Anal. Chem. 1993, 65, 1017-1022
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Quantitative Microdialysis under Transient Conditions R. J. Olson and J. B. Justice, Jr.' Department of Chemistry, Emory University, Atlanta, Georgia 30322
A mlcrodlalysls method lo descrlbed whlch allows for the quantltatlve determlnatlon of extracellular analyte concentratlon under tramlent condltlons. The method provldes the extracellular concentratlonand the In VIVO probe recovery as a functlon of tlme. The technlque k based on the method for steady-state condltlons but dmers In the use of a betweengroup rather than a wlthln-group dedgn. Followlng cocaine and amphetamine admlnlstratlon, a slgnlflcantly greater Increase In extracellular DA was found than was esthnated from the dlalysate udng conventlonalmlcrodlalyslsmethods. The dlrcrepancy Is due to mkrodlalysls probe recovery decreadng concurrently wlth the Increase observed In extracehlarDAfokwingcocalne and amphetaminelnJecUon& The method estimates the extracellular concentratlon lnda pendently of any changes In recovery.
INTRODUCTION Microdialysis has gained wide recognition as a valuable tool for sampling the extracellular space of living tissue.112 In this technique, a dialysis probe is placed in a local brain region and is perfused continuously with a solution which closely resembles the fluid present in the extracellular space. An analyte present in higher concentration on one side of the dialysis membrane than the other will diffuse down ita concentrationgradient, moving either into or out of the probe. Thus, the probe may be operated either in the delivery mode by adding a substance to the perfusate which is not present extracellularly, or in the sampling mode where the perfusate is devoid of an endogenous compound. A majority of the literature focuses on the sampling mode of microdialysis. In order to extract the most informationfrom microdialysis samples, it is useful to draw some relationship between the concentrationof analytein the dialysate and ita concentration in vivo. Early techniques estimated the dialysis probe efficiency in vitro and used this value for in vivo calculations.3 This method proved to be insufficient because transport characteristics, and thus probe efficiency, differ for solution and tis8ue.a The approach described by LGnnroth et al.933 *Addrese correspondence to this author at the Department of Chemistry, Emory University, 1515 Pierce Dr., Atlanta, GA 30322. Telefax: (404)727-6632. (1) Robinson, T. E.; Justice, J. B., Jr. Microdialysis in the Neurosciences; Elsevier Science Puslishers: New York, 1991. (2) Rollema, H.; Westerink, B.; Drijfhout, W. J. Monitoring Molecules in Neuroscience: Proceedings of the 5th International Conference on in Viuo Methods; Krips Repro, Meppel: The Netherlands, 1991. (3) Ungerstedt, U.; Harrera-Marschitz, M.; Jungnelius, U., Sttihle, L.; Toeaman,U.; Zetteratrom,T. Advances inD0pamineResearch;Pergamon Press: New York, 1982. (4) Benveniste, H.; Hansen, A. J.; Ottosen, N. S. J. Neurochem. 1989, 52, 1741-1750. (5) Nicholaon, C.; Phillips, J. M. J. Physiol. 1981, 321, 225-257. (6) Bungay, P. M.; Morrison, P. F.; Dedrick, R. L. Life Sci. 1990, 46, 105-119. ~ . . (7) Lindefors, N.; Amberg, G.; Ungerstedt, U. J.Pharmacol. Methods 1989,22, 141-156. (8) Morrison, P. F.; Bungay, P. M.; Hsiao, J. K.; Ball, B. A.; Mefford, I. N.; Dedrick, R. L. J. Neurochem. 1991,57, 103-119. (9) Lonnroth, P.; Janason, P. A.; Smith, U. Am. J. Physiol. 1987,253, E228-E231.
