Simon Waldbaum1, F Edward Dudek. 1. Department of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State University, Fort Collins, Colorado, USA.
Abstract
PURPOSE: The paired-pulse technique has been widely used as a convenient but indirect measure of "inhibition" in hippocampal circuits of normal and epileptic animals. Most investigators have used a single paired-pulse protocol, whereas others have utilized repetitive paired pulses. This study investigated which parameters influence results from paired-pulse tests, focusing on the repetitive paired-pulse technique; it aims to assess how this technique may be used in an unbiased and quantitative manner across animal preparations for comparisons of control and experimental epileptic animals. METHODS: The perforant path was stimulated while field potentials were recorded from the granule cell layer under isoflurane anesthesia. Paired-pulse suppression was analyzed as a function of stimulation intensity and interpulse interval and frequency. RESULTS: Paired-pulse suppression was greater with increased stimulus intensity and decreased interpulse interval (20-100 ms). During repetitive protocols, stimulation frequencies <or=1.0 Hz produced paired-pulse suppression similar to single paired-pulse responses, but caused more paired-pulse suppression between 1.0 and 4.0 Hz at all but the lowest intensities. The amplitude of the population spike produced by the conditioning pulse increased progressively during stimulation at higher frequencies (1.0-4.0 Hz). DISCUSSION: The single paired-pulse technique is highly dependent on stimulation parameters, as is the repetitive paired-pulse protocol, which is more variable. To generate reliable, consistent, and unbiased data in comparisons of control and experimental epileptic groups, all parameters should be specified and controlled across experiments. Paired-pulse suppression is susceptible to alterations in many mechanisms, and, therefore, represents a circuit response rather than an assay of gamma-aminobutyric acid (GABA)ergic inhibition in epilepsy research.
PURPOSE: The paired-pulse technique has been widely used as a convenient but indirect measure of "inhibition" in hippocampal circuits of normal and epileptic animals. Most investigators have used a single paired-pulse protocol, whereas others have utilized repetitive paired pulses. This study investigated which parameters influence results from paired-pulse tests, focusing on the repetitive paired-pulse technique; it aims to assess how this technique may be used in an unbiased and quantitative manner across animal preparations for comparisons of control and experimental epileptic animals. METHODS: The perforant path was stimulated while field potentials were recorded from the granule cell layer under isoflurane anesthesia. Paired-pulse suppression was analyzed as a function of stimulation intensity and interpulse interval and frequency. RESULTS: Paired-pulse suppression was greater with increased stimulus intensity and decreased interpulse interval (20-100 ms). During repetitive protocols, stimulation frequencies <or=1.0 Hz produced paired-pulse suppression similar to single paired-pulse responses, but caused more paired-pulse suppression between 1.0 and 4.0 Hz at all but the lowest intensities. The amplitude of the population spike produced by the conditioning pulse increased progressively during stimulation at higher frequencies (1.0-4.0 Hz). DISCUSSION: The single paired-pulse technique is highly dependent on stimulation parameters, as is the repetitive paired-pulse protocol, which is more variable. To generate reliable, consistent, and unbiased data in comparisons of control and experimental epileptic groups, all parameters should be specified and controlled across experiments. Paired-pulse suppression is susceptible to alterations in many mechanisms, and, therefore, represents a circuit response rather than an assay of gamma-aminobutyric acid (GABA)ergic inhibition in epilepsy research.