RESUMEN
Amyloid-ß (Aß)-peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aß precursor protein (APP) by ß-site APP-cleaving enzyme (BACE). Using live-cell imaging of APP and BACE labeled with pH-sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α-secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual-color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Membrana Celular/metabolismo , Células HeLa , Humanos , Transporte de Proteínas , ProteolisisRESUMEN
Lithium is an effective mood-stabilizer in the treatment of bipolar affective disorder. While glycogen synthase kinase 3-mediated and inositol depletion-dependent effects of lithium have been described extensively in literature, there is very little knowledge about the consequences of lithium treatment on vesicle recycling and neurotransmitter availability. In the present study we have examined acute and chronic effects of lithium on synaptic vesicle recycling using primary hippocampal neurons. We found that exocytosis of readily releasable pool vesicles as well as recycling pool vesicles was unaffected by acute and chronic treatment within the therapeutic range or at higher lithium concentrations. Consistent with this observation, we also noticed that the network activity and number of active synapses within the network were also not significantly altered after lithium treatment. Taken together, as lithium treatment does not affect synaptic vesicle release at even high concentrations, our data suggest that therapeutic effects of lithium in bipolar affective disorder are not directly related to presynaptic function.
Asunto(s)
Antimaníacos/farmacología , Hipocampo/citología , Litio/farmacología , Neuronas/citología , Terminales Presinápticos/efectos de los fármacos , Ácidos/farmacología , Compuestos de Amonio/farmacología , Animales , Supervivencia Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Bloqueadores de los Canales de Sodio/farmacología , Sinapsinas/genética , Sinapsinas/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Tetrodotoxina/farmacología , Factores de Tiempo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Concurrent imaging of spectrally distinct fluorescence probes has become an important method for live-cell microscopy experiments in many biological disciplines. The technique enables the identification of a multitude of causal relationships. However, interactions between fluorescent dyes beyond an obvious overlap of their fluorescent spectra are often neglected. Here we present the effects of the well-established fluorescent dyes FM®2-10 or FM®1-43 on the recently introduced pH-dependent probe CypHer™5E. Spectrophotometry as well as live-cell fluorescence microscopy revealed that both FM dyes are effective quenchers of CypHer™5E. Control experiments indicated that this effect is reversible and not due to bleaching. We conclude that, in general, parallel measurements of both dyes are possible, with low FM dye concentrations. Nevertheless, our results implicate that special care has to be taken in such dual colour experiments especially when analysing dynamic CypHer™5E signals in live-cell microscopy.
Asunto(s)
Carbocianinas/química , Colorantes Fluorescentes/química , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Animales , Hipocampo/citología , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Imagen Molecular , Neuronas/citología , Ratas , Ratas Wistar , Espectrometría de Fluorescencia , Vesículas Sinápticas/metabolismoRESUMEN
Hippocampal neurons in tissue culture develop functional synapses that exhibit considerable variation in synaptic vesicle content (20-350 vesicles). We examined absolute and fractional parameters of synaptic vesicle exocytosis of individual synapses. Their correlation to vesicle content was determined by activity-dependent discharge of FM-styryl dyes. At high frequency stimulation (30 Hz), synapses with large recycling pools released higher amounts of dye, but showed a lower fractional release compared to synapses that contained fewer vesicles. This effect gradually vanished at lower frequencies when stimulation was triggered at 20 Hz and 10 Hz, respectively. Live-cell antibody staining with anti-synaptotagmin-1-cypHer 5, and overexpression of synaptopHluorin as well as photoconversion of FM 1-43 followed by electron microscopy, consolidated the findings obtained with FM-styryl dyes. We found that the readily releasable pool grew with a power function with a coefficient of 2/3, possibly indicating a synaptic volume/surface dependency. This observation could be explained by assigning the rate-limiting factor for vesicle exocytosis at high frequency stimulation to the available active zone surface that is proportionally smaller in synapses with larger volumes.
