Monitoring Dopamine in Vivo by Microdialysis Sampling and On-Line

Sep 6, 2006 - Microdialysis sampling was coupled on-line to micellar electrokinetic chromatography (MEKC) to monitor extracellular dopamine concentrat...
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Monitoring Dopamine in Vivo by Microdialysis Sampling and On-Line CE-Laser-Induced Fluorescence Minshan Shou,† Carrie R. Ferrario,‡ Kristin N. Schultz,† Terry E. Robinson,§ and Robert T. Kennedy*†,§,|

Department of Chemistry, Neuroscience Program, Department of Psychology, and Department of Pharmacology, 930 North University Avenue, University of Michigan, Ann Arbor, Michigan 48109

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 µL/min and the dialysate mixed on-line with 6 mM naphthalene-2,3dicarboxaldehye and 10 mM potassium cyanide pumped at 0.12 µL/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-β-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. Dopamine is an important neurotransmitter in the central nervous system involved in many functions including reward* Corresponding author. Phone: 734-615-4363. E-mail: [email protected]. † Department of Chemistry. ‡ Neuroscience Program. 10.1021/ac0608218 CCC: $33.50 Published on Web 09/06/2006

© 2006 American Chemical Society

related behavior, movement, and mood.1-3 Alterations in neuronal release and uptake of dopamine have been implicated in psychiatric disorders4 and addiction.5 As a result of the biological significance of dopamine, it is important to be able to monitor its concentration in the brain on behaviorally relevant time scales. Fast-scan cyclic voltammetry (FSCV) can monitor dopamine concentration changes with subsecond temporal resolution,6 making it extremely powerful for short-term measurements; however, the electrodes are limited by their inability to determine basal concentrations and to measure changes that occur over times longer than ∼90 s.7 Microdialysis sampling coupled with high-performance liquid chromatography with electrical chemical detection (HPLC-EC) complements FSCV by allowing estimation of basal dopamine concentrations and monitoring over longer intervals (hours).8 Temporal resolution for dopamine monitoring by microdialysis with HPLC-EC is usually 10-20 min, which is too low for many behavioral experiments; however, sampling intervals of 1-2 min have been achieved, allowing better correlation of behavior and concentration changes.9,10 When such high temporal resolution §

Department of Psychology. Department of Pharmacology. (1) Roitman, M. F.; Stuber, G. D.; Phillips, P. E.; Wightman, R. M.; Carelli, R. M. J. Neurosci. 2004, 24, 1265-1271. (2) Bassareo, V.; Di Chiara, G. Neuroscience 1999, 89, 637-641. (3) Castro, S. L.; Zigmond, M. J. Brain Res. 2001, 901, 47-54. (4) Evans, A. H.; Lees, A. J. Curr. Opin. Neurol. 2004, 17, 393-398. (5) Hyman, S. E.; Malenka, R. C. Nat. Rev. Neurosci. 2001, 2, 695-703. (6) Phillips, P. E.; Stuber, G. D.; Heien, M. L.; Wightman, R. M.; Carelli, R. M. Nature 2003, 422, 573-574. (7) Heien, M. L.; Khan, A. S.; Ariansen, J. L.; Cheer, J. F.; Phillips, P. E.; Wassum, K. M.; Wightman, R. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10023-10028. (8) Plock, N.; Kloft, C. Eur. J. Pharm. Sci. 2005, 25, 1-24. (9) Jenkins, W. J.; Becker, J. B. Eur. J. Neurosci. 2003, 18, 1997-2001. (10) Bradberry, C. W.; Rubino S. R. Neuropsychopharmacology 2004, 29, 676685. |

