Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Producción CientíficaSusceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 h, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.2018/02/02Ministerio de Economía y Competitividad (BFU2012-37146)Junta de Castilla y Leòn (BIO103/VA45/11, VA145U13 and BIO/VA33/13

Changes in expression of hypothalamic releasing hormone receptors in individual rat anterior pituitary cells during maturation, puberty and senescence

Producción CientíficaAnterior pituitary (AP) is formed by five different cell types, each one producing a different AP hormone whose secretion is regulated by a specific hypothalamic-releasing hormone (HRH). On the other hand, a significant number of AP cells express multiple HRH receptors (multiresponsive cells). Plastic changes in expression of HRH receptors in individual AP cells are involved in critical endocrine events. Here we have characterized the changes in functional responses to CRH, LHRH, TRH, and GHRH in individual AP cells throughout the whole life span of the rat. To this end, calcium responses to the HRHs were followed by single-cell imaging in freshly dispersed AP cells prepared from rats of different ages (0–540 postnatal days). Three different cell pools were identified: 1) monoresponsive cells, holding a single class of HRH receptor; 2) multiresponsive cells; and 3) nonresponsive cells. The relative abundance of each pool changed with age. Nonresponsive cells were abundant at birth, multiresponsive cells were abundant at puberty, and monoresponsive cells dominated at senescence. The relative abundance of each HRH receptor changed largely with age but not gender. In addition, the contribution of monoresponsive and multiresponsive cells to responses to each HRH changed very much with age. Thus, the anterior pituitary shows large changes in cell populations typed by functional responses to HRHs during maturation, puberty, and senescence.Fondo de Investigaciones Sanitarias (grant FIS 03/1231)Ministerio de Ciencia, Innovación y Universidades (grants BFI 2001-2073 and BFU-2004-02765/BFI

Aging Enables Ca2+ Overload and Apoptosis Induced by Amyloid- Oligomers in Rat Hippocampal Neurons: Neuroprotection by Non-Steroidal Anti-Inflammatory Drugs and R-Flurbiprofen in Aging Neurons

Producción CientíficaThe most important risk factor for Alzheimer’s disease (AD) is aging. Neurotoxicity in AD has been linked to dyshomeostasis of intracellular Ca2+ induced by small aggregates of the amyloid- peptide 1-42 (A 42 oligomers). However, how aging influences susceptibility to neurotoxicity induced by A 42 oligomers is unknown. In this study, we used longterm cultures of rat hippocampal neurons, a model of neuronal in vitro aging, to investigate the contribution of aging to Ca2+ dishomeostasis and neuron cell death induced by A 42 oligomers. In addition, we tested whether non-steroidal antiinflammatory drugs (NSAIDs) and R-flurbiprofen prevent apoptosis acting on subcellular Ca2+ in aged neurons.We found that A 42 oligomers have no effect on young hippocampal neurons cultured for 2 days in vitro (2 DIV). However, they promoted apoptosis modestly in mature neurons (8 DIV) and these effects increased dramatically after 13 DIV, when neurons display many hallmarks of in vivo aging. Consistently, cytosolic and mitochondrial Ca2+ responses induced by A 42 oligomers increased dramatically with culture age. At low concentrations, NSAIDs and the enantiomer R-flurbiprofen lacking antiinflammatory activity prevent Ca2+ overload and neuron cell death induced by A 42 oligomers in aged neurons. However, at high concentrations R-flurbiprofen induces apoptosis. Thus, A 42 oligomers promote Ca2+ overload and neuron cell death only in aged rat hippocampal neurons. These effects are prevented by low concentrations of NSAIDs and R-flurbiprofen acting on mitochondrial Ca2+ overload.Ministerio de Economía y Competitividad (BFU2012- 627 37146)Junta de Castilla y León (VA145U13, 625 BIO/VA33/13, BIO103/VA45/11

Rapid changes in anterior pituitary cell phenotypes in male and female mice after acute cold stress

Producción CientíficaThe anterior pituitary (AP) is made of five different cell types. The relative abundance and phenotype of AP cells may change in different physiological situations as an expression of pituitary plasticity. Here, we analyze in detail the phenotype of mouse corticotropes and the effects of acute cold stress on AP cell populations. The hormone content and the expression of hypothalamic-releasing hormone (HRH) receptors in all the five AP cell types were studied in the male and female mice at rest and after a 30-min cold stress. Expression of HRH receptors was evidenced by imaging the single-cell cytosolic Ca2+ responses in fura-2-loaded cells. Hormone contents were studied by multiple, simultaneous immunofluorescence of all the five hormones. Corticotropes displayed a striking sexual dimorphism, even in the resting condition. Male corticotropes showed the orthodox phenotype. They were monohormonal, storing only ACTH, and monoreceptorial, responding only to CRH. In contrast, female corticotropes were made of about equal parts of orthodox cells and multifunctional cells, which co-stored additional AP hormones and expressed additional HRH receptors. Cold stress did not modify the number of ACTH containing cells, but, according to immunostaining, it increased the relative abundance of other AP cell types at the expense of the pool of cells storing no hormones. Cold stress also modified the response to CRH and other HRHs. Most of these phenotypical changes presented a strong sexual dimorphism. These results indicate that pituitary plasticity is even larger than previously thought.Fondo de Investigaciones Sanitarias (grant FIS03/1231)Ministerio de Ciencia, Innovación y Universidades (grants BFU-2004-02765/BFI, BFU2005-02078 and BFU2007-60157

Marine heterocyclic compounds that modulate intracellular calcium signals: Chemistry and synthesis approaches

Producción CientíficaIntracellular Ca2+ plays a pivotal role in the control of a large series of cell functions in all types of cells, from neurotransmitter release and muscle contraction to gene expression, cell proliferation and cell death. Ca2+ is transported through specific channels and transporters in the plasma membrane and subcellular organelles such as the endoplasmic reticulum and mitochondria. Therefore, dysregulation of intracellular Ca2+ homeostasis may lead to cell dysfunction and disease. Accordingly, chemical compounds from natural origin and/or synthesis targeting directly or indirectly these channels and proteins may be of interest for the treatment of cell dysfunction and disease. In this review, we show an overview of a group of marine drugs that, from the structural point of view, contain one or various heterocyclic units in their core structure, and from the biological side, they have a direct influence on the transport of calcium in the cell. The marine compounds covered in this review are divided into three groups, which correspond with their direct biological activity, such as compounds with a direct influence in the calcium channel, compounds with a direct effect on the cytoskeleton and drugs with an effect on cancer cell proliferation. For each target, we describe its bioactive properties and synthetic approaches. The wide variety of chemical structures compiled in this review and their significant medical properties may attract the attention of many different researchers.Ministerio de Ciencia e Innovación - (grant RTI2018-099298-B-100)Junta de Castilla y León - (grant VA294-P18

Stability and multiscroll attractors of control systems via the abscissa

"We present an approach to generate multiscroll attractors via destabilization of piecewise linear systems based on Hurwitz matrix in this paper. First we present some results about the abscissa of stability of characteristic polynomials from linear differential equations systems; that is, we consider Hurwitz polynomials. The starting point is the Gauss–Lucas theorem, we provide lower bounds for Hurwitz polynomials, and by successively decreasing the order of the derivative of the Hurwitz polynomial one obtains a sequence of lower bounds. The results are extended in a straightforward way to interval polynomials; then we apply the abscissa as a measure to destabilize Hurwitz polynomial for the generation of a family of multiscroll attractors based on a class of unstable dissipative systems (UDS) of affine linear type.

Calcium dynamics and resting transcriptional activity regulates prolactin gene expression

Producción CientíficaResearch on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca2+ release, followed by sustained Ca2+ influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca2+]i increase. In addition, transcriptional responses were related to the rate of Ca2+ entry but not Ca2+ release. Finally, cells lacking transcriptional responses (but showing [Ca2+]i rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca2+ induced by TRH (but not the Ca2+ release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.National Institutes of Health (grant DK-38215)Fondo de Investigaciones Sanitarias (grant FIS 01/0769

Stability and Multiscroll Attractors of Control Systems via the Abscissa

We present an approach to generate multiscroll attractors via destabilization of piecewise linear systems based on Hurwitz matrix in this paper. First we present some results about the abscissa of stability of characteristic polynomials from linear differential equations systems; that is, we consider Hurwitz polynomials. The starting point is the Gauss–Lucas theorem, we provide lower bounds for Hurwitz polynomials, and by successively decreasing the order of the derivative of the Hurwitz polynomial one obtains a sequence of lower bounds. The results are extended in a straightforward way to interval polynomials; then we apply the abscissa as a measure to destabilize Hurwitz polynomial for the generation of a family of multiscroll attractors based on a class of unstable dissipative systems (UDS) of affine linear type

Cumplimiento de las recomendaciones de prevención secundaria de enfermedad coronaria en pacientes sometidos a revascularización coronaria percutánea en el Hospital Universitario Fundación Santa Fe de Bogotá

Marco de referenciadiferentes ensayos clínicos han mostrado que el efecto de las revascularizaciones y los procedimientos de angioplastia en el pronóstico del paciente, tiene sólo un efecto a corto plazo, mientras que la prevención secundaria (cambio de estilo de vida y tratamiento intensivo) mejora el pronóstico a largo plazo.Objetivoevaluar el cumplimiento de las recomendaciones para prevención cardiovascular secundaria, de los pacientes sometidos a revascularización coronaria percutánea en el hospital universitario Fundación Santa Fe de Bogotá, entre los años 2008 y 2010.Materiales y métodosestudio descriptivo, ambispectivo, cuya población estuvo conformada por 332 pacientes sometidos a intervención coronaria percutánea atendidos entre enero de 2008 a diciembre de 2010 en el servicio de hemodinamia del Hospital Universitario Fundación Santa Fe de Bogotá. La recolección de la información se realizó mediante revisión de las historias clínicas de los pacientes y vía telefónica. Las metas de control se basaron en las recomendaciones de la Asociación Americana del Corazón y el Colegio Americano de Cardiología.Resultados332 pacientes fueron sometidos a revascularización coronaria percutánea y 4 fueron excluidos del estudio por falta de datos. La presentación clínica más frecuente fue la angina inestable (32,93%). Sólo se contactaron 172 (52,43%) pacientes, de los cuales 77,4% eran hombres y 22,6% mujeres. Únicamente 6 (3,5%) cumplían con el 100% de las recomendaciones para la prevención secundaria de enfermedad coronaria y 17% cumplía menos del 50% de éstas.Conclusionesla experiencia en la Fundación Santa Fe de Bogotá muestra un pobre cumplimiento de las metas de prevención secundaria de enfermedad coronaria.Contextdifferent clinical trials have shown that the effect of revascularization and angioplasty procedures in the prognosis of the patient has only a short-term effect, while secondary prevention (lifestyle change and intensive treatment) improves long-term prognosis.Objectiveto assess compliance with the recommendations for secondary prevention of cardiovascular disease in patients undergoing percutaneous coronary revascularization at the University Hospital Fundación Santa Fe de Bogotá, between 2008 and 2010.Materials and methodsa descriptive ambispective study, whose population was conformed by 332 patients undergoing percutaneous coronary intervention seen between January 2008 and December 2010 in the service of hemodynamics of the University Hospital Fundación Santa Fe de Bogotá. The data collection was conducted by reviewing the medical records of the patients and by telephone. Control targets were based on the recommendations of the American Heart Association and the American College of Cardiology.Results332 patients underwent percutaneous coronary revascularization. 4 were excluded from the study due to lack of data. The most common clinical presentation was unstable angina (32.93%). Only 172 patients (52.43%) were contacted. From these, 77.4% were male and 22.6% female. Only 6 (3.5%) met 100% of the recommendations for secondary prevention of coronary heart disease and 17% met less than 50% of these recommendations.Conclusionsthe experience in the Fundación Santa Fe de Bogotá shows a poor compliance with the goals of secondary prevention of coronary disease