Anal. Chem. 1984, 56,573-575
LITERATURE CITED Martinez, R. I. Int. J. Chem. Kinet. 1980, 12, 771-775. Stephens, E. R. A&. Environ. Sci. Techno/. 1989, 1 , 119-146. Grosjean, D.; McMurry, P. H. “Secondary Organic Aerosol Formation: Homogeneous and Heterogeneous Chemical Pathways”; ERT Document No. P-A098-04, National Technical Information Service PB-82262262, Springfleid, VA, 1962. Grosjean, D. Environ. Sci. Techno/. 1983, 17, 13-19. Gay, B. W., Jr.; Noonan, R. C.; Bufalini, J. J.; Hanst, P. L. Environ. Sci. Technoi. 1978, 10, 82-85. Joseph, D. W.; Spicer, C. W. Anal. Chem. 1978, 50, 1400-1403. Groslean, D. Anal. Lett. 1982, 15, 785-796. Fung, K.; Grosjean, D. Anal. Chem. 1981, 53, 168-171. Vanderzanden, J. W.; Birks, J. W. Chem. Phys. Len. 1982, 88, 109-1 13. Hanst, P. L. Adv. Envlron. Sci. Techno/. 1971, 2 , 91-213. Cox, R. A,; Roffey, M. J. Environ. Sci. Techno/. 1977, 1 7 , 901-906. Hoidren, M. W.; Rasmussen, R. A. Envlron. Sci. Technoi. 1978, 70, 195-187. Nieboer, H.; VanHam, J. Atmos. Environ. 1978, 70, 115-120. Lonneman, W. A. Envlron. Sci. Techno/. 1977, 1 1 , 194-196. Watanabe, I.; Stephens, E. R. Environ. Sci. Techno/. 1978, 12, 222-223. Nieisen, T.; Hansen, A. M.; Thomsen, E. L. Atmos. Environ. 1982, 16, 2447-2450. Penkett, S.A.; Sandalls, F. J.; Loveiock. J. E. Atmos. Environ. 1975, 9, 139-140. Sandalls, F. J.; Penkett, S.A.; Jones, B. M. R. “Preparation of Peroxy-
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acetylnitrate and Its Determination in the Atmosphere”; HMSO: London, 1974; AERE Report R-7807. Lee, Y.-N.; Schwartz, S.E. J. Phys. Chem. 1981, 85, 840-848. U S . Environmental Protection Agency. “Comparability of nine methods for monitoring NOp in the ambient air”, Report EPA-650/4-74-012, Washington, DC, March 1974. Cox, R. A. J. Photochem. 1974, 3 , 175-188. Uselman, W. M.;Levine, S.2.; Chan, W. H.; Calvert, J. G.; Shaw, J. H. I n “Nitrogenous Air Pollutants: Chemical and Biological Implications”; GrosJean, D., Ed.; Ann Arbor Science Publishers: Ann Arbor, MI, 1979; pp 17-54. Lee, Y. P.; Stimpfie, R. M.; Perry, R. A.; Mucha, J. A.; Evanson, K. M.; Jenning, D. A.; Howard, C. J. Int. J. Chem. Kinet. 1982, 74, 71 1-732. Boneiii, J. E.; Greenberg, J. P.; Lazrus, A. L.; Spencer, J. E.; Sedlacek, W. A. Atmos. Environ. 1978, 12, 1591-1594. Berg, W. W.; Winchester, J. W. J. Geophys. Res. 1917, 82, 5945-5953. Aoki, T.; Munemori, M. Anal. Chem. 1983, 55, 209-212. Joiiey, R. L. Prepr. Pap. Nati. Meet., Div. Envlron. Chem., Am. Chem. SOC.1982, 68-71. Oliver, B. G.; Carey, J. H. Environ. Sci. Techno/. 1977, 1 1 , 893-895. Fung, K.; Grosjean, D. Presented at the Pacific Conference on Chemistry and Spectroscopy, Pasadena, CA, Oct 26-28, 1983.
