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The Journal of Neuroscience, May 15, 1998, 18(10):3639-3649
Defective Motor Behavior and Neural Gene Expression in
RII -Protein Kinase A Mutant Mice
Eugene P.
Brandon1,
Sheree F.
Logue4,
Monique
R.
Adams1,
Ming
Qi1,
Sean P.
Sullivan1,
Alvin M.
Matsumoto2,
Daniel M.
Dorsa1, 3,
Jeanne M.
Wehner4,
G. Stanley
McKnight1, and
Rejean L.
Idzerda1
Departments of 1 Pharmacology, 2 Medicine
and the Geriatric Research Education and Clinical Center of the
Veterans Affairs Puget Sound Health Care System, and
3 Psychiatry and Behavioral Science, School of Medicine,
University of Washington, Seattle, Washington 98195, and
4 Institute for Behavioral Genetics, University of
Colorado, Boulder, Colorado 80309
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ABSTRACT |
Motor behavior is modulated by dopamine-responsive neurons in the
striatum, where dopaminergic signaling uses G-protein-coupled pathways,
including those that result in the activation of cAMP-dependent protein
kinase (PKA). The RII isoform of PKA is highly enriched in the
striatum, and targeted disruption of the RII gene in mice leads to a
dramatic reduction in total PKA activity in this region. Although the
mutant mice show typical locomotor responses after acute administration
of dopaminergic drugs, they display abnormalities in two
experience-dependent locomotor behaviors: training on the rotarod task
and locomotor sensitization to amphetamine. In addition, amphetamine
induction of fos is absent, and the basal expression of
dynorphin mRNA is reduced in the striatum. These results demonstrate that motor learning and the regulation of neuronal gene expression require RII PKA, whereas the acute locomotor effects of dopaminergic drugs are relatively unaffected by this PKA deficiency.
Key words:
cAMP-dependent protein kinase; PKA; knock-out; mouse; striatum; dopamine; amphetamine; locomotion; rotarod; sensitization; fos; dynorphin
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INTRODUCTION |
The basal ganglia modulate motor
output from the CNS and have been implicated in several movement
disorders, including Parkinson's disease, Huntington's disease, and
tardive dyskinesia (Albin et al., 1989 ). The nigrostriatal dopaminergic
projection, which degenerates in Parkinson's disease, plays a critical
role in the modulation of basal ganglia function in humans. Similarly,
dopaminergic activity in the striatum modulates motor functions in
rodents (Gage et al., 1983; Walsh and Wagner, 1992; Emerich et al.,
1993; Zhou and Palmiter, 1995).
The psychomotor stimulants, cocaine and amphetamine, are believed to
exert their locomotor activating properties primarily by increasing
dopaminergic neurotransmission in the striatum. Lesions of the
mesostriatal dopaminergic projection abolish amphetamine-enhanced locomotion in rodents (Creese and Iversen, 1975; Koob et al., 1981).
Both cocaine and amphetamine increase synaptic dopamine levels by
affecting dopamine transporter function (Groves and Rebec, 1976;
McMillen, 1983; Sulzer et al., 1995; Giros et al., 1996). A
sensitization phenomenon occurs with repeated amphetamine administration, whereby each sequential administration of drug results
in an incrementally greater physiological response (Robinson and
Becker, 1986; Kalivas and Stewart, 1991). Sensitization requires activation of D1- and D2-like dopamine receptors (Ujike et al., 1989;
Vezina and Stewart, 1989) and depends on protein synthesis (Karler et
al., 1993). Recently, mice have been developed that have alterations in
their dopaminergic function. Mice genetically deficient in dopamine
show extreme hypoactivity unless rescued with L-dopa (Zhou
and Palmiter, 1995). Conversely, mice lacking the dopamine transporter
are hyperactive and are unresponsive to the locomotor-activating
effects of amphetamine and cocaine (Giros et al., 1996). Mice lacking
the various dopamine receptors (D1R, D2R, D3R, D4R) exhibit a range of
locomotor defects (Drago et al., 1994; Xu et al., 1994a,b; Baik et al.,
1995; Accili et al., 1996; Rubinstein et al., 1997). Collectively,
these mutants demonstrate a critical role for dopaminergic signaling in
the regulation of motor output. In some cases, changes in the
expression of striatal peptides that are believed to modulate motor
output (such as dynorphin, substance P, and enkephalin) have been
correlated with the motor defects.
In the striatum, distinct populations of efferent medium spiny neurons
express D1- and D2-like receptors (Gerfen et al., 1990; Hersch et al.,
1995), although recent data suggest the possibility that some neurons
express both receptor subtypes (Surmeier et al., 1996). D1-like
receptors generally are thought to act via Gs-type proteins
to increase the production of cAMP by adenylyl cyclase and thus
activate the cAMP-dependent protein kinase, PKA. Conversely, D2-like
receptors are Gi/Go-coupled and can
inhibit cyclase activity, activate an inward rectifying
K+ channel, or inhibit Ca2+
channels (Stoof and Kebabian, 1981; Greif et al., 1995; Surmeier et
al., 1995). Thus, PKA likely serves as one of the important effector
molecules in dopamine responsive neurons.
In the mouse there are six PKA subunit isoform genes. Each PKA
holoenzyme consists of two homodimeric regulatory (R) subunits and two
catalytic (C) subunits; the type of holoenzyme is defined by its
regulatory subunits (i.e., RII-PKA is
RII2C2). In situ hybridization analysis suggests that, of the regulatory subunits (RI , RI, RII, and RII), RII has the highest expression
in the striatal complex, including the caudoputamen, nucleus accumbens, and islands of Calleja (Cadd and McKnight, 1989). To distinguish which
effects of dopamine depend on RII-PKA and which are likely to rely
on other G-protein-mediated signaling pathways, we have created mice
with a targeted disruption of the predominant striatal regulatory
subunit, RII. The RII knock-out mice are severely deficient in
striatal PKA activity and exhibit changes in gene expression and
locomotor behavior.
