Estudios del rol modulador de ghrelina sobre la actividad neuroendocrina: circuitos neuronales implicados en la activación de las neuronas CRF hipofisiotrópicas

En la presente Tesis Doctoral estudiamos los circuitos neuronales que median los efectos de la ghrelina sobre las neuronas CRF hipofisiotrópicas del NPV y el eje HHA. Esperamos que el esclarecimiento de estos circuitos pueda ayudar a entender cómo funcionan y cómo se integran la homeostasis energética y el estrés. Así como también, contribuir al desarrollo de futuras terapias que intenten manipular las acciones de la ghrelina.Facultad de Ciencias Exacta

Brain circuits mediating the orexigenic action of peripheral ghrelin: narrow gates for a vast kingdom

The nervous and endocrine systems act together to regulate all physiological processes essential for the body homeostasis control. Given the strict communication restrictions that the brain-blood barrier (BBB) imposes, the interplay between these two systems requires a variety of delicate anatomical interfaces and physiological mechanisms that guarantee the precise function of the neuroendocrine system as a whole. The study of the mechanisms by which hormones act in the brain in order to regulate specific neuronal populations is a research topic rather neglected. Our group studies the neuronal circuitries and molecular mechanisms by which the stomach-produced hormone ghrelin regulates appetite and other physiological functions. A clear notion of the brain targets of peripheral ghrelin is essential for the comprehensive understanding of the physiological role of this hormone. Ghrelin is called "the hunger hormone" since it is the only known orexigenic peptide hormone. The target for ghrelin orexigenic actions is the brain, which contains a variety of ghrelin-responsive nuclei; however, several evidences suggest that the accessibility of peripheral ghrelin to the brain is strikingly low. Here, we briefly summarize the current knowledge in this topic and discuss this intriguing neuroendocrinological issue.Instituto Multidisciplinario de Biología Celula

Is ghrelin synthesized in the central nervous system?

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.Instituto Multidisciplinario de Biología Celula

Fasting enhances pyroglutamyl peptidase II activity in tanycytes of the mediobasal hypothalamus of male adult rats

Fasting down-regulates the hypothalamus-pituitary-thyroid (HPT) axis activity through a reduction of TRH synthesis in neurons of the parvocellular paraventricular nucleus of the hypothalamus (PVN). These TRH neurons project to the median eminence (ME), where TRH terminals are close to the cytoplasmic extensions of β2 tanycytes. Tanycytes express pyroglutamyl peptidase II (PPII), the TRH-degrading ectoenzyme that controls the amount of TRH that reaches the anterior pituitary. We tested the hypothesis that regulation of ME PPII activity is another mechanism by which fasting affects the activity of the HPT axis. Semiquantitative in situ hybridization histochemistry data indicated that PPII and deiodinase 2 mRNA levels increased in tanycytes after 48 hours of fasting. This increase was transitory, followed by an increase of PPII activity in the ME, and a partial reversion of the reduction in PVN pro-TRH mRNA levels and the number of TRH neurons detected by immunohistochemistry. In fed animals, adrenalectomy and corticosterone treatment did not change ME PPII activity 72 hours later. Methimazole-induced hypothyroidism produced a profound drop in tanycytes PPII mRNA levels, which was reverted by 3 days of treatment with T4. The activity of thyroliberinase, the serum isoform of PPII, was increased at most fasting time points studied. We conclude that delayed increases in both the ME PPII as well as the thyroliberinase activities in fasted male rats may facilitate the maintenance of the deep down-regulation of the HPT axis function, despite a partial reactivation of TRH expression in the PVN

Is ghrelin synthesized in the central nervous system?

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.Instituto Multidisciplinario de Biología Celula

Sperm nuclear study in the clinical practice

Introducción: Durante la espermatogénesis las células germinales masculinas sufren un complejo proceso de remodelamiento nuclear en el cual la cromatina espermática reemplaza cerca del 85% de sus histonas testiculares por proteínas pequeñas y fuertemente básicas denominadas protaminas. Este proceso es necesario no sólo para generar un núcleo hidrodinámico que permita el transporte de todo el genoma masculino a través del tracto reproductivo, sino también para proteger la información genética del ataque de nucleasas, especies reactivas del oxígeno (ROS) y mutágenos, que podrían inducir daño en el ADN. La disminución en el contenido de protaminas está ligada a una condensación incompleta o aberrante de la cromatina espermática y a una pobre integridad del ADN. Esta situación generaría un estado de ´susceptibilidad de la cromatina´ al ataque de agentes endógenos y exógenos que podrían generar altos niveles de fragmentación en el ADN espermático. Objetivo: Evaluar la relación entre los niveles de empaquetamiento de la cromatina espermática, el grado de daño en el ADN y la presencia de marcadores apoptóticos en espermatozoides eyaculados. Metodología: Se analizaron 213 muestras de pacientes que consultaron por infertilidad y 86 donantes fértiles. La compactación de la cromatina fue determinada por Cromomicina A3 (CMA3) y Azul de Anilina (AA), la fragmentación del ADN por el test de TUNEL con epifluorescencia y los niveles de apoptosis por Caspasa 3-Activa. Los datos se analizaron mediante tablas de contingencia 2x2 (Epidat 3.1), análisis de correlación de Pearson y test de Student (SPSS 16.0 para Windows; SPSS Inc., Chicago, IL). Resultados: Se encontró una correlación positiva entre los niveles de fragmentación espermática y el grado de compactación de la cromatina. De modo inverso, los valores de Caspasa 3-Activa correlacionaron negativamente con TUNEL. Además, se determinó el riesgo relativo de presentar valores alterados de TUNEL, en aquellas muestras con valores alterados de CMA3 y AA. Por otra parte, se encontró que algunos de los parámetros seminales básicos se encuentran disminuidos al momento que los valores de TUNEL y CMA3 y AA estan alterados. Conclusión: A lo largo de este trabajo se demostró la importancia que tienen los métodos de diagnósticos clásicos, como CMA3 y AA, para determinar la susceptibilidad de los espermatozoides al daño en el ADN, y como los parámetros seminales básicos están relacionados con estos niveles de daño. La importancia del estudio profundo de la gameta masculina reside en proveer nuevos conocimientos en el abordaje de los tratamientos en reproducción asistida.Introduction. During spermatogenesis, germinal cells suffer a complex nuclear remodeling in which 85% of testicular histones are replaced with small and basic proteins named protamines. This process is important and necessary to confer hydrodynamic shape and genetic information protection during transportation through the female tract. A poor content of protamines is related to incomplete or aberrant chromatin condensation and poor DNA integrity. This state could generate a “susceptibility state of chromatin” to endogenous and exogenous agents’ attack that could enhances DNA fragmentation. Aim. To study the relationship between chromatin remodeling, DNA damage and apoptotic status of sperm samples. Methodology. Semen samples of 213 patients and 86 sperm donors were analyzed for chromatin packaging using Chromomycin A3 (CMA3) and Aniline Blue (AB), DNA fragmentation through TUNEL assay and apoptotic levels through the identification of active-Caspase-3 by immunocytochemistry. Data were analyzed with contingency tables (2x2) (Epidat 3.1), Pearson´s correlation coefficient and Student´s t-test (SPSS 16.0 for Windows; SPSS Inc., Chicago, IL).Results. When abnormal chromatin package is present, a positive correlation with high levels of DNA fragmentation is observed. The relative risk of showing high levels of DNA fragmentation on altered chromatin samples was determined. In addition to that, we found that motility and sperm concentration were diminished when TUNEL were abnormal.Conclusions. We have shown the relationship between abnormal chromatin packing and DNA fragmentation. Likewise, we observed that some parameters such motility and sperm concentration are related with this damage. Our findings could be useful tools during the clinical search of the underlying male factor.Fil: Cabral, Noelia Edith. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico. Departamento de Estudios Biomédicos y Biotecnológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Faut, Monica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez Torres, maría Agustina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico. Departamento de Estudios Biomédicos y Biotecnológicos; ArgentinaFil: Rawe, Vanesa Yanina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentin

Neuroanatomical and functional characterization of CRF neurons of the amygdala using a novel transgenic mouse model

The corticotropin-releasing factor (CRF)-producing neurons of the amygdala have been implicated in behavioral and physiological responses associated with fear, anxiety, stress, food intake and reward. To overcome the difficulties in identifying CRF neurons within the amygdala, a novel transgenic mouse line, in which the humanized recombinant Renilla reniformis green fluorescent protein (hrGFP) is under the control of the CRF promoter (CRF-hrGFP mice), was developed. First, the CRF-hrGFP mouse model was validated and the localization of CRF neurons within the amygdala was systematically mapped. Amygdalar hrGFP-expressing neurons were located primarily in the interstitial nucleus of the posterior limb of the anterior commissure, but also present in the central amygdala. Secondly, the marker of neuronal activation c-Fos was used to explore the response of amygdalar CRF neurons in CRF-hrGFP mice under different experimental paradigms. C-Fos induction was observed in CRF neurons of CRF-hrGFP mice exposed to an acute social defeat stress event, a fasting/refeeding paradigm or lipopolysaccharide (LPS) administration. In contrast, no c-Fos induction was detected in CRF neurons of CRF-hrGFP mice exposed to restraint stress, forced swimming test, 48-h fasting, acute high-fat diet (HFD) consumption, intermittent HFD consumption, ad libitum HFD consumption, HFD withdrawal, conditioned HFD aversion, ghrelin administration or melanocortin 4 receptor agonist administration. Thus, this study fully characterizes the distribution of amygdala CRF neurons in mice and suggests that they are involved in some, but not all, stress or food intake-related behaviors recruiting the amygdala

Fasting induces remodeling of the orexigenic projections from the arcuate nucleus to the hypothalamic paraventricular nucleus, in a growth hormone secretagogue receptor–dependent manner

Objective: Arcuate nucleus (ARC) neurons producing Agouti-related peptide (AgRP) and neuropeptide Y (NPY; ARCAgRP/NPY neurons) are activated under energy-deficit states. ARCAgRP/NPY neurons innervate the hypothalamic paraventricular nucleus (PVH), and ARC→PVH projections are recognized as key regulators of food intake. Plasma ghrelin levels increase under energy-deficit states and activate ARCAgRP/NPY neurons by acting on the growth hormone secretagogue receptor (GHSR). Here, we hypothesized that activation of ARCAgRP/NPY neurons in fasted mice would promote morphological remodeling of the ARCAgRP/NPY→PVH projections in a GHSR-dependent manner.Methods: We performed 1) fluorescent immunohistochemistry, 2) imaging of green fluorescent protein (GFP) signal in NPY-GFP mice, and 3) DiI axonal labeling in brains of ad libitum fed or fasted mice with pharmacological or genetic blockage of the GHSR signaling and then estimated the density and strength of ARCAgRP/NPY→PVH fibers by assessing the mean fluorescence intensity, the absolute area with fluorescent signal, and the intensity of the fluorescent signal in the fluorescent area of the PVH.Results: We found that 1) the density and strength of ARCAgRP/NPY fibers increase in the PVH of fasted mice, 2) the morphological remodeling of the ARCAgRP/NPY→PVH projections correlates with the activation of PVH neurons, and 3) PVH neurons are not activated in ARC-ablated mice. We also found that fasting-induced remodeling of ARCAgRP/NPY→PVH fibers and PVH activation are impaired in mice with pharmacological or genetic blockage of GHSR signaling.Conclusion: This evidence shows that the connectivity between hypothalamic circuits controlling food intake can be remodeled in the adult brain, depending on the energy balance conditions, and that GHSR activity is a key regulator of this phenomenon.Fil: Cabral, Agustina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Fernandez, Gimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Tolosa, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Rey Moggia, María de Los Ángeles. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de La Plata; ArgentinaFil: Calfa, Gaston Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: de Francesco, Pablo Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin