D1 Dopamine Receptor Activation Reduces GABAA Receptor ...

D 1 Dopamine Receptor Activation Reduces GABA A Receptor 

Currents in Neostriatal Neurons Through a PKA/DARPP-32/PP1 

Signaling Cascade 

JORGE FLORES-HERNANDEZ, 1 SALVADOR HERNANDEZ, 1 GRETCHEN L. SNYDER, 2 ZHEN YAN, 2 

ALLEN A. FIENBERG, 2 STEPHEN J. MOSS, 3 PAUL GREENGARD 2 , AND D. JAMES SURMEIER 1 

1 Department of Physiology and Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611; 

2 Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York City, New York 10021; and 3 Medical 

Research Council/Laboratory of Molecular Cell Biology and Department of Pharmacology, University College, London 

WC1E 6BT, United Kingdom 

Flores-Hernandez, Jorge, Salvador Hernandez, Gretchen L. Snyder, 

Zhen Yan, Allen A. Fienberg, Stephen J. Moss, Paul Greengard, 

and D. James Surmeier. D 1 dopamine receptor activation 

reduces GABA A receptor currents in neostriatal neurons through a 

PKA/DARPP-32/PP1 signaling cascade. J. Neurophysiol. 83: 

2996–3004, 2000. Dopamine is a critical determinant of neostriatal 

function, but its impact on intrastriatal GABAergic signaling is poorly 

understood. The role of D 1 dopamine receptors in the regulation of 

postsynaptic GABA A receptors was characterized using whole cell 

voltage-clamp recordings in acutely isolated, rat neostriatal medium 

spiny neurons. Exogenous application of GABA evoked a rapidly 

desensitizing current that was blocked by bicuculline. Application of 

the D 1 dopamine receptor agonist SKF 81297 reduced GABA-evoked 

currents in most medium spiny neurons. The D 1 dopamine receptor 

antagonist SCH 23390 blocked the effect of SKF 81297. Membranepermeant 

cAMP analogues mimicked the effect of D 1 dopamine 

receptor stimulation, whereas an inhibitor of protein kinase A (PKA; 

Rp-8-chloroadenosine 3,5 cyclic monophosphothioate) attenuated 

the response to D 1 dopamine receptor stimulation or cAMP analogues. 

Inhibitors of protein phosphatase 1/2A potentiated the modulation by 

cAMP analogues. Single-cell RT-PCR profiling revealed consistent 

expression of mRNA for the 1 subunit of the GABA A receptor—a 

known substrate of PKA—in medium spiny neurons. Immunoprecipitation 

assays of radiolabeled proteins revealed that D 1 dopamine 

receptor stimulation increased phosphorylation of GABA A receptor 

1/3 subunits. The D 1 dopamine receptor-induced phosphorylation 

of 1/3 subunits was attenuated significantly in neostriata from 

DARPP-32 mutants. Voltage-clamp recordings corroborated these 

results, revealing that the efficacy of the D 1 dopamine receptor modulation 

of GABA A currents was reduced in DARPP-32-deficient 

medium spiny neurons. These results argue that D 1 dopamine receptor 

stimulation in neostriatal medium spiny neurons reduces postsynaptic 

GABA A receptor currents by activating a PKA/DARPP-32/protein 

phosphatase 1 signaling cascade targeting GABA A receptor 1 

subunits. 

INTRODUCTION 

Disordered neostriatal dopaminergic signaling is a central 

determinant of a variety of psychomotor illnesses including 

Parkinson’s disease, schizophrenia, and drug abuse (Grace et 

The costs of publication of this article were defrayed in part by the payment 

of page charges. The article must therefore be hereby marked “advertisement” 

in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 

al. 1998; Hornykiewcz 1973; Koob et al. 1997; Wise 1998). 

Although dopamine is known to modulate voltage-dependent 

ion channels in neostriatal neurons (e.g., Surmeier et al. 1992, 

1995), its regulation of classical neurotransmission is less 

clearly defined (Calabresi et al. 1993; Cepeda et al. 1998; Kita 

et al. 1995; Mercuri et al. 1985; Nicola and Malenka 1998; Yan 

and Surmeier 1997). This is particularly true of GABAergic 

signaling. Inhibitory GABAergic synaptic input to neostriatal 

neurons is thought to be entirely of intrinsic origin, arising 

either from recurrent collaterals of GABAergic medium spiny 

projection neurons or from GABAergic interneurons (Kita 

1993; Wilson and Groves 1980). Only a handful of studies 

have attempted to determine how dopamine influences this 

intrastriatal GABAergic pathway. Most of those studies have 

focused on dopamine’s actions at D 1 dopamine receptors. For 

example, D 1 dopamine receptor stimulation increases GABA 

release (Harsing and Zigmond 1997). However, D 1 dopamine 

receptor agonists appear to be ineffective in modulating 

GABAergic synaptic potentials in dorsal neostriatal neurons, in 

spite of the fact that they inhibit GABAergic signaling in the 

nucleus accumbens (Nicola and Malenka 1998; cf. Calabresi et 

al. 1993). 

At face value, the inability of D 1 dopamine receptor agonists 

to modulate GABAergic synaptic potentials in medium spiny 

neurons is surprising. D 1 dopamine receptors are expressed by 

the majority of medium spiny neurons (Gerfen 1992; Surmeier 

et al. 1996). These receptors positively couple to adenylyl 

cyclase, resulting in the stimulation of protein kinase A (PKA) 

(Stoof and Kebabian 1984; Walaas and Greengard 1991). PKA 

has been shown to modulate GABA A receptor-mediated currents 

in both heterologous and native expression systems 

(Smart 1997). 

