 2%-10% of the measured trait variation and, taken together, the loci can account for over 25% of a trait's total population variation. A replicate population (N = 378) was used to confirm initially observed loci for one trait (femur length), and, when the two groups were merged, the combined population demonstrated increased power to detect loci. In contrast to human population studies, our mouse genome-wide searches find loci that individually explain a larger fraction of the observed variation. Also, the additive effects of our detected mouse loci more closely match the predicted genetic component of variation. The genetic loci discovered are logical candidates for components of the genetic networks having evolutionary conservation with human biology. PMID:22588897

  18. Learning new color names produces rapid increase in gray matter in the intact adult human cortex

    PubMed Central

    Kwok, Veronica; Niu, Zhendong; Kay, Paul; Zhou, Ke; Mo, Lei; Jin, Zhen; So, Kwok-Fai; Tan, Li Hai

    2011-01-01

    The human brain has been shown to exhibit changes in the volume and density of gray matter as a result of training over periods of several weeks or longer. We show that these changes can be induced much faster by using a training method that is claimed to simulate the rapid learning of word meanings by children. Using whole-brain magnetic resonance imaging (MRI) we show that learning newly defined and named subcategories of the universal categories green and blue in a period of 2 h increases the volume of gray matter in V2/3 of the left visual cortex, a region known to mediate color vision. This pattern of findings demonstrates that the anatomical structure of the adult human brain can change very quickly, specifically during the acquisition of new, named categories. Also, prior behavioral and neuroimaging research has shown that differences between languages in the boundaries of named color categories influence the categorical perception of color, as assessed by judgments of relative similarity, by response time in alternative forced-choice tasks, and by visual search. Moreover, further behavioral studies (visual search) and brain imaging studies have suggested strongly that the categorical effect of language on color processing is left-lateralized, i.e., mediated by activity in the left cerebral hemisphere in adults (hence “lateralized Whorfian” effects). The present results appear to provide a structural basis in the brain for the behavioral and neurophysiologically observed indices of these Whorfian effects on color processing. PMID:21464316

  19. Colocalization properties of elementary Ca(2+) release signals with structures specific to the contractile filaments and the tubular system of intact mouse skeletal muscle fibers.

    PubMed

    Georgiev, Tihomir; Zapiec, Bolek; Förderer, Moritz; Fink, Rainer H A; Vogel, Martin

    2015-12-01

    Ca(2+) regulates several important intracellular processes. We combined second harmonic generation (SHG) and two photon excited fluorescence microscopy (2PFM) to simultaneously record the SHG signal of the myosin filaments and localized elementary Ca(2+) release signals (LCSs). We found LCSs associated with Y-shaped structures of the myosin filament pattern (YMs), so called verniers, in intact mouse skeletal muscle fibers under hypertonic treatment. Ion channels crucial for the Ca(2+) regulation are located in the tubular system, a system that is important for Ca(2+) regulation and excitation-contraction coupling. We investigated the tubular system of intact, living mouse skeletal muscle fibers using 2PFM and the fluorescent Ca(2+) indicator Fluo-4 dissolved in the external solution or the membrane dye di-8-ANEPPS. We simultaneously measured the SHG signal from the myosin filaments of the skeletal muscle fibers. We found that at least a subset of the YMs observed in SHG images are closely juxtaposed with Y-shaped structures of the transverse tubules (YTs). The distances of corresponding YMs and YTs yield values between 1.3 μm and 4.1 μm including pixel uncertainty with a mean distance of 2.52±0.10 μm (S.E.M., n=41). Additionally, we observed that some of the linear-shaped areas in the tubular system are colocalized with linear-shaped areas in the SHG images.

  20. Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells.

    PubMed

    Bulut-Karslioglu, Aydan; De La Rosa-Velázquez, Inti A; Ramirez, Fidel; Barenboim, Maxim; Onishi-Seebacher, Megumi; Arand, Julia; Galán, Carmen; Winter, Georg E; Engist, Bettina; Gerle, Borbala; O'Sullivan, Roderick J; Martens, Joost H A; Walter, Jörn; Manke, Thomas; Lachner, Monika; Jenuwein, Thomas

    2014-07-17

    Heterochromatin is required to restrict aberrant expression of retrotransposons, but it remains poorly defined due to the underlying repeat-rich sequences. We dissected Suv39h-dependent histone H3 lysine 9 trimethylation (H3K9me3) by genome-wide ChIP sequencing in mouse embryonic stem cells (ESCs). Refined bioinformatic analyses of repeat subfamilies indicated selective accumulation of Suv39h-dependent H3K9me3 at interspersed repetitive elements that cover ∼5% of the ESC epigenome. The majority of the ∼8,150 intact long interspersed nuclear elements (LINEs) and endogenous retroviruses (ERVs), but only a minor fraction of the >1.8 million degenerate and truncated LINEs/ERVs, are enriched for Suv39h-dependent H3K9me3. Transcriptional repression of intact LINEs and ERVs is differentially regulated by Suv39h and other chromatin modifiers in ESCs but governed by DNA methylation in committed cells. These data provide a function for Suv39h-dependent H3K9me3 chromatin to specifically repress intact LINE elements in the ESC epigenome.

  1. Exogenous spatial attention: Evidence for intact functioning in adults with autism spectrum disorder

    PubMed Central

    Grubb, Michael A.; Behrmann, Marlene; Egan, Ryan; Minshew, Nancy J.; Heeger, David J.; Carrasco, Marisa

    2013-01-01

    Deficits or atypicalities in attention have been reported in individuals with autism spectrum disorder (ASD), yet no consensus on the nature of these deficits has emerged. We conducted three experiments that paired a peripheral precue with a covert discrimination task, using protocols for which the effects of covert exogenous spatial attention on early vision have been well established in typically developing populations. Experiment 1 assessed changes in contrast sensitivity, using orientation discrimination of a contrast-defined grating; Experiment 2 evaluated the reduction of crowding in the visual periphery, using discrimination of a letter-like figure with flanking stimuli at variable distances; and Experiment 3 assessed improvements in visual search, using discrimination of the same letter-like figure with a variable number of distractor elements. In all three experiments, we found that exogenous attention modulated visual discriminability in a group of high-functioning adults with ASD and that it did so in the same way and to the same extent as in a matched control group. We found no evidence to support the hypothesis that deficits in exogenous spatial attention underlie the emergence of core ASD symptomatology. PMID:24326863

  2. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    PubMed Central

    Webb, Carol F.; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-01-01

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. PMID:26111446

  3. Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord.

    PubMed

    Abdulhaleem, Felemban Athary M; Song, Xiaohong; Kawano, Rie; Uezono, Naohiro; Ito, Ayako; Ahmed, Giasuddin; Hossain, Mahmud; Nakashima, Kinichi; Tanaka, Hideaki; Ohta, Kunimasa

    2015-05-01

    Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell-adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M-AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M-AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH-/-) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M-AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal.

  4. Whole-Cell Patch-Clamp Recording of Mouse and Rat Inner Hair Cells in the Intact Organ of Corti.

    PubMed

    Goutman, Juan D; Pyott, Sonja J

    2016-01-01

    Whole-cell patch clamping is a widely applied method to record currents across the entire membrane of a cell. This protocol describes application of this method to record currents from the sensory inner hair cells in the intact auditory sensory epithelium, the organ of Corti, isolated from rats or mice. This protocol particularly outlines the basic equipment required, provides instructions for the preparation of solutions and small equipment items, and methodology for recording voltage-activated and evoked synaptic currents from the inner hair cells.

  5. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    SciTech Connect

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  6. Tissue tropism of recombinant coxsackieviruses in an adult mouse model.

    PubMed

    Harvala, Heli; Kalimo, Hannu; Bergelson, Jeffrey; Stanway, Glyn; Hyypiä, Timo

    2005-07-01

    Recombinant viruses, constructed by exchanging the 5' non-coding region (5'NCR), structural and non-structural protein coding sequences were used to investigate determinants responsible for differences between coxsackievirus A9 (CAV9) and coxsackievirus B3 (CBV3) infections in adult mice and two cell lines. Plaque assay titration of recombinant and parental viruses from different tissues from adult BALB/c mice demonstrated that the structural region of CBV3 determined tropism to the liver tissue due to receptor recognition, and the 5'NCR of CBV3 enhanced viral multiplication in the mouse pancreas. Infection with a chimeric virus, containing the structural region from CBV3 and the rest of the genome from CAV9, and the parental CBV3 strain, caused high levels of viraemia in adult mice. The ability of these viruses to infect the central nervous system suggested that neurotropism is associated with high replication levels and the presence of the CBV3 capsid proteins, which also enhanced formation of neutralizing antibodies. Moreover, the appearance of neutralizing antibodies correlated directly with the clearance of the viruses from the tissues. These results demonstrate potential pathogenicity of intraspecies recombinant coxsackieviruses, and the complexity of the genetic determinants underlying tissue tropism.

  7. Intact Pericellular Matrix of Articular Cartilage Is Required for Unactivated Discoidin Domain Receptor 2 in the Mouse Model

    PubMed Central

    Xu, Lin; Polur, Ilona; Servais, Jacqueline M.; Hsieh, Sirena; Lee, Peter L.; Goldring, Mary B.; Li, Yefu

    2011-01-01

    Increased expression of the discoidin domain receptor 2 (DDR2) results from its interaction with collagen type II. This induces expression of matrix metalloproteinase (MMP)-13, leading to osteoarthritis (OA). To investigate the impact of the pericellular matrix of chondrocytes on DDR2, we generated a mouse model with inducible overexpression of DDR2 in cartilage. Conditional overexpression of DDR2 in mature mouse articular cartilage was controlled via the cartilage oligomeric matrix protein promoter using the Tet-Off-inducible system. Doxycycline was withdrawn at 1 month of age, and knee joints were examined at 2, 3, and 4 months of age. Microsurgery was performed on 3-month-old transgenic mice overexpressing DDR2 to destabilize the medial meniscus, and serial paraffin sections were examined at 2, 4, 8, and 12 weeks after surgery. DDR2 expression increased in the knee joints of transgenic mice. However, the increased DDR2 did not induce MMP-13 expression. No OA-like changes were observed in the transgenic mice at the age of 4 months. When transgenic mice were subjected to destabilizing of the medial meniscus, we observed accelerated progression to OA, which was associated with DDR2 activation. Therefore, conditionally overexpressing DDR2 in the mature articular cartilage of mouse knee joints requires activation to induce OA, and altered biomechanical stress can accelerate the onset of cartilage loss and progression to OA in transgenic mice. PMID:21855682

  8. Functional properties of K+ currents in adult mouse ventricular myocytes

    PubMed Central

    Brouillette, Judith; Clark, Robert B; Giles, Wayne R; Fiset, Céline

    2004-01-01

    Although the K+ currents expressed in hearts of adult mice have been studied extensively, detailed information concerning their relative sizes and biophysical properties in ventricle and atrium is lacking. Here we describe and validate pharmacological and biophysical methods that can be used to isolate the three main time- and voltage-dependent outward K+ currents which modulate action potential repolarization. A Ca2+-independent transient outward K+ current, Ito, can be separated from total outward current using an ‘inactivating prepulse’. The rapidly activating, slowly inactivating delayed rectifier K+ current, IKur, can be isolated using submillimolar concentrations of 4-aminopyridine (4-AP). The remaining K+ current, Iss, can be obtained by combining these two procedures: (i) inactivating Ito and (ii) eliminating IKur by application of low concentration of 4-AP. Iss activates relatively slowly and shows very little inactivation, even during depolarizations lasting several seconds. Our findings also show that the rate of reactivation of Ito is more than 20-fold faster than that of IKur. These results demonstrate that the outward K+ currents in mouse ventricles can be separated based on their distinct time and voltage dependence, and different sensitivities to 4-AP. Data obtained at both 22 and 32°C demonstrate that although the duration of the inactivating prepulse has to be adapted for the recording temperature, this approach for separation of K+ current components is also valid at more physiological temperatures. To demonstrate that these methods also allow separation of these K+ currents in other cell types, we have applied this same approach to myocytes from mouse atria. Molecular approaches have been used to compare the expression levels of different K+ channels in mouse atrium and ventricle. These findings provide new insights into the functional roles of IKur, Ito and Iss during action potential repolarization. PMID:15272047

  9. An anatomic gene expression atlas of the adult mouse brain.

    PubMed

    Ng, Lydia; Bernard, Amy; Lau, Chris; Overly, Caroline C; Dong, Hong-Wei; Kuan, Chihchau; Pathak, Sayan; Sunkin, Susan M; Dang, Chinh; Bohland, Jason W; Bokil, Hemant; Mitra, Partha P; Puelles, Luis; Hohmann, John; Anderson, David J; Lein, Ed S; Jones, Allan R; Hawrylycz, Michael

    2009-03-01

    Studying gene expression provides a powerful means of understanding structure-function relationships in the nervous system. The availability of genome-scale in situ hybridization datasets enables new possibilities for understanding brain organization based on gene expression patterns. The Anatomic Gene Expression Atlas (AGEA) is a new relational atlas revealing the genetic architecture of the adult C57Bl/6J mouse brain based on spatial correlations across expression data for thousands of genes in the Allen Brain Atlas (ABA). The AGEA includes three discovery tools for examining neuroanatomical relationships and boundaries: (1) three-dimensional expression-based correlation maps, (2) a hierarchical transcriptome-based parcellation of the brain and (3) a facility to retrieve from the ABA specific genes showing enriched expression in local correlated domains. The utility of this atlas is illustrated by analysis of genetic organization in the thalamus, striatum and cerebral cortex. The AGEA is a publicly accessible online computational tool integrated with the ABA (http://mouse.brain-map.org/agea). PMID:19219037

  10. RELATIONSHIP BETWEEN INSULIN-RESISTANCE PROCESSING SPEED AND SPECIFIC EXECUTIVE FUNCTION PROFILES IN NEUROLOGICALLY-INTACT OLDER ADULTS

    PubMed Central

    Frazier, Darvis T.; Bettcher, Brianne M.; Dutt, Shubir; Patel, Nihar; Mungas, Dan; Miller, Joshua; Green, Ralph; Kramer, Joel H.

    2016-01-01

    Objective This study investigated the relationship between insulin-resistance and constituent components of executive function in a sample of neurologically-intact older adult subjects using the homeostasis model assessment (HOMA-IR) and latent factors of working memory, cognitive control and processing speed derived from confirmatory factor analysis. Low-density lipoprotein (LDL), mean arterial pressure (MAP), along with body mass index (BMI) and white matter hypointensity (WMH) were used to control for vascular risk factors, adiposity and cerebrovascular injury. Methods The study included 119 elderly subjects recruited from the University of California, San Francisco Memory and Aging Center. Subjects underwent neuropsychological assessment, fasting blood draw and brain magnetic resonance imaging (MRI). Partial correlations and linear regression models were used to examine the HOMA-IR-executive function relationship. Results Pearson correlation adjusting for age showed a significant relationship between HOMA-IR and working memory (rp=−.18, p=.047), a trend with cognitive control (rp=−.17, p=.068), and no relationship with processing speed (rp=.013, p=.892). Linear regression models adjusting for demographic factors (age, education and gender), LDL, MAP, BMI and WMH indicated that HOMA-IR was negatively associated with cognitive control (r=−.256; p=.026) and working memory (r=−.234; p=.054). Conclusions These results suggest a greater level of peripheral insulin-resistance is associated with decreased cognitive control and working memory. After controlling for demographic factors, vascular risk, adiposity and cerebrovascular injury, HOMA-IR remained significantly associated with cognitive control, with working memory showing a trend. These findings substantiate the insulin-resistance-executive function hypothesis and suggest a complex interaction, demonstrated by the differential impact of insulin-resistance on processing speed and specific aspects of

  11. Optimal clearance of Sporothrix schenckii requires an intact Th17 response in a mouse model of systemic infection.

    PubMed

    Ferreira, Lucas Souza; Gonçalves, Amanda Costa; Portuondo, Deivys Leandro; Maia, Danielle Cardoso Geraldo; Placeres, Marisa Campos Polesi; Batista-Duharte, Alexander; Carlos, Iracilda Zeppone

    2015-08-01

    The discovery of Th17 cells, along with many other Th cell subsets in the recent years, has expanded the Th1/Th2 paradigm that had persisted since its proposition by Mosmann in 1986. Defined by the characteristic expression of the transcription factor retinoic-related orphan receptor γt (RORγt) and production of IL-17A (IL-17), Th17 cells are powerful inducers of tissue inflammation with a recognized role against extracellular bacteria and fungi. Despite this, the interest in their study came from the pivotal role they play in the development and maintenance of major chronic inflammatory conditions such as multiple sclerosis, rheumatoid arthritis and Crohn's disease, hence they have been the target of promising new anti-Th17 therapies. Accordingly, the identification of opportunistic pathogens whose clearance relies on the Th17 response is of huge prophylactic importance. As shown here for the first time, this applies to Sporothrix schenckii, a thermo-dimorphic fungus and the causative agent of sporotrichosis. Our results show that both Th17 and Th1/Th17 mixed cells are developed during the S. schenckii systemic mice infection, which also leads to augmented production of IL-17 and IL-22. Also, by using an antibody-mediated IL-23 depletion model, we further demonstrate that optimal fungal clearance, but not survival, depends on an intact Th17 response.

  12. Immunochemical detection of arylamine N-acetyltransferase during mouse embryonic development and in adult mouse brain.

    PubMed

    Stanley, L A; Copp, A J; Pope, J; Rolls, S; Smelt, V; Perry, V H; Sim, E

    1998-11-01

    Arylamine N-acetyltransferases (NATs) are important in susceptibility to xenobiotic-induced disorders (e.g., drug-induced autoimmune disease, bladder cancer), but their role in endogenous metabolism is yet to be elucidated. The discovery that human NAT1 acts upon p-aminobenzoylgluatamate (p-ABG) to generate p-acetamidobenzoylglutamate (p-AABG), a major urinary metabolite of folic acid, suggests that human NAT1 may play a role in folic acid metabolism and hence in the normal development of the neural tube. In this study we examined the distribution of NAT in neuronal tissue from adult mice and embryos. Immunohistochemical staining of the adult mouse cerebellum revealed NAT2 (the mouse homologue of human NAT1) expression in the cell bodies and dendrites of Purkinje cells and in the neuroglia of the molecular layer. In embryos, NAT2 was detected in developing neuronal tissue on days 9.5, 11.5, and 13.5. It was expressed intensely in the nerual tube around the time of closure. The level of expression subsequently declined in the neuroepithelium but increased in glial cells. In addition, NAT2 was detected in the developing heart and gut. These findings demonstrate that the embryo itself expresses an enzyme which is involved in the metabolism of folic acid, so that the role played by both mother and embryo must be considered when examining the role of folic acid in embryonic development. These findings imply that polymorphisms in NAT genes could play a role in determining susceptibility to neural tube defects (NTD) and orofacial clefting, developmental disorders which can be prevented by dietary administration of folic acid. PMID:9839355

  13. Visualizing form and function in organotypic slices of the adult mouse parotid gland

    PubMed Central

    Warner, Jennifer D.; Peters, Christian G.; Saunders, Rudel; Won, Jong Hak; Betzenhauser, Matthew J.; Gunning, William T.; Yule, David I.; Giovannucci, David R.

    2008-01-01

    An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function. PMID:18669626

  14. Expression of dominant negative cadherin in the adult mouse brain modifies rearing behavior.

    PubMed

    Edsbagge, Josefina; Zhu, Shunwei; Xiao, Min-Yi; Wigström, Holger; Mohammed, Abdul H; Semb, Henrik

    2004-03-01

    The cadherin superfamily of cell-cell adhesion molecules (CAM) are crucial regulators of morphogenesis and axonal guidance during development of the nervous system and have been suggested to play important roles in neural plasticity of the brain. To study the latter, we created a mouse model that expressed a dominant negative classical cadherin in the brain of adult mice. The mice were tested for spontaneous motor activity and exploratory behavior in the open field, anxiety in the plus-maze, and spatial learning and memory in the water-T maze. Mice expressing the dominant negative cadherin displayed reduced rearing behavior, but no change in motor activity, in the open field, indicating deficits in exploratory behavior. In the water maze, animals expressing the mutant cadherin showed normal escape latencies and were indistinguishable from control littermates. Similarly, LTP in hippocampal slices of cadherin mutant and control mice were indistinguishable. These findings demonstrate intact spatial learning in mice expressing a dominant negative cadherin but altered rearing behavior, suggesting the involvement of classical cadherins in mechanisms mediating rearing behavior.

  15. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

    SciTech Connect

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.

    2008-03-10

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen gene expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

  16. Purinergic signaling promotes proliferation of adult mouse subventricular zone cells.

    PubMed

    Suyama, Satoshi; Sunabori, Takehiko; Kanki, Hiroaki; Sawamoto, Kazunobu; Gachet, Christian; Koizumi, Schuichi; Okano, Hideyuki

    2012-07-01

    In adult mammalian brains, neural stem cells (NSCs) exist in the subventricular zone (SVZ), where persistent neurogenesis continues throughout life. Those NSCs produce neuroblasts that migrate into the olfactory bulb via formation of transit-amplifying cells, which are committed precursor cells of the neuronal lineage. In this SVZ niche, cell-cell communications conducted by diffusible factors as well as physical cell-cell contacts are important for the regulation of the proliferation and fate determination of NSCs. Previous studies have suggested that extracellular purinergic signaling, which is mediated by purine compounds such as ATP, plays important roles in cell-cell communication in the CNS. Purinergic signaling also promotes the proliferation of adult NSCs in vitro. However, the in vivo roles of purinergic signaling in the neurogenic niche still remain unknown. In this study, ATP infusion into the lateral ventricle of the mouse brain resulted in an increase in the numbers of rapidly dividing cells and Mash1-positive transit-amplifying cells (Type C cells) in the SVZ. Mash1-positive cells express the P2Y1 purinergic signaling receptor and infusion of the P2Y1 receptor-specific antagonist MRS2179 decreased the number of rapidly dividing bromodeoxyuridine (BrdU)-positive cells and Type C cells. Moreover, a 17% reduction of rapidly dividing BrdU-positive cells and a 19% reduction of Mash1-positive cells were observed in P2Y1 knock-out mice. Together, these results suggest that purinergic signaling promotes the proliferation of rapidly dividing cells and transit-amplifying cells, in the SVZ niche through the P2Y1 receptor. PMID:22764232

  17. Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover.

    PubMed

    Akiyama, K; Vickroy, T W; Watson, M; Roeske, W R; Reisine, T D; Smith, T L; Yamamura, H I

    1986-03-01

    (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems. PMID:3005550

  18. PPARγ mRNA in the adult mouse hypothalamus: distribution and regulation in response to dietary challenges

    PubMed Central

    Liu, Yang; Huang, Ying; Lee, Syann; Bookout, Angie L.; Castorena, Carlos M.; Wu, Hua; Gautron, Laurent

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARγ signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARγ-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARγ mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARγ mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis (VOLT), and the subfornical organ. Within the hypothalamus, PPARγ was present at moderate levels in the suprachiasmatic nucleus (SCh) and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARγ was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARγ mRNA expression was upregulated in the SCh in response to fasting. Double in situ hybridization further demonstrated that PPARγ was primarily expressed in neurons rather than glia. Collectively, our observations provide a comprehensive map of PPARγ distribution in the intact adult mouse hypothalamus. PMID:26388745

  19. Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy

    PubMed Central

    Hammer, Karin P.; Hohendanner, Felix; Blatter, Lothar A.; Pieske, Burkert M.; Heinzel, Frank R.

    2015-01-01

    Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart. Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 μm by 315 μm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length). Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4 hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities. Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling. PMID:25628569

  20. Single adult rabbit and rat cardiac myocytes retain the Ca2+- and species-dependent systolic and diastolic contractile properties of intact muscle

    PubMed Central

    1986-01-01

    The systolic and diastolic properties of single myocytes and intact papillary muscles isolated from hearts of adult rats and rabbits were examined at 37 degrees C over a range of stimulation frequencies and bathing [Ca2+]o (Cao). In both rabbit myocytes and intact muscles bathed in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted in a positive staircase of twitch performance. During stimulation at 2 min-1, twitch performance also increased with increases in Cao up to 20 mM. In the absence of stimulation, both rabbit myocytes and muscles were completely quiescent in less than 15 mM Cao. Further increases in Cao caused the appearance of spontaneous asynchronous contractile waves in myocytes and in intact muscles caused scattered light intensity fluctuations (SLIF), which were previously demonstrated to be caused by Ca2+-dependent spontaneous contractile waves. In contrast to rabbit preparations, intact rat papillary muscles exhibited SLIF in 1.0 mM Cao. Two populations of rat myocytes were observed in 1 mM Cao: approximately 85% of unstimulated cells exhibited low-frequency (3-4 min-1) spontaneous contractile waves, whereas 15%, during a 1-min observation period, were quiescent. In a given Cao, the contractile wave frequency in myocytes and SLIF in intact muscles were constant for long periods of time. In both intact rat muscles and myocytes with spontaneous waves, in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted, on the average, in a 65% reduction in steady state twitch amplitude. Of the rat myocytes that did not manifest waves, some had a positive, some had a flat, and some had a negative staircase; the average steady state twitch amplitude of these cells during stimulation at 120 min-1 was 30% greater than that at 6 min-1. In contrast to rabbit preparations, twitch performance during stimulation at 2 min-1 saturated at 1.5 mM Cao in both intact rat muscles and in the myocytes with spontaneous waves. We

  1. CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

    PubMed

    Cronan, Mark R; Rosenberg, Allison F; Oehlers, Stefan H; Saelens, Joseph W; Sisk, Dana M; Jurcic Smith, Kristen L; Lee, Sunhee; Tobin, David M

    2015-12-01

    Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ. PMID:26449262

  2. The impact of sleep on neuropsychological performance in cognitively intact older adults using a novel in-home sensor based sleep assessment approach

    PubMed Central

    Seelye, Adriana; Mattek, Nora; Howieson, Diane; Riley, Thomas; Wild, Katherine; Kaye, Jeffrey

    2015-01-01

    The relationship between recent episodes of poor sleep and cognitive testing performance in healthy cognitively intact older adults is not well understood. In this exploratory study, we examined the impact of recent sleep disturbance, sleep duration, and sleep variability on cognitive performance in 63 cognitively intact older adults using a novel unobtrusive in-home sensor based sleep assessment methodology. Specifically, we examined the impact of sleep the night prior, the week prior, and the month prior to a neuropsychological evaluation on cognitive performance. Results showed that mildly disturbed sleep the week prior and month prior to cognitive testing was associated with reduced working memory on cognitive evaluation. One night of mild sleep disturbance was not associated with decreased cognitive performance the next day. Sleep duration was unrelated to cognition. In-home, unobtrusive sensor monitoring technologies provide a novel method for objective, long-term, and continuous assessment of sleep behavior and other everyday activities that might contribute to decreased or variable cognitive performance in healthy older adults. PMID:25642948

  3. Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion.

    PubMed

    Launay, Pierre-Serge; Godefroy, David; Khabou, Hanen; Rostene, William; Sahel, Jose-Alain; Baudouin, Christophe; Melik Parsadaniantz, Stéphane; Reaux-Le Goazigo, Annabelle

    2015-10-01

    Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.

  4. Sox2 and Jagged1 Expression in Normal and Drug-Damaged Adult Mouse Inner Ear

    PubMed Central

    Campbell, Sean; Taylor, Ruth R.; Forge, Andrew; Hume, Clifford R.

    2007-01-01

    Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear. PMID:18157569

  5. The influence of intracellular pH on contraction, relaxation and [Ca2+]i in intact single fibres from mouse muscle.

    PubMed

    Westerblad, H; Allen, D G

    1993-07-01

    1. The effects of intracellular pH (pHi) on myoplasmic free calcium concentration ([Ca2+]i) and contractile performance were studied in intact single fibres dissected from mouse skeletal muscle. Indo-1 was used to measure [Ca2+]i and pHi was altered by changing perfusate CO2. 2. Tetanic tension was decreased at acidic pHi and increased at alkaline pHi whereas the rate of mechanical relaxation was showed at both acidic and alkaline pHi. Resting and tetanic [Ca2+]i were increased at acidic pHi and decreased at alkaline pHi while the final rate of decline of [Ca2+]i after a tetanus was markedly slowed at acid pHi but only marginally accelerated at alkaline pHi. 3. Steady-state [Ca2+]i-tension curves were constructed from measurements of tetani at different stimulus frequencies. The curves at acid pHi showed a reduced maximum Ca(2+)-activated tension and a reduced Ca2+ sensitivity, and curves at alkaline pHi showed the opposite changes. 4. Two methods were devised to determine the contribution of [Ca2+]i to the rate of relaxation. In one method the instantaneous tension was plotted as a function of instantaneous [Ca2+]i throughout a tetanus and compared with the steady-state [Ca2+]i-tension relation. In a second method the [Ca2+]i signal during a tetanus was converted to a Ca(2+)-derived tension record by means of the steady-state [Ca2+]i-tension relation and this Ca(2+)-derived tension was then compared to the true tension. 5. The sarcoplasmic reticulum (SR) pump function was analysed by plotting -d[Ca2+]i/dt against [Ca2+]i during the final slow decline of [Ca2+]i after a tetanus. This analysis shows that the Ca2+ uptake by the SR is a third- or fourth-power function of [Ca2+]i and that acidosis substantially slows the rate of SR Ca2+ pumping. 6. In conclusion, the slowing of relaxation at acidic pHi could be attributed to a slowing of cross-bridge detachment rather than the observed slowing of the rate of uptake of Ca2+. Conversely the slowing of relaxation in

  6. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    SciTech Connect

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  7. Of Mice and Men-Warning: Intact Versus Castrated Adult Male Mice as Xenograft Hosts Are Equivalent to Hypogonadal Versus Abiraterone Treated Aging Human Males, Respectively

    PubMed Central

    Sedelaar, J.P. Michiel; Dalrymple, Susan S.; Isaacs, John T.

    2014-01-01

    BACKGROUND Immune deficient male mice bearing human prostate cancer xenografts are used to evaluate therapeutic response to novel androgen ablation approaches and the results compared to surgical castration based upon assumption that testosterone microenvironment in intact and castrated adult male mice mimics eugonadal and castrated aging adult human males. METHODS To test these assumptions, serum total testosterone (TT) and free testosterone (FT) were determined longitudinally in groups (n > 20) of intact versus castrated adult male nude, NOG, and immune competent C57BL/6 mice. RESULTS In adult male mice, TT and FT varies by 30- to 100-fold within the same animal providing a microenvironment that is only equivalent to hypogonadal, not eugonadal, adult human males (TT is 1.7 ± 1.2 ng/ml [5.8 ± 4.1 nM] in nude and 2.5 ± 1.3 ng/ml [8.7 ± 4.4 nM] in NOG mice versus >4.2 ng/ml [14.7 nM] in eugonadal humans). This was confirmed based upon enhanced growth of androgen dependent human prostate cancer xenografts inoculated into mice supplemented with exogenous testosterone to elevate and chronically maintain serum TT at a level (5 ng/ml [18 nM]) equivalent to a 50-year-old eugonadal human male. In castrated mice, TT and FT range from 2 to 20 pg/ml (7–70 pM) and <0.8 pg/ml (<2.6 pM), respectively, which is equivalent to castrate resistant prostate cancer (CRPC) patients treated with abiraterone. This was confirmed based upon the inability of another CYP17A1 inhibitor, ketoconazole, to inhibit the growth of CRPC xenografts in castrated mice. CONCLUSIONS Adult male mice supplemented with testosterone mimic eugonadal human males, while unsupplemented animals mimic standard androgen ablation and castrated animals mimic abiraterone treated patients. These studies confirm what is claimed in Robert Burns’ poem “To a Mouse” that “The best laid schemes of mice and men/often go awry. PMID:23775398

  8. Localization of PPAR isotypes in the adult mouse and human brain

    PubMed Central

    Warden, Anna; Truitt, Jay; Merriman, Morgan; Ponomareva, Olga; Jameson, Kelly; Ferguson, Laura B.; Mayfield, R. Dayne; Harris, R. Adron

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. PPAR agonists have well-documented anti-inflammatory and neuroprotective roles in the central nervous system. Recent evidence suggests that PPAR agonists are attractive therapeutic agents for treating neurodegenerative diseases as well as addiction. However, the distribution of PPAR mRNA and protein in brain regions associated with these conditions (i.e. prefrontal cortex, nucleus accumbens, amygdala, ventral tegmental area) is not well defined. Moreover, the cell type specificity of PPARs in mouse and human brain tissue has yet to be investigated. We utilized quantitative PCR and double immunofluorescence microscopy to determine that both PPAR mRNA and protein are expressed ubiquitously throughout the adult mouse brain. We found that PPARs have unique cell type specificities that are consistent between species. PPARα was the only isotype to colocalize with all cell types in both adult mouse and adult human brain tissue. Overall, we observed a strong neuronal signature, which raises the possibility that PPAR agonists may be targeting neurons rather than glia to produce neuroprotection. Our results fill critical gaps in PPAR distribution and define novel cell type specificity profiles in the adult mouse and human brain. PMID:27283430

  9. Oligodendrogenesis in the fornix of adult mouse brain; the effect of LPS-induced inflammatory stimulation.

    PubMed

    Fukushima, Shohei; Nishikawa, Kazunori; Furube, Eriko; Muneoka, Shiori; Ono, Katsuhiko; Takebayashi, Hirohide; Miyata, Seiji

    2015-11-19

    Evidence have been accumulated that continuous oligodendrogenesis occurs in the adult mammalian brain. The fornix, projection and commissure pathway of hippocampal neurons, carries signals from the hippocampus to other parts of the brain and has critical role in memory and learning. However, basic characterization of adult oligodendrogenesis in this brain region is not well understood. In the present study, therefore, we aimed to examine the proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) and the effect of acute inflammatory stimulation on oligodendrogenesis in the fornix of adult mouse. We demonstrated the proliferation of OPCs and a new generation of mature oligodendrocytes by using bromodeoxyuridine and Ki67 immunohistochemistry. Oligodendrogenesis of adult fornix was also demonstrated by using oligodendrocyte transcription factor 2 transgenic mouse. A single systemic administration of lipopolysaccharide (LPS) attenuated proliferation of OPCs in the fornix together with reduced proliferation of hippocampal neural stem/progenitor cells. Time course analysis showed that a single administration of LPS attenuated the proliferation of OPCs during 24-48 h. On the other hand, consecutive administration of LPS did not suppress proliferation of OPCs. The treatment of LPS did not affect differentiation of OPCs into mature oligodendrocytes. Treatment of a microglia inhibitor minocycline significantly attenuated basal proliferation of OPCs under normal condition. In conclusion, the present study indicates that continuous oligodendrogenesis occurs and a single administration of LPS transiently attenuates proliferation of OPCs without changing differentiation in the fornix of the adult mouse brains.

  10. Regeneration and characterization of adult mouse hippocampal neurons in a defined in vitro system.

    PubMed

    Varghese, Kucku; Das, Mainak; Bhargava, Neelima; Stancescu, Maria; Molnar, Peter; Kindy, Mark S; Hickman, James J

    2009-02-15

    Although the majority of human illnesses occur during adulthood, most of the available in vitro disease models are based upon cells obtained from embryonic/fetal tissues because of the difficulties involved with culturing adult cells. Development of adult mouse neuronal cultures has a special significance because of the abundance of transgenic disease models that use this species. In this study a novel cell culture method has been developed that supports the long-term survival and physiological regeneration of adult mouse hippocampal cells in a serum-free defined environment. In this well-defined, controlled system, adult mouse hippocampal cells survived for up to 21 days in culture. The cultured cells exhibited typical hippocampal neuronal morphology and electrophysiological properties after recovery from the trauma of dissociation, and stained positive for the expected neuronal markers. This system has great potential as an investigative tool for in vitro studies of adult diseases, the aging brain or transgenic models of age-associated disorders. PMID:18955083

  11. Ascl3 marks adult progenitor cells of the mouse salivary gland.

    PubMed

    Rugel-Stahl, Anastasia; Elliott, Marilyn E; Ovitt, Catherine E

    2012-05-01

    The Ascl3 transcription factor marks a subset of salivary gland duct cells present in the three major salivary glands of the mouse. In vivo, these cells generate both duct and secretory acinar cell descendants. Here, we have analyzed whether Ascl3-expressing cells retain this multipotent lineage potential in adult glands. Cells isolated from mouse salivary glands were cultured in vitro as non-adherent spheres. Lineage tracing of the Ascl3-expressing cells within the spheres demonstrates that Ascl3+ cells isolated from adult glands remain multipotent, generating both duct and acinar cell types in vitro. Furthermore, we demonstrate that the progenitor cells characterized by Keratin 5 expression are an independent population from Ascl3+ progenitor cells. We conclude that the Ascl3+ cells are intermediate lineage-restricted progenitor cells of the adult salivary glands.

  12. A comprehensive transcriptomic analysis of infant and adult mouse ovary.

    PubMed

    Pan, Linlin; Gong, Wei; Zhou, Yuanyuan; Li, Xiaonuan; Yu, Jun; Hu, Songnian

    2014-10-01

    Ovary development is a complex process involving numerous genes. A well-developed ovary is essential for females to keep fertility and reproduce offspring. In order to gain a better insight into the molecular mechanisms related to the process of mammalian ovary development, we performed a comparative transcriptomic analysis on ovaries isolated from infant and adult mice by using next-generation sequencing technology (SOLiD). We identified 15,454 and 16,646 transcriptionally active genes at the infant and adult stage, respectively. Among these genes, we also identified 7021 differentially expressed genes. Our analysis suggests that, in general, the adult ovary has a higher level of transcriptomic activity. However, it appears that genes related to primordial follicle development, such as those encoding Figla and Nobox, are more active in the infant ovary, whereas expression of genes vital for follicle development, such as Gdf9, Bmp4 and Bmp15, is upregulated in the adult. These data suggest a dynamic shift in gene expression during ovary development and it is apparent that these changes function to facilitate follicle maturation, when additional functional gene studies are considered. Furthermore, our investigation has also revealed several important functional pathways, such as apoptosis, MAPK and steroid biosynthesis, that appear to be much more active in the adult ovary compared to those of the infant. These findings will provide a solid foundation for future studies on ovary development in mice and other mammals and help to expand our understanding of the complex molecular and cellular events that occur during postnatal ovary development. PMID:25251848

  13. A comprehensive transcriptomic analysis of infant and adult mouse ovary.

    PubMed

    Pan, Linlin; Gong, Wei; Zhou, Yuanyuan; Li, Xiaonuan; Yu, Jun; Hu, Songnian

    2014-10-01

    Ovary development is a complex process involving numerous genes. A well-developed ovary is essential for females to keep fertility and reproduce offspring. In order to gain a better insight into the molecular mechanisms related to the process of mammalian ovary development, we performed a comparative transcriptomic analysis on ovaries isolated from infant and adult mice by using next-generation sequencing technology (SOLiD). We identified 15,454 and 16,646 transcriptionally active genes at the infant and adult stage, respectively. Among these genes, we also identified 7021 differentially expressed genes. Our analysis suggests that, in general, the adult ovary has a higher level of transcriptomic activity. However, it appears that genes related to primordial follicle development, such as those encoding Figla and Nobox, are more active in the infant ovary, whereas expression of genes vital for follicle development, such as Gdf9, Bmp4 and Bmp15, is upregulated in the adult. These data suggest a dynamic shift in gene expression during ovary development and it is apparent that these changes function to facilitate follicle maturation, when additional functional gene studies are considered. Furthermore, our investigation has also revealed several important functional pathways, such as apoptosis, MAPK and steroid biosynthesis, that appear to be much more active in the adult ovary compared to those of the infant. These findings will provide a solid foundation for future studies on ovary development in mice and other mammals and help to expand our understanding of the complex molecular and cellular events that occur during postnatal ovary development.

  14. A Randomized Trial to Measure the Impact of a Community-Based Cognitive Training Intervention on Balance and Gait in Cognitively Intact Black Older Adults

    PubMed Central

    Smith-Ray, Renae L.; Makowski-Woidan, Beth; Hughes, Susan L.

    2015-01-01

    Introduction Fall prevention is important for maintaining mobility and independence into old age. Approaches for reducing falls include exercise, tai chi, and home modifications; however, causes of falling are multifactorial and include not just physical but cognitive factors. Cognitive decline occurs with age, but older adults with the greatest declines in executive function experience more falls. The purpose of this study was twofold: to demonstrate the feasibility of a community-based cognitive training program for cognitively intact Black older adults and to analyze its impact on gait and balance in this population. Method This pilot study used a pretest/posttest randomized trial design with assignment to an intervention or control group. Participants assigned to the intervention completed a computer-based cognitive training class that met 2 days a week for 60 min over 10 weeks. Classes were held at senior/community centers. Primary outcomes included balance as measured by the Berg Balance Scale (BBS), 10-meter gait speed, and 10-meter gait speed under visuospatial dual-task condition. All measures were assessed at baseline and immediately post-intervention. Results Participants were community-dwelling Black adults with a mean age of 72.5 and history of falls (N = 45). Compared to controls, intervention participants experienced statistically significant improvements in BBS and gait speed. Mean performance on distracted gait speed also improved more for intervention participants compared to controls. Conclusion Findings from this pilot randomized trial demonstrate the feasibility of a community-based cognitive training intervention. They provide initial evidence that cognitive training may be an efficacious approach toward improving balance and gait in older adults known to have a history of falls. PMID:25274713

  15. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org).

  16. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org). PMID:22832508

  17. Primary monolayer culture of adult mouse hepatocytes -- a model for the study of hepatotropic viruses.

    PubMed

    Arnheiter, H

    1980-01-01

    Primary monolayer cultures of adult mouse hepatocytes isolated by collagenase perfusion of the liver in situ were exposed to 2 hepatotropic viruses, an avian influenza A virus adapted to grow in mouse liver in vivo and a herpes simplex type I virus. Influenza virus infection led to lysis ofindividual hepatocytes and total monolayer destruction within 18 to 120 hours after infection according to the virus dose used. Virus replication was evidenced by assaying hepatocyte supernates for hemagglutinin and infectivity, by immunofluorescent staining and by electron microscopy. Herpes virus infection resulted in polykaryocyte formation followed by nuclear pycnosis and cell lysis. Virus replication was assayed by titration of supernate infectivity.

  18. Fluoxetine increases plasticity and modulates the proteomic profile in the adult mouse visual cortex

    PubMed Central

    Ruiz-Perera, L.; Muniz, M.; Vierci, G.; Bornia, N.; Baroncelli, L.; Sale, A.; Rossi, F.M.

    2015-01-01

    The scarce functional recovery of the adult CNS following injuries or diseases is largely due to its reduced potential for plasticity, the ability to reorganize neural connections as a function of experience. Recently, some new strategies restoring high levels of plasticity in the adult brain have been identified, especially in the paradigmatic model of the visual system. A chronic treatment with the anti-depressant fluoxetine reinstates plasticity in the adult rat primary visual cortex, inducing recovery of vision in amblyopic animals. The molecular mechanisms underlying this effect remain largely unknown. Here, we explored fluoxetine effects on mouse visual cortical plasticity, and exploited a proteomic approach to identify possible candidates mediating the outcome of the antidepressant treatment on adult cortical plasticity. We showed that fluoxetine restores ocular dominance plasticity in the adult mouse visual cortex, and identified 31 differentially expressed protein spots in fluoxetine-treated animals vs. controls. MALDITOF/TOF mass spectrometry identification followed by bioinformatics analysis revealed that these proteins are involved in the control of cytoskeleton organization, endocytosis, molecular transport, intracellular signaling, redox cellular state, metabolism and protein degradation. Altogether, these results indicate a complex effect of fluoxetine on neuronal signaling mechanisms potentially involved in restoring plasticity in the adult brain. PMID:26205348

  19. Effects of CB1 receptor agonism and antagonism on behavioral fear and physiological stress responses in adult intact, ovariectomized, and estradiol-replaced female rats.