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has had more success in addressing the problem of quantitation and has been applied to the measurement of basal extracellular dopamine" and the effect of chronic cocaine on dopamine.12 Decreased recovery due to loss of release and uptake sites has been reported,13and the problem of circadian variation has been noted for the application of the method.14 The method has also been applied to the measurement of exogenous sub~tances.~5 This technique involves the serial perfusion of several different concentrations of analyte through the probe so that the extracellular concentration is bracketed. When the concentrationof analyte perfused through the probe is greater than ita extracellular concentration, there will be a net loss of analyte from the probe. Likewise, when the perfusate concentration is less than the extracellular concentration, there will be a net gain of analyte to the probe. When the concentration of analyte perfused through the probe is equal to the extracellular concentration,there will be no net diffusion of the analyte into or out of the probe. This concentration is the zero point and corresponds to the extracellular concentration of analyte. In order to find the extracellular concentration, the difference between the concentration of analyte perfused through the probe and that obtained in the dialysate (Ci, C o d is plotted for each perfusion concentration (Ci,), and a linear regression is performed. Probe efficiency, or recovery, is the slope of the regression &e, and the extracellular concentration is determined from ita intersection with Ci, Cout = 0. This method has an advantage over previous methods in that it estimates extracellular concentration independently of probe recovery. However, it is timeconsuming and applies only to steady-state conditions. Although applicable to studies involving chronic effecta of drugs on basal states, the method is not useful in studies which monitor the response of extracellular analyte to drug administration. Currently, brain microdialysis for transient conditions is performed by analyzing the concentration of neurotransmitter in the dialysate while perfusing with a solution devoid of the neurotransmitter (typically artifcial cerebrospinal fluid). Therefore,the recovery of the probe is afactor in the analysis. While a great deal of information has been generated to characterize the probe under steady-state conditions, little is known about the dynamics of recovery under transient conditions.198 Recently,Parsons et al.13showed that recovery in brain tissue can be altered independently of extracellular concentration. This work suggested that in vivo probe recovery may be dependent on active neurotransmitter processes such as uptake and release. A consequence of such (10) Lonnroth, P.; Janason, P. A.; Fredholm, B. B.; Smith, U. Am. J. Physiol. 1989,256, E25eE255. (11) Parsons,L. H.; Justice, J.B., Jr.J.Neurochem. 1992,58,212-218. (12) Parsons, L. H.; Smith, A. D.; Justice, J. B., Jr. Synapse 1991,9,
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(13) Parsons, L. H.; Smith, A. D.; Justice, J. B., Jr. J.Neurosci.Methods 1991,40, 139-147. (14) Smith, A. D.; Olson, R. J.; Justice, J. B., Jr. J. Neurosci. Methods 1992,44, 33-41. (15) Menacherry, S.;Huburt, W.; Justice, J. B., Jr. Anal. Chem. 1992, 64,577-583. 0 1993 American Chemlcal Society
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a dependence is that t h e measurement of dialysate concentration may not accurately reflect changes in t h e extracellular concentration. In t h e present study, a method is described which allows for quantitative microdialysis under transient conditions. T h e protocol is based on t h e method for steady state, b u t differs in t h e use of a between-group rather t h a n a within-group design. In other words, three or four subjects are perfused, each with a single concentration of analyte, rather t h a n perfusing a single subject with multiple concentrations of analyte. T h e concentrations bracket t h e extracellular concentration of t h e analyte of interest. T h e d a t a from t h e subjects may then be combined a t each time point for regression as in the steady-state experiment. This design provides t h e extracellular concentration of analyte a n d t h e probe recovery with respect t o time. It has particular application to studies which evaluate t h e response of neurotransmitters to drug challenge. T h e technique is demonstrated with three drugs which are widely used in microdialysis experiments; cocaine, amphetamine, a n d haloperidol.