Asunto(s)
Hipocampo/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Artefactos , Endocitosis , Exocitosis , Colorantes Fluorescentes/metabolismo , Hipocampo/citología , Hipocampo/ultraestructura , Cinética , Microscopía Fluorescente , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Ratas Wistar , Propiedades de Superficie , Vesículas Sinápticas/ultraestructura , Factores de TiempoRESUMEN
Spastin is a microtubule severing ATPase that regulates intracellular and axonal transport of vesicles. Intracellular vesicle trafficking was analyzed in differentiated SH-SY5Y-neuroblastoma cells, transfected with spastin wild-type and three spastin mutations (ΔN, K388R, S44L) to investigate spastin-mediated effects on the velocity of vesicles, stained with LysoTracker Red®. The vesicle velocity varied considerably between mutations and detailed analysis revealed up to five distinct velocity classes. Microtubule severing by overexpressed wild-type spastin caused reduced vesicle velocity. S44L and ΔN mutations, which were functionally impaired, showed similar velocities as control cells. K388R-transfected cells exhibited an intermediate velocity profile. The results support the idea that spastin mutations not only alter axonal transport, but in addition regulate intracellular trafficking in the cell soma as well.
Asunto(s)
Adenosina Trifosfatasas , Transporte Axonal/fisiología , Vesículas Citoplasmáticas/metabolismo , Mutación , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Transporte Biológico/fisiología , Línea Celular Tumoral , Genes Reporteros , Humanos , Lisosomas/metabolismo , EspastinaRESUMEN
Cargo movement along axons and dendrites is indispensable for the survival and maintenance of neuronal networks. Key parameters of this transport such as particle velocities and pausing times are often studied using kymograph construction, which converts the transport along a line of interest from a time-lapse movie into a position versus time image. Here we present a method for the automatic analysis of such kymographs based on the Hough transform, which is a robust and fast technique to extract lines from images. The applicability of the method was tested on simulated kymograph images and real data from axonal transport of synaptophysin and tetanus toxin as well as the velocity analysis of synaptic vesicle sharing between adjacent synapses in hippocampal neurons. Efficiency analysis revealed that the algorithm is able to detect a wide range of velocities and can be used at low signal-to-noise ratios. The present work enables the quantification of axonal transport parameters with high throughput with no a priori assumptions and minimal human intervention.
Asunto(s)
Axones/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Quimografía/métodos , Movimiento , Algoritmos , Animales , Automatización , Benchmarking , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sinapsis/metabolismo , Sinaptofisina/metabolismo , Toxina Tetánica/metabolismo , Factores de TiempoRESUMEN
Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.
Asunto(s)
Amantadina/farmacología , Amitriptilina/farmacología , Cinarizina/farmacología , Flavoxato/farmacología , Lisosomas/efectos de los fármacos , Amantadina/química , Amantadina/metabolismo , Aminas , Amitriptilina/química , Amitriptilina/metabolismo , Cationes , Línea Celular Tumoral , Cinarizina/química , Cinarizina/metabolismo , Simulación por Computador , Retroalimentación Fisiológica , Flavoxato/química , Flavoxato/metabolismo , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lisosomas/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Tamaño de los Orgánulos , PermeabilidadRESUMEN
Effects of the antidepressant fluoxetine on stimulation-dependent synaptic vesicle exocytosis were examined in cultured primary hippocampal neurons. Synaptic vesicles were fluorescently labeled in vitro with FM1-43, washed and subsequently destained in two consecutive cycles. Exocytosis was triggered by electric field stimulation and imaged by fluorescence microscopy. In control preparations, the second staining-destaining cycle caused a significant reduction of relative fluorescence loss, number of active synapses and half-decay time (t(50)). These fatigue effects were largely prevented by short-term administration of 1 microM fluoxetine, which was present before and during the second stimulation cycle. Fluoxetine concentrations above 10 microM inhibited exocytosis almost completely but showed no other toxic effects on neurons. Stressed neurons, grown under hyperosmotic conditions, were even more fatigue-protected by fluoxetine. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recovery of neurotransmission from exhausting stimuli in healthy and in hyperosmotically stressed neurons as well.
