Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.

Alkanethiols spontaneously assembles from solution or vapour on oxide free metal surfaces resulting in a close-packed molecular stuctures with a high degree of orientation and molecular order. In this study, inkjet printing technique is used to immobilize monolayers of alkanethiols on gold electrodes. The quality of the inkjetted monolayers are analyzed by electrochemical methods, i.e. cyclic voltammetry and electrochemical impedance spectroscopy, and by Polarization Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS) which show a similar molecular quality to those produced by immersion technique, the standard technique. The kinetic and mass transfer behaviours of micro-scale structures of inkjetted monolayers, e.g. bands and dots array electrodes, are explored by electrochemical methods. The microscale inkjetted structures of monolayers are of interest in the fields of microelectronic devices (e.g. chemical and biosensors) and optoelectronic devices. Taking benefits from multichannel existing in the printhead, mixtures of SAMs are demonstrated. Mixing of monolayers differing in functional groups provides a model surface to study interface phenomena at molecular level such as ion permeation, selective chemical binding, and electron transfer kinetic.

This thesis reports the design and fabrication of a low-cost reliable microelectrode array sensing platform and its application toward water quality monitoring, including heavy metal ion detection. Individually addressable microelectrodes were designed in a planar array on a nonconductive glass substrate by a photolithography method. The size, shape, composition, and functionality of the microelectrodes were theoretically explored in order to maximize performance. The microelectrode array sensing platform was proven and characterized in the K3Fe(CN)6 electrochemical standard using cyclic voltammetry. The sensor platform exhibited well defined voltammograms and had increased sensitivity relative to a commercially available microelectrode of similar size. Feasibility for application to heavy metal ions, copper and lead, detection in aqueous solutions was demonstrated utilizing the electrochemical method of anodic stripping voltammetry. Well defined voltammograms for the copper and lead ions were obtained with individual microelectrodes of the sensor platform, and compared against the similar sized commercially available microelectrode; increased sensitivity was observed.

We have applied an electrophysiologic technique (Reuss L.(1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cell to nystatin. Intracellular TMA+ activity (αiTMA) was measured with TMA+ -sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions αiTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased αiTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(αiTMA)O/(αiTMA)4min] - 1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less than predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 × 286 mosmol (control), cell water volume increased by a factor of 1.44 ± 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 ± 0.02, P< 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 ± 0.04 to 1.24 ± 0.09, P < 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of the K+ -channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.

yes / Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Epilepsy is a disease affecting millions of people worldwide. Despite over 50 years of research, the mechanisms that generate and sustain ictal discharges, a key neural hallmark of seizures, remain unknown. While once thought to be caused by hypersynchronous neuronal firing, we now recognize that the activity underlying ictal discharges is much more complex. With the development of microelectrode arrays (MEAs) suitable for use in humans, it is possible to observe neural activity at fine spatiotemporal scales in human patients with epilepsy. However, the diversity of seizure characteristics and limited patient population has led to a number of conflicting observations and theories. The purpose of this work is to elucidate mechanisms underlying ictal discharges in humans by applying statistical analyses and computational modeling to MEA recordings from human patients with epilepsy. We approach this aim in two projects. In the first project, we unify two seemingly conflicting theories surrounding cortical sources of ictal discharges. According to the ictal wavefront theory, ictal discharges are seeded at an expanding narrow front of high neuronal firing that delineates the boundary between regions of cortex with compromised functionality, and surrounding territory where the seizure is observable in electrical recordings, but cortical function remains intact. A second theory posits that discharges are predominantly seeded from a stationary localized cortical source. The two theories are based on observations from MEA recordings of seizures in two different small cohorts of patients. In this project, we analyze and model the discharge propagation patterns in a combined dataset from both cohorts. We show that discharges are seeded at the ictal wavefront in addition to other–possibly stationary–locations. In the second project, we characterize spatiotemporal patterns in the secondary transients of complex ictal discharges. Electrographic recordings of ictal discharges often have complex waveforms. Existing analyses focus on the spatiotemporal dynamics of the first, high-amplitude transient. In this project, we establish that ictal discharges often comprise multiple transients separated by ≈60 ms. Surprisingly, and contrary to our initial hypothesis, we find that individual transients within a complex discharge may propagate with different speeds, suggesting that different mechanisms are involved in the propagation of different transients.

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No description available.

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