CCK-8S) of Protein Phosphorylation in the Neostriatum (Forskonln/N-Methyl-D-Aspartic Acid/Glutamate) GRETCHEN L

Home , Neuropeptide

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11277-11281, December 1993 Neurobiology Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum (forskonln/N-methyl-D-aspartic acid/glutamate) GRETCHEN L. SNYDER*, GILBERTO FISONE*, PATRIZIA MORINOt, VIDAR GUNDERSEN*, OLE PETTER OTTERSEN*, TOMAS HOKFELTt, AND PAUL GREENGARD*§ *Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY 10021; tDepartment of Histology and Neurobiology, Karolinska Institute, S-10401, Stockholm, Sweden; and *Department of Anatomy, University of Oslo, Blindern, N-0317 Oslo, Norway Contributed by Tomas Hokfelt, August 16, 1993

ABSTRACT Despite physiological evidence that cholecys- rons, apparently through a mechanism that involves the tokinin (CCK) is an excitatory neurotransmitter in the brain, release of an excitatory neurotransmitter and activation of little is known about its mechanism of action. CCK immuno- NMDA receptors. reactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phos- MATERIALS AND METHODS phorylation ofa dopamine- and cAMP-regulated 32-kDa phos- Materials. RPMI 1640 balanced salt solution, bovine serum phoprotein (DARPP-32) in striatal neurons. The effect of albumin, and 3-isobutylmethylxanthine were obtained from CCK-8S is prevented by antagonists of CCKB and N-methyl- Sigma; forskolin was from Calbiochem; NMDA and (+)-MK- D-aspartate receptors. Our results support a model in which 801 hydrogen maleate (MK-801) were from Research Bio- CCK-8S, originating from CCK or CCK/glutamate cortico- chemicals; CCK-8S was from Bachem; CI-988 was from J. striatal neurons, promotes the release of an excitatory neuro- Hughes; cAMP, RIA, and ECL Western blotting detection transmitter that causes the dephosphorylation and inactivation kits were from Amersham; and goat anti-mouse horseradish of DARPP-32, a potent protein phosphatase inhibitor, thereby peroxidase-linked antibody was from Pierce. Nitrocellulose modulating neuronal excitability. filters (pore size, 0.2 jum) were obtained from Schleicher & Schuell. A monoclonal antibody purification kit (Affi-Gel Numerous peptides are widely distributed in the brain (1), MAPSII) was obtained from Bio-Rad. and many of them coexist with classic transmitters in the Preparation and Incubation ofStriatal Slices. Male Sprague- same neurons (2). It was proposed that peptides may act as Dawley rats (150-200 g) were sacrificed by decapitation. neurotransmitters (2) but, with few exceptions (3-5), it has Brains were rapidly removed and placed in 50 ml of cold, been difficult to identify their physiological role in the central oxygenated RPMI 1640 medium. Striata were dissected on an nervous system. The gut hormone cholecystokinin (CCK) (6) ice-cold surface and placed in 5 ml of fresh, cold RPMI 1640 is present in the brain (7) mainly in the form of its sulfated medium. Coronal slices (350 ,um) were prepared from each C-terminal octapeptide (CCK-8S) (8). It has a wide distribu- striatum by using a Mcllwain tissue chopper (Brinkmann) and tion as shown by radioimmunoassay (9), immunohistochem- pooled; individual slices were placed (four per tube) in 5-nl istry (10, 11), and in situ hybridization (12). CCK-8S, like conical, polypropylene incubation tubes containing 1 ml of actions on neurons ice-cold, fresh RPMI 1640 medium. This solution was then glutamate, exerts excitatory target (13, replaced with 2 ml of fresh, oxygenated, Mg2+-free buffer 14) and there is evidence not only for glutamatergic (15) but containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.3 also for CCKergic (16-19) corticostriatal pathways in the mM CaCl2, 25 mM NaHCO3, 11.7 mM glucose, and 0.5 mM brain. 