Fast cyclic voltammetry: measurement of dopamine in the presence of its biological precursors and metabolites

125

J. Electroanal Chem., 283 (1990) 125-133 Elsevier Sequoia S .A ., Lausanne - Printed in The Netherlands

Fast cyclic voltammetry : measurement of dopamine in the presence of its biological precursors and metabolites Sepehr Hafizi, Zygmunt L. Kruk and Jonathan A. Stamford Deportment of Pharmacology, The London Hospital Medical College, Turner Street, London El 2AD (Great Britain) (Received 28 September 1989; in revised form 29 November 1989)

ABSTRACT In vivo voltammetry is widely used to measure dopamine concentrations in the extracellular fluid of the animal brain . However, a potentially serious analytical problem is the presence in vivo of other compounds that may interfere with electrochemical measurements of dopamine . The present study examined the effects of physiological concentrations of various biological precursors and metabolites of dopamine on the detection of the parent compound with fast cyclic voltammetry at carbon fibre microelectrodes. No compound affected the potentials of the oxidation or reduction peaks for dopamine . Homovaoillic acid caused an apparent increase in the sensitivity of the electrodes to dopamine while ascorbic acid decreased the height of the reduction peak . Effects were small and we conclude that physiological levels of precursors and metabolites do not constitute a hazard to the voltammetric detection of dopamine in vivo using fast cyclic voltammetry .

INTRODUCTION

In vivo voltammetry is an increasingly popular method for the measurement of transmitter release in the brain [1] . Fortuitously, of the various small molecules that serve as transmitters, a few (primarily the catecholamines and indoleamines) are oxidizable within the usable potential range of solid electrodes and thus theoretically detectable by electrochemical methods [2] . Of particular interest is dopamine (DA : 3,4-dihydroxyphenylethylamine) which is a transmitter in the caudate nucleus and limbic system, areas of dysfunction in Parkinson's Disease and schizophrenia respectively [3,4] . The pathophysiological importance of DA has made it a subject of much research, directed particularly at drugs which can enhance or inhibit its release in vivo . In vivo voltammetry allows repetitive readings to be taken without the need to sacrifice the animal [5] . * To whom correspondence should be addressed. 0022-0728/90/$03.50

' 1990 - Elsevier Sequoia S .A .

1 26

Our particular focus is upon the measurement of DA release in response to electrical stimulation of the input nerves in the median forebrain bundle (MFB) [6]. Using fast cyclic voltametry (FCV) at carbon fibre microelectrodes (CFMs) we have measured stimulated DA release in vivo with sub-second time resolution [7] . With charging-current subtraction (8] it is possible to extract faradaic information from current signals with a charging to faradaic current ratio greater than 100 :1 . The brain milieu is however as complex an "electrochemical cell" as one can envisage, and thus the analytical challenge to in vivo electrochemists is the detection of DA in the presence of the other compounds that may confound its selective measurement 19]The extracellular fluid (ECF) of the brain contains high levels of ascorbic acid (AA) [10] . Often this constitutes a serious source of interference since in the presence of AA, DA is catalytically regenerated from its quinone (DOQ), resulting in an artificially amplified DA oxidation current [11] . To a large degree this problem is obviated with FCV by the high voltage scan rates and microscopic geometry of the carbon fibre working electrode (12] . However, the caudate nucleus contains many other compounds that might compromise accurate detection of DA in vivo . Not surprisingly, brain regions with high levels of DA also have higher concentrations of its precursors and metabolites [13] . Since such compounds have structural similarities to DA it is conceivable that they might alter the response of CFMs to DA. Since one cannot remove potential contaminants from the ECF it is necessary to quantify their effect upon DA measurement in vitro in order to extrapolate to the in vivo situation . The objective of the present study was to establish whether physiological levels of metabolites and precursors interfered with the detection of DA by FCV . AA and uric acid were also investigated . Although not metabolically involved with DA, both compounds are present in vivo [14] and constitute potential analytical hazards . EXPERIMENTAL

Electrodes Carbon fibre microelectrodes were made as previously described [15] . Borosilicate glass tubes (2 tnm o.d . ; 10 cm length; Clark Electromedical Instruments GC 200-10) were placed in acetone while single carbon fibres were inserted . Filled glass tubes were pulled in a vertical electrode puller and the protruding fibre was cut to the required length (50-100 g,m) using ratchet-mounted jewellers forceps. Figure 1 shows a scanning electron micrograph of the tip of a finished electrode . The reference electrode was a silver/ silver chloride (Ag/AgCI) disc with a physiological (0 .9% w/v) NaCl solution salt bridge . The auxiliary electrode was a gold pin . Chemicals