The preferred substrate for PKA in the GABA A receptor 

oligomer is the subunit. But of the three cloned subunits 

found in neurons (1–3), only 1 and 3 are efficiently phosphorylated 

by PKA (McDonald et al. 1998). Moreover, the 

functional consequences of 1 and 3 subunit phosphorylation 

are qualitatively different. In heterologous systems, phosphorylation 

of 1 subunits results in diminished GABA A receptor 

currents, whereas phosphorylation of 3 subunits leads to 

increased currents (McDonald et al. 1998). This suggests that 

by controlling the expression of subunits, their assembly into 

2996 0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society

D 1 MODULATION OF GABA A RECEPTORS 

2997 

functional receptors or their accessibility, neurons can regulate 

the consequences of PKA activation. An example of how this 

type of regulation might work has been reported in the hippocampus 

where PKA diminished GABAergic miniature inhibitory 

postsynaptic currents (mIPSCs) in CA1 pyramidal 

neurons that express 1 subunits, whereas PKA had no effect 

on mIPSCs in granule cells that express primarily 2 subunits 

(Poisbeau et al. 1999). Although it is clear that medium spiny 

neurons express GABA A receptors, the contribution of 1, 2, 

and 3 subunits to these channels has not been studied. 

To provide a more thorough examination of their regulation 

by D 1 dopamine receptors, GABA A receptors in acutely isolated, 

voltage-clamped medium spiny neurons were stimulated 

with exogenous GABA. These experiments revealed a consistent 

decrement in GABA-evoked currents after D 1 dopamine 

receptor stimulation. These observations then were pursued 

with a combination of molecular, biochemical, and electrophysiological 

techniques to determine the signaling pathway 

linking D 1 dopamine receptors to GABA A receptors. 

METHODS 

Electrophysiological methods 

ACUTE-DISSOCIATION PROCEDURE. Neostriatal neurons from adult 

(4 wk) rats were acutely dissociated using procedures similar to 

those previously described (Song et al. 1998; Surmeier et al. 1996; 

Yan and Surmeier 1997). In brief, rats were anesthetized with methoxyflurane 

and decapitated; brains were removed quickly, iced, and 

then blocked for slicing. The blocked tissue was cut into 400-m 

slices with a Vibroslice (Campden Instruments, London) while bathed 

in a low-Ca 2 (100 M), N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic 

acid] (HEPES)-buffered salt solution [containing (in mM) 

140 Na isethionate, 2 KCl, 4 MgCl 2 , 0.1 CaCl 2 , 23 glucose, and 15 

HEPES, pH 7.4, 300–305 mosm/l]. Slices then were incubated for 

1–6 h at room temperature (20–22°C) in a NaHCO 3 -buffered saline 

bubbled with 95% O 2 -5% CO 2 [which contained (in mM) 126 NaCl, 

2.5 KCl, 2 CaCl 2 , 2 MgCl 2 , 26 NaHCO 3 , 1.25 NaH 2 PO 4 ,1 pyruvic 

acid, 0.2 ascorbic acid, 0.1 N G -nitro-L-arginine, 1 kynurenic acid, and 

10 glucose, pH 7.4 with NaOH, 300–305 mosm/l]. All reagents 

were obtained from Sigma Chemical (St. Louis, MO). Slices then 

were transferred into the low-Ca 2 buffer and regions of the dorsal 

neostriatum dissected and placed in an oxygenated Cell-Stir chamber 

(Wheaton, Millville, NJ) containing pronase (Sigma protease Type 

XIV, 1–3 mg/ml) in HEPES-buffered Hank’s balanced salt solution 

(HBSS, Sigma Chemical) at 35°C. Dissections were limited to tissue 

rostral to the anterior commissure. After 20–40 min of enzyme 

digestion, tissue was rinsed three times in the low-Ca 2 , HEPESbuffered 

saline and mechanically dissociated with a graded series of 

fire-polished Pasteur pipettes. The cell suspension then was plated into 

a 35-mm Lux petri dish mounted on the stage of an inverted microscope 

containing HEPES-buffered HBSS saline. 

In some experiments, cultured neostriatal neurons were used. Cultures 

were generated as previously described from E18 Sprague- 

Dawley rat pups (Surmeier et al. 1988). After 3 days in vitro, cultures 

were transferred to defined media consisting of Neurobasal media 

supplemented with B-27, Pen/Strep, L-glutamine (0.5 mM; Life Technologies), 

brain derived neurotrophic factor (BDNF, 50 nM; Promega), 

and glial derived neurotrophic factor (GDNF, 30 nM; Promega). 

The media was partially replaced (50%) every 4 days 

thereafter. Cultures were used for recording 10–14 days after plating. 

WHOLE CELL RECORDINGS. Recordings of GABA-activated currents 

employed standard techniques (Yan and Surmeier 1997). Electrodes 

were pulled from Corning 7052 glass and fire-polished before 

use. For acutely isolated neurons, the internal solution consisted of (in 

mM) 180 N-methyl-D-glucamine (NMG), 40 HEPES, 4 MgCl 2 , 5 1,2 

bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA), 

12 phosphocreatine, 2 Na 2 ATP, 0.2 Na 3 GTP, and 0.1 leupeptin, pH 

7.2–3 with H 2 SO 4 , 265–270 mosm/l. The external solution consisted 

of (in mM) 135 NaCl, 20 CsCl, 1 MgCl 2 , 10 HEPES, 0.001 TTX, 0.5 

BaCl 2 , and 10 glucose, pH 7.3 with NaOH, 300–305 mosm/l. For 

cultured neurons, the internal solution consisted of (in mM) 30 CsCl, 

80 K 2 SO 4 , 10 HEPES, 6 MgCl 2 , 3.5 BAPTA, 12 phosphocreatine, 2 

Na 2 ATP, 0.2 Na 3 GTP, and 0.1 leupeptin, pH 7.2–3 with H 2 SO 4 , 

265–270 mosm/l. The external solution consisted of (in mM) 140 

NaCl, 2.8 KCl, 1 MgCl 2 , 10 HEPES, 0.001 TTX, 0.5 CaCl 2 , and 10 

glucose, pH 7.3 with NaOH, 300–305 mosm/l. 

