A closed flow chamber for long-term multichannel recording and optical monitoring.

An autoclavable chamber and associated medium circulation system has been constructed to provide a stable environment for mammalian cultures for long periods of time. The chamber was specifically designed for (a) multichannel electrophysiological recording from monolayer networks with photoetched multielectrode matrices, (b) for microscope observations of networks in the living state and (c) for the manipulation of such networks with laser cell surgery. The chamber components can be assembled under sterile conditions in less than 30 s to minimize pH and osmolarity stress to the monolayer cultures. An open chamber version provides a constant medium bath for pharmacological studies. The closed chamber version, attached to a 10-ml medium reservoir and a peristaltic pump, has so far provided constant conditions for continual recording over an 8-day period. Flow characteristics within the closed chamber, optical properties, pH maintenance, and schemes for drug addition are described.

Stimulation of monolayer networks in culture through thin-film indium-tin oxide recording electrodes.

Monolayer networks, obtained from murine spinal cord tissue and grown on a matrix of 64 photo-etched, indium-tin oxide (ITO) microelectrodes, can be electrically stimulated through such thin-film recording electrodes. Multichannel coordinated network activity can be evoked and spontaneous network activity can be modified by generation of additional, multichannel bursting. Although single pulses through 1 electrode may trigger network responses, networks generally react best to short trains of pulses. Response thresholds approximate standard physiological strength/duration relationships. Repetitive stimulation trains often generate network activity patterns akin to epileptiform activity. The ITO conductors remain stable for recording under warm saline for long periods of time (maximum test period: 8 months). However, most electrodes show a reduction in impedance after several thousand stimulus pulses. Electrode breakdown in the form of ITO oxidation and loss of light transmittance occurs before hydrolysis is observed but requires a combination of voltage levels and pulse lengths beyond that needed for effective network stimulation.