Sub-quantal release is not dominant during prolonged depolarization in adrenal chromaffin cells.

Exocytosis, which mediates important functions like synaptic transmission and stress responses, has been postulated to release all transmitter molecules in the vesicle in the "all-or-none" quantal hypothesis. Challenging this hypothesis, amperometric current recordings of catecholamine release propose that sub-quantal or partial transmitter release is dominant in various cell types, particularly chromaffin cells. The sub-quantal hypothesis predicts that fusion pore closure (kiss-and-run fusion), the cause of sub-quantal release, is dominant, and blocking pore closure increases quantal size.

Fluorescent Sensor for the Visualization of Amino Acid Neurotransmitters in Neurons Based on an SAr Reaction.

Glutamate is an important excitatory neurotransmitter, while GABA is an inhibitory neurotransmitter. However, direct and accurate visualization of these important signaling agents by a chemical sensor is still very challenging. Here, a novel coumarin-based fluorescent sensor for the selective labeling and imaging of amino acids in neurons has been developed.

Synthesis of a Near-Infrared Fluorescent Probe for Imaging Catecholamines via a Tandem Nucleophilic Aromatic Substitution.

A near-infrared (NIR) fluorescent probe NS667 was developed using a novel synthetic strategy by integrating an electron-rich 1,2,3,4-tetrahydroquinoxaline (THQ) into the scaffold from NS510, which binds to catecholamines with high affinity. The fluorophore core was constructed with a tandem nucleophilic aromatic substitution. Upon binding to catecholamines, the fluorescence of this probe shifted, with the emission in the NIR region.

A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine's Effect on Cellular Amino Acids via Inhibiting Cathepsin L.

Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction of nutrient balance can result in human diseases such as diabetes. Much remains to be discovered about how cells transport, store, and utilize amino acids due to limited research tools.

A High-Affinity Fluorescent Sensor for Catecholamine: Application to Monitoring Norepinephrine Exocytosis.

A fluorescent sensor for catecholamines, NS510, is presented. The sensor is based on a quinolone fluorophore incorporating a boronic acid recognition element that gives it high affinity for catecholamines and a turn-on response to norepinephrine. The sensor results in punctate staining of norepinephrine-enriched chromaffin cells visualized using confocal microscopy indicating that it stains the norepinephrine in secretory vesicles.

Estimating amperometric spike parameters resulting from quantal exocytosis using curve fitting seeded by a matched-filter algorithm.

Background: Quantal exocytosis of oxidizable neurotransmitters can be detected as spikes of amperometric current using electrochemical microelectrodes. Measurements of spike parameters indicate the maximal transmitter flux, flux duration, and amount of transmitter released from individual vesicles. Automated analysis algorithms need to reject spikes that overlap in time.

A matched-filter algorithm to detect amperometric spikes resulting from quantal secretion.

Background: Electrochemical microelectrodes located immediately adjacent to the cell surface can detect spikes of amperometric current during exocytosis as the transmitter released from a single vesicle is oxidized on the electrode surface. Automated techniques to detect spikes are needed in order to quantify the spike rate as a measure of the rate of exocytosis.

New Method: We have developed a Matched Filter (MF) detection algorithm that scans the data set with a library of prototype spike templates while performing a least-squares fit to determine the amplitude and standard error.

Electrochemical measurement of quantal exocytosis using microchips.

Carbon-fiber electrodes (CFEs) are the gold standard for quantifying the release of oxidizable neurotransmitters from single vesicles and single cells. Over the last 15 years, microfabricated devices have emerged as alternatives to CFEs that offer the possibility of higher throughput, subcellular spatial resolution of exocytosis, and integration with other techniques for probing exocytosis including microfluidic cell handling and solution exchange, optical imaging and stimulation, and electrophysiological recording and stimulation. Here we review progress in developing electrochemical electrode devices capable of resolving quantal exocytosis that are fabricated using photolithography.

Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode.

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis.

Selective catecholamine recognition with NeuroSensor 521: a fluorescent sensor for the visualization of norepinephrine in fixed and live cells.

A method for the selective labeling and imaging of catecholamines in live and fixed secretory cells is reported. The method integrates a tailored approach using a novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that can exploit the high concentration of neurotransmitters and acidic environment within secretory vesicles for the selective recognition of norepinephrine and dopamine. The utility of the method was demonstrated by selectively labeling and imaging norepinephrine in secretory vesicles such that discrimination between norepinephrine- and epinephrine-enriched populations of chromaffin cells was observed.

Fabrication of two-layer poly(dimethyl siloxane) devices for hydrodynamic cell trapping and exocytosis measurement with integrated indium tin oxide microelectrodes arrays.

The design, fabrication and test of a microfluidic cell trapping device to measure single cell exocytosis were reported. Procedures on the patterning of double layer template based on repetitive standard photolithography of AZ photoresist were investigated. The replicated poly(dimethyl siloxane) devices with 2.

