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increasing fraction off carbonic acid is ionized as the solution becomes more dilute ("6.8% in 0.1 mM carbonic acid, for example). A second point is that the high concentration of K+ on the exchange column ("4.2 M) pushes the ion-exchange equilbrium to the right. For 0.1 mM carbonic acid, it can be calculated that only a few theoretical plates would be needed for complete conversion of H&03 to K+ and HC03-. Strong acids will also undergo ion exchange in the enhancement columns. Since the eluent is simply water, the enhancement columns will last a long time before regeneration or replacement is necessary unless the samples analyzed have a very large amount of strong acids or their salts. It is possible that ion-exchange membrane reactors, which can be continuously regenerated, could be used in place of the enhancement columns. The results of our work suggest that it is also possible to determine weak bases such as NH3 and (the ions of such bases) by ion-exclusion chromatography and conductivity detection by using the enhancement technique. This will be the subject of another report.
ACKNOWLEDGMENT We wish to thank A. Miyanaga and M. Unno of TSK Mfg. Co., Ltd., for the gift of the plastic column used in this research.
LITERATURE CITED (1) Symanski. J. S.; Bruckenstein, S. Anal. Chem. 1986, 58, 1766. (2) Syrnanski, J. S.; Bruckenstein, S.Anal. Chem. 1988, 58, 1771. (3) Tanaka, K; Ishizuka, T.; Sunahara, H. J. Chromafogr. 1979, 774, 153. (4) Turkelson, V. T.; Richards, M. Anal. Chem. 1978, 50, 1420.
(5) (6) (7) (8)
Application Note 25; Dionex Corp.: Sunnyvale, CA. Sept. 1980 Tanaka, K.; Fritz, J. S. J. Chromafog. 1986, 367, 151. Tanaka, K. Bunseki Kagaku 1981, 30, 358. Tanaka, K.; Ishizuka, T. Wafer Res. 1982, 76. 719.
RECEIVED for review August 19, 1986. Accepted November 17, 1986. This research was supported by the International Supporting Research and Development Program at Los Alamos National Laboratory, which is operated by the University of California.
Rapid Sampling and Determination of Extracellular Dopamine in Vivo William H. Church and Joseph B. Justice, Jr.*
Department of Chemistry, Emory University, Atlanta, Georgia 30322
Rapki sampling and analysis of the neurotransmitter dopamlne present In the extracellular fluid of rat braln have been achieved by use of mlcrodldy&. Concentrated 0.5-pL samples are obtalned by uslng a perfusion flow rate of 210 nL/ min. A 5mln analysls Is performed with a smallbore (1.0 mm Ld.) llquld chromatographic column and electrochemlcal detection. The retentlon t h e for dopamlne is 3 mln. The tlme scale of the analysls Is appropriate for the study of acute pharmacological and behavioral manlpulations. The chromatographic system Is evaluated on the bask of detectlon llmlt and dynamic response to concentratlon changes In the sampled medlum. The performance of the system is characterIred In vitro and in vivo. The system can routinely measure 20 nM dopamine in 0.5-pL samples.
Traditional methods for studying neurotransmitter systems and their relationship to behavior have relied on the use of pharmacological manipulations and selective lesioning techniques. These methods usually involve sacrifice of the animal and subsequent dissection of brain tissue for neurochemical analysis. Information obtained from tissue content data concerning the dynamics of neurochemical activity is often ambiguous. Recently, methods have been developed which allow sampling and analysis of compounds in the extracellular fluid of intact brain regions (1). The advantages of sampling the extracellular fluid have been reviewed (2). Perfusion of brain structures using push/pull cannulae has proved to be a powerful technique for investigating neurochemical processes (3, 4). The development of the dialysis cannula has further enhanced the utility of this technique for the study of brain chemistry (5, 6). This method has been applied very effectively to the study of the nigrostriatal dopamine system of 0003-2700/87/0359-07 12$0 1.50/0
the rat brain (7-9). When this method is combined with high-performance liquid chromatography and electrochemical detection, a sensitive and selective means of monitoring electroactive compounds within the brain is achieved (10-14). Due to the low nanomolar concentration of dopamine (approximately 20 nM) (15)and other neurotransmitters in the extracellular fluid, relatively large volumes of perfusate must be collected in order for these compounds to be detected by conventional chromatographic techniques. Typically, 40 pL of perfusate is collected at a perfusion rate of 2 pL/min, resulting in an analysis every 20 min. In order to obtain a more complete temporal profile of the dynamics of extracellular dopamine, a higher sampling rate is required. With the advent of smallbore packed chromatographic columns, it became apparent that their application in dialysis work would be advantageous. The ability to incorporate small (C1 pL) sample volumes and their utility in fast analysis (16) makes them particularly suited for sample-limited situations that frequently occur in biological studies. We report here the development and application of an on-line microdialysis/smallbore liquid chromatographic system which samples and analyzes the extracellular fluid from the striatum of rats at 5-min intervals.