3-isobutylmethylxanthine. The tubes were transferred to a Medium-sized spiny neurons, which comprise the largest 30°C water bath and incubated under constant oxygenation cell population of the striatum, are excited by glutamate with 95% 02/5% CO2 for 30 min. Drug solutions were added acting through N-methyl-D-aspartate (NMDA)-type recep- to slices for 5 min. After drug treatment, tissue slices were tors (20, 21). These neurons are highly enriched in dopamine- rapidly transferred to polypropylene tubes, immediately fro- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) zen on dry ice, and stored at -70°C until assayed. (22). Dopamine, by acting on D1 receptors and increasing Immunoblotting. Frozen tissue samples were sonicated in cAMP, stimulates phosphorylation of DARPP-32 by cAMP- an aliquot of boiling 1% SDS and boiled for an additional 10 dependent protein kinase, converting it into a potent inhibitor min. Small aliquots of the homogenate were retained for of protein phosphatase 1 (23). Conversely, glutamate, by protein determination by the method of Lowry et al. (26) acting on NMDA receptors, and increasing calcium influx, using bovine serum albumin as a standard. Equal quantities stimulates the calcium/calmodulin-dependent protein phos- (250 ig) of each sample were loaded onto 12% acrylamide phatase calcineurin leading to dephosphorylation and inac- gels and proteins were separated by SDS/PAGE (27) and tivation ofDARPP-32 (24). These and other data indicate that then transferred to nitrocellulose membranes (0.2 ,im) by the the antagonistic effects of dopamine and glutamate on the method of Towbin et al. (28). The membranes were then excitability of striatal neurons may be expressed, at least in immunoblotted using a monoclonal antibody (mAB-23) part, through alterations in the state of phosphorylation of (1:100 dilution) (25) that selectively detects the phosphory- DARPP-32. We report here biochemical and immunocyto- lated form ofDARPP-32. In some experiments, a monoclonal chemical evidence that CCK, like glutamate, is able to antibody (C24-5a) (1:2000 dilution) (29) generated against regulate the phosphorylation of DARPP-32 in striatal neu- Abbreviations: DARPP-32, dopamine- and cAMP-regulated 32-kDa The publication costs ofthis article were defrayed in part by page charge phosphoprotein; CCK-8S, cholecystokinin octapeptide (sulfated); payment. This article must therefore be hereby marked "advertisement" NMDA, N-methyl-D-aspartic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 11277 Downloaded by guest on September 27, 2021 11278 Neurobiology: Snyder et al. Proc. Natl. Acad. Sci. USA 90 (1993) DARPP-32, which is not phosphorylation state specific, was Postembedding Electron Micrographic Analysis of Gluta- used to estimate the total amount of DARPP-32 in samples. mate-like Immunoreactivity. Slices of rat striatum (30) were Antibody binding was revealed by incubation with goat incubated in oxygenated Krebs' solution, fixed in a mixture anti-mouse horseradish peroxidase-conjugated IgG (1:4000 of formalin/glutaraldehyde, treated with osmium tetroxide, dilution) and the ECL Western blotting detection system. dehydrated, and embedded in Durcupan (Fluka). Ultrathin Chemiluminescence was detected by autoradiography. Au- sections were stained by a postembedding immunogold toradiographic signals were then quantified by densitometry method (31). The grids were incubated with a rabbit antise- using a Bio-Rad model 620 video densitometer and Bio-Rad rum to glutamate (glutamate 03; 1:500) (32) to which were 1-D analyst software. Statistical significance of the data was added, to remove traces of crossreactivity, the amino acid- determined by one-directional Student's t test. glutaraldehyde complexes aspartate-G (100 ,uM) and gluta- cAMP Radjoimmunoassay. Striatal slices, prepared and mine-G (300 MM). After a brief rinse in polyethylene glycol, treated as for the phosphorylation experiments, were