The objective of the proposed project is to determine the effect of clinically relevant concentrations of ethanol on the firing threshold of small central neurons. Although ethanol has known anesthetic properties, the mechanisms of the action of ethanol on the firing threshold of small central neurons has never been accurately measured. Computer simulation experiments predict that the threshold of granule cells should be increased with ethanol. Preliminary experiments in the hippocampus using the strength-duration curve have also shown that ethanol can increase threshold at clinically relevant doses (50 mM) at a wide range of pulse width and amplitude. The mechanism for the increase in threshold is not clear, but computer simulations show that a decrease in membrane capacitance produced by ethanol could be responsible for this change in threshold. Both a voltage and a current threshold contribute to the observed firing threshold in membrane. These thresholds have traditionally been measured with the strength-duration curve which relates the amplitude to the pulse width of the current just necessary to fire the cell. This method, however, is very insensitive to large changes in threshold when both the voltage and the current thresholds have been modified. Synaptic thresholds have also been estimated by measuring the amplitude of an EPSP just necessary to generate a spike. Ethanol has been should to increase this value of the threshold as well. However, this value is only an estimate and is highly dependent on the rise time of the membrane voltage and has no direct relationship to the true value of the voltage threshold. It is therefore proposed to study the effect of ethanol on the firing threshold of granule cells at various clinically relevant concentrations (20 to 100 mM) by measuring the current- voltage relationship of the membrane using voltage clamp techniques. Both the voltage and the current threshold can be obtained with this technique and the synaptic threshold can also be directly measured. The analysis of the difference between the direct firing threshold and the synaptic threshold is important for the determination of the locus of action of ethanol. The measurements will be made using the in-vitro hippocampal preparation technique. This preparation is particularly useful since it allows direct access to the neurons and the ethanol solution can be applied directly to the tissue. The threshold parameters will also be measured during the acute tolerance phase. Acute tolerance (adaptation during the course of a single exposure) has been reported to share the same mechanisms with chronic tolerance (adaptation during the repeated exposure to ethanol). Therefore, the determination of the ethanol effects during this acute tolerance period will provide information about the process leading to the development of the long-term effects of ethanol.
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