Activity-Induced Notch Signaling in Neurons
Requires Arc/Arg3.1 and Is Essential
for Synaptic Plasticity in Hippocampal Networks
Lavinia Alberi,1,2,7,* Shuxi Liu,1,2Yue Wang,5Ramy Badie,1,2Constance Smith-Hicks,2,3Jing Wu,3Tarran J. Pierfelice,1,2
Bagrat Abazyan,4Mark P. Mattson,3,5Dietmar Kuhl,6Mikhail Pletnikov,4Paul F. Worley,2,3and Nicholas Gaiano1,2,3,*
1Institute for Cell Engineering, Neuroregeneration Program
2Department of Neurology
3Solomon H. Snyder Department of Neuroscience
4Department of Psychiatry and Behavioral Sciences
Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
5Laboratory of Neuroscience, National Institute of Aging, Baltimore, MD 21224, USA
6Institute for Molecular and Cellular Cognition, Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf,
Hamburg 20251, Germany
7Present address: Department of Medicine/Anatomy, University of Fribourg, Rte Albert Gockel 1, 1700 Fribourg, Switzerland
*Correspondence: firstname.lastname@example.org (L.A.), email@example.com (N.G.)
Notch signaling in the nervous system has been most
studied in the context of cell fate specification.
However, numerous studies have suggested that
Notch also regulates neuronal morphology, synaptic
plasticity, learning, and memory. Here we show that
Notch1 and its ligand Jagged1 are present at the
synapse, and that Notch signaling in neurons occurs
in response to synaptic activity. In addition, neuronal
Notch signaling is positively regulated by Arc/Arg3.1,
an activity-induced gene required for synaptic plas-
ticity. In Arc/Arg3.1 mutant neurons, the proteolytic
activation of Notch1 is disrupted both in vivo and
in vitro. Conditional deletion of Notch1 in the postnatal
hippocampus disrupted both long-term potentiation
(LTP) and long-term depression (LTD), and led to defi-
cits in learning and short-term memory. Thus, Notch
signaling is dynamically regulated in response to
neuronal activity, Arc/Arg3.1 is a context-dependent
plasticity that contributes to memory formation.
Notch receptors and ligands are highly conserved transmem-
brane proteins that are expressed in the developing mammalian
et al., 2002). The function of Notch signaling in the nervous
system has been most studied in the context of neural stem/
progenitor cell regulation, and neuronal/glial cell fate specifica-
tion (Louvi and Artavanis-Tsakonas, 2006). However, numerous
reports have suggested that Notch also plays a role in neuronal
differentiation (Breunig et al., 2007; Eiraku et al., 2005; Redmond
et al., 2000; Sestan et al., 1999), neuronal survival (Lu ¨tolf et al.,
2002; Saura et al., 2004), and neuronal plasticity (Costa et al.,
2003; de Bivort et al., 2009; Ge et al., 2004; Matsuno et al.,
While studies in both vertebrates and invertebrates suggest
that Notch signaling regulates neuronal plasticity, learning, and
memory, it remains unclear where and how Notch is activated
it interacts with known plasticity genes. Here we provide
activity, and that this signaling is heavily dependent upon the
activity-regulated plasticity gene Arc/Arg3.1 (Arc hereafter)
(Chowdhury et al., 2006; Link et al., 1995; Lyford et al., 1995;
Shepherd et al., 2006). Furthermore, disruption of Notch1 in
CA1 of the postnatal hippocampus reveals that Notch signaling
is required to maintain spine density and morphology, as well
as to regulate synaptic plasticity and memory formation.
Notch1 Is Present at the Synapse and Is Induced
by Neuronal Activity
Using an antibody that recognizes the active form of Notch1
(NICD1, S3 fragment), we found Notch1 present in the cell
soma and dendrites of neurons in many regions of the brain,
including the cerebral cortex and hippocampus (Figure 1A and
data not shown). We also found that NICD1 and the activity-
induced protein Arc were present in manyof the same cells, sug-
gesting that Notch1 signaling occurs in active neurons. Indeed,
most EGFP+ neurons in a transgenic Notch reporter (TNR)
mouse line (Mizutani et al., 2007) expressed Arc (e.g., 73% of
EGFP+ cells in the cortex; see Figure 3A). In cultured neurons,
Notch1 was enriched in the dendrites and cell soma, while the
ligand Jagged1 (Jag1) was enriched in axons (Figures 1B and
S1A, available online). Notch1, NICD1, and Jag1 colocalized
with synaptic proteins (Figures 1C–1E and S1), and NICD1 was
enriched in synaptosomal fractions derived from cortical
extracts (Figure 1F).
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Increased neuronal activity after treatment with NMDA
(Figures 2A and 2B) or bicuculline (Figure 2C) led to higher
NICD1 levels, while treatment with the NMDA receptor blocker
AP5 led to reduced NICD1 levels (Figure 2B). Neuronal activity
also increased Notch1 protein levels (Figures 2C–2E, see also
Figure 3E), including the preprocessed form of the receptor
(Figures 2D and 2E), and Jag1 expression (Figure 2F). Activity-
induced Notch1 expression occurred in the presence of the
transcriptional inhibitor actinomycin-D, suggesting that pre-ex-
isting Notch1 transcript is translated in response to synaptic
activity (Figures 2D and 2E).
To test the effect of synaptic activity on Notch expression and
processing further, we used acute hippocampal slices. The
Schaffer collateral pathway was activated to induce LTP (Fig-
ure 2I), and increased Arc expression was observed in both
CA3 and CA1 neurons (Figure 2G). In addition, somal Notch1
expression was increased in CA3 and CA1 (6.1-fold by pixel
count, n = 6, p < 0.02) (Figure 2G), as was NICD1 staining (Fig-
ure 2H and data not shown). The increase in Notch expression
in CA1 could be reduced by AP5 (Figure S2).
Neuronal Notch Signaling Occurs In Vivo in Response
We next evaluated Notch expression and signaling in response
to neuronal network activity in vivo after exploration of a novel
environment. This behavioral paradigm activates specific
ensembles of hippocampal pyramidal neurons that can be
identified by expression of Arc (Guzowski et al., 1999). TNR
mice were allowed to explore a novel environment for 5 min,
and were sacrificed 1.5 or 8 hr later. Consistent with prior work
(Ramı ´rez-Amaya et al., 2005), the number of Arc+ hippocampal
CA1 neurons was increased ?3-fold at 1.5 hr, and ?2-fold at
8 hr (Figure S3A). In addition, the number of Notch1+ CA1
neurons was elevated at both time points (?3-fold, Figures S3A
and S3C), as was EGFP expression (indicative of Notch activity)
at 8 hr (Figures S3B and S3C). Notably, nearly all (94%–97%) of
the Arc+ neurons also had Notch1 signal in the nucleus (e.g.,
see Figure 3C), indicating that Arc induction and Notch signaling
occur in the same neuronal networks in response to exploration.
at 1.5 hr, and 29% at 8 hr). Thus, the temporal dynamics of
Notch1 and Arc may be different, with Notch1 persisting longer
than Arc, or not all neuronal Notch signaling occurs in Arc+
Neuronal Notch Signaling Is Disrupted in Arc Mutants
Both Notch signaling (Fortini and Bilder, 2009; Vaccari et al.,
2008) and Arc function (Chowdhury et al., 2006; Shepherd
et al., 2006) engage Dynamin-mediated endocytosis, raising
Figure 1. Notch1 Is Present at the Synapse in Mature Neurons
(A) The somatosensory cortex is shown. Arc and NICD1 are both present in the soma (arrows) and apical dendrites of layer V neurons. (B) DIV21 (21 days in vitro)
axonal processes (arrowheads). (C) Notch1 colocalizes in dendritic spines with the synaptic protein PSD95 (see also Figure S1). (D) Jag1 colocalizes with the
synaptic protein Synapsin I. (E) The activated form of Notch-1 (NICD1) colocalizes with PSD95 in DIV21 neurons. (F) Subcellular fractionation of adult mouse
cortices reveals that S3 fragment of Notch1 (asterisk) is enriched in synaptosomal fractions (P2, washed P20, and membranes P3), as compared to the cyto-
plasmic fraction (S2). Scale bars = 75 mm (A), 25 mm (B and E), and 5 mm (C and D).
thepossibility that theymight interact. Thus,weexamined Notch
activity in the adult brain of Arc mutants using the TNR mouse
line. Of 15 Arc mutants, 12 (80%) had reduced EGFP expression
(Notch activity) throughout the cerebral cortex as compared to
22 nonmutants (Figures 3A and 3B). Arc mutants also had
reduced NICD1 levels, consistent with less Notch signaling in
the absence of Arc (Figure 3B).
To test if Arc is required for Notch pathway recruitment in
response to network activity in vivo, we compared Notch1
expression in the hippocampus of wild-type and Arc mutants
after exploration of a novel environment. In controls, we
observed elevated expression of both Arc and Notch1, the latter
of which was localized to both the cell soma and the nucleus, in
CA1 (not shown) and CA3 (Figure 3C). In contrast, no change
Figure 2. Notch Signaling Occurs in Neurons in Response to Activity
(A) NICD1 is increased in cultured hippocampal neurons after NMDA treatment. Relative NICD1 signal levels with (n = 4) or without (n = 3) treatment (right) are
while NMDA receptor blockade (AP5) decreased NICD1 (4.3-fold, n = 3, p < 0.05) and Arc (10.0-fold, n = 3, p < 0.01). EDTA treatment was used a positive control
to activate Notch1(Rand et al., 2000). (C) Treatment of hippocampal neurons with bicuculline increased Arc and Notch1 S3 fragment levels (2.9-fold at 4 hr, n = 5,
p < 0.001) (asterisk). (D) Western blot (WB) showing that full-length (pre-S1 cleavage) Notch1 protein levels increase in response to bicuculline, even with the
transcriptional inhibitor actinomycin-D (Act-D). (E) Quantification of four experiments shows that the expression of full-length Notch1 (normalized to b-actin) is
substantially increased after bicuculline treatment, with or without Act-D. ns, not significant. b-tub, b-tubulin; b-act, b-actin. (F) Quantitative RT-PCR of hippo-
campal cultures treated with bicuculline for 4 hr shows that Jag1 expression was increased in response to increased neuronal activity (*p < 0.04, n = 4). (G) Three
and one-half hours after LTP induction in the CA1 region of acute hippocampal slices from adult mice, both Arc and Notch1 protein levels were elevated in the
soma of CA3 (arrow) and CA1 (arrowhead) neurons. (H) Immunohistochemistry (IHC) revealed increased NICD1 in CA1 in response to LTP. (I) Plot of field excit-
atory postsynaptic potential (fEPSP)in hippocampal slices. Mean values from four animals are shown. Scale bars = 50mm.Standard deviation is shown in (A), (E),
in Notch1 expression or subcellular localization was observed in
Arc mutants (Figure 3D).
Arc Regulates the Proteolytic Processing of Notch1
neuronal cultures. In the absence of Arc there was a reduction in
the S3 cleaved form of Notch1 (NICD1) (Figure 3E), indicating
that Arc positively regulates the g-secretase-mediated cleavage
of Notch1 in neurons. Treatment with bicuculline led to elevated
Notch1 and NICD1 levels in control neurons, but not in Arc
mutant neurons (Figure 3E), indicating that Arc is required for
the activity-mediated recruitment of neuronal Notch signaling.
No change in Jag1 expression was observed in Arc mutant
Figure 3. Neuronal Notch Signaling Is Disrupted in Arc Mutants In Vivo
(A) Five-week-old Arc mutant and wild-type animals containing the TNR transgene were examined to determine the impact of Arc disruption on the Notch
pathway in vivo. Arc mutants had reduced EGFP expression, indicating reduced Notch activation (somatosensory cortex is shown). Scale bar = 70 mm. (B)
Western blot of cell lysates derived from the cerebral cortex of Arc knockout (KO) animals revealed reduced NICD1 generation (asterisk) and EGFP expression.
Two different exposures of the S3 band are shown. NICD1 band intensity (normalized to b-tubulin) was compared between six wild-type and six Arc KO animals
(**p < 0.01). Standard deviation is shown. (C) In 5-week-old wild-type animals, exploration of a novel environment resulted in a rapid increase in Arc and Notch1
expression in CA1 (not shown) and CA3 (Notch1 signal intensity for cage control and 45 min after exploration was 6.8 ± 2.6 arbitrary units [a.u.], and 21.5 ±
5.2 a.u., respectively; n = 3 each, p < 0.01). Much of the Notch1 protein was in the cell soma and nucleus, consistent with active Notch1 signaling. (D) No increase
in Notch1 expression was observed in Arc KO animals after exploration (cage control and 45 min after exploration was 11.2 ± 3.3 a.u. and 13.4 ± 4.8 a.u., respec-
tively; n = 3 each). (E) Western blot analysis from Arc KO and wild-type hippocampal neuronal cultures revealed that, in the absence of Arc, Notch processing
is reduced; the S3 band (asterisk) is nearly absent, unlike the S1 band (upper). (F) Western blot of Arc mutant hippocampal cultures infected with Sindbis virus
expressing either full-length Arc or a nonfunctional form lacking residues 91–100 (Δ) (Chowdhury et al., 2006). (G) Arc and Dynamin coimmunoprecipitate with
Notch1 from cortical protein preparations. (H) Notch1 coimmunoprecipitates with Arc from protein lysates generated from neuronal cultures. This interaction
was not detected in Arc KO cultures. Scale bars = 50 mm.
cultures (Figure S4), in line with the idea that receptor process-
ing, and not ligand availability, is defective in mutant cells.
In an effort to rescue Notch1 processing in Arc mutant cells,
we used Sindbis virus to introduce functional or nonfunctional
Arc into mutant neurons in vitro. Restoration of Arc expression
rescued Notch1 processing (2.9-fold increase, n = 3, p <
0.001) (Figure 3F), suggesting that the Notch1 cleavage defect
in Arc mutant neurons is not caused by aberrant neuronal differ-
entiation. A form of Arc lacking the ability to bind Endophilin and
participate in endocytic trafficking (D91–100) (Chowdhury et al.,
2006) was unable to restore Notch1 processing in Arc mutant
neurons (Figure 3F).
Next, we found that Arc and Dynamin coimmunoprecipitated
with Notch1 in protein preparations from adult cortical extracts
(Figure 3G). In addition, Notch1 coimmunoprecipitated with
Arc in protein extracts from wild-type, but not Arc mutant,
cortical tissue (Figure 3H). Thus, Arc-mediated Dynamin-driven
endocytosis of Notch1 may be important for activity-dependent
Notch signaling in neurons. Interestingly, Arc is not required for
Notch activation in embryonic forebrain progenitors (Figure S5),
indicating that Arc regulates Notch in a context-dependent
Conditional Deletion of Notch1 in the Postnatal
neuronal ensembles after spatial exploration, we next tested the
function of Notch in such ensembles. To conditionally knock out
Notch1 in the postnatal hippocampus, we crossed Notch1flox/flox
(Radtke et al., 1999) mice with the CamKII-cre (T29-1) driver line
(Tsien et al., 1996), and Notch1 deletion was confirmed at both
the mRNA and protein levels (Figure S6 and Figure 4A, respec-
tively; n = 6 each). Golgi-Cox staining of CA1 pyramidal neurons
revealed that loss of Notch1 postnatally did not affect dendritic
length (Figures 4B and 4C). However, spine density on
secondary and tertiary dendrites was reduced (Figures 4D and
4F), and spine morphologies were altered (Figures 4E and 4F).
To test the role of Notch in synaptic plasticity, the electrophys-
iological properties of Notch1 conditional knockout (cKO)
animals were tested using hippocampal slices and field
Figure 4. Loss of Notch Function in CA1 Affects Neuronal Morphology and Plasticity
(A) IHC shows that in Notch1 cKO mice, Notch1 expression is reduced in the CA1 region of the hippocampus (arrowheads). cKO animals had increased Notch1
expression in astrocytes (arrow). Inset scale bar = 25 mm. Note that these mice were exposed to a novel environment to increase Notch1 expression. (B) Golgi-
impregnated CA1 pyramidal neurons reveal no difference in gross dendritic morphology between Notch1 cKOs and controls. (C) Notch1 cKO CA1 neurons have
comparable lengths of apical and basal dendrites. (D) In Notch1 cKOs spine density of CA1 dendrites is reduced 16% (p < 0.001). (E) In Notch1 cKOs the number
of mushroom spinesis 25% reduced on CA1 pyramidal dendrites, and the number of thin spinesis 40% increased (p < 0.001). (F) Images of Golgi-stained control
and Notch1 cKO dendritic spines. Scale bar in (F) = 10 mm. (G) Notch1 cKO (closed circles) has normal basal transmission as compared to controls (open circles).
(H) The paired pulse facilitation (PPF) curve is the same in Notch1 cKO and control slices. (I and J) LTP and LTD were reduced in the Notch1 cKO slices (p < 0.01
each). Standard deviation is shown in (C)–(E).
recordings. Basal transmission was the same for mutants and
controls (10–11 slices) (Figure 4G), and the paired pulse facilita-
tion (PPF) protocol revealed that Notch1 cKO slices had presyn-
aptic strength comparable to that of controls (Figure 4H).
However, when we induced LTP in the Schaffer collateral
pathway, the magnitude of LTP in the CA1 region was uniformly
higher in controls (188.5 ± 23.1, n = 6) than in Notch1 cKO slices
(140.9 ± 20.6, n = 5, p < 0.05) (Figure 4I). Similarly, after low-
frequency stimulation, LTD in CA1 was uniformly reduced in
(70.0 ± 11.5, n = 5, p < 0.05) (Figure 4J). Thus, Notch1 influences
the magnitude of both the potentiation and depression of
Notch1 cKO Mice Display Deficits in Learning
and Acquisition of New Memory
Next we performed behavioral tests to evaluate the cognitive
abilities of Notch1 cKO mice. During novel object recognition
testing, mutants initially had a lower novel object preference
than controls, and the next day, in contrast to controls, mutants
had no preference (Figure 5A). Similarly, in a social interaction
test, unlike controls, Notch1 cKO mice did not interact more
with a new subject (Figure 5B), although like controls, mutants
preferred a subject to an object (not shown). In Y-maze testing,
Figure 5. Notch1 Conditional Ablation Causes
Deficits in Memory Acquisition
(A) While both control and Notch1 cKO animals spent
more time exploring a novel object 25 min after exposure
to two identical objects (83.2% ± 3.5% preference versus
68.8% ±3.9%,respectively,p<0.001), 24hrlater,Notch1
cKOs did not display any preference for the novel object
(52.8% ± 4.5%, p = 0.4) in contrast to controls (65.4% ±
3.6%, p < 0.01) (n = 12 for both Notch1 cKO and WT).
(B)Notch1 cKOsshowednopreference foranovelsubject
(48.3% ± 4.1%, p = 0.6) in contrast to controls (60.2% ±
3.8%, p < 0.01) (n = 13 Notch1 cKO and n = 16 control).
(C and D) Despite normal alternation in a Y-maze, Notch1
cKO mice failed to show a robust preference for a previ-
ously hidden arm, while controls did (n = 12 Notch1
cKO, p < 0.07; n = 15 control; p < 0.022). (E and F) The
average time to find the platform in a Morris water maze
was higher on day two, three, and four for Notch1 cKO
animals (p < 0.05 for each time point). Similar results
were obtained with reversal learning, where the platform
was placed in the opposite quadrant (F). A repeated-
measure ANOVA was used to assess statistical signifi-
cance in (E) and (F). Twenty-four hours after the last of
five training sessions for both initial and reversal learning,
both Notch1 cKOs and controls spent more time in the
target quadrant, indicating comparable memory retrieval
after repetitive learning (n = 14, Notch1 cKO and n = 18,
WT) (Figures S6A and S6B). Standard error is shown.
Notch1 cKO mice chose alternating arms at
the same frequency as controls (not shown),
but showed no preference for a previously
hidden arm (Figures 5C and 5D).
Next, spatial reference memory was investi-
gated using the Morris water maze. Perfor-
mance improved over 5 days of learning in
was greater in the mutants (Figure 5E, p < 0.01), despite the
average swim speed being comparable (p = 0.4). A learning
deficit was also seen in the Notch1 cKO mice when subjected
to reversal learning (Figure 5F). In both cases, 24 hr after the
last learning session, mutant and control mice spent more time
in the target quadrant (Figures S7A and S7B). Thus, Notch1
cKO mice can learn using spatial cues, although they do so
more slowly than wild-types.
In line with the previous report on the Notch1+/?mice (Costa
et al., 2003), we could not detect any difference between Notch1
cKO and controls in contextual fear-conditioning 24 hr after
a shock was delivered (Figure S7C). In addition, Notch1 cKO
mutant mice displayed normal motor coordination (rotarod
test), motor activity (open field test), and anxiety levels (elevated
plus maze) (Figure S7C).
Notch1 Is at the Synapse and Can Be Activated
in Response to Neuronal Activity
We have shown that Notch1 colocalizes with PSD95 in cultured
neurons, and that the transcriptionally active form of the
receptor, NICD1, is present at the synapse. In addition, we
have shown that Jag1 is present in axons, localizes to synapses,
and is upregulated in response to neuronal activity. Stimulation
of neurons in culture, in hippocampal slices, or in vivo after
exposure to a novel environment all lead to increased Notch1
expression and signaling. The notion that activity-dependent
g-secretase-mediated Notch receptor activation can occur at
g-secretase activity cleaves EphA4 in response to neuronal
activity (Inoue et al., 2009).
Activity-Induced Neuronal Notch Signaling Requires Arc
The activity-regulated neuronal Notch signaling we have identi-
fied both in vitro and in vivo is heavily dependent upon Arc. In
Arc mutant neurons we observe a drastic reduction in the S3
cleaved form of Notch1, indicating that the g-secretase-medi-
ated processing is disrupted in the absence of Arc function.
Furthermore, our rescue and coimmunoprecipitation experi-
ments indicate that the role of Arc in mediating Notch1 activation
requires its association with Endophilin, and that Arc exists in
a protein complex with Notch1 and Dynamin. Thus, in addition
to its role in AMPA receptor trafficking (Chowdhury et al., 2006;
Shepherd et al., 2006), Arc appears to regulate synaptic plas-
ticity through interactions with the Notch pathway.
Notch1 Ablation Affects Neuronal Morphology,
Plasticity, and Memory Acquisition
We next probed the potential function of activity-induced Notch
signaling by conditionally deleting Notch1 in CA1 of the adult
hippocampus.This modelisanimprovement overthe Notch1+/–,
CBF1+/–(Costa et al., 2003) and Notch1 antisense mice (Wang
et al., 2004), because deletion occurs after development is
complete. Ablation of Notch1 in pyramidal CA1 neurons affects
and depression reduced. Our LTP result is consistent with
reduced potentiation resulting from decreased Notch1 expres-
sion (Wang et al., 2004), or conditional g-secretase disruption
(via ablation of Presenilin 1/2) (Saura et al., 2004). However,
our LTD result differs from those in previous studies, the former
of which found enhanced LTD, and the latter of which found no
change in LTD. This incongruence can be explained by the fact
that the previous studies were confounded by possible develop-
mental defects (Wang et al., 2004), and by lack of specificity with
respect to Notch signaling (Saura et al., 2004).
memory processes in hippocampal networks, we tested the
Notch1 cKO mice using numerous behavioral paradigms. In
the absence of Notch1, learning and rapid memory retrieval of
itive learning is not. A function for Notch in rapid processing is
consistent with the increase in Notch activation in hippocampal
networks that occurs shortly after sensory input.
In summary, we have shown that Notch signaling is highly
dynamic in mature neurons, and that it is induced in response
to neuronal activity both in vitro and in vivo. In addition, we
have identified the activity-regulated gene Arc as a context-
dependent regulator of Notch signaling, and have shown that
Arc is required for the g-secretase-mediated activation of
disruption we have shown that Notch1 is required for normal
spine morphology, synaptic plasticity, and memory processing.
All mice were maintained in accordance with the Institutional Animal Care
and Use Committee (IACUC) at Johns Hopkins University School of
Medicine. Generation of Arc mutant mice has been previously described
(Plath et al., 2006). Notch1 cKO and wild-type littermate control (Notch1flox/+,
Notch1flox/flox, and CamKII-Cre) mice were obtained by crossing Notch1flox/flox
mice on a CD1 background to the CamKII-Cre (T29-1) mouse line on a
C57BL6/129 background (Tsien et al., 1996).
For novel spatial exploration, cage control mice (t = 0 hr) were killed directly
from their home cages, whereas the experimental mice performed a 5 min
exploration session, and were returned to their home cage prior to analysis
at the given time point. Novel object recognition was done accordingly to
a published protocol (Bevins and Besheer, 2006). In the Y-maze mice were
videotaped and scored for time spent in each arm and number of entries in
each arm using the StopWatch Plus software. The social interaction testing
was carried out in three sessions using a three-chambered box with openings
between the chambers. The Morris water maze test was done according to
a published protocol (Vorhees and Williams, 2006). Details for all behavioral
tests are provided in the Supplemental Information.
Cell Culture and In Vitro Manipulation
plated on poly-L-lysine-coated 60 mm dishes or 18 mm glass coverslips.
Neurons were exposed to pharmacological manipulations after 14 days
in vitro (DIV). For Sindbis virus infection, the pSinRep5 vector (Invitrogen)
was used to generate viruses expressing either full-length Arc or a nonfunc-
tional form with residues 91–100 deleted (Chowdhury et al., 2006).
Subcellular Fractionation, Immunoprecipitation, and Western Blot
Synaptosomal fractions were prepared as previously described (Blackstone
et al., 1992). Standard western blot protocols were used. Details regarding
fractionation, immunoprecipitation, and western blot protocols are provided
in the Supplemental Information. Quantitation of individual protein bands
was done using ImageJ software. Values were averaged between experi-
ments, and Student’s t test was used to compare samples.
Antibodies, Immunostaining, and Image analysis
A complete list of the antibodies used can be found in the Supplemental Infor-
in ice-cold acetone-methanol (1:1) at –20?C for 10 min. The immunostainings
with rabbit anti-Arc and anti-Notch1 antibodies were performed using the TSA
fluorescence amplification kit (Perkin Elmer). ImageJ software (NIH) was used
to quantify fluorescence intensity of immunostainings with NICD1 (Figure 2A),
EGFP (Figure S3B), and Notch1 (see legend for Figures 3C and 3D). Student’s
t test was used to determine p values.
Golgi-Cox Staining and Spine Imaging and Analysis
Golgi-Cox staining (FD NeuroTechnologies) was performed according to the
manufacturer’s instructions. Dendrite and spine lengths/widths were mea-
sured using Reconstruct software by the Neural Systems Laboratory (http://
www.bu.edu/neural/Reconstruct.html). Spine length and width data were
analyzed using the Kolmogorov-Smirnov statistical test.
Hippocampal Slice Preparation and Electrophysiology
Transverse hippocampal slices (350 mm) were prepared from Notch1 cKO
and control mice, and maintained in artificial cerebrospinal fluid at
room temperature. Data were collected using an Axopatch 1D amplifier
(Molecular Device); signals were filtered at 2 kHz, digitized at 10 kHz, and Download full-text
analyzed using pCLAMP 8 software (Molecular Device).
Supplemental Information for this article includes Supplemental Experimental
Procedures andseven Supplemental Figures and can befound withthisarticle
online at doi:10.1016/j.neuron.2011.01.004.
The authors thank Jason Shepherd, Richard Flannery, Marlin Dehoff, Vera
Goh, and Keejung Yoon for technical and intellectual input during the course
of this project. We also thank Ted Dawson and Jay Baraban for critically
reading the manuscript. Funding for this work came from the Institute for
Cell Engineering at Johns Hopkins University (N.G.), a NARSAD Young Inves-
tigator Award (N.G), the James S. McDonnell Foundation (N.G.), and the
National Institute of Mental Health (P.F.W.).
Accepted: January 11, 2011
Published: February 9, 2011
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