| 1. |
- Zhang, B, et al.
(författare)
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Temporal Resolution in Electrochemical Imaging on Single PC12 Cells Using Amperometry and Voltammetry at Microelectrode Arrays
- 2011
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Ingår i: ANALYTICAL CHEMISTRY. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 83:2, s. 571-577
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Tidskriftsartikel (refereegranskat)abstract
- Carbon-fiber-microelectrode arrays (MEAs) have been utilized to electrochemically image neurochemical secretion from individual pheochromocytoma (PC12) cells. Dopamine release events were electrochemically monitored from seven different locations on single PC12 cells using alternately constant-potential amperometry and fast-scan cyclic voltammetry (FSCV). Cyclic voltammetry, when compared to amperometry, can provide excellent chemical resolution; however, spatial and temporal resolution are both compromised. The spatial and temporal resolution of these two methods have been quantitatively compared and the differences explained using models of molecular diffusion at the nanogap between the electrode and the cell. A numerical simulation of the molecular flux reveals that the diffusion of dopamine molecules and electrochemical reactions both play important roles in the temporal resolution of electrochemical imaging. The simulation also reveals that the diffusion and electrode potential cause the differences in signal crosstalk between electrodes when comparing amperometry and FSCV.
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| 2. |
- Bergman, Jenny, et al.
(författare)
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Co-detection of dopamine and glucose with high temporal resolution
- 2018
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Ingår i: Catalysts. - : MDPI AG. - 2073-4344. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal activity and brain glucose metabolism are tightly coupled, where triggered neurotransmission leads to a higher demand for glucose. To better understand the regulation of neuronal activity and its relation to high-speed metabolism, development of analytical tools that can temporally resolve the transients of vesicular neurotransmitter release and fluctuations of metabolites such as glucose in the local vicinity of the activated neurons is needed. Here we present an amperometric biosensor design for rapid co-detection of glucose and the neurotransmitter dopamine. The sensor is based on the immobilization of an ultra-thin layer of glucose oxidase on to a gold-nanoparticle-covered carbon fiber microelectrode. Our electrode, by altering the potential applied at the sensor surface, allows for the high-speed recording of both glucose and dopamine. We demonstrate that, even though glucose is electrochemically detected indirectly through the enzymatic product and the electroactive dopamine is sensed directly, when exposing the sensor surface to a mixture of the two analytes, fluctuations in glucose and dopamine concentrations can be visualized with similar speed and at a millisecond time scale. Hence, by minimizing the enzyme coating thickness at the sensor surface, dual detection of glucose and dopamine can be realized at the same sensor surface and at time scales necessary for monitoring fast metabolic alterations during neurotransmission. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
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| 3. |
- Kim, Angela, et al.
(författare)
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Arginine-vasopressin mediates counter-regulatory glucagon release and is diminished in type 1 diabetes.
- 2021
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Ingår i: eLife. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Insulin-induced hypoglycemia is a major treatment barrier in type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (8 to 4 mM) has no significant effect on glucagon secretion in the perfused mouse pancreas or in isolated mouse islets (in vitro), and yet associates with dramatic increases in plasma glucagon. The identity of the systemic factor(s) that elevates circulating glucagon remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Alpha-cells express high levels of the vasopressin 1b receptor (V1bR) gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or V1bR. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. AVP injection increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP/copeptin in humans and this hormone stimulates glucagon secretion from human islets. In patients with T1D, hypoglycemia failed to increase both copeptin and glucagon. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.
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| 4. |
- Kurczy, Michael, 1980, et al.
(författare)
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Composition Based Strategies for Controlling Radii in Lipid Nanotubes
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Nature routinely carries out small-scale chemistry within lipid bound cells and organelles. Liposome–lipid nanotube networks are being developed by many researchers in attempt to imitate these membrane enclosed environments, with the goal to perform small-scale chemical studies. These systems are well characterized in terms of the diameter of the giant unilamellar vesicles they are constructed from and the length of the nanotubes connecting them. Here we evaluate two methods based on intrinsic curvature for adjusting the diameter of the nanotube, an aspect of the network that has not previously been controllable. This was done by altering the lipid composition of the network membrane with two different approaches. In the first, the composition of the membrane was altered via lipid incubation of exogenous lipids; either with the addition of the low intrinsic curvature lipid soy phosphatidylcholine (soy-PC) or the high intrinsic curvature lipid soy phosphatidylethanolamine (soy-PE). In the second approach, exogenous lipids were added to the total lipid composition during liposome formation. Here we show that for both lipid augmentation methods, we observed a decrease in nanotube diameter following soy-PE additions but no significant change in size following the addition of soy-PC. Our results demonstrate that the effect of soy-PE on nanotube diameter is independent of the method of addition and suggests that high curvature soy-PE molecules facilitate tube membrane curvature.
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| 5. |
- Mellander, Lisa J., et al.
