Differential pulse voltammetry in brain tissue. I. Detection of 5-hydroxyindoles in the rat striatum

Brain Research, 223 (1981) 287-298 Elsevier/North-Holland Biomedical Press

287

D I F F E R E N T I A L PULSE V O L T A M M E T R Y IN BRAIN TISSUE. I. D E T E C T I O N OF 5 - H Y D R O X Y I N D O L E S IN T H E RAT S T R I A T U M

CESPUGLIO R., FARADJI H., PONCHON J. L., BUDA M., RIOU F., GONON F., PUJOL J. F. and JOUVET M. Department of Experimental Medicine, University Claude Bernard, 8, Avenue Rockefeller, 69008 Lyon (France)

(Accepted March 5th, 1981) Key words: differential pulse voltammetry - - oxidation potential -- peak 3 -- electrochemical

detection - - 5-hydroxyindoles- - striatum.

SUMMARY In vitro, differential pulse voltammetry combined with electrochemically treated carbon fiber electrodes enabled detection, in different solution of 5-hydroxyindole compounds, of an oxidation peak 3 at +300 mV. In vivo, a striatal peak 3 was also recorded at this potential. Electrolytic or 5,7dihydroxytryptamine lesions interrupting the medial forebrain bundle (MFB) were followed by a decrease of 65 ~o and 64 ~ in peak height, but not elimination of the peak. Biochemical determinations were significantly correlated to the peak 3 measurements. The existence of peak 3 as well as hydroxyindole compounds in blood suggested a blood contamination under the experimental conditions employed. This possibility is confirmed both by the complete disappearance of striatal peak 3 in animals with the MFB lesioned and surgically prepared a week before recordings, and by biochemical measurements in parachlorophenylalanine-treated or perfused (phosphate-buffered saline solution) animals.

INTRODUCTION Previous studies using electrochemical techniques have shown that the determination of catecholamines, indolamines, their metabolites and ascorbic acid was possible in vitro14,16,1a,24,28. An important field was also opened by preliminary work that demonstrated the possibility of in vivo monitoring, especially in brain tissuell, 15, 16 and cerebrospinal fluid 21. The most important limitations, at the moment, in the use of these techniques are, however, the sensitivity, the selectivity and the life time of electrodes when introduced into brain tissue4, t6. 0006-8993/81/0000-0000/$ 2.50 © Elsevier/North-Holland Biomedical Press

288 The use of pyrolytic carbon fiber (PCF) electrodes electrochemically treated together with differential pulse voltammetry (DPV) limits these inconveniences and allows separation, as previously shown 16, of oxidation peaks of ascorbic acid (AA) and catechols. With a potential scanning of--100 mV to +200 mV, two separate oxidation peaks were obtained in the rat striatum: peak 1 (--50 mV) dependent upon brain AA and peak 2 ( + 100 mV) dependent upon extracellular dihydroxyphenylacetic acid (DOPAC) 8. By use of a potential sweep above -1-300 mV, an oxidation peak 3 (+300 mV), corresponding to the potential where 5-hydroxyindole compounds are oxidized, has also been demonstrated15. The results obtained by Marsden is and Marsden et al. (1979) 19 suggest a possible detection at this potential of extracellular serotonin (5HT). In this study we have attempted to demonstrate, by the use of PCF electrodes electrochemically treated, and DPV that the oxidation peak 3 in rat striatum depends solely on 5-hydroxyindoles. MATERIALSAND METHODS OFA male rats (200 g, 1FFA-CREDO) were used in this study, and all DPV recordings were performed in the striatum of anaesthetized animals (chloral hydrate 400 mg/kg i.p.). DPV required the use of 3 electrodes: reference (Ag/AgCI, TACUSSEL) and auxilliary electrodes (platinium, diameter: 150 #m)both placed on the skull, frontal area of the cortical bone and a working electrode, made according to a previously described technique 24, located in the striatum. The active part of this last electrode was a pyrolytic carbon fiber (diameter: 8 #m; length : 500 #m, ref. AGT/F 10,000; Carbon Lorraine Genevilliers, France). Before use, it was electrochemically treated in a saline solution with a triangular current (0.3 V, 70 Hz, 20 s; 0-2.5 V, 20 Hz, 20 s; 0-1.5 V, 70 Hz, 20 s). The in vitro response of the working electrode in separate solutions of variable concentrations of 5-hydroxytryptophan (5-HTP), 5hydroxytryptamine (5-HT), 5-hydroxyindolacetic acid (5-HIAA) and other indoles was measured with a 'PRG5' polarograph (TACUSSEL) (Fig. 1). The following parameters were used: potential range (PR) --0.05 to +0.45 V/reference (for 5hydroxyindoles); -4).05 to +0.95 V/reference (for indoles); scanning speed 20 mV/s; pulses of 50 mV in amplitude and 28 ms of duration; frequency: 5 Hz. In vivo, electrochemical measurements, with the same parameters as in vitro (potential explored: --0.05 to +0.45 V/reference) were effected every minute. Before and after each experiment the response of the electrode was calibrated in a 5 #M 5-HIAA solution. The height of peak 3 was measured as shown in Fig. 2. Unilateral electrolytic lesions (n = 13) of the medial forebrain bundle (MFB) were performed (anterior (A): 3.4; lateral (L): 1.5; vertical (V): + 4 - - De Grootl°; 2 mA; 15 s), as were neurotoxic lesions (n = 11) of the same structure after desmethylimipramine (DMI; 25 mg/kg) using 5,7-dihydroxytryptamine (5,7-DHT, a specific 5HT neurotoxin; 5/zg/0.4 #1)2. Transections of the MFB (n = 4) were also effected (width (w): 3 mm; A: 3.4 - - Koenig and Klippel lz) and for these transected animals