    PubMed

    Simone, J J; Malivoire, B L; McCormick, C M

    2015-10-15

    There is growing interest in the development of cannabis-based therapies for the treatment of fear and anxiety disorders. There are a few studies, but none in females, of the effects of the highly selective cannabinoid receptor type 1 (CB1) agonist, arachidonyl 2'-chlorethylamide (ACEA), on behavioral fear. In experiment 1 involving gonadally-intact females, ACEA (either 0.1 or 0.01 mg/kg) was without effect in the elevated plus maze (EPM), and the lower dose decreased anxiety in the open field test (OFT). AM251 increased anxiety in the EPM and decreased locomotor activity in the OFT. Twenty-four hours after fear conditioning, neither ACEA nor AM251 affected generalized fear or conditioned fear recall. AM251 and 0.1 mg/kg ACEA impaired, and 0.01 mg/kg ACEA enhanced, within-session fear extinction. AM251 increased plasma corticosterone concentrations after the fear extinction session, whereas ACEA was without effect. Based on evidence that estradiol may moderate the effects of CB1 receptor signaling in females, experiment 2 involved ovariectomized (OVX) rats provided with 10-μg 17β-Estradiol and compared with OVX rats without hormone replacement (oil vehicle). Irrespective of hormone treatment, AM251 increased anxiety in the EPM, whereas ACEA (0.01 mg/kg) was without effect. Neither hormone nor drug altered anxiety in the OFT, but estradiol increased and AM251 decreased distance traveled. After fear conditioning, AM251 decreased generalized fear. Neither hormone nor drug had any effect on recall or extinction of conditioned fear, however, ACEA and AM251 increased fear-induced plasma corticosterone concentrations. Further, when results with intact rats were compared with those from OVX rats, gonadal status did not moderate the effects of either AM251 or ACEA, although OVX displayed greater anxiety and fear than did intact rats. Thus, the effects of CB1 receptor antagonism and agonism in adult female rats do not depend on ovarian estradiol. PMID:26311003

  20. Effects of CB1 receptor agonism and antagonism on behavioral fear and physiological stress responses in adult intact, ovariectomized, and estradiol-replaced female rats.

    PubMed

    Simone, J J; Malivoire, B L; McCormick, C M

    2015-10-15

    There is growing interest in the development of cannabis-based therapies for the treatment of fear and anxiety disorders. There are a few studies, but none in females, of the effects of the highly selective cannabinoid receptor type 1 (CB1) agonist, arachidonyl 2'-chlorethylamide (ACEA), on behavioral fear. In experiment 1 involving gonadally-intact females, ACEA (either 0.1 or 0.01 mg/kg) was without effect in the elevated plus maze (EPM), and the lower dose decreased anxiety in the open field test (OFT). AM251 increased anxiety in the EPM and decreased locomotor activity in the OFT. Twenty-four hours after fear conditioning, neither ACEA nor AM251 affected generalized fear or conditioned fear recall. AM251 and 0.1 mg/kg ACEA impaired, and 0.01 mg/kg ACEA enhanced, within-session fear extinction. AM251 increased plasma corticosterone concentrations after the fear extinction session, whereas ACEA was without effect. Based on evidence that estradiol may moderate the effects of CB1 receptor signaling in females, experiment 2 involved ovariectomized (OVX) rats provided with 10-μg 17β-Estradiol and compared with OVX rats without hormone replacement (oil vehicle). Irrespective of hormone treatment, AM251 increased anxiety in the EPM, whereas ACEA (0.01 mg/kg) was without effect. Neither hormone nor drug altered anxiety in the OFT, but estradiol increased and AM251 decreased distance traveled. After fear conditioning, AM251 decreased generalized fear. Neither hormone nor drug had any effect on recall or extinction of conditioned fear, however, ACEA and AM251 increased fear-induced plasma corticosterone concentrations. Further, when results with intact rats were compared with those from OVX rats, gonadal status did not moderate the effects of either AM251 or ACEA, although OVX displayed greater anxiety and fear than did intact rats. Thus, the effects of CB1 receptor antagonism and agonism in adult female rats do not depend on ovarian estradiol.

  1. Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics

    PubMed Central

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T.; Sorensen, Staci A.; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M.; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-01-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

  2. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

    PubMed

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T; Sorensen, Staci A; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-02-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

  3. Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes

    PubMed Central

    Nickerson, John M.; Goodman, Penny; Chrenek, Micah A.; Johnson, Christiana J.; Berglin, Lennart; Redmond, T. Michael.; Boatright, Jeffrey H.

    2013-01-01

    Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 microliters in the human eye and less than 1 microliter in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past ten years (1). PMID:22688698

  4. Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles.

    PubMed

    Patel, Shreya; Peretz, Jackye; Pan, Yuan-Xiang; Helferich, William G; Flaws, Jodi A

    2016-02-15

    Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36μM) for 18-96h. Every 24h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96h, and the expression of cell cycle regulators at 18h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. PMID:26792615

  5. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  6. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system.

    PubMed

    Marques, Sueli; Zeisel, Amit; Codeluppi, Simone; van Bruggen, David; Mendanha Falcão, Ana; Xiao, Lin; Li, Huiliang; Häring, Martin; Hochgerner, Hannah; Romanov, Roman A; Gyllborg, Daniel; Muñoz-Manchado, Ana B; La Manno, Gioele; Lönnerberg, Peter; Floriddia, Elisa M; Rezayee, Fatemah; Ernfors, Patrik; Arenas, Ernest; Hjerling-Leffler, Jens; Harkany, Tibor; Richardson, William D; Linnarsson, Sten; Castelo-Branco, Gonçalo

    2016-06-10

    Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra(+) oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra(+) population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS. PMID:27284195

  7. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

    PubMed

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  8. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system.

    PubMed

    Marques, Sueli; Zeisel, Amit; Codeluppi, Simone; van Bruggen, David; Mendanha Falcão, Ana; Xiao, Lin; Li, Huiliang; Häring, Martin; Hochgerner, Hannah; Romanov, Roman A; Gyllborg, Daniel; Muñoz-Manchado, Ana B; La Manno, Gioele; Lönnerberg, Peter; Floriddia, Elisa M; Rezayee, Fatemah; Ernfors, Patrik; Arenas, Ernest; Hjerling-Leffler, Jens; Harkany, Tibor; Richardson, William D; Linnarsson, Sten; Castelo-Branco, Gonçalo

    2016-06-10

    Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra(+) oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra(+) population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS.

  9. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.

    PubMed

    van Praag, H; Kempermann, G; Gage, F H

    1999-03-01

    Exposure to an enriched environment increases neurogenesis in the dentate gyrus of adult rodents. Environmental enrichment, however, typically consists of many components, such as expanded learning opportunities, increased social interaction, more physical activity and larger housing. We attempted to separate components by assigning adult mice to various conditions: water-maze learning (learner), swim-time-yoked control (swimmer), voluntary wheel running (runner), and enriched (enriched) and standard housing (control) groups. Neither maze training nor yoked swimming had any effect on bromodeoxyuridine (BrdU)-positive cell number. However, running doubled the number of surviving newborn cells, in amounts similar to enrichment conditions. Our findings demonstrate that voluntary exercise is sufficient for enhanced neurogenesis in the adult mouse dentate gyrus.

  10. Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve

    PubMed Central

    Lang, Hainan; Xing, Yazhi; Brown, LaShardai N.; Samuvel, Devadoss J.; Panganiban, Clarisse H.; Havens, Luke T.; Balasubramanian, Sundaravadivel; Wegner, Michael; Krug, Edward L.; Barth, Jeremy L.

    2015-01-01

    The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration. PMID:26307538

  11. Histology and Ultrastructure of Transitional Changes in Skin Morphology in the Juvenile and Adult Four-Striped Mouse (Rhabdomys pumilio)

    PubMed Central

    Stewart, Eranée; Ajao, Moyosore Salihu

    2013-01-01

    The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin. PMID:24288469

  12. Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue.

    PubMed

    Okonogi, Noriyuki; Nakamura, Kazuhiro; Suzuki, Yoshiyuki; Suto, Nana; Suzue, Kazutomo; Kaminuma, Takuya; Nakano, Takashi; Hirai, Hirokazu

    2014-07-01

    Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV-GFP mice, aged 8-12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

  13. Light scattering properties vary across different regions of the adult mouse brain.

    PubMed

    Al-Juboori, Saif I; Dondzillo, Anna; Stubblefield, Elizabeth A; Felsen, Gidon; Lei, Tim C; Klug, Achim

    2013-01-01

    Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.

  14. Light Scattering Properties Vary across Different Regions of the Adult Mouse Brain

    PubMed Central

    Stubblefield, Elizabeth A.; Felsen, Gidon

    2013-01-01

    Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue. PMID:23874433

  15. Light scattering properties vary across different regions of the adult mouse brain

    NASA Astrophysics Data System (ADS)

    Al-Juboori, Saif I.

    Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.

  16. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation.

    PubMed

    Korogod, Natalya; Petersen, Carl C H; Knott, Graham W

    2015-01-01

    Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions. PMID:26259873

  17. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation

    PubMed Central

    Korogod, Natalya; Petersen, Carl CH; Knott, Graham W

    2015-01-01

    Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions. DOI: http://dx.doi.org/10.7554/eLife.05793.001 PMID:26259873

  18. De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation

    PubMed Central

    Chen, Wanqiu; Sun, Zhengda; Han, Zhenying; Jun, Kristine; Camus, Marine; Wankhede, Mamta; Mao, Lei; Arnold, Tom; Young, William L.; Su, Hua

    2014-01-01

    Background and Purpose In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor (VEGF) stimulation induces brain AVMs (bAVMs) in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal VEGF stimulation is sufficient to induce bAVM in the adult mouse. Methods Focal angiogenesis was induced in the brain of eight-week-old Pdgfb-iCreER;Alk12f/2f mice by injection of adeno-associated viral vectors expressing VEGF (AAV-VEGF). Two weeks later, EC-Alk1 deletion was induced by tamoxifen (TM) treatment. Vascular morphology was analyzed, and EC proliferation and Dysplasia Index (number of vessels with diameter >15μm per 200 vessels) were quantified10 days after TM administration. Results Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of TM-treated Pdgfb-iCreER;Alk12f/2f mice. Induced bAVMs were marked by increased Dysplasia Index (P<0.001), and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. Conclusions Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo bAVM. PMID:24457293

  19. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells.

    PubMed

    Wang, X; Al-Dhalimy, M; Lagasse, E; Finegold, M; Grompe, M

    2001-02-01

    The emergence of cells with hepatocellular properties in the adult pancreas has been described in several experimental models. To determine whether adult pancreas contains cells that can give rise to therapeutically useful and biochemically normal hepatocytes, we transplanted suspensions of wild-type mouse pancreatic cells into syngeneic recipients deficient in fumarylacetoacetate hydrolase and manifesting tyrosinemia. Four of 34 (12%) mutant mice analyzed were fully rescued by donor-derived cells and had normal liver function. Ten additional mice (29%) showed histological evidence of donor-derived hepatocytes in the liver. Previous work has suggested that pancreatic liver precursors reside within or close to pancreatic ducts. We therefore performed additional transplantations using either primary cell suspensions enriched for ducts or cultured ducts. Forty-four mutant mice were transplanted with cells enriched for pancreatic duct cells, but only three of the 34 (9%) recipients analyzed displayed donor-derived hepatocytes. In addition, 28 of the fumarylacetoacetate hydrolase-deficient mice were transplanted with cultured pancreatic duct cells, but no donor-derived hepatocytes were observed. Our results demonstrate for the first time that adult mouse pancreas contains hepatocyte progenitor cells capable of significant therapeutic liver reconstitution. However, contrary to previous reports, we were unable to detect these cells within the duct compartment. PMID:11159194

  20. Human tau expression reduces adult neurogenesis in a mouse model of tauopathy.

    PubMed

    Komuro, Yutaro; Xu, Guixiang; Bhaskar, Kiran; Lamb, Bruce T

    2015-06-01

    Accumulation of hyperphosphorylated and aggregated microtubule-associated protein tau (MAPT) is a central feature of a class of neurodegenerative diseases termed tauopathies. Notably, there is increasing evidence that tauopathies, including Alzheimer's disease, are also characterized by a reduction in neurogenesis, the birth of adult neurons. However, the exact relationship between hyperphosphorylation and aggregation of MAPT and neurogenic deficits remains unclear, including whether this is an early- or late-stage disease marker. In the present study, we used the genomic-based hTau mouse model of tauopathy to examine the temporal and spatial regulation of adult neurogenesis during the course of the disease. Surprisingly, hTau mice exhibited reductions in adult neurogenesis in 2 different brain regions by as early as 2 months of age, before the development of robust MAPT pathology in this model. This reduction was found to be due to reduced proliferation and not because of enhanced apoptosis in the hippocampus. At these same time points, hTau mice also exhibited altered MAPT phosphorylation with neurogenic precursors. To examine whether the effects of MAPT on neurogenesis were cell autonomous, neurospheres prepared from hTau animals were examined in vitro, revealing a growth deficit when compared with non-transgenic neurosphere cultures. Taken together, these studies provide evidence that altered adult neurogenesis is a robust and early marker of altered, cell-autonomous function of MAPT in the hTau mouse mode of tauopathy and that altered adult neurogenesis should be examined as a potential marker and therapeutic target for human tauopathies.

  1. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  2. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    SciTech Connect

    Sun, Li; Wu, Zhou; Baba, Masashi; Peters, Christoph; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2010-08-27

    Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy

  3. A novel type of self-beating cardiomyocytes in adult mouse ventricles

    SciTech Connect

    Omatsu-Kanbe, Mariko; Matsuura, Hiroshi

    2009-04-10

    This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.

  4. Expression of Quaking RNA-Binding Protein in the Adult and Developing Mouse Retina

    PubMed Central

    Aono, Kentaro; Kawashima, Togo; Inoue, Kiyoshi; Ku, Li; Feng, Yue; Koike, Chieko

    2016-01-01

    Quaking (QKI), which belongs to the STAR family of KH domain-containing RNA-binding proteins, functions in pre-mRNA splicing, microRNA regulation, and formation of circular RNA. QKI plays critical roles in myelinogenesis in the central and peripheral nervous systems and has been implicated neuron-glia fate decision in the brain; however, neither the expression nor function of QKI in the neural retina is known. Here we report the expression of QKI RNA-binding protein in the developing and mature mouse retina. QKI was strongly expressed by Müller glial cells in both the developing and adult retina. Intriguingly, during development, QKI was expressed in early differentiating neurons, such as the horizontal and amacrine cells, and subsequently in later differentiating bipolar cells, but not in photoreceptors. Neuronal expression was uniformly weak in the adult. Among QKI isoforms (5, 6, and 7), QKI-5 was the predominantly expressed isoform in the adult retina. To study the function of QKI in the mouse retina, we examined quakingviable(qkv) mice, which have a dysmyelination phenotype that results from deficiency of QKI expression and reduced numbers of mature oligodendrocytes. In homozygous qkv mutant mice (qkv/qkv), the optic nerve expression levels of QKI-6 and 7, but not QKI-5 were reduced. In the retina of the mutant homozygote, QKI-5 levels were unchanged, and QKI-6 and 7 levels, already low, were also unaffected. We conclude that QKI is expressed in developing and adult Müller glia. QKI is additionally expressed in progenitors and in differentiating neurons during retinal development, but expression weakened or diminished during maturation. Among QKI isoforms, we found that QKI-5 predominated in the adult mouse retina. Since Müller glial cells are thought to share properties with retinal progenitor cells, our data suggest that QKI may contribute to maintaining retinal progenitors prior to differentiation into neurons. On the other hand, the expression of QKI in

  5. Comparison of two methods for calculating the frictional properties of articular cartilage using a simple pendulum and intact mouse knee joints.

    PubMed

    Drewniak, Elizabeth I; Jay, Gregory D; Fleming, Braden C; Crisco, Joseph J

    2009-08-25

    In attempts to better understand the etiology of osteoarthritis, a debilitating joint disease that results in the degeneration of articular cartilage (AC) in synovial joints, researchers have focused on joint tribology, the study of joint friction, lubrication, and wear. Several different approaches have been used to investigate the frictional properties of articular cartilage. In this study, we examined two analysis methods for calculating the coefficient of friction (micro) using a simple pendulum system and BL6 murine knee joints (n=10) as the fulcrum. A Stanton linear decay model (Lin micro) and an exponential model that accounts for viscous damping (Exp micro) were fit to the decaying pendulum oscillations. Root mean square error (RMSE), asymptotic standard error (ASE), and coefficient of variation (CV) were calculated to evaluate the fit and measurement precision of each model. This investigation demonstrated that while Lin micro was more repeatable, based on CV (5.0% for Lin micro; 18% for Exp micro), Exp micro provided a better fitting model, based on RMSE (0.165 degrees for Exp micro; 0.391 degrees for Lin micro) and ASE (0.033 for Exp micro; 0.185 for Lin micro), and had a significantly lower coefficient of friction value (0.022+/-0.007 for Exp micro; 0.042+/-0.016 for Lin micro) (p=0.001). This study details the use of a simple pendulum for examining cartilage properties in situ that will have applications investigating cartilage mechanics in a variety of species. The Exp mu model provided a more accurate fit to the experimental data for predicting the frictional properties of intact joints in pendulum systems.

  6. Comparison of two methods for calculating the frictional properties of articular cartilage using a simple pendulum and intact mouse knee joints.

    PubMed

    Drewniak, Elizabeth I; Jay, Gregory D; Fleming, Braden C; Crisco, Joseph J

    2009-08-25

    In attempts to better understand the etiology of osteoarthritis, a debilitating joint disease that results in the degeneration of articular cartilage (AC) in synovial joints, researchers have focused on joint tribology, the study of joint friction, lubrication, and wear. Several different approaches have been used to investigate the frictional properties of articular cartilage. In this study, we examined two analysis methods for calculating the coefficient of friction (micro) using a simple pendulum system and BL6 murine knee joints (n=10) as the fulcrum. A Stanton linear decay model (Lin micro) and an exponential model that accounts for viscous damping (Exp micro) were fit to the decaying pendulum oscillations. Root mean square error (RMSE), asymptotic standard error (ASE), and coefficient of variation (CV) were calculated to evaluate the fit and measurement precision of each model. This investigation demonstrated that while Lin micro was more repeatable, based on CV (5.0% for Lin micro; 18% for Exp micro), Exp micro provided a better fitting model, based on RMSE (0.165 degrees for Exp micro; 0.391 degrees for Lin micro) and ASE (0.033 for Exp micro; 0.185 for Lin micro), and had a significantly lower coefficient of friction value (0.022+/-0.007 for Exp micro; 0.042+/-0.016 for Lin micro) (p=0.001). This study details the use of a simple pendulum for examining cartilage properties in situ that will have applications investigating cartilage mechanics in a variety of species. The Exp mu model provided a more accurate fit to the experimental data for predicting the frictional properties of intact joints in pendulum systems. PMID:19632680

  7. Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus

    PubMed Central

    Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Koyama, Hiroyuki; Hosoda, Kiminori; Kangawa, Kenji; Nakao, Kazuwa

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A—positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability. PMID:26849804

  8. Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries?

    PubMed Central

    2012-01-01

    positive immuno staining on germ cell nest-like clusters and at places primordial follicles appeared connected through oocytes. Conclusions The results of the present study show that gonadotropin (PMSG) treatment to adult mouse leads to increased pluripotent stem cell activity in the ovaries, associated with increased meiosis, appearance of several cohorts of PF and their assembly in close proximity of OSE. This was found associated with the presence of germ cell nests and cytoplasmic continuity of oocytes in PF. We have earlier reported that pluripotent ovarian stem cells in the adult mammalian ovary are the VSELs which give rise to slightly differentiated OGSCs. Thus we propose that gonadotropin through its action on pluripotent VSELs augments neo-oogenesis and PF assembly in adult mouse ovaries. PMID:23134576

  9. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

    PubMed Central

    Keighren, Margaret A.; Flockhart, Jean H.

    2016-01-01

    ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

  10. Fingolimod induces neurogenesis in adult mouse hippocampus and improves contextual fear memory.

    PubMed

    Efstathopoulos, P; Kourgiantaki, A; Karali, K; Sidiropoulou, K; Margioris, A N; Gravanis, A; Charalampopoulos, I

    2015-11-24

    Fingolimod (FTY720) was the first per os administered disease-modifying agent approved for the treatment of relapsing-remitting multiple sclerosis. It is thought that fingolimod modulates the immune response by activating sphingosine-1 phosphate receptor type 1 (S1P1) on lymphocytes following its in vivo phosphorylation. In addition to its immune-related effects, there is evidence that fingolimod exerts several other effects in the central nervous system, including regulation of the proliferation, survival and differentiation of various cell types and their precursors. In the present study, we have investigated the effect of fingolimod on the production of new neurons in the adult mouse hippocampus and the association of this effect with the ability for pattern separation, an established adult neurogenesis-dependent memory function. Immunofluorescence analysis after chronic administration of a physiologic dose of fingolimod (0.3 mg kg(-1)) revealed a significant increase in both the proliferation and the survival of neural progenitors in the area of dentate gyrus of hippocampus, compared with control animals. These effects were replicated in vitro, in cultures of murine hippocampal neural stem/precursor cells that express S1P1 receptor, suggesting cell-autonomous actions. The effects of fingolimod on neurogenesis were correlated to enhanced ability for context discrimination after fear conditioning. Since impairment of adult hippocampal neurogenesis and memory is a common feature of many neuropsychiatric conditions, fingolimod treatment may be beneficial in therapeutic armamentarium of these disorders.

  11. Localization and regulation of PML bodies in the adult mouse brain.

    PubMed

    Hall, Małgorzata H; Magalska, Adriana; Malinowska, Monika; Ruszczycki, Błażej; Czaban, Iwona; Patel, Satyam; Ambrożek-Latecka, Magdalena; Zołocińska, Ewa; Broszkiewicz, Hanna; Parobczak, Kamil; Nair, Rajeevkumar R; Rylski, Marcin; Pawlak, Robert; Bramham, Clive R; Wilczyński, Grzegorz M

    2016-06-01

    PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.

  12. A mouse model of adult-onset anaemia due to erythropoietin deficiency.

    PubMed

    Yamazaki, Shun; Souma, Tomokazu; Hirano, Ikuo; Pan, Xiaoqing; Minegishi, Naoko; Suzuki, Norio; Yamamoto, Masayuki

    2013-01-01

    Erythropoietin regulates erythropoiesis in a hypoxia-inducible manner. Here we generate inherited super-anaemic mice (ISAM) as a mouse model of adult-onset anaemia caused by erythropoietin deficiency. ISAM express erythropoietin in the liver but lack erythropoietin production in the kidney. Around weaning age, when the major erythropoietin-producing organ switches from the liver to the kidney, ISAM develop anaemia due to erythropoietin deficiency, which is curable by administration of recombinant erythropoietin. In ISAM severe chronic anaemia enhances transgenic green fluorescent protein and Cre expression driven by the complete erythropoietin-gene regulatory regions, which facilitates efficient labelling of renal erythropoietin-producing cells. We show that the majority of cortical and outer medullary fibroblasts have the innate potential to produce erythropoietin, and also reveal a new set of erythropoietin target genes. ISAM are a useful tool for the evaluation of erythropoiesis-stimulating agents and to trace the dynamics of erythropoietin-producing cells. PMID:23727690

  13. Expression profiling of long noncoding RNAs in neonatal and adult mouse testis.

    PubMed

    Sun, Jin; Wu, Ji

    2015-09-01

    In recent years, advancements in genome-wide analyses of the mammalian transcriptome have revealed that long noncoding RNAs (lncRNAs) is pervasively transcribed in the genome and an increasing number of studies have demonstrated lncRNAs as a new class of regulatory molecules are involved in mammalian development (Carninci et al. (2005); Fatica and Bozzoni (2014)), but very few studies have been conducted on the potential roles of lncRNAs in mammalian testis development. To get insights into the expression patterns of lncRNA during mouse testis development, we investigated the lncRNAs expression profiles of neonatal and adult mouse testes using microarray platform and related results have been published (Sun et al., PLoS One 8 (2013) e75750.). Here, we describe in detail the experimental system, methods and validation for the generation of the microarray data associated with our recent publication (Sun et al., PLoS One 8 (2013) e75750.). Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE43442. PMID:26217809

  14. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

    PubMed

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E; Lai, Courteney; Humphries, R Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sor(tm1(Cre/ERT)Nat)/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  15. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

    PubMed Central

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E.; Lai, Courteney; Humphries, R. Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sortm1(Cre/ERT)Nat/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  16. Chronic morphine induces premature mitosis of proliferating cells in the adult mouse subgranular zone.

    PubMed

    Mandyam, Chitra D; Norris, Rebekah D; Eisch, Amelia J

    2004-06-15

    The birth of cells with neurogenic potential in the adult brain is assessed commonly by detection of exogenous S phase markers, such as bromodeoxyuridine (BrdU). Analysis of other phases of the cell cycle, however, can provide insight into how external factors, such as opiates, influence the cycling of newly born cells. To this end, we examined the expression of two endogenous cell cycle markers in relation to BrdU: proliferating cell nuclear antigen (PCNA) and phosphorylated histone H3 (pHisH3). Two hours after one intraperitoneal BrdU injection, BrdU-, PCNA-, and pHisH3-immunoreactive (IR) cells exhibited similar distribution in the adult mouse subgranular zone (SGZ). Quantitative analysis within the SGZ revealed a relative abundance of cells labeled for PCNA > BrdU > pHisH3. Similar to our reports in rat SGZ, chronic morphine treatment decreased BrdU- and PCNA-IR cells in mouse SGZ by 28 and 38%, respectively. We also show that pHisH3-IR cells are influenced by chronic morphine to a greater extent (58% decrease) than are BrdU- or PCNA-IR cells. Cell cycle phase analysis of SGZ BrdU-IR cells using triple labeling for BrdU, PCNA, and pHisH3 revealed premature mitosis in chronic morphine-treated mice. These results suggest that morphine-treated mice have a shorter Gap2/mitosis (G(2)/M) phase when compared to sham-treated mice. These findings demonstrate the power of using a combination of exogenous and endogenous cell cycle markers and nuclear morphology to track proliferating cells through different phases of the cell cycle and to reveal the regulation of cell cycle phase by chronic morphine. PMID:15160390

  17. Retinal lesions induce fast intrinsic cortical plasticity in adult mouse visual system.

    PubMed

    Smolders, Katrien; Vreysen, Samme; Laramée, Marie-Eve; Cuyvers, Annemie; Hu, Tjing-Tjing; Van Brussel, Leen; Eysel, Ulf T; Nys, Julie; Arckens, Lutgarde

    2016-09-01

    Neuronal activity plays an important role in the development and structural-functional maintenance of the brain as well as in its life-long plastic response to changes in sensory stimulation. We characterized the impact of unilateral 15° laser lesions in the temporal lower visual field of the retina, on visually driven neuronal activity in the afferent visual pathway of adult mice using in situ hybridization for the activity reporter gene zif268. In the first days post-lesion, we detected a discrete zone of reduced zif268 expression in the contralateral hemisphere, spanning the border between the monocular segment of the primary visual cortex (V1) with extrastriate visual area V2M. We could not detect a clear lesion projection zone (LPZ) in areas lateral to V1 whereas medial to V2M, agranular and granular retrosplenial cortex showed decreased zif268 levels over their full extent. All affected areas displayed a return to normal zif268 levels, and this was faster in higher order visual areas than in V1. The lesion did, however, induce a permanent LPZ in the retinorecipient layers of the superior colliculus. We identified a retinotopy-based intrinsic capacity of adult mouse visual cortex to recover from restricted vision loss, with recovery speed reflecting the areal cortical magnification factor. Our observations predict incomplete visual field representations for areas lateral to V1 vs. lack of retinotopic organization for areas medial to V2M. The validation of this mouse model paves the way for future interrogations of cortical region- and cell-type-specific contributions to functional recovery, up to microcircuit level. PMID:26663520

  18. Generation of a novel mouse model that recapitulates early and adult onset glycogenosis type IV.

    PubMed

    Akman, H Orhan; Sheiko, Tatiana; Tay, Stacey K H; Finegold, Milton J; Dimauro, Salvatore; Craigen, William J

    2011-11-15

    Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the glycogen branching enzyme (GBE). The diagnostic feature of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with variable tissue involvement and age of disease onset. Absence of enzyme activity is lethal in utero or in infancy affecting primarily muscle and liver. However, residual enzyme activity (5-20%) leads to juvenile or adult onset of a disorder that primarily affects muscle as well as central and peripheral nervous system. Here, we describe two mouse models of GSD IV that reflect this spectrum of disease. Homologous recombination was used to insert flippase recognition target recombination sites around exon 7 of the Gbe1 gene and a phosphoglycerate kinase-Neomycin cassette within intron 7, leading to a reduced synthesis of GBE. Mice bearing this mutation (Gbe1(neo/neo)) exhibit a phenotype similar to juvenile onset GSD IV, with wide spread accumulation of PG. Meanwhile, FLPe-mediated homozygous deletion of exon 7 completely eliminated GBE activity (Gbe1(-/-)), leading to a phenotype of lethal early onset GSD IV, with significant in utero accumulation of PG. Adult mice with residual GBE exhibit progressive neuromuscular dysfunction and die prematurely. Differently from muscle, PG in liver is a degradable source of glucose and readily depleted by fasting, emphasizing that there are structural and regulatory differences in glycogen metabolism among tissues. Both mouse models recapitulate typical histological and physiological features of two human variants of branching enzyme deficiency. PMID:21856731

  19. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  20. Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

    PubMed

    Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

    2016-07-29

    Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

  1. Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice

    PubMed Central

    Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

    2016-01-01

    Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes. PMID:26923756

  2. Patterns and dynamics of subventricular zone neuroblast migration in the ischemic striatum of the adult mouse

    PubMed Central

    Zhang, Rui L; Chopp, Michael; Gregg, Sara R; Toh, Yier; Roberts, Cindi; LeTourneau, Yvonne; Buller, Benjamin; Jia, Longfei; Davarani, Siamak P Nejad; Zhang, Zheng G

    2009-01-01

    The migratory behavior of neuroblasts after a stroke is poorly understood. Using time-lapse microscopy, we imaged migration of neuroblasts and cerebral vessels in living brain slices of adult doublecortin (DCX, a marker of neuroblasts) enhanced green fluorescent protein (eGFP) transgenic mice that were subjected to 7 days of stroke. Our results show that neuroblasts originating in the subventricular zone (SVZ) of adult mouse brain laterally migrated in chains or individually to reach the ischemic striatum. The chains were initially formed at the border between the SVZ and the striatum by neuroblasts in the SVZ and then extended to the striatum. The average speed of DCX-eGFP-expressing cells within chains was 28.67±1.04 μm/h, which was significantly faster (P < 0.01) than the speed of the cells in the SVZ (17.98±0.57 μm/h). Within the ischemic striatum, individual neuroblasts actively extended or retracted their processes, suggestive of probing the immediate microenvironment. The neuroblasts close to cerebral blood vessels exhibited multiple processes. Our data suggest that neuroblasts actively interact with the microenvironment to reach the ischemic striatum by multiple migratory routes. PMID:19436318

  3. Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium

    PubMed Central

    Rodriguez, Steve; Sickles, Heather M.; DeLeonardis, Chris; Alcaraz, Ana; Gridley, Thomas; Lin, David M.

    2008-01-01

    Notch receptors are expressed in neurons and glia in the adult nervous system, but why this expression persists is not well-understood. Here we examine the role of the Notch pathway in the postnatal mouse main olfactory system, and show evidence consistent with a model where Notch2 is required for maintaining sustentacular cell function. In the absence of Notch2, the laminar nature of these glial-like cells is disrupted. Hes1, Hey1, and Six1, which are downstream effectors of the Notch pathway, are down-regulated, and cytochrome P450 and Glutathione S-transferase (GST) expression by sustentacular cells is reduced. Functional levels of GST activity are also reduced. These disruptions are associated with increased olfactory sensory neuron degeneration. Surprisingly, expression of Notch3 is also down-regulated. This suggests the existence of a feedback loop where expression of Notch3 is initially independent of Notch2, but requires Notch2 for maintained expression. While the Notch pathway has previously been shown to be important for promoting gliogenesis during development, this is the first demonstration that the persistent expression of Notch receptors is required for maintaining glial function in adult. PMID:18155189

  4. Temporal profiles of synaptic plasticity-related signals in adult mouse hippocampus with methotrexate treatment.

    PubMed

    Yang, Miyoung; Kim, Juhwan; Kim, Sung-Ho; Kim, Joong-Sun; Shin, Taekyun; Moon, Changjong

    2012-07-25

    Methotrexate, which is used to treat many malignancies and autoimmune diseases, affects brain functions including hippocampal-dependent memory function. However, the precise mechanisms underlying methotrexate-induced hippocampal dysfunction are poorly understood. To evaluate temporal changes in synaptic plasticity-related signals, the expression and activity of N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, extracellular signal-regulated kinase 1/2, cAMP responsive element-binding protein, glutamate receptor 1, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor were examined in the hippocampi of adult C57BL/6 mice after methotrexate (40 mg/kg) intraperitoneal injection. Western blot analysis showed biphasic changes in synaptic plasticity-related signals in adult hippocampi following methotrexate treatment. N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, and glutamate receptor 1 were acutely activated during the early phase (1 day post-injection), while extracellular signal-regulated kinase 1/2 and cAMP responsive element-binding protein activation showed biphasic increases during the early (1 day post-injection) and late phases (7-14 days post-injection). Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression increased significantly during the late phase (7-14 days post-injection). Therefore, methotrexate treatment affects synaptic plasticity-related signals in the adult mouse hippocampus, suggesting that changes in synaptic plasticity-related signals may be associated with neuronal survival and plasticity-related cellular remodeling.

  5. Stroke Increases Neural Stem Cells and Angiogenesis in the Neurogenic Niche of the Adult Mouse

    PubMed Central

    Zhang, Rui Lan; Chopp, Michael; Roberts, Cynthia; Liu, Xianshuang; Wei, Min; Nejad-Davarani, Siamak P.; Wang, Xinli; Zhang, Zheng Gang

    2014-01-01

    The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction. PMID:25437857

  6. Expression of Npas4 mRNA in Telencephalic Areas of Adult and Postnatal Mouse Brain

    PubMed Central

    Damborsky, Joanne C.; Slaton, G. Simona; Winzer-Serhan, Ursula H.

    2015-01-01

    The transcription factor neuronal PAS domain-containing protein 4 (Npas4) is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON), piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu), septum and basolateral amygdala nucleus (BLA), basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5), transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission. PMID:26633966

  7. High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord

    PubMed Central

    Beaudet, Marie-Josée; Yang, Qiurui; Cadau, Sébastien; Blais, Mathieu; Bellenfant, Sabrina; Gros-Louis, François; Berthod, François

    2015-01-01

    Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases. PMID:26577180

  8. Doublecortin (DCX) is not Essential for Survival and Differentiation of Newborn Neurons in the Adult Mouse Dentate Gyrus

    PubMed Central

    Dhaliwal, Jagroop; Xi, Yanwei; Bruel-Jungerman, Elodie; Germain, Johanne; Francis, Fiona; Lagace, Diane C.

    2016-01-01

    In the adult brain, expression of the microtubule-associated protein Doublecortin (DCX) is associated with neural progenitor cells (NPCs) that give rise to new neurons in the dentate gyrus. Many studies quantify the number of DCX-expressing cells as a proxy for the level of adult neurogenesis, yet no study has determined the effect of removing DCX from adult hippocampal NPCs. Here, we use a retroviral and inducible mouse transgenic approach to either knockdown or knockout DCX from adult NPCs in the dentate gyrus and examine how this affects cell survival and neuronal maturation. Our results demonstrate that shRNA-mediated knockdown of DCX or Cre-mediated recombination in floxed DCX mice does not alter hippocampal neurogenesis and does not change the neuronal fate of the NPCs. Together these findings show that the survival and maturation of adult-generated hippocampal neurons does not require DCX. PMID:26793044

  9. Adult plasticity in the subcortical auditory pathway of the maternal mouse.

    PubMed

    Miranda, Jason A; Shepard, Kathryn N; McClintock, Shannon K; Liu, Robert C

    2014-01-01

    Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system - motherhood - is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered. PMID:24992362

  10. Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine.

    PubMed

    Schreiber, Rainer; Faria, Diana; Skryabin, Boris V; Wanitchakool, Podchanart; Rock, Jason R; Kunzelmann, Karl

    2015-06-01

    Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca(2+) and opening of apical Cl(-) channels. In infants and young animals, but not in adults, Ca(2+)-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca(2+)-dependent Cl(-) channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca(2+)-dependent Cl(-) secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca(2+)-activated Cl(-) secretion. In contrast, Ano10 is essential for Ca(2+)-dependent Cl(-) secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl(-) secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca(2+) increase, while the essential and possibly only apical Cl(-) channel is CFTR. These results define a new role of Ano1 for intestinal Ca(2+)-dependent Cl(-) secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

  11. Rhythmic Ganglion Cell Activity in Bleached and Blind Adult Mouse Retinas

    PubMed Central

    Menzler, Jacob; Channappa, Lakshmi; Zeck, Guenther

    2014-01-01

    In retinitis pigmentosa – a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor’s dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the

  12. Adult plasticity in the subcortical auditory pathway of the maternal mouse.

    PubMed

    Miranda, Jason A; Shepard, Kathryn N; McClintock, Shannon K; Liu, Robert C

    2014-01-01

    Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system - motherhood - is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered.

  13. Vasoactive intestinal peptide antagonist treatment during mouse embryogenesis impairs social behavior and cognitive function of adult male offspring.

    PubMed

    Hill, Joanna M; Cuasay, Katrina; Abebe, Daniel T

    2007-07-01

    Vasoactive intestinal peptide (VIP) is a regulator of rodent embryogenesis during the period of neural tube closure. VIP enhanced growth in whole cultured mouse embryos; treatment with a VIP antagonist during embryogenesis inhibited growth and development. VIP antagonist treatment during embryogenesis also had permanent effects on adult brain chemistry and impaired social recognition behavior in adult male mice. The neurological deficits of autism appear to be initiated during neural tube closure and social behavior deficits are among the key characteristics of this disorder that is more common in males and is frequently accompanied by mental retardation. The current study examined the blockage of VIP during embryogenesis as a model for the behavioral deficits of autism. Treatment of pregnant mice with a VIP antagonist during embryonic days 8 through 10 had no apparent effect on the general health or sensory or motor capabilities of adult offspring. However, male offspring exhibited reduced sociability in the social approach task and deficits in cognitive function, as assessed through cued and contextual fear conditioning. Female offspring did not show these deficiencies. These results suggest that this paradigm has usefulness as a mouse model for aspects of autism as it selectively impairs male offspring who exhibit the reduced social behavior and cognitive dysfunction seen in autism. Furthermore, the study indicates that the foundations of some aspects of social behavior are laid down early in mouse embryogenesis, are regulated in a sex specific manner and that interference with embryonic regulators such as VIP can have permanent effects on adult social behavior.

  14. Quantitative Expression Profile of Distinct Functional Regions in the Adult Mouse Brain

    PubMed Central

    Nagano, Mamoru; Uno, Kenichiro D.; Tsujino, Kaori; Hanashima, Carina; Shigeyoshi, Yasufumi; Ueda, Hiroki R.

    2011-01-01

    The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems. PMID:21858037

  15. Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease

    PubMed Central

    Ambade, Aditya; Satishchandran, Abhishek; Gyongyosi, Benedek; Lowe, Patrick; Szabo, Gyongyi

    2016-01-01

    AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD). METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA). RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice. CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC. PMID:27122661

  16. Neurotoxic effects of ochratoxin A on the subventricular zone of adult mouse brain.

    PubMed

    Paradells, Sara; Rocamonde, Brenda; Llinares, Cristina; Herranz-Pérez, Vicente; Jimenez, Misericordia; Garcia-Verdugo, Jose Manuel; Zipancic, Ivan; Soria, Jose Miguel; Garcia-Esparza, Ma Angeles

    2015-07-01

    Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner. PMID:25256750

  17. LRRK2 is expressed in areas affected by Parkinson's disease in the adult mouse brain.

    PubMed

    Simón-Sánchez, Javier; Herranz-Pérez, Vicente; Olucha-Bordonau, Francisco; Pérez-Tur, Jordi

    2006-02-01

    The leucine-rich repeat kinase 2 (LRRK2) gene was recently found to have multiple mutations that are causative for autosomal dominant inherited Parkinson's disease (PD). Previously, we used Northern blot analysis to show that this gene was expressed in the cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe and caudate putamen. However, a more comprehensive map of LRRK2 mRNA localization in the central nervous system is still lacking. In this study we have mapped the distribution of the mRNA encoding for LRRK2 using nonradioactive in situ hybridization. We detected a moderate expression of this PD-related gene throughout the adult B2B6 mouse brain. A stronger hybridization signal was observed in deep cerebral cortex layers, superficial cingulate cortex layers, the piriform cortex, hippocampal formation, caudate putamen, substantia nigra, the basolateral and basomedial anterior amygdala nuclei, reticular thalamic nucleus and also in the cerebellar granular cell layer. Given that LRRK2 mRNA is highly enriched in motor systems and also is expressed in other systems, we may conclude that mutations in LRRK2 may affect several motor and nonmotor structures that may play an important role in the development of PD.

  18. Neurotoxic effects of ochratoxin A on the subventricular zone of adult mouse brain.

    PubMed

    Paradells, Sara; Rocamonde, Brenda; Llinares, Cristina; Herranz-Pérez, Vicente; Jimenez, Misericordia; Garcia-Verdugo, Jose Manuel; Zipancic, Ivan; Soria, Jose Miguel; Garcia-Esparza, Ma Angeles

    2015-07-01

    Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner.

  19. Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors

    PubMed Central

    Belgard, T. Grant; Montiel, Juan F.; Wang, Wei Zhi; García-Moreno, Fernando; Ponting, Chris P.; Molnár, Zoltán

    2013-01-01

    The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14–27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676–12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates. PMID:23878249

  20. Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.

    PubMed

    Belgard, T Grant; Montiel, Juan F; Wang, Wei Zhi; García-Moreno, Fernando; Margulies, Elliott H; Ponting, Chris P; Molnár, Zoltán

    2013-08-01

    The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates.

  1. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

    PubMed

    Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  2. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    PubMed Central

    Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  3. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle

    PubMed Central

    1981-01-01

    Intact cardiac cells from the adult rat or rabbit ventricle were isolated by enzymatic digestion with a progressive increase of the [free Ca2+] in the solution. These cells were electrically stimulated in the presence of 2.50 mM free Ca2+, and a twitch of maximum amplitude was elicited by the positive inotropic interventions that were found to be optimum. Then the cells were chemically skinned, and the maximum tension induced by a saturating [free Ca2+] was used as a reference to express the tension developed during the twitch of the intact cells. The myoplasmic [free Ca2+] reached during the twitch was inferred from the tension-pCa curve. In mechanically skinned cells of the same animal species, the myoplasmic [free Ca2+] reached during Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR) was inferred by two methods using (a) the tension-pCa curve and (b) a direct calibration of the transients of aequorin bioluminescence. The induction of a maximum Ca2+ release from the SR required a larger Ca2+ preload of the SR and a higher [free Ca2+] trigger in the rabbit than in the rat skinned cells. However, the results obtained with the two methods of inference of the myoplasmic [free Ca2+] suggest that in both animal species a maximum myoplasmic [free Ca2+] of pCa approximately 5.40 was reached during both the optimum Ca2+-induced release of Ca2+ from the SR of the skinned cells and the optimum twitch of the intact cells. This was much lower than the [free Ca2+] necessary for the full activation of the myofilaments (pCa approximately 4.90). PMID:6796647

  4. A Randomized Trial to Measure the Impact of a Community-Based Cognitive Training Intervention on Balance and Gait in Cognitively Intact Black Older Adults

    ERIC Educational Resources Information Center

    Smith-Ray, Renae L.; Makowski-Woidan, Beth; Hughes, Susan L.

    2014-01-01

    Introduction: Fall prevention is important for maintaining mobility and independence into old age. Approaches for reducing falls include exercise, tai chi, and home modifications; however, causes of falling are multifactorial and include not just physical but cognitive factors. Cognitive decline occurs with age, but older adults with the greatest…

  5. Dynamic expression of TrkB receptor protein on proliferating and maturing cells in the adult mouse dentate gyrus

    PubMed Central

    Donovan, Michael H.; Yamaguchi, Masahiro; Eisch, Amelia J.

