Non-DA prefronto-cortical efferents modulate D1 receptors in the nucleus accumbens

Brain Research, 305 (1984) 43- 50 Elsevier

43

BRE 10152

N o n - D A Prefronto-Cortical Efferents Modulate D 1 Receptors in the Nucleus Accumbens MICHEL REIBAUD, Gt~RARD BLANC, JEANNE-MARIE STUDLER, JACQUES GLOWINSKI and JEAN-POL TASSIN Chaire de Neuropharmacologie, INSERM U.114, Groupe NB, Coll~gede France, 11 place Marcelin Berthelot, 75231 Paris cedex 05 (France) (Accepted December 6th, 1983) Key words: D a receptors - - nucleus accumbens - - cortical lesions - - glutamate uptake

Bilateral injections of 6-OHDA in the ventral mesencephalic tegmentum (VMT) destroy the DA afferents of the nucleus accumbens but do not induce any denervation supersensitivity of the D l receptors in the nucleus accumbens. Because other non-DA afferent fibers might also regulate D~ receptors in that structure, bilateral ablations of the prefrontal cortex were performed. This lesion induced a 55% decrease of [3H]glutamicacid high-affinity uptake activity in the nucleus accumbens. When the prefronto-cortical ablation was performed simultaneously with the bilateral injection of 6-OHDA in the VMT, a marked hypersensitivity of DA-sensitive adenylate cyclase activity was observed in the nucleus accumbens (+52% increase of Vma×and a twofold decrease of the Kapp for DA) while the ablation of the prefrontal cortex alone induced only a ÷ 14% (P < 0.01) increase of D 1receptors in that structure. These results indicate that the regulation of D 1 receptors in the nucleus accumbens is not solely dependent on the presynaptic DA innervation and that other non-DA fibers, such as those of the cortico-nucleus accumbens pathway, might contribute to it. INTRODUCTION Most of the dopaminergic (DA) innervation of cortical and subcortical structures arises from n e u r o n a l populations which are distributed in the ventral mesencephalic t e g m e n t u m (VMT) and the substantia nigra (SN)3. All areas of projection of the different ascending D A systems contain postsynaptic D A receptors coupled to an adenylate cyclase (D 1 receptors) 4,9,10,12,15,16,21,28 whose topographical distributions closely resemble those of D A nerve terminals4,21,24. Data obtained in several laboratories have suggested that the destruction of D A afferents induces a hypersensitivity of postsynaptic D A receptors16,17, 27. However, recently we have demonstrated that afterents other than D A fibers could also contribute to the regulation of D 1 receptor sensitivity. Indeed, the characteristics of D 1 receptors in the rat prefrontal

cortex were not affected following bilateral injections of 6-hydroxydopamine ( 6 - O H D A ) into the VMT leading to the destruction of not only D A , but also noradrenergic (NA), ascending pathways. Conversely, bilateral electrolytic lesions of the V M T which led to a degeneration of D A cortical afferents but preserved the cortical N A innervation induced an important increase in the cortical DA-sensitive adenylate cyclase activity (corresponding to an increased n u m b e r of D 1 receptors25). These data st~ggested that the N A innervation plays a permissive role in the development of D 1 receptor hypersensitivity associated with the destruction of D A afferent fibers. Moreover, surprisingly, in these experiments neither chemical nor electrolytic lesions of the V M T affected D 1 receptor sensitivity in the nucleus accumbens, although in both cases D A levels were reduced to 5% of the control values in this subcortical structure25. The latter results led us to propose that non-

* M. Reibaud is a research fellow from Rh6ne Poulenc Sant6. Correspondence: J. P. Tassin, Chaire de Neuropharmacologie, INSERM U.114, Groupe NB, Coll6ge de France, 11 place Marcelin Berthelot, 75231, Paris cedex 05, France. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

44 D A afferents were also involved in the regulation of the DA-sensitive adenylate cyclase activity in the nucleus accumbens. More explicitly, we suggested that a modification of the activity of the descending cortico-nucleus accumbens pathway induced by the VMT lesions prevented the expected denervation supersensitivity of D 1 receptors in the nucleus accumbens 23,25. Such a hypothesis is substantiated by electrophysiologicalS,26, anatomical2,13 and biochemicaP 8A9 studies which indicate that the mesocorticoprefrontal D A neurons control the activity of cortical branched cells innervating several sub-cortical structures and that the nucleus accumbens receives glutamatergic afferents from the prefrontal cortex 29. The present investigation was undertaken to verify the hypothesis mentioned above. For this purpose, DA-sensitive adenylate cyclase was estimated in the nucleus accumbens following bilateral ablation of the prefrontal cortex in rats either with or without bilateral 6-OHDA-induced degeneration of nucleus accumbens D A innervation. The efficacy of the cortical ablations was first evaluated by measuring changes in [3H]glutamic acid high affinity uptake activity in subcortical structures. It will be shown that the expected D 1 receptor supersensitivity in the nucleus accumbens resulting from DA denervation only occurs following ablation of the prefrontal cortex. MATERIALS AND METHODS

a rate of 0.3/A/min using a motor-driven 5/~1 Hamilton microsyringe connected to a polyethylene tube attached to a stainless steel cannula of 0.2 mm diameter. The cannula was implanted at the following coordinates: --3.9 mm to bregma, 0.5 mm lateral to midline and 8.7 mm under the calvarium and was kept in place for 3 min after the injection. (2) Bilateral ablation of the prefrontal cortex A 4 × 4 mm piece of skull centered 4.0 mm in front of the bregma was removed with a dental drill and placed in physiological serum. The prefrontal cortex was aspirated with a vacuum pump connected to a 0.9 mm diameter stainless steel cannula which was moved along the following coordinates: (a) anterior to bregma: 2.45-5.45 mm at an angle of 4° in the posteroanterior direction: (b) _+ 0.5 mm lateral to midline at an angle of 10°; and (c) 1 mm under the calvarium on the proximal side of the bregma (2.45 mm to 3.45 mm anterior to bregma) and 2 mm under the calvarium on the distal side of the bregma. Histological controls of this lesion are presented in Fig. 1. Following the bilateral prefrontal cortex aspiration, the skull piece was put back in place, the animals received an intraperitoneal injection of 3 ml distilled water and were kept at 37 °C until recovery from anesthesia. Sham-operated animals had the skull piece cut out but no lesion.

Animals and surgery

(3) Simultaneous bilateral injections of 6- OHDA into the V M T and ablations of the prefrontal cortex

Male Sprague-Dawley rats (Charles River) weighing 280-300 g were used. They were individually housed in a thermo-regulated room (22 °C), with a 12 h dark-light cycle (dark 19.00-07.00 h). Animals were anesthetized by an intraperitoneal injection of sodium thiopental (Pentothal, Abbott) (50 mg/kg, 10 mg/ml). Operations were performed with a stoelting stereotaxic apparatus (Krieg model 51200; incisors bar, 3.4 mm above the interaural line).

When both types of lesions were performed, the bilateral aspiration of the prefrontal cortex was first achieved and the animal was then placed in another stereotaxic apparatus in order to receive 6-OHDA micro-injections into the VMT.

(1) 6-OHDA lesions of the V M T 6-OHDA (4 ktg in 1/zl) was dissolved extemporaneously in the presence of ascorbic acid (1 mg/ml) in an isotonic saline solution neutralized to pH 4.5. The injections were performed bilaterally in the VMT at

Biochemical procedures (1) Tissue sampling Rats were sacrificed by decapitation between 11.00 h and 14.00 h, 6-12 weeks following the lesions. Brains were rapidly removed and the frontal half was fixed with sodium chloride (9%) on a Leitz Wetzler microtome stage refrigerated to - - 7 °C using a Leitz Kryomat. When the tissue was frozen (about 15 min), serial sections (500/~m thick) were

45

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Fig. 1. Histological control of bilateral ablation of the prefrontal cortex. Slices were 40 ~um thick and every fourth slice was retained. The beginning of the striatum (9800Bm plane in the atlas of K6nig and KlippeP 1) was taken as a landmark.

made using the beginning of the striatum as a landmark (the 9800/~m frontal plane according to the atlas of K6nig and Klippe111). Slices were placed with paint brushes on the specimen holder covered with aluminum foil and microdiscs of tissue were taken as described below. (2) High-affinity [3H]glutamic acid uptake activity (a) Purification of L-[3,4-3H]glutamic acid. An acidified solution of L-[3,4-3H]glutamic acid (42.3 Ci/mmol, NEN chemicals) was poured on an H +Dowex column (height: 20 mm, diameter: 4 mm). The column was washed with 1 ml of 0.01 N HC1 and the purified [3H]glutamic acid was eluted with 0.1 M Tris-HCl, pH 7.4 in a 0.5 ml peak. (b) Estimation of [3H]glutamic acid high-affinity uptake activity. The method used was adapted according to the method previously described for the estimation of [3H]DA uptake activity2L Microdiscs

of tissues were punched out with a cooled stainless steel cylindric tube sharpened at its extremity (0.9 mm diameter) and blown into 1.5 ml Eppendorf plastic tubes containing 15 ,ul of 0.25 M sucrose. They were directly homogenized for 10 s with a piston made of dental cement (Stellon from Detrey) using a 1.5 ml Eppendorf tube as a matrix. Immediately after homogenization, 400/~1 of a physiological medium (in mM: glucose 5; NaC1 136; KC1 5.6; MgC12 1.2; NaH2PO4 1.2; CaCI: 2.2; NaHCO3 16.2) were added (partly to rinse the piston). After 5 min of preincubation at 25 °C, 10/d of [3H]glutamic acid (final concentration 10-7 M) were added and the incubation was continued for 3 min. The incubation was stopped by cooling the tubes in a water bath at 0 °C. Tubes were then centrifuged for 30 min at 20,000 g, the pellets were washed twice with 400/A of sodium chloride (0.9%) and finally suspended in 50/d of Triton X-100 (1%). Radioactivity was measured by transferring

46 this suspension to vials containing 10 ml of scintillation medium. Blanks were obtained by omitting Na2HPO 4 and NaC1 in the preincubation m e d i u m and by replacing them by sucrose (final concentration 280 m M ) and Tris-PO4, pH: 7.4 (final concentration 1.2 mM). High-affinity glutamic acid uptake activity was expressed in pmol/min/mg protein. (4) Linearity of the assay. In all cases, high affinity [3H]glutamic acid uptake activity into crude synaptosomes was linear for at least 3 rain and the quantity of [3H]glutamic acid i n c o r p o r a t e d into the h o m o g e n a t e s was always less than 1% of total radioactivity. (3) DA-sensitive adenylate cyclase activity Six microdiscs of tissue d i a m e t e r (0.9 mm) were punched out bilaterally in the nucleus accumbens (planes 9600/~m and 9100/~m of the atlas of KOnig and Klippe111), blown into 200/A of a 2 m M Tris-maleate (pH 7.2), 2 m M E G T A (pH 7.2), 300 m M sucrose solution and gently h o m o g e n i z e d in a P o t t e r - E l v e j hem apparatus (10 strokes). A d e n y l a t e cyclase activity was m e a s u r e d by conversion of [a-32P]ATP into [a-32p]cyclic A M P . The experimental conditions were similar to those described before 4. The purification of [a-32p]cyclic

A M P was done according to Salomon et al. 20. A d enylate cyclase activities were expressed in p m o l of cyclic A M P produced/min/mg protein. (4) Estimation o f D A contents Fifty/A aliquots taken from the sucrose h o m o g e n ates p r e p a r e d to measure adenylate cyclase activity, were p o u r e d into 100/~1 of a 0.1 N perchloric acid, 0.01 N thioglycolic acid solution. A f t e r ultra-sonication and centrifugation, pellets were k e p t to estimate protein content by the m e t h o d of Lowry 14 and catechols contained in the supernatants were a d s o r b e d and purified on alumina micro-columns 7. Eluates were dried under vacuum and a radioenzymatic procedure using tritiated S-adenosyl methionine ([3H]SAM, A m e r s h a m , spec. act. 8-15 Ci/mmol) as a methyl d o n o r was p e r f o r m e d . [3H]Methoxytyramine was extracted and s e p a r a t e d by silica-gel chromatography 8.

Statistical analysis Results o b t a i n e d in lesioned rats were c o m p a r e d with their respective control values by the Student's ttest. RESULTS

TABLE I

Effects of bilateral ablation of the pre-frontal cortex on high-@ finity [3H]glutamic acid uptake activity in different cortical and subcortical structures

(1) Effects o f bilateral ablation of the prefrontal.cortex on the high-affinity ff H]glutamic acid uptake activity in subcortical structures

Six weeks after the lesion, animals were killed and their brain was frozen at --7 °C. Microdiscs (diameter 0.9 mm) from different areas were punched out on 500 ~m thick coronal slices. After homogenization the crude synaptosomes were incubated for 3 min in the presence of 10-7 M [3H]glutamic acid. Resuits are the mean + S.E.M.

As already p o i n t e d out, several reports have suggested that neurons which originate in the prefrontal cortex and project to subcortical structures including the nucleus accumbens, are glutamatergicl,6, 29. The estimation of [3H]glutamic acid u p t a k e sites in the nucleus accumbens s e e m e d therefore a reliable index to quantify the extent of destruction of cortico-nucleus accumbens fibers elicited by the bilateral prefronto-cortical ablation. In fact, a 55% decrease in [3H]glutamic acid high-affinity u p t a k e activity was observed 6 weeks following the lesions (Table I). A s expected, the nucleus accumbens was not the only structure affected by the bilateral cortical ablation. A n important decrease of [3H]glutamic acid u p t a k e was also noted in the rhinal cortex, the rostral part of the striatum, the lateral h a b e n u l a and the m e d i o - d o r sal nucleus of the thalamus. Conversely, no signifi-

Structure

ff H]Glutamic acid high-affinity uptake activity (pmol/min/mg protein) Controls

Lesioned

Nucleus accumbens Rostral striatum Caudal striatum Lateral habenula Rhinal cortex Medio-dorsalthalamus Superior colliculus Substantia grisea

0.65 + 0.04 0.58 +__0.02 0.30 + 0.08 0.31 + 0.04 0.56 _+0.05 0.56 + 0.04 0.19 + 0.02 0.23 + 0.03

0.29 + 0.30 + 0.23 + 0.14 + 0.38 + 0.45 + 0.18 + 0.15 +

0.03 0.03 0.03 0.02 0.03 0.04 0.02 0.02

(--55%**) (---48%**) (n.s.) (--55%**) (--32%**) (--20%*) (n.s.) (--35%*)

* P < 0.05 and ** P < 0.001 when compared with sham-operated animals, ns, not significant.

47

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Fig. 2. Effects of (1) 6-OHDA VMT lesion, (2) bilateral ablation of the prefrontal cortex and (3) both types of lesions, on D 1receptors and DA levels in the nucleus accumbens. Six to twelve weeks after the lesion(s), animals were decapitated, brains were removed and 6 microdiscs (diameter 0.9 mm) were punched out from the nucleus accumbens. After homogenization, aliquots were taken for the estimation of DA-sensitive adenylate cyclase activity and DA endogenous levels. Results are the means of data obtained with groups of at least 6 rats. Mean values + S.E.M. of sham-operated animals were: D A levels 78 + 6 ng/mg protein; basal adenylate cyclase activity: 33 + 2 pmol cyclic AMP per min per mg protein; DA (10-4 M) stimulated adenylate cyclase activity: 160 + 12 pmol cyclic AMP/min/mg protein. The basal adenylate cyclase activities were not affected in either group of lesioned animals *P < 0.05; • P < 0.01 and **P < 0.001 (Student's t-test when compared with respective values obtained in sham-operated animals).

cant effect was observed in the caudal part of the striatum and in the superior colliculus (Table I). (2) Effects of." (1) bilateral 6-OHDA injections in the