R~~~~~~~for review July 26, 1982, R~~~~~~~~~~September 2, 1983. Accepted Novmeber 7, 1983.
CORRESPONDENCE In Vivo Monitoring of Dopamine Release in the Rat Brain with Differential Normal Pulse Voltammetry Sir: Chemical neurotransmission is achieved by release of neurotransmitter substances from nerve terminals into the synaptic cleft where they partidy diffuse into the extracellular fluid. Therefore in vivo monitoring of extracellular neurotransmitter concentrations inside the mammalian brain is of pride interest. Several neurotransmitters such as dopamine (DA) are easily oxidizable and many studies (for review see ref 1and 2) have focused on the following question: Is in vivo electrochemistry capable of monitoring spontaneous DA release in the rat striatum (the striatum is a brain region densely innervated by dopaminergic terminals)? Up to now the answer to this question was no; it has been reported that extracellular DA concentration was below the detection limit (50 nM) (1-5). Moreover, such monitoring was complicated by the fact that ascorbic acid (AA) and 3,4-dihydroxyphenylaceticacid (DOPAC) which are easily oxidizable, are present in the extracellular space in much higher concentrations (1-6). Separation of AA from catechols such as DA and DOPAC was achieved by means of treated carbon fiber electrodes ( 4 , 5 ) . Unfortunately, with these electrodes, DOPAC and DA oxidize at almost the same potential. Therefore, in the present study, in an attempt to monitor DA alone, DOPAC was eliminated from the brain by inhibiting its synthesis. This was achieved by pargyline pretreatment of the rats (5). In order to measure mammalian extracellular DA concentration, we employed, in conjunction with our treated carbon fiber electrodes (7),a promising electrochemical technique developed by Osteryoung’s group (for review on electrochemical techniques see ref 8). This technique combines the advantages of differential pulse voltammetry (DPV) and of normal pulse voltammetry (NPV). Due to its sensitivity and selectivity, DPV has been widely used. With DNPV, as well as with DPV, the oxidation current is differentiated by means of a measuring pulse (see Figure 1). The advantages of NPV 0003-2700/84/0356-0573$0 1.5010
for in vivo electrochemistry have been recently pointed out (3): Since the electrode is a t a resting potential during the greater part of the scan, it minimizes the filming of the electrode surface by electrogenerated products and, therefore, it improves the stability of the response. As with NPV, but unlike DPV, the DNPV scan is entirely pulsed (Figure 1). With this technique we were able to detect very low DA concentrations (detection limit 5 nM) and to monitor from the striatum of pargyline-treated rats a catechol peak due to oxidation of the DA released in the extracellular fluid by dopaminergic nerve endings.