Recordings were obtained with an Axon Instruments 200 patchclamp 

amplifier that was controlled and monitored with a PC 486 

clone running pCLAMP (v. 6.0) with a DigiData 1200 series interface 

(Axon Instruments, Foster City, CA). Electrode resistances were 

typically 2–4 M in the bath. After seal rupture, series resistance 

(4–10 M) was compensated (70–90%) and periodically monitored. 

Drugs were applied with a gravity-fed “sewer pipe” system. The array 

of application capillaries (ca. 150 m ID) was positioned a few 

hundred micrometers from the cell under study. Solution changes 

were made by altering the position of the array with a microprocessorcontrolled 

DC drive system (Newport-Klinger, Irvine, CA). Solution 

changes were complete within 1 s. The membrane potential was 

held at 0 mV in recordings from acutely isolated neurons; the membrane 

potential was held at 80 mV in recordings from cultured 

neurons. GABA (0.1–2,000 M) was applied briefly (3–5 s) every 

minute 

Dopamine receptor ligands—dopamine, SKF-81297 and R()- 

SCH-23390 (RBI, Natick, MA)—were made up as concentrated 

stocks in deoxygenated water containing 0.1% sodium metabisulfite. 

Solutions were protected from ambient light. Second-messenger reagents 

Sp-8-chloroadenosine 3:5-cyclic monophosphothioate (Sp- 

Cl-cAMPS), Rp-8-chloroadenosine 3,5 cyclic monophosphothioate 

(Rp-Cl-cAMPS), 3,6 dichloro-1-b-D-ribofuranosylbenzimidazole 

3,5 cyclic monophosphothioate (Sp-5,6-DCl-cBIMPS), 8-(4-chlorophenylthio) 

guanosine-3,5 monophosphate (8-pCPT-cGMP) (Biolog 

Life Sciences, La Jolla, CA) were made up as concentrated stocks in 

water or dimethyl sulfoxide and stored at 70°C. Stocks were thawed 

and diluted immediately before use. 

STATISTICAL METHODS. Data analyses were performed with Axo- 

Graph (Axon Instruments, ver. 2.0) and SYSTAT (Chicago, IL). Box 

plots were used for graphic presentation of the data because of the 

small sample sizes (Tukey 1977). The box plot represents the distribution 

as a box with the median as a central line and the hinges as the 

edges of the box (the hinges divide the upper and lower halves of the 

distributions in half). The inner fences (shown as a line originating 

from the edges of the box) run to the limits of the distribution 

excluding outliers (defined as points that are 1.5 times the interquartile 

range beyond the interquartiles) (Tukey 1977); outliers are shown as 

asterisks or circles. Nonparametric statistical tests (Kruskal-Wallis 

ANOVA, Mann-Whitney U test) were used because of the small 

sample sizes and the uncertain sampling distributions. 

Single-neuron RNA harvest and RT-PCR analysis 

Methods similar to those previously described were used (Song et 

al. 1998; Tkatch et al. 1998; Yan and Surmeier 1997). Briefly, after 

recording, neostriatal neurons were lifted up into a stream of control 

solution and aspirated into the electrode by negative pressure. Electrodes 

contained 5 l of sterile recording solution (see preceding 

text). The capillary glass used for making electrodes had been autoclaved 

and heated to 150°C for 2 h. Sterile gloves were worn during 

the procedure to minimize RNase contamination. After aspiration, the 

electrode was broken and contents ejected into a 0.5-ml Eppendorf 

tube containing 5 l diethyl pyrocarbonate (DEPC)-treated water, 1

2998 FLORES-HERNANDEZ ET AL. 

l RNasin (28 U/l), 1 l dithiothreitol (DTT; 0.1 M), and 1 l of 

oligo(dT) (0.5 g/l) primer. The mixture was heated to 70°C for 10 

min and incubated on ice for 1 min. Single-strand cDNA was synthesized 

from the cellular mRNA by adding SuperScript II RT (1 l, 

200 U/l) and buffer (4 l, 5 first strand buffer: 250 mM Tris-HCl, 

375 mM KCl, 15 mM MgCl 2 ), DTT (1 l, 0.1 M) and mixed dNTPs 

(1 l, 10 mM). The mixture (20 l) was incubated for 50 min in a 

42°C water bath. The reaction was terminated by heating the mixture 

to 70°C for 15 min and then icing. The RNA strand in the RNA-DNA 

hybrid then was removed by adding 1 l RNase H (2 U/l), and the 

solution was incubated for 20 min at 37°C. All reagents except for 

RNasin (Promega, Madison, WI) were obtained from Life Technologies 

(Grand Island, NY). The cDNA from the reverse transcription 

(RT) of RNA in single neostriatal neurons was subjected to polymerase 

chain reactions (PCR) to detect the expression of various mRNAs. 

PCR amplification was carried out with a thermal cycler (MJ 

Research, Watertown, MA) with thin-walled plastic tubes. Conventional 

45-cycle PCR amplification was used for the detection of ChAT 

mRNA. Reaction mixtures contained 2–2.5 mM MgCl 2 , 0.5 mM of 

each of the dNTPs, 0.8–1 M primers, 2.5 U Taq DNA polymerase 

(Promega), 5 l 10 buffer (Promega), and 1–2 l cDNA template 

made from the single cell RT reaction (see preceding text). The 

thermal cycling program for these PCR amplifications was: 94°C for 

1 min, 58°C for 1 min, and 74°C for 1.5 min. To detect GABA A 

receptor subunit mRNAs (1–4, 1–3) in single cells, degenerate and 

“nested” primers were employed. In the first step, degenerate primers 

targeting the conserved regions of all the subunits were mixed with 

one-fourth of the single cell cDNA template and 30 rounds of amplification 

were performed. The same procedure was used to amplify 

subunit cDNA. In the second step, an aliquot (2 l) of diluted (1:10) 

first-round PCR product was used as the template for 40-cycle PCR 

with subunit-specific nested primers. These primers were positioned 

inside the region spanned by the degenerate primers. The primer sets 

used have been published previously (Surmeier et al. 1996; Yan and 

Surmeier 1997). 

