Anal. Chem. 1986, 58, 1033-1036
1033
Effect of Electrocatalytic and Nucleophilic Reactions on Fast Voltammetric Measurements of Dopamine at Carbon Fiber Microelectrodes Jonathan A. Stamford Department of Pharmacology, The London Hospital Medical College, Turner Street, London El ZAD, UK Eiectrocataiytic and nucleophilic reactions constitute a potentially maJor hazard to the accurate voltammetric measurement of doparnine in vivo. The extent of such interactions was assessed in vltro using high-speed cyclic voltammetry at carbon fiber mlcroelectrodes. Both ascorbic acid and giutathione decreased the height of the dopamine-o -quinone reduction peak but did not affect dopamine oxidation peak height. The effect was dependent on the electrode dimensions and voltage scan rate. Although the reactions seem to occur in vivo, they do not interfere with accurate measurement of dopamine at high-voltage scan rates.
Dopamine ((3,4-dihydroxyphenyl)ethylamine,DA) is a neurotransmitter in the mammalian central nervous system (CNS). It is present a t high concentration in the caudateputamen complex ( 1 ) where it is released from the terminal synapses of the nigrostriatal pathway ( 2 ) . Electrical stimulation of the axons of this pathway has been shown to release DA into the extracellular fluid of the caudate-putamen (3), which can be monitored by voltammetric methods (4-6). Such studies have yielded much information on the dynamics of DA release and uptake. In some experiments untreated carbon fiber microelectrodes were used to measure the DA released by high-intensity stimulation. These electrodes allow very high temporal resolution of DA concentration changes, but have a detection threshold around 1pM ( 4 , 5 ) . Other studies have used more sensitive electrically pretreated carbon fiber electrodes to measure DA release following lowintensity stimulations, which raise extracellular levels of DA into the high nanomolar concentration range (6). Such techniques have, however, a poorer time resolution because of adsorption of DA onto the electrode surface. This study concerns the use of untreated carbon fiber electrodes. A major problem of in vivo voltammetric techniques is response selectivity (7, 8). The mammalian CNS contains, in addition to DA, other electroactive neurotransmitters (e.g., noradrenaline and 5-hydroxytryptamine) and other readily oxidizable compounds (ascorbic acid (9),glutathione (IO),uric acid (11),et al.). Judicious placement of the working electrode into certain brain regions can reduce the interference of the neurotransmitters. The caudate-putamen, for example, has much less noradrenaline and 5-hydroxytryptamine than DA (12,131. The interference of the nonneurotransmitters is not so amenable to this strategy. The compounds are ubiquitous throughout the CNS, and their extracellular concentrations often exceed those of the neurotransmitters by at least an order of magnitude. The mere presence of such compounds complicates the measurement of neurotransmitters. In addition, there is good evidence that the concentration of some of these chemicals can change in response to pharmacological manipulations ( 1 4 , 1 5 ) ,thus confusing in vivo voltammetric data still further. Of particular interest is the specific problem of measurement of dopamine in the presence of ascorbic acid (AA) or glutathione (GSH). High-speed cyclic voltammetry (HSCV) has been used with carbon fiber microelectrodes successfully
to measure the DA released in vivo by ionophoresis (16)and by electrical stimulation of the nigrostriatal nerves ( I 7). Pharmacological studies have indicated that DA is the sole contributor to the voltammogram obtained on stimulation (18). However, such studies do not rule out the possibilitiy that the DA concentration measured is “amplified” by electrocatalysis of AA oxidation (19)or by a nucleophilic reaction with GSH. Although catechol-mediated electrocatalysis of AA oxidation is used in vitro as an analytical aid (20-22), in vivo its occurrence is undesirable. The observation of electrocatalysis depends on the relative potentials at which AA and DA oxidize. When AA oxidizes at a lower anodic voltage than DA (23), electrocatalysis cannot happen, whereas shifting AA oxidation to higher potentials allows it to occur. The reaction requires the simultaneous presence, in the vicinity of the electrode