Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore

or fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.National Institutes of Health GM-3885

Oxygen sensing by ion channels and chemotransduction in single glomus cells

We have monitored cytosolic [Ca2+] and dopamine release in intact fura-2-loaded glomus cells with microfluoroimetry and a polarized carbon fiber electrode. Exposure to low PO2 produced a rise of cytosolic [Ca2+] with two distinguishable phases: an initial period (with PO2 values between 150 and approximately 70 mm Hg) during which the increase of [Ca2+] is very small and never exceeds 150-200 nM, and a second phase (with PO2 below approximately 70 mm Hg) characterized by a sharp rise of cytosolic [Ca2+]. Secretion occurs once cytosolic [Ca2+] reaches a threshold value of 180 +/- 43 nM. The results demonstrate a characteristic relationship between PO2 and transmitter secretion at the cellular level that is comparable with the relation described for the input (O2 tension)output (afferent neural discharges) variables in the carotid body. Thus, the properties of single glomus cells can explain the sensory functions of the entire organ. In whole-cell, patch-clamped cells, we have found that in addition to O2-sensitive K+ channels, there are Ca2+ channels whose activity is also regulated by PO2. Ca2+ channel activity is inhibited by hpoxia, although in a strongly voltage-dependent manner. The average hypoxic inhibition of the calcium current in 30% +/- 10% at -20 mV but only 2% +/- 2% at +30 mV. The differential inhibition of K+ and Ca2+ channels by hypoxia helps to explain why the secretory response of the cells is displaced toward PO2 values (below approximately 70 mm Hg) within the range of those normally existing in arterial blood. These data provide a conceptual framework for understanding the cellular mechanisms of O2 chemotransduction in the carotid body

Monitoring synaptic function at the neuromuscular junction of a mouse expressing synaptopHluorin

We monitored presynaptic exocytosis and vesicle recycling at neuromuscular junctions of transgenic mice expressing synaptopHluorin (spH), using simultaneous optical and electrophysiological recordings. Synaptic transmission was indistinguishable from that in wildtype controls. Fluorescence rose during and decayed monotonically after stimulus trains to the nerve, with amplitudes and decay times increasing with the amount of stimulation. The relatively large size of synaptic terminals allowed us to examine the spatial profile of fluorescence changes. We identified hot spots of exocytosis, which were stable with repeated trains. Photobleach experiments showed that spH freshly exposed by nerve stimulation was not preferentially retrieved by compensatory endocytosis; instead, most retrieved spH preexisted in the surface membrane. Finally, we compared fluorescence and electrical [summed end-plate potentials (EPPs)] estimates of exocytosis, which diverged during repeated trains, as fluorescence exceeded summed EPPs, although the average amplitude of miniature EPPs was unchanged. This might reflect exocytosis of spH-containing, acetylcholine-free (“empty”) vesicles or other organelles during intense stimulation

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholaminefree granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis

Entrenamiento basado en simulación durante el grado para el diagnóstico de estenosis mitral. Clínica, imágenes y toma de decisiones

Dada la importancia del conocimiento de la estenosis mitral, se desarrollan simulaciones para el entrenamiento de su diagnóstico durante el grado. Se preparan varios supuestos clínicos de alta fidelidad y diferentes niveles de dificultad y se adaptan a maniquí SimMan, incluyendo información de anamnesis, signos e imágenes reales anonimizadas. Una vez probados los casos, son utilizados en sesiones teórico-prácticas para reforzar lo aprendido en clase. Los alumnos presentan dificultades para reconocer la patología basándose en la clínica y la información recabada durante la anamnesis. No obstante, mantienen alto grado de sospecha clínica que no confirman hasta la visualización de las imágenes. El alumnado es capaz de reconocer el cuadro gracias a las imágenes complementarias, pero no de elaborar un juicio clínico previo. Creemos que se debe reforzar el reconocimiento de signos, haciendo hincapié en que las pruebas complementarias sirven para confirmar una presunción clínica que previamente han debido de elaborar ellos mismos

The HERC1 ubiquitin ligase regulates presynaptic membrane dynamics of central synapses

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efciency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics.Junta de Andalucía BIO-209MINECO-DGICYT BFU2015-64536-RJunta de Andalucía BIO-122MINECO-DGICYT BFU2015-64536-RMINECO-AEI/FEDER, UE, BFU2016-80295-

Cardiac surgery simulation: The future of teaching in our specialty?

Introducción: La simulación como herramienta docente se está estableciendo como una prioridad en los currículos de nuestra especialidad en determinados países, principalmente anglosajones, y cada vez se están desarrollando escenarios más sofisticados y completos. Métodos: Se ha fomentado la creación de un equipo multidisciplinar con profesores de universidad, estudiantes de Medicina y médicos con el objetivo de mejorar la docencia del Grado en Medicina y valorar su potencial exportación a la formación especializada en Cirugía Cardiovascular. Resultados: Los alumnos de Medicina participantes demostraron una clara mejoría en la asimilación de información, así como en sus habilidades clínico-prácticas, conforme se fueron estableciendo escenarios de simulación. Del mismo modo, esta herramienta permitió una evaluación objetiva tanto de los resultados in situ como de la evolución temporal de los mismos. Conclusiones: Dado el rendimiento positivo en estudiantes de Medicina, y debido a la cada vez mayor dificultad en la formación de residentes en nuestros hospitales (cuestiones ético-legales, escasez de tiempo de formación, complejidad de casos), se debería plantear la creación de un grupo de Simulación a nivel nacional para valorar la inclusión de la misma en el currículo de la especialidad.Introduction: Simulation is becoming a powerful teaching tool of our specialty in certain countries, particularly in English-speaking ones, in which sophisticated scenarios are being increasingly developed. Methods: We have encouraged the creation of a multidisciplinary team with university professors, medical students, and physicians in order to improve teaching in the School of Medicine and to assess the potential use of the developed teaching techniques into specialised training in Cardiovascular Surgery. Results: Medical students showed a clear improvement in the assimilation of information and skills in clinical practice when they were setting up scenarios. Similarly,this tool allowed an objective assessment of the results in situ as well as over time. Conclusions: Given the positive performance by medical students, and because ofthe increasing difficulty in training residents in our hospitals (ethical and legal issues, shortage of patients, complexity of cases), Simulation groups should be created at national level to assess the inclusion of this into the curriculum of the specialt

Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons

Background: Mast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions. Methods: Calcium imaging was used to simultaneously record changes in intracellular calcium [Ca2+]i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat. Results: Bidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca2+]i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca2+]i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS). Conclusions: MC-hippocampal neuron interaction affects neuronal [Ca2+]i and exocytosis signaling through a NMDAR-dependent mechanism.Spanish Ministry of Economy and Competitivityco-funded by the European Social Fund and the Junta de Andalucía (BIO-209 and BIO-236

Simulación en cirugía cardíaca: ¿el futuro de la docencia en nuestra especialidad?

Introducción: La simulación como herramienta docente se está estableciendo como una prioridad en los currículos de nuestra especialidad en determinados países, principalmente anglosajones, y cada vez se están desarrollando escenarios más sofisticados y completos. Métodos: Se ha fomentado la creación de un equipo multidisciplinar con profesores de universidad, estudiantes de Medicina y médicos con el objetivo de mejorar la docencia del Grado en Medicina y valorar su potencial exportación a la formación especializada en Cirugía Cardiovascular. Resultados: Los alumnos de Medicina participantes demostraron una clara mejoría en la asimilación de información, así como en sus habilidades clínico-prácticas, conforme se fueron estableciendo escenarios de simulación. Del mismo modo, esta herramienta permitió una evaluación objetiva tanto de los resultados in situ como de la evolución temporal de los mismos. Conclusiones: Dado el rendimiento positivo en estudiantes de Medicina, y debido a la cada vez mayor dificultad en la formación de residentes en nuestros hospitales (cuestiones ético-legales, escasez de tiempo de formación, complejidad de casos), se debería plantear la creación de un grupo de Simulación a nivel nacional para valorar la inclusión de la misma en el currículo de la especialidad