Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
EMBO Rep ; 23(8): e54361, 2022 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-35735260

RESUMEN

The striatum is a subcortical brain region responsible for the initiation and termination of voluntary movements. Striatal spiny projection neurons receive major excitatory synaptic input from neocortex and thalamus, and cyclic nucleotides have long been known to play important roles in striatal function. Yet, the precise mechanism of action is unclear. Here, we combine optogenetic stimulation, 2-photon imaging, and genetically encoded scavengers to dissect the regulation of striatal synapses in mice. Our data show that excitatory striatal inputs are tonically depressed by phosphodiesterases (PDEs), in particular PDE1. Blocking PDE activity boosts presynaptic calcium entry and glutamate release, leading to strongly increased synaptic transmission. Although PDE1 degrades both cAMP and cGMP, we uncover that the concentration of cGMP, not cAMP, controls the gain of striatal inputs. Disturbing this gain control mechanism in vivo impairs motor skill learning in mice. The tight dependence of striatal excitatory synapses on PDE1 and cGMP offers a new perspective on the molecular mechanisms regulating striatal activity.


Asunto(s)
Cuerpo Estriado , Sinapsis , Animales , Cuerpo Estriado/metabolismo , Ácido Glutámico/metabolismo , Ratones , Neuronas/metabolismo , Sinapsis/fisiología , Transmisión Sináptica , Tálamo/metabolismo
2.
J Cell Sci ; 132(16)2019 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-31371487

RESUMEN

The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.


Asunto(s)
Dendritas/metabolismo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismo , Miosina Tipo V/metabolismo , Animales , Dendritas/genética , Femenino , Hipocampo/citología , Ratones , Proteínas de Microfilamentos/genética , Miosina Tipo V/genética , Ratas , Ratas Wistar
3.
Glia ; 64(11): 1962-71, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27462832

RESUMEN

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.


Asunto(s)
Astrocitos/metabolismo , Glucosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacología , Animales , Animales Recién Nacidos , Biotinilación , Encéfalo/citología , Encéfalo/diagnóstico por imagen , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosamina/análogos & derivados , Glucosamina/farmacología , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factor I del Crecimiento Similar a la Insulina/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estimulación Física , Transporte de Proteínas/genética , ARN Mensajero/metabolismo , Transfección , Vibrisas/fisiología
4.
J Neurosci ; 34(38): 12738-44, 2014 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-25232111

RESUMEN

Experience-dependent plasticity of synaptic transmission, which represents the cellular basis of learning, is accompanied by morphological changes in dendritic spines. Astrocytic processes are intimately associated with synapses, structurally enwrapping and functionally interacting with dendritic spines and synaptic terminals by responding to neurotransmitters and by releasing gliotransmitters that regulate synaptic function. While studies on structural synaptic plasticity have focused on neuronal elements, the structural-functional plasticity of astrocyte-neuron relationships remains poorly known. Here we show that stimuli inducing hippocampal synaptic LTP enhance the motility of synapse-associated astrocytic processes. This motility increase is relatively rapid, starting <5 min after the stimulus, and reaching a maximum in 20-30 min (t(1/2) = 10.7 min). It depends on presynaptic activity and requires G-protein-mediated Ca(2+) elevations in astrocytes. The structural remodeling is accompanied by changes in the ability of astrocytes to regulate synaptic transmission. Sensory stimuli that increase astrocyte Ca(2+) also induce similar plasticity in mouse somatosensory cortex in vivo. Therefore, structural relationships between astrocytic processes and dendritic spines undergo activity-dependent changes with metaplasticity consequences on synaptic regulation. These results reveal novel forms of synaptic plasticity based on structural-functional changes of astrocyte-neuron interactions.


Asunto(s)
Astrocitos/fisiología , Espinas Dendríticas/fisiología , Plasticidad Neuronal/fisiología , Potenciales de Acción/fisiología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Calcio/metabolismo , Femenino , Hipocampo/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Corteza Somatosensorial/fisiología , Transmisión Sináptica/fisiología
5.
J Comput Neurosci ; 34(1): 27-37, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22692349

RESUMEN

Chromaffin cells have been widely used to study neurosecretion since they exhibit similar calcium dependence of several exocytotic steps as synaptic terminals do, but having the enormous advantage of being neither as small or fast as neurons, nor as slow as endocrine cells. In the present study, secretion associated to experimental measurements of the exocytotic dynamics in human chromaffin cells of the adrenal gland was simulated by using a model that combines stochastic and deterministic approaches for short and longer depolarizing pulses, respectively. Experimental data were recorded from human chromaffin cells, obtained from healthy organ donors, using the perforated patch configuration of the patch-clamp technique. We have found that in human chromaffin cells, secretion would be mainly managed by small pools of non-equally fusion competent vesicles, slowly refilled over time. Fast secretion evoked by brief pulses can be predicted only when 75% of one of these pools (the "ready releasable pool" of vesicles, abbreviated as RRP) are co-localized to Ca²âº channels, indicating an immediately releasable pool in the range reported for isolated cells of bovine and rat (Álvarez and Marengo, J Neurochem 116:155-163, 2011). The need for spatial correlation and close proximity of vesicles to Ca²âº channels suggests that in human chromaffin cells there is a tight control of those releasable vesicles available for fast secretion.


Asunto(s)
Células Cromafines/fisiología , Exocitosis/fisiología , Modelos Biológicos , Dinámicas no Lineales , Glándulas Suprarrenales/citología , Biofisica , Calcio/metabolismo , Células Cultivadas , Estimulación Eléctrica , Humanos , Procesos Estocásticos
6.
FASEB J ; 26(1): 346-54, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21917987

RESUMEN

In the present study, we have electrophysiologically characterized native nicotinic acetylcholine receptors (nAChRs) in human chromaffin cells of the adrenal gland as well as their contribution to the exocytotic process. α-Conotoxin AuIB blocked by 14 ± 1% the acetylcholine (ACh)-induced nicotinic current. α-Conotoxin MII (α-Ctx MII) exhibited an almost full blockade of the nicotinic current at nanomolar concentrations (IC(50)=21.6 nM). The α6*-preferring α-Ctx MII mutant analogs, α-Ctx MII[H9A,L15A] and α-Ctx MII[S4A,E11A,L15A], blocked nAChR currents with an IC(50) of 217.8 and 33 nM, respectively. These data reveal that nAChRs in these cells include the α6* subtype. The washout of the blockade exerted by α-conotoxin BuIA (α-Ctx BuIA; 1 µM) on ACh-evoked currents was slight and slow, arguing in favor of the presence of a ß4 subunit in the nAChR composition. Exocytosis was almost fully blocked by 1 µM α-Ctx MII, its mutant analogs, or α-Ctx BuIA. Finally, the fluorescent analog Alexa Fluor 546-BuIA showed distinct staining in these cells. Our results reveal that α6ß4* nAChRs are expressed and contribute to exocytosis in human chromaffin cells of the adrenal gland, the main source of adrenaline under stressful situations.


Asunto(s)
Médula Suprarrenal/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Receptores Nicotínicos/fisiología , Acetilcolina/farmacología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Células Cromafines/efectos de los fármacos , Conotoxinas/farmacología , Exocitosis/efectos de los fármacos , Colorantes Fluorescentes/farmacología , Humanos , Antagonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Compuestos de Quinolinio/farmacología
7.
J Pharmacol Exp Ther ; 342(2): 263-72, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22537772

RESUMEN

The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca(2+) channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca(2+) measurements performed with mitochondrial ratiometric pericam demonstrated that Ca(2+) uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.


Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Canales de Calcio/metabolismo , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Exocitosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Ácido gamma-Aminobutírico/análogos & derivados , Glándulas Suprarrenales/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Bovinos , Humanos , Isoleucina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Neurotransmisores/metabolismo , Pregabalina , Ácido gamma-Aminobutírico/farmacología
8.
J Neurochem ; 116(1): 105-21, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21054386

RESUMEN

This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.


Asunto(s)
Potenciales de Acción/fisiología , Canales de Calcio Tipo L/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Animales , Células Cultivadas , Células Cromafines/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
9.
Front Mol Neurosci ; 14: 635820, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33762909

RESUMEN

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

10.
Cell Mol Neurobiol ; 30(8): 1407-15, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21107679

RESUMEN

Chromaffin cells are neuroendocrine cells mainly found in the medulla of the adrenal gland. Most existing knowledge of these cells has been the outcome of extensive research performed in animals, mainly in the cow, cat, mouse and rat. However, some insight into the physiology of this neuroendocrine cell in humans has been gained. This review summarizes the main findings reported in human chromaffin cells under physiological or disease conditions and discusses the clinical implications of these results.


Asunto(s)
Células Cromafines/fisiología , Células Cromafines/trasplante , Enfermedad , Médula Suprarrenal/citología , Médula Suprarrenal/embriología , Médula Suprarrenal/trasplante , Gránulos Cromafines/metabolismo , Humanos
11.
Nat Commun ; 11(1): 5083, 2020 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-33033259

RESUMEN

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but long-term depression (LTD) was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both long-term potentiation (LTP) and LTD are possible.