EXPERIMENTAL SECTION Chemicals. Dopamine (DA),3,4-dihydroxyphenylaceticacid (DOPAC),and ascorbic acid (AA) were dissolved in phosphate buffered saline (PBS) solution (KCl, 0.2 g/L; NaCl, 0.8 g/L; NazHP04.2Hz0,1.44 g/L; KHZPO4,0.2 g/L; pH 7.4). In order to prevent spontaneous oxidation of DA, AA was always present in DA solutions. However, the DA and DOPAC response did not depend on the AA concentration (between 50 and 500 wM). Treated Carbon Fiber Electrodes. Their preparation has been described elsewhere (9). Some of them have been furnished by Solea Tacussel Co. (72, rue d’hlsace, 69100 Villeurbanne, France). They were electrochemically treated according to a procedure slightly modified from that previously described (7): An anodic potential of a triangular wave form (initial potential, 0 V; amplitude 2.9 V; frequency 70 Hz) was applied for 20 s. Then, a continuous potential (-0.8 V) was applied for 5 s. Finally a continuous potential (+1.5 V) was applied for 5 s. During this treatment electrodes were immersed in the PBS solution. Apparatus. Electrochemical treatments as well as automatic measurementswith DPV or with DNPV were performed by means of a new apparatus (“Biopulse”,Solea Tacussel)specially designed for this purpose. Reference and auxiliary electrodes were as previously described (7). DNPV parameters are defined in Figure 1 and their values were T = 0.5 s, t l = 70 ms, t z = 30 ms, A V = 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 3, MARCH 1984
DNPV
!Potential
IN VITRO DNPV
LI I
STRIATUM (Pargyline + Chloral hydrate)
t l It2
0
+85mV
c U
Figure 1. Potential-time function for DNPV. The current is sampled for 10 rns just before (in A) and at the end (in 9)of the rneasurlng pulse. The difference of these currents (le - iA) is recorded for every pulse. DOPAC 2 pM ~ AA 200 pM
AA (200
pM) +
DA
(nM)
~
8 C 2
.zz
-0.2
0
co.2
0
-0.2
0
+0.2
v
Figure 3. Voltammograms recorded in vitro from a PBS solution containing AA (200 pM) and DA (20 nM) and from the striatum of an , 2 h before anesthetized rat which has been pretreated with pargyline recording. In vitro tests were performed before (continuous line) and after (dashed line) the in vivo implantation. AMPHETAMINE 2mg/kg ( n : 5 )
+55mv
0
4
3
+85mV
c
2
w
il -0.2
0
c0.2
0
+0.2
0
to.2
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0
+0.2
v
Figure 2. Voltammograms recorded from various solutions by means of DNPV and treated carbon fiber electrodes. 30 mV, Av = 2 mV, and Pi = -200 mV for in vitro experiments and Pi = -240 mV for in vivo ones. In Figure 3 the DPV parameters were as follows: pulse modulation; amplitude 30 mV; duration, 30 ms; period, 0.5 s; speed of the linear sweep, 4 mV/s. Animals. Male rata (300 f 30 g) were pretreated by pargyline (75 mg/kg, i.p.) and anesthetized with chloral hydrate (400 mg/kg, i.p.) 1 h after the pargyline injection. They were placed in a stereotaxic apparatus and kept anesthetized with additional chloral injections as needed. Carbon fiber electrodes were implanted in the striatum through a hole in the skull. The dura mater was gingerly resected. The cortex surface was carefully cleaned and wetted with physiological medium before the carbon fiber electrode penetration. Auxiliary and reference electrodes were in contact with the skull by means of a semiliquidjunction. For drug experiments (Figure 4) the results were expressed as the percentage of the mean control value calculated by averaging the six absolute values of the DA peak height obtained during the last 10 min before the injection.
RESULTS AND DISCUSSION In Vitro Measurement of AA, DOPAC, and DA by DNPV. As compared to DPV, within the same experimental conditions, DNPV improved the sensitivity of treated carbon fiber electrodes for DOPAC and for DA but not for AA; the amplitude of this gain varied from 1to 1.8 depending on the choice of the DNPV parameters (data not shown). In this respect the main parameter is the initial potential (Pi). In fact, when Pi = 0 V, DNPV did not improve the sensitivity for DA or DOPAC. Parameters have been chosen in order to discriminate against the charging current and to obtain the maximal sensitivity. Figure 2 shows that although DA and DOPAC cannot be separated they peak at distinct potentials (DOPAC at +55 f 10 mV and DA at +85 f 5 mV). Our treated electrodes were about 50 times more sensitive for DOPAC than for AA and 100 times more sensitive for DA than for DOPAC. Every time the AA or the DOPAC concentrations were changed, a stable response was immediately obtained, while this was not the case of DA. In fact, as previously reported (7)it took 3 to 6 min to obtain a stable DA response.
L SALINE
AMPH.
Tim. (min.)
