High-repetition fast-scan cyclic voltammetry and chronoamperometry were used to quantify and characterize the kinetics of dopamine and dopamine-o-quinone adsorption and desorption at carbon-fiber microelectrodes. A flow injection analysis system was used for the precise introduction and removal of a bolus of electroactive substance on a sub-second time scale to the disk-shaped surface of a microelectrode that was fabricated from a single carbon fiber (Thornel type T650 or P55). Pretreatment of the electrode surfaces consisted of soaking them in purified isopropyl alcohol for a minimum of 10 min, which resulted in S/N increasing by 200-400% for dopamine above that for those that were soaked in reagent grade solvent. Because of adsorption, high scan rates (2,000 V/s) are shown to exhibit equivalent S/N ratios as compared to slower, more traditional scan rates. In addition, the steady-state response to a concentration bolus is shown to occur more rapidly when cyclic voltammetric scans are repeated at short intervals (4 ms). The new methodologies allow for more accurate determinations of the kinetics of neurotransmitter release events (10-500 ms) in biological systems. Brain slice and in vivo experiments using T650 cylinder microelectrodes show that voltammetrically measured uptake kinetics in the caudate are faster using 2,000 V/s and 240 Hz measurements, as compared to 300 V/s and 10 Hz.

Terminal activity causes an increase in local cerebral blood flow that can be quantified by measuring the accompanying increase in tissue oxygen. Alkaline pH changes can also follow neuronal activation. The purpose of these studies was to determine whether these changes in extracellular oxygen and pH correlate. Fast-scan cyclic voltammetry was used to detect changes in dopamine, pH and oxygen levels simultaneously in the caudate-putamen after electrical stimulation of the substantia nigra in anesthetized rats. The biphasic increases in oxygen and pH followed similar time courses, and were delayed a few seconds from the immediate release and uptake of dopamine. The changes following administration of neurotransmitter receptor antagonists as well as agents that modulate blood flow were identical for oxygen and pH. Two distinct mechanisms were identified that give rise to the oxygen and pH changes: blood vessel dilatation caused by nitric oxide synthesis after muscarinic receptor activation and adenosine receptor activation. We conclude that changes in blood flow accompanying terminal activity cause alkaline pH shifts by the rapid removal of carbon dioxide, a component of the extracellular brain buffering system. Keywords: cerebral blood flow, dopamine, pH, striatum, functional magnetic resonance imaging. The brain depends on regulation of the circulation to maintain an adequate energy supply. Control of local blood flow by a variety of endogenous substances has been demonstrated. For example, the neurotransmitters GABA and glutamate are vasodilators in hippocampal slices (Fergus and Lee 1997;Lovick et al. 1999), and dopamine neurons directly innervate intraparenchymal vessels in the cortex causing vasoconstriction (Krimer et al. 1998). Blood flow is also regulated by metabolic byproducts such as adenosine, K + or H + (Sandor 1999). Classically, cerebral blood flow was determined by measuring the rate of hydrogen clearance at a platinum microelectrode after hydrogen formation in the brain (Young 1980). More recently, Lowry et al. (1997) have shown that regional increases in tissue oxygen, measured electrochemically, parallel the increases in cerebral blood flow measured by hydrogen clearance during behavioral activation. An increase in tissue oxygen occurs because oxygen utilization rates, although accelerated, are lower than oxygen delivery rates owing to vasodilatation (Fox and Raichle 1986). This increase in oxygen after terminal activation is the basis for the brain imaging techniques positron emission tomography, which measures blood flow, and blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI), which measures blood oxygenation (Raichle 1998).Cerebral blood flow is important not only in the delivery of metabolic nutrients but also in the removal of carbon dioxide, a byproduct of oxidative metabolism and component of the brain buffering system. The enzyme carbonic anhydrase catalyzes the hydration of carbon dioxide to produce carbonic acid, which can dissociate to bicarbonat...

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Insulin vesicles contain a chemically rich mixture of cargo that includes ions, small molecules, and proteins. At present, it is unclear if all components of this cargo escape from the vesicle at the same rate or to the same extent during exocytosis. Here, we demonstrate through real-time imaging that individual rat and human pancreatic ␤-cells secrete insulin in heterogeneous forms that disperse either rapidly or slowly. In healthy pancreatic ␤-cells maintained in culture, most vesicles discharge insulin in its fastrelease form, a form that leaves individual vesicles in a few hundred milliseconds. The fast-release form of insulin leaves vesicles as rapidly as C-peptide leaves vesicles. Healthy ␤-cells also secrete a slow-release form of insulin that leaves vesicles more slowly than C-peptide, over times ranging from seconds to minutes. Individual ␤-cells make vesicles with both forms of insulin, though not all vesicles contain both forms of insulin. In addition, we confirm that insulin vesicles store their cargo in two functionally distinct compartments: an acidic solution, or halo, and a condensed core. Thus, our results suggest two important features of the condensed core: 1) It exists in different states among the vesicles undergoing exocytosis and 2) its dissolution determines the availability of insulin during exocytosis. Diabetes 55:600 -607, 2006 I nsufficient insulin secretion in the face of insulin resistance leads to the disease type 2 diabetes (1). The mechanism responsible for insufficient insulin secretion remains unclear. Before it is possible to understand why insulin secretion fails to compensate for insulin resistance during the progression of type 2 diabetes (2), it is essential to understand insulin secretion at all levels of biological complexity, ranging from the whole pancreas within a living animal to the pancreatic ␤-cell and its fundamental unit of secretion, the insulin secretory vesicle.Insulin vesicles contain a chemically complex mixture of ions, small molecules, and proteins (3,4). Among the ions, there are large amounts of two divalent cations: zinc and calcium (5). Both of these ions are believed to play important roles in the regulated secretion of insulin (6). Among the vesicle proteins, insulin-related peptides comprise ϳ75% of the mass (7). The remaining 25% of the protein mass includes more than a hundred different membrane and cargo proteins. Although our understanding of regulated secretion of insulin has progressed greatly in recent years, the mechanisms that control the trafficking, processing, and secretion of insulin vesicle cargo are still matters of debate and extensive research (8).Previously, we (9) and others (10 -13) have studied secretion from single insulin vesicles by imaging fluorescently tagged proteins consisting of a vesicle cargo protein linked to a fluorescent protein. The fluorescently tagged vesicle cargo proteins, hereafter referred to as fluorescent cargo proteins, have provided a valuable and powerful tool for studies of single vesicle exocytosis. H...

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Described is an improved data acquisition system for fast-scan cyclic voltammetry (FSCV). The system was designed to significantly diminish noise sources that were identified in previously recorded FSCV measurements for the detection of neurotransmitters. Minimized noise is necessary to observe the low concentrations of neurotransmitters that are physiologically important. The system was based on a high-speed, 16-bit AD/DA acquisition board that allowed high scan rates and better resolved the small faradaic currents which remained after background subtraction. Irregularities that occur when independent timing sources are used for generation of the voltage waveform and collection of the current can create large noise artifacts near the voltage limits during FSCV. These were eliminated by the use of a single acquisition board that generated the voltage waveform and collected the current. Noise from frequency drift of the power line was eliminated through the use of a phase-locked loop. To demonstrate the improved performance of the system, data were collected using carbon-fiber microelectrodes in a flow injection analysis system and in brain slices. This new data acquisition system performed significantly better than another system previously used in our laboratory without these features. The improved detection limits of the new system allowed clearly resolved current spikes featuring pre-release "feet" to be recorded adjacent to individual mast cells following chemical stimulation. When combined with false-color plots, the low-noise system facilitated identification of dopamine release in a freely moving animal.

We compared secretion kinetics for four different fluorescent cargo proteins, each targeted to the lumen of insulin secretory vesicles. Upon stimulation, individual vesicles displayed one of four distinct patterns of fluorescence change: i), disappearance, ii), dimming, iii), transient brightening, or iv), persistent brightening. For each fusion protein, a different pattern of fluorescence change dominated. Furthermore, we demonstrated that the dominant pattern depends upon both i), the specific choice of fluorescent protein, and ii), the sequence of amino acids linking the cargo protein to the fluorescent protein. Thus, in beta-cells, experiments involving fluorescent cargo proteins for the study of exocytosis must be interpreted carefully, as design of a fluorescent cargo protein determines secretion kinetics at exocytosis.

In healthy individuals, plasma insulin levels oscillate in both fasting and fed states. Numerous studies of isolated pancreata and pancreatic islets support the hypothesis that insulin oscillations arise because the underlying rate of insulin secretion also oscillates; yet, insulin secretion has never been observed to oscillate in individual pancreatic ␤-cells. Using expressed fluorescent vesicle cargo proteins and total internal reflection fluorescence (TIRF) microscopy, we demonstrate that glucose stimulates human pancreatic ␤-cells to secrete insulin vesicles in short, coordinated bursts of ϳ70 vesicles each. Randomization tests and spectral analysis confirmed that the temporal patterns of secretion were not random, instead exhibiting alternating periods of secretion and rest, recurring with statistically significant periods of 15-45 s. Although fluorescent vesicles arrived at the plasma membrane before, during, and after stimulation, their rate of arrival was significantly slower than their rate of secretion, so that their density near the plasma membrane dropped significantly during the cell's response. To study in greater detail the vesicle dynamics during cyclical bursts of secretion, we applied trains of depolarizations once a minute and performed simultaneous membrane capacitance measurements and TIRF imaging. Surprisingly, young fluorescent insulin vesicles contributed at least half of the vesicles secreted in response to a first train, even though young vesicles were vastly outnumbered by older, nonfluorescent vesicles. For subsequent trains, young insulin vesicles contributed progressively less to total secretion, whereas capacitance measurements revealed that total stimulated secretion did not decrease. These results suggest that in human pancreatic ␤-cells, young vesicles are secreted first, and only then are older vesicles recruited for secretion.