    2008-01-01

    Brain-derived neurotrophic factor (BDNF) is implicated in regulation of adult hippocampal neurogenesis, presumably via its primary receptor, TrkB, but controversy exists about how BDNF affects neurogenesis (e.g. proliferation vs. survival/differentiation). This controversy arises, in part, due to the lack of information about if and when TrkB is expressed on adult neural precursors in vivo. Using multiple methods to analyze proliferating and maturing cells in the adult mouse subgranular zone (SGZ), we find that the proportion of proliferating cells that are TrkB-IR is low and it remains low for at least one week following BrdU labeling, but increases as neuroblasts mature. Use of the nestin-GFP transgenic mouse revealed the likelihood of being TrkB-IR increased with presumed maturity of the cell type. Stem-like cells, which rarely divide, were likely to express TrkB. However, early progenitors and late progenitors, which are still in the cell cycle had rare TrkB expression. Immature neuroblasts, however, were more likely to express TrkB, especially as their morphology became more mature. Taken together, these findings emphasize that expression of TrkB protein is closely linked to progression towards neuronal maturity. This provides evidence that maturing cells but not proliferating cells in the adult mouse SGZ have the molecular machinery necessary to respond directly to BDNF. Furthermore, these findings lay critical groundwork for further exploration of the role of BDNF-TrkB signaling in regulation of adult hippocampal neurogenesis. PMID:18240316

  6. Endoglin Deficiency in Bone Marrow Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse after VEGF Stimulation

    PubMed Central

    Choi, Eun-Jung; Walker, Espen J.; Degos, Vincent; Jun, Kristine; Kuo, Robert; Pile-Spellman, John; Su, Hua; Young, William L.

    2013-01-01

    Background and Purpose Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng+/−) mice. We hypothesized that Eng+/− BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. Methods BM transplantation was performed using adult wild-type (WT) and Eng+/− mice as donors/recipients. An adeno-associated viral vector expressing VEGF (AAV-VEGF) was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 μm/100 vessels), and BMDCs in the angiogenic foci were analyzed. Results The dysplasia index of WT/Eng+/− BM mice was higher than WT/WT BM mice (p<0.001) and was similar to Eng+/−/Eng+/− BM mice (p=0.2). Dysplasia in Eng+/− mice was partially rescued by WT BM (p<0.001). WT/WT BM and WT/Eng+/− BM mice had similar numbers of BMDCs in the angiogenic foci (p=0.4), most of which were CD68+. Eng+/− monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. Conclusions ENG-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations. PMID:23306322

  7. Fructose metabolism in the adult mouse optic nerve, a central white matter tract.

    PubMed

    Meakin, Paul J; Fowler, Maxine J; Rathbone, Alex J; Allen, Lynne M; Ransom, Bruce R; Ray, David E; Brown, Angus M

    2007-01-01

    Our recent report that fructose supported the metabolism of some, but not all axons, in the adult mouse optic nerve prompted us to investigate in detail fructose metabolism in this tissue, a typical central white matter tract, as these data imply efficient fructose metabolism in the central nervous system (CNS). In artificial cerebrospinal fluid containing 10 mmol/L glucose or 20 mmol/L fructose, the stimulus-evoked compound action potential (CAP) recorded from the optic nerve consisted of three stable peaks. Replacing 10 mmol/L glucose with 10 mmol/L fructose, however, caused delayed loss of the 1st CAP peak (the 2nd and 3rd CAP peaks were unaffected). Glycogen-derived metabolic substrate(s) temporarily sustained the 1st CAP peak in 10 mmol/L fructose, as depletion of tissue glycogen by a prior period of aglycaemia or high-frequency CAP discharge rendered fructose incapable of supporting the 1st CAP peak. Enzyme assays showed the presence of both hexokinase and fructokinase (both of which can phosphorylate fructose) in the optic nerve. In contrast, only hexokinase was expressed in cerebral cortex. Hexokinase in optic nerve had low affinity and low capacity with fructose as substrate, whereas fructokinase displayed high affinity and high capacity for fructose. These findings suggest an explanation for the curious fact that the fast conducting axons comprising the 1st peak of the CAP are not supported in 10 mmol/L fructose medium; these axons probably do not express fructokinase, a requirement for efficient fructose metabolism.

  8. Genetic influences on exercise-induced adult hippocampal neurogenesis across 12 divergent mouse strains

    PubMed Central

    Clark, Peter J.; Kohman, Rachel A.; Miller, Daniel S.; Bhattacharya, Tushar K.; Brzezinska, Weronika J.; Rhodes, Justin S.

    2011-01-01

    New neurons are continuously born in the hippocampus of several mammalian species throughout adulthood. Adult neurogenesis represents a natural model for understanding how to grow and incorporate new nerve cells into pre-existing circuits in the brain. Finding molecules or biological pathways that increase neurogenesis has broad potential for regenerative medicine. One strategy is to identify mouse strains that display large versus small increases in neurogenesis in response to wheel running so the strains can be contrasted to find common genes or biological pathways associated with enhanced neuron formation. Therefore, mice from 12 different isogenic strains were housed with or without running wheels for 43 days to measure the genetic regulation of exercise-induced neurogenesis. The first 10 days mice received daily injections of BrdU to label dividing cells. Neurogenesis was measured as the total number of BrdU cells co-expressing NeuN mature neuronal marker in the hippocampal granule cell layer by immunohistochemistry. Exercise increased neurogenesis in all strains, but the magnitude significantly depended on genotype. Strain means for distance run on wheels, but not distance traveled in cages without wheels, were significantly correlated with strain mean level of neurogenesis. Further, certain strains displayed greater neurogenesis than others for a fixed level of running. Strain means for neurogenesis under sedentary conditions were not correlated with neurogenesis under runner conditions suggesting that different genes influence baseline versus exercise-induced neurogenesis. Genetic contributions to exercise-induced hippocampal neurogenesis suggest that it may be possible to identify genes and pathways associated with enhanced neuroplastic responses to exercise. PMID:21223504

  9. Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

    PubMed Central

    Gelain, Fabrizio; Bottai, Daniele; Vescovi, Angleo; Zhang, Shuguang

    2006-01-01

    Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology. PMID:17205123

  10. Modulation of phase durations, phase variations, and temporal coordination of the four limbs during quadrupedal split-belt locomotion in intact adult cats.

    PubMed

    D'Angelo, Giuseppe; Thibaudier, Yann; Telonio, Alessandro; Hurteau, Marie-France; Kuczynski, Victoria; Dambreville, Charline; Frigon, Alain

    2014-10-15

    Stepping along curvilinear paths produces speed differences between the inner and outer limb(s). This can be reproduced experimentally by independently controlling left and right speeds with split-belt locomotion. Here we provide additional details on the pattern of the four limbs during quadrupedal split-belt locomotion in intact cats. Six cats performed tied-belt locomotion (same speed bilaterally) and split-belt locomotion where one side (constant side) stepped at constant treadmill speed while the other side (varying side) stepped at several speeds. Cycle, stance, and swing durations changed in parallel in homolateral limbs with shorter and longer stance and swing durations on the fast side, respectively, compared with the slow side. Phase variations were quantified in all four limbs by measuring the slopes of the regressions between stance and cycle durations (rSTA) and between swing and cycle durations (rSW). For a given limb, rSTA and rSW were not significantly different from one another on the constant side whereas on the varying side rSTA increased relative to tied-belt locomotion while rSW became more negative. Phase variations were similar for homolateral limbs. Increasing left-right speed differences produced a large increase in homolateral double support on the slow side, while triple-support periods decreased. Increasing left-right speed differences altered homologous coupling, homolateral coupling on the fast side, and coupling between the fast hindlimb and slow forelimb. Results indicate that homolateral limbs share similar control strategies, only certain features of the interlimb pattern adjust, and spinal locomotor networks of the left and right sides are organized symmetrically. PMID:25031257

  11. Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

    PubMed Central

    Carreira, Vinicius S.; Fan, Yunxia; Kurita, Hisaka; Wang, Qin; Ko, Chia-I; Naticchioni, Mindi; Jiang, Min; Koch, Sheryl; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Xia, Ying; Rubinstein, Jack; Puga, Alvaro

    2015-01-01

    The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease. PMID:26555816

  12. Comprehensive Analysis of Neonatal versus Adult Unilateral Decortication in a Mouse Model Using Behavioral, Neuroanatomical, and DNA Microarray Approaches

    PubMed Central

    Yoshikawa, Akira; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Shioda, Seiji

    2014-01-01

    Previously, studying the development, especially of corticospinal neurons, it was concluded that the main compensatory mechanism after unilateral brain injury in rat at the neonatal stage was due in part to non-lesioned ipsilateral corticospinal neurons that escaped selection by axonal elimination or neuronal apoptosis. However, previous results suggesting compensatory mechanism in neonate brain were not correlated with high functional recovery. Therefore, what is the difference among neonate and adult in the context of functional recovery and potential mechanism(s) therein? Here, we utilized a brain unilateral decortication mouse model and compared motor functional recovery mechanism post-neonatal brain hemisuction (NBH) with adult brain hemisuction (ABH). Three analyses were performed: (1) Quantitative behavioral analysis of forelimb movements using ladder walking test; (2) neuroanatomical retrograde tracing analysis of unlesioned side corticospinal neurons; and (3) differential global gene expressions profiling in unlesioned-side neocortex (rostral from bregma) in NBH and ABH on a 8 × 60 K mouse whole genome Agilent DNA chip. Behavioral data confirmed higher recovery ability in NBH over ABH is related to non-lesional frontal neocortex including rostral caudal forelimb area. A first inventory of differentially expressed genes genome-wide in the NBH and ABH mouse model is provided as a resource for the scientific community. PMID:25490135

  13. Comparative proteomic analysis of mouse livers from embryo to adult reveals an association with progression of hepatocellular carcinoma.

    PubMed

    Lee, Nikki P Y; Leung, Kar-wai; Cheung, Nicole; Lam, Brian Y; Xu, Michelle Z; Sham, Pak C; Lau, George K; Poon, Ronnie T P; Fan, Sheung Tat; Luk, John M

    2008-05-01

    To identify potential oncofetal biomarkers that distinguish hepatocellular carcinoma (HCC) from healthy liver tissues, we compared and analyzed the proteomic profiles of mouse livers at different developmental stages. Fetal (E13.5, E16.5), newborn (NB), postnatal (3-week) and adult (3-month) livers were isolated and profiled by 2-D PAGE. Statistical analysis using linear regression and false discovery rate (FDR) revealed that 361 protein spots showed significant changes. Unsupervised hierarchical tree analysis segregated the proteins into fetal, NB, and postnatal-adult clusters. Distinctive protein markers were identified by MALDI-TOF/MS and the corresponding mRNA profiles were further determined by Q-PCR. Fetal markers (hPCNA, hHSP7C, hHEM6) and postnatal-adult markers (hARGI1, hASSY, hBHMT, hFABPL) were selected for testing against a panel of seven human hepatocyte/HCC cell lines and 59 clinical specimens. The fetal proteins were found to be overexpressed in the metastatic HCC cell lines and the tumor tissues, whereas the postnatal-adult proteins were expressed in non-tumor tissues and normal hepatocytes. This "Ying-Yang" pattern, as orchestrated by distinct fetal and adult markers, is hypothesized to indicate the progressive change of the liver from a growing, less-differentiated organ into a functional metabolic center. Thus, embryogenesis and tumorigenesis share certain oncofetal markers and adult "hepatic" phenotypes are lost in HCC.

  14. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.

    PubMed

    Gritti, A; Parati, E A; Cova, L; Frolichsthal, P; Galli, R; Wanke, E; Faravelli, L; Morassutti, D J; Roisen, F; Nickel, D D; Vescovi, A L

    1996-02-01

    It has been established that the adult mouse forebrain contains multipotential (neuronal/glial) progenitor cells that can be induced to proliferate in vitro when epidermal growth factor is provided. These cells are found within the subventricular zone of the lateral ventricles, together with other progenitor cell populations, whose requirements for proliferation remain undefined. Using basic fibroblast growth factor (bFGF), we have isolated multipotential progenitors from adult mouse striatum. These progenitors proliferate and can differentiate into cells displaying the antigenic properties of astrocytes, oligodendrocytes, and neurons. The neuron-like cells possess neuronal features, exhibit neuronal electrophysiological properties, and are immunoreactive for GABA, substance P, choline acetyl-transferase, and glutamate. Clonal analysis confirmed the multipotency of these bFGF-dependent cells. Most significantly, subcloning experiments demonstrated that they were capable of self-renewal, which led to a progressive increase in population size over serial passaging. These results demonstrate that bFGF is mitogenic for multipotential cells from adult mammalian forebrain that possess stem cell properties. PMID:8558238

  15. Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

    PubMed

    Al Alam, Denise; Danopoulos, Soula; Schall, Kathy; Sala, Frederic G; Almohazey, Dana; Fernandez, G Esteban; Georgia, Senta; Frey, Mark R; Ford, Henri R; Grikscheit, Tracy; Bellusci, Saverio

    2015-04-15

    Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

  16. PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain

    PubMed Central

    Liu, Mali; Wu, Guoxin; Baysarowich, Jennifer; Kavana, Michael; Addona, George H.; Bierilo, Kathleen K.; Mudgett, John S.; Pavlovic, Guillaume; Sitlani, Ayesha; Renger, John J.; Hubbard, Brian K.; Fisher, Timothy S.; Zerbinatti, Celina V.

    2010-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates hepatic low-density lipoprotein receptor (LDLR) levels in humans. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including the very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2) and the β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are all highly expressed in the CNS and have been implicated in Alzheimer's disease. To better understand the role of PCSK9 in regulating these additional target proteins in vivo, their steady-state levels were measured in the brain of wild-type, PCSK9-deficient, and human PCSK9 overexpressing transgenic mice. We found that while PCSK9 directly bound to recombinant LDLR, VLDLR, and apoER2 protein in vitro, changes in PCSK9 expression did not alter the level of these receptors in the mouse brain. In addition, we found no evidence that PCSK9 regulates BACE1 levels or APP processing in the mouse brain. In conclusion, our results suggest that while PCSK9 plays an important role in regulating circulating LDL cholesterol levels by reducing the number of hepatic LDLRs, it does not appear to modulate the levels of LDLR and other membrane-bound proteins in the adult mouse brain. PMID:20453200

  17. Activation of CB1 inhibits NGF-induced sensitization of TRPV1 in adult mouse afferent neurons.

    PubMed

    Wang, Z-Y; McDowell, T; Wang, P; Alvarez, R; Gomez, T; Bjorling, D E

    2014-09-26

    Transient receptor potential vanilloid 1 (TRPV1)-containing afferent neurons convey nociceptive signals and play an essential role in pain sensation. Exposure to nerve growth factor (NGF) rapidly increases TRPV1 activity (sensitization). In the present study, we investigated whether treatment with the selective cannabinoid receptor 1 (CB1) agonist arachidonyl-2'-chloroethylamide (ACEA) affects NGF-induced sensitization of TRPV1 in adult mouse dorsal root ganglion (DRG) afferent neurons. We found that CB1, NGF receptor tyrosine kinase A (trkA), and TRPV1 are present in cultured adult mouse small- to medium-sized afferent neurons and treatment with NGF (100ng/ml) for 30 min significantly increased the number of neurons that responded to capsaicin (as indicated by increased intracellular Ca(2 +) concentration). Pretreatment with the CB1 agonist ACEA (10nM) inhibited the NGF-induced response, and this effect of ACEA was reversed by a selective CB1 antagonist. Further, pretreatment with ACEA inhibited NGF-induced phosphorylation of AKT. Blocking PI3 kinase activity also attenuated the NGF-induced increase in the number of neurons that responded to capsaicin. Our results indicate that the analgesic effect of CB1 activation may in part be due to inhibition of NGF-induced sensitization of TRPV1 and also that the effect of CB1 activation is at least partly mediated by attenuation of NGF-induced increased PI3 signaling.

  18. Targeted deletion of Vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse

    PubMed Central

    Nordenankar, Karin; Bergfors, Assar

    2015-01-01

    Background Anxiety is a natural emotion experienced by all individuals. However, when anxiety becomes excessive, it contributes to the substantial group of anxiety disorders that affect one in three people and thus are among the most common psychiatric disorders. Anxiolysis, the reduction of anxiety, is mediated via several large groups of therapeutical compounds, but the relief is often only temporary, and increased knowledge of the neurobiology underlying anxiety is needed in order to improve future therapies. Aim We previously demonstrated that mice lacking forebrain expression of the Vesicular glutamate transporter 2 (Vglut2) from adolescence showed a strong anxiolytic behaviour as adults. In the current study, we wished to analyse if removal of Vglut2 expression already from mid-gestation of the mouse embryo would give rise to similar anxiolysis in the adult mouse. Methods We produced transgenic mice lacking Vglut2 from mid-gestation and analysed their affective behaviour, including anxiety, when they had reached adulthood. Results The transgenic mice lacking Vglut2 expression from mid-gestation showed certain signs of anxiolytic behaviour, but this phenotype was not as prominent as when Vglut2 was removed during adolescence. Conclusion Our results suggest that both embryonal and adolescent forebrain expression of Vglut2 normally contributes to balancing the level of anxiety. As the neurobiological basis for anxiety is similar across species, our results in mice may help improve the current understanding of the neurocircuitry of anxiety, and hence anxiolysis, also in humans. PMID:25857802

  19. A generational study of glyphosate-tolerant soybeans on mouse fetal, postnatal, pubertal and adult testicular development.

    PubMed

    Brake, Denise G; Evenson, Donald P

    2004-01-01

    The health safety of transgenic soybeans (glyphosate-tolerant or Roundup Ready) was studied using the mammalian testis (mouse model) as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a transgenic soybean or a non-transgenic (conventional) diet through gestation and lactation. After weaning, the young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63 and 87 days after birth, three male mice and an adult reference mouse were killed, the testes surgically removed, and the cell populations measured by flow cytometry. Multi-generational studies were conducted in the same manner. The results showed that the transgenic foodstuffs had no effect on macromolecular synthesis or cell growth and differentiation as evidenced by no differences in the percentages of testicular cell populations (haploid, diploid, and tetraploid) between the transgenic soybean-fed mice and those fed the conventional diet. Additionally, there were no differences in litter sizes and body weights of the two groups. It was concluded that the transgenic soybean diet had no negative effect on fetal, postnatal, pubertal or adult testicular development.

  20. Chronic serotonin-norepinephrine reuptake transporter inhibition modifies basal respiratory output in adult mouse in vitro and in vivo

    PubMed Central

    Warren, Kelly A.; Solomon, Irene C.

    2012-01-01

    Respiratory disturbances are a common feature of panic disorder and present as breathing irregularity, hyperventilation, and increased sensitivity to carbon dioxide. Common therapeutic interventions, such as tricyclic (TCA) and selective serotonin reuptake inhibitor (SSRI) antidepressants, have been shown to ameliorate not only the psychological components of panic disorder but also the respiratory disturbances. These drugs are also prescribed for generalized anxiety and depressive disorders, neither of which are characterized by respiratory disturbances, and previous studies have demonstrated that TCAs and SSRIs exert effects on basal respiratory activity in animal models without panic disorder symptoms. Whether serotonin-norepinephrine reuptake inhibitors (SNRIs) have similar effects on respiratory activity remains to be determined. Therefore, the current study was designed to investigate the effects of chronic administration of the SNRI antidepressant venlafaxine (VHCL) on basal respiratory output. For these experiments, we recorded phrenic nerve discharge in an in vitro arterially-perfused adult mouse preparation and diaphragm electromyogram (EMG) activity in an in vivo urethane-anesthetized adult mouse preparation. We found that following 28-d VHCL administration, basal respiratory burst frequency was markedly reduced due to an increase in expiratory duration (TE), and the inspiratory duty cycle (TI/Ttot) was significantly shortened. In addition, post-inspiratory and spurious expiratory discharges were seen in vitro. Based on our observations, we suggest that drugs capable of simultaneously blocking both 5-HT and NE reuptake transporters have the potential to influence the respiratory control network in patients using SNRI therapy. PMID:22871263

  1. The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

    PubMed Central

    Neal, Alice; Boldrin, Luisa; Morgan, Jennifer Elizabeth

    2012-01-01

    Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. PMID:22662253

  2. A physiologically based pharmacokinetic model for atrazine and its main metabolites in the adult male C57BL/6 mouse

    SciTech Connect

    Lin Zhoumeng; Fisher, Jeffrey W.; Ross, Matthew K.; Filipov, Nikolay M.

    2011-02-15

    Atrazine (ATR) is a chlorotriazine herbicide that is widely used and relatively persistent in the environment. In laboratory rodents, excessive exposure to ATR is detrimental to the reproductive, immune, and nervous systems. To better understand the toxicokinetics of ATR and to fill the need for a mouse model, a physiologically based pharmacokinetic (PBPK) model for ATR and its main chlorotriazine metabolites (Cl-TRIs) desethyl atrazine (DE), desisopropyl atrazine (DIP), and didealkyl atrazine (DACT) was developed for the adult male C57BL/6 mouse. Taking advantage of all relevant and recently made available mouse-specific data, a flow-limited PBPK model was constructed. The ATR and DACT sub-models included blood, brain, liver, kidney, richly and slowly perfused tissue compartments, as well as plasma protein binding and red blood cell binding, whereas the DE and DIP sub-models were constructed as simple five-compartment models. The model adequately simulated plasma levels of ATR and Cl-TRIs and urinary dosimetry of Cl-TRIs at four single oral dose levels (250, 125, 25, and 5 mg/kg). Additionally, the model adequately described the dose dependency of brain and liver ATR and DACT concentrations. Cumulative urinary DACT amounts were accurately predicted across a wide dose range, suggesting the model's potential use for extrapolation to human exposures by performing reverse dosimetry. The model was validated using previously reported data for plasma ATR and DACT in mice and rats. Overall, besides being the first mouse PBPK model for ATR and its Cl-TRIs, this model, by analogy, provides insights into tissue dosimetry for rats. The model could be used in tissue dosimetry prediction and as an aid in the exposure assessment to this widely used herbicide.

  3. Sex differences in adrenocortical structure and function. XVI. Stereological and karyometric studies on the cortex of the suprarenal gland of intact adult male and female Mongolian gerbils (Meriones unguiculatus).

    PubMed

    Malendowicz, L K

    1984-10-01

    The histological structure of intact adult male and female Mongolian gerbil (Meriones unguiculatus) suprarenal cortex has been described, and quantitative stereological and karyometric studies were performed. The weight of the gland relative to body weight was higher in female than in male gerbils, but the volume of the gland was similar in both sexes. The relative volume of the zona reticularis was higher in the female, while no sex difference was observed in the absolute volume of all suprarenal components (expressed in mm3). In all cortical zones, average cell volume was higher in the female; the nuclear volume of the zona glomerulosa and zona reticularis were also higher in the female. In the zona fasciculata, some large cells with large nuclei (greater than 210 micrometers 3) were observed. These are probably polyploid cells and have not been described in other species. The suprarenal cortex of the male gerbil contained more parenchymal cells than that of the female, the difference being dependent upon variation in the number of cells in the zona fasciculata and zona reticularis. Despite these structural differences, gland homogenates from male and female animals secreted similar amounts of cortisol and the plasma levels in the two sexes were the same.

  4. Repair of liver mediated by adult mouse liver neuro-glia antigen 2-positive progenitor cell transplantation in a mouse model of cirrhosis

    PubMed Central

    Zhang, Hongyu; Siegel, Christopher T.; Shuai, Ling; Lai, Jiejuan; Zeng, Linli; Zhang, Yujun; Lai, Xiangdong; Bie, Ping; Bai, Lianhua

    2016-01-01

    NG2-expressing cells are a population of periportal vascular stem/progenitors (MLpvNG2+ cells) that were isolated from healthy adult mouse liver by using a “Percoll-Plate-Wait” procedure. We demonstrated that isolated cells are able to restore liver function after transplantation into a cirrhotic liver, and co-localized with the pericyte marker (immunohistochemistry: PDGFR-β) and CK19. Cells were positive for: stem cell (Sca-1, CD133, Dlk) and liver stem cell markers (EpCAM, CD14, CD24, CD49f); and negative for: hematopoietic (CD34, CD45) and endothelial markers (CD31, vWf, von Willebrand factor). Cells were transplanted (1 × 106 cells) in mice with diethylnitrosamine-induced cirrhosis at week 6. Cells showed increased hepatic associated gene expression of alpha-fetoprotein (AFP), Albumin (Alb), Glucose-6-phosphatase (G6Pc), SRY (sex determining region Y)-box 9 (Sox9), hepatic nuclear factors (HNF1a, HNF1β, HNF3β, HNF4α, HNF6, Epithelial cell adhesion molecule (EpCAM), Leucine-rich repeated-containing G-protein coupled receptor 5-positive (Lgr5) and Tyrosine aminotransferase (TAT). Cells showed decreased fibrogenesis, hepatic stellate cell infiltration, Kupffer cells and inflammatory cytokines. Liver function markers improved. In a cirrhotic liver environment, cells could differentiate into hepatic lineages. In addition, grafted MLpvNG2+ cells could mobilize endogenous stem/progenitors to participate in liver repair. These results suggest that MLpvNG2+ cells may be novel adult liver progenitors that participate in liver regeneration. PMID:26905303

  5. MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells.

    PubMed

    Wissink, Erin M; Smith, Norah L; Spektor, Roman; Rudd, Brian D; Grimson, Andrew

    2015-11-01

    Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection. PMID:26416483

  6. MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells

    PubMed Central

    Wissink, Erin M.; Smith, Norah L.; Spektor, Roman; Rudd, Brian D.; Grimson, Andrew

    2015-01-01

    Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection. PMID:26416483

  7. Daily rhythms of core temperature and locomotor activity indicate different adaptive strategies to cold exposure in adult and aged mouse lemurs acclimated to a summer-like photoperiod.

    PubMed

    Terrien, Jeremy; Zizzari, Philippe; Epelbaum, Jacques; Perret, Martine; Aujard, Fabienne

    2009-07-01

    Daily variations in core temperature (Tc) within the normothermic range imply thermoregulatory processes that are essential for optimal function and survival. Higher susceptibility towards cold exposure in older animals suggests that these processes are disturbed with age. In the mouse lemur, a long-day breeder, we tested whether aging affected circadian rhythmicity of Tc, locomotor activity (LA), and energy balance under long-day conditions when exposed to cold. Adult (N = 7) and aged (N = 5) mouse lemurs acclimated to LD14/10 were exposed to 10-day periods at 25 and 12 degrees C. Tc and LA rhythms were recorded by telemetry, and caloric intake (CI), body mass changes, and plasma IGF-1 were measured. During exposure to 25 degrees C, both adult and aged mouse lemurs exhibited strong daily variations in Tc. Aged animals exhibited lower levels of nocturnal LA and nocturnal and diurnal Tc levels in comparison to adults. Body mass and IGF-1 levels remained unchanged with aging. Under cold exposure, torpor bout occurrence was never observed whatever the age category. Adult and aged mouse lemurs maintained their Tc in the normothermic range and a positive energy balance. All animals exhibited increase in CI and decrease in IGF-1 in response to cold. The decrease in IGF-1 was delayed in aged mouse lemurs compared to adults. Moreover, both adult and aged animals responded to cold exposure by increasing their diurnal LA compared to those under Ta = 25 degrees C. However, aged animals exhibited a strong decrease in nocturnal LA and Tc, whereas cold effects were only slight in adults. The temporal organization and amplitude of the daily phase of low Tc were particularly well preserved under cold exposure in both age groups. Sexually active mouse lemurs exposed to cold thus seemed to prevent torpor exhibition and temporal disorganization of daily rhythms of Tc, even during aging. However, although energy balance was not impaired with age in mouse lemurs after cold exposure

  8. POD Nanoparticles Expressing GDNF Provide Structural and Functional Rescue of Light-induced Retinal Degeneration in an Adult Mouse

    PubMed Central

    Read, Sarah P; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-01-01

    Peptide for ocular delivery (POD) is a novel cationic cell-penetrating peptide (CPP) which, when conjugated with polyethylene glycol (PEG-POD), can deliver plasmid DNA to the retinal pigment epithelium (RPE) of adult murine retina. PEG-POD nanoparticles containing an expression cassette for glial cell line–derived neurotrophic factor (PEG–POD~GDNF) were investigated for their ability to inhibit light-induced photoreceptor apoptosis. PEG-POD~GDNF, control nanoparticles, or buffer were injected into the subretinal space of adult murine retina and retinal degeneration induced by blue light. Animals injected with PEG-POD~GDNF showed a significant reduction (3.9–7.7 fold) in apoptosis relative to control-injected animals. The thickness of the outer nuclear layer (ONL) of the superior retina of PEG-POD~GDNF-injected eyes was significantly greater (23.6–39.3%) than control-injected retina 14 days post-light treatment. PEG-POD~GDNF-injected eyes showed a 27–39% greater functional response relative to controls, as measured by electroretinogram (ERG) 7 days post-light treatment. This is one of only two studies demonstrating histological and functional rescue of a mouse model of retinal degeneration following nonviral administration of a transgene into adult retina. Although rescue is short lived for clinical application, this study represents an important step in the development of nonviral gene therapy for retinal diseases. PMID:20700110

  9. (Photosynthesis in intact plants)

    SciTech Connect

    Not Available

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  10. Chronic Social Stress Affects Synaptic Maturation of Newly Generated Neurons in the Adult Mouse Dentate Gyrus

    PubMed Central

    Chen, Chien-Chung; Huang, Chiung-Chun

    2016-01-01

    Background: Chronic stress has been found to suppress adult neurogenesis, but it remains unclear whether it may affect the maturation process of adult-born neurons. Here, we examined the influence of chronic social defeat stress on the morphological and electrophysiological properties of adult-born dentate granule cells at different developmental stages. Methods: Adult C57BL/6 mice were subjected to 10 days of chronic social defeat stress followed by a social interaction test 24 hours after the last defeat. Defeated mice were segregated into susceptible and unsusceptible subpopulations based on a measure of social interaction test. Combining electrophysiology with retrovirus-mediated birth-dating and labeling, we examined the impact of chronic social defeat stress on temporal regulation of synaptic plasticity of adult-born dentate granule cells along their maturation. Results: Chronic social defeat stress decreases the survival and dendritic complexity of adult-born dentate granule cells. While chronic social defeat stress doesn’t alter the intrinsic electrophysiological properties and synaptic transmission of surviving adult-born dentate granule cells, it promotes the developmental switch in synaptic N-methyl-D-aspartate receptors from predominant GluN2B- to GluN2A-containing receptors, which transform the immature synapse of adult-born dentate granule cells from one that exhibits enhanced long-term potentiation to one that has normal levels of long-term potentiation. Furthermore, chronic social defeat stress increases the level of endogenous repressor element-1 silencing transcription factor mRNA in adult-born dentate granule cells, and knockdown of the repressor element-1 silencing transcription factor in adult-born dentate granule cells rescues chronic social defeat stress-induced morphological deficits and accelerated developmental switch in synaptic N-methyl-D-aspartate receptor subunit composition. Conclusions: These results uncover a previously

  11. A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia.

    PubMed

    Simon, Camille; Chagraoui, Jalila; Krosl, Jana; Gendron, Patrick; Wilhelm, Brian; Lemieux, Sébastien; Boucher, Geneviève; Chagnon, Pierre; Drouin, Simon; Lambert, Raphaëlle; Rondeau, Claude; Bilodeau, Annie; Lavallée, Sylvie; Sauvageau, Martin; Hébert, Josée; Sauvageau, Guy

    2012-04-01

    In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.

  12. Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease.

    PubMed

    Kim, Hye Yun; Kim, Hyunjin V; Yoon, Jin H; Kang, Bo Ram; Cho, Soo Min; Lee, Sejin; Kim, Ji Yoon; Kim, Joo Won; Cho, Yakdol; Woo, Jiwan; Kim, YoungSoo

    2014-12-12

    Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aβ. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aβ-related damages.

  13. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia

    PubMed Central

    Zhang, Qifeng; Smethurst, Elizabeth; Segonds-Pichon, Anne; Schrewe, Heinrich; Wakelam, Michael J. O.

    2016-01-01

    Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA), a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO) mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA) regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction. PMID:27658289

  14. Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease

    PubMed Central

    Kim, Hye Yun; Kim, Hyunjin V.; Yoon, Jin H.; Kang, Bo Ram; Cho, Soo Min; Lee, Sejin; Kim, Ji Yoon; Kim, Joo Won; Cho, Yakdol; Woo, Jiwan; Kim, YoungSoo

    2014-01-01

    Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aβ. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aβ-related damages. PMID:25502280

  15. Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

    PubMed

    Detloff, P J; Lewis, J; John, S W; Shehee, W R; Langenbach, R; Maeda, N; Smithies, O

    1994-10-01

    We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

  16. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse.

    PubMed

    Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

    2016-03-01

    Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood-brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus.

  17. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse.

    PubMed

    Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

    2016-03-01

    Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood-brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

  18. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

    PubMed Central

    Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

    2015-01-01

    Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

  19. RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

    PubMed Central

    Draper, Julia E.; Sroczynska, Patrycja; Tsoulaki, Olga; Leong, Hui Sun; Fadlullah, Muhammad Z. H.; Miller, Crispin; Kouskoff, Valerie; Lacaud, Georges

    2016-01-01

    The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to

  20. PPARβ/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone

    PubMed Central

    Bernal, Carolina; Araya, Claudia; Palma, Verónica; Bronfman, Miguel

    2015-01-01

    The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARβ/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARβ/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARβ/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARβ/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARβ/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis. PMID:25852474

  1. PPARβ/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone.

    PubMed

    Bernal, Carolina; Araya, Claudia; Palma, Verónica; Bronfman, Miguel

    2015-01-01

    The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARβ/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARβ/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARβ/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARβ/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARβ/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis. PMID:25852474

  2. PPARβ/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone.

    PubMed

    Bernal, Carolina; Araya, Claudia; Palma, Verónica; Bronfman, Miguel

    2015-01-01

    The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARβ/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARβ/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARβ/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARβ/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARβ/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis.

  3. The physics of intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter; Griffiths, D. J.; Albee, A. L.

    1994-01-01

    The ability to capture projectiles intact at hypervelocities in underdense media open a new area of study in physics. Underdense material behaves markedly different than solid, liquid, or gas upon hypervelocity impact. This new phenomenon enables applications in science that would either not be possible or would be very costly by other means. This phenomenon has been fully demonstrated in the laboratory and validated in space. Even more interesting is the fact that this hypervelocity intact capture was accomplished passively. A better understanding of the physics of intact capture will lead to improvements in intact capture. A collection of physical observations of this phenomenon is presented here.

  4. MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults.

    PubMed

    Wefers, Benedikt; Hitz, Christiane; Hölter, Sabine M; Trümbach, Dietrich; Hansen, Jens; Weber, Peter; Pütz, Benno; Deussing, Jan M; de Angelis, Martin Hrabé; Roenneberg, Till; Zheng, Fang; Alzheimer, Christian; Silva, Alcino; Wurst, Wolfgang; Kühn, Ralf

    2012-01-01

    MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype.Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as a new valuable approach, allowing it to discriminate between gene function in the adult and the developing postnatal brain. PMID:22529971

  5. Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

    SciTech Connect

    Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.; Wilson, Amber; Xia, Linghui; Yu, Hong; Webster, Keith A.

    2010-06-11

    Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

  6. Deficits in Adult Neurogenesis, Contextual Fear Conditioning, and Spatial Learning in a Gfap Mutant Mouse Model of Alexander Disease

    PubMed Central

    Paylor, Richard; Messing, Albee

    2013-01-01

    Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations in GFAP lead to the fatal neurodegenerative disorder, Alexander disease (AxD), which is characterized by cytoplasmic protein aggregates known as Rosenthal fibers along with variable degrees of leukodystrophy and intellectual disability. The mechanisms by which mutant GFAP leads to these pleiotropic effects are unknown. In addition to astrocytes, GFAP is also expressed in other cell types, particularly neural stem cells that form the reservoir supporting adult neurogenesis in the hippocampal dentate gyrus and subventricular zone of the lateral ventricles. Here, we show that mouse models of AxD exhibit significant pathology in GFAP-positive radial glia-like cells in the dentate gyrus, and suffer from deficits in adult neurogenesis. In addition, they display impairments in contextual learning and spatial memory. This is the first demonstration of cognitive phenotypes in a model of primary astrocyte disease. PMID:24259590

  7. HETEROTOPICALLY TRANSPLANTED CVO NEURAL STEM CELLS GENERATE NEURONS AND MIGRATE WITH SVZ CELLS IN THE ADULT MOUSE BRAIN

    PubMed Central

    Bennett, Lori B.; Cai, Jingli; Enikolopov, Grigori; Iacovitti, Lorraine

    2010-01-01

    Production of new neurons throughout adulthood has been well characterized in two brain regions, the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ [3]. The present study demonstrates that NSCs derived from two astrogliogenic CVOs, the median eminence and organum vasculosum of the lamina terminalis of the Nestin-GFP mouse, possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs, following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice, migrate caudally to the SVZ and express early neuronal markers (TUC-4, PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU+ cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively, these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ, they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain. PMID:20298755

  8. Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart.

    PubMed

    Malliaras, Konstantinos; Zhang, Yiqiang; Seinfeld, Jeffrey; Galang, Giselle; Tseliou, Eleni; Cheng, Ke; Sun, Baiming; Aminzadeh, Mohammad; Marbán, Eduardo

    2013-02-01

    Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3-4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation.

  9. Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling.

    PubMed

    Yu, Zhi-Bin; Wei, Hongguang; Jin, J-P

    2012-07-01

    Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.

  10. Space research with intact organisms

    NASA Technical Reports Server (NTRS)

    Phillips, Robert W.; Haddy, Francis J.

    1992-01-01

    Effects of space exposure on intact organisms are briefly reviewed, and examples of future experiments that might provide new information on the role of gravity in the evolution of life are suggested. It is noted that long term experiments with intact plant and animals for studying gravitational thresholds will provide important new insights.

  11. Loss of sensory input causes rapid structural changes of inhibitory neurons in adult mouse visual cortex.

    PubMed

    Keck, Tara; Scheuss, Volker; Jacobsen, R Irene; Wierenga, Corette J; Eysel, Ulf T; Bonhoeffer, Tobias; Hübener, Mark

    2011-09-01

    A fundamental property of neuronal circuits is the ability to adapt to altered sensory inputs. It is well established that the functional synaptic changes underlying this adaptation are reflected by structural modifications in excitatory neurons. In contrast, the degree to which structural plasticity in inhibitory neurons accompanies functional changes is less clear. Here, we use two-photon imaging to monitor the fine structure of inhibitory neurons in mouse visual cortex after deprivation induced by retinal lesions. We find that a subset of inhibitory neurons carry dendritic spines, which form glutamatergic synapses. Removal of visual input correlates with a rapid and lasting reduction in the number of inhibitory cell spines. Similar to the effects seen for dendritic spines, the number of inhibitory neuron boutons dropped sharply after retinal lesions. Together, these data suggest that structural changes in inhibitory neurons may precede structural changes in excitatory circuitry, which ultimately result in functional adaptation following sensory deprivation.

  12. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  13. The Bulk of Autotaxin Activity Is Dispensable for Adult Mouse Life.

    PubMed

    Katsifa, Aggeliki; Kaffe, Eleanna; Nikolaidou-Katsaridou, Nefeli; Economides, Aris N; Newbigging, Susan; McKerlie, Colin; Aidinis, Vassilis

    2015-01-01

    Autotaxin (ATX, Enpp2) is a secreted lysophospholipase D catalysing the production of lysophosphatidic acid, a pleiotropic growth factor-like lysophospholipid. Increased ATX expression has been detected in a number of chronic inflammatory diseases and different types of cancer, while genetic interventions have proven a role for ATX in disease pathogenesis. Therefore, ATX has emerged as a potential drug target and a large number of ATX inhibitors have been developed exhibiting promising therapeutic potential. However, the embryonic lethality of ATX null mice and the ubiquitous expression of ATX and LPA receptors in adult life question the suitability of ATX as a drug target. Here we show that inducible, ubiquitous genetic deletion of ATX in adult mice, as well as long-term potent pharmacologic inhibition, are well tolerated, alleviating potential toxicity concerns of ATX therapeutic targeting. PMID:26569406

  14. The Bulk of Autotaxin Activity Is Dispensable for Adult Mouse Life

    PubMed Central

    Katsifa, Aggeliki; Kaffe, Eleanna; Nikolaidou-Katsaridou, Nefeli; Economides, Aris N.; Newbigging, Susan; McKerlie, Colin; Aidinis, Vassilis

    2015-01-01

    Autotaxin (ATX, Enpp2) is a secreted lysophospholipase D catalysing the production of lysophosphatidic acid, a pleiotropic growth factor-like lysophospholipid. Increased ATX expression has been detected in a number of chronic inflammatory diseases and different types of cancer, while genetic interventions have proven a role for ATX in disease pathogenesis. Therefore, ATX has emerged as a potential drug target and a large number of ATX inhibitors have been developed exhibiting promising therapeutic potential. However, the embryonic lethality of ATX null mice and the ubiquitous expression of ATX and LPA receptors in adult life question the suitability of ATX as a drug target. Here we show that inducible, ubiquitous genetic deletion of ATX in adult mice, as well as long-term potent pharmacologic inhibition, are well tolerated, alleviating potential toxicity concerns of ATX therapeutic targeting. PMID:26569406

  15. Neonatal tissue injury reduces the intrinsic excitability of adult mouse superficial dorsal horn neurons.

    PubMed

    Li, J; Baccei, M L

    2014-01-01

    Tissue damage during the neonatal period evokes long-lasting changes in nociceptive processing within the adult spinal cord which contribute to persistent alterations in pain sensitivity. However, it remains unclear if the observed modifications in neuronal activity within the mature superficial dorsal horn (SDH) following early injury reflect shifts in the intrinsic membrane properties of these cells. Therefore, the present study was undertaken to identify the effects of neonatal surgical injury on the intrinsic excitability of both GABAergic and presumed glutamatergic neurons within lamina II of the adult SDH using in vitro patch clamp recordings from spinal cord slices prepared from glutamic acid decarboxylase-green fluorescent protein (Gad-GFP) mice. The results demonstrate that hindpaw surgical incision at postnatal day (P) 3 altered the passive membrane properties of both Gad-GFP and adjacent, non-GFP neurons in the mature SDH, as evidenced by decreased membrane resistance and more negative resting potentials in comparison to naïve littermate controls. This was accompanied by a reduction in the prevalence of spontaneous activity within the GABAergic population. Both Gad-GFP and non-GFP neurons displayed a significant elevation in rheobase and decreased instantaneous firing frequency after incision, suggesting that early tissue damage lowers the intrinsic membrane excitability of adult SDH neurons. Isolation of inward-rectifying K(+) (K(ir)) currents revealed that neonatal incision significantly increased K(ir) conductance near physiological membrane potentials in GABAergic, but not glutamatergic, lamina II neurons. Overall, these findings suggest that neonatal tissue injury causes a long-term dampening of intrinsic firing across the general population of lamina II interneurons, but the underlying ionic mechanisms may be cell-type specific.

  16. Odour enrichment increases adult-born dopaminergic neurons in the mouse olfactory bulb.

    PubMed

    Bonzano, Sara; Bovetti, Serena; Fasolo, Aldo; Peretto, Paolo; De Marchis, Silvia

    2014-11-01

    The olfactory bulb (OB) is the first brain region involved in the processing of olfactory information. In adult mice, the OB is highly plastic, undergoing cellular/molecular dynamic changes that are modulated by sensory experience. Odour deprivation induces down-regulation of tyrosine hydroxylase (TH) expression in OB dopaminergic interneurons located in the glomerular layer (GL), resulting in decreased dopamine in the OB. Although the effect of sensory deprivation is well established, little is known about the influence of odour enrichment on dopaminergic cells. Here we report that prolonged odour enrichment on C57BL/6J strain mice selectively increases TH-immunopositive cells in the GL by nearly 20%. Following odour enrichment on TH-green fluorescent protein (GFP) transgenic mice, in which GFP identified both mature TH-positive cells and putative immature dopaminergic cells expressing TH mRNA but not TH protein, we found a similar 20% increase in GFP-expressing cells, with no changes in the ratio between TH-positive and TH-negative cells. These data suggest that enriched conditions induce an expansion in the whole dopaminergic lineage. Accordingly, by using 5-bromo-2-deoxyuridine injections to label adult-generated cells in the GL of TH-GFP mice, we found an increase in the percentage of 5-bromo-2-deoxyuridine-positive dopaminergic cells in enriched compared with control conditions, whereas no differences were found for calretinin- and calbindin-positive subtypes. Strikingly, the fraction of newborn cells among the dopaminergic population doubled in enriched conditions. On the whole, our results demonstrate that odour enrichment drives increased integration of adult-generated dopaminergic cells that could be critical to adapt the OB circuits to the environmental incoming information.

  17. Characterizing Newly Repopulated Microglia in the Adult Mouse: Impacts on Animal Behavior, Cell Morphology, and Neuroinflammation

    PubMed Central

    Elmore, Monica R. P.; Lee, Rafael J.; West, Brian L.; Green, Kim N.

    2015-01-01

    Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the

  18. Characterizing newly repopulated microglia in the adult mouse: impacts on animal behavior, cell morphology, and neuroinflammation.