Products were visualized by staining with ethidium bromide and 

analyzed by electrophoresis in 2% agarose gels. All products were 

sequenced using a dye termination procedure and found to match the 

published sequences. Care was taken to ensure that the PCR signal 

arose from cellular mRNA. In addition to the controls noted above 

(e.g., primers that span splice sites), negative controls for contamination 

from extraneous and genomic DNA were run for every batch of 

neurons. To ensure that genomic DNA did not contribute to the PCR 

products, neurons were aspirated and processed in the normal manner 

except that the reverse transcriptase was omitted. Contamination from 

extraneous sources was checked by replacing the cellular template 

with water. Both controls were consistently negative in these experiments. 

Preparation, radioactive labeling, and treatment of 

neostriatal slices 

Male C57BL/6 mice (8–12 wk of age) were killed by decapitation. 

The brain was removed rapidly from the skull and transferred to an 

ice-cold surface where it was blocked then mounted to the cutting 

surface of a Vibratome (TPI). Coronal sections (400 m) of the brain 

were cut and pooled in 10 ml of ice-cold, oxygenated calcium-free, 

phosphate-free Krebs bicarbonate buffer containing the following 

components (in mM): 125 NaCl, 4 KCl, 26 NaHCO 3, 1.5 MgSO 4 , 0.5 

EGTA, and 10 glucose (pH 7.4). Slices of neostriatum were dissected 

from these coronal sections under a dissecting microscope. The slices 

were pooled in a dish of cold buffer and transferred individually to 

4-ml polypropylene centrifuge tubes containing 2 ml of fresh buffer at 

4°C. The Krebs bicarbonate buffer then was replaced with fresh 

solution. The tubes were connected to an oxygenation manifold supplying 

a 95%O 2 -5%CO 2 mix and maintained in a 30°C water bath. 

After 15 min the buffer was replaced with a fresh phosphate-free, 

oxygenated Krebs bicarbonate buffer containing 1.5 mM CaCl 2 and 

lacking EGTA. A 2.0-mCi aliquot of [ 32 P]orthophosphoric acid (Du- 

Pont NEN; specific activity 8,500–9,120 Ci/mmol) was added to each 

tube, and the tissue was preincubated for 60 min. The radioactive 

buffer then was removed, and tissue sections were rinsed twice with 

2 ml of fresh buffer. The tissue was incubated in the absence or 

presence of test substances, as indicated. At the end of the incubation, 

the buffer was rapidly aspirated, and the tissue slices were immediately 

frozen in liquid nitrogen and stored at 80°C until assayed. 

Immunoprecipitation and analysis of [ 32 P]phosphate-labeled 

GABA A receptor subunit 

[ 32 P]phosphate-labeled tissue slices were sonicated in 150 lof1% 

sodium dodecyl sulfate (SDS) containing NaF (50 mM) and 1 mM 

EGTA added as phosphatase inhibitors, and a cocktail of protease 

inhibitors, including 25 mM benzamidine, 100 M phenylmethylsulfoxide, 

20 g/ml chymostatin, 20 g/ml pepstatin A, 5 g/ml leupeptin, 

and 5 g/ml antipain (Peptide International). To this homogenate 

were added 5 volumes of Buffer A, composed of 20 mM 

Tris/HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 

0.2% bovine serum albumen (BSA) and the cocktail of phosphatase 

and protease inhibitors described above. Aliquots of the homogenate 

(10 l) were retained for the determination of total [ 32 P]phosphate 

incorporation into trichloroacetic acid-precipitated protein. A 10 mg 

aliquot of preswollen Protein A-Sepharose CL-4B (Pharmacia Biotech) 

was added to each sample and the mixture agitated for 30 min 

at 4°C. The Sepharose beads were pelleted by centrifugation for 15 s 

at 2,000 rpm in a tabletop microcentrifuge. The supernatant was 

transferred to tubes containing 2.5 g of an antiserum generated 

against a peptide sequence contained in both the 1 and 3 subunits 

of the GABA A receptor. The samples were mixed for 2hat4°C, then 

transferred to fresh 1.5-ml Eppendorf tubes containing 10 mg preswollen 

Protein A-Sepharose Cl-4B beads and mixed for 1hat4°C. 

The beads were pelleted by centrifugation and washed once with 1 ml 

of Buffer A; three times with 1 ml of a buffer containing 20 mM 

Tris/HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 

0.1% SDS, and 0.2% BSA; three times with a buffer containing 20 

mM Tris/HCl, pH 8.0, 500 mM NaCl, 0.5% Triton X-100, and 0.2% 

BSA; and once with 1 ml of a buffer containing 50 mM Tris/HCl, pH 

8.0. After the final wash the beads were resuspended in 50 l ofa 

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer 

composed of 50 mM Tris/HCl (pH 6.7), 10% glycerol, 2% SDS, 10% 

2-mercaptoethanol, and 0.01% bromphenol blue. The tubes were 

vortexed vigorously and the beads were centrifuged. The recovered 

proteins were separated on 10% acrylamide gels. The gels were dried, 

and [ 32 P]phosphate incorporation was quantified using a PhosphorImager 

400B and ImageQuant software from Molecular Dynamics. 

