1Department of Physiology and Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611; 2Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York City, New York 10021; and 3Medical Research Council/Laboratory of Molecular Cell Biology and Department of Pharmacology, University College, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Flores-Hernandez, Jorge,
Salvador Hernandez,
Gretchen L. Snyder,
Zhen Yan,
Allen A. Fienberg,
Stephen J. Moss,
Paul Greengard, and
D. James Surmeier.
D1 Dopamine Receptor Activation Reduces
GABAA 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 D1 dopamine
receptors in the regulation of postsynaptic GABAA 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 D1 dopamine receptor
agonist SKF 81297 reduced GABA-evoked currents in most medium spiny
neurons. The D1 dopamine receptor antagonist SCH 23390 blocked the effect of SKF 81297. Membrane-permeant cAMP analogues
mimicked the effect of D1 dopamine receptor stimulation, whereas an inhibitor of protein kinase A (PKA; Rp-8-chloroadenosine 3',5' cyclic monophosphothioate) attenuated the response to
D1 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 GABAA
receptor
a known substrate of PKA
in medium spiny neurons.
Immunoprecipitation assays of radiolabeled proteins revealed that
D1 dopamine receptor stimulation increased phosphorylation
of GABAA receptor
1/
3 subunits. The D1
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 D1 dopamine receptor modulation of
GABAA currents was reduced in DARPP-32-deficient medium
spiny neurons. These results argue that D1 dopamine
receptor stimulation in neostriatal medium spiny neurons reduces
postsynaptic GABAA receptor currents by activating a
PKA/DARPP-32/protein phosphatase 1 signaling cascade targeting
GABAA receptor
1 subunits.
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INTRODUCTION |
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Disordered neostriatal dopaminergic signaling is a
central determinant of a variety of psychomotor illnesses including
Parkinson's disease, schizophrenia, and drug abuse (Grace et
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 D1 dopamine receptors. For
example, D1 dopamine receptor stimulation
increases GABA release (Harsing and Zigmond 1997
). However, D1 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 D1 dopamine
receptor agonists to modulate GABAergic synaptic potentials in medium
spiny neurons is surprising. D1 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 GABAA receptor-mediated
currents in both heterologous and native expression systems
(Smart 1997
).
The preferred substrate for PKA in the GABAA
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 GABAA
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
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 GABAA
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 D1 dopamine receptors, GABAA 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 D1 dopamine receptor stimulation. These observations then were pursued with a combination of molecular, biochemical, and electrophysiological techniques to determine the signaling pathway linking D1 dopamine receptors to GABAA receptors.
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METHODS |
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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-µm
slices with a Vibroslice (Campden Instruments, London) while bathed in
a low-Ca2+ (100 µM),
N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic
acid] (HEPES)-buffered salt solution [containing (in mM) 140 Na
isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 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 NaHCO3-buffered
saline bubbled with 95% O2-5%
CO2 [which contained (in mM) 126 NaCl, 2.5 KCl,
2 CaCl2, 2 MgCl2, 26 NaHCO3, 1.25 NaH2PO4,1 pyruvic acid, 0.2 ascorbic acid, 0.1 NG-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-Ca2+ 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-Ca2+,
HEPES-buffered 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.
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 MgCl2, 5 1,2 bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), 12 phosphocreatine, 2 Na2ATP, 0.2 Na3GTP, and 0.1 leupeptin, pH = 7.2-3 with
H2SO4, 265-270 mosm/l. The
external solution consisted of (in mM) 135 NaCl, 20 CsCl, 1 MgCl2, 10 HEPES, 0.001 TTX, 0.5 BaCl2, 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 K2SO4, 10 HEPES, 6 MgCl2, 3.5 BAPTA, 12 phosphocreatine, 2 Na2ATP, 0.2 Na3GTP, and 0.1 leupeptin, pH = 7.2-3 with
H2SO4, 265-270 mosm/l. The
external solution consisted of (in mM) 140 NaCl, 2.8 KCl, 1 MgCl2, 10 HEPES, 0.001 TTX, 0.5 CaCl2, and 10 glucose, pH = 7.3 with NaOH,
300-305 mosm/l.