EXPERIMENTAL SECTION Chromatography. A schematic diagram of the microdialysis/smaUbore liquid chromatography system is illustrated in Figure la. Mobile phase delivery was performed with a LC-5000 precision syringe pump (ISCO Co., Lincoln, NE). Samples were injected by use of a 7413 internal sample loop injection valve configured with a 0.5 p L sample loop (Rheodyne, Inc., Cotati, CA). Injections were made with an air actuator and a locally constructed electronic counter. A 1.0 mm i.d. X 10 cm column was packed with CI8 5 pm packing (Phase Sep, Norwalk, CT) using the procedure of Myer and Hartwick (17). The smallbore column was connected directly to the injection valve and the stainless 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
’
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Figure 1. (a) Diagram of the entire system showlng on-line connection of the dialysis probe and the position of the chromatographic system with respect to the animal test chamber. (b) Schematic diagram of the dialysis cannula. The dialysis probe is 320 pm in diameter and has an active length of 4 mm. Arrows indicate direction of perfusate flow.
steel half of the detector cell block with commercially available fittings (Sci-Con, Winter Park, FL). A 0.5-pm stainless steel frit with a Kel-F ring was placed between the column and the detector block. No frit was used at the inlet to the column due to difficulty in removing the frit from the injection valve port for cleaning. We felt it was necessary to eliminate all connecting tubing associated with the column to decrease extracolumn band broadening which can degrade the performance of the chromatographic column. This is particularly important when using columns of reduced internal diameter and small injection volumes, as the dead volume associated with the system can easily approach 50% of the injected sample volume. The effective volume of the electrochemical cell was reduced from 1.2 to 0.25 pL by replacing the commercial spacer with a 20 pm thick sheet of cellophane. A 1.5 mm X 16 mm channel defined the flow path over the electrode. Electrochemical detection was carried out with a LC-3 amperometric detector (BioAnalytical systems, West Lafayette, IN), a glassy carbon working electrode, and a downstream Ag/AgC1 reference electrode. Oxidation of electroactive compounds was performed at a potential of +0.65 V. Chromatograms were recorded on a Perkin-Elmer strip chart recorder and stored after analog to digital conversion using a microcomputer. The mobile phase used to achieve the selective retention of dopamine was 0.05 M sodium phosphate buffer, 0.1 mM EDTA, 2.2 mM sodium octyl sulfate, and 5.0 mM triethylamine with 15% methanol. The pH was adjusted to 5.6 with 6 M HCl. The flow rate was 150 pL/min with a pressure drop of 1800 psi.
