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The Journal of Neuroscience, June 15, 2002, 22(12):5188-5197
Phosphodiesterase 1B Knock-Out Mice Exhibit Exaggerated Locomotor
Hyperactivity and DARPP-32 Phosphorylation in Response to Dopamine
Agonists and Display Impaired Spatial Learning
Tracy M.
Reed1, 3,
David
R.
Repaske2, *,
Gretchen L.
Snyder4,
Paul
Greengard4, and
Charles V.
Vorhees1, *
Divisions of 1 Developmental Biology and
2 Endocrinology, Children's Hospital Research Foundation,
Cincinnati, Ohio 45229, 3 Department of Biology, College of
Mount St. Joseph, Cincinnati, Ohio 45233, and 4 Laboratory
of Molecular and Cellular Neuroscience, Rockefeller University, New
York, New York 10021
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ABSTRACT |
Using homologous recombination, we generated mice
lacking phosphodiesterase-mediated (PDE1B) cyclic
nucleotide-hydrolyzing activity. PDE1B / mice
showed exaggerated hyperactivity after acute
D-methamphetamine administration. Striatal slices from
PDE1B/ mice exhibited increased
levels of phospho-Thr34 DARPP-32 and
phospho-Ser845 GluR1 after dopamine D1 receptor
agonist or forskolin stimulation. PDE1B/ and
PDE1B+/ mice demonstrated Morris maze
spatial-learning deficits. These results indicate that enhancement of
cyclic nucleotide signaling by inactivation of PDE1B-mediated cyclic
nucleotide hydrolysis plays a significant role in dopaminergic function
through the DARPP-32 and related transduction pathways.
Key words:
phosphodiesterases; DARPP-32; dopamine-stimulated
locomotor activity; spatial learning and memory; Morris water maze; methamphetamine; SKF81297; forskolin
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INTRODUCTION |
Calcium/calmodulin-dependent
phosphodiesterases (CaM-PDEs) are members of one of 11 families of PDEs
(Soderling et al., 1999 ;Yuasa et al., 2001) and comprise the only
family that acts as a potential point of interaction between the
Ca2+ and cyclic nucleotide signaling
pathways. The three known CaM-PDE genes, PDE1A-C, are expressed within
the CNS. PDE1A is expressed throughout the brain, with higher levels in
CA1-CA3 and cerebellum and a low level in the striatum (Borisy et al.,
1992; Yan et al., 1994). PDE1B is expressed predominantly in the
striatum, dentate gyrus, olfactory tract, and cerebellum, regions
having high levels of dopaminergic innervation (Furuyama et al., 1994;
Polli and Kincaid, 1994; Yan et al., 1994). PDE1C is expressed
primarily in olfactory epithelium, cerebellar granule cells, and
striatum (Yan et al., 1995, 1996).
The expression of CaM-PDEs in the striatum and evidence indicating that
cyclic nucleotides and calcium are principal second messengers of the
signal transduction pathways in the striatum (Altar et al., 1990)
suggest that CaM-PDEs may play a role in motor control (Traficante et
al., 1976; Tsou et al., 1993; Drago et al., 1994; Konradi et al., 1994;
Polli and Kincaid, 1994). Furthermore, NMDA receptor activation results
in increased intracellular Ca2+
concentrations (Kotter, 1994; Greengard et al., 1999) that activate effectors such as calmodulin-dependent kinase-II (CaMKII) and calcineurin and have the potential to activate CaM-PDEs. Dopamine D1 or
D2 receptor activation leads to adenylyl cyclase activation or
inhibition, respectively (Traficante et al., 1976; Monsma et al., 1990;
Cunningham and Kelley, 1993; Miserendino and Nestler, 1995).
Intracellular concentrations of cGMP also are increased after dopamine
D1 receptor activation and are unchanged or inhibited after D2 receptor
activation (Altar et al., 1990). Cyclic nucleotides activate PKA- or
PKG-dependent protein kinases that phosphorylate downstream signaling
elements such as DARPP-32 (dopamine- and cAMP-regulated phosphoprotein,
Mr 32 kDa) and cAMP-responsive element-binding protein (CREB). These signaling pathways are
downregulated by PDEs by hydrolysis of the cyclic nucleotides to their
5'-monophosphates. Calcium-regulated PDEs are therefore potential
interfaces between dopamine- and glutamate-regulated signaling pathways.
Long-term potentiation (LTP) and long-term depression (LTD), the major
forms of plasticity associated with hippocampally mediated learning and
memory, also are regulated by cyclic nucleotides and CaM signal
transduction cascades, as demonstrated by disruptions that occur in
response to changes in protein kinase A (PKA) (Skoulakis et al., 1993;
Abel et al., 1997), CaMKII (Bach et al., 1995; Mayford et al., 1995;
Malenka and Nicoll, 1999), CREB (Bourtchuladze et al., 1994; Yin et
al., 1994; Guzowski and McGaugh, 1997), and calcineurin (Mansuy et al.,
1998). Drosophila mutants including dunce, a
cAMP-specific PDE mutant (Qui and Davis, 1993), rutabaga, an
adenylyl cyclase mutant (Livingstone et al., 1984), and a PKA mutant
(Skoulakis et al., 1993) all have shown altered intracellular levels of
cAMP or an altered cAMP signaling pathway with concomitant olfactory-conditioning deficits (Davis et al., 1995). cGMP also has
been implicated in learning and memory through the nitric oxide
retrograde signal transduction pathway (Gally et al., 1990; Garthwaite,
1991).
A physiological role for the PDEs in the brain has not been
established, although PDE4 inhibitors have shown clinical efficacy as
antidepressants (Houslay et al., 1998). The localization of PDE1 family
members within the striatum, hippocampus, and olfactory tract as well
as their ability to interact with two essential signal transduction
pathways suggests a potential role for CaM-PDEs in motor control,
learning, and olfaction. To investigate the role of one of these, we
generated mice in which PDE1B was rendered inactive by targeted
disruption of the catalytic domain and characterized the mice via tests
related to the functions associated with regions in which PDE1B is
expressed (locomotor activity for striatum, spatial learning for
dentate gyrus, and olfactory orientation for olfactory tract).
