Nicotinic effect of acetylcholine on the release of newly synthesized [3H]dopamine in rat striatal slices and cat caudate nucleus

Brain Research, 106 (1976) 117 13l

117

~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

N I C O T I N I C E F F E C T OF A C E T Y L C H O L I N E ON T H E RELEASE OF N E W L Y S Y N T H E S I Z E D [ 3 H ] D O P A M I N E IN R A T S T R I A T A L SLICES A N D CAT CAUDATE NUCLEUS

M. F. G I O R G U I E F F , M. L. LE FLOC'H, T. C. WESTFALL*, J. GLOWINSK1 AND M. J. BESSON

Groupe NB, I.N.S.E.R.M. U 114, Colldge de France, 11, place Marcelin Berthelot, Paris' 5e (France) (Accepted September 8th, 1975)

SUMMARY

The effect of acetylcholine (ACh), carbachol and nicotinic blocking agents on the release of newly synthesized [3H]dopamine ([~H]DA) was studied in vitro on rat striatal slices and in vivo on the cat caudate nucleus. In the latter case, the animals were anaesthetized with halothane; in some experiments an 'enc6phale isol6' preparation was used to eliminate anaesthesia. Rat striatal slices placed in a superfusion chamber were continuously superfused with L-[3,5-3H]tyrosine. A cup placed on the ventricular surface of the cat caudate nucleus similarly allowed a continuous superfusion of the structure with the 3H amino acid. In both cases the quantities of [3H]DA contained in serial superfusate fractions were estimated; the drugs were always added in superfusing medium. In vitro ACh (10 -5 M) and carbachol (10 -5 M)enhanced the release of [3H]DA (90 ~'/o)- Similar results were obtained in vivo in anaesthetized cats. The effect of ACh (10 5 M ) w a s more pronounced (125~) in presence of eserine (10 -4 M ) t h a n with ACh alone (65 ~o). ACh was also effective in unanaesthetized cats. The ACh effect on [3H]DA release was reproducible within the same experiment both in vitro and in vivo. This allowed to test the effect of anticholinergic agents on the ACh induced release of [3H]DA. In vivo hexamethonium (10 -4 M, 10 '~M) partially blocked the release of [3H]DA induced by ACh (10 -5 M) alone; the effect was not seen when ACh was added in the presence of eserine (10 -4 M). Both in vivo and in vitro the prior introduction of mecamylamine into the superfusing medium antagonized the stimulating effect of ACh (10 5M) on [3H]DA release. The effects of this nicotinic blocking agent were seen with various concentrations (10-6; 10 5; 10 4M) in the in vitro experiments. * On Sabbatical year as a Macy Faculty Scholar and IUPHAR Fellow from Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Va. U.S.A.

118 These data suggest that the release of DA from dopaminergic terminals can be regulated by cholinergic presynaptic receptors exhibiting nicotinic characteristics The respective role of nicotinic and muscarinic receptors in the release of DA is discussed. From these results, it can be assumed that cholinergic and anticholinergic agents may act on the metabolism of DA in the nigrostriatal DA neurones through their effect on cholinergic presynaptic receptors.

INTRODUCTION

Recent data strongly suggest that the nigro-neostriatal dopaminergic pathway excerts a tonic inhibitory effect on the activity of cholinergic interneurones present in the caudate putamen complex. This was shown by measuring the release of acetylcholine (ACh) in the caudate nucleus of the cat 4,35 and by estimating the rate of the transmitter utilization19, 26 or turnover 37 in the striatum of the rat after surgical, neurophysiological or pharmacological manipulation of dopaminergic transmission. Briefly, there was an increase in both the release and turnover of ACh after the interruption or blockade of dopaminergic transmission whereas an opposite effect was observed following its facilitation. There is also information which indicates that cholinergic mechanisms could be involved in the control of dopamine (DA) metabolism in the dopamlnergic pathway. Indeed, the peripheral injection of cholinergic agents such as oxotremorine activated the synthesis ~3 and utilization14,27, '-'9 of striatal DA whereas muscarinic blockers such as atropine decreased the rate of utilization of the transmitter a,5,15,21-23.~0. Moreover, anticholinerglc agents were shown to reduce the stimulation of DA turnover induced by neuroleptics2,15, 30. These data were interpreted as the result of alterations of cholinergic transmissions within the striatum; effects which could influence the activity of dopammergic neurones via the striatonigral loop. Other cholinergic systems seem to be involved in the control of the dopaminergic pathway. For instance micromjectlons of ACh or of carbachol into the substantia nigra of the rat reduced the synthesis and utilization of DA in striatal dopaminergic terminals 22" atropine exerted the opposite effectS,22. Etectrophysiological recordings made in the substantia nigra suggested that these effects were mediated through inhibitory interneurones sensitive to cholinergic and anticholinergm agentsL The presence of high levels of ACh and of choline acetyltransferase in the substantia nigra ~6 is consistent with the hypothesis of an indirect cholinergic control of the dopaminergic pathway at this level. However, cholinergic and anticholinergic drugs could also act directly on dopaminergic terminals. This was first suggested by earlier experiments which revealed that ACh increased the release of [aH]DA previously synthesized from L-[3,5 8H]tyrosine in the isolated striatum 6. More recently Westfall reported that nicotine stimulated the release of [aH]DA previously taken up in striatal slices of the rat~9; this facilitation was decreased by hexamethonium and ACh 39. The effect of ACh on the nicotine induced release of [3H]DA could be prevented by prior administration

