Differential pulse voltammetry in brain tissue: III mapping of the rat serotoninergic raphe nuclei by electrochemical detection of 5-HIAA

Brain Research, 270 (1982) 4.% 54

45

Elsevier

D i f f e r e n t i a l P u l s e V o l t a m m e t r y in B r a i n T i s s u e : III M a p p i n g o f t h e R a t Serotoninergic Raphe Nuclei by Electrochemical Detection of 5-HIAA

F R A N C E S C O CRESPI*, R A Y M O N D CESPUGLIO and MICHEL JOUVET

Department de Mbdecine Exp&imentale, 1NSERM U 52, Universitk Claude Bernard, 8, A venue Rockefeller, 69373 Lyon Cedex 2 (France) (Accepted November 23rd, 1982)

Key words: differential pulse voltammetry - oxidation potential - peak 3 - electrochemical detection 5-hydroxyindoles - raphe nuclei

Differential pulse voltammetry using a new type of carbon fiber electrode, electrochemically treated, is described. The working electrode contains 3 pyrolytic carbon fibers, and passes more current, thus giving a greater sensitivity (with the PRG5 Tacussel polarographic system) than the original monofiber electrodes. It is now possible to investigate brain areas where the monofiber electrodes, working near the limit of PRG5 sensitivity, showed too small a signal. These electrodes have, in addition, better mechanical resistance and can be used (after trypsin cleaning and further electrochemical treatment) for several experiments. Electrochemical measurements made in the nucleus raphe dorsalis before and after treatment with p-chlorophenylalanine, reserpine, clorgyline and clorgyline followed by reserpine, suggest that as in the striatum 5-hydroxyindolacetic acid (5-HIAA) is mainly responsible for peak 3. The map of the raphe system made with this technique is well correlated with the serotoninergic system of the raphe: the highest peak heights are recorded in the raphe dorsalis. INTRODUCTION

Electrochemical detection in vitro and in the central nervous system of catecholamines, 5-hydroxyindoleamines, their metabolites and ascorbic acid (AA) has been already demonstrated by several authors ~6~19-24.27.29.33. However these authors, using amperometric techniques, did not obtain well separated peaks in the electrochemical measurements. The use of differential pulse voltammetry (DPV) performed with carbon paste electrodes 16or carbon fiber electrodes6.W3.29 permitted better separation of the peaks due to different substances. The use of pyrolytic carbon fiber electrodes, electrochemically treated 6~°.~3 enabled us to detect in vitro and in vivo 3 separate oxidation peaks: peak 1 at - 5 0 mV; peak 2 at + 100 mV and peak 3 at + 300 mV. Following pharmacological analysis it appeared that, in the striatum,

these signals depend respectively on extracellular AA, dihydroxyphenylacetic acid (DOPAC) 6 and 5-hydroxyindoleacetic acid (5-HIAA) ~°.24.25. DPV techniques now permit the detection of specific compounds in brain tissue. In this work an attempt has been made to obtain a better in vivo performance of the pyrolytic carbon fiber electrodes by modifying the construction, and the resultant electrode has been used to detect peak 3 in the raphe system. Pharmacological analysis was done at this level using: (i)p-chlorophenylalanine (PCPA), which blocks 5-HT synthesis ~7, (similar experiments have been performed in striatum8.9.25). (ii) reserpine, which gives an increase in extracellular 5-HT and 5HIAA concentrations31; and (iii) clorgyline, which is a monoamine oxidase inhibitor ~4,35, its action involving an intracellular 5-HT accumulation with a great loss in extracellular 5-HIAA concentration. Finally, the topography of raphe

* To whom correspondence should be addressed at: Instituto di Ricerche Farmacologiche Mario Negri Via Eritrea 62, 20157 Milano, Italy. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

46 nuclei obtained with the DPV technique has been compared with the distribution of indolaminergic cells in the raphe system3L MATERIALS A N D M E T H O D S

OFA male rats (200 g, IFFA Credo France) anesthetized with chloral-hydrate (400 mg/kg i.p.) were used in this study. All the animals were held in a stereotaxic apparatus, and the K6nig and Klippel coordinates were used for placing electrodes .8. Reference (Ag-AgC1, Tacussel, France) and auxiliary (platinum, diameter 150/~m) electrodes were made and placed on the cortical bone following the method previously described ~°.~3-~8. The working electrodes used were obtained by a modification of the original design. They were made with 2, 3, 5, 8, 10 and about 20 carbon fibers as opposed to a single carbon fiber in the original studies ~°,t3,29. The pyrolytic carbon fibers (diameter = 8 ~m) were inserted into a glass pipette following the described method 29. The length of the carbon fibers protruding from the pipette was about 500 ~m. Before use, these electrodes were electrochemically treated in a phosphate buffered saline solution (PBS) with a triangular voltage ((~3 V, 70 Hz, 20 s; 0-2.5 V, 70 Hz, 20 s; 0- 1.5 V, 70 Hz, 20 s) and tested in a 5 #mol 5HIAA solution, the treatment and measurement being performed with a 'PRG5' polarograph (Tacussel, France). The potential waveform applied to the working electrode was as follows: square pulses 50 mV amplitude, 28 ms duration, frequency 5 Hz, superimposed on a ramp rising from -0.05 to +0.45 V, at 20 mV/s. In vivo and in vitro, one such sweep was applied once every 5 rain, giving a single electrochemical measurement. Before and after each experiment the response of the electrode was calibrated in a 5 #mol 5-HIAA solution. The same electrode was used for several experiments (up to 10). Before each new in vivo experiment it was cleaned in a trypsin solution (10% in PBS) and again treated following the electrical parameters described above. In vitro, using a 5/~mol 5-HIAA solution, a di-

rect comparison between the old monocarbon fiber working electrodes and the new modified working electrodes was made. The same comparison between all the electrodes used was done in vivo, in the raphe dorsalis nucleus. Following this comparison we decided to use working electrodes equipped with 3 pyrolytic carbon fibers.

Pharmacological identification ofpeak 3 In the RDN of rat, pharmacological investigations were made by systemic treatment with: (1) saline (NaC1 0.9% i.p.), n = 4; (2)p-chlorophenylalanine (PCPA (Sigma)) 400 mg/kg i.p. daily for 2 days, n = 5; (3) reserpine (Sigma) 10 mg/kg i.p., n = 4; and (4) clorgyline (Sigma) 10 mg/kg i.p., n = 4; after 3 h 50 min these animals received reserpine as in point 3. Our modified electrodes were also used to establish maps of peak 3 in 4 nuclei of the raphe system. The following coordinates were used: raphe dorsalis nucleus (RDN), A = 5.2; L = 0; V = 5.0; raphe centralis nucleus (RCN), A = 3.3; L = 0; V -- 2.0; raphe pontis nucleus ( R P N ) , A = 1.8; L = 0; V = 1.8; and raphe magnus nucleus (RMN), P -- 0.6; L = 0; V = 0.3. In each nucleus, recordings were made at several different A-P positions, and the RDN was also explored in 2 lateralities (see Figs. 3-6). The same electrode was placed in each of these 4 raphe nuclei successively, to obtain and to compare their peak 3 heights. Finally, at the end of each experiment and for histological control, an electrolytic lesion (5 mA, 3 min) was made in each nucleus of the raphe system. This lesion was made with the working electrode in the position where the maximal peak 3 height had been recorded. The brain was then rapidly removed for histological processing (cresyl violet). RESULTS

(A) Electrode characteristics All results were obtained using our modified working electrodes with 3 pyrolytic carbon fibers. Compared to the mono carbon fiber elec-

47 trodes and to modified electrodes having 5 or 10 or about 20-carbon fibers, it permits measurement in vitro and in vivo with a better resolved peak at 280-300 mV. The electrochemical response obtained in either experimental situation with a 3-fiber electrodes is in the range of 25 125 nA, where the response obtained with a monofiber electrode would be only around 5 nA, the limit of sensitivity of 'PRG5 Tacussel' system. Thus with the new electrodes, detection of smaller signals is possible. The 3-fiber electrodes, compared to the monofiber electrodes, are less fragile at the active tip (500 ~m long), and last longer in each experimental study (10- 12 h compared to the 6-8 h of the monofiber electrodes). In addition, these 3-fiber electrodes can be reused several times after trypsin cleaning and a repeat electrochemical treatment (see Materials and Methods). No deterioration was observed in their electrochemical characteristics after this treatment, although monofiber electrodes physically deteriorate during a second electrochemical treatment.

(B) Pharmacological modifications of the peak 3 recorded in the RDN In this nucleus, with the modified electrodes

A

used, the exploration of the oxidation potential in the range o f - 0 . 0 5 to + 0.45 V shows two peaks. The first signal is probably dependent upon extracellular concentration of AA and catechols6.13: we have called this signal:peak 1 + peak 2'. However the precise identification of this signal and the separation of the putative compounds constituting it remain to be done. The other peak (peak 3) occurs at about + 300 mV.

Here we have analyzed the modification in the peak height induced by several pharmacological treatments.

(1) p-chlorophenylalanine (PCPA) treatment. In PCPA treated rats (n = 5) and in comparison with an untreated group of animals (n = 4), the same 3 fiber electrode was used to measure peak 3 heights. The peak 3 height of the treated animals is about 70% less than that of the control animals. This difference is significant (P 0.001 ) (Fig. 1). (2) Reserpine treatment. 20-30 min after reserpine administration, peak 3 height increases by 20% in comparison with an untreated group of animals (NaC1 0.9%). Control values are again reached 90 min after the drug administration (Fig. 2). (3) Clorgyline treatment. Treatment with clor-

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48

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Fig. 2. Left: voltammograms obtained with modified working electrodes implanted in RDN of rats, treated with: A, saline (0.6 ml i.p.), peak 3 is stable over a period of 4 h; B, reserpine (10 mg/kg i.p.), peak 3 increased transiently; and C, clorgyline (10 mg/kg i.p.), peak 3 decreases. Reserpine (10 mg/kg i.p.) after clorgyline produces no change. Right: plots of mean ÷ S.D. peak 3 heights from 4 experiments as illustrated on the left.

gyline (IMAO) induced a rapid large decrease in the peak 3 height (70-80%); this decrease is maximal 2 h after clorgyline. At this point, an injection of reserpine is not followed by a significant change in the peak 3 height (Fig. 2). (C) Peak 3 map in the raphe system (1) Individual maps of the RDN, RCN, R P N and RMN. In the RDN, RPN and RMN, the coordinates at which the largest peak heights were recorded are well correlated with the anatomical topography of these nuclei. However, for RCN, the highest peak 3 was recorded 500 ~m up from the histological theoretical center of this nucleus. These comparisons were made after histologi-

cal verification of the microlesions made at the points of maximum peak 3 heights in each nucleus (Figs. 3-6). (2) Peak 3 height: inter-raphe nuclei comparison. The direct comparison of the peak 3 heights recorded in the 4 raphe nuclei studied (RDN, RCN, RPN and RMN) demonstrated a signficant superiority of the peak 3 recorded in the RDN (see Fig. 7). No significant difference was found between RCN, RPN and RMN. The differences observed has been established from the position where the highest peak 3 was recorded in RCN, RPN and RMN, and in position B 5 in RDN (see Fig. 3). Calculations from lesion positions suggest that recording positions B 1 and B 2 for RDN were in the third ventricle.

49

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Fig. 3. Map of raphe dorsalis nucleus (RDN). Right part of picture: A-C, anterior-posterior electrode tracks; D-E, lateral tracks. For each track several DPV measurements were made: 7 for A, 9 for B, 7 for C, 5 for E and 5 for D, shown as successive points on the lines and expressed by progressive numeration in the plots. Plots show the mean _+ S.D. of peak 3 heights obtained from 5 experiments. In the lower part of picture there is an example of the peaks obtained on track in one animal. The histological picture (bottom left) shows the electrical lesion obtained by the working electrode in position 5 of track B. Arrows show this lesion, tim, fasciculus longitudinalis medialis. V, third ventricle. DISCUSSION

In this work, an improvement in DPV technique was obtained with the modified working electrodes equiped with 3 pyrolytic carbon fibers. These electrodes show better longevity, less mechanical fragility and greater sensitivity in DPV detection by virtue of the larger current passed, compared to monofiber electrodes which could only be used at the limit of the sensitivity range of PRG5 Tacussel system. The disproportionate increase in sensitivity of the 3-fib-

er over the monofiber electrodes is still a matter for speculation; in view of the fact that a second electrochemical treatment almost invariably physically damages a monofiber electrode, perhaps the first treatment has already weakened it. A 3-fiber electrode will be subjected to a lower current density per fiber than a monofiber one submitted to the same voltage for treatment. With our modified electrode, it is now possible to explore the brain areas less rich in 5-HT cells than the raphe system, where the monofiber electrodes are not sensitive enough. The limits of this technique remain in the modest lifetime

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51 Moreover, it is possible to conclude that the signal obtained by DPV measurements in the raphe system depends essentially on the extracellular 5-HIAA, since treatment with reserpine subsequent to a clorgyline injection does not affect the height of peak 3.

of the electrodes (about 10 h before cleaning and retreatment is necessary). Our results with PCPA treatment suggest that the peak observed at + 300 mV in RDN depends on the indoleamine content, since peak 3 as well as the 5-HT level, decreases in RDN after PCPA treatment 17.

Raphe Pontis map by DPV technique

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Fig. 7. Comparison of peak 3 heights between the 4 raphe nuclei. A: voltammograms obtained using the same modified electrode in RDN, RCN, RPN and RMN of the same rat (p. 3 = peak 3). B: plots of mean --- S.D. peak 3 heights from 5 experiments ( ~ peak 3 height significantly different from the other 3 nuclei, P ~ 0.01).

Others have shown that in the rat striatum, peak 3 recorded seems to be mainly dependent upon extracellular 5-HIAA, according to the pharmacological analysis done in this area 2.9. Indeed, the striatum is well innervated with serotoninergic terminals 1,7,34,most of these proceeding from the RDN 4.~1,22,26,3°.31,while the raphe nuclei contain essentially the cell bodies 2,5,12.15,2s,32. The DPV results and the histological observations permit us to demonstrate a variation in 5-HIAA distribution within a single raphe nucleus, as well as between the 4 raphe nuclei studied. The map of the raphe system, prepared in this work, demonstrates that the maximal peak 3 height is always obtained when the working electrode is located in the site containing the largest concentration of serotoninergic cell bodies2.12'28"32. This result together with the pharmacological data obtained, demonstrates that DPV provides a good means to detect serotonergic innervation in brain areas. REFERENCES 1 Aghajanian, G. K. and Bloom, F. E., Localization oftritiated serotonin in rat brain by electron-microscopic autoradiography, J. Pharmacol. exp. Ther., 156 (1967) 2330. 2 Aghajanian, G. K., Kuhar, M. J. and Roth, R. H., Serotonin-containing neuronal perikarya and terminals: differential effects P-chlorophenylalanine, Brain Research, 54

The highest peak 3 detected in the RDN, compared to the one obtained in the RCN, RPN and RMN, favors again this hypothesis: indeed histologicaP5 and biochemical works2,3,5,15.28have already described that the largest aggregation of serotoninergic cell bodies is located in the RDN. The results presented here have been obtained in acute experiments. We are now making efforts to further prolong the lifetime of the electrodes in order to permit measurements in chronic animals. ACKNOWLEDGEMENTS

This work was supported by INSERM U 52 (CRL 80.60.2), CNRS (L.A. 162) and DRET (Grant 80.175). F.C. was a recipient of a fellowship from the European Economic Community and 'Ministero del Lavoro, Italia'. We thank Dr. Alex Milne for help in the preparation of this paper.

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