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Development of remote stimulation techniques for neuronal tissues represents a challenging goal. Among the potential methods, mechanical stimuli are the most promising vectors to convey information non-invasively into intact brain tissue. In this context, selective mechano-sensitization of neuronal circuits would pave the way to develop a new cell-type-specific stimulation approach. We report here, for the first time, the development and characterization of mechano-sensitized neuronal networks through the heterologous expression of an engineered bacterial large-conductance mechanosensitive ion channel (MscL). The neuronal functional expression of the MscL was validated through patch-clamp recordings upon application of calibrated suction pressures. Moreover, we verified the effective development of in-vitro neuronal networks expressing the engineered MscL in terms of cell survival, number of synaptic puncta and spontaneous network activity. The pure mechanosensitivity of the engineered MscL, with its wide genetic modification library, may represent a versatile tool to further develop a mechano-genetic approach.This article has an associated First Person interview with the first author of the paper.
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Proteínas de Escherichia coli/genética , Canales Iónicos/genética , Mecanotransducción Celular/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Supervivencia Celular/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Activación del Canal Iónico/genética , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/metabolismo , Técnicas de Placa-Clamp , Cultivo Primario de Células , Ingeniería de Proteínas/métodos , Ratas , TransfecciónRESUMEN
The past decade has seen an intensive effort to achieve optical imaging resolution beyond the diffraction limit. Apart from the Pendry-Veselago negative index superlens, implementation of which in optics faces challenges of losses and as yet unattainable fabrication finesse, other super-resolution approaches necessitate the lens either to be in the near proximity of the object or manufactured on it, or work only for a narrow class of samples, such as intensely luminescent or sparse objects. Here we report a new super-resolution microscope for optical imaging that beats the diffraction limit of conventional instruments and the recently demonstrated near-field optical superlens and hyperlens. This non-invasive subwavelength imaging paradigm uses a binary amplitude mask for direct focusing of laser light into a subwavelength spot in the post-evanescent field by precisely tailoring the interference of a large number of beams diffracted from a nanostructured mask. The new technology, which--in principle--has no physical limits on resolution, could be universally used for imaging at any wavelength and does not depend on the luminescence of the object, which can be tens of micrometres away from the mask. It has been implemented as a straightforward modification of a conventional microscope showing resolution better than λ/6.
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In this paper, we report the characterization of 'Hi-Spot' cultures formed by the re-aggregation of dissociated postnatal CNS tissue grown at an air-liquid interface. This produces a self-organised, dense, organotypic cellular network. Western blot, immunohistochemical, viral transfection and electron microscopy analyses reveal neuronal and glial populations, and the development of a synaptic network. Multi-electrode array recordings show synaptically driven network activity that develops through time from single unit spiking activity to global network bursting events. This activity is blocked by tetanus toxin and modified by antagonists of glutamatergic and GABAergic receptors suggesting tonic activity of excitatory and inhibitory synaptic signaling. The tissue-like properties of these cultures has been further demonstrated by their relative insensitivity to glutamate toxicity. Exposure to millimolar concentrations of glutamate for hours is necessary to produce significant excitotoxic neuronal death, as in vivo. We conclude that 'Hi-Spots' are biological analogues of CNS tissue at a level of complexity that allows for detailed functional analyses of emergent neuronal network properties.
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Encéfalo/citología , Red Nerviosa/citología , Neuroglía/citología , Neuronas/citología , Potenciales de Acción , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Muerte Celular/efectos de los fármacos , Ácido Glutámico/toxicidad , Inmunohistoquímica , Microscopía Confocal , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Ratas , Ratas Wistar , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica/efectos de los fármacos , Toxina Tetánica/farmacología , Técnicas de Cultivo de Tejidos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Microscopy with extreme ultraviolet (EUV) light can provide many advantages over optical, hard x-ray or electron-based techniques. However, traditional EUV sources and optics have large disadvantages of scale and cost. Here, we demonstrate the use of a laboratory-scale, coherent EUV source to image biological samples-mouse hippocampal neurons-providing quantitative phase and amplitude transmission information with a lateral resolution of 80 nm and an axial sensitivity of ~1 nm. A comparison with fluorescence imaging of the same samples demonstrated EUV imaging was able to identify, without the need for staining or superresolution techniques, <100-nm-wide and <10-nm-thick structures not observable from the fluorescence images. Unlike hard x-ray microscopy, no damage is observed of the delicate neuron structure. The combination of previously demonstrated tomographic imaging techniques with the latest advances in laser technologies and coherent EUV sources has the potential for high-resolution element-specific imaging within biological structures in 3D.
