Neurobiology of Learning and Memory 103 (2013) 39–49

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Neurobiology of Learning and Memory
journal homepage: www.elsevier.com/locate/ynlme

Disrupting Jagged1–Notch signaling impairs spatial memory
formation in adult mice
Derya Sargin a,b,c,d, Leigh C.P. Botly a,b,c,d, Gemma Higgs a,d, Alexander Marsolais a,d, Paul W. Frankland a,b,c,d,Sean E. Egan e,f, Sheena A. Josselyn a,b,c,d,⇑aProgram in Neurosciences & Mental Health, Hospital for Sick Children, 555 University Ave, Toronto, ON, Canada M5G 1X8
Department of Psychology, University of Toronto, Toronto, ON, Canada M5G 1X8
Department of Physiology, University of Toronto, Toronto, ON, Canada M5G 1X8
dInstitute of Medical Sciences, University of Toronto, Toronto, ON, Canada M5G 1X8eProgram in Developmental and Stem Cell Biology, Hospital for Sick Children, 101 College St., Rm 14-309, Toronto, ON, Canada M5G 1L7
f
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8bca r t i c l e

i n f oArticle history:
Received 15 January 2013
Revised 20 March 2013
Accepted 20 March 2013
Available online 6 April 2013
Keywords:
Notch
Jagged1
Spatial memory formation
Hippocampus
Water maze
Adult micea b s t r a c t
It is well-known that Notch signaling plays a critical role in brain development and growing evidence
implicates this signaling pathway in adult synaptic plasticity and memory formation. The Notch1 receptor is activated by two subclasses of ligands, Delta-like (including Dll1 and Dll4) and Jagged (including
Jag1 and Jag2). Ligand-induced Notch1 receptor signaling is modulated by a family of Fringe proteins,
including Lunatic fringe (Lfng). Although Dll1, Jag1 and Lfng are critical regulators of Notch signaling,
their relative contribution to memory formation in the adult brain is unknown. To investigate the rolesof these important components of Notch signaling in memory formation, we examined spatial and fear
memory formation in adult mice with reduced expression of Dll1, Jag1, Lfng and Dll1 plus Lfng. We also
examined motor activity, anxiety-like behavior and sensorimotor gating using the acoustic startle
response in these mice. Of the lines of mutant mice tested, we found that only mice with reduced Jag1
expression (mice heterozygous for a null mutation in Jag1, Jag1+/ ) showed a selective impairment in spatial memory formation. Importantly, all other behavior including open ﬁeld activity, conditioned fear
memory (both context and discrete cue), acoustic startle response and prepulse inhibition, was normal
in this line of mice. These results provide the ﬁrst in vivo evidence that Jag1–Notch signaling is criticalfor memory formation in the adult brain.
Crown Copyright Ó 2013 Published by Elsevier Inc. All rights reserved.

1. Introduction
The Notch pathway is a highly-conserved ubiquitous signaling
system which plays a fundamental and well-studied role in cell–
cell communication [reviewed in (Artavanis-Tsakonas, Rand, &Lake, 1999; Guruharsha, Kankel, & Artavanis-Tsakonas, 2012;
Kopan, 2012)]. Notch genes encode single-pass transmembrane
receptor proteins. Drosophila have a single Notch gene (del Amo
et al., 1993), C. elegans have two Notch genes [LIN-12, GLP-1])(Austin & Kimble, 1989), while mammals have four [Notch1–4](del Amo et al., 1993; Lardelli, Dahlstrand, & Lendahl, 1994;
Uyttendaele et al., 1996). In mammals, there are two subclassesof Notch ligands, Delta-like (Dll1 and Dll4, referred to as Delta in
Drosophila) and Jagged (Jag1 and Jag2, referred to as Serrate in Drosophila) [reviewed in (Bray, 2006)]. The ability of these ligands toactivate Notch signaling is modulated by glycosylation mediated,⇑ Corresponding author at: Program in Neurosciences & Mental Health, Hospitalfor Sick Children, 555 University Ave, Toronto, ON, Canada M5G 1X8.
E-mail address: sheena.josselyn@sickkids.ca (S.A. Josselyn).

