Regional differences in the regulation of acetylcholine release upon D2 dopamine and N-methyl-d-aspartate receptor activation in rat nucleus accumbens and neostriatum

Brain Research, 566 (1991) 1-7 © 1991 Elsevier Science Publishers iLV. All rights tese,-ved. 0006-8993/91/$03.50 BRES 17049

Research Reports

Regional differences in the regulation of acetylcholine release upon D2 dopamine and N-methyl-i)-aspartate receptor activation in rat nucleus accumbens and neostriatum J . M . L . Henselmans 1 and J.C. Stoof 2 Departments of SAnatomy and 2Neurology, Medical Faculty, Free University, Amsterdam (The Netherlands) (Accepted 21 May 1991)

Key words: Acetylcholine release; Da dopamine receptor; N-Methyl-D-aspartate receptor; Neostriatum; Nucleus accumbens

The effect of D 2 dopami~ae receptor activation on either the electrically, or N-methyl-D-aspartate induced release of radiolabeled acetylcholine (ACh) was investigated in different areas of the nucleus accumbens and the neostriatum of rats, by using a superfusion technique. Sequential slices of 100/~m were chopped along either a rostrocaudal, mediolateral or dorsoventral axis. In every slice the effect of a supramaximal concentration of the selective D 2 receptor agonist quinpirole on the release of ACh was measured. In the entire neostriatum the release of ACh was reduced by approximately 70% in the presence of quinpirole. By contrast, in the nucleus accumbens, a gradual decrease in the inhibitory effect of quinpirole on the release of ACh was observed along both the rostral-to-caudal and the lateral-to-medial axes. Whereas in the rostrolateral part a 50% inhibition could be observed, in the caudomedial part no significant inhibition could be detected. Also the N-methyl-D-asp~rtate induced release of ACh was smaller in the caudomedial part as compared to the rostrolateral part of the nucleus accumbens. It is ~oneluded that the nucleus accumbens is a very heterogenous structure with respect to the regulation of the release of ACh by D2 dopamine and N-methyl-o-amartate receptor activation. INTRODUCTION

The mammalian striatum receives afferents from cortical, thalamic, limbic and mesencephalic structures, whereas efferents are directed mainly to the pallidal complex and the mesencephalon. The striatal output neurons, which are medium sized spiny neurons, account for the large majority of striatal neurons. Strategically positioned between these neurons large aspiny neurons can be observed which represent, at least in part, the striatal interneurons. Although they comprise only 1-2% of the striatal neurons, the striatal cholinergic interneurons apparently play an important role in the transfer of information from the inputs of the striatum, to the output neurons e'3°'36. The importance of this cholinergic system is strongly illustrated in Parkinson's disease, in which the nigrostriatal dopaminergic projection is severely damaged. Drug therapy in this disorder can be achieved by using anti-cholinergics as well as dopaminomimetics. This suggests a delicate balance between the cholinergic and dopaminergic systems in the striatum. The existence of such a balance is supported by the findings that dopamine (DA) is able, both in vitro and in vivo, to inhibit the release of acetylcholine (ACh) in the

striatum through activation of a D 2 dopamine receptor subtype n't4'45. In contrast with the inhibitory effect of DA, released from nerve terminals of cells originating in the mesencephalon, glutamate, which serves as a neurotn~nsmitter in the striatal afferents derived from telencephalic structures 9'ts'ts, enhances the ACh release in the striatum. This effect is mediated by the N-methyl-Daspartate (NMDA) receptor subtype 31'41. Thus, the activity of the cholinergic interneurons it', the striatum is regulated by an excitatory glutamatergic and Bn inhibitory dopaminergic input. In a previous study from our laboratory the effect of D2 DA receptor activation on the release of ACh was investigated in the striatum of the lizard Gekko gecko and the rat 4e. It appeared that in the striatum of Gekko the selective D2 DA receptor agonist quinpirole (LY 171555) was unable to inhibit the release of ACh to any significant extent. Interestingly, activation of D2 receptors in neostriatal tissue of the rat induced an almost complete inhibition of the release of ACh, whereas under similar conditions in the nucleus accumbens ACh release was inhibited for only 50% (see also ref. 50). In these studies, the neostriatum or nucleus accumbens were dissected as a whole and randomly chopped in small ds.

Correspondence: J.C. Stool, Department of Neurology, Medical Faculty, Free University, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Fax: (31) (20) 6462228.

sue slices. Thi': could imply that the 50% inhibition found in the nucleus accumbens reflects the average percentage of inhibition, leaving open the possibility that regional differences occur. In the light of these findings, and recent imw_unohistochemicai 4'47"49, electrophysiological 5'st and behavioral 7'2s studies, indicating that 'the striatum' is in fact a very heterogeneous structure, it is hypothesized that the nucleus accumbens is also inhomogenous with respect to the D 2 receptor mediated inhibition of the release of A C h . Therefore, the present stud,~ describes a detailed investigation of the interaction between dopaminergic and cholinergic elements in different regions of the nucleus accumbens and the neostriatum of the rat. In addition, effects of N M D A on the release of A C h were investigated in these brain regions, since recent studies suggest that glutamate m a y also regulate (via N M D A receptors) D A mediated functions in the striatum 39'43. For instance, frontal decortication prevents depression of the activity of cholinergic neurons by quinpirole and other dopaminomimetics, suggesting that the effect of D A on the A C h release is modulated by neocortically derived glutamate ~°. MATERIALS AND METHODS Male Wistar rats (weighing 140-160 g) were sacrificed by decapitation. Their brains were quickly removed and transferred to a l~ebs.Ringer bicarbonate (KRB) medium (composition in raM: NaC! 121, KCI 1.9, KH2PO4 1.2, CaCI 2 1.2, NaHCO~ 25, MgSO,, 1.2 and glucose 10) and equilibrated with 95% O2 and 5q, CO2. Experiments in which the influence of NMDA on the ACh release was investigated were performed in a magnesium-free KRB,

Dissection and incubation procedure for the study of ACh release from sequential slices Brains were cut into two hemispheres and from one hemisphere either the neostriatum - or the nucleus accumbens - was carefully dissected as a whole. Subsequently, the tissue was transferred to a Mcliwain tissue chopper and sequer, t.lai ~li~-es of 100/~m were obtained in either a rostrocaudal, med;olateral, dorsoventral or in a dorsolateral to ventromedial direction. Under a dissection microscope, remaining parts from adherent smletures were carefully removed from each slice in order to avoid their interference with the signal in the superfusion experiment. The slices were put one by one in each of the wells of a Nunclon microwell plate containing 100 ~! KRB-medium per well. Subsequently, the microwell plate was placed in a Dubnoff metabolic shaker and the tissue was preincubated during 15 rain at 37 °C. Following the preincubation, the KRB-medium was replaced by 100 ~1 KRB-medium containing 0.5 ~Ci/ml [3H]choline and the incubation was continued during a period of 15 min, after which each of the slices was put in one of the chambers of a superfusion apparatus.

Dissection and incubation procedure for the study of ACh release from pooled minislices In order to obtain minislices, either the neostriata or the nuclei accumbens of both hemispheres of four rats were minced by passing them twice, in two perpendicular directions, through the Mcllwain tissue chopper (micrometer setting 300 ~m), The minislices were pooled and transferred to a Dubnoff metabolic shaker for a preincubation period of 15 min in KRB-medium at 37 °C, followed

by an incubation period of 15 min in the presence of 0.5/~Ci/ml [t4C]choline and 1.25/~Ci/ml [3H]dopamine. At the end of the incubation period a 10 ~tl aliquot of the pooled minislices suspension was put in each of the chambers of a 24-chamber superfusion apr~aratus.

Superfusion procedure Superfusion with KRB medium was set at 0.25 ml/min and after 45 rain of presuperfusion, 7 successive 15-min fractions were collected. During the first 10 min of the second (first stimulation -S1) and fifth (second stimulation = $2) fraction either 1 mM NMDA was present in the s~ ~rfusion medium or biphasic electrical block pulses (2 ms, 1 Hz, 24 mA) were applied. If used, quinpirole was added 5 min before the fourth fraction was collected. At the end of the experiment the tissue was superfused during a period of 7 rain with 0.1 N HCI in order to collect the remaining radioactivity. The amount of radioactivity present in each of the superfusion fractions and in the HCI extract was estimated by liquid scintillation spectroscopy. In order to correct for differences in the amount of tissue present in each of the superfusion chambers, data for each chamber were expressed as fractional rates. The fractional rate is defined as the amount of radioactivity present in a particular collection period divided by the sum of the total amount of radioactivity present in that particular collection period, subsequent collection periods and in the HCi extract. The increase in the fractio.~! rate induced by depolarization was defined as the difference bet~veen the fractional rates of the stimulated fraction and the average of the preceding and subsequent non-stitnulated fractions.