119 of atropine 4°. It was thus postulated that presynaptic receptors of both the muscarinic and nicotinic type were present on dopaminergic nerve terminals of the rat striatum as previously observed in peripheral noradrenergic terminals 28. The present study was undertaken to further analyze the mechanisms involved in the effects of ACh, cholinergic agonists and antagonists on the release of [3H]DA newly synthesized from L-[3,5-3H]tyrosine. For this purpose two experimental models were used. Changes in transmitter release were studied in vivo in the caudate nucleus of the cat using a cup technique and in vitro in striatal slices of the rat using a microsuperfusion chamber. MATERIALS A N D M E T H O D S

Cat experiments

Cats of both sexes (2-3 kg) were placed into Perspex boxes and anaesthetized with a mixture of air-oxygen (1.5-0.5 v/v) containing 5 ~ halothane. A cannula was introduced into the trachea and the halothane concentration reduced to 2.5 ~ . The halothane anaesthesia was maintained during all the experiments. In some cases, the 'enc6phale isol6' preparation was used and animals were prepared according to the technique described by Puizillout et al. ~2. In these experiments, the animals were placed on artificial respiration after section of the medulla at C1-C2. In both preparations, the ventilation was controlled by monitoring the CO~ content in the expired air with an automatic analyzer. The percentage of CO2 was maintained at 3.5-3.8 ~ by adjusting either the proportion of air-oxygen (anaesthetized cats) or the volume of inspired air ('enc6phale isol6' cats). The femoral blood pressure was continuously monitored with a Statham transducer connected to a recorder. In anaesthetized cats, the halothane concentration was adjusted during the experiments to maintain the mean arterial blood pressure at 60-70 mm Hg. Rectal temperature was maintained at 37 °C with a thermoregulated heating blanket. In all cases the animals were first fixed in a Horsley-Clark apparatus, and a surgical decortication was done to expose the dorsoventricular surface of the caudate nucleus; the head was then maintained immobilized with two iron bars placed on the stereotaxic apparatus and finally fixed with acrylic resin in two points of the skull: the frontal sinus and the parietal bone3L This fixation avoided nociceptive contention ; in these conditions the 'enc6phale isol6' cats presented normal slow wave and paradoxical sleep iv. A cup was placed on the exposed surface of the caudate nucleus and an oxygenated physiological medium (in m M : NaCI 126 ; Na HCO3 27.5 ; K C12.4; K HzPO 4 0.5 ; CaCI2 1.1; MgCI2 0.83; Na2SO4 0.5; glucose 5, adjusted to pH 7.3 with a mixture O2-CO2 9 5 - 5 ~ v/v) containing 50 #Ci/ml of purified L-[3,5-3H]tyrosine (Radiochemical Center Amersham, or CEA Gif/Yvette 50 Ci/mmole) was continuously introduced into the cup at a constant rate (6 ml/h) as previously described 8. The superfusate was collected in serial fractions (10 min each) in cooled tubes containing o~ thioglycolic acid, 6 ~ ; DA 0.005/o, 100 #1 of a protective solution ( E D T A 0.27o; o/. tyrosine, 0.002 °/o). Three ml of ethanol were added to each fraction, and the samples were kept at - - 1 8 °C until the determination of [3H]DA.