Asunto(s)
Espinas Dendríticas/metabolismo , Retículo Endoplásmico/metabolismo , Sinapsis/metabolismo , Animales , Hipocampo/metabolismo , Potenciación a Largo Plazo , Miosina Tipo V/metabolismo , Ratas Wistar , Imagen de Lapso de Tiempo
12.
Nat Commun ; 11(1): 2464, 2020 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-32424147

RESUMEN

Information within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. Here we introduce SynTagMA to tag active synapses in a user-defined time window. Upon 395-405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.


Asunto(s)
Imagenología Tridimensional , Sinapsis/fisiología , Potenciales de Acción , Animales , Axones/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Fluorescencia , Hipocampo/citología , Luz , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Ratas Sprague-Dawley , Ratas Wistar , Sinaptofisina/metabolismo , Factores de Tiempo
13.
Nat Commun ; 9(1): 4440, 2018 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-30361563

RESUMEN

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.


Asunto(s)
Neuronas/metabolismo , Ingeniería de Proteínas/métodos , Animales , Anticuerpos/metabolismo , Fluorescencia , Células HeLa , Humanos , Luz , Proteínas Luminiscentes/metabolismo , Ratones , Neuronas/citología , Ratas Wistar , Corteza Visual/metabolismo , Pez Cebra/metabolismo
14.
J Neurochem ; 103(6): 2281-90, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17883397

RESUMEN

The whole-cell secretory response evoked by acetylcholine (ACh) in human chromaffin cells was examined using a new protocol based on quickly switching from the voltage-clamp to the current-clamp (CC) configuration of the patch-clamp technique. Our experiments revealed that Ca(2+) entry through the nicotinic receptor at hyperpolarized membrane potentials contributed as much to the exocytosis (100.4 +/- 27.3 fF) evoked by 200 ms pulses of ACh, as Ca(2+) flux through voltage-dependent Ca(2+) channels at depolarized membrane potentials. The nicotinic current triggered a depolarization event with a peak at +49.3 mV and a 'plateau' phase that ended at -23.9 mV, which was blocked by 10 mumol/L mecamylamine. When a long ACh stimulus (15 s) was applied, the nicotinic current at the end of the pulse reached a value of 15.45 +/- 3.6 pA, but the membrane potential depolarization still remained at the 'plateau' stage until withdrawal of the agonist. Perfusion with 200 mumol/L Cd(2+) during the 15 s ACh pulse completely abolished the plasma membrane depolarization at the end of the pulse, indicating that Ca(2+) entry through Ca(2+) channels contributed to the membrane potential depolarization provoked by prolonged ACh pulses. These findings also reflect that voltage-dependent Ca(2+) channels were recruited by the small current flowing through the desensitized nicotinic receptor to maintain the depolarization. Finally, muscarinic receptor activation triggered a delayed exocytotic process after prolonged ACh stimulation, dependent on Ca(2+) mobilization from the endoplasmic reticulum. In summary, we show here that nicotinic and muscarinic receptors contribute to the exocytosis of neurotransmitters in human chromaffin cells, and that the nicotinic receptor plays a key role in several stages of the stimulus-secretion coupling process in these cells.


Asunto(s)
Médula Suprarrenal/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Neurotransmisores/metabolismo , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/fisiología , Acetilcolina/metabolismo , Acetilcolina/farmacología , Médula Suprarrenal/citología , Adulto , Anciano , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafines/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Exocitosis/efectos de los fármacos , Femenino , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Persona de Mediana Edad , Técnicas de Placa-Clamp , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos
15.
Eur J Pharmacol ; 796: 115-121, 2017 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-27988286

RESUMEN

The present study was performed to evaluate the Cav1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Cav1.2 and Cav1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Cav1.2 and Cav1.3 subtypes, but not Cav1.1 or Cav1.4. Electrophysiological experiments were conducted to investigate the contribution of Cav1 channels to the exocytotic process and cell excitability. Cav1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3µM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (-34mV), which elicits 10.4mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Cav1 channels in exocytosis and cell excitability.


Asunto(s)
Potenciales de Acción , Caveolina 1/metabolismo , Células Cromafines/citología , Células Cromafines/metabolismo , Exocitosis , Potenciales de Acción/efectos de los fármacos , Calcio/metabolismo , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Humanos , Isradipino/farmacología , Nifedipino/farmacología
16.
Diabetes ; 66(1): 64-74, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27999108

RESUMEN

Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes.