Values for [ 32 P]phosphate content were normalized for the total 

[ 32 P]phosphate incorporated into TCA-precipitable protein. 

RESULTS 

D 1 dopamine receptor stimulation reduces GABA-evoked 

currents 

The application of GABA (100 M) evoked a rapidly desensitizing 

current in medium spiny neurons voltage clamped 

at 0 mV. The current was blocked by bicuculline (100 M) and 

reversed near the Cl reversal potential (data not shown), 

implicating GABA A receptors. As shown in Fig. 1A, the D 1 

dopamine receptor agonist SKF 81297 (1 M) reversibly decreased 

GABA-evoked currents in the majority of medium 

spiny neurons (20/28) initially sampled (n 20, P 0.05, 

Kruskal-Wallis ANOVA). On average, peak whole cell currents 

evoked by GABA (100 M) were reduced by 18% by


2999 

cyclase, exogenous application of membrane permeant cAMP 

analogues should mimic the receptor-driven modulation. To 

test this hypothesis, Sp-Cl-cAMPS (100 nM) was perfused 

during whole cell recording. As shown in Fig. 2A, Sp-ClcAMPS 

decreased GABA-evoked currents in a manner similar 

to the D 1 dopamine receptor agonist (n 12, P 0.05, 

Kruskal-Wallis ANOVA). The median decrease in peak 

evoked current was similar (22%) to that of SKF 81297 (Fig. 

2B). The membrane permeant cGMP analogue 8-pCPT-cGMP 

(100 M) had no effect on GABA-evoked currents, arguing 

that the modulation was PKA specific (n 7, P 0.05, 

Kruskal-Wallis ANOVA). 

FIG. 1. D 1 dopamine receptor agonists reduce GABA-evoked currents. A: 

whole cell voltage-clamp recordings from an acutely isolated medium spiny 

neuron showing currents evoked by GABA (100 M) application in the 

absence (control) and presence of SKF 81297 (1 M). Response was reversed 

on washing off the D 1 dopamine receptor agonist. Right: box plot summarizing 

the reduction in peak GABA-evoked currents produced by SKF 81297 (n 

21). As shown, the coapplication of the D 1 dopamine receptor antagonist SCH 

23390 (1 M) with SKF 81297 significantly attenuated the modulation (n 9, 

P 0.05, Kruskal-Wallis ANOVA). B: application of SKF 81297 also 

reversibly decreased inward GABA-evoked currents in cultured striatal neurons. 

SKF 81297 (0.1–1 M). The kinetics of the evoked currents 

were not noticeably altered by D 1 dopamine receptor agonists. 

Coapplication of the D 1 dopamine receptor antagonist SCH 

23390 (1 M) blocked the reduction in evoked currents produced 

by SKF 81297 (n 9, P 0.05, Kruskal-Wallis 

ANOVA; Fig. 1A). To verify that the D 1 receptor-mediated 

modulation was not a consequence of dissociation, cultured 

neostriatal neurons also were studied. In these recordings, the 

Cl reversal potential was near 0 mV, and the cell was held at 

80 mV. As shown in Fig. 1B, the D 1 receptor agonist SKF 

81297 (1 M) also reversibly reduced the inward currents 

evoked by GABA (100 M) in these neurons. More than 

one-half of the cultured neurons responded to SKF 81297 

(11/17). In the responsive subset, SKF 81297 reduced the 

bicuculline-sensitive GABA-evoked currents by an average of 

22 4% (mean SD). 

Activation of D 1 dopamine receptors in medium spiny neurons 

leads to the stimulation of adenylyl cyclase and the 

elevation of cytosolic cyclic AMP (cAMP) (Stoof and Kebabian 

1984). If the modulation of GABA A receptors by D 1 

dopamine receptors was mediated by stimulation of adenylyl 

FIG. 2. cAMP analogues mimic the D 1 dopamine receptor modulation of 

GABA-evoked currents and protein phosphatase 1/protein phosphatase 2A 

(PP1/PP2A) inhibition enhances the modulation. A: whole cell voltage-clamp 

recordings from an acutely isolated medium spiny neuron showing currents 

evoked by GABA (100 M) application in the absence (control) and presence 

of the protein kinase A (PKA) agonist Sp-8-chloroadenosine 3:5-cyclic 

monophosphothioate (Sp-Cl-cAMPS; 10 M). Response was reversed on 

washing. B: box plot summarizing the reduction in peak GABA-evoked 

currents produced by SKF 81297 (n 21), Sp-Cl-cAMPS (n 12) and the 

PKG agonist 8-(4-chlorophenylthio) guanosine-3,5 monophosphate (8- 

pCPT-cGMP; n 7). Modulation was similar with the cAMP analogue and the 

receptor agonist but 8-pCPT-cGMP failed to significantly modulate the currents 

(P 0.05, Kruskal-Wallis ANOVA). C: whole cell voltage-clamp 

recordings from an acutely isolated medium spiny neuron showing currents 

evoked by GABA (100 M) application in the absence (control) and presence 

of 3,6 dichloro-1-b-D-ribofuranosylbenzimidazole 3,5 cyclic monophosphothioate 

(Sp-5,6-DCl-cBIMPS; 100 nM) alone and the PKA agonist Sp-5,6- 

DCl-cBIMPS (100 nM) plus calyculin A (100 nM). Response was reversed on 

washing. D: box plot summarizing the reduction in peak GABA-evoked 

currents produced by Sp-5,6-DCl-cBIMPS (n 5) and Sp-5,6-DCl-cBIMPS 

plus calyculin A (n 9). Modulation by Sp-5,6-DCl-cBIMPS in the presence 

of calyculin A was significantly larger than in the presence of Sp-5,6-DClcBIMPS 

alone (P 0.05, Kruskal-Wallis ANOVA).