soni- the grids were'incubated for 2 hr with a goat anti-rabbit IgG cated in 4 mM Tris/EDTA buffer and assayed for cAMP coupled to colloidal gold particles (diameter, 10 nm; 1:20; using an Amersham cAMP RIA kit. Janssen). The sections were finally counterstained and ex- Immunocytochemical Preembedding Electron Microscopic amined as described above. In each of these experimental Analysis of CCK-like Immunoreactivity. Rats were perfused groups adsorption controls were included. with 1% paraformaldehyde followed by a mixture of 1% Immunofluorescence Microscopic Analysis ofAspartate-Hlke paraformaldehyde and 2.5% glutaraldehyde. The brains were Immunoreactivity. Sections (14 ,um) from formalin-fixed rat postfixed and cut in a vibrating microtome at 40 t&m. Sections brains were processed for the indirect immunofluorescence were treated with 1% sodium borohydrate for 1 min and then technique-i.e., incubated with a polyclonal rabbit an'tiserum left overnight in 20%o sucrose solution. After freeze-thaw to aspartate (1:400) (33), followed by fluorescein isothiocy- treatment, sections were incubated for 48 hr with a mono- anate-conjugated goat anti-rabbit antibodies (1:80) and ex- clonal antibody to gastrin-CCK (28.2; 1:5000; J. Walsh, amination in a'Nikon Microphot-FX microscope. personal communication), followed by a biotinylated donkey anti-mouse IgG (1:200; 2 hr) and avidin-biotin-peroxidase complex (1:100; 2 hr; Vector ABC kit). Peroxidase activity RESULTS was visualized by using diaminobenzidine (Sigma) as chro- Effect of CCK-8S on the Phosphorylation of DARPP-32. mogen. Sections were then treated with osmium tetroxide Forskolin, a drlig that activates adenylyl cyclase and in- (1%), dehydrated, and embedded'in Epon. Stained areas of creases cAMP levels in brain slices, increased the phosphor- striatum were selected under the light microscope and cut. ylation of DARPP-32 severalfold in striatal slices. CCK-8S Ultrathin sections, counterstained'with uranyl acetate and decreased the forskolin-stimulated phosphorylation of lead citrate, were examined in a JEOL x 1200 electron DARPP-32 by approximately one'-half (Fig. 1 A and C). microscope. CCK-8S alone did not significantly affect the phosphoryla-

co Cl) at A Cl) B C) C-) C) FIG. 1. CCK-8S abolishes forskolin-induced z Z z phosphorylation of DARPP-32 in striatal J slices. o o 0 0 0 a: e Cl) a: U) ,e C) Slices were incubated for 5 min in the absence FC- U) Cl) CI H- 0 c: 0 0 0 0 (control) or presence of forskolin (10 MM), C) Li- 0eV -N LM and/or CCK-8S (1 pM). (A) Phosphorylated kDa kDa DARPP-32 was detected at a molecular mass of -32 kDa using a phosphospecific antibody. (B) 49- 49- Total DARPP-32 was detected in the same samples as shown in A using a monoclonal antibody against DARPP-32 (C24-5a). The positions of 33- ._ ... _ ..,. 33- 4-O* the phospho-DARPP-32 and DARPP-32 immunoreactive bands in A and B are indicated by 24- 24- arrows. (C) Effect of various concentrations of CCK-8S on forskolin-induced phosphorylation of DARPP-32. Striatal slices were incubated with forskolin (FORSK) (10 pM) alone or to- gether with the indicated concentrations of CCK-8S (1 nM to 10 uM) for 5 min. CONTR, Phospho DARPP-32 DARPP-32 control. The amount of phosphorylated DARPP-32 for each condition is expressed as a 175 D percentage of phospho-DARPP-32 in the presence of forskolin alone. Data are presented as 150- means ± SEM for 3-10 experiments. (Different I from forskolin alone: *, P < 0.05; t, P < 0.001; 125- a-Zful; one-tailed t test.) (D) CI-988, a selective CCKB 100- receptor antagonist, prevents the effect of CLa- c- CCK-8S on forskolin-induced DARPP-32 phosphorylation. Striatal slices were preincubated 75- with 0 CI-988 (1 MAM) for 10 min and then incu- fL 50- FRS FORSK bated for an additional 5 min in the absence a- 0-8 i :BS (control) or presence of forskolin (10 