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Dopamine Enhancement of NMDA Currents in Dissociated Medium-

Sized Striatal Neurons: Role of D1 Receptors and DARPP-32

JORGE FLORES-HERNANDEZ,1

CARLOS CEPEDA,1

ELIZABETH HERNANDEZ-ECHEAGARAY1

,CHRISTOPHER R. CALVERT,1 EVE S. JOKEL,1 ALLEN A. FIENBERG,2

PAUL GREENGARD,2 AND MICHAEL S. LEVINE1

1Mental Retardation Research Center, University of California, Geffen School of Medicine, Los Angeles, California 90095;

and 2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021

Received 13 May 2002; accepted in final form 1 August 2002

Flores-Hernandez, Jorge, Carlos Cepeda, Elizabeth Hernandez-Echeagaray, Christopher R. Calvert, Eve S. Jokel, Allen A. Fien-berg, Paul Greengard, and Michael S. Levine.Dopamine enhance-ment of NMDA currents in dissociated medium-sized striatal neurons:role of D1 receptors and DARPP-32. J Neurophysiol88: 3010 3020,2002; 10.1152/jn.00361.2002. Dopamine (DA), via activation of D1

receptors, enhances N-methyl-D-aspartate (NMDA)-evoked responsesin striatal neurons. The present investigation examined further theproperties of this enhancement and the potential mechanisms bywhich this enhancement might be effected. Dissociated medium-sizedstriatal neurons were obtained from intact rats and mice or mutantmice lacking the DA and cyclic adenosine 3,5 monophosphate(cAMP)-regulated phosphoprotein of MR 32,000 (DARPP-32).NMDA (101,000 M) induced inward currents in all neurons. Inacutely dissociated neurons from intact rats or mice, activation of D1receptors with the selective agonist, SKF 81297, produced a dose-dependent enhancement of NMDA currents. This enhancement wasreduced by the selective D1 receptor antagonist SKF 83566. Quinpi-role, a D2 receptor agonist alone, produced small reductions ofNMDA currents. However, it consistently and significantly reducedthe enhancement of NMDA currents by D1 agonists. In dissociated

striatal neurons, in conditions that minimized the contributions ofvoltage-gated Ca2 conductances, the D1-induced potentiation wasnot altered by blockade of L-type voltage-gated Ca2 conductances incontrast to results in slices. The DARPP-32 signaling pathway has animportant role in D1 modulation of NMDA currents. In mice lackingDARPP-32, the enhancement was significantly reduced. Furthermore,okadaic acid, a protein phosphatase 1 (PP-1) inhibitor, increasedD1-induced potentiation, suggesting that constitutively active PP-1attenuates D1-induced potentiation. Finally, activation of D1 recep-tors produced differential effects on NMDA and gamma aminobutyricacid (GABA)-induced currents in the same cells, enhancing NMDAcurrents and inhibiting GABA currents. Thus simultaneous activationof D1, NMDA, and GABA receptors could predispose medium-sizedspiny neurons toward excitation. Taken together, the present findingsindicate that the unique potentiation of NMDA receptor function by

activation of the D1 receptor signaling cascade can be controlled bymultiple mechanisms and has major influences on neuronal function.

I N T R O D U C T I O N

Dopamine (DA) modulates responses evoked by activationof glutamate receptors in medium-sized spiny striatal neurons

(Cepeda et al. 1993; Levine et al. 1996a,b; Price et al. 1999;Yan et al. 1999). Our laboratory has shown in striatal slices thatthe direction of this modulation depends on both the glutamatereceptor subtype subject to DA modulation and the DA recep-tor subtype preferentially activated (Cepeda and Levine 1998;

Cepeda et al. 1993; Levine et al. 1996a,b). Activation of D1family receptors enhances responses, in particular those medi-ated by N-methyl-D-aspartate (NMDA) receptors, whereas ac-tivation of D2 family receptors reduces responses, in particularthose mediated by non-NMDA receptors (Cepeda et al. 1993;Levine et al. 1996b).

We have been studying the mechanisms involved in theenhancement of NMDA responses by D1 receptors. One pos-sibility is that DA modulation of voltage-gated Ca2 conduc-tances contributes to D1 enhancement of NMDA receptor-mediated responses because previous studies have shown thatactivation of D1 receptors enhances L-type Ca2 currents(Hernandez-Lopez et al. 1997; Surmeier et al. 1995). Weprovided support for this alternative by demonstrating that

blockade of L-type Ca2 channels in striatal slices markedlyreduced the D1-mediated potentiation of responses due toNMDA receptors (Cepeda et al. 1998a). However, blockade ofL-type Ca2 channels did not completely abolish the enhance-ment, suggesting that other mechanisms contribute to the mod-ulation (Cepeda et al. 1998a).

Another mechanism by which DA could modulate NMDAreceptors is by changing the state of phosphorylation of theNMDA receptor. DA alters cyclic adenosine 3,5 monophos-phate (cAMP) production. Activation of D1 receptors increasesformation of cAMP and stimulates protein kinase A (PKA).Stimulation of the cAMP-PKA cascade with forskolin en-hances NMDA-evoked responses (Blank et al. 1997; Colwell

and Levine 1995). Furthermore, D1 receptor activation andforskolin increase NMDAR1 subunit phosphorylation througha synergistic mechanism involving increased phosphorylationand decreased dephosphorylation of the receptor subunit (Ra-jadhyaksha et al. 1998; Snyder et al. 1998). The DA andcAMP-regulated phosphoprotein of M

R 32,000 (DARPP-32)

(Ouimet et al. 1984; Walaas and Greengard 1984) is partlyresponsible for these effects (Fienberg et al. 1998). DARPP-32

Address for reprint requests: M. S. Levine, Mental Retardation ResearchCtr., 760 Westwood Plaza NPI58-258, University of California, Los Angeles,CA 90095 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 88: 30103020, 2002;

10.1152/jn.00361.2002.

