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The Journal of Neuroscience, July 15, 1998, 18(14):5301-5310
A Complex Program of Striatal Gene Expression Induced by
Dopaminergic Stimulation
Joshua D.
Berke1, 2,
Ronald F.
Paletzki3,
Gabriel J.
Aronson3,
Steven
E.
Hyman1, and
Charles R.
Gerfen3
1 Molecular Plasticity Section, National Institute of
Neurological Disorders and Stroke, Bethesda, Maryland 20892, 2 Program in Neuroscience, Harvard University, Boston,
Massachusetts 02115, and 3 Laboratory of Systems
Neuroscience, National Institute of Mental Health, Bethesda, Maryland
20892
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ABSTRACT |
Dopamine acting in the striatum is necessary for normal movement
and motivation. Drugs that change striatal dopamine neurotransmission can have long-term effects on striatal physiology and behavior; these
effects are thought to involve alterations in gene expression. Using
the 6-hydroxydopamine lesion model of Parkinson's disease and
differential display PCR, we have identified a set of more than 30 genes whose expression rapidly increases in response to stimulation of
striatal dopamine D1 receptors. The induced mRNAs include
both novel and previously described genes, with diverse time courses of
expression. Some genes are expressed at near-maximal levels within 30 min, whereas others show no substantial induction until 2 hr or more
after stimulation. Some of the induced genes, such as CREM, CHOP, and
MAP kinase phosphatase-1, may be components of a homeostatic response
to excessive stimulation. Others may be part of a genetic program
involved in cellular and synaptic plasticity. A very similar set of
genes is induced in unlesioned animals by administration of the
psychostimulant cocaine or the antipsychotic eticlopride, although in
distinct striatal cell populations. In contrast to some previously
described early genes, most of the novel genes are not induced in
cortex by apomorphine, indicating specificity of induction. Thus we
have identified novel components of a complex, coordinated genetic
program that is induced in striatal cells in response to various
dopaminergic manipulations.
Key words:
dopamine; striatum; CREB; differential display PCR; immediate-early genes; neuronal plasticity; addiction
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INTRODUCTION |
The mesostriatal dopamine system
plays a key role in normal motor function (Albin et al., 1989 ) and
associative learning (Schultz et al., 1997). Destruction of midbrain
dopamine cells causes Parkinson's disease, and long-term dopamine
replacement therapy with L-DOPA is associated with
dyskinesias, hallucinations, and fluctuations in therapeutic response
(Chase et al., 1993). The use of dopamine antagonists as antipsychotics
can result in tardive dyskinesia, while drugs such as cocaine that
increase striatal dopamine release can cause dependency and addiction
(for review, see Hyman, 1996).
Many of the persistent consequences of dopaminergic drugs appear to
result from changes in striatal physiology, including altered
sensitivity to neurotransmitters. For example, cocaine administration
leads to altered responsiveness of striatal medium spiny neurons to
dopamine (Henry and White, 1991). Dopaminergic drugs rapidly cause
changes in gene expression in striatal neurons (Robertson et al., 1989;
Dragunow et al., 1990; Graybiel et al., 1990); these gene expression
changes may be responsible for the alterations in striatal physiology.
One example is dynorphin gene expression, which is progressively
induced in dorsal striatum by cocaine. This may be a compensatory
adaptation contributing to the aversive aspects of withdrawal by
decreasing dopamine release (Steiner and Gerfen, 1993; Cole et al.,
1995; Shippenberg and Rea, 1997).
Dopamine acts in the striatum through the D1 and
D2 subfamilies of G-protein-coupled receptors.
D1 receptors are coupled to GS/Golf and increase cAMP production,
whereas D2 receptors are coupled to
Gi/Go and decrease cAMP. D1
receptor activation appears to be necessary for striatal gene induction
by cocaine or L-DOPA (Young et al., 1991; Morelli et al.,
1993; Steiner and Gerfen, 1995). This induction occurs selectively in
the 40-50% of striatal neurons whose dopamine receptors are
predominantly of the D1 type and project to the substantia
nigra pars reticulata (Gerfen et al., 1990, 1995; Robertson et al.,
1990). Increased cAMP can activate protein kinase A, which
phosphorylates the transcription factor cAMP response element-binding
protein (CREB). D1 stimulation of striatal cells also
appears to cause changes in CREB phosphorylation and c-fos
expression by increasing intracellular calcium, entering through either
voltage-sensitive calcium channels or NMDA receptors (Keefe and Gerfen,
1996; Konradi et al., 1996).