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measurements are performed off-line, they require collection, storage, and analysis of microliter-volume samples. At high temporal resolution, this can become inconvenient (60 samples/h for 1-min temporal resolution). Microdialysis with on-line HPLCEC methods has been developed for dopamine, alleviating this problem; however, temporal resolution is at best 5 min because of the limits on separation speed.11,12 Furthermore, achieving 5 min or better temporal resolution often involves optimization for dopamine to the exclusion of monitoring other analytes, especially for on-line measurements where the separation must be performed rapidly. Capillary electrophoresis (CE) has proven to be a useful alternative to HPLC for coupling to microdialysis sampling.13,14 The high mass sensitivity and low sample volume requirements of CE allow frequent sampling and therefore improved temporal resolution.15-18 Indeed, temporal resolution of a few seconds has been achieved, allowing measurements to be made on a behaviorally relevant time scale.19-22 These properties also make CE compatible with lower microdialysis perfusion flow rates that increase recovery. The higher recovery increases dialysate concentrations, thus improving sensitivity, and allows for quantitative monitoring.23 Finally, CE is compatible with high-speed, highresolution separations making it feasible to perform on-line monitoring of multiple neurotransmitters with high temporal resolution.24,25 CE with both electrochemical15 and laser-induced fluorescence (LIF) detection26 has been coupled off-line to microdialysis for dopamine measurements. For LIF detection, dialysate is collected in fractions and derivatized, using an automated system, with naphthalene-2,3-dicarboxaldehye (NDA) to form fluorescent products. This method is extremely sensitive, with detection limits of 0.3 nM, and has been used to achieve temporal resolution of 10 s for dopamine.27 While this is a powerful method, it has some limitations. It requires on-column preconcentration and a 5-min (11) Feenstra, M. G.; Botterblom, M. H. Brain Res. 1996, 742, 17-24. (12) Chaurasia, C. S.; Chen, C. E.; Ashby, C. R., Jr. J. Pharm. Biomed. Anal. 1999, 19, 413-422. (13) Kennedy, R. T.; Watson, C. J.; Haskins, W. E.; Powell, D. H.; Strecker, R. E. Curr. Opin. Chem. Biol. 2002, 6, 659-665. (14) Davies, M. I.; Cooper, J. D.; Desmond, S. S.; Lunte, C. E.; Lunte, S. M. Adv. Drug Delivery Rev. 2000, 45, 169-188. (15) Qian, J.; Wu, Y.; Yang, H.; Michael, A. C. Anal. Chem. 1999, 71, 44864492. (16) Tucci, S.; Rada, P.; Sepulveda, M. J.; Hernandez, L. J. Chromatogr., B 1997, 694, 343-349. (17) Robert, F.; Bert, L.; Lambas-Senas, L.; Denoroy, L.; Renaud, B. J. Neurosci. Methods 1996, 70, 153-162. (18) Lada, M. W.; Vickroy, T. W.; Kennedy, R. T. Anal. Chem. 1997, 69, 45604565. (19) Venton, B. J.; Robinson, T. E.; Kennedy, R. T. J. Neurochem. 2006, 96, 236-246. (20) Silva, E.; Hernandez, L.; Quinonez, B.; Gonzalez, L. E.; Colasante, C. Neuroscience 2004, 124, 395-404. (21) Presti, M. F.; Watson, C. J.; Kennedy, R. T.; Yang, M.; Lewis, M. H. Pharmacol. Biochem. Behav. 2004, 77, 501-507. (22) Lena, I.; Parrot. S.; Deschaux, O.; Muffat-Joly, S.; Sauvinet, V.; Renaud, B.; Suaud-Chagny, M. F.; Gottesmann, C. J. Neurosci. Res. 2005, 81, 891899. (23) Lada, M. W.; Kennedy, R. T. Anal. Chem. 1996, 68, 2790-2797. (24) Bowser, M. T.; Kennedy, R. T. Electrophoresis 2001, 22, 3668-3676. (25) Zhou, S. Y.; Zuo, H.; Stobaugh, J. F.; Lunte, C. E.; Lunte, S. M. Anal. Chem. 1995, 67, 594-599. (26) Robert, F.; Bert, L.; Denoroy, L.; Renaud, B. Anal. Chem. 1995, 67, 18381844. (27) Bert, L.; Parrot, S.; Robert. F.; Desvignes, C.; Denoroy, L.; Suaud-Chagny, M. F.; Renaud, B. Neuropharmacology 2002, 43, 825-835.

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separation, making it inconvenient for sustained high temporal resolution monitoring. For a 1-h experiment, at least 30 h of analysis time would be required if samples were collected at 10-s intervals. A second limitation is that the separation conditions are such that only catecholamines can be detected. This limitation can be circumvented by analyzing the same sample with different separation conditions. Such an approach, which takes advantage of the small sample requirement of CE, has allowed amino acids and catecholamines to be monitored in one microdialysis experiment.28 In the present study, we report a method that uses microdialysis coupled on-line with CE-LIF for monitoring dopamine. Like previous uses of CE for monitoring dopamine, this method takes advantage of the high sensitivity of CE to achieve good sampling frequency (as fast as 60 s). Unlike previous uses of CE for dopamine in microdialysis, this method is on-line allowing the convenience of automated operation and the ability to monitor dopamine during an in vivo experiment. The system is compatible with low flow rates that enhance recovery and improve sensitivity for dopamine. Furthermore, the separation conditions are such that several other amino acids are also resolved, allowing multianalyte detection. The method is demonstrated to be useful for correlating drug-induced behavior and dopamine changes in vivo. EXPERIMENTAL SECTION Materials and Solutions. All chemicals from commercial sources were used as received without further purification. Amino acid standards, dopamine hydrochloride, norepinephrine hydrochloride, nomifensine maleate, R-methyl-p-tyrosine (AMPT), sodium tetraborate, sodium dodecyl sulfate (SDS), (2-hydroxypropyl)β-cyclodextrin (HP-β-CD), and EDTA were from Sigma-Aldrich (St. Louis, MO). KCN was from Fisher Scientific (Chicago, IL). NDA was from Molecular Probes (Eugene, OR). Cocaine was from the National Institute on Drug Abuse (NIDA, Bethesda, MD). Fused-silica capillaries were from Polymicro Technology (Phoenix, AZ). Microdialysis probes with a 4-mm sampling length were made in-house using cellulose hollow fiber (18 000 MW cutoff, Spectrum Lab Inc., Rancho Dominguez, CA) as described previously.24 Artificial cerebral spinal fluid (aCSF) consisted of 145 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.2 mM CaCl2, and 0.5 mM NaH2PO4, adjusted to pH 7.4. Unless stated otherwise, all analytes were dissolved in aCSF. NDA was dissolved in a 50:50 mixture (v/v) of acetonitrile and 80 mM borate at pH 9.2 at the concentrations specified. KCN and 500 mM EDTA was dissolved in 80 mM borate at pH 9.2. Fluorescence Intensity Measurement. To determine the effect of pH on fluorescence of the NDA-dopamine derivative, 1 µM dopamine solution was mixed with 6 mM NDA and 10 mM KCN at 10:1:1 ratio (v/v/v) in a sample vial. Derivatization was allowed to proceed under the ambient temperature for 5 min. The derivative was diluted 10-fold by 30 mM sodium phosphate buffer at different pHs. The emission spectrum from 460 to 560 nm was recorded with 442-nm excitation on a FluoroMax-2 fluorometer (Instrument S. A. Inc., Edison, NJ). (28) Parrot, S.; Sauvinet, V.; Riban, V.; Depaulis, A.; Renaud, B.; Denoroy, L. J. Neurosci. Methods 2004, 140, 29-38.