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MATERIALS AND METHODS |
Gene targeting. Two overlapping clones, TRC2 and TRC4
corresponding to exons 2-13 of the PDE1B gene, were obtained by
screening an I-129/SvJ  Dash II mouse genomic library as described
previously (Reed et al., 1998). A 0.8 kb XbaI fragment
containing exons 4 and 5 and a small portion of the multiple cloning
site of pBluescript II KS( ) (Stratagene, La Jolla, CA) was blunt
end-ligated into the BamHI site of the targeting vector. A 5 kb AccI/KpnI fragment containing genomic DNA 3'
of exon 9, including 2.4 kb of the noncoding sequence 3' to exon 13, was blunt end-ligated into the ClaI site of the targeting
vector (see Fig. 1A). The targeting vector backbone pGKKOV has been described previously (Li et al., 1996). This vector is
derived from pBluescript II SK(+), with a herpes simplex
virus-thymidine kinase selectable marker and a PGK-HPRT
selectable marker inserted into the KpnI and
HindIII sites, respectively, of the multiple cloning site.
The orientation of inserts was determined by sequencing.
The targeting vector (50 µg) was linearized with NotI and
electroporated into 9.3 × 106 E14TG2a
embryonic stem (ES) cells. ES cells were plated on ten 100 cm tissue
culture plates (Fisher Scientific, Houston, TX) on 2.2 × 106 mitomycin C-treated mouse embryonic fibroblasts in 10 ml of DMEM per plate containing 15% ES Cell Qualified fetal calf serum
(Invitrogen, San Diego, CA), 0.3 mM
L-glutamine, 75,000 U of Pen/Strep, 60 U of
leukemia inhibitory factor, and 0.009%  -mercaptoethanol supplemented with 2 µM Cytovene and 1× hypoxanthine
aminopterin thymidine (HAT) (Life Technologies) for the
selection process. After 5 d of selection, 302 ES cell clones were
selected and expanded in 24-well plates. Clones were screened by
Southern blot analysis. Cells from two positive clones were injected
into C57BL/6 blastocysts and implanted into pseudopregnant females.
Chimeric offspring were bred to C57BL/6 mice (Charles River,
Wilmington, MA) to produce PDE1B+/
offspring that were bred to C57BL/6 mice for two additional generations to give a total of three backcross breedings. The offspring resulting from interbreeding the third generation backcross were used for behavioral testing.
Southern blot analysis. Southern blot analysis was performed
as described previously (Reed et al., 1998) on StuI or
NdeI/KpnI-digested DNA that was purified from ES
cells or tail biopsies. Briefly, prehybridization and
hybridization were performed at 65°C in 1% SDS, 1 M NaCl, and 10% dextran sulfate. Blots were
screened with a 347 bp PCR-generated probe corresponding to mouse PDE1B
exon 3 and 164 bp of 5' and 3' flanking intron sequences, and the probe was outside of the targeted region of the PDE1B gene. PCR primers used
were 5'-GACACTAAGTGGGTATAGCTGGGT-3' and 5'-GTGGGAATAAGTCTCAGGGTAGG-3'. Then 25 ng of probe was radiolabeled with
32P-dCTP by random priming with the
Prime-It II Kit (Stratagene), boiled with 5 mg salmon sperm DNA in 500 µl of water for 5 min, and added to 30 ml of hybridization buffer.
Blots were hybridized for 12-16 hr; the final posthybridization wash
was in 2× SSC plus 1% SDS at 65°C. Homologous recombination events
resulted in size shifts from 3.7 to 2.2 kb (StuI) (see Fig.
1A,B) and from 3.7 to 10.6 kb
(NdeI/KpnI) (see Fig. 1A).
Northern blot analysis. Total brain RNA was isolated via the
TriReagent method (Molecular Research Center, Cincinnati, OH). RNA (10 µg) was fractionated on a 1% denaturing agarose gel and transferred
for 24 hr onto a Biotrans nylon membrane (ICN Biochemicals, Aurora, OH)
by using 20× SSC. The blot was prehybridized in 10 ml of solution
consisting of 50% formamide, 50 mM
NaPO4, 5× SSC, 5× Denhardt's, 0.5% SDS, and
1% glycine with 250 µl of 10 mg/ml salmon sperm DNA at 42°C for 4 hr. The blot was hybridized with 32P-labeled probe K-17, a 348 bp mouse
partial PDE1B cDNA corresponding to the central catalytic domain,
as described previously (Repaske et al., 1992). The probe was boiled
for 5 min with 100 µl of 10 mg/ml salmon sperm DNA, added to 10 ml of
the hybridization buffer of 50% formamide, 20 mM
NaPO4, 5× SSC, 1× Denhardt's, 0.5% SDS, and
10% dextran sulfate, and incubated at 42°C for 21 hr. The final
posthybridization wash was in 0.1× SSC plus 0.1% SDS at 55°C.
Subjects. Animals used for behavioral analysis were the
offspring of the third generation backcrossing
(PDE1B/, PDE1B+/, mice and
their WT littermates), and those for biochemistry were the offspring of
third and fourth generation backcrossing. Mice for behavioral analysis
were earmarked on postnatal day 7 (P7) and weighed weekly, beginning on
P7 and ending on P112. Tail biopsies were obtained on P21 or P42.
Litters were weaned on P28. Mice were housed, same gender, two to four
mice per cage. Individual mice were excluded from testing if there were
no same-gender littermates or if visible ocular defects were present.
One experimenter performed all procedures and was blinded to genotypes
until the end of the experiment.
Olfactory orientation. On P9, P11, and P13, all offspring in
each litter were tested individually for olfactory orientation to their
home cage scent as described previously (Acuff-Smith et al., 1992). The
apparatus consists of a 12 × 38 cm enclosed runway flanked with
30 × 38 cm enclosed chambers containing equal amounts of either
home bedding or clean bedding. The runway floor was marked every 2.5 cm. The home bedding for each litter was changed on P7. The bedding was
not changed again until after testing was completed (P14) and was used
on each test day and returned to the home cage. Before individual
testing the pups were removed from the dam and placed in a holding cage
on a heating pad to maintain body temperature. During the 1 min/d test
period each mouse was placed in the center of the runway, and head
position was scored every 10 sec by using runway positions of +1 to +7 for movement toward the home bedding and 1 to 7 for movement toward
the clean bedding. Individual scores were summed for each test day.
