RESUMEN
Advanced 3D imaging modalities, such as micro-computed tomography (micro-CT), have been incorporated into the high-throughput embryo pipeline of the International Mouse Phenotyping Consortium (IMPC). This project generates large volumes of raw data that cannot be immediately exploited without significant resources of personnel and expertise. Thus, rapid automated annotation is crucial to ensure that 3D imaging data can be integrated with other multi-dimensional phenotyping data. We present an automated computational mouse embryo phenotyping pipeline that harnesses the large amount of wild-type control data available in the IMPC embryo pipeline in order to address issues of low mutant sample number as well as incomplete penetrance and variable expressivity. We also investigate the effect of developmental substage on automated phenotyping results. Designed primarily for developmental biologists, our software performs image pre-processing, registration, statistical analysis and segmentation of embryo images. We also present a novel anatomical E14.5 embryo atlas average and, using it with LAMA, show that we can uncover known and novel dysmorphology from two IMPC knockout lines.
Asunto(s)
Embrión de Mamíferos/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Femenino , Imagenología Tridimensional/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/fisiología , Fenotipo , Programas InformáticosRESUMEN
Basigin is a well-known extracellular stimulator of fibroblasts and may confer resistance to apoptosis of fibroblasts in vitro under some pathological status, but its exact function in fibroblasts and the underlying mechanism remain poorly understood. The systematic Basigin gene knockout leads to the perinatal lethality of mice, which limits the delineation of its function in vivo. In this study, we generated a fibroblast-specific Basigin knock-out mouse model and demonstrated the successful deletion of Basigin in fibroblasts. The fibroblast-specific deletion of Basigin did not influence the growth, fertility and the general condition of the mice. No obvious differences were found in the size, morphology, and histological structure of the major organs, including heart, liver, spleen, lung and kidney, between the knockout mice and the control mice. The deletion of Basigin in fibroblasts did not induce apoptosis in the tissues of the major organs. These results provide the first evidence that the fibroblast-specific Basigin knock-out mice could be a useful tool for exploring the function of Basigin in fibroblasts in vivo.
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Basigina/genética , Fibroblastos/metabolismo , Ratones Noqueados , Animales , Apoptosis , Basigina/deficiencia , Basigina/metabolismo , Femenino , Fertilidad , Fibroblastos/citología , Eliminación de Gen , Marcación de Gen , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Ratones Noqueados/crecimiento & desarrollo , Ratones Noqueados/fisiología , Ratones TransgénicosRESUMEN
Loss-of-function mutations in the progranulin gene (GRN) are a common cause of familial frontotemporal lobar degeneration (FTLD). A high degree of heterogeneity in the age-of-onset, duration of disease, and clinical presentation of FTLD, even among families carrying the same GRN mutation, suggests that additional modifying genes may be important to pathogenesis. Progranulin-knockout mice display subtle behavioral abnormalities and progressive neuropathological changes, as well as altered dendritic morphology and synaptic deficits in the hippocampus. In this study we evaluated multiple neuropathological endpoints in aged progranulin knockout mice and their wild-type littermates on two different genetic backgrounds: C57Bl/6 and 129/SvImJ. We find that in most brain regions, both strains are susceptible to progranulin-mediated neuropathological phenotypes, including astrogliosis, microgliosis, and highly accelerated deposition of the aging pigment lipofuscin. Neuroinflammation due to progranulin deficiency is exaggerated in the B6 strain and present, but less pronounced, in the 129 strain. Differences between the strains in hippocampal neuron counts and neuronal morphology suggest a complex role for progranulin in the hippocampus. We conclude that core progranulin-mediated neurodegenerative phenotypes are penetrant on multiple inbred mouse strains, but that genetic background modulates progranulin's role in neuroinflammation and hippocampal biology.