On-Line Reaction and Separation. The on-line system is similar to that described previously.29 Unless stated otherwise, the dialysis probe was perfused at 0.3 µL/min with a CMA/102 microdialysis syringe pump (CMA, Boston, MA). The outlet from the probe, which was a 100-µm-outer diameter (o.d.) by 40-µminner diameter (i.d.) by 7-cm-long fused-silica capillary, was connected to one arm of a 0.25-mm-bore PEEK microvolume cross (Valco Inc. Houston, TX). The NDA and KCN solutions were perfused into two arms of the cross at 0.12 µL/min each. The reaction mixture flowed out of the fourth arm of the cross into a 100-µm-i.d. by 360-µm-o.d. reaction capillary. The length of the reaction capillary was 32 cm and allowed for 230-250-s reaction time (determined to be the minimal time for complete reaction) at the flow rates used. The outlet of the reaction capillary was coupled on-line to the electrophoresis capillary using a flow-gated interface.30 The electrophoresis capillary was 10-µm i.d. by 360-µm o.d. with a total length of 16 cm and an inlet to detection window length of 14.5 cm. A 850 V/cm electrical field was applied from a 1000R CZE power supply (Spellman, Plainview, NY). The separation buffer consisted of 30 mM NaH2PO4, 6.5 mM SDS, and 2 mM HP-β-CD adjusted to pH 7.45 by 1 M NaOH. Capillaries were rinsed for 2 min with 0.1 M NaOH and 10 min with separation buffer once per day prior to experiments. Injections were performed at 2 kV for 200 ms. Data were collected and analyzed using software written in-house.31 The LIF detector was an Axioskop 20 fluorescence microscope (Carl Zeiss, Hanover, MD). The 413-nm line of a 14-mW CL-2000 diode-pumped crystal laser (CrystaLaser, Reno, NV) was used as the excitation source. A 20% neutral density filter and a 415 ( 10 nm excitation filter (Chroma, Rockingham, VT) were placed in the excitation beam path to attenuate background noise. The laser beam was reflected by a 480-nm dichroic mirror through the objective and focused onto the capillary through a 40×, 1.30 numerical aperture Fluar oil immersion lens (Carl Zeiss, Hanover, MD). The emitted fluorescence was collected by the objective, passed through the dichroic mirror and filtered with a 490 ( 10 nm emission filter. The light was spatially filtered using the diaphragm on the photometer. Data were collected at 500 Hz and low-pass filtered at 100 Hz using an RC filter on the photometer system (DCP-5, CRG Electronics, Houston, TX). Animal Surgery and Sampling. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 200-250 g were anesthetized with intraperitoneal (i.p.) injections of ketamine (65 mg/kg) and domitor (0.5 mg/kg). The surgery was performed on a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). The microdialysis probe was implanted anterior +1.0 mm, lateral +2.6 mm, and ventral -8.0 mm from the Bregma into the striatum. The probe was equilibrated by perfusing aCSF for 2 h to allow neurotransmitter concentrations to equilibrate. Basal electropherogams were collected for 45-60 min to ensure neurotransmitter levels were stable. After obtaining stable baseline values, the perfusion solution was switched to nomifensine, AMPT, or cocaine (all dissolved in aCSF) while continuously recording electropherograms to test the (29) Shou, M.; Smith, A. D.; Shackman, J. G.; Peris, J.; Kennedy, R. T. J. Neurosci. Methods 2004, 138, 189-197. (30) Hooker, T. F.; Jorgenson, J. W. Anal. Chem. 1997, 69, 4134-4142. (31) Shackman, J. G.; Watson, C. J.; Kennedy, R. T. J. Chromatogr., A 2004, 1040, 273-282.

