In vivo Monitoring of Serotonin in the Striatum of Freely-Moving Rats with One-Minute Temporal Resolution by Online Microdialysis-Capillary High Performance Liquid Chromatography at Elevated Temperature and Pressure

摘要

Online monitoring of serotonin in striatal dialysate from freely moving rats was carried out for more than sixteen hours at one-minute time resolution using microdialysis coupled online to a capillary HPLC system operating at about 500 bar and 50 °C. Several aspects of the system were optimized towards robust, in vivo online measurements. A two-loop, eight-port rotary injection valve demonstrated better consistency of continuous injections than the more commonly used two-loop, ten-port valve. A six-port loop injector for introducing stimulating solutions (stimulus injector) was placed inline between the syringe pump and microdialysis probe. We minimized solute dispersion by using capillary tubing (75 µm i.d., 70 cm long) for the probe inlet and outlet. In vitro assessment of concentration dispersion during transport with 30-s time resolution showed that the dispersion standard deviation for serotonin was well within the desired system temporal resolution. Each 30- or 60-second measurement reflects the integral of the true time response over the measurement time. We have accounted for this mathematically in determining the concentration dispersion during transport. The delay time between a concentration change at the probe and its detection is seven minutes. The timing of injections from the stimulus injector and the cycle time for the HPLC monitoring of the flow stream was controlled. The electrochemical detector contained a 13 µm spacer to minimize detector dead volume. During in vivo experiments, retention time and separation efficiency were stable and reproducible. There was no statistically significant change over 5.5 hours in the electrochemical detector sensitivity factor for serotonin. Dialysate serotonin concentrations change significantly in response to a 120 mM K⁺ stimulus. Release of serotonin evoked by a ten-minute, 120 mM K⁺ stimulation, but not for other K⁺ stimuli, exhibited a reproducible, oscillating profile of dialysate serotonin concentration vs. time. Infusion of fluoxetine, a serotonin uptake inhibitor, increased dialysate serotonin concentrations and stimulated release magnitude. Transient serotonin increases were observed in response to the stress associated with unexpected handling. This system is simple, efficient, reliable, and suitable for study of serotonin neurochemistry associated with emotion and behavior.