Proc. Natl. Acad. Sci. USAVol. 89, pp. 11214-11218, December 1992Neurobiology

Distinct presynaptic control of dopamine release in striosomal- andmatrix-enriched areas of the rat striatum by selective agonists ofNK1, NK2, and NK3 tachykinin receptors

(sftosme/autora phc lan)

LEON TREMBLAY, MARIE-LOU KEMEL, MARCEL DESBAN, CHRISTIAN GAUCHY, AND JACQUES GLOWINSKILaboratoire de Neuropharmacologie, Institut National de la Sante et de la Recherche Medicale Unit6 114, College de France, 11, Place Marcelin Berthelot,75231 Paris Cedex 05, France

Communicated by Solomon Snyder, June 18, 1992

ABSTRACT Using a sensitive in vitro microperfuslonmethod, the effects of selective and potent agils of NK1,NK2, and NK3 tachykinin receptors {[ProSP, [Lys5,MeLeue,NklJNKA-(4-10), and [Pro7]NKB, respectively} on the pre-synaptic control of doine relase were investgted instriosomal-enriched (area rich in [3Hnlxone bi mg sites)and matrix-enriched areas of the rat riatum. Marked differ-ences could be demonstrated as follows: (a) when used at 0.1F&M, the NK1 agonist simulated the release of [Hjdopaminecontinuously synthesized from [3H1tyrosine in both compart-ments, while the NK2 and NK3 agon e d the releaseof (3Hldopamlne only In the matrix; (it) the stimuat effectof the NK3 agonist was less pronounced than those of the NK1and NK2 agonists; (ii) the NK1 agonist-evoked _weretetrodotoxin (1 FM) sensitive, while those of the NK2 and NK3agonits were, respectively, partially and totally tetrodotoxin

int; (iv) specific receptors are involved in these responsessince the stiulatory effects ofthe NK1 and NK2 agoni were,respectively, blocked by potent antagonists ofNK1 (RP-67580;1 FM) and NK2 (SR-48968; 1 FM) receptors, while theseantagonists did not affect the NK3 agonist-evoked response; (v)the i t simulator effect of the NK1 agonist was partiallyreduced under local blockade ofcholinergic to in thematrix but not in the striosomal-enriched area. Interestingly,this study aiso revealed mismatches between autoradlegraphicdata and receptor-mediated responses, since NK2 binding sitescould not be observed in the staum while NK3 but not NK1binding sites were visualized in the striosomal-enriched area.

In several species, including humans, the striatum is dividedinto two main anatomical compartments, the striosomes (orpatches) and the matrix, which can be distinguished byseveral biochemical markers such as acetylcholinesterase (1,2) and ,u opiate binding sites (3) found in large amounts in thematrix and striosomes, respectively. These compartmentsalso differ in some of their cortical, thalamic, or mesenceph-alic afferents and by the target structures oftheir efferents (4,5). According to studies performed in the cat and rat, distinctpopulations of mesencephalic dopaminergic neurons inner-vate the striosomes and the matrix (6, 7). This has led us toinvestigate the presynaptic regulation of dopamine (DA)release in striosomal- and matrix-enriched areas by an invitro superfusion procedure (8). Marked differences could bedemonstrated between these compartments in the pattern oramplitude of the acetylcholine- and N-methyl-D-aspartate-evoked release of DA (8, 9).The present study was undertaken to determine whether

additional functional differences between striosomes andmatrix could be shown by examining the effects of specific

agonists ofNK1, NK2, and NK3 tachykinin receptors on thepresynaptic regulation of DA release in striosomal- andmatrix-enriched areas of the rat striatum. This choice wasmade for several reasons: (i) substance P (SP), neurokinin A(NKA), and neurokinin B (NKB), the endogenous ligands ofNK1, NK2, and NK3 receptors are present in the striatum(10, 11), and NK1, NK3, but not NK2 binding sites have beenshown in this structure (12). (ii) SP-containing neurons havebeen visualized in both compartments, but fibers enriched inSP immunoreactivity are particularly dense in striosomes(13). (iii) According to in situ hybridization studies, a slightlylarger proportion of tachykinin-containing neurons is presentin striosomes than in the matrix (14) and the rate oftachykininsynthesis is higher in striosomal neurons (15). (iv) mRNA forNKB precursors are expressed in striatopallidal neurons (16),which are all located in the matrix (17). (v) SP stimulates therelease of DA through a tetrodotoxin (TTX)-sensitive pro-cess from whole striatal slices of rat (18).