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Fig. 1. Calibration curves (oxidation current vs concentration) obtained in vitro in solutions with variable concentrations of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT) and 5hydroxyindolacetic acid (5-HIAA). Treated pyrolytic carbon fibers (active length = 500/zm) have been used. For 5-HTP and 5-HT the oxidation current measured is linear in a 1/~M range and for 5H I A A the oxidation current is linear for solutions within a 20 /~M range. It appears (5-HTP, 5-HT, 5-HIAA together) that the electrodes used are 6-8 times less sensitive to 5-HIAA than to 5-HTP. IN VITRO ,(

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Fig. 2. Differential pulse voltammograms obtained in vitro or in vivo in the rat striatum. In vitro: the use of treated pyrolytic carbon fibers enables peak 1 and 2 to be obtained with solutions of respectively AA (50 #M) and DOPAC (5/zM). Peak 3 is produced with solutions of 5-HTP, 5-HT and 5-HIAA; in this figure peak 3 has been obtained in 1 #M 5-HT solution, though a signal of similar amplitude is produced with a 6 #M 5-HIAA solution; dotted line represents the voltammogram = PBS; broken vertical line indicates that peak 3 has been recorded separately from peaks 1 and 2; h is the measured height of peak 3. In vivo: voltammograms obtained from the rat striatum, following unilateral transection of the MFB (A = 3.5; L = 0.5) 10 days before the recording session. On the intact side (A), peaks 1 and 2 associated with AA and DOPAC and peak 3 are present. Peak 3 has been recorded separately from peak 1 and 2 (broken vertical line). On the lesioned side (B) and 10 days after the lesion, peak 2 and 3 are absent. Surgery necessary for the electrode penetration was performed one week before the recording session. Peak 3 may be partially restored by producing a haemorrhage at the cortical level (dots). AA, ascorbic acid; DOPAC, dihydroxyphenylacetic acid; A, anterior; L, lateral; MFB, medial forebrain bundle; PBS, phosphate-buffered saline solution. For other abbreviations see Fig. 1.

290 the surgery necessary for the electrode penetration was performed one week before the electrochemical recordings. For all animals the recording sessions began 10-20 days after lesions. In control and lesioned rats the presence of oxidation peaks 1 and 2 was checked, and in this instance electrodes were electrochemically treated in the same way except for the triangular current applied during the treatment (0-3 V, 70 Hz, 20 s) and the PR explored (--0.1 to +0.2 V/reference). At the end of each experimental session lesions were verified histologically. The variations of the height of oxidation peak 3 in parachlorophenylalanine(PCPA) treated animals (n = 6; 400 mg/kg × 2/24 h. I.P. 48 h after treatment) were also measured and compared to an untreated control group (n = 6). Peak 3 was also detected in the blood obtained after an intentional haemorrhage at the cortical level (n = 4). In all animals 5-hydroxyindole compounds were measured in the striatum. Furthermore, radioenzymatic (RE) assay of 5-HT was employed according to the technique described by Saavedra et al. z6, and a high-performance liquid chromatography (HPLC) assay of 5-HT and 5-HIAA adapted according to a modification of the method described by Grafeo and Karger in 19769 and Neekeers and Meek in 197620. For these assays different microdissections of the striatum were used.