120

Rat striatal slice experiments Charles River (Sprague-Dawley) male rats (weighing 200-250 g) were sacrificed by decapitation. The brain was rapidly removed from the skull and kept at 4 "C. Striata were dissected with glass manipulators and chopped with a Mctlwain appa,-atus in slices of 0.7 mm thick. Slices were suspended in a physiological medium, and two slices (approximatively 20 mg of tissue) were then transferred in a sFecialty designed glass superfusion chamber (internal volume of the chamber 150 /~l) placed in a water bath (37 '-'C). The slices were maintained between two platinium mesh wires and superfused by a continuous flow (0,2 ml/min) of the oxygenated physiological medium containing L-[3,5-aH]tyrosine (50/~Ci/ml). Superfusates were collected in serial fractions (2.5 min or 5 min) into iced tubes containing 100/~l of the protective solution described above and 2 ml of ethanol. The samples were kept at --18 °C until the determination of [3H]DA. Biochemical determinations" Purification of L-/3,5-3H/tyrosine. To ensure low blanks for [3H]DA determinations, L-[3,5-3H]tyrosine was purified before each experiment. The [aH]tyrosine Solution, adjusted to pH 8.4 with sodium hydroxyde (0.1 N), was passed on three successive alumina columns (I C0 mg of alumina), the pH of the effluent being readjusted to 8.4 before each passage through the alumina. The effluent of the last alumina column was adjusted to pH 2 with hydrochloric acid (2 N) and passed onto a Dowex 50 W X4 (H ~) column (2 cm high, 0.5 cm diameter) to retain [~H]tyrosine. The resin was washed with 2 ml of hydrochloric acid (0.1 N), then [3H]tyrosine was eluted with a sodium chloride solution (0.9~) added in successive 2 ml fractions to determine the elution pattern of the labelled amino acid. [3H]Tyrosine was generally eluted in the third fraction and then added to a known volume of physiological medium to obtain a final concentration of about 50/~Ci of [~H]tyrosine/ml. Separation of ,f'~H ] D A from l!3H / tyrosine and 3H metabolites. [3H]DA in superfusates was isolated using the technique described by Cheramy et al. a3 with some modifications. Superfusate fractions containing the protective solution (100 #l) and ethanol (2 or 3 ml) were centrifuged. Water (5 ml) containing Triton X-t00 (1 ~o) was added to each supernatant which was adjusted to pH 6.9 with a NaH2PO4-K2HPO4 buffer (0.01 M, pH 7.2) and then passed on an Amberlite column (Amberlite CG 50 H + type il, 200-400 mesh, purified and buffered with a NaHzPO4-K2HPO4 solution 0.2 M, pH 6.1 ; 2.5 cm high, 0.5 cm diameter) to separate 3H amines ([aH]DA and aH aminated metabolites) from [3H]tyrosine. An aliquot (50/A) was taken on the Amberlite effluent to measure the total radioactivity mainly represented by [3H]tyrosine. The Amberlite column was washed with 5 ml of ethanol-water (3.5 v/v) then with 5 ml of water containing Triton X-100 (1 ~o). The aH amines were eluted with 5 ml of 0.2 N acetic acid (Triton X-100 1%o) in tubes containing 500/zl of a protective solution (EDTA 2 ~Uo-thioglycolic acid 0.6 ~/o). Finally [3H]DA was separated from the [3H]O-methylated amine by adsorption on activated alumina. Each Amberlite eluate adjusted at pH 8.4 with a Tris buffer

121 (0.5 M) was passed on an alumina column (100 mg, 0.5 cm diameter). The alumina columns were washed with 5 ml of CH3COONa solution (0.2 M, pH 8.4, containing Triton X-100 1%o) and then with 5 ml of tritonned water (1%/o). [3H[DA was eluted with 1 ml of hydrochloric acid (0.2 N) containing Triton X-100 (1~o). Eluates were collected in counting vials, and the radioactivity estimated by liquid scintillation spectrometry after the addition of 10 ml of a toluene-Triton (3-1.5 v/v), PPO (0.4 ~), POPOP (0.01 ~o) solution. The addition of Triton X-100 (1~o) at each step of the separation considerably diminished the contamination due to [3H]tyrosine. In these conditions the blank was very low (0.5-1 times above the background of the scintillation counter) and thus allowed a precise estimation of the low quantities of [aH]DA released in superfusate fractions. (For example, quantities of [3H]DA corresponding to 0.1 nCi were 4-5 times above the blank value.) All data were corrected for the recovery of [3H]DA (70~o). Statistical analysis of all data was performed using Student's t-test. A value of P << 0.05 was considered statistically significant. Substances used in the present study were: L-[3,5-3H]tyrosine (CEA Gif/Yvette, Radiochemical Center, Amersham), acetylcholine chloride (Merck), carbachol hydrochloride (Sigma), choline hydrochloride (Merck), eserine salicylate (Rh6ne Poulenc), hexamethonium bromide (Sigma) and mecamylamine hydrochloride (Sigma).

EFFECT OF ACh(10-SM) ON3HOA RELEASE FROM RAT STRIATAL SLICES

I

I

I

20 I

I

I

I

40 I

I

min I

¢

E ~.s

"~

Z Z

_}

//

a¢

Z

4-

// //

"z AChI0-5M

Fig. I. Effect of ACh on the spontaneous releaseof (3H]DA from rat striatal slices. Rat striatal slices placed in a superfusion chamber were superfused at 37 °C with an oxygenated physiological medium, containing L-[3,5-3H]tyrosine (50 /~Ci/ml, 1 ml/5 rain). [3H]DA released into the superfusate was measured in serial fractions (5 rain each). Fifteen experiments with similar levels of spontaneous [3H]DA release were selected and the mean value ± S.E.M. of [3H]DA present in the fractions just before, during and after ACh (10 ~M) introduction into the chamber calculated.