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MATERIALS AND METHODS |
Gene disruption and genotyping. The disruption of the
RII gene in embryonic stem (ES) cells has been described (Brandon et al., 1995a). Chimeras were bred to obtain heterozygotes, which were
interbred to produce homozygous mutant and wild-type littermates. All
experiments used these F1 (50% C57BL/6 and 50% 129SvJ) mice or the
offspring generated from breeding each genotype, and they were age- and
gender-matched. ES cells and mice were genotyped by genomic Southern
blot and/or PCR analysis, using standard techniques.
Northern blot analysis. Tissue was homogenized in 8 M guanidine hydrochloride and 25 mM sodium
acetate, and RNA was precipitated with 0.6 vol of ethanol. After
centrifugation, the RNA pellet was resuspended in 8 M
guanidine hydrochloride and 25 mM sodium acetate, extracted
with a 50:50 mixture of phenol and chloroform, and then reprecipitated
with 0.6 vol of ethanol. Northern blot analysis was performed
essentially as described (Mosley et al., 1989), using a
32P-riboprobe synthesized from a 357 base pair cDNA
fragment that encodes a C-terminal portion of RII.
Western blot analysis and kinase assays. Protein was
prepared from various brain regions by Dounce homogenization in PBS
containing (in mM) 250 sucrose, 1 EGTA, 4 EDTA, 4 DTT, and
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) plus
0.5% Triton X-100, 2 µg/ml leupeptin, 3 µg/ml aprotinin, and 0.2 mg/ml soybean trypsin inhibitor, followed by sonication and
centrifugation for 10 min at 12,000 × g at 2°C.
Supernatant proteins (40 µg/lane) were resolved by 10% SDS-PAGE and
transferred onto nitrocellulose (Schleicher & Schuell, Keene, NH).
Blots were pretreated overnight at room temperature in blocking buffer
(5% BSA, 0.2% Tween-20, and 0.01% sodium azide) and probed with
antisera to murine PKA subunits as described (Cummings et al., 1996).
Kinase activity in the tissue supernatants was assayed as described
(Clegg et al., 1987), using Kemptide as a substrate in the presence or
absence of 5 µM cAMP, each in the presence or absence of
40 µg/ml PKI peptide inhibitor. Three mice of each genotype were
assayed, and the results were averaged. Activity is reported as units
(pmol per min) per milligram of protein after nonspecific kinase
activity is subtracted (the kinase activity not inhibited by PKI). The
half-maximal activation constants (Ka)
were determined in mutant and wild-type striatum as described (Cadd et
al., 1990). Striata from three mice of each genotype were pooled and
then assayed in triplicate. The high concentration of PKA in the
homogenates exceeds the cAMP concentration that should yield
half-maximal activation, making an accurate Ka
comparison difficult with cAMP, so a cyclic nucleotide with lower
affinity, cIMP, was used instead.
Rotarod performance. The rotarod apparatus (Ugo Basile,
Varese, Italy) was used in two different acceleration modes, gradually increasing either from 4 to 30 rpm ("slow" speed) or from 5 to 35 rpm ("fast" speed) over the course of 5 min. Mice were placed on
the apparatus, and rotation was initiated. Latency to fall was recorded
automatically. Trials were given within the last 4 hr of the light
phase of the 12 hr light/dark cycle, 10-25 min apart. Ten trials were
given on the first day and four on the second. Mice that stayed on the
rotarod for >300 sec were considered complete responders; their
latencies were recorded as 300 sec. At the slow speed, 6 of 17 wild-type mice reached this criterion, and no mutants did. At the fast
speed only 1 of 11 mutants reached this criterion, and none of the wild
types did.
Locomotor activity. Mice were placed in the darkened testing
room 30 min before testing. Locomotor activity was assessed in an
automated open field arena illuminated by a bright white light. The
number of infrared photobeam interruptions in each perpendicular axis
was recorded and totalled for each session. The arena was cleaned with
75% ethanol after each trial. For drug studies, mice were injected
intraperitoneally with drug or saline 15 min before testing and placed
in a holding cage until testing. Amphetamine and cocaine test sessions
were 15 min; those for quinpirole and SKF38393 were 30 min.
Paw print assay. Age-matched (~15 weeks) male mice were
tested during the last 6 hr of the light phase of the light/dark cycle. The back paws of each mouse were dipped into ink, and the animals were
placed at the entry of a dark tunnel (9.2 × 6.3 × 35.5 cm). Footprints were recorded on a clean sheet of white paper placed on the
floor of the tunnel. Stride length was determined by measuring the
distance between each step on the same side of the body. For each
animal, the six strides closest to the center of the paper were
measured. This excluded strides at the beginning and end of the tunnel
where the animal was initiating and terminating movement. Average
stride length was calculated. The length of the shortest of these
strides was subtracted from the length of the longest to determine the
range in stride length for each subject.
Grooming. Mice were housed individually for 24 hr before the
experiment. Trials were given within the last 4 hr of the light phase
of the light/dark cycle. Mice were injected intraperitoneally, and then
food and water were removed from the cage. Each mouse was observed once
per minute, and its activity was recorded (activities included
locomotion, grooming, nibbling on bedding or feces, digging in bedding,
and stationary/sleeping). The proportion of each type of activity
observed from 15 to 115 min after injection was determined and
analyzed.