effect of local application of these substances on dopamine in the dialysate. An unpaired t-test was applied to analyze whether the drug caused a significant change of dopamine basal level. Freely Moving Animal Experiments. In vivo experiment protocol was approved by University of Michigan Unit for Laboratory Animal Medicine. Rats were individually housed (14: 10 light/dark cycle) with food and water continually available. After acclimation to the animal colony, rats were anesthetized using a mixture of ketamine and xylazine (100 and 10 mg/kg administered i.p.). A catheter for administering drug dosage intravenously (i.v.) was implanted in the right jugular vein using procedures described previously.32 A guide cannula (Plastics One, Roanoke, VA) was implanted in the brain at anterior +1.0 mm, lateral +2.0 mm, and ventral -4.0 mm from Bregma. Rats were allowed 4-6 days to recover before the experiment began. During this time, catheters were flushed with 0.1 mL of gentamicin (50 mg/kg in 0.9% sterile saline solution) daily. In the evening prior to an in vivo measurement session, the rat was placed in a Raturn testing bowl (BAS, Lafayette, IN) at 6-7 p.m. and tethered to the counterbalanced arm equipped with a commutator. The testing bowl automatically rotated to the opposite direction of the animal movement when the commutator turned 90°. A microdialysis probe with 4-mm effective length was implanted into the striatum through the guide cannula and perfused with aCSF overnight at 0.1 µL/min. After overnight perfusion, the flow rate was increased to 0.3 µL/min. The dialysate from probe outlet was delivered to the reaction cross via a 60cm-long, 75-µm-i.d., 360-µm-o.d. capillary, and electropherograms were monitored until peak heights stabilized. To test the effect of cocaine injection, electropherograms were continuously collected 15 min for basal level, 20 min following saline infusion, and 6070 min following i.v. infusion of a bolus 2 mg/kg cocaine. The i.v. administration of cocaine was as previously described.33 A video camera was mounted above the Raturn to record animal behavior during the experiment. Behavioral Measurements. The behavioral response to cocaine was assessed off-line by viewing the video of the test session. Behavior was rated once every 90 s using an eight-point scale,34 in which a score of 1 ) asleep, 2 ) inactive, 3 ) normal in place activity, 4 ) normal, alert, active, 5 ) hyperactive, 6 ) slow patterned stereotypy, 7 ) fast patterned stereotypy, and 8 ) restricted (focused) stereotypy. RESULTS AND DISCUSSION On-Line Reaction. Microdialysis with on-line NDA derivatization and CE-LIF analysis has been successfully used previously for in vivo amino acid monitoring.25,29 To maximize sensitivity for dopamine, which was present at much lower concentrations than the amino acids, it was necessary to use lower microdialysis sampling flow rates to enhance the recovery and lower reagent flow rates to avoid excessive dilution of the dialysate. When flow rates were reduced to 0.3 µL/min sampling and 0.24 µL/min reagent flow from 1.0 µL/min sampling and 2.0 µL/min reagent flow rates, we frequently observed precipitate formation in the flow-gated interface, which led to irreproducible flow rates and (32) Weeks J. R. Methods in Psychobiology; Academic Press: London, 1972; Vol. 2, pp 155-168. (33) Samaha A. N.; Li Y.; Robinson T. E. J. Neurosci. 2002, 22, 3244-3250. (34) Ellinwood, E. H., Jr.; Balster, R. L. Eur J. Pharmacol. 1974, 28, 35-41.

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Figure 2. Effect of pH on fluorescence intensity of NDA derivative of dopamine. A 1 µM dopamine standard was derivatized in a sample vial at room temperature, and the derivative was further diluted 10fold by phosphate buffer at different pHs. Fluorescence intensity was monitored by a fluorometer with excitation at 442 nm. The emission spectra were scanned from 460 to 560 nm.

Figure 1. Electropherograms demonstrating the detection limit of dopamine sampled by microdialysis and analyzed by CE-LIF. Traces show the peak for dopamine when sampling 10 and 2 nM dopamine (detection limit) and blank solution. Dopamine was sampled by a 4-mm-length microdialysis probe (at 37 °C) and derivatized on-line by mixing with NDA and cyanide. The flow rate was 0.3 µL/min for aCSF.