MATERIALS AND METHODSAutoradographic Experiments. Male Sprague-Dawley rats

(250-300 g; Charles River Breeding Laboratories) were killedby decapitation and their brains were rapidly removed andfrozen in isopentane at -400C. Series of saggital sections (20jum thick) taken at lateral (L = 4.7-4.0; matrix enriched) ormedial (L = 3.0-2.0; striosome enriched) levels of the brainwere cut with a cryostat at -200C and then mounted ontogelatin-coated glass slides. Alternate sections were used foreither [3H]naloxone or one of the 125I-labeled tachykininligands. Binding experiments with [3H~naloxone (56.1 Ci/mmol; 1 Ci = 37 GBq; Amersham) were performed asdescribed (19). Tachykinin binding experiments were per-formed as described by Saffroy et al. (12). 125I-labeled Bol-ton-Hunter SP (1251-BHSP; 200,000 cpm/mI = 65 pM; NEN),125I-NKA (275,000 cpm/ml = 100 pM; NEN), and 125I1Bolton-Hunter eledoisin (1251-BHE; 200,000 cpm/ml = 65pM; NEN) were used to label NK1, NK2, and NK3 recep-tors, respectively, with nonspecific binding being determinedby adding corresponding selective agonists at a concentrationof 1 A&M, [Pro9ISP, [Lys5,MeLeu9,Nle'0]NKA-(4-10), and[ProI]NKB for NK1, NK2, and NK3 receptors, respectively.Autoradiographic films were developed for 40 sec, 2 min, or3 min for experiments made with either 125I-BHSP, 1251-BHE,or 125I-NKA, respectively.[3HJDA Releae. As described (8,9), male Sprague-Dawley

rats (250-300 g; Charles River Breeding Laboratories) werekilled and their brains were rapidly removed and cooled.Thick saggital slices (1.0-1.3 mm) were cut with a Vibratome

Abbreviations: SP, substance P; BHE, Bolton-Hunter eledoisin;BHSP, Bolton-Hunter SP; DA, dopamine; NKA, neurokinin A;NKB, neurokinin B; TTX, tetrodotoxin.

11214

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) 11215

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FIG. 1. Comparison of localization of [3H]naloxone, NK1, and NK3 tachykinin binding sites in the rat striatum. The two saggital planes (L= 2.5 and L = 4.5 for medial and lateral sections, respectively) were those used in release experiments. (Upper and Lower) Areas enrichedin striosomes (s) and matrix (mx), respectively. Adjacent brain sections were used for comparison of localization of [3H]naloxone, 125I-BHSP,and 125I-BHE binding. (Left) Photomicrographs of autoradiograms obtained from [3H]naloxone binding revealing the striosomes (dark areas),125I-BHSP binding for the NK1 receptors, and 125I-BHE binding for the NK3 receptors. (Right) Drawing of the superposition of areas enrichedin [3H]naloxone binding (delimited by heavy lines) and in tachykinin binding (hatched areas). ac, Superposition ofareas enriched in [3H]naloxone(a) and in 125I-BHSP (c) binding; bd, superposition of areas enriched in [3H]naloxone (b) and in 125I-BHE (d) binding; ef and eg, superpositionof areas enriched in [3H]naloxone binding (e) and of regions enriched in 125I-BHSP (f) and 17-I-BHE (g) binding respectively. -*, Caudorostralaxis; R, rostral. (Bar = 0.5 mm.)

and placed in a superfusion chamber in continuously renewedartificial cerebrospinal fluid (34TC; saturated with O2/CO2;95:5, vol/vol). Onto each of the selected striosomal- ormatrix-enriched areas, a microsuperfusion device was placedvertically, and cerebrospinal fluid containing L-[3,5-3H]tyro-sine (60 MCi/ml) and bovine serum albumin (0.02%) wascontinuously delivered (30 Al/min) through each device (seeFigs. 1 and 2). After a prelabeling period (40 min), the releaseof newly synthesized [3H]DA was estimated in successive5-min superfusate fractions, [3H]DA being separated from[3H]tyrosine and 3H metabolites by ion-exchange chroma-tography and alumina adsorption.

