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Estimation of chronic dopamine release from the caudate nucleus of theMacaca mulatta
Brain Research, 77 (1974) 257-268 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
257
ESTIMATION OF CHRONIC DOPAMINE RELEASE FROM THE CAUDATE NUCLEUS OF THE MA C A C A MULATTA
CHRISTIAN GAUCHY, BERNARD BIOULAC, ANDRI~ CHERAMY, MARIE-JO BESSON, JACQUES GLOWINSKI AND JEAN-DIDIER VINCENT
Groupe NB (INSERM U.114), Coll~ge de France, Paris 5e and Laboratoire de Neurophysiologie, Facult~ de M~decine, Bordeaux (France) (Accepted April 16th, 1974)
SUMMARY
Using a cup permanently implanted on the antero-lateral surface of the caudate nucleus, attempts were made to study the spontaneous and D-amphetamine induced release of [aH]dopamine (DA), which was continuously synthesised from L-[3,5-aH]tyrosine in the unanaesthetised Macaca mulatta placed in a restraining chair with freedom of movement of the head and members. The spontaneous release of [aH]DA from dopaminergic terminals was detected during the continuous superfusion of the tissue with [3H]~rnino acid in 8 monkeys. In most cases [aH]DA release reached a steady state level within 30 min after the onset of superfusion. Similar results were obtained when the experiments with [aH]tyrosine were carried out on 3 successive days. In 2 of the cases examined, the quantity of spontaneously released [aH]DA during the last 24 or 48 h of a 5-6 day experiment was markedly increased as compared to that observed during previous days; this effect did not appear to be related to changes in the specific activity of tyrosine in the superfusing medium. In 3 monkeys D-amphetamine (10 -5 M) increased the level of [aH]DA release by 15-20 times that of the normal spontaneous release of the transmitter, the effect was less pronounced in 2 other animals exhibiting a higher level of spontaneous [aH]DA release. The amphetamine effect could be detected every day in longitudinal studies. For example, in one animal the ratio of the amphetamine-induced [aH]DA release to spontaneous [3H]DA release varied only from 10 to 15 during a 5 day experiment. This new approach should make possible long term studies on the regulatory processes involved in DA release from dopaminergic terminals of the nigro-neostriatal system in the unanaesthetised primate.
258
C. GAUCHY el a[.
INTRODUCTION
The importance of the nigro-neostriatal dopaminergic system in extrapyramidal functions has been clearly demonstrated11. Whereas many reports have been devoted to the analysis of dopamine (DA) metabolism in the caudate nucleus of various mammals, demonstration of the in vivo release of DA has only been made in a few cases. The release of endogenously synthesised DA from dopaminergic terminals has been examined particularly in the cat. Different approaches were used: DA or labelled DA synthesised from [14C]- or [SH]tyrosine were estimated in perfusates of the lateral ventricles6,1s 20,24, in superfusates of a push-pull cannula inserted into the caudate nucleus 15,16,2° or into the putamen 17, and in superfusates of a cup disposed at the surface of the caudate nucleus1, 2,5. Studying the primate offers several advantages when compared to the cat, since extrapyramidal disorders can be induced in monkeys by lesions or drugs 13. However, only a preliminary attempt has been made to study the release of DA from the caudate nucleus in Macaca mulatta: using a push-pull cannula, Defeudis et al. 7 failed to detect any release of [14C]DA after labelling of dopaminergic terminals with [14C]tyrosine, although [14C]catecholamines could be found in tissues. In a complementary study2~, these authors observed a release of both [14C]DA and [14C]noradrenaline during a 1 h local superfusion of the tissue with [14C]DOPA. In this experiment, release of [14C]DA may not be specific since [14C]DOPA can be decarboxylated into serotoninergic terminals and in pericytes as well as into dopaminergic terminals. Therefore, in the present study we attempted to demonstrate the spontaneous and evoked release of [aH]DA endogenously synthesised from L-[3,5-aH]tyrosine in the caudate nucleus ofMacaca mulatta. Furthermore, in employing the cup technique used in the acute cat preparation x,2,5 we have extended this methodology to study the release of DA in a chronic preparation. The principle of this methodology is to place a permanent cup upon the external surface of the caudate nucleus and to superfuse the tissue with a sterile physiological medium. During the experiment, [ZH]tyrosine of high specific activity is introduced continuously into the cup and the newly synthesised and simultaneously released [3H]DA is analysed in successive collected fractions of superfusates. In the present study, the spontaneous release of the transmitter was examined daily in various monkeys for periods lasting as long as 6 days. Furthermore, the release capacity of dopaminergic terminals was tested each day by measuring their responsiveness to amphetamine*. METHODS
Preparation of the animals Eight adult female monkeys (Macaca mulatta) weighing from 4 to 4.5 kg were used in this study: Valentine (No. 0), Nina (No. 1), King-Kong (No. 2), Zoe (No. 3),
* A preliminaryreport was made at the FederationProceedings4.
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CHRONIC DOPAMINE RELEASE IN THE MONKEY
Justine (No. 4), Sigma (No. 5), Severine (No. 6) and Proserpine (No. 7). Each animal was previously conditioned to a restraining chair for primates allowing free movement of the head and members. A first surgical operation was made under Nembutal® (40 mg/kg, i.p.). After fixation of the animal in a stereotaxic apparatus, a plastic platform was attached to the surface of the skull with stainless steel bolts. This platform was designed to allow the introduction and the chronic fixation of the superfusing cup during the second surgery. Wires for EEG, EMG, EKG, eyes and respiratory movements were soldered to pin-connectors cemented to the platform. A silicone rubber cannula was placed in the right atrium, by passing it through the internal jugular vein. This cannula was inserted under the skin of the neck and fixed to the cranial platform. After recovery from surgery, the monkeys were placed in their restraining chair for about 10 days. In the second surgery performed under ketamine (Ketalar®) (15 mg/kg, i.m.) and halothane (Fluothane®) anaesthesia, the animals were kept in their restraining chairs and their heads were temporarily immobilised according to Evarts technique 8. Following a small cortical resection, a specially designed cup, driven with the aid of a tridimensional manipulator, was placed on the antero-lateral surface of the left caudate nucleus as indicated in Fig. 1. In order to limit neurological disorders the cup was introduced through the prefrontal cortex (Fig. 1). Under these experimental conditions the lateral ventricle remains intact. Superfusion of the tissue with artificial cerebrospinal fluid was started as soon as the cup was in the correct position and then the cup was permanently cemented to the platform. The animals' heads were then
Superfusates
Superfusing medium 02 + C02
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Fig. 1. Schematic representation of cup localisation at the surface of the caudate nucleus. The anterior and lateral coordinates are those of the stereotaxic atlas of Snider and Lee 22. cd, candate nucleus; pl, sealed plastic platform; put, putamen.
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liberated and isotopic experiments were begun only after their complete recovery from the anaesthesia. Both surgical steps were performed under strict aseptic conditions. The surface of the caudate nucleus was superfused continuously with oxygenated, warmed (37 °C), sterile physiological medium (in mM: NaCI, 126.5; NaHCGz, 27.5; KCI, 2.4; KH2PO4, 0.5; CaCI2, 1.1; MgCI2, 0.83; NazSO4, 0.5; glucose, 5.9; adjusted to pH 7.4 with an O2-CO2--95:5--mixture), at a rate of 6 ml/h using a peristaltic pump. The superfusing medium was introduced via a small stainless steel tube sealed to the cup 1 mm above the surface of the exposed tissue. The volume of the physiological medium in the cup was kept constant (3130/zl) by continuous suction with a peristaltic pump through a second stainless steel tube placed 8 mm above the tissue. Moreover, the medium was continuously agitated and oxygenated in the cup with a light stream of sterile O2-COz (95:5). Superfusion with the physiological medium was kept constant in order to maintain the nervous tissues in good condition. Polygraphic recordings were made regularly, particularly during [ZH]DA release experiments to check the animals' states. Shortly following the onset of superfusion the animals took food and water which were provided ad libitum. To prevent oedema andinfection, furosemide (Lasilix®) (0.1305 g per animal) and cefalotin (Keflin®) (10 mg/kg) were respectively injected twice and once a day intramuscularly. At the end of the experiments, the animals were sacrificed by an injection of Nembutal. Brains were immediately removed and kept in formalin (10 ~,,) for histological controls. Behavioural and polygraphic observations Neurological examination was performed periodically. No major clinical disturbance was observed after recovery from the cortical surgery. The only transient disorder was a hypokinesia or mild and regressive contralateral paresis. The monkeys maintained a good appetite and were able to eat by themselves. On the basis of the EEG, nucchal electromyography, eyes and body movements, we were able to judge whether the animal was sleeping, in quiet waking or aroused. Both normal EEG patterns and cycling sleep-waking organisation confirmed the lack of central injury. Since our main purpose was first directed towards establishing the technique and improving chronical methodology, the correlation between [ZH]DA release and behaviour was not systematically attempted. {aHj DA release experiments and biochemical determinations Release experiments were carried out once or twice a day for periods lasting as long as 6 days. For this purpose, L-[3,5-3H]tyrosine (40 Ci/mmoles, Radiochemical Centre, Amersham, England or CEA, 91 Gif sur Yvette, France and previously purified by adsorption on Alumina and ion-exchange chromatography on Dowex AG 50 WX4) was dissolved in the physiological mediumL Fifteen minute serial fractions of superfusates were collected in cooled tubes containing 100 /~I of a stabilising solution (ascorbic acid 1~/o: EDTA 1.5%: DA 0.001%) and 3 ml of ethanol. D-Amphetamine sulphate (10 -~ M)was dissolved in the
CHRONIC DOPAMINE RELEASE IN THE MONKEY
261
superfusing medium and introduced for 15 min into the cup once or twice every day. Superfusate fractions were weighed for volume determination and adjusted to pH 6.7; they were then kept at --17 °C for one night. Following the centrifugation of the samples, total radioactivity of each fraction was estimated on a 20 /zl aliquot. [SH]DA was separated from [ZH]tyrosine and [3H]metabolites by ion exchange chromatography on Amberlite CG 50 and by adsorption chromatography on Alumina as described previously3. The eluates (1 ml) from the Alumina column were diluted in 10 ml of Instagel® (Packard Instrument S.A.) and the radioactivity was then measured by liquid scintillation counting. [3H]DA and [aH]tyrosine recoveries in Alumina column eluates were 70 and < 0.0001 ~o respectively. In all experiments, blanks represented by [3H]tyrosine contamination were subtracted and data were corrected for recoveries. In some experiments the identity of [aH]catecholamines was checked on pooled eluates of Alumina column by paper chromatography after acetylation of the amines and organic extraction of the acetylated derivatives14. The radiochromatogram indicated the presence of 2 peaks of radioactivity corresponding to [aH]DA and [3H]noradrenaline; [aH]DA represented more than 85 ~ of the total [3H]catecholamines recovered on the radiochromatogram; therefore, [aH]catecholamines eluted from the Alumina columns were considered as [aH]DA in all experiments. In some cases, the level of tyrosine found in superfusate fractions was estimated. The amino acid was isolated from Amberlite effluents by ion-exchange chromatography on Dowex AG 50 WX4 as described by Javoy et al. 12, and estimated by the spectrofluorimetric method of Udenfriend23. RESULTS
Spontaneous andamphetamine evoked release of [3H]dopamine during the first day of superfusion The spontaneous release of [ZH]DA was studied in 5 monkeys 3 h following the onset of superfusion when the animals had completely recovered from anaesthesia. As an example, results obtained with Severine are illustrated in Fig. 2. [ZH]DA could be detected in superfusates in the first 15 min period of superfusion with [SH]tyrosine. [3H]DA release increased in the second fraction and then remained almost constant. In this experiment, the radioactivity present as [3H]DA in superfusates was about 2 times that of the blank values due to [SH]tyrosine. The introduction of amphetamine (10 -5 M) into the superfusing medium increased the release of [3H]DA by 15 times. After removal of the drug, [3H]DA levels returned slowly to those of spontaneous release. The qualitative pattern of the effects observed was quite comparable from one animal to the next: spontaneous and evoked release of the transmitter could be detected in all cases. Small quantitative variations in [3H]DA spontaneous release occurred from one animal to another. For example, [ZH]DA spontaneous release was almost identical in Severine, Justine and Zoe but was much more prono~ficed in Proserpine (maximal level about 11 times blank values) and Sigma (maximal level 8 times blank
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Fig. 2. Effect of amphetamine on [aH]dopamine release from the caudate nucleus of the Macaca mulatta. Release of [~H]DA immediately after the onset of superfusion of the caudate nucleus with L-[3,5-3H]tyrosine (60/~Ci/ml; 6 ml/h) in 15 min serial fractions. D-Amphetamine (10 -5 M) was added into the superfusing medium during a single 15 min fraction.
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I Amph. 10-5M Fig. 3. Comparison of spontaneous release and amphetamine induced release of [aH]DA in various monkeys during the first day of superfusion. Each experimental point represents the amount of [SH]DA released during a 15 rain fraction on the first day of superfusion in Sigma, Proserpine, Justine, Severine and Zoe. The black square corresponds to the 15 rain fraction during which o-amphetamine (10 -5 M) was added. As in Fig. 2, the caudate nucleus was superfused continuously with L-[3,5-aH]tyrosine (60/zCi/ml at a rate of 6 ml/h).
263
CHRONIC DOPAMINE RELEASE IN THE MONKEY TABLE I
MEAN VALUE OF SPONTANEOUS AND D-AMPHETAMINE INDUCED [3H]DA RELEASE DURING THE FIRST EXPERIMENTAL DAY IN THE VARIOUS MONKEYS Each value represents the m e a n i S.E.M. of data corresponding to the quantity of radioactivity (nCi/l 5 min fraction) recovered in successively collected fractions. The n u m b e r in brackets corresponds to the n u m b e r of fractions used to establish the mean. SR = spontaneous [aH]DA release (blank value deducted); bc = blank value; SR/bc = ratio of the [8H]DA spontaneous release to the blank value; ER = [3HIDA release evoked by amphetamine (10 -5 M ) ; ER/SR = ratio of evoked [aH]DA release over spontaneous [aHIDA release.
SR
bc
SR
ER
be
Proserpine Zoe Justine Sigma Severine
3.41 i 0.16 (7) 0.32 ± 0.01 (3) 0.82 i 0.19 (5) 2.37 4- 0.09 (6) 1.04 i 0.17 (3) 0.76 4- 0.08 (6) 5.98 ~: 0.70 (4) 0.75 ± 0.02 (5) 0.50 4- 0.06 (4) 0.50 4- 0.07 (4)
10.66 0.35 1.38 7.97 1.00
ER SR
13.0 (1) 18.8 (1) 16.6 (1) 26.3 (1) 7.6 (l)
3.8 22.9 16.0 4.4 15.3
values) (Fig. 3 and Table I). Sigma was the only monkey in which the spontaneous release of [aH]DA increased continuously as a function of time. The effect of amphetamine was particularly striking in Severine, Zoe and Justine since [aH]DA levels reached from 15 to 23 times the mean spontaneous release of the [all]amine estimated in the fractions preceding amphetamine addition. Surprisingly enough this effect was relatively less pronounced in Proserpine and Sigma (about 4 times), animals in which [SH]DA spontaneous release was particularly elevated (Fig. 3 and Table 1).
Spontaneous and amphetamine evoked release of [aH]dopamine as a function of the number of days following the onset of superfusion The spontaneous release of [aH]DA as well as the amphetamine effect could be observed for a few days in various monkeys. Zoe and Justine were the first 2 monkeys in which we attempted to study the evolution of the [SH]DA spontaneous and evoked release as a function of time. Positive results were obtained for 3 days with both animals. As a result of improvements made in the chronic superfusing method from one experiment to the next, the [aH]DA release experiments were enabled to be progressively prolonged. Indeed, the best results were obtained with the last 3 monkeys (Sigma, Severine and Proserpine) who were studied for 4, 5 and 6 days, respectively. Whereas technical incidents were responsible for the sacrifice of the first 2 animals, this was not the ease in the last 3 experiments which were interrupted as previously programmed. The results obtained with Severine and Proserpine concerning the evolution of [SH]DA spontaneous and amphetamine induced release as well as the fluctuations of other parameters, examined in these experiments, will be described in detail. The average spontaneous release of [SH]DA was calculated every day in serial 15 rain fractions. As indicated in Fig. 4, small variations in [SH]DA spontaneous release were observed from day to day in each animal during the first 3 and 5 days for
264
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Fig. 4. Estimation of various parameters in tong term release experiments carried out on Severine and Proserpine. The figure represents from the top to the bottom: (1) the change in the volume of the superfusate, collected in serial fractions (as a function of the number of days), the volume of superfusing fluid introduced into the cup being 1.5 ml; (2) the output of tyrosine as well as the specific activity of tyrosine present in the superfusate; (3) the difference between the amount of pH]tyrosine introduced into the cup and the amount of [SH]tyrosine recovered in the superfusate at the end of the 15 min fraction (clearance of pH]tyrosine expressed in percent of the amount originally introduced); (4) the quantity of [SH]DA released; (5) the ratio of [aH]DA released to the specific activity of tyrosine into the superfusate. Each value represents the mean of the data obtained from 15 min serial fractions collected during each daily experiment. These parameters correspond to the spontaneous release of the transmitter. As before, L-[3,5-3H]tyrosine was introduced continuously into the cup at a rate of 6 ml/h (60 #Ci/ml).
Severine and Proserpine, respectively. A marked increase in the mean release o f p H ] D A was noticed in the last 48 h (Severine) and 24 h (Proserpine) of the experiments. This effect did not seem to be related to changes in the volumes o f the superfusates. The small increase in the mean volume of superfusates, observed during the first 3 and 5 days, when compared to the volume o f superfusing fluid introduced during each t 5 min period into the cup may be due to an excretion o f interstitial fluid. High levels of tyrosine were found initially in superfusates (Fig. 4); they seem to decrease as a function o f time and were almost identical at the end of the experiments to the concentration o f the [3H]amino acid introduced into the cup (Fig. 4). At the same time, the clearance o f the [SH]amino acid from the superfusing fluid (difference between [ZH]tyrosine introduced into the cup and [aH]tyrosine found in superfusates), increased as a function o f the number o f days. Therefore, the mean specific activity o f
265
CHRONIC DOPAMINE RELEASE IN THE MONKEY
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II Amph 10- 5M Fig. 5. Comparison of the effect of amphetamine on [aH]DA release in Severine as a function of the number of days. The [3H]DA release experiment was carried out as described in Fig. 3. Each curve represents the spontaneous release and the D-amphetamine (10 -5 M) induced release obtained during the 5 successive days of the experiment. TABLE II MEAN VALUE OF SPONTANEOUS AND D-AMPHETAMINE INDUCED
[3H]DA
RELEASE D U R I N G THE 5 SUCCES-
SIVE DAYS OF THE EXPERIMENT CARRIED OUT ON SEVERINE
The various parameters were calculated and expressed as indicated in Table I. SR
be
SR
ER
bc
Day 1 Day2 Day 3 Day 4 Day 5
0.50 0.54 0.78 2.92 1.89
± 0.06 (4) ~: 0.07 (5) -4-0.07 (7) ± 0.35 (6) i 0.21 (6)
0.50 ± 0.07 (4) 0.36±0.04(3) 1.19 ± 0.01 (3) 1.25 4- 0.09 (4) 1.59 ± 0.13 (4)
1.0 1.5 0.7 2.3 1.2
ER SR
7.6 (1) 8.2(1) 8.6 (1) 27.0 (1) 22.1 (1)
15.3 15.1 11.0 9.2 11.7
t y r o s i n e in the collected superfusates increased slowly d u r i n g the first days a n d very quickly d u r i n g the last 24 o r 48 h (Fig. 4). W h e n tyrosine specific activity in superfusates was t a k e n into a c c o u n t in o r d e r to d e t e r m i n e the v a r i a t i o n s in [ 3 H ] D A release ( [ 3 H ] D A release/specific activity o f t y r o s i n e in collected superfusates), the e v o l u t i o n o f the p a t t e r n o f [ a H ] D A release was n o t qualitatively affected (Fig. 4). T h e e v o k e d release o f [ a H ] D A o b s e r v e d in Severine, for each e x p e r i m e n t a l d a y following a m p h e t a m i n e a d d i t i o n into the cup, is illustrated in Fig. 5 a n d T a b l e II.
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The absolute quantity of [3H]DA released during amphetamine addition was maximal on the 4th and 5th days and the ratio of 'evoked' to 'spontaneous' [3H]DA release decreased slightly as a function of the number of days. No appreciable differences were observed in the effect of amphetamine when the application of the drug was repeated a second time each experimental day. DISCUSSION
The results obtained demonstrate for the first time a spontaneous and Damphetamine induced release of DA endogenously synthesised from its natural precursor in the caudate nucleus of the primate. These data could be observed repeatedly for 5-6 days in unanaesthetised animals exhibiting a normal behaviour in their restraining chairs. The use of the cup technique and the continuous superfusion of the tissue with L-[3,5-3H]tyrosine offers several advantages which have already been discussed extensively in previous studies on DA release from the cat caudate nucleus1, 2,5. Briefly, the cup method allows the superfusion of a relatively large surface area of tissue restricted to a single structure. Moreover, [3H]tyrosine ensures a specific labelling of dopaminergic terminals and facilitates the detection of minute amounts of the [~H]transmitter in the superfusate. Indeed, [ZH]DA can only be formed in dopaminergic terminals which selectively contain tyrosine hydroxylaselO; quantities of transmitter as small as 5-10 pg can easily be estimated when tyrosine of high specific activity is used. Furthermore, as previously shown, newly synthesised [3H]DA is preferentially available to be used in spontaneous and drug-induced release processes 1. A few precautions must be taken to ensure the success of a chronic superfusion in an animal able to move its head and members: sterile conditions must be maintained throughout the experiment; the cup must be permanently sealed on the plastic platform as soon as possible to avoid any damage to the tissues during movements; the superfusion with the physiological oxygenated medium must start immediately and should not be interrupted to keep the tissue in good condition; and finally, constant observation of the animal is required to minimise technical as well as physiological incidents. We have previously shown in experiments with cats that the spontaneous release of newly synthesised [3H]DA depends on nerve activity: [3H]DA release was immediately blocked after introduction of tetrodotoxin 1 into the cup or following the transection of the nigro-neostriatal pathway 2. The spontaneous release of [3H]DA may also depend on nerve impulses in Macaca mulatta. In most animals, the spontaneous release of [3H]DA reached a maximal level shortly (less than 30 min) after the onset of [3H]tyrosine introduction into the cup. This differs slightly from results obtained on cats in which the spontaneous release of [3H]DA reached a steady state level only 90 min after beginning the [3H]amino acid superfusion. This difference could be attributed to the site of superfusion: the ependyma of the ventricular surface superfused in the cat could limit the diffusion of the [3H]amino acid in tissues. Moreover, in contrast to the monkeys, the cats were immo-
CHRONIC DOPAMINE RELEASE IN THE MONKEY
267
bilised with gallamine and locally anaesthetised. In some cases, variations in the levels of spontaneous released [aH]DA could be seen from one monkey to the next; they may be related to slight differences in the sites of superfusion or in the states of the animals. In all cases, even though the daily labelling experiment lasted as long as 9 h, no marked variations in the amount of [aH]DA spontaneously released could be seen in 15 min serial collected fractions. Small fluctuations were detected from day to day in the mean spontaneous release of the labelled transmitter during the first 3 experimental days. We cannot yet explain the processes involved in the abrupt rise in [aH]DA spontaneous release noted in the last 24 or 48 h during the 5-6 day experiment carried out on Severine and Proserpine. This phenomenon was also apparent when the changes in tyrosine specific activity in the superfusate were taken into account in expressing the results (Fig. 5). However, modifications in the specific activity of tyrosine localised in tissues or in the pool involved in DA synthesis cannot be excluded. It is unlikely that these effects are related to daily rhythmic fluctuations of plasma levels of endogenous or labelled tyrosine since they were not observed during the first 3 experimental days. The effect of amphetamine on [SH]DA release was examined to determine the sensitivity as a function of time of dopaminergic terminals to the drug. As discussed in previous cat studies 1, amphetamine acts mainly by a direct releasing effect on dopaminergic terminals; its inhibitory action on the DA reuptake process is probably also involved. Furthermore, after labelling of dopaminergic terminals of the cat caudate nucleus with exogenous [SH]DA it has been shown that the amphetamine effect on DA release is partially dependent on nerve activity: a reduced action of the drug was observed shortly following interruption of the nigro-neostriatal pathway28. In most of our experiments, the introduction of amphetamine into the cup greatly stimulated [aH]DA release. During the first experimental day, the release induced by amphetamine reached from 15-20 times that of the mean spontaneous release in 3 of the 5 monkeys studied. The longitudinal studies, made in a few cases, indicated that the amphetamine response was almost identical from one day to the next for a period lasting as long as 5 days. These observations illustrate the reproducibility of results obtained by this approach. The similar reactivity of dopaminergic terminals to amphetamine observed from one day to the next provides further proof of the quality of the preparation as a function of time. The approach described in this study should make possible investigations concerning long term changes in the activity of the nigro-neostriatal dopaminergic system induced by lesions or chronic pharmacological treatments in unanaesthetised, free-moving animals. ACKNOWLEDGEMENTS
This research was supported by grants from INSERM (ATP No. 71-4525 15), DGRST (Contract No. 71 73208), CNRS (ATP No. 4207), DRME (Contract No. 73061) and the Socirt6 des Usines Rhrne-Poulenc. We would like to thank Floreal Rodriguez for his skillful technical assistance.
268
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REFERENCES 1 BESSON, M. J., CHERAMY,A., FELTZ, P., AND GLOWINSKI, J., Dopamine: spontaneous and druginduced release from the caudate nucleus in the cat, Brain Research, 32 (1971) 407-424. 2 BESSON, M. J., CHERAMY,A., GAUCHY, C., AND GLOWINSKI,J., In vivo continuous estimation of aH-dopamine synthesis and release in the cat caudate nucleus. Effects of tt-methyl-p-tyrosine and of transection of the nigro-neostriatal pathway, Arch. PharmacoL, 278 (1973) 101-105. 3 BESSON,M. J., CHERAMY,A., GAUCHY,C., AND MUSACCHIO,J., Effects of some psychotropic drugs on tyrosine hydroxylase activity in different structures of the rat brain, Europ. J. Pharmacol., 22 (1973) 181-186. 4 BIOULAC, B., GAUCHY, C., CHERAMY, A., BESSON, M. J., GLOWINSKI, J., AND VINCENT, J.-D., Chronic estimation of DA release from the caudate nucleus of the Macaca mulatta, Fed. Proc., 32, 3 (1973) Abstract 3372. 5 CHERAMY,A., GAUCHY, C., GLOWINSKI,J., AND BESSON,M. J., In vivo activation by benzatropine of dopamine release and synthesis in the caudate nucleus, Europ. J. Pharmacol., 21 (1973) 246-248. 6 CHIUEH, C. C., AND MOORE, K. E., Release of endogenously synthesized catecholamines from the caudate nucleus by stimulation of the nigro-striatal pathway and by the administration of Damphetamine, Brain Research, 50 (1973) 221-225. 7 DEEEUDIS,F. V., DELGADO,J. M. R., AND ROTH, R. H., Content and release of amino acids and catecholamines in monkey brain, Nature (Lond.), 233 (1969) 74-75. 8 EVARTS,E. V., A technic for recording activity of subcortical neurons in moving animals, Electroenceph, clin. Neurophysiol., 24 0968) 83-86. 9 GAUCHY,C., AGID, Y., GLOWINSKI,J., AND CHERAMY, A., Acute effects of morphine on dopamine synthesis and release and tyrosine metabolism in the rat striatum, Europ. J. Pharmacol., 22 (1973) 311-319. l0 GOLDSTEIN,M., ANAGNOSTE,B., BATTISTA,A. F., OWEN, W. S., AND NAKATANI,S., Studies of amines in the striatum in monkeys with nigral lesions: the disposition, biosynthesis and metabolites of 3H-dopamine and 14C-serotonin in the striatum, J. Neurochem., 16 (1969) 645-653. 11 HORNYK1EWICZ,O., Dopamine and extrapyramidal motor function and dysfunction. In Neurotransmitters, Res. Publ. Ass. nerv. ment. Dis., 1972. 12 JAvoY, F., AGID, Y., BOUVET,D., AND GLOWINSKI,J., Changes in neostriatal DA metabolism after carbachol or atropine microinjections into the substantia nigra, Brain Research, 68 (1974) 253-260. 13 LAROCHELLE,L., BEDARD,P., POmlER, L. J., AND SOURKES,T. L., Correlative neuroanatomical and neuropharmacological study of tremor and catatonia in the monkey, Neuropharmacol., l0 (1971) 273-288. 14 LAVERTY,R., AND SHARMAN,D. F., The estimation of small quantities of 3,4-dihydroxyphenylethylamine in tissues, Brit. J. PharmacoL, 24 (1965) 538-548. 15 MCKENZIE, G. M., AND SZERB, J. C., The effect of dihydroxyphenylalanine, pheniprazine and Damphetamine on the in vivo release of dopamine from the caudate nucleus, J. Pharmacol. exp. Ther., 162 (1968) 302-308. 16 MCLENNAN, H., The release of acetylcholine and 3-hydroxytyramine from the caudate nucleus, J. Physiol. (Lond.), 174 (1964) 152-161. 17 MCLENNAN, H., The release of dopamine from the putamen, Experientia (Basel), 21 0965) 725-726. 18 PORTIG, P. J., SHARMAN, D. F., AND VOGT, M., Release by tubocurarine of dopamine and homovanillic acid from the superfused caudate nucleus, J. Physiol. (Lond.), 194 (1968) 565-572. 19 PORTIG,P. J., AND VOGT, M., Release into the cerebral ventricles of substances with possible transmitter function in the caudate nucleus, J. Physiol. (Lond.), 204 (1969) 687-715. 20 RIDDELL, D., AND SZERB, J. C., The release in vivo of dopamine synthesized from labelled precursors in the caudate nucleus of the cat, J. Pharmacol., 18 (1971) 989-1006. 21 ROTH, R. H., ALLIKMETS,L., AND DELGADO,J. M. R., Synthesis and release of noradrenaline and dopamine from discrete region of monkey brain, Arch. int. Pharmacodyn., 181 (1969) 273-282. 22 SNIDER, R. S., AND LEE, J. C., A Stereotaxic Atlas of the Monkey Brain, Univ. of Chicago Press, Chicago, Ill., 1961. 23 UDENFRIEND, S., Fluorescence Assay in Biology and Medicine, Academic Press, New York, 1962, 130-134 24 VON VOIGTLANDERAND MOORE,K. E., Involvement of nigro-striatal neurons in the in vivo release of dopamine by amphetamine, amantadine and tyramine, J. Pharmacol. exp. Ther., 184 (1973) 542-552.
257
ESTIMATION OF CHRONIC DOPAMINE RELEASE FROM THE CAUDATE NUCLEUS OF THE MA C A C A MULATTA
CHRISTIAN GAUCHY, BERNARD BIOULAC, ANDRI~ CHERAMY, MARIE-JO BESSON, JACQUES GLOWINSKI AND JEAN-DIDIER VINCENT
Groupe NB (INSERM U.114), Coll~ge de France, Paris 5e and Laboratoire de Neurophysiologie, Facult~ de M~decine, Bordeaux (France) (Accepted April 16th, 1974)
SUMMARY
Using a cup permanently implanted on the antero-lateral surface of the caudate nucleus, attempts were made to study the spontaneous and D-amphetamine induced release of [aH]dopamine (DA), which was continuously synthesised from L-[3,5-aH]tyrosine in the unanaesthetised Macaca mulatta placed in a restraining chair with freedom of movement of the head and members. The spontaneous release of [aH]DA from dopaminergic terminals was detected during the continuous superfusion of the tissue with [3H]~rnino acid in 8 monkeys. In most cases [aH]DA release reached a steady state level within 30 min after the onset of superfusion. Similar results were obtained when the experiments with [aH]tyrosine were carried out on 3 successive days. In 2 of the cases examined, the quantity of spontaneously released [aH]DA during the last 24 or 48 h of a 5-6 day experiment was markedly increased as compared to that observed during previous days; this effect did not appear to be related to changes in the specific activity of tyrosine in the superfusing medium. In 3 monkeys D-amphetamine (10 -5 M) increased the level of [aH]DA release by 15-20 times that of the normal spontaneous release of the transmitter, the effect was less pronounced in 2 other animals exhibiting a higher level of spontaneous [aH]DA release. The amphetamine effect could be detected every day in longitudinal studies. For example, in one animal the ratio of the amphetamine-induced [aH]DA release to spontaneous [3H]DA release varied only from 10 to 15 during a 5 day experiment. This new approach should make possible long term studies on the regulatory processes involved in DA release from dopaminergic terminals of the nigro-neostriatal system in the unanaesthetised primate.
258
C. GAUCHY el a[.
INTRODUCTION
The importance of the nigro-neostriatal dopaminergic system in extrapyramidal functions has been clearly demonstrated11. Whereas many reports have been devoted to the analysis of dopamine (DA) metabolism in the caudate nucleus of various mammals, demonstration of the in vivo release of DA has only been made in a few cases. The release of endogenously synthesised DA from dopaminergic terminals has been examined particularly in the cat. Different approaches were used: DA or labelled DA synthesised from [14C]- or [SH]tyrosine were estimated in perfusates of the lateral ventricles6,1s 20,24, in superfusates of a push-pull cannula inserted into the caudate nucleus 15,16,2° or into the putamen 17, and in superfusates of a cup disposed at the surface of the caudate nucleus1, 2,5. Studying the primate offers several advantages when compared to the cat, since extrapyramidal disorders can be induced in monkeys by lesions or drugs 13. However, only a preliminary attempt has been made to study the release of DA from the caudate nucleus in Macaca mulatta: using a push-pull cannula, Defeudis et al. 7 failed to detect any release of [14C]DA after labelling of dopaminergic terminals with [14C]tyrosine, although [14C]catecholamines could be found in tissues. In a complementary study2~, these authors observed a release of both [14C]DA and [14C]noradrenaline during a 1 h local superfusion of the tissue with [14C]DOPA. In this experiment, release of [14C]DA may not be specific since [14C]DOPA can be decarboxylated into serotoninergic terminals and in pericytes as well as into dopaminergic terminals. Therefore, in the present study we attempted to demonstrate the spontaneous and evoked release of [aH]DA endogenously synthesised from L-[3,5-aH]tyrosine in the caudate nucleus ofMacaca mulatta. Furthermore, in employing the cup technique used in the acute cat preparation x,2,5 we have extended this methodology to study the release of DA in a chronic preparation. The principle of this methodology is to place a permanent cup upon the external surface of the caudate nucleus and to superfuse the tissue with a sterile physiological medium. During the experiment, [ZH]tyrosine of high specific activity is introduced continuously into the cup and the newly synthesised and simultaneously released [3H]DA is analysed in successive collected fractions of superfusates. In the present study, the spontaneous release of the transmitter was examined daily in various monkeys for periods lasting as long as 6 days. Furthermore, the release capacity of dopaminergic terminals was tested each day by measuring their responsiveness to amphetamine*. METHODS
Preparation of the animals Eight adult female monkeys (Macaca mulatta) weighing from 4 to 4.5 kg were used in this study: Valentine (No. 0), Nina (No. 1), King-Kong (No. 2), Zoe (No. 3),
* A preliminaryreport was made at the FederationProceedings4.
259
CHRONIC DOPAMINE RELEASE IN THE MONKEY
Justine (No. 4), Sigma (No. 5), Severine (No. 6) and Proserpine (No. 7). Each animal was previously conditioned to a restraining chair for primates allowing free movement of the head and members. A first surgical operation was made under Nembutal® (40 mg/kg, i.p.). After fixation of the animal in a stereotaxic apparatus, a plastic platform was attached to the surface of the skull with stainless steel bolts. This platform was designed to allow the introduction and the chronic fixation of the superfusing cup during the second surgery. Wires for EEG, EMG, EKG, eyes and respiratory movements were soldered to pin-connectors cemented to the platform. A silicone rubber cannula was placed in the right atrium, by passing it through the internal jugular vein. This cannula was inserted under the skin of the neck and fixed to the cranial platform. After recovery from surgery, the monkeys were placed in their restraining chair for about 10 days. In the second surgery performed under ketamine (Ketalar®) (15 mg/kg, i.m.) and halothane (Fluothane®) anaesthesia, the animals were kept in their restraining chairs and their heads were temporarily immobilised according to Evarts technique 8. Following a small cortical resection, a specially designed cup, driven with the aid of a tridimensional manipulator, was placed on the antero-lateral surface of the left caudate nucleus as indicated in Fig. 1. In order to limit neurological disorders the cup was introduced through the prefrontal cortex (Fig. 1). Under these experimental conditions the lateral ventricle remains intact. Superfusion of the tissue with artificial cerebrospinal fluid was started as soon as the cup was in the correct position and then the cup was permanently cemented to the platform. The animals' heads were then
Superfusates
Superfusing medium 02 + C02
~
(~ Superfusates
pl
A. 22
Lat.8
Fig. 1. Schematic representation of cup localisation at the surface of the caudate nucleus. The anterior and lateral coordinates are those of the stereotaxic atlas of Snider and Lee 22. cd, candate nucleus; pl, sealed plastic platform; put, putamen.
260
c. GAUCHY el a].
liberated and isotopic experiments were begun only after their complete recovery from the anaesthesia. Both surgical steps were performed under strict aseptic conditions. The surface of the caudate nucleus was superfused continuously with oxygenated, warmed (37 °C), sterile physiological medium (in mM: NaCI, 126.5; NaHCGz, 27.5; KCI, 2.4; KH2PO4, 0.5; CaCI2, 1.1; MgCI2, 0.83; NazSO4, 0.5; glucose, 5.9; adjusted to pH 7.4 with an O2-CO2--95:5--mixture), at a rate of 6 ml/h using a peristaltic pump. The superfusing medium was introduced via a small stainless steel tube sealed to the cup 1 mm above the surface of the exposed tissue. The volume of the physiological medium in the cup was kept constant (3130/zl) by continuous suction with a peristaltic pump through a second stainless steel tube placed 8 mm above the tissue. Moreover, the medium was continuously agitated and oxygenated in the cup with a light stream of sterile O2-COz (95:5). Superfusion with the physiological medium was kept constant in order to maintain the nervous tissues in good condition. Polygraphic recordings were made regularly, particularly during [ZH]DA release experiments to check the animals' states. Shortly following the onset of superfusion the animals took food and water which were provided ad libitum. To prevent oedema andinfection, furosemide (Lasilix®) (0.1305 g per animal) and cefalotin (Keflin®) (10 mg/kg) were respectively injected twice and once a day intramuscularly. At the end of the experiments, the animals were sacrificed by an injection of Nembutal. Brains were immediately removed and kept in formalin (10 ~,,) for histological controls. Behavioural and polygraphic observations Neurological examination was performed periodically. No major clinical disturbance was observed after recovery from the cortical surgery. The only transient disorder was a hypokinesia or mild and regressive contralateral paresis. The monkeys maintained a good appetite and were able to eat by themselves. On the basis of the EEG, nucchal electromyography, eyes and body movements, we were able to judge whether the animal was sleeping, in quiet waking or aroused. Both normal EEG patterns and cycling sleep-waking organisation confirmed the lack of central injury. Since our main purpose was first directed towards establishing the technique and improving chronical methodology, the correlation between [ZH]DA release and behaviour was not systematically attempted. {aHj DA release experiments and biochemical determinations Release experiments were carried out once or twice a day for periods lasting as long as 6 days. For this purpose, L-[3,5-3H]tyrosine (40 Ci/mmoles, Radiochemical Centre, Amersham, England or CEA, 91 Gif sur Yvette, France and previously purified by adsorption on Alumina and ion-exchange chromatography on Dowex AG 50 WX4) was dissolved in the physiological mediumL Fifteen minute serial fractions of superfusates were collected in cooled tubes containing 100 /~I of a stabilising solution (ascorbic acid 1~/o: EDTA 1.5%: DA 0.001%) and 3 ml of ethanol. D-Amphetamine sulphate (10 -~ M)was dissolved in the
CHRONIC DOPAMINE RELEASE IN THE MONKEY
261
superfusing medium and introduced for 15 min into the cup once or twice every day. Superfusate fractions were weighed for volume determination and adjusted to pH 6.7; they were then kept at --17 °C for one night. Following the centrifugation of the samples, total radioactivity of each fraction was estimated on a 20 /zl aliquot. [SH]DA was separated from [ZH]tyrosine and [3H]metabolites by ion exchange chromatography on Amberlite CG 50 and by adsorption chromatography on Alumina as described previously3. The eluates (1 ml) from the Alumina column were diluted in 10 ml of Instagel® (Packard Instrument S.A.) and the radioactivity was then measured by liquid scintillation counting. [3H]DA and [aH]tyrosine recoveries in Alumina column eluates were 70 and < 0.0001 ~o respectively. In all experiments, blanks represented by [3H]tyrosine contamination were subtracted and data were corrected for recoveries. In some experiments the identity of [aH]catecholamines was checked on pooled eluates of Alumina column by paper chromatography after acetylation of the amines and organic extraction of the acetylated derivatives14. The radiochromatogram indicated the presence of 2 peaks of radioactivity corresponding to [aH]DA and [3H]noradrenaline; [aH]DA represented more than 85 ~ of the total [3H]catecholamines recovered on the radiochromatogram; therefore, [aH]catecholamines eluted from the Alumina columns were considered as [aH]DA in all experiments. In some cases, the level of tyrosine found in superfusate fractions was estimated. The amino acid was isolated from Amberlite effluents by ion-exchange chromatography on Dowex AG 50 WX4 as described by Javoy et al. 12, and estimated by the spectrofluorimetric method of Udenfriend23. RESULTS
Spontaneous andamphetamine evoked release of [3H]dopamine during the first day of superfusion The spontaneous release of [ZH]DA was studied in 5 monkeys 3 h following the onset of superfusion when the animals had completely recovered from anaesthesia. As an example, results obtained with Severine are illustrated in Fig. 2. [ZH]DA could be detected in superfusates in the first 15 min period of superfusion with [SH]tyrosine. [3H]DA release increased in the second fraction and then remained almost constant. In this experiment, the radioactivity present as [3H]DA in superfusates was about 2 times that of the blank values due to [SH]tyrosine. The introduction of amphetamine (10 -5 M) into the superfusing medium increased the release of [3H]DA by 15 times. After removal of the drug, [3H]DA levels returned slowly to those of spontaneous release. The qualitative pattern of the effects observed was quite comparable from one animal to the next: spontaneous and evoked release of the transmitter could be detected in all cases. Small quantitative variations in [3H]DA spontaneous release occurred from one animal to another. For example, [ZH]DA spontaneous release was almost identical in Severine, Justine and Zoe but was much more prono~ficed in Proserpine (maximal level about 11 times blank values) and Sigma (maximal level 8 times blank
262
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=m AMPH.10-5M
Fig. 2. Effect of amphetamine on [aH]dopamine release from the caudate nucleus of the Macaca mulatta. Release of [~H]DA immediately after the onset of superfusion of the caudate nucleus with L-[3,5-3H]tyrosine (60/~Ci/ml; 6 ml/h) in 15 min serial fractions. D-Amphetamine (10 -5 M) was added into the superfusing medium during a single 15 min fraction.
L
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/ l c 9
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, ZOE
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I Amph. 10-5M Fig. 3. Comparison of spontaneous release and amphetamine induced release of [aH]DA in various monkeys during the first day of superfusion. Each experimental point represents the amount of [SH]DA released during a 15 rain fraction on the first day of superfusion in Sigma, Proserpine, Justine, Severine and Zoe. The black square corresponds to the 15 rain fraction during which o-amphetamine (10 -5 M) was added. As in Fig. 2, the caudate nucleus was superfused continuously with L-[3,5-aH]tyrosine (60/zCi/ml at a rate of 6 ml/h).
263
CHRONIC DOPAMINE RELEASE IN THE MONKEY TABLE I
MEAN VALUE OF SPONTANEOUS AND D-AMPHETAMINE INDUCED [3H]DA RELEASE DURING THE FIRST EXPERIMENTAL DAY IN THE VARIOUS MONKEYS Each value represents the m e a n i S.E.M. of data corresponding to the quantity of radioactivity (nCi/l 5 min fraction) recovered in successively collected fractions. The n u m b e r in brackets corresponds to the n u m b e r of fractions used to establish the mean. SR = spontaneous [aH]DA release (blank value deducted); bc = blank value; SR/bc = ratio of the [8H]DA spontaneous release to the blank value; ER = [3HIDA release evoked by amphetamine (10 -5 M ) ; ER/SR = ratio of evoked [aH]DA release over spontaneous [aHIDA release.
SR
bc
SR
ER
be
Proserpine Zoe Justine Sigma Severine
3.41 i 0.16 (7) 0.32 ± 0.01 (3) 0.82 i 0.19 (5) 2.37 4- 0.09 (6) 1.04 i 0.17 (3) 0.76 4- 0.08 (6) 5.98 ~: 0.70 (4) 0.75 ± 0.02 (5) 0.50 4- 0.06 (4) 0.50 4- 0.07 (4)
10.66 0.35 1.38 7.97 1.00
ER SR
13.0 (1) 18.8 (1) 16.6 (1) 26.3 (1) 7.6 (l)
3.8 22.9 16.0 4.4 15.3
values) (Fig. 3 and Table I). Sigma was the only monkey in which the spontaneous release of [aH]DA increased continuously as a function of time. The effect of amphetamine was particularly striking in Severine, Zoe and Justine since [aH]DA levels reached from 15 to 23 times the mean spontaneous release of the [all]amine estimated in the fractions preceding amphetamine addition. Surprisingly enough this effect was relatively less pronounced in Proserpine and Sigma (about 4 times), animals in which [SH]DA spontaneous release was particularly elevated (Fig. 3 and Table 1).
Spontaneous and amphetamine evoked release of [aH]dopamine as a function of the number of days following the onset of superfusion The spontaneous release of [aH]DA as well as the amphetamine effect could be observed for a few days in various monkeys. Zoe and Justine were the first 2 monkeys in which we attempted to study the evolution of the [SH]DA spontaneous and evoked release as a function of time. Positive results were obtained for 3 days with both animals. As a result of improvements made in the chronic superfusing method from one experiment to the next, the [aH]DA release experiments were enabled to be progressively prolonged. Indeed, the best results were obtained with the last 3 monkeys (Sigma, Severine and Proserpine) who were studied for 4, 5 and 6 days, respectively. Whereas technical incidents were responsible for the sacrifice of the first 2 animals, this was not the ease in the last 3 experiments which were interrupted as previously programmed. The results obtained with Severine and Proserpine concerning the evolution of [SH]DA spontaneous and amphetamine induced release as well as the fluctuations of other parameters, examined in these experiments, will be described in detail. The average spontaneous release of [SH]DA was calculated every day in serial 15 rain fractions. As indicated in Fig. 4, small variations in [SH]DA spontaneous release were observed from day to day in each animal during the first 3 and 5 days for
264
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Fig. 4. Estimation of various parameters in tong term release experiments carried out on Severine and Proserpine. The figure represents from the top to the bottom: (1) the change in the volume of the superfusate, collected in serial fractions (as a function of the number of days), the volume of superfusing fluid introduced into the cup being 1.5 ml; (2) the output of tyrosine as well as the specific activity of tyrosine present in the superfusate; (3) the difference between the amount of pH]tyrosine introduced into the cup and the amount of [SH]tyrosine recovered in the superfusate at the end of the 15 min fraction (clearance of pH]tyrosine expressed in percent of the amount originally introduced); (4) the quantity of [SH]DA released; (5) the ratio of [aH]DA released to the specific activity of tyrosine into the superfusate. Each value represents the mean of the data obtained from 15 min serial fractions collected during each daily experiment. These parameters correspond to the spontaneous release of the transmitter. As before, L-[3,5-3H]tyrosine was introduced continuously into the cup at a rate of 6 ml/h (60 #Ci/ml).
Severine and Proserpine, respectively. A marked increase in the mean release o f p H ] D A was noticed in the last 48 h (Severine) and 24 h (Proserpine) of the experiments. This effect did not seem to be related to changes in the volumes o f the superfusates. The small increase in the mean volume of superfusates, observed during the first 3 and 5 days, when compared to the volume o f superfusing fluid introduced during each t 5 min period into the cup may be due to an excretion o f interstitial fluid. High levels of tyrosine were found initially in superfusates (Fig. 4); they seem to decrease as a function o f time and were almost identical at the end of the experiments to the concentration o f the [3H]amino acid introduced into the cup (Fig. 4). At the same time, the clearance o f the [SH]amino acid from the superfusing fluid (difference between [ZH]tyrosine introduced into the cup and [aH]tyrosine found in superfusates), increased as a function o f the number o f days. Therefore, the mean specific activity o f
265
CHRONIC DOPAMINE RELEASE IN THE MONKEY
25
5EVERINE
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DAY I
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DAY 2
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DAY
4
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II Amph 10- 5M Fig. 5. Comparison of the effect of amphetamine on [aH]DA release in Severine as a function of the number of days. The [3H]DA release experiment was carried out as described in Fig. 3. Each curve represents the spontaneous release and the D-amphetamine (10 -5 M) induced release obtained during the 5 successive days of the experiment. TABLE II MEAN VALUE OF SPONTANEOUS AND D-AMPHETAMINE INDUCED
[3H]DA
RELEASE D U R I N G THE 5 SUCCES-
SIVE DAYS OF THE EXPERIMENT CARRIED OUT ON SEVERINE
The various parameters were calculated and expressed as indicated in Table I. SR
be
SR
ER
bc
Day 1 Day2 Day 3 Day 4 Day 5
0.50 0.54 0.78 2.92 1.89
± 0.06 (4) ~: 0.07 (5) -4-0.07 (7) ± 0.35 (6) i 0.21 (6)
0.50 ± 0.07 (4) 0.36±0.04(3) 1.19 ± 0.01 (3) 1.25 4- 0.09 (4) 1.59 ± 0.13 (4)
1.0 1.5 0.7 2.3 1.2
ER SR
7.6 (1) 8.2(1) 8.6 (1) 27.0 (1) 22.1 (1)
15.3 15.1 11.0 9.2 11.7
t y r o s i n e in the collected superfusates increased slowly d u r i n g the first days a n d very quickly d u r i n g the last 24 o r 48 h (Fig. 4). W h e n tyrosine specific activity in superfusates was t a k e n into a c c o u n t in o r d e r to d e t e r m i n e the v a r i a t i o n s in [ 3 H ] D A release ( [ 3 H ] D A release/specific activity o f t y r o s i n e in collected superfusates), the e v o l u t i o n o f the p a t t e r n o f [ a H ] D A release was n o t qualitatively affected (Fig. 4). T h e e v o k e d release o f [ a H ] D A o b s e r v e d in Severine, for each e x p e r i m e n t a l d a y following a m p h e t a m i n e a d d i t i o n into the cup, is illustrated in Fig. 5 a n d T a b l e II.
266
~:. G A U C H Y ~'l a[.
The absolute quantity of [3H]DA released during amphetamine addition was maximal on the 4th and 5th days and the ratio of 'evoked' to 'spontaneous' [3H]DA release decreased slightly as a function of the number of days. No appreciable differences were observed in the effect of amphetamine when the application of the drug was repeated a second time each experimental day. DISCUSSION
The results obtained demonstrate for the first time a spontaneous and Damphetamine induced release of DA endogenously synthesised from its natural precursor in the caudate nucleus of the primate. These data could be observed repeatedly for 5-6 days in unanaesthetised animals exhibiting a normal behaviour in their restraining chairs. The use of the cup technique and the continuous superfusion of the tissue with L-[3,5-3H]tyrosine offers several advantages which have already been discussed extensively in previous studies on DA release from the cat caudate nucleus1, 2,5. Briefly, the cup method allows the superfusion of a relatively large surface area of tissue restricted to a single structure. Moreover, [3H]tyrosine ensures a specific labelling of dopaminergic terminals and facilitates the detection of minute amounts of the [~H]transmitter in the superfusate. Indeed, [ZH]DA can only be formed in dopaminergic terminals which selectively contain tyrosine hydroxylaselO; quantities of transmitter as small as 5-10 pg can easily be estimated when tyrosine of high specific activity is used. Furthermore, as previously shown, newly synthesised [3H]DA is preferentially available to be used in spontaneous and drug-induced release processes 1. A few precautions must be taken to ensure the success of a chronic superfusion in an animal able to move its head and members: sterile conditions must be maintained throughout the experiment; the cup must be permanently sealed on the plastic platform as soon as possible to avoid any damage to the tissues during movements; the superfusion with the physiological oxygenated medium must start immediately and should not be interrupted to keep the tissue in good condition; and finally, constant observation of the animal is required to minimise technical as well as physiological incidents. We have previously shown in experiments with cats that the spontaneous release of newly synthesised [3H]DA depends on nerve activity: [3H]DA release was immediately blocked after introduction of tetrodotoxin 1 into the cup or following the transection of the nigro-neostriatal pathway 2. The spontaneous release of [3H]DA may also depend on nerve impulses in Macaca mulatta. In most animals, the spontaneous release of [3H]DA reached a maximal level shortly (less than 30 min) after the onset of [3H]tyrosine introduction into the cup. This differs slightly from results obtained on cats in which the spontaneous release of [3H]DA reached a steady state level only 90 min after beginning the [3H]amino acid superfusion. This difference could be attributed to the site of superfusion: the ependyma of the ventricular surface superfused in the cat could limit the diffusion of the [3H]amino acid in tissues. Moreover, in contrast to the monkeys, the cats were immo-
CHRONIC DOPAMINE RELEASE IN THE MONKEY
267
bilised with gallamine and locally anaesthetised. In some cases, variations in the levels of spontaneous released [aH]DA could be seen from one monkey to the next; they may be related to slight differences in the sites of superfusion or in the states of the animals. In all cases, even though the daily labelling experiment lasted as long as 9 h, no marked variations in the amount of [aH]DA spontaneously released could be seen in 15 min serial collected fractions. Small fluctuations were detected from day to day in the mean spontaneous release of the labelled transmitter during the first 3 experimental days. We cannot yet explain the processes involved in the abrupt rise in [aH]DA spontaneous release noted in the last 24 or 48 h during the 5-6 day experiment carried out on Severine and Proserpine. This phenomenon was also apparent when the changes in tyrosine specific activity in the superfusate were taken into account in expressing the results (Fig. 5). However, modifications in the specific activity of tyrosine localised in tissues or in the pool involved in DA synthesis cannot be excluded. It is unlikely that these effects are related to daily rhythmic fluctuations of plasma levels of endogenous or labelled tyrosine since they were not observed during the first 3 experimental days. The effect of amphetamine on [SH]DA release was examined to determine the sensitivity as a function of time of dopaminergic terminals to the drug. As discussed in previous cat studies 1, amphetamine acts mainly by a direct releasing effect on dopaminergic terminals; its inhibitory action on the DA reuptake process is probably also involved. Furthermore, after labelling of dopaminergic terminals of the cat caudate nucleus with exogenous [SH]DA it has been shown that the amphetamine effect on DA release is partially dependent on nerve activity: a reduced action of the drug was observed shortly following interruption of the nigro-neostriatal pathway28. In most of our experiments, the introduction of amphetamine into the cup greatly stimulated [aH]DA release. During the first experimental day, the release induced by amphetamine reached from 15-20 times that of the mean spontaneous release in 3 of the 5 monkeys studied. The longitudinal studies, made in a few cases, indicated that the amphetamine response was almost identical from one day to the next for a period lasting as long as 5 days. These observations illustrate the reproducibility of results obtained by this approach. The similar reactivity of dopaminergic terminals to amphetamine observed from one day to the next provides further proof of the quality of the preparation as a function of time. The approach described in this study should make possible investigations concerning long term changes in the activity of the nigro-neostriatal dopaminergic system induced by lesions or chronic pharmacological treatments in unanaesthetised, free-moving animals. ACKNOWLEDGEMENTS
This research was supported by grants from INSERM (ATP No. 71-4525 15), DGRST (Contract No. 71 73208), CNRS (ATP No. 4207), DRME (Contract No. 73061) and the Socirt6 des Usines Rhrne-Poulenc. We would like to thank Floreal Rodriguez for his skillful technical assistance.
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c. GAUCHY ~'t a/'.
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