MLM), CL FOS *OS _ONT .0 | CCK-8S (1 uM), and CI-988 as indicated. The 25- OCKT amount of phosphorylated DARPP-32 for each 0O condition is expressed as a percentage of phospho-DARPP-32 in the presence of forskolin - + + 4 Data are as means ± GCKS OCK8S OC(8S CCK8S CK0(S alone. presented SEM for I rli 10 nM lffl nM 1 KM 10 pM three experiments. (Different from forskolin alone; *, P < 0.05; one-tailed t test). Downloaded by guest on September 27, 2021 Neurobiology: Snyder et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11279 tion state of DARPP-32 (Fig. 1A). CCK-8S (1 F.M) did not decrease the total amount of DARPP-32 detected in either A 125- untreated or forskolin-treated striatal slices (Fig. 1B), nor did it significantly affect the level of cAMP in striatal slices T treated with forskolin (10.0 ± 2.8 pmol of cAMP per mg of g 100- protein in forskolin-treated slices as compared with 11.3 + 4.6 pmol of cAMP per mg of protein in slices incubated with 75 - forskolin and CCK-8S; n = 4). The decrease in the forskolin- CL T induced phosphorylation ofDARPP-32 produced by CCK-8S CK was blocked by pretreatment of striatal slices with CI-988 (1 ,uM), a selective nonpeptide antagonist ofthe CCKB receptor 0 50 (Fig. 1D). a Comparison ofEffects ofNMDA and CCK-8S on Phosphor- ° 25

ylation of DARPP-32. The activation of NMDA-type gluta- 'C a- - - mate receptors in striatum has been demonstrated to reduce the forskolin-induced phosphorylation of DARPP-32 in me- 0- dium-sized spiny neurons (24). We examined the possibility that CCK-8S might reduce the forskolin-induced phosphorylation of DARPP-32 by a mechanism involving the activation ofglutamate receptors. MK-801 (100 ,uM), a competitive antagonist of the NMDA-type glutamate receptor, abolished B 1 25]i the effect of CCK-8S on the forskolin-induced phosphorylation of DARPP-32. MK-801 did not affect the basal level of 0 I phosphorylated DARPP-32 (data not shown) nor did it sig- (N~ 1 00- nificantly affect the amount ofphospho-DARPP-32 measured 0~ r in forskolin-treated slices (Fig. 2A). NMDA decreased forskolin-induced phosphorylation of a DARPP-32 in striatal slices (Fig. 2B). CCK-8S produced no additional reduction in the forskolin-stimulated phosphorylation of DARPP-32 beyond that seen in the presence of Q- NMDA alone (Fig. 2B). Preembedding Electron Microscopic Analysis of CCK-like IL Immunoreactivity. Sections of the caudate nucleus, stained with CCK antibody and included in resin, were first analyzed in the light microscope. CCK-positive punctate elements were seen all over the striatum. Medial areas, close to the lateral ventricle, with patches of CCK-like immunoreactivity, were selected for sectioning. Electron microscopic analysis of these areas revealed numerous immunoreactive ter- FIG. 2. Effect of CCK-8S on DARPP-32 phosphorylation in- minals, often in close association with the fiber bundles ofthe volves activation of glutamate receptors. (A) MK-801, an NMDA internal capsule. Some of these stained terminals were rela- receptor antagonist, prevents the reduction by CCK-8S of forskolin- tively small and filled with numerous densely packed, spher- induced DARPP-32 phosphorylation. Striatal slices were preincu- ical vesicles (Fig. 3A). They made a distinct, straight asym- bated with MK-801 (100 uM) for 10 min and then incubated for an metric contact with a small spine (Fig. 3A). additional 5 min with the specified combinations of forskolin (10 Postembedding Electron Microscopic Analysis of Gluta- ,uM), CCK-8S (1 ,uM), and MK-801 as indicated. The amount of mate-like A high concentration of gold phosphorylated DARPP-32 for each condition is expressed as a Immunoreactivity. percentage ofphospho-DARPP-32 in the presence offorskolin alone. particles was found over terminals filled with small, round Data are presented as means ± SEM for three experiments. (*, P < vesicles and with straight, asymmetric contacts to spines 0.05 compared to control; t, P < 0.05 compared to forskolin alone; (Figs. 3B and 4). Other types of terminals were less enriched t, P < 0.05 compared to forskolin and CCK-8S; one-tailed t test.) (B) in gold particles (Fig. 3B). Furthermore, postsynaptic spines, Nonadditivity ofeffects of NMDA and CCK-8S on forskolin-induced dendritic elements, and glial structures were poorly labeled. phosphorylation of DARPP-32. Striatal slices were incubated in the Immunofluorescent Microscopic Analysis of Aspartate-like absence (control) or presence of the indicated combinations of Immunoreactivity. A few strongly aspartate-immunoreactive forskolin (10 ,uM), CCK-8S (1 ,uM), and NMDA (100 ,uM) for 5 min. cell bodies could be seen throughout the caudatoputamen. The amount of phosphorylated DARPP-32 for each condition is They were medium-sized, round, and often with immunore- expressed as a percentage of phospho-DARPP-32 in the presence of active fine and forskolin alone. Data are presented as means ± SEM for four processes arising from the cell body (Fig. 3 C experiments. (Different from forskolin alone: *, P < 0.05; t, P < D). Some smaller, weakly immunoreactive cell bodies could 0.01; one-tailed t test.) also be seen. In addition, and often associated with stained cell bodies, small networks of aspartate-positive, varicose fiber plexuses were observed (Fig. 3D). None of the immu- (22), a phosphoprotein that, when phosphorylated by cAMP- noreactive structures described in the three experimental dependent protein kinase, functions as a potent inhibitor of groups could be observed after incubation with control sera. protein phosphatase 1 (23). Previous studies demonstrated that dopamine, acting on D1 receptors, stimulates adenylyl cyclase, elevates cAMP, and activates cAMP-dependent DISCUSSION protein kinase, leading to phosphorylation of DARPP-32 (35, Medium-sized spiny neurons comprise the major output 36). Glutamate, acting on NMDA receptors, increases cal- pathway from striatum to substantia nigra. These neurons are cium influx into neurons and stimulates calcium-dependent inhibited by dopamine acting through Di-type receptors and enzymes including the calcium/calmodulin-dependent phos- are excited by glutamate acting through NMDA-type recep- phatase (i.e., calcineurin), leading to dephosphorylation of tors (20,21). These neurons are highly enriched in DARPP-32 DARPP-32 (24). These and other data indicate that the Downloaded by guest on September 27, 2021 11280 Neurobiology: Snyder et al. Proc. Natl. Acad. Sci. USA 90 (1993)

FIG. 3. (A) Electron micrograph of a section stained with CCK antiserum. An immunoreactive terminal contains small densely packed vesicles and makes a distinct, straight asymmetric synaptic contact (arrowheads) with a small spine (star). An adjacent terminal is unstained (arrow). (Bar = 500 nm.) (B) Electron micrograph of a striatal slice stained with a glutamate antiserum. A small terminal containing densely packed round vesicles is enriched with gold particles. It makes straight, asymmetric contact (arrowheads) with two small spines (stars). Another terminal (arrow) with clear and less densely packed vesicles is almost completely devoid of gold particles. (Bar = 200 nm.) (C and D) Immunofluorescence micrographs of striatal sections stained with an antiserum to aspartate. (C) A medium-sized, strongly immunofluorescent cell body with fine positive fibrous profiles is seen. (Bar = 50 gm.) (D) Several immunoreactive varicose, fibrous elements form a sparse network. (Bar = 50 Am.) antagonistic effects of dopamine and glutamate on the activ- that the peptide selectively reduced the proportion of ity of striatal neurons may, in part, be expressed through the DARPP-32 in the phosphorylated form rather than increasing state of phosphorylation of