David W. Barnett

Electrophysiology of Stimulus-Secretion Coupling in Human β-Cells

Herein, we review the applicability to human β-cells of an electrophysiologically based hypothesis of the coupling of glucose metabolism to insulin secretion. According to this hypothesis, glucose metabolism leads to the generation of intracellular intermediates (including ATP), which leads to closure of ATP-sensitive K+ channels. Channel closure results in membrane depolarization, the onset of electrical activity, and voltage-dependent Ca2+ entry. The resultant rise in cytosolic Ca2+ leads to Ca2+-dependent exocytosis of insulin granules. We found that most of the published experimental evidence for human β-cells supports this hypothesis. In addition, we present three other emerging lines of evidence in support of this hypothesis for human islet β-cells: 1) the effects of pH1-altering maneuvers on insulin secretion and electrical activity; 2) preliminary identification of LVA and HVA single Ca2+ channel currents; and 3) validation of the feasibility of Cm measurements to track insulin granule exocytosis. On the basis of this last new line of evidence, we suggest that combinations of Cm measurements and electrical activity/membrane current measurements may help define the roles of diverse electrical activity patterns, displayed by human β-cells, in stimulus-induced insulin secretion.

Stimulus-Secretion Coupling in β-Cells of Transplantable Human Islets of Langerhans: Evidence for a Critical Role for Ca2+ Entry

With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis of glucose transduction and a Ca2+ hypothesis of depolarization-secretion coupling, both previously proposed for rodent islet β-cells. We report that several features of human β-cell physiology are well accounted for by these hypotheses. With whole-islet perifusion, we demonstrated that insulin secretion induced by glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release induced by these secretagogues is enhanced by the dihydropyridine Ca2+ channel agonist BAYk8644 and depressed by the dihydropyridine Ca2+-channel antagonist nifedipine. All of the aforementioned secretagogues provoke increases in cytosolic free Ca2+, which are dependent on extracellular Ca2+ and are altered by the dihydropyridine drugs. Individual β-cells in the islet display diminished resting membrane conductance, graded depolarization, and complex electrical patterns, including bursts of action potentials in response to stimulatory concentrations of glucose or tolbutamide. Individual islet β-cells display voltage-dependent Ca2+ currents that are activated at membrane potentials traversed during the excursion of the action potential. In most cells, the Ca2+ currents are enhanced by BAYk8644 and depressed by nifedipine at concentrations that have parallel effects on secretagogue-induced increases in cytosolic Ca2+ and insulin secretion. These survey studies should provide the basis for more detailed investigations of the relationship of voltage-dependent ionic currents to electrical activity patterns and of electrical activity patterns to granule exocytosis in single human β-cells.

Sleep-stage scoring in the rat using a support vector machine.

Analysis and classification of sleep stages is a fundamental part of basic sleep research. Rat sleep stages are scored based on electrocorticographic (ECoG) signals recorded from electrodes implanted epidurally and electromyographic (EMG) signals from the temporalis or nuchal muscle. An automated sleep scoring system was developed using a support vector machine (SVM) to discriminate among waking, nonrapid eye movement sleep, and paradoxical sleep. Two experts scored retrospective data obtained from six Sprague-Dawley rodents to provide the training sets and subsequent comparison data used to assess the effectiveness of the SVM. Numerous time-domain and frequency-domain features were extracted for each epoch and selectively reduced using statistical analyses. The SVM kernel function was chosen to be a Gaussian radial basis function and kernel parameters were varied to examine the effectiveness of optimization methods. Tests indicated that a common set of features could be chosen resulted in an overall agreement between the automated scores and the expert consensus of greater than 96%.

Optimization of Microelectrode Design for Cortical Recording Based on Thermal Noise Considerations

Intracortical microelectrode recordings of neural activity show great promise as control signals for neuroprosthetic applications. However, faithful, consistent recording of single unit spiking activity with chronically implanted silicon-substrate microelectrode arrays has proven difficult. Many approaches seek to enhance the long-term performance of microelectrode arrays by, for example, increasing electrode biocompatibility, decreasing electrode impedance, or improving electrode interface properties through application of small voltage pulses. The purpose of this study was to use computational models to optimize the design of microelectrodes. We coupled detailed models of the neural source signal, silicon-substrate microelectrodes, and thermal noise to define the electrode contact size that maximized the signal-to-noise ratio (SNR). Model analysis combined a multi-compartment cable model of a layer V cortical pyramidal neuron with a 3D finite element model of the head and microelectrode to define the amplitude and time course of the recorded signal. A spatially-lumped impedance model was parameterized with in vitro and in vivo spectroscopy data and used to define thermal noise as a function of electrode contact size. Our results suggest that intracortical microelectrodes with a contact size of ~380 mum2 will provide an increased SNR in vivo and improve the long-term recording capabilities of silicon-substrate microelectrode arrays

α‐Latrotoxin increases spontaneous and depolarization‐evoked exocytosis from pancreatic islet β‐cells

α‐Latrotoxin (α‐LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential‐evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of α‐LT on single human, mouse and canine β‐cells. In isolated and combined measurements, α‐LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca2+, into the micromolar range, that are dependent on extracellular Ca2+; (ii) large conductance non‐selective cation channels; and (iii) Ca2+‐dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, α‐LT potentiates depolarization‐induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca2+. These results strongly support the hypothesis that α‐LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) α‐LT inserts into the plasma membrane to form Ca2+ permeable channels and promote Ca2+ entry thereby triggering Ca2+‐dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, α‐LT augments depolarization‐evoked exocytosis probably by second messenger‐induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca2+‐sensitive pool of insulin granules activated by global cytosolic Ca2+ concentrations in the range of ∼1 μm.