122 TABLE l EFFECT OF A C H AND CARBACHOL ON

[3HIDA

RELEASE FROM RAT STR[ATAL SLICES

Rat striatal slices placed in a superfusion chamber (2~chamber) (37 °C) were continuously superfused with an oxygenated physiological medium containing L-[3,5~3H]tyrosine (50/~Ci/ml, I ml/5 min). The release of [3H]DA was measured in serial fractions (5 min). ACh, carbachol or choline were applied during 2 fractions (10 rain). The data are the mean value ± S,E.M. of the quantity of [3H]DA released just before and during the application of the drugs, The mean was calculated by taking the two fractions before and the two fractions during the drug application of all experiments (n -~ total number of fractions).

3H/ DA released(nCi) /fraction (5 min) Before ACh, 10-°M(n 12) ACh, 10-SM(n = 32) Carbachol, 10-~M(n - 15) Choline, 10-SM(n - - 6 )

0.27 0.69 0.58 0.58

± ~: ~: ~

% change

During 0,02 0.08 0.07 0.04

0.42 1.26 1.13 0.31

~ ± ± ~t

0.03 0.14 0.12 0.01

! 55** ! 83* ! 95** n.s.

* P < 0.01. ** P < 0.001.

n.s., not significant.

RESULTS

Effects of A Ch and carbachol on the release of [3H]DA synthesized from L-/3,5-3H tyrosine in striatal slices of the rat When rat striatal slices were continuously superfused with L-[3,5-ZH]tyrosme (50 #Cijml: 1 ml/5 rain), the quantity of [3H]DA released (outflow) in 2.5 min fraction increased progressively and reached a steady-state level after 15-20 min of superfusion. This pattern was seen in all experiments but variations in the mean value of [ZH]DA released were detected from one experiment to another (0.02 nCi2 nCi/fraction). Cholinergic agents were applied when the steady-state was reached. In 15 experiments, the addition of ACh (10-SM) to the superfusing medium during two fractions of 5 rain each enhanced the release of [SH]DA by an average of 9 0 ~ (mean of [ZH]DA content in the two fractions) when compared to the mean of the spontaneous level determined during the two preceding fractions (Table 1, Fig. 1). A smaller concentration of ACh (10-6M) enhanced [ZH]DA release to 55 ~ (Table l). In most cases after cessation of the ACh (10-SM) application, the release of [ZH]DA remained higher than before the ACh addition. In some experiments when a second addition of ACh (10-SM) was made 25 min after the first one, the per cent change in [3H]DA release was similar to that observed during the first application. This was seen even though the level of the spontaneous release before the second application was higher than before the first one (Table II). Choline (10-SM) was without effect while carbachol (10-SM), a cholinergic agonist not hydrolyzed by cholinesterases, stimulated [ZH]DA release as did ACh (10-SM) (Table I).

123 TABLE II EFFECT OF REPEATED APPLICATION OF A C H o N

[3H]DA RELEASE in vivo

AND

in vitro

These experiments were carried out as described previously (Figs. 1 and 2) on the caudate nucleus of the cat (in vivo) or on striatal slices of the rat (in vitro). ACh alone or in the presence of eserine was applied twice in the same experiment. These applications lasted for 20 min and 7.5 min for the in vivo and in vitro experiments respectively. The second ACh application was made about 90 and 20 min after the first one in cat and rat experiments, respectively. The table illustrates data obtained from individual experiments; they correspond to [3H]DA found in two (cat) or three (rat) pooled fractions before and during ACh application. These fractions were of 10 min each in the cat and 2.5 min each in the rat experiments. ~, increased release in percentage when compared to the spontaneous release.