injections. We hypothesized that the precipitates were MgHPO4 and CaHPO4 that formed upon mixing the basic reagent solutions, aCSF (containing divalent cations), and electrophoresis buffer containing phosphate ions. Supporting this hypothesis, we found that addition of 50 mM EDTA (mixture of 500 mM EDTA and 10 mM KCN at a 1: 9 volume ratio), which chelates the Mg2+ and Ca2+, to the CN- reagent solution prevented clogs from forming even at the lowest flow rates tested. Under these conditions, it was possible to use even lower flow rates; however, with the syringe pumps used in these experiments, lower flow rates resulted in pulsations in flow that caused unacceptable peak height variations. The detection limit for the NDA derivative of dopamine was ∼0.6 nM based on the concentration required to obtain a signalto-noise ratio of 3 in electropherograms, with signal being peak height and noise the root-mean-square of the noise in the trace. Because some dilution is necessary to add reagents (1.8-fold in this case), the detection limit for dopamine in a sample stream was ∼1 nM. This detection limit is ∼5-fold higher that HPLC-EC; however, the low volume requirement of the CE makes this method compatible with low dialysis flow rate, which increases recovery and helps offset this difference. More importantly, the LOD is sufficient to monitor dopamine in many brain regions. Using dialysis probes with 4-mm length, at 0.3 µL/min in a stirred solution at 37 °C the recovery was 68 ( 16% (mean ( SD, n ) 19). As a result, the detection limit for dopamine in a sampled solution was ∼2 nM (Figure 1). (Although this experiment allows an estimate the LOD from sampled solutions, it cannot be extrapolated to an in vivo detection limit because in vivo recovery may differ from in vitro recovery.)35 Amino acids were not the focus of this study; however, we also evaluated the detection limits 6720 Analytical Chemistry, Vol. 78, No. 19, October 1, 2006

for serine, taurine, glycine, glutamate, and aspartate. We found that they were 5-15 nM (sampled) depending on the amino acid, which is 3-6-fold better than previously reported for on-line measurements with NDA.24,25,29 The improvements over previous results are attributed to the use of lower flow rates, for enhanced recovery, and less dilution of sample. The relative standard deviation (RSD) in peak area of multiple injections (15-20 analyses of 40 nM dopamine in aCSF, with or without probe) was 4-7%. The linearity of dopamine signal was tested between from 5 to 100 nM, which yielded a correlation coefficient of 0.993. Separation Conditions. Micellar electrokinetic chromatography with SDS has previously been used to resolve 17 NDA amino acid derivatives in dialysate in 30 s,29 making it an attractive starting point for the development of a rapid dopamine assay. This prior work used 30 mM SDS in 15 mM phosphate buffer at pH 8.0 as the separation medium and an electric field of 1.3 kV/cm applied across a 10-cm-long by 10-µm-i.d. separation capillary. In preliminary experiments for the analysis of dopamine, these conditions were found to be unacceptable because the NDA derivative of dopamine had a significant drop in fluorescence above pH 7.5 (Figure 2) and it coeluted with fluorescent interfering peaks found in the blank. (The identity of these peaks was not determined.) To improve sensitivity and resolution of dopamine, the pH was lowered to 7.4 and the capillary lengthened to 12 cm. To further improve resolution, the SDS concentration was varied between 3 and 30 mM while monitoring the separation of dopamine from a fluorescent byproduct (unidentified) and norepinephrine (a closely related compound that is a potential interfering substance). As shown in Figure 3, resolution reached a maximum when the SDS concentration was just below the nominal critical micellar concentration (8 mM for SDS), suggesting that the derivatives were strongly partitioned into the micellar phase. Further, in vitro tests with other amino acids showed that dopamine was also resolved from the common amino acids under these conditions. Although dopamine was resolved from all tested compounds in vitro at this SDS concentration, its peak overlapped with other compounds present in dialysate collected from live animals (Figure (35) Cano-Cebrian, M. J.; Zornoza, T.; Polache, A.; Granero, L. Curr. Drug Metab. 2005, 6, 83-90.

Figure 3. Effect of SDS concentration on the resolution (Rs) between dopamine and noradrenaline (DA/NA) and DA and an unknown (DA/UN) background peak in in vitro tests.

Figure 5. Pharmacologic verification of the dopamine signal. (a) Comparison of electropherograms collected before (solid trace) and 5 min after (dashed trace) 5 µM nomifensine was added to the dialysis perfusion solution (trace representative of 6 experiments). (b) Comparison of electropherograms collected before (solid trace) and 70 min after 300 µM AMPT was added to the dialysis perfusion solution (trace representative of 5 experiments). For both experiments, the dopamine migration time was identified by standards. Traces are offset for clarity.

Figure 4. Effect of HP-β-CD on the resolution of dopamine (DA) from in vivo samples. Electropherograms of dialysate were collected in vivo using 30 mM phosphate, 6.5 mM SDS, and 0 (a) or 2 (b) mM HP-β-CD at pH 7.45 as the separation buffer. After collecting basal electropherograms (solid trace), dopamine was added to the dialysis perfusion solution (dashed trace) to verify the migration time of dopamine.

4a). HP-β-CD, a commonly used additive36 in CE, was utilized to shift the unidentified interfering species away from dopamine. (The NDA dopamine derivative is nearly neutral so its migration behavior is minimally affected by neutral HP-β-CD). At 2 mM HP(36) Ueda, T.; Mitchell, R.; Kitamura, F.; Metcalf, T. J. Chromatogr. 1992, 593, 265-274.