VMT, (2) bilateral ablation of the prefrontal cortex, and (3) both types of lesions, on D 1 receptor sensitivity and DA levels in the nucleus accumbens Confirming previous results 25, despite the almost total destruction of afferent D A fibers ( D A levels being decreased by 90%) no significant change in D 1 receptor characteristics was detected 10 weeks after 6 - O H D A VMT lesions (Fig. 2). Although D A levels in the nucleus accumbens remained unchanged following bilateral ablation of the prefrontal cortex, a slight but significant increase (+14%, P < 0.01) in the maximal activity of the DA-sensitive adenylate cyclase was observed in this structure. Finally, when the two types of lesions were performed simultaneously, the maximal activity of the DA-sensitive adenylate cyclase was markedly enhanced in the nucleus accumbens (+52%, P < 0.001) (Fig. 2), this phenomenon being associated with a decrease of D A levels (--93%) similar of that observed in 6-OHDA-le-

sioned rats. Further experiments indicated that the hypersensitivity of D1 receptors induced by the combined destruction of ascending D A neurons and prefrontal cortex, did not only correspond to an increased number of D1 receptors, but also to a twofold decrease of the K~., value as revealed by an Eadie-Hofstee plot of the data (Fig. 3). DISCUSSION

Several investigations ]have been made to demonstrate changes in DA-sensitive adenylate cyclase activity following denervation of D A afferent fibersl2,16,17,28. Although most of these studies were performed on the nigro-striatal D A pathway, results differ from one laboratory to another. This variability could be related to the selectivity of the procedure used to destroy the D A fibers. Our own data obtained in the rat prefrontal cortex substantiate this statement since D 1 receptor hypersensitivity in this structure was shown to be dependent on the type of lesion madeZL In other words, concomitant destruction of neuronal fibers other than the D A fibers un-

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c A M P f o r m e d in presence of D A ( p m o l e s / m i n . x m g protein) / DA (pM) Fig. 3. Effects of simultaneous bilateral ablation of the prefrontal cortex and bilateral injection of 6 - O H D A in the V M T on the kinetic characteristics of the DA-sensitive adenylate cyclase of the nucleus accumbens. Experimental conditions were identical to those described in Fig. 2, except that D A concentrations varied from 3.16 × 10 -7 M to 10-4 M. Each point corresponds to the m e a n of 3 determinations. Variations of values in 5 different s h a m - o p e r a t e d animals were within 8%. (1) and (2) correspond to two different animals. A10 represents the medial mesencephalic group of D A cell bodies located in the V M T (Swedish nomenclature).

der investigation may prevent the expected change in DA-sensitive adenylate cyclase activity in the target area. In the nucleus accumbens, neither electrolytic nor chemical destruction of D A afferents could significantly modify D 1 receptor sensitivity, suggesting that DA denervation-induced supersensitivity of D1 receptors was prevented by the concomitant destruction of neuronal systems other than D A afferents. This is indeed the case since all ascending D A neurons, including the mesocortico-prefrontal D A neurons, are destroyed following electrolytic or 6O H D A lesions of the VMT. Moreover, we have previously shown that D 1 receptor supersensitivity in the nucleus accumbens occurs only in rats with VMT 6O H D A lesions performed in the presence of desmethylimipramine, a condition which prevents the destruction of ascending NA fibers passing near the VMT, but which also partially protects the mesocortico-prefrontal D A neurons 25. The latter results suggest that the mesocortico-prefrontal D A neurons could play a role in the regulation of D 1 receptors in the nucleus accumbens fibers, since these D A neurons have been shown to exert an inhibitory influence on cortical cellsS. According to this hypothesis, acti-

vation of these cortical cells prior to 6 - O H D A or electrolytic lesions of the VMT destroying all ascending D A fibers (including the mesocortico-prefrontal D A neurons), could prevent the development of the expected D 1 receptor supersensitivity in the nucleus accumbens (Fig. 4). These assumptions led us to examine the effect of bilateral ablation of the prefrontal cortex on the nucleus accumbens DA-sensitive adenylate cyclase activity, both in normal rats and in animals with 6 - O H D A lesions in the VMT. The bilateral ablations of the prefrontal cortex induced an important reduction of [3H]glutamic acid high-affinity uptake activity in several subcortical structures including the nucleus accumbens. Since [3H]glutamic acid high-affinity uptake provides an index of glutamatergic innervation, our results are in agreement with previous anatomical and biochemical observations indicating that numerous subcortical structures are innervated by glutamatergic neurons originating from the prefrontal cortex 2,6,13,29. Moreover, the decrease in [3H]glutamic acid uptake observed in the rostral but not in the caudal part of the striatum revealed the selectivity of these ablations, since it is known that the prefrontal cortex innervates only the anterior medial part of the striatum 2. The

49

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Fig. 4 Effects of lesions of the VMT and the prefrontal cortex on the cortico-nucleus accumbens fibers and the DA-sensitive adenylate cyclase activity in the nucleus accumbens. (1) 6-OHDA lesions of the VMT destroy both cortical and nucleus accumbens DA afferents. The cortico-nucleus accumbens pathway is disinhibited. There is no change in the DA-sensitive adenylate cyclase activity. (2) 6-OHDA VMT + prefrontal cortex ablation lesions: the DA afferents have disappeared but the cortico-nucleus accumbens pathway is also destroyed. There is a +52% increase in the DA-sensitive adenylate cyclase activity. slight but significant increase in D A - s e n s i t i v e adenylate cyclase maximal activity observed in the nucleus accumbens 10 weeks after the bilateral ablations of the prefrontal cortex in rats without 6 - O H D A V M T lesions provided the first indication for a role of cortico-nucleus accumbens fibers in the regulation of D 1 receptor sensitivity. This was further d e m o n s t r a t e d in rats with bilateral 6 - O H D A V M T lesions. As previously shown 25, 6 - O H D A V M T lesions were without effect on D1 receptor sensitivity. However, a m a r k e d increase in the D A - s e n s i t i v e adenylate cyclase activity was seen in 6 - O H D A - l e s i o n e d rats in which bilateral ablations of the prefrontal cortex had been simultaneously p e r f o r m e d . Complementary experiments indicated that the enhanced DA-sensitive adenylate cyclase activity found in the nucleus accumbens was due both to an increased

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n u m b e r and to an increased affinity of the D 1 receptors. Therefore, the expression of D 1 r e c e p t o r supersensitivity following D A denervation in the nucleus accumbens seems to be d e p e n d e n t on the activity of cortico-nucleus accumbens fibers. M o r e explicitly, our data reveal that the suppression of this prefrontal cortical control induced either by destruction of the corresponding neurons (this study, Fig. 4) or by inhibition of their activity (tonic inhibitory influence of the mesocortico-prefronml D A neurons 25) is a prerequisite for the occurrence of D A denervation-induced supersensitivity of D1 receptors in the nucleus accumbens. H o w e v e r , since the descending prefronto-cortical neurons innervate several subcortical structures it cannot be completely excluded that neurons other than the cortico-nucleus accumbens fibers are contributing to the effect observed. Electrophysiological studies have allowed to demonstrate that m o n o a m i n e r g i c neurons can m o d u l a t e the sensitivity of heterologous receptors on a target cell. F o r example, D A released from dendrites in the SN can reduce the inhibitory effect of G A B A on the activity of nigro-thalamic G A B A e r g i c neurons 30 and N A released from nerve terminals in the cerebellum facilitates the inhibitory effect of G A B A on the activity of cerebellar Purkinje cells 31. Conversely, in the present biochemical study we have p r o v i d e d evidence for the control of m o n o a m i n e r g i c r e c e p t o r sensitivity in the nucleus accumbens by heterologous, probably glutamatergic, fibers originating in the prefrontal cortex. ACKNOWLEDGEMENTS A u t h o r s wish to thank M o n i q u e Saffroy and Marcel D e s b a n for the histological procedures. This research was s u p p o r t e d by grants from R h 6 n e - P o u l e n c Sant6, D R E T (81.050) and I N S E R M .

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