To gain a more complete picture of dopamine-related changes in striatal
gene expression, we used differential display PCR (DDPCR). We took
advantage of the rat 6-hydroxydopamine lesion model of Parkinson's
disease, because the dopamine supersensitive state shows very robust
CREB phosphorylation and induction of gene expression response after
D1 receptor stimulation (Robertson et al., 1989; Cole et
al., 1994). The specificity of this model was increased further by
giving a continuous infusion of a D2 agonist to diminish
lesion-induced alterations in striatopallidal neurons (Gerfen et al.,
1990). Gene expression was compared between the normal and
6-OHDA-lesioned striata of animals that were given either saline or a
D1 agonist and killed 1 or 24 hr later. In this paper we
describe a large set of known and novel genes that are rapidly induced
by D1 stimulation, compare their time courses of
expression, and examine their responses to other drugs.
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MATERIALS AND METHODS |
Animals. For all experiments, male Sprague Dawley
rats (Taconic, NY), weighing ~200 gm, were used. Rats were housed in
groups of three in a temperature-controlled room on a 12 hr light/dark schedule, with free access to food and water.
Dopamine depletions. These were performed as described
(Gerfen et al., 1995). Briefly, 16 µg of 6-hydroxydopamine (in 2 µl of 0.02% ascorbic acid) was infused into the right medial forebrain bundle/rostral substantia nigra at anteroposterior (AP) +3.5 mm, mediolateral (ML) +1.5 mm, dorsoventral (DV) +2.0 mm (relative to
interaural zero). Animals were then left for 3 weeks to allow time for
dopamine receptor supersensitivity to develop. At 3 weeks after the
lesion, animals used for differential display and initial in
situ confirmation of differential expression were implanted with
an osmotic pump (Alza Pharmaceuticals, Palo Alto, CA). The pump was
filled with quinpirole, set to administer a dose of 1 mg · kg 1 · d1,
and then implanted subcutaneously in the back. Animals were injected
with saline or SKF38393 5 d later, without removing the osmotic
pump.
Pharmacological treatments. Acute drug treatments were given
intraperitoneally, dissolved in 0.02% ascorbic acid. All drugs were
obtained from Sigma [(St. Louis, MO) cocaine HCl, 6-hydroxydopamine, apomorphine] or RBI [(Natick, MA) SKF38393, quinpirole,
eticlopride].
RNA isolation. Animals were killed with CO2 and
decapitation. Brains were removed and rapidly chilled in ice-cold
saline, and the striata were dissected out between approximately +11 mm and +9 mm AP [relative to intra-aural zero, using the atlas of Paxinos
and Watson (1986)]. Tissue was frozen in tubes chilled on dry ice and
stored at  80°C and then homogenized using a Polytron (Kinematica/Brinkmann Instruments) in TriReagent with Microcarrier gel
(Molecular Research) following the manufacturer's instructions. RNA
was further purified by incubation with DNase I (Promega, Madison, WI)
and phenol/chloroform, chloroform/isoamylalcohol extractions. After UV
spectrophotometric quantitation, RNA integrity and quality were
verified by running 1 µg on a 1% native agarose gel.
Differential display RT-PCR. For each condition to be
tested, striata from two separate animals were used for reverse
transcription. RNA (2 µg) was mixed with 0.5 µl of RNAsin
(Promega), 0.4 nmol of dNTP (Boehringer Mannheim, Indianapolis, IN), 4 µl of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase
buffer (Promega), and 4 pmol of 3' primer in 19 µl of total volume.