Determination of brain amphetamine levels. Animals were
injected intraperitoneally with D-amphetamine (5 mg/kg) and
killed 15 min later. Brains were removed, weighed, and homogenized in ice-cold 0.05 M sodium borate, pH 10; methamphetamine was
added as an internal standard. Homogenates were centrifuged at
10,000 × g at 10°C for 10 min. Drugs were extracted
from the supernatants with an equal volume of ethyl acetate, followed
by centrifugation at 1000 × g at 10°C for 10 min.
The extract was dried under a stream of nitrogen and resuspended in a
50:50 mixture of acetonitrile/1-chlorobutane. Samples (1 µl) were
analyzed by gas chromatography in a Hewlett-Packard 5890 with an HP-5
column (25 m × 0.32 mm) and a nitrogen/phosphorous detector.
Split (50:1) 1 µl injections were analyzed. The injection port was
heated to 250°C, and samples were chromatographed at an initial
temperature of 150°C and ramped at 10°C per minute to 260°C and
held for 5 min. The detector was maintained at 275°C. The flow rate
was 1.2 ml/min helium. Data were collected and analyzed on an HP3396
integrator, and areas under the curve were determined by using an
amphetamine standard curve (5-95 ng/µl; r = 0.99); recoveries were calculated on the basis of the internal standard. Data
are reported as micrograms of amphetamine per gram of brain wet
weight.
Hormone analyses. Glucocorticoid levels were assayed in
blood plasma, using a rat corticosterone-3H kit (ICN
Biomedicals, Cleveland, OH). Dopamine was assayed in striatal
homogenates by HPLC with electrochemical detection as described
(Liebmann and Matsumoto, 1990).
Dopamine transporter assay. The striatum was dissected and
homogenized, and synaptosomes were prepared as described (Grady et al.,
1992) with modifications. After centrifugation at 12,000 × g, the pellet was resuspended in buffer containing (in
mM) 128 NaCl, 2.4 KCl, 3.2 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 HEPES, pH 7.4, 10 dextrose, 1 ascorbate, and 0.01 pargyline. Aliquots
were incubated with or without various concentrations of
D-amphetamine (10 9 to
103 M), along with 5 µCi of
3H-dopamine (0.1 µM; specific activity = 48 Ci/mmol). The assay was terminated by the addition of ice-cold
buffer and then filtered over GF/C filters with washing. Radioactivity
was determined in a scintillation counter, and the results were
calculated per milligram of protein.
Receptor binding. Dopamine D1 and D2 receptor binding was
performed as described (Bouthenet et al., 1991) with modifications. D1
receptors were assayed with 125I-labeled SCH23982 (DuPont)
and D2 receptors were labeled with 125I-sulpiride
(Amersham, Arlington Heights, IL). Sections were incubated for 45 min
at room temperature with 250 µl of (in mM) 50 Tris-HCl buffer, pH 7.4, 120 NaCl, 5 KCl, 1 CaCl2, 5.7 ascorbic acid, and 0.01 8-hydroxyquinoline plus 3 nM
[125I] drug. Then the slides were washed in fresh
buffer, dipped in deionized water, and dried promptly. Nonspecific
binding was defined as residual binding in the presence of 3 mM fluphenazine. Labeled slides were stored overnight at
4°C in the presence of a desiccant and then apposed for 30 min to
Hyperfilm -Max, along with plastic standards containing known
concentrations of 125I (Amersham). Similar results were
obtained in two independent experiments.
In situ hybridization. In situ hybridization
was performed as described (Ward and Dorsa, 1996), using
35S-labeled antisense riboprobes for dynorphin,
c-fos, or RII. The specificity of the probes was
established by hybridization with labeled sense probes. Brain sections
from different groups were matched anatomically by bright-field
microscopy. For quantitative analysis autoradiograms were analyzed with
a microcomputer-based image analysis system (MCID, Imaging Research,
St. Catherine's, Ontario, Canada) to determine OD, and the dynorphin
image was colorized for illustration. For both c-fos and
dynorphin, samples of the size indicated in Figure 7A were
taken from coronal slices between the genu of the corpus collosum and
the decussation of the anterior commissure. An average value for each
region was determined for each mouse (~20 samples per region per
mouse); the averages shown in Figures 6B and
7B represent averages of the values determined for each
mouse. For the data shown in Figure 7B, values from the
septum of each slice were subtracted before averaging; this region
showed no specific signal over cells on emulsion-coated slides and thus
was considered nonspecific background.
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RESULTS |
Mice lacking RII are fertile and long-lived
Gene targeting was performed by standard techniques (Capecchi,
1989). The mutation eliminated the entire coding region of the first
exon of the RII gene, including the translation start site (Fig.
1A). Germ
line-competent chimeras were bred to produce heterozygous mice.
Wild-type, heterozygous, and homozygous mutant offspring from crosses
of heterozygotes (Fig. 1B) were produced at the
predicted Mendelian frequency, indicating that no embryonic lethality
is associated with the mutation. Homozygous mutant mice expressed no
detectable mRNA for RII, as determined by Northern blot analysis
(Fig. 1C) and in situ analysis (Fig.
1D). Figure 1D also illustrates the
high expression of RII mRNA in the striatum of normal mice.
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Figure 1.
Targeted disruption of RII. A,
Genomic locus, targeting vector, and predicted structure of targeted
locus. The targeting vector replaces the coding region of exon 1 of the
RII gene with a neomycin resistance cassette (neo).
Restriction enzyme sites shown include the following: A,
AatII; E, EcoRI;
H, HindIII; R,
RsrI. The probe fragment used to identify disrupted
alleles in ES cells and mice is shown. B, Genomic
Southern blot of tail DNA from offspring of a cross of heterozygotes.
