Viral vector-mediated overexpression of estrogen receptor-alpha in striatum enhances the estradiol-induced motor activity in female rats and estradiol-modulated GABA release.

Classical estrogen receptor-signaling mechanisms involve estradiol binding to intracellular nuclear receptors [estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta)] to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K(+)-evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERalpha located on the membrane of medium spiny GABAergic neurons. This experiment examined whether overexpression of ERalpha in the striatum would enhance the effect of estradiol on rotational behavior and the K(+)-evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERalpha cDNA (AAV.ERalpha) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERalpha in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared with controls and exhibited behavioral sensitization of contralateral rotations induced by a low-dose of amphetamine. ERalpha overexpression also enhanced the inhibitory effect of estradiol on K(+)-evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior.

Time-resolved microdialysis for in vivo neurochemical measurements and other applications.

Monitoring changes in chemical concentrations over time in complex environments is typically performed using sensors and spectroscopic techniques. Another approach is to couple sampling methods, such as microdialysis, with chromatographic, electrophoretic, or enzymatic assays. Recent advances of such coupling have enabled improvements in temporal resolution, multianalyte capability, and automation. In a sampling and analysis method, the temporal resolution is set by the mass sensitivity of the analytical method, analysis time, and zone dispersion during sampling. Coupling methods with high speed and mass sensitivity to microdialysis sampling help to reduce some of these contributions to yield methods with temporal resolution of seconds. These advances have been primarily used in monitoring neurotransmitters in vivo. This review covers the problems associated with chemical monitoring in the brain, recent advances in using microdialysis for time-resolved in vivo measurements, sample applications, and other potential applications of the technology such as determining reaction kinetics and process monitoring.

Monitoring dopamine in vivo by microdialysis sampling and on-line CE-laser-induced fluorescence.

Microdialysis sampling was coupled on-line to micellar electrokinetic chromatography (MEKC) to monitor extracellular dopamine concentration in the brains of rats. Microdialysis probes were perfused at 0.3 microL/min and the dialysate mixed on-line with 6 mM naphthalene-2,3-dicarboxaldehye and 10 mM potassium cyanide pumped at 0.12 microL/min each into a reaction capillary. The reaction mixture was delivered into a flow-gated interface and separated at 90-s intervals. The MEKC separation buffer consisted of 30 mM phosphate, 6.5 mM SDS, and 2 mM HP-beta-CD at pH 7.4, and the electric field was 850 V/cm applied across a 14-cm separation distance. Analytes were detected by laser-induced fluorescence excited using the 413-nm line of a 14-mW diode-pumped laser. The detection limit for dopamine was 2 nM when sampling by dialysis. The basal dopamine concentration in dialysates collected from the striatum of anesthetized rats was 18 +/- 3 nM (n = 12). The identity of the putative dopamine peak was confirmed by showing that dopamine uptake inhibitors increased the peak and dopamine synthesis inhibitors eliminated the peak. The utility of this method for behavioral studies was demonstrated by correlating dopamine concentrations in vivo and with psychomotor behavior in freely moving rats following the intravenous administration of cocaine. Over 60 additional peaks were detected in the electropherograms, suggesting the potential for monitoring many other substances in vivo by this method.

Systematic error in automated in-tube solid-phase microextraction.

The widely employed configuration for automated in-tube solid-phase microextraction (SPME) involves modification of a commercial liquid chromatographic autosampler into an automated extraction device. This popular configuration is demonstrated to result in an inherent systematic error in the quantitation of analyte in a given matrix. The source of error is traced to the accumulation of analyte in the extraction and the pre-extraction segment (i.e., sample loop, metering valve and tubing prior to the metering valve) of the autosampler where the analyte comes in contact with the residual mobile phase. This results in cross-contamination due to sample/mobile phase mixing. The quantity of analyte accumulated in these segments is shown to consistently increase with the increasing number of draw/eject cycles. As a result of the accumulation, the amount of analyte recorded leads to inaccurate quantitative information, leading to overestimation of the limit of detection and limit of quantitation, when automated in-tube SPME is employed as an approach for sample enrichment. Insertion of a 100-microl air plug prior to extraction step was able to significantly minimize sample/mobile phase mixing of analyte with the residual mobile phase in the pre-extraction and extraction step, thus minimizing the systematic error.