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is phosphorylated by cAMP-dependent PKA on a single thre-onine residue, Thr34, and transformed into a potent inhibitor ofprotein phosphatase-1 (PP-1) (Hemmings et al. 1984). In turn,PP-1 regulates the phosphorylation state of many neurotrans-mitter receptors and voltage-gated ion channels (Greengard etal. 1999). Recent evidence supports a role for PP-1 in regulat-ing -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) receptor channels in medium-sized striatal neurons(Yan et al. 1999).

The present study was designed to further examine theproperties of, and the mechanisms underlying D1 modulationof NMDA currents. We used dissociated neurons becauseadequate voltage clamp can more easily be obtained than fromneurons in slices. In our previous study using striatal slices(Cepeda et al. 1998a), interpretation of the findings was com-plicated by space-clamp limitations in cells with extendedprocesses. In addition, we examined the contribution ofDARPP-32 and PP-1 to the modulation.

M E T H O D S

Animals

All procedures were carried out in accordance with the NationalInstitutes of Healths Guide for Care and Use of Laboratory Animalsand were approved by the Institutional Animal Care and Use Com-mittee at the University of California at Los Angeles. Experimentsused Sprague-Dawley rats (21 40 days old; n 78), DARPP-32knockout [148 6 (SE) days of age, 137166 age range, n 6] andwild-type (WT) mice of the same strain as the DARPP-32 knockouts(145 5 days of age,134 164 age range, n 8). The mice weregenerated at the Rockefeller University and shipped to UCLA. Thegeneration and characterization of these mice have been described(Fienberg and Greengard 2000; Fienberg et al. 1998).

Dissociation procedure

Striatal neurons from rats or DARPP-32 knockout and WT micewere acutely dissociated using procedures slightly modified fromthose previously described (Bargas et al. 1994; Flores-Hernandez etal. 2000; Surmeier et al. 1995). Briefly, rats and mice were anesthe-tized with Halothane, perfused transcardially, and decapitated. Brainswere quickly removed and placed in ice-cold, low-Ca2 sucrosesolution that contained (in mM) 250 sucrose, 2.5 KCl, 0.1 CaCl

2, 1

Na2

HPO4

, 4 MgSO4

, 11 glucose, and 15 N-[2-hydroxyethyl]pipera-zine-N-[2-ethanesulfonic acid] (HEPES), pH 7.4, 300 305mOsm/l. Brain blocks were cut to obtain 400-m coronal slices usinga DSK microslicer (Osaka, Japan) while bathed in the same solution.Slices were then incubated for 1 6 h at room temperature (20 22C)in a NaHCO3buffered Earles balanced salts solution (EBSS) bubbledwith 95% O2-5% CO2and supplemented with (in mM) 1 pyruvic acid,0.005 glutathione, 0.1 NG-nitro-L-arginine, and 1 kynurenic acid,pH 7.4 with NaOH, 300 305 mOsm/l. After 1 h incubation, aslice was placed in low-Ca2 isethionate solution containing (in mM)140 Na isethionate, 2 KCl, 2 MgCl2, 0.1 CaCl2, 23 glucose, and 15HEPES, pH 7.4, 300 305 mOsm/l, bubbled with O2 and supple-mented as described in the preceding text. With the aid of a micro-scope, the dorsal striatum was dissected. Dissections were limited tothe tissue rostral to the anterior commissure to reduce the possibilityof contamination from the globus pallidus.

The tissue was placed in an oxygenated cell-stir chamber (Wheaton,Millville, NJ) containing Papain (12 mg/ml, Calbiochem) in aHEPES-buffered Hanks balanced salt solution (HBSS, Sigma Chem-ical) at 35C bubbled with O2and supplemented as before, pH 7.4with NaOH, 300 305 mOsm/l. After 20 40 min of enzyme digestion,

tissue was rinsed three times with the low-Ca2-isethionate solutionand mechanically dissociated with a graded series of fire-polishedPasteur pipettes. The cell suspension was then plated into a 35-mmLux petri dish mounted on the stage of an upright fixed-stage micro-scope (Zeiss Axioskop, Thornwood, NY) containing HEPES-bufferedHBSS saline.

Whole cell recordings

Whole cell recordings employed standard techniques (Bargas et al.1994). Electrodes were pulled from Corning 8250 glass (A-M Sys-tems, Carlsborg, WA) and heat polished prior to use. The internalsolution consisted of (in mM) 175 N-methyl-D-glucamine (NMDG),40 HEPES, 2 MgCl

2, 10 ethylene glycol-bis(-aminoethyl ether)-N,

N,N,N-tetraacetic acid (EGTA), 12 phosphocreatine, 2 Na2ATP, 0.2Na3GTP, and 0.1 leupeptin, pH 7.27.3 with H2SO4, 265270mOsm/l. The external solution consisted of (in mM) 127 NaCl, 20CsCl, 5 BaCl

2, 2 CaCl

212 glucose, 10 HEPES, 0.001 tetrodotoxin,

and 0.02 glycine, pH 7.3 with NaOH, 300 305 mOsm/l. Bariumwas used as the main charge carrier through Ca2 channels and tobetter block K conductances. Addition of 2 mM Ca2 to the externalsolution helped preserve the physiological internal environment andalso to favor processes dependent on activation of second messengers(Greengard et al. 1999). For example, previous studies have shownthat NMDA receptor channel function is regulated by endogenousCa2-dependent phosphatases (Lieberman and Mody 1994). Onepotential concern is the anomalous mole-fraction effect, one of theproperties of multi-ion channels. For example, for Ca2 channels,when Ba2 and Ca2 are combined, the current produced is lowerthan with either cation alone (Hille 2001). This effect would furtherreduce the contribution of Ca2 conductances to D1-induced modu-lation. To determine if this combination of ions affected NMDAresponses, we tested solutions with Ba2 alone versus the combina-tion of Ba2 and Ca2 on currents evoked by NMDA and found nodifference in the evoked NMDA current.