DARPP-32. Since, in the stria- the degradation of the protein. In addition, CCK-8S did not tum, the excitatory amino acid glutamate is presumably inhibit the formation of cAMP by forskolin in striatal slices. released from terminals of corticostriatal neurons (15), and This suggests that the reduction in DARPP-32 phosphoryla- since the excitatory neuropeptide CCK-8S (13, 14) is also tion produced by the peptide may be mediated through present in corticostriatal neurons (16-19), it was ofinterest to activation ofphosphatase(s) rather than inhibition ofadenylyl study the effect of this peptide on phosphorylation of cyclase. DARPP-32. In the present study, it was also shown that the CCK-8S- The present study shows that CCK-8S, a neuropeptide that induced reduction in DARPP-32 phosphorylation was pre- is highly concentrated in striatum, reduces the forskolin- induced phosphorylation of DARPP-32 in striatal slices. The decrease in DARPP-32 phosphorylation by CCK-8S is most likely mediated by activation of CCKB receptors since the CORTEX effect is blocked by pretreatment of slices with CI-988, a selective antagonist ofthis receptor (37). These observations are consistent with the high abundance ofCCKB receptors in most regions of the rodent brain, including striatum (38). Incubation of untreated or forskolin-treated slices with CCK-8S did not decrease total DARPP-32 levels, indicating

300* n=1 4 's 250 T E STRIATUM _L 200 0

0. n=1 7 OUTPUT 1 5000 n=1 4 50 0* p_ A B C D FIG. 4. Levels of glutamate-like immunoreactivity in different SUBSTANTIA NIGRA DA cell profiles in striatal slices. A, type III terminals (corticostriatal); B, other types ofterminals (miscellaneous, pooled); C, postsynaptic dendrites; D, postsynaptic spines. For the correlation of particle FIG. 5. CCK may regulate phosphorylation of DARPP-32 in densities a digitizer was used to record the areas of cell profiles and medium-sized spiny neurons by stimulating release ofglutamate from the data were analyzed by a computer program (MORFOREL) (34). corticostriatal nerve terminals and/or aspartate from terminals of Particle numbers were fed into the computer after manual counting. striatal interneurons. It is possible that CCK and glutamate are Data represent means + SEM for n proffles (numbers over columns) colocalized in corticostriatal neurons, in which case CCK-induced corrected for particle density over empty resin (1.5 particles per release of glutamate would occur via autoreceptors. The effects of ,um2). Data in A were significantly different, statistically, from data CCK on aspartate-containing interneurons might occur on dendrites, in B, C, and D (P < 0.001; Student's t test). soma, or nerve terminals. For further explanation see text. Downloaded by guest on September 27, 2021 Neurobiology: Snyder et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11281 vented by MK-801, an NMDA antagonist, and that the inhib- UCLA, DDC Antibody Core, National Institutes ofHealth GrantDK itory effects of NMDA and CCK-8S on DARPP-32 phosphor- 17294 (Drs. J. Walsh and H. C. Wong). ylation were not additive. These data strongly suggest that 1. Snyder, S. H. (1980) Science 209, 976-983. CCK-8S decreases the phosphorylation ofDARPP-32 through 2. Hokfelt, T., Johansson, O., Ljungdahl, A, Lundberg, J. M. & Schultzberg, M. (1980) Nature (London) 284, 515-521. an NMDA receptor-mediated mechanism. The effects of 3. Magistretti, P. J., Morrison, J. H., Showmaker, W. J. Sapin, V. & CCK-8S on DARPP-32 phosphorylation may be mediated by Bloom, F. E. (1981) Proc. Natl. Acad. Sci. USA 77, 6535-6539. effects on corticostriatal glutamatergic nerve terminals. The 4. Weisskopf, M. G., Zalutsky, R. A. & Nicoll, R. A. (1993) Nature (Lon- don) 362, 423-427. results of the present study show that CCK is present in nerve 5. Wagner, J.