β-CD, dopamine appeared to be nearly baseline resolved from adjacent peaks (Figure 4b), but at higher and lower HP-β-CD concentrations (1 and 3 mM), dopamine was only partially resolved from the other compounds (data not shown). Although dopamine was resolved under these conditions, dayto-day and capillary-to-capillary variation of EOF prevented reliable dopamine resolution. More consistent resolution was obtained by using a longer separation capillary (16 cm), a higher phosphate buffer concentration (30 mM), and a lower applied voltage (13.5 kV). With these conditions, the migration time for dopamine was 81.6 ( 3.3 s (mean ( SD, n ) 26, different days and capillaries) and within 1 day had an RSD of 0.06-0.11% (110-170 injections). Peak efficiencies were between 200 000 and 250 000 theoretical plates calculated using peak width at half-height. Electropherograms were typically acquired at 90-s intervals; although 60 s was possible by overlapping injections. In Vivo Characterization of Dopamine. The basal concentration of dopamine in dialysate samples from anesthetized rats, measured 2 h after probe implantation with a perfusion flow rate of 0.3 µL/min, was 18 ( 3 nM (calculated from peak area, mean ( SEM, n ) 12). This concentration is close to a reported value37 (37) Sauvinet, V.; Parrot, S.; Benturquia, N.; Bravo-Moraton, E.; Renaud, B.; Denoroy, L. Electrophoresis 2003, 24, 3187-3196.

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Figure 6. Temporal resolution of the system. Electropherograms were collected at 11-s intervals while probes were perfused at 0.3 µL/min. The probe was switched from 100 to 200 nM dopamine at the first arrow and back to 100 nM dopamine at the second arrow. Arrow positions have been corrected for the dead time of the system, which was 420 s.

and agrees with previous measurements of dopamine dialysate concentrations, which are between 5 and 40 nM depending on the microdialysis flow rate, probe length, brain region, and state of consciousness.38-40 Quantitative estimates of brain extracellular concentrations by no-net-flux microdialysis are typically 5-20 nM in this brain region.41,42 The similarity to that measured here may be attributed to the high recovery with low flow rates. Because of the complexity of the electropherogram, pharmacological characterization was performed to ensure that the dopamine peak was properly identified and that dopamine was not coeluting with other substances. Dopamine uptake inhibitors, known to increase dopamine concentrations in extracellular space, were perfused into the striatum through the dialysis probe to test the effect on the putative dopamine peak. Infusion of 5 µM nomifensine, a dose known to cause a substantial increase in dopamine,27 caused the peak to increase rapidly (Figure 5a) to 644 ( 57% of basal (mean ( SEM, t ) 16.6, p < 0.01, n ) 6 rats) within 8 min. The magnitude of the dopamine increase is comparable to27 or slightly higher43,44 than previously reported values for comparable doses. (Larger increases in dopamine with uptake inhibition are not surprising in view of the low dialysis flow rates used here. Previous studies have indicated the dialysis probe recovery can decrease with uptake inhibitors, but this effect is less significant at low flow rates, like those used here.45). The perfusion of cocaine, another dopamine uptake blocker, at 20 µM, a concentration shown to cause a significant effect,45 also gener(38) Pettit, H. O.; Justice, J. B. Pharmacol. Biochem. Behav. 1989, 34, 899904. (39) Westerink, B. H.; De Vries, J. B. J. Neurochem. 1988, 51, 683-687. (40) Hamilton, M. E.; Mele, A.; Pert, A. Brain Res. 1992, 597, 1-7. (41) Parrot, S.; Bert, L.; Mouly-Badina, L.; Sauvinet, V.; Colussi-Mas, J.; LambasSenas, L.; Robert, F.; Bouilloux, J. P.; Suaud-Chagny, M, F.; Denoroy, L.; Renaud, B. Cell. Mol. Neurobiol. 2003, 23, 793-804. (42) Sam, P. M.; Justice, J. B., Jr. Anal. Chem. 1996, 68, 724-728. (43) Mazei, M. S.; Pluto, C. P.; Kirkbride, B.; Pehek, E. A. Brain Res. 2002, 936, 58-67. (44) Del Arco, A.; Mora, F. Brain Res. Bull. 2002, 57, 623-630. (45) Smith, A. D.; Justice, J. B. J. Neurosci. Methods 1994, 54, 75-82.