3' primers were "one-base-anchored" and of the form GGGCG AAGCT
TTTTT TTTTT M, where M is A, C, or G. These primers include a
HindIII restriction site (Liang et al., 1994) and partial T7
polymerase sites. The reaction was heated at 65°C for 5 min, and then
at 37°C for 10 min. MMLV reverse transcriptase (1 µl; Promega) was
then added, and the reaction was left at 37°C for an additional 50 min. Finally the reaction was heated to 95°C for 5 min and then
placed on ice. Of this RT reaction, 1.1 µl was mixed with 1.1 µl of
10× PCR buffer (Boehringer Mannheim), 0.22 nmol of dNTP, 2.2 pmol of
3' primer as above, 2.2 pmol of 5' primer, 0.14 µl of
33P-dATP (catalog #NEG612H; New England Nuclear, Boston,
MA), and 0.11 µl of Taq polymerase (Boehringer Mannheim)
in a total volume of 11 µl. 5' primers were of the form GACAC TATAG
AATTC NNNNN NN, where N is any base. These primers include an
EcoRI restriction site and a partial SP6 polymerase site.
Thermal cycling was performed in an MJ Research PTC-200 96 V cycler
under the following conditions (modified from Zhao et al., 1995):
94°C, 2 min; 4 cycles of 94°C, 15 sec, 40°C, 4 min, 72°C, 3 min; 29 cycles of 92°C, 15 sec, 55°C, 1 min, 72°C, 1.5 min + 3 sec/cycle; 68°C for 5 min; 4°C, hold. To each PCR reaction 2.6 µl
of 5× DNA dye was added, and 1.5 µl samples were loaded onto a 61 cm
gel in a Genomyx-LR sequencer (Genomyx Corp., Foster City, CA). The gel
was 340 µm thick and was cast from 4.5% HR-1000 gel (Genomyx). Gels
were run for 16 hr at 50°C, 1000 V, 100 W, then dried onto the glass
plate, washed, and exposed to Biomax-MR film (Eastman Kodak, Rochester,
NY) for 2-4 d. Using this protocol we obtained a consistent band
pattern ranging from ~500 bp to ~2 kb in size. In most cases, a 100 bp DNA ladder (Life Technologies, Gaithersburg, MD) was radioactively labeled and run alongside the DDPCR samples to allow accurate estimation of band size. In total, approximately 150 primer
combinations were used for each of the four conditions (see Fig. 1);
each primer combination produced several hundred bands.
Differentially expressed bands were excised from the gel and
reamplified in the presence of 0.4 µl of Expand High Fidelity polymerase mix (Boehringer Mannheim), 4 µl of 10× HiFi buffer 2 (Boehringer Mannheim), 1.6 nmol of dNTP, and 16 pmol of each reamp
primers in a total volume of 40 µl. Reamp primers were CGCGC GTAAT
ACGAC TCACT ATAGG GCGAA GCTTT TTTTT TTT, and CATAC GATTT AGGTG ACACT
ATAGA ATTC. These complete the RNA polymerase sites on either side of
the reamplified DDPCR band. Of this reaction, 32 µl was mixed with 8 µl of DNA dye and run out on a 1% agarose gel with ethidium bromide.
Where a band of the predicted size was apparent, it was excised and
purified using Qiaquick columns (Qiagen Inc., Valencia, CA) into 30 µl of water, and 2 µl was used as a template for in
vitro transcription as described below. In situ
hybridization was performed (see below) using tissue sections from
animals treated in the same way as the DDPCR RNA samples. Genes whose
differential expression was confirmed with in situ hybridization were cycle-sequenced, either directly using the reamp
primers described above or by previous subcloning into pGEM-7z (Promega) or pCRII-TOPO (Invitrogen, Carlsbad, CA).
Sequence analysis. Sequence data were compared with GenBank
at the National Library of Medicine, using the blastn algorithm on the
"nr" nonredundant database, at their online website. In some cases
(e.g., fosB), sequences gave no match to rat sequences but extremely
close matches to known mouse sequences; these were assumed to be the
rat homologs of the mouse genes. Sequences that gave no matches were
designated as novel sequences. In one case (ania-7) the sequence of the
DDPCR fragment contained a repeat sequence found in many genes. In this
instance, oligo probes were designed to the unique portion of the
sequence and used to reconfirm differential expression. Novel sequences
were further compared with the GenBank dbEST database. We found that
ania-6 and ania-11 each had several close matches to mouse and human
expressed sequence tags; the others did not. All novel gene fragments
presented in this paper were subcloned, and the in situ
hybridization shown uses probes from individual, sequenced colonies.
Sequences were submitted to GenBank under the following accession
numbers: ania1-6, AF030086-91; ania7-12, AF050659-64.