DNA was digested with HindIII and probed with the
fragment shown in A. The wild-type allele yields a 4.7 kb band, and the disrupted allele yields a 3.0 kb band. Genotypes are
indicated above each lane: wild type (+/+), heterozygous (+/ ), and
homozygous mutant (/). C, Northern blot of brain
total RNA (10 µg) from wild-type (+/+) and homozygous mutant (/)
mice, probed with a 350 bp riboprobe specific for RII. The migration
of RII mRNA is indicated. D, In situ
hybridization of wild-type (WT) and homozygous
mutant (RII ko) brain slices, using the
RII-specific riboprobe. Expression is high in the cortex and
striatum and is low in the globus pallidus.
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RII is normally present at high levels in adipose tissue, brain, and
hematopoietic tissues (fetal liver and bone marrow). RII also is
expressed in reproductive cells, where it is regulated by hormones that
activate PKA, suggesting an important reproductive function (Jahnsen et
al., 1986; Oyen et al., 1988). However, RII mutant mice exhibited
normal fertility, as evidenced by both pregnancy rate and litter size.
The mice were morphologically normal in all tissues examined except the
adipose, where they showed a reduction in fat accumulation (Cummings et
al., 1996).
RII is the major isoform of PKA in the striatum
Although both in situ analysis of RII mRNA
expression (Cadd and McKnight, 1989) and immunohistochemical analysis
of RII protein expression (Ludvig et al., 1990; Glantz et al., 1992) have been performed on mouse brain, the relative contribution of RII
holoenzyme to the total PKA complement in any given region of the brain
has not been determined previously. Western blots comparing equal
amounts of protein from various regions of the mouse brain showed that
RII expression is highest in the striatum, lower in other brain
regions, and nearly undetectable in the cerebellum (Fig.
2A). Kinase assays were
performed to determine the amount of cAMP-dependent activity remaining
in various brain regions of RII knock-out mice (Fig. 2C).
In the whole brain, total cAMP-stimulated activity was reduced by
~50%, as compared with wild-type mice, similar to the reduction
observed in isolated cortex. A modest decline was detected in
cerebellum, whereas a 75% reduction in activity was evident in the
striatum. Thus, RII-PKA comprises the majority of PKA in the normal
mouse striatum.
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Figure 2.
RII is the major PKA isoform in the striatum.
A, Western blot of several regions of wild-type mouse
brain probed with RII-specific antiserum. Lanes (from
left to right), cblm,
cerebellum; bstm, brainstem; hpth,
hypothalamus; hppc, hippocampus; cllc,
colliculi; mdbr, midbrain; strm,
striatum; nctx, neocortex. B, Western
blot analysis of PKA subunit isoform levels in striatum, using
homogenates from three wild-type (+/+) and three RII knock-out
(/) mice. Blots were probed with antibodies to the indicated PKA
subunits. C, Kinase assay with homogenates of the
indicated brain regions from wild-type (+/+) and mutant (/) mice.
Phosphorylation of the PKA substrate Kemptide was assayed in the
presence (Total) or absence
(Basal) of 5 µM cAMP. Error bars
represent SEM. D, PKA activation curves with striatal
homogenates from wild-type (wt) and
RII/ (mutant) mice. Half-maximal
activation (dotted lines) was achieved at ~4
µM cIMP in wild-type mice and 1 µM cIMP in
mutants. cIMP was used instead of cAMP because of its lower affinity
for PKA R subunits (see Materials and Methods).
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Previous studies suggested that loss of RII might lead to
compensatory changes in other PKA subunits. A compensatory increase in
RI has been detected in multiple systems in which the PKA system has
been perturbed (Amieux et al., 1997), including the brown adipose
tissue of the RII mutants (Cummings et al., 1996) and the brain of
RI null mutants (Brandon et al., 1995b). In striatum from RII
mutants, elevated levels of both RI and RI were found, with a
smaller increase in the RII isoform level (Fig.
2B). However, the increases in these R subunits did
not compensate fully for the loss of RII, leaving the unbound C
subunits susceptible to proteolysis. Thus, the catalytic subunits of
PKA (C and C) were reduced dramatically in the mutant striatum, as predicted by the kinase assays. All of these changes in subunit levels are likely to result from altered protein stability, because mRNA levels for the subunits are unchanged in the RII mutants (data
not shown) (Amieux et al., 1997).
The loss of RII and increases in RI and RI indicate a shift in
isoform prevalence from type II to type I PKA in mutant striatum. A
comparison of PKA activation curves (Fig. 2D)
demonstrated that the PKA remaining in mutant striatum was activated at
an approximately fourfold lower cyclic nucleotide concentration than in
wild type. This is consistent with the increased sensitivity of type I
kinase to activation, as compared with type II (Cummings et al.,
1996).
RII knock-out mice are impaired in the rotarod task
Because PKA is one of the potential downstream effectors of
dopamine receptor activation, we sought to determine whether motor output was disrupted in these mice. We used the accelerating rotarod task to measure the mouse's ability to coordinate movement under challenging conditions (Dunham and Miya, 1957). The task measures how
long a mouse can stay on a rotating horizontal rod as its speed of
rotation is increased. Performance of this task involves both the
cerebellum and the striatum and has been shown both to require dopamine
and to evoke its release in the striatum (Bertolucci et al., 1990;
Emerich et al., 1993; Lalonde et al., 1995). Mice were placed on the
rod; rotation was initiated and then accelerated from 5 to 35 rpm over
the course of 5 min. As shown in Figure 3A, wild-type mice have
difficulty with the task at first but within a few trials improve their
performance significantly. In contrast, RII mutants show some
acquisition but never attain the same competence with this task as
wild-type mice. We considered the possibility that the RII knock-out
mice require a longer consolidation period to learn the rotarod task.
This phenotype might be expected if the RII-PKA isoform plays a
significant role in short-term motor learning, but not in long-term
memory formation, i.e., that which might require novel gene expression or protein synthesis (Goelet et al., 1986). Thus, the mice were tested
again 1 d later to determine whether their latencies improved. Only slight improvement by the mutants was seen on the second day (Fig.