Asunto(s)
Encéfalo/patología , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Agresión/fisiología , Animales , Encéfalo/fisiopatología , Conducta Exploratoria/fisiología , Femenino , Gliosis/genética , Gliosis/patología , Gliosis/fisiopatología , Granulinas , Péptidos y Proteínas de Señalización Intercelular/genética , Estudios Longitudinales , Masculino , Aprendizaje por Laberinto/fisiología , Ratones de la Cepa 129/fisiología , Ratones Endogámicos C57BL/fisiología , Ratones Noqueados/fisiología , Actividad Motora/fisiología , Neuroinmunomodulación/fisiología , Neuronas/patología , Tamaño de los Órganos , Progranulinas , Reconocimiento en Psicología/fisiología , Caracteres Sexuales , Especificidad de la Especie , Análisis de SupervivenciaRESUMEN
Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84-178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2null/null mice. Finally, we set up a tamoxifen administration protocol that allows an efficient, time-specific inactivation of brain serotonin synthesis. On the whole, we generated a suitable genetic tool to investigate how serotonin depletion impacts on time-specific events during central nervous system development and adulthood life.
Asunto(s)
Química Encefálica/fisiología , Ratones Noqueados/fisiología , Serotonina/análisis , Triptófano Hidroxilasa/fisiología , Alelos , Animales , Química Encefálica/genética , Femenino , Genotipo , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Triptófano Hidroxilasa/efectos de los fármacosRESUMEN
Estrogens, predominantly 17ß-estradiol, exert diverse effects throughout the body in both normal and pathophysiology, during development and in reproductive, metabolic, endocrine, cardiovascular, nervous, musculoskeletal and immune systems. Estrogen and its receptors also play important roles in carcinogenesis and therapy, particularly for breast cancer. In addition to the classical nuclear estrogen receptors (ERα and ERß) that traditionally mediate predominantly genomic signaling, the G protein-coupled estrogen receptor GPER has become recognized as a critical mediator of rapid signaling in response to estrogen. Mouse models, and in particular knockout (KO) mice, represent an important approach to understand the functions of receptors in normal physiology and disease. Whereas ERα KO mice display multiple significant defects in reproduction and mammary gland development, ERß KO phenotypes are more limited, and GPER KO exhibit no reproductive deficits. However, the study of GPER KO mice over the last six years has revealed that GPER deficiency results in multiple physiological alterations including obesity, cardiovascular dysfunction, insulin resistance and glucose intolerance. In addition, the lack of estrogen-mediated effects in numerous tissues of GPER KO mice, studied in vivo or ex vivo, including those of the cardiovascular, endocrine, nervous and immune systems, reveals GPER as a genuine mediator of estrogen action. Importantly, GPER KO mice have also demonstrated roles for GPER in breast carcinogenesis and metastasis. In combination with the supporting effects of GPER-selective ligands and GPER knockdown approaches, GPER KO mice demonstrate the therapeutic potential of targeting GPER activity in diseases as diverse as obesity, diabetes, multiple sclerosis, hypertension, atherosclerosis, myocardial infarction, stroke and cancer.
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Ratones Noqueados/genética , Receptores Acoplados a Proteínas G/genética , Animales , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunidad , Ratones , Ratones Noqueados/inmunología , Ratones Noqueados/fisiología , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Obesidad/genética , Obesidad/inmunología , Obesidad/metabolismo , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The genetic manipulation of the laboratory mouse has been well developed and generated more and more mouse lines for biomedical research. To advance our science exploration, it is necessary to share genetically modified mouse lines with collaborators between institutions, even in different countries. The transfer process is complicated. Significant paperwork and coordination are required, concerning animal welfare, intellectual property rights, colony health status, and biohazard. Here, we provide a practical example of importing a transgenic mice line, Dynamin 1 knockout mice, from Yale University in the USA to Perking University in China for studying cell secretion. This example including the length of time that required for paper work, mice quarantine at the receiving institution, and expansion of the mouse line for experiments. The procedure described in this paper for delivery live transgenic mice from USA to China may serve a simple reference for transferring mouse lines between other countries too.