in part, by a family of sugar transferases termed Fringe proteins(Brückner, Perez, Clausen, & Cohen, 2000; Fleming, Gu, & Hukriede,
1997; Klein & Arias, 1998; Moloney et al., 2000a,b; Panin, Papayannopoulos, Wilson, & Irvine, 1997). In mammals, there are three
Fringe genes: Lunatic fringe [Lfng], Manic fringe [Mfng] and Radical
fringe [Rfng] (Cohen et al., 1997; Johnston et al., 1997). Lfng canboth enhance Notch1 signaling induced by Dll1 and suppress
Notch1 signaling induced by Jag1 (Hicks et al., 2000).
Canonical Notch signaling is initiated by a Notch ligand, expressed on the surface of one cell, binding to the extracellular domain of a Notch receptor that is located on the surface of aneighboring cell. In this way, Notch signaling is somewhat uniquein that signaling is restricted to neighboring cells. Once activated,
the Notch receptor undergoes proteolytic cleavage and the soluble(and active) Notch intracellular domain (NICD) is released. NICD
then translocates to the nucleus where it regulates target gene
expression by associating with the central DNA binding transcription factor RBP-J (Kao et al., 1998; Schroeter, Kisslinger, & Kopan,
1998; Struhl & Adachi, 1998). RBP-J normally represses gene1074-7427/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nlm.2013.03.001

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D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

expression, but when bound to NICD, gene expression is activated(Jarriault et al., 1995; Kato et al., 1997).
Although traditionally studied in cell fate speciﬁcation during
development, the Notch signaling pathway is increasingly recognized to play an important role in the adult nervous system (Yoon& Gaiano, 2005). Notch is expressed by neurons in the adult mousebrain where it is present at particularly high levels in the hippocampus (Berezovska, Xia, & Hyman, 1998). In addition, the Notch
ligands, Jag1 and Dll1, are also expressed in the hippocampus(Breunig, Silbereis, Vaccarino, Sestan, & Rakic, 2007; Stump et al.,2002). Several studies implicate Notch signaling in adult synaptic
plasticity, learning, and memory [reviewed in (Costa, Drew, & Silva,
2005)]. For instance, increasing Notch function enhances long-term
memory formation, whereas disrupting Notch inhibits memory
formation in Drosophila across several paradigms (Ge et al., 2004;
Matsuno, Horiuchi, Tully, & Saitoe, 2009; Presente, Boyles, Serway,
de Belle, & Andres, 2004). Consistent with this, long-term potentiation (LTP), argued to be a cellular correlate of memory formation,is impaired in mice with reduced Notch1 protein in the hippocampus [produced by expressing antisense directed against Notch1mRNA] (Wang et al., 2004). Importantly, this impairment is reversed by application of the ligand Jag1 (Wang et al., 2004). Furthermore, although homozygous deletion of Notch1 isembryonically lethal (Swiatek, Lindsell, del Amo, Weinmaster, &Gridley, 1994), heterozygous Notch1+/ mice develop normally,
but show speciﬁc deﬁcits in spatial memory formation (Costa,
Honjo, & Silva, 2003). This ﬁnding was recently conﬁrmed and extended in mutant mice in which Notch1 is conditionally knocked
out in adult forebrain neurons (Alberi et al., 2011).
These ﬁndings suggest that Notch1 signaling regulates memory
formation in adult mice. However, the potential roles of the different Notch1 ligands and Fringe proteins are unknown. Here we
examined learning and memory in several lines of mice with reduced levels of the Notch1 ligands Dll1 and Jag1, as well as a critical modulator of Notch receptor-ligand afﬁnity, Lfng. In each case,
we used mice that were heterozygous for a null mutation andfound that reduced expression of Jag1 (but not Dll1 or Lfng) produced deﬁcits in spatial memory formation while sparing fear
memory formation. Importantly, Jag1+/ mice showed normal motor activity, anxiety-like behavior, and sensorimotor gating, suggesting that the spatial memory impairment was speciﬁc.

