| 1. |
- Abbas, Abdul-Karim, 1959, et al.
(författare)
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Bicarbonate-sensitive cysteine induced elevation of extracellular aspartate and glutamate in rat hippocampus in vitro
- 1997
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Ingår i: Neurochemistry International. - 0197-0186. ; 30:3, s. 253-259
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Tidskriftsartikel (refereegranskat)abstract
- The effect of different concentrations of cysteine (0.125, 0.25, 0.5 and 1 mM) on the net efflux of endogenous amino acids was studied by the incubation of rat hippocampal slices. Addition of cysteine (1 mM) in bicarbonate containing low K+ medium (5 min) selectively increased the basal net efflux of glutamate and aspartate by 370% and 396%, respectively. High K+ media (50 mM) containing cysteine (1 mM) evoked the net efflux of glutamate and aspartate by 1 454% and 1 019%, respectively. The corresponding effects in control slices without cysteine were 669% and 404%, respectively. No changes were observed on the concentrations of GABA, glutamine and taurine. The cysteine oxidation products, cysteine sulfinate (0.5 μM) and cystine (0.25 mM) were without effects. The effect of cysteine (0.5 mM) was dramatically reduced in media with no added bicarbonate/CO2. Thus, cysteine in a bicarbonate-sensitive manner selectively increases the extracellular concentration of excitotoxic amino acids in adult rat brain in vitro, possibly by interfering with the carrier-mediated glutamate uptake/ release
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| 2. |
- Ahemaiti, Aikeremu, 1984, et al.
(författare)
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A multifunctional pipette for localized drug administration to brain slices
- 2013
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Ingår i: Journal of Neuroscience Methods. - : Elsevier BV. - 0165-0270 .- 1872-678X. ; 219:2, s. 292-296
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study. (C) 2013 Elsevier B.V. All rights reserved.
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| 3. |
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| 4. |
- Ahemaiti, Aikeremu, 1984, et al.
(författare)
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Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices
- 2015
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Ingår i: Journal of Neuroscience Methods. - : Elsevier BV. - 0165-0270 .- 1872-678X. ; 241, s. 132-136
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Tidskriftsartikel (refereegranskat)abstract
- Background: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. New method: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. Results: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. Comparison with existing method: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. Conclusions: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices.
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| 5. |
- Jansson, Erik, 1984, et al.
(författare)
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Effect of cholesterol depletion on the pore dilation of TRPV1
- 2013
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Ingår i: Molecular Pain. - : SAGE Publications. - 1744-8069. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
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| 6. |
- Shijun, Xu, 1984, et al.
(författare)
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A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells
- 2015
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:1, s. 381-387
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Tidskriftsartikel (refereegranskat)abstract
- Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent a-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.
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| 7. |
- Trkulja, Carolina, 1984, et al.
(författare)
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Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:42, s. 14875-14882
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Tidskriftsartikel (refereegranskat)abstract
- Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.
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