Agonists [Pro9]SP, [Lys5,MeLeu9,Nle1°0NKA-(4-10), and[Pro7]NKB were synthesized and purified by S. Lavielle andG. Chassaing (University Paris 6). The NK1 (RP-67580) andNK2 (SR-48968) antagonists were generous gifts from Rhone-Poulenc Rorer and Sanofi Recherche, respectively.

RESULTSDistribution of Tachykinin Binding Sites in Striatal Com-

partments. As described (3, 9, 14, 20), striosomes or patcheswere distinguished from the matrix by [3H]naloxone bindingon rat striatal sections and autoradiography, striosomes and

matrix being, respectively, rich and poor in [3H]naloxonebinding sites (Fig. 1). For comparison with biochemical data,attempts were made to define the distribution of tachykininbinding sites in striatal compartments using 1251-BHSP, 125I-NKA, and 1251-BHE as ligands for NK1, NK2, and NK3receptors, respectively, and saggital sections taken at later-alities (L, 2.5 and L, 4.5) identical to those used in the releaseexperiments. Confirming previous results, NK2 binding siteswere not observed in the striatum. As revealed by compar-ison with [3H]naloxone binding, NK1 binding sites weredistributed in all parts of the striatum but predominantly inthe matrix. In fact, the thin border zone enriched in[3H]naloxone binding in the lateral section was completelydevoid of 125I-BHSP binding sites and little or no 125I-BHSPbinding was detected in more medially located striosomes(Fig. 1). In contrast to NK1 binding sites, NK3 binding siteswere found in both matrix and striosomal compartments butonly in some parts of the striatum (Fig. 1). High densities ofNK1 and NK3 binding sites were particularly observed in themost rostromedial and lateral parts of the striatum in zonesselected to superfuse striosomal- and matrix-enriched areas,respectively.

Effects of Selective Tachykinin Agonists on SpontaneousRelease of Newly Synthesized [3HJDA in Striosomal- and

Table 1. Concentration response relationships for selective NK1, NK2, and NK3 tachykinin agonists on release of[3H]DA in striosomal- and matrix-enriched areas

Striosomal-enriched area Matrix-enriched areaAgonist 0.01 PM 0.1 AM 1 JAM 0.01 AM 0.1 AM 1 UM

[Pro91SP 122.1* + 5.5 134.9* ± 3.6 133.7* ± 6.3 114.9* + 4.1 143.7* ± 7.5 140.4* ± 7.0[Lys5,MeLeu9,Nle'0]-NKA-(4-10) 97.8 + 3.9 %.2 + 3.1 108.2 + 5.9 102.7 ± 5.1 135.7* + 5.1 129.2* ± 8.6

[Pro7INKB 100.3 + 4.4 100.1 + 1.5 103.9 ± 5.3 95.5 ± 3.7 114.3* ± 3.0 115.6* ± 3.8Agonists were applied during the last 25 min of the superfusion experiments. The release of [3H]DA was estimated and

is expressed as indicated in Fig. 2; then calculations were made to determine the mean release of [3HJDA during the last25 min ofthe superfusion experiments performed without (control) or with increasing concentrations ofeach agonist. Resultsare means + SEM of data obtained in 8-12 experiments.*P < 0.01 when compared with control values.