STATISTICAL METHODS.
Data analyses were performed with AxoGraph (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 µl 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 µl diethyl pyrocarbonate (DEPC)-treated water, 1 µl
RNasin (28 U/µl), 1 µl dithiothreitol (DTT; 0.1 M), and 1 µl of
oligo(dT) (0.5 µg/µl) 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 µl,
200 U/µl) and buffer (4 µl, 5× first strand buffer: 250 mM
Tris-HCl, 375 mM KCl, 15 mM MgCl2), DTT (1 µl,
0.1 M) and mixed dNTPs (1 µl, 10 mM). The mixture (20 µl) 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 µl
RNase H (2 U/µl), 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 MgCl2, 0.5 mM of
each of the dNTPs, 0.8-1 µM primers, 2.5 U Taq DNA
polymerase (Promega), 5 µl 10× buffer (Promega), and 1-2 µl 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
GABAA 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 µl) 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 µm) 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 NaHCO3, 1.5 MgSO4, 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%O2-5%CO2 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 CaCl2 and lacking EGTA. A
2.0-mCi aliquot of [32P]orthophosphoric acid
(DuPont 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
[32P]phosphate-labeled GABAA receptor subunit
[32P]phosphate-labeled tissue slices
were sonicated in 150 µl of 1% 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 µM phenylmethylsulfoxide, 20 µg/ml chymostatin, 20 µg/ml
pepstatin A, 5 µg/ml leupeptin, and 5 µg/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 µl) were retained for the determination of total
[32P]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 µg of an antiserum generated against a peptide
sequence contained in both the 1 and
3 subunits of the
GABAA receptor. The samples were mixed for 2 h at 4°C, then transferred to fresh 1.5-ml Eppendorf tubes containing
10 mg preswollen Protein A-Sepharose Cl-4B beads and mixed for 1 h
at 4°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 µl of a
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
[32P]phosphate incorporation was quantified
using a PhosphorImager 400B and ImageQuant software from Molecular
Dynamics. Values for [32P]phosphate content
were normalized for the total [32P]phosphate
incorporated into TCA-precipitable protein.
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RESULTS |
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D1 dopamine receptor stimulation reduces GABA-evoked currents
The application of GABA (100 µM) evoked a rapidly desensitizing
current in medium spiny neurons voltage clamped at 0 mV. The current
was blocked by bicuculline (100 µM) and reversed near the
Cl reversal potential (data not shown),
implicating GABAA receptors. As shown in Fig.
1A, the
D1 dopamine receptor agonist SKF 81297 (1 µM)
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 µM) were reduced by 18% by
SKF 81297 (0.1-1 µM). The kinetics of the evoked currents were not
noticeably altered by D1 dopamine receptor
agonists. Coapplication of the D1 dopamine receptor antagonist SCH 23390 (1 µM) blocked the reduction in evoked
currents produced by SKF 81297 (n = 9, P < 0.05, Kruskal-Wallis ANOVA; Fig. 1A).
To verify that the D1 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
D1 receptor agonist SKF 81297 (1 µM) also
reversibly reduced the inward currents evoked by GABA (100 µM) 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).
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Activation of D1 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 GABAA
receptors by D1 dopamine receptors was mediated by stimulation of adenylyl 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-Cl-cAMPS decreased GABA-evoked currents in a manner similar to the
D1 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 µM) had no effect on GABA-evoked currents, arguing that the
modulation was PKA specific (n = 7, P > 0.05, Kruskal-Wallis ANOVA).
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A major cellular target of cAMP is the regulatory subunit of PKA. To determine whether the cellular effects of the D1 agonist and cAMP analogues were mediated by PKA, the membrane permeant PKA inhibitor Rp-Cl-cAMPS was employed. Application of Rp-Cl-cAMPS (5 µM) significantly reduced the response to SKF 81297 (median response = 8%, n = 4, P < 0.05, Kruskal-Wallis ANOVA) and to Sp-Cl-cAMPS (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
In the GABAA receptor oligomer, the
preferred targets of PKA are subunits. In heterologous systems, PKA
has been shown to reduce GABAA receptor-mediated
currents by phosphorylating a serine residue (S409) in the
1 subunit
(McDonald 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
D1 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 D1 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.