2. Materials and methods
2.1. Mice
All experimental procedures were conducted in accordancewith Canadian Council on Animal Care (CCAC) guidelines and approved by the Animal Care Committee at the Hospital for Sick Children. Adult male and female C57Bl/6NTac mice (3–4 months old)
were used for all experiments. Mice were housed 3–5 animals
per cage on a 12 h light/dark cycle and provided with food andwater ad libitum.
All lines of mice were heterozygous for a null mutation encoding each gene of interest. Generation of a Dll1+/ was described previously (Hrabe de Angelis, McIntyre II, & Gossler, 1997). Lfng lossof-function allele (Lfng ) was generated by deletion of exon 2 (Xuet al., 2010). Double heterozygous mice (Dll1+/ /Lfng+/ ) were generated by crossing Dll1+/ and Lfng+/ single heterozygous mice.
Jag1 allele was produced by replacement of a portion of the extracellular domain (the last 3 EGFL repeats and the CR domain) as wellas the entire intracellular domain with a transmembrane domain
linked to b-geo (Xu et al., 2012). For all experiments, mice heterozygous for the desired mutation (Dll1+/ , Lfng+/ , Dll1+/ /Lfng+/ ,Jag1+/ ) were generated from wild-type (WT) X heterozygous(HET) parents, and their respective WT littermates were used ascontrols. Mice were handled for 7 days (2–4 min) prior to the startof behavioral testing. Multiple behavioral tests were conducted on
all mice in the following test order: open ﬁeld, Morris water maze,contextual and cued fear conditioning, acoustic startle response(habituation), prepulse inhibition of the acoustic startle reﬂex
and threshold for an acoustic stimulus to elicit a startle reﬂex.

2.2. Morris water maze
The Morris water maze was used to assess spatial memory formation (Morris, 1984). A circular tank (diameter 120 cm, depth
50 cm) was ﬁlled with warm water (28 ± 1 °C, depth 40 cm) madeopaque by the addition of non-toxic tempura paint. A circular escape platform (diameter 10 cm) was submerged 0.5 cm below
the water surface. The platform was located in a ﬁxed position
throughout training. Mice were trained to locate the platformusing extra-maze cues placed 1 m from the pool perimeter.
Mice were trained with one of two training protocols. In the intense training protocol, mice received 2 blocks of 3 trials per day,for 6 training days. In the weak training protocol, mice received
3 blocks of 4 trials per day for 3 training days. Trials were initiatedby placing the mouse into the pool facing the wall at one of four
randomly assigned start positions. Mice were allowed to swim until they found the platform, or until 60 s elapsed. Mice that failed toﬁnd the platform within 60 s were guided to the platform. Aftereach training trial, the mouse remained on the platform for 15 s.
A probe test (during which the platform was removed and miceplaced into the pool for 60 s) was conducted at the end of training
to assess spatial memory formation. Mice that formed a memory of
the spatial location of the platform tended to swim in the area ofthe pool in which the platform was previously located. The probe
test for the intense training protocol was performed on day 7,
24 h after completion of training. The probe test for the weak training protocol was conducted 60 min after the ﬁnal training trial. A
visible version of the water maze task was performed on one lineof mice. In this version, the platform was marked with a local
cue throughout the 3-d training protocol.
The swimming paths were acquired by an overhead video camera and analyzed using an automated tracking system (Actimetrics,
Wilmette, IL). Escape latency and swim speed during training wererecorded for each mouse. The percentage of time mice spent in theTarget Zone (a 20 cm radius zone centered on the former platform
location) versus the average of three other equivalent zones of thepool (but located in the other 3 quadrants, Other Zone) in the probe
tests was quantiﬁed and used as our index of spatial memory
formation.