3A). With further testing, mice of both genotypes maintained performances similar to those observed on day 2, with the latencies for
mutants remaining consistently below those of wild-type mice (data not
shown). In a second set of experiments, mice were tested at a less
challenging rate of acceleration to determine whether this might
facilitate their acquisition of this task. The wild-type mice showed a
more rapid acquisition and increased their average latencies at this
lower rate, but the RII mutants were still dramatically impaired
(Fig. 3B). These data show that the ability to coordinate
motor output is disrupted by the absence of RII. The defect in motor
coordination is specific for the loss of the RII subunit of PKA,
because mice lacking the RI subunit isoform showed no such decrement
(data not shown).
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Figure 3.
Impaired performance of RII knock-out mice on
the rotarod task. A, RII/
(open squares; n = 11) and wild-type
control mice (closed squares; n = 13) were tested for their ability to stay on the accelerating rotarod.
Ten trials were conducted on the first day and four on the second day
(error bars represent SEM). ANOVA for repeated measures revealed a
significant effect of trials 1-10 on day 1 in both wild-type
(F(9,108) = 7.62; p < 0.001) and RII knock-out mice (F(9,90) = 5.11; p < 0.001) as well as a significant effect
of genotype (F(1,22) = 14.07, p < 0.002 on day 1;
F(1,22) = 10.24, p < 0.005 on day 2). B, When tested at a lower rate of
acceleration, RII/ (open squares;
n = 18) mice were impaired significantly, as
compared with wild-type control mice (closed squares;
n = 17; F(1,33) = 60.76, p < 0.001 on day 1;
F(1,33) = 26.90, p < 0.001 on day 2).
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Poor performance on the rotarod task can result from cerebellar
dysfunction, although we considered this to be unlikely in the RII
mutants because little if any RII normally is expressed in the
cerebellum. To assess cerebellar function, we performed a paw print
assay in which the hind paws of the mice were inked, allowing their
tracks to be recorded as they walked through a tunnel. Typical tracks
from wild-type and mutant mice are shown in Figure
4A, revealing no
obvious differences. Analyses of mean stride length and mean range in
stride length also showed no abnormalities in the mutants (Fig.
4B). A standard open-field test in a novel setting
was used to examine spontaneous horizontal locomotor activity because
this has been shown to be greater in mice with vestibular deficits. No
difference in photobeam breaks was observed between the mutant and
wild-type mice (Fig. 4C). These data demonstrate that the
absence of RII does not produce cerebellar defects manifested as
balance or locomotor abnormalities.
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Figure 4.
RII mutants have no obvious cerebellar or
locomotor behavioral defects. A, Hind paw footprints
were recorded, and representative prints of a wild-type (+/+) and
RII mutant (/) mouse are shown. B, Mean stride
length and mean range in stride length in the paw print assay were
similar in both wild-type (+/+; n = 10) and RII
mutant (/; n = 9) mice. C,
Locomotor activity in response to a novel environment was recorded in
an open-field arena, and similar responses were observed in wild-type
(+/+; n = 10) and RII mutant (/;
n = 9) mice. Error bars represent SEM in all
panels.
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RII knock-out mice exhibit typical acute behavioral responses to
dopaminergic agents but increased sensitization to chronic
amphetamine
Acute administration of an indirect dopaminergic agonist
(amphetamine or cocaine) causes increased horizontal locomotion in mice. Figure 5A demonstrates
that a high dose of amphetamine or cocaine evoked acute locomotor
responses that were similar in wild-type and RII mutant mice. Under
some treatment regimens, repeated administration of amphetamine
produces a sensitization phenomenon whereby each sequential
administration of drug results in a greater response than that seen
previously (Robinson and Becker, 1986; Kalivas and Stewart, 1991). We
found that, whereas the wild-type mice displayed modest sensitization
to amphetamine, the RII mutant mice displayed significantly greater
sensitization, particularly at low doses. Figure 5B depicts
the responses to two different doses of amphetamine administered for
5 d.
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Figure 5.
Response of RII mutants to dopaminergic agents.
A, Acute locomotor responses. Wild-type (solid
bars) and RII/ (stippled
bars) mice were treated with saline (n = 5 WT and 5 KO), a 10 mg/kg dose of D-amphetamine
(amph; n = 5 WT and 6 KO), or a 20 mg/kg dose of cocaine (n = 6 WT and 9 KO).
Locomotor activity was determined in an open field by recording
photobeam breaks. Error bars represent SEM. B, Enhanced
sensitization to low-dose amphetamine. Wild-type (solid
bars) and RII/ (stippled
bars) mice were treated with saline (n = 5 WT and 5 KO), a 2.5 mg/kg dose of D-amphetamine
(n = 5 WT and 6 KO), or a 5 mg/kg dose of
D-amphetamine (n = 6 WT and 6 KO) for 5 d,
and locomotion was determined. Locomotor responses to three other
doses were determined also (data not shown). When saline treatment was
compared with 1.0, 2.5, 5.0, and 10 mg/kg doses of amphetamine,
repeated measures ANOVA revealed a significant effect of genotype
(F(1,42) = 41.07; p < 0.001). C, D1 agonist-induced grooming behavior.
Wild-type (solid bars) and RII/
(stippled bars) mice were treated with saline
(n = 8 WT and 8 KO) or a 5.0 mg/kg dose of SKF81297
(n = 18 WT and 18 KO). Each mouse was observed for
grooming and other activities; the percentage of time spent grooming is
reported as mean ± SEM. Grooming increased significantly with
SKF81297 in both genotypes (p < 0.02), but
mutants responded somewhat less than wild types
(p = 0.05). D, Acute
locomotor responses to D1 and D2 agonists. Wild-type (solid
bars) and RII/ (stippled
bars) mice were treated with saline (n = 8 WT and 11 KO), an 8.0 mg/kg dose of SKF38393 (n = 5 WT and 7 KO), or a 2.5 mg/kg dose of quinpirole
(quin; n = 5 WT and 5 KO), and
locomotor responses were determined.