A Ch

IaH]DA released (nCi) First A Ch application

Cat

10-SM 10 ~M ÷ eserine 10 4M

Rat

10-SM

Second A Ch application

Before

During

%

Before

During

%

5.23 2.52 4.92 3.42 1.36

7.53 4.17 12.93 4.62 3.29

÷ ÷ ÷ ÷ ÷

44 65 214 35 142

3.45 2.34 4.08 4.06 1.69

5.81 3.90 12.64 6.11 3.78

÷ + t + +

0.50 1.18

0.86 2.65

÷ 72 + 124

1.14 2.04

1.96 4.99

68 67 210 50 121

+ 72 t 144

In vivo effects of ACh and carbachol on the release of ( Z H j D A synthesized from L-[3,5 -3] tyrosine in the caudate nucleus of the cat The effects o f A C h were e x a m i n e d in a n a e s t h e t i z e d ( h a l o t h a n e ) or u n a n a e s t h e t ized cats ('enc6phale isol6' p r e p a r a t i o n ) using the cup technique described in M e t h ods. In b o t h types o f p r e p a r a t i o n s , the s p o n t a n e o u s release o f [3H]DA increased progressively d u r i n g the first 90 rain a n d then reached a steady state level d u r i n g c o n t i n u o u s superfusion o f the c a u d a t e nucleus with L-[3,5-3H]tyrosine. As in rat striatal slices, the level of s p o n t a n e o u s release was different f r o m one a n i m a l to another. A C h (10-SM) was a d d e d to the superfusing m e d i u m 2 h after the onset o f superfusion in the absence or presence o f eserine (10-4M). H a l o t h a n e anaesthesia b l o c k e d the release o f ACh2a; in such a n a e s t h e t i z e d animals, A C h (10-SM) increased the release o f newly synthesized [3H]DA by 65 % in the absence o f eserine (Table I l l ) . Eserine (10 4M) alone did n o t affect [3H]DA release, b u t when A C h (10-SM) was a d d e d s i m u l t a n e o u s l y with eserine ( 1 0 - a M ) the release o f [3H]DA was m o r e pron o u n c e d (127%) (Table III, Fig. 2A) t h a n w i t h A C h (10-SM) alone. The results c o n c e r n i n g the effects o f A C h alone or in presence o f eserine are the m e a n o f changes in [3H]DA release detected in various cat e x p e r i m e n t s in which the effects of A C h were e x a m i n e d one or several times. The per cent changes in [3H]DA release were similar when A C h (10-SM) plus eserine (10-4M) were a p p l i e d at two different times in a single e x p e r i m e n t (Table II). H o w e v e r i m p o r t a n t v a r i a t i o n s in the intensity o f the A C h (10-SM) plus eserine ( 1 0 - 4 M ) effect were observed f r o m one a n i m a l to a n o t h e r (20 % - 6 0 0 ~ ) . These differences did n o t a p p e a r to be related to the s p o n t a n e o u s

124 TABLE 111 EFFECT OF A C H AND CARBACHOt, ON THE

[:~H]DARELEASE

FROM THE CAT CAUDATE NUCLEUS

The caudate nucleus was superfused continuously with L-[3,5-ZH]tyrosine(50/zCi/ml, 1 ml/10 min) in halothane anaesthetized cats. The release of [~H]DA was measured in serial fractions (10 rain). ACh, ACh plus eserine or carbachol were applied during two fractions (20 min). Data are the mean i S.E.M. of the quantity of [3H]DA released just before and during the application of drugs. The mean was calculated by taking the two fractions preceding the drug application and the two fractions corresponding to the period during which cholinergic agents were introduced into the cup in all experiments, n - total number of fractions: %, increased release in percentage when compared to the spontaneous release. :~H/ D.4 released ( nCi) /fraction (10 rain)

ACh, 10-~M(n - 14) ACh 10-SM ÷ eserine, 10-4M(n -- 20) Carbachol, 10 ~M(n 10)

% change

BeJore

During

0.80 ~: 0.10

1.32 2_ 0.19

~ 65*

1.96 ± 0.19 0.27~,: 0.03

4.46 -2:0.45 0.53 5L:0.08

i 127"** + 96**

* P < 0.05. ** P < 0.01. *** P <."0.001. level of [3H]DA release. In most cases the spontaneous release of [3H]DA returned to its original level following a 20 min application of ACh (10-SM) plus eserine (10-4M). Fig. 2 illustrates the effect of a longer application (60 min) of ACh (10-SM) plus eserine (10-4M). After an initial marked stimulation of [3H]DA release seen during the first two 10 min fractions, the quantity of [aH]DA declined in the following fractions but remained higher than the spontaneous release measured before ACh application. In anaesthetized animals, carbachol (10-SM) stimulated the release of [~H]DA in a similar way to that observed with ACh (10-SM) plus eserine (10-4M)(Table Iit). The stimulating effect of ACh (10-SM) plus eserine (10-4M) on [3H]DA release could also be easily detected in two experiments made on unanaesthetized animals ('enc6phale isol6' preparation). Effects o f nicotinic blockers on the A Ch induced [aH] D A release in cat caudate nucleus and in rat striatal slices The effects on the ACh induced [3H]DA release of hexamethonium and mecamylamine (two peripheral nicotinic blocking agents) were examined in vivo (cat caudate nucleus) or in vitro (rat striatal slices). For this purpose, the effects of A c h on [3H]DA release were analyzed successively in the same experiment in the absence and presence of the blocking agent. The anticholinergic drugs were added continuously to the superfusing medium before and during the second application of ACh. As already mentioned, the spontaneous release of [SH]DA did not always return to its original base line level following the first application of ACh. However the ACh stimulating effect on [aH]DA release was reproducible when expressed as per cent

125 EFFECT OF ACh (10"5M) ON3H-DA RELEASE FROM THE CAT CAUDATE NUCLEUS

®

® ,

, 18.o . . . . .