    PubMed

    Elmore, Monica R P; Lee, Rafael J; West, Brian L; Green, Kim N

    2015-01-01

    Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the

  19. Expression pattern of STOP lacZ reporter gene in adult and developing mouse brain.

    PubMed

    Couégnas, Alice; Schweitzer, Annie; Andrieux, Annie; Ghandour, M Said; Boehm, Nelly

    2007-05-15

    Stable tubulin-only polypeptide (STOP) proteins are microtubule-associated proteins responsible for microtubule stabilization in neurons. STOP null mice show apparently normal cerebral anatomy but display synaptic defects associated with neuroleptic-sensitive behavioral disorders. STOP null mice have therefore been proposed as an animal model for the study of schizophrenia. In the present study, the expression pattern of STOP gene in developing and adult brain has been examined by using lacZ gene inserted in the STOP locus, as a reporter gene. beta-Galactosidase (beta-gal) immunostaining was confined to neuronal cells and projections. Strong labeling was observed in the whole olfactory system, cortical layer VII, hippocampus, hypothalamus, cerebellum, habenula, fasciculus retroflexus, and interpeduncular nucleus in adults. Additionally, ventral thalamic nucleus, clusters of positive cells in striatum, and Cajal-Retzius cells of cortical layer I were labeled in young mice. The strong expression of STOP lacZ reporter gene observed in brain is confined to areas that may be involved in the schizophrenia-related symptoms observed in STOP-deficient mice.

  20. Differential genomic imprinting regulates paracrine and autocrine roles of IGF2 in mouse adult neurogenesis

    PubMed Central

    Ferrón, S. R.; Radford, E. J.; Domingo-Muelas, A.; Kleine, I.; Ramme, A.; Gray, D.; Sandovici, I.; Constancia, M.; Ward, A.; Menheniott, T. R.; Ferguson-Smith, A. C.

    2015-01-01

    Genomic imprinting is implicated in the control of gene dosage in neurogenic niches. Here we address the importance of Igf2 imprinting for murine adult neurogenesis in the subventricular zone (SVZ) and in the subgranular zone (SGZ) of the hippocampus in vivo. In the SVZ, paracrine IGF2 is a cerebrospinal fluid and endothelial-derived neurogenic factor requiring biallelic expression, with mutants having reduced activation of the stem cell pool and impaired olfactory bulb neurogenesis. In contrast, Igf2 is imprinted in the hippocampus acting as an autocrine factor expressed in neural stem cells (NSCs) solely from the paternal allele. Conditional mutagenesis of Igf2 in blood vessels confirms that endothelial-derived IGF2 contributes to NSC maintenance in SVZ but not in the SGZ, and that this is regulated by the biallelic expression of IGF2 in the vascular compartment. Our findings indicate that a regulatory decision to imprint or not is a functionally important mechanism of transcriptional dosage control in adult neurogenesis. PMID:26369386

  1. Competition and Homeostasis of Excitatory and Inhibitory Connectivity in the Adult Mouse Visual Cortex

    PubMed Central

    Saiepour, M. Hadi; Chakravarthy, Sridhara; Min, Rogier; Levelt, Christiaan N.

    2015-01-01

    During cortical development, synaptic competition regulates the formation and adjustment of neuronal connectivity. It is unknown whether synaptic competition remains active in the adult brain and how inhibitory neurons participate in this process. Using morphological and electrophysiological measurements, we show that expressing a dominant-negative form of the TrkB receptor (TrkB.T1) in the majority of pyramidal neurons in the adult visual cortex does not affect excitatory synapse densities. This is in stark contrast to the previously reported loss of excitatory input which occurs if the exact same transgene is expressed in sparse neurons at the same age. This indicates that synaptic competition remains active in adulthood. Additionally, we show that interneurons not expressing the TrkB.T1 transgene may have a competitive advantage and obtain more excitatory synapses when most neighboring pyramidal neurons do express the transgene. Finally, we demonstrate that inhibitory synapses onto pyramidal neurons are reduced when TrkB signaling is interfered with in most pyramidal neurons but not when few pyramidal neurons have this deficit. This adjustment of inhibitory innervation is therefore not a cell-autonomous consequence of decreased TrkB signaling but more likely a homeostatic mechanism compensating for activity changes at the population level. PMID:25316336

  2. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9

    PubMed Central

    Carroll, Kelli J.; Makarewich, Catherine A.; McAnally, John; Anderson, Douglas M.; Zentilin, Lorena; Liu, Ning; Giacca, Mauro; Bassel-Duby, Rhonda; Olson, Eric N.

    2016-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart. PMID:26719419

  3. A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland.

    PubMed

    Catalán, Marcelo A; Kondo, Yusuke; Peña-Munzenmayer, Gaspar; Jaramillo, Yasna; Liu, Frances; Choi, Sooji; Crandall, Edward; Borok, Zea; Flodby, Per; Shull, Gary E; Melvin, James E

    2015-02-17

    Activation of an apical Ca(2+)-activated Cl(-) channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl(-) current and Cl(-) efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl(-) channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A(-/-)). Ca(2+)-dependent salivation was abolished in Tmem16A(-/-) mice, demonstrating that Tmem16A is obligatory for Ca(2+)-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A(-/-) mice in response to the β-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr(∆F508/∆F508)) or ClC-2 (Clcn2(-/-)) Cl(-) channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl(-) channel. Indeed, Cl(-) channel blockers abolished fluid secretion, indicating that Cl(-) channel activity is critical for IPR-stimulated secretion. These data suggest that β-adrenergic-induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A(-/-) mice identify Tmem16A as the Cl(-) channel essential for muscarinic, Ca(2+)-dependent fluid secretion in adult mouse salivary glands.

  4. Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse

    PubMed Central

    Zampieri, Alexandre; Champagne, Julien; Auzemery, Baptiste; Fuentes, Ivanna; Maurel, Benjamin; Bienvenu, Frédéric

    2015-01-01

    We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed “Tandem-HTRF”, can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material. PMID:26503526

  5. Build a better mouse: directly-observed issues in computer use for adults with SMI.

    PubMed

    Black, Anne C; Serowik, Kristin L; Schensul, Jean J; Bowen, Anne M; Rosen, Marc I

    2013-03-01

    Integrating information technology into healthcare has the potential to bring treatment to hard-to-reach people. Individuals with serious mental illness (SMI), however, may derive limited benefit from these advances in care because of lack of computer ownership and experience. To date, conclusions about the computer skills and attitudes of adults with SMI have been based primarily on self-report. In the current study, 28 psychiatric outpatients with co-occurring cocaine use were interviewed about their computer use and opinions, and 25 were then directly observed using task analysis and think aloud methods as they navigated a multi-component health informational website. Participants reported low rates of computer ownership and use, and negative attitudes towards computers. Self-reported computer skills were higher than demonstrated in the task analysis. However, some participants spontaneously expressed more positive attitudes and greater computer self-efficacy after navigating the website. Implications for increasing access to computer-based health information are discussed.

  6. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer

    PubMed Central

    Ou, Jingxing; Vijayasarathy, Camasamudram; Ziccardi, Lucia; Chen, Shan; Zeng, Yong; Marangoni, Dario; Pope, Jodie G.; Bush, Ronald A.; Wu, Zhijian; Li, Wei; Sieving, Paul A.

    2015-01-01

    Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor–depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1–signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology. PMID:26098217

  7. Gestational ketogenic diet programs brain structure and susceptibility to depression & anxiety in the adult mouse offspring

    PubMed Central

    Sussman, Dafna; Germann, Jurgen; Henkelman, Mark

    2015-01-01

    Introduction The ketogenic diet (KD) has seen an increase in popularity for clinical and non-clinical purposes, leading to rise in concern about the diet's impact on following generations. The KD is known to have a neurological effect, suggesting that exposure to it during prenatal brain development may alter neuro-anatomy. Studies have also indicated that the KD has an anti-depressant effect on the consumer. However, it is unclear whether any neuro-anatomical and/or behavioral changes would occur in the offspring and persist into adulthood. Methods To fill this knowledge gap we assessed the brain morphology and behavior of 8-week-old young-adult CD-1 mice, who were exposed to the KD in utero, and were fed only a standard-diet (SD) in postnatal life. Standardized neuro-behavior tests included the Open-Field, Forced-Swim, and Exercise Wheel tests, and were followed by post-mortem Magnetic Resonance Imaging (MRI) to assess brain anatomy. Results The adult KD offspring exhibit reduced susceptibility to anxiety and depression, and elevated physical activity level when compared with controls exposed to the SD both in utero and postnatally. Many neuro-anatomical differences exist between the KD offspring and controls, including, for example, a cerebellar volumetric enlargement by 4.8%, a hypothalamic reduction by 1.39%, and a corpus callosum reduction by 4.77%, as computed relative to total brain volume. Conclusions These results suggest that prenatal exposure to the KD programs the offspring neuro-anatomy and influences their behavior in adulthood. PMID:25642385

  8. Contribution of Bone Marrow Hematopoietic Stem Cells to Adult Mouse Inner Ear: Mesenchymal Cells and Fibrocytes

    PubMed Central

    Lang, Hainan; Ebihara, Yasuhiro; Schmiedt, Richard A.; Minamiguchi, Hitoshi; Zhou, Daohong; Smythe, Nancy; Liu, Liya; Ogawa, Makio; Schulte, Bradley A.

    2008-01-01

    Bone marrow (BM)-derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP+ BM cells also were transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP+ cells was performed 3-20 months after transplant. GFP+ cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP+ neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP+ cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP+ cells in the spiral ligament expressed immunoreactive Na, K-ATPase or the Na-K-Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP+ cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs. PMID:16538683

  9. Comparative Analysis of the Expression Profile of Wnk1 and Wnk1/Hsn2 Splice Variants in Developing and Adult Mouse Tissues

    PubMed Central

    Shekarabi, Masoud; Lafrenière, Ron G.; Gaudet, Rébecca; Laganière, Janet; Marcinkiewicz, Martin M.; Dion, Patrick A.; Rouleau, Guy A.

    2013-01-01

    The With No lysine (K) family of serine/threonine kinase (WNK) defines a small family of kinases with significant roles in ion homeostasis. WNK1 has been shown to have different isoforms due to what seems to be largely tissue specific splicing. Here, we used two distinct in situ hybridization riboprobes on developing and adult mouse tissues to make a comparative analysis of Wnk1 and its sensory associated splice isoform, Wnk1/Hsn2. The hybridization signals in developing mouse tissues, which were prepared at embryonic day e10.5 and e12.5, revealed a homogenous expression profile with both probes. At e15.5 and in the newborn mouse, the two probes revealed different expression profiles with prominent signals in nervous system tissues and also other tissues such as kidney, thymus and testis. In adult mouse tissues, the two expression profiles appeared even more restricted to the nervous tissues, kidney, thymus and testis, with no detectable signal in the other tissues. Throughout the nervous system, sensory tissues, as well as in Cornu Ammonis 1 (CA1), CA2 and CA3 areas of the hippocampus, were strongly labeled with both probes. Hybridization signals were also strongly detected in Schwann and supporting satellite cells. Our results show that the expression profiles of Wnk1 isoforms change during the development, and that the expression of the Wnk1 splice variant containing the Hsn2 exon is prominent during developing and in adult mouse tissues, suggesting its important role in the development and maintenance of the nervous system. PMID:23451271

  10. Analysis of chaperone mRNA expression in the adult mouse brain by meta analysis of the Allen Brain Atlas.

    PubMed

    Tebbenkamp, Andrew T N; Borchelt, David R

    2010-10-28

    The pathology of many neurodegenerative diseases is characterized by the accumulation of misfolded and aggregated proteins in various cell types and regional substructures throughout the central and peripheral nervous systems. The accumulation of these aggregated proteins signals dysfunction of cellular protein homeostatic mechanisms such as the ubiquitin/proteasome system, autophagy, and the chaperone network. Although there are several published studies in which transcriptional profiling has been used to examine gene expression in various tissues, including tissues of neurodegenerative disease models, there has not been a report that focuses exclusively on expression of the chaperone network. In the present study, we used the Allen Brain Atlas online database to analyze chaperone expression levels. This database utilizes a quantitative in situ hybridization approach and provides data on 270 chaperone genes within many substructures of the adult mouse brain. We determined that 256 of these chaperone genes are expressed at some level. Surprisingly, relatively few genes, only 30, showed significant variations in levels of mRNA across different substructures of the brain. The greatest degree of variability was exhibited by genes of the DnaJ co-chaperone, Tetratricopeptide repeat, and the HSPH families. Our analysis provides a valuable resource towards determining how variations in chaperone gene expression may modulate the vulnerability of specific neuronal populations of mammalian brain.

  11. Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles

    PubMed Central

    Esquerdo, Mariona; Grau-Bové, Xavier; Garanto, Alejandro; Toulis, Vasileios; Garcia-Monclús, Sílvia; Millo, Erica; López-Iniesta, Ma José; Abad-Morales, Víctor; Ruiz-Trillo, Iñaki; Marfany, Gemma

    2016-01-01

    Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders. PMID:26934049

  12. Astrocytic adaptation during cerebral angiogenesis follows the new vessel formation induced through chronic hypoxia in adult mouse cortex

    NASA Astrophysics Data System (ADS)

    Masamoto, Kazuto; Kanno, Iwao

    2014-03-01

    We examined longitudinal changes of the neuro-glia-vascular unit during cerebral angiogenesis induced through chronic hypoxia in the adult mouse cortex. Tie2-GFP mice in which the vascular endothelial cells expressed green fluorescent proteins (GFP) were exposed to chronic hypoxia, while the spatiotemporal developments of the cortical capillary sprouts and the neighboring astrocytic remodeling were characterized with repeated two-photon microscopy. The capillary sprouts appeared at early phases of the hypoxia adaptation (1-2 weeks), while the morphological changes of the astrocytic soma and processes were not detected in this phase. In the later phases of the hypoxia adaptation (> 2 weeks), the capillary sprouts created a new connection with existing capillaries, and its neighboring astrocytes extended their processes to the newly-formed vessels. The findings show that morphological adaptation of the astrocytes follow the capillary development during the hypoxia adaptation, which indicate that the newly-formed vessels provoke cellular interactions with the neighboring astrocytes to strengthen the functional blood-brain barrier.

  13. An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

    PubMed Central

    Franco, Joy A.; Kloefkorn, Heidi E.; Hochman, Shawn; Wilkinson, Katherine A.

    2014-01-01

    Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice. PMID:25285602

  14. Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

    PubMed

    Hemmer, Kathrin; Zhang, Mingyue; van Wüllen, Thea; Sakalem, Marna; Tapia, Natalia; Baumuratov, Aidos; Kaltschmidt, Christian; Kaltschmidt, Barbara; Schöler, Hans R; Zhang, Weiqi; Schwamborn, Jens C

    2014-09-01

    Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications. PMID:25241741

  15. The transformation of synaptic to system plasticity in motor output from the sacral cord of the adult mouse.

    PubMed

    Jiang, Mingchen C; Elbasiouny, Sherif M; Collins, William F; Heckman, C J

    2015-09-01

    Synaptic plasticity is fundamental in shaping the output of neural networks. The transformation of synaptic plasticity at the cellular level into plasticity at the system level involves multiple factors, including behavior of local networks of interneurons. Here we investigate the synaptic to system transformation for plasticity in motor output in an in vitro preparation of the adult mouse spinal cord. System plasticity was assessed from compound action potentials (APs) in spinal ventral roots, which were generated simultaneously by the axons of many motoneurons (MNs). Synaptic plasticity was assessed from intracellular recordings of MNs. A computer model of the MN pool was used to identify the middle steps in the transformation from synaptic to system behavior. Two input systems that converge on the same MN pool were studied: one sensory and one descending. The two synaptic input systems generated very different motor outputs, with sensory stimulation consistently evoking short-term depression (STD) whereas descending stimulation had bimodal plasticity: STD at low frequencies but short-term facilitation (STF) at high frequencies. Intracellular and pharmacological studies revealed contributions from monosynaptic excitation and stimulus time-locked inhibition but also considerable asynchronous excitation sustained from local network activity. The computer simulations showed that STD in the monosynaptic excitatory input was the primary driver of the system STD in the sensory input whereas network excitation underlies the bimodal plasticity in the descending system. These results provide insight on the roles of plasticity in the monosynaptic and polysynaptic inputs converging on the same MN pool to overall motor plasticity.

  16. Selective depression of nociceptive responses of dorsal horn neurones by SNC 80 in a perfused hindquarter preparation of adult mouse.

    PubMed

    Cao, C Q; Hong, Y G; Dray, A; Perkins, M N

    2001-01-01

    -nociceptive dorsal horn neurones were not inhibited by SNC 80 at a dose of up to 10 microM (n=5). These data demonstrate that delta-opioid receptor modulate nociceptive, but not non-nociceptive, transmission in spinal dorsal horn neurones of the adult mouse. The potentiation of neuronal activity by HS 378 may reflect an autoregulatory role of the endogenous delta-opioid in nociceptive transmission in mouse. PMID:11731107

  17. Peptidergic influences on proliferation, migration, and placement of neural progenitors in the adult mouse forebrain.

    PubMed

    Stanic, Davor; Paratcha, Gustavo; Ledda, Fernanda; Herzog, Herbert; Kopin, Alan S; Hökfelt, Tomas

    2008-03-01

    Neural progenitor proliferation, differentiation, and migration are continually ongoing processes in the subventricular zone (SVZ) and rostral migratory stream (RMS) of the adult brain. There is evidence that peptidergic systems may be involved in the molecular cascades regulating these neurogenic processes, and we examined a possible influence of neuropeptide Y (NPY) and cholecystokinin (CCK) systems in cell proliferation and neuroblast formation in the SVZ and RMS and generation of interneurons in the olfactory bulb (OB). We show that NPY and the Y1 and Y2 receptor (R) proteins are expressed in and surrounding the SVZ and RMS and that Y1R is located on neuroblasts in the anterior RMS. Mice deficient in Y1Rs or Y2Rs have fewer Ki-67-immunoreactive (ir) proliferating precursor cells and doublecortin-ir neuroblasts in the SVZ and RMS than WT mice, and less calbindin-, calretinin-, and tyrosine hydroxylase-ir interneurons in the OB. Mice lacking CCK1Rs have fewer proliferating cells and neuroblasts than normal and a shortage of interneurons in the OB. These findings suggest that both NPY and CCK through their receptors help to regulate the proliferation of precursor cells, the amount of neuroblast cells in the SVZ and RMS, and influence the differentiation of OB interneurons.

  18. Morphological analysis of activity-reduced adult-born neurons in the mouse olfactory bulb.

    PubMed

    Dahlen, Jeffrey E; Jimenez, Daniel A; Gerkin, Richard C; Urban, Nathan N

    2011-01-01

    Adult-born neurons (ABNs) are added to the olfactory bulb (OB) throughout life in rodents. While many factors have been identified as regulating the survival and integration of ABNs into existing circuitry, the understanding of how these factors affect ABN morphology and connectivity is limited. Here we compare how cell intrinsic [small interfering RNA (siRNA) knock-down of voltage gated sodium channels Na(V)1.1-1.3] and circuit level (naris occlusion) reductions in activity affect ABN morphology during integration into the OB. We found that both manipulations reduce the number of dendritic spines (and thus likely the number of reciprocal synaptic connections) formed with the surrounding circuitry and inhibited dendritic ramification of ABNs. Further, we identified regions of ABN apical dendrites where the largest and most significant decreases occur following siRNA knock-down or naris occlusion. In siRNA knock-down cells, reduction of spines is observed in proximal regions of the apical dendrite. This suggests that distal regions of the dendrite may remain active independent of Na(V)1.1-1.3 channel expression, perhaps facilitated by activation of T-type calcium channels and NMDA receptors. By contrast, circuit level reduction of activity by naris occlusion resulted in a global depression of spine number. Together, these results indicate that ABNs retain the ability to develop their typical overall morphological features regardless of experienced activity, and activity modulates the number and location of formed connections.

  19. Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis.

    PubMed

    Ostrowski, Stephen M; Wright, Margaret C; Bolock, Alexa M; Geng, Xuehui; Maricich, Stephen M

    2015-07-15

    Merkel cells are mechanosensitive skin cells whose production requires the basic helix-loop-helix transcription factor Atoh1. We induced ectopic Atoh1 expression in the skin of transgenic mice to determine whether Atoh1 was sufficient to create additional Merkel cells. In embryos, ectopic Atoh1 expression drove ectopic expression of the Merkel cell marker keratin 8 (K8) throughout the epidermis. Epidermal Atoh1 induction in adolescent mice similarly drove widespread K8 expression in glabrous skin of the paws, but in the whisker pads and body skin ectopic K8+ cells were confined to hair follicles and absent from interfollicular regions. Ectopic K8+ cells acquired several characteristics of mature Merkel cells in a time frame similar to that seen during postnatal development of normal Merkel cells. Although ectopic K8+ cell numbers decreased over time, small numbers of these cells remained in deep regions of body skin hair follicles at 3 months post-induction. In adult mice, greater numbers of ectopic K8+ cells were created by Atoh1 induction during anagen versus telogen and following disruption of Notch signaling by conditional deletion of Rbpj in the epidermis. Our data demonstrate that Atoh1 expression is sufficient to produce new Merkel cells in the epidermis, that epidermal cell competency to respond to Atoh1 varies by skin location, developmental age and hair cycle stage, and that the Notch pathway plays a key role in limiting epidermal cell competency to respond to Atoh1 expression.

  20. Running increases neurogenesis without retinoic acid receptor activation in the adult mouse dentate gyrus.

    PubMed

    Aberg, Elin; Perlmann, Thomas; Olson, Lars; Brené, Stefan

    2008-01-01

    Both vitamin A deficiency and high doses of retinoids can result in learning and memory impairments, depression as well as decreases in cell proliferation, neurogenesis and cell survival. Physical activity enhances hippocampal neurogenesis and can also exert an antidepressant effect. Here we elucidate a putative link between running, retinoid signaling, and neurogenesis in hippocampus. Adult transgenic reporter mice designed to detect ligand-activated retinoic acid receptors (RAR) or retinoid X receptors (RXR) were used to localize the distribution of activated RAR or RXR at the single-cell level in the brain. Two months of voluntary wheel-running induced an increase in hippocampal neurogenesis as indicated by an almost two-fold increase in doublecortin-immunoreactive cells. Running activity was correlated with neurogenesis. Under basal conditions a distinct pattern of RAR-activated cells was detected in the granule cell layer of the dentate gyrus (DG), thalamus, and cerebral cortex layers 3-4 and to a lesser extent in hippocampal pyramidal cell layers CA1-CA3. Running did not change the number of RAR-activated cells in the DG. There was no correlation between running and RAR activation or between RAR activation and neurogenesis in the DG of hippocampus. Only a few scattered activated retinoid X receptors were found in the DG under basal conditions and after wheel-running, but RXR was detected in other areas such as in the hilus region of hippocampus and in layer VI of cortex cerebri. RAR agonists affect mood in humans and reduce neurogenesis, learning and memory in animal models. In our study, long-term running increased neurogenesis but did not alter RAR ligand activation in the DG in individually housed mice. Thus, our data suggest that the effects of exercise on neurogenesis and other plasticity changes in the hippocampal formation are mediated by mechanisms that do not involve retinoid receptor activation.

  1. Intact capture of hypervelocity particles

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    1986-01-01

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  2. Intact capture of hypervelocity particles

    NASA Astrophysics Data System (ADS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  3. Deep-brain magnetic stimulation promotes adult hippocampal neurogenesis and alleviates stress-related behaviors in mouse models for neuropsychiatric disorders

    PubMed Central

    2014-01-01

    Background Repetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation (DMS) is an effective therapy for various neuropsychiatric disorders including major depression disorder. The molecular and cellular mechanisms underlying the impacts of rTMS/DMS on the brain are not yet fully understood. Results Here we studied the effects of deep-brain magnetic stimulation to brain on the molecular and cellular level. We examined the adult hippocampal neurogenesis and hippocampal synaptic plasticity of rodent under stress conditions with deep-brain magnetic stimulation treatment. We found that DMS promotes adult hippocampal neurogenesis significantly and facilitates the development of adult new-born neurons. Remarkably, DMS exerts anti-depression effects in the learned helplessness mouse model and rescues hippocampal long-term plasticity impaired by restraint stress in rats. Moreover, DMS alleviates the stress response in a mouse model for Rett syndrome and prolongs the life span of these animals dramatically. Conclusions Deep-brain magnetic stimulation greatly facilitates adult hippocampal neurogenesis and maturation, also alleviates depression and stress-related responses in animal models. PMID:24512669

  4. Intact capture of cosmic dust

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1991-01-01

    The focus of this development effort is to capture dust particles at hypervelocities intact and unmelted in order to preserve volatile organics. At the same time, the capture process must minimize any organic elemental or compound contamination to prevent any compromise of exobiological analyses. Inorganic silicate aerogel has been developed as a successful capture medium to satisfy both requirements of intact capture and minimal organic contamination. Up to 6 km/s, silicate projectiles from a few microns up to 100 microns have been captured intact without any melting and with minimal loss of mass. Carbon in silicate aerogel can be reduced to less than 1 part in 1000 and hydrogen 3 parts in 1000 when baked in air. Under controlled inert gas environments, additional hydrocarbon reduction can be achieved.

  5. The transformation of synaptic to system plasticity in motor output from the sacral cord of the adult mouse.

    PubMed

    Jiang, Mingchen C; Elbasiouny, Sherif M; Collins, William F; Heckman, C J

    2015-09-01

    Synaptic plasticity is fundamental in shaping the output of neural networks. The transformation of synaptic plasticity at the cellular level into plasticity at the system level involves multiple factors, including behavior of local networks of interneurons. Here we investigate the synaptic to system transformation for plasticity in motor output in an in vitro preparation of the adult mouse spinal cord. System plasticity was assessed from compound action potentials (APs) in spinal ventral roots, which were generated simultaneously by the axons of many motoneurons (MNs). Synaptic plasticity was assessed from intracellular recordings of MNs. A computer model of the MN pool was used to identify the middle steps in the transformation from synaptic to system behavior. Two input systems that converge on the same MN pool were studied: one sensory and one descending. The two synaptic input systems generated very different motor outputs, with sensory stimulation consistently evoking short-term depression (STD) whereas descending stimulation had bimodal plasticity: STD at low frequencies but short-term facilitation (STF) at high frequencies. Intracellular and pharmacological studies revealed contributions from monosynaptic excitation and stimulus time-locked inhibition but also considerable asynchronous excitation sustained from local network activity. The computer simulations showed that STD in the monosynaptic excitatory input was the primary driver of the system STD in the sensory input whereas network excitation underlies the bimodal plasticity in the descending system. These results provide insight on the roles of plasticity in the monosynaptic and polysynaptic inputs converging on the same MN pool to overall motor plasticity. PMID:26203107

  6. Early social enrichment rescues adult behavioral and brain abnormalities in a mouse model of fragile X syndrome.

    PubMed

    Oddi, Diego; Subashi, Enejda; Middei, Silvia; Bellocchio, Luigi; Lemaire-Mayo, Valerie; Guzmán, Manuel; Crusio, Wim E; D'Amato, Francesca R; Pietropaolo, Susanna

    2015-03-13

    Converging lines of evidence support the use of environmental stimulation to ameliorate the symptoms of a variety of neurodevelopmental disorders. Applying these interventions at very early ages is critical to achieve a marked reduction of the pathological phenotypes. Here we evaluated the impact of early social enrichment in Fmr1-KO mice, a genetic mouse model of fragile X syndrome (FXS), a major developmental disorder and the most frequent monogenic cause of autism. Enrichment was achieved by providing male KO pups and their WT littermates with enhanced social stimulation, housing them from birth until weaning with the mother and an additional nonlactating female. At adulthood they were tested for locomotor, social, and cognitive abilities; furthermore, dendritic alterations were assessed in the hippocampus and amygdala, two brain regions known to be involved in the control of the examined behaviors and affected by spine pathology in Fmr1-KOs. Enrichment rescued the behavioral FXS-like deficits displayed in adulthood by Fmr1-KO mice, that is, hyperactivity, reduced social interactions, and cognitive deficits. Early social enrichment also eliminated the abnormalities shown by adult KO mice in the morphology of hippocampal and amygdala dendritic spines, namely an enhanced density of immature vs mature types. Importantly, enrichment did not induce neurobehavioral changes in WT mice, thus supporting specific effects on FXS-like pathology. These findings show that early environmental stimulation has profound and long-term beneficial effects on the pathological FXS phenotype, thereby encouraging the use of nonpharmacological interventions for the treatment of this and perhaps other neurodevelopmental diseases.

  7. Early Social Enrichment Rescues Adult Behavioral and Brain Abnormalities in a Mouse Model of Fragile X Syndrome

    PubMed Central

    Oddi, Diego; Subashi, Enejda; Middei, Silvia; Bellocchio, Luigi; Lemaire-Mayo, Valerie; Guzmán, Manuel; Crusio, Wim E; D'Amato, Francesca R; Pietropaolo, Susanna

    2015-01-01

    Converging lines of evidence support the use of environmental stimulation to ameliorate the symptoms of a variety of neurodevelopmental disorders. Applying these interventions at very early ages is critical to achieve a marked reduction of the pathological phenotypes. Here we evaluated the impact of early social enrichment in Fmr1-KO mice, a genetic mouse model of fragile X syndrome (FXS), a major developmental disorder and the most frequent monogenic cause of autism. Enrichment was achieved by providing male KO pups and their WT littermates with enhanced social stimulation, housing them from birth until weaning with the mother and an additional nonlactating female. At adulthood they were tested for locomotor, social, and cognitive abilities; furthermore, dendritic alterations were assessed in the hippocampus and amygdala, two brain regions known to be involved in the control of the examined behaviors and affected by spine pathology in Fmr1-KOs. Enrichment rescued the behavioral FXS-like deficits displayed in adulthood by Fmr1-KO mice, that is, hyperactivity, reduced social interactions, and cognitive deficits. Early social enrichment also eliminated the abnormalities shown by adult KO mice in the morphology of hippocampal and amygdala dendritic spines, namely an enhanced density of immature vs mature types. Importantly, enrichment did not induce neurobehavioral changes in WT mice, thus supporting specific effects on FXS-like pathology. These findings show that early environmental stimulation has profound and long-term beneficial effects on the pathological FXS phenotype, thereby encouraging the use of nonpharmacological interventions for the treatment of this and perhaps other neurodevelopmental diseases. PMID:25348604

  8. Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis

    PubMed Central

    Delp, Emili E.; Swamynathan, Sudha; Kao, Winston W.; Swamynathan, Shivalingappa K.

    2015-01-01

    Purpose. In previous studies, conditional disruption of Klf4 in the developing mouse ocular surface from embryonic day 10 resulted in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells, revealing the importance of Klf4 in ocular surface maturation. Here, we use spatiotemporally regulated ablation of Klf4 to investigate its functions in maintenance of adult corneal epithelial homeostasis. Methods. Expression of Cre was induced in ternary transgenic (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse corneal epithelium by doxycycline administered through intraperitoneal injections and drinking water, to generate corneal epithelium–specific deletion of Klf4 (Klf4Δ/ΔCE). Corneal epithelial barrier function was tested by fluorescein staining. Expression of selected Klf4-target genes was determined by quantitative PCR (QPCR), immunoblotting, and immunofluorescent staining. Results. Klf4 was efficiently ablated within 5 days of doxycycline administration in adult Klf4Δ/ΔCE corneal epithelium. The Klf4Δ/ΔCE corneal epithelial barrier function was disrupted, and the basal cells were swollen and rounded after 15 days of doxycycline treatment. Increased numbers of cell layers and Ki67-positive proliferating cells suggested deregulated Klf4Δ/ΔCE corneal epithelial homeostasis. Expression of tight junction proteins ZO-1 and occludin, desmosomal Dsg and Dsp, basement membrane laminin-332, and corneal epithelial–specific keratin-12 was decreased, while that of matrix metalloproteinase Mmp9 and noncorneal keratin-17 increased, suggesting altered Klf4Δ/ΔCE corneal epithelial cell identity. Conclusions. Ablation of Klf4 in the adult mouse corneas resulted in the absence of characteristic corneal epithelial cell differentiation, disrupted barrier function, and squamous metaplasia, revealing that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis. PMID:26047041

  9. A new genus and species of demodecid mites from the tongue of a house mouse Mus musculus: description of adult and immature stages with data on parasitism.

    PubMed

    Izdebska, J N; Rolbiecki, L

    2016-06-01

    The study of the parasitofauna of the house mouse Mus musculus (Rodentia: Muridae) Linnaeus is particularly important owing to its multiple relationships with humans - as a cosmopolitan, synanthropic rodent, bred for pets, food for other animals or laboratory animal. This article proposes and describes a new genus and species of the parasitic mite based on adult and immature stages from the house mouse. Glossicodex musculi gen. n., sp. n. is a medium-sized demodecid mite (adult stages on average 199 µm in length) found in mouse tissue of the tongue. It is characterized by two large, hooked claws on each tarsus of the legs; the legs are relatively massive, consisting of large, non-overlapping segments. The palps consist of three slender, clearly separated, relatively narrow segments, wherein their coxal segments are also quite narrow and spaced. Also, segments of the palps of larva and nymphs are clearly isolated, and on the terminal segment, trident claws that resemble legs' claws can be found. On the ventral side, in immature stages, triangular scuta, topped with sclerotized spur, can be also observed. Glossicodex musculi was noted in 10.8% of mice with a mean infection intensity of 2.2 parasites per host.

  10. Purification of oogonial stem cells from adult mouse and human ovaries: an assessment of the literature and a view toward the future.

    PubMed

    Woods, Dori C; White, Yvonne A R; Tilly, Jonathan L

    2013-01-01

    Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonatal and adult mouse ovaries by 4 independent groups using multiple strategies. Two of these groups also reported that isolated mouse OSCs, once transplanted back into ovaries of adult female mice, differentiate into fully functional eggs that ovulate, fertilize, and produce healthy embryos and offspring. Arguably, one of the most significant advances in this emerging field was provided by a new research study published this year, which reported the successful isolation and functional characterization of OSCs from ovaries of reproductive age women. Two commentaries on this latest work, one cautiously supportive and one highly skeptical, were published soon afterward. This article evaluates the current literature regarding postnatal oogenesis in mammals and discusses important next steps for future work on OSC biology and function.

  11. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

    PubMed

    Broutier, Laura; Andersson-Rolf, Amanda; Hindley, Christopher J; Boj, Sylvia F; Clevers, Hans; Koo, Bon-Kyoung; Huch, Meritxell

    2016-09-01

    Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol. PMID:27560176

  12. No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle.

    PubMed

    Blaauw, Bert; Del Piccolo, Paola; Rodriguez, Laura; Hernandez Gonzalez, Victor-Hugo; Agatea, Lisa; Solagna, Francesca; Mammano, Fabio; Pozzan, Tullio; Schiaffino, Stefano

    2012-08-01

    The presence and role of functional inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP(3)Rs is too low and the kinetics of Ca(2+) release from IP(3)R is too slow compared with ryanodine receptors to contribute to the Ca(2+) transient during excitation-contraction coupling. However, it has been suggested that IP(3)-dependent Ca(2+) release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP(3)-dependent Ca(2+) release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca(2+) transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP(3), or (c) photolysis of membrane-permeant caged IP(3), no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP(3)-IP(3)R system does not appear to affect global calcium levels in adult mouse skeletal muscle.

  13. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

    PubMed

    Broutier, Laura; Andersson-Rolf, Amanda; Hindley, Christopher J; Boj, Sylvia F; Clevers, Hans; Koo, Bon-Kyoung; Huch, Meritxell

    2016-09-01

    Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

  14. Monitoring Intact Viruses Using Aptamers

    PubMed Central

    Kumar, Penmetcha K. R.

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  15. Monitoring Intact Viruses Using Aptamers.

    PubMed

    Kumar, Penmetcha K R

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  16. Language and Williams syndrome: how intact is "intact"?

    PubMed

    Karmiloff-Smith, A; Grant, J; Berthoud, I; Davies, M; Howlin, P; Udwin, O

    1997-04-01

    It has been claimed that Williams syndrome (WS), a rare neurodevelopmental disorder, is characterized by serious cognitive deficits alongside intact language. The syndrome is often used as a prime example of the modularity of an innate faculty for morphosyntactic rules. We challenge this claim and hypothesize that morphosyntax, although surprisingly good given WS level of mental retardation, is by no means intact. We make an initial test of this hypothesis through an analysis of the receptive language of a group of English-speaking WS individuals on a standardized morphosyntactic test. We then present an experimental study of expressive language that examines grammatical gender assignment in French-speaking WS patients. Despite a Verbal Mental Age selected to be higher than the chronological age of the young control group, these people with WS continue even in adulthood to show clear-cut deficits in their production of an aspect of morphosyntax that normal children acquire effortlessly very early. The results of the 2 studies, one focusing on receptive language and the other on expressive language, challenge the notion that comprehension and use of morphosyntactic rules in WS individuals are intact. The Within-domain dissociations regarding the use of grammatical gender assignment across several sentence clements and their difficulties in understanding embedded sentences-two quintessentially linguistic skills-suggest that we must rethink the notion of spared, modular, language capacities in Williams syndrome. We conclude that WS language follows a different path to normal acquisition and may turn out to be more like second language learning. PMID:9180000

  17. Glycosphingolipid patterns in primary mouse kidney cultures

    SciTech Connect

    Lyerla, T.A.; Gross, S.K.; McCluer, R.H.

    1986-12-01

    Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D-valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture. The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl- and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male-specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells. Neutral glycosphingolipids were labeled in 2-week-old cultures using (/sup 3/H)palmitate. The (/sup 3/H)palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D-valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.

  18. Mouse genetic differences in voluntary wheel running, adult hippocampal neurogenesis and learning on the multi-strain-adapted plus water maze.

    PubMed

    Merritt, Jennifer R; Rhodes, Justin S

    2015-03-01

    Moderate levels of aerobic exercise broadly enhance cognition throughout the lifespan. One hypothesized contributing mechanism is increased adult hippocampal neurogenesis. Recently, we measured the effects of voluntary wheel running on adult hippocampal neurogenesis in 12 different mouse strains, and found increased neurogenesis in all strains, ranging from 2- to 5-fold depending on the strain. The purpose of this study was to determine the extent to which increased neurogenesis from wheel running is associated with enhanced performance on the water maze for 5 of the 12 strains, chosen based on their levels of neurogenesis observed in the previous study (C57BL/6 J, 129S1/SvImJ, B6129SF1/J, DBA/2 J, and B6D2F1/J). Mice were housed with or without a running wheels for 30 days then tested for learning and memory on the plus water maze, adapted for multiple strains, and rotarod test of motor performance. The first 10 days, animals were injected with BrdU to label dividing cells. After behavioral testing animals were euthanized to measure adult hippocampal neurogenesis using standard methods. Levels of neurogenesis depended on strain but all mice had a similar increase in neurogenesis in response to exercise. All mice acquired the water maze but performance depended on strain. Exercise improved water maze performance in all strains to a similar degree. Rotarod performance depended on strain. Exercise improved rotarod performance only in DBA/2 J and B6D2F1/J mice. Taken together, results demonstrate that despite different levels of neurogenesis, memory performance and motor coordination in these mouse strains, all strains have the capacity to increase neurogenesis and improve learning on the water maze through voluntary wheel running.

  19. Mouse genetic differences in voluntary wheel running, adult hippocampal neurogenesis and learning on the multi-strain-adapted plus water maze

    PubMed Central

    Merritt, Jennifer; Rhodes, Justin S.

    2014-01-01

    Moderate levels of aerobic exercise broadly enhance cognition throughout the lifespan. One hypothesized contributing mechanism is increased adult hippocampal neurogenesis. Recently, we measured the effects of voluntary wheel running on adult hippocampal neurogenesis in 12 different mouse strains, and found increased neurogenesis in all strains, ranging from 2 to 5 fold depending on the strain. The purpose of this study was to determine the extent to which increased neurogenesis from wheel running is associated with enhanced performance on the water maze for 5 of the 12 strains, chosen based on their levels of neurogenesis observed in the previous study (C57BL/6J, 129S1/SvImJ, B6129SF1/J, DBA/2J, and B6D2F1/J). Mice were housed with or without a running wheels for 30 days then tested for learning and memory on the plus water maze, adapted for multiple strains, and rotarod test of motor performance. The first 10 days, animals were injected with BrdU to label dividing cells. After behavioral testing animals were euthanized to measure adult hippocampal neurogenesis using standard methods. Levels of neurogenesis depended on strain but all mice had a similar increase in neurogenesis in response to exercise. All mice acquired the water maze but performance depended on strain. Exercise improved water maze performance in all strains to a similar degree. Rotarod performance depended on strain. Exercise improved rotarod performance only in DBA/2J and B6D2F1/J mice. Taken together, results demonstrate that despite different levels of neurogenesis, memory performance and motor coordination in these mouse strains, all strains have the capacity to increase neurogenesis and improve learning on the water maze through voluntary wheel running. PMID:25435316

  20. Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain.

    PubMed

    Kosi, Nina; Alić, Ivan; Kolačević, Matea; Vrsaljko, Nina; Jovanov Milošević, Nataša; Sobol, Margarita; Philimonenko, Anatoly; Hozák, Pavel; Gajović, Srećko; Pochet, Roland; Mitrečić, Dinko

    2015-02-01

    The nucleolar protein 2 gene encodes a protein specific for the nucleolus. It is assumed that it plays a role in the synthesis of ribosomes and regulation of the cell cycle. Due to its link to cell proliferation, higher expression of Nop2 indicates a worse tumor prognosis. In this work we used Nop2(gt1gaj) gene trap mouse strain. While lethality of homozygous animals suggested a vital role of this gene, heterozygous animals allowed the detection of expression of Nop2 in various tissues, including mouse brain. Histochemistry, immunohistochemistry and immunoelectron microscopy techniques, applied to a mature mouse brain, human brain and on mouse neural stem cells revealed expression of Nop2 in differentiating cells, including astrocytes, as well as in mature neurons. Nop2 was detected in various regions of mouse and human brain, mostly in large pyramidal neurons. In the human, Nop2 was strongly expressed in supragranular and infragranular layers of the somatosensory cortex and in layer III of the cingulate cortex. Also, Nop2 was detected in CA1 and the subiculum of the hippocampus. Subcellular analyses revealed predominant location of Nop2 within the dense fibrillar component of the nucleolus. To test if Nop2 expression correlates to cell proliferation occurring during tissue regeneration, we induced strokes in mice by middle cerebral artery occlusion. Two weeks after stroke, the number of Nop2/nestin double positive cells in the region affected by ischemia and the periventricular zone substantially increased. Our findings suggest a newly discovered role of Nop2 in both mature neurons and in cells possibly involved in the regeneration of nervous tissue.

  1. Adult Neurogenesis in the Female Mouse Hypothalamus: Estradiol and High-Fat Diet Alter the Generation of Newborn Neurons Expressing Estrogen Receptor α

    PubMed Central

    Yang, Jane; Nettles, Sabin A.; Byrnes, Elizabeth M.

    2016-01-01

    Estrogens and leptins act in the hypothalamus to maintain reproduction and energy homeostasis. Neurogenesis in the adult mammalian hypothalamus has been implicated in the regulation of energy homeostasis. Recently, high-fat diet (HFD) and estradiol (E2) have been shown to alter cell proliferation and the number of newborn leptin-responsive neurons in the hypothalamus of adult female mice. The current study tested the hypothesis that new cells expressing estrogen receptor α (ERα) are generated in the arcuate nucleus (ARC) and the ventromedial nucleus of the hypothalamus (VMH) of the adult female mouse, hypothalamic regions that are critical in energy homeostasis. Adult mice were ovariectomized and implanted with capsules containing E2 or oil. Within each hormone group, mice were fed an HFD or standard chow for 6 weeks and treated with BrdU to label new cells. Newborn cells that respond to estrogens were identified in the ARC and VMH, of which a subpopulation was leptin sensitive, indicating that the subpopulation consists of neurons. Moreover, there was an interaction between diet and hormone with an effect on the number of these newborn ERα-expressing neurons that respond to leptin. Regardless of hormone treatment, HFD increased the number of ERα-expressing cells in the ARC and VMH. E2 decreased hypothalamic fibroblast growth factor 10 (Fgf10) gene expression in HFD mice, suggesting a role for Fgf10 in E2 effects on neurogenesis. These findings of newly created estrogen-responsive neurons in the adult brain provide a novel mechanism by which estrogens can act in the hypothalamus to regulate energy homeostasis in females. PMID:27679811

  2. Adult Neurogenesis in the Female Mouse Hypothalamus: Estradiol and High-Fat Diet Alter the Generation of Newborn Neurons Expressing Estrogen Receptor α

    PubMed Central

    Yang, Jane; Nettles, Sabin A.; Byrnes, Elizabeth M.