A major cellular target of cAMP is the regulatory subunit of 

PKA. To determine whether the cellular effects of the D 1 

agonist and cAMP analogues were mediated by PKA, the 

membrane permeant PKA inhibitor Rp-Cl-cAMPS was employed. 

Application of Rp-Cl-cAMPS (5 M) significantly 

reduced the response to SKF 81297 (median response 8%, 

n 4, P 0.05, Kruskal-Wallis ANOVA) and to Sp-ClcAMPS 

(median response 12%, n 4, P 0.05, Kruskal- 

Wallis ANOVA). 

To test for the involvement of protein phosphatase 1 (PP1) 

and protein phosphatase 2A (PP2A), the membrane permeant 

inhibitor calyculin A (100 nM) was applied. In the presence of 

the cAMP analogue Sp-5,6-DCl-cBIMPS (100 nM), calyculin 

A further reduced the GABA-evoked currents (Fig. 2C), increasing 

the median modulation to near 38% (n 4, P 0.05, 

Kruskal-Wallis ANOVA, Fig. 2D). 

Medium spiny neurons express 1 subunit mRNA 

n the GABA A receptor oligomer, the preferred targets of 

PKA are subunits. In heterologous systems, PKA has been 

shown to reduce GABA A receptor-mediated currents by phosphorylating 

a serine residue (S409) in the 1 subunit (Mc- 

Donald et al. 1998). On the other hand, phosphorylation of 3 

subunits by PKA increases GABA-evoked currents. 2 subunits 

do not appear to be regulated by PKA in situ (McDonald 

et al. 1998). In light of these findings, our results predict that 

medium spiny neurons that are responsive to D 1 dopamine 

receptor agonists should express 1 subunits. To test this 

hypothesis, single-cell RT-PCR experiments were performed 

(Yan and Surmeier 1997). Neurons expressing mRNA for the 

releasable peptide substance P (SP) previously have been 

shown to express D 1 dopamine receptor mRNA (Gerfen 1992; 

Surmeier et al. 1996). 1 mRNA was detected in all 11 

SP-expressing neurons tested. 3 mRNA was found in 7 of 

these 11. In contrast, 2 mRNA was not detected in medium 

spiny neurons. In Fig. 3A, the PCR amplicons from one of 

these neurons are shown. 

D 1 dopamine receptor stimulation increases phosphorylation 

of GABA A 1/3 subunits 

To determine whether PKA phosphorylated subunits in 

situ, immunoprecipitation experiments were performed. Neostriatal 

slices from C57BL/6 mice were preincubated with 

[ 32 P]orthophosphate, washed, and incubated with the adenylyl 

cyclase activator, forskolin (50 M). GABA A receptor subunits 

were immunoprecipitated using an antibody that recognized 

both the 1 and 3 subunits. Forskolin treatment increased 

the phosphorylation of a protein band of M r 55 kDa 

by 843 143% (n 5, P 0.05 vs. control, Mann-Whitney 

U test). This protein band was verified to be a subunit 

because its appearance was selectively blocked by preabsorption 

of the antibody with a fusion protein representing a sequence 

common to both 1 and 3 subunits (Fig. 3B). Incubation 

of neostriatal slices with the D 1 receptor agonist, 

SKF81297 increased receptor phosphorylation by 250% (Fig. 

3C). 

FIG. 3. Medium spiny neurons express GABA A receptor 1/3 subunits 

and D 1 dopamine receptor stimulation increases their phosphorylation. A, top: 

gel showing the scRT-PCR amplicons derived from a neuron expressing SP 

mRNA but not ENK mRNA. A, bottom: gel showing scRT-PCR GABA A 

receptor amplicons generated from the medium spiny neuron used in A. Note 

that both 1 and 3, but not 2, mRNA were detectable in this neuron. In a 

sample of 11 neurons, all 11 had detectable 1. Seven had detectable levels of 

3 mRNA, and none had detectable 2 mRNA. B: autoradiogram showing 

phosphorylation of GABA A receptor 1/3 subunits in mouse [ 32 P]-labeled 

neostriatal slices. Slices were incubated in the absence (control) or presence of 

forskolin (Fsk; 50 M, 5 min). Forskolin treatment increased the phosphorylation 

of a protein band associated with the 1/3 subunits of the GABA A 

receptor (4). Some forskolin-treated slices were immunoprecipitated with 

antiserum that had been preabsorbed with a GST-linked fusion protein representing 

sequences common to both the 1 and 3 subunits (1/3 fusion, 1 

g). Other forskolin-treated slices were immunoprecipitated with an unrelated 

hexahistidine fusion protein representing a region of the third transmembrane 

loop of the N-methyl-D-aspartate receptor subunit, NR1 (NR1 fusion, 1 g; 

provided by Drs. Lit-Fui Li and Richard L. Huganir, The Johns Hopkins 

University). C: treatment of prelabeled slices with the D 1 receptor agonist 

SKF81297 (1 M, 5 min) increased the phosphorylation of the receptor (1). 

In 4 experiments, summarized in the bottom panel, the phosphorylation state of 

subunits was increased 2- to 3-fold in response to treatment with SKF81297 

in wild-type neostriatal slices but not in those from DARPP-32 knockout mice 

(*P 0.05, compared with wild-type control, Mann-Whitney U test). 

Disruption of DARPP-32 diminishes the efficacy of D 1 

stimulation 

DARPP-32 is a key inhibitor of PP1 in medium spiny 

neurons that express D 1 dopamine receptors (Greengard et al. 

1999). Phosphorylation of DARPP-32 by PKA increases its 

inhibition of PP1 activity. Deletion mutations of DARPP-32 

have been shown to blunt PKA-mediated phosphorylation of


3001 

other cellular proteins (Fienberg et al. 1998). As shown in Fig. 