Locomotor activity. On P50, P51, or P52, locomotor activity
was measured in a 30.5 × 30.5 cm Digiscan apparatus containing 16 photo detector-LED pairs along the x-axis and 16 pairs
along the y-axis (model rxy2z, Accuscan Electronics,
Columbus, OH). Prechallenge activity was recorded in 3 min intervals
over a 1 hr period. Mice were challenged with a subcutaneous injection of 1 mg/kg D-methamphetamine (METH)-HCl (free
base) in saline to yield an injection volume of 5 ml/kg body weight;
activity was recorded for an additional 2 hr at 3 min intervals.
Recording was done during the light cycle. Horizontal activity and
total distance moved (in centimeters) were measured, as were regional movements (center vs periphery).
Morris maze. On P50 all animals within a litter were
administered four timed trials in a 15 × 244 cm straight water
channel with a wire ladder at one end. The channel was constructed of PVC material and filled with 27-29°C water to a depth of 35 cm. Subjects were placed in the channel at the opposite end from the ladder
and given a maximum of 60 sec to find the ladder and escape. These
trials were used to measure swimming proficiency and motivation to
escape from water before the Morris water maze trials. Two litters of
mice were excluded from further behavioral testing because of failure
to swim after repeated placements in the water.
The Morris water maze was used with modifications for mice (Upchurch
and Wehner, 1988). The maze was a circular stainless steel perimeter
122 cm in diameter that was surrounded by an outer tank 162 cm in
diameter. The inner perimeter was covered with flat white paint. The
clear acrylic platform was 10 × 10 cm and submerged 1 cm below
the surface of the water. White tempera paint was added to the water to
camouflage the platform. Water temperature was maintained at
27-29°C. Litters were divided into two groups to balance for
platform position. Testing began on P51 and continued for 18 d,
with 6 d each for hidden platform acquisition and reversal (with
the platform moved to the opposite quadrant), followed by cued
learning. For hidden platform acquisition the platform was placed in
the SE or NW quadrant; start positions (N, S, E, and W) were altered on
every trial in a quasi-random sequence such that each cardinal position
was used only once per day. Four trials were given each day with a
maximum time limit of 1 min and intertrial intervals of 30 sec. Mice
not finding the platform in 1 min were placed on the platform for the
30 sec intertrial interval. Probe trials (1 min) were given on day 3 before the acquisition trials and day 6 after the last acquisition
trial with the platform removed; probe trials were begun from novel
start locations. For the reversal phase the same procedure was followed
with the platform shifted to the opposite quadrant. Probe trials for
mice tested with the NW platform site for reversal were not used for
statistical analysis because of a procedural error. For cued learning,
black curtains were drawn around the maze to obscure distal cues, and
the platform was marked by using a black solid cylinder (5 × 7 cm) mounted on a 14 cm brass rod above the platform. Four trials per
day were given, with the platform and start positions randomly located for each trial. Mice were placed in a holding cage for the 30 sec
intertrial interval while the platform was positioned for the next
trial. Data were recorded for acquisition and reversal phases with a
video tracking system (San Diego Instruments, San Diego, CA). Cued
acquisition latencies were recorded with a hand-held timer and observed
on a closed circuit monitor.
Preparation and treatment of neostriatal slices. Male
PDE1B/ and WT mice (8-12 weeks of
age) were decapitated. The brain was transferred rapidly to an ice-cold
surface, blocked, and fixed to the cutting surface of a Vibratome (Ted
Pella, Redding, CA) maintained at 4°C. The brain was submersed in
cold, oxygenated (95% O2/5%
CO2) Krebs' bicarbonate buffer of the following
composition (in mM): 125 NaCl, 5 KCl, 26 NaHCO3, 1.5 CaCl2, 1.5 MgSO4, and 10 glucose, pH 7.4. Coronal mouse
brain sections (400 µm in thickness) were cut and pooled in cold
buffer. Striatal or nucleus accumbens slices were cut from the coronal
sections under a dissecting microscope. Individual slices were
preincubated in fresh buffer for 15 min at 30°C; the buffer was
replaced, and preincubation continued for an additional 30 min. At the
end of this second preincubation period the buffer was replaced with
Krebs' buffer or buffer containing test substances for 5 min. Slices
were frozen immediately in liquid nitrogen and stored at 80°C until assayed.
Immunoblotting for phospho-DARPP-32 and phospho-GluR1.
Frozen tissue samples were sonicated in 1% SDS. Small aliquots of
the homogenate were retained for protein determination by the BCA protein assay method (Pierce, Rockford, IL), using bovine serum albumin
as a standard. Equal amounts of protein (50 µg) were loaded onto 10%
acrylamide gels, separated by SDS/PAGE (Laemmli, 1970), and transferred
to nitrocellulose membranes (0.2 µm; Schleicher & Schuell, Keene, NH)
by the method of Towbin et al. (1979). Membranes were immunoblotted
with the following antibodies: an antiserum detecting the
Ser845-phosphorylated form of GluR1
(Kameyama et al., 1998); an antiserum (PharMingen, San Diego, CA)
detecting the C-terminal region of GluR1, irrespective of
phosphorylation state; mAb 23 (Snyder et al., 1992), a phosphorylation
state-specific monoclonal antibody detecting a DARPP-32 peptide
containing phospho-Thr34, the site
phosphorylated by PKA; or C24-5a, a monoclonal antibody detecting
DARPP-32 irrespective of phosphorylation state (Hemmings and Greengard,
1986).
Antibody binding was revealed by incubation with either a goat
anti-rabbit horseradish peroxidase-linked IgG or a goat anti-mouse horseradish peroxidase-linked IgG (Pierce) and the enhanced
chemiluminescence (ECL) immunoblotting detection system (Amersham
Biosciences, Arlington Heights, IL). Chemiluminescence was detected by
autoradiography with DuPont NEN autoradiography film (Boston, MA), and
bands were quantified by analysis of scanned images by NIH 1.52 Image
software (Bethesda, MD). Because the linear range for quantitation of
ECL signals by densitometry is limited, several film exposures were obtained for each set of samples to insure that the signals were within
a density range that allowed for accurate quantitation.
