Abstract
In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 μm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis.
Highlights
Vesicles play an important role in synaptic signaling during neuronal transmission, as they are the major organelles for the storage and release of neurotransmitters.[1−3] Quantification of intravesicular neurotransmitter content and understanding the dynamic release process is vital for studying the mechanism of neurotransmission and malfunction in neurodegenerative diseases
Electrochemical strategies based on micro/nanoelectrodes and multielectrode arrays have been developed for quantitative measurements of intravesicular content and real-time monitoring of their release dynamics.[4−7] Our group has recently developed a technique, vesicle impact electrochemical cytometry (VIEC), allowing quantification of the catecholamine content inside single adrenal chromaffin vesicles as they adsorb and rupture on a 33-μm-diameter diskshaped carbon electrode.[8]
We present a novel method for visualization of the opening dynamics of single mammalian vesicles and quantitative measurement of their content using a combination of VIEC with Confocal laser scanning microscopy (CONF) imaging on a gold-modified Indium tin oxide (ITO) microelectrode
Summary
Vesicles play an important role in synaptic signaling during neuronal transmission, as they are the major organelles for the storage and release of neurotransmitters.[1−3] Quantification of intravesicular neurotransmitter content and understanding the dynamic release process is vital for studying the mechanism of neurotransmission and malfunction in neurodegenerative diseases. The pattern for the insulated area was defined on top of the MEAs by UV lithography (KS MA6, Suss MicroTec) with a second chrome mask showing the probe area and the contact pad design and was subsequently baked at 65 °C for 1 min and 95 °C for 3 min on a hot plate It was developed with SU-8 developer mr-Dev 600 (Micro Resist Technology) for 2 + 2 min with a mild shaking. 1 mL of vesicle solution was placed on the Au/ITO microelectrode device in a polylactic acid (PLA) chamber (1 × 2.5 × 0.5 cm) and let stand still for 5 min so that the vesicles adsorb onto the electrode surface.
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