Generation of probe templates for known genes. For most of
the known genes tested, 450-1000 bp templates were obtained by PCR
amplification from rat cDNA (Clontech, Palo Alto, CA, or Life Technologies), followed by subcloning into pCRII-TOPO and sequencing to
confirm identity. In some cases, 45-50 mer oligo probes were designed
from GenBank sequences (c-jun: GGTCG CAACC CAGTC CATCT TGTGT ACCCT
TGGCT TCAGT ACTCG GACAC; junB: ACTGG GCGCA GGCGG GCAGG CCAGA GTCCA
GTGTG TGAGC TGCGC C; COX-2: GAACA TCACG GGCTC CGCCA CCTTC CTACG CCAGC
AATCT GACGT; nur77: GTGGT CACGC GGTCC TGGGC TCGTT GCTGG TGTTC CATAT
TGAGC) and checked with blastn as above to avoid cross-hybridization.
In all cases oligonucleotides were from Life Technologies. GenBank
accession numbers used to design probes were as follows: arc U19866;
egr-3 U12428; narp S82649; rheb U08227; fra-2 U18982; tPA M31197; c-fos
X06769; nur77 U17254; c-jun X12761; junB X54686; junD D26307; COX-2
S67722.
Probe labeling and in situ hybridization.
Procedures were as described in Gerfen et al. (1995). Briefly, RNA
probes were produced by in vitro transcription using SP6 or
T7 polymerases, in the presence of 35-S-UTP (New England Nuclear) or
digoxygenin (Boehringer Mannheim). Oligonucleotide probes were
end-labeled using terminal deoxynucleotidyl transferase (Boehringer
Mannheim) and 35S-dATP (New England Nuclear).
35S-labeled probes were hybridized overnight
either singly or in combination with digoxygenin-labeled enkephalin or
D1 receptor probes. Each slide had three 12 µm coronal
tissue sections from rostral, middle, and caudal striatum (~10.7,
9.2, and 8.0 mm anterior to the interaural line). After washing,
digoxygenin label was detected using alkaline phosphatase-conjugated
Fab fragment (Boehringer Mannheim) and visualized with nitroblue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate toluidine salts.
Slides were placed on Biomax MR film (Kodak) and allowed to expose for
3-30 d. Slides were then dipped in emulsion (Amersham or Kodak),
exposed for 1-3 months, and developed using standard darkroom
techniques.
Image analysis. Images were captured from film using a light
table and CCD camera, together with National Institutes of Health Image
software (W. Rasband, National Institute of Mental Health). For
quantitation of gene induction, average gray values from an area of
dorsal striatum of fixed size were measured on both rostral and middle
striatal sections and averaged. For each section, the average gray
value of a portion of overlying white matter (corpus callosum) was
subtracted, as background correction.
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RESULTS |
We obtained RNA for DDPCR from rats treated in the following
manner. First we performed unilateral 6-hydroxydopamine lesions, causing destruction of the nigrostriatal dopamine pathway. To further
isolate a D1-supersensitive response, 3 weeks after the lesion we implanted a subcutaneous osmotic pump that continuously supplied quinpirole, a D2 agonist (1 mg · kg1 · d1). Five
days later the animals were given injections of either saline or
SKF38393 (5 mg/kg, i.p.), a selective D1 agonist. As shown
in Figure 1, we compared the unlesioned
and lesioned striata of saline-injected animals with striata from
lesioned animals given SKF38393 and killed 1 hr and 24 hr later.
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Figure 1.
A fragment of a typical differential display film.
This portion of the film has an approximate size range of 600-1500 bp
and is 30 cm long. The conditions used in the screen are as follows.
Conditions 1 and 2 are the unlesioned
(left) and lesioned (right) striata,
respectively, from animals given saline injections and killed 1 hr
later. Conditions 3 and 4 are
the lesioned striata from animals given SKF38393 (5 mg/kg)
and killed after 1 and 24 hr, respectively. For each condition we used
RNA from two different animals for reverse transcription, and each
reverse transcription reaction was used for two duplicate PCR
reactions, for a total of four lanes per condition for each primer
combination. Arrows mark examples of differentially
expressed bands; the white spots at the edge of these
bands are pinholes used to orient the film over the dried gel for band
excision. The upper of the two arrows
indicates an 850 bp fragment of ania-6 mRNA.