Statistical methods. Data were analyzed via ANOVA (general
linear model). For data that had repeated measure components, split plot ANOVAs were used, with day and trial treated as within-subject factors in the analyses (Kirk, 1995). Data were averaged within a
litter for each genotype having more than one subject of the same
gender (Holson and Pearce, 1992). Significant interactions were
analyzed further by using simple-effect ANOVA. A posteriori group comparisons were performed by the method of Duncan. Analyses for
time-dependent interactions were performed on activity data, using
trend analyses by orthogonal decomposition.
 2 was used to analyze whether the
proportion of mice of each genotype matched predicted Mendelian ratios.
Neostriatal slice data were analyzed by a Mann-Whitney U
test, as indicated, with significance defined as p < 0.05.
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RESULTS |
Generation of PDE1B-deficient mice
To generate mice deficient in PDE1B activity, we constructed a
targeting vector to disrupt the sequences encoding the PDE1B catalytic
domain in mouse ES cell genomic DNA by homologous recombination (Fig.
1A). Of the 302 ES cell
colonies that survived HAT and Cytovene selection, 26 were analyzed by
Southern blot with a probe corresponding to PDE1B exon 3 and flanking
intron sequence. The disrupted gene was present in nine of these clones
(data not shown).
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Figure 1.
Targeting strategy for PDE1B and
Southern and Northern blot analysis of offspring of the third
generation backcrossing. A, Schematic of a portion of
the endogenous PDE1B gene and the result of homologous recombination
with the targeting vector. A 3.1 kb portion of the PDE1B gene encoding
the central catalytic domain was disrupted by replacement with the 2.9 kb HPRT gene. Disruption resulted in a size shift in the
StuI fragment from 3.7 to 2.2 kb and a size shift in an
NdeI/KpnI fragment from 3.7 to 10.6 kb.
The bars above the PDE1B gene represent genomic DNA
sequences included in the targeting vector. The bars
below the PDE1B gene indicate the restriction fragments used for
Southern blot analysis of ES cell and mouse genomic DNA.
Numbered thick vertical lines represent exons.
Thin vertical lines indicate restriction enzyme sites.
S, StuI; N,
NdeI; X, XbaI;
A, AccI; K,
KpnI. B, Southern blot of offspring of
third generation backcross mice of mouse genomic DNA digested with
StuI demonstrating wild-type
(W), heterozygous
(H), and null (N)
genotypes. The 2.2 kb StuI fragment was generated by
homologous recombination with the targeting vector. All blots were
probed with a PDE1B exon 3 probe. C, Brain total RNA
from mice of each genotype was analyzed by Northern blot by hybridizing
with K-17, a cDNA probe corresponding to the central catalytic domain
(Repaske et al., 1992). The expected ~3.0 kb transcript is observed
in the WT and PDE1B+/ mice only
(top). The methylene blue-stained ribosomal bands on the
same Northern blot demonstrate equal RNA loading
(bottom).
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Two recombinant ES cell lines were microinjected into embryonic day 3.5 (E3.5) C57BL/6 blastocysts and implanted in pseudopregnant females.
Twenty-eight chimeras were generated (17 males and 11 females); eight
(29%) from one ES cell line demonstrated germline transmission when
bred to C57BL/6 mice. Third backcross
PDE1B+/ mice were interbred, resulting
in 178 wild-type (WT; 27%), 309 PDE1B+/
(47%), and 157 PDE1B/ (24%) mice and
13 mice that died before being genotyped (2%). These proportions did
not differ significantly from the expected Mendelian ratio of 1:2:1 by
2 analysis. A Southern blot
demonstrating F4 genotypes is shown in Figure
1B.
The physical appearance and general behavior of
PDE1B/ and
PDE1B+/ mice were identical to those of
their WT littermates. There were no significant differences in either
preweaning or postweaning weights among the three genotypes. There was
a small, but significant, increase in the number of
PDE1B/ (6%) and
PDE1B+/ (6%) mice that died within 1 week after birth compared with WT mice (2%), as determined by
Fisher's test for uncorrelated proportions. A Northern blot of total
brain RNA probed with K-17, a 348 bp mouse partial PDE1B cDNA within
the targeted domain (Repaske et al., 1992), demonstrated a reduction in
PDE1B mRNA in the heterozygote and the absence of PDE1B mRNA in the
null (Fig. 1C).
To characterize the phenotype of the
PDE1B/ mice, we performed a series of
tests designed to assess functions known to be associated with the
brain regions in which PDE1B is expressed most abundantly. These were
locomotor activity and DARPP-32 phosphorylation for striatal function,
spatial learning for dentate gyrus function, and olfactory orientation
for olfaction.
Locomotor activity
We studied both exploratory and D-METH stimulated
activity in the PDE1B/,
PDE1B+/, and WT mice. A significant main
effect of genotype was observed on horizontal locomotor activity for
both the 60 min prechallenge exploratory activity
(p < 0.0003) and 120 min postchallenge
(p < 0.0001) periods (Fig.
2). Furthermore, a significant genotype by interval interaction was observed for both prechallenge
(p < 0.03) and postchallenge
(p < 0.0001) horizontal activity. Simple-effect ANOVAs and post hoc tests showed that prechallenge
PDE1B+/ locomotor activity was
comparable with that of WT mice (Fig. 2). However,
PDE1B/ mice explored more, especially
during the first 30 min. From 30 to 60 min the
PDE1B/ mice habituated toward WT
levels but remained slightly more active. Simple-effect ANOVAs and
post hoc group comparisons on post-METH challenge horizontal
activity showed that the PDE1B+/ mice
were not significantly more active than WT mice.
PDE1B/ mice, on the other hand, were
significantly more active than WT mice. The hyperactivity lasted for
~90 min after METH treatment.
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Figure 2.
Horizontal activity. Shown is the mean ± SEM
horizontal activity for PDE1B/,
PDE1B+/, and WT mice (males and females combined).
An arrow indicates the time of methamphetamine
administration (1 mg/kg). Counts represent the total number of photo
beam interruptions per 3 min interval. PDE1B/
mice were hyperactive compared with WT mice both before
(F(2,120) = 8.55; p < 0.0003) and after (F(2,120) = 11.18;
p < 0.0001) methamphetamine challenge.