(författare)
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Amperometric post spike feet reveal most exocytosis is via extended kiss-and-run fusion
- 2012
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 2:artikel nr 907, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- The basis for communication between nerve cells lies in the process of exocytosis, the fusion of neurotransmitter filled vesicles with the cell membrane resulting in release of the signaling molecules. Even though much is known about this process, the extent that the vesicles are emptied upon fusion is a topic that is being debated. We have analyzed amperometric peaks corresponding to release at PC12 cells and find stable plateau currents during the decay of peaks, indicating closing of the vesicle after incomplete release of its content. Using lipid incubations to alter the amount of transmitter released we were able to estimate the initial vesicular content, and from that, the fraction of release. We propose a process for most exocytosis events where the vesicle partially opens to release transmitter and then closes directly again, leaving the possibility for regulation of transmission within events.
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| 6. |
- Mellander, Lisa J., et al.
(författare)
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Electrochemical Probes for Spatial Detection of Exocytosis and Vesicles
- 2010
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 11:13, s. 2756-2763
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Tidskriftsartikel (refereegranskat)abstract
- Unraveling the mechanistic details of neurotransmitter exocytosis is arguably among the most important molecular problems in neuroscience today. Investigations at single cells, particularly with electrochemical methods, have given unique chemical and biological insight into this process at the fundamental level. The rapid response time (submillisecond) of microelectrodes makes them well suited for monitoring the dynamic process of exocytosis. We review here recent developments in electrochemical techniques to spatially and simultaneously detect exocytosis across a single cell and to measure the transmitter content of single vesicles removed from cells. The former method is used to demonstrate dynamic heterogeneity in release across a cell, and in the latter work comparison is made between vesicle content and release to conclude that only a fraction of the transmitter is released during full exocytosis.
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| 7. |
- Mellander, Lisa J.
(författare)
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The Mechanisms of Exocytosis Studied in Cells and Models with Amperometry
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Exocytosis is the universal basis for neuronal communication, allowing the controlled release of neurotransmitter molecules from the presynaptic cell. Exocytosis has been widely studied in many systems. However, the details of this vital process are not fully understood. Exocytosis is quantal in its nature in that the neurotransmitter is released in packages. These packages were initially assumed to be comprised of the entire vesicular content. More recently, this simplistic view has been replaced with a more complex one with several different suggested modes of release. Vesicles can, in addition to the irreversible complete collapse of the vesicle into the cell membrane, fuse transiently with the cell membrane in a process that has been termed kiss-and-run. Transient fusion has the advantage of allowing the vesicle not to release its entire content but instead potentially control the fraction of neurotransmitter molecules it lets out. To elucidate the details of the exocytotic process, it is necessary to resolve individual release events. Amperometric detection at micrometer sized electrodes is an excellent tool for this purpose, providing quantitative and detailed kinetic information about single release events. In this thesis I have employed amperometry to study the mechanisms of exocytosis at PC12 cells and in an artificial cell model. In paper I, detection of exocytosis using amperometry and fast scan cyclic voltammetry at a microelectrode array is compared and evaluated. In paper II, amperometric detection of exocytosis is compared at a disk and a ring-shaped microelectrode, and the results are used to evaluate the diffusion coefficient of dopamine in the extracellular matrix surrounding the cell. In paper III, I define and explore a new feature of amperometric peaks recorded at PC12 cells, the post spike foot. This feature is used to show that changing the lipid composition of the cell membrane can alter the fraction of neurotransmitter released per event. In paper IV the influence of membrane lipid composition on lipid nanotube dimensions is studied using amperometry at a lipid nanotube-liposome network. It has been demonstrated that sorting of membrane lipids based on structure occurs and a dynamic method for controlling lipid nanotube diameter has been established. The same experimental setup is used in paper V, but here it is applied to PC12 cell plasma membrane vesicles and used to study the membrane dynamics of exocytosis. In this system it has been demonstrated that release occurs though two distinct mechanisms; full and partial distension of the initial fusion pore.
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| 8. |
- Mellander, Lisa J., et al.
(författare)
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Two modes of exocytosis in an artificial cell
- 2014
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.
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| 9. |
- Najafinobar, Neda, 1985, et al.
(författare)
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Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6:Article number: 33702
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Tidskriftsartikel (refereegranskat)abstract
- Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.
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| 10. |
- Nguyen, Tho D. K., et al.
(författare)
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Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells
- 2022
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Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:3, s. 4831-4842
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Tidskriftsartikel (refereegranskat)abstract
- For decades, "all-or-none"and "kiss-and-run"were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically. © 2022 The Authors. Published by American Chemical Society.
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| 11. |
- Ren, Lin, et al.
(författare)
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The evidence for open and closed exocytosis as the primary release mechanism
- 2016
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Ingår i: Quarterly Reviews of Biophysics. - : Cambridge University Press (CUP). - 0033-5835 .- 1469-8994. ; 49
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Tidskriftsartikel (refereegranskat)abstract
- Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss- and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.
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