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Figure 7. Correlation of dopamine (DA) concentration, behavior, and expected cocaine level in awake animals. Dopamine in dialysate (a) and psychomotor behavior rating (b) changes after intravenous administration of 2 mg/kg cocaine or saline at time 0.0 s. Dopamine is expressed as the percent of basal recorded for 5 min before the infusion of cocaine. Error bars are ( SEM (n ) 3 rats total). (c) Predicted cocaine concentration following injection calculated using pharmacokinetic parameters described previously.48 (d) Overlay of all three parameters normalized so their maximal value during the experiment is 1.0 and minimal value during the experiment is 0.0 to highlight temporal correlation.

ated a rapid rise of dopamine (420 ( 48%, t ) 10.3, p < 0.01, mean ( SEM, n ) 3 rats, data not shown) as expected. We next evaluated the effect of AMPT, a tyrosine hydroxylase inhibitor that blocks catecholamine synthesis46,47 and depletes dopamine in dialysate samples when perfused through the probe at high micromolar concentrations.45 After a 1-h perfusion with 300 µM AMPT, the dopamine peak was decreased by 84 ( 5% (mean ( SEM, t ) 60.7, p < 0.01, n ) 5 rats) as shown in Figure 5b. These results support the conclusion that the peak detected is primarily due to dopamine. Temporal Resolution. The temporal resolution of the system was measured by rapidly moving the probe between two vials containing stirred solutions of dopamine (100 and 200 nMm respectively) at 37 °C while recording electropherograms. Electropherograms were acquired at 11-s intervals (10-s separation time plus injection and voltage ramping time, which was 1.2 s) to better monitor the concentration changes. This acquisition rate was possible because only dopamine was being measured and overlapping injections could be used. Using these conditions, we found that approximately the concentration change (up to 90% of the maximal change) required four electropherograms corresponding to ∼45 s at 0.3 µL/min (see Figure 6). From the results, we conclude that the temporal resolution for monitoring will be the electropherogram collection interval of 60-90 s. Dopamine Response to Intravenous Cocaine Infusion. As a demonstration of the utility of this method for behavioral studies, we monitored the in vivo response of dopamine to an i.v. infusion of saline or 2 mg/kg cocaine (n ) 3) while monitoring the psychomotor responses in freely moving rats. As shown in Figure 7, infusion of cocaine evoked increases in dopamine (Figure 7a) (46) Ayala, C. A.; Jaffe, E. H. Neuropharmacology 1993, 32, 1401-1409. (47) Sauve, Y.; Reader, T. A. Neurochem. Res. 1988, 13, 807-815.

Figure 8. Sample electropherogram collected from the striatum of an anesthetized rat illustrating peak capacity of the method. (a) Full scale plot with amino acids identified by migration time matching marked. (b) Expanded scale for electropherogram collected from control samples (derivatization reagents sampling aCSF in vitro, lower trace) and in vivo samples (upper trace) at 10-fold higher gain illustrating smaller peaks detected. Fluorescence units are equivalent in the plots. Peaks that were consistently observed in vivo and not observed in control samples were counted. Numbers for off-scale peaks are not shown. Peak 55 is dopamine.

and psychomotor behavior rating (Figure 7b) that coincided with each other, increased at the same rate, peaked at the same time, and had similar decays (see overlay plot in Figure 7d). Infusions of saline had no effect on the dopamine response or psychomotor ratings (Figure 7). This result is not surprising given that cocaine is thought to exert its psychomotor activating effects primarily through actions on dopamine neurotransmission in the brain. The one discrepancy in this plot is a decrease in psychomotor rating while the dopamine levels remain elevated beyond 8 min. This effect was not explored but could be due to dopamine receptor desensitization. Using a pharmacokinetic model of cocaine distribution48 (Figure 7c), we also compared changes of both behavioral response and dopamine concentrations in dialysate to that of cocaine concentration expected in the brain (Figure 7d). Again, the time to peak and decays are quite similar within the temporal resolution of the measurements. Given that increased efflux of dopamine is thought to be a direct effect of cocaine, this result is not unexpected although observing such correlations is difficult at lower temporal resolution. The excellent temporal correspondence between dialysate dopamine, behavior, and expected cocaine concentration in brain indicates that this method can be used to correlate changes in drug-induced behavior with in vivo neurochemistry. This conclusion is in agreement with previous studies that have shown that microdialysis with high sampling frequency and CE analysis can record dynamic changes in amino acids or catecholamines that

correlate to behavior.20-22 This conclusion is in contrast to that reached in a previous study that suggested that processes inherent to microdialysis sampling, such as diffusion through tissue and across the probe membrane, would prevent a good correlation between dynamic release of dopamine and behavior.49 This prior study used microdialysis sampling at 20-min intervals and behavioral rating at 10-min intervals following i.p. injection of the dopamine uptake inhibitor GBR 12909. It was found that the dopamine measurements lagged the behavioral changes by 30 min. (In contrast, an assay of dopamine uptake using microelectrodes taken at 10-min intervals had no lag.) In our study though, which should be a much more demanding test of dynamic responsiveness because of the rapid onset of neurochemical and behavioral responses to i.v. infusion of cocaine (3 min to peak in this case), we found no discrepancy between the onset of the behavioral response and the increase in extracellular dopamine levels. Furthermore, the timing of the change in behavior and dopamine were both in agreement with the predicted changes in brain concentration of cocaine. It seems likely then that collection of 20-min fractions in the GBR 12909 study prevented good correlation, rather than an effect inherent to microdialysis. Indeed, in a previous study,19 we demonstrated that sampling frequency can have dramatic effects on the ability to correlate amino acid changes to behavior. Multianalyte Resolution. The peak capacity of the separation, determined by dividing the separation window of 60 s by the

(48) Pan, H. T.; Menacherry, S.; Justice, J. B., Jr. J. Neurochem. 1991, 56, 12991306.