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Neurobiology: Tremblay et aL

11216 Neurobiology: Tremblay et al.

Matrix- e Are. At 0.1 or 1 ,uM, the selective NK1agonist [Pro9jSP markedly stimulated the release of pH]DAin both the striosomal- and matrix-enriched areas (Table 1).These effects appeared immediately, lasted throughout thepeptide application, and were of similar amplitude, withmaximal responses being seen at the end of the treatment(Fig. 2). Although less pronounced, a stimulatory effect oftheNK1 agonist was already observed at 0.01 ,uM in bothsuperfused areas (Table 1).

In contrast to what was observed with [Pro9]SP, whateverthe concentration used (0.01-1 ILM), the selective NK2{[Lys5,MeLeu9,Nle10JNKA-(4-10)} and NK3 (UPro7JNKB)agonists, were without effect on PH]DA release in thestriosomal-enriched area (Fig. 2, Table 1). However, stimu-latory responses occurred with both agonists in the matrix-enriched area (Table 1). The [Lys5,MeLeu?,Nle10]NKA44-10)-evoked release of [3HIDA increased with time and

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reached a maximal amplitude similar to that observed with[Pro91SP, while the [Pro7NKB-evoked response was lesspronounced (Fig. 2, Table 1).

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of (Iro'SP wd [L.WqMeLe9',euJ A(4) on te Re-eans of pHDA in the Mati-Enriched Are Selective and

potent nonpeptide NK1 and NK2 antagonists (RP-67580 andSR-48568, respectively) recently available (21, 22) were usedto determine the specificity of the [ProPJSP- and [Lys5,MeLeu9,Nle1O1NKA-(4-10)-evoked release of PH]DA in thematrix-enriched area (Fig. 3). When used at 1 AM, bothRP-67580 and SR-48968 were without effect on the sponta-neous release of [3H1DA. The selective NK1 antagonistprevented the [Pro9]SP (0.1 AM)-evoked release of [3HJDAwhile the stimulatory effect of [Lys5,MeLeu',Nle10]NKA-(4-10) (0.1 u&M) was not affected. The NK2 antagonistabolished the response induced by the NK2 agonist but onlysightly reduced the stimulatory effect of the NK1 agonist.Neither RP-67580 nor SR-48968 antagonized the [Pro7INKB-evoked release of [3H]DA, indicating that this NK3 agonistacts through specific receptors (Fig. 3).

Differential Inluence of TTX on the StI ltoy Effects ofNK1, NK2, NK3 Tachyk A on the Rele of

(3HDA. TTX, which blocks voltage-dependent sodium chan-nels, is generally used to distinguish direct (TTX resistant)from indirect (TTX sensitive) presynaptic regulation of DArelease mediated by neurotransmitters. In agreement withour observations obtained on whole striatal slices (18), TTX(1 AtM) completely abolished the [Pro'JSP-evoked release of[3H]DA in both the striosomal- and matrix-enriched areas,

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FIG. 2. Effects of selective NK1, NK2, and NK3 tachykininagonists on the release of [3HIDA in striosomal- and matrix-enrichedareas of rat striatum. As reported (9) and as described in Materialsand Methods, medial and lateral saggital slices were superfused in aspecially designed chamber. Microsuperfusion devices were appliedvertically with light pressure onto the selected striosomal-enriched(L = 2.5, center of rectangle a) and matrix-ennrched (L = 4.5, centerof rectangle e) areas, respectively, rich and poor in [3H~naloxonebinding sites illustrated in Fig. 1. Volume of area superfused corre-sponds only to 0.6 mm3 (8). Selective agonists of NK1, NK2, andNK3 receptors, [Pro9]SP, [Lys5,MeLeu9,Nle10jNKA-(4-10), and[Pro7INKB, respectively, were applied at a concentration of0.1 F&Mfor the last 25 min of the experiment, 65 min after the onset oft3H]tyrosine superfusion. In each experiment, [3HIDA recovered insuccessive 5-min fractions was expressed as a percentage of themean spontaneous release of PHIDA, determined from estimationsmade in the five fractions that preceded the onset of tachykininapplication. Results are expressed as means + SEM ofdata obtainedfrom 8-16 experiments. o, Control; *, tachykinin agonist experi-ments. *, P < 0.01 when compared with respective control values(eight experiments).