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The great majority of the differentially expressed genes we observed
showed acute, but not persistent, upregulation after D1
stimulation. Some examples are shown in Figure 1. This rapidly induced
yet transient genetic response is the subject of the rest of this
paper.
Identification of D1-induced genes
More than 30 genes are rapidly induced by selective D1
stimulation in the supersensitive rat striatum. Some examples are shown in Figure 2. Of the genes found in the
screen, some (e.g., zif268) were expected because
of previous studies; others (e.g., CHOP) are known genes not previously
known to be regulated under these circumstances, and others have no
matching sequences in GenBank. Two other novel gene fragments had
consistent but weak inductions and are not shown. In parallel with the
differential display screen, we also examined the expression pattern of
a number of genes whose induction was either expected because of
previous work or had been described as immediate-early genes in
brain under other conditions. Some of these are shown in Figure
2.
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Figure 2.
Examples of genes acutely induced by SKF38393.
Each image is of a coronal rat brain section through the middle
striatum, after in situ hybridization with a probe to
the gene indicated. All of the brain sections are from animals given
unilateral 6-hydroxydopamine lesions and then injected with SKF38393 (5 mg/kg) and killed 2 hr later (except tPA, shown at 4 hr
after SKF38393). The lesioned side is shown on the left
in each case. The genes designated "ania-1,"
"ania-2," etc., have no named matches in GenBank. Genes
were designated as previously known on the basis of matches to the
following accession numbers: homer U92079; CHOP (= GADD153) U36994;
krox20 (= egr-2) U78102; preprotachykinin M34184; CREM M60285; fosB
X14897; zif268 (= NGFIA/egr-1) M18416; MKP-1 (= CL100)
S81478.
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To confirm that these genes are indeed being induced in D1
receptor-bearing striatonigral neurons, in some cases we performed double-label in situ hybridization. As an example, Figure
3 shows that the novel gene ania-4 is
selectively induced in cells expressing the D1 receptor but
not enkephalin (a marker of striatopallidal neurons). All the genes we
have so far examined in this way (including arc, c-fos,
zif268, ania-1, ania-3, ania-4, and ania-6; data not shown) have also shown selective induction in
D1-receptor-expressing cells after SKF38393.
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Figure 3.
Selective gene induction in
D1-receptor-bearing striatal neurons. Double-label
in situ hybridization was performed with probes to the
gene ania-4 (silver grains) and either D1
receptor (A) or enkephalin
(B). The field in each case is taken from a brain
section treated as in Figure 2 (i.e., 2 hr after SKF38393, 5 mg/kg),
with additional hybridizations using digoxygenin-labeled probes,
visualized by a dark reaction product. A, Induced ania-4
mRNA colocalizes with D1 receptor mRNA. B,
Induced ania-4 mRNA does not colocalize with mRNA for enkephalin, a
marker of D2-receptor-bearing striatopallidal neurons.
Scale bars, ~50 µm.
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Time courses of gene induction
As an initial functional comparison of the many induced genes, we
examined in detail their time course of induction after D1
stimulation in the lesioned striatum. Although most induced genes show
maximal induction ~2 hr after stimulation, overall they show striking
differences in time course; some examples are illustrated in Figure
4A. Some genes, such as
c-fos and ania-5, are highly upregulated within 30 min,
whereas others, such as ania-4 and ania-6, take 2 hr to show
substantial increases. After they reach peak induction, mRNAs can
either be rapidly degraded or show persistent expression (compare
ania-4, ania-6). Figure 4B shows the quantitation of
the images in Figure 4A, measuring the optical
density from the dorsal striatum for four animals per time point.
Although D1-receptor-expressing cells throughout the
striatum can respond with this diverse set of genes, there are regional
differences in the time course of expression. Most genes appear to be
induced more rapidly in ventral striatum and persist longer in dorsal
striatum.
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Figure 4.
Five examples of distinct temporal patterns of
gene expression after SKF38393 (5 mg/kg) given to 6-OHDA lesioned
animals. Animals were killed at either 0, 0.5, 1, 2, 4, or 8 hr after
injection of SKF38393, as indicated. A, Representative
in situ hybridization images from each time point.
Lesioned side is on the left as in Figure 2.
B, Bar charts of average gray value measurements from
the dorsal striatum. For each gene, sections from the same four animals
were measured at each time point. The x-axis indicates
average gray value, in arbitrary units. Error bars indicate SEM.