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PURPOSE: Blunt aortic injuries (BAI) have historically been considered an indication for emergent surgical intervention. Nevertheless, the observation that the outcome of the concomitant traumatic injuries has a major impact on prognosis and the rise of thoracic endovascular aortic repair (TEVAR) as an effective therapy for BAI have significantly changed in recent years the treatment algorithm of this condition. Our objective was to identify findings associated with the aortic injury which would be the best predictor of prognosis, with the objective of guiding the decision-making process for selecting the optimal timing of aortic repair. METHODS: We reviewed blunt aortic injuries from 3 Level I Trauma Centers from July 2008 to December 2016. We analyzed overall and BAI-related 30-day mortality in relation to: hemodynamics, timing of treatment, TEVAR vs open repair, and aortic injury grade as defined by the Society for Vascular Surgery. Based on computed tomographic angiography (CT scan) imaging, we selected the radiologic aortic findings most indicative of high mortality risk, which we defined as "Radiographic Severe Injury" (RSI): (1) total/partial aortic transection, (2) active contrast extravasation, or (3) the association of 2 of more of the following: contained contrast extravasation > 10 mm, periaortic hematoma, and/or mediastinal hematoma with thickness > 10 mm, or significant left pleural effusion. RESULTS: Of a total of 76 consecutive patients, 50 (66%) underwent immediate repair, 24 (31%) delayed aortic repair, and 2 (3%) died prior to repair. 58 patients (76%) had TEVAR, while 16 (24%) had open repair. Overall mortality was 18% and BAI-related mortality was 13%. In BAI-related mortalities, 70% of patients had RSI. Patients with high risk of overall mortality had hypotension and tachycardia (SBP < 100, HR ≥ 100), high ISS, and required vasopressors. Factors only associated with BAI-related mortality included RSI. CONCLUSION: CT scan findings suggestive of RSI are predictive of mortality associated with BAI. Radiologic assessment of the severity of the aortic injury with characterization for the presence of RSI may represent the key factors to determine the optimal timing of treatment of the aortic injury and guide the overall treatment strategy. LEVEL OF EVIDENCE: IV.
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Aorta/lesiones , Traumatismo Múltiple/diagnóstico por imagen , Heridas no Penetrantes/diagnóstico por imagen , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Aorta/diagnóstico por imagen , Aorta/cirugía , Toma de Decisiones Clínicas , Procedimientos Endovasculares/métodos , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/mortalidad , Traumatismo Múltiple/cirugía , Traumatismo Múltiple/terapia , Radiografía , Estudios Retrospectivos , Medición de Riesgo , Tomografía Computarizada por Rayos X , Centros Traumatológicos/estadística & datos numéricos , Heridas no Penetrantes/mortalidad , Heridas no Penetrantes/cirugía , Heridas no Penetrantes/terapia , Adulto JovenRESUMEN
Brain injury due to seizures results in transiently increased cell proliferation and neurogenesis in the subgranular zone of the adult dentate gyrus. In contrast, the immature postnatal brain appears to be more resistant to cell death after seizure-induced brain injury and paradoxically reacts to seizures by reducing SGZ proliferation. Organotypic hippocampal slice cultures are a useful paradigm for modelling the early postnatal hippocampus. We have investigated the temporal relationship between cell death and cell proliferation after kainate in the granule cell layer of rat organotypic hippocampal slice cultures equivalent to post natal day 11 animals. We found stable numbers and densities of mature thionine stained cells in the granule cell layer over 72 h in control cultures grown in defined medium. We also found a slowly declining cell proliferation rate over the same time period under control conditions. We report evidence of early cell death in the granule cell layer after just 2 h exposure to 5 microM kainate, followed by a significant decrease in cell proliferation in the granule cell layer at 24 h. In contrast to control conditions, cell proliferation rose significantly in the kainate exposed cultures by 72 h back to levels seen at 2 h. There were no significant changes in cell labelling with antibody to activated caspase-3 between kainate treated and control cultures at any time point examined. Our results suggest that kainate-induced injury in the early postnatal hippocampus damages precursor cells contributing to a reduction in granule layer cell proliferation.
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Proliferación Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Ácido Kaínico/farmacología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Hipocampo/fisiología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.
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Músculo Esquelético/metabolismo , Neuronas/metabolismo , Intoxicación por Organofosfatos/metabolismo , Compuestos Organofosforados/toxicidad , Compuestos de Piridinio/toxicidad , Receptores Nicotínicos/metabolismo , Acetilcolinesterasa/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Línea Celular , Cobayas , Humanos , Músculo Esquelético/patología , Neuronas/patologíaRESUMEN
An event-driven framework is used to construct a physiologically motivated large-scale model of the piriform cortex containing in the order of 10(5) neuron-like computing units. This approach is based on a hierarchically defined highly abstract neuron model consisting of finite-state machines. It provides computational efficiency while incorporating components which have identifiable counterparts in the neurophysiological domain. The network model incorporates four neuron types, and glutamatergic excitatory and GABA(A) and GABA(B) inhibitory synapses. The spatio-temporal patterns of cortical activity and the temporal and spectral characteristics of simulated electroencephalograms (EEGs) are studied. In line with previous experimental and compartmental work, 1) shock stimuli elicit EEG profiles with either isolated peaks or damped oscillations, the response type being determined by the intensity of the stimuli, and 2) temporally unpatterned input generates EEG oscillations supported by model-wide waves of excitation.
