Amperometry methods for monitoring vesicular quantal size and regulation of exocytosis release

Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells

A functioning artificial secretory cell

We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane

Co-detection of dopamine and glucose with high temporal resolution

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 visu alized 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

Generation of interconnected vesicles in a liposomal cell model

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca2+ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca2+ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems

Counting the number of enzymes immobilized onto a nanoparticle-coated electrode

To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic

The real catecholamine content of secretory vesicles in the CNS revealed by electrochemical cytometry

Resolution of synaptic vesicle neurotransmitter content has mostly been limited to the study of stimulated release in cultured cell systems, and it has been controversial as to whether synaptic vesicle transmitter levels are saturated in vivo. We use electrochemical cytometry to count dopamine molecules in individual synaptic vesicles in populations directly sampled from brain tissue. Vesicles from the striatum yield an average of 33,000 dopamine molecules per vesicle, an amount considerably greater than typically measured during quantal release at cultured neurons. Vesicular content was markedly increased by L-DOPA or decreased by reserpine in a time-dependent manner in response to in vivo administration of drugs known to alter dopamine release. We investigated the effects of the psychostimulant amphetamine on vesicle content, finding that vesicular transmitter is rapidly depleted by 50% following in vivo administration, supporting the "weak base hypothesis'' that amphetamine reduces synaptic vesicle transmitter and quantal size

Mechanics of lipid bilayer junctions affecting the size of a connecting lipid nanotube

In this study we report a physical analysis of the membrane mechanics affecting the size of the highly curved region of a lipid nanotube (LNT) that is either connected between a lipid bilayer vesicle and the tip of a glass microinjection pipette (tube-only) or between a lipid bilayer vesicle and a vesicle that is attached to the tip of a glass microinjection pipette (two-vesicle). For the tube-only configuration (TOC), a micropipette is used to pull a LNT into the interior of a surface-immobilized vesicle, where the length of the tube L is determined by the distance of the micropipette to the vesicle wall. For the two-vesicle configuration (TVC), a small vesicle is inflated at the tip of the micropipette tip and the length of the tube L is in this case determined by the distance between the two interconnected vesicles. An electrochemical method monitoring diffusion of electroactive molecules through the nanotube has been used to determine the radius of the nanotube R as a function of nanotube length L for the two configurations. The data show that the LNT connected in the TVC constricts to a smaller radius in comparison to the tube-only mode and that tube radius shrinks at shorter tube lengths. To explain these electrochemical data, we developed a theoretical model taking into account the free energy of the membrane regions of the vesicles, the LNT and the high curvature junctions. In particular, this model allows us to estimate the surface tension coefficients from R(L) measurements

Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis

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