    2016-01-01

    Estrogens and leptins act in the hypothalamus to maintain reproduction and energy homeostasis. Neurogenesis in the adult mammalian hypothalamus has been implicated in the regulation of energy homeostasis. Recently, high-fat diet (HFD) and estradiol (E2) have been shown to alter cell proliferation and the number of newborn leptin-responsive neurons in the hypothalamus of adult female mice. The current study tested the hypothesis that new cells expressing estrogen receptor α (ERα) are generated in the arcuate nucleus (ARC) and the ventromedial nucleus of the hypothalamus (VMH) of the adult female mouse, hypothalamic regions that are critical in energy homeostasis. Adult mice were ovariectomized and implanted with capsules containing E2 or oil. Within each hormone group, mice were fed an HFD or standard chow for 6 weeks and treated with BrdU to label new cells. Newborn cells that respond to estrogens were identified in the ARC and VMH, of which a subpopulation was leptin sensitive, indicating that the subpopulation consists of neurons. Moreover, there was an interaction between diet and hormone with an effect on the number of these newborn ERα-expressing neurons that respond to leptin. Regardless of hormone treatment, HFD increased the number of ERα-expressing cells in the ARC and VMH. E2 decreased hypothalamic fibroblast growth factor 10 (Fgf10) gene expression in HFD mice, suggesting a role for Fgf10 in E2 effects on neurogenesis. These findings of newly created estrogen-responsive neurons in the adult brain provide a novel mechanism by which estrogens can act in the hypothalamus to regulate energy homeostasis in females.

  3. Long-term treatment with L-DOPA or pramipexole affects adult neurogenesis and corresponding non-motor behavior in a mouse model of Parkinson's disease.

    PubMed

    Chiu, W-H; Depboylu, C; Hermanns, G; Maurer, L; Windolph, A; Oertel, W H; Ries, V; Höglinger, G U

    2015-08-01

    Non-motor symptoms such as hyposmia and depression are often observed in Parkinson's disease (PD) and can precede the onset of motor symptoms for years. The underlying pathological alterations in the brain are not fully understood so far. Dysregulation of adult neurogenesis in the dentate gyrus of the hippocampus and the olfactory bulb has been recently suggested to be implicated in non-motor symptoms of PD. However, there is so far no direct evidence to support the relationship of non-motor symptoms and the modulation of adult neurogenesis following dopamine depletion and/or dopamine replacement. In this study, we investigated the long-term effects of l-DOPA and pramipexole, a dopamine agonist, in a mouse model of bilateral intranigral 6-OHDA lesion, in order to assess the impact of adult neurogenesis on non-motor behavior. We found that l-DOPA and pramipexole can normalize decreased neurogenesis in the hippocampal dentate gyrus and the periglomerular layer of the olfactory bulb caused by a 6-OHDA lesion. Interestingly, pramipexole showed an antidepressant and anxiolytic effect in the forced swim test and social interaction test. However, there was no significant change in learning and memory function after dopamine depletion and dopamine replacement, respectively.

  4. Long-term treatment with L-DOPA or pramipexole affects adult neurogenesis and corresponding non-motor behavior in a mouse model of Parkinson's disease.

    PubMed

    Chiu, W-H; Depboylu, C; Hermanns, G; Maurer, L; Windolph, A; Oertel, W H; Ries, V; Höglinger, G U

    2015-08-01

    Non-motor symptoms such as hyposmia and depression are often observed in Parkinson's disease (PD) and can precede the onset of motor symptoms for years. The underlying pathological alterations in the brain are not fully understood so far. Dysregulation of adult neurogenesis in the dentate gyrus of the hippocampus and the olfactory bulb has been recently suggested to be implicated in non-motor symptoms of PD. However, there is so far no direct evidence to support the relationship of non-motor symptoms and the modulation of adult neurogenesis following dopamine depletion and/or dopamine replacement. In this study, we investigated the long-term effects of l-DOPA and pramipexole, a dopamine agonist, in a mouse model of bilateral intranigral 6-OHDA lesion, in order to assess the impact of adult neurogenesis on non-motor behavior. We found that l-DOPA and pramipexole can normalize decreased neurogenesis in the hippocampal dentate gyrus and the periglomerular layer of the olfactory bulb caused by a 6-OHDA lesion. Interestingly, pramipexole showed an antidepressant and anxiolytic effect in the forced swim test and social interaction test. However, there was no significant change in learning and memory function after dopamine depletion and dopamine replacement, respectively. PMID:25839898

  5. HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse

    PubMed Central

    Lim, Shu Ly; Geoghegan, Joel; Hempfling, Anna-Lena; Bergmann, Martin; Goodnow, Christopher C.; Ormandy, Christopher J.; Wong, Lee; Mann, Jeff; Scott, Hamish S.; Jamsai, Duangporn; Adelson, David L.

    2015-01-01

    piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. PMID:26496356

  6. Purification of neural precursor cells reveals the presence of distinct, stimulus-specific subpopulations of quiescent precursors in the adult mouse hippocampus.

    PubMed

    Jhaveri, Dhanisha J; O'Keeffe, Imogen; Robinson, Gregory J; Zhao, Qiong-Yi; Zhang, Zong Hong; Nink, Virginia; Narayanan, Ramesh K; Osborne, Geoffrey W; Wray, Naomi R; Bartlett, Perry F

    2015-05-27

    The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however, whether these stimuli regulate the same or different precursor populations remains unknown. Here, we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population, which comprises the majority of neurosphere-forming precursors, there are two distinct subpopulations of quiescent precursor cells, one directly activated by high-KCl depolarization, and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus, and show that the NE-responsive precursors are selectively regulated by GABA, whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally, based on RNAseq analysis by deep sequencing, we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors, which may give rise to neural progeny with different functional capacity.

  7. HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.

    PubMed

    Lim, Shu Ly; Qu, Zhi Peng; Kortschak, R Daniel; Lawrence, David M; Geoghegan, Joel; Hempfling, Anna-Lena; Bergmann, Martin; Goodnow, Christopher C; Ormandy, Christopher J; Wong, Lee; Mann, Jeff; Scott, Hamish S; Jamsai, Duangporn; Adelson, David L; O'Bryan, Moira K

    2015-10-01

    piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. PMID:26496356

  8. On-Going Frontal Alpha Rhythms Are Dominant in Passive State and Desynchronize in Active State in Adult Gray Mouse Lemurs

    PubMed Central

    Rahman, Anisur; Lamberty, Yves; Bordet, Regis; Richardson, Jill C.; Forloni, Gianluigi; Drinkenburg, Wilhelmus; Lopez, Susanna; Aujard, Fabienne; Babiloni, Claudio; Pifferi, Fabien

    2015-01-01

    The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8–12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7–9 Hz) during passive state. During active state, there was a reduction in alpha power density (8–12 Hz) and an increase of power density at slow frequencies (1–4 Hz). Relative EMG activity was related to EEG power density at 2–4 Hz (positive correlation) and at 8–12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology. PMID:26618512

  9. On-Going Frontal Alpha Rhythms Are Dominant in Passive State and Desynchronize in Active State in Adult Gray Mouse Lemurs.

    PubMed

    Infarinato, Francesco; Rahman, Anisur; Del Percio, Claudio; Lamberty, Yves; Bordet, Regis; Richardson, Jill C; Forloni, Gianluigi; Drinkenburg, Wilhelmus; Lopez, Susanna; Aujard, Fabienne; Babiloni, Claudio; Pifferi, Fabien

    2015-01-01

    The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8-12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7-9 Hz) during passive state. During active state, there was a reduction in alpha power density (8-12 Hz) and an increase of power density at slow frequencies (1-4 Hz). Relative EMG activity was related to EEG power density at 2-4 Hz (positive correlation) and at 8-12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology. PMID:26618512

  10. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

    PubMed

    Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD. PMID:27136348

  11. Adult mouse motor units develop almost all of their force in the subprimary range: a new all-or-none strategy for force recruitment?

    PubMed

    Manuel, Marin; Heckman, C J

    2011-10-19

    Classical studies of the mammalian neuromuscular system have shown an impressive adaptation match between the intrinsic properties of motoneurons and the contractile properties of their motor units. In these studies, the rate at which motoneurons start to fire repetitively corresponds to the rate at which individual twitches start to sum, and the firing rate increases linearly with the amount of excitation ("primary range") up to the point where the motor unit develops its maximal force. This allows for the gradation of the force produced by a motor unit by rate modulation. In adult mouse motoneurons, however, we recently described a regime of firing ("subprimary range") that appears at lower excitation than what is required for the primary range, a finding that might challenge the classical conception. To investigate the force production of mouse motor units, we simultaneously recorded, for the first time, the motoneuron discharge elicited by intracellular ramps of current and the force developed by its motor unit. We showed that the motor unit developed nearly its maximal force during the subprimary range. This was found to be the case regardless of the input resistance of the motoneuron, the contraction speed, or the tetanic force of the motor unit. Our work suggests that force modulation in small mammals mainly relies on the number of motor units that are recruited rather than on rate modulation of individual motor units.

  12. Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

    SciTech Connect

    Jung, Yoon Hee

    2010-03-10

    This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W{sup v} mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

  13. Adult mouse motor units develop almost all of their force in the subprimary range: a new all-or-none strategy for force recruitment?

    PubMed

    Manuel, Marin; Heckman, C J

    2011-10-19

    Classical studies of the mammalian neuromuscular system have shown an impressive adaptation match between the intrinsic properties of motoneurons and the contractile properties of their motor units. In these studies, the rate at which motoneurons start to fire repetitively corresponds to the rate at which individual twitches start to sum, and the firing rate increases linearly with the amount of excitation ("primary range") up to the point where the motor unit develops its maximal force. This allows for the gradation of the force produced by a motor unit by rate modulation. In adult mouse motoneurons, however, we recently described a regime of firing ("subprimary range") that appears at lower excitation than what is required for the primary range, a finding that might challenge the classical conception. To investigate the force production of mouse motor units, we simultaneously recorded, for the first time, the motoneuron discharge elicited by intracellular ramps of current and the force developed by its motor unit. We showed that the motor unit developed nearly its maximal force during the subprimary range. This was found to be the case regardless of the input resistance of the motoneuron, the contraction speed, or the tetanic force of the motor unit. Our work suggests that force modulation in small mammals mainly relies on the number of motor units that are recruited rather than on rate modulation of individual motor units. PMID:22016552

  14. S100A6 (calcyclin) is a novel marker of neural stem cells and astrocyte precursors in the subgranular zone of the adult mouse hippocampus.

    PubMed

    Yamada, Jun; Jinno, Shozo

    2014-01-01

    S100A6 (calcyclin), an EF-hand calcium binding protein, is considered to play various roles in the brain, for example, cell proliferation and differentiation, calcium homeostasis, and neuronal degeneration. In addition to some limbic nuclei, S100A6 is distributed in the rostral migratory stream, one of the major neurogenic niches of the adult brain. However, the potential involvement of S100A6 in adult neurogenesis remains unclear. In this study, we aimed to elucidate the role of S100A6 in the other major neurogenic niche, the subgranular zone of the dentate gyrus in the adult mouse hippocampus. Immunofluorescent multiple labeling showed that S100A6 was highly expressed in neural stem cells labeled by sex determining region Y-box 2, brain lipid-binding protein protein and glial fibrillary acidic protein. S100A6+ cells often extended a long process typical of radial glial morphology. In addition, S100A6 was found in some S100β+ astrocyte lineage cells. Interestingly, proliferating cell nuclear antigen was detected in a fraction of S100A6+/S100β+ cells. These cells were considered to be lineage-restricted astrocyte precursors maintaining mitotic potential. On the other hand, S100A6 was rarely seen in neural lineage cells labeled by T-box brain protein 2, doublecortin, calretinin and calbindin D28K. Cell fate-tracing experiment using BrdU showed that the majority of newly generated immature astrocytes were immunoreactive for S100A6, while mature astrocytes lacked S100A6 immunoreactivity. Administration of S100 protein inhibitor, trifluoperazine, caused a reduction in production of S100β+ astrocyte lineage cells, but had no impact on neurogenesis. Overall, our data provide the first evidence that S100A6 is a specific marker of neural stem cells and astrocyte precursors, and may be especially important for generation of astrocytes in the adult hippocampus.

  15. Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

    SciTech Connect

    Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F. )

    1990-02-01

    ({sup 125}I)Iodomouse GH (({sup 125}I)iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse.

  16. Methods in laboratory investigation. Autoradiographic demonstration of the specific binding and nuclear localization of 3H-dexamethasone in adult mouse lung.

    PubMed

    Beer, D G; Cunha, G R; Malkinson, A M

    1983-12-01

    This report describes the first autoradiographic demonstration of specific nuclear localization of 3H-dexamethasone in different cell types of the lung. Adult mouse lung tissue was incubated in vitro for 90 minutes with 17 nM 3H-dexamethasone in the presence or absence of various nonradioactive steroids. After extensive washing to remove any nonspecifically bound ligand, the specimens were processed for autoradiography using the thaw-mount method. In the absence of competing steroids, silver grains were localized in the nuclei of alveolar type II cells, bronchiolar and arteriolar smooth muscle cells, fibroblasts, and endothelial cells of the pulmonary vasculature. No significant nuclear concentration of label was observed in the bronchiolar epithelium, however. The specificity of 3H-dexamethasone labeling was demonstrated by incubating 17 nM 3H-dexamethasone with a 600-fold excess of either unlabeled dexamethasone, estrogen, dihydrotestosterone, or progesterone. These autoradiographic binding and steroid competition studies were confirmed by quantifying with liquid scintillation counting the specific 3H-dexamethasone binding in nuclear and cytosolic fractions prepared from lung tissues that had undergone identical incubation and washing procedures as those for autoradiography. These results demonstrate that many cell types in adult lung are targets for glucocorticoids and may respond to physiologic concentrations of this hormone.

  17. RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro.

    PubMed

    Soldati, Chiara; Caramanica, Pasquale; Burney, Matthew J; Toselli, Camilla; Bithell, Angela; Augusti-Tocco, Gabriella; Stanton, Lawrence W; Biagioni, Stefano; Buckley, Noel J; Cacci, Emanuele

    2015-08-01

    Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate. PMID:25691247

  18. Isoform-Specific Modulation of Inflammation Induced by Adenoviral Mediated Delivery of Platelet-Derived Growth Factors in the Adult Mouse Heart

    PubMed Central

    Ylä-Herttuala, Seppo; Betsholtz, Christer; Andrae, Johanna

    2016-01-01

    Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFRα and PDGFRβ) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRα agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFRα-positive fibroblasts, PDGFRβ agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirus-induced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses. PMID:27513343

  19. Identification of a Sustained Neurogenic Zone at the Dorsal Surface of the Adult Mouse Hippocampus and Its Regulation by the Chemokine SDF-1

    PubMed Central

    Belmadani, Abdelhak; Ren, Dongjun; Bhattacharyya, Bula J.; Rothwangl, Katharina B.; Hope, Thomas J.; Perlman, Harris; Miller, Richard J.

    2015-01-01

    We identified a previously unknown neurogenic region at the dorsal surface of the hippocampus; (the “subhippocampal zone,” SHZ) in the adult brain. Using a reporter mouse in which SHZ cells and their progeny could be traced through the expression of EGFP under the control of the CXCR4 chemokine receptor promoter we observed the presence of a pool of EGFP expressing cells migrating in direction of the dentate gyrus (DG), which is maintained throughout adulthood. This population appeared to originate from the SHZ where cells entered a caudal migratory stream (aCMS) that included the fimbria, the meninges and the DG. Deletion of CXCR4 from neural stem cells (NSCs) or neuroinflammation resulted in the appearance of neurons in the DG, which were the result of migration of NSCs from the SHZ. Some of these neurons were ectopically placed. Our observations indicate that the SHZ is a neurogenic zone in the adult brain through migration of NSCs in the aCMS. Regulation of CXCR4 signaling in these cells may be involved in repair of the DG and may also give rise to ectopic granule cells in the DG in the context of neuropathology. PMID:25656357

  20. Identification of a sustained neurogenic zone at the dorsal surface of the adult mouse hippocampus and its regulation by the chemokine SDF-1.

    PubMed

    Belmadani, Abdelhak; Ren, Dongjun; Bhattacharyya, Bula J; Rothwangl, Katharina B; Hope, Thomas J; Perlman, Harris; Miller, Richard J

    2015-11-01

    We identified a previously unknown neurogenic region at the dorsal surface of the hippocampus; (the "subhippocampal zone," SHZ) in the adult brain. Using a reporter mouse in which SHZ cells and their progeny could be traced through the expression of EGFP under the control of the CXCR4 chemokine receptor promoter we observed the presence of a pool of EGFP expressing cells migrating in direction of the dentate gyrus (DG), which is maintained throughout adulthood. This population appeared to originate from the SHZ where cells entered a caudal migratory stream (aCMS) that included the fimbria, the meninges and the DG. Deletion of CXCR4 from neural stem cells (NSCs) or neuroinflammation resulted in the appearance of neurons in the DG, which were the result of migration of NSCs from the SHZ. Some of these neurons were ectopically placed. Our observations indicate that the SHZ is a neurogenic zone in the adult brain through migration of NSCs in the aCMS. Regulation of CXCR4 signaling in these cells may be involved in repair of the DG and may also give rise to ectopic granule cells in the DG in the context of neuropathology.

  1. RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro.

    PubMed

    Soldati, Chiara; Caramanica, Pasquale; Burney, Matthew J; Toselli, Camilla; Bithell, Angela; Augusti-Tocco, Gabriella; Stanton, Lawrence W; Biagioni, Stefano; Buckley, Noel J; Cacci, Emanuele

    2015-08-01

    Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate.

  2. Synergistic and additive effects of enriched environment and lithium on the generation of new cells in adult mouse hippocampus.

    PubMed

    Schaeffer, Evelin L; Cerulli, Fabiana G; Souza, Hélio O X; Catanozi, Sergio; Gattaz, Wagner F

    2014-07-01

    Hippocampal atrophy is reported in several neuropathological disorders. The hippocampal dentate gyrus (DG) is a brain region where adult neurogenesis constitutively occurs. There are some reports suggesting the ability of endogenous neurogenesis to initiate neuronal repair in the hippocampus in response to neuropathological conditions, but its capacity to compensate for neuronal loss is limited. Among strategies to enhance adult hippocampal neurogenesis are enriched environment and lithium. This study aimed to assess whether both strategies could interact to potentiate the generation of new cells in the adult DG. Healthy adult male C57BL/6 mice were divided into four treatment groups for 28 days: control, lithium, enriched environment, enriched environment plus lithium. The animals were injected with BrdU (cell proliferation marker) shortly before the start of the treatments and killed 28 days later for analysis of newly generated cells. Two-way ANOVA followed by post hoc test revealed a significant synergistic interaction between enriched environment and lithium in the total number of BrdU(+) cells in the entire DG (p = 0.019), a trend towards significant synergistic interaction in the dorsal DG (p = 0.075), and a significant additive effect in the ventral DG (p = 0.001). These findings indicate that the combination of enriched environment and lithium has both synergistic and additive effects on the generation of new cells in the healthy adult DG (these effects being possibly segregated along the dorso-ventral axis of the hippocampus), and suggest that it might be worth investigating whether this combination would have a similar effect in neuropathological conditions.

  3. Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

    PubMed

    Wen, Qing; Zheng, Qiao-Song; Li, Xi-Xia; Hu, Zhao-Yuan; Gao, Fei; Cheng, C Yan; Liu, Yi-Xun

    2014-12-15

    Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development.

  4. Direct measurement of hormone-induced acidification in intact bone.

    PubMed

    Belinsky, G S; Tashjian, A H

    2000-03-01

    Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR. Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2'-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.

  5. Effects of maternal L-tryptophan depletion and corticosterone administration on neurobehavioral adjustments in mouse dams and their adolescent and adult daughters.

    PubMed

    Zoratto, Francesca; Berry, Alessandra; Anzidei, Francesca; Fiore, Marco; Alleva, Enrico; Laviola, Giovanni; Macrì, Simone

    2011-08-01

    Major depressive disorder (MDD), a pathology characterized by mood and neurovegetative disturbances, depends on a multi-factorial contribution of individual predisposition (e.g., diminished serotonergic transmission) and environmental factors (e.g., neonatal abuse or neglect). Despite its female-biased prevalence, MDD basic research has mainly focused on male rodents. Most of present models of depression are also devalued due to the fact that they typically address only one of the aforementioned pathogenetic factors. In this paper we first describe the basic principles behind mouse model development and evaluation and then articulate that current models of depression are intrinsically devalued due to poor construct and/or external validity. We then report a first attempt to overcome this limitation through the design of a mouse model in which the genetic and the environmental components of early risk factors for depression are mimicked together. Environmental stress is mimicked through the supplementation of corticosterone in the maternal drinking water while biological predisposition is mimicked through maternal access to an L-tryptophan (the serotonin precursor) deficient diet during the first week of lactation. CD1 dams and their offspring exposed to the L-tryptophan deficient diet (T) and to corticosterone (80mg/l; C) were compared to animal facility reared (AFR) subjects. T and C mice served as intermediate reference groups. Adolescent TC offspring, compared to AFR mice, showed decreased time spent floating in the forced-swim test and increased time spent in the open sectors of an elevated 0-maze. Adult TC offspring showed reduced preference for novelty, decreased breakpoints in the progressive ratio operant procedure and major alterations in central BDNF levels and altered HPA regulation. The route of administration and the possibility to control the independent variables predisposing to depressive-like symptoms disclose novel avenues towards the development

  6. Intrathecal application of cyproheptadine impairs locomotion in intact rats.

    PubMed

    Majczyński, Henryk; Cabaj, Anna; Górska, Teresa

    In intact adult rats, cyproheptadine, a 5-HT2 antagonist, administered intrathecally at the midlumbar segments was found to impair hindlimb locomotor movements during overground locomotion. These effects were dose-dependent; they varied from transient complete hindlimb paraplegia seen at doses of 300 microg/20 microl, to short-lasting trunk instability at doses of 100 microg/20 microl. After the return of overground locomotion, transient abduction of one of the hindlimbs was observed in some animals. These findings demonstrate that the blockade of 5-HT2 receptors affects locomotion in intact rats. Our results provide support for the hypothesis of serotonergic involvement in rat locomotion, which, so far, has been based mainly on the effects of 5-HT2 agonists on the recovery of locomotion in spinal rats.

  7. Secretion of Shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor

    PubMed Central

    Zhao, Hu; Feng, Jifan; Seidel, Kerstin; Shi, Songtao; Klein, Ophir; Sharpe, Paul; Chai, Yang

    2014-01-01

    Mesenchymal stem cells (MSCs) are typically defined by their in vitro characteristics, and as a consequence the in vivo identity of MSCs and their niches are poorly understood. To address this issue, we used lineage tracing in a mouse incisor model and identified the neurovascular bundle (NVB) as an MSC niche. We found that NVB sensory nerves secrete Shh protein, which activates Gli1 expression in periarterial cells that contribute to all mesenchymal derivatives. These periarterial cells do not express classical MSC markers used to define MSCs in vitro. In contrast, NG2+ pericytes represent an MSC subpopulation derived from Gli1+ cells; they express classical MSC markers and contribute little to homeostasis but are actively involved in injury repair. Likewise, incisor Gli1+ cells but not NG2+ cells exhibit typical MSC characteristics in vitro. Collectively, we demonstrate that MSCs originate from periarterial cells and are regulated by Shh secretion from a NVB. PMID:24506883

  8. Exploratory investigations of hypervelocity intact capture spectroscopy.

    PubMed

    Tsou, P; Griffiths, D J

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  9. Exploratory investigations of hypervelocity intact capture spectroscopy

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Griffiths, D. J.

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  10. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability....

  11. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  12. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  13. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  14. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  15. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability....

  16. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  17. Effects of organisational oestradiol on adult immunoreactive oestrogen receptors (alpha and beta) in the male mouse brain.

    PubMed

    Kudwa, A E; Harada, N; Honda, S-I; Rissman, E F

    2007-10-01

    Steroid hormones act on developing neural circuits that regulate the hypothalamic-pituitary-gonadal axis and are involved in hormone-sensitive behaviours. To test the hypothesis that developmental exposure to oestradiol (E(2)) organises the quantity of adult oestrogen receptors (ERalpha and ERbeta), we used male mice with a targeted mutation of the aromatase enzyme gene (ArKO) and their wild-type (WT) littermates. These mice are unable to aromatise testosterone to E(2), but still express both ERalpha and beta. To evaluate adult responsiveness to E(2), gonadectomised males were implanted with Silastic capsules containing E(2), or an empty implant, 5 days prior to sacrifice. Immunoreactivity for ERalpha and ERbeta was quantified in the caudal ventromedial nucleus (VMN) and the medial preoptic area (POA). Regardless of genotype, adult treatment with E(2) reduced ERalpha-immunoreactive (ir) and ERbeta-ir cell numbers in the POA, as well as ERbeta-ir, but not ERalpha-ir, cell numbers in the VMN. Genotype, and thus endogenous exposure to E(2), produced opposite effects on ER expression in the two brain areas. In the VMN, ArKO males had more ERalpha-ir and ERbeta-ir cells than did WT males. In the POA, ArKO males had fewer ERalpha-ir and ERbeta-ir cells than did WT males. Thus, numbers of immunoreactive neurones containing both ERs in the adult ArKO male were enhanced in the POA, but decreased in the VMN, and most likely these patterns were established during the developmental critical period. Furthermore, although both ERalpha and beta-ir cell numbers are altered by the disruption of the aromatase gene, ERbeta is altered in a more robust and region-specific manner.

  18. Suppression of c-Kit signaling induces adult neurogenesis in the mouse intestine after myenteric plexus ablation with benzalkonium chloride.

    PubMed

    Tamada, Hiromi; Kiyama, Hiroshi

    2016-01-01

    Adult neurogenesis rarely occurs in the enteric nervous system (ENS). In this study, we demonstrated that, after intestinal myenteric plexus (MP) ablation with benzalkonium chloride (BAC), adult neurogenesis in the ENS was significantly induced in c-kit loss-of-function mutant mice (W/W(v)). Almost all neurons and fibers in the MP disappeared after BAC treatment. However, 1 week after ablation, substantial penetration of nerve fibers from the non-damaged area was observed in the MP, longitudinal muscle and subserosal layers in both wildtype and W/W(v) mice. Two weeks after BAC treatment, in addition to the penetrating fibers, a substantial number of ectopic neurons appeared in the subserosal and longitudinal muscle layers of W/W(v) mice, whereas only a few ectopic neurons appeared in wildtype mice. Such ectopic neurons expressed either excitatory or inhibitory intrinsic motor neuron markers and formed ganglion-like structures, including glial cells, synaptic vesicles and basal lamina. Furthermore, oral administration of imatinib, an inhibitor of c-Kit and an anticancer agent for gastrointestinal stromal tumors, markedly induced appearance of ectopic neurons after BAC treatment, even in wildtype mice. These results suggest that adult neurogenesis in the ENS is negatively regulated by c-Kit signaling in vivo. PMID:27572504

  19. Impaired adult hippocampal neurogenesis and its partial reversal by chronic treatment of fluoxetine in a mouse model of Angelman syndrome.

    PubMed

    Godavarthi, Swetha K; Dey, Parthanarayan; Sharma, Ankit; Jana, Nihar Ranjan

    2015-09-01

    Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe cognitive and motor deficits, caused by the loss of function of maternally inherited Ube3a. Ube3a-maternal deficient mice (AS model mice) recapitulate many essential features of AS, but how the deficiency of Ube3a lead to such behavioural abnormalities is poorly understood. Here we have demonstrated significant impairment of adult hippocampal neurogenesis in AS mice brain. Although, the number of BrdU and Ki67-positive cell in the hippocampal DG region was nearly equal at early postnatal days among wild type and AS mice, they were significantly reduced in adult AS mice compared to wild type controls. Reduced number of doublecortin-positive immature neurons in this region of AS mice further indicated impaired neurogenesis. Unaltered BrdU and Ki67-positive cells number in the sub ventricular zone of adult AS mice brain along with the absence of imprinted expression of Ube3a in the neural progenitor cell suggesting that Ube3a may not be directly linked with altered neurogenesis. Finally, we show that the impaired hippocampal neurogenesis in these mice can be partially rescued by the chronic treatment of antidepressant fluoxetine. These results suggest that the chronic stress may lead to reduced hippocampal neurogenesis in AS mice and that impaired neurogenesis could contribute to cognitive disturbances observed in these mice.

  20. Suppression of c-Kit signaling induces adult neurogenesis in the mouse intestine after myenteric plexus ablation with benzalkonium chloride

    PubMed Central

    Tamada, Hiromi; Kiyama, Hiroshi

    2016-01-01

    Adult neurogenesis rarely occurs in the enteric nervous system (ENS). In this study, we demonstrated that, after intestinal myenteric plexus (MP) ablation with benzalkonium chloride (BAC), adult neurogenesis in the ENS was significantly induced in c-kit loss-of-function mutant mice (W/Wv). Almost all neurons and fibers in the MP disappeared after BAC treatment. However, 1 week after ablation, substantial penetration of nerve fibers from the non-damaged area was observed in the MP, longitudinal muscle and subserosal layers in both wildtype and W/Wv mice. Two weeks after BAC treatment, in addition to the penetrating fibers, a substantial number of ectopic neurons appeared in the subserosal and longitudinal muscle layers of W/Wv mice, whereas only a few ectopic neurons appeared in wildtype mice. Such ectopic neurons expressed either excitatory or inhibitory intrinsic motor neuron markers and formed ganglion-like structures, including glial cells, synaptic vesicles and basal lamina. Furthermore, oral administration of imatinib, an inhibitor of c-Kit and an anticancer agent for gastrointestinal stromal tumors, markedly induced appearance of ectopic neurons after BAC treatment, even in wildtype mice. These results suggest that adult neurogenesis in the ENS is negatively regulated by c-Kit signaling in vivo. PMID:27572504

  1. Impaired adult hippocampal neurogenesis and its partial reversal by chronic treatment of fluoxetine in a mouse model of Angelman syndrome.

    PubMed

    Godavarthi, Swetha K; Dey, Parthanarayan; Sharma, Ankit; Jana, Nihar Ranjan

    2015-09-01

    Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe cognitive and motor deficits, caused by the loss of function of maternally inherited Ube3a. Ube3a-maternal deficient mice (AS model mice) recapitulate many essential features of AS, but how the deficiency of Ube3a lead to such behavioural abnormalities is poorly understood. Here we have demonstrated significant impairment of adult hippocampal neurogenesis in AS mice brain. Although, the number of BrdU and Ki67-positive cell in the hippocampal DG region was nearly equal at early postnatal days among wild type and AS mice, they were significantly reduced in adult AS mice compared to wild type controls. Reduced number of doublecortin-positive immature neurons in this region of AS mice further indicated impaired neurogenesis. Unaltered BrdU and Ki67-positive cells number in the sub ventricular zone of adult AS mice brain along with the absence of imprinted expression of Ube3a in the neural progenitor cell suggesting that Ube3a may not be directly linked with altered neurogenesis. Finally, we show that the impaired hippocampal neurogenesis in these mice can be partially rescued by the chronic treatment of antidepressant fluoxetine. These results suggest that the chronic stress may lead to reduced hippocampal neurogenesis in AS mice and that impaired neurogenesis could contribute to cognitive disturbances observed in these mice. PMID:26231800

  2. Anxiety- rather than depression-like behavior is associated with adult neurogenesis in a female mouse model of higher trait anxiety- and comorbid depression-like behavior.

    PubMed

    Sah, A; Schmuckermair, C; Sartori, S B; Gaburro, S; Kandasamy, M; Irschick, R; Klimaschewski, L; Landgraf, R; Aigner, L; Singewald, N

    2012-01-01

    Adult neurogenesis has been implicated in affective disorders and the action of antidepressants (ADs) although the functional significance of this association is still unclear. The use of animal models closely mimicking human comorbid affective and anxiety disorders seen in the majority of patients should provide relevant novel information. Here, we used a unique genetic mouse model displaying higher trait anxiety (HAB) and comorbid depression-like behavior. We demonstrate that HABs have a lower rate of hippocampal neurogenesis and impaired functional integration of newly born neurons as compared with their normal anxiety/depression-like behavior (NAB) controls. In HABs, chronic treatment with the AD fluoxetine alleviated their higher depression-like behavior and protected them from relapse for 3 but not 7 weeks after discontinuation of the treatment without affecting neurogenesis. Similar to what has been observed in depressed patients, fluoxetine treatment induced anxiogenic-like effects during the early treatment phase in NABs along with a reduction in neurogenesis. On the other hand, treatment with AD drugs with a particularly strong anxiolytic component, namely the neurokinin-1-receptor-antagonist L-822 429 or tianeptine, increased the reduced rate of neurogenesis in HABs up to NAB levels. In addition, challenge-induced hypoactivation of dentate gyrus (DG) neurons in HABs was normalized by all three drugs. Overall, these data suggest that AD-like effects in a psychopathological mouse model are commonly associated with modulation of DG hypoactivity but not neurogenesis, suggesting normalization of hippocampal hypoactivity as a neurobiological marker indicating successful remission. Finally, rather than to higher depression-related behavior, neurogenesis seems to be linked to pathological anxiety. PMID:23047242

  3. Characterization of Aromatase Expression in the Adult Male and Female Mouse Brain. I. Coexistence with Oestrogen Receptors α and β, and Androgen Receptors

    PubMed Central

    Stanić, Davor; Dubois, Sydney; Chua, Hui Kheng; Tonge, Bruce; Rinehart, Nicole; Horne, Malcolm K.; Boon, Wah Chin

    2014-01-01

    Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, including the bed nucleus of the stria terminalis, olfactory tubercle, medial amygdaloid nucleus and medial preoptic area, with the densest distributions of EGFP-positive cell bodies in the bed nucleus and medial amygdala. Differences between male and female mice were apparent, with the density of EGFP-positive cell bodies and fibres being lower in some brain regions of female mice, including the bed nucleus and medial amygdala. EGFP-positive cell bodies in the bed nucleus, lateral septum, medial amygdala and hypothalamus co-expressed oestrogen receptor (ER) α and β, or the androgen receptor (AR), although single-labelled EGFP-positive cells were also identified. Additionally, single-labelled ERα−, ERβ- or AR-positive cell bodies often appeared to be surrounded by EGFP-immunoreactive nerve fibres/terminals. The widespread distribution of EGFP-positive cell bodies and fibres suggests that aromatase signalling is common in the mouse brain, and that locally synthesised brain oestrogens could mediate biological effects by activating pre- and post-synaptic oestrogen α and β receptors, and androgen receptors. The higher number of EGFP-positive cells in male mice may indicate that the autocrine and paracrine effects of oestrogens are more prominent in males than females. PMID:24646567

  4. Influence of age on mechanical properties of healing fractures and intact bones in rats.

    PubMed

    Ekeland, A; Engesoeter, L B; Langeland, N

    1982-08-01

    Mechanical properties of fractured and intact femora have been studied in young and adult, male rats. A standardized, closed, mid-diaphyseal fracture was produced in the left femur, the right femur serving as control. The fracture was left to heal without immobilization. At various intervals, both fractured and intact femora were loaded in torsion until failure. The fractured femora regained the mechanical properties of the contralateral, intact bones after about 4 weeks in young and after about 12 weeks in adult rats. For intact bones, both the ultimate torsional moment (strength) and the torsional stiffness increased with age of the animals, whereas the ultimate torsional angle remained unchanged. For bone as a material, however, the ultimate torsional stress (strength) and the modulus of rigidity (stiffness) increased with age only in young rats, being almost constant in the adult animals. The various biomechanical parameters of the healing fractures did not reach those of the contralateral, intact bones simultaneously. The torsional moment required to twist a healing femoral fracture 20 degrees (0.35 radians), a deformation close to what an intact femur can resist, proved to be a functional and simple measure of the degree of fracture repair in rats.

  5. p53 E3 ubiquitin protein ligase homolog regulates p53 in vivo in the adult mouse eye lens

    PubMed Central

    Jaramillo-Rangel, Gilberto; Ortega-Martínez, Marta; Sepúlveda-Saavedra, Julio; Saucedo-Cárdenas, Odila; Montes-de-Oca-Luna, Roberto

    2013-01-01

    Purpose p53 is a transcription factor that plays an important role in preventing cancer development. p53 participates in relevant aspects of cell biology, including apoptosis and cell cycle control and must be strictly regulated to maintain normal tissue homeostasis. p53 E3 ubiquitin protein ligase homolog (Mdm2) is an important negative regulator of p53. The purpose of this study was to determine if Mdm2 regulates p53 in vivo in the adult lens. Methods We analyzed mice expressing human p53 transgene (Tgp53) selectively in the lens in the presence or absence of Mdm2. Mice with the required genotypes were obtained by crossing transgenic, mdm2+/−, and p53−/− mice. Eye phenotype and lens histology and ultrastructure were analyzed in adult mice. Results In a wild-type genetic background (mdm2+/+), lens damage and microphthalmia were observed only in mice homozygous for Tgp53 (t/t). However, in an mdm2 null background, just one allele of Tgp53 (mdm2−/−/Tgp53t/0 mice) was sufficient to cause lens damage and microphthalmia. Furthermore, Mdm2 in only one allele was sufficient to rescue these deleterious effects, since the mdm2+/−/Tgp53t/0 mice had eye size and lens morphology similar to the control mice. Conclusions Mdm2 regulates p53 in the adult lens in vivo. This information may have relevance for analyzing normal and pathological conditions of the lens, and designing cancer therapies targeting Mdm2–p53 interaction. PMID:24339722

  6. Tissue inhibitor of metalloproteinases-2 is expressed in the interstitial matrix in adult mouse organs and during embryonic development.

    PubMed Central

    Blavier, L; DeClerck, Y A

    1997-01-01

    Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a member of a family of inhibitors of matrix-degrading metalloproteinases. A better insight into the role of this inhibitor during development and in organ function was obtained by examining the temporospatial expression of TIMP-2 in mice. Northern blot analysis indicated high levels of TIMP-2 mRNA in the lung, skin, reproductive organs, and brain. Lower levels of expression were found in all other organs with the exception of the liver and gastrointestinal tissue, which were negative of these tissues with complete absence of TIMP-2 mRNA in the epithelium. In the testis, TIMP-2 was present in the Leydig cells, and in the brain, it was expressed in pia matter and in neuronal tissues. TIMP-2 expression in the placenta increased during late gestation and was particularly abundant in spongiotrophoblasts In mouse embryo (day 10.5-18.5), TIMP-2 mRNA was abundant in mesenchymal tissues that surrounded developing epithelia and maturing skeleton. The pattern of expression significantly differs from that observed with TIMP-1 and TIMP-3, therefore, suggesting specific roles for each inhibitor during tissue remodeling and development. Images PMID:9285822

  7. Bioluminescence imaging of mitochondrial Ca2+ dynamics in soma and neurites of individual adult mouse sympathetic neurons

    PubMed Central

    Núñez, Lucía; Senovilla, Laura; Sanz-Blasco, Sara; Chamero, Pablo; Alonso, María T; Villalobos, Carlos; García-Sancho, Javier

    2007-01-01

    Changes in the cytosolic Ca2+ concentration ([Ca2+]c) are essential for triggering neurotransmitter release from presynaptic nerve terminals. Calcium-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER) may amplify the [Ca2+]c signals and facilitate neurotransmitter release in sympathetic neurons. In adrenal chromaffin cells, functional triads are formed by voltage-operated Ca2+ channels (VOCCs), CICR sites and mitochondria. In fact, mitochondria take up most of the Ca2+ load entering the cells and are essential for shaping [Ca2+]c signals and exocytosis. Here we have investigated the existence of such functional triads in sympathetic neurons. The mitochondrial Ca2+ concentration ([Ca2+]m) in soma and neurites of individual mouse superior cervical ganglion (SCG) neurons was monitored by bioluminescence imaging of targeted aequorins. In soma, Ca2+ entry through VOCCs evoked rapid, near millimolar [Ca2+]m increases in a subpopulation of mitochondria containing about 40% of the aequorin. Caffeine evoked a similar [Ca2+]m increase in a mitochondrial pool containing about 30% of the aequorin and overlapping with the VOCC-sensitive pool. These observations suggest the existence of functional triads similar to the ones described in chromaffin cells. In neurites, mitochondria were able to buffer [Ca2+]c increases resulting from activation of VOCCs but not those mediated by caffeine-induced Ca2+ release from the ER. The weaker Ca2+ buffering by mitochondria in neurites could contribute to facilitate Ca2+-induced exocytosis at the presynaptic sites. PMID:17234693

  8. Genomic Recombination Leading to Decreased Virulence of Group B Streptococcus in a Mouse Model of Adult Invasive Disease

    PubMed Central

    Teatero, Sarah; Lemire, Paul; Dewar, Ken; Wasserscheid, Jessica; Calzas, Cynthia; Mallo, Gustavo V.; Li, Aimin; Athey, Taryn B.T.; Segura, Mariela; Fittipaldi, Nahuel

    2016-01-01

    Adult invasive disease caused by Group B Streptococcus (GBS) is increasing worldwide. Whole-genome sequencing (WGS) now permits rapid identification of recombination events, a phenomenon that occurs frequently in GBS. Using WGS, we described that strain NGBS375, a capsular serotype V GBS isolate of sequence type (ST)297, has an ST1 genomic background but has acquired approximately 300 kbp of genetic material likely from an ST17 strain. Here, we examined the virulence of this strain in an in vivo model of GBS adult invasive infection. The mosaic ST297 strain showed intermediate virulence, causing significantly less systemic infection and reduced mortality than a more virulent, serotype V ST1 isolate. Bacteremia induced by the ST297 strain was similar to that induced by a serotype III ST17 strain, which was the least virulent under the conditions tested. Yet, under normalized bacteremia levels, the in vivo intrinsic capacity to induce the production of pro-inflammatory cytokines was similar between the ST297 strain and the virulent ST1 strain. Thus, the diminished virulence of the mosaic strain may be due to reduced capacity to disseminate or multiply in blood during a systemic infection which could be mediated by regulatory factors contained in the recombined region. PMID:27527222

  9. Alteration of SLP2-like immunolabeling in mitochondria signifies early cellular damage in developing and adult mouse brain.

    PubMed

    Morozov, Yury M; Sun, Yu-Yo; Kuan, Chia-Yi; Rakic, Pasko

    2016-01-01

    Mitochondria play a critical role in various pathways of regulated cell death. Here we propose a novel method for detection of initial derangement of mitochondria in degenerating and dying neuronal cells. The method is based on our recent finding that antibodies directed against the cannabinoid type 1 receptor (CB1) also bind the mitochondrial stomatin-like protein 2 (SLP2) that belongs to an inner mitochondrial membrane protein complex. It is well established that SLP2 regulates mitochondrial biogenesis and respiratory functions. We now show that anti-CB1 antibodies recognize conformational epitopes but not the linear amino acid sequence of SLP2. In addition we found that anti-CB1 serum mostly labels swollen mitochondria with early or advanced stages of pathology in mouse brain while other proteins of the complex may mask epitopes of SLP2 in the normal mitochondria. Although neurons and endothelial cells in healthy brains contain occasional immunopositive mitochondria detectable with anti-CB1 serum, their numbers increase significantly after hypoxic insults in parallel with signs of cellular damage. Moreover, use of electron microscopy suggests relocation of SLP2 from its normal functional position in the inner mitochondrial membrane into the mitochondrial matrix in pathological cells. Thus, SLP2-like immunolabeling serves as an in situ histochemical target detecting early derangement of mitochondria. Anti-CB1 serum is crucial for this purpose because available anti-SLP2 antibodies do not provide selective labeling of mitochondria in the fixed tissue. This new method of detecting mitochondrial dysfunction can benefit the in vitro research of human diseases and developmental disorders by enabling analysis in live animal models.