Asunto(s)
Astrocitos/metabolismo , Glucosa/metabolismo , Animales , Transporte Biológico/fisiología , Transportador de Glucosa de Tipo 1/metabolismo , Glucógeno/metabolismo , Inmunoensayo , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ácido Láctico/metabolismo , Masculino , Ratones , Neuronas/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Tomografía de Emisión de Positrones
17.
Med Image Anal ; 19(1): 87-97, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25299432

RESUMEN

Dendritic spines may be tiny in volume, but are of major importance for neuroscience. They are the main receivers for excitatory synaptic connections, and their constant changes in number and in shape reflect the dynamic connectivity of the brain. Two-photon microscopy allows following the fate of individual spines in brain slice preparations and in live animals. The diffraction-limited and non-isotropic resolution of this technique, however, makes detection of such tiny structures rather challenging, especially along the optical axis (z-direction). Here we present a novel spine detection algorithm based on a statistical dendrite intensity model and a corresponding spine probability model. To quantify the fidelity of spine detection, we generated correlative datasets: Following two-photon imaging of live pyramidal cell dendrites, we used serial block-face scanning electron microscopy (SBEM) to reconstruct dendritic ultrastructure in 3D. Statistical models were trained on synthetic fluorescence images generated from SBEM datasets via point spread function (PSF) convolution. After the training period, we tested automatic spine detection on real two-photon datasets and compared the result to ground truth (correlative SBEM data). The performance of our algorithm allowed tracking changes in spine volume automatically over several hours. Using a second fluorescent protein targeted to the endoplasmic reticulum, we could analyze the motion of this organelle inside individual spines. Furthermore, we show that it is possible to distinguish activated spines from non-stimulated neighbors by detection of fluorescently labeled presynaptic vesicle clusters. These examples illustrate how automatic segmentation in 5D (x, y, z, t, λ) allows us to investigate brain dynamics at the level of individual synaptic connections.


Asunto(s)
Dendritas/ultraestructura , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Células Piramidales/ultraestructura , Algoritmos , Animales , Inteligencia Artificial , Región CA1 Hipocampal , Humanos , Aumento de la Imagen/métodos , Microscopía Electrónica de Rastreo/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
18.
Curr Drug Targets ; 14(11): 1220-4, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23621508

RESUMEN

Astrocytes, classically considered as supportive cells for neurons without a direct role in brain information processing, are emerging as relevant elements in brain physiology through their ability to regulate neuronal activity and synaptic transmission and plasticity. In relation to the key role of astrocyte-neuron interactions in synaptic physiology, accumulating evidence suggests that dysfunctions of neuron-astrocyte signaling may be linked to the pathology of various neurological and neurodegenerative diseases. In this article, we summarize the evidence supporting the importance of astrocyte-neuron communication in synaptic physiology, which have led to reveal astrocytes as integral elements of synaptic function. We also discuss how this novel view of astrocytic functions on brain physiology is prompting us to reconsider the possible astrocytic roles in brain diseases, and specifically in depression.


Asunto(s)
Astrocitos/fisiología , Encéfalo/fisiología , Comunicación Celular , Neuronas/fisiología , Sinapsis/fisiología , Animales , Astrocitos/efectos de los fármacos , Encéfalo/fisiopatología , Humanos , Trastornos del Humor/fisiopatología , Neurotransmisores/fisiología , Transmisión Sináptica
19.
Front Cell Neurosci ; 7: 51, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23658537

RESUMEN

In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo.

20.
Br J Pharmacol ; 165(4): 908-21, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21790533

RESUMEN

BACKGROUND AND PURPOSE: Expression of α7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells. EXPERIMENTAL APPROACH: To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors. KEY RESULTS: The nicotinic current provoked by 300µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4ms rise time) and inactivated component (8.5 ± 0.4ms time constant) (termed α7), which was blocked by α-Bgtx or MLA, followed by a slower component (non-α7). α7 nAChR currents were dissected by blocking the non-α7 nAChR current component of the ACh and choline response with the α6* nAChR blocker α-conotoxin (α-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an α7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. α7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1mM) and PNU-120596 (10µM), potentiated responses that were blocked by α-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited α7 nAChR currents, using choline in the presence of α-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists. CONCLUSIONS AND IMPLICATIONS: Our electrophysiological recordings of pure α7 nAChR currents elicited by rapid application of agonists demonstrated that functional α7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.


Asunto(s)
Células Cromafines/fisiología , Exocitosis/fisiología , Receptores Nicotínicos/fisiología , Acetilcolina/farmacología , Aconitina/análogos & derivados , Aconitina/farmacología , Adolescente , Adulto , Anciano , Bungarotoxinas/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/fisiología , Adulto Joven , Receptor Nicotínico de Acetilcolina alfa 7
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA