Real-time control of electrical stimulation: A novel tool for compound screening and electrophysiologic analysis

Whole cell assays for compound detection and drug screening have become an increasingly attractive approach that achieves a functional response without the traditional high costs of animal studies. Cardio-active pharmaceutical screening is particularly well-suited to cell-based electrical assays, as myocytes have the ability to generate their own electrical signature - the cardiac action potential. However, several subtleties complicate the design of systems relying on spontaneous action potentials, including the need for strict environmental control and variability of contraction properties. Electrical stimulation affords the investigator a means by which to elicit propagated action potentials that might otherwise be absent. Furthermore, stimulation itself confers certain advantages which are absent in spontaneous cultures, including the ability to control the beat rate of the culture precisely (and with it rate-dependent biologic processes), the ability to assess excitability via stimulation threshold, and the ability to assess the refractory period. Traditional techniques using manual or computer-controlled open-loop stimulators can be slow, accuracy is limited, transient effects are missed, and pacing itself may generate unobserved physiological responses.;This dissertation presents an electrical stimulation system that utilizes a closed-loop algorithm to measure and track stimulation threshold in real time, extending the capabilities currently offered by stimulation. Recording of electrical field potentials evoked by stimulation through electrodes of the same microelectrode array is made possible by stimulus artifact extraction algorithms that allow detection of APs which could otherwise be obscured. A novel closed-loop algorithm that is tolerant of the high quantization and low data rates inherent in this cell-based system was designed to enable feedback using the efficacy of stimulation, although feedback of any measurable physiological parameter is possible. The hybrid hardware/software stimulation system, along with temperature control circuitry and a custom fluidic perfusion system, comprise a desktop hardware suite that is easily transportable and flexible, enabling convenient cell-level analysis in other laboratories and with other electrical cell types. The system is characterized using various physiologic stimuli, including temperature variation, ion channel block, electrolyte disturbance, and beta-adrenergic receptor stimulation and blockade.