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FIG. 3. Effects of selective NK1 and NK2 tachykinin antagonistson evoked release of P3HIDA induced by NK1, NK2, and NK3agonists in matrix-enriched areas. Tachykinin agonists (0.1 atM) wereapplied during the last 25 min ofsuporfusion and, when included, theNK1 (RP-67580) and NK2 (SR48968) nonpeptide tachykinin antag-onists were added at 1 F&M into the superfusion medium from theonset of pH]tyrosine supefusion. Antagonists di not affect spon-taneous release of [3HJDA. In each experiment, the release of

[3H]DA was estimated and is expressed as indiated in Fig. 2. Thencalculations were made to determine the mean release of [3HIDAduring the lest 25 min ofthe superfusion experiments performed withor without the agonists in the absence or presence ofeach antagonist.Results are means SEM of data obtained in 8-12 experimentsexpressed as a percentage of corresponding control values. *, P <0.01 when compared with data obtained with the agonists but withoutthe antagonists (open bars); A, P < 0.01 when compared with dataobtained in the presence of the antagonists alone.

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Proc. Nad. Acad. Sci. USA 89 (1992)

Proc. NatL. Acad. Sci. USA 89 (1992) 11217

indicating that local circuits are involved in the effects of theNK1 agonist (Fig. 4). In contrast, TTX only partially reducedthe stimulatory effect of [Lys5,MeLeu9,Nle'0]NKA-(4-10)on pH]DA release in the matrix-enriched area (Fig. 4), theTTX-resistant evoked response of the NK2 agonist beingobserved only 15 min after the onset of the agonist applica-tion. The [Pro7]NKB-evoked release of [3H]DA occurring inthe matrix-enriched area was not affected by TTX, suggest-ing that the effect of the NK3 agonist is mediated by NK3receptors located on dopaminergic nerve terminals. Finally,in the presence of TTX, neither NK2 nor NK3 tachykininagonist modified the release of PH]DA in the striosomal-enriched area.Partl Blckade ofthe [Pr'SP-Evoked Relese of [3HJDA in

the Matrix-E dArea Under Interrupton of CTransmission. Recently, we have reported that [Pro9JSP butnot the NK2 orNK3 selective agonists stimulate the release ofacetylcholine from striatal slices (23). Therefore, complemen-tary experiments were performed to determine whether theTTX-sensitive indirect stimulatory effect of [Pro9]SP on[3H]DA release observed in both striatal compartments couldbe related to the activation of cholinergic interneurons. Theblockade of cholinergic transmission by the combined appli-cation of atropine (1 ,uM) and pempidine (10 FM), antagonistsof muscarinic and nicotinic receptors, respectively, was with-out effect in the striosomal-enriched area {[Pro9JSP (0.1 puM)= 134.9% of control spontaneous release ± 3.6 SEM; n = 8;[Pro9ISP + atropine and pempidine = 131.8% ± 4.2%; n = 9;P > 0.05, not significant} but reduced by more than half the[ProP]SP-evoked release of [3H]DA in the matrix-enrichedarea {[ProP]SP (0.1 ,uM) = 143.7% of control spontaneousrelease ± 7.5%; n = 12; [ProljSP + atropine and pempidine =120.8% ± 4.4%; n = 10; P < 0.01}.

DISCUSSIONSeveral neurotransmitters contained in afferent fibers orneurons of the striatum including amino acids, amines, and

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FiG. 4. Differential sensitivity to TTX of the responses inducedby NK1, NK2, orNK3 tachykinin agonists. When added, 1 LMTTXwas included in the superfusion medium from the onset of pH]ty-rosine superfusion. Results, which are expressed as described in Fig.3, are means ± SEM of data obtained in 8-12 experiments. *, P <0.01 when compared with data obtained with the correspondingagonist but without TTX (open bars); A, P < 0.01 when comparedwith data obtained with corresponding control values (TTX in theabsence of the agonist).