Quantification of noise sources for amperometric measurement of quantal exocytosis using microelectrodes.

Electrochemical microelectrodes are commonly used to record amperometric spikes of current that result from oxidation of transmitter released from individual vesicles during exocytosis. Whereas the exquisite sensitivity of these measurements is well appreciated, a better understanding of the noise sources that limit the resolution of the technique is needed to guide the design of next-generation devices. We measured the current power spectral density (S(I)) of electrochemical microelectrodes to understand the physical basis of dominant noise sources and to determine how noise varies with the electrode material and geometry.

Modes of exocytosis and electrogenesis underlying canine biphasic insulin secretion.

Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion.

Automated targeting of cells to electrochemical electrodes using a surface chemistry approach for the measurement of quantal exocytosis.

Here we describe a method to fabricate a multi-channel high-throughput microchip device for measurement of quantal transmitter release from individual cells. Instead of bringing carbon-fiber electrodes to cells, the device uses a surface chemistry approach to bring cells to an array of electrochemical microelectrodes. The microelectrodes are small and "cytophilic" in order to promote adhesion of a single cell whereas all other areas of the chip are covered with a thin "cytophobic" film to block cell attachement and facilitate movement of cells to electrodes.

Post-CMOS fabrication of Working Electrodes for On-Chip Recordings of Transmitter Release.

The release of neurotransmitters and hormones from secretory vesicles plays a fundamental role in the function of the nervous system including neuronal communication. High-throughput testing of drugs modulating transmitter release is becoming an increasingly important area in the fields of cell biology, neurobiology, and neurology. Carbon-fiber amperometry, provides high-resolution measurements of amount and time course of transmitter release from single vesicles, and their modulation by drugs and molecular manipulations.

A microfluidic cell trap device for automated measurement of quantal catecholamine release from cells.

Neurons and endocrine cells secrete neurotransmitter and hormones in discrete packets in a process called quantal exocytosis. Electrochemical microelectrodes can detect spikes in current resulting from the oxidation of individual quanta of transmitter only if the electrodes are small and directly adjacent to release sites on the cell. Here we report development of a microchip device that uses microfluidic traps to automatically target individual or small groups of cells to small electrochemical electrodes.

Preferential cell attachment to nitrogen-doped diamond-like carbon (DLC:N) for the measurement of quantal exocytosis.

Electrochemical measurement of transmitter or hormone release from individual cells on microchips has applications both in basic science and drug screening. High-resolution measurement of quantal exocytosis requires the working electrode to be small (cell-sized) and located in immediate proximity to the cell. We examined the ability of candidate electrode materials to promote the attachment of two hormone-secreting cell types as a mechanism for targeting cells for to recording electrodes with high precision.

Electrochemical Properties of Carbon Nanoparticles Entrapped in Silica Matrix.

Carbon-based electrode materials have been widely used for many years for electrochemical charge storage, energy generation, and catalysis. We have developed an electrode material with high specific capacitance by entrapping graphite nanoparticles into a sol-gel network. Films from the resulting colloidal suspensions were highly porous due to the removal of the entrapped organic solvents from sol-gel matrix giving rise to high Brunauer-Emmett-Teller (BET) specific surface areas (654 m(2)/g) and a high capacitance density ( approximately 37 F/g).

Magnetron sputtered diamond-like carbon microelectrodes for on-chip measurement of quantal catecholamine release from cells.

Carbon electrodes are widely used in electrochemistry due to their low cost, wide potential window, and low and stable background noise. Carbon-fiber electrodes (CFE) are commonly used to electrochemically measure "quantal" catecholamine release via exocytosis from individual cells, but it is difficult to integrate CFEs into lab-on-a-chip devices. Here we report the development of nitrogen doped diamond-like carbon (DLC:N) microelectrodes on a chip to monitor quantal release of catecholamines from cells.

Phosphorylation of SNAP-25 at Ser187 mediates enhancement of exocytosis by a phorbol ester in INS-1 cells.

Activation of diacylglycerol (DAG) signaling pathways with phorbol esters dramatically enhances Ca2+-triggered exocytosis from both endocrine cells and neurons, however the relevant targets of DAG are controversial. A possible effector mechanism for this signaling pathway is phosphorylation of SNAP-25 (25 kDa synaptosome-associated protein) at Ser187 by PKC. Here, we investigated the role of Ser187 in the enhancement of exocytosis by the phorbol ester PMA (phorbol 12-myristate 13-acetate).

Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes.

Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (approximately 20 microm by 20 microm).

Vesicle pool heterogeneity at hippocampal glutamate and GABA synapses.

Glutamate and GABA are the major fast excitatory and inhibitory neurotransmitters, respectively, in the CNS. Although glutamate and GABA have clearly distinct postsynaptic actions, we are just beginning to appreciate that presynaptic differences between glutamatergic and GABAergic neurons may contribute to distinct functions of these transmitter systems. We therefore probed possible differences between the functional synaptic vesicle populations of glutamatergic and GABAergic neurons.