The neuronal response to a Ca2?stimulus is a complex process involving direct Ca2?/calmodulin (CaM) actions as well as secondary
cytoplasm and the nucleus to control gene expression. To dissect the role of nuclear from cytoplasmic Ca2?/CaM signaling in memory
formation, we generated transgenic mice that express a dominant inhibitor of Ca2?/CaM selectively in the nuclei of forebrain neurons
and only after the animals reach adulthood. These mice showed diminished neuronal activity-induced phosphorylation of cAMP re-
sponse element-binding protein, reduced expression of activity-induced genes, altered maximum levels of hippocampal long-term
potentiation, and severely impaired formation of long-term, but not short-term, memory. Our results demonstrate that nuclear Ca2?/
solidation of short-term to long-term synaptic plasticity and
memory in Drosophila (Yin et al., 1994, 1995), Aplysia (Kandel,
2001), and mice (Bourtchuladze et al., 1994; Kida et al., 2002;
Pittenger et al., 2002). Although the critical role of CREB family
transcription factors in long-term memory (LTM) seems to be
conserved across species, the second messenger signaling path-
ways required are less clear. In Aplysia, the formation of LTM is
mediated by serotonin stimulation of cAMP and direct CREB
activation by protein kinase A (Kandel, 2001). In Drosophila,
genetic screens for learning and memory identified mutants,
term memory (STM) into LTM in invertebrates is mediated by
the direct activation of cAMP and phosphorylation of CREB by
cAMP-dependent protein kinase (Kandel, 2001). In mammals, a
STM to LTM is less clear.
Excitatory activity in neurons leads to alterations in gene ex-
pression that are mediated by increases in intracellular Ca2?
(Bading, 2000; West et al., 2001). This activity-dependent gene
expression is thought to play a critical role in stabilizing forms of
neuronal plasticity that may underlie the formation of LTM.
Ca2?signaling to the nucleus has been studied extensively in
dingham et al., 1997, 2001; Deisseroth et al., 1998) as well as
ways such as cAMP (Chetkovich et al., 1991) and MAP kinase
of these three signaling pathways can regulate CREB (Shaywitz
and Greenberg, 1999), and it has been difficult to determine the
relative contribution of each separate pathway to the transcrip-
tional events required for stabilization of behavioral memory.
Extensive pharmacological and genetic data demonstrate be-
havioral effects from altering both the MAP kinase and cAMP
iments, it has not been possible to dissociate nuclear from cyto-
plasmic and acute from developmental roles of these complex
signaling pathways. In addition, in most cases, the memory im-
pairments seen in genetic experiments do not parallel the effects of
either protein synthesis inhibitors or CREB inhibition, suggesting
that these pathways alone do not account for the activation of the
The direct nuclear Ca2?signaling pathway has been studied
expressing a competitive inhibitor of CaM only in neuronal nu-
clei. We found that, although these mice had intact STM, their
LTM was severely impaired across multiple behavioral tasks.
Moreover, we demonstrate that these phenotypes reflect deficits
Generation of CaM binding polypeptide transgenic mice
The CaM binding polypeptide (CaMBP) gene from the vector pSVL-
10858 • TheJournalofNeuroscience,December1,2004 • 24(48):10858–10867
(Mayford et al., 1996) to create pKL107. In pKL107, the CaMBP gene is
downstream of the tetO promoter and flanked by artificial introns and a
3?-polyadenylation signal. The CaMBP transgene was purified away
from vector sequences and microinjected into B6/D2 F2embryos.
Founder animals were crossed to mice carrying the tetracycline transac-
tivator (tTA) under control of the calcium–calmodulin-dependent pro-
tein kinase II? (CaMKII?) promoter (Mayford et al., 1996). Double
transgenic males (carrying both the tetO-CaMBP and CaMKII?-tTA
used in the experiments. Experiments used 10- to 24- week-old mice for
behavior studies and 8- to 16-week-old mice for electrophysiology. All
mice were kept on a doxycycline (Dox) diet (40 ?g of Dox per 1 gm of
of age. To activate gene expression, Dox was removed from the diet 2–4
weeks before starting an experiment.
In situ hybridization
Brains were dissected and rapidly frozen in OCT embedding compound
(Sakura, Tokyo, Japan). Parasagittal cryostat sections (20 ?m) were
mounted onto microscope slides, fixed in 4% paraformaldehyde (PFA)
for 10 min, and frozen at ?80°C until use. Slices were hybridized as
described previously (Mayford et al., 1995) to an35S-labeled antisense
of the CaMBP transcript (Abel et al., 1997). The sections were washed
twice for 10 min at room temperature in 2? SSC and then twice for 60
min at 60°C in 0.2? SSC. Slides were exposed to autoradiographic film
(Biomax MR; Kodak, Rochester, NY) for 2–4 weeks.
Hippocampi were dissected and homogenized on ice. Nuclear and cyto-
plasmic fractions were obtained with the NE-PER and TE-PER extrac-
tion reagents (Pierce, Rockford, IL), and protease inhibitors were in-
cluded as recommended by the manufacturer. Protein concentrations
were quantified using Pierce Micro BCA protein assay reagent and then
diluted to 1 ?g/?l with PBS containing protease inhibitors. The samples
were aliquoted and stored at ?80°C until use. For Western blots, pro-
teins were separated by SDS-PAGE (12% polyacrylamide; Amresco, So-
lon, OH) and blotted onto nitrocellulose membranes (Protran; Schlei-
apparatus (Trans-Blot SD; Bio-Rad, Hercules, CA). Membranes were
stained with Ponceau S solution, rinsed and incubated in blocking solu-
primary antibodies were used at the dilution of 1:1000 except for anti-
(1:20,000). Monoclonal anti-CaM and polyclonal anti-Fos were ob-
tained from Upstate Biotechnology (Lake Placid, NY); polyclonal anti-,
total-, and phospho-MAPK were from Cell Signaling Technology (Bev-
erly, MA); the nuclear loading control monoclonal anti-neuronal nuclei
anti-Syntaxin 13 was from Synaptic Systems (Goettingen, Germany);
and monoclonal anti-?Tubulin and anti-?Actin were from Sigma (Mil-
anti-rabbit horseradish peroxidase-conjugated IgG (1:5000 and
1:10,000, respectively; Pierce). Signal detection by chemiluminescence
was performed with Super Signal according to the instructions of the
For CaMBP, mice were perfused transcardially with 4% PFA. The brains
were removed and postfixed in PFA for 6 hr and then placed in 20%
sucrose solution overnight; on the following day, the brains were frozen
in OCT, and coronal sections (6 ?m) were mounted on slides. Sections
1 hr with 5% normal goat serum in PBS, and then incubated overnight
al., 1995). Sections were subsequently washed and incubated with a cya-
nine 3 (Cy3)-conjugated goat anti-rabbit IgG (1:500; Jackson Immu-
noResearch, West Grove, PA). Slides were rinsed and counterstained
with 2 ?M Syto13 (Molecular Probes, Eugene, OR), rinsed again, and
mounted in Slowfade Light antifade mounting medium (Molecular
Probes). Fluorescent images were taken with a confocal laser-scanning
?m-thick paraffin-embedded brain sections. Antigen retrieval was per-
formed at 95°C for 30 min in a Tris-EDTA buffer. The immunostaining
protocol was similar to the CaMBP protocol, except that the rabbit anti-
CaM antibody (Zymed Laboratories, San Francisco, CA) was used at
1:100 dilution and the secondary antibody was Cy2 conjugated. Nuclear
counterstaining was performed with 10 ?M BoPro (Molecular Probes).
incubation with Cy3-coniugated goat anti-rabbit IgG, and counter-
stained with 4?,6?-diamidino-2-phenylindole (DAPI) 48 hr after trans-
fection (Wang et al., 1995).
For CREB immunostaining as well as for MAPK and Fos Western
blotting, seizure was induced by intraperitoneal injection of pentyle-
netetrazole (50 mg/kg body weight) (Morgan et al., 1987). Mice were
taken from their home cages, injected, and placed into an empty mouse
cage for observation. Mice were killed 10 min after seizure onset. Only
mice that developed generalized tonic–clonic seizure were used in the
present study. Brains were dissected and prepared as described. Coronal
sections (12 ?m) were taken on a cryostat, mounted on slides, fixed for 30
min in 4% PFA, and washed in PBS. Slides were incubated in blocking
munoResearch) for 2–3 hr. Slides were rinsed again, mounted in Slowfade
Light antifade mounting medium (Jackson ImmunoResearch), and cover-
slipped. Sections were visualized on a confocal microscope, and the signal
verse hippocampal slices essentially as described previously (Mayford et
al., 1995). Recording was in an interface chamber at 30°C with constant
perfusion of artificial CSF (ACSF) (in mM: 124 NaCl, 4.4 KCl, 1.2
bipolar stimulating electrode and low-resistance glass recording elec-
of CA1. Stimuli were delivered at intensities that evoked an fEPSP slope
0.02 Hz. The baseline synaptic response was collected for 30 min before
the tetanus. Long-term potentiation (LTP) was induced by one train of
100 Hz for 1 sec or by three trains of 100 Hz for 1 sec at a 5 min inter-
stimulus interval. The same stimulus intensities were used for tetaniza-
used with the numbers of tested animals as n.
Morris water maze. The water-maze apparatus consisted of a fiberglass
tank that was 122 cm in diameter and 61 cm high with white sides and
cm diameter) was hidden ?1 cm beneath the water surface. The water
tank was surrounded by a circular white plastic curtain 70 cm from the
edge of the tank. Four visual cues (50 ? 76 cm) consisting of simple
black-and-white geometric patterns were evenly spaced on the curtain
and were 50 cm above the rim on the tank at their lower edge. For the
visible platform task, the platform was marked with a red flag and its
position was changed for each trial. Every mouse received three trials
each day for 4 d, in which the average time to reach the platform was
measured. For the hidden platform task, the flag was removed and the
platform remained in the same position throughout training for each
randomly determined start locations (W, West; S, South; E, East; N,
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