*p < 0.05; **p < 0.01. n = +/+, 71; +/, 114; /, 49.
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To determine whether the stimulated response of the
PDE1B/ mice was qualitatively as well
as quantitatively different from that of WT mice, we used trend
analysis, using orthogonal decomposition. PDE1B/ to WT comparisons showed
significant linear, quadratic, and cubic trends for the genotype by
interval interaction (p < 0.03), whereas the
PDE1B+/ to WT interactions showed no
significant differences. In the PDE1B/
to WT comparison, the quadratic trend best fit the data, revealing that
the PDE1B/ mice not only were more
active but also had a larger rate of change (greater slope) than the WT
mice in response to METH. This can be seen in Figure 2 by the sharper
rise in activity over the same time period in
PDE1B/ mice after METH than occurred
in WT mice.
The related measure of total distance followed the same pattern as
horizontal activity. As with horizontal activity, there was a
significant main effect of genotype both prechallenge
(p < 0.001) and postchallenge
(p < 0.0002).
PDE1B/ mice traveled greater distances
compared with WT mice in the prechallenge (p < 0.004) and postchallenge periods (p < 0.0002; data not shown). A genotype by interval interaction also was observed for both prechallenge (p < 0.0001) and
postchallenge (p < 0.0001), and the pattern was
comparable with that seen for horizontal activity. One difference was
that a genotype by sex interaction was obtained during the prechallenge
period (p < 0.05). As can be seen in Figure 3,
PDE1B/ females accounted for this
effect on total distance; males showed no pre-METH differences. After
METH challenge both PDE1B/ males and
females showed greater stimulated hyperactivity than WT mice.
PDE1B+/ males showed an intermediate
response to METH, suggesting a gene-dosage effect. Analyses of
regional changes in activity showed the same significant main effects
and interactions as seen with total distance.
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Figure 3.
Sex differences in total distance. Shown is the
mean ± SEM distance traveled by males (top) and
females (bottom) in the activity apparatus in 3 min
blocks. The arrow indicates time of methamphetamine
administration (1 mg/kg). Female PDE1B/ mice,
but not male PDE1B/ mice, were hyperactive
compared with WT mice during the pre-methamphetamine challenge period
(F(2,120) = 3.07; p < 0.05); this sex difference did not continue into the postchallenge
period. n = +/+, 71; +/, 114; /, 49.
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Phosphorylation of PKA substrates in
PDE1B/ mice
Based on the locomotor activity differences in
PDE1B/ mice that used the indirect
dopamine agonist methamphetamine, we investigated the possibility that
these mice would exhibit enhanced protein phosphorylation in response
to activation of adenylyl cyclase. Forskolin (10 µM), a
direct activator of adenylyl cyclase, was used to increase cAMP levels
in striatal slices from WT and PDE1B/
mice (Fig. 4A).
Forskolin treatment induced a significantly greater fold increase in
levels of phospho-Thr34 DARPP-32 in
striatal slices from PDE1B/ mice
(65.7 ± 20-fold over untreated control) than from WT mice (21.9 ± 6-fold over untreated control) (p < 0.05, null compared with WT mice) (Fig. 4B). To
investigate PKA-mediated protein phosphorylation further, we
used a dopamine agonist. Striatal slices prepared from male WT
and PDE1B/ mice were treated with the
selective agonist of dopamine D1-type receptors, SKF81297 (10 µM), for 10 min. The effect of this treatment on phosphorylation of DARPP-32 at Thr34
and the GluR1 AMPA receptor at Ser845, two
substrates regulated via activation of D1 receptors and PKA (Greengard
et al., 1999), was measured by using phosphorylation state-specific
antibodies (Fig. 4C,D). In striatal slices from WT mice, D1
agonist treatment increased the levels of
phospho-Thr34 DARPP-32 by 4.7 ± 2.3-fold and phospho-Ser845 GluR1 by
4.4 ± 0.8-fold (Fig. 4D). In contrast, in
striatal slices from PDE1B/ mice,
there were significantly greater increases in levels of both
phospho-Thr34 (15.8 ± 5-fold over
control) and phospho-Ser845 (13.1 ± 3.2-fold over control) (Fig. 4D)
(p < 0.05; Mann-Whitney U test). An
enhanced D1-mediated phosphorylation at both
Thr34 and
Ser845 also was observed in nucleus
accumbens slices from PDE1B/ compared
with WT mice (data not shown). No significant difference was detected
in basal levels of phospho-Thr34 or
phospho-Ser845 or in total levels of
DARPP-32 or GluR1 in untreated striatal (Fig. 4C) or
accumbens (data not shown) slices from WT and
PDE1B/ mice.
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Figure 4.
Phosphorylation of PKA-mediated substrates in WT
and PDE1B/ mice. A, Immunoblot
showing level of phospho-Thr34-DARPP-32 in control
(Con) striatal slices and in slices treated with the
adenylyl cyclase activator forskolin (For) from WT and
PDE1B/ mice. B, Fold increase in
phospho-Thr34-DARPP-32 in response to forskolin
(*p < 0.05 compared with WT forskolin;
Mann-Whitney U test; n = 3 mice per
treatment group). C, Immunoblot showing levels of
phospho-Thr34 and total DARPP-32
(left) and phospho-Ser845 and total
GluR1 (right) in untreated (Con) and
D1-agonist-treated (D1) striatal slices from WT and
PDE1B/ mice. D, Mean ± SEM
increase in the levels of phospho-Thr34-DARPP-32
(left) and phospho-Ser845-GluR1
(right) on incubation with D1 agonist
(*p < 0.05 compared with untreated control slices;
 p < 0.05 compared with D1-treated WT
slices; Mann-Whitney U test; n = 3 mice per treatment group).
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Olfactory orientation
No significant differences were found among the three genotypes in
olfactory orientation scores (data not shown).
Learning and memory
In straight channel trials no significant differences were found
among the three genotypes, thereby demonstrating equal swimming ability
and motivation to escape (data not shown). Similarly, there were no
main effects or interactions found for cued learning when proximal cues
were present and distal cues were removed from the Morris water maze
(data not shown).