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Potenciales de Acción/fisiología , Corteza Cerebral/fisiología , Modelos Neurológicos , Red Nerviosa/fisiología , Redes Neurales de la Computación , Neuronas/fisiología , Simulación por Computador , Estimulación Eléctrica/métodos , Electroencefalografía/métodos , Electroencefalografía/estadística & datos numéricos , Campos Electromagnéticos , Electrofisiología/métodos , Humanos , Interneuronas/fisiología , Red Nerviosa/anatomía & histología , Vías Olfatorias/fisiología , Células Piramidales/fisiología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Hypothermia has been demonstrated to be an effective neuroprotective strategy in a number of models of ischaemic and excitotoxic neurodegeneration in vitro and in vivo. Reduced glutamate release and free radical production have been postulated as potential mechanisms underlying this effect but no definitive mechanism has yet been reported. In the current study, we have used oxygen-glucose deprivation in organotypic hippocampal slice cultures as an in vitro model of cerebral ischaemia. When assessed by propidium iodide fluorescence, reducing the temperature during oxygen-glucose deprivation to 31-33 degrees C was significantly neuroprotective but this effect was lost if the initiation of hypothermia was delayed until the post-insult recovery period. The neuroprotective effects of hypothermia were associated with a significant decrease in both nitric oxide production, as assessed by 3-amino-4-aminomethyl-2',7'-difluorofluorescein fluorescence, and superoxide formation. Further, hypothermia significantly attenuated NMDA-induced nitric oxide formation in the absence of hypoxia/hypoglycaemia. We conclude that the neuroprotective effects of hypothermia are mediated through a reduction in nitric oxide and superoxide formation and that this effect is likely to be downstream of NMDA receptor activation.
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Isquemia Encefálica/metabolismo , Radicales Libres/metabolismo , Hipocampo/metabolismo , Hipotermia Inducida , Animales , Animales Recién Nacidos , Hipoxia de la Célula/fisiología , Citoprotección/fisiología , Agonistas de Aminoácidos Excitadores/farmacología , Colorantes Fluorescentes , Glucosa/metabolismo , Hipocampo/efectos de los fármacos , Técnicas In Vitro , N-Metilaspartato/farmacología , Degeneración Nerviosa/prevención & control , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Superóxidos/metabolismo , Temperatura , Factores de TiempoRESUMEN
We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5-Diaminofluoroscein diacetate, DAF-2 DA. Following DAF-2 loading, ganglia showed cell-specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L-NAME prevented much of the initial development of DAF-2 fluorescence, whereas the inactive isomer D-NAME had no effect. Washout of preincubated L-NAME caused increased cell-specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L-arginine also resulted in an increase of DAF-2 fluorescence in a cell-specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF-2 fluorescence increases in most cells. We therefore presume that the cell-specific pattern of DAF-2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF-2 staining pattern and previously published studies of NOS activity are discussed.
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Fluoresceína/análisis , Ganglios Sensoriales/química , Óxido Nítrico/análisis , Óxido Nítrico/biosíntesis , Animales , Astacoidea , Femenino , Fluorescencia , Ganglios Sensoriales/citología , Ganglios Sensoriales/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismoRESUMEN
The location of sarco/endoplasmic-reticulum calcium ATPase (SERCA) retention/retrieval motifs in the sequence of the SERCA1 has been investigated by examining the subcellular location in COS-7 cells of enhanced-green-fluorescent-protein-tagged calcium-pump chimaeras. These chimaeras have been constructed from the fast-twitch SERCA1 and the plasma-membrane calcium ATPase PMCA3. The N-terminal, central and C-terminal segments of these calcium pumps were exchanged between SERCA1 and PMCA3. The segments exchanged correspond to residues 1-211, 212-711 and 712-994 of SERCA1, and residues 1-264, 265-788 and 789-1159 of PMCA3 respectively. Only chimaeras containing the N-terminal segment of SERCA1 were located in the endoplasmic reticulum (ER), whereas chimaeras containing the N-terminal segment from PMCA3 were able to escape from the ER and enter the endomembrane pathway en route for the plasma membrane. Co-localization of SERCA1 in COS-7 cells with the ER/Golgi-intermediate compartment marker ERGIC53 indicates that SERCA1 is maintained in the ER by a process of retrieval. These results indicate that the N-terminal region of SERCA1, containing transmembrane helices M1 and M2, contains an ER-retrieval signal.