713
Cannula Construction. The design of the microdialysis cannula is illustrated in Figure Ib. Two lengths of fused silica (0.150 mm 0.d. X 0.025 mm id.) were inserted into a 7.0 mm length of dialysis tubing (320 pm 0.d.; Cuprophan hollow fibers, Enka Glanztoff AG, West Germany) which had been sealed at one end with polymide sealing resin (Alltech Associates, Deerfield, IL). The distance between the ends of the fused silica tubes was 4 mm and constituted the active length of the dialysis probe. The membrane was sealed at the top with the polymide resin. The probe was inserted into a threaded cannula base and secured with epoxy. Sampling. Artificial cerebrospinal fluid (CSF) was prepared by adding 7.5 g of NaCI, 0.20 g of KCl, 0.15 g of CaCl,, and 0.19 g of MgCl, to 1 L of distilled water. Flow through the cannula was achieved by use of a Harvard syringe pump modified to simultaneously push and pull CSF through the probe. The perfusion rate was set at 210 nL/min. The inflow fused silica tube (push side) was connected to a 250-pL Hamilton gas-tight syringe. A 50 cm outflow fused silica tube (pull side) was connected directly from the cannula to the injection valve by inserting in. stainless the end of the fused silica into a short piece of steel connecting tubing (0.007 in. i.d.). The fused silica was secured flush with the end of the stainless steel tubing using epoxy. The connection to the injection valve was made with an Upchurch Finger-tight ferrule (Sci-Con). Characterization of the dialysis cannula was carried out prior to and after each in vivo experiment. The recovery of each cannula was determined by comparing the peak heights of standard solutions collected using the cannula to those injected directly. Standard curves were constructed from solutions of 10,50, 100, 200, and 300 nM dopamine. The standards were made from a stock solution which was 2 pM in dopamine and contained ascorbic acid, uric acid, the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and serotonin (5-HT) and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in concentrations which reflected their extracellular concentrations after dilution with artificial CSF. System Performance. Stability. The ability of the system to maintain sensitivity over the duration of a typical experiment was investigated. Calibration curves were constructed as described above. The stability of the system was evaluated based on the reproducibility of the calibration curves. Response. The ability of the system to follow discrete changes in the sampling medium was examined. The time required for a sample to travel from the dialysis probe to the injection valve following a 1-min step change in the external concentration was established, as well as the response of the system to step changes of 1-, 2 - , 5-, and 10-min duration. Step changes in external concentration were created by removing the cannula from a beaker containing 50 nM DA and placing it into a beaker containing 150 nM DA for the appropriate time interval. The cannula was then removed, quickly washed with artificial CSF, and replaced in the solution of lower concentration. I n Vivo Experiments. Male Sprague-Dawley rats weighing 200-300 g were anesthetized with chloral hydrate (400 mg/kg) and the microdialysis probe stereotaxically implanted in the anterior striatum (AP, +1.5; ML, +2.4; 6.0 mm below dura) (18). Haloperidol was made up in 0.9% saline and administered at doses of 0.05 and 2.0 mg/ kg. The doses were calculated as the free base and injected in a volume of 1mL/kg body weight. Apomorphine was made up in saline solution with 1%acetic acid and administered at a dose of 0.4 mg/kg. The dose was calculated as the free base and injected in a volume of 1 mL/kg body weight. Materials. HPLC grade methanol and triethylamine were purchased from Fisher Scientific (Pittsburgh, PA). Sodium octyl sulfate was obtained from Kodhk (Rochester, NY). Dopamine, DOPAC, HVA, serotonin, and 5HIAA were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were general purpose grade and obtained from Fisher Scientific. Deionized water was distilled twice in glass in the laboratory and was used in all solutions. R E S U L T S A N D DISCUSSION Smallbore columns offer two advantages over conventional columns which make their use ideal for the analysis of brain perfusates. The increase in mass sensitivity seen with columns
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of reduced diameters is very useful in sample-limited applications. It allows for the analysis of smaller sample volumes without a loss in detection limit. Secondly, the ability to generate high linear mobile phase velocities facilitates fast separations without excessive solvent consumption. The use of a smallbore column in the present study was motivated by the desire to analyze nanomolar concentrations of dopamine in small sample volumes and a t a high sampling frequency. We were also interested in lowering the detection limit of our system so that behaviorally induced changes in extracellular dopamine could be monitored. Sampling. In order to accurately track fluctuations in the concentrations of neurotransmitters in the brain, it is necessary to sample the extracellular fluid a t as high a frequency as possible. One way to achieve a fast sampling rate is to collect the same volume of perfusate in a shorter time. This can be done by perfusing a t high perfusion rates. While a higher perfusion rate is a viable alternative in many kinds of experiments, the exchange properties of the dialysis membrane lead to excessively dilute samples as perfusion rate is increased. This is the result of a decrease in exchange across the dialysis membrane as perfusion rate increases (19). High perfusion rates (>1 pL/min) can also create artificial concentration gradients within the extracellular fluid which may alter neuronal activity. In addition these gradients may introduce error in the measurements. An alternate approach to increasing the sampling rate is to decrease the amount of sample collected per unit time. At