In acquisition of a hidden platform in the Morris water maze, both
PDE1B+/ and
PDE1B/ mice had significantly longer
path length than WT mice (group main effect; p < 0.0002) (Fig. 5A). A
significant day by genotype by platform interaction also was found
(p < 0.04). Although WT mice tested with the
platform in both quadrants had shorter path lengths than
PDE1B+/ or
PDE1B/ mice, the interaction occurred
because the effect was larger on days 3-6 with the platform in the SE
quadrant. A significant trial by genotype by sex effect on path length
was found also; the contribution of gender was small and not
instructive.
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Figure 5.
Spatial learning in the Morris water maze.
A, Path length (mean ± SEM) to reach the platform
for mice of each genotype. PDE1B/ and
PDE1B+/ mice showed significantly increased path
length compared with WT mice (F(2,112) = 9.19; p < 0.0002). B, Cumulative
distance from the platform (mean ± SEM) for mice of each
genotype. PDE1B/ and
PDE1B+/ mice showed a significantly increased
cumulative distance compared with WT mice
(F(2,112) = 8.38; p < 0.0004). Data are shown for the average of four trials per day
averaged across all 6 d. **p < 0.01 compared
with wild-type mice. n = +/+, 55; +/, 101; /,
66.
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|
The same pattern was observed for cumulative distance from target
during acquisition (Fig. 5B). Cumulative distance from the target measures the animal's distance from the platform every 55 msec.
PDE1B+/ and
PDE1B/ mice had significantly longer
cumulative distances than WT mice (group main effect; p < 0.0004). A significant day by genotype by platform interaction
(p < 0.05) was found in which WT to
PDE1B/ and
PDE1B+/ comparisons were again largest
on days 3-6 for the SE quadrant. In addition, a trial by genotype by
sex interaction (p < 0.01) was observed on this
measure. As before, the gender contribution was minor.
For latency an almost identical pattern was seen (data not shown). As
with the other measures, the effect was larger for one goal quadrant
than the other (SE). PDE1B/ and
PDE1B+/ mice had significantly longer
latencies than WT mice on days 3 (p < 0.03) and
4 (p < 0.01), respectively. A trial by genotype by sex interaction (p < 0.004) was found, and
as before the contribution of sex was minor.
Learning curves for path length are shown in Figure
6. The patterns for cumulative distance
and latency were comparable with those for path length. As can be seen,
all groups showed similar performance on day 1, indicating that there
were no preexisting performance differences among the genotypes. On
subsequent days WT mice showed steady improvement, acquiring shorter
paths to the goal on each successive day.
PDE1B/ and
PDE1B+/ mice, on the other hand, showed
less improvement than WT mice, resulting in significant group
differences on test days 3-6. Even on day 6 after 24 trials,
PDE1B/ and
PDE1B+/ mice did not perform as well as
WT mice.
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Figure 6.
Spatial learning in the Morris water maze:
acquisition of hidden platform task. Data are shown for the average of
four trials per day for each of the three genotypes.
PDE1B/ and PDE1B+/ mice
showed significantly increased path length compared with WT mice on
days 3-6 (genotype by day by platform interaction;
F(10,560) = 2.01; p < 0.04). *p < 0.05 and **p < 0.01 compared with WT mice. Filled diamonds, +/+
(n = 55); shaded squares, +/
(n = 101); open triangles, /
(n = 66).
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Probe trial measurements of path length and time in the target zone
were analyzed two ways: by annuli and quadrants. First, three annuli
were defined. The target annulus was the one demarcated by the inner
and outer edges of the platform, the outer annulus was from the outside
boundary of the target annulus to the side-wall, and the inner annulus
was from the inside boundary of the target annulus to the center.
Second, probe trial data were analyzed by dividing the maze into four
equal quadrants. Overall, PDE1B/
and PDE1B+/ mice swam significantly less
in the target annulus than WT mice (p < 0.04)
(Fig. 7A). A significant
genotype by platform position interaction was found in both the target
annulus (p < 0.04) and the outer annulus
(p < 0.05).
PDE1B/ and
PDE1B+/ mice swam less in the target
annulus than WT mice, especially those tested with the platform in the
SE quadrant (p < 0.01). Conversely,
PDE1B/ and
PDE1B+/ mice swam significantly more in
the outer annulus than WT mice (p < 0.01) (Fig.
7B). No significant difference was found in path length or
time within the inner annulus or in platform site crossings. A
significant interaction of day by genotype by platform position (p < 0.01) was found for average distance from
the target in which PDE1B+/ mice were
significantly farther from the target site than WT mice
(p < 0.007). No differences in time or distance
in the target quadrant were found.
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Figure 7.
Spatial memory in the Morris water maze:
probe trial performance. Path length (mean ± SEM) is expressed as
a percentage of total swim time in the target annulus
(A) and the outer annulus
(B) averaged across the two probe trials and
gender. *p < 0.01 compared with wild-type
mice.
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For reversal testing, no significant differences were found for the
measures of path length or cumulative distance. A trial by genotype
interaction (p < 0.003) was observed for
latency in which the PDE1B/ mice had
significantly longer latencies than WT mice on the last trial of each
day (p < 0.01). No significant differences were found on reversal probe trials.
| |
DISCUSSION |
We generated mice deficient in PDE1B by disruption of the portion
of the PDE1B gene that encodes the central catalytic domain (Fig. 1).
PDE1B mRNA is undetectable in the
PDE1B/ mouse, and there was no
apparent upregulation of the normal allele in the
PDE1B+/ mouse (Fig. 1C). We
have demonstrated that mice lacking PDE1B activity have significantly
increased spontaneous and METH-induced locomotor activity compared with
WT and PDE1B+/ mice.
PDE1B/ mice have alterations in
behavior that are likely a consequence of the disruption of striatal
pathways involved in the regulation of locomotor activity as
demonstrated by the changes in phosphorylation levels of DARPP-32 and
GluR1. PDE1B/ mice are hyperactive in
comparison to WT and PDE1B+/ mice
measured as either horizontal activity (Fig. 2) or total distance (Fig.