2~o

~

~

rain

I

~7

~S i i./

E

g

i., / //./

3

m

/

LL

2

S AChlO-5M + EserinelO-4M

V//////A ACh 10"5M +

Eserine IO~M

Fig. 2. Effect of ACh in presence of eserine on the spontaneous release of [3H]DA from the cat caudate nucleus. The ventricular surface of the caudate nucleus was superfused continuously with an oxygenated physiological medium containing L-[3,5-3H]tyrosine (50 /~Ci/ml, 1 ml/10 min) in cats anaesthetized with halothane. The release of newly synthesized [3H]DA was measured in serial superfusate fractions (10 min). In A, ACh (10-SM) and eserine (10-4M) were added to the superfusing medium during two fractions (20 min). Data correspond to the mean value ± S.E.M. of I 1 applications of ACh performed in 8 cat experiments in which the level of the spontaneous release of [3H]DA was comparable. B illustrates data obtained in one cat experiment in which ACh (10 5M) plus eserine (10 4M) was added to the superfusing medium during 60 min.

o f the [3H]DA c o n t e n t f o u n d in the two or three fractions which preceeded the A C h a p p l i c a t i o n . T o t a k e in a c c o u n t these factors, results were expressed in three different ways. A C h effects were c o m p a r e d (1) by calculating the change in [3H]DA release in per cent o f the release detected before each A C h a p p l i c a t i o n , (2) by m e a s u r i n g the total a m o u n t o f [3H]DA released d u r i n g each A C h a p p l i c a t i o n , a n d (3) by calculating the difference between the total a m o u n t o f [3H]DA release d u r i n g and before each A C h a p p l i c a t i o n (Table IV). The e x p e r i m e n t s with h e x a m e t h o n i u m were only carried o u t in the anaesthetized zat. The d r u g alone in c o n c e n t r a t i o n o f 10 5M or 10 4M i n d u c e d a progressive increase in [~H]DA release when a d d e d c o n t i n u o u s l y to the superfusing m e d i u m . This effect lasted for a b o u t 1 h a n d the A C h effect was tested when [3H]DA release ret u r n e d to a level similar or even lower t h a n t h a t detected in fractions p r e c e d i n g