-J., Terman, G. W. & Chaukin, C. (1993) Nature (London) endings in the striatum that resemble the so-called type III 363, 451-454. axospinous terminals arising from corticostriatal neurons (39). 6. Mutt, V. & Jorpes, E. (1968) Eur. J. Biochem. 6, 392-397. 7. Vanderhaegen, J.-J., Signeau, J. C. & Gepts, W. (1975) Nature (London) Moreover, in parallel experiments terminals with similar mor- 257, 604-605. phological characteristics were shown to be enriched in glu- 8. Dockray, G. J. (1976) Nature (London) 264, 568-570. tamate. Whether glutamate and CCK-8S are present in the 9. Beinfeld, M. C., Meyer, D. K., Eskay, R. L., Jensen, R. T. & Brown- stein, M. J. (1981) Brain Res. 212, 51-57. same or separate corticostriatal neurons remains to be estab- 10. Innis, R. B. & Snyder, S. H. (1980) Proc. Natl. Acad. Sci. USA 77, lished. In the former case, CCK-8S would act on autorecep- 6917-6921. tors, and in the latter case it would act on heteroreceptors of 11. Vanderhaeghen, J.-J., Lostra, F., Vierendeels, G., Gilles, C., Deschep- has been shown to per, C. & Verbanck, P. (1981) Peptides 2, 81-88. glutamatergic terminals. In fact, CCK-8S 12. Ingram, S. M., Kraus, R. G., Baldino, I. F., Skeen, L. C. & Lewis, facilitate the release of other classic neurotransmitters such as M. E. (1989) J. Comp. Neurol. 287, 260-272. dopamine (40-42). 13. Dodd, J. & Kelly, J. S. (1981) Brain Res. 205, 337-350. CCK could activate a ofCCK 14. Hill, R. G. & Boden, P. (1989) in The Neuropeptide Cholecystokinin Alternatively, subpopulation (CCK), eds. Hughes, J., Dockray, G. & Woodruff, G. (Halsted, New receptors on aspartate- and/or glutamate-containing inter- York), pp. 186-194. neurons (43). In fact, the present results demonstrate a 15. Streit, P. (1984) in Cerebral Cortex, Vol. 2, Functional Properties of of interneurons. The Cortical Cells, eds. Jones, E. G. & Peters, A. (Plenum, New York), pp. population aspartate-immunoreactive 119-143. case for interneurons being responsible for the effect of 16. Meyer, D. K., Beinfeld, M. C., Oertel, W. H. & Brownstein, M. J. CCK-8S on DARPP-32 phosphorylation is supported by the (1982) Science 215, 187-188. observation that a majority of CCKB receptors appear to 17. Meyer, D. K. & Protopapas, Z. (1985) Ann. N.Y. Acad. Sci. 448, intrinsic striatal whereas few 133-143. reside on neurons, receptors 18. Burgunder, J.-M. & Young, W. S. (1990) J. Comp. Neurol. 300, 26-46. appear to be localized to terminals of cortical or nigral origin 19. Morino, P., Herrera-Marschitz, M., Meana, J. J., Ungerstedt, U. & (38) and by the report that CCK-8S stimulates aspartate Hokfelt, T. (1992) Neurosci. Lett. 148, 133-136. release from striatal slices (44), presumably from interneu- 20. Calabresi, P., Mercuri, M., Stanzione, P., Stefani, A. & Bernardi, G. rons. like as an at (1987) Neuroscience 20, 757-771. Aspartate, acting, glutamate, agonist 21. Chiodo, L. A. & Berger, T. W. (1986) Brain Res. 375, 198-203. NMDA receptors (45), would be expected to inhibit the 22. Ouimet, C., Miller, P. E., Hemmings, H. C., Jr., Walaas, S. I. & phosphorylation of DARPP-32 in medium-sized spiny neu- Greengard, P. (1984) J. Neurosci. 4, 111-124. rons. Since residual effects of CCK-8S are sometimes ob- 23. Hemmings, H. C., Jr., Greengard, P., Tung, H. Y. L. & Cohen, P. (1984) in individual even in the of Nature (London) 310, 503-505. served experiments presence high 24. Halpain, S., Girault, J.-A. & Greengard, P. (1990) Nature (London) 343, concentrations of MK-801, we cannot exclude the additional 369-372. possibility that a population of CCKB receptors located on 25. Snyder, G. L., Girault, J.-A., Chen, J., Czernik, A. J., Kebabian, J. W., medium-sized spiny neurons also contribute to the inhibition Nathanson, J. A. & Greengard, P. (1992) J. Neurosci. 12, 3071-3083. 