3, D 1 dopamine receptor agonists significantly increased radiolabeled 

phosphate incorporation into 1/3 subunits in slices 

from wild-type mice but failed to do so in DARPP-32 knockout 

mice. 

Functional assays then were performed to determine whether 

the DARPP-32 mutation altered the ability of D 1 agonists to 

modulate GABA-evoked currents. In wild-type mouse neostriatal 

neurons, as in the rat neurons described in the preceding 

text, SKF 81297 (1 M) decreased GABA A receptor-mediated 

currents. In neostriatal neurons from DARPP-32 knockout 

mice, 1 M SKF 81297 reduced currents by a similar amount. 

However, at lower concentrations of SKF 81297 (200 nM), the 

modulation of GABA-evoked currents was reduced dramatically 

in medium spiny neurons from DARPP-32 knockout 

mice compared with that seen in wild-type neurons (Fig. 4, A 

and B). A statistical summary of these experiments is shown in 

Fig. 4C. These results clearly indicate that DARPP-32 increases 

the efficacy of D 1 dopamine receptor stimulation in 

modulating GABA A receptors. 

DISCUSSION 

D 1 dopamine receptor activation triggers a PKA-dependent 

modulation of GABA A receptors 

FIG. 4. Disruption of DARPP-32 decreases the efficacy of the D 1 dopamine 

receptor modulation of GABA currents. A: in murine (as in rat) neostriatal 

medium spiny neurons, SKF 81297 produced a robust modulation of GABA A 

currents. B: in DARPP-32 mutant neurons, the modulation produced by 200 

nM SKF 81297 was significantly smaller than that produced in wild-type 

neurons at the same agonist concentration. C: statistical summary showing D 1 

dopamine receptor modulation (expressed as percent reduction in GABAevoked 

currents) in wild-type (n 7) and DARPP-32 mutant neurons (n 7) 

at 200 nM SKF 81297. Also shown are the responses to a high (1 M) agonist 

concentration in wild-type (n 2) and mutant neurons (n 4). Note that the 

lower SKF 81297 dose was saturating in wild-type neurons. However, in 

DARPP-32 mutants, this dose only evoked 25% of the maximal response seen 

in wild-type neurons (P 0.05, Kruskal-Wallis ANOVA). 

The data presented demonstrate that D 1 dopamine receptor 

stimulation reduces GABA A receptor-evoked currents in 

neostriatal medium spiny neurons. This reversible modulation 

was effectively antagonized by SCH 23390 at D 1 dopamine 

receptor-specific concentrations. As expected of a 

G s/olf -linked D 1 dopamine receptor signaling pathway (Stoof 

and Kebabian 1984), the modulation was mimicked by 

cAMP analogues. A primary cellular target of cAMP is the 

regulatory subunit of the PKA holoenzyme. An inhibitor of 

PKA (Rp-Cl-cAMPS) effectively reduced the consequences 

of receptor stimulation as well as bath application of cAMP 

analogues, implicating PKA-mediated protein phosphorylation 

in the modulation. The enhancement of the D 1 dopamine 

receptor-mediated modulation by inhibition of PP1/ 

PP2A with calyculin A added strength to the inference that 

protein phosphorylation was involved. These observations 

are consistent with previous studies showing PKA-mediated 

reductions in recombinant and native GABA A receptor currents 

(Heuschneider and Schwartz 1989; Moss et al. 1992; 

Poisbeau et al. 1999; Porter et al. 1990; Schwartz et al. 

1991). As expected from recombinant studies showing that 

PKA phosphorylation of 1 subunits reduces evoked currents 

(McDonald et al. 1998), single-cell RT-PCR experiments 

revealed that essentially all medium spiny neurons 

expressed GABA A 1 subunit mRNA. Radiolabeling/immunoprecipitation 

studies confirmed that 1/3 subunit protein 

was phosphorylated in striatal neurons after D 1 dopamine 

receptor stimulation, suggesting that the 1 subunit was 

the obligate target of D 1 dopamine receptor-stimulated 

PKA. 

In addition to 1 subunit mRNA, neostriatal medium 

spiny neurons frequently had detectable levels of 3 subunit 

mRNA. The lower detection probability of 3 subunit 

mRNA suggests that it was present in lower copy number 

than 1 subunit mRNA (Song et al. 1998; Tkatch et al. 

1998). If this difference was preserved at the protein level, 

1-containing GABA A receptors would be the predominant 

oligomer. This inference is in agreement with the reduction


in current amplitudes produced by D 1 dopamine receptor 

agonists. However, phosphorylation of 3 subunits may be 

evident in other circumstances. Preliminary experiments 

using higher concentrations of the D 1 dopamine receptor 

agonist SKF 81297 (10 M) or cAMP analogues (e.g., 

Sp-Cl-cAMPS, 100 M) have revealed an enhancement of 

GABA-evoked currents in medium spiny neurons. It is 

possible that in this circumstance, 3 subunits are phosphorylated 

by PKA, leading to the increment in current amplitude 

(McDonald et al. 1998). Subunit-specific phosphoantibodies 

will be required to test this hypothesis. 

Disruption of DARPP-32 diminishes the efficacy of D 1 

dopamine receptor coupling to GABA A receptors 

Null mutation of DARPP-32 significantly attenuated the 

phosphorylation of presumptive 1 subunits after D 1 dopamine 

receptor stimulation. This observation is in accord with previous 

studies showing a functional attenuation of D 1 dopamine 

receptor signaling in DARPP-32 mutants (Fienberg et al. 