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Cruzamiento/métodos , Técnicas de Genotipaje/métodos , Ratones Noqueados/genética , Comités de Atención Animal , Animales , China , Dinamina I/genética , Ciencia de los Animales de Laboratorio/métodos , Ciencia de los Animales de Laboratorio/normas , Ratones , Ratones Noqueados/fisiología , UniversidadesRESUMEN
Intestinal absorption is an essential step in the therapeutic use of most orally administered drugs and often mediated by enterocyte transmembrane transporters. Here we discuss several of these drug transport systems and knockout mouse models to study them. These studies showed that Multidrug resistance-associated protein 2 (Mrp2) can limit intestinal drug absorption. Organic cation transporter n1 (Octn1) and Octn2 might also facilitate intestinal drug absorption, although direct in vivo evidence is lacking. On the other hand, intestinal uptake of drugs is facilitated by the Equilibrative nucleoside transporter 1 (Ent1), Mrp3 and possibly Mrp4. No significant role in intestinal absorption for Oct1 and Oct2 or for Organic anion-transporting polypeptides (Oatp) 1a and 1b was found so far.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Transporte Biológico/fisiología , Absorción Intestinal/fisiología , Proteínas de Transporte de Membrana/metabolismo , Ratones Noqueados/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/fisiología , Ratones , Ratones Noqueados/genética , Ratones Noqueados/fisiología , Modelos AnimalesRESUMEN
An imbalanced immune system has long been known to influence a variety of mood disorders including anxiety, obsessive-compulsive disorders and depression. In this study, we sought to model the impact of an immunocompromised state on these emotional behaviors using RAG-1â»/â» mice, which lack T and B cells. We also investigated the relative contribution of CD4⺠or CD8⺠T cells to these manifestations using RAG-1â»/â»/OT-II and RAG-1â»/â»/OT-I transgenic mice, respectively. Our results show that RAG-1â»/â» mice present a significant increase in digging and marble-burying activities compared with wild-type mice. Surprisingly, these anxiety-like behaviors were significantly reverted in RAG-1â»/â»/OT-II but not RAG-1â»/â»/OT-I transgenic mice. Immunodepletion experiments with anti-CD4 or anti-CD8 in C57/BL6 mice or repopulation studies in RAG-1â»/â» mice did not reproduce these findings. Microarray analysis of the brain of RAG-1â»/â» and RAG-1â»/â»/OT-II mice revealed a significantly different gene fingerprint, with the latter being more similar to wild-type mice than the former. Further analysis revealed nine main signaling pathways as being significantly modulated in RAG-1â»/â» compared with wild-type mice. Taken together, these results suggest that life-long rather than transient immunodeficient conditions influence the emotional behaviors in mice. Most interestingly, these effects seem to correlate with a specific absence of CD4⺠rather than CD8⺠T cells. Validation of these findings in man might provide new clues on the mechanism by which early life immune modulation might impact mood response in adults and provide a further link between immune and emotional well-being.