Recordings were obtained with an Axon Instruments 200A patch-clamp amplifier and controlled with a Pentium clone running pClamp(v. 8.01) with a DigiData 1200 series interface (Axon Instruments,Foster City, CA). Electrode resistance was typically 2 4 M in thebath. After seal rupture, series resistance (20 M) was compensated(70 90%) and periodically monitored. Recordings were made frommedium-sized cells that had short (75m) proximal dendrites (Fig.1). Unless otherwise noted, the cell membrane potential was held at40 mV. This holding potential was used because it reduced activa-tion of Ca2 channels and allowed examination in the same cell ofboth NMDA and GABA currents.

Drug application

Drugs were applied with a gravity-fed three-pipe system. Thearray of application capillaries (ca. 150m ID) was positioned a fewhundred micrometers from the cell under study. Solution changeswere performed by changing the position of the array with a DC drivesystem controlled by a SF-77B perfusion system (Warner Instruments,Hamden, CT) synchronized by pClamp. The solution changes werecomplete within 100 ms. Generally, NMDA (11,000 M) wasapplied for 3 s every 1520 s.

Drugs were prepared as concentrated stocks in deoxygenated water.DA receptor ligands used were SKF 81297 (D1 agonist), SKF 83566(D1 antagonist), and quinpirole (D2 agonist; RBI, Natick, MA). DAreceptor agonist solutions were protected from ambient light. Otherdrugs used were nifedipine, an L-type Ca2 channel antagonist(Sigma), the NMDA receptor antagonist 2-amino-phosphonovalerate(AP5, Tocris, Baldwin, MO), Ro 32 0432 [a cell permeable proteinkinase C (PKC) inhibitor], okadaic acid (PP-1/2A inhibitor), andnorokadaone (an inactive form of okadaic acid). The latter three drugswere obtained from Calbiochem (La Jolla, CA).

3011D1 POTENTIATION OF NMDA RESPONSES

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Statistical analyses

Values in the figures and text are presented as means SE.Dose-response curves werefit with a sigmoid function (Origin, Mi-crocal Software, Northampton, MA). Differences among group means

were assessed with appropriatet-tests or ANOVAs for independent orrepeated measures. Welchs approximation to the t-test for unequalvariances was used when group variances were not homogeneous(Welch 1947). For post hoc evaluations using ANOVAs, the Bonfer-roni t-test was used because this test is one of the more conservativeapproaches using multiple comparisons.

R E S U L T S

Experiments were performed only on medium-sized neurons(Fig. 1). Acutely dissociated medium-sized neurons in both ratsand mice were easy to distinguish from large-sized putativecholinergic cells based on somatic cross-sectional size andmembrane capacitance. In most cells in rats and mice, theinitial dendritic segments were preserved and in some cases

secondary branches showed spines. No attempt was made tofurther identify and separate the medium-sized cells into dif-ferent subpopulations of projection cells or projection cells andinterneurons. Because the medium-sized interneurons repre-sent only a small percentage of cells in the striatum, weassumed that most recordings were obtained from projectionneurons.

NMDA currents in striatal neurons from rats

Application of NMDA for 3 s induced inward currents in allneurons tested (Fig. 2A). As pointed out in the preceding text,neurons were held at 40 mV to inactivate most Ca2 con-ductances and Mg2 was removed from the bath to maximizethe response to NMDA. To establish a concentration-responserelationship, NMDA was applied at multiple concentrations in13 neurons (1, 10, 50, 100, 200, and 1000 M; not all neuronswere tested with each concentration; Fig. 2A). Small, non- orslowly desensitizing currents began to be observed at 10 Mand increased in amplitude in a concentration-dependent man-ner. As concentration increased, two types of responses oc-curred. Neurons displayed a fast, desensitizing componentwhich appeared at 50 M and at higher concentrations ofNMDA. Other neurons displayed an initial peak but they did

not desensitize as rapidly. Because both types of responseswere modulated approximately equally by application of D1agonists, data were pooled. Concentration-response functionswere generated for peak NMDA currents by normalizing thepeak amplitude to the saturating concentration (1,000 M).There was a statistically significant increase in peak currentwith concentration (F 24.2, df 5/50, P 0.001). Theresulting bestfit sigmoidal concentration-response function forthe peak current had an EC50of 28 M (Fig. 2B1) and a Hillcoefficient of 1.23. Normalized peak current density was com-puted by dividing by cell capacitance (Alzheimer et al. 1993)and was best fit by a sigmoidal function with comparableEC50s and Hill coefficients (Fig. 2B2). Because current andcurrent density functions for responses induced by NMDAwere similar and there was no significant variation in neuroncapacitance across different experimental groups within rats ormice, we will present only data for evoked currents. As istypical, NMDA currents were blocked by AP5 (Fig. 2A2). At

10M AP5, the peak current induced by 100M NMDA wasreduced by 38 8% (n 5), at 50M AP5 the reduction was72 12% (n 5), and at 100 M AP5 it was 81 11% (n 7; F 14.8, df 3/14,P 0.01).

Activation of D1 receptors enhances NMDA currents

Based on the concentration-response functions we chose 100M of NMDA as the concentration to examine modulation byDA receptor agonists because it was above the EC50 for thepeak currents and produced consistent responses in each neu-ron examined. To establish a concentration-response relation-ship, the selective D1 receptor agonist SKF 81297 was appliedat multiple concentrations in 11 neurons (1, 10, and 100 nMand 1 and 10 M; not all neurons were tested with eachconcentration; Fig. 3A). Application of SKF 81297 signifi-cantly enhanced peak NMDA currents in a concentration-dependent fashion (F 9.91, df 4/22,P 0.001; Fig. 3B).The enhancement had an EC50of 188 nM and a Hill coefficientof 0.59. In addition, there was a concentration-dependent in-crease in the decay time constant (F 3.63, df 5/26, P 0.025), indicating that the decay of the peak current wassignificantly slower in the presence of the D1 agonist. Thelargest increases in decay time constants were 36 10 and

FIG. 1. Examples of medium-sized neurons, with patch electrodes attached, acutely dissociated from a rat (A) and a DARPP-32knockout mouse (B).