[ZH]DA in vivo AND

in vitro

Rat

Cat

Cat

10-4M 10-aM 10-SM 10-SM t0-6M 10-6M 10-GM 10 ~'M

t0 4M lO-4M

10 5M lO-SM IO-aM 10 4M

Cholinergic blocker

IO-~M

10-~M 10-~M 10-sM 10 ~ M

10-SM

10 ~ M 10-SM

10 5M 10-~M eserine 10-4M

10-ZM 10-SM 10 ~M + eserine 10 4 M

ACh

0.80 1.31 2.07 2.13 1.50 3.15 2.77 2.91

I 1.23

7.60 0.45 0.48 0.94 1.09 0.80 1.85 1.39 1.38

12.61

7.03

2.78

6.32

2.92 3.32 0.43

During

1.97 2.56 0.31

Before

First A Ch application

0.35 0.83 1.13 1,04 0.70 1.30 1.38 1.53

3.63

6.29

4.25

0:95 0.76 0.12

A

77 i 170 -~- 120 + 94 T 87 @ 70 : 99 - III

-~- 48

99

-~ 153

~ 48 +- 30 ~ 40

During

2.99

2.42 2.20 0.33

0.67 0.80 1:23 1.66 1.24 2.70 2.44 3.36

13.31

8.61

0.81 0.94 1.34 1.70 t.47 3.04 3.08 3.86

15.64

11.09

Mecamylamine

1.24

1.95 2.17 0.28

Hexamethonium

°o change Before

Second A Ch application

0.15 0.14 0.11 0.04 0.23 0.34 0.64 0.50

2.33

2.48

1.75

0.47 0.03 0.05

A

+ 22 ~ 17 ! 9 ~ 2 ! 18 -- 13 ~ 27 15

i 17

~ 29

~ 141

24 0 T 18

% change

64

70

8

--50 100 55

.... 71 ...... 90 92 97 79 - 81 72 86

-

% bk)cl, ade

Experiments were carried o u t as described in Figs. 1 a n d 2. A C h alone or in presence o f eserine was applied twice in the s a m e e x p e r i m e n t ; the second application being m a d e a b o u t 90 m i n and 20 rain after the first one in cat a n d rat experiments, respectively. Cholinergic blockers were a d d e d c o n t i n u o u s ly prior to (60 rain cat a n d 15 min rat) and d u r i n g the second A C h application. T h e table illustrates data obtained f r o m individual experiments. They correspond to the quantity of [3H]DA f o u n d in two (cat) or three (rat) pooled fractions before a n d during A C h application. T h e s e fractions were o f 10 min each in the cat a n d of 2.5 rain each in the rat. ?',, increase of [aH]DA overflow above the level o f the s p o n t a n e o u s release; °o change, increased release in percentage when c o m p a r e d to the s p o n t a n e o u s release; % blockade, inhibition in percentage o f the A C h effect in presence of the cholinergic blocking agent.

BLOCKING EFFECTS OF HEXAMETHONIUM AND MECAMYLAMINE ON THE A C H INDUCED RELEASE OF

T A B L E IV G',

127 EFFECT OF HECAMYLAHINE ON ACh INDUCED RELEASE 0F3H-DA IN RAT STRIATAL SLICES

q

2p

3o ~p so

@ ~o

A u~ ~.0

~.3

~

m AChI0"~

~"~'0"SM MECAMYLAMINEI0"5M

Fig. 3. Effect of mecamylamine on the ACh induced release of [3H]DA from striatal slices of the rat. Striatal slices (2/chamber) were superfused continuously with L-[3,5-3H]tyrosine (50 /~Ci/ml, 0.5 ml/2.5 min) and [SH]DA estimated in serial superfusate fractions. ACh (10 5M) was added twice to the superfusion medium. The second ACh application was made in the absence (upper part) or presence (lower part) of mecamylamine (10 ~M).

hexamethonium application. At this time, the ACh (10 5M) effect was markedly reduced in the presence of hexamethonium (10-SM or 10-4M) (Table IV); the blocking action of hexamethonium was not observed when ACh (10-SM) was applied in the presence of eserine (10-aM). Both the in vivo and in vitro application of mecamylamine (10-4M)resulted in a slight increase in [3H]DA release. The anticholinergic agent markedly decreased the ACh (10-SM) induced release of [3H]DA in both types of preparation. The inhibitory effect of mecamylamine in the cat preparation was still seen when ACh (10-SM) was added in the presence of eserine, although the effect was less pronounced as compared to ACh alone. As shown by experiments carried out in vitro in smaller concentrations (10-SM, 10 6M) mecamylamine did not affect the spontaneous release of [3H]DA and exerted a more important inhibitory effect on the ACh induced release of [3H]DA (Table IV, Fig. 3). DISCUSSION

The analysis of the effects of cholinergic and anticholinergic agents on the release of [3H]DA from dopaminergic terminals of the nigrostriatal system were made in all cases during continuous superfusion of the tissues with L-[3,5-3H]tyrosine. This procedure offers some advantages when compared to previously used methods which consisted in the estimation of the release of labelled DA recently taken up in

128 tissues. First, the labelled newly synthesized transmitter originates specifically from dopaminergic terminals in which the tyrosine hydroxylase is localized. Secondly, in most cases the newly formed amine is preferentially released from nerve terminals! s,~'~. Thirdly, some differences were already seen between the pattern of release of the labelled amine endogenously synthesized from its precursor or previously taken up in catecholaminergic neurones 8,11. For instance, in earlier studies it was not possible to detect the ACh induced release of [3H]DA previously taken up in the isolated striatum of the rat 6. This is in contrast with that observed with the newly synthesized transmitter. Finally, a close relationship occurs between release and synthesis processes in rapid regulatory mechanisms 9,;~s. The in vivo cat preparation was selected in order to analyze the effects of cholinergic and anticholinergic agents in intact dopaminergic neurones in which nerve firing is present. Animals were anaesthetized with halothane, an anaesthetic agent known to increase nerve impulse flow in dopaminergic neurones 1° and to abolish ACh release in the caudate nucleus ~4. The 'enc6phale isol6' preparation was used in some experiments to avoid the possible interferences of anaesthesia on the effects observed; moreover, in the unanaesthetized cat, ACh is released in the caudate nucleus '~4. Experiments were also carried out in vitro using striatal slices of the rat. In this simplified complementary model, it can be assumed that cholinergic agents are acting on dopaminergic terminals. With these two preparations and using similar analytical methods, it could be shown that the labelled amine isolated from superfusates was almost entirely represented by [3H]DA (95~)s. ACh (10-SM) produced an immediate and marked increase in the outflow of [3H]DA both in the in vivo and in vitro experiments. These changes were most likely related to modifications in the transmitter release. The ACh effect was seen regardless of the spontaneous level of release, and was very reproducible when two applications of ACh were given in the same experiment. This reproducibility allowed the comparison of the effect of ACh in the absence and presence of anticholinergic agents in the same experiment. Since ACh was present in the medium which continuously superfused the tissue it was not necessary to simultaneously add a cholinesterase inhibitor to observe the ACh effect in the rat striatum or the cat caudate nucleus. It was observed in the cat, however, that although eserine was ineffective by itself, it reinforced the ACh effect. Carbachol (10-SM) stimulated [aH]DA release in a similar way to that observed with ACh (10-~M) and thus provides additional evidence for the specificity of the ACh effect. In most cases, the spontaneous release of [3H]DA did not return to its original level after the short application (10 min in vitro, 20 min in vivo) of either ACh or carbachol. It is possible that a sustained change in membrane permeability could be partly responsible for this phenomenon. In some experiments, when hexamethonium or mecamylamine were added in relatively high concentration (t0-~M), they progressively increased the outflow of the labelled transmitter; these effects, which were less pronounced with smaller concentrations, could be partly dependent on the drugs action on inactivating processes of the transmitter. The stimulating effect of ACh on [aH]DA release could be related to an ac-