Asunto(s)
Conducta Animal/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Emociones/fisiología , Proteínas de Homeodominio/fisiología , Animales , Corticosterona/sangre , Citocinas/sangre , Citometría de Flujo , Huésped Inmunocomprometido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/chicken ß-actin (CBA)-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for green fluorescent protein (GFP)-treated mice. ssAAV9/CBA-MECP2-treated mice also showed significant improvement in the phenotype severity score, in locomotor function, and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type (WT) mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary (sc) adeno-associated virus (AAV) vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously (IV) into juvenile (4-5 weeks old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2-4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética , Ratones Noqueados/fisiología , Síndrome de Rett/terapia , Tasa de Supervivencia , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Masculino , Ratones , Ratones Noqueados/genética , Fenotipo , Síndrome de Rett/genéticaRESUMEN
AIMS: The establishment of a genetic knockout murine model of glutaric acidemia type I (GAI) with complete loss of glutaryl-CoA dehydrogenase (GCDH) activity has been used to investigate the pathological mechanisms underlying neurological symptoms in this disorder. However, very little has been reported on the neurobehavior of GCDH deficient mice (Gcdh(-/-)). MAIN METHODS: In the present study we evaluated physical (body and weight gain) and neuromotor development (appearance of coat, upper incisor eruption, eye-opening day, motor coordination, muscular strength and climbing), as well as cognitive behavior (inhibitory avoidance) in Gcdh(-/-), as compared to wild type (WT) mice. KEY FINDINGS: We found that Gcdh(-/-) mice did not differ in body and weight gain, appearance of coat, upper incisor eruption, motor coordination and muscular strength, but had a significant delayed eye opening, implying a mild impairment of neurodevelopment in these animals. Furthermore, the climbing behavior was significantly higher in Gcdh(-/-) as compared to WT mice, suggesting an altered dopaminergic function. Finally, Gcdh(-/-) mice presented a deficit of short- and long-term memories in the inhibitory avoidance task. SIGNIFICANCE: Although it is difficult to extrapolate the present findings to the human condition, our present data are particularly interesting in view of the psychomotor/mental delay that occurs in a significant number of GAI patients with no previous history of acute encephalopathy with striatum destruction. Strict and early treatment possibly associated with novel therapies seems therefore important to prevent learning/memory disabilities in GAI patients.
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Glutaril-CoA Deshidrogenasa/deficiencia , Animales , Reacción de Prevención/fisiología , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Glutaril-CoA Deshidrogenasa/genética , Masculino , Ratones , Ratones Noqueados/genética , Ratones Noqueados/crecimiento & desarrollo , Ratones Noqueados/fisiología , Fuerza Muscular/genética , Fuerza Muscular/fisiología , Umbral del Dolor/fisiología , Equilibrio Postural/genética , Equilibrio Postural/fisiología , Desempeño Psicomotor/fisiologíaRESUMEN
AIMS: Serotonin transporter knockout (5-HTT KO) mice exhibit elevated basal extracellular serotonin, increased depressive-like behaviors and increased rapid eye movement sleep. Because abnormalities of circadian rhythms are associated with mood disorders, we tested the hypothesis that 5-HTT KO mice would have altered circadian rhythmicity. MAIN METHODS: Homecage locomotor activity was recorded in wild-type (WT) and KO mice under a standard 12:12 light-dark cycle. After 4weeks of recording, mice received a one-hour light pulse at circadian time (CT) 14 and then were kept under constant darkness for 3weeks. KEY FINDINGS: There were no significant differences in amplitude, period, acrophase or total home cage locomotor activity between WT and KO mice during the 12:12 light-dark cycle or during constant darkness. The mean phase delay to a CT 14 light pulse was significantly attenuated in KO compared to WT mice. SIGNIFICANCE: Acute increases in serotonin have been reported to attenuate photic phase shifts. The current study demonstrates that this effect is maintained in the face of a lifelong absence of 5-HTT.
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Ritmo Circadiano/fisiología , Ratones Noqueados/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Animales , Ritmo Circadiano/genética , Líquido Extracelular/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Actividad Motora/genética , Actividad Motora/fisiología , Fotoperiodo , Serotonina/análisis , Serotonina/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Factores de TiempoRESUMEN
STUDY OBJECTIVE: Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. DESIGN: Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. MEASUREMENTS AND RESULTS: NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. CONCLUSIONS: Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders.
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Afecto/fisiología , Receptores de Neurotensina/fisiología , Sueño/fisiología , Animales , Ansiedad/genética , Depresión/genética , Electroencefalografía , Electromiografía , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/fisiología , Actividad Motora/fisiología , Sitios de Carácter Cuantitativo , Privación de Sueño/fisiopatologíaRESUMEN
To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains.