surface, of dopamine-0-quinone (DOQ) and reduced AA. The reaction also depends on the irreversibility of AA oxidation. Dehydroascorbate (DHA), the oxidation product of AA, hydrates rapidly in aqueous solution and cannot be reduced (24). The first-order rate constant for this reaction is 1.42 X lo3 s-l (25). Interestingly at 1000 V s-l it has been reported that a small DHA reduction peak can be detected at mercury microelectrodes (26). However, at the carbon fiber microelectrodes used in this study, no reduction current was observed. When DA is oxidized by the working electrode to DOQ (eq 1) (27),some DOQ chemically oxidizes AA to DHA (eq a), at the same time reducing itself back to DA (28). Thus, in the presence of AA, DA may be repetitively oxidized leading to an amplified DA oxidation current (29). GSH can also react with DOQ by nucleophilic attack at the 6 position of the catechol ring (eq 4) (27). The product of this reaction is voltammetrically indistinguishable from DA (30) and oxidizes to its corresponding quinone (eq 5 ) . This report describes the electrocatalytic and nucleophilic reactions of AA and GSH with DA. The influences of electrode size and voltage scan rate were also investigated. Uric acid was also examined briefly. However, no effect on DA oxidation or reduction characteristics was observed at a uric acid concentration M) 10-20 times greater than the physiological extracellular level in rat striatum (31). The interference with DA oxidation is, therefore, not discussed.
EXPERIMENTAL SECTION Electrodes. Carbon fiber microelectrodes were constructed as previously described (32). Borosilicate glass tubes (2 h m o.d., 10 cm length; Clark Electromedical Instruments GC 200-10) were placed with one end in a petri dish filled with acetone. The acetone completely filled the tubes by capillarity. Individual carbon fibers (10cm length, 8 pm diameter; Courtaulds Grafil XA-S) were floated down the tubes, and the fluid was drained off (some work used 4-pm-diameterfibers). In addition to making the filling process easier, the acetone also removed the bonding resin from the carbon fiber surface, which otherwise impeded electron transfer. Once filled, the ends of the tubes were sheathed in Heatshrink (RS Products), placed in a vertical electrode puller (Model 2001, SRI Instruments). The furnace temperature setting‘ was typically
0003-2700/86/0358-1033$01.50/0a 1986 American Chemical Society
1034
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
O 0
x
y
'
HO
+
Figure 1. Carbon fiber microelectrode. Photomontage of sequential scanning electron micrographs showing the etched conical tip of a single fiber encased in glass sheath.
0
0
O$:
0
0 0 HO ""QH
__I
+,,
O 0
n
R +R2SH 1
O
-
2
:
no
.ZH*
a ZC-
8090 of maximum, and the magnetic puller was set to 6090. Under
these conditions the glass contracted around the carbon ring fiber to produce a tight seal. Once pulled, the electrodes were examined under a microscope to ensure that the glass-bcarbon seal was adequate. Electrical connection with the carbon fiber was made with thin (0.5 mm diameter) plastic-insulated copper wires whose ends had been stripped of insulation and dipped into electridy conductivesilver paint (RS Products). The carbon fiber protruding beyond the glaas was then etched electrochemically in concentrated acid dichromate solution (33) to a conical tip (51, lengthdiameter ratio, Figure 1). The electrodes were washed in distilled water and stored in phosphate buffered saline (PBS, pH 7.4) until use. Silver-silver chloride (Ag/AgCI) reference electrodes were used throughout. The electrodes were constructed from 4-cm lengths of Teflon-coated silver wire stripped of covering for 2 em at one end and coated in AgCl by anodizing at +1.5 V in 1.0 M HCI for 5 min until a light gray Ag/CI coating appeared. The electrodes were washed in distilled water. Salt bridges consisted of 2-an-long plastic pipet tips (Eppendofl tilled with phpiolOgical(O.9% w/v) NaCl solution. A small packing of cotton wool at the tip prevented fluid leakage. The auxiliary electrode consisted of a 4an length of platinum wire (0.5 mm diameter, Goodfellows Metals, Ltd.). Chemicals. DA hydrochloride, sodium 1-ascorbate, and reduced glutathione were obtained from the Sigma Chemical Co., Poole, Dorset, and used as received. All other chemicals were of standard reagent grade. Solutions were prepared in PBS (0.15 M, pH 7.4) immediately prior to use. Voltammetry. Voltammetry was performed in still solutions at 20-22 O C . A conventional three-electrode locally constructed
potentiostat was used throughout. Because of the high-voltage scan rates being used, a Faraday cage was not needed for accurate current measurements. The input voltage to the potentiostat consisted of 1'/* cycles of a triangular wave form scanning from -1.0 to +1.0 V vs. Ag/AgCI. The initial scan direction was cathodic. The 1'/2cycle wave form was derived from experiments in vivo where the initial cathodic scan was used to test for the presence of oxidized species in rat brain extracellular fluid. To enable direct comparison with in vivo data, the same wave form was used in this study (see ref 18). The frequency of the triangular wave form was adjusted to give voltage scan rates of 30, 300, or 3000 V sd. The output from the potentiostat, in the form of a current vs. time signal, was displayed on a digital storage oscilloscope (Nicolet Explorer 3 series). This had the facility to store multiple wave forms and to perform electronic subtractions of one wave form from another. Because of the high-voltage scan rates, a charging current was seen that was substantially larger than the faradaic current. Therefore, to examine purely faradaic current, a background signal wave form was stored in one channel of the oscillmpe. Background current signals were recorded in PBS alone M) or GSH MI. DA or with the addition of AA M) was then added to the cell. The test wave form containing dopamine redox information was stored in the other channel. The pure DA faradaic current was derived by subtraction of the background current from the test current signal. Some experiments used a Gould OS 4100 oscilloscope. In one experiment, to study the concentration dependence of nucleophilic addition, DA (5 X lo* M) w88 used. The effect of AA or GSH was examined over a tenfold range (5 X lo4 to 5 x M). The peak oxidation and reduction current for DA ( i , and iw), anodic and cathodic peak potentials (Ep8and Ew), and peak charging currents (ieh) could be determined directly from the oscilloscope by use of the on-screen numerics (Nicolet Explorer 3). Wave forms were also stored on magnetic disk for subsequent analysis. R E S U L T S AND DISCUSSION DA oxidation a t etched carbon fiber microelectrodes in PBS is a quasi-reversible process (34). At 300 V. s-', E,. is 459 + 14 mV and E, is at -77 21 mV vs. Ag/AgCI. This peak separation is much greater than the 58/n mV predicted for a c l a s s i d y reversible redox system. The peak separation also increases as the voltage scan rate rises. For a revenible system this would not be seen (35). T h e DA redox process at these electrodes shows evidence of slight reactant adsorption. A plot of i p S / W 2against V (voltage scan rate) has a slight gradient (not shown). For a classical diffusion-controlled process the oxidation current should be proportional to VI2(34,36),and thus the graph of i,/V'/2 vs. V should parallel the x axis. Also, the sharp decrease in iw/ipabetween 300 and 3000 V s-' (0.76 to 0.55) supports a reactant adsorption mechanism (35), although kinetic control of the reaction rate may also he a determinant a t this very high scan rate (37). Interestingly, the oxidation of AA at these electrodes appears to be diffusion controlled. Plots of ip./ V/*vs. V are parallel to the x axis (not shown). It has also been shown by other groups that sharp oxidation peaks for catechols are
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
Table I. Influence of Voltage Scan Rate on Nucleophilic and Catalytic Reactions
Table 11. Influence of Electrode Size on Catalytic and Nucleophilic Reactionsu reaction at the following electrode diameters
reaction (nA) at the following voltage scan rates 300 V s-l 3000 V s-l 30 V s-l DA ,i + AA
M) M)
+ GSH DO$ i,
+ AA + GSH
M) M) M)
11.75 f 0.85" 60.38 f 8.30 239.3 f 39.8 10.33 f 1.29 58.60 f 7.58 229.5 f 41.3 (87.9%) (97.1%) (95.9%) 12.33 f 1.00 55.75 f 7.58 232.5 f 36.2 (104.9%) (92.3%) (97.2%) 8.90 f 0.46 46.15 f 4.71 130.5 f 22.6 6.13 f l . l O b 39.23 f 4.8gC 128.8 f 19.6 (98.7%) (68.9%) (85.0%) 3.90 f 0.54' 23.78 f 7.22' 83.3 f 18.1b (63.8%) (43.8%) (51.5%)
*
a Means f standard error of mean (n = 4). P vs. controls in PBS alone (paired t test).
1035
8 Irm
4 Irm
ich, nA (peak charging current) 372.5 f 17.ObtC 128.0 f 12.4 DA ipc/$, % 77.9 f 4.6' 67.1 f 3.2 DA ipa/Lchi % 16.6 f 3.0d 25.8 f 4.9 Aipc DOQ, % (+ AA, M) -15.4 f 3.5e -5.5 f 7.2 Ai,, DOQ, % (+ GSH, M) -51.5 f 10.5c -7.4 f 5.2
"All parameters measured at 300 V s-l, DA M). bMeans f standard error of means (n = 4). c P < 0.01 (student's t test). d P < 0.02 (Student's test). e NS, not significant.
< 0.05. 'P < 0.01
frequently adsorption-controlled, whereas AA peaks a t the same electrodes often retain diffusion control (29,36,38,39). The relative oxidation peak positions of DA, AA, and GSH determine the possible significance of a catalytic or nucleophilic reaction. The AA EOxis typically very variable at carbon electrodes and subject to small changes in the nature of the electrode surface (28). The position often reflects the age of the electrode and is frequently independent of the catechol E,, (40). Under the present experimental protocol AA oxidizes about 100 mV later than DA and is thus available for catalysis. GSH is not electroactive over the potential range studied. A slight rise in current toward the anodic scan reversal point (+1V) is occasionally seen, consistent with previous data (7). This, of course, means that the AA and GSH reactions have different time scales. The electrocatalysis of AA oxidation by DA should occur up to the voltage where AA is oxidized directly by the electrode (eq 3) (25),whereas the nucleophilic reaction of GSH with DA (30) is not terminated by the oxidation of GSH and thus continues until direct reduction of DOQ by the electrode. Most studies of electrocatalysis have concentrated on the influence on i, However, it is obvious that any reaction which regenerates the reduced electroactive species (eq 2) or a similar structure (eq 4) should also affect .,i Table I shows that GSH and AA exert no effect on i, a t any value of V. The effect of AA and GSH on DA oxidation current is to broaden the "tail" of the oxidation peak. Integrated DA oxidation current is consequently elevated by AA and particularly by GSH. Conversely, i, is consistently decreased by AA and GSH. In both cases the percentage decrease in i, is greater a t lower M, the effect of GSH is greater than that values of V. At of AA, irrespective of V. Table I1 shows the effect of electrode size on the catalytic and nucleophilic phenomena. The nucleophilic reaction of DA with GSH is dramatically reduced at the smaller electrode. The effect of AA also seems to be reduced, but not significantly. Thus i t is clear that the influence of electrocatalytic and nucleophilic phenomena on DA oxidation is determined by the voltage scan rate (V) and the electrode size. The decrease in i, for DOQ is proportional to the square root of the concentration of AA or GSH, supporting previous data (29). Figure 2 illustrates the case for GSH. Microelectrodes exhibit enhanced mass transport characteristics (41,421. The natural consequence of this is that for ratio is decreased the DA-DOQ redox couple, the i,,/i,, compared with the values seen a t larger electrodes because a smaller fraction of the DOQ remains at the electrode surface to be reduced back to DA. This is further supported by the data of Table 11. The ipc/iparatios are significantly smaller a t the 4-pm-diameter electrodes than at the 8-pm versions.
I 0
50
100
m d Flgure 2. Effect of glutathione on DOQ reduction current: DOQ reduction height (expressed as percentage of the value in phosphate buffered saline alone) for a 5 X M DA solution, plotted against the square root of the glutathione concentration. Each point is the mean f standard error of the mean (n = 3). The line is a regression plot (correlation coefficient = 0.995).
I t is also noteworthy that the ipa/ich value for the 4-pm electrodes is higher than for the 8-bm equivalents. This is consistent with a model of accelerated mass transport with decreasing electrode size. A similar phenomenon has been observed with chronoamperometry at carbon fiber electrodes where current on the reverse step is almost entirely nonfaradaic (43, 44). The enhanced mass transport also reduces the fraction of DOQ that, on reduction, returns to the electrode surface to be oxidized again (28). Thus, the effects of catalytic and nucleophilic reactions are attenuated. The influence of electrocatalysis and nucleophilic substitution is also determined by the time allowed for the process. At 3000 V s@the period between the DA oxidation and reduction peaks is much shorter than at 30 V s-l, and consequently, the influence of the catalytic and nucleophilic reactions is much reduced. The first-order rate constant for nucleophilic substitution of GSH into DOQ is between 208 and 480 s-l (27,30). Thus, a t the highest voltage scan rates the kinetics of the reaction may be insufficiently fast for it to occur in the time between DA oxidation and reduction. By use of these principles, it is clear that the amplification of DA oxidation by AA or GSH should be directly related to the size of the measuring electrode and also to the voltage scan rate. Slow voltage scans a t relatively large electrodes show much greater (several fold) enhancement of DA oxidation current (29). As stated earlier, the etched electrodes used in this study show a small degree of reactant adsorption, though much less than some other treated carbon fiber electrodes (23,45). This itself would be expected to enhance the catalytic and nucleophilic reactions. Electrodes with highly adsorbed or chemically attached catalysts (21, 22, 46, 47) show more
1036
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
I
I
20nA
N-(N-L-y-glutamyl-L-cysteiny1)glycine = glutathione.
20nA
n
Registry No. DA, 51-61-6;AA, 50-81-7;GSH, 70-18-8;DOQ, 50673-96-6.
LITERATURE CITED
EOPP
V
I
I
IO
-
IN VITRO
0
VI
Ag/AgCI
+
1
IO
EOpp I
10
V
-
I
0
VI
Ag/AgCI
+
1
1.0
IN VIVO
Flgure 3. Cyclic voltammograms of DA in vitro and in vivo: two background current-corrected high-speed cyclic voltammograms of DA (300 V s-' scan rate). The trace on the left shows the response from an electrode in a solution of DA (2 X lon5M) in phosphate buffered saline. On the right is a voltammogram from the same electrode implanted in the caudate nucleus of an anaesthetized rat in which DA release was evoked by electrical stimulation. I n vivo voltammetric parameters used were as in vitro. Experimental conditions (surgery, stimulation, etc.) were as described in ref 18. Note the decreased ipc/ipa ratio in vivo.
pronounced reactions than when the catalyst is freely diffusible. When implanted into brain tissue, etched microelectrodes rapidly lose much of the DA adsorption and behave more like unetched microelectrodes (unpublished observations). Unetched carbon fiber microelectrodes show good diffusion control, and these electrodes are finding increasing favor for rapid voltammetric measurements in vivo (48,49). With such electrodes the catalytic reaction is likely to be even further minimized. The experiments described here use M AA or GSH. These concentrations are representative of cerebral levels (10, 50). At these concentrations, despite the effects on the reduction peak, the oxidation peak height is unaffected. The extracellular levels of AA may, in fact, be much lower than M (23, 51). It has recently been shown that, 3 h after intraperitoneal administration of AA (1.76 g/kg), the striatal extracellular AA concentration does not exceed M (49). The extracellular level of GSH may also be considerably lower than the whole tissue levels, since GSH is considered to be mainly intracellular and GSH depletion is used as an index of cell injury. Figure 3 shows that catalytic and/or nucleophilic phenomena probably do occur in vivo and affect the reduction peak for DOQ. The scan, on the left, in PBS shows a higher ipc/ipa ratio than the comparable scan in vivo. Although measurements of DA concentration based on i, values are likely to be accurate, it is clear that the ipc/i,, ratio for the DA-DOQ couple cannot be used quantitatively to define reaction reversibility in vivo.
CONCLUSIONS Electrocatalytic and nucleophilic interferences on DA oxidation can be detected a t carbon fiber microelectrodes in vitro. The effects of AA and GSH are detectable mainly on the DOQ reduction peak. In both cases the effects are decreased by fast-voltage scan rates and small electrodes. No effect of GSH or AA on peak DA oxidation current is detectable. Thus, measurements of DA concentration using fast cyclic voltammetry in vivo are unlikely to be affected by AA or GSH concentrations (loT3M) in the high physiological/ pharmacological range.
ACKNOWLEDGMENT I wish to thank Julian Millar and Selwyn Mable for design and construction of the voltammetric apparatus and Mary Stock for secretarial assistance. Redox equations for the reactions discussed in the text are as follows: R1 = CH2CH2NH2= ethylamine and R2SH =
Paikovits, M. I n "Neurobiology of Dopamine"; Horn, A. S . , Korf, J.. Westerink, B. H. C.. Eds.; Academic Press: London, 1979; pp 343-356. Anden, N. E.; Fuxe, K.; Hamberger, B.; Hokfelt, T. Acta Physiol. Scand. 1966, 67, 306-312. Riddell, D.; Szerb, J. C. J . Neurochem. 1971, 18, 989-1006. Ewing, A. G.; Bigelow, J. C.: Wightman, R. M. Science (Washington, D.C.) 1983, 221, 169-171. Stamford, J. A.; Kruk, 2. L.; Millar, J.; Wightman, R. M. Neurosci. Lett. 1984, 51, 133-138. Gonon, F. G.; Buda, M. Neuroscience (NY) 1958, 14, 765-774. Adams, R. N.; Marsden, C. A. "Handbook of Psychopharmacology"; Plenum: New York, 1962; Chapter 1. Stamford, J. A. Brain Res. Rev. 1985, IO, 119-135. Milby, K.; Oke, Pi.; Adams, R. N. Neurosci. Lett. 1982, 28, 15-20. Martin, H.; McIlwain, H. Biochem. J . 1959, 7 1 , 275-280. Crespi, F.; Sharp, T.; Maidment, N.;Marsden, C. A. Neurosci. Lett. 1983, 43, 203-207.. Oke, A.; Keller, R.; Adams, R. N. Brain Res. 1978, 148, 245-250. Saavedra, J. M. Fed. Proc. Fed. Am. SOC. Exp. Biol. 1977, 36, 2134-2 141. Wilson, R. L.; Wightman, R. M. Brain Res. 1985, 339, 219-226. Mueller, K.; Palmour, R.; Andrews, C.; Knott, P. J. Brain Res. 1985, 335, 231-235.~ Millar, J.; Armstrong James, M.; Kruk, 2. L. Brain Res. 1981, 205, 419-424. Kruk, 2. L.; Millar, J.: Stamford, J. A. Br. J . Pharmacol. 1985, 84, 199P. Millar, J.; Stamford, J. A.; Kruk, 2. L.; Wightman, R. M. Eur. J . Pharmacol. 1985, 109, 341-348. Neweli, G. A.; Coihoun. E. H. SOC.Neurosci. Abstr. 1982, 8 , 252.72. Evans, J. F.; Kuwana, T. J . Nectroanal. Chem. 1977, 80, 409-416. Ueda, C.; Tse, D. C. S.; Kuwana, T. Anal. Chem. 1982, 54, 850-856. Stutts, K. D.; Wightman, R. M. Anal. Chem. 1983, 54, 1576-1579. Gonon, F.; Buda, M.; Cespuglio, R.; Jouvet, M.; Pujoi, J.-F. Brain Res. 1981, 223, 69-80. Lechlen, A.; Vaienta, P.; Nurnberg, H. W.; Patriarche, G. J. Fresenius' 2'. Anal. Chem. 1982, 311, 105-108. Perone, S. P.; Kretiow, W. J. Anal. Chem. 1966, 38, 1760-1763. Wehmeyer, K. R.; Wightman, R. M. Anal. Chem. 1985, 57, 1969- 1993. Sternson, A. W.; McCreery, R.; Feinberg, 6.:Adams, R. N. Electroanal. Chem. Interfacial Electrochem. 1973, 46, 313-321. Dayton, M. A.; Ewing, A. G.; Wightman, R. M. Anal. Chem. 1980, 52, 2392-2396. Kovach, P. M.; Ewing, A. G.; Wilson, R. L.; Wightman, R. M. J . Neurosci. Methods 1984, IO, 215-227. Tse, D. C. S.; McCreery, R. L.; Adams, R. N. J . Med. Chem. 1976, 19, 37-39. Zetterstrom, T.; Sharp, T.; Marsden, C. A,; Ungerstedt, U. J . Neurochem. 1983, 41, 1769-1773. Armstrong James, M.; Millar, J. J . Neurosci. Methods 1979, 1 , 279-287. Armstrong James, M.; Fox, K.; Millar, J. J . Neurosci. Methods 1980, 2 , 431-432. Bond, A. M. "Modern Polarographic Methods in Analytical Chemistry"; Marcel Dekker: New York and Basel, 1980. Schachterle, S. D.; Perone, S. P. Anal. Chem. 1981, 53, 1672-1678. Stutts, K. J.; Kovach, P. M.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1983, 54, 1632-1634. Howeii, J. 0.; Wightman, R. M. Anal. Chem. 1984, 56, 524-528. Gonon, F.; Fombarlet, C.; Buda, M.; Pujol, J.-F. Anal. Chem. 1981, 5 3 , 1386-1389. Falat, L.; Cheng, H-Y. Anal. Chem. 1982, 5 4 , 2108-2111. Plotsky, P. M. Brain Res. 1982, 235, 179-184. Wightman, R. M. Anal. Chem. 1981, 53, 1125A-1130A. Galus, Z.;Schenk, J. 0.; Adams. R. N. J . Electroanal. Chem. 1982, 135, 1-11. Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1980, 52, 946-950. Ewing, A. G.; Dayton, M. A.; Wightman, R. M. Anal. Chem. 1981, 5 3 , 1842-1 847. Marcenac, F.; Gonon, F. G. Anal. Chem. 1985, 57, 1778-1779. Degrand, C.; Miller, L. L. J . Am. Chem. SOC. 1980, 102?5728-5732. Zak, J.; Kuwana, T. J . Am. Chem. SOC. 1982, 104, 5514-5515. Kuhr, W. G.; Ewing, A. G.; Caudill, W. L.; Wightman, R. M. J . Neurochem. 1984, 43, 560-569. Stamford, J. A,; Kruk, 2. L.; Millar, J. Neurosci. Lett. 1985. 60, 357-362. Adlard, B. P. F.; De Souza, S. W.; Moon, S. J . Neurochem. 1973, 2 1 , R77-RR1 -. . - - . . Ewing, A. G.; Wightman, R. M.; Dayton, M. A. Brain Res. 1982, 249, 361-370.
RECEIVED for review October 17, 1985. Accepted December 3, 1985.