perfusion rates less than 2 pL/min, less material is collected per minute but the sample volume is smaller. Because the exchange across the dialysis membrane increases nonlinearly with slower perfusion rates, relatively concentrated samples are obtained. However, there is a lower limit to perfusion rate imposed by the microdialysis sampling technique. It has been shown that at perfusion rates less than 100 nL/min, sample dispersion within the fused silica tubing increases dramatically as a function of perfusion rate (20). In addition, total displacement of mobile phase from the sample loop must occur between injections to ensure injection of undiluted samples. With these restrictions in mind, a perfusion rate of 210 nL/min was chosen. At this perfusion rate the exchange across the dialysis membrane is 80-85% and dispersion in the fused silica tubing is minimal. For a 5-min sampling interval a total of 1.05 pL of perfusate travels through the sample loop between injections. When a 0.5 pL sample loop is used, 0.55 pL of perfusate flows through the loop between injections to displace the mobile phase. Also, at this perfusion rate, the concentration gradient generated within the extracellular fluid is less than gradients induced by higher perfusion rates. Over any given time period, a perfusion rate of 210 nL/min removes only about one-third of the material removed when using a perfusion rate of 2 pL/min (for sample recoveries of 80% and 20%, respectively). System Performance. The chromatographic conditions have been adjusted so that dopamine eluted in 2-4 min while all other detectable electroactive compounds present in the perfusate eluted in the solvent front. Figure 2 shows a chromatogram of a standard solution of 300 pM ascorbic acid, 5 pM each DOPAC, HVA, and 5-HIAA, 100 nM 5-HT, and 50 nM dopamine and a chromatogram of a brain perfusate collected from the anterior striatum of an anesthetized rat. Dopamine was well-resolved from the solvent front and produced an easily quantifiable signal a t resting physiological concentrations. When neurochemical experiments are conducted, it is usually necessary to monitor the animal over a period of several hours. The ability of the chromatographic system to produce a constant response over the course of an experiment
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The standard represents a 50 nM dopamine solution. (over 100 perfusate injections) was tested by comparing a calibration curve generated prior to an in vivo experiment to a calibration curve generated after termination of the experiment. There was no evidence of degradation in the sensitivity of the system (precalibration, slope = 0.830, r > 0.99, n = 5; postcalibration, slope = 0.826, r > 0.99, n = 5 ) . The detection limit for dopamine was 2.0 fmol ( S I N = 2) both before and after the in vivo experiment. This detection limit enables the quantitation of dopamine down to an extracellular concentration of 5 nM. The recovery characteristics of the dialysis cannula decreased only slightly after use in vivo. Recovery was 84% before the experiment and 82% following 5 h of sampling in the brain. The stability of the system is due in large part to the filtration of the perfusate by the dialysis membrane. A major concern in sampling is that the signal generated at the detector accurately represents changes occurring in vivo and that sample integrity is maintained. The response of the sampling and chromatographic systems to abrupt changes in extracellular concentrations must be known if the system is to be used to relate the data to behavior or pharmacokinetics. Therefore, the time required for a sample to move from the dialysis cannula to the injection valve was determined, as was the response of the system to step changes of varying duration. The travel time of a sample from the dialysis probe to the injection valve was determined by using a 1-min step change. Travel time was definded as the time between the initiation of a step change in concentration in the sampling medium and the injection of that sample. Theoretically, if the 1-min sample was contained entirely within the sample loop at the time of injection, a response of 210/500 of 42% of the maximum response should be obtained. The 210/500 represents that fraction of the sample loop (500 nL) which would be occupied by a sample plug 1min wide at a perfusion rate of 210 nL/min.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
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Maximum response was defined as the limiting response observed for repeated injections of continuously perfused 150 nM DA solution. The results of this experiment indicated that the travel time required for a sample to move from the dialysis cannula to the injection loop when connected by a 50 cm length of fused silica was 7 min. For a travel time of 5 min, a response of 18% of the maximum was seen indicating injection of only the leading portion of the sample plug. After a 10-min interval between the step change in the medium and injection, the observed response was 18% of the maximum also and represents injection of the trailing edge of the sample plug. The contributions of the various components of the sampling system to this travel time were calculated to verify this time delay. At a perfusion rate of 210 nL/min, 2.4 min was required to fill the 0.5 pL sample loop. The volumes of the cannula and the fused silica connecting the cannula and the injection valve were 0.11 pL (31 s) and 0.08 p L (23 s), respectively. The volume present a t the fused silica-injection valve interface was slightly less than 0.5 pL and contributed significantly to the travel time. The flow of the perfusion pump was tested as fluctuations in perfusion flow would effect sample travel time. The flow generated by the pump was accurate within 5 % . The diffusion time across the dialysis membrane for dopamine in a CSF solution was experimentally determined to be 20 s a t 25 "C. The contribution of the sampling components (dialysis cannula, fused silica line, and injection