Asunto(s)
Ratones Noqueados/fisiología , Canales de Potasio con Entrada de Voltaje/genética , Animales , Western Blotting , Química Encefálica/genética , Calbindinas , Colorantes , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Proteínas de Interacción con los Canales Kv/genética , Canal de Potasio Kv.1.1/genética , Ratones , Ratones Noqueados/genética , Fibras Musgosas del Hipocampo/metabolismo , Terminales Presinápticos/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Canales de Potasio Shal/genética , Regulación hacia Arriba/fisiologíaRESUMEN
A-type (atrial) natriuretic peptide (ANP) levels in the heart and plasma were examined by immunohistochemistry, electron microscopy, and radioimmunoassay (RIA) in angiotensin II type 1a receptor knockout (Agtr1a KO) mice. Additionally, the ANP mRNA level in the heart was measured using a real-time polymerase chain reaction (PCR) assay. The blood pressure in Agtr1a KO mice was significantly lower than that in wild-type (WT) mice. The number of ANP granules and ANP immunoreactivity in the auricular cardiocytes were significantly lower in Agtr1a KO mice than in WT mice. Ultrastructurally, the ventricular cardiocytes in Agtr1a KO mice occasionally had ANP-like granules, which were not present in WT mice. The plasma, auricular, and ventricular ANP and plasma cyclic guanosine monophosphate (cGMP) concentrations were significantly higher in Agtr1a KO mice than in WT mice. The ANP mRNA levels of the auricular and ventricular cardiocytes in the Agtr1a KO mice were almost twice as large as those in WT mice. The present data suggest that a notable increase in the ANP biosynthesis and release in the heart of Agtr1a KO mice may account for the reduction in blood pressure together with the lack of an AGTR1A receptor in this model.
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Factor Natriurético Atrial/biosíntesis , Hipotensión/genética , Ratones Noqueados/fisiología , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Factor Natriurético Atrial/genética , Presión Sanguínea/genética , Modelos Animales de Enfermedad , Expresión Génica , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Hipotensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , ARN Mensajero/metabolismo , Receptor de Angiotensina Tipo 1/deficiencia , Receptor de Angiotensina Tipo 1/genéticaRESUMEN
BACKGROUND AND PURPOSE: Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7(-/-) mice (Pfizer KO). EXPERIMENTAL APPROACH: Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS: RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS: Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.
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Ratones Noqueados/fisiología , Receptores Purinérgicos P2X7/fisiología , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , ARN Mensajero/genética , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/metabolismo , Alineación de Secuencia , Bazo/metabolismoRESUMEN
PURPOSE: We evaluated the ability of the ketogenic diet (KD) to improve thresholds to flurothyl-induced seizures in two mouse lines with Scn1a mutations: one that models Dravet syndrome (DS) and another that models genetic (generalized) epilepsy with febrile seizures plus (GEFS+). METHODS: At postnatal day 21, mouse models of DS and GEFS+ were fasted for 12-14 h and then placed on either a 6:1 (fats to proteins and carbohydrates) KD or a standard diet (SD) for 2 weeks. At the end of the 2-week period, we measured thresholds to seizures induced by the chemiconvulsant flurothyl. Body weight, ß-hydroxybutyrate (BHB) levels, and glucose levels were also recorded every 2 days over a 2-week period in separate cohorts of mutant and wild-type mice that were either on the KD or the SD. KEY FINDINGS: Mice on the KD gained less weight and exhibited significantly higher BHB levels compared to mice on the SD. It is notable that thresholds to flurothyl-induced seizures were restored to more normal levels in both mouse lines after 2 weeks on the KD. SIGNIFICANCE: These results indicate that the KD may be an effective treatment for refractory patients with SCN1A mutations. The availability of mouse models of DS and GEFS+ also provides an opportunity to better understand the mechanism of action of the KD, which may facilitate the development of improved treatments.
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Dieta Cetogénica , Epilepsia Generalizada/dietoterapia , Ácido 3-Hidroxibutírico/sangre , Animales , Convulsivantes/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Epilepsia Generalizada/genética , Canales Epiteliales de Sodio/genética , Flurotilo/farmacología , Ratones , Ratones Noqueados/genética , Ratones Noqueados/fisiología , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Aumento de PesoRESUMEN
Based on electrophysiological, neurochemical, genetic and neuropharmacological approaches it is currently accepted that serotonin (5-HT) functions to promote waking (W) and to inhibit rapid-eye movement sleep (REMS). The serotonin-containing neurons of the dorsal raphe nucleus (DRN) provide part of the serotonergic innervation of the telencephalon, diencephalon, mesencephalon and rhombencephalon of laboratory animals and man. The DRN has been subdivided into several clusters on the basis of differences in cellular morphology, expression of other neurotransmitters and afferent and efferent connections. These differences among subpopulations of 5-HT neurons may have important implications for neural mechanisms underlying 5-HT modulation of sleep and waking. The DRN contains 5-HT and non-5-HT neurons. The latter express a variety of substances including dopamine, γ-aminobutyric acid (GABA) and glutamate. In addition, nitric oxide and a number of neuropeptides have been characterized in the DRN. Available evidence tends to indicate that non-5-HT cells contribute to the regulation of the activity of 5-HT neurons during the sleep-wake cycle through local circuits and/or their mediation of the effects of afferent inputs. Mutant mice that do not express 5-HT(1A) or 5-HT(1B) receptor exhibit greater amounts of REMS than their wild-type couterparts. 5-HT(2A) and 5-HT(2C) receptor knockout mice show a significant increase of W and a reduction of slow wave sleep that is related, at least in part, to the increased release of norepinephrine and dopamine. A normal circadian sleep pattern is observed in 5-HT(7) receptor knockout mice; however, the mutants spend less time in REMS. Local microinjection of 5-HT(1B), 5-HT(2A/2C), 5-HT(3) and 5-HT(7) receptor agonists into the DRN selectively suppresses REMS in the rat. In contrast, microinjection of 5-HT(1A) receptor agonists promotes REMS. Similarly, local administration of the melanin-concentrating hormone or the GABA(A) receptor agonist muscimol produces an increase of REMS in the rat. Presently, there are no data on the effect of local infusion into the DRN of noradrenergic, dopaminergic, histaminergic, orexinergic and cholinergic agonists on sleep variables in laboratory animals.
Asunto(s)
Núcleos del Rafe/fisiología , Receptores de Serotonina/fisiología , Sueño REM/fisiología , Vigilia/fisiología , Animales , Humanos , Ratones , Ratones Noqueados/fisiología , Neuronas/fisiología , Neuronas Aferentes/fisiología , Núcleos del Rafe/anatomía & histología , Ratas , Serotonina/fisiología , Sueño/fisiologíaAsunto(s)
Nivel de Alerta/fisiología , Histamina/metabolismo , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Locomoción/fisiología , Motivación/fisiología , Neuropéptidos/metabolismo , Animales , Trastornos de Somnolencia Excesiva/genética , Trastornos de Somnolencia Excesiva/metabolismo , Trastornos de Somnolencia Excesiva/fisiopatología , Histidina Descarboxilasa/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados/fisiología , Neuropéptidos/genética , OrexinasRESUMEN
SPAK/STK39 is a mammalian protein kinase involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both central and the peripheral nervous systems. We have previously shown that disruption of the gene encoding SPAK by homologous recombination in mouse embryonic stem cells results in viable mice that lack expression of the kinase. With the exception of reduced fertility, these mice do not exhibit an overt adverse phenotype. In the present study, we examine the neurological phenotype of these mice by subjecting them to an array of behavioral tests. We show that SPAK knockout mice displayed a higher nociceptive threshold than their wild-type counterparts on the hot plate and tail flick assays. SPAK knockout mice also exhibited a strong locomotor phenotype evidenced by significant deficits on the rotarod and decreased activity in open-field tests. In contrast, balance and proprioception was not affected. Finally, they demonstrated an increased anxiety-like phenotype, spending significantly longer periods of time in the dark area of the light/dark box and increased thigmotaxis in the open-field chamber. These results suggest that the kinase plays an important role in CNS function, consistent with SPAK regulating ion transport mechanisms directly involved in inhibitory neurotransmission.