3012 FLORES-HERNANDEZ ET AL.



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34 12% at 1- and 10-M concentrations of SKF 81297,respectively. Because the greatest modulation occurred for D1agonist concentrations of 1 and 10 M, alterations in D1modulation in subsequent experiments used one or both ofthese concentrations to examine the properties of the modula-tion.

To provide evidence for specificity, in a separate group of

neurons, the effects of SKF 81297 (1 M) were examined inthe presence of the selective D1 receptor antagonist SKF 83566(1 M;n 15 cells; Fig. 4A). Alone at this concentration, SKF83566 had little net effect on the NMDA current (4.4 2.3%average decrease with both increases and decreases in peakNMDA current occurring). Higher concentrations of the D1antagonist alone produced more consistent decreases in peakNMDA current and were thus not used. The magnitude of the

D1-induced enhancement was reduced from 22 2.3 (range of42.5 to 14% increase in the presence of SKF 81297 alone;all cells displayed increases) to 15 2.8% by the antagonist(all but 1 cell showed decreases in agonist-induced potentiationin the presence of the antagonist;t 2.81, df 14,P 0.02;Fig. 4B).

Activation of D2 receptors reduced the enhancement ofNMDA currents produced by D1 receptor activation

We have shown previously that activation of D2 DA recep-tors can decrease responses mediated by activation of NMDAreceptors using current-clamp recordings in striatal slices (Ce-peda et al. 1993; Levine and Cepeda 1998). However, theseeffects could have been mediated by pre- and/or postsynaptic

FIG

. 2. N-methyl-D

-aspartate (NMDA) dose-response relationships in rat medium-sized stri-atal neurons.A1: a representative set of whole-cell currents evoked by 6 different concentrationsof NMDA in the same cell. For these and othertraces, NMDA was applied at the horizontalline, and the voltage was held at 40 mV inMg2-free solution.A2: whole- cell inward cur-rents evoked by NMDA (100 M) reduced bythe receptor antagonist 2-amino-phosphono-valerate (AP5, 50 M).B, 1 and 2: peak cur-rents and peak current densities with best fitfunctions plotted. Concentrations on the ab-scissa are log coordinates.

FIG. 3. NMDA currents are potentiated by applica-tion of the full D1 dopamine (DA) receptor agonist SKF81297 in a concentration-dependent manner in neuronsfrom rats. A: examples of potentiation of NMDA (100

M) current by four concentrations (10 and 100 nMand 1 and 10 M) of SKF 81297 in a single neuron.Each pair of responses represent the control responseand then the response in the presence of the D1 agonistat each concentration (4). Data are not shown in A forthe 1 nM concentration shown graphically in B. B:concentration-response functions of the mean percentmodulation for peak currents plotted with a best-fitfunction. Concentrations on the abscissa are log coor-dinates.




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actions of D2 receptors as we have previously shown a pre-synaptic action of this receptor subtype using D2 receptor-deficient mice (Cepeda et al. 2001). To determine if activationof D2 receptors alters NMDA currents, we applied the D2

agonist quinpirole (10 M) in a separate group of neurons (n11). Alone, quinpirole produced slight and variable changes inmean peak amplitude of NMDA current (average 4 2%reduction with both increases and decreases in peak NMDAcurrent occurring). We then applied quinpirole in the presenceof SKF 81297 to determine if it affected the enhancement ofNMDA currents (Fig. 4C). In this group of neurons, SKF81297 (1 M) produced a 25 4% enhancement of theNMDA response (t 3.38, df 10, P 0.01). Whenquinpirole was then applied in the presence of the D1 agonist,the D1-induced enhancement of the mean peak NMDA currentwas reduced by 50% to 12 2% (all cells showed areduction in the agonist-induced potentiation; t 5.35, df

10,P

0.001; Fig. 4D

).

Role of L-type Ca2 conductances

In medium-sized spiny neurons and in cortical pyramidalcells L-type Ca2 conductances play a role in the enhancementof NMDA currents by activation of D1 receptors (Cepeda et al.1998a; Wang and ODonnell 2001). Previously, we demon-strated that in the striatal slice blockade of L-type Ca2 con-ductances markedly reduced D1-mediated potentiation ofNMDA currents, suggesting that Ca2 conductances in den-drites contributed to the effects of the D1 agonist (Cepeda et al.

1998a). Here we demonstrate in dissociated striatal neurons, inconditions that minimize the contribution of voltage-gatedCa2 conductances [most of the dendrites have been removed,holding potential was 40 mV, which inactivates L-type Ca2

conductances (Hille 2001) and better space clamp can beobtained], blockade of L-type Ca2 conductances had almostno effect on D1-induced modulation. Application of SKF81297 (10 M,n 7) (Fig. 4E,left) produced a 42.7 13.6%increase in the peak NMDA current (Fig. 4F, left bar graph).Application of the L-type Ca2 channel antagonist nifedipine(10 M) alone produced inconsistent effects on NMDA cur-rents (6.7 6.9% reduction in mean peak current with bothincreases and decreases in peak NMDA current occurring; Fig.4E, right). When SKF 81297 (10 M) was applied in thepresence of nifedipine the modulation induced by the D1agonist also was not affected (47.6 15.5%; Fig. 4F,right bargraph). Thus the increase in NMDA current induced by acti-

vation of NMDA receptors is not dependent on activation ofL-type Ca2 currents in acutely isolated striatal neurons underthe present conditions indicating that other mechanisms con-tribute to the modulation.