26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951)J. of DARPP-32 phosphorylation. Biol. Chem. 193, 265-275. The results of the present study indicate that CCK-8S 27. Laemmli, 0. (1970) Nature (London) 227, 680-685. stimulates the release of an excitatory amino acid from cor- 28. Towbin, H., Staehlin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA ticostriatal terminals and/or from striatal interneurons, 76, 4350-4354. NMDA on striatal DARPP-32- 29. Hemmings, H. C., Jr., & Greengard, P. (1986)J. Neurosci. 6, 1469-1481. thereby activating receptors 30. Storm-Mathisen, J., Ottersen, 0. P., Fu-ling, T., Gundersen, V., Laake, containing medium-sized spiny neurons (Fig. 5). It was shown J. H. & Nordbo, G. (1986) Med. Biol. 64, 127-132. previously that activation of NMDA receptors on these neu- 31. Ottersen, 0. P. (1989) Anat. Embryol. 180, 1-15. rons results in dephosphorylation of DARPP-32 (24). Our 32. Ji, Z., Aas, J. E., Laake, J., Walberg, F. & Ottersen, 0. P. (1991) J. and results one model which Comp. Neurol. 301, 296-310. present previous suggest by 33. Hepler, J. R., Toomin, C. S., McCarthy, K. D., Conti, F., Battaglia, G., CCK-8S may regulate neuronal excitability: the dephosphor- Rustioni, A. & Petrusz, P. (1988) J. Histochem. Cytochem. 36, 13-22. ylation of DARPP-32, by abolishing its ability to act as an 34. Blackstad, T. W., Karagulle, T. & Ottersen, 0. P. (1990) Comp. Biol. inhibitor of protein phosphatase 1 (23), leads to dephosphor- Med. 20, 15-34. the ion 35. Walaas, S. I., Aswad, D. W. & Greengard, P. (1983) Nature (London) ylation and activation of electrogenic pump Na+,K+- 301, 69-71. ATPase (46) and thereby to an alteration of membrane poten- 36. Walaas, S. I. & Greengard, P. (1984) J. Neurosci. 4, 84-98. tial and neuronal excitability. The data provide further support 37. Hughes, J., Boden, P., Costall, B., Domeney, A., Kelly, E., Horwell, for the hypothesis that DARPP-32 is a final common pathway D. C., Hunter, J. C., Pinnock, R. D. & Woodruff, G. N. (1990) Proc. the actions not of neurotransmitters Natl. Acad. Sci. USA 87, 6728-6732. integrating only multiple 38. Beresford, I. J. M., Hall, M. D., Clark, C. R., Hill, R. G., Hughes, J. & but also of neuropeptides on neostriatal neurons. Sirinathsinghji, D. J. S. (1987) Neuropeptides 10, 109-136. 39. Hassler, R., Chung, J. W., Rinne, U. & Wagner, A. (1978) Exp. Brain We thank Dr. A. Rustioni for a generous supply of the aspartate Res. 31, 67-80. antiserum and Dr. J. Hughes, Parke Davis, NRC (Cambridge, U.K.) 40. Vickroy, T. W., Bianchi, B. R., Kerwin, J. F., Kopecka, H. & Nadzen, for a generous donation of CI-988. This work was funded by United A. M. (1988) Eur. J. Pharmacol. 152, 371-372. States Public Health Service Grants MH-40899 to P.G. and MH- 41. Altar, C. A. & Boyar, W. C. (1989) Brain Res. 483, 321-326. 43230 to T.H. G.L.S. is the recipient of a research grant from the 42. Marshall, F. H., Barnes, S., Hughes, J., Woodruff, G. N. & Hunter, American Parkinson Disease Association and funding from Elf- J. C. (1991) J. Neurochem. 56, 917-922. Aquitaine (Montpellier, France). G.F. is the recipient of a postdoc- 43. Nicklas, W. J., Duvoisin, R. C. & Berl, S. (1979) Brain Res. 167, toral fellowship from the Swedish Natural Science Research Council 107-117. 44. Barnes, S., Whistler, H. L., Hughes, J., Woodruff, G. N. & Hunter, (NR-B-PD 06633-301). The work was further supported by the J. C. (1991) J. Neurochem. 56, 1409-1416. Swedish Medical Research Council (04X-2887), Marianne och Mar- 45. Roberts, P. J., Storm-Mathisen, J. & Bradford, H. F., eds. (1985) Ex- cus Wallenbergs Stiftelse, Environmental Protection Agency (CR- citatory Amino Acids (Macmillan, London). 819506) (P.G.), and Wenner-Gren Center Foundation (P.M.). The 46. Bertorello, A. M., Aperia, A., Walaas, S. I., Nairn, A. C. & Greengard, CCK monoclonal antibody was generously provided by CURE/ P. (1991) Proc. Natl. Acad. Sci. USA 88, 11359-11362. Downloaded by guest on September 27, 2021