1998). Phosphorylation of DARPP-32 by PKA effectively inhibits 

PP1 and dephosphorylation of phosphoproteins targeted 

by PKA (Greengard et al. 1999). The physiological studies 

presented here argue that the loss of DARPP-32 lowered the 

efficacy of the D 1 dopamine receptor agonist but did not reduce 

the maximum functional effect. These results suggest that at 

low levels of D 1 dopamine receptor stimulation, phospho- 

DARPP-32 inhibition of PP1 effectively enhances PKA-mediated 

phosphorylation of GABA A receptor 1 subunits. However, 

at higher levels of receptor stimulation and PKA 

activation, PP1 must compete less effectively with PKA at the 

1 subunit, making phosphoDARPP-32 inhibition of PP1 a 

less significant factor. This shift in balance would explain the 

relatively modest consequences of exogenous PP1/PP2 inhibitors 

at higher agonist or cAMP analogue concentrations. It 

recently was demonstrated that cdk5-induced phosphorylation 

of DARPP-32 at thr-75 converts DARPP-32 into a PKA inhibitor 

(Bibb et al. 1999). It will be interesting to determine 

whether that phenomenon contributes to the phenotype of the 

DARPP-32 knockout mice seen in the present study. The 

ability of elevated doses of D 1 receptor agonist to overcome the 

signaling deficit in DARPP-32-deficient mice has been observed 

with other targets (Fienberg et al. 1998; Greengard et al. 

1999). It is unclear whether this feature will generalize to other 

D 1 dopamine receptor/PKA signaling targets such as L-type 

Ca 2 channels (Surmeier et al. 1995), AMPA receptors (Yan et 

al. 1999), or N-methyl-D-aspartate receptors (Snyder et al. 

1998). 

Reconciliation with previous studies 

How can our results be reconciled with the reported 

inability of D 1 dopamine receptor stimulation to modulate 

inhibitory postsynaptic potentials in dorsal striatal neurons 

(Nicola and Malenka 1998)? There are several potential 

explanations. One is that the modulation described here 

depends on a reduction in the affinity of GABA A receptors 

for GABA. If this was the case, the D 1 receptor modulation 

may not be evident at the saturating concentrations of 

GABA thought to be achieved at synapses (Jones and Westbrook 

1995). Another possibility is that the small sample 

size in the previously reported study did not include medium 

spiny neurons that express D 1 dopamine receptors. There 

were no controls for this possibility, and the D 1 receptor 

expressing group constitutes only 60% of all medium 

spiny neurons (Gerfen 1992; Surmeier et al. 1996). A third 

possibility is that GABA A receptors modulated by the D 1 

dopamine receptor signaling cascade are at specific 

GABAergic synapses or are extrajunctional. In contrast to 

local synaptic stimulation, bath application of GABA would 

effectively activate all GABA A receptors, providing a more 

robust (if less physiological) test. In support of this thesis, 

there is compelling evidence that the subunit composition of 

GABA A receptors can be site specific (Nusser et al. 1996– 

1998; Somogyi et al. 1996) and that subunits can serve a 

targeting function (Connolly et al. 1996). It is also true that 

studies using different means of activating GABAergic input 

to medium spiny neurons have led to the conclusion that 

D 1 dopamine receptors are capable of modulating GABAergic 

responsiveness at synaptic sites (Calabresi et al. 1993; 

Kita et al. 1995; Mercuri et al. 1985). 

Functional implications 

The attenuation of intrastriatal GABAergic inhibition of 

medium spiny neurons could significantly alter their activity 

patterns and signal processing. In vivo, blockade of ongoing 

GABAergic inhibition of medium spiny neurons significantly 

elevates basal activity (Nisenbaum and Berger 1992). D 1 dopamine 

receptor-mediated suppression of GABAergic inhibition 

arising from GABAergic interneurons (Kita 1993; Koos 

and Tepper 1999) would substantially enhance the ability of 

cortical activity to evoke spiking in medium spiny neurons. D 1 

dopamine receptor-mediated modulation of intrinsic voltagedependent 

conductances suppresses the responses to cortical 

inputs at negative membrane potentials but enhances the ability 

of glutamatergic inputs to evoke activity during sojourns in the 

depolarized up-state (Calabresi et al. 1987; Galarraga et al. 

1997; Hernandez-Lopez et al. 1997; Schiffmann et al. 1995; 

Surmeier and Kitai 1997; Surmeier et al. 1992, 1995). In the 

depolarized up-state, near spike threshold, D 1 receptor-mediated 

alterations in perisomatic GABAergic efficacy would have 

a profound impact on spike generation and timing (Koos and 

Tepper 1999). 

The dopaminergic suppression of GABA A receptor currents 

also may help unravel the paradoxical role of recurrent axon 

collaterals of medium spiny neurons. These recurrent collaterals 

form a dense intrastriatal network that is known from 

anatomic studies to target medium spiny neurons (Wilson and 

Groves 1980). This network often has been postulated to 

provide a functionally important feedback inhibition within the 

striatum (Beiser and Houk 1998; Wickens et al. 1995). However, 

a direct test of this hypothesis has failed to find any 

evidence of recurrent inhibition mediated by these collaterals 

(Jaeger et al. 1994). These observations could be reconciled if 

the response to GABA at these sites was suppressed by ambient 

D 1 dopamine receptor tone and was only functional in the 

absence of substantial dopaminergic tone as found in Parkinson’s 

disease.


3003 

This work was supported by National Institutes of Health Grants 

NS-34696 to D. J. Surmeier and MH-40899/DA-10044 to P. Greengard. 

Much of this work was performed at the University of Tennessee, Memphis, 

TN. 

Present address of J. Flores-Hernandez: Dept. of Psychiatry, UCLA, 760 

Westwood Plaza, Los Angeles, CA 90024-1749. 

Address for reprint requests: D. J. Surmeier, Dept. of Physiology/NUIN, 

Northwestern Univ. Medical School, 320 E. Superior St., Chicago, IL 

60611. 

Received 30 November 1999; accepted in final form 26 January 2000. 

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