Asunto(s)
Exocitosis/efectos de los fármacos , Fluoxetina/farmacología , Hipocampo/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Animales , Animales Recién Nacidos , Células Cultivadas , Estimulación Eléctrica , Exocitosis/fisiología , Hipocampo/metabolismo , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Ratas , Ratas Wistar , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.
Asunto(s)
Axones/metabolismo , Quimografía/métodos , Animales , Cinética , Transporte de Proteínas , Ratas , Reproducibilidad de los Resultados , Sinaptofisina/metabolismoRESUMEN
There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce "kiss-and-run" exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential "kiss-and-run" and "full-collapse" exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release.
RESUMEN
BACKGROUND: Different pools and functions have recently been attributed to spontaneous and evoked vesicle release. Despite the well-established function of evoked release, the neuronal information transmission, the origin as well as the function of spontaneously fusing synaptic vesicles have remained elusive. Recently spontaneous release was found to e.g. regulate postsynaptic protein synthesis or has been linked to depressive disorder. Nevertheless the strength and cellular localization of this release form was neglected so far, which are both essential parameters in neuronal information processing. FINDINGS: Here we show that the complete recycling pool can be turned over by spontaneous trafficking and that spontaneous fusion rates critically depend on the neuronal localization of the releasing synapse. Thereby, the distribution equals that of evoked release so that both findings demonstrate a uniform regulation of these fusion modes. CONCLUSIONS: In contrast to recent works, our results strengthen the assumption that identical vesicles are used for evoked and spontaneous release and extended the knowledge about spontaneous fusion with respect to its amount and cellular localization. Therefore our data supported the hypothesis of a regulatory role of spontaneous release in neuronal outgrowth and plasticity as neurites secrete neurotransmitters to initiate process outgrowth of a possible postsynaptic neuron to form a new synaptic connection.
Asunto(s)
Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Compartimento Celular , Exocitosis , Hipocampo/metabolismo , Humanos , Fusión de MembranaRESUMEN
In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval.
Asunto(s)
Hipocampo/metabolismo , Concentración de Iones de Hidrógeno , Sinapsis/metabolismo , Potenciales de Acción , Animales , Endocitosis , Hipocampo/fisiopatología , Ratas , Ratas WistarRESUMEN
Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer™5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.
Asunto(s)
Antidepresivos/farmacología , Fluoxetina/farmacología , Hipocampo/efectos de los fármacos , Sinapsis/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Animales , Animales Recién Nacidos , Células Cultivadas , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Hipocampo/citología , Hipocampo/metabolismo , Ratas , Ratas Wistar , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca2+ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2-/-/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb. Immunohistochemistry and western blotting confirmed this pattern and revealed expression of EFhd2 during neuronal maturation. In cortical neurons, EFhd2 was detected in neurites marked by MAP2 and co-localized with pre- and post-synaptic markers. Approximately one third of EFhd2 associated with a biochemically isolated synaptosome preparation. There, EFhd2 was mostly confined to the cytosolic and plasma membrane fractions. Both synaptic endocytosis and exocytosis in primary hippocampal EFhd2-/- neurons were unaltered but transport of synaptophysin-GFP containing vesicles was enhanced in EFhd2-/- primary hippocampal neurons, and notably, EFhd2 inhibited kinesin mediated microtubule gliding. Therefore, we found that EFhd2 is a neuronal protein that interferes with kinesin-mediated transport.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Cinesinas/metabolismo , Neuritas/metabolismo , Animales , Transporte Axonal , Células Cultivadas , Hipocampo/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas , Sinaptosomas/metabolismoRESUMEN
FM dyes are an established tool to analyze synaptic vesicle pools. However, quantitative measurements using FM dyes are typically based on the re-release properties of previously labelled vesicles, which might vary depending on the experimental setup. An FM dye protocol independent of the previous labelling of vesicle membrane has not been applied for quantitative measurements of individual synaptic vesicles before. We therefore analyzed the direct staining of newly exocytosed vesicle membrane with FM dyes in cultured rat hippocampal neurons. In the presence of FM 1-43, stimulation-induced synaptic activity led to a stable fluorescence increase. The quantal release of synaptic vesicles was preserved and its amplitude correlated highly with the exocytic dye loss induced by a subsequent stimulation. Thus, the method presented here provides a tool for the pool-independent measurement of synaptic vesicle exocytosis.