Dopamine hydrochloride (DA), sodium-l-ascorbate (AA), L ,8-3,4-dihydroxyphenyalamine (L-DOPA), 3-methoxytyramine hydrochloride (3-MT), 4-hydroxy-3-



127

Carbon fibre

G

Fig . 1 . A carbon fibre microelectrode . Scanning electron micrograph montage of a carbon fibre mieroelectrode of the type used in the present study . The carbon fibre tip is shown protruding from the glass insulation on the right hand side .

methoxyphenylacetic acid (HVA), 3,4-dihydroxyphenylaeetic acid (DOPAC), Lt_yrosine hydrochloride (L-TYR) and uric acid (UA) were obtained from Sigma Chemical Co., Poole, Dorset . Stock solutions were made in 0.01 M hydrochloric acid with further dilutions being made in citrate + phosphate buffer (Na 2HPO, 12 .9 g/l. citric acid 1 .055 g/l : pH 7 .4) . All reagents were of Analar grade and were used as received . Voltammetry

Voltammetry was carried out at 22°C in citrate phosphate buffer using a conventional three-electrode potentiostat (16] . A 1i cycle triangular waveform scanning from -1 .0 V to + 1 .0 V vs . Ag/AgCI, with initial scan from 0 to -1 .0 V, comprised the input voltage to the potentiostat . A voltage scan rate of 300 V s -- ' was used . Scans were repeated at intervals of 0 .6 s throughout the experiments . Data analysis

The potentiostat output (in the form of a current vs time signal) was displayed on a digital storage oscilloscope (Nicolet 3091) and a microcomputer (Tandon Plus) via a CED 1400 analogue-digital converter (Cambridge Electronics Design) . The multi-channel signal averager program (Cambridge Electronics Design) on the Tandon Plus allowed the storage and averaging of 100 sweeps in a single file . Current signals were recorded in citrate + phosphate buffer alone or DA (1, 2, 5, 10, 20 µM), the current values being the average of 100 scans at each concentration . Faradaic current for DA was determined by computerised subtraction of the signals in buffer (consisting solely of the charging current) . To study the effect of the DA metabolites on voltammetric measurement of DA . background current signals were recorded in citrate + phosphate buffer alone or with the addition of DOPAC (10µM), AA (200 µM), UA (10 µM), L-DOPA (1 µM), L-TYR (20 µM), 3-MT (1 µM), HVA (5 µM), or in a mixture of all of the

1 28

aforementioned chemicals . DA (10 µM) was then added to the cell and its faradaic current determined, as above, by subtraction of the averaged background currents . All faradaic signals were stored on floppy disks for subsequent analysis. The peak oxidation and reduction current for DA (i o„ and irea ) were determined using the microcomputer (Tandon Plus) . Anodic and cathodic peak potentials (E., and E,) and charging currents (i ch) were determined on the oscilloscope (Nicolet 3091). Statistics

The redox parameters for DA alone or in the presence of other compounds were compared using a paired t-test . RESULTS AND DISCUSSION

These experiments address a problem pertinent to in vivo voltammetric research . During the past five years FCV has been used to measure the release of DA by short stimulations of the input neurones [7] . A "background" signal is stored before the stimulation and subtracted from scans taken during the stimulus train . Since the charging current is constant, the difference is solely the faradaic current of the compound released by stimulation . In the caudate nucleus the chemical released is DA, confirmed by various chemical and pharmacological means [6]. The "background" signal is primarily charging current with a small amount of faradaic information for other electroactive species present in ECF . The concentrations of these compounds change slowly relative to the rate of DA release during stimulation . Over the time scale of the DA measurements, their concentration is essentially constant . The analytical question is, therefore, whether fixed concentrations of these compounds can alter the faradaic current obtained for DA . We have attempted to mimic, as closely as possible, the situation in vivo . The concentrations used in this study are towards the top of the range found in the rat brain : AA : 200 IM, UA: 10 µM, L-TYR : 20 pM, L-DOPA : 1 µM, 3-MT : 1 µM, DOPAC: 10 gM, and HVA : 5 pM [10,13,17-20] . Initially, the electrodes were calibrated in DA at 1-20 pM, this being representative of the peak levels obtained during in vivo experiments [6] . Figure 2 shows a typical background-current corrected cyclic voltammogram for DA . On first inspection, the voltammogram seems to show features characteristic of an adsorbed reactant. However this is largely a consequence of the very high voltage scan rates used (300 V/s) . At lower (100 mV/s) scan rates, carbon fibre microelectrodes show the plateau behaviour indicative of a diffusion-controlled process [11] . Only when the scan rate is taken to the extremes used in this study does the relatively symmetrical peak-shaped voltammogram develop to the extent seen here . Even so, extension of the anodic scan to potentials above + 1000 mV vs . Ag/AgCI enables a plateau to be observed . With a surface-confined process this would not be the case . Furthermore, the rapid response of the electrodes to changes in DA concentration seen in vivo is inconsistent with a strongly adsorbed species . Current showed good

1 29 4ed v 'Redox 'ox

ratio'

[ed

U E ied -,ono

0

1

,ooo

E aoo /mvtvsAg/AgCI)

Fig . 2 . Redox characteristics of dopamine . A cyclic voltammogram of DA (10 fAM) in citrate- phosphate buffer, pH 7 .4, 22 ° C . The charging current has been removed from the signal by digital subtraction of the current obtained in buffer without DA. Er , is the applied voltage . E0, and E, ed are the potentials of the oxidation and reduction peaks respectively. iox and i r,d are the currents at the oxidation and reduction peaks respectively . The ratio of i,rd to i,„ is here called the "redox ratio" .

Linearity with concentration on both oxidation (r > 0 .987) and reduction peaks (r > 0 .989) as expected for a diffusion-controlled process . 10 AM DA was used for subsequent experiments since this fell in the middle of the concentration range . The effects of the metabolites and precursors were examined upon several aspects of the faradaic signal for DA . Figure 2 shows the parameters measured : oxidation and reduction peak heights (iox and i r,d respectively) and the potentials at which they occur (E0x and Er d ) . The "redox ratio" (i,/i ox j was also measured. Table I summarises the redox characteristics of DA under the conditions of the present study. Eox and Erod showed considerable separation (approx 650 mV) . The value, much greater than the 28 mV predicted for a classically reversible two-electron redox couple, is consistent with a quasi-reversible process [21] . This infers kinetic rather than purely thermodynamic control of the peak positions [22], a TABLE I Redox characteristics for DA (10 µM) in citrate +phosphate buffer, pH . .4,220C Means±s .e.m. of five 7 electrodes . Voltammetric parameters . Scan rate : 300 V/s, scan range - 1 to 1 V vs. Ag/AgCI Parameter

Mean +s .em .

F,,/mV vs . Ag/AgCI Ernd /mV vs . Ag/AgCI i ° ,/nA i,°d /nA Redoxratio/r% 10 3 i°x i ;t

468 .2±14.5 -170.5±12 .2 52.1± 7 .4 34.6± 4.5 69.1± 6 .5 38 .9± 1 .1

13 0 TABLE 2 Effects of various compounds on dopamine redox potentials . All values relative to the peak potentials for DA alone . (Means ± s .e.m ., n = 4/5 .) "Mixture" comprises all of the compounds listed separately below . No values were significantly different from those of DA alone (paired i-test) Compound Mixture Precursors L-TYR

L-DOPA Metabolites 3-MT DOPAC HVA

Concentration/µM

20 1

1 10

5

AE,,,/mV

dE,ra /mV

+11 .9±8.2

+0 .2± 6 .5

+3.6±4.0 -6.7±7 .4

+2 .5+ 1 .8 +6 .1± 2 .3

+0.9±8.1 +2.1±5 .6 +1 .2±4.2

-15 .1±10 .1 +5 .3± 4 .4 -2 .1± 8 .1

-2.5±8.7 +5 .7±3.0

-13 .1±

Miscellaneous

CA

10

AA

200

+5 .6+ 5 .0 5 .2

consequence of the high scan rate, as previously reported for etched carbon fibre electrodes [12] . The oxidation peak height was 52 .1 ± 7 .4 nA . The degree of variation was due to differences in electrode size . When this was taken into account by correcting i,,,, for ic ,,, the variation was reduced showing that oxidation current is proportional to electrode surface area. Reduction peaks were about 70% of the oxidation current values . In vivo DA concentration is typically measured with a sample-and-hold circuit which monitors the current at the DA Eox on the scan [6] . Since ieh is constant, any changes in current on successive scans are taken to be due to DA release . This method, however, assumes that the DA Eax does not change during an experiment since any shifts would mean that a pre-set sample-and-hold gave an inaccurate value of DA concentration . Table 2 shows the effects of the various precursors, metabolites and miscellaneous compounds on the redox potentials for DA . Initially a solution, comprising all of the compounds at their physiological concentration, was tested . The mixture had no significant effect on DA E ox or E.d . Since the effect might be the sum of negative and positive shifts, each component was tested individually . None caused a significant shift in the redox potentials of DA . Thus the sample-and-hold measurement method is not compromised by metabolites at physiological concentration . However, the actions of the various neurochemicals upon peak heights were more complex (Table 3) . Again the mixture was tested first . Whereas no effect on DA i s was observed, a modest though not significant decrease in fi red occurred . The redox ratio showed a significant (22 .5%) decrease (relative to the value for DA alone) . As before, individual components of the mixture were then assessed alone . Only HVA

13 1 TABLE 3 Effects of various compounds on dopamine peak heights . All values are mean ±s .e.m . (n = 4/5) . Values are expressed as percentage of those for DA alone Compound

Concentration/

% of values for DA alone

NM

DA i„x

Mixture

DA i,

103 .9±1 .3

83 .7+7 .4

Redox ratio 78 .5-6 .4

Precursors L-TYR L-DOPA

20 1

100.3±0.6 103 .4±2 .6

101 .5±0 .3" 103 .4±1 .6

101 .2-0 .5 100 .1-1 .0

Metabolites 3-MT DOPAC HVA

1 10 5

95 .9±2 .2 100 .0±1 .5 104.7±1 .0 "

95 .1±3 .7 101 .6±1 .1 111 .0±1 .9 "

99 .3-3 .8 101 .5-_0 .9 106 .0+1 .4 1

102 .1±0 .9 100 .8±0 .5

102 .4+1 .0 75 .3-5 .2"

100 .3+0 .9 74 .7+5 .2"

Miscellaneous UA AA

10 200

P < 0 .05. " P < 0 .01 vs. DA alone (paired i-test).

had a significant effect, causing a small increase (4 .7%) in i„ x . This was matched by an 11% increase in i fed and a 6% rise in the DA redox ratio . L-TYR had no effect on DA i 0% but caused a significant increase in i,,d . The small size (1 .5%) and absence of significant change in the redox ratio make it likely that the significance is artefactual . The causes of the HVA effect are unclear . HVA has been reported to adsorb heavily at carbon surfaces resulting in electrode "poisoning" [23], although the effect seen here is a sensitisation of the electrodes to DA . One possible explanation is that HVA, being acidic, associates with basic sites on the electrode surface which would otherwise repel DA, a cation . This matches the facilitation or inhibition of electron transfer by surface charge alteration previously described for other carbon surfaces [24] . Further support is obtained from the 3-MT data . At 1 µM, 3-MT seems to cause a slight (not significant) decrease in DA sensitivity . At 10 µM (albeit a non-physiological level), this was much clearer with a 15% drop in sensitivity to DA . Since 3-MT is basic and also adsorbs onto the electrode, a net increase in surface positivity might lead to a reduced attraction for DA . Further studies are necessary to establish this. The clearest effect on DA redox characteristics was observed with AA . While having no effect on the oxidation peak, the DA i,,,d and redox ratio were decreased by about 25% . This effect is similar to that previously reported at etched carbon fibre electrodes [12] and is evidence of electrocatalysis . Interestingly, whereas at etched electrodes a 15% decrease in DA i fed was observed at 1 mM AA, the present study shows a greater effect at a lower AA concentration .



1 32

-1000 E app

0

1000

JrnV(vs Ag/AgGD

Fig . 3. Concentration dependent effect of ascorbic acid on the redox properties of dopamine . A series of superimposed charging-current corrected cyclic voltammograms for DA (10 AM) in citrate+ phosphate buffer, pH 7 .4, 22 ° C. In each case the faradaic current for DA alone is obtained by digital subtraction of scans obtained in buffer containing AA at the concentrations shown . The AA faradaic current is not included .

Figure 3 shows a series of superimposed cyclic voltammograms for DA in the presence of AA . No effect on DA i°x was observed although the "tail" of the peak was raised . However, a concentration-dependent decrease in i d was seen over the 100-800 tM AA range . Thus measurements of DA concentration based upon peak i,,, are unaffected although iced values are altered by changes in AA concentration . Nevertheless, even drugs known to release AA in vivo have only modest effects upon ECF AA concentrations [25,26] and changes of the size used here to illustrate the response in vitro are unlikely to occur in vivo. In conclusion, the study has shown that it is possible to measure DA accurately on the basis of i, . Physiological levels of DA precursors and metabolites do not alter DA peak positions and have little or no action upon peak heights . The slight electrode sensitising effect of HVA is largely counteracted by the opposing action of 3-MT . The electrocatalytic action of AA means that increases in AA concentration can decrease the DA reduction current although changes in the physiological range are unlikely to have a significant effect . We conclude that fast voltammetric measurements of DA in vivo are unlikely to be affected by the presence of physiological levels of these neurochemicals . ACKNOWLEDGEMENTS

This research was funded, in part, by The Wellcome Trust of Great Britain . J .A . Stamford is a Royal Society University Research Fellow . REFERENCES I J.A . Stamford, Brain Res. Rev., 10 (1985) 119 . 2 R .N . Adams and C.A . Marsden in L.L Iversen, S .D. Iversen and S .H . Snyder (Eds.), Handbook of Psychopharmacology, Vol . 15, Plenum Press, New York, 1982, Ch . I-

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