3). These changes occurred in response to a novel environment (initial
exploration) and in a more pronounced manner in response to METH
challenge. However, the METH-induced response was distinct, as
indicated by the fact that the exploratory differences had dissipated
in large part by the time the METH was administered. The postchallenge
interval was characterized by two phases of response. The first phase
preceded the onset of effects of METH (first 9 min) when the
PDE1B/ mice showed a larger rise in
activity than WT, showing that even handling and injection induced a
greater response in PDE1B/ mice than
in WT mice. Then activity decreased in all groups, followed by a large
increase beginning ~24 min after injection. The
PDE1B/ mice were already more active
by this time. As the METH effect increased, the
PDE/ mice responded with larger
increases in activity than WT or PDE+/
mice. Although not quantified, no signs of stereotypy were noted among
any of the three genotypes. However, with higher doses of METH, a shift
to stereotypy would be expected, accompanied by a reduction in
horizontal locomotion. It is worth noting that the present experiment
did not include groups challenged with saline after the first hour to
assess the locomotor habituation that would be expected during hours
2-3 of testing. Such controls will be needed in future experiments
with this model to characterize fully the response of the
PDE1B/ mice to dopaminergic agonists.
Locomotor activity has been used extensively to test for the effects of
neurotoxins and psychostimulants (Rafales, 1986). Psychostimulants such
as cocaine and amphetamines have been shown to affect locomotor
activity by dopaminergic pathways (Traficante et al., 1976). These
substances bind to the dopamine transporter (DAT), resulting in
increased extracellular DA and subsequent increased DA interaction with
D1 receptors. Increased D1 receptor interactions result in activating
adenylyl cyclase activity, which increases intracellular levels of cAMP
(Giros et al., 1996). Increased intracellular levels of cAMP activate
PKA, which phosphorylates intracellular proteins, including DARPP-32
and CREB (Cunningham and Kelley, 1993; Konradi et al., 1994;
Miserendino and Nestler, 1995). DA binding to D1 receptors also
increases intracellular levels of cGMP via an unknown mechanism that
leads to the activation of protein kinase G (PKG) and the
phosphorylation of DARPP-32 and other proteins (Altar et al., 1990;
Tsou et al., 1993). Disruptions of these pathways cause changes in
levels of locomotor activity as shown in mice lacking DA, DA receptors,
DAT, or DARPP-32. Mice that lack an active tyrosine hydroxylase gene,
the rate-limiting enzyme in DA synthesis, or D1 receptors were
hypoactive (Drago et al., 1994) even in response to cocaine (Xu et al.,
1994) and exhibited decreased rearing (Zhou and Palmiter, 1995). Mice
lacking D2 receptors also had reduced levels of activity and reduction in controlled motor movement (Balk et al., 1995). Conversely, mice
lacking DAT demonstrated increased levels of locomotor activity that
were unaltered by cocaine or METH challenge (Giros et al., 1996).
DARPP-32 null mice exhibited greater sensitivity to repeated injections
of cocaine but did not have significantly different levels of locomotor
activity in response to acute cocaine administration (Fienberg et al.,
1998; Hiroi et al., 1999).
PDE1B/ mice demonstrated increased
levels of activity in comparison with WT mice and even greater
increases in activity with an acute METH challenge. Removing PDE1B
cyclic nucleotide-hydrolyzing ability presumably increased the
magnitude and prolonged the duration of the D1 receptor-generated
increase in cyclic nucleotides and their downstream phosphorylation
signaling. Therefore, although we found increased activity as seen in
the DAT-knock-out mice (Giros et al., 1996), the effect in
PDE1B/ mice was mediated
postsynaptically rather than presynaptically as in the DAT-KO mouse.
Our mice differed dramatically from DAT-KO mice in response to
sympathomimetic challenge, in that DAT-KO mice show blunted
responses to stimulants whereas PDE1B-KO mice show exaggerated responses.
As reviewed recently (Greengard et al., 1999), the phosphorylation
state of DARPP-32 plays a central role in the cellular and molecular
responses within medium spiny striatal neurons. Glutamate and D2 DA
receptors increase intracellular Ca2+,
thereby activating calcineurin. Calcineurin dephosphorylates DARPP-32,
resulting in disinhibition of protein phosphatase-1 (PP-1) (Kotter,
1994; Greengard et al., 1999). Conversely, DA D1 receptors, adenosine
receptors, and nitric oxide (NO) pathways increase intracellular cyclic
nucleotide concentrations that activate PKA and PKG to phosphorylate
DARPP-32 and promote the inhibition of PP-1, thereby having the reverse
effect (Greengard et al., 1999). PP-1 regulates the activity of
neurotransmitter receptors and voltage-gated ion channels, thereby
affecting striatal synaptic transmission (Surmeier et al., 1995; Yan
and Surmeier, 1997; Snyder et al., 1998). PDE1B lowers intracellular
levels of cAMP and cGMP by hydrolyzing them in response to
Ca2+ activation (Krinks et al., 1984;
Erneux et al., 1985), thus terminating the signaling cascade. The
present study provides evidence that PDE1B is a regulator of the
phosphorylation state of DARPP-32 (Fig. 4).
Spatial learning deficits in PDE1B/
and PDE1B+/ mice were seen also. The
effect was comparable in PDE1B/ mice
that had no detectable PDE1B and in
PDE1B+/ mice that had ~50% of the
normal adult PDE1B level (Figs. 5, 6).
PDE1B+/ and
PDE1B/ mice swam greater distances to
reach the platform than WT mice and were further away from the platform
on average than WT mice, suggesting that they used a less efficient
search strategy. This is supported by the probe trial data in that
PDE1B+/ and
PDE1B/ mice swam less than WT mice in
the target annulus and more in the outer annulus (Fig. 7). Decreases in
target annulus swimming combined with increases in outer annulus
swimming have been shown to reflect impaired spatial ability in rats
(Saucier et al., 1996). Learning differences among genotypes were not
attributable to visual impairments or motivational deficits, because
performances in the straight channel and cued platform version of the
maze were not different among the groups.
It has been shown previously that rats with striatal lesions are
impaired in Morris maze spatial learning (D'Hooge and De Deyn, 2001).