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In Figure 5A we show
quantifications for the time course of expression of other genes. The
figure is roughly organized so that more rapidly induced genes are on
the left of more slowly induced genes, and genes with a relatively
prolonged induction are below those with a more rapid decay rate. A
number of genes, such as ania-5, ania-7, ania-8, and krox-20, show an
apparent dip at the 1 hr time point. Note that the dip in ania-5 was
seen in two separate experiments, each with four animals per time point (Figs. 4B, 5) and is statistically significant
(one-tailed t tests, p < 0.001, comparing 1 hr time point with 0.5 and 2 hr time points). All of the mRNAs that
show a dip at 1 hr appear to be rapidly degraded (Fig. 5A).
The dip therefore may be the result of transient desensitization of the
D1 signaling pathway, in combination with rapid mRNA
degradation.
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Figure 5.
A, Time course examples for genes
reaching peak induction within 2 hr. B, Preprodynorphin
and narp take longer to reach peak induction. Graphs are quantitations
from tissue sections at the same time points as in Figure 4, except for
a few genes with rapid decay rates where data are shown from an
experiment in which animals were killed at 0, 0.5, 1, 1.5, 2, and 4 hr
after SKF38393 (5 mg/kg). The y-axis in each case is
average gray value in dorsal striatum (in arbitrary units), and the
x-axis is hours after SKF38393. In most cases
n = 4 animals per time point; for a few cases
n = 2 or 3. Error bars indicate SEM. Where no error
bar is visible, the error was smaller than the graph symbol.
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Other genes, such as narp and preprodynorphin, take longer to reach
peak induction; these are shown in Figure 5B. All genes investigated return close to baseline within 24 hr, including ania-4,
egr-3, rheb, narp, and preprotachykinin (data not shown).
Gene induction after other dopaminergic manipulations
Many classes of drugs can cause changes in striatal gene
expression. To what extent are these different pharmacological stimuli engaging a similar genetic program? We examined the profile of gene
expression 1 hr after injecting cocaine, apomorphine, or eticlopride.
The psychostimulant cocaine blocks presynaptic dopamine, norepinephrine, and serotonin reuptake transporters, causing prolonged availability of these neurotransmitters in the synaptic space. Apomorphine is a mixed D1/D2 agonist,
and eticlopride is a D2 antagonist with antipsychotic
properties. Inductions in response to these drugs are bilateral. The
animals were not previously given 6-OHDA lesions or any other
treatment. Figure 5 shows some examples. Of the genes we tested with a
strong striatal response to SKF38393, all also showed a strong striatal
response to cocaine and eticlopride, with cocaine consistently
producing a more medial induction pattern in the striatum than
eticlopride.
The striatal cells responding to cocaine in each case are presumed to
be D1-receptor-bearing striatonigral cells (Ruskin and Marshall, 1994; Steiner and Gerfen, 1995), whereas those responding to
eticlopride are presumed to be D2-receptor-bearing
striatopallidal cells (Robertson et al., 1992). We performed
double-label in situ hybridization on tissue sections of
eticlopride- or cocaine-treated animals and confirmed that the novel
gene ania-1 is induced in the expected cells (data not shown). It
appears that cocaine and eticlopride can induce highly similar genetic
programs in distinct cellular populations.
Different brain areas may have distinct sets of inducible genes.
Apomorphine, like other dopamine agonists, causes a relatively modest
induction of gene expression in the striatum of normal (unlesioned)
animals; however, it can cause considerable gene induction in the
cortex. Figure 6 shows that most of the
novel genes we have found using striatum as source material show little cortical response to apomorphine, compared with c-fos, another previously described early gene, MAP kinase phosphatase-1 (MKP-1; also
called CL100), and the novel gene ania-3.
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Figure 6.
The striatal response to cocaine and eticlopride
uses a similar set of genes. All brain sections shown are from normal
(i.e., unlesioned) animals given the indicated drug and killed 1 hr
later. All drugs were given intraperitoneally at the following doses:
cocaine 25 mg/kg, apomorphine 2 mg/kg, eticlopride 1 mg/kg.