(49) Budygin, E. A.; Kilpatrick, M. R.; Gainetdinov, R. R.; Wightman, R. M. Neurosci. Lett. 2000, 281, 9-12.

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Figure 10. Overlaid electropherograms of dialysate from before (solid trace) and cocaine during administration (dashed trace). The 20 µM cocaine was perfused into the striatum of an anesthetized rat via reverse dialysis at 0.3 µL/min. Two unidentified peaks (indicated by arrows) appeared simultaneously with the dopamine (DA) increase. Trace was taken 180 s after the initial dopamine increase. Traces are representative of data from 3 animals.

Figure 9. Effect of PDC perfusion on in vivo dialysate concentration of dopamine (DA), glutamate (Glu), and aspartate (Asp). Plots show the concentration of dopamine and amino acids in dialysate as a percentage of basal level (taken before the application of drug) as PDC at 2 (solid circle) and 10 mM (open circle) was perfused into the striatum of anesthetized rats via reverse dialysis at 0.3 µL/min. The arrow indicates the time point where the drug reached the brain. Data points are mean ( SEM (n ) 4). For 10 mM PDC, the maximal signals were for glutamate and aspartate were off-scale so the data are indicative of the minimal changes that occurred.

average peak width of 0.8 s at 4σ, was 74, suggesting the potential to monitor many other compounds besides dopamine. Indeed, as shown in Figure 7b, 62 peaks that are not associated with reagent are detected in dialysate samples. Matching migration times and using standard addition methods revealed that several of the peaks correspond to known amino acids (Figure 8a) including the neurotransmitters glycine, taurine, glutamate, and aspartate as well as citrulline, histidine, serine, and glutamine. (GABA is another neuroactive amino acid, but it was not resolved.) It will be of interest to confirm the identity of these compounds and other species that are detected because, in principle, they could be 6724 Analytical Chemistry, Vol. 78, No. 19, October 1, 2006

monitored simultaneously with dopamine providing a route to studying the interaction of these neurotransmitter and metabolite systems. Preliminary experiments were performed to demonstrate the potential utility of this system for multianalyte measurements. In one test, we monitored dopamine, glutamate, and aspartate while infusing PDC through the dialysis probe. PDC is a glutamate uptake inhibitor that is expected to increase excitatory amino acid efflux in the brain. Such changes are recorded at 2 and 10 mM PDC as shown in Figure 9; however, when the inhibitor is increased to 10 mM, a substantial increase in dopamine is also recorded. This increase in dopamine suggests a positive interaction between excitatory amino acids and dopaminergic neurons when the concentration of glutamate and aspartate are sufficiently elevated.27,44,50 Thus, in this example, the interaction of neurotransmitters can be monitored. These data also illustrate that an important issue to be resolved, however, is the dynamic range of the measurement. In this example, the large change in glutamate resulted in its signal being off-scale at the settings need to detect dopamine. Even without identification of the peaks, the pattern of the amine metabolome that is detected by this method may provide useful information on effects of drugs or other experimental manipulations. An example is illustrated in Figure 10, which compares electropherograms of dialysate before and after 20 µM cocaine administration by reverse dialysis. In addition to the expected increase in dopamine, two other peaks (one at 76.5 s and one at 87.5 s) showed consistent and substantial increases in signal. The earlier eluting peak had a migration time similar to that of norepinepherine, which may also be released by cocaine; however, its identity was not confirmed because the basal concentration was too low to detect. The migration time of the later eluting peak does not match any of the 20 common amino acids and is not due to cocaine or impurities in the drug. Further investigation of these signals may reveal previously unknown effects of cocaine. (50) Del Arco, A.; Mora, F. J. Neural. Transm. 2005, 112, 97-109.

CONCLUSION We have demonstrated a microdialysis method with an automated on-line reaction/separation scheme capable of monitoring dopamine in brains of freely moving animals at 60-90-s intervals. This temporal resolution, previously achieved by offline CE methods,15,26 fills an important niche between the millisecond measurements possible with carbon fiber microelectrodes and the longer term measurements possible with microdialysis and HPLC. The utility of the temporal resolution is revealed in the correlation to drug-induced behaviors made possible by this method. The use of on-line measurements makes the method convenient relative to previous off-line microdialysis methods that

achieved similar temporal resolution. Furthermore, the high peak capacity suggests the potential to simultaneously monitor other transmitters and metabolites. ACKNOWLEDGMENT This research is supported by NIH (NS38476).

Received for review May 3, 2006. Accepted August 22, 2006. AC0608218

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