2.3. Contextual and cued fear conditioning
During training, mice were placed in a Med Associates (St. Albans, VT) chamber (24  30  21 cm) located in a soundproof
room and allowed to explore the environment for 2 min. Mice were
presented with a tone (85 dB, 2800 Hz) for 30 s, that co-terminatedwith a foot shock [0.5 mA, 1 s]. 24 h after training, context fear
memory was assessed by placing mice in the training context
and freezing was assessed for 5 min. 24 h later, discrete cue fear
memory was assessed by placing mice in a novel context. 2 min later the tone was played for 3 min. Percentage of time mice spent
freezing (lack of movement except respiration (Fanselow, 1980))
was recorded by a video camera, assessed using FreezeFrame andFreezeView (ActiMetrics) software, and used as our index of fear
memory.

D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

2.4. Open ﬁeld
The open ﬁeld test was used to measure overall motor activity
and anxiety-like behavior. Each mouse was placed in the middle
of a Plexiglas box (45  45  20 cm), evenly lit from above, and allowed to explore the environment for 15 min. The position of themouse was recorded by an overhead video camera and analyzed
using LimeLight 2 software (ActiMetrics). This software divided
the open ﬁeld into 3 zones; Zone 1 = the outermost perimeter ofthe box, Zone 2 = the middle zone, and Zone 3 = center zone. The
overall distance travelled and percentage of time spent in each of
the three zones was assessed.
2.5. Auditory startle response
The acoustic startle reﬂex was tested using an SR-LAB startle
testing system (San Diego Instruments, CA, USA). Mice were placedin a Plexiglas testing cylinder (3.2 cm internal diameter). Acoustic
startle stimuli and prepulse stimuli were delivered via a high-frequency speaker, placed 15 cm from the testing cylinder. Background white noise was generated by a standard speaker. The
testing cylinder was mounted on a sensor platform. A piezoelectric
accelerometer, attached to the base of the sensor platform, detected and transduced cage movements that were then digitizedby and stored in a computer. Startle amplitude was taken as the
maximal response that occurred in 100 ms after presentation of
the startle stimulus. The sound levels for background noise andstartle/prepulse stimuli were calibrated with a digital sound level
meter. The speakers, testing cylinder and sensor platform werehoused within a sound-attenuated chamber.
2.5.1. Habituation of the acoustic startle response
Mice were placed in the testing cylinder and 5 min later, presented with 80 startle pulses of 120 dB each (15 s interstimulusinterval, ISI).
2.5.2. Threshold of the acoustic startle response
The following day, the startle threshold for each mouse wasdetermined. Following a 5-min acclimation period, mice were presented with a total of 99 trials (15 s ISI). There were 11 trial types:
no stimulus (NS), and 10 types of startle trials in which the intensity of the startle stimulus randomly varied from 75 to 120 dB(with 5 dB increments). The startle stimuli were 40 ms noise bursts
with a rise/fall time of less than 1 ms. The 11 trial types (NS, startle
stimuli) were presented in a pseudorandom order such that eachtrial type was presented once within a block of 11 trials. Startle
threshold was deﬁned as the minimal intensity at which responding was signiﬁcantly greater than in the NS trials.
2.5.3. Prepulse inhibition of the acoustic startle response
Prepulse inhibition refers to the phenomenon in which a response to a strong startling stimulus is weakened or inhibited if
preceded by a weaker stimulus. A reduction or inhibition of a startle response by a prepulse is thought to reﬂect the ability of anorganism to temporarily change responding to changing circumstances. A disruption of prepulse inhibition is thought to reﬂect
an inability to ﬁlter non-relevant stimuli.
24 h after determining the startle threshold, mice were tested
for prepulse inhibition of the startle response. Following a 5-min
acclimation period, mice were presented with 20 habituation trials(120 dB, ISI 15 s). In the prepulse inhibition phase, mice were presented with a total of 90 trials. Three prepulse intensities were
tested: 70, 75 and 80 dB. Prepulse stimuli were 20 ms in duration
with a rise/fall time of less than 1 ms. For each prepulse intensity,
there were three types of trials: prepulse alone, prepulse + startle
stimulus, and startle stimulus alone. In the prepulse + startle stim-