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DISCUSSION |
A complex set of dopamine-induced genes
We wished to find genes whose expression was potentially
responsible for the altered cellular and behavioral responses observed after exposure to dopaminergic drugs. We chose to use D1
stimulation in unilaterally 6-OHDA-lesioned rats because this model
gives very robust gene expression, allows direct side-to-side
comparison within each animal, and is directly relevant to the clinical
phenomenon of L-DOPA-induced dyskinesias. Using
approximately 150 primer combinations, we found that more than 30 genes
can be rapidly and transiently induced in this system; of these,
approximately half had not been described previously. Thus the striatal
response to D1 stimulation involves a complex program of
gene expression.
Because our DDPCR screen did not find all of the genes we know to be
induced by dopamine, it is reasonable to assume that other novel genes
remain to be found. Although it is difficult to estimate the total
number of induced genes, we note that of the known genes we expected to
find, about half appeared in the screen. This implies that the total
number of inducible genes is probably more than 50. One limitation of
the present study is that the DDPCR screen only examined 1 and 24 hr
time points after D1 stimulation. There may be additional
genes that show no detectable increase within the first hour but are
subsequently induced and return to baseline within 24 hr. We note that
the technique did display great sensitivity, detecting increases at the
1 hr time point of a CREM gene and several novel genes that are barely
visible with in situ hybridization. We did not observe any
genes that are downregulated by D1 stimulation. We cannot be certain whether this is because of a limitation in our use of the
DDPCR technique or whether it reflects the reality of few or no rapidly
downregulated genes.
The 6-OHDA-lesioned striatum displays increased dopamine responsiveness
compared with the unlesioned striatum, and this can be increased
further by D1 stimulation (Juncos et al., 1989; Morelli et
al., 1989). Yet compared with the many genes induced within 1 hr of
dopamine stimulation, our DDPCR screen found very few genes with
altered expression caused by 6-OHDA lesion alone or at 24 hr after
dopamine stimulation. It is possible that the altered dopamine
sensitivity associated with these states is achieved through
persistently altered expression of a small number of genes. Alternatively, it is possible that substantial changes in gene expression are not required.
Induction time courses are diverse but transient
We have demonstrated that a large number of striatal genes are
rapidly induced by dopamine stimulation and return to baseline within
24 hr. The transience of this genetic response to dopamine is striking.
It may be that a brief burst of gene upregulation is enough to alter
cellular physiology, through persistent modification of proteins
(Lisman and Goldring, 1988), through production of persistent proteins
[such as chronic Fras (Hope et al., 1994)], or by causing structural
changes such as alterations in cellular morphology or formation of new
synapses (Robinson and Kolb, 1997). These types of explanation may
account for the transient sensitivity to blockers of gene expression of
late-phase hippocampal long-term potentiation [L-LTP (Nguyen et al.,
1994)]. The set of genes necessary for L-LTP may overlap with those we
have observed, because D1 receptors have been directly
implicated in hippocampal long-term potentiation (Frey et al., 1991;
Huang and Kandel, 1995), and the cAMP/PKA/CREB pathway has been shown
to be necessary for L-LTP and other memory models (for review, see
DeZazzo and Tully, 1995). It has not yet been determined which genes
downstream of CREB are critical for memory formation.
The large number of D1-induced striatal mRNAs do not form a
single discrete wave of gene expression, but rather show great variation in both the rapidity with which they are induced and their
relative persistence (Fig. 5A,B). Because many of the most rapidly induced genes are transcription factors, they may be regulating some of the later genes. It may be possible to learn more about specific genes by blocking transcription and/or translation at specific
times after stimulation. We note, however, that a delay does not
necessarily indicate dependence on new protein synthesis. Although both
c-fos and dynorphin mRNAs are believed to respond to
phosphorylation of CREB in striatum (Cole et al., 1995),
c-fos is induced within 30 min after SKF38393, whereas
dynorphin does not show substantial induction in the lesioned striatum
until ~4 hr (Fig. 5B).
A common genetic program induced by distinct drug classes
Psychostimulants and antipsychotic drugs can lead to behavioral
abnormalities both in humans and in animal models, including stereotypies and dyskinesias. Dyskinesias are also observed with the
use of dopamine agonists in therapy for Parkinson's disease. We have
observed that all of these treatments induce very similar sets of genes
(Figs. 2, 6), despite quite different mechanisms of action. Although
cocaine and D1 agonists are causing gene induction in
striatonigral cells, and eticlopride is inducing genes in
striatopallidal cells, the similarity of the induced genes suggests
that similar adaptations are taking place in each case.