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One explanation for the increased responsiveness to amphetamine of the
RII knock-out mice could be altered pharmacokinetics (Camp et al.,
1994). Therefore, amphetamine levels in the brains of mutant and
wild-type mice 15 min after drug (5.0 mg/kg) administration were
analyzed by gas chromatography. The mice were not significantly different (6.85 ± 1.09 µg/gm for 12 mutants and 5.99 ± 1.03 µg/gm for 9 wild-type mice; p = 0.55).
Hyperresponsiveness to amphetamine also has been seen in rodents with
elevated glucocorticoid levels (Pauly et al., 1993). When stressed by
amphetamine administration (5.0 mg/kg), RII mutant mice actually
have lower plasma corticosterone levels than wild-type control mice
(291.4 ± 41.2 ng/ml for wild-type mice, n = 15;
173.2 ± 36.0 ng/ml for RII mutants, n = 14;
all at 15 min after drug administration), ruling out high
glucocorticoids as an explanation for the increased responsiveness of
the mutants.
To investigate whether the loss of RII impacts both D2 and D1
dopaminergic pathways, we determined the locomotor responsiveness of
mutant mice to specific agonists. The D1R agonist SKF38393 activated
horizontal locomotion in both mutant and wild-type control mice at
several doses (8.0 mg/kg is shown in Fig. 5D). A second D1R-mediated acute behavior also was examined; the D1R agonist SKF81297
increased grooming significantly in both genotypes (Fig. 5C). As observed by other investigators (Picetti et al.,
1997), a low dose of the D2/D3R agonist quinpirole suppressed
locomotion in wild-type mice; RII mutants responded similarly (Fig.
5D). Whether this suppression is attributable to quinpirole
acting on D3 receptors or on presynaptic D2 receptors is a matter of debate at present. These data demonstrate that acute locomotor responses to dopaminergic agonists are normal. Thus, locomotor defects
do not provide a simple explanation for the poor rotarod performance of
the mutants.
Besides the obvious loss of PKA in the striatum, what other components
of the dopaminergic pathways might be altered in the mutant mice? As
shown in Table 1, no significant
difference in the level of dopamine was found in the striatum. D1-like
receptor levels were measured in various regions of the caudoputamen,
and although the value for the dorsolateral region is smaller in the knock-out mice, as compared with wild types, this difference did not
reach statistical significance (p = 0.07).
Similarly, the D2-like receptor levels were normal. The function of the
dopamine transporter was investigated by measuring dopamine uptake into synaptosomes, and no differences were observed in either basal transporter function or in the inhibition of dopamine uptake by amphetamine.
RII-PKA is required for the induction of c-fos in
the dorsomedial striatum by amphetamine
To begin to ascertain whether gene regulatory events also might be
altered in the RII mutant mice, we examined the ability of
amphetamine to induce c-fos mRNA in the striatum. The
c-fos promoter contains a Ca2+/CRE
element (Sheng et al., 1990), which is responsive to cAMP, and thus
expression of c-fos provides a reasonable indicator of endogenous PKA-mediated gene induction (Sassone-Corsi et al., 1988).
Basal levels of c-fos mRNA were low in the striata of both
RII knock-out and wild-type mice. As shown in Figure
6, 1 hr after the administration of
amphetamine the wild-type mice exhibited a significant induction of
c-fos mRNA in the dorsomedial region of the striatum. In
striking contrast, the RII mutant mice almost entirely lacked this
induction. Induction of c-fos in the cortex remained intact.
The lack of striatal c-fos induction is specific for the
RII mutants because mice lacking the RI subunit isoform showed
normal induction of c-fos in this region (data not shown).
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|
Figure 6.
RII knock-out mice lack the induction of
c-fos mRNA in the dorsomedial striatum by amphetamine.
A, In situ hybridization for
c-fos mRNA is shown as dark-field photomicrographs.
Representative images from dorsomedial striatum demonstrate that
c-fos mRNA expression is induced in wild-type mice
(WT) 1 hr after intraperitoneal injection of 10 mg/kg D-amphetamine (bottom panels) but is
not induced in RII/ mutants (RII
KO). Images from saline-treated animals of each genotype are
shown also (top panels). Scale bar, 50 µm.
B, Densitometry measurements from several forebrain
regions of wild-type (solid bars) or
RII/ (stippled bars) mice treated
with saline () or 10 mg/kg D-amphetamine (+).
RII/ mice do not induce c-fos in the
dorsomedial (DM Str) region, as compared with wild-type
mice (p < 0.001). Other regions shown are
dorsolateral striatum (DL Str) and cingulate cortex
(Cing Cx). Error bars represent SEM
(n = 3-6 mice for each condition).
|
|
Reduced dynorphin mRNA in RII mutants
Dynorphin, a  opioid receptor agonist that is expressed by
striatal neurons, is believed to affect motor function and
sensitization to psychostimulants (Thompson et al., 1990; Angulo and
McEwen, 1994; Heidbreder et al., 1995). Psychostimulants have been
shown to induce dynorphin mRNA in the striatum of the rat (Steiner and Gerfen, 1993; Jaber et al., 1995; Wang et al., 1995), and induction is
believed to be regulated mainly by the PKA-CREB pathway (Douglass et
al., 1994; Cole et al., 1995). We were unable to measure a significant
induction of dynorphin mRNA after chronic amphetamine administration in
either wild-type or mutant dorsolateral striatum with our drug
treatment paradigm (data not shown). However, constitutive expression
of dynorphin mRNA was reduced in the mutant striatum, particularly in
the dorsolateral region, where expression was ~30% of normal (Fig.
