Odor, drug and toxin analysis with neuronal networks in vitro: extracellular array recording of network responses.

Neurons, by virtue of intrinsic electrophysiological mechanisms, represent transducers that report the dynamics of cell death, receptor-ligand interactions, alterations in metabolism, and generic membrane perforation processes. In cell culture, mammalian neurons form fault-tolerant, spontaneously active systems with great sensitivity to their chemical environment and generate response profiles that are often concentration- and substance-specific. Changes in action potential patterns are usually detected before morphological changes and cell damage occur, which provides sensitivity and reversibility. Such biological systems can be used to screen rapidly for novel pharmacological substances, toxic agents, and for the detection of certain odorants. Existing simple culture preparations can already be employed effectively for the detection of chemical compounds. So far, three strategies have been investigated in pilot experiments: (1) Substance-dependent major changes in spontaneous native activity patterns. All synaptically active agents (e.g. glutamate, strychnine, N-methyl D-aspartic acid) as well as metabolic poisons generate such changes. (2) Substance-dependent changes in network oscillations via disinhibition. The regularized, oscillatory activity is altered by synaptically and metabolically active substances, ion channel blockers, and toxins. (3) Detection of paroxysmal responses indicating major, pathological membrane currents in large subpopulation of cells. We have explored these three strategies via 64 channel array recordings using spontaneously active murine spinal cord cultures. The glycine receptor blocker strychnine reliably generated increased multichannel bursting at 5-20 nM and regular, coordinated bursting above 5 microM. During biculline-induced network oscillations many compounds alter oscillation frequencies or terminate activity in a substance-specific manner. Finally, the gp120 protein of the AIDS virus (at 1 microgram/ml) produces massive, unique paroxysmal discharges that may last as long as 2 min. These results indicate that cultured neuronal networks are practical systems that can be used for the detection and identification of a great variety of chemical substances. The concept of dynamic fingerprinting to identify specific compounds is discussed.

Distribution-free graphical and statistical analysis of serial dependence in neuronal spike trains.

Two-dimensional 'joint' interval distributions of sequential interspike (ISIs) are a commonly used tool in neuronal spike train analysis. We present and evaluate here a modification of the joint interval plot using ranked ISIs. This modification provides clearer graphical evaluation of serial dependence in ISI sequences, a distribution-free basis for isolating changes in serial dependence across experimental treatments from changes in ISI distributions, and a basis for unambiguous statistical tests of serial dependence and stationarity. To validate this method and illustrate the advantages of its use we have applied it to both single-neuron spike trains recorded from cultured mammalian spinal cord neurons and artificial spike trains generated by stochastic models with defined burst envelopes and serial dependencies.

The use of neuronal networks on multielectrode arrays as biosensors.

Mammalian spinal neuronal networks growing on arrays of photoetched electrodes in culture provide a highly stable system for the long-term monitoring of multichannel, spontaneous or evoked electrophysiological activity. In the absence of the homeostatic control mechanisms of the central nervous system, these networks show remarkable sensitivities to minute chemical changes and mimic some of the properties of sensory tissue. These sensitivities could be enhanced by receptor up-regulation and altered by the expression of unique receptors. The fault-tolerant spontaneous network activity is used as a dynamic platform on which large changes in activity signify detection of chemical substances. We present strategies for the expression of novel supersensitivities to foreign molecules via genetic engineering that involves the grafting of ligand binding cDNA onto truncated native receptor DNA and the subsequent expression of such chimeric receptors.

Potassium and calcium channel dependence of bursting in cultured neuronal networks.

Increases in extracellular potassium concentrations reliably increase burst rates in cultured fetal murine spinal cord networks. This effect could be mimicked by either blocking voltage-gated potassium conductances or facilitating excitatory synaptic interactions, but not by blocking specific calcium-dependent potassium conductances or tonic depolarization. Spontaneous bursting in cultured networks is apparently dependent on potassium currents and intracellular calcium levels, but not on the pharmacologically characterized calcium-dependent potassium conductances.

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.

An inexpensive, nondedicated, automated pupillometric measurement system.

A new component pupillometric system is presented. This system utilizes a commercially available microcomputer digital analyzer to measure and record pupillary diameter from a videotape record. The principal advantages of this system over the conventional electronic pupillometer are its low cost, portability, and the nondedicated nature of its components. This provides the psychophysiological investigator with a much more flexible and adaptable pupillometric system than has heretofore been available.