valve) to sample dispersion must be minimized if integrity of the individual sample is to be maintained. T o test the system's ability to maintain sample integrity, the response to step changes in the concentration of the perfusion medium was investigated. The observed responses for step changes of varying time durations are shown in Figure 3. The horizontal bars indicate the time during which the 150 pM DA solution was perfused. The step changes were initiated 5 min prior to injection. This was an arbitrary choice since when applying this technique in vivo the initiation of changes in the concentration of the extracellular fluid will be random with respect to time. The step durations can be classified into two groups-samples whose initial volumes are less than the injection volume (1-and 2-min durations at 210 nL/min) and samples whose initial volumes are larger than the injection volume (5- and 10-min durations). For the first group, sample integrity is maintained if the step change is contained within one injection. Subsequent injections should produce the base-line response. Consider the 1-min period of increased concentration. A response corresponding to 18% of the maximum signal was observed only in the sample injected following the step change. The maximum response was determined by perfusing the higher concentration solution until a steady response (maximum response) was achieved. The next sample had returned to base
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Figure 5. Extracellular dopamine concentrations following pharmacological manipulatlons: (A) 2.0 mg/kg haloperidol (ip); (6)0.05 mg/kg haloperidol (lp); (C) control (saline injectlon); (D) 0.4 mg/kg apomorphine. Arrow indicates time of injection.
line. The response seen for the 2-min change was similar to that for the 1-min change. A response above base line was obtained only for the injection following the step change. The results indicate that fluctuations which occur for less than one sampling interval will be represented by a single increase in response with the following responses returning to base line. For step changes representing the second group, more than one injection will contain part of the sample. The 5-min step change represents an initial sample size of 1.05 pL. A response was obtained in two injections with the largest signal generated by the second injection. A signal representing 18% of the maximum was observed for the first injection and one that was 72% of the maximum was observed for the second injection. The 10 min step change showed similar response characteristics. Starting with an initial volume of 2.1 pL, the sample was contained in three injections. The respective responses were 18%, 91%, and 80% of the maximum. As seen in the above results, on-line dialysis perfusion retains sample integrity and the system is capable of accurately responding to step changes. The response characteristics will also be affected by the perfusion rate at which the sample is collected as well as the size of the sample loop, any associated dead volume, and the sampling interval. In Vivo Experiments. A typical chromatogram obtained in vivo is shown in Figure 4. This particular series was obtained from the striatum of a freely moving rat. To verify the ability of the system to monitor changes of dopamine concentrations in the extracellular fluid of the rat brain, pharmacological studies were undertaken in anesthetized rats. Drugs whose effects on the dopaminergic system are welldocumented and that would produce either an increase or a
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decrease in extracellular dopamine levels were chosen. Figure 5 shows the results of the pharmacological studies. Dopamine levels are expressed as a percent of the preinjection control value. The mean value of the six samples immediately prior to drug injection constituted the control value. The arrow indicates the time of drug injection. Curve C represents a control animal which received a saline injection. Curves A and B represent the change in the extracellular concentration of dopamine following the administration of the neuroleptic drug haloperidol at two different doses (2.0 and 0.05 mg/kg, respectively). Haloperidol, a dopamine antagonist, is known to block dopamine receptors in the striatum and thereby increase the level of extracellular dopamine. The 2.0 mg/kg ip dose showed an onset of the drug effect 15 min after injection. The extracellular dopamine concentration increased to 1.2 fiM or 1700% above predrug base-line level 45 min after drug administration and remained elevated at this level for the duration of the experiment. The 0.05 mg/kg ip dose showed an onset of the drug effect 30 min after injection. The increase in extracellular dopamine peaked at 36 nM or 125% above predrug levels 60 min following drug administration. A slow decrease toward predrug levels was then observed for the remainder of the experiment. Curve D represents the changes in extracellular dopamine seen following the administration of 0.4 mg/kg apomorphine. Apomorphine is a dopamine agonist which decreases the release of dopamine by interacting directly with postsynaptic dopamine receptors. The onset of the drug effect was detected 25 min after injection. Apomorphine decreased extracellular dopamine to 8 nM or 20% of predrug levels 10 min later. The dopamine concentration remained at this level for the next 45 min and then briefly fell to nondetectable levels for 10 min. An increase back toward basal levels was observed 3 h after injection of the drug. The experiments reported here demonstrate that the microdialysis/smallbore chromatography system is capable of monitoring the extracellular dynamics of the dopaminergic system on a time scale relevant to the study of acute pharmacokinetics. The present system is currently being applied
to studies involving behaving animals. Use of this system in anesthetized and freely moving animals will provide useful information in the study of neurochemistry, pharmacology, and behavior.
ACKNOWLEDGMENT The authors thank L. Phebus for donating the dialysis tubing.
LITERATURE CITED Marsden, C. A. Ed. I n Measurement of Neurotransmitter Release I n Vivo; Why: New York, 1984. Zetterstrom, T. I n PharmacologicalAnalysis of Central Dopaminergic Neurotransmkslon Using a Novel In Vivo Braln Perfusion Method; Karolinska Institute: Sweden, 1986; pp 6-7. Glowinski, J.; Cheramy, A.; Giorguiff, M. F. I n The Neurobiology of Dopamine; Academic: New York, 1979; Vol. 14; Chapter 15. Loullis, C. C.; Hingtgen, J. N.; Aprison M. H. Pharmacol. Biochem. Behav. 1980, 12, 959-963. Ungerstedt, U; F'ycock, C. Bull Schweiz. Akad. Med. Wiss. 1974, 1278, 1-5. Tossman, U.; Ungerstedt, U. Acta Physioi. Scand. 1986, 128, 9-14. Clemens, J. A.; Phebus, L. A. Life Sci. 1984, 35, 671-677. Ungerstedt, U. I n Measurement of NeurotransmitterRelease I n Vivo; Wiley: New York. 1984 pp 81-105. Blakeiy, R. D.; Wages, S.A.; Justice, J. E., Jr.; Herndon, J. G.; Neili, D. 8. Braln Res. 1984, 308, 1-8. Zetterstrom, T.; Sharp, T.; Marsden, C. A.; Ungerstedt, U. J . Neurochem. 1983, 41, 1769-1773. Zetterstrom, T.; Ungerstedt, U. Eur. J. Pharmacol. 1984, 9 7 , 29-36. Zetterstrom, T.; Sharp, T.; Ungerstedt, U. Eur. J . Pharmacol. 1984, 106, 27-37. Imperato, A.; Di Chmra, G. J. Neurosci. 1984 4 , 966-977. Imperato, A.; Di Chiara, G. J. Neurosci. 1985, 5, 297-306. Church, W. H.; Justice, J. B., Jr. Tenth International Symposium on COIUmn Liquid Chromatography, San Francisco. CA, 1986; Abstract 2310. Scott, R. P. W.; Kucera, P.; Munroe, M. J . Chromatogr. 1979, 186, 475-487. Myer, R. F.; Hartwick, A., Jr. Anal. Chem. 1984, 5 6 , 2211-2214. Pellegrino, L.; Pellegrho, A.; Cushman, A. A Stereotaxic Atlas of the Rat Brain; Plenum: New York, 1979. Johnson, R. D.; Justice, J. B., Jr. Brain Res. Buil. 1983, 10, 567-571. Wages, S . A.; Church, W. H.; Justice, J. B., Jr. Anal. Chem., 1986, 5 8 , 1649-1856.
RECEIVED for review August 25,1986. Accepted November 1, 1986. This work was supported by NSF Grant BNS8509576.