Therefore, it is possible that the impaired Morris maze learning seen
in PDE1B/ and
PDE1B+/ mice may be the result of the
striatal phenotype of these mice. Alternatively, the Morris maze
deficits could be the result of the deficiency of PDE1B function in the
dentate gyrus, the other area of high expression of this enzyme and an
area closely associated with spatial learning (D'Hooge and De Deyn,
2001). A third possibility is that the Morris maze deficits could be
the product of the dual effect of PDE1B disruption in the striatum and
the dentate gyrus. These dual neuroanatomical substrates for learning
might explain why both PDE1B/ and
PDE1B+/ mice were affected in the Morris
maze, but only PDE1B/ mice were
affected on locomotion inasmuch as locomotion has only one region of
PDE1B involvement compared with at least two regions for spatial learning.
The dual region effect also may explain why
PDE1B/ and
PDE1B+/ mice are not entirely the same
as animals with striatal lesions in terms of Morris maze performance.
Rats with striatal lesions (especially dorsomedial lesions) exhibit
increased thigmotaxis (Devan et al., 1996, 1999; Devan and White, 1999;
D'Hooge and De Deyn, 2001) reminiscent of the pattern seen here in
mice with disrupted PDE1B function on probe trials. However, some
studies find that rats with striatal lesions exhibit increased
thigmotaxis only on early acquisition trials and on probe trials (Devan
et al., 1996, 1999; Devan and White, 1999). Rats with striatal lesions also are reported to be impaired on cued learning (Devan et al., 1996,
1999; Devan and White, 1999), whereas mice with disrupted PDE1B are
not. However, rats with quinolinate-induced medial striatal lesions
show impaired spatial acquisition in the Morris maze in the absence of
changes in cued learning (Furtado and Mazurek, 1996). Taken together,
these findings suggest that the combined disruption of PDE1B function
in the striatum and dentate gyrus best accounts for the Morris water
maze learning and memory deficits seen in our mice.
An alternative explanation for why the
PDE1B/ and
PDE1B+/ mice differ in the pattern of
phenotypic changes between the tests of locomotor activity and learning
may arise from the influence of other PDEs. For example, expression of
PDE1C in the striatum may have sufficient reserve capacity to
compensate for deletion of one allele of PDE1B in
PDE1B+/ mice and allow these mice to
have locomotor responses comparable with WT mice. However, PDE1C
expression has not been reported in dentate gyrus, thereby precluding
compensation in PDE1B+/ mice in terms of
spatial learning.
The results of these experiments support the conclusion that regulation
of intracellular cyclic nucleotide concentration is important in the
cellular processes that underlie learning and memory. The alterations
observed in learning in PDE1B/ and
PDE1B+/ mice suggest that PDE1B may be
involved in these underlying cellular processes.
Changes in cyclic nucleotide regulation have resulted in learning
deficits in olfactory discrimination paradigms as demonstrated by
Drosophila mutants and also have resulted in deficits in
spatial learning and memory as demonstrated in CREB mutant mice
(Livingstone et al., 1984; Qui and Davis, 1993; Skoulakis et al., 1993;
Bourtchuladze et al., 1994; Yin et al., 1994; Guzowski and McGaugh,
1997). Mutations in CaMKII (Bach et al., 1995; Mayford et al., 1995)
and alterations in the level of expression of calcineurin (Mansuy et
al., 1998) have implicated Ca2+ and CaM
signal cascades in spatial learning and memory tasks. PDE1B regulation
by both CaMKII (Skoulakis et al., 1993) and calcineurin has been
demonstrated in vitro (Sharma and Wang, 1985, 1986), suggesting a possible mechanism by which the learning deficits observed
in the PDE1B mutants may occur.
Studies in rodents and humans have demonstrated that CaM-PDEs are
expressed during periods of hippocampal synaptogenesis (Ludvig et al.,
1991; Lal et al., 1999). The hippocampus has been implicated extensively in processes involved in long-term memory processing and
storage (Jarrard, 1993; Zola-Morgan and Squire, 1993). Processes necessary for spatial navigation do not become functional until 1 month
of age in rats, a period of time shortly after hippocampal development
(Schenk, 1985). It is possible that CaM-PDEs serve a role in
establishing the synaptic connections necessary for spatial navigation.
Although CaM-PDEs hydrolyze both cAMP and cGMP, in vitro
studies have demonstrated a lower Km
for cGMP as substrate (Yan et al., 1995). The NO-cGMP signaling
pathway also has been implicated in LTD in the dentate gyrus (Wu et
al., 1998) and corticostriatal pathway (Engels et al., 1995). In both
systems an inhibitor of cGMP PDEs induced LTD (Engels et al., 1995; Wu
et al., 1998). More recently, Ca2+/CaM activation of NO
synthase was shown to modulate CA1 synaptic potentiation through NO at
postsynaptic sites (Ko and Kelly, 1999). CaM-PDEs may be predicted to
be an intermediary between the cyclic nucleotide/NO and
Ca2+/CaM signaling pathways. Indeed, PDE1B
degradation of elevated cGMP levels resulting indirectly from
stimulation of a Ca2+/CaM-dependent NO
synthase was observed in cytosolic fractions of crude rat brain
synaptosomes (Mayer et al., 1993).
We found no significant deficit in
PDE1B/ or
PDE1B+/ in a test of neonatal olfactory
orientation. This task is dependent on mice being able to detect the
smell of their home cage bedding. Because we did not observe any
differences in this task, it is possible that PDE1B does not play a
direct role in early postnatal odorant orientation. However, it may be
involved in other processes that were not assessed by this task such as
relaying or processing of discrete odorant information to other brain regions.
| |
FOOTNOTES |
Received Oct. 31, 2001; revised March 5, 2002; accepted March 26, 2002.
*
D.R.R. and C.V.V. contributed equally to this work.
This work was supported by National Institutes of Health Grants T32
ES07051 (T.M.R.), RO1 DA06733 (C.V.V.), and MH40899 and DA10044 (P.G.
and G.L.S.) and by funds from the Children's Hospital Research
Foundation (D.R.R.). We thank Dr. Richard L. Huganir for providing the
phospho-Ser845 GluR1 antibody. The excellent
technical assistance of Stacey Galdi is gratefully acknowledged. We
also thank Drs. Kenn Holmback and Rebecca Muraoka for technical
guidance with the ES cell culture.
Correspondence should be addressed to Dr. Charles V. Vorhees, Division
of Developmental Biology, Children's Hospital Research Foundation,
3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: charles.vorhees{at}chmcc.org.
| |
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ABSTRACT
INTRODUCTION
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