neuropeptides have already been shown to be involved indirect (TTX resistant) and/or indirect (TTX sensitive) reg-ulation ofDA release from nerve terminals of the nigrostriataldopaminergic neurons. However, in spite of the presence oftachykinins in collaterals of the populations of striatal effer-ent neurons, which innervate the substantia nigra and/or theinternal or external pallidum, very little is known about therole of these peptides in these processes. Using new selectiveagonists of NK1, NK2, and NK3 receptors and recentlydiscovered potent and selective NK1 and NK2 antagonists,we show in the present study that presynaptic regulation ofDA release in the rat striatum involves not only NK1 but alsoNK2 and NK3 receptors. Moreover, depending on the typeof tachykinin receptors involved, this regulation is eitherTTX resistant or TTX sensitive and, interestingly, is some-times restricted to a single striatal compartment.LaW Circuits Contribute to the SP-Evoked Fneltto of

DA Release In Both Striosomal- and Mtrix-E ed Areas.The stimulation of NK1 receptors with [ProISP, a selectiveand potent NK1 agonist increased the release of newlysynthesized [3H]DA in both striosomal- and matrix-enrichedareas of the striatum. Confirming that NK1 receptors areinvolved in these responses, the [Pro9]SP-evoked release ofPHIDA in the matrix was abolished in the presence ofRP-67580, the NK1 antagonist, while SR48968, the NK2antagonist, showed little blockade. Curiously, as revealed bythe respective distributions of [3H]naloxone (a striosomalmarker) and 125I-BHSP binding, NK1 binding sites seem to bepredominantly located in the matrix. This is surprising sincethe [Pro9JSP-evoked release of [3H]DA occurred in bothstriatal compartments. In addition, SP-containing neuronsare present in both the striosomes and the matrix and SPimmunoreactive fibers seem to be more abundant in strio-somes. This discrepancy could be related to the widelyobserved (but not yet explained) mismatch between thelocalizations of endogeneous peptides and their correspond-ing binding sites reported in several studies and already notedfor tachykinins in other brain areas (24).The [Pro9JSP-evoked release of[3H]DA wasTTX sensitive

in both striatal compartments, indicating that local circuitsmay contribute to these processes. The interruption of cho-linergic transmission with both atropine and pempidine re-duced the [Pro9JSP-evoked release of [3H]DA in the matrixbut not in the striosomal-enriched area. This selective inter-vention of cholinergic interneurons in the matrix can easily beexplained by the following observations: (i) the cell bodies ofcholinergic interneurons are mainly located inthe matrix (25),(ii) these cholinergic interneurons express mRNA for NK1receptors (26), (iii) [Pro9]SP, but not NK2 or NK3 agonists,stimulates the release of acetylcholine from rat striatal slices(23), and acetylcholine has been shown to stimulate presyn-aptically the release ofDA through muscarinic and nicotinicreceptors (8).

Selective Ftatlry Control of DA Release in the MatrixUnderStimulonoMNK Receptors. The lack ofNK2 bindingsites in the brain and particularly in the striatum in spite ofthepresence of NKA precursor molecules and NKA in thisstructure could suggest that central NK2 receptors differ bytheir structure and pharmacological properties from periph-eral NK2 receptors. In fact, NK2 receptors have been clonedfrom peripheral tissues but to our knowledge have not yetbeen cloned from the brain (27).

According to studies performed on the rat duodenum, aperipheral tissue particularly rich in NK2 binding sites,[Lys5,MeLeu9,Nle10JNKA- 10) is the most potent and se-lective NK2 agonist available (28). This NK2 agonist stimu-lated the release of pHTDA in the matrix-enriched area withan amplitude similar to that observed with [Pro9lSP. How-ever, in contrast to what was found with the NK1 agonist,[Lys5,MeLeu9,Nle'0]NKA-(4-10) was without effect in the

Neurobliology: Tremblay et al.