  10. Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

    PubMed

    Nakatsuka, Ryusuke; Iwaki, Ryuji; Matsuoka, Yoshikazu; Sumide, Keisuke; Kawamura, Hiroshi; Fujioka, Tatsuya; Sasaki, Yutaka; Uemura, Yasushi; Asano, Hiroaki; Kwon, A-Hon; Sonoda, Yoshiaki

    2016-01-01

    Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

  11. Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex123

    PubMed Central

    Billeh, Yazan N.; Bernard, Amy; de Vivo, Luisa; Honjoh, Sakiko; Mihalas, Stefan; Ng, Lydia; Koch, Christof

    2016-01-01

    Abstract Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25–P30, ≥50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments. PMID:27351022

  12. No effect of running and laboratory housing on adult hippocampal neurogenesis in wild caught long-tailed wood mouse

    PubMed Central

    Hauser, Thomas; Klaus, Fabienne; Lipp, Hans-Peter; Amrein, Irmgard

    2009-01-01

    Background Studies of adult hippocampal neurogenesis (AHN) in laboratory rodents have raised hopes for therapeutic interventions in neurodegenerative diseases and mood disorders, as AHN can be modulated by physical exercise, stress and environmental changes in these animals. Since it is not known whether cell proliferation and neurogenesis in wild living mice can be experimentally changed, this study investigates the responsiveness of AHN to voluntary running and to environmental change in wild caught long-tailed wood mice (Apodemus sylvaticus). Results Statistical analyses show that running had no impact on cell proliferation (p = 0.44), neurogenesis (p = 0.94) or survival of newly born neurons (p = 0.58). Likewise, housing in the laboratory has no effect on AHN. In addition, interindividual differences in the level of neurogenesis are not related to interindividual differences of running wheel performance (rs = -0.09, p = 0.79). There is a correlation between the number of proliferating cells and the number of cells of neuronal lineage (rs = 0.63, p < 0.001) and the number of pyknotic cells (rs = 0.5, p = 0.009), respectively. Conclusion Plasticity of adult neurogenesis is an established feature in strains of house mice and brown rats. Here, we demonstrate that voluntary running and environmental changes which are effective in house mice and brown rats cannot influence AHN in long-tailed wood mice. This indicates that in wild long-tailed wood mice different regulatory mechanisms act on cell proliferation and neurogenesis. If this difference reflects a species-specific adaptation or a broader adaptive strategy to a natural vs. domestic environment is unknown. PMID:19419549

  13. Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex.

    PubMed

    Billeh, Yazan N; Rodriguez, Alexander V; Bellesi, Michele; Bernard, Amy; de Vivo, Luisa; Funk, Chadd M; Harris, Julie; Honjoh, Sakiko; Mihalas, Stefan; Ng, Lydia; Koch, Christof; Cirelli, Chiara; Tononi, Giulio

    2016-01-01

    Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25-P30, ≥ 50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments. PMID:27351022

  14. Impaired hippocampal plasticity and altered neurogenesis in adult Ube3a maternal deficient mouse model for Angelman syndrome.

    PubMed

    Mardirossian, Sandrine; Rampon, Claire; Salvert, Denise; Fort, Patrice; Sarda, Nicole

    2009-12-01

    Angelman syndrome (AS) is a severe neurodevelopmental disorder characterized by mental retardation, seizures and sleep disturbances. It results from lack of the functional maternal allele of UBE3A gene. Ube3a maternal-deficient mice (Ube3a m-/p+), animal models for AS, are impaired in hippocampal-dependent learning tasks as compared with control (Ube3a m+/p+) mice. We first examined the basal expression of immediate early genes which expression is required for synaptic plasticity and memory formation. We found that basal expression of c-fos and Arc genes is reduced in the DG of Ube3a maternal deficient mice compared to their non-transgenic littermates. We then examined whether adult hippocampal neurogenesis, which likely serves as a mechanism toward brain plasticity, is altered in these transgenic mice. Neurogenesis occurs throughout life in mammalian dentate gyrus (DG) and recent findings suggest that newborn granule cells are involved in some forms of learning and memory. Whether maternal Ube3a deletion is detrimental on hippocampal neurogenesis is unclear. Herein, we show, using the mitotic marker Ki67, the birthdating marker 5-bromo-2'-dexoyuridine (BrdU) and the marker doublecortin (DCX) to respectively label cell proliferation, cell survival or young neuron production, that the Ube3a maternal deletion does not affect the proliferation nor the survival of newborn cells in the hippocampus. In contrast, using the postmitotic neuronal marker (NeuN), we show that Ube3a maternal deletion is associated with a lower fraction of BrdU+/NeuN+ newborn neurons among the population of surviving new cells in the hippocampus. Collectively, these findings suggest that some aspects of adult neurogenesis and plasticity are affected by Ube3a deletion and may contribute to the hippocampal dysfunction observed in AS mice.

  15. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure

    PubMed Central

    Chater-Diehl, Eric J.; Castellani, Christina A.; Alberry, Bonnie L.; Singh, Shiva M.

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD. PMID:27136348

  16. New mouse model of acute adult T-cell leukemia generated by transplantation of AKT, BCLxL, and HBZ-transduced T cells.

    PubMed

    Kasugai, Yumiko; Yoshida, Noriaki; Ohshima, Koichi; Matsuo, Keitaro; Seto, Masao; Tsuzuki, Shinobu

    2016-08-01

    Adult T-cell leukemia/lymphoma (ATL) develops in human T-cell leukemia virus type 1 (HTLV-1) carriers. Although the HTLV-1-encoded HBZ gene is critically involved, HBZ alone is insufficient and additional, cooperative "hits" are required for the development of ATL. Candidate cooperative hits are being defined, but methods to rapidly explore their roles in ATL development in collaboration with HBZ are lacking. Here, we present a new mouse model of acute type ATL that can be generated rapidly by transplanting in vitro-induced T cells that have been retrovirally transduced with HBZ and two cooperative genes, BCLxL and AKT, into mice. Co-transduction of HBZ and BCLxL/AKT allowed these T cells to grow in vitro in the absence of cytokines (Flt3-ligand and interleukin-7), which did not occur with any two-gene combination. Although transplanted T cells were a mixture of cells transduced with different combinations of the genes, tumors that developed in mice were composed of HBZ/BCLxL/AKT triply transduced T cells, showing the synergistic effect of the three genes. The genetic/epigenetic landscape of ATL has only recently been elucidated, and the roles of additional "hits" in ATL pathogenesis remain to be explored. Our model provides a versatile tool to examine the roles of these hits, in collaboration with HBZ, in the development of acute ATL. PMID:27223899

  17. Effects of neuregulin-1 administration on neurogenesis in the adult mouse hippocampus, and characterization of immature neurons along the septotemporal axis

    PubMed Central

    Mahar, Ian; MacIsaac, Angus; Kim, John Junghan; Qiang, Calvin; Davoli, Maria Antonietta; Turecki, Gustavo; Mechawar, Naguib

    2016-01-01

    Adult hippocampal neurogenesis is associated with learning and affective behavioural regulation. Its diverse functionality is segregated along the septotemporal axis from the dorsal to ventral hippocampus. However, features distinguishing immature neurons in these regions have yet to be characterized. Additionally, although we have shown that administration of the neurotrophic factor neuregulin-1 (NRG1) selectively increases proliferation and overall neurogenesis in the mouse ventral dentate gyrus (DG), likely through ErbB3, NRG1’s effects on intermediate neurogenic stages in immature neurons are unknown. We examined whether NRG1 administration increases DG ErbB3 phosphorylation. We labeled adultborn cells using BrdU, then administered NRG1 to examine in vivo neurogenic effects on immature neurons with respect to cell survival, morphology, and synaptogenesis. We also characterized features of immature neurons along the septotemporal axis. We found that neurogenic effects of NRG1 are temporally and subregionally specific to proliferation in the ventral DG. Particular morphological features differentiate immature neurons in the dorsal and ventral DG, and cytogenesis differed between these regions. Finally, we identified synaptic heterogeneity surrounding the granule cell layer. These results indicate neurogenic involvement of NRG1-induced antidepressant-like behaviour is particularly associated with increased ventral DG cell proliferation, and identify novel distinctions between dorsal and ventral hippocampal neurogenic development. PMID:27469430

  18. Long-chain n-3 PUFAs from fish oil enhance resting state brain glucose utilization and reduce anxiety in an adult nonhuman primate, the grey mouse lemur.

    PubMed

    Pifferi, Fabien; Dorieux, Olène; Castellano, Christian-Alexandre; Croteau, Etienne; Masson, Marie; Guillermier, Martine; Van Camp, Nadja; Guesnet, Philippe; Alessandri, Jean-Marc; Cunnane, Stephen; Dhenain, Marc; Aujard, Fabienne

    2015-08-01

    Decreased brain content of DHA, the most abundant long-chain n-3 polyunsaturated fatty acid (n-3 LCPUFA) in the brain, is accompanied by severe neurosensorial impairments linked to impaired neurotransmission and impaired brain glucose utilization. In the present study, we hypothesized that increasing n-3 LCPUFA intake at an early age may help to prevent or correct the glucose hypometabolism observed during aging and age-related cognitive decline. The effects of 12 months' supplementation with n-3 LCPUFA on brain glucose utilization assessed by positron emission tomography was tested in young adult mouse lemurs (Microcebus murinus). Cognitive function was tested in parallel in the same animals. Lemurs supplemented with n-3 LCPUFA had higher brain glucose uptake and cerebral metabolic rate of glucose compared with controls in all brain regions. The n-3 LCPUFA-supplemented animals also had higher exploratory activity in an open-field task and lower evidence of anxiety in the Barnes maze. Our results demonstrate for the first time in a nonhuman primate that n-3 LCPUFA supplementation increases brain glucose uptake and metabolism and concomitantly reduces anxiety. PMID:26063461

  19. The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult

    PubMed Central

    Zhang, Bin; Arun, Gayatri; Mao, Yuntao S.; Lazar, Zsolt; Hung, Gene; Bhattacharjee, Gourab; Xiao, Xiaokun; Booth, Carmen J.; Wu, Jie; Zhang, Chaolin; Spector, David L.

    2012-01-01

    SUMMARY Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3′-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates Malat1 is not essential for mouse pre- and post-natal development. Furthermore, depletion of Malat1 does not impact global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis regulatory role of Malat1 gene transcription. PMID:22840402

  20. Membrane structure in isolated and intact myelins.

    PubMed Central

    Inouye, H; Karthigasan, J; Kirschner, D A

    1989-01-01

    The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions. PMID:2752082

  1. Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium.

    PubMed

    Doyle, Kharen L; Karl, Tim; Hort, Yvonne; Duffy, Liesl; Shine, John; Herzog, Herbert

    2008-05-01

    While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.

  2. A Western diet ecological module identified from the 'humanized' mouse microbiota predicts diet in adults and formula feeding in children.

    PubMed

    Siddharth, Jay; Holway, Nicholas; Parkinson, Scott J

    2013-01-01

    The interplay between diet and the microbiota has been implicated in the growing frequency of chronic diseases associated with the Western lifestyle. However, the complexity and variability of microbial ecology in humans and preclinical models has hampered identification of the molecular mechanisms underlying the association of the microbiota in this context. We sought to address two key questions. Can the microbial ecology of preclinical models predict human populations? And can we identify underlying principles that surpass the plasticity of microbial ecology in humans? To do this, we focused our study on diet; perhaps the most influential factor determining the composition of the gut microbiota. Beginning with a study in 'humanized' mice we identified an interactive module of 9 genera allied with Western diet intake. This module was applied to a controlled dietary study in humans. The abundance of the Western ecological module correctly predicted the dietary intake of 19/21 top and 21/21 of the bottom quartile samples inclusive of all 5 Western and 'low-fat' diet subjects, respectively. In 98 volunteers the abundance of the Western module correlated appropriately with dietary intake of saturated fatty acids, fat-soluble vitamins and fiber. Furthermore, it correlated with the geographical location and dietary habits of healthy adults from the Western, developing and third world. The module was also coupled to dietary intake in children (and piglets) correlating with formula (vs breast) feeding and associated with a precipitous development of the ecological module in young children. Our study provides a conceptual platform to translate microbial ecology from preclinical models to humans and identifies an ecological network module underlying the association of the gut microbiota with Western dietary habits.

  3. Melatonin attenuates methamphetamine-induced inhibition of neurogenesis in the adult mouse hippocampus: An in vivo study.

    PubMed

    Singhakumar, Rachen; Boontem, Parichart; Ekthuwapranee, Kasima; Sotthibundhu, Areechun; Mukda, Sujira; Chetsawang, Banthit; Govitrapong, Piyarat

    2015-10-01

    Methamphetamine (METH), a highly addictive psychostimulant drug, is known to exert neurotoxic effects to the dopaminergic neural system. Long-term METH administration impairs brain functions such as cognition, learning and memory. Newly born neurons in the dentate gyrus of the hippocampus play an important role in spatial learning and memory. Previous in vitro studies have shown that METH inhibits cell proliferation and neurogenesis in the hippocampus. On the other hand, melatonin, a major indole secreted by the pineal gland, enhances neurogenesis in both the subventricular zone and dentate gyrus. In this study, adult C57BL/6 mice were used to study the beneficial effects of melatonin on METH-induced alterations in neurogenesis and post-synaptic proteins related to learning and memory functions in the hippocampus. The results showed that METH caused a decrease in neuronal phenotypes as determined by the expressions of nestin, doublecortin (DCX) and beta-III tubulin while causing an increase in glial fibrillary acidic protein (GFAP) expression. Moreover, METH inhibited mitogen-activated protein kinase (MAPK) signaling activity and altered expression of the N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B as well as calcium/calmodulin-dependent protein kinase II (CaMKII). These effects could be attenuated by melatonin pretreatment. In conclusion, melatonin prevented the METH-induced reduction in neurogenesis, increase in astrogliogenesis and alteration of NMDA receptor subunit expression. These findings may indicate the beneficial effects of melatonin on the impairment of learning and memory caused by METH.

  4. Function-triggering antibodies to the adhesion molecule L1 enhance recovery after injury of the adult mouse femoral nerve.

    PubMed

    Guseva, Daria; Loers, Gabriele; Schachner, Melitta

    2014-01-01

    L1 is among the few adhesion molecules that favors repair after trauma in the adult central nervous system of vertebrates by promoting neuritogenesis and neuronal survival, among other beneficial features. In the peripheral nervous system, L1 is up-regulated in Schwann cells and regrowing axons after nerve damage, but the functional consequences of this expression remain unclear. Our previous study of L1-deficient mice in a femoral nerve injury model showed an unexpected improved functional recovery, attenuated motoneuronal cell death, and enhanced Schwann cell proliferation, being attributed to the persistent synthesis of neurotrophic factors. On the other hand, transgenic mice over-expressing L1 in neurons led to improved remyelination, but not improved functional recovery. The present study was undertaken to investigate whether the monoclonal L1 antibody 557 that triggers beneficial L1 functions in vitro would trigger these also in femoral nerve repair. We analyzed femoral nerve regeneration in C57BL/6J mice that received this antibody in a hydrogel filled conduit connecting the cut and sutured nerve before its bifurcation, leading to short-term release of antibody by diffusion. Video-based quantitative analysis of motor functions showed improved recovery when compared to mice treated with conduits containing PBS in the hydrogel scaffold, as a vehicle control. This improved recovery was associated with attenuated motoneuron loss, remyelination and improved precision of preferential motor reinnervation. We suggest that function-triggering L1 antibodies applied to the lesion site at the time of injury over a limited time period will not only be beneficial in peripheral, but also central nervous system regeneration. PMID:25393007

  5. Despite strong behavioral disruption, Delta9-tetrahydrocannabinol does not affect cell proliferation in the adult mouse dentate gyrus.

    PubMed

    Kochman, Linda J; dos Santos, Angela Amancio; Fornal, Casimir A; Jacobs, Barry L

    2006-10-01

    Marijuana is a widely abused illicit drug known to cause significant cognitive impairments. Marijuana has been hypothesized to target neurons in the hippocampus because of the abundance of cannabinoid receptors present in this structure. While there is no clear evidence of neuropathology in vivo, suppression of brain mitogenesis, and ultimately neurogenesis, may provide a sensitive index of marijuana's more subtle effects on neural mechanisms subserving cognitive functions. We examined the effects of different doses and treatment regimens of Delta(9)-tetrahydrocannabinol (THC), the main active ingredient in marijuana, on cell proliferation in the dentate gyrus of adult male mice. Following drug treatment, the thymidine analog 5-bromo-2'-deoxyuridine (BrdU; 200 mg/kg, i.p.) was administered two hours prior to sacrifice to assess cell proliferation, the first step in neurogenesis. Administration of THC produced dose-dependent catalepsy and suppression of motor activity. The number of BrdU-labeled cells was not significantly changed from vehicle control levels following either acute (1, 3, 10, 30 mg/kg, i.p.), sequential (two injections of 10 or 30 mg/kg, i.p., separated by 5 h), or chronic escalating (20 to 80 mg/kg, p.o.; for 3 weeks) drug administration. Furthermore, acute administration of the potent synthetic cannabinoid receptor agonist R-(+)-WIN 55,212-2 (WIN; 5 mg/kg, i.p.) also had no significant effect on cell proliferation. These findings provide no evidence for an effect of THC on hippocampal cell proliferation, even at doses producing gross behavioral intoxication. Whether marijuana or THC affects neurogenesis remains to be explored.

  6. Subchronic Inhalation of Soluble Manganese Induces Expression of Hypoxia-associated Angiogenic Genes in Adult Mouse Lungs

    PubMed Central

    Bredow, Sebastian; Falgout, Melanie M.; March, Thomas H.; Yingling, Christin M.; Malkoski, Stephen P.; Aden, James; Bedrick, Edward J.; Lewis, Johnnye L.; Divine, Kevin K.

    2007-01-01

    Although the lung constitutes the major exposure route for airborne manganese (Mn), little is known about the potential pulmonary effects and the underlying molecular mechanisms. Transition metals can mimic a hypoxia-like response, activating the hypoxia inducible factor-1 (HIF-1) transcription factor family. Through binding to the hypoxia-response element (HRE) these factors regulate expression of many genes, including vascular endothelial growth factor (VEGF). Increases in VEGF, an important biomarker of angiogenesis, have been linked to respiratory diseases, including pulmonary hypertension. The objective of this study was to evaluate pulmonary hypoxia-associated angiogenic gene expression in response to exposure of soluble Mn(II) and to assess the genes' role as intermediaries of potential pulmonary Mn toxicity. In vitro, 0.25 mM Mn(II) altered morphology and slowed the growth of human pulmonary epithelial cell lines. Acute doses between 0.05 and 1 mM stimulated VEGF promoter activity up to 3.7-fold in transient transfection assays. Deletion of the HRE within the promoter had no effect on Mn(II)-induced VEGF expression but decreased cobalt [Co(II)]-induced activity 2-fold, suggesting that HIF-1 may not be involved in Mn(II)-induced VEGF gene transcription. Nose-only inhalation to 2 mg Mn(II)/m3 for 5 days at 6h/day produced no significant pulmonary inflammation but induced a 2-fold increase in pulmonary VEGF mRNA levels in adult mice and significantly altered expression of genes associated with murine angiogenesis. These findings suggest that even short-term exposures to soluble, occupationally relevant Mn(II) concentrations may alter pulmonary gene expression in pathways that ultimately could affect the lungs' susceptibility to respiratory disease. PMID:17467022

  7. Phenotypic characterization of a Csf1r haploinsufficient mouse model of adult-onset leukodystrophy with axonal spheroids and pigmented glia (ALSP).

    PubMed

    Chitu, Violeta; Gokhan, Solen; Gulinello, Maria; Branch, Craig A; Patil, Madhuvati; Basu, Ranu; Stoddart, Corrina; Mehler, Mark F; Stanley, E Richard

    2015-02-01

    Mutations in the colony stimulating factor-1 receptor (CSF1R) that abrogate the expression of the affected allele or lead to the expression of mutant receptor chains devoid of kinase activity have been identified in both familial and sporadic cases of ALSP. To determine the validity of the Csf1r heterozygous mouse as a model of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) we performed behavioral, radiologic, histopathologic, ultrastructural and cytokine expression studies of young and old Csf1r+/- and control Csf1r+/+ mice. Six to 8-month old Csf1r+/- mice exhibit cognitive deficits, and by 9-11 months develop sensorimotor deficits and in male mice, depression and anxiety-like behavior. MRIs of one year-old Csf1r+/- mice reveal lateral ventricle enlargement and thinning of the corpus callosum. Ultrastructural analysis of the corpus callosum uncovers dysmyelinated axons as well as neurodegeneration, evidenced by the presence of axonal spheroids. Histopathological examination of 11-week-old mice reveals increased axonal and myelin staining in the cortex, increase of neuronal cell density in layer V and increase of microglial cell densities throughout the brain, suggesting that early developmental changes contribute to disease. By 10-months of age, the neuronal cell density normalizes, oligodendrocyte precursor cells increase in layers II-III and V and microglial densities remain elevated without an increase in astrocytes. Also, the age-dependent increase in CSF-1R+ neurons in cortical layer V is reduced. Moreover, the expression of Csf2, Csf3, Il27 and Il6 family cytokines is increased, consistent with microglia-mediated inflammation. These results demonstrate that the inactivation of one Csf1r allele is sufficient to cause an ALSP-like disease in mice. The Csf1r+/- mouse is a model of ALSP that will allow the critical events for disease development to be determined and permit rapid evaluation of therapeutic approaches. Furthermore

  8. A Small Motor Cortex Lesion Abolished Ocular Dominance Plasticity in the Adult Mouse Primary Visual Cortex and Impaired Experience-Dependent Visual Improvements.

    PubMed

    Pielecka-Fortuna, Justyna; Kalogeraki, Evgenia; Greifzu, Franziska; Löwel, Siegrid

    2015-01-01

    It was previously shown that a small lesion in the primary somatosensory cortex (S1) prevented both cortical plasticity and sensory learning in the adult mouse visual system: While 3-month-old control mice continued to show ocular dominance (OD) plasticity in their primary visual cortex (V1) after monocular deprivation (MD), age-matched mice with a small photothrombotically induced (PT) stroke lesion in S1, positioned at least 1 mm anterior to the anterior border of V1, no longer expressed OD-plasticity. In addition, in the S1-lesioned mice, neither the experience-dependent increase of the spatial frequency threshold ("visual acuity") nor of the contrast threshold ("contrast sensitivity") of the optomotor reflex through the open eye was present. To assess whether these plasticity impairments can also occur if a lesion is placed more distant from V1, we tested the effect of a PT-lesion in the secondary motor cortex (M2). We observed that mice with a small M2-lesion restricted to the superficial cortical layers no longer expressed an OD-shift towards the open eye after 7 days of MD in V1 of the lesioned hemisphere. Consistent with previous findings about the consequences of an S1-lesion, OD-plasticity in V1 of the nonlesioned hemisphere of the M2-lesioned mice was still present. In addition, the experience-dependent improvements of both visual acuity and contrast sensitivity of the open eye were severely reduced. In contrast, sham-lesioned mice displayed both an OD-shift and improvements of visual capabilities of their open eye. To summarize, our data indicate that even a very small lesion restricted to the superficial cortical layers and more than 3mm anterior to the anterior border of V1 compromised V1-plasticity and impaired learning-induced visual improvements in adult mice. Thus both plasticity phenomena cannot only depend on modality-specific and local nerve cell networks but are clearly influenced by long-range interactions even from distant brain regions.

  9. A Small Motor Cortex Lesion Abolished Ocular Dominance Plasticity in the Adult Mouse Primary Visual Cortex and Impaired Experience-Dependent Visual Improvements.

    PubMed

    Pielecka-Fortuna, Justyna; Kalogeraki, Evgenia; Greifzu, Franziska; Löwel, Siegrid

    2015-01-01

    It was previously shown that a small lesion in the primary somatosensory cortex (S1) prevented both cortical plasticity and sensory learning in the adult mouse visual system: While 3-month-old control mice continued to show ocular dominance (OD) plasticity in their primary visual cortex (V1) after monocular deprivation (MD), age-matched mice with a small photothrombotically induced (PT) stroke lesion in S1, positioned at least 1 mm anterior to the anterior border of V1, no longer expressed OD-plasticity. In addition, in the S1-lesioned mice, neither the experience-dependent increase of the spatial frequency threshold ("visual acuity") nor of the contrast threshold ("contrast sensitivity") of the optomotor reflex through the open eye was present. To assess whether these plasticity impairments can also occur if a lesion is placed more distant from V1, we tested the effect of a PT-lesion in the secondary motor cortex (M2). We observed that mice with a small M2-lesion restricted to the superficial cortical layers no longer expressed an OD-shift towards the open eye after 7 days of MD in V1 of the lesioned hemisphere. Consistent with previous findings about the consequences of an S1-lesion, OD-plasticity in V1 of the nonlesioned hemisphere of the M2-lesioned mice was still present. In addition, the experience-dependent improvements of both visual acuity and contrast sensitivity of the open eye were severely reduced. In contrast, sham-lesioned mice displayed both an OD-shift and improvements of visual capabilities of their open eye. To summarize, our data indicate that even a very small lesion restricted to the superficial cortical layers and more than 3mm anterior to the anterior border of V1 compromised V1-plasticity and impaired learning-induced visual improvements in adult mice. Thus both plasticity phenomena cannot only depend on modality-specific and local nerve cell networks but are clearly influenced by long-range interactions even from distant brain regions

  10. Effects of cyclophosphamide and acrolein in organoid cultures of mouse limb bud cells grown in the presence of adult rat hepatocytes.

    PubMed

    Ghaida, J; Merker, H J

    1992-01-01

    The effects were evaluated of cyclophosphamide (CPA) and its metabolite, acrolein, on chondrogenesis in organoid cultures of mouse limb bud mesenchymal cells co-cultured with non-enzymatically isolated adult rat hepatocytes. The studies were conducted with or without the simultaneous addition of 2-mercaptoethanesulphonic acid sodium (mesna) or glutathione (GSH). Alcian blue binding assay and light and electron microscopic techniques were used. Increasing concentrations of the two compounds (bioactivated CPA, 18-180 mum; acrolein, 50-500 mum) led to a dose-dependent inhibition of chondrogenesis associated with cellular dedifferentiation and/or cytotoxicity. Addition of mesna (1 mm) or GSH (1 mm) partially protected the cultures against CPA and acrolein. However, the protective effect depended on the dose of CPA or acrolein used. A higher protection was observed with mesna than with GSH, and the effect was more pronounced with acrolein than with CPA. The morphological findings suggested that CPA and acrolein acted by different mechanisms. Bioactivated CPA primarily inhibited the differentiation process, whereas acrolein exhibited a high cytotoxic activity affecting particularly monolayer cells that normally grow on the periphery of the cultures. These findings suggest that acrolein possesses a specific mode of action directed towards this type of cell. This could be explained by the specific shape and/or behaviour of the cells (i.e. cytoskeletal arrangement, proliferation rate, migration activity, intercellular communication pattern, etc.). The results demonstrated that the cell system used was suitable for the performance of cytotoxicity and teratogenicity studies such as those conducted with CPA and acrolein.

  11. Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: a study in the adult mouse brain.

    PubMed

    Fukaya, M; Watanabe, M

    2000-10-30

    Postsynaptic density (PSD)-95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits. PMID:11027400

  12. Calorie restriction and susceptibility to intact pathogens

    PubMed Central

    2008-01-01

    Long-term calorie restriction (CR) causes numerous physiological changes that ultimately increase mean and maximum lifespan of most species examined to date. One physiological change that occurs with CR is enhanced immune function, as tested using antigens and mitogens to stimulate an immune response. Fewer studies have used intact pathogen exposure to test whether the enhanced capacity of the immune response during CR actually decreases susceptibility of hosts to their pathogens. So far, studies using intact bacteria, virus, and helminth worm exposure indicate that, despite similar or enhanced immune system function, CR hosts are more susceptible to infection by intact pathogens than their fully fed counterparts. Long-term CR studies that examine susceptibility to a variety of parasite taxa will help determine if direct CR or CR mimetics will be beneficial to people living in pathogen-rich environments. PMID:19424864

  13. Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney.

    PubMed

    Georgas, Kylie; Rumballe, Bree; Wilkinson, Lorine; Chiu, Han Sheng; Lesieur, Emmanuelle; Gilbert, Thierry; Little, Melissa H

    2008-11-01

    The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

  14. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  15. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  16. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  17. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  18. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  19. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    EPA Science Inventory

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  20. 46 CFR 178.320 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements. 178.320 Section 178.320... TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.320 Intact stability... stability proof test in accordance with § 178.330 of this part in the presence of a Coast Guard...

  1. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements-general. 178.310 Section... (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.310 Intact stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  2. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements-general. 178.310 Section... (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.310 Intact stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  3. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-general. 178.310 Section... (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.310 Intact stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  4. Feasible pickup from intact ossicular chain with floating piezoelectric microphone

    PubMed Central

    2012-01-01

    Objectives Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Methods Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. Results The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. Conclusions It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size. PMID:22353161

  5. Small Particles Intact Capture Experiment (SPICE)

    NASA Technical Reports Server (NTRS)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  6. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  7. Silica Aerogel Captures Cosmic Dust Intact

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1994-01-01

    The mesostructure of silica aerogel resembles stings of grapes, ranging in size from 10 to 100 angstrom. This fine mesostructure transmits nearly 90 percent of incident light in the visible, while providing sufficiently gentle dissipation of the kinetric energy of hypervelocity cosmic dust particles to permit their intact capture. We introduced silica aerogel in 1987 as capture medium to take advantage of its low density, fine mesostruicture and most importantly, its transparency, allowing optical location of captured micron sized particles.

  8. Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation

    PubMed Central

    Sriraman, Kalpana; Anand, Sandhya; Bhutda, Smita

    2015-01-01

    This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin−/CD45−, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy. PMID:25779995

  9. Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation.

    PubMed

    Sriraman, Kalpana; Bhartiya, Deepa; Anand, Sandhya; Bhutda, Smita

    2015-07-01

    This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin-/CD45-, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy.

  10. Measuring mitochondrial function in intact cardiac myocytes

    PubMed Central

    Dedkova, Elena N.; Blatter, Lothar A.

    2011-01-01

    Mitochondria are involved in cellular functions that go beyond the traditional role of these organelles as the power plants of the cell. Mitochondria have been implicated in several human diseases, including cardiac dysfunction, and play a role in the aging process. Many aspects of our knowledge of mitochondria stem from studies performed on the isolated organelle. Their relative inaccessibility imposes experimental difficulties to study mitochondria in their natural environment – the cytosol of intact cells – and has hampered a comprehensive understanding of the plethora of mitochondrial functions. Here we review currently available methods to study mitochondrial function in intact cardiomyocytes. These methods primarily use different flavors of fluorescent dyes and genetically encoded fluorescent proteins in conjunction with high-resolution imaging techniques. We review methods to study mitochondrial morphology, mitochondrial membrane potential, Ca2+ and Na+ signaling, mitochondrial pH regulation, redox state and ROS production, NO signaling, oxygen consumption, ATP generation and the activity of the mitochondrial permeability transition pore. Where appropriate we complement this review on intact myocytes with seminal studies that were performed on isolated mitochondria, permeabilized cells, and in whole hearts. PMID:21964191

  11. Multi-View Intact Space Learning.

    PubMed

    Xu, Chang; Tao, Dacheng; Xu, Chao

    2015-12-01

    It is practical to assume that an individual view is unlikely to be sufficient for effective multi-view learning. Therefore, integration of multi-view information is both valuable and necessary. In this paper, we propose the Multi-view Intact Space Learning (MISL) algorithm, which integrates the encoded complementary information in multiple views to discover a latent intact representation of the data. Even though each view on its own is insufficient, we show theoretically that by combing multiple views we can obtain abundant information for latent intact space learning. Employing the Cauchy loss (a technique used in statistical learning) as the error measurement strengthens robustness to outliers. We propose a new definition of multi-view stability and then derive the generalization error bound based on multi-view stability and Rademacher complexity, and show that the complementarity between multiple views is beneficial for the stability and generalization. MISL is efficiently optimized using a novel Iteratively Reweight Residuals (IRR) technique, whose convergence is theoretically analyzed. Experiments on synthetic data and real-world datasets demonstrate that MISL is an effective and promising algorithm for practical applications. PMID:26539856

  12. Calcium Signaling in Intact Dorsal Root Ganglia

    PubMed Central

    Gemes, Geza; Rigaud, Marcel; Koopmeiners, Andrew S.; Poroli, Mark J.; Zoga, Vasiliki; Hogan, Quinn H.

    2013-01-01

    Background Ca2+ is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca2+ signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca2+ recording techniques use high K+ or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. Methods Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. Results Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca2+ transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Aβ-type neurons. Unitary Ca2+ transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca2+ transients in C-type neurons, but increased transients in Aβ-type neurons. Conclusions Refined observation of Ca2+ signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state. PMID:20526180

  13. True navigation in migrating gulls requires intact olfactory nerves.

    PubMed

    Wikelski, Martin; Arriero, Elena; Gagliardo, Anna; Holland, Richard A; Huttunen, Markku J; Juvaste, Risto; Mueller, Inge; Tertitski, Grigori; Thorup, Kasper; Wild, Martin; Alanko, Markku; Bairlein, Franz; Cherenkov, Alexander; Cameron, Alison; Flatz, Reinhard; Hannila, Juhani; Hüppop, Ommo; Kangasniemi, Markku; Kranstauber, Bart; Penttinen, Maija-Liisa; Safi, Kamran; Semashko, Vladimir; Schmid, Heidi; Wistbacka, Ralf

    2015-01-01

    During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances. PMID:26597351

  14. True navigation in migrating gulls requires intact olfactory nerves

    PubMed Central

    Wikelski, Martin; Arriero, Elena; Gagliardo, Anna; Holland, Richard A.; Huttunen, Markku J.; Juvaste, Risto; Mueller, Inge; Tertitski, Grigori; Thorup, Kasper; Wild, Martin; Alanko, Markku; Bairlein, Franz; Cherenkov, Alexander; Cameron, Alison; Flatz, Reinhard; Hannila, Juhani; Hüppop, Ommo; Kangasniemi, Markku; Kranstauber, Bart; Penttinen, Maija-Liisa; Safi, Kamran; Semashko, Vladimir; Schmid, Heidi; Wistbacka, Ralf

    2015-01-01

    During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances. PMID:26597351

  15. True navigation in migrating gulls requires intact olfactory nerves.

    PubMed

    Wikelski, Martin; Arriero, Elena; Gagliardo, Anna; Holland, Richard A; Huttunen, Markku J; Juvaste, Risto; Mueller, Inge; Tertitski, Grigori; Thorup, Kasper; Wild, Martin; Alanko, Markku; Bairlein, Franz; Cherenkov, Alexander; Cameron, Alison; Flatz, Reinhard; Hannila, Juhani; Hüppop, Ommo; Kangasniemi, Markku; Kranstauber, Bart; Penttinen, Maija-Liisa; Safi, Kamran; Semashko, Vladimir; Schmid, Heidi; Wistbacka, Ralf

    2015-01-01

    During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances.

  16. Quantitative proteomic analysis of intact plastids.

    PubMed

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described. PMID:24136541

  17. Quantitative proteomic analysis of intact plastids.

    PubMed

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  18. 50 CFR 622.276 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated,...

  19. 50 CFR 622.381 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.381 Section 622... Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia... head and fins intact. Such fish may be eviscerated, gilled, and scaled, but must otherwise...

  20. 50 CFR 622.276 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated,...

  1. 7 CFR 160.29 - Containers to remain intact.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Containers to remain intact. 160.29 Section 160.29... STANDARDS FOR NAVAL STORES Analysis, Inspection, and Grading on Request § 160.29 Containers to remain intact... the containers holding such naval stores remain intact as sampled until the analysis,...

  2. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  3. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  4. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  5. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  6. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  7. Induction of Overt Menstruation in Intact Mice

    PubMed Central

    Rudolph, Marion; Döcke, Wolf-Dietrich; Müller, Andrea; Menning, Astrid; Röse, Lars; Zollner, Thomas Matthias; Gashaw, Isabella

    2012-01-01

    The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply. PMID:22412950

  8. An Empirical Failure Criterion for Intact Rocks

    NASA Astrophysics Data System (ADS)

    Peng, Jun; Rong, Guan; Cai, Ming; Wang, Xiaojiang; Zhou, Chuangbing

    2014-03-01

    The parameter m i is an important rock property parameter required for use of the Hoek-Brown failure criterion. The conventional method for determining m i is to fit a series of triaxial compression test data. In the absence of laboratory test data, guideline charts have been provided by Hoek to estimate the m i value. In the conventional Hoek-Brown failure criterion, the m i value is a constant for a given rock. It is observed that using a constant m i may not fit the triaxial compression test data well for some rocks. In this paper, a negative exponent empirical model is proposed to express m i as a function of confinement, and this exercise leads us to a new empirical failure criterion for intact rocks. Triaxial compression test data of various rocks are used to fit parameters of this model. It is seen that the new empirical failure criterion fits the test data better than the conventional Hoek-Brown failure criterion for intact rocks. The conventional Hoek-Brown criterion fits the test data well in the high-confinement region but fails to match data well in the low-confinement and tension regions. In particular, it overestimates the uniaxial compressive strength (UCS) and the uniaxial tensile strength of rocks. On the other hand, curves fitted by the proposed empirical failure criterion match test data very well, and the estimated UCS and tensile strength agree well with test data.

  9. Recollections of Parent Characteristics and Attachment Patterns for College Women of Intact vs. Non-Intact Families

    ERIC Educational Resources Information Center

    Kilmann, Peter R.; Carranza, Laura V.; Vendemia, Jennifer M. C.

    2006-01-01

    This study contrasted offsprings' attachment patterns and recollections of parent characteristics in two college samples: 147 females from intact biological parents and 157 females of parental divorce. Secure females from intact or non-intact families rated parents positively, while insecure females rated parents as absent, distant, and demanding.…

  10. Postnatal day 7 ethanol treatment causes persistent reductions in adult mouse brain volume and cortical neurons with sex specific effects on neurogenesis.

    PubMed

    Coleman, Leon G; Oguz, Ipek; Lee, Joohwi; Styner, Martin; Crews, Fulton T

    2012-09-01

    Ethanol treatment on postnatal day seven (P7) causes robust brain cell death and is a model of late gestational alcohol exposure (Ikonomidou et al., 2000). To investigate the long-term effects of P7 ethanol treatment on adult brain, mice received either two doses of saline or ethanol on P7 (2.5 g/kg, s.c., 2 h apart) and were assessed as adults (P82) for brain volume (using postmortem MRI) and cellular architecture (using immunohistochemistry). Adult mice that received P7 ethanol had reduced MRI total brain volume (4%) with multiple brain regions being reduced in both males and females. Immunohistochemistry indicated reduced frontal cortical parvalbumin immunoreactive (PV + IR) interneurons (18-33%) and reduced Cux1+IR layer II pyramidal neurons (15%) in both sexes. Interestingly, markers of adult hippocampal neurogenesis differed between sexes, with only ethanol treated males showing increased doublecortin and Ki67 expression (52 and 57% respectively) in the dentate gyrus, consistent with increased neurogenesis compared to controls. These findings suggest that P7 ethanol treatment causes persistent reductions in adult brain volume and frontal cortical neurons in both males and females. Increased adult neurogenesis in males, but not females, is consistent with differential adaptive responses to P7 ethanol toxicity between the sexes. One day of ethanol exposure, e.g. P7, causes persistent adult brain dysmorphology.

  11. Oligodeoxynucleotide Probes for Detecting Intact Cells

    NASA Technical Reports Server (NTRS)

    Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

    2004-01-01

    A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

  12. NIH mouse study finds gut microorganisms may determine cancer treatment outcome

    Cancer.gov

    An intact gut commensal microbiota, which is a population of microorganisms living in the intestine, is required for optimal response to cancer therapy, according to a mouse study by scientists at the National Cancer Institute (NCI)

  13. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  14. Protein methylation reactions in intact pea chloroplasts

    SciTech Connect

    Niemi, K.J. )

    1989-04-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.

  15. Drilling to gabbro in intact ocean crust.

    PubMed

    Wilson, Douglas S; Teagle, Damon A H; Alt, Jeffrey C; Banerjee, Neil R; Umino, Susumu; Miyashita, Sumio; Acton, Gary D; Anma, Ryo; Barr, Samantha R; Belghoul, Akram; Carlut, Julie; Christie, David M; Coggon, Rosalind M; Cooper, Kari M; Cordier, Carole; Crispini, Laura; Durand, Sedelia Rodriguez; Einaudi, Florence; Galli, Laura; Gao, Yongjun; Geldmacher, Jörg; Gilbert, Lisa A; Hayman, Nicholas W; Herrero-Bervera, Emilio; Hirano, Nobuo; Holter, Sara; Ingle, Stephanie; Jiang, Shijun; Kalberkamp, Ulrich; Kerneklian, Marcie; Koepke, Jürgen; Laverne, Christine; Vasquez, Haroldo L Lledo; Maclennan, John; Morgan, Sally; Neo, Natsuki; Nichols, Holly J; Park, Sung-Hyun; Reichow, Marc K; Sakuyama, Tetsuya; Sano, Takashi; Sandwell, Rachel; Scheibner, Birgit; Smith-Duque, Chris E; Swift, Stephen A; Tartarotti, Paola; Tikku, Anahita A; Tominaga, Masako; Veloso, Eugenio A; Yamasaki, Toru; Yamazaki, Shusaku; Ziegler, Christa

    2006-05-19

    Sampling an intact sequence of oceanic crust through lavas, dikes, and gabbros is necessary to advance the understanding of the formation and evolution of crust formed at mid-ocean ridges, but it has been an elusive goal of scientific ocean drilling for decades. Recent drilling in the eastern Pacific Ocean in Hole 1256D reached gabbro within seismic layer 2, 1157 meters into crust formed at a superfast spreading rate. The gabbros are the crystallized melt lenses that formed beneath a mid-ocean ridge. The depth at which gabbro was reached confirms predictions extrapolated from seismic experiments at modern mid-ocean ridges: Melt lenses occur at shallower depths at faster spreading rates. The gabbros intrude metamorphosed sheeted dikes and have compositions similar to the overlying lavas, precluding formation of the cumulate lower oceanic crust from melt lenses so far penetrated by Hole 1256D.

  16. Drilling to gabbro in intact ocean crust.

    PubMed

    Wilson, Douglas S; Teagle, Damon A H; Alt, Jeffrey C; Banerjee, Neil R; Umino, Susumu; Miyashita, Sumio; Acton, Gary D; Anma, Ryo; Barr, Samantha R; Belghoul, Akram; Carlut, Julie; Christie, David M; Coggon, Rosalind M; Cooper, Kari M; Cordier, Carole; Crispini, Laura; Durand, Sedelia Rodriguez; Einaudi, Florence; Galli, Laura; Gao, Yongjun; Geldmacher, Jörg; Gilbert, Lisa A; Hayman, Nicholas W; Herrero-Bervera, Emilio; Hirano, Nobuo; Holter, Sara; Ingle, Stephanie; Jiang, Shijun; Kalberkamp, Ulrich; Kerneklian, Marcie; Koepke, Jürgen; Laverne, Christine; Vasquez, Haroldo L Lledo; Maclennan, John; Morgan, Sally; Neo, Natsuki; Nichols, Holly J; Park, Sung-Hyun; Reichow, Marc K; Sakuyama, Tetsuya; Sano, Takashi; Sandwell, Rachel; Scheibner, Birgit; Smith-Duque, Chris E; Swift, Stephen A; Tartarotti, Paola; Tikku, Anahita A; Tominaga, Masako; Veloso, Eugenio A; Yamasaki, Toru; Yamazaki, Shusaku; Ziegler, Christa

    2006-05-19

    Sampling an intact sequence of oceanic crust through lavas, dikes, and gabbros is necessary to advance the understanding of the formation and evolution of crust formed at mid-ocean ridges, but it has been an elusive goal of scientific ocean drilling for decades. Recent drilling in the eastern Pacific Ocean in Hole 1256D reached gabbro within seismic layer 2, 1157 meters into crust formed at a superfast spreading rate. The gabbros are the crystallized melt lenses that formed beneath a mid-ocean ridge. The depth at which gabbro was reached confirms predictions extrapolated from seismic experiments at modern mid-ocean ridges: Melt lenses occur at shallower depths at faster spreading rates. The gabbros intrude metamorphosed sheeted dikes and have compositions similar to the overlying lavas, precluding formation of the cumulate lower oceanic crust from melt lenses so far penetrated by Hole 1256D. PMID:16627698

  17. Distribution of immunoreactive glutamine synthetase in the adult human and mouse brain. Qualitative and quantitative observations with special emphasis on extra-astroglial protein localization.