Asunto(s)
Exocitosis/fisiología , Colorantes Fluorescentes , Hipocampo/metabolismo , Neuronas/metabolismo , Coloración y Etiquetado/métodos , Vesículas Sinápticas/metabolismo , Animales , Hipocampo/ultraestructura , Procesamiento de Imagen Asistido por Computador , Neuronas/ultraestructura , Ratas , Ratas Wistar , Vesículas Sinápticas/ultraestructuraRESUMEN
To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.
Asunto(s)
Endocitosis/fisiología , Hipocampo/citología , Hipocampo/fisiología , Sinapsis/fisiología , Potenciales de Acción/fisiología , Animales , Estimulación Eléctrica , Endosomas/metabolismo , Endosomas/ultraestructura , Hipocampo/ultraestructura , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sinapsis/ultraestructuraRESUMEN
Antipsychotic drugs are effective for the treatment of schizophrenia. However, the functional consequences and subcellular sites of their accumulation in nervous tissue have remained elusive. Here, we investigated the role of the weak-base antipsychotics haloperidol, chlorpromazine, clozapine, and risperidone in synaptic vesicle recycling. Using multiple live-cell microscopic approaches and electron microscopy of rat hippocampal neurons as well as in vivo microdialysis experiments in chronically treated rats, we demonstrate the accumulation of the antipsychotic drugs in synaptic vesicles and their release upon neuronal activity, leading to a significant increase in extracellular drug concentrations. The secreted drugs exerted an autoinhibitory effect on vesicular exocytosis, which was promoted by the inhibition of voltage-gated sodium channels and depended on the stimulation intensity. Taken together, these results indicate that accumulated antipsychotic drugs recycle with synaptic vesicles and have a use-dependent, autoinhibitory effect on synaptic transmission.
Asunto(s)
Antipsicóticos/metabolismo , Antipsicóticos/farmacología , Neuronas/efectos de los fármacos , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Aminas/metabolismo , Animales , Animales Recién Nacidos , Biofisica , Células Cultivadas , Simulación por Computador , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Microdiálisis , Microscopía Electrónica , Modelos Neurológicos , Neuroblastoma/patología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Serotonina/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Sinapsis/metabolismo , Sinapsis/ultraestructura , Vesículas Sinápticas/metabolismo , Tetrodotoxina/farmacología , Factores de Tiempo , TransfecciónRESUMEN
Multiprobe high pressure measurements require electrical leads in the sample chamber. Compared to conventional wire-based techniques, metallic tracks patterned onto the anvil surface improve reliability and ease of use, and enable novel and more demanding measurements under high pressure. We have developed new anvil designs based on sputter-deposited tracks on alumina and moissanite anvils. These anvils allow convenient and reliable measurements of electrical transport properties or of the magnetic susceptibility under hydrostatic conditions, as demonstrated by test measurements on Pb and Ca(3)Ru(2)O(7).
RESUMEN
In many instances of signal and image processing, it is indispensable to precisely distinguish scattered peaks from a background, e.g., camera signals in microscopy. Here we addressed the detection of Gaussian signals in simulated line profiles (LP) comparable with e.g., fluorescence microscopy data. In a first step, we measured the applicability of histogram-based global background estimation. We find that the method is valid for typical scattered Gaussian signals if they are averagely separated by interpeak distances of 5.5 standard deviations. This enabled us to design global background determination-based peak detection (GBPD). GBPD was compared with two local background determination-based signal detection methods that had been designed for analysis of electrophysiological data and microscopy images, respectively. We were able to prove via receiver-operator characteristic (ROC) comparisons of signal-to-noise ratio (SNR), interpeak distance, and filtering behavior that, when applicable, GBPD brings advantages in knowledge needed a priori, performance at any SNR, controllability and spatial resolution.