11218 Neurobiology: Tremblay et al.

striosomal-enriched area. Confirming previous results, nospecific binding of 125I-NKA (a NK2 ligand) could be ob-served in the striatum or in other brain structures. Never-theless, tachykinin receptors closely related to peripheralNK2 receptors seem to be involved in the response found inthe matrix since the stimulatory effect of the NK2 agonist onthe release of [3H]DA was abolished in the presence of theNK2 antagonist SR-48568 and not affected by the NK1antagonist RP-67580. Some of these receptors seem to belocated on dopaminergic nerve terminals, since the responseevoked by the NK2 agonist was partially TTX resistant.Altogether, these results suggest that SP and NKA, which arecolocalized in numerous striatal efferent neurons (29) and arevery likely released locally from their collaterals, act differ-ently on the release ofDA in the striatum. This is reminiscentof results obtained in either the cat or rat indicating that (i)pars compacta neurons are more sensitive toNKA than to SP(30), (ii)NKA stimulates the dendritic release ofDA while SPexerts the opposite effect (31), and, finally, (iii) when appliedin low concentrations in the substantia nigra, NKA and SPstimulate the release ofDA in the ipsilateral caudate nucleusthrough different processes (31, 32). In addition, differentbehavioral responses were observed when either NKA or SPwas injected into the substantia nigra or the ventral tegmentalarea, which is also densely innervated by a neuronal pathwayrich in tachykinins (33, 34).

Direct Presynaptic Facilitatory Control ofDA Release in theMatrix Under Stimulation of NK3 Receptors. While neuronsrich in SP and NKA are found in both striatal compartments(14), NKB-containing neurons that project mainly to theglobus pallidus (16) seem to be exclusively located in thematrix (17). However, as shown with 1251-BHE, NKB bindingsites seem to be distributed in both striosomal- and matrix-enriched areas selected for our superfusion experiments.Nevertheless, the NK3 agonist [Pro7]NKB stimulated therelease of[3H]DA only in the matrix-enriched area, this effectbeing less pronounced than those observed with either theNK1 or the NK2 agonist. Unfortunately, selective NK3antagonists are not yet available. However, the [Pro7]NKB-evoked response seen in the matrix seems to be mediated byNK3 receptors, since it was affected neither by RP-67580 norby SR-48968. In addition, the weak stimulatory effect of[Pro7]NKB on the release of[3H]DA persisted in the presenceof TTX, suggesting that, in the matrix, part of the NK3receptors are located on dopaminergic nerve terminals.

Previously, we have shown in the cat or rat that N-methyl-D-aspartate and acetylcholine stimulate the release of[3H]DA through TTX-resistant processes in both striatalcompartments. In addition to these direct presynaptic facil-itatory regulations ofDA release, both N-methyl-D-aspartateand acetylcholine TTX-sensitive responses mediated by localcircuits could be demonstrated, but only in the matrix,providing evidence for functional differences between striatalcompartments (8, 9). Our present results extend these find-ings since presynaptic facilitations ofDA release induced byNK2 and NK3 agonists were only found in the matrix, theseeffects being partially (NK2 agonist) or totally (NK3 agonist)TTX resistant. Moreover, the stimulation of NK1 receptorsby the selective NK1 agonist was associated with a markedfacilitation of [3H]DA release in both striatal compartments.These effects are indirect and cholinergic interneurons areinvolved in the NK1 agonist-evoked response, again only inthe matrix. Several reports have indicated that dopaminergicneurons that innervate the striatum interact with striatalefferent tachykinin-containing neurons (4). Our findings dem-onstrate that reciprocal regulation occurs between these

neuronal systems, these interactions being particularly com-plex in the matrix.

The authors wish to thank J. C. Beaujouan, S. Lavielle, and G.Chassaing for very helpful discussions. This research was supportedby grants from Caisse Nationale de I 'Assurance Maladie desTravailleurs salaries, Direction des Recherches, Etudes et Tech-niques (90.078), Human Frontiers, Rhone Poulenc Rorer, and Fondsde la Recherche en Sante du Quebec (fellowship to L.T.).

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science 23, 223-242.7. Gerfen, C. R., Herkenham, M. & Thibault, J. (1987) J. Neu-

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