Effect of haloperidol and sulpiride on dopamine metabolism in nucleus accumbens and olfactory tubercle: A study by in vivo voltammetry

0306-4522/85 $3.00+ 0.00 PergamonPressLtd 0 1985IBRO

Neuroscience Vol. 14,No. 3, pp. 775-782,1985 Printed in Great Britain

EFFECT OF HALOPERIDOL AND SULPIRIDE ON DOPAMINE METABOLISM IN NUCLEUS ACCUMBENS AND OLFACTORY TUBERCLE: A STUDY BY IN WV0 VOLTAMMETRY A. LOUILOT,M. BUDA*, F. GONON*, H. SIMON,M. LE MOAL and J. F. PUJOL* INSERM U.259, Laboratoire de Psychobiologie des Comportements Adaptatifs, rue Camille Saint Saens, 33077 Bordeaux Cedex, France;* INSERM U.171, Groupe de Recherche de Neurochimie Fonctionnelle, 1 Avenue Georges Clbmenceau, 69230 St Genis-Laval, France Abstract-Differential pulse voltammetry used with electrochemically pretreated carbon fibre microelectrodes enables separation between the two peaks corresponding to the ascorbic acid and catechol oxidation currents. The effects of haloperidol and sulpiride on the 3,4-dihydroxyphenylacetic acid peak recorded in the nucleus accumbens and olfactory tubercle of rats were studied. Chloral hydrate anaesthetized preparations and chronic preparations were used. A microdevice was designed to implant electrodes in freely moving rats. Voltammograms were recorded every minute in each structure in acute preparations and every 2 min in chronic preparations. In acute preparations haloperidol induced a similar dose-dependent increase in the catechol oxidation peak in both structures. Sulpiride at all doses only induced an increase in the olfactory tubercle. In chronic preparations haloperidol and sulpiride had even larger effects on the 3,4-dihydroxyphenylacetic acid peak in both regions. In these preparations sulpiride induced a significant increase in nucleus accumbens. The effects induced by haloperidol in the two regions were greater than those induced by sulpiride. The main conclusions of this study are that the results of voltammetry agree with biochemical results on the effects of haloperidol and sulpiride on dopamine metabolism. An interaction of chloral hydrate with the effects of the two neuroleptics was also observed.

The pharmacological profiles of drugs on specific biological or behavioral assays are characterized in terms of time-course and dose-response evaluations. However, it is more difficult to evaluate the neurochemical effects of psychotropic drugs. The postmortem measures generally used do not allow a direct approach to the dynamic processes related to the mechanisms of action of these agents. Biochemical neuropharmacologists have recently developed new push-pull or dialysis techniques which circumvent these difficulties and allow continuous measurement to be carried out. In vivo electrochemical techniques present a new and valuable approach to study of these problems. Differential pulse voltammetry (DPV) used in combination with electrochemically pretreated pyrolytic carbon fibre microelectrodes enables continuous monitoring in limited brain regions of alterations in dopamine (DA) metabolism.2~5~6*20 The ascorbic acid and catechol oxidation currents can be clearly separated with this technique.‘*5 Pharmacological investigations demonstrate that under normal conditions the main contributor to the catechol current is the presynaptic metabolite of DA, 3,4_dihydroxyphenylacetic acid (DOPAC).@

The DPV technique can be used for quantitative evaluation of the dose-dependent alterations of regional DOPAC concentrations induced by psychotropic drugs. In a recent study using DPV in acute animal preparations after (+) and (-) amphetamine administration, a different reactivity of the DA terminals within the nucleus accumbens (ACC) and the olfactory tubercle (TWO)‘” was observed. Postmortem biochemical methods had demonstrated that antipsychotic drugs induced differential regional changes of DA metabolism.‘s~‘g*23.24 The present study was carried out to investigate the effects of a classical neuroleptic (haloperidol) and an atypical neuroleptic (sulpiride) on DA metabolism in two areas of the ventral striatum:” the ACC and TUO. Dose-response measurements were conducted in anaesthetized animals which permitted the recording of signals from the two regions simultaneously. However, it has been recently reported that anaesthetics alter the reactivity of neurons to psychoactive drugs in several brain regions.g.15.21For that reason a microdevice designed to implant working electrodes in freely moving rats was used. The effects of the two antipsychotic drugs were tested in these conditions.

Please address all correspondence to Dr. A. Louilot.

Electrochemical

EXPERIMENTALPROCEDURES ACC, nucleus accumbens; DA, dopamine; DOPAC, 3:4-dihydroxyphenylacetic acid; DPV, differential pulse voltammetry; TUO, olfactory tubercle.

Abbreviations:

775

procedure

A classical three-electrode system with working, reference and auxilliary electrodes was used. Differential pulse voltammetry coupled with electrochemically pretreated carbon

776

A. Louilot

fibre microelectrodes was employed. Microelectrode preparation and electrochemical treatment procedures have been described previous!y.8,‘6 For acute animal preparations the reference electrode was a conventional Ag/AgC! electrode (Tacusse! Villeurbanne, France) and the auxilliary electrode was a platinum wire. For chronic animal preparations the reference electrode was an AgCl-coated silver wire obtained from a Teflon-coated silver wire (Ref. AGIOT, Medwire Corp., Mt. Vernon, NY);’ the auxilliary electrode was a small, stainless steel screw. The voltammetric apparatus (PRGS Tacusse! Villeurbanne, France) was set up with the following parameters: scan rate, 10 mV/s; voltage range, 0, +200 mV vs the Ag/AgCl reference electrode; pulse modulation amplitude, 50 mV; pulse modmation period, 0.2 s; pulse modulation duration, 48 ms. Differential pulse voltammograms were recorded every minute alternately in each structure, ACC and TWO for anaesthetized acute rats, and every 2 min for unanaesthetized chronic rats. Working electrodes were disconnected between recordings. The height of the catechol peak which appeared in vitro at -t 100 mV, was measured as described previous!y.6 Drugs and saline were injected at the end of the control period of 30min during which the signal varied less than 10%. For each experiment the mean of the 15 peak heights measured during the control period was computed and represented the 100% value of the catechol peak height. Before and after each experiment the response of the carbon fibre microelectrode was calibrated in solutions which contained different concentrations of DOPAC. In some cases, the working electrode implantation site was visualized by’an electrolytic lesion obtained by passing a current through the microelectrode and verified according to the atlas of Konig and K!ippe!.13 A typical lesion was shown in previous work.lJ Animals

(Merck) and saline were injected i.p. in a volume of 1ml/kg body weight. Doses were always expressed as the drug weight. None of the drugs gave an electrochemical vitro, in the potential range of 0, + 200 mV.

which

took approx.

30-60s.

Drugs

Haloperido! (Haldol, Laboratoires Lebrun), sulpiride (Dogmatil, Laboratoires Delagrange), chlora! hydrate

signal in

A

and surgery

Male Sprague-Dawley rats (Iffa-Credo, France) weighing 300-450 g were used under acute and chronic conditions. For both preparations anaesthesia was produced by chloral hydrate (400 mg/kg i.p.). The animals were placed in a stereotaxic apparatus (incisor bar 3 mm below interaural zero). The stereotaxic co-ordinates for the two recording regions were for ACC (+ IO.6 mm anterior to the interaural line, 1.5 mm lateral to the midline, 7 mm below the cortical surface), and for TUO (+ 9.9, + 2.2 and 9 mm, respectively). For acute preparations two working electrodes were implanted in the same animal, one in each recording region. A saline bridge formed the electrical contact between the reference electrode, the auxilliary electrode and the skull bone. The animals were kept anaesthetized throughout the experiments by injections of chloral hydrate (100 mg/kg) as required. For chronic preparations, a micromanipulator was devised to implant electrodes in unanaesthetized and unrestrained rats. Four stainless steel screws were inserted in the skull bone; the auxilliary electrode was screwed in to touch the dura mater; the reference electrode was carefully slid between the dura mater and the bone. Only one micromanipulator (Fig. 1) was implanted per rat, i.e. only one region was recorded at a time. The micromanipulator was stereotaxically implanted as a simple electrode and was cemented on the skull surface with acrylic dental cement. Then the three electrodes were connected to a small threepin-plug. This plug was connected to the voltammetric apparatus by means of a rotating connector (Air Precision, Paris, France) and a flexible cable. The surgery was followed by a recovery and habituation period of a few days (4-6). To change the working electrode the rat was held in one hand,

et al.

B

1UTl Fig. 1. Micromanipulator: schematic design. The system made in Duralumin is composed of one sheath (B) and one electrode holder (A). Once fixed on the electrode holder (A} with dental cement, the working electrode (4) was moved down as follows: the electrode holder (A) was placed over the upper part of the sheafh (B), [the siicbng cylinder (5) completely masking the working electrode (411 the whole sliding cylinder was introduced into the sheath body [8) and the piston (3) maintained in the upper position; the piston (3) then was moved down in the sliding cyymder (5) until the vertica4 guide (2) butted the plate (7). The down-stroke was wntinued by the sliding of the vertical guide (2) in the opening of the plate (7) and the fina position was attained by screwing the r&r&eter screw (I)~on the t.hRod (SW pm per revo4ution) (6); at that time the workmg etectrode (4) was careMy guided by the guide can&a (10) ptaviously fixed on the sheath bottom (9) with acryfie detita4 cement. Once positioned, the working e4ectrode (4-lc&&t&d beyond the guide cannula (10) by 1 or 2 mm. The workirrg &e&rode was changed by operating in inverse order. When imphurted the microtuar&tWvr was protected by a two-part .ptaatic cover. The lower part was previously cemertW to the sheath (B) with dental cement and the upper part placed at the end of the implantation.

Voltammet~c

3A NaCI

study of neuroleptics

0.9 % (ip) n=6

o--a

TUO ACC

inj

t I 0

I TIME

08

NaCI

I 1

I

(h.)

0.9 % ACC

41

0

4

TUO

Pig. 2(A). Catechol peak height measured from DP voltammograms recorded from the nucleus accumbens (ACC) and the olfactory tubercle (IWO) of anaesthetized saline controls, For each experiment the results were expressed as the percentage (mean f SEM) of the mean pre-injection value calculated by averaging the 15 absolute vaiues of the peak heights obtained during the control period (30 min pre-injection period). n represents the number of treated rats. (B) Typical vohammograms recorded from the ACC and TUO. The working electrodes were implanted in the ACC and TUO of the same rat. The electrochemical signal was detected, between 0, -t 200 mV vs the reference electrode every minute using alternate electrodes, in each structure. The arrow indicates the time of the i.p. injection.

778

HALOPERIDOL r

~-~ O.O5mg/kgGp)

n:6

O.lmglkg(ip)

n.6

0.2mgIkg(ip)

i

1 -6.5

finj I 0

I I

1 1

I 2

TIME iiiz&Fq

(h.) /I

Fig. 3. Catechol peak height measured from DP voltammograms recorded from the ACC and TUO of anaesthetized rats treated with haloperidol. For comments, see legend of Fig. 2. Data analysis and statistics Results were expressed as percentages (mean + SEM). For acute preparations results of each structure were submitted to a two-factor analysis of variance (ANOVA), with drugs and saline being the independent factor and time the repeated measure. In order to compare the effects of haloperidol and sulpiride on the peak height in the ACC and TUO dose-response curves were established 2 h after drug injection. In this study the increase of the peak heights induced by haloperidol and sulpiride in the ACC and TUO were evaluated by comparison with the respective decreases observed in saline controls at the same time. Differences between ACC and TUO values were analysed with ANOVA. For chronic preparations results were only analysed qualitatively.

HALOPERIDOL

(2h.post-injj

RESULTS Acute preparations

In anaesthetized control animals peak height decreased in ACC more than in TUO (Fig. 2A,B). Two hours after the injection of saline the peak heights were 79 f 5% (n = 6) in ACC and 95 + 4% (n = 6) in TUO (Fig. 2A). At all doses used, haloperidol increased the catechol peak height in both structures (Fig. 3), with a slightly larger increase in TUO. The maximum in-

Dose mg/kg

tip)

Fig. 4. Effect of haloperidol on @echo1 pealE h@bt measured in the ACC and ‘IWO, CX+Z#W@~&&the r%$Wivt saline control values 2 h at& the in&e&n. I&ta~Its are means with SEM of data obtained on 5-8 animals and are expressed as a percentage of saline controls (see text).

Voltammetric study of neuroleptics

-4

SULPIRIDE

l2Smgkg(id

179

25mglkgW

n-5

TUO ACC nr5

tinj

SanglWip)

n55

100mglkgf ipI

TIME

.)

n&

1

(h-1

Fig. 5. Catechol peak height measured from DP voltammograms recorded from the ACC and TUO of anaesthetized rats treated with sulpiride. For comments, see legend of Fig. 2.

creases in both structures were observed after the 0.5 mg/kg injection, and reached 175 f 8% (n = 5) and 202 + 4% (n = 5) of their initial values in ACC and TUO, respectively. The dose effect was statistically significant: F(5,32) = 18.1, P < 0.001 in ACC; F(5,32) = 19.2, P < 0.001 in TUO. The increase in peak height above the respective saline control values, 2 h after injections was similar in both regions studied (Fig. 4); F(1,54) = 0.137, N.S. Sulpiride increased the catechol peak height only in the TUO (Fig. 5). The maximum increase was observed after the 25 mg/kg injection, when the peak height reached 158 + 11% (n = 5) of its initial value. The dose effect was statistically significant F(4,22) = 5.8, P < 0.01 in TUO, but not significant in ACC

F(4,22) = 0.9, N.S. Statistical differences between the ACC and TUO were observed when peak height variations above respective control values were compared 2 h after the injection (Fig. 6) F(1,34) = 16, P < 0.001. Chronic preparations

The doses used for haloperidol and sulpiride were those that produced the greatest increase in peak height in acute experiments. In control (saline), freely moving rats, peak heights remained relatively stable in both the ACC and TUO during the 2 h following the saline injection (Fig. 7A,B). Administration of 0.5 mg/kg haloperidol caused a rapid increase in peak height in the ACC (Fig. 7C).

780

A.

SULPIRIDE

-I,

0

0

’

12.5

(2 h. post

-ini)

1 25

Dose mglkg

Louilot et al.

-

TUO

D---C]

ACC

I

I

So

mo

1

($1

Fig. 6. Effect of sulpiride on catechol peak height measured in the ACC and TUO, compared with the respective saline control values 2 h after the injection. Results are means with SEM of data obtained on 5-6 animals and expressed as a percentage. of saline controls.

The height reached 287 + 36% (n = 3) of its initial value 2 h after the injection. Sulpiride (25 mg/kg) injection produced an increase in peak height in both the ACC and TUO (Fig. 7D,E). The maximum increases, observed 2 h after the injection, were 164 f 8% (n = 4) in the ACC and 210 f 26% (n = 3) in the TUO. DISCUSSION

The present study was a quantitative investigation of the effects on DA metabolism of a classical neuroleptic (haloperidol) and an atypical neuroleptic (sulpiride) in the ACC and TUO by DPV. We also evaluated the interaction between chloral hydrate and the antipsychotics agents on DA metabolism using this technique. The results obtained in this study showed that haloperidol induced statistically significant and dosedependent increases of the catechol peak height in both structures. Similar results were obtained after administration of sulpiride in the TUO. These increases in peak height as measured by DPV, correspond qualitatively to increased levels of DOPAC in the ACC and TUO, which have been reported using biochemical method~.~~‘~~‘**~ Furthermore the present results show that haloperidol had a greater effect than sulpiride which also agrees with the biochemical analysis carried out by Scatton et ai.” the haIopecidd However, comparison of dose-response curves, obtained using DPV from acute anaesthetized preparations, with biochemical

results in awake animals” showed that the peak height increases were relatively smaller than the increases in DOPAC levels. A similar difference was observed with sulpiride. In addition no change of catechol peak height was seen at any dose in the ACC of anaesthetized animals. These results could be explained by an interaction of chloral hydrate with the neuroleptic drugs. In fact, the results obtained from chronic preparations show that the increases induced by the two antipsychotic agents were much higher, and agreed better with the biochemical results, especially for the action of sulpiride in the ACC. Similar differences between anaesthetized and nonanaesthetized preparations have been reported using the DPV technique in the striatum after administration of 0.5 mg/kg haloperido16 The progressive decline of the catechol peak height observed in acute anaesthetized preparations after saline administration may well be related to the effects of chloral hydrate, since such a decrease was not seen in chronic preparations. The results from acute and chronic experiments using DPV showed that haloperidol induced similar effects on DA metabolism in the two structures, with sulpiride having a greater effect in the TUO than in the ACC. This latter result agrees with biochemical studies,” and might be explained by a preferential blockade of DA receptors in the TUO by sulpiride. Indeed it has been shown that low doses of sulpiride do not displace [3H]spiperone in the ACC.” Considering that sulpiride is reported to be a specific antagonist of D2-dopamine receptors”.” and that the increase in DOPAC levels induced by neuroleptics is consecutive to the blockade of DA-receptors** it is suggested that the preferential action of sulpiride in the TUO may be explained by the existence of different D2-receptor populations in the two brain regions. In other respects, it is of interest to note that halothane anaesthesia prevents sulpiride-induced increases in firing from substantia nigra DA cells.‘5 This result could be paralleled with the lack of increase in the catechol peak height observed in the ACC in chloral hydrate-anaesthetized animals. Since it has been suggested that general anaesthetics interfere with conformational changes of proteins immersed in the cellular membrane,4 it is hypothesized that chloral hydrate acts in such a way and indirectly prevents the normal drug-receptor interaction in the ACC and TUO. In any case, these results provide evidence of an interaction between anaesthetics and antipsychotic drugs and emphasize the importance of using nonanaesthetized preparations. Since the response characteristics of a pretreated microelectrode in vim change after 4-5 h of recording, i.e. the electrochemical signal tends to become distorted,6,7 a system allowing the implantation of newly treated microelectrodes into the same animal is needed. A system was used in the present study which allowed us to avoid having to anaesthetize or restrain animal&’

781

Voltammetric study of neuroleptics TUO o-4ACC

TIME

(h.1

TIME

(h.)

Fig. 7. Catechol peak height measured from DP voltammograms recorded from the ACC and TUO of freely moving rats treated with saline, haloperidol and sulpiride. For each experiment the results were expressed as the percentage (mean f SEM) of the mean pre-injection value calculated by averaging the 15 absolute values of the peak heights obtained during the control period (30 min pre-injection period). The working electrodes were implanted in the ACC or TUO of different rats. The electrochemical signal was detected between 0, +200 mV vs the reference electrode every 2 min. The arrows indicate the time of the i.p. injection and n represents the number of animals.

while implanting new microelectrodes. Moreover, compared to the previously described technique’ the advantage of the present system is that changing the carbon fibre electrode is more convenient and rapid and does not require any restraint to the head of the rat. Summary

The present results obtained with DPV show that the two antipsychotic drugs, haloperidol and sulpiride, induce catechol increases in peak heights that

are fully comparable to the increases in DOPAC levels that have been measured in biochemical studies. Haloperidol was found to have similar effects in both structures whereas sulpiride had a greater effect in TUO than in ACC. Chloral hydrate was also found to interfere with the action of antipsychotics drugs on DA metabolism. Acknowledgements-This

work was supported by INSERM (Unites 171 et 259) and CNRS (LA 162) grants. The authors thank Mlle C. Sparks for typing the manuscript.

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782

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(‘1 u/.

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19 September 1984)