In vivo voltammetric determination of the kinetics of dopamine metabolism in the rat

Neuroscience Letters, 56 (1985) 365-369

365

Elsevier Scientific Publishers Ireland Ltd.

NSL 03327

IN VIVO V O L T A M M E T R I C D E T E R M I N A T I O N O F T H E KINETICS O F D O P A M I N E M E T A B O L I S M IN T H E RAT

A D R I A N C. M I C H A E L I, JOSEPH B. JUSTICE, Jr. t,* and D A R R Y L B. N E I L L 2

Departments of l Chemistry and ePsychology, Emory University, Atlanta, G A (U.S.A.) (Received December 12th, 1984; Revised version received February I lth, 1985; Accepted March 5th, 1985)

Key words." dopamine

3,4-dihydroxyphenylacetic acid - turnover - striatum - voltammetry - electrical stimulation - rat

In vivo voltammetry at carbon paste electrodes placed in the striatum of chloral hydrate-anesthetized rats was used to monitor changes in extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) following lmin periods of electrical stimulation of the ascending nigro-striatal pathways. Statistical analysis of resultant changes in D O P A C allows simultaneous determination of the rate constants for the turnover of both dopamine (DA) (0.046/min) and D O P A C (0.053/min). The data demonstrate that there are substantial temporal differences between neuronal release of D A and changes in striatal D O P A C levels. This should be considered when metabolite measurements are used as an index of neurotransmitter activity.

The metabolic pathway by which dopamine (DA) is catabolized following release from nerve terminals of the striatum has been the subject of extensive studies [4, 7, 12-15]. It has been demonstrated that conversion to 3,4-dihydroxyphenylacetic acid (DOPAC) is quantitatively the main route of DA degradation [12, 14]. The rate constant for the conversion of DA to DOPAC has been inaccessible despite its importance in the understanding of dopaminergic dynamics. In this study we have developed a method by which this rate constant can be measured directly in vivo. We report here on the change in striatal extracellular DOPAC monitored by in vivo voltammetry following brief electrical stimulation of the medial forebrain bundle (MFB) of rats. Our results indicate that there is considerable delay in the appearance of DOPAC in the extraceilular fluid (ECF) following stimulated DA release. Released DA is rapidly cleared from ECF following short periods of electrical stimulation [6]. Therefore, the changes in extracellular DA and DOPAC following stimulation occur at different times. This has enabled us to evaluate directly the rate constants for the appearance and clearance of DOPAC in striatal ECF. The technique for the present study has been chronoamperometry [1] in conjunction with small carbon electrodes. A short electrolysis time and a small electrode surface area minimize the perturbational properties of in vivo voltammetry while allow-

*Author for correspondence.

366 ing a high sampling rate and sensitivity to neurochemical events [10]. An indication that these electrodes do not substantially alter the neurochemical environment of brain tissue is provided by the observation that the use of either 6- or 60-s sampling intervals does not affect the temporal features of the change of the in vivo signal following electrical stimulation. Male Sprague-Dawley rats were maintained under chloral hydrate anesthesia throughout the experiment. Bipolar stimulating electrodes (Plastic Products, Roanoke, VA) were placed in the MFB using the coordinate system of Ewing et al. [5] (2.2 mm posterior to bregma; 1.6 mm lateral; 7.6 mm below dura; vertical placement was individually optimized in each experiment, see below). Carbon paste working electrodes were prepared in glass capillaries of 20-40 ~m o.d. and placed in the anterior portion of the ipsilateral striatum (2.4 m m anterior to bregma; 2.5 mm lateral; 4.0 m m below dura). Reference electrodes consisted of silver wire coated with AgCI and inserted directly into brain tissue. Chloride in the ECF of brain tissue completed the reference electrode. Stainless-steel wire served as the control electrode. Voltammetric recording was initiated immediately following electrode placement. Electrochemistry was performed using a computer-controlled potentiostat-amplitier system [11]. One second chronamperometry at 6- or 60-s intervals was used continuously throughout the experiment. The resting electrode potential was - 100 mV versus the reference described above. The pulse potential was 500 mV versus reference. Two to 3 h were allowed for the oxidation current to reach a baseline signal, following which stimulation sessions were administered. If the stimulation did not result in a change of the recorded oxidation current within 30 min, the stimulation electrode was lowered 0.2 m m and the stimulation repeated. No more than two relocations of the stimulating electrode were needed. Electrical stimulation was performed with a Grass SD-9 stimulator in conjunction with a Grass CCU-1 constant current unit (stimulation parameters: 100 Hz square waves, 1 ms/pulse, 300 ms/train. 1 train/s for 1 rain). Fig. 1A shows an example of an electrochemical experiment performed in the striaturn of an anesthetized rat. At the point indicated, al-min electrical stimulation session was administered. Following stimulation there was a marked increase Jn the chronoamperometric signal that maximized at approximately 20 rain after stimulation and returned to baseline 60 rain after stimulation. We concluded that the observed increase in signal is due to changes in D O P A C because pargyline administration (100 mg/kg; n = 3 ) abolished the result of stimulation (Fig. IB) thus monoamines are not contributors; ~-methyl-p-tyrosine (~-MPT) (200 mg/kg, n--3) also abolished the stimulation effect (data not shown), thus 5-hydroxyindoleacetic acid is not a significant contributor; and homovanillic acid is not significantly oxidized at the applied potential used here. The pargyline and ~-MPT results also indicate that ascorbic acid and uric acid do not contribute to the increase following stimulation of MFB. This is supported by the observation that the compound released during stimulation is voltametrically identical to DA and not to ascorbic acid [5, 9] and by the observation of Blakely et al. [2] that increases in extracellular D O P A C are not necessarily coupled to increases in ascorbic acid.

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Fig. 1. In vivo chronoamperometric oxidation current recorded at 1-min intervals from the striatum of an anesthetized rat versus the time in minutes from stimulation. A: result observed with no drug treatment. B: result observed followingpretreatment with 100 mg/kg pargyline. A and B were recorded from the same electrode in the same rat in a continuous in vivo recording session. The baseline current in A is 0.50 nA which decayed for 3 h followingpargyline injection to a baseline current of 0.29 nA at the time of stimulation in B.

In Fig. 1 there is an increase in the chronoamperometric current immediately following stimulation that is apparently due to DA. The increase is only slightly larger than baseline noise, thus no specific characterization of this signal has been made. When 1-s chronoamperometry is used, the combined removal of D A from the vicinity of the electrode by both tissue reuptake mechanisms and the electrode surface reaction preclude direct monitoring of released DA. Chronoamperometric pulses of 100-ms duration provide a more sensitive D A measurement if the necessary instrumental requirements are considered [3] (unpublished observations). From the results and discussion above it appears that the change in signal following electrical stimulation is due to DOPAC. These data should, therefore, be able to provide kinetic information concerning the appearance and clearance of D O P A C in ECF. To this end, the change from baseline of the voltammetric signal after stimulation has been analysed by non-linear regression. The assumption of first order kinetics has been applied to a number of studies concerned with D A metabolism [4, 13-15]. With this assumption one may write a model for the appearance and clearance of D O P A C in ECF as: D O P A C (t) = A[exp ( - k ] t ) - exp ( - k2t)] which espressed the increase in extracellular D O P A C following electrical stimulation as a function of time in terms of two rate constants, kl and k2, and an amplitude factor, A. k] and k2 represent the rate constants of D O P A C appearance and clearance, respectively. Fig. 2 shows the result obtained from the regression analysis when the data from 6 stimulations were averaged. As seen, there is excellent agreement between the

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Fig. 2. The change from baseline of in vivo chronoamperometric oxidation current versus time in minutes after stimulation. Data points are the average of data from 6 stimulations in 5 rats. Four of the stimulations were at a 6-s sampling interval and 2 at a 60-s interval. The sampling rate did not change the nature of the observed response, therefore data points from the sessions at 60-s intervals were combined with every 10th point from sessions at 6-s intervals. Prior to averaging, the data were not normalized by electrode calibration, therefore the response units are arbitrary. The solid line is the result of fitting the model (see text) to these data. The equation of the line is: f(t)= 1097 [exp(-0.046t)-exp(-0.053t)]. See text for parameter confidence intervals. experimental data and the model, implying that the assumption o f first order kinetics is valid. The value o f the amplitude parameter, A, in arbitrary response units is 1097 (950/o non-linear confidence range o f 1070 to 1123). The response units used in Fig. 2 are arbitrary for the following reasons. The amplitude o f the change in oxidation current following stimulation was variable because o f differences in electrode calibration, placement o f both the voltammetric and stimulating electrodes in vivo and the actual change in D O P A C concentration. F o r the purposes o f the present study, no attempt at estimating a change in D O P A C concentration from the change in oxidation current has been made. Hence, each mean in Fig. 2 represents the combination o f data that have not been normalized yielding arbitrary units. Despite the differences in the signal amplitude, the temporal features o f the response to stimulation were very similar in all cases. This is reflected by the confidence intervals o f the two rate parameters (below). The value o f k2, the rate constant for the clearance o f D O P A C , has been the subject o f m a n y studies [4, 13-15]. The value o f k2 measured here is 0.053 l/rain (95% non-linear confidence range o f 0.0529 to 0.0533/min) and is in g o o d agreement with reported values of 0.056/min by Dedek et al. [4] and 0.056/ min by Westerink et al. [13]. These authors used m o n o a m i n e oxidase inhibition and followed the subsequent change in tissue content o f D O P A C . The value o f the rate constant for the appearance o f D O P A C , kl, determined in this study is 0.0457/min (95% non-linear confidence range o f 0.0455 to 0.0459/min). A l t h o u g h the turnover o f D A has been equated to the turnover o f D O P A C [12, 14], no rate constant for this step has previously been reported. In vivo voltammetry only monitors c o m p o u n d s in ECF, therefore these rate constants must include the effects o f mass transport o f D O P A C from the intracellular c o m p a r t m e n t to the extracellular space. The similarity o f k2 for extracellular D O P A C and total tissue D O P A C [4, 13] indicates that there is a rapid equilibration between

369

intra- and extracellular DOPAC. Therefore, mass transport of DOPAC from the intracellular environment is not likely to be the rate determining step in the appearance of DOPAC in ECF. We interpret kl as the in vivo rate constant of enzymatic DA metabolism in the striatum. Therefore, kl and k2 should be equivalent to the rate constants for the turnover of D A and DOPAC, respectively. Most studies of dopaminergic activity have used metabolite measurements as indices of neurotransmitter activity since it has not been possible to monitor the endogenous transmitter directly. However, one must recognize the differences between the time-course of transmitter release and the change in metabolite concentrations in vivo. This is clearly demonstrated in the data reported above, in which a 1-min period of elevated D A release results in a change in DOPAC concentration that lasts on the order of an hour. This work was supported by NSF Grant BNS 8210773 and the Emory University Research Fund. 1 Adams, R.N. and Marsden, C.A., Electrochemical detection methods for monoamine measurements in vitro and in vivo. In L.L. Iversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook of Psychopharmacology, Vol. 15, Plenum Press, New York, 1982, pp. 1-74. 2 Blakely, R.D., Wages, S.A., Justice, J.B., Herndon, J.G. and Neill, D.B., Neuroleptics increase striatal catecholamine metabolites but not ascorbic acid in dialyzed perfusate, Brain Res., 308 (1984) 1-8. 3 Dayton, M.A., Ewing, A.G. and Wightman, R.M., Diffusion processes measured at microvoltammetric electrodes in brain tissue, J. Electroanal. Chem., 146 (1983) 189 200. 4 Dedek, J., Baumes, R., Tien-Duc, N., Gomeni, R. and Korf, J., Turnover of free and conjugated (sulphonyloxy) dihydroxyphenylacetic acid and homovanillic acid in rat striatum, J. Neurochem., 33 (1979) 687 695. 5 Ewing, A.G., Bigelow, J.C. and Wightman, R.M., Direct in vivo monitoring of dopamine released from two striatal compartments in the rat, Science, 221 (1983) 169-171. 6 Ewing, A.G. and Wightman, R.M., Monitoring the stimulated release of dopamine with in vivo voltammetry. II, clearance of released dopamine from extracellular fluid, J. Neurochem., 43 (1984) 570577. 7 Korf, J., Grasdijk, L. and Westerink, B.H.C., Effects of electrical stimulation of the nigrostriatal pathway of the rat on dopamine metabolism, J. Neurochem., 26 (1976) 579-584. 8 Kovach, P.M., Ewing, A.G., Wilson, R.L. and Wightman, R.M., In vitro comparison of the selectivity of electrodes for in vivo electrochemistry, J. Neurosci. M eth., l0 (1984) 215 227. 9 Kuhr, W.G., Ewing, A.G., Caudill, W.L. and Wightman, R.M., Monitoring the stimulated release of dopamine with in vivo voltammetry. I. Characterization of the response observed in the caudate nucleus of the rat, J. Neurochem., 43 (1984) 560-569. 10 Lindsay, W.S., Justice, J.B. and Salamone, J., Simulation studies of in vivo electrochemistry, Computers Chem., 4 (1979) 19-26. 11 Lindsay, W.S., Kizzort, B.L., Justice, J.B., Salamone, J.D. and Neill, D.B., Microcomputer controlled multielectrode system for in vivo electrochemistry, Chem. Biomed. and Environ. Instrument. 10 (1980) 311 330. 12 Westerink, B.H.C., Further studies on the sequence of dopamine metabolism in the rat brain, Europ. J. Pharmacol., 56 (1979) 313-322. 13 Westerink, B.H.C., Bosker, F.J. and Wirix, E., Formation and metabolism of dopamine in nine areas of the rat brain: modifications by haloperidol, J. Neurochem., 42 (1984) 1321 1327. 14 Westerink, B.H.C. and Korf, J., Turnover of acid dopamine metabolites in the striatal and mesolimbic tissue of the rat brain, Europ. J. Pharmacol., 37 (1976) 249-255. 15 Wilk, S., Watson, E. and Travis, B., Evaluation of dopamine metabolism in rat striatum by a gas chromatographic technique, Europ. J. Pharmacol., 30(1975) 238-243.