EXPERIMENTAL SECTION Materials. All chemicals were obtained from Sigma (St. Louis, MO). Nembutal was obtained from Abbott Laboratories (Chicago, IL). Guide cannula, dummy cannula, skull screws, cranioplastic cement, and mounting swivels were obtained from Plastics One (Roanoke, VA). Fused Silica (100 pm o.d., 40 pm i d . ) was obtained from Polymicro Technologies (Phoenix, AZ). Cellulose dialysis membrane (6000 MW cutoff) was purchased from Spectrum (Los Angeles, CA). Artificial cerebrospinal fluid (CSF) consisted of 149 mM NaC1, 2.8 mM KCl, 1.2 mM CaC12,1.2 mM MgC12, 0.25 mM ascorbic acid, and 5.4 mM D-glucose, p H 7.4. Dopamine (DA) solutions were prepared daily by serial dilution of a 10 pM stock solution in CSF to 10, 20, and 40 mM concentrations. P r o b e Construction. Two lengths of fused silica (110 and 120 cm) were inserted into a 4-mm tube of cellulose dialysis membrane which was sealed at one end with polyimide resin (Alltech, Waukegan, IL). The silica capillaries were adjusted so that the difference between the two lengths was 2 or 4 mm, depending upon the size of the targeted brain region. Probes of 2 mm were used in the nucleus accumbens, and probes of 4 mm were used in the striatum. The base of the tube was then sealed to the capillaries with polyimide resin leaving a 2- or 4-mm active area for diffusion to occur.L6The lengths of silica were protected with plastic tubing and inserted into a spring so that animal rotation would not result in the breaking of the probe's perfusion and collection lines. The longest length of silica was then inserted intoa 26 gauge needle (Becton Dickinson, Rutherford, NJ) which was later connected to a 500-pL Hamilton syringe for perfusion of the probe. S u b j e c t s a n d Surgery. Male wistar rats (250-275 g; Harlan Sprague-Dawley Inc., Indianapolis, IN) were group housed in a temperature-controlled room and were maintained on a 12:12-h lightidark cycle (lights on a t 7 am). Food and water were available ad libitum. A minimum of 4 days was allowed between time of delivery and surgery. For all surgeries, rats were anesthetized with Nembutal (50 mgikg, ip) and were placed in a stereotaxic device. The site of incision was then cleaned with betadine solution (Purdue Frederick, Norwalk, CT) and a 20-gauge stainless steel guide cannula was placed in the skull at appropriate coordinates to position it just above the desired brain region (AP +3.4, LR +1.5 from bregma, DV -6.0 from dura for nucleus accumbens; AP +2.7, LR -2.7 from bregma, DV -2.7 from dura for striatum; incisor bar set a t +5.0 mm).': The guide cannula was secured to the skull using screws and cranioplastic cement. A dummy cannula was then inserted to prevent clogging of the tube prior to the experiment. Following surgery, animals were injected with penicillin (60 000 units) in (16)Wages, S. A.; Church, W. H.; Justice, J. B., Jr. Anal. Chern. 1986, 58,
order to prevent bacterial infection and were supplied with a postoperative analgesic. A recovery period of 5-7 days was allowed prior to experimentation. All surgical procedures were performed in accordance with recommendations given by Emory University Institutional Animal Use and Care Committee. Following the experiment, animals were given a lethal dose of chloral hydrate and were perfused transcardially with saline and 1 0 5 formalin. The brains were removed and stored in 10% formalin. At a later date, brains were sliced with a freezing microtome and were stained so that probe placement could be verified by comparison with a map.15 Microdialysis. On the evening prior to the experiment, animals were weighed and placed in test boxes. Probes were connected to 500-pL Hamilton syringes which were then attached to Harvard syringe pumps (Model 2274) and were perfused at a rate of 0.2 pL/min with artificial CSF. Probes 2 mm long were used in nucleus accumbens while 4-mm probes were used in the striatum. After the probes were checked for leaks, they were inserted through the guide cannula into the desired brain region and were perfused overnight (approximately 8 h). The following morning, the perfusion medium was changed to either 0, 10, 20, or 40 nM DA, and the flow rate was gradually increased to 0.6 pL/min. Following an equilibration period of approximately 30 min, samples were collected into microcentrifuge tubes every 10 min and were frozen on dry ice. After 7-10 samples were collected for baseline data, a challenge injection of either cocaine (20 mgikg, i.p.), amphetamine (1.25 mgikg, ip), or haloperidol (1mgikg, ip) was administered. Samples were then collected and frozen at regular intervals for 80,120,and 180 min, respectively. Thawed samples and standards were injected in 0.5-pL aliquots onto a microbore HPLC column (0.5 mm i.d.; 10 cm length; 5 pm C-18 stationary phase). The mobile phase consisted of 27.2 mM sodium phosphate buffer with 10% MeOH (vol/vol), 4.9 mM triethylamine, 0.13 mM disodium ethylenediaminetetraacetate, 0.99 mM sodium octyl sulfate, p H 5.75. The flow rate was 33 pLimin using an ISCO LC-5000 syringe pump. Detection of DA was accomplished using an EG&G Princeton Applied Research amperometric detector (EG&G Model 400) with working (Model MF-100) and reference (Model