129 tivation of cholinergic presynaptic receptors of the nicotinic type localized on dopaminergic terminals. Indeed the release of [aH]DA induced by ACh was markedly reduced both in vivo and in vitro by a prior application of various concentrations of mecamylamine, a well known blocker of peripheral nicotinic receptors a6. Moreover, hexamethonium, another nicotinic blocker, partially prevented the stimulating effect of ACh in the anaesthetized cat preparation. Hexamethonium has been shown to selectively block the ACh or nicotine induced release of noradrenaline in peripheral it and central noradrenergic terminals a9 as well as of [aH]DA previously taken up in striatal slices of the rat 39. Finally, it has also been observed that a small concentration of mecamylamine (10 6M) prevented the ACh induced release of newly synthesized [aH]5-HT in hypothalamic slices ~°. Direct evidence of the occurrence of cholinergic receptors of the nicotinic type has recently been provided by binding studies of labelled nicotinic agents in tissue homogenates 3a,34. These data led us to postulate the involvement of presynaptic cholinergic receptors with nicotinic characteristics in the control of the release of DA from dopaminergic terminals. However, as revealed by the experiments performed first in peripheral noradrenergic neurones, the action of ACh on the release of catecholamines appears complex. Indeed, in addition to its stimulatory effect, ACh also seems to exert an inhibitory action on the transmitter release by acting on muscarinic receptors 2s. Presynaptic muscarinic receptors may also be involved in the control of NA release from central noradrenergic terminals TM a9. Furthermore, as already noted, ACh inhibited the nicotine induced release of [3H]DA in striatal slices 39, the latter effect being prevented by atropine 4°. This strongly supports the possibility that there are presynaptic receptors of both the nicotinic and muscarinic type on dopaminergic nerve terminals. If this is the case, ACh could exert two opposite effects on the release of the transmitter. Preliminary in vitro data, obtained by examining the release of [3H]DA from striatal slices continuously superfused with L-[3,5-3H]tyrosine, provide further arguments for this statement. In these experiments, the K + (50 mM) induced release of [3H]DA was reduced in the presence of small concentrations of ACh (10-6M). In addition, scopolamine (10 5M) not only prevented the ACh inhibitory effect but unmasked the nicotinic facilitatory action of ACh. In other words, in the presence of scopolamine the K + induced release of [aH]DA was potentiated by ACh (10-6M) (unpublished observations). This duality in the effects of ACh may explain some of the individual variations in the stimulating action of ACh on DA release seen from one animal to another in our experiments. The occurrence of presynaptic cholinergic receptors on various types of aminergic terminals is puzzling. It remains to be established whether or not these receptors play a functional role in the regulation of transmitter release. The anatomical organization of the striatum is such that this could be the case for the nigrostriatal dopaminergic neurones. The numerous cholinergic interneurones localized in this structure could control or modulate in some way the regulation of DA release at a presynaptic level. In any case, the changes in DA release induced by the peripheral injections of cholinergic and anticholinergic agents could be partly related to a direct effect of these drugs on dopaminergic terminals as well as an indirect action

130 i n v o l v i n g c h o l i n e r g i c n e u r o n a l p a t h w a y s i m p l i c a t e d in the r e g u l a t i o n o f the n i g r o s t r i a t a l D A system. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y G r a n t s f r o m I N S E R M

(75.5.153.6) U S P H S N S

10260 a n d les U s i n e s C h i m i q u e s R h 6 n e - P o u l e n c .

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