Fig. 3. Typical medial forebrain bundle (MFB) lesion (denoted by arrows). Direct photography (without histological treatment) of a frontal brain cut (600/~rn).

291 In perfused (n = 5; PBS, 5 °C, 1 rain) and non-perfused (n - 5) animals, 5-HT and 5-HIAA concentrations in the blood were also measured by HPLC. HCLO4 4 M (1/10 v/v) containing fl-mercaptoethanol (0.5 M) was added to heparinized blood and after centrifugation (15 min, 4 °C) and neutralization (potassium acetate) the supernatant was analyzed directly. RESULTS

In vitro The 5-hydroxyindole compounds such as 5-HTP, 5-HT, 5-HIAA and 5hydroxytryptophol (5-HTol) had an oxidation peak at about 280-300 mV. Different curves (oxidation current versus concentration) were obtained in solutions with varying concentrations of 5-HTP, 5-HT and 5-HIAA. It appeared that the oxidation current measured with 5-HTP and 5-HT varied linearly with concentrations between 0.1 and 1/zM, while with 5-HIAA the oxidation current was linear in solutions varying from 0.1 to 20 #M (Fig. 1). The indoles tested, such as tryptophan (TRY), tryptamine (TRYne), indolacetic acid (IAA) and melatonin had an oxidation peak at 680-700 mV, whereas solutions of AA and DOPAC had oxidation peaks at about --50 mV and + 100 mV respectively. In vivo In the striatum, the exploration of the oxidation current for a potential sweep between --200 mV and +200 mV revealed the existence of two successive peaks, 1 and 2, appearing at --50 mV and + 100 mV s. This exploration, followed by a potential sweep between +200 mV and +450 mV, indicated oxidation peak 3 at +300 mV (Fig. 2; in vivo, A). The mean height of this latter peak was 2.96 ± 0.8 nA (n = 10) corresponding to a current in vitro obtained in a 5 #M 5-HIAA solution (Fig. 2, in vitro). Electrolytic lesions of the MFB Unilateral electrolytic lesions of the MFB (Fig. 3) induced in the ipsilateral striatum an average 65 ~ decrease in the height of peak 3. Peak 2 was absent when the lesions of the MFB were complete (Fig. 2, in vivo, B) though peak 1 remained. The RE biochemical measurements demonstrated an 86 ~ decrease in striatal 5-HT on the lesioned side when compared with the intact side and HPLC measurements similarly indicated a decrease of 85 ~ in 5-HT and 84 ~ in 5-HIAA. On the intact side the 5-HT and 5-HIAA amounts measured by HPLC were, respectively, 498 q- 45 ng/g and 1271 _-k 64 ng/g (Table I; Fig. 4). 5,7-dihydroxytryptamine (5,7-DHT) lesions Unilateral 5,7-DHT lesions of the MFB resulted in a 64 ~o decrease in the height of peak 3 on the lesioned side; peak 2 was present but varied in height and peak 1 was also always present. RE measurement showed a decrease in striatal 5-HT of 80 ~ on

292 TABLE I

Variations of the peak 3 height measured in the striatum by differential pulse voltammetry (DP V) a.l?er electrolytic lesions of the MFB and variations of the 5-HT and 5-HIAA striatal concentrations induced by these lesions Biochemical measurements were effected by radioenzymatic technique (RE) and high-performance liquid chromatography (HPLC). IS, intact side; LS, lesioned side; IS/LS ~, percentage difference; m, mean; ND, not detectable; for other abbreviations see Figs. 1 and 2. m values are followed by the standard error of the mean (S.E.M.). The biochemical results are expressed in ng of 5- HT and 5-HIAA/g of tissue and in percentages. Differences are significant at P < 0.001. * values excluded for the mean calculation. -4-adequate lesion ; --, inadequate lesion.

5-HT (RE) (rig~g)

5-HT (HPLC) (ng/g) 5-HIAA (HPLC)

IS

IS

LS

IS

LS

328 217" 566 627 475 535 570 615 247 193 608 562 646 498i45 1004- 9

45 212" 151 ND 263 114 125 ND 114 64 ND ND ND 73 :k24 15:k 5

955 946* 1046 1558 1359 1303 1473 1283 1228 909 1616 1262 1256 1271 -4- 64 100,4, 5

131 84 688* 1* 172 75 149 64 502 33 278 43 332 69 217 63 441 50 115 48 183 100 81 66 81 85 207--41 16± 3

84

65

(ng/g) LS

Elec- 566 46 troly- 200* 196" tic 243 56 lesions 263 23 (n = 13) 330 83 176 46 440 23 80 ND 266 103 163 36 430 ND 166 43 743 70 m 322±55 44 -4-9 100,4,17 14.4.3 -~ ~

~6

85

DPV Anatomy ( % IS/LS) (MFB lesions)

--4-4-

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the lesioned side a n d H P L C measurements d e m o n s t r a t e d a decrease in 5 - H T of 81 a n d in 5 - H I A A o f 83 ~ . O n the intact side the 5-HT a n d 5 - H I A A concentrations using H P L C were, respectively, o f 457 4- 58 ng/g a n d 1424 4- 62 ng/g (Table II, Fig. 4).

Parachrlorophenylalanine ( P C P A ) treatment Striatal peak 3 height in P C P A - t r e a t e d rats, when c o m p a r e d with a n untreated g r o u p of animals, showed a decrease of 72 %. 5 - H T a n d 5 - H I A A a m o u n t s were n o t detectable in this nucleus using the H P L C technique (50 /,1 v o l u m e injected for analysis), t h o u g h R E measurements d e m o n s t r a t e d a measurable decrease of 88 (control: 622 4- 66 ng/g; P C P A : 73 4- 8 ng/g) (Table III, Fig. 5). P e a k 3 in blood Electrochemical m e a s u r e m e n t s in b l o o d d e m o n s t r a t e d the existence at a + 3 0 0 m V potential o f a peak 3 with a n amplitude which m a y be u p to twice as great as that of the striatal peak 3 (Fig. 6). Biochemical measurements of b l o o d 5 - H T a n d 5 - H I A A c o n c e n t r a t i o n s in control a n d P C P A - t r e a t e d rats are shown in Table IV. It can be seen

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Fig. 4. Correlations between the percentage decrease of peak 3 measured by DPV 10-20 days after electrolytic lesions (L) (black dots) or 5,7-DHT lesions (open dots) and the percentage decrease of 5H I A A and 5-HT measured by HPLC. Linear correlation (straight lines) are drawn; coefficients of correlation (r) are" (A) L: 0.83; 5,7-DHT: 0.54; (B) L: 0.84; 5,7-DHT: 0.53; (C) L: 0.84; 5,7-DHT: 0.54; (D) L: 0.90; 5,7-DHT: 0.53. Abscissae: high-performance liquid chromatography (HPLC) or radio enzymatic (RE) 5-HT measurements; ordinate: differential pulse voltammetry (DPV); 5,7D H T : 5,7-dihydroxytryptamine. For other abbreviations see Figs. 1 and 2. For the formulations considered (A, B, C, D), when r < 0.5 only a minor portion of the total variation is linearly related (r = 0.1 then 4 % ; r = 0.9 then 80%). TABLE II

Variations of the peak 3 height measured in the striatum by D P V after 5,7-DHT lesions and variations of the 5-HT and 5-H1AA striatal concentrations induced by the same lesions Differences are significant at P < 0.01. For abbreviations see Table I and Figs. 1, 2, 3, 4.

5-HT (RE) (ng/g)

5-HT (HPLC) (ng/g)

5-HIAA (HPLC) (ng/g)

IS

LS

IS

LS

IS

LS

6 143 126 123 40 43 120 113 56 46 81 4- 15 204- 3

304 410 304 608 395 289 365 563 867 456 4574-58 1004-12

122 152 106 258 ND ND 106 76 76 ND 895:25 194- 5

1216 1624 1535 1243 1651 1094 1461 1542 1569 1311 14244-62 1004- 4

203 570 407 686 81 47 231 74 108 81 2484-73 174- 5

5,7343 D H T 393 lesions 233 (n=10)413 440 420 366 520 573 286 m 3984-32 % 1004- 8 :~ ~

80

81

83

D P V (% IS/LS) 70 75 60 50 62 65 60 70 78 60

64 4- 2

294 TABLE 1II Striatal variations in 5-HTconcentrations aJter P C P A treatment measured by the R E technique

Differences are significant at P <: 0.001. For abbreviations see Figs. 1,6 and Table I. Control ( R E ) (n -- 5)

P C P A ( R E ) (n = 6)

593 381 687 706 743

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Fig. 5. Variations induced in the peak 3 height after parachlorophenylalanine (PCPA) treatment (48 h after 400 mg/kg x 2/24 h). A: control (C) peaks 1 and 2 are not separated and gave a single peak (1 + 2); peak 2 is, however, well resolved; PCPA-treated (Tr); there is a 72 ~ decrease in the peak 3 height after treatment• In this experiment the working electrode was successively located in the striatum of the control rat, then into treated rat and finally returned to the original control rat. Abscissae: 04).5 V. B : mean variations induced in peak 3 height by PCPA (n = 6). For other abbreviations see Fig. 5.

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Fig. 6. Voltammograms obtained from posterior striatum and blood. In the striatum, peaks 1, 2 and 3 are present following a single sweep in the potential range--200 mV ÷ 400 mV though separation of peaks 1 and 2 remains only during the first 5 or 10 measurements. This absence of stability in peaks 1 and 2 produces a progressive shift of these signals until they become united. Abscissae: voltage for both striatum and blood. Note also that peak 3 is present in blood at the same oxidation potential as in the brain tissue. For abbreviations see Fig. 2.

295 TABLE IV 5-HTandS-H1AA plasmatic concentrations in controlandPCPA-treatedanimals

PCPA induces a 70% decrease in 5-HT concentrations, 5-HIAA is not detectable in these treated animals (50 #1 injected for HPLC analysis). Differences are significant at P < 0.002. For abbreviations see Fig. 1 and Tables I and II. 5 - H T (ng/ml) (HPLC 50 i~1)

5-HIAA (ng/ml) (HPLC 50 i~1)

Control (n = 5)

PCPA (n = 5)

Control (n = 5)

PCPA (n = 5)

233 358 688 436 269 m=3964-82 5 % = 70

59 131 185 77 136 1174-22

3 13 15 14 11 11 4-2

ND ND ND ND ND

that 70 ~o of 5-HT remained after PCPA treatment and although very small amounts of 5-HIAA were present in controls (11 -I- 2 #g/g), 5-HIAA was not detectable after PCPA treatment. Transections interrupting the MFB Unilateral transections interrupting the MFB together with the surgery necessary for the electrode penetration was performed one week before the recording sessions. In these conditions it was possible to suppress peak 3 completely on the lesioned side and then restore it with a haemorrhage at the cortical level (3 rats). Peak 2, however, was always absent and peak 1 was present at all times after the transections (Fig. 2, in vivo, B). 5-HT and 5-HIAA

c o n c e n t r a t i o n s in the s t r i a t u m o f p e r f u s e d rats

Striatal 5 - H T a n d 5 - H I A A c o n c e n t r a t i o n s o f perfused an d n o n - p e r f u s e d animals were measured. The differences were 16 ~o for 5 - H T an d 25 ~ f o r 5 - H I A A (Table V). In co n t r o l animals the striatal c o n c e n t r a t i o n s o f 5 - H T an d 5 - H I A A were, respectively, 401 :k 53 ng/g a n d 717 -4- 69 ng/g. TABLE V 5-HTand S-HIA A striatal concentrations measured with or without brain perfusion ( PBS, 5 ° C)

For abbreviations see earlier tables and figures. 5 - H T (ng/g)

5-HIAA (ng/g)

no H P L C Control (n = 5)

HPLC no H P L C Perfused (n = 5) Control (n = 5)

366 266 422 588 366 m~401 ±53 ~ = 16

255 455 333 222 400 333 ± 4 3

708 583 789 942 565 7174-69

HPLC Perfused (n = 5)

403 592 529 511 628 532 4- 47 25

296 DISCUSSION In vitro, at the potential +300 mV, four 5-hydroxyindoles are found to be oxidized (5-HTP, 5-HIAA and 5-HTol). Two of these (5-HTP and 5-HIAA) constitute the principal metabolic steps of 5-HT synthesis and degradation 1,~,v. 5-HT transformation into 5-hydroxyindole acetaldehyde is, however, not considered, as this product is rapidly metabolized by monoamine oxydases to form 5-HIAA 25,27 and the minor pathways of 5-HT catabolism to melatonin21, bufotenin17 and 5-HTol a,6 are at concentrations too low to affect readings reported here. The question arises then whether in vivo the electrochemical signal obtained at +300 mV depends only on 5-hydroxyindoles. 5-hydroxyindoles in the rat striatum originate in the anterior raphe system passing by way of the MFB 2. Electrolytic destruction of this bundle, which completely suppresses the peak 2 (DOPAC) 8, only reduces peak 3 by 65 ~ and the 5-HT and 5HIAA concentrations in the striatum by 85 ~ and 84 ~, respectively. Now, 5,7-DHT is generally accepted as a specific neurotoxin for 5-HT neurons 2. Indeed after injection of 5,7-DHT into the MFB, peak 2 remained and peak 3 was decreased by 64 ~. The 5-HT and 5-HIAA concentrations in the striatum after 5,7DHT are also decreased similarly to those after electrolytic lesions (5-HT: 81 ~ ; 5HIAA: 83 ~). 5-HT synthesis blocking by PCPA ~2 causes a large decrease in 5-HT and 5HIAA and also considerably reduces peak 3. Analogous results have been obtained by Marsden et al. 19 using chronoamperometry or cyclic voltammetry. Thus, results obtained after both lesion and PCPA, suggest that it is the hydroxyindoles that contribute most to peak 3, though in each experimental situation a small peak 3 remained together with small concentrations of 5-HT or 5-HIAA. It seems hardly likely that these residual concentrations originate in the rostral raphe system since the injection of 5,7-DHT into the MFB blocks the anterograde axonal transport into the striatum of labelled proline injected into the dorsal raphe nucleus2. The existence of 5-hydroxyindoles~a,a° as well as the presence of peak 3 in blood, suggested the existence of blood contamination under the acute experimental conditions employed. Indeed, suppressing this phenomenon by performing the surgery necessary for the electrode penetration one week before the recording session suppressed the striatal peak 3 on the transected side. Further, after intentional haemorrhage, peak 3 could once again be detected. In PCPA-treated rats, the existence of a small peak 3 as well as small amounts of 5-HT (12~o) could also be explained by blood contamination since after such treatment some 70 ~o of the 5-HT remains in the blood. Finally, blood contamination in these electrochemical recordings is confirmed by the biochemical results obtained with PBS perfused animals showing that the differences between perfused and non perfused animals are of the order of 16 ~ for 5HT and 25 ~ for 5-HIAA. These values are close to the percentages of 5-HT and 5HIAA remaining after electrolytic or 5,7-DHT lesions. Further, in view of the biochemical results, it appears that the 5-HIAA

297 concentration on the control side o f lesioned animals is double that f o u n d in perfused animals. These results are in agreement with those obtained by Nieoullon et al. 22, who showed an interdependence o f the nigrostriatal dopaminergic systems on the two sides of the brain by lesioning the substantia nigra and monitoring the D A release in both striata. The results reported here also suggest the existence o f a regulating outflow mechanism in the raphe-striata interconnections. In conclusion, these results strongly imply that the striatal peak 3 is dependent u p o n 5-hydroxyindoles present in this nucleus. It cannot o f course be completely excluded that an u n k n o w n p r o d u c t oxidizable at + 3 0 0 mV and detectable as 5-HT and 5 - H I A A m a y be present in blood or in brain tissue. A n identification o f the hydroxyindole c o m p o u n d oxidizable at + 3 0 0 mV mainly responsible for peak 3 is now needed; it is to this subject that part II is addressed. ACKNOWLEDGEMENTS This work was supported by I N S E R M U 52 ( C R L 80.60.2), C N R S (L.A. 162) and D R E T (Grant 80.175). We thank A. McRae-Degueurce for help in performing R E assays.

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