RE11 electrodes from Bioanalytical Systems Inc. and an applied potential of 0.700 V versus AgiAgC1. This system eluted DA in 6 min. Quantitation of samples was accomplished through the use of a standard curve constructed from standards which had been frozen in 6-pL aliquots in microcentrifuge tubes. D a t a Analysis a n d Statistics. Data for the groups receiving 0, 10,20,and 40 n M DA were used to construct a graph for each time point by plotting DAi, - DA,,,, the difference between the concentration of DA perfused through the probe (DAi,) and that obtained in the dialysate (DA,,,) versus DAi, (Figure 2). Linear regression yielded the slope (recovery) and intercept (-DA,,, at 0 DA,,). To calculate the extracellular concentration, the equation for linear regression, y = mx + h was solved for x a t the point of no net flux, that is when y = 0. Thus, xy=o is -b/m, where m is recovery, h is -DA,,, at 0 DAi,, and x,=o is the extracellular DA concentration. Error for probe recovery was obtained directly from the regression statistics. Error for extracellular concentration was calculated by propagation of errors from the errors associated with the y-intercept and slope.18 The equation used for this calculation was SEM,,, = + (S,/m)2]1i2 where SEM,,, is the standard error of the mean of the extracellular concentration, Sb is the standard deviation of the y-intercept, b is the y-intercept, S , is the standard deviation of the probe recovery, and m is the probe recovery. In all cases, error is reported as the standard error of the mean (SEM). During the course of the study, 627 samples were collected. Of these, four samples were lost to chromatography problems and two were outliers (defined as values which had residuals lying three or more standard deviations from the mean of the residuals). In order to maintain the continuity of the time course graphs,
1649-1656.
(17) Pellegrino, L.; Pellegrino, A.; Cushman, A. A stereotaxic atlas of the rat brain; Plenum Press: New York, 1979.
(18)Young, H. D. Statistical T r e a t m e n t McGraw-Hill: New York, 1962; Chapter 13.
of
E x p e r i m e n t a l Data;
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Time (min) Flgure 1. Tlme course of dlalysate DA ( D b ) for three groups of rats (n = 4 per group) recehrlng either 0 nM DA,,, (open circles), 10 nM DA ,, (filled clrcles),or 40 nM DAh (open triangles). Results were obtalned wing 2-mm probes In the nucleus accumbens flowing at a rate of 0.6 Wmln. SampleswerecoHectedevery10min. Atterthreeconsecuthre samples were collected which varied less than 10% from each other, an Injectlon of cocaine (20 mg/kg. lp) was administered. Error bars are standard error of the mean (SEM).
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RESULTS AND DISCUSSION
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Cocaine. Figure 1 shows the time course of DLUtwith 2-mm probes in the nucleus accumbens for three groups of rata (n= 4 per group)receiving a challenge injection of cocaine (20mg/kg, ip). The data from these groups were combined at each sampled time interval and were plotted in the same manner as the experimentfor steady-state conditions. Linear regression provided the extracellular concentrationand probe recovery at each time point. Plots for the baseline data and the first five samples following cocaine are shown in Figure 2. During baseline, the extracellular concentration was 5.3 f 0.2 nM, and probe recovery was 48 f 0.9% (ISEM). These values are in agreement with previous reports of basal extracellular DA and recovery in the nucleus accumbensusing a within-subjects design under steady-state conditions.11-13 As expected from theoretical ~onsiderations~J9 the recovery in this experiment is lower than in previous reportall-l3 due to the higher flow rate employed using this design (0.6 rL/ min versus 0.2 pL/min). Following cocaine administration, the extracellular DA increased and recovery decreased. Both effecta were maximal approximately 20 min after drug (Figure 3). At this point, the extracellular concentration had increased to 42.3 f 3.1 nM and recovery had fallen by almost half (29I 6.1%; p < 0.01). Both extracellular DA and recovery returned to near baseline values approximately 60 min following the drug. As in conventional microdialysisexperimenta, differences in drug pharmacokinetics between subjects alter the time course of the response so that the error is usually larger during the maximum effect of drug. This was seen in the extracellular concentration and the recovery in the present experiment as well. The decrease in probe recovery following cocaine is consistent with previous studies which suggest that in vivo microdialyeisrecovery is affected by active processes.*J3These processes include any mechanism which provides a source or a sink for the chemical species in the extracellular fluid. Examples are synthesis and metabolism, release and uptake, and transport into and out of blood vessels. For catecholamine neurotransmitters, these processes are primarily release and uptake. Administration of 6-hydroxydopamine(SOHDA), ~
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(19)Johnson, R. D.; Justice, J. B., Jr. Brain Res. Bull. 1983,10,567571.
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Flgure 2. Regression lines showlng recovery and extracellular Concentration for samples collected dwlng basellne (panel A) and following cocalne (20 mglkg, Ip) for l m l n Intervals at 10,20,30,40 and 50 mln (panels 8-F, respectively). The dmerence between DA,, and D%, was plottedagainst DA,,for the threegroupsat each sampkd time Interval (n = 4 per group). Unear regression on the comblned data (n = 12) provldedthe extracellular concentration andthe recovery for each sampiedtime Interval. Extracellularconcentratlmls lndlcated by the dotted verllcal line. The average extracellular concentratbn 0.2 nM. The extracellular concentration durlng baseline was 5.2 obtained 10,20,30,40. and 50 mln following cocaine was 19.4 f 0.9, 42.3 f 3.1,28.0 f 1.8,24.7 f 0.2, and 13.5 f 0.7 nM, respecthrely. Recovery is the slope of the regressed Ilne. Average recovery for baseline samples was 48.3 f 0.9%. Recovery obtalned 10,20,30, 40, and 50 rnln followlngthedrug was34 f 3%,29 f 6%, 41 f 6%, 38 f 0.6%, and 43 30%, respectively. Error bars are SEMs.
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Time (min) Flgure 3. Time course of extracellular DA (open ckc4s) and probe recovery (fllled circles) In the nucleus accumbens folbwlng cocalne (20 mg/kg, Ip). Recovery and extracellular DA were calculated as shown In Flgure 2. Error bars are SEMs.
a neurotoxinwhich selectively destroys DA cells, also resulted in a reduced recovery of DA. Destroying DA cells reduces the number of release and uptake sites in the vicinity of the probe. This reduction of sites does not change the extracellular concentration, but reduces probe recovery because it reduces the exchange of DA between the extracellular fluid and DA nerve terminals. There are fewer DA nerve terminals to remove excess DA from the extracellular fluid when DA is supplied by the probe. Also, there are fewer DA nerve terminals to supply DA to the extracellular fluid when
ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993
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T i m e (rnin) Flgure 4. (Panel A, Top): Comparison of the time course of dialysate DA ( D b ) for the group receiving 0 nM DA,, (open circles; n = 4) and extracellular DA (filled circles; n = 12) in the nucleus accumbens following cocaine (20 mglkg, ip). Error bars are SEW. (Panel 6, Bottom): Comparison of the percent baseline of D& for the group receiving 0 nM DA,, (open circles; n = 4) and extracellular DA (filled circles; n = 12) following cocaine (20 mglkg, Ip). depleted of DA by the probe. Cocainehas the pharmacological action of reducing uptake of DA into the nerve terminal by blocking the DA transporter. As with 6-OHDA, the concentration gradient of DA near the probe (and thus the flux of DA to the probe) is altered due to reduced uptake andlor release. The change in recovery has implications for conventional microdialysis experiments in which the D k U tfor the 0 nM group is used to estimate changes occuring in extracellular DA. Figure 4a compares the time course of extracellular DA and D k U tfor the 0 nM group followingcocaine. Extracellular DA rose from 5.3 f 0.2 to 42.3 f 3.1 nM while D k U tfor the 0 nM group rose from 2.4 f 0.2 to 13.3 f 4.4 nM (ASEM). As expected from the observation of decreased recovery, the result obtained from the conventional microdialysis experiment significantly underestimates the increase of extracellular DA. Data obtained in microdialysis experiments are often reported as precent of baseline. Figure 4b compares D k U t for the 0 nM group and extracellular DA during the time course of cocaine, both expressed as percent baseline. At the point of maximum change (20 min following the drug), DLUt had risen to 560% of baseline while extracellularconcentration had risen to 804 7%. These results were similar to previous reporta of D b U tfollowing cocaine.20tZ1 Amphetamine. The experiment was also repeated in the nucleus accumbens using a challenge injection of D-amphetamine sulfate (AMPH; 1.25 mg/kg, ip). The time course of D k u tis shown for the three groups in Figure 5a. Following amphetamine, DLUtrose for all groups, peaking between 30 and 40 min after the drug. (20)Pettit, H.0.; Pan, H.-T.; Parsons, L. H.; Justice, J. B., Jr. J. Neurochem. 1990,55, 798-804. (21)Carboni,E.; Imperato, A.; Perezzani, L.; Di Chiara, G. Neuroscience 1989,28, 653-661.
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Time (min) Flgure 6. (PanelA, Top): Time course of dialysate DA ( D L ) for three groups of rats (n = 5 for 0 nM group, n = 4 for 10 and 40 nM groups) receiving elther 0 nM DCG, (open circles), 10 nM DAk, (filled circles), and 40 nM DA,,, (open Mangles). Results were obtained using 2-mm probes in the nucleus accumbens flowing at a rate of 0.6 pL/min.
SampleswerecoHectedevery10min. Afte~rthreeconsecuthresamples werecoliectedwhlchvarlediessthan 10% fromeachother,aninJectktn of amphetamine (1.25 mg/kg, Ip) was administered. Error bars are SEMs. (Panel 6, Bottom): Time course of extracellular DA (open circles) and probe recovery (filled circles) in the nucleus accumbens following amphetamine (1.25 mglkg, ip). Recovery and extracellular DA were calculated as shown in Figure 2. Average baseilne recovery and extracellular DA were 38 f 1% and 6.8 f 0.3 nM, respecthrely. Forty minutes following the drug, recovery had decreased to 24 f 4% and extracellular DA had increased to 58.0 f 2.7 nM. Error bars are SEMs.
As with the cocaine study, data were combined at each time interval, and linear regression was performed to obtain the extracellular concentration and recovery as a function of time (Figure 5b). Basal extracellular DA was 6.8 f 0.3 nM and recoverywas 38 f 1% Forty minutes after amphetamine administration, extracellular DA increased to 58.0 f 2.7 nM and probe recovery decreased by almost half to 24 f 4 % (p < 0.002). Recovery had not returned to baseline values when collections were halted 120 min following the drug. The primary action of amphetamine is to stimulate the release of DA from dopaminergicnerve terminals. One might expect that the concentration gradient and the flux of DA to the probe would increase. However, the increased release of DA by amphetamine is thought to occur by exchangediffusion via the uptake carrierazZThe net effect of amphetamine displacing DA from the terminal and competing for the uptake carrier is a reduction in the rate of exchange of DA between the intracellular and extracellularcompartments. A reduction in extracellular-intracellular exchangewould lead to a reduction in recovery.8 This interpretation is consistent with the 6-OHDA and cocaine results. It is possible that other actions of amphetamine, which include uptake inhibition and decreased metabolism, also contributed to the observation^.^^^^^^ Metabolismof DA to 3-methoxytyramine
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(22)Fischer, J. F.; Cho, A. K. J. Pharrnacol. Exp. Ther. 1979, 208, 203-209.
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Time (rnin) Figuro 6. (Panel A, Top): Comparlson of the time course of dialysate DA ( D h ) for the group receiving 0 nM D& (open circles; n = 5) and extracellular DA (filled circles; n = 13) In the nucleus accumbens followlng AMPH (1.25 mg/kg, Ip). Error bars are SEMs. (Panel B, Bottom): Comparlson of the percent baseline of D h for the group receiving 0 nM DA,,, (open circles; n = 5) and extracellular DA (filled circles; n = 13) following AMPH (1.25 mg/kg, lp).
(3-MT) is another mode of loss from the extracellular compartment and could thus affect recovery. However, inhibition of DA metabolism to 3-MT or DOPAC has no effect on recovery.26 A comparison of conventional microdialysis data (DLUt for the 0 nM group) and extracellular DA for the time course of amphetamine is shown in Figure 6. DLUtrose from 2.4 f 0.3 during baseline to 15.7 f 3.4 nM while extracellular DA rose from 6.8 f 0.3 to 58.0 f 2.7 nM 30 rnin following the drug. As with the cocaine data, the dialysate concentration underestimated the changes occurring extracellularly. Figure 6b shows the same data when expressed as percent baseline. Thirty minutes following the drug, DLUthad risen to 661 % and extracellular DA had risen to 862 % of baseline. These results are similar to those found by other investigabrs.23.24,27
Haloperidol. The method was also demonstrated using a challenge injection of haloperidol, a DA receptor antagonist which stimulates release of DA.2a31 The experiment was conducted using 4-mmmicrodialysis probes in the striatum at a flow rate of 0.6 pL/min. Figure 7a shows the time course of DLUtfor four groups of rats receiving either 0, 10,20, or (23) Pehek, E. A,; Schechter, M. D.; Yamamoto,B. K. Neuropharmacology 1990,29, 1171-1176. (24) Robinson, T. E.; Camp, D. M. Neuropsychopharmacology 1990, 3, 163-173. (25) Kalix, P.; Braenden, 0. Pharmacol. Reu. 1986,37, 149-164. (26) Smith, A. D.; Justice, J. B., Jr., unpublished work. (27) Sharp, T.; Zetterstram, T.; Ljungberg, T.; Ungerstedt, U. Brain Res. 1987,401, 322-330. (28) Moghaddam, B.; Bunney, B. S.J . Neurochem. 1990,54, 17551760. (29) Westerink, B. H. C.; Hofsteed, R. M.; Tuntler, J.; de Vries, J. B. J.Neurochem. 1989,52, 722-729.
(30) Brown, E. E.; Damsma, G.; Cumming, P.; Fibiger, H. C. J. Neurochem. 1991,57,701-707. (31) Kihara, T.; Ikeda, M.; Mataushita,A. Brain Res. 1990,519,44-49.
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Time (rnin) Figure 7. (Panel A, lop): Time course of dialysate DA ( D h ) for four groups of rats (n = 5 per group) receiving either 0 nM DA,, (open chcles), 10 nM DAh (filled circles), 20 nM DA,, (open triangles), or 40 nM DAh (filled triangles). Results were obtained uslng 4-mm probes Inthe striatum flowingat a rate of 0.6 pL/mln. Samples were collected every 15 mln. After three consecutive samples were collected which varied less than 10% from each other, an Injection of haloperidol (1 mg/kg, lp) was administered. (Panel B, Bottom): Tlme course of extracellular DA (open circles)and probe recovery (filled circles) In the striatumfollowing haloperidol(1 mg/kg, Ip). Recoveryand extracellular DA were calculatedas shown In F w e 2. Error bars are SEMs. Average baseline recoveryand extracellular DA were 76 & 2% and 5.9 f 1.1 nM, respectively. Extracellular DA rose to 12.4 f 0.7 nM 60 mln following the drug while recovery remalnedunchanged. Error bars are SEMs.
40 nM DAi,. Following three baseline points, an injection of haloperidol (1 mg/kg, ip) was administered. Samples were collected every 15 min. Recovery and extracellular concentration were calculated for each sampled time interval as described above and are shown in Figure 7b. During baseline, the average recovery was 76.0 f 2.0%,and the extracellular concentration was 5.9 f 1.1 nM. The recovery is higher here than in the amphetamine and cocaineexperiments due to the longer probe length (4 mm versus 2 mm) employed in this study.'g Forty-five minutes after haloperidol administration, extracellular DA rose to 12.4 f 0.7 nM and recovery did not change (75 f 7% p C 0.05, n.s.1. Extracellular DA did not return to baseline within 180 min, at which time the collection of samples was stopped. Figure 8a compares extracellular DA with DLUtfor the 0 nM group. D k U trose from 4.2 f 0.7 to 8.9 f 1.4 nM 45 rnin following the drug. The difference between the two groups is not as large as with the studies using cocaine and amphetamine because recovery remained unchanged throughout the time course of the experiment. Figure 8b compares the data from the two groups when expressed as percent baseline. D k U t for the 0 nM group rose to 212%, and extracellular DA rose to 210% of baseline 45 rnin following the drug. This experiment demonstrates the method for transient conditions using four groups of five subjects (n = 20) rather than three groups of four subjects (n = 12). The error
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Time (Min) Flgwo 6. (Panel A, Top): Comparison of the time course of D%, for the group receivlng 0 M DAh (open cirdes; n = 5) to exiracellular DA (filled ckcbs; n = 20) In the strletum folbwhrg haloperkkl(1 mg/kg, ip). Error bars are SEW. (Panel B, Bottom): Comparison of the percent basellne of D%, for the group receMng 0 nM DAh (open circles; n = 5) and extracellular DA (filled ckcles; n = 20) following haloperidol (1 rnglkg, ip). associated with predrug extracellular DA and recovery is similar to the values obtained using the three-group design. This indicates that the use of three groups of animals is probably sufficientfor conductingthe experiment. Less error was observed in extracellular DA and recovery during the region of maximum change after haloperidol challenge than was seen following amphetamine or cocaine challenges. This may be due to the increased number of animals or to the smaller increase of DA induced by haloperidol as compared to cocaine or amphetamine. These experiments were designed to monitor extracellular DA under transient conditions induced by drug challenge. Although we did not observe a change in recovery following haloperidol administration, it is not appropriate to conclude that haloperidol will not alter recovery under other experimental parameters. It is possible that the experimental conditions which were employed were not appropriate for detecting a change in slope. The longer probe length used in this experiment produced a high recovery (76%). Conditions of high recovery are less susceptible to change from the active processes which can otherwise alter recovery.8Thus, as recovery approaches 100?6 ,active processes such as release and uptake will have less effect and become necessarily smaller, bounded above by the equilibriumconditionof 100% and below by recovery in the absence of any active processes. This implies that conditions of high recovery may be more (32) Parsons,L. H.; Schad, C.; Justice, J. B., Jr. J. Neurochern. 1992, in press.
suitable for estimating changes in extracellular concentration for conventional experiments which involve the use of drugs known to affect recovery. Conditions of high recovery may be reached by increasing probe length or by decreasing the flow rate. Probe length is limited by the size of the structure under study. Flow rate considerations for sampling interval and sample size have been discussed previously.ls Summary. A method is presented which allows the quantitative measurement of the extracellular concentration of analytes under transient conditions. The method also provides the in vivo recovery as a function of time. The present approach extends a method which has been shown to be applicable in the steady state to the more widely useful conditions of transient response to drug challenge. There are several advantages to this approach. One is that the concentration is determined independently of any change in recovery. Also, the addition of the analyte of interest to the perfusate offers the analytical advantage of increasing the signal while quantitating low levels of neurotransmitters and other compounds in the extracellular fluid. Another benefit of the approach is that one of the experimentalgroups corresponds to the conventional microdialysis method, providing these data as part of the results. The major disadvantage of the method compared to other techniques is that more subjects are required. In one of the experiments, four groups of five animals were used (n = 20). However, comparison of the magnitudes of error in the extracellular concentration and recovery demonstrate that three groups of four animals (n = 12) are adequate. Thus, the addition of two more perfusion concentrations to the conventional experiment (perfusion with 0 DA) provides significantly more information in microdialysis than may be obtained perfusing with the 0 nM concentration alone. The method also provides the in vivo recovery of the probe as a function of time. The data obtained in this experiment are consistent with previous reports which suggest a possible dependenceof probe recovery on active processes in the tissue surrounding the probe. The recovery may change over time in pharmacological studies of drugs which affect release and uptake. Following the administrationof drugswhich decrease recovery, the change in dialysate concentrationobtained from conventional microdialysis experiments underestimates the change in extracellular concentration. Other pharmacological agents may produce no change or may increase recovery. The effect on recovery can be minimized by operating the probe at near equilibriumconditions (Le.,slow perfusion flow rates) which minimize perturbation of the neuronal environment. However, experiments, in which information concerning the dynamics of active processes is desired,12J3@ are best conducted under conditions where recovery is maximally affected.
ACKNOWLEDGMENT The authors gratefully acknowledge Azhar Nizam for providing assistance with statistics. We also thank Benjamin Liem and Irfan Imami for their help with this research. J.B.J. acknowledges support from the NIDA Research Scientist Development Award KO2 DA00179. This work was supported by NSF Grant BNS9111617.
RECEIVED for review August 19, 1992. Accepted for publication December 31, 1992.