Modulation of NMDA currents was reduced in DARPP-32knockout mice

To provide information about the mechanism underlying D1potentiation of NMDA currents, we examined D1 modulationin DARPP-32 knockout mice. Although these mice were olderthan the rats, our goal was to examine mechanisms underlying

FIG. 4. A: example of antagonism of SKF 81297-induced enhancement of NMDA current by the spe-cific D1 receptor antagonist SKF 83566 (1 M) in aneuron from a rat. Left: the response to NMDA (100M).Middle: the enhancement by application of SKF81297. Right: attenuation of the enhancement in thepresence of SKF 83566. - - -, helps visualize thechanges.B: bar graphs showing mean percent modu-lation of the peak NMDA current by SKF81297 alone

(left bar) and reduction of mean percent SKF 81297modulation by SKF 83566 (right bar; SE). Asteriskindicates that the decrease is statistically significant(see text for statistics). C: example of decreases ofSKF 81297-induced enhancement of NMDA currentby the specific D2 receptor agonist quinpirole (10M) in a neuron from a rat. Left: the response toNMDA (100 M). Middle: the increase produced bySKF 81297 (1 M). Right: quinpirole reduces theenhancement produced by SKF 81297 in the sameneuron. - - -, helps visualize the changes. D: bargraphs showing mean percent modulation of the peakNMDA current (SE) by SKF 81297 alone (left bar)and the decrease in the mean percent modulation(SE) produced by quinpirole (right bar). Asteriskindicates the decrease is statistically significant (seetext for statistics). E: blockade of L type Ca2 cur-

rents does not reduce the modulation of NMDA cur-rents by SKF 81297 in neurons from rats. Left: re-sponse to NMDA and its potentiation in the presenceof SKF 81297 (10 M). Right: nifedipine alone (10M) produces little change in the NMDA current anddoes not alter the increase produced by SKF 81297. F:bar graphs showing that percent modulations of theNMDA current in the presence or absence of nifedi-pine were similar.




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the modulation that are more easily done in mice in which

genetic alterations can be taken advantage of. Thus we did not

directly compare data obtained from mice and rats in this study

because of the age difference. The role of DARPP-32 in the

modulation of striatal neuronal excitability has been well doc-

umented (Fienberg and Greengard 2000; Fienberg et al. 1998).

In DARPP-32 knockout mice, the inhibition of PP-1 byDARPP-32 should be absent, and we would predict that D1potentiation of NMDA-induced responses would be reduced.NMDA currents were examined in 25 neurons from WT and 23neurons from DARPP-32 knockout mice (Fig. 5, A and B).

There was a slight reduction in the mean peak amplitude of theNMDA current in neurons obtained from DARPP-32 knockout compared with WT mice (442 51 vs. 370 47 pA,respectively) that was not statistically significant, however. Weexamined the enhancement of NMDA currents by the D1agonist at two concentrations, 1 and 10 M (n 12 cells inWTs;n 14 cells in knockouts). In WT mice, the increases inNMDA current produced by application of SKF81297 were20.3 2.7 and 32.4 4.6% at 1 and 10 M, respectively (Fig.5, A, top, and B). In DARPP-32 knockout mice, the corre-sponding increases were 15.2 1.7 and 16.9 3.2%, respec-tively (Fig. 5A, bottom, and B). At both concentrations, theenhancement of the NMDA current was greater in WT than inDARPP-32 knockout mice (t 5.69, df 11,P 0.01;t3.66, df 13,P 0.01 for 1 and 10 M concentrations of theD1 agonist, respectively; Fig. 5B). Thus D1-induced potentia-tion is significantly reduced in the DARPP-32 knockout micebut is not absent, suggesting that the DARPP-32 protein is partof a series of mechanisms that contributes to D1 potentiation ofNMDA currents.

To further examine the role of PP-1 in the enhancement ofNMDA current, we applied the PP-1 inhibitor okadaic acid(Greengard et al. 1999) at 100 nM, a concentration that blocksPP-1 (Caporaso et al. 2000; Ishihara et al. 1989). Alone,okadaic acid produced a small but inconsistent increase in the

NMDA current in WT mice (3.4 2.8%, n 4). In the

presence of the D1 agonist (1 M), okadaic acid significantlyincreased the D1-induced potentiation from 20.3 2.7 to

29.9 3.2% in the WT mice (t 6.31, df 4,P 0.01; Fig.

5,Cand D). In DARPP-32 knockout mice, alone okadaic acid

also produced a small inconsistent change in NMDA (7.0

1.9%, n 8). In the presence of the D1 agonist (1 M),okadaic acid significantly increased the potentiation from15.2 1.7 to 28.3 2.9% (t 6.25, df 7, P 0.001;Fig. 5D).

We next tested the same concentration of okadaic acid in

striatal neurons (n 6) obtained from rats. Alone okadaic acidproduced inconsistent effects on the peak NMDA current(4.3 1.1%). Application of 1 M SKF 81297 induced a14.8 1.7% increase in the NMDA (100 M) current. In thepresence of the D1 agonist, okadaic acid significantly increasedthe potentiation to 22.2 2.7% (t 3.43, df 5,P 0.05).Application of the inactive form of okadaic acid, norokadaone(100 nM) produced no change in D1-induced potentiation[14.5 2.8 vs. 11.7 3.2% (n 3) before and duringapplication of norokadaone, respectively].

