14 research outputs found
Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity
Signals recorded from neurons with extracellular planar sensors have a wide
range of waveforms and amplitudes. This variety is a result of different
physical conditions affecting the ion currents through a cellular membrane. The
transmembrane currents are often considered by macroscopic membrane models as
essentially a homogeneous process. However, this assumption is doubtful, since
ions move through ion channels, which are scattered within the membrane.
Accounting for this fact, the present work proposes a theoretical model of
heterogeneous membrane conductivity. The model is based on the hypothesis that
both potential and charge are distributed inhomogeneously on the membrane
surface, concentrated near channel pores, as the direct consequence of the
inhomogeneous transmembrane current. A system of continuity equations having
non-stationary and quasi-stationary forms expresses this fact mathematically.
The present work performs mathematical analysis of the proposed equations,
following by the synthesis of the equivalent electric element of a
heterogeneous membrane current. This element is further used to construct a
model of the cell-surface electric junction in a form of the equivalent
electrical circuit. After that a study of how the heterogeneous membrane
conductivity affects parameters of the extracellular electrical signal is
performed. As the result it was found that variation of the passive
characteristics of the cell-surface junction, conductivity of the cleft and the
cleft height, could lead to different shapes of the extracellular signals
High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria
Recently developed technologies have enabled multi-well measurement of O2 consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1–10 µg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples