Bruin Research, 548 (1991) 25~ 259 © 1991 Elsevier Science Publishers B.V. 0006-8993/91i$03.50 A DONIS 0006899391165820

256

BRES 16582

Long-term depression in striatal dopamine release monitored by in vivo voltammetry in free moving rats J.H. Abraini, L. Raharison and J.C. Rostain Laboratoire de biologie des hautes pressions, CNRS URA 1330, Facult~ de mddecine nord, Marseille (France)

(Accepted 4 December 1990) Key words: In vivo voltammetry; Long-term recording; Dopamine; Striatum; Intracerebroventricular injection

It was proposed to monitor in free moving rats, by in vivo voltammetry, the effects of intracerebroventricular (i.c.v.) administration of drugs known to act on the synthesis of dopamine (DA), using an original muitifiber carbon electrode which enables without-discontinuity long-term recordings in extraceUular DA release. Results show that i.c.v, administration of a-methyl-p-tyrosine, 7-butyrolactone, and apomorphine induced long-term depression in striatal DA release, over periods of time of more than 24 h. These results are in agreement with the dopaminergic hypothesis; and we conclude that i.c.v administration of drugs and the use of the multifiber carbon electrode constitutes a valuable tool to monitor DA metabolism in chronically implanted animals. INTRODUCTION T h e m a j o r i t y of in vivo studies dealing with the mechanisms of activation of synthesis in dopaminergic n e u r o n s have b e e n p e r f o r m e d using push-pull, dialysis and v o l t a m m e t r y techniques which are c o m m o n l y used n o w a d a y s to m o n i t o r directly neurotransmitter brain monoamines. D e v e l o p m e n t of long-term e x p e r i m e n t s able to monitor extraceUular release of d o p a m i n e ( D A ) in unanesthetized animals could be of great value for further studies on the m o d u l a t i o n of D A synthesis. In this study, we have m o n i t o r e d long-term effects of drugs, known to act on D A synthesis and T H activity such as a - m e t h y l - p - t y r o s i n e ( A M P T ) , a T H inhibitor and b l o c k e r of D A synthesis, 7-butyrolactone ( G B L ) , a m o d u l a t o r of d o p a m i n e r g i c neuronal firing rate, and a p o m o r p h i n e , a D1/D2 r e c e p t o r agonist. Drugs were administered intracerebroventricularly (i.c.v.), and extracellular striatal D A release was rec o r d e d using differential pulse v o l t a m m e t r y (DPV) and an original multifiber carbon electrode d e v e l o p e d by Forni 5, derived from the one of G o n o n and co-workers 9. The working carbon electrode that we used has been d e m o n s t r a t e d to be selective for D A by in vitro and in vivo e x p e r i m e n t s 4"6'7 as c o m p a r e d to other e n d o g e n o u s c o m p o u n d s , such as ascorbic acid ( A A ) , uric acid ( U A ) , n o r e p i n e p h r i n e ( N E ) , epinephrine (E), and 3,4-dihy-

droxyphenylacetic acid ( D O P A C ) , in spite of the fact that these c o m p o u n d s slightly alter the a m p l i t u d e of the D A electrochemical response 4"6. F u r t h e r m o r e , :as comp a r e d to similar existing m e t h o d s 8'9'19, this device presents the advantage of enabling long-term voltammetric recordings 7, and of being suitable for use, in chronically i m p l a n t e d animals, for very long periods of time exceeding several months 4'6. MATERIALS AND METHODS Animals and drug treatment

Male Sprague-Dawley rats (n = 16) weighing 300-350 g at time of surgery were used. Rats were housed at 21 + 0.5 °C in individual home cages under a 12-12 h light-dark cycle (lights on from 07.00 to 19.00 h), with free access to food and water. The following drugs were delivered in 10 pl phosphate-buffered saline solution (PBS) and injected i.c.v, through a stainless steel cannula, using micro-syringe (ref. 705 RN, Hamilton, U.S.A.), catheter and cannula: 10-6 M, i.e. 90/~g/kg GBL (Aldrich Chemie, ref. B-10-3608) (n = 4); 10 7 M, i.e. 50 /~g/kg AMPT (Sigma Chemical, ref. M-8753) (n = 4); and 10-8 M, i.e. 10 ~g/kg apomorphine (Research Biochemical, ref. D-4) (n = 4). Before pharmacological investigations, basal fluctuations of DA release were recorded during a 24-h period (n = 4). The animals were injected at 11.00 h. Surgery and voltammetric recordings

Differential pulse voltammetry was used according to the method developed by several authors L9'1°'14"15. Voltammetric measurements were performed on unrestrained awake animals using a PRG5 polarograph (Tacussel, France), and a classical 3-electrode potentiostatic system with reference, auxiliary, and working electrodes. As described previouslys , the working carbon electrode was made

Correspondence: J.H. Abraini, Laboratoire de biologie des hautes pressions, Facult6 de m6decine nord, Boulevard Dramard, 13326 Marseille c6dex 15, France.

257 ba~eline

from a rigid rod of 10,000 carbon fibers (ref. A G T 4F 10,000, Carbon Lorraine, France) sharpened at one extremity to reduce the external diameter of the electrode from 1 mm to about 50/~m at the tip. The entire electrode was encased using an insulating resin and the tip was exposed using an abrasive disc to shape the active surface of the electrode. Before use, the working electrodes were electrochemically pretreated by applying a triangular wave potential of 0-3 V, 70 Hz, 20 s; 0-2 V, 70 Hz, 20 s; and 0-1 V, 70 Hz, 15 ss. Before being implanted the working carbon electrodes were calibrated in vitro. Solutions of 1 0 - 3 M DOPAC, 1 0 - 3 M AA, 1 0 - 3 M uric acid (UA) were used to check no oxidative response was recorded in these compounds. DA at 10-s M was used to check the DA electrochemical response occurred, as described previously4'6, at around 160 mV vs (range: 150 mV-170 mV). Working carbon electrode and stainless steel cannula were stereotaxically and respectively implanted, according to the atlas of K6nig and Klippel H, in the caudate nucleus (A: 8.62; L: 2; H: 1.4) and the right lateral ventricle (A: 5.91; L: 1.4; H: 2) of the animals, under general anesthesia (pentobarbital sodium 30 mg/kg i.p. and ketamine 100 mg/kg i.m.). The reference and auxiliary electrodes (stainless steel screws) were fixed to the bone. The electrodes were attached to a miniconnector and electrodes, connector, and cannula were held in place with dental cement (ref. resin cement, Ivoclar, Switzerland). After surgery, the animals were allowed to recover one week before being submitted to the pharmacological investigations. For voltammetric recordings, the animals were connected to the polarograph through a flexible cable and a swivel connector; and the polarograph was set up to the following parameters: scan rate 10 mV/s or 20 mV/s; voltage range 0 to 500 mV vs or 0 to 1000 mV vs; pulse modulation amplitude 50 mV; pulse modulation duration 48 ms; pulse period 0.2 s. Voltammograms were recorded every 3 min. Extracellular DA release was quantitated automatically by measuring the height of the DA oxidation peak, using a computerized device. Results were compared to the mean of the DA peaks recorded during a 2-h control period before injection, taken as the 100% value.

120

Statistical methods

d u r i n g t h e d a r k p e r i o d , a n d less t h a n - 4 %

Non-parametric tests were used such as Kruskal-Wallis test (one-way ANOVA), Mann-Whitney U-test (U-test) and median value + the 25th-75th percentiles. For each hour of recording, the U-test was used to compare data obtained in injected animals during drug experiments to the corresponding data (same time hour) recorded in non-injected animals during control experiments TM.

light p e r i o d .

120 baselim

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12 14 16 18 20 22 IX) 02 04 06 08 I0

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Fig. 2. Development of the extracellular D A release recorded from the striatum of free-moving rats after administration of GBL at a dose of 90/~g/kg. Y-axis: extracellular DA release expressed as a percentage difference from control value (120 min); median values + 25-75 percentiles were obtained from n = 4 rats. X-axis: time from 08.00 to 11.00 h next day. U-test: **P < 0.02, *P < 0.05 vs control experiments (dotted line).

RESULTS

Control experiments B a s a l v a l u e s in e x t r a c e l l u l a r D A r e l e a s e w e r e r e c o r d e d during a 24-h period from 08.00 to 08.00 h the next day (Fig. 1). D A r e l e a s e w a s f o u n d t o b e h i g h e r d u r i n g t h e dark period than during the light period. Nevertheless, the maximal fluctuations observed during the 24-h period o f r e c o r d i n g w e r e less t h a n + 7 %

from the mean value during the

Effects of drugs Effect of G B L on the extracellular D A release. A d m i n i s t r a t i o n o f G B L at 9 0 / ~ g / k g w a s f o u n d t o i n d u c e a

120i baselim /~PT

108

80

60

40 08 10 12 14 16 18 20 22 08 02 04 06 08 10 Fig. 1. Control experiments: basal values in extracellular D A release recorded from the caudate nucleus of free-moving rats during a 24-h period of control. Y-axis: extracellular DA release expressed as a percentage difference from the 24-h mean value, median values + 25-75 percentiles were obtained from n = 4 rats. X-axis: time from 08.00 to 08.00 h next day. Extracellular DA release increased during the dark period of the nycthohemeral.

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0~ 10 1;p 14 16 18 20 22 08 0~ 04 06 08 10 h Fig. 3. Development of the extracellular D A release recorded from the striatum of free-moving rats after AMPT administration at a dose of 50 ]~g/kg. Y-axis: extracellular D A release expressed as a percentage difference from control value (120 min); median values + 25-75 percentiles were obtained from n = 4 rats. X-axis: time from 08.00 to 11.00 h next day. U-test: **P < 0.02, *P < 0.05 vs control experiments (dotted line).

258 120

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DISCUSSION

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08 10 12 14 16 18 20 22 00 02 IN h Fig. 4. Development of the extracellular DA release recorded from the striatum of free-moving rats after 10 #g/kg apomorphine administration. Y-axis: extracellular DA release expressed as a percentage difference from control value (120 min); median values + 25-75 percentiles were obtained from n = 4 rats. X-axis: time from 08.00 to 04.00 h next day. U-test: **P < 0.02, *P < 0.05 vs control experiments (dotted line).

significant decrease in extracellular D A release (U-test, P < 0.02) (Fig. 2). After drug administration, the decline in extracellular D A release was observed during the following 20 h, with a maximal decrease of 16%, with the 25th-75th percentiles o f - 1 3 % and - 2 9 % , as compared to the values obtained during the 2-h period of control before drug administration. Twenty hours after drug administration, striatal release of dopamine was found to progressively return to control values. Effect of A M P T on the extracellular DA release. Injection of AMPT, a T H inhibitor, delivered at a dose of 50 #g/kg, resulted in a rapid and significant decrease in extracellular D A release (U-test, P < 0.02) which was detectable 1 h after injection (Fig. 3). A progressive decline in extracellular D A release was observed during the following 17 h; and a steady state was reached from 17 to 21 h after A M P T administration, with a maximal decrease of 34% (25th-75th percentiles: - 1 9 % and - 5 3 % ) followed by an increase in extracellular DA release. Decreased dopamine began to return to normal 21 h after A M P T administration. Effect of apomorphine on the extraceUular DA release. Apomorphine administered at a dose of 10/~g/kg was found to cause a significant decrease in extracellular D A release (U-test, P < 0.02), detectable 30 min after drug injection (Fig. 4). A progressive decline was observed during the following 11 h, until a maximal decrease of 35% (25th-75th percentiles: - 1 5 % and - 6 5 % ) as compared to the 2-h period of control before drug administration. Twelve hours after apomorphine administration, a rapid increase of the electrochemical D A response was found, which began to return to control value.

In the present study, we used the i.c.v, route of injection because we need this technique for future hyperbaric experiments (because of technical data such as administration of drug in free-moving animals through a closed chamber). Moreover, i.c.v, drug administration enables one to avoid the additional factors of the well known pharmacological phenomenon of the 'first pass effect' which occurs by the systemic administration of drugs. Nevertheless, this technique also presents major disadvantages, particularly the inability to make ready comparisons with clinical data. The results of the present study confirm and extend previous data suggesting that the multifiber carbon electrode developed by Forni 5 is selective for DA, and enables long-term recordings in extracellular D A release 7. I.c.v. administration of G B L was found to decrease extracellular D A release. These results are in agreement with previous data in which G B L administration was found to induce similar e f f e c t s 4'13. Since it has been suggested that GBL acts as y-hydroxybutyrate 4, a similar compound which has been demonstrated to decrease the firing rate of the dopaminergic neurons 2, the GBLinduced D A decrease correlates with the dopaminergic hypothesis according to which neuronal firing rate modulates DA synthesis 16']8. Administration of AMPT was found to decrease extracellular D A release. Similar decreases in D A metabolism resulting from AMPT administration have previously been reported by several other authors 4"9'12. After the administration of apomorphine, a D1/D 2 receptor agonist, extracellular D A voltammograms decreased. This decrease was found to be similar to those shown in other previous studies 3"4. That apomorphine is a short-lasting D A agonist was confirmed by the fact that its effect was shorter than those obtained with AMPT and GBL. The presently reported changes induced by GBL, AMPT, and apomorphine in extracellular D A release are therefore in excellent agreement with the results of other previous studies in which voitammetry was used to investigate D A synthesis. However, i.c.v, administration of drugs was found to induce long-term depression in D A release as compared to systemic administration. Indeed, in previous experiments performed with systemic administration and the same type of electrode that we used, apomorphine and G B L were found to decrease D A levels for only 3-4 h a . These long-term effects could be due to the fact that, using this route of injection, drugs are stored in the cerebrospinai fluid and thus would act longer.

259 In conclusion, the validity of the method that we used

valuable tool for investigating D A release in chronically

to m o n i t o r extraceUular D A release in chronically implanted animals is further proved by the stability of the

implanted animals, and could be used to m o n i t o r the

D A basal values recorded as a function of time, during a 24-h period of control. The present study shows and confirms that the multifiber carbon electrode that we

effects on D A metabolism of various types of experiments such as behavioral and learning experiments, or drug post-effect studies.

used, enables without-discontinuity long-term recordings of extracellular D A release. This electrode constitutes a

Acknowledgements. This work was supported by Grant DRET 87-168.

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