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D1 dopamine receptor stimulation increases
phosphorylation of GABAA 1/
3 subunits
To determine whether PKA phosphorylated subunits in situ,
immunoprecipitation experiments were performed. Neostriatal slices from C57BL/6 mice were preincubated with
[32P]orthophosphate, washed, and incubated with
the adenylyl cyclase activator, forskolin (50 µM).
GABAA 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 ~Mr 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
D1 receptor agonist, SKF81297 increased receptor phosphorylation by 250% (Fig. 3C).
Disruption of DARPP-32 diminishes the efficacy of D1 stimulation
DARPP-32 is a key inhibitor of PP1 in medium spiny neurons that
express D1 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 other cellular proteins
(Fienberg et al. 1998
). As shown in Fig. 3,
D1 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 D1 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 µM) decreased GABAA receptor-mediated currents. In neostriatal neurons from DARPP-32 knockout mice, 1 µM 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 D1 dopamine receptor stimulation in modulating GABAA receptors.
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DISCUSSION |
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D1 dopamine receptor activation triggers a PKA-dependent modulation of GABAA receptors
The data presented demonstrate that D1
dopamine receptor stimulation reduces GABAA
receptor-evoked currents in neostriatal medium spiny neurons. This
reversible modulation was effectively antagonized by SCH 23390 at
D1 dopamine receptor-specific concentrations. As
expected of a Gs/olf-linked
D1 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
D1 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 GABAA 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 GABAA
1 subunit mRNA.
Radiolabeling/immunoprecipitation studies confirmed that
1/
3
subunit protein was phosphorylated in striatal neurons after D1 dopamine receptor stimulation, suggesting that
the
1 subunit was the obligate target of
D1 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
GABAA receptors would be the predominant
oligomer. This inference is in agreement with the reduction in current
amplitudes produced by D1 dopamine receptor agonists. However, phosphorylation of
3 subunits may be evident in
other circumstances. Preliminary experiments using higher
concentrations of the D1 dopamine receptor
agonist SKF 81297 (10 µM) or cAMP analogues (e.g., Sp-Cl-cAMPS, 100 µM) 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 D1 dopamine receptor coupling to GABAA receptors
Null mutation of DARPP-32 significantly attenuated the
phosphorylation of presumptive 1 subunits after
D1 dopamine receptor stimulation. This
observation is in accord with previous studies showing a functional
attenuation of D1 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 D1 dopamine
receptor agonist but did not reduce the maximum functional effect.
These results suggest that at low levels of D1
dopamine receptor stimulation, phosphoDARPP-32 inhibition of PP1
effectively enhances PKA-mediated phosphorylation of
GABAA 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
D1 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 D1 dopamine receptor/PKA signaling targets such as L-type Ca2+ 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 D1 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 GABAA receptors for
GABA. If this was the case, the D1 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 D1 dopamine receptors. There were no
controls for this possibility, and the D1
receptor expressing group constitutes only ~60% of all medium spiny
neurons (Gerfen 1992
; Surmeier et al.
1996
). A third possibility is that GABAA
receptors modulated by the D1 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
GABAA receptors, providing a more robust (if less
physiological) test. In support of this thesis, there is compelling
evidence that the subunit composition of GABAA
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 D1 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). D1
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.
D1 dopamine receptor-mediated modulation of
intrinsic voltage-dependent 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,
D1 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 GABAA 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
D1 dopamine receptor tone and was only functional
in the absence of substantial dopaminergic tone as found in
Parkinson's disease.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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Address for reprint requests: D. J. Surmeier, Dept. of Physiology/NUIN, Northwestern Univ. Medical School, 320 E. Superior St., Chicago, IL 60611.
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.
Received 30 November 1999; accepted in final form 26 January 2000.
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REFERENCES |
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