A recent report demonstrated a functional role for PKC inD1 modulation of NMDA-induced responses in nucleus ac-cumbens neurons (Chergui and Lacey 1999). Ro 32 0432, aselective PKC inhibitor, blocked D1-induced potentiation ofNMDA-induced responses. To determine if PKC contributedto D1-induced potentiation of NMDA responses in striatalneurons, we examined the effects of Ro 32 0432 in the dis-sociated cells. In the presence of Ro 32 0432 (5 M) alone,NMDA-induced peak currents were essentially unchanged(3.6 3.0%,n 8). SKF 81297 (1 M) induced a 16.6 2.2% (n 11) increase in the peak response. When Ro32 0432 was combined with SKF 81297, the potentiationincreased (23.9 4.3%, n 11). Therefore in dissociatedstriatal neurons antagonism of PKC does not appear to blockD1-induced potentiation.

FIG. 5. A, top: the current response to NMDAalone and its enhancement by SKF 81297 (1 M) ina wild-type (WT) mouse. Bottom: the current re-sponse to NMDA and the smaller enhancement pro-duced by SKF 81297 in a DARPP-32 knockoutmouse.B: bar graphs showing mean percent modu-lation (SE) produced by 1 and 10 M SKF 81297in WT and DARPP-32 knockout mice. Both de-creases in modulation were statistically significant in

the DARPP-32 knockout mice (asterisks; see text forstatistics).C: okadaic acid (OA), a protein phospha-tase 1 (PP-1) inhibitor, enhances D1 modulation ofNMDA currents in neurons from WT andDARPP-32 knockout mice. Left: trace from a WTmouse shows the response to NMDA (100 M).

Middle: trace shows the increase produced by SKF81297 (1M).Right: trace shows that OA (100 nM)increased the enhancement produced by SKF 81297in the same neuron. Dashed horizontal line helpsvisualize the changes. D: bar graphs showing meanpercent modulation of the peak NMDA current(SE) by SKF 81297 alone and in the presence ofOA in WT mice (left 2 bars) and in the DARPP-32knock out mice (right 2 bars). Asterisks indicate theincreases produced by OA were statistically signifi-cant (see text for statistics).




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D1 agonists produced differential effects on NMDA andGABA currents in the same neurons

A previous study (Flores-Hernandez et al. 2000) demon-strated that activation of D1 receptors reduces GABA currentsin medium-sized striatal neurons. To determine if GABA andNMDA currents are modulated in the same neurons by activa-tion of D1 receptors, we examined the effects of the D1 agoniston both types of currents. GABA (100 M, 3 s) was applied

similarly to NMDA at a holding potential of40 mV. Theconcentration of GABA chosen was the same as used in aprevious study (Flores-Hernandez et al. 2000). As demon-strated in that study, GABA induced a rapid peak outwardcurrent followed by a steady-state outward current (Fig. 6A,top). The mean amplitude of the peak current was 484 50 pA(n 13). When SKF 81297 (10 M) was applied, the peakcurrent was significantly reduced by 29.7 3.8% (t 4.29,df 12, P 0.001; Fig. 6B, left bar). In the same neurons,when SKF 81297 (10 M) was applied, NMDA (100 M)currents were significantly enhanced by 24.3 5.8% (t 6.96,df 12, P 0.001; Fig. 6A, bottom, and B, right bar). Todetermine if there was a relationship between D1 modulationof GABA and NMDA currents, the percent of modulations foreach amino acid were correlated. There was a low negativePearson product-moment correlation coefficient (R0.285),suggesting a moderate relationship between the ability of D1receptor activation to modulate both NMDA and GABA cur-rents. This effect was not statistically significant, however.Thus a net effect of activation of D1 receptors at this holdingpotential is to enhance excitation not only by increasingNMDA current but also by reducing GABA-mediated currents.

D I S C U S S I O N

A number of interesting findings have emerged from theseexperiments. D1 receptor activation enhances NMDA recep-

tor-mediated currents in dissociated medium-sized spiny neu-rons in a concentration-dependent manner. This effect is re-duced by application of a D1 receptor antagonist. We haveshown previously that the D1-induced potentiation of NMDA

responses is also blocked in D1 receptor-deficient mice (Levineet al. 1996a). While D2 receptor activation had little direct

effect on postsynaptic NMDA currents in the present experi-

ments, it reduced the D1-induced potentiation, indicating that

D1 receptors needed to be activated before D2 receptor effects

would be observed. In this preparation, the D1-induced poten-

tiation is independent of L-type voltage-gated Ca2 conduc-

tances, as in our experimental conditions, these channels were

inactivated at membrane potentials of40 mV and additionalblockade of L-type channels did not significantly alter theenhancement. The present findings demonstrate that theDARPP-32 signaling pathway also has an important role in the

modulation of NMDA currents by D1 receptors. In DARPP-32

knockout mice the enhancement was significantly reduced.Furthermore, okadaic acid, a PP-1/2A inhibitor, increases D1-

induced potentiation suggesting that constitutively active PP-

1/2A attenuated D1-induced potentiation. Thus phosphoryla-

tion of NMDA receptor subunits by activation of the D1

signaling cascade is a potential mechanism contributing to the

modulation demonstrated in these experiments (Greengard et

al. 1999). Finally, activation of D1 receptors produces differ-

ential effects on NMDA and GABA currents in the same cells,enhancing NMDA currents and inhibiting GABA currents.Virtually all neurons examined displayed potentiation in

response to application of D1 agonists. This is at variance withthe hypothesis that D1 and D2 receptors are segregated ontodifferent populations of medium-sized spiny striatal neurons(Gerfen 1992). This hypothesis remains controversial (Sur-meier et al. 1993). For example, a previous study using single-cell reverse transcriptase coupled with PCR demonstrated thatalmost all striatal neurons displayed subtypes of both D1 andD2 family receptors, either the D1 or D5 receptor and one ormore of the D2, D3, or D4 subtypes (Surmeier et al. 1996).More recently, additional evidence for co-localization of D1and D2 receptors on medium-sized striatal spiny neurons has

been provided (Aizman et al. 2000). However, it should bepointed out that our studies, at least in rats, used youngeranimals, and there may be maturational differences in DAreceptor segregation that occur after early developmental pe-

FIG. 6. A, top: currents activated by GABA (100 M) alone (left), in the presence of SKF 81297 (10 M;middle), and GABA(100 M) alone again (wash; right). Bottom: currents activated by NMDA (100 M) alone (left), in the presence of SKF 81297(10M;middle), and NMDA (100M) alone again (Wash; right) in the same neuron from a rat. Note that SKF 81297 attenuatesGABA-induced currents while it enhances NMDA-induced currents. B: bar graphs showing that application of SKF 81297 inhibitsGABA currents while concomitantly enhancing NMDA currents in the same group of neurons. Asterisks indicate the decreases andincreases in modulation were statistically significant (see text for statistics).




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riods. Based on the evidence cited in the preceding text, it isnot particularly surprising that virtually all neurons displayedD1-induced potentiation and that it could be reduced by a D2family agonist. What did vary from neuron-to-neuron was themagnitude of the potentiation or its attenuation, a variable thatcould reflect the density of D1 or D2 family receptors ex-pressed by specific neurons or the degree to which dendriticprocesses remained after the dissociation. Although there wasa clear dose-response relationship within individual neurons(see Fig. 3), the degree of modulation at the higher concentra-tions of the D1 agonist varied.

Multiple mechanisms for D1 receptor modulation of NMDAreceptors

Multiple mechanisms are capable of mediating the D1 ago-nist-induced potentiation of NMDA-evoked responses. Elimi-nation of each mechanism alone decreases the potentiation butdoes not eliminate it, suggesting these mechanisms operateindependently but synergistically.

One of the mechanisms involves the D1/cAMP/PKA cas-cade. Forskolin, an activator of this cascade enhances NMDA

responses (Blank et al. 1997; Colwell and Levine 1995). Fur-thermore, D1 receptor activation increases NR1 subunit phos-phorylation via the DARPP-32 signaling cascade (Fienberg etal. 1998; Rajadhyaksha et al. 1998; Snyder et al. 1998).DARPP-32 when phosphorylated by PKA acts as a potentinhibitor of PP-1. Mutant mice that lack DARPP-32 have asignificant reduction in D1-induced NMDA receptor modula-tion. Although potentiation still exists, increasing the concen-tration of the D1 agonist does not increase the potentiation asmuch in the knock out as it does in the WT. Okadaic acid, aPP-1/2A inhibitor further increased potentiation providing ad-ditional evidence for the importance of this cascade. Thefinding that okadaic acid restored D1 modulation in theDARPP-32 mutant suggests the system remains intact with

little compensatory change in the mutant mice. We also ob-served that NMDA currents were slightly decreased in theDARPP-32 knockout mice. While this effect was not statisti-cally significant, possibly DARPP-32 plays a small constitu-tively active role in maintaining the levels of excitability ofNMDA receptors and/or in their state of phosphorylation. Insummary, our evidence indicates that increased NMDA recep-tor phosphorylation (probably of the NR1 subunit) mediated byactivation of PKA and inhibition of PP-1/2A can contribute tothe increase in membrane current (Greengard et al. 1999).

In the current study, we have presented data from both ratsand mice, albeit of different ages. We purposely did not makedirect comparisons because of the age differences. Althoughwe used younger rats, our developmental studies indicate thatby 3 wk of age, responses to glutamate receptor agonists anddopaminergic modulation are relatively mature (Cepeda et al.1998a,b; Colwell et al. 1998; Hurst et al. 2001). However, inboth species, D1-induced potentiation was observed and atleast the effects of okadaic acid were similar, suggesting thatcomparable mechanisms may underlie the potentiation.

Another mechanism contributing to D1 potentiation ofNMDA responses involves L-type Ca2 conductances. Block-ade of L-type Ca2 conductances reduces the enhancement ofNMDA currents in striatal slices (Cepeda et al. 1998a). Thereis good evidence that D1 receptor activation enhances L-type

Ca2 currents in medium-sized striatal neurons possibly via

direct phosphorylation of the channel by PKA and inhibition of

PP-1 (Surmeier et al. 1995), causing increased intracellular

Ca2. Although we could not rule out space-clamp limitations

in our previous study in the slice (Cepeda et al. 1998a), in the

present experiments, we attempted to minimize the contribu-

tion of voltage-gated conductances to determine if the enhance-

ment occurred independent of their modulation. In dissociated

cells using a holding potential of 40 mV, which wouldfurther decrease the influences of L-type conductances, theaddition of the L-type Ca2 channel antagonist nifedipine did

not alter D1-induced potentiation. These findings indicate thatthe role of L-type Ca2 channels was minimized in dissociated

cells. In addition, most of the dendrites were removed by the

dissociation procedure. L-type Ca2 conductances have a more

important role when dendrites are present (Cepeda et al.

1998a). In fact, L-type Ca2 channels are present on dendritic

spines and interact with NMDA receptors (Carlin et al. 2000;

Segal 1995).

A recent study demonstrated in cells from nucleus accum-

bens slices that PKC inhibition blocked the enhancement of

NMDA currents by D1 agonists (Chergui and Lacey 1999).However, in the present study, we demonstrate that this effectdoes not occur in dissociated striatal neurons, indicating thatsignaling mechanisms may differ depending on neuronal loca-tion or the type of preparation.

Other mechanisms that can contribute to the enhancement ofNMDA responses by D1 agonists include modulation of ionicpumps and regulation of intracellular Ca2 stores (Bertorello etal. 1990; Mahan et al. 1990) as we have reviewed previously(Cepeda and Levine 1998). A recent report provided evidencefor a unique D1-NMDA receptor interaction in the control ofmembrane trafficking (Dunah and Standaert 2001). D1 recep-tor activation caused rapid movement of NMDA receptor sub-units between intracellular and postsynaptic sites. Conversely,

activation of NMDA receptors recruits functional D1 receptorsfrom the interior of the cell to the plasma membrane, inparticular dendritic spines, without affecting the distribution ofD2 receptors, and this effect is abolished by incubation of cellsin Ca2-free medium (Scott et al. 2002). Finally, there is recentevidence that there is direct protein-protein coupling betweenDA receptors and subunits of GABAA and NMDA receptors(Liu et al. 2000a,b). Overall, these multiple mechanisms permita high degree of synergy and redundancy in signaling throughactivation of D1 and NMDA receptors.