7). Expression in the dorsomedial region was also lower. These data suggest that constitutive dynorphin gene
expression depends on PKA, which is reduced dramatically in the
mutants. In view of the current literature, we speculate that the loss
of PKA-regulated expression of genes such as dynorphin might underlie
the increased sensitization to amphetamine that is seen in the RII
mutant.
View larger version (84K):
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|
Figure 7.
Dynorphin mRNA is reduced in RII knock-out
mice. A, Dynorphin mRNA is expressed in a mainly normal
pattern in RII/ mice (RII KO) but
at reduced levels in the dorsolateral and dorsomedial regions.
Wild-type (WT) brain is shown for comparison.
Ovals indicate the regions sampled for the densitometric
analysis shown in B. B, Quantitative
densitometry of three regions of the striatum from wild-type
(solid bars; n = 7) and
RII/ mice (stippled bars;
n = 7) shows that no significant difference is
observed in the ventrolateral striatum, but reductions are seen in the
dorsolateral (p < 0.001) and dorsomedial
(p < 0.002) regions. Error bars represent
SEM. DL, Dorsolateral; DM, dorsomedial;
Str, striatum; VL, ventrolateral.
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| |
DISCUSSION |
Regulatory subunits serve three recognized roles in the modulation
of overall PKA activity in cells. First, they bind to and inhibit
catalytic subunits when cAMP levels are low, and they respond to
increases in cAMP by releasing the active C subunits. Second, the type
II subunits (RII and RII) bind to a family of anchoring proteins
(AKAPs) in the cell and consequently can specifically tether the PKA
holoenzyme near potential substrates. Third, the interaction of R with
C stabilizes both molecules against proteolysis, maintaining functional
levels of PKA in the cell. Most cells express multiple R subunit
isoforms; therefore, absence of a specific R subunit isoform might be
expected to create a surplus of free C subunits, which could stabilize
other R isoforms in the cell. This altered complement of R isoforms
within the cell might change the subcellular localization of PKA or
change its sensitivity to cAMP. If other R subunits within the cell are unable to compensate, a loss of C subunit is expected because of
proteolysis.
Disruption of the RII subunit in mice leads to dramatic changes in
the PKA system in tissues that express high levels of this subunit,
such as adipose and brain. In white and brown adipose tissue the loss
of RII is compensated for mainly by a concomitant increase in the
RI subunit and assembly of a type I kinase with increased
sensitivity to activation by cAMP (Cummings et al., 1996; Amieux et
al., 1997). The physiological outcome of this change is brown adipose
activation, leading to increased metabolic rate and a lean phenotype.
In brain tissues we find much less compensation by RI and a greater
loss of total PKA activity because of C subunit degradation (see Fig.
1). The region of the brain that expresses the highest levels of
RII, the striatum, suffers the greatest loss of total PKA activity,
with only ~25% of the kinase activity present in wild-type mice,
whereas brain regions that express very little RII, such as the
cerebellum, maintain normal kinase levels as expected.
Within the medium spiny neurons of the striatum, RII protein is
localized in dendritic and perikaryal regions (Ludvig et al., 1990;
Glantz et al., 1992), suggesting possible roles in both immediate
responses to dopaminergic transmission and longer term gene induction
events associated with cAMP signaling. PKA anchoring proteins such as
AKAP150 are coexpressed in striatal neurons and likely are essential
for the subcellular distribution of RII (Glantz et al., 1992; Rubin,
1994; Faux and Scott, 1996). The functional significance of the
coexpression has not been demonstrated directly for striatal neurons,
but in hippocampal neurons the anchoring of PKA via AKAPs is important
for AMPA/kainate receptor modulation (Rosenmund et al., 1994). Despite
the total loss of RII in our mutant mice, the levels of AKAP150
remain undisturbed in brain extracts (A. Sikorski and S. McKnight,
unpublished data). Thus not only are the RII mutant mice PKA
deficient, but they also have lost the major anchored form of PKA
expressed in the striatum, and their phenotype may reflect a
combination of the kinase deficiency and the absence of correct
subcellular localization.
Gene induction in striatal neurons is inhibited, but acute motor
responses are normal
The striatum is a major target for the neuromodulator, dopamine,
and some of the actions of dopamine are thought to be mediated by
changes in cAMP and the subsequent regulation of PKA activity. We
examined whether dopaminergic signaling in the striatum was affected in
RII mutant mice, leading to abnormalities in downstream effects on
gene activation and behavior. Despite the complete loss of the major
form of PKA in the striatum (RII), the mutant mice continue to
demonstrate normal acute locomotor responses to amphetamine and cocaine
with rapid and robust increases in horizontal locomotor activity. These
drugs act by increasing the levels of released dopamine in the synapse,
stimulating both D1- and D2-like receptors to increase locomotor
activity. In our studies, quinpirole, a D2/D3 agonist, acts to inhibit
motor activity when given alone, and this inhibition was not affected
by the loss of RII. Administration of a D1R agonist caused a modest
increase in motor activity, and the wild-type and RII mutant mice
were again indistinguishable. This normal acute responsiveness of the RII-deficient mice was surprising given previous studies in rats demonstrating the modulation of cocaine-induced locomotion by cAMP
analogs infused into the nucleus accumbens (Miserendino and Nestler,
1995). Together, these studies suggest either that the low level of
remaining PKA in the RII mutants is sufficient for coupling dopamine
receptor occupancy to neuronal modulation or that other signal
transduction pathways are primarily responsible for acute dopaminergic
locomotor responses.