Radiochemicals and drugs [14C]Choline {spec. act. 55 mCi/mM), [3H]dopamine (spec. act. 48 Ci/mM) and 13H]choline (spec. act. 15 Ci/mM), were purchased from the Radiochemical Centre (Amersham). Quinpirole was a generous gift from Eli-Lilly and NMDA was purchased from Sigma.

RESULTs;

A Ch release from sequential slices Neostvlatum, In Fig. I the effect of the selective D2 receptor agonist quinpirole (in a concentration of 1 ~tM) on the electrically-evoked release of radiolabeled ACh in the neostriatum is demonstrated per sequentially dissected 100/~m slice. The effects are presented from tissue dissected along rostrocaudal, mediolateral, dorsoventral and dorsolateral-to-ventromedial axes (Fig. 1AD). The geometry of the neostriatum enabled us to dissect 24 100-/~m slices along both rostrocaudal and dorsolateral-to-ventromediai axes. Only 13 slices could be dissected along the medial-to-lateral and dorsal-to-ventral axes. The observations reveal that the inhibitory effect of D2 receptor stimulation on the ACh release is approximately 70% in every 100/~m tissue slice, independent of the direction of dissection. Nucleus accumbens. The geometry of the nucleus accumbens enabled us to dissect 12 sequential slices of 100 /~m each along the rostrocaudal axis, whereas along the mediolateral axis not more than 10 slices of 100/~m could be obtained. In this brain region a rostrai-to-caudal decrease in the effect of 1 /~M quinpirole on the electrically-evoked release of ACh was found (Fig. 2A).

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Fig. 1. Electrically induced release of [3H]ACh from the sequentially dissected neostriatal (100/~m) tissue slices in the absence (triangles) and presence (circles) of 1/~M quinpirole. The release of [3H]ACh is expressed as the ra*.io between the fractional rates of the second (S2) and the first (Sl) electrical stimulation. Values (average from 3-8 different cxperiments) are given for the individual tissue slices dissected along the rostrocaudai (panel A, n = 4), mediolateral (panel B, n == 8), ventrodorsal (panel C, n = 5), or dorsolateral-to-ventromedial (panel D, n - 3) axis. S.E.M. values were too small to be depicted in the figure (<9%). Average SI ± S.E.M. values of the first of the sequentially dissected slices in panels A, B, C and D were 11.50 ± 1.81, 10.50 ± 1.49, 10.50 ± 0.69 and 9.68 ± 1.45, respectively. Data points are fitted by means of a linear regression line, and are depicted as dotted lines.

In the e x t r e m e rostral part of the nucleus accumbens the release was inhibited/or approximately 55%, This percentage gradually decreased towards no inhibition at all

in its most caudal part. Investigation of the medial-tolateral axis showed an increase in the inhibition percentage, the inhibition .being medially approximately 10~

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Fig. 2. Electrically induced release of [3H]ACh from sequentially dissected nucleus accumbens (100/~m) tissue slices in the absence (triangles) and presence (circles) of 1/~M quinpirole. The ,'•lease of [JH]ACh is expressed as the ratio between the fractional rates of the second ($2) and the first (S1) electrical stimulation. Values (average from 3-7 different experiments) are given for the individual tissue slices dissected along the rostrocaudal (panel A, n = 7) - and mediolateral (panel B, n = 3) axis. S,E.M. values were too small to be depicted in the figure (<8%). Average S1 +- S.E.M. values of the first of the seque,tially dissected slices in panels A and B were 5.62 - 0.72 and 4.94 +0.87, respectively. Data points are fitted by means of a linear regression line, and are depicted as dotted lines. For further experimental details see Materials and Methods.

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Fig. 3. A: the ~,ffect of NMDA (in a concentration of 1 mM) on the release of [t4C]ACh from pooled tissue slices dissected from the neostriatum (NS), h e rostrolateral part (ARL) or the caudomedial part (ACM) of the nucleus accumbens. The release of [t4C]ACh is presented as the fraction~! rate of the first stimulation (S1). Values represent means --. S.E.M. of 3 different experiments in which each experimental condition was ,,,died in 4 superfusion chambers (n = 12). B: the NMDA (in a concentration of 1 mM) induced release of [t4C]ACh from pooled tissue slices dissected from the neostriatum (NS), the rostrolateral part (ARL) or the caudomedial part (ACM) of the nucleus accumbens in the absence (open bars) and presence (hatched bars) of 1/~M quinpirole. The release of [14C]ACh is expressed as the ratio between the fractional rates of the second ($2) and the first (SI) NMDA induced stimulation. Values represent means - S.E.M. of 3 different experiments in which each experimental condition was studied in 4 superfusion chambers. *P < 0.001 versus control, two-tailed Student's t-test (n ffi 12).

and laterally approximately 50% (Fig. 2B).