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ulus trial, onset of the prepulse preceded onset of the startle stimulus by 100 ms. All startle stimuli were presented in a
pseudorandom sequence with the constraint that each stimulus
intensity occur only once in each consecutive four-trial block.
The percentage prepulse inhibition (%PPI) was calculated permouse for each of the three prepulse conditions using the formula%PPI = [1 – (startle amplitude elicited by prepulse + startle stimulus)/(startle amplitude elicited by startle stimulus alone)]  100.
A high %PPI score indicates more sensorimotor gating.
2.6. Statistical analyses
Data were analyzed with 1 and 2-way analyses of variance (ANOVAs) using Statistica (Statsoft) software. For water maze, we analyzed escape latency during training and the time spent in Target
versus Other Zones during water maze probe trials. For fear conditioning, we compared the time spent freezing in the context and to
the tone. Newman–Keuls post hoc tests were performed as
appropriate.
3. Results
3.1. Spatial memory formation is impaired in Jag1+/ mice
To assess spatial learning and memory in adult Dll1+/ , Lfng+/ ,Dll1+/ /Lfng+/ and Jag1+/ mice, we used the hidden platform version of the Morris water maze, a task critically dependent on hippocampal function (Morris, 1984). We compared performance ofthe mutant mice to their respective WT littermates. Mice weretrained to ﬁnd a hidden platform submerged below the water surface, located in a ﬁxed position throughout training (6 days of
training with 2 blocks of 3 trials/day). We examined spatial memory formation in a probe test conducted at the end of training during which the platform was removed from the pool.
3.1.1. Intense water maze training protocol (6 days)
3.1.1.1. Training. All groups, regardless of line (Dll1, Lfng, Dll1/Lfng,
Jag1) or genotype (WT, HET), showed decreased latencies to ﬁnd
the platform over the course of training (Fig. 1, left panel). This
interpretation was supported by the results of a series of ANOVAs
conducted on each line of mice [with between-groups factor Genotype (HET, WT) and within-groups factor Days (6)] (Table S1).
Brieﬂy, in the Dll1 line, both Dll1+/ mice (HET) and their WT littermate controls showed decreased latencies to ﬁnd the platform overtraining days, although Dll1+/ mice showed longer escape latencies overall than WT mice (Fig. 1B). In the Lfng line, both HET
and WT mice showed decreased escape latencies over training
days that did not differ (Fig. 1C). A similar pattern of results wasobserved in the Dll1/Lfng and Jag1 lines of mice; both Dll1+/ /Lfng+/ and Jag1+/ mice showed a decrease in latency over days
that did not differ from WT littermate mice (Fig. 1D and E). Therefore, in general, all mice, regardless of line or genotype, located theplatform faster over training days (with the exception of Dll1+/
which showed a decrease in latency over days, but were slower
than their WT littermates). Importantly, these various mutationsin the Notch signaling pathway did not affect swim speed or thigmotaxis during training [no effect of Genotype (HET versus WT) in
each line, data not shown].
3.1.1.2. Probe test. To assess spatial memory formation, we conducted a probe test (during which the platform was removed fromthe pool) 24 h after the ﬁnal training trial. We quantiﬁed theamount of time mice spent searching in the target zone (20 cm radius, centered on the location of the platform during training; 11%of the total pool surface) versus the time spent in an average of42

D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

Fig. 1. Spatial memory formation is impaired in Jag1+/ mice only. (A) Schematic representation of the water maze pool showing Target (T) and Other (O) zones. (B–E) