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1). A series of 28 repetitive measurements of 2.5 X lo-’ M Pb(I1) over an unbroken period of 3 h yielded highly reproducible peaks with a relative standard deviation of 1.8%, a mean current of 1.83 FA, and a range of 1.75-1.87 pA (conditions, as in the standard additions experiment). The above data illustrate that the electrolyte presence in the detector compartment swamps the effect of variable amounts of electrolyte inadvertently present in the sample. For example, each standard addition of Pb(I1) increased the nitric acid content of the sample by approximately 10“ M (the latter being used for preparing the metal ion stock solution). Similarly, the highly stable response demonstrates that small changes in the composition of the detector solution due to the continuous sample inflow (e.g., increased content of Hg(I1) and associated nitric acid or some dilution) do not affect the performance. The renewable surface of the in situ plated mercury f i i electrode makes it especially suitable for long-run monitoring. In conclusion, the use of a large volume wall-jet detector--with a deaerated electrolytic solution at the detector compartment-permits trace metal analyses of electrolyte-free nondeaerated samples. This would facilitate analyses of nonaqueous matrices and result in increased speed and simplicity of on-line stripping measurements, as well as reduced contamination risks. Such operation is not feasible with the commonly used low-dead volume detectors. It has been shown recently that microelectrodes can be used to obtain undistorted cyclic voltammograms in highly resistive solutions (16). We are presently evaluating this approach for trace metal stripping measurements in such solutions. The present wall-jet detector possesses the additional distinct advantage of using
nondeaerated samples. Unlike the medium-exchange procedure (7, 17), where the stripping step is performed after transferring a more favorable solution to the cell, the favorable detector solution-in the present design-is “active” (with respect to the cell conductivity) during both the deposition and stripping steps. This is important for measurements in electrolyte-free solutions, as conductive medium is essential in both steps.
LITERATURE CITED Vydra, F.; Stulik, K.; Julakova, E. “Electrochemical Strlpping Analysls”; Wlley: New York, 1976. Wang, J. “Stripping Analysis: Prlnclples, Instrumentation and Applicatlons”; VCH Publishers: Deerfield Beach, FL, 1985. Zirino, A.; Lieberman, S. H.; Clavell, C. Envlron. Scl. Techno/. 1978, 12, 73. Wang, J. Am. Lab. (Fairfhld, Conn.) 1983, 7 , 14. Anderson, L.; Jagner, D.; Jsefson, M. Anal. Chem. 1982, 5 4 , 1371. Wang, J.; Dewaid, H. D.; Greene, B. Anal. Chim. Acta 1983, 146, 45. Hu, A.; Dessy, R. E.; Granell, A. Anal. Chem. 1983, 55, 320. Elbicki. J. M.; Morgan, D. M.; Weber, S. G. Anal. Chem. 1984, 56, 978. Gunaslngham, H.; Fleet, B. Anal. Chem. 1983, 55, 1409. Gunaslngham, H.; Tay, 0. T.; Ang, K. P. Anal. Chem. 1984, 56, 2422. Wang, J.; Dewald. H. D. Anal. Len. 1983, 16, 925. Wang, J.; Ariel, M. Anal. Chim. Acta 1978, 99, 89. Hanekamp, H. B.; Voogt, W. H.; Bos, P.; Frei, R. W. Anal. Chlm. Acta 1980, 118, 81. Wang, J.; Dewald, H. D. Anal. Chem. 1983, 55, 933. Copeland, T. R.; Christie, J. H.; Skogerboe, R. K.; Osteryoung, R. Anal. Chem. 1973, 4 5 , 995. Bond, A. M.; Fleischmann, M.; Robinson, J. J. Nectroanal. Chem. 1984, 168, 299. Wang, J.; Greene, B. Water Res. W83, 17, 1635.
RECEIVED for review January 28, 1985. Accepted March 25, 1985. This work was supported in part by the National Institutes of Health, Grant No. GM30913-01A1.
Fast in Vivo Monitoring of Dopamine Release in the Rat Brain with Differential Pulse Amperometry Franpoise Marcenac and Franpois Gonon* Inserm U 171, H6pital Ste. EugBnie, Pauillon 4 H, 1, Avenue Georges ClBmenceau, 69230 St. Genis Laual, France In an attempt to study dopaminergic neurotransmission in the brain, electrochemical techniques have already been applied to monitor in vivo spontaneous as well as evoked dopamine (DA) release from dopaminergic nerve terminals (1-7). Wightman and his group combined chronoamperometry (5) or fast cyclic voltammetry (3) with untreated carbon fiber microelectrodes to monitor evoked DA release after the electrical stimulation of the dopaminergic neuronal pathway. With these techniques the extracellular DA concentration was monitored every 0.25 to every 6 s (2,3). However, due to their low sensitivity (detection limit around 5 pM) their application was, up to now, restricted to the measurement of DA release, provided that it was strongly stimulated by high-frequency electrical stimulations. In order to improve the selectivity of chronoamperometry, Marsden et al. (4) suggested the use of double-step chronoamperometry. However, no applications of this technique have already been reported. In our group we recently combined a new technique, differential normal pulse voltammetry (DNPV) with electrochemically treated carbon fiber electrodes and we were able to detect from the striatum of pargyline-treated rats a signal due to the spontaneous DA release (1,6). Moreover, DNPV allowed us to record the effect of low-frequency electrical stimulations (1). Unfortunately, with that technique, DNP voltammograms were recorded every 1 win. Thus, in an attempt to monitor the kinetics of rapid phenomena, we developed a new technique that is as selective and sensitive as
DNPV but much faster: differential pulse amperometry (DPA). DPA was directly derived from DNPV. It consisted of an unlimited series of dual pulses identical with that used in DNPV but at a constant potential (Figure 1). Like with DNPV, the oxidation current was differentiated during a measuring pulse. The parameter values were chosen like those of DNPV, except for the final potential, that was set at a value slightly under the oxidation potential of DA (+80 mV vs. the Ag/AgCl reference electrode (6)). With DPA, we were able to detect very low variations of DA concentrations,to measure these concentrations every 0.4 s and thus to monitor kinetics of electrically evoked DA release under “physiological”conditions from the striatum of pargyline-treated and anaesthetized rats.
EXPERIMENTAL SECTION Reagents. Dopamine hydrochloride (Sigma) and ascorbic acid (AA) (Merck) were dissolved in a phosphate buffered saline (PBS) solution (KC1,0.2 g/L; NaC1,0.8 g/L; NazHPO4-2Hz0,1.44 g/L; KH2P04,0.2 g/L; pH 7.4). In order to prevent spontaneous oxidation of DA, AA was always added into the solutions before the addition of DA. However, the DA response did not depend on the AA concentration (between 50 and 300 pM). Electrochemical Procedures. The carbon fiber electrodes were prepared as previously described (7). Before each experiment, they were electrochemically treated ( I , 6). Electrochemical treatments, as well as DPA measurements, were performed by
0003-2700/65/0357-1778$01.50/00 1985 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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-m .-m v)
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a D I
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Flgure 1. Potential-time function for DPA. The current is sampled for 10 ms just before (in A) and at the end (in B) of the measuring pulse. The difference of the currents ( i s - i A ) is recorded for every pulse.
I
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Flgure 2. Time course of the DA signal response to variations of DA
and AA concentrations. The values indicated in the figure correspond to the final DA and AA concentrations in the PBS solution. means of a new apparatus (Biopulse, Solea Tacussel, Villeurbanne, France). A classical three-electrode system was used (7). DPA parameters are defined in Figure 1 and their values were T = 0.4 s, t l = 70 ms, t 2 = 40 ms, V = 50 mV, Pi = -240 mV, and Pf= +60 mV. Animals. Male rats (300 & 30 g) were pretreated with pargyline (75 mg/kg, i.p.), in order to pharmacologically suppress the contribution of 3,4-dihyroxyphenylacetic acid (DOPAC) to the DA oxidation signal (1,6). They were anaesthetized with urethane (1.2g/kg, i.p.) 2 h after the pargyline injection. During the whole experiment,body temperature was monitored by a rectal probe and automatically adjusted to 37.5 “C by a homeothermic blanket. Electrochemically treated carbon fiber electrodes were implanted into the striatum as previously described ( I ) . Auxiliary and reference electrodes were placed on the skull surface. The nigro-striatal pathway was electrically stimulated by means of a bipolar concentric electrode as previously described ( I ) . Briefly, bursted stimulations lasted 40 s and consisted of 25 pulse trains 1.2s long (period, 1.6 e ) . Inside the pulse train the frequency of the current pulses was 20 Hz, the pulse duration 0.3 ms, and the pulse amplitude 250 wA.
RESULTS AND DISCUSSION In Vitro Measurements of Variations of DA Concentrations. It appears from Figure 2 that an appropriate choice of the initial and final potentials allowed us to measure low variations of DA concentrations (detection limit, 5 nM) independently of AA concentration variations (in a range of 100 pM). Moreover, the amplitude of the DA response was linearly dependent of the DA concentration (between 5 and 200 nM). Therefore, when combined with electrochemically treated carbon fiber electrodes, DPA is as selective and sensitive as DNPV (6). In Vivo Measurements of Evoked DA Release. As previously described (6) on our treated electrodes, DOPAC and DA oxidize at almost the same potential. Therefore, in order to monitor a pure signal due to DA, DOPAC was eliminated in vivo by inhibiting its synthesis. For this purpose, the rats were pretreated with a monoamine oxidase inhibitor (pargyline). To monitor DA release, this pretreatment represents a limitation that has been discussed elsewhere (I,6).
stimulation
,DA
lOOnM
4 -0 2 4 min Figure 3. Effect of electrical stimulation of the DA neuronal pathway on the DA release measured in the strlatum of a pargyline-treated and anaesthetized rat. This effect Is then mimicked in vitro with the same electrode. The values of the concentrations Indicated in the figure correspond to the final concentrations. 2
0
Electrical stimulations of the DA neuronal pathway for 40 s induced an immediate and large increase of the extracellular DA concentration in the striatum of pargyline-treated and anaesthetized rats (Figure 3). This increase lasted as long as the electrical stimulation did and the DA signal returned to the base line 1.2 min after the end of the stimulation. As compared with in vitro calibration after the in vivo experiment, it appears that during a “standard” stimulation, the DA concentration reached about 150 nM. However, it was relatively awkward to compare in vivo and in vitro results, since the electrode response-time seemed to be longer in vitro than in vivo. So, the in vivo evoked DA release might be underestimated when compared to in vitro results. Moreover, it appears from Figure 2 that the time response of the electrode was between 1 and 2 min. In fact, as it has been previously described (6, B), DA adsorbs to our electrodes and the adsorption and desorption processes are at the origin of this relatively slow time response. In conclusion, this study shows that DPA made possible the rapid and sensitive measurement of low DA concentrations without any overlap by AA. In fact, it measures a differentiated current due to DA oxidation and, thus, the contribution of other oxidizable compounds such as AA was eliminated by the differentiation provided that the difference between their oxidation potentials and the DA were sufficient (that is more than 100 mV). Therefore, among the numerous electrochemical techniques that have been brought into play to monitor in vivo oxidizable compounds from the mammalian brain (for review, see ref 4),DPA combines the performances of the most sensitive and selective ones with the rapidity of measurement of the fastest ones. Thus, DPA might be a potent tool for in vivo neurochemistry as well as for other purposes. Registry No. Dopamine, 51-61-6. LITERATURE CITED (1) Gonon, F. G.; Buda, M. Neuroscience (Oxford),1885, 14, 765-774. (2) Kuhr, W. G.; Ewlng. A. G.; Caudill, W. L.; Wlghtman, R. M. J . Neurochem. 1884, 43, 560-569. Stamford,J. A.; Kruk, 2. L.; Mlllar, J.; Wightman. R. M. Neurosci. Lett. 1984, 51, 133-138. (4) Marsden, C. A.; Brazell, M. P.; Maidment, N. T. In “Measurement of Neurotransmitter Release in Vlvo”; Marsden, C. A., Ed.; Wiley: ChiChester. .. . 1984: rr 127-151 ._.. - - , oo (5) Wightman, R. M.; Strope, E.; Plotsky, P.; Adams, R. N. Brain Res. 1078. 159. 55-68. (6) a n o n , F.’G.; Navarre, F.; Buda, M. J. Anal. Chem. 1984, 5 6 , 573-575. (7) a n o n , F. 0.; Buda. M.; Pujol, J. F. In ”Measurement of Neurotransmitter Release In Vlvo”; Marsden, C. A., Ed., Wiiey: Chichester, 1984; pp 153-171. (8) anon, F. G.; Fombarlet, C. M.; Buda, M. J.; Pujol. J. F. Anal. Chem. 1881, 53. 1386-1389. (3)
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RECEIVED for review January 15,1985. Accepted April 1,1985. This work was supported by INSERM (U 171), CNRS (LA 162),Universit6 Claude Bernard (U.E.R. Lyon Nord).