ACh release from pooled minislices In the next set of experiments tissue slices from the rostrolateral or caudomedial parts of the nuclei accumbens of 4 different animals were pooled. For comparison also tissue slices from the neostriatum were included, The release of ACh was evoked by applying 1 mM NMDA. During the first stimulation with NMDA (S1), a fractionei rate of 10,0% was observed in neostriatal tissue, whereas in the rostrolateral and caudomedial parts of the accumbens the observed values were 8.2 and 5.0, respectively (Fig. 3A). The NMDA-evoked release of ACh from neostriatal tissue was inhibited for approximately 70% by 1 #M quinpirole. In the rostrolateral part of the nucleus accumbens this percentage amounted to 40% and no inhibition could be observed in the caudomedial part (Fig. 3B). DISCUSSION

It is generally accepted that the depolarization.evoked release of radioactivity from striatal tissue, previously incubated in the presence of radioactive choline, reflects the release of A C h t2. Moreover, it has been extensively documented that inhibition of the release of ACh from striatal tissue by quinpirole is mediated by a D 2 dopam i n e receptor t2.t4.45. The neostriatum has been reported to be a rather heterogeneous structure for dopaminergic and for cholin-

ergic markers. For instance, rostrocaudal and mediolateral gradients were found in the density of the dopaminergic innervation, the D2 receptor binding and the dopaminergic uptake system t'4'13.lT'2s''m. In studies using choline acetyitransferase immunocytochemistry as a marker for cholinergic cells a rather homogeneous distribution was found for the cell bodies, the nerve fibers and the neuropillg'~s'42; however, a lateromedial gradient has been reported for the muscarinic receptor binding37 and the cholinergic uptake system 47. Nevertheless, we observed a striking similarity in the effect of D2 receptor activation on the ACh release over the different neostriatal axes. By contrast, in the nucleus accumbens a gradual decrease in the effect of D2 receptor activation on the ACh release was found along the rostrocaudal and the lateromedial axes. As can be predicted from these results, the most profound differences were observed between the rostrolaterai and caudomedial parts of the accumbens. These findings might be explained by the fact that in the nucleus accumbens of the rat clear differences in cholinergic and dopaminergic nerve fiber density exist between a central 'core region' and a more peripheral 'shell' region2t'36'49, Characteristic features of the shell region, which is most prominent in the caudal and medial part of the nucleus accumbens, are the high density of cholinergic cells and cholinergic as well as dopaminergic fibers36'49. The core region shows a lower density of cholinergic and dopaminergic fibers. The regional distribution of the D 2 receptors in the nucleus accumbens has so far not been described. The only clear

observation is that the average number of these receptors in the nucleus accumbens seems to be lower than in the n e ~ t a a t u m 2'3'34. Since the shell region expands along the ro:,tral-to-caudal as well as the lateral-to-medial axis, it may be that the differences we observed in the extent to which the release of ACh was inhibited by quinpirole in the different regions of the accumbens, reflect shell versus core differences. By inference, especially with respect to the De mediated inhibition of the ACh release, the core region seems to be more related to the neostriatum than the shell region. This hypothesis seems compatible with the observed effects of NMDA on the release of ACh, since in the rostrolateral part of the nucleus accumbens the effect of NMDA receptor activation on the ACh release was similar to that found in the neostriatum, but significantly larger than in the caudomedial part. Apparently, the shell resion may be looked upon as having a markedly different organization of glutamatergic and dopaminergic inputs in relation to the cholinergic system. In this context it must be noted that shell and core region of the nucleus accumbens differ markedly in their neurochemical composition and their input-output relations e~'22''9. An interesting difference between core and shell is that the former receives its main cortical inputs from the neocortex, whereas the cortical inputs of the latter are primarily derived from allocortical regions~ i.e. the ventral subiculum 2°, of the hippocampal formati<,.~13s, However, both these cortical afferent systems are assumed to be glutamatergic 9'~5'~6'4°, The absence of a significant effect of D-2 receptor activation1 on the ACh release in the caudomedial part of the nucleus accumbens shows much similarity with the observations in the striatum of the lizard Gekko gecko. In the striatum of this species D2 receptor activation does not inhibit of the release of ACh 4~ in spite of the presence of a D A input to the striatum 44. In this reptile

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Acknowledgements. The authors wish to thank Drs. H.J. Oroenewegen and P.V. Hoogland for critically reading the manuscript and Mr. D. de Jong for preparing the photomicrographs.

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