In dissociated cells in the present study, we found that D2receptor activation significantly reduced the enhancement ofNMDA currents produced by the D1 agonist. This effect can-not be easily explained by the actions of the D2 agonist alone.The D2 agonist produced a 4% average reduction alone but a13% reduction in the presence of the D1 agonist. Although it ispossible that the dissociation procedure disrupts aspects of D2receptor function, an enabling effect of D1 activation on D2function has been reported previously for nucleus accumbensneurons (Wachtel and White 1995; White 1987). Thus it ap-pears that D1 receptors may need to be activated beforepostsynaptic D2-mediated effects become apparent. In the cellsthat co-localize D1 and D2 family receptors (Surmeier et al.1996), the antagonism of D1 effects by D2 receptors mayprovide a protective mechanism against excessive excitation




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induced when NMDA and D1 receptors are simultaneouslyactivated (Cepeda et al. 1998b).

The potentiation in the NMDA current caused by the D1agonist was reduced by co-application of the D1 antagonistSKF 83566, suggesting that the potentiation was specific toactivation of D1 receptors. The decrease in average potentia-tion could not be accounted for by the effects of the D1antagonist alone at the 1 M concentration. Previous studieshave shown that D1 receptor-mediated effects, at least onGABA currents, can be blocked by D1 antagonists in bothdissociated and cultured striatal neurons (Flores-Hernandez etal. 2000), although in acutely isolated neurons, there is evi-dence that D1 receptor activation reduces specific K conduc-tances via allosteric regulation of the channels rather than viacoupling to adenylyl cyclase (Nisenbaum et al. 1998).

DA modulation of responses mediated by activation ofglutamate and GABA receptors

There is considerable evidence for DA modulation of glu-tamate- and GABA-evoked responses in striatum, nucleus ac-cumbens, and cerebral cortex (Blank et al. 1997; Cepeda et al.

1992, 1993, 1998a; Chergui and Lacey 1999; Colwell andLevine 1995; Flores-Hernandez et al. 2000; Galarraga et al.1997; Gonon and Sundstrom 1996; Harvey and Lacey 1997;Hernandez-Lopez et al. 1997; Hsu et al. 1995; Levine et al.1996a,b; Price et al. 1999; Seamans et al. 2001; Wang andODonnell 2001; Yan et al. 1999; Zheng et al. 1999; but seeCalabresi et al. 1995; Nicola and Malenka 1998). The differ-ential effects of D1 receptor activation on NMDA and GABAresponses in the same neuron represent an important mecha-nism for amplification of synaptic input. Previous studies havedemonstrated that activation of GABA receptors substantiallyreduces responses mediated by glutamate receptor activationby a combination of pre- and postsynaptic mechanisms (Cala-bresi et al. 1991). If DA is released simultaneously and D1

receptors are activated, there will be a simultaneous attenuationof the GABAergic response and a potentiation of the glutama-tergic response, effectively amplifying corticostriatal inputs.Thus simultaneous activation of D1 and NMDA receptorswould predispose medium-sized spiny neurons toward a moredepolarized membrane potential, especially when their mem-brane potential is in the up-state (Wilson and Kawaguchi1996). If GABA receptors are activated in this condition, theinhibitory response would be attenuated by D1 effects, eitherprolonging the enhancement or further potentiating excitatoryinputs. Interestingly, under these conditions, if DA also acti-vates D2 receptors, the amplification will be reduced.

Functional implications

The findings indicate that D1 receptor activation enhancesNMDA currents, in part through a signaling cascade involvingDARPP-32 and PP-1. Taken together with our previous evi-dence that Ca2 currents can contribute to the modulation ofNMDA responses, these results show that multiple pathwaysappear to be involved in the modulation of NMDA responsesby D1 receptors. There are important implications of the D1-NMDA receptor interactions. At a morphological level, theenhancement of NMDA responses by activation of D1 recep-tors could effectively stabilize and reinforce corticostriatal

synaptic connections via interactions in spines. For example,PP-1 regulated by DARPP-32 is particularly abundant in thepostsynaptic density fraction (Shields et al. 1985; Sim et al.1994) and is enriched in dendritic spines (Ouimet et al. 1995),where most excitatory inputs converge. PP-1 interacts withdifferent targeting proteins such as spinophillin and neurabinand may control spine morphology, dynamics, and synapticefficacy (Harris and Kater 1994). As pointed out in the pre-ceding text, there is now evidence that D1 receptor activationalters trafficking of NMDA subunits (Dunah and Standaert2001) and NMDA receptor activation recruits D1 receptorsinto the membrane (Scott et al. 2002). At the functional level,D1-NMDA receptor interactions appear to be involved in stri-atal synaptic plasticity. They support the induction of long-term potentiation (Charpier and Deniau 1997; Kerr and Wick-ens 2001), and such potentiation is absent in DARPP-32-deficient mice (Calabresi et al. 2000). In conclusion, it is clearthat the unique potentiation of NMDA receptor function byactivation of the D1 receptor signaling cascade has majorinfluences on neuronal function.

The authors acknowledge D. Crandall and C. Gray for preparation of the

illustrations.This work was supported by National Institutes of Health Grants NS-33538and NS-35649 to M. S. Levine and MH-40899 and DA-10044 to P. Greengard,by National Association Research in Schizophrenia and Depression to M. S.Levine, and by Office of Naval Research Grant N00149810436 to M. S.Levine.

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