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In Vivo Monitoring of Dopamine by Microdialysis with 1 min Temporal Resolution Using Online Capillary Liquid Chromatography with Electrochemical Detection.

ABSTRACT: Microdialysis is often applied to understanding brain function. Because neurotransmission involves rapid events, increasing the temporal resolution of in vivo measurements is desirable. Here, we demonstrate microdialysis with online capillary liquid chromatography for the analysis of 1 min rat brain dialysate samples at 1 min intervals. Mobile phase optimization involved adjusting the pH, buffer composition, and surfactant concentration to eliminate interferences with the dopamine peak. By analyzing electrically evoked dopamine transients carefully synchronized with the switching of the online LC sample valve, we demonstrate that our system has both 1 min sampling capabilities and bona fide 1 min temporal resolution. Evoked DA transients were confined to single, 1 min brain dialysate samples. After uptake inhibition with nomifensine (20 mg/kg i.p.), responses to electrical stimuli of 1 s duration were detected.

SUBMITTER: Gu H

PROVIDER: S-EPMC4835028 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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In Vivo Monitoring of Dopamine by Microdialysis with 1 min Temporal Resolution Using Online Capillary Liquid Chromatography with Electrochemical Detection.

Gu Hui H Varner Erika L EL Groskreutz Stephen R SR Michael Adrian C AC Weber Stephen G SG

Analytical chemistry 20150526 12

Microdialysis is often applied to understanding brain function. Because neurotransmission involves rapid events, increasing the temporal resolution of in vivo measurements is desirable. Here, we demonstrate microdialysis with online capillary liquid chromatography for the analysis of 1 min rat brain dialysate samples at 1 min intervals. Mobile phase optimization involved adjusting the pH, buffer composition, and surfactant concentration to eliminate interferences with the dopamine peak. By analy ...[more]

PMID: 25970591

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Project description:Recently, our laboratory has demonstrated the technical feasibility of monitoring dopamine at 1 min temporal resolution with microdialysis and online liquid chromatography. Here, we monitor dopamine in the rat striatum during local delivery of high potassium/low sodium or nomifensine in awake-behaving rats. Microdialysis probes were implanted and perfused continuously with or without dexamethasone in the perfusion fluid for 4 days. Dexamethasone is an anti-inflammatory agent that exhibits several positive effects on the apparent health of the brain tissue surrounding microdialysis probes. Dopamine was monitored 1 or 4 days after implantation under basal conditions, during 10 min applications of 60 mM or 100 mM K+, and during 15 min applications of 10 μM nomifensine. High K+ and nomifensine were delivered locally by adding them to the microdialysis perfusion fluid using a computer-controlled, low-dead-volume six-port valve. Each day/K+/dexamethasone combination elicited specific dopamine responses. Dexamethasone treatment increased dopamine levels in basal dialysates (i.e., in the absence of K+ or nomifensine). Applications of 60 mM K+ evoked distinct responses on days one and four after probe implantation, depending upon the presence or absence of dexamethasone, consistent with dexamethasone's ability to mitigate the traumatic effect of probe implantation. Applications of 100 mM K+ evoked dramatic oscillations in dopamine levels that correlated with changes in the field potential at a metal electrode implanted adjacent to the microdialysis probe. This combination of results indicates the role of spreading depolarization in response to 100 mM K+. With 1 min temporal resolution, we find that it is possible to characterize the pharmacokinetics of the response to the local delivery of nomifensine. Overall, the findings reported here confirm the benefits arising from the ability to monitor dopamine via microdialysis at high sensitivity and at high temporal resolution.

Project description:Online monitoring of serotonin in striatal dialysate from freely moving rats was carried out for more than 16 h at 1 min 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 toward 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, 10-port valve. A six-port loop injector for introducing stimulating solutions (stimulus injector) was placed in-line between the syringe pump and microdialysis probe. We minimized solute dispersion by using capillary tubing (75 ?m inside diameter, 70 cm long) for the probe inlet and outlet. In vitro assessment of concentration dispersion during transport with a 30 s time resolution showed that the dispersion standard deviation for serotonin was well within the desired system temporal resolution. Each 30 or 60 s 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 7 min. The timing of injections from the stimulus injector and the cycle time for the HPLC monitoring of the flow stream were controlled. The electrochemical detector contained a 13 ?m 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 h 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 10 min, 120 mM K(+) stimulation, but not for other K(+) stimuli, exhibited a reproducible, oscillating profile of dialysate serotonin concentration versus 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 the study of serotonin neurochemistry associated with emotion and behavior.

Project description:Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35 kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15-50 cm long were packed with 1.7 µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35 kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200 mg/mL) while sonicating the column resulted in 6-34% increase in peak capacity for 50 cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50 cm long columns operated at 35 kpsi provided a 20-95% increase in chromatographic peak capacity compared with 15 cm columns operated at 15 kpsi. Analysis times up to 4 h were evaluated, generating peak capacities up to 410 ± 5 (n = 3, measured at 4σ) and identifying 480 ± 85 lipids (n = 2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.

Dataset Information

In Vivo Monitoring of Dopamine by Microdialysis with 1 min Temporal Resolution Using Online Capillary Liquid Chromatography with Electrochemical Detection.

Publications

In Vivo Monitoring of Dopamine by Microdialysis with 1 min Temporal Resolution Using Online Capillary Liquid Chromatography with Electrochemical Detection.

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