As with D1 agonists in the dopamine-depleted striatum,
induction of c-fos by either psychostimulants or
antipsychotics appears to require CREB (Konradi et al., 1994; Konradi
and Heckers, 1995). The common induction of CREB in striatal neurons
may account for the similar program of gene induction that we have
observed. Other transcription factors may also be involved, because
SKF38393 in lesioned striatum has been shown to increase AP-1 binding
(Huang and Walters, 1996), as have psychostimulants and antipsychotics (Nguyen et al., 1992). This increase in AP-1 binding is consistent with
the increases in fos and jun family member mRNAs. At the present time,
however, there are no proven target genes that are upregulated in
striatum by AP-1.
Specificity of gene induction
It is possible that the set of genes reported here merely
represents a general neuronal response to strong stimulation. One reason to think that this is not the case is the variation in response
to apomorphine. At the dose shown in Figure 6, a number of mRNAs are
strongly induced in cortex, including c-fos, MKP-1, and
ania-3. Others, including most of the novel genes reported here, do not
show a substantial apomorphine response. Thus the expression of the set
of genes discussed in this paper is not coupled in a fixed manner;
i.e., they do not represent a common response to any stimulation that
induces c-fos.
We note that most induced genes described previously in brain were
initially found outside brain or in other brain regions such as
hippocampus (Nedivi et al., 1993; Yamagata et al., 1993). The use of a
striatum-based screen in the present study may account for the large
number of novel genes found and their relative striatal specificity of
induction.
Genetic change and neuronal function
The first genes known to show rapid induction in response to
dopaminergic drugs were transcription factors, such as
zif268, krox20, and members of the AP-1 family (Cole et al.,
1992). These are presumably involved in regulating the expression of
other genes. Over the last several years Worley and colleagues
(Yamagata et al., 1994; Lyford et al., 1995; Tsui et al., 1996;
Brakeman et al., 1997), using subtractive hybridization methods in
seizure models, have uncovered genes (including arc, homer, narp, and rheb) that may have more direct effects on cellular physiology. At the
present time we do not know the functions of the novel gene sequences
(ania-1 through ania-12) reported here. Although it is possible that
some of these novel sequences represent previously unreported parts of
known genes, most of them do not show a spatial and temporal expression
profile in common with the known genes we have studied. Our current
efforts are directed toward discovery and manipulation of their
corresponding full-length mRNAs.
Several of the induced genes may be acting as homeostatic responses to
activation of signal transduction pathways. Among these are MKP-1
[also called CL100 (Takano et al., 1995)], which dephosphorylates MAP
kinase, CREM, some forms of which are inhibitory versions of CREB
(Stehle et al., 1993), and CHOP, which is an inhibitor/modulator of the
transcription factor C/EBP (Ron and Habener, 1992). The induction of
the mRNAs for these genes potentially represents a compensatory
response to activation of their cognate proteins. The MAP kinase and
C/EBP pathways have been implicated in memory formation in other
systems (Alberini et al., 1994; Martin et al., 1997). To our knowledge,
no one has yet examined the role of MAP kinases or C/EBP activation in
dopamine-induced striatal gene induction.
Why do striatal cells respond to dopaminergic drugs with this diverse
set of induced genes? There are several forms of neuronal adaptation
taking place, including alterations in sensitivity to neurotransmitters
and changes in membrane properties. The challenge is to relate these
adaptations to specific changes in gene expression. We have shown that
the sets of genes potentially involved is large. The task of sorting
out the mechanisms by which they affect physiology will require
extensive efforts by a number of laboratories.
| |
FOOTNOTES |
Received March 23, 1998; revised May 4, 1998; accepted May 7, 1998.
This work was supported by the Intramural Programs of the National
Institute of Mental Health and the National Institute of Neurological
Disorders and Stroke. We thank Alex Cummins, Ron Harbaugh, Jim Nagle,
Erica Olsen, and Erin Perry for technical assistance.
Correspondence should be addressed to Joshua Berke, National Institute
of Neurological Disorders and Stroke, 36/4C-24, 36 Convent Drive,
Bethesda, MD 20892-4135.
| |
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Top
Abstract
Introduction