The RII mutant mice are deficient in their ability to modulate gene
expression in both D1R and D2R neurons. The induction of
c-fos mRNA in the dorsomedial striatum is nearly absent in the mutant mice (see Fig. 6), and the basal level of expression of
dynorphin (a specific product of D1R neurons) is decreased in the
mutant mice as well (see Fig. 7). These defects in gene expression are
not limited to D1R neurons, because recently we have shown that the
haloperidol-mediated induction of both c-fos and neurotensin
mRNA is absent in the dorsolateral striatum of RII mutant mice
(Adams et al., 1997). Haloperidol has D2R antagonist activity and would
be expected to elicit an increase in cAMP and PKA activity in
D2R-containing neurons by blocking the inhibitory effect of D2R on
adenylyl cyclase. We postulate that neither D1R nor D2R neurons in
RII mutant mice are fully functional in eliciting PKA-mediated gene
induction because of their reduced PKA levels. Evidence for a graded
response to cAMP levels is suggested by studies in Aplysia
sensory neurons demonstrating that a robust activation of neurons is
required to cause translocation of the C subunit into the nucleus and
gene induction (Bacskai et al., 1993). Recent experiments performed
with rat striatal cultures indicate that sustained phosphorylation by
PKA of cAMP response element binding protein (CREB) or related
transcription factors is essential for CRE-mediated gene expression
(Bito et al., 1996; Liu and Graybiel, 1996), consistent with our
findings in the RII mutants.
Sensitization to amphetamine and other psychostimulants requires
protein synthesis and, very likely, changes in gene expression. Neuropeptides such as dynorphin may play an inhibitory role in sensitization (Heidbreder et al., 1995), and, as we show in Figure 7,
dynorphin expression in the striatum is decreased in the RII mutants. Although the locomotor response to acute administration of
high-dose amphetamine is very similar in mutant and control mice,
chronic treatment with low-dose amphetamine resulted in exaggerated
sensitization in the RII mutants (see Fig. 5). This altered
responsiveness may result, at least in part, from the observed defects
in basal and drug-induced gene expression.
Complex locomotor behavior
A model of striatal circuitry has been proposed previously (Albin
et al., 1989; DeLong, 1990; Graybiel, 1990; Gerfen, 1992). In this
model D1R-containing GABAergic striatal neurons primarily contribute to
a direct pathway with output to the substantia nigra pars reticulata
(SNr). The GABAergic D2R neurons form an indirect pathway via the
globus pallidus and subthalamic nucleus that ultimately results in a
positive glutamatergic output at the SNr. The inhibitory (GABAergic)
output of the direct pathway is integrated with the positive output
from the indirect pathway by the SNr for which the output to the motor
thalamus provides an inhibitory modulation of locomotor activity.
Despite extensive studies the mechanisms by which dopamine modulates
the output of striatal projection neurons remain unresolved, although
it appears that dopamine affects the ability of these cells to respond
to other inputs as opposed simply to evoking excitatory or inhibitory
potentials itself (Bargas and Galarraga, 1995; Surmeier et al., 1995).
Complex motor behaviors therefore may involve dopaminergic-mediated
changes in the activity of the different striatal neurons, which then
are deciphered by the SNr and integrated into a signal that is returned
to the motor cortex. Although this is certainly an oversimplification
of the neural pathways involved in modulating motor behavior, it
provides a framework for analyzing phenotypic changes in locomotion in the RII-mutant mice.
What is the role of striatal PKA in the complex regulation of locomotor
behavior? We observed no obvious impairment in the normal exploratory
movement of RII mutant mice (see Fig. 4); however, when challenged
on a rotarod apparatus, a more complicated locomotor task, the RII
mutants displayed a severe deficit (see Fig. 3). This deficit could be
attributable to a variety of factors. Cerebellar defects have been
shown to affect performance in this task (Lalonde et al., 1995), but
given the absence of any significant change in PKA activity in the
RII knock-out cerebellum, this seemed unlikely. The paw print
assessment (see Fig. 4) confirmed the absence of any detectable
cerebellar abnormality. Earlier studies have indicated that striatal
manipulations also can affect rotarod performance (Emerich et al.,
1993), and dopamine metabolism has been found to be increased in the
striatum during the performance of this task (Bertolucci et al., 1990).
We postulate that the rotarod requires increased, temporally modulated
activity of dopaminergic signaling and that PKA is important in the
acquisition of this behavioral task. It is proposed that PKA-mediated
gene expression events contribute to the "motor learning" that
occurs during successive rotarod trials and that the deficit in gene
expression in the RII mutants compromises such learning.
In conclusion, the RII mutant mice display discrete defects in motor
behavior that correlate with a severe loss of striatal PKA activity and
loss of PKA-mediated gene expression. Our observations support and
extend the current thinking on the role of striatal dopamine in motor
behavior and suggest that the RII mutants should be particularly
useful for further studies on the mechanisms of dopaminergic modulation
of gene expression and behavior.
| |
FOOTNOTES |
Received Jan. 8, 1998; revised Feb. 27, 1998; accepted Feb. 27, 1998.
This research was supported by National Institutes of Health Grant
DA-10156 (J.M.W.), Research Scientist Development Award AA-00141
(S.F.L.), Training Grant GM-07108 (E.P.B.), and GM-32875 (G.S.M.). We
thank Kirstin Gerhold, Kelly Millett, and Thong Su for excellent
technical assistance. We also thank Dr. James Douglass for providing
the dynorphin cDNA, Dr. Alan Unis for dopamine receptor analysis, and
Dr. Carol Quaife for assistance with photomicroscopy. We are grateful
to Drs. Richard Palmiter and Eric Kandel for insightful comments on
this manuscript.
Correspondence should be addressed to Dr. G. Stanley McKnight,
Department of Pharmacology, University of Washington, Box 357750, Seattle, WA 98195.
| |
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Top
Abstract
Introduction
Gene