Effects of power frequency electromagnetic fields on melatonin and sleep in the rat

Background Studies investigating the effect of power frequency (50–60 Hz) electromagnetic fields (EMF) on melatonin synthesis in rats have been inconsistent with several showing suppression of melatonin synthesis, others showing no effect and a few actually demonstrating small increases. Scant research has focused on the ensuing sleep patterns of EMF exposed rats. The present study was designed to examine the effects of extremely low power frequency electromagnetic fields (EMF) on the production of melatonin and the subsequent sleep structure in rats. Methods Eighteen male Sprague-Dawley rats were exposed to a 1000 milligauss (mG) magnetic field for 1 month. Urine was collected for the final 3 days of the exposure period for analysis of 6-sulphatoxymelatonin, the major catabolic product of melatonin found in urine. Subsequent sleep was analyzed over a 24-hour period. Results Melatonin production was mildly increased in exposed animals. Although there were no statistically significant changes in sleep structure, exposed animals showed slight decreases in REM (rapid eye movement) sleep as compared to sham (non-exposed) animals. Conclusions Power frequency magnetic fields induced a marginally statistically significant increase in melatonin levels in exposed rats compared to control. Subsequent sleep analysis indicated little effect on the sleep architecture of rats, at least not within the first day after 1 months continuous exposure. Varying results in the literature are discussed and future research suggested.

Electrical activity and exocytotic correlates of biphasic insulin secretion from β-cells of canine islets of Langerhans

Biphasic insulin secretion in response to glucose, consisting of a transient first phase followed by a progressive second phase, is a well described in pancreatic islets. Using single canine β-cells we have compared the time courses of electrical activity and insulin granule exocytosis to biphasic insulin secretion. Short trains of action potentials, similar those found during first phase insulin secretion, trigger phasic exocytosis from a small pool of insulin granules, likely an immediately releasable pool docked near voltage activated Ca2+ channels. In contrast, plateau depolarizations to between −35 and −20 mV resembling those during second phase insulin secretion, trigger tonic exocytosis from a larger pool of insulin granules, likely a highly Ca2+-sensitive pool farther from Ca2+ channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine β-cells two distinct components of exocytosis, tuned to two components of electrical activity, may contribute significantly to biphasic insulin secretion.

Ion channels underlying stimulus-exocytosis coupling and its cell-to-cell heterogeneity in β-cells of transplantable porcine islets of Langerhans

Given the growing interest in porcine islets as model tissue for studying the pathogenesis of human diabetes mellitus and its treatment by transplantation, we investigated stimulus-exocytosis coupling in single porcine β-cells using patch clamp electrophysiology, Ca2+ imaging, capacitance tracking and amperometry. We establish that porcine β-cells display several features prominently seen in β-cells from human islets of Langerhans. These include: (i) wide heterogeneity of electrical responsiveness to glucose; (ii) dependence of action potential activity on voltage-dependent Na+ as well as high voltage activated Ca2+ current; (iii) heterogeneity of time course of depolarization-evoked insulin granule exocytosis; and (iv) the dependence of vigorous single cell electrical activity and insulin granule exocytosis on the presence of agents that enhance cytosolic cAMP concentration. These findings promote the usefulness of porcine β-cells as a model for studying β-cell function in large mammals, including humans, as well as an appropriate source of tissue for xenotransplantation.

Phasic and tonic modes of depolarization-exocytosis coupling in β-cells of porcine islets of Langerhans

In response to depolarizations that open voltage dependent Ca2+ channels single porcine β-cells display heterogeneous time courses of exocytosis. Some cells display phasic exocytosis that is triggered by individual or short burst of action potentials typically characteristic of glucose-induced electrical activity or brief voltage clamp depolarization. Other cells, singularly or additionally, display tonic exocytosis that (i) is triggered during prolonged (up to seconds-long) depolarizations to voltages (-30 to -20 mV), and (ii) coincides with rises in global cytosolic [Ca2+] > 500 nM. We suggest that tonic exocytosis (i) likely results from a recently described pool of granules that is more Ca2+ sensitive and less co-localized with voltage-sensitive Ca2+ entry channels than that contributing to phasic exocytosis and (ii) helps tune exocytosis to glucose-induced electrical activity when the latter consists of spike activity followed by intervals of plateau depolarization to nearly -20 mV.

Optimization of multi-frequency techniques used for cell membrane capacitance estimation

Measurements of cell membrane capacitance serve as an indicator of cell membrane surface area and thus have traditionally been used in stimulus-secretion coupling to monitor exocytosis and endocytosis of secretory vesicles. In order to accurately monitor secretion, high-resolution methods of tracking small (10/sup -15/ F) changes in baseline capacitance must be utilized. Most presently used techniques require assumptions that are not appropriate under all recording conditions or suffer from a low signal-to-noise ratio (SNR). This paper proposes to improve upon current estimation techniques by developing an optimized multi-frequency sinusoidal stimulus waveform for accurate capacitance estimations. The waveform is designed by jointly optimizing the phase and voltage of individual frequency components so as to minimize the theoretical bound on the variance of the capacitance estimate, while maintaining a preselected limit on the overall stimulus magnitude.