Figure 4. Effect of saline and amphetamine treatments on the DA peak helght recorded every 2 mln by DNPV from the striatum of pargyline-treated rats. Values were expressed as percent of the DA peak height before lnjectlons (mean f standard error of means, n determinations). This response time did not depend on the fact that scans were recorded or were not but did depend on the DA concentration in the solution: The higher this concentration was, the longer it took. However, a linear response was obtained for DA (from 5 to 50 nM) in the presence of a high AA concentration. This delayed response for DA but not for AA and DOPAC suggests that DA was adsorbed on the treated carbon fiber. Therefore, it is likely that the DA response corresponded to an artifically high DA concentration at the electrode surface. In Vivo Measurement of DA Release. Figure 3 shows that in the striatum of a pargyline-treated rat a catechol peak appearing at +85 mV is still detectable. Electrodes were calibrated in vitro for DA before and after the in vivo implantation. Unfortunately, after the in vivo experiment a loss in sensitivity (30 to 50%) was observed (Figure 3). The amplitude of this loss depended on the quality of the surgical approach and on the duration of the in vivo recording. Since the in vivo DA peak height was stable for 1 h (Figure 4), it is likely that this loss occurred during the penetration or during the removal of the electrode from the brain. Due to this loss, exact estimation of the presumed DA peak height in terms of DA concentration was not possible. However, Figure 3 as well as other experiments in which the electrode
Anal. Chem. 1984. 56. 575-578
was calibrated after the in vivo experiment suggested that this height corresponded to a DA concentration between 15 and 25 nM. As regards in vitro experiments, DNPV, as compared with DPV, represents a moderate improvement; but in vivo, in an attempt to monitor DA release, it represents a decisive one (Figure 3). It is likely that, in vivo, the diffusion of DA in the extracellular space is hindered. This limits the DA amount which can reach the electrode surface. In these conditions it is likely that a DPV scan led to a DA depletion in the vicinity of the electrode. On the other hand, since with DNPV the electrode is at a resting potential during four-fifths of the scan duration, this minimizes the number of DA molecules getting oxidized during one scan and, thus, the use of DNPV instead of DPV results in a large increase in the in vivo DA signal. Moreover, as expected from studies employing NPV (3),DNPV signals were more stable than DPV ones. In fact the DPV signal disappeared after a few seans while DNPV ones were stable for more than 1 h (Figure 4). When recorded from the striatum of a pargyline-treated rat, DNPV allowed us to record a peak at +85 mV. This peak was attributed to DA rather than to a residual DOPAC contribution since it appears at the same potential as DA and since it is greatly increased by amphetamine injections (Figure 4). In fact, numerous studies demonstrated that DA release is strongly stimulated by amphetamine while the DOPAC level is simultaneously decreased (4-6). Our conclusion has been reinforced by additional arguments (10): This presumed DA peak was completely suppressed following a specific degeneration of striatal dopaminergic terminals, it was decreased by injections of dopaminergic agonists and increased by antagonists; these drug effects were much more rapid than the effect of the same drugs on the striatal DOPAC level. As estimated here from in vitro calibrations, striatal DA concentration in the extracellular space was between 15 and 25 nM. This concentration has been recently estimated to be 54 nM by means of perfusion with dialysis tubing implanted in the striatum of a normal rat (6). Although these two techniques are very different these estimations are in agreement; this suggests that pargyline treatment did not induce a dramatic change in the extracellular DA concentration. It has been also observed that striatal DA level, as measured post-mortem, is moderately increased (+25 % ) by pargyline (11). However, these estimations question that recently reported by Ewing et al. (12):In their electrochemical study the extracellular DA concentration was measured following electrical stimulation of the nigrostriatal pathway and estimated to be 35.5 pM. This seems in contrast with a previous report by the same authors who showed that their technique
575
was not sensitive enough for monitoring the amphetamine stimulated DA release (3). In conclusion, this study demonstrates that in vivo electrochemical monitoring of spontaneous DA release is feasible providing that the DOPAC contribution to the catechol peak is pharmacologically suppressed by inhibition of the DOPAC synthesis. In order to reach this goal, DNPV appears to be superior to other already used techniques. Moreover, it points out that DNPV used in combinationwith treated carbon fiber electrodes makes the detection of some molecules such as catecholamines possible within nanomolar concentrations. ACKNOWLEDGMENT We thank the Solea Tacussel Company which designed the new apparatus used in this study accordingly to our specifications. Registry No. Dopamine, 51-61-6. LITERATURE CITED Adams, R. N.; Marsden, C. A. I n “Handbook in Psychopharmacology”; Iversen, L. L., Iversen, S. D., Snyder, S.H., Eds.; Plenum Press: New York, 1963; Vol. 15, pp 1-74. a n o n F.; Cespugllo R.; Buda M.; Pujol J. F. In “Methods in Blogenic Amine Research”; Parvez, S.,Nagatsu, T., Nagatsu, I., Parvez, H., Eds.; Elsevier: Amsterdam, 1983. Ewing, A. G.; Dayton, M. A.; Wightman, R. M. Anal. Chem. 1981, 53, 1842-1847. Gonon, F.; Buda, M.; Cespugllo, R.; Jouvet, M.; Pujol. J. F. Nature (London) 1980, 286, 902-904. Gonon, F.; Buda, M.; Cespugllo, R.; Jouvet, M.; Pujol, J. F. Brain Res. 1981, 223, 69-80. ZetterstrBm, T.; Sharp, T.; Marsden, C. A.; Ungerstedt, U. J . Neurochem., In press. Gonon, F.; Fombarlet, C. M.; Buda, M. J.; PuJol, J. F. Anal. Chem. 1061, 53, 1386-1389. Borman, S. A. Anal. Chem. 1982, 54, 698A-705A. Gonon, F.; Buda, M.; Pujol, J. F. I n “Measurement of Neurotransmitter Release”; Mardsen, C. A., Ed.; Wiley: London, In press. Gonon, F.; Buda, M.; Pujol, J. F. 5th International Catecholamine Symposium, Gijteborg, 1983; Abstract No. 177 (Supplement to Progress in Neuro-Psychopharmacology and Blologlcal Psychiatry). Westerink. B. H. C. Eur. J . Pharmacal. 1979. 56, 313-322. Ewing, A. G.; Blgelow, J. C.; Wightman, R. M. Science 1983, 227, 169-1 71.
’
Present address: Laboratolre de Chimie Organlque et CIn(tlque, ICPI, rue du Plat, 65002 Lyon, France.
F. G. Gonon* Florence Navarre’ M. J. Buda INSERM U 171 H8pital Ste. Eug6nie Pavillon 4 H 69230 St. Genis Laval, France
RECEIVED for review August 4,1983. Accepted November 1, 1983.
Variations in Electron-Transfer Rate at Polished Glassy Carbon Electrodes Exposed to Air Sir: Glassy carbon has found applications as an anode and as a cathode in numerous electroanalytical methods (1).This material is not inert, however, and redox reactions of functional groups on glassy carbon surfaces have been observed (1-9). In order to obtain reproducibleelectroanalyticalresults, a wide variety of pretreatment procedures have been recommended. Specific techniques range from polishing the electrode surface with alumina or other abrasives of submicron 0003-2700/84/0356-0575$01.50/0
particle size (1-3, 7-12) to polishing followed by electrochemical ( I , 6,12)or chemical (4)pretreatments. Ultrasonic cleaning (5,12,13) has been used to remove traces of polishing materials from the surface. That such a variety of pretreatments have proliferated is related to a relatively poor understanding of the nature and reactivity of the electrode surface before and after pretreatment. In this report, we provide evidence that polishing of glassy carbon produces a @ 1984 American Chemlcal Soclely