    PubMed

    Bernstein, Hans-Gert; Bannier, Jana; Meyer-Lotz, Gabriela; Steiner, Johann; Keilhoff, Gerburg; Dobrowolny, Henrik; Walter, Martin; Bogerts, Bernhard

    2014-11-01

    Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

  18. Gsx2 controls region-specific activation of neural stem cells and injury-induced neurogenesis in the adult subventricular zone

    PubMed Central

    López-Juárez, Alejandro; Howard, Jennifer; Ullom, Kristy; Howard, Lindsey; Grande, Andrew; Pardo, Andrea; Waclaw, Ronald; Sun, Yu-Yo; Yang, Dianer; Kuan, Chia-Yi; Campbell, Kenneth; Nakafuku, Masato

    2013-01-01

    Neural stem cells (NSCs) reside in widespread regions along the lateral ventricle and generate diverse olfactory bulb (OB) interneuron subtypes in the adult mouse brain. Molecular mechanisms underlying their regional diversity, however, are not well understood. Here we show that the homeodomain transcription factor Gsx2 plays a crucial role in the region-specific control of adult NSCs in both persistent and injury-induced neurogenesis. In the intact brain, Gsx2 is expressed in a regionally restricted subset of NSCs and promotes the activation and lineage progression of stem cells, thereby controlling the production of selective OB neuron subtypes. Moreover, Gsx2 is ectopically induced in damaged brains outside its normal expression domains and is required for injury-induced neurogenesis in the subventricular zone (SVZ). These results demonstrate that mobilization of adult NSCs is controlled in a region-specific manner and that distinct mechanisms operate in continuous and injury-induced neurogenesis in the adult brain. PMID:23723414

  19. Gsx2 controls region-specific activation of neural stem cells and injury-induced neurogenesis in the adult subventricular zone.

    PubMed

    López-Juárez, Alejandro; Howard, Jennifer; Ullom, Kristy; Howard, Lindsey; Grande, Andrew; Pardo, Andrea; Waclaw, Ronald; Sun, Yu-Yo; Yang, Dianer; Kuan, Chia-Yi; Campbell, Kenneth; Nakafuku, Masato

    2013-06-01

    Neural stem cells (NSCs) reside in widespread regions along the lateral ventricle and generate diverse olfactory bulb (OB) interneuron subtypes in the adult mouse brain. Molecular mechanisms underlying their regional diversity, however, are not well understood. Here we show that the homeodomain transcription factor Gsx2 plays a crucial role in the region-specific control of adult NSCs in both persistent and injury-induced neurogenesis. In the intact brain, Gsx2 is expressed in a regionally restricted subset of NSCs and promotes the activation and lineage progression of stem cells, thereby controlling the production of selective OB neuron subtypes. Moreover, Gsx2 is ectopically induced in damaged brains outside its normal expression domains and is required for injury-induced neurogenesis in the subventricular zone (SVZ). These results demonstrate that mobilization of adult NSCs is controlled in a region-specific manner and that distinct mechanisms operate in continuous and injury-induced neurogenesis in the adult brain.

  20. Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb.

    PubMed

    Liang, Yajie; Li, Kaizhen; Riecken, Kristoffer; Maslyukov, Anatoliy; Gomez-Nicola, Diego; Kovalchuk, Yury; Fehse, Boris; Garaschuk, Olga

    2016-07-01

    The behavior of adult-born cells can be easily monitored in cell culture or in lower model organisms, but longitudinal observation of individual mammalian adult-born cells in their native microenvironment still proves to be a challenge. Here we have established an approach named optical cell positioning system for long-term in vivo single-cell tracking, which integrates red-green-blue cell labeling with repeated angiography. By combining this approach with in vivo two-photon imaging technique, we characterized the in vivo migration patterns of adult-born neurons in the olfactory bulb. In contrast to the traditional view of mere radial migration of adult-born cells within the bulb, we found that juxtaglomerular cells switch from radial migration to long distance lateral migration upon arrival in their destination layer. This unique long-distance lateral migration has characteristic temporal (stop-and-go) and spatial (migratory, unidirectional or multidirectional) patterns, with a clear cell age-dependent decrease in the migration speed. The active migration of adult-born cells coincides with the time period of initial fate determination and is likely to impact on the integration sites of adult-born cells, their odor responsiveness, as well as their survival rate.

  1. A diet enriched in polyphenols and polyunsaturated fatty acids, LMN diet, induces neurogenesis in the subventricular zone and hippocampus of adult mouse brain.

    PubMed

    Valente, Tony; Hidalgo, Juan; Bolea, Irene; Ramirez, Bartolomé; Anglés, Neus; Reguant, Jordi; Morelló, José Ramón; Gutiérrez, Cristina; Boada, Mercè; Unzeta, Mercedes

    2009-01-01

    At present it is widely accepted that there are at least two neurogenic sites in the adult mammalian brain: the subventricular zone (SVZ) of lateral ventricles and the subgranular zone (SGZ) of the hippocampus dentate gyrus. The adult proliferation rate declines with aging and is altered in several neurodegenerative pathologies including Alzheimer's disease. The aim of this work was to study whether a natural diet rich in polyphenols and polyunsaturated fatty acids (LMN diet) can modulate neurogenesis in adult mice and give insight into putative mechanisms. Results with BrdU and PCNA demonstrated that the LMN fed mice had more newly generated cells in the SVZ and SGZ, and those with DCX (undifferentiated neurons) and tyrosine hydroxylase, calretinin, and calbindin (differentiated neurons) immunostainings and western blots demonstrated a significant effect on neuronal populations, strongly supporting a positive role of the LMN diet on adult neurogenesis. In primary rat neuron cultures, the LMN cream dramatically protected against damage caused by both hydrogen peroxide and Abeta(1-42), demonstrating a potent antioxidant effect that could play a major role in the normal adult neurogenesis and, moreover, the LMN diet could have a significant effect combating the cognitive function decline during both aging and neurodegenerative diseases such as Alzheimer's disease.

  2. Teaching basic neurophysiology using intact earthworms.

    PubMed

    Kladt, Nikolay; Hanslik, Ulrike; Heinzel, Hans-Georg

    2010-01-01

    Introductory neurobiology courses face the problem that practical exercises often require expensive equipment, dissections, and a favorable student-instructor ratio. Furthermore, the duration of an experiment might exceed available time or the level of required expertise is too high to successfully complete the experiment. As a result, neurobiological experiments are commonly replaced by models and simulations, or provide only very basic experiments, such as the frog sciatic nerve preparation, which are often time consuming and tedious. Action potential recordings in giant fibers of intact earthworms (Lumbricus terrestris) circumvent many of these problems and result in a nearly 100% success rate. Originally, these experiments were introduced as classroom exercises by Charles Drewes in 1978 using awake, moving earthworms. In 1990, Hans-Georg Heinzel described further experiments using anesthetized earthworms. In this article, we focus on the application of these experiments as teaching tools for basic neurobiology courses. We describe and extend selected experiments, focusing on specific neurobiological principles with experimental protocols optimized for classroom application. Furthermore, we discuss our experience using these experiments in animal physiology and various neurobiology courses at the University of Bonn. PMID:23494516

  3. Ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb.

    PubMed

    Doyle, Wayne I; Hammen, Gary F; Meeks, Julian P

    2014-01-01

    The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm(3)) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

  4. Ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb.

    PubMed

    Doyle, Wayne I; Hammen, Gary F; Meeks, Julian P

    2014-01-01

    The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm(3)) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior. PMID:25145699

  5. Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

    PubMed Central

    Doyle, Wayne I.; Hammen, Gary F.; Meeks, Julian P.

    2014-01-01

    The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior. PMID:25145699

  6. Response of adult mouse uterus to early disruption of estrogen receptor-alpha signaling is influenced by Krüppel-like factor 9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is a well-acknowledged early event in tumor initi...

  7. Positive youth development, life satisfaction, and problem behaviors of adolescents in intact and non-intact families in Hong Kong.

    PubMed

    Shek, Daniel T L; Leung, Hildie

    2013-01-01

    This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families. PMID:24400264

  8. Positive Youth Development, Life Satisfaction, and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

    PubMed Central

    Shek, Daniel T. L.; Leung, Hildie

    2013-01-01

    This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families. PMID:24400264

  9. Adult glucocorticoid exposure leads to transcriptional and DNA methylation changes in nuclear steroid receptors in the hippocampus and kidney of mouse male offspring.

    PubMed

    Petropoulos, Sophie; Matthews, Stephen G; Szyf, Moshe

    2014-02-01

    Synthetic glucocorticoids (sGCs) are commonly prescribed for the management of inflammatory and endocrine disorders. However, nothing is known regarding the effects of sGC on adult germline methylome and whether these effects can be transmitted to the next generation. We hypothesized that administration of sGC to adult male mice alters DNA methylation in mature sperm and modifies the transcription and methylation of steroid receptors in male F1 offspring. Adult C57BL/6 males (n = 10/group) were injected on five consecutive days with 1 mg/kg sGC (i.e., dexamethasone) or vehicle and euthanized 35 or 60 days after initial treatment or bred with control females (60 days postinitial treatment; n = 5/group). A significant increase in global non-CpG methylation was observed in F0 sperm 60 days following sGC treatment. In the hippocampus and kidney of Postnatal Day 50 (PND50) and PND240 male offspring derived from fathers exposed to sGC, significant differences in mineralocorticoid receptor (Nr3c2; Mr), estrogen alpha receptor (Nr3a1; Ers1), and glucocorticoid receptor (Nr3c1; Gr) expression were observed. Furthermore, significant demethylation in regulatory regions of Mr, Gr, and Esr1 was observed in the PND50 kidney derived from fathers exposed to sGC. This is the first demonstration that paternal pharmacological exposure to sGC can alter the expression and DNA methylation of nuclear steroid receptors in brain and somatic tissues of offspring. These findings provide proof of principle that adult male exposure to sGC can affect DNA methylation and gene expression in offspring, indicating the possibility that adult experiences that evoke increases in endogenous glucocorticoid (i.e., stress) might have similar effects.

  10. Transient Suppression of Dbx1 PreBötzinger Interneurons Disrupts Breathing in Adult Mice

    PubMed Central

    Vann, Nikolas C.; Pham, Francis D.; Hayes, John A.; Kottick, Andrew; Del Negro, Christopher A.

    2016-01-01

    Interneurons derived from Dbx1-expressing precursors located in the brainstem preBötzinger complex (preBötC) putatively form the core oscillator for inspiratory breathing movements. We tested this Dbx1 core hypothesis by expressing archaerhodopsin in Dbx1-derived interneurons and then transiently hyperpolarizing these neurons while measuring respiratory rhythm in vitro or breathing in vagus-intact adult mice. Transient illumination of the preBötC interrupted inspiratory rhythm in both slice preparations and sedated mice. In awake mice, light application reduced breathing frequency and prolonged the inspiratory duration. Support for the Dbx1 core hypothesis previously came from embryonic and perinatal mouse experiments, but these data suggest that Dbx1-derived preBötC interneurons are rhythmogenic in adult mice too. The neural origins of breathing behavior can be attributed to a localized and genetically well-defined interneuron population. PMID:27611210

  11. Transient Suppression of Dbx1 PreBötzinger Interneurons Disrupts Breathing in Adult Mice.

    PubMed

    Vann, Nikolas C; Pham, Francis D; Hayes, John A; Kottick, Andrew; Del Negro, Christopher A

    2016-01-01

    Interneurons derived from Dbx1-expressing precursors located in the brainstem preBötzinger complex (preBötC) putatively form the core oscillator for inspiratory breathing movements. We tested this Dbx1 core hypothesis by expressing archaerhodopsin in Dbx1-derived interneurons and then transiently hyperpolarizing these neurons while measuring respiratory rhythm in vitro or breathing in vagus-intact adult mice. Transient illumination of the preBötC interrupted inspiratory rhythm in both slice preparations and sedated mice. In awake mice, light application reduced breathing frequency and prolonged the inspiratory duration. Support for the Dbx1 core hypothesis previously came from embryonic and perinatal mouse experiments, but these data suggest that Dbx1-derived preBötC interneurons are rhythmogenic in adult mice too. The neural origins of breathing behavior can be attributed to a localized and genetically well-defined interneuron population. PMID:27611210

  12. Experimental serpentinization of intact dunite cores

    NASA Astrophysics Data System (ADS)

    Luhmann, A. J.; Tutolo, B. M.; Kong, X. Z.; Bagley, B. C.; Schaen, A. T.; Saar, M. O.; Seyfried, W. E., Jr.

    2014-12-01

    Serpentinization in ultramafic-hosted hydrothermal systems, such as Lost City, produces relatively cool and alkaline fluids that support diverse ecosystems. To simulate serpentinization in such systems, we conducted single-pass, flow-through experiments on dunite cores cut out of a sample from Jackson County, North Carolina. Experimental seawater prepared using laboratory-grade reagents and standards was pumped through a core at 150ºC and 150 bar pore-fluid outlet pressure at a flow rate of 0.01 ml/min. An additional experiment will be conducted at 200ºC. At 150ºC, permeability decreased by 2.3 times with reaction progress over the course of the 36 day experiment. Fluid-rock reaction generally produced CO2, H2, CH4, and CO concentrations of 100 μmol/kg, up to 40 μmol/kg, 2 μmol/kg, and less than 1 μmol/kg, respectively. Outlet fluid chemistry was relatively stable, except for initial peaks in Al, Ba, Fe, Mn, and Si. pH of outlet fluids increased with reaction progress, but it was always lower (6.9-7.4) than the initial seawater (7.8). X-ray computed tomography scans were/will be collected for both pre- and post-experimental cores. The combination of flow-through experiments on solid, intact rock cores cut out of natural samples and X-ray tomography permits visualization and quantification of mineralogical changes and flow path evolution during serpentinization. This approach further permits physical and chemical processes to be documented on a fine scale to better understand feedbacks between chemical reactions and flow fields, with implications for ultramafic-hosted hydrothermal systems.

  13. Gestational and Lactational Exposure to Atrazine via the Drinking Water Causes Specific Behavioral Deficits and Selectively Alters Monoaminergic Systems in C57BL/6 Mouse Dams, Juvenile and Adult Offspring

    PubMed Central

    Krishna, Saritha; Ye, Xiaoqin; Filipov, Nikolay M.

    2014-01-01

    Atrazine (ATR) is one of the most frequently detected pesticides in the U.S. water supply. This study aimed to investigate neurobehavioral and neurochemical effects of ATR in C57BL/6 mouse offspring and dams exposed to a relatively low (3 mg/l, estimated intake 1.4 mg/kg/day) concentration of ATR via the drinking water (DW) from gestational day 6 to postnatal day (PND) 23. Behavioral tests included open field, pole, grip strength, novel object recognition (NOR), forced swim, and marble burying tests. Maternal weight gain and offspring (PND21, 35, and 70) body or brain weights were not affected by ATR. However, ATR-treated dams exhibited decreased NOR performance and a trend toward hyperactivity. Juvenile offspring (PND35) from ATR-exposed dams were hyperactive (both sexes), spent less time swimming (males), and buried more marbles (females). In adult offspring (PND70), the only behavioral change was a sex-specific (females) decreased NOR performance by ATR. Neurochemically, a trend toward increased striatal dopamine (DA) in dams and a significant increase in juvenile offspring (both sexes) was observed. Additionally, ATR exposure decreased perirhinal cortex serotonin in the adult female offspring. These results suggest that perinatal DW exposure to ATR targets the nigrostriatal DA pathway in dams and, especially, juvenile offspring, alters dams’ cognitive performance, induces sex-selective changes involving motor and emotional functions in juvenile offspring, and decreases cognitive ability of adult female offspring, with the latter possibly associated with altered perirhinal cortex serotonin homeostasis. Overall, ATR exposure during gestation and lactation may cause adverse nervous system effects to both offspring and dams. PMID:24913803

  14. Body condition and habitat use by Hermann's tortoises in burnt and intact habitats

    PubMed Central

    Lecq, S.; Ballouard, J.-M.; Caron, S.; Livoreil, B.; Seynaeve, V.; Matthieu, L.-A.; Bonnet, X.

    2014-01-01

    In Mediterranean regions, fires threaten terrestrial tortoises. Nevertheless, varying proportions of adults survive fire; these surviving individuals can play a central role for population recovery. The regions devastated by fire often include important habitat of Hermann's tortoises (Testudo hermanni hermanni), so assessing the ability of survivors to persist is essential for conserving the species. Body-condition indices provide an integrative estimate of how well individuals cope with environmental variations and impacts, including fires. Between 2002 and 2009, we monitored Hermann's tortoises in intact and burnt habitats in southeastern France. In summer 2003, a strong fire ravaged half of the surveyed zone, providing an opportunity to compare body condition of tortoises between intact and burnt areas over time. Six years later, the impact of fire on vegetation was still marked; large trees were abundant in the intact area, whereas open shrub vegetation prevailed in the burnt area. In both areas, the mean body condition of tortoises fluctuated over time; however, there were no differences between the two areas. A radio-tracking experiment demonstrated that individuals from each area were residents, and not vagrants commuting between areas. We also assessed changes in body condition and microhabitat use in radio-tracked individuals. We found no significant differences between the tortoises living in the burnt and intact areas, despite subtle differences in habitat use. In conclusion: (i) surviving tortoises in an area ravaged by fire can maintain their body condition like individuals living in an intact area, and thus, individuals from burnt areas should not be translocated to supposedly better areas; and (ii) depopulated burnt areas are likely to be appropriate for population-augmentation programmes. PMID:27293640

  15. 50 CFR 622.186 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but...

  16. 50 CFR 622.186 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but...

  17. 50 CFR 622.247 - Landing golden crab intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing golden crab intact. 622.247... ATLANTIC Golden Crab Fishery of the South Atlantic Region § 622.247 Landing golden crab intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that golden crab on that vessel...

  18. 50 CFR 622.247 - Landing golden crab intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing golden crab intact. 622.247... ATLANTIC Golden Crab Fishery of the South Atlantic Region § 622.247 Landing golden crab intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that golden crab on that vessel...

  19. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  20. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  1. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  2. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  3. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  4. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  5. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  6. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  7. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  8. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations...

  9. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  10. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  11. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  12. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  13. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  14. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations...

  15. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  16. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  17. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  18. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  19. 50 CFR 622.455 - Landing spiny lobster intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing spiny lobster intact. 622.455... ATLANTIC Spiny Lobster Fishery of Puerto Rico and the U.S. Virgin Islands § 622.455 Landing spiny lobster intact. (a) A Caribbean spiny lobster in or from the Caribbean EEZ must be maintained with head...

  20. 50 CFR 622.455 - Landing spiny lobster intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing spiny lobster intact. 622.455... ATLANTIC Spiny Lobster Fishery of Puerto Rico and the U.S. Virgin Islands § 622.455 Landing spiny lobster intact. (a) A Caribbean spiny lobster in or from the Caribbean EEZ must be maintained with head...

  1. 50 CFR 622.10 - Landing fish intact--general.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH... landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified... intact. (a) Finfish in or from the Gulf EEZ or Caribbean EEZ, except as specified in paragraphs (b)...

  2. 50 CFR 622.10 - Landing fish intact--general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH... landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified... intact. (a) Finfish in or from the Gulf EEZ or Caribbean EEZ, except as specified in paragraphs (b)...

  3. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  4. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  5. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact longitudinal stability. 172.095 Section 172.095... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be shown by design calculations to have a longitudinal metacentric height (GM) in feet (meters) in...

  6. RNAi silencing of P/Q-type calcium channels in Purkinje neurons of adult mouse leads to episodic ataxia type 2.

    PubMed

    Salvi, Julie; Bertaso, Federica; Mausset-Bonnefont, Anne-Laure; Metz, Alexandra; Lemmers, Céline; Ango, Fabrice; Fagni, Laurent; Lory, Philippe; Mezghrani, Alexandre

    2014-08-01

    Episodic ataxia type-2 (EA2) is a dominantly inherited human neurological disorder caused by loss of function mutations in the CACNA1A gene, which encodes the CaV2.1 subunit of P/Q-type voltage-gated calcium channels. It remains however unknown whether the deficit of cerebellar CaV2.1 in adult is in direct link with the disease. To address this issue, we have used lentiviral based-vector RNA interference (RNAi) to knock-down CaV2.1 expression in the cerebellum of adult mice. We show that suppression of the P/Q-type channels in Purkinje neurons induced motor abnormalities, such as imbalance and ataxic gait. Interestingly, moderate channel suppression caused no basal ataxia, while β-adrenergic activation and exercise mimicked stress induced motor disorders. Moreover, stress-induced ataxia was stable, non-progressive and totally abolished by acetazolamide, a carbonic anhydrase inhibitor used to treat EA2. Altogether, these data reveal that P/Q-type channel suppression in adult mice supports the episodic status of EA2 disease.

  7. Isolation of Radial Glia-Like Neural Stem Cells from Fetal and Adult Mouse Forebrain via Selective Adhesion to a Novel Adhesive Peptide-Conjugate

    PubMed Central

    Markó, Károly; Kőhidi, Tímea; Hádinger, Nóra; Jelitai, Márta; Mező, Gábor; Madarász, Emília

    2011-01-01

    Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes. PMID:22163310

  8. Tests for intact and collapsed magnetofossil chains

    NASA Astrophysics Data System (ADS)

    Egli, R.

    2012-04-01

    first time. These results match measurements obtained previously on magnetosome-rich sediments in smallest details, showing that the identification of distinct intact and collapsed chain structures is possible. On the other hand, these results show that caution should be used when interpreting sediment hysteresis properties as mixtures of single domain (SD), multidomain (MD), and superparamagnetic (SP) particles; because some collapsed chain structures closely mimic SD-MD-SP mixing trends in a Day plot, although being made only of SD particles.

  9. Identification and localization of estrogen receptor alpha- and beta-positive cells in adult male and female mouse intestine at various estrogen levels.

    PubMed

    Kawano, Naoko; Koji, Takehiko; Hishikawa, Yoshitaka; Murase, Kunihiko; Murata, Ikuo; Kohno, Shigeru

    2004-05-01

    Although estrogen is implicated in the regulation of mammalian intestinal function, the presence and the distribution of estrogen receptor (ER)-positive cells in the intestine are still controversial. The present study was designed to localize ERalpha- and ERbeta-expressing cells in female and male mouse intestines immunohistochemically under various estrogen conditions, especially in female mice, ovariectomized as well at various phases of the estrous cycle. Western blot analysis detected both ERalpha (66-kDa band) and ERbeta (56-kDa band). Immunohistochemical staining of paraffin-embedded sections after antigen-retrieval treatment with autoclaving revealed staining for ERalpha in submucosal interstitial cells, and double staining identified these cells as a subtype of intestinal macrophages. The number of these cells varied according to the estrous cycle phase. Administration of 17beta-estradiol to ovariectomized mice resulted in a significant increase in the number of ERalpha-positive macrophages. On the other hand, the nuclei of nerve cells in Auerbach and Meissner plexuses were positive for both ERalpha and ERbeta, but the number of positive nerve cells was not affected by estrogen. Our results indicate that estrogen and estrogenic compounds may exert their actions on the intestine in two ways; one is through interstitial macrophages and the other is through intestinal neurons.

  10. Vascular-derived TGF-β increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain

    PubMed Central

    Pineda, Jose R; Daynac, Mathieu; Chicheportiche, Alexandra; Cebrian-Silla, Arantxa; Sii Felice, Karine; Garcia-Verdugo, Jose Manuel; Boussin, François D; Mouthon, Marc-André

    2013-01-01

    Neurogenesis decreases during aging and following cranial radiotherapy, causing a progressive cognitive decline that is currently untreatable. However, functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover, we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures, irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly, the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice, prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging. PMID:23526803

  11. Mapping of neurotrophins and their receptors in the adult mouse brain and their role in the pathogenesis of a transgenic murine model of bovine spongiform encephalopathy.

    PubMed

    Marco-Salazar, P; Márquez, M; Fondevila, D; Rabanal, R M; Torres, J M; Pumarola, M; Vidal, E

    2014-05-01

    Neurotrophins are a family of growth factors that act on neuronal cells. The neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3, -4 and -5. The action of neurotrophins depends on two transmembrane-receptor signalling systems: (1) the tropomyosin-related kinase (Trk) family of tyrosine kinase receptors (Trk A, Trk B and Trk C) and (2) the p75 neurotrophin receptor (p75(NTR)). The interaction between neurotrophic factors and their receptors may be involved in the mechanisms that regulate the differential susceptibility of neuronal populations in neurodegenerative diseases. The aim of the present study was to evaluate the role of neurotrophins in the pathogenesis of bovine spongiform encephalopathy (BSE) using a transgenic mouse overexpressing bovine prnp (BoTg 110). Histochemistry for Lycopersicum esculentum agglutinin, haematoxylin and eosin staining and immunohistochemistry for the abnormal isoform of the prion protein (PrP(d)), glial fibrillary acidic protein (GFAP), NGF, BDNF, NT-3 and the receptors Trk A, Trk B, Trk C and p75(NTR) was performed. The lesions and the immunolabelling patterns were assessed semiquantitatively in different areas of the brain. No significant differences in the immunolabelling of neurotrophins and their receptors were observed between BSE-inoculated and control animals, except for p75(NTR), which showed increased expression correlating with the distribution of lesions, PrP(d) deposition and gliosis in the BSE-inoculated mice.

  12. Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics.

    PubMed

    Daynac, Mathieu; Morizur, Lise; Kortulewski, Thierry; Gauthier, Laurent R; Ruat, Martial; Mouthon, Marc-André; Boussin, François D

    2015-01-01

    Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies. PMID:26436641

  13. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    NASA Technical Reports Server (NTRS)

    Barker, Ed; Abercromby, Kira J.; Abell, Paul

    2009-01-01

    A key objective of NASA s Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the 0.6 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectra is flat and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar panels with a strong band gap feature near 1 micron. The two spacecraft are spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but with fewer

  14. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    NASA Astrophysics Data System (ADS)

    Albercromby, Kira J.; Abell, Paul; Barker, Ed

    2009-03-01

    A key objective of NASA's Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the ~ 0.65 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectrum has a slight slope and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar cells with a strong band gap feature near 1 micron. The two spacecraft were spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but

  15. Disruption of the ErbB signaling in adolescence increases striatal dopamine levels and affects learning and hedonic-like behavior in the adult mouse.

    PubMed

    Golani, Idit; Tadmor, Hagar; Buonanno, Andres; Kremer, Ilana; Shamir, Alon

    2014-11-01

    The ErbB signaling pathway has been genetically and functionally implicated in schizophrenia. Numerous findings support the dysregulation of Neuregulin (NRG) and epidermal growth factor (EGF) signaling in schizophrenia. However, it is unclear whether alterations of these pathways in the adult brain or during development are involved in the pathophysiology of schizophrenia. Herein we characterized the behavioral profile and molecular changes resulting from pharmacologically blocking the ErbB signaling pathway during a critical period in the development of decision making, planning, judgments, emotions, social cognition and cognitive skills, namely adolescence. We demonstrate that chronic administration of the pan-ErbB kinase inhibitor JNJ-28871063 (JNJ) to adolescent mice elevated striatal dopamine levels and reduced preference for sucrose without affecting locomotor activity and exploratory behavior. In adulthood, adolescent JNJ-treated mice continue to consume less sucrose and needed significantly more correct-response trials to reach the learning criterion during the discrimination phase of the T-maze reversal learning task than their saline-injected controls. In addition, JNJ mice exhibited deficit in reference memory but not in working memory as measured in the radial arm maze. Inhibition of the pathway during adolescence did not affect exploratory behavior and locomotor activity in the open field, social interaction, social memory, and reversal learning in adult mice. Our data suggest that alteration of ErbB signaling during adolescence resulted in changes in the dopaminergic systems that emerge in pathological learning and hedonic behavior in adulthood, and pinpoints the possible role of the pathway in the development of cognitive skills and motivated behavior. PMID:25451700

  16. Effects of intraoperative electron irradiation in the dog on cell turnover in intact and surgically-anastomosed aorta and intestine

    SciTech Connect

    Sindelar, W.F.; Morrow, B.M.; Travis, E.L.; Tepper, J.; Merkel, A.B.; Kranda, K.; Terrill, R.

    1983-04-01

    Adults dogs were subjected to laparotomy and intraoperative electron irradiation after division and reanastomosis of aorta or after construction of a blind loop of small intestine having a transverse suture line and an end-to-side anastomosis. Dogs received intraoperative irradiation of both intact and anastomosed aorta or intestine in doses of 0, 2000, 3000, or 4500 rad. Animals were sacrificed at seven days or three months following treatment. At 24 hours prior to sacrifice, dogs received 5 mCi tritiated thymidine intravenously. Irradiated and non-irradiated segments of aorta and small intestine, including intact and anastomotic regions, were analyzed for tritiated thymidine incorporation and were subjected to autoradiography. Incorporation studies showed diminution in tritiated thymidine uptake by irradiated portions of aorta and small intestine, in both intact and anastomotic regions. Autoradiograms revealed that irradiated areas of intact or anastomotic aorta or intestine had diminished labeling of stromal cells, suggesting a lowered cell proliferative capacity of irradiated tissue compared to non-irradiated portions. Inflammatory cells showed similar labeling indices in irradiated and non-irradiated tissues, both intact and surgically-manipulated, suggesting that irradiation does not significantly affect a subsequent local inflammatory response. Radiation-induced decreases in tritiated thymidine incoporation in irradiated aorta and small intestine were generally more marked at seven days than at three months following irradiation, suggesting that radiation-induced depression of cell turnover rates decreases with time.

  17. Vesta is not an intact protoplanet

    NASA Astrophysics Data System (ADS)

    Consolmagno, G.; Turrini, D.; Golabek, G.; Svetsov, V.; Sirono, S.; Tsiganis, K.

    2014-07-01

    The Dawn mission was designed to explore ''remnant intact protoplanets from the earliest epoch of solar system formation'' [1]. However, models of Vesta composed of an iron core, olivine mantle, and HED crust in chondritic proportions cannot match the joint constraints from Dawn [1] of Vesta's density, core size, and the extremely limited presence of exposed olivine on its surface. Vesta has a mean density of 3456 kg/m3 and its surface composition is well matched by howardites. The Dawn gravity data suggest a nickel-iron core of radius 110 km and density 7500--7800 kg/m3. The Rheasilvia impact basin, formed within a pre-existing large basin, Veneneia, should have excavated material from a depth of 50 km to 80 km or more below Vesta's surface [2]. If the howardite crust were thinner than 50--80 km, a significant amount of olivine-rich material, derived from depth, would have been exposed within this basin; models suggest that olivine would also be distributed both on Vesta's surface and in space as meteorite-source Vestoids. Such olivine is rare on Vesta, among the Vestoids, or in our meteorite collection. Vesta's density is similar to an L chondrite, but the Na and K abundances in Vesta are strongly depleted compared to chondrites and the average metal content of an L chondrite, 8.4% by mass, would give a core radius less than 90 km. A 110 km radius metallic core, via the Dawn data, represents 15% of Vesta's mass. The Mg/Al ratio in cosmic abundances is about 10:1, but roughly 1:1 within the eucrites; thus if Vesta started with cosmic abundances, the eucrites can only represent 10% of the parent body total mass. Likewise the 10 x chondritic rare earth trace elements (REE) abundance seen in most eucrites demands that, regardless of formation mechanism, these basalts were crystallized from a melt representing 10% of the mass of the source region [3]. Thus the howardite crust of a chondritic HED parent body, mixing all the available eucritic and diogenitic material

  18. Germline ablation of dermatan-4O-sulfotransferase1 reduces regeneration after mouse spinal cord injury.

    PubMed

    Rost, S; Akyüz, N; Martinovic, T; Huckhagel, T; Jakovcevski, I; Schachner, M

    2016-01-15

    Chondroitin/dermatan sulfate proteoglycans (CSPGs/DSPGs) are major components of the extracellular matrix. Their expression is generally upregulated after injuries to the adult mammalian central nervous system, which is known for its low ability to restore function after injury. Several studies support the view that CSPGs inhibit regeneration after injury, whereas the functions of DSPGs in injury paradigms are less certain. To characterize the functions of DSPGs in the presence of CSPGs, we studied young adult dermatan-4O-sulfotransferase1-deficient (Chst14(-/-)) mice, which express chondroitin sulfates (CSs), but not dermatan sulfates (DSs), to characterize the functional outcome after severe compression injury of the spinal cord. In comparison to their wild-type (Chst14(+/+)) littermates, regeneration was reduced in Chst14(-/-) mice. No differences between genotypes were seen in the size of spinal cords, numbers of microglia and astrocytes neither in intact nor injured spinal cords after injury. Monoaminergic innervation and re-innervation of the spinal cord caudal to the lesion site as well as expression levels of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were similar in both genotypes, independent of whether they were injured and examined 6weeks after injury or not injured. These results suggest that, in contrast to CSPGs, DSPGs, being the products of Chst14 enzymatic activity, promote regeneration after injury of the adult mouse central nervous system.

  19. 56. POWDER MAGAZINE, VIEW OF INTACT WOOD SHEATHING ON THE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    56. POWDER MAGAZINE, VIEW OF INTACT WOOD SHEATHING ON THE SOUTHWEST REAR VENTILATION PASSAGE. (SHEATHING HELP CONTROL HUMIDITY AND DECREASE DANGER OF MAETAL STRIKING STONE AND SPARKING.) - Fort Monroe, Fortress, Hampton, Hampton, VA

  20. The Impact of Long-Term Exposure to Space Environment on Adult Mammalian Organisms: A Study on Mouse Thyroid and Testis

    PubMed Central

    Masini, Maria Angela; Albi, Elisabetta; Barmo, Cristina; Bonfiglio, Tommaso; Bruni, Lara; Canesi, Laura; Cataldi, Samuela; Curcio, Francesco; D'Amora, Marta; Ferri, Ivana; Goto, Katsumasa; Kawano, Fuminori; Lazzarini, Remo; Loreti, Elisabetta; Nakai, Naoya; Ohira, Takashi; Ohira, Yoshinobu; Palmero, Silvio; Prato, Paola; Ricci, Franco; Scarabelli, Linda; Shibaguchi, Tsubasa; Spelat, Renza; Strollo, Felice; Ambesi-Impiombato, Francesco Saverio

    2012-01-01

    Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis. In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10−7M and 10−8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains. In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. −90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules. Space-induced changes of structure and function of thyroid and testis/epididymis could be

  1. The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

    PubMed

    Masini, Maria Angela; Albi, Elisabetta; Barmo, Cristina; Bonfiglio, Tommaso; Bruni, Lara; Canesi, Laura; Cataldi, Samuela; Curcio, Francesco; D'Amora, Marta; Ferri, Ivana; Goto, Katsumasa; Kawano, Fuminori; Lazzarini, Remo; Loreti, Elisabetta; Nakai, Naoya; Ohira, Takashi; Ohira, Yoshinobu; Palmero, Silvio; Prato, Paola; Ricci, Franco; Scarabelli, Linda; Shibaguchi, Tsubasa; Spelat, Renza; Strollo, Felice; Ambesi-Impiombato, Francesco Saverio

    2012-01-01

    Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7)M and 10(-8)M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be

  2. Griffonia simplicifolia isolectin B4 identifies a specific subpopulation of angiogenic blood vessels following contusive spinal cord injury in the adult mouse.

    PubMed

    Benton, Richard L; Maddie, Melissa A; Minnillo, Danielle R; Hagg, Theo; Whittemore, Scott R

    2008-03-01

    After traumatic spinal cord injury (SCI), disruption and plasticity of the microvasculature within injured spinal tissue contribute to the pathological cascades associated with the evolution of both primary and secondary injury. Conversely, preserved vascular function most likely results in tissue sparing and subsequent functional recovery. It has been difficult to identify subclasses of damaged or regenerating blood vessels at the cellular level. Here, adult mice received a single intravenous injection of the Griffonia simplicifolia isolectin B4 (IB4) at 1-28 days following a moderate thoracic (T9) contusion. Vascular binding of IB4 was maximally observed 7 days following injury, a time associated with multiple pathologic aspects of the intrinsic adaptive angiogenesis, with numbers of IB4 vascular profiles decreasing by 21 days postinjury. Quantitative assessment of IB4 binding shows that it occurs within the evolving lesion epicenter, with affected vessels expressing a temporally specific dysfunctional tight junctional phenotype as assessed by occludin, claudin-5, and ZO-1 immunoreactivities. Taken together, these results demonstrate that intravascular lectin delivery following SCI is a useful approach not only for observing the functional status of neovascular formation but also for definitively identifying specific subpopulations of reactive spinal microvascular elements.

  3. Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence☆

    PubMed Central

    Dorà, Natalie J.; Hill, Robert E.; Collinson, J. Martin; West, John D.

    2015-01-01

    The limbal epithelial stem cell (LESC) hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC) hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks. PMID:26554513

  4. Mrp4, a new mitogen-regulated protein/proliferin gene; unique in this gene family for its expression in the adult mouse tail and ear.

    PubMed

    Fassett, J T; Hamilton, R T; Nilsen-Hamilton, M

    2000-05-01

    Mitogen-regulated proteins (also known as proliferin; mrp/plf) are nonclassical members of the PRL/GH family. They are expressed at high levels during midgestation when they are thought to induce angiogenesis and uterine growth. There are between four and six mrp/plf genes, and three different complementary DNAs have been cloned. Here we identify a fourth mrp/plf gene (mrp4) that we have cloned and characterized. MRP4 is 91% identical in amino acid sequence with the other MRP/PLF proteins but is missing two glycosylation sites that are present in the other forms. Consistent with the loss of two of three glycosylation sites, the expressed form of MRP4 has a lower apparent molecular weight compared with other MRP/PLFs. In vivo, mrp4 is expressed in the placenta and the adult skin. Expression of mrp4 messenger RNA peaks in the placenta on day 12. In the skin, mrp4 expression is specific to the ears and tails of mice. Our results suggest that, as well as having growth and angiogenic effects during pregnancy, the MRP/PLFs may have functions in nonreproductive tissues. Unique among the members of the mrp/plf family for its expression in the hair follicles of the tail and ear, MRP4 is expected to have a singular role in the growth and development of these follicles.

  5. A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

    PubMed

    Loers, Gabriele; Cui, Yi-Fang; Neumaier, Irmgard; Schachner, Melitta; Skerra, Arne

    2014-06-15

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions.

  6. A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

    PubMed

    Loers, Gabriele; Cui, Yi-Fang; Neumaier, Irmgard; Schachner, Melitta; Skerra, Arne

    2014-06-15

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions. PMID:24673421

  7. Blocking the postpartum mouse dam's CB1 receptors impairs maternal behavior as well as offspring development and their adult social-emotional behavior.

    PubMed

    Schechter, Michal; Pinhasov, Albert; Weller, Aron; Fride, Ester

    2012-01-15

    Maternal care is the newborns' first experience of social interaction, which affects their development and social competence throughout life. For the first time, we investigated the involvement of the endocannabinoid system (ECS) in mother-infant interaction in mice. We found that blocking the dam's CB1 receptors (CB1R) by the antagonist/inverse agonist rimonabant (SR141716) during postpartum days 1-8 affected maternal behavior as well as the social and emotional characteristics of the offspring as adults. Pups of rimonabant treated dams (RTD) had lower body weight during the first week of life and emitted fewer ultrasonic vocalizations (USVs) than vehicle treated dams (VTD). RTD crouched less over their pups and exhibited delayed pup retrieval. In Y-maze preference tests conducted at weaning age, females and males of both groups preferred their dam over milk. Males and females of RTD preferred dam over pup and pup over milk as opposed to the control group. At the age of 2.5 months, males of RTD displayed less motor activity. In the social behavior test, RTD male and female offspring were both more active, showing higher levels of active social interaction and rearing. These results indicate that the ECS is crucial for establishment of maternal behavior during the first postpartum week, with a long-term impact on the offspring's socio-emotional development. PMID:22020200

  8. In vivo robotics: the automation of neuroscience and other intact-system biological fields

    PubMed Central

    Kodandaramaiah, Suhasa B.; Boyden, Edward S.; Forest, Craig R.

    2013-01-01

    Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to impact neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience, and present a concrete example with our recent automation of in vivo whole cell patch clamp electrophysiology of neurons in the living mouse brain. PMID:23841584

  9. In vivo robotics: the automation of neuroscience and other intact-system biological fields.

    PubMed

    Kodandaramaiah, Suhasa B; Boyden, Edward S; Forest, Craig R

    2013-12-01

    Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to influence neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience and present a concrete example with our recent automation of in vivo whole-cell patch clamp electrophysiology of neurons in the living mouse brain.

  10. Cardiac Myocyte Alternans in Intact Heart: Influence of Cell-Cell Coupling and β-Adrenergic Stimulation

    PubMed Central

    Hammer, Karin P.; Ljubojevic, Senka; Ripplinger, Crystal M.; Pieske, Burkert M.; Bers, Donald M.

    2015-01-01

    Background Cardiac alternans are proarrhythmic and mechanistically link cardiac mechanical dysfunction and sudden cardiac death. Beat-to-beat alternans occur when beats with large Ca2+ transients and long action potential duration (APD) alternate with the converse. APD alternans are typically driven by Ca2+ alternans and sarcoplasmic reticulum (SR) Ca2+ release alternans. But the effect of intercellular communication via gap junctions (GJ) on alternans in intact heart remains unknown. Objective We assessed the effects of cell-to-cell coupling on local alternans in intact Langen-dorff-perfused mouse hearts, measuring single myocyte [Ca2+] alternans synchronization among neighboring cells, and effects of β-adrenergic receptor (β-AR) activation and reduced GJ coupling. Methods and Results Mouse hearts (C57BL/6) were retrogradely perfused and loaded with Fluo-8 AM to record cardiac myocyte [Ca2+] in situ with confocal microscopy. Single cell resolution allowed analysis of alternans within the intact organ during alternans induction. Carbenoxolone (25 μM), a GJ inhibitor, significantly increased the occurrence and amplitude of alternans in single cells within the intact heart. Alternans were concordant between neighboring cells throughout the field of view, except transiently during onset. β-AR stimulation only reduced Ca2+ alternans in tissue that had reduced GJ coupling, matching effects seen in isolated myocytes. Conclusions Ca2+ alternans among neighboring myocytes is predominantly concordant, likely because of electrical coupling between cells. Consistent with this, partial GJ uncoupling increased propensity and amplitude of Ca2+ alternans, and made them more sensitive to reversal by β-AR activation, as in isolated myocytes. Electrical coupling between myocytes may thus limit the alternans initiation, but also allow alternans to be more stable once established. PMID:25828762

  11. Intact skull chronic windows for mesoscopic wide-field imaging in awake mice

    PubMed Central

    Silasi, Gergely; Xiao, Dongsheng; Vanni, Matthieu P.; Chen, Andrew C. N.; Murphy, Timothy H.

    2016-01-01

    Background Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex. New Method We describe a reproducible and simple procedure for chronic through-bone wide-field imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months. Results The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30 minutes. A single set-screw provides a stable means of attachment for mesoscale assessment without obscuring the cortical field of view. Comparison with Existing Methods We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice. Conclusions We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors. PMID:27102043

  12. Non-invasive optical imaging of tumor growth in intact animals

    NASA Astrophysics Data System (ADS)

    Lu, Jinling; Li, Pengcheng; Luo, Qingming; Zhu, Dan

    2003-12-01

    We describe here a system for rapidly visualizing tumor growth in intact rodent mice that is simple, rapid, and eminently accessible and repeatable. We have established new rodent tumor cell line -- SP2/0-GFP cells that stably express high level of green fluorescent protein (GFP) by transfected with a plasmid that encoded GFP using electroporation and selected with G418 for 3 weeks. 1 x 104 - 1x107 SP2/0-GFP mouse melanoma cells were injected s.c. in the ears and legs of 6- to 7-week-old syngeneic male BALB/c mice, and optical images visualized real-time the engrafted tumor growth. The tumor burden was monitored over time by cryogenically cooled charge coupled device (CCD) camera focused through a stereo microscope. The results show that the fluorescence intensity of GFP-expressing tumor is comparably with the tumor growth and/or depress. This in vivo optical imaging based on GFP is sensitive, external, and noninvasive. It affords continuous visual monitoring of malignant growth within intact animals, and may comprise an ideal tool for evaluating antineoplastic therapies.

  13. Plasticity of Intact Rubral Projections Mediates Spontaneous Recovery of Function after Corticospinal Tract Injury

    PubMed Central

    Siegel, Chad S.; Fink, Kathren L.; Strittmatter, Stephen M.

    2015-01-01

    Axons in the adult CNS fail to regenerate after injury, and therefore recovery from spinal cord injury (SCI) is limited. Although full recovery is rare, a modest degree of spontaneous recovery is observed consistently in a broad range of clinical and nonclinical situations. To define the mechanisms mediating spontaneous recovery of function after incomplete SCI, we created bilaterally complete medullary corticospinal tract lesions in adult mice, eliminating a crucial pathway for voluntary skilled movement. Anatomic and pharmacogenetic tools were used to identify the pathways driving spontaneous functional recovery in wild-type and plasticity-sensitized mice lacking Nogo receptor 1. We found that plasticity-sensitized mice recovered 50% of normal skilled locomotor function within 5 weeks of lesion. This significant, yet incomplete, spontaneous recovery was accompanied by extensive sprouting of intact rubrofugal and rubrospinal projections with the emergence of a de novo circuit between the red nucleus and the nucleus raphe magnus. Transient silencing of this rubro–raphe circuit in vivo via activation of the inhibitory DREADD (designer receptor exclusively activated by designer drugs) receptor hM4di abrogated spontaneous functional recovery. These data highlight the pivotal role of uninjured motor circuit plasticity in supporting functional recovery after trauma, and support a focus of experimental strategies on enhancing intact circuit rearrangement to promote functional recovery after SCI. PMID:25632122

  14. Lymph flow pattern in the intact thoracic duct in sheep.

    PubMed

    Onizuka, M; Flatebø, T; Nicolaysen, G

    1997-08-15

    1. To study the lymph flow dynamics in the intact thoracic duct, we applied an ultrasound transit-time flow probe in seven anaesthetized and four unanaesthetized adult sheep (approximately 60 kg). In unanaesthetized non-fasting animals we found that lymph flow in the thoracic duct was always regular pulsatile (pulsation frequency, 5.2 +/- 0.8 min-1) with no relation to heart or respiratory activity. At baseline the peak level of the thoracic duct pulse flow was 11.6-20.7 ml min-1 with a nadir of 0-3.6 ml min-1. Mean lymph flow was 5.4 +/- 3.1 ml min-1. The flow pattern of lymph in the thoracic duct was essentially the same in the anaesthetized animals. 2. In both the anaesthetized and unanaesthetized animals, the lymph flow response to a stepwise increase in the outflow venous pressure showed interindividual variation. Some were sensitive to any increase in outflow venous pressure, but others were resistant in that lymph flow did not decrease until outflow venous pressure was increased to higher levels. This resistance was also observed in the high lymph flow condition produced by fluid infusion in the anaesthetized animal and mechanical constriction of the caudal vena cava in the unaesthetized animals. Pulsation frequency of the thoracic duct flow initially increased and then decreased with a stepwise increase in the outflow venous pressure. This initial increase might be a compensatory response to maintain lymph flow against elevated outflow venous pressure. 3. To test the effect of long-term outflow venous pressure elevation in unanaesthetized sheep, outflow venous pressure was increased by inflation of a cuff around the cranial vena cava for 1, 5 or 25 h. The cuff was inflated to a level where lymph flow was reduced. Lymph flow remained low or decreased further during the entire cuff-inflation period. We calculated the lymph debt caused by the outflow venous pressure elevation and the amount 'repaid' when venous pressure returned to normal. Lymph debt for 25 h

  15. Lymph flow pattern in the intact thoracic duct in sheep.

    PubMed Central

    Onizuka, M; Flatebø, T; Nicolaysen, G

    1997-01-01

    1. To study the lymph flow dynamics in the intact thoracic duct, we applied an ultrasound transit-time flow probe in seven anaesthetized and four unanaesthetized adult sheep (approximately 60 kg). In unanaesthetized non-fasting animals we found that lymph flow in the thoracic duct was always regular pulsatile (pulsation frequency, 5.2 +/- 0.8 min-1) with no relation to heart or respiratory activity. At baseline the peak level of the thoracic duct pulse flow was 11.6-20.7 ml min-1 with a nadir of 0-3.6 ml min-1. Mean lymph flow was 5.4 +/- 3.1 ml min-1. The flow pattern of lymph in the thoracic duct was essentially the same in the anaesthetized animals. 2. In both the anaesthetized and unanaesthetized animals, the lymph flow response to a stepwise increase in the outflow venous pressure showed interindividual variation. Some were sensitive to any increase in outflow venous pressure, but others were resistant in that lymph flow did not decrease until outflow venous pressure was increased to higher levels. This resistance was also observed in the high lymph flow condition produced by fluid infusion in the anaesthetized animal and mechanical constriction of the caudal vena cava in the unaesthetized animals. Pulsation frequency of the thoracic duct flow initially increased and then decreased with a stepwise increase in the outflow venous pressure. This initial increase might be a compensatory response to maintain lymph flow against elevated outflow venous pressure. 3. To test the effect of long-term outflow venous pressure elevation in unanaesthetized sheep, outflow venous pressure was increased by inflation of a cuff around the cranial vena cava for 1, 5 or 25 h. The cuff was inflated to a level where lymph flow was reduced. Lymph flow remained low or decreased further during the entire cuff-inflation period. We calculated the lymph debt caused by the outflow venous pressure elevation and the amount 'repaid' when venous pressure returned to normal. Lymph debt for 25 h

  16. p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

    PubMed

    Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

    2015-03-01

    The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development.

  17. Vulnerability of ecosystems to climate change moderated by habitat intactness.

    PubMed

    Eigenbrod, Felix; Gonzalez, Patrick; Dash, Jadunandan; Steyl, Ilse

    2015-01-01

    The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network.

  18. Vulnerability of ecosystems to climate change moderated by habitat intactness.

    PubMed

    Eigenbrod, Felix; Gonzalez, Patrick; Dash, Jadunandan; Steyl, Ilse

    2015-01-01

    The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network. PMID:25059822

  19. Effects of different concentration and duration time of isoflurane on acute and long-term neurocognitve function of young adult C57BL/6 mouse

    PubMed Central

    Liu, Jianhui; Wang, Peijun; Zhang, Xiaoqing; Zhang, Wei; Gu, Guojun

    2014-01-01

    Postoperative cognitive dysfunction (POCD) is a decline in cognitive performance after a surgery with anaesthesia. The exact reasons of surgery and/or anaesthesia resulting in POCD are unclear. The aim of this study is to investigate the effects of different concentration and duration time of isoflurane anaesthesia on cognitive performance and cellular mechanisms involved in learning and memory function. In present work, young adult male C57BL/6 mice (age: 8 weeks) were anaesthetized by different concentration isoflurane in 100% oxygen for different duration time (Mice in group I1 received 0.7% isoflurane 0.5 h, mice in I2 received 0.7% isoflurane 2 h, mice in I3 received 1.4% isoflurane 2 h, and mice in I4 received 1.4% isoflurane 4 h). Non-anaesthetized mice served as control group (I0). Spatial learning was assessed at 10 days post-anesthesia in Morris water maze (MWM). Hippocampal protein expressions of activated caspase 3, NMDA receptor subunit NR2B, and extracellular-signal regulated kinase (ERK) 1/2 were evaluated 24 hours and 2 weeks post anesthesia. Protein expression of activated caspase3 was detected acute elevated in I3 (24 h post-anesthesia) and acute and long-term elevated in I4 (24 hours and 2 weeks post-anesthesia). There was no significant difference between I1, I2 and control group. Protein expressions of NR2B showed an acute and long-term increasement in I1 and I2, decreasement in I4, and an acute decline, then returned to normal in I3 compared to control group. The ratio of phosopho-ERK1/2 to total-ERK showed an acute increasement in I1 and I2, then came to normal 2 weeks post anesthesia compared to control group, meanwhile, we detected an acute and long-term decline in I3 and I4. In MWM test, mice in I1 and I2 showed cognitive improvement, mice in I3 showed similar to control group, while mice in I4 demonstrated cognitive impairment, which were approximately corresponding to the changes of protein expression of NR2B and activation of ERK1

  20. Simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo.

    PubMed

    Manuel, Marin; Marin, Manuel; Heckman, C J

    2012-12-05

    recordings of motoneurons in vivo(2-4 ), including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice(5,6). However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry(7-9), or reproducting human disease(10,11), we expect this technique to be an essential tool for the study of spinal motor system.

  1. Children computer mouse use and anthropometry.

    PubMed

    Hughes, Erin E; Johnson, Peter W

    2012-01-01

    Studies have shown that increased computer use among adults in occupational settings is associated with the development of cumulative trauma disorders; however, the need to address how adult-sized mice and keyboards are affecting children is becoming increasingly important as both access to and use of computers is increasing among today's youth. To address the potential mismatch that exists between child stature and computer input device size and activation force, we have applied existing, age-specific, anthropometric data to elements of device design, including mouse size (length, width, height, switch location), and mouse-button activation forces. Trends supported the development of smaller computer input devices with lower activation forces for smaller statured individuals including children. Distinct and consistent trends in size delineations were seen across gender and age groups-trends that correlate well with grades and schooling in the United States education system . Three to four mouse sizes would be recommended: a mouse sized for adult and high school males; one for adult and high school females and junior high males; one for elementary school children, aged 6 to 10 years; and possibly a mouse for the smallest users who are less than six years old. PMID:22316827

  2. Immunization with a Double-Mutant (R192G/L211A) of the Heat-Labile Enterotoxin of Escherichia coli Offers Partial Protection against Campylobacter jejuni in an Adult Mouse Intestinal Colonization Model.

    PubMed

    Albert, M John; Haridas, Shilpa; Ebenezer, Mathew; Raghupathy, Raj; Khan, Islam

    2015-01-01

    We have previously shown that antibodies to cholera toxin (CT) reacted with the major outer membrane proteins (MOMPs) from Campylobacter jejuni strains on Western blot. Further, oral immunization with CT significantly protected against challenge with C. jejuni in an adult mouse colonization model of infection. CT and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are structurally and functionally related. LT and its mutants including the double-mutant LT (R192G/L211A) (dmLT), are powerful mucosal adjuvants. Unlike LT which is reactogenic, dmLT has been shown to be safe for human use. In the current study, we determined whether rabbit anti-dmLT antibodies reacted with MOMPs from C. jejuni strains and whether immunization with dmLT would afford protection against C. jejuni. On Western blot, the MOMPs from C. jejuni 48 (Penner serotype O:19), C. jejuni 75 (O:3) and C. jejuni 111 (O:1,44) were probed with rabbit antibodies to dmLT or LT-E112K (a non-toxic LT mutant), which showed a lack of reaction. Adult BALB/c mice were orally immunized with dmLT and orally challenged with C. jejuni 48 or 111. Protection from colonization with the challenge bacteria was studied by enumerating Campylobacter colonies in feces daily for 9 days. Vaccination produced robust serum and stool antibody responses to dmLT and no antibody responses to C. jejuni MOMP. Vaccinated mice showed reduced colonization and excretion of both challenge strains compared to control mice. However, the differences were not statistically significant. The protective efficacy of the dmLT vaccine varied from 9.1% to 54.5%. The lack of cross-reaction between the MOMP and dmLT suggests that protection is not mediated by cross-reacting antibodies, but may be due to activation of innate immunity. As dmLT is safe for humans, it could be incorporated into a C. jejuni vaccine to enhance its efficacy. PMID:26540197

  3. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APP{sub swe}/PS1{sub {Delta}E9} transgenic mouse model of Alzheimer's disease

    SciTech Connect

    Tang, Jun; Song, Min; Wang, Yanyan; Fan, Xiaotang; Xu, Haiwei; Bai, Yun

    2009-07-31

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP{sub swe}/PS1{sub {Delta}E9} mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP{sub swe}/PS1{sub {Delta}E9} transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  4. "Sightblind": perceptual deficits in the "intact" visual field.

    PubMed

    Bola, Michał; Gall, Carolin; Sabel, Bernhard A

    2013-01-01

    Unilateral visual cortex lesions caused by stroke or trauma lead to blindness in contralateral visual field - a condition called homonymous hemianopia. Although the visual field area processed by the uninjured hemisphere is thought to be "intact," it also exhibits marked perceptual deficits in contrast sensitivity, processing speed, and contour integration. Such patients are "sightblind" - their blindness reaches far beyond the primary scotoma. Studies showing perceptual deficits in patients' intact fields are reviewed and implications of these findings are discussed. It is concluded that consequences of partial blindness are greater than previously thought, since perceptual deficits in the "intact" field likely contribute to subjective vision loss in patients with visual field defect. This has important implications for vision diagnosis and rehabilitation. PMID:23805126

  5. [Application of capillary electrophoresis in analysis of intact mammalian cells].

    PubMed

    Zhang, Lu; Qu, Feng; Lou, Beilei

    2012-02-01

    Cell is the basic structural and functional unit of human body. The research of cells' structure, function and behavior is very important. Capillary electrophoresis (CE) is a powerful tool for the separation and analysis, the application of which in cell analysis has progressed significantly. In this paper, the developments of CE applied in the intact mammalian cell analysis are reviewed, which consist of cell population and single cell analysis. The erythrocyte, boar sperm, HeLa cells, SH-SY5Y cells, Caco-2 cells, K562 cells and rat cerebellar granule cells are involved in this review. The methods and conditions for the intact mammalian cell analysis are summarized. In addition, the problems caused by the breakage, aggregation, sedimentation, adsorption and electrophoretic heterogeneity of the cell in the intact mammalian cell analysis by CE are discussed, and the corresponding solutions are introduced. Also, the future research trends are presented. Forty nine papers in all are reviewed.

  6. Cloning the laboratory mouse.

    PubMed

    Wakayama, T; Yanagimachi, R

    1999-06-01

    A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two-cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1% of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?

  7. A Pharmacokinetic Study Comparing Eslicarbazepine Acetate Administered Orally as a Crushed or Intact Tablet in Healthy Volunteers.

    PubMed

    Sunkaraneni, Soujanya; Kharidia, Jahnavi; Schutz, Ralph; Blum, David; Cheng, Hailong

    2016-07-01

    The relative bioequivalence of crushed versus intact eslicarbazepine acetate (ESL) tablets (800 mg) administered orally in healthy adults was evaluated in an open-label, randomized, 2-period crossover study with a 5-day washout between treatments. Sample blood levels of eslicarbazepine and (R)-licarbazepine were determined; pharmacokinetic parameters were derived for eslicarbazepine. Bioequivalence was established if the 90% confidence intervals (CIs) for the geometric mean treatment ratios of eslicarbazepine AUC(0-∞) and Cmax were within the prespecified 80%-125% range. Twenty-seven subjects in the intent-to-treat population (n = 28) completed both treatment periods. Eslicarbazepine exposure measures were similar for crushed versus intact ESL tablets: average Cmax , 11 700 versus 11 500 ng/mL; AUC(0-∞) , 225 000 versus 234 000 ng·h/mL; AUC(0-last) , 222 000 versus 231 000 ng·h/mL, respectively. Geometric least squares mean ratios (90%CIs) comparing eslicarbazepine exposure measures were within the 80%-125% range (Cmax , 102.63% [97.07%-108.51%]; AUC(0-∞) , 96.72% [94.36%-99.13%]; AUC0-last , 96.69% [94.24%-99.21%]). In conclusion, ESL administered orally as a crushed tablet sprinkled on applesauce, or intact were bioequivalent in healthy subjects. Eslicarbazepine bioavailability was not significantly altered by crushing, indicating that ESL tablets can be administered intact or crushed. PMID:27249205

  8. Perceived parental control processes, parent-child relational qualities and psychological well-being of Chinese adolescents in intact and non-intact families in Hong Kong.

    PubMed

    Shek, Daniel T L; Lee, Tak Yan

    2007-01-01

    This paper examines whether Chinese adolescents' perceptions (N = 3,017) of parental behavioral control (parental knowledge, expectation, monitoring, discipline, and demandingness as well as parental control based on indigenous Chinese concepts), parental psychological control, parent-child relational qualities (perceived parental trust, child's trust of the parents, child's readiness to communicate with the parents, and child's satisfaction with parental control), and adolescent psychological well-being (hopelessness, mastery, life satisfaction and self-esteem) differed in intact and non-intact families. Results showed that relative to non-intact families, parental behavioral control processes were higher and parent-child relational qualities were better in intact families. In contrast, parental psychological control was higher in non-intact families than in intact families. Finally, the psychological well-being of adolescents in non-intact families was poorer than that of adolescents in intact families. PMID:17593768

  9. Fostering Activities of Daily Living by Intact Nursing Home Residents

    ERIC Educational Resources Information Center

    Blair, Charles E.; Glaister, Judy; Brown, Alston; Phillips, Carolyn

    2007-01-01

    We assessed effectiveness of four education programs in providing nursing assistants with ability to produce a therapeutic milieu supportive of intact residents' activities of daily living, positive self-esteem and mood: (1) a combination of Orem's Systems of Nursing Care and Skinner's Applied Behavioral Analysis, (2) Applied Behavioral Analysis,…

  10. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements. 174.145 Section 174.145 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES...) The righting arm curve must be positive to at least 60 degrees. (f) For the purpose of this...

  11. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... show that 2 inches (50mm) of positive metacentric height can be maintained by each tankship when it...

  12. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... show that 2 inches (50mm) of positive metacentric height can be maintained by each tankship when it...

  13. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... show that 2 inches (50mm) of positive metacentric height can be maintained by each tankship when it...

  14. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements. 174.145 Section 174.145 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... stability requirements. (a) In each condition of loading and operation, each vessel must be shown by...

  15. 46 CFR 174.145 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements. 174.145 Section 174.145 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... stability requirements. (a) In each condition of loading and operation, each vessel must be shown by...

  16. 46 CFR 174.045 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units §...

  17. 50 CFR 622.381 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., king mackerel, and Spanish mackerel in or from the Gulf, Mid-Atlantic, or South Atlantic EEZ, except as specified for king mackerel and Spanish mackerel in paragraph (b) of this section, must be maintained with... intact through offloading ashore, as specified in this section. (b) Cut-off (damaged) king or...

  18. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  19. Intact human holo-transferrin interaction with oxaliplatin.

    PubMed

    Zhao, Yuan-Yuan; Mandal, Rupasri; Li, Xing-Fang

    2005-01-01

    We report the interaction of intact human holo-transferrin (holo-Tf) with oxaliplatin (an anticancer drug), and the characterization of a complex composed of (1:1) intact holo-Tf and the parent oxaliplatin molecule using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS). The molecular weight of this complex was determined to be 80,077 Da, which was an increase of 397 mass units compared to the protein alone (79,680 Da), suggesting that a parent drug molecule was bound to the intact protein. We further examined the interaction between the intact protein and oxaliplatin using size-exclusion high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS). The protein complex and free oxaliplatin were separated by HPLC and quantitatively determined by simultaneous monitoring of both 195Pt and 56Fe using ICPMS. The HPLC/ICPMS detected both Pt and Fe signals at retention time of 2.6 min, identifying the protein-drug complex. The Fe signal at 2.6 min did not change with the increase in incubation time of the reaction mixture containing holo-Tf and oxaliplatin, while the Pt signal at the same retention time increased over time, further demonstrating that the formation of this complex does not affect the protein-bound Fe. The binding constant of the (1:1) intact human holo-Tf-oxaliplatin complex was determined to be 7.7x10(5) M-1. Both nanoESI-MS and HPLC/ICPMS results support that the holo-Tf and parent oxaliplatin molecules form complexes through non-covalent binding, suggesting that holo-Tf may be a useful carrier for oxaliplatin delivery.

  20. Microinjection of follicle-enclosed mouse oocytes

    PubMed Central

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    2011-01-01

    Summary The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment. PMID:19085139

  1. Microinjection of Follicle-Enclosed Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

  2. Water permeability of primary mouse keratinocyte cultures grown at the air-liquid interface

    SciTech Connect

    Cumpstone, M.B.; Kennedy, A.H.; Harmon, C.S.; Potts, R.O.

    1989-04-01

    In order to study the development of the epidermal permeability barrier in vitro, tritiated water (HTO) flux was measured across murine keratinocytes cultured at the air-liquid interface. Using a micro-diffusion technique, it was shown that air-liquid cultures form areas where the water diffusion is comparable to that of intact neonatal mouse skin. When water permeability is measured over a large area of the culture surface, however, significantly higher flux is obtained. These results show that under the culture conditions used, areas of water barrier comparable to intact neonatal mouse skin coexist with regions of less complete barrier formation.

  3. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

    ERIC Educational Resources Information Center

    Schoonenboom, Judith

    2016-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

  4. Intact brain cells: a novel model system for studying opioid receptor binding

    SciTech Connect

    Rogers, N.F.; El-Fakahany, E.E.

    1985-07-29

    The use of a novel tissue preparation to study opioid receptor binding in viable, intact cells derived from whole brains of adult rats is presented. Mechanically dissociated and sieved cells, which were not homogenized at any stage of the experimental protocol, and iso-osmotic physiological buffer were used in these experiments. This system was adapted in order to avoid mechanical and chemical disruption of cell membranes, cytoskeletal ultrastructure or receptor topography by homogenization or by the use of nonphysiological buffers, and to mimic in vivo binding conditions as much as possible. Using (/sup 3/H)naloxone as the radioligand, the studies showed saturable and stereospecific high-affinity binding of this opioid antagonist in intact cells, which in turn showed consistently high viability. (/sup 3/H)Naloxone binding was also linear over a wide range of tissue concentrations. This technique provides a very promising model for future studies of the binding of opioids and of many other classes of drugs to brain tissue receptors in a more physiologically relevant system than those commonly used to date.

  5. Mink-mouse hybridomas that secrete mink immunoglobulin G.

    PubMed

    Galakhar, N L; Djatchenko, S N; Fomicheva, I I; Mechetina, L V; Taranin, A V; Belousov, E S; Nayakshin, A M; Baranov, O K

    1988-11-25

    Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.

  6. Immunizing adult female mice with a TcpA-A2-CTB chimera provides a high level of protection for their pups in the infant mouse model of cholera.

    PubMed

    Price, Gregory A; Holmes, Randall K

    2014-12-01

    Vibrio cholerae expresses two primary virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP). CT causes profuse watery diarrhea, and TCP (composed of repeating copies of the major pilin TcpA) is required for intestinal colonization by V. cholerae. Antibodies to CT or TcpA can protect against cholera in animal models. We developed a TcpA holotoxin-like chimera (TcpA-A2-CTB) to elicit both anti-TcpA and anti-CTB antibodies and evaluated its immunogenicity and protective efficacy in the infant mouse model of cholera. Adult female CD-1 mice were immunized intraperitoneally three times with the TcpA-A2-CTB chimera and compared with similar groups immunized with a TcpA+CTB mixture, TcpA alone, TcpA with Salmonella typhimurium flagellin subunit FliC as adjuvant, or CTB alone. Blood and fecal samples were analyzed for antigen-specific IgG or IgA, respectively, using quantitative ELISA. Immunized females were mated; their reared offspring were challenged orogastrically with 10 or 20 LD50 of V. cholerae El Tor N16961; and vaccine efficacy was assessed by survival of the challenged pups at 48 hrs. All pups from dams immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture survived at both challenge doses. In contrast, no pups from dams immunized with TcpA+FliC or CTB alone survived at the 20 LD50 challenge dose, although the anti-TcpA or anti-CTB antibody level elicited by these immunizations was comparable to the corresponding antibody level achieved by immunization with TcpA-A2-CTB or TcpA+CTB. Taken together, these findings comprise strong preliminary evidence for synergistic action between anti-TcpA and anti-CTB antibodies in protecting mice against cholera. Weight loss analysis showed that only immunization of dams with TcpA-A2-CTB chimera or TcpA+CTB mixture protected their pups against excess weight loss from severe diarrhea. These data support the concept of including both TcpA and CTB as immunogens in development of an effective multivalent

  7. Metabolic impact of adult-onset, isolated, growth hormone deficiency (AOiGHD) due to destruction of pituitary somatotropes.

    PubMed

    Luque, Raul M; Lin, Qing; Córdoba-Chacón, José; Subbaiah, Papasani V; Buch, Thorsten; Waisman, Ari; Vankelecom, Hugo; Kineman, Rhonda D

    2011-01-19

    Growth hormone (GH) inhibits fat accumulation and promotes protein accretion, therefore the fall in GH observed with weight gain and normal aging may contribute to metabolic dysfunction. To directly test this hypothesis a novel mouse model of adult onset-isolated GH deficiency (AOiGHD) was generated by cross breeding rat GH promoter-driven Cre recombinase mice (Cre) with inducible diphtheria toxin receptor mice (iDTR) and treating adult Cre(+/-),iDTR(+/-) offspring with DT to selectively destroy the somatotrope population of the anterior pituitary gland, leading to a reduction in circulating GH and IGF-I levels. DT-treated Cre(-/-),iDTR(+/-) mice were used as GH-intact controls. AOiGHD improved whole body insulin sensitivity in both low-fat and high-fat fed mice. Consistent with improved insulin sensitivity, indirect calorimetry revealed AOiGHD mice preferentially utilized carbohydrates for energy metabolism, as compared to GH-intact controls. In high-fat, but not low-fat fed AOiGHD mice, fat mass increased, hepatic lipids decreased and glucose clearance and insulin output were impaired. These results suggest the age-related decline in GH helps to preserve systemic insulin sensitivity, and in the context of moderate caloric intake, prevents the deterioration in metabolic function. However, in the context of excess caloric intake, low GH leads to impaired insulin output, and thereby could contribute to the development of diabetes.

  8. Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors.

    PubMed

    Watson, Deborah J; Passini, Marco A; Wolfe, John H

    2005-01-01

    Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors. PMID:15703488

  9. Less Daily Computer Use is Related to Smaller Hippocampal Volumes in Cognitively Intact Elderly

    PubMed Central

    Silbert, Lisa C.; Dodge, Hiroko H.; Lahna, David; Promjunyakul, Nutta-on; Austin, Daniel; Mattek, Nora; Erten-Lyons, Deniz; Kaye, Jeffrey A.

    2016-01-01

    Background: Computer use is becoming a common activity in the daily life of older individuals and declines over time in those with mild cognitive impairment (MCI). The relationship between daily computer use (DCU) and imaging markers of neurodegeneration is unknown. Objective:The objective of this study was to examine the relationship between average DCU and volumetric markers of neurodegeneration on brain MRI. Methods: Cognitively intact volunteers enrolled in the Intelligent Systems for Assessing Aging Change study underwent MRI. Total in-home computer use per day was calculated using mouse movement detection and averaged over a one-month period surrounding the MRI. Spearman’s rank order correlation (univariate analysis) and linear regression models (multivariate analysis) examined hippocampal, gray matter (GM), white matter hyperintensity (WMH), and ventricular cerebral spinal fluid (vCSF) volumes in relation to DCU. A voxel-based morphometry analysis identified relationships between regional GM density and DCU. Results: Twenty-seven cognitively intact participants used their computer for 51.3 minutes per day on average. Less DCU was associated with smaller hippocampal volumes (r = 0.48, p = 0.01), but not total GM, WMH, or vCSF volumes. After adjusting for age, education, and gender, less DCU remained associated with smaller hippocampal volume (p = 0.01). Voxel-wise analysis demonstrated that less daily computer use was associated with decreased GM density in the bilateral hippocampi and temporal lobes. Conclusions: Less daily computer use is associated with smaller brain volume in regions that are integral to memory function and known to be involved early with Alzheimer’s pathology and conversion to dementia. Continuous monitoring of daily computer use may detect signs of preclinical neurodegeneration in older individuals at risk for dementia. PMID:26967228

  10. Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals*

    PubMed Central

    Hammond, Dean E.; Claydon, Amy J.; Simpson, Deborah M.; Edward, Dominic; Stockley, Paula; Hurst, Jane L.

    2016-01-01

    Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole, Myodes glareolus. The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days−1; liver 0.136 days−1; heart, 0.054 days−1, and skeletal muscle, 0.035 days−1). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054. PMID:26839000

  11. An In Vitro Intact Globe Expansion Method for Evaluation of Cross-linking Treatments

    PubMed Central

    Mattson, Matthew S.; Huynh, Joyce; Wiseman, Meredith; Coassin, Marco; Kornfield, Julia A.; Schwartz, Daniel M.

    2010-01-01

    Purpose. To measure the tissue mechanical response to elevated intraocular pressure (IOP) using intact globe expansion of rabbit eyes. This method examined rabbit kit (2–3 weeks old) eyes as a model for weakened tissue and evaluated riboflavin/UVA and glyceraldehyde cross-linking treatments. Methods. The ocular shape of enucleated eyes was photographed during a 24-hour period while a controlled IOP was imposed (either low IOP = 22 mm Hg or high IOP = 85 mm Hg). Untreated controls consisted of kit eyes tested at both low- and high IOP and adult eyes tested at high IOP. Treated kit eyes (dextran controls, riboflavin/UVA treatment of the cornea, and glyceraldehyde treatment of the entire globe) were tested at high IOP. Results. Low IOP elicited negligible creep of the sclera and very gradual creep of the cornea. In contrast, high IOP induced up to an 8% strain in the sclera and a 15% strain in the cornea of rabbit kit eyes. The expansion of adult eyes was less than one third that of kit eyes at the same, high IOP. Riboflavin/UVA treatment of corneas reduced expansion compared with that in both dextran-treated and untreated control corneas. Glyceraldehyde treatment prevented expansion of the cornea and sclera. Conclusions. The intact globe expansion method (GEM) imposes a loading geometry comparable to in vivo conditions and can quantify changes in mechanical stability as a function of testing conditions (e.g., IOP, tissue maturation, and therapeutic cross-linking) with small sample sizes and small variability. Rabbit kit eyes provide a model of weak tissue suitable for screening treatments that strengthen the cornea and sclera. PMID:20071684

  12. Memory deficits with intact cognitive control in the methylazoxymethanol acetate (MAM) exposure model of neurodevelopmental insult.

    PubMed

    O'Reilly, Kally C; Perica, Maria I; Fenton, André A

    2016-10-01

    Cognitive impairments are amongst the most debilitating deficits of schizophrenia and the best predictor of functional outcome. Schizophrenia is hypothesized to have a neurodevelopmental origin, making animal models of neurodevelopmental insult important for testing predictions that early insults will impair cognitive function. Rats exposed to methylazoxymethanol acetate (MAM) at gestational day 17 display morphological, physiological and behavioral abnormalities relevant to schizophrenia. Here we investigate the cognitive abilities of adult MAM rats. We examined brain activity in MAM rats by histochemically assessing cytochrome oxidase enzyme activity, a metabolic marker of neuronal activity. To assess cognition, we used a hippocampus-dependent two-frame active place avoidance paradigm to examine learning and spatial memory, as well as cognitive control and flexibility using the same environment and evaluating the same set of behaviors. We confirmed that adult MAM rats have altered hippocampal morphology and brain function, and that they are hyperactive in an open field. The latter likely indicates MAM rats have a sensorimotor gating deficit that is common to many animal models used for schizophrenia research. On first inspection, cognitive control seems impaired in MAM rats, indicated by more errors during the two-frame active place avoidance task. Because MAM rats are hyperactive throughout place avoidance training, we considered the possibility that the hyperlocomotion may account for the apparent cognitive deficits. These deficits were reduced on the basis of measures of cognitive performance that account for motor activity differences. However, though other aspects of memory are intact, the ability of MAM rats to express trial-to-trial memory is delayed compared to control rats. These findings suggest that spatial learning and cognitive abilities are largely intact, that the most prominent cognitive deficit is specific to acquiring memory in the MAM

  13. Memory deficits with intact cognitive control in the methylazoxymethanol acetate (MAM) exposure model of neurodevelopmental insult.

    PubMed

    O'Reilly, Kally C; Perica, Maria I; Fenton, André A

    2016-10-01

    Cognitive impairments are amongst the most debilitating deficits of schizophrenia and the best predictor of functional outcome. Schizophrenia is hypothesized to have a neurodevelopmental origin, making animal models of neurodevelopmental insult important for testing predictions that early insults will impair cognitive function. Rats exposed to methylazoxymethanol acetate (MAM) at gestational day 17 display morphological, physiological and behavioral abnormalities relevant to schizophrenia. Here we investigate the cognitive abilities of adult MAM rats. We examined brain activity in MAM rats by histochemically assessing cytochrome oxidase enzyme activity, a metabolic marker of neuronal activity. To assess cognition, we used a hippocampus-dependent two-frame active place avoidance paradigm to examine learning and spatial memory, as well as cognitive control and flexibility using the same environment and evaluating the same set of behaviors. We confirmed that adult MAM rats have altered hippocampal morphology and brain function, and that they are hyperactive in an open field. The latter likely indicates MAM rats have a sensorimotor gating deficit that is common to many animal models used for schizophrenia research. On first inspection, cognitive control seems impaired in MAM rats, indicated by more errors during the two-frame active place avoidance task. Because MAM rats are hyperactive throughout place avoidance training, we considered the possibility that the hyperlocomotion may account for the apparent cognitive deficits. These deficits were reduced on the basis of measures of cognitive performance that account for motor activity differences. However, though other aspects of memory are intact, the ability of MAM rats to express trial-to-trial memory is delayed compared to control rats. These findings suggest that spatial learning and cognitive abilities are largely intact, that the most prominent cognitive deficit is specific to acquiring memory in the MAM

  14. Radioactive satellites - Intact reentry and breakup by debris impact

    NASA Technical Reports Server (NTRS)

    Anz-Meador, P. D.; Potter, A. E., Jr.

    1991-01-01

    There is a substantial mass of radioactive material in nuclear reactors or radioisotope thermal generators (RTGs) in orbit about the earth. This paper examines the reentry of intact nuclear fuel cores and RTGs and the fragmentation and subsequent radioactive debris cloud deposition and evolution resulting from the impact of orbital debris upon an orbiting reactor, fuel core, or RTG. To assess the intact reentry, decay rates and a predicted decay date using historical and projected orbital decay data, are estimated. The current NASA debris environment model is utilized to estimate impact rates and debris cloud evolution of a fragmentation event. Results of these analyses are compared and concepts are tendered which would tend to minimize the radiological debris hazard to personnel and structures both on the earth's surface and in low earth orbit.

  15. Structural determination of intact proteins using mass spectrometry

    DOEpatents

    Kruppa, Gary; Schoeniger, Joseph S.; Young, Malin M.

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  16. Effect of antimycin a on photosynthesis of intact spinach chloroplasts.

    PubMed

    Schacter, B Z; Gibbs, M; Champigny, M L

    1971-10-01

    Low concentrations (0.5-10 mum) of antimycin A were shown to increase the rate of CO(2) fixation, O(2) evolution and inorganic phosphate esterification in intact spinach (Spinacia oleracea) chloroplasts. The increase was highest when the light intensity was saturating. Stimulation was independent of the bicarbonate concentration and was accompanied by an enhancement in the synthesis of glycerate 3-phosphate with a decrease in dihydroxyacetone phosphate. The antibiotic decreased the Michaelis constant of the chloroplast but not of ribulose 1,5-diphosphate carboxylase for bicarbonate. It was suggested that antimycin A is affecting that portion (outer envelope) of the intact chloroplast which contains the enzyme mechanism for controlling the pace of CO(2) fixation.

  17. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  18. Crisscross heart with dextrocardia and intact interventricular septum

    PubMed Central

    Muneer, P. Kader; Kalathingathodika, Sajeer; Chakanalil, Govindan Sajeev; Sony, Manuel M.

    2014-01-01

    Crisscross heart is a rare congenital heart disease characterized by a twisted atrioventricular connection, as a result of rotation of the ventricular mass along its long axis. We report an asymptomatic 48-year-old woman referred to us for evaluation of a cardiac murmur. Further evaluation showed situs solitus, dextrocardia with normal atrioventricular and ventriculoarterial connection, and a crisscross relation of the atrioventricular valves. Unlike the usual case of crisscross heart, our patient had an intact ventricular septum. PMID:24701094

  19. Assay of nitrogenase activity in intact plant systems.

    PubMed

    Jain, M K; Vlassak, K

    1975-01-01

    Nitrogenase activity was assayed in intact system of Cichorium intybus, a non-leguminous commercially cultivated crop, Dahlia pinnata and Helianthus annus, and Taraxacum officinale, a common weed plant. The assay was made in fabricated cylinders which could accomodate pot with plants. In such kind of assay along with rhizosphere microflora, the nitrogen fixed by phyllosphere nitrogen fixing microflora could also be accounted, which otherwise was difficult to be accounted for. PMID:1211718

  20. Using NMR to study full intact wine bottles

    NASA Astrophysics Data System (ADS)

    Weekley, A. J.; Bruins, P.; Sisto, M.; Augustine, M. P.

    2003-03-01

    A nuclear magnetic resonance (NMR) probe and spectrometer capable of investigating full intact wine bottles is described and used to study a series of Cabernet Sauvignons with high resolution 1H NMR spectroscopy. Selected examples of full bottle 13C NMR spectra are also provided. The application of this full bottle NMR method to the measurement of acetic acid content, the detection of complex sugars, phenols, and trace elements in wine is discussed.

  1. Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

    PubMed

    Gaub, S; Fisher, S E; Ehret, G

    2016-02-01

    Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits.

  2. Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

    PubMed Central

    Ng, Kiaw Kiaw; Motoda, Yoko; Watanabe, Satoru; Sofiman Othman, Ahmad; Kigawa, Takanori; Kodama, Yutaka; Numata, Keiji

    2016-01-01

    In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology. PMID:27100681

  3. Rheumatoid synovial cells from intact joints. Morphology, growth, and polykaryocytosis.

    PubMed

    Clarris, B J; Fraser, J R; Moran, C J; Muirden, K D

    1977-08-01

    Synovial cell lines were isolated by instillation of trypsin or chymotrypsin into intact knee joints of patients with persistent rheumatoid effusions resistant to conventional therapy. Morphology and growth in the primary phase were compared with rheumatoid cells isolated from excised synovium and nonrheumatoid synovial cells obtained from intact joints of cadavers or amputated limbs. Cell populations from all sources included varying proportions of macrophage-like and fibroblast-like cells, with only 1-3% multinucleated cells. In medium supplemented with calf serum alone, rheumatoid cells from intact joints showed negligible changes in morphology. However, in the presence of nonrheumatoid, autologous rheumatoid or homologous rheumatoid serum a rapid increase occurred in size of the macrophage-like cells and numbers of polykaryocytes, including some giant syncytial cells. These effects were directly proportional to serum concentration and were identical in fresh or heat-inactivated serum. In most of these rheumatoid cell lines no multiplication occurred, regardless of serum type or concentration. In rheumatoid synovial cells from excised synovium, human serum induced both polykaryocytosis and rapid growth of fibroblasts. Nonrheumatoid synovial cells grew rapidly but few polykaryocytes developed, mostly with less than 6 nuclei. Evidence of viral infection in rheumatoid synovial cells was sought by electron microscopy after stimulation of polykaryocytosis by human serum. In one of the cultures many cells were found with intranuclear particles possessing characteristics of the adenovirus group. PMID:901027

  4. A chamber for applying pressure to roots of intact plants.

    PubMed

    Gee, G W

    1973-11-01

    A chamber was designed to apply up to 20 bars pressure to roots of intact plants. The unique features of this chamber are a split top arrangement to permit enclosing roots of intact plants within the chamber, a circulation coil to control temperature of rooting media, and a valve arrangement to permit changing solution without disturbing the plant. Changes in transpiration in response to changes in the pressure applied to roots of intact pepper plants illustrate one use of the equipment. Well watered plants at low light (0.05 langley/min) were observed to exude water from the leaf margins when 5 bars pressure was applied to the roots. When roots were cut off, a 1 bar pressure caused exudation. Plants with cooled roots or plants in dry soil did not exude water when as much as 6 bars pressure was applied. Transient response of transpiration rates to sudden application and release of pressure was observed in pepper and bean plants but not in rhododendron. The magnitude of this transient response was highly dependent upon light intensity and CO(2) concentration in the aerial environment.

  5. Capillary Zone Electrophoresis-Mass Spectrometry of Intact Proteins.

    PubMed

    Domínguez-Vega, Elena; Haselberg, Rob; Somsen, Govert W

    2016-01-01

    Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS detection. This chapter focuses on important practical considerations when applying CE-MS for the analysis of intact proteins. Technological aspects with respect to the use of CE-MS interfaces and application of noncovalent capillary coatings preventing protein adsorption are treated. Critical factors for successful protein analysis are discussed and four typical CE-MS systems are described demonstrating the characterization of different types of intact proteins by CE-MS. These methodologies comprise the use of sheath-liquid and sheathless CE-MS interfaces, and various types of noncovalent capillary coatings allowing efficient and reproducible protein separations. The discussion includes the analysis of lysozyme-drug conjugates and the therapeutic proteins human growth hormone, human interferon-β-1a, and human erythropoietin. PMID:27473479

  6. A chamber for applying pressure to roots of intact plants.

    PubMed

    Gee, G W

    1973-11-01

    A chamber was designed to apply up to 20 bars pressure to roots of intact plants. The unique features of this chamber are a split top arrangement to permit enclosing roots of intact plants within the chamber, a circulation coil to control temperature of rooting media, and a valve arrangement to permit changing solution without disturbing the plant. Changes in transpiration in response to changes in the pressure applied to roots of intact pepper plants illustrate one use of the equipment. Well watered plants at low light (0.05 langley/min) were observed to exude water from the leaf margins when 5 bars pressure was applied to the roots. When roots were cut off, a 1 bar pressure caused exudation. Plants with cooled roots or plants in dry soil did not exude water when as much as 6 bars pressure was applied. Transient response of transpiration rates to sudden application and release of pressure was observed in pepper and bean plants but not in rhododendron. The magnitude of this transient response was highly dependent upon light intensity and CO(2) concentration in the aerial environment. PMID:16658586

  7. Sphingolipid metabolism in organotypic mouse keratinocyte cultures

    SciTech Connect

    Madison, K.C.; Swartzendruber, D.C.; Wertz, P.W.; Downing, D.T. )

    1990-12-01

    Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine.

  8. Children do not exhibit ambiguity aversion despite intact familiarity bias

    PubMed Central

    Li, Rosa; Brannon, Elizabeth M.; Huettel, Scott A.

    2015-01-01

    The phenomenon of ambiguity aversion, in which risky gambles with known probabilities are preferred over ambiguous gambles with unknown probabilities, has been thoroughly documented in adults but never measured in children. Here, we use two distinct tasks to investigate ambiguity preferences of children (8- to 9-year-olds) and a comparison group of adults (19- to 27-year-olds). Across three separate measures, we found evidence for significant ambiguity aversion in adults but not in children and for greater ambiguity aversion in adults compared to children. As ambiguity aversion in adults has been theorized to result from a preference to bet on the known and avoid the unfamiliar, we separately measured familiarity bias and found that children, like adults, are biased towards the familiar. Our findings indicate that ambiguity aversion emerges across the course of development between childhood and adolescence, while a familiarity bias is already present in childhood. PMID:25601848

  9. Mouse Curve Biometrics

    SciTech Connect

    Schulz, Douglas A.

    2007-10-08

    A biometric system suitable for validating user identity using only mouse movements and no specialized equipment is presented. Mouse curves (mouse movements with little or no pause between them) are individually classied and used to develop classication histograms, which are representative of an individual's typical mouse use. These classication histograms can then be compared to validate identity. This classication approach is suitable for providing continuous identity validation during an entire user session.

  10. Building a Brainier Mouse.

    ERIC Educational Resources Information Center

    Tsien, Joe Z.

    2000-01-01

    Describes a genetic engineering project to build an intelligent mouse. Cites understanding the molecular basis of learning and memory as a very important step. Concludes that while science will never create a genius mouse that plays the stock market, it can turn a mouse into a quick learner with a better memory. (YDS)

  11. Optical clearing based cellular-level 3D visualization of intact lymph node cortex

    PubMed Central

    Song, Eunjoo; Seo, Howon; Choe, Kibaek; Hwang, Yoonha; Ahn, Jinhyo; Ahn, Soyeon; Kim, Pilhan

    2015-01-01

    Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellular-level structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex. PMID:26504662

  12. Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs1,2,3

    PubMed Central

    Epp, Jonathan R.; Niibori, Yosuke; (Liz) Hsiang, Hwa-Lin; Mercaldo, Valentina; Deisseroth, Karl

    2015-01-01

    Abstract The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology—anatomical and functional—connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy. PMID:26464982

  13. A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

    PubMed Central

    Suzuki, Takuo; Miyazaki, Chihiro; Ishii-Watabe, Akiko; Tada, Minoru; Sakai-Kato, Kumiko; Kawanishi, Toru; Kawasaki, Nana

    2015-01-01

    Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies. PMID:25891896

  14. A case of mixed transcortical aphasia with intact naming.

    PubMed

    Heilman, K M; Tucker, D M; Valenstein, E

    1976-09-01

    Altholgh Lichtheim recognized that Wernicke's 'reflex arch' (primary auditory area, to Wernicke's area, to Broca's area, to primary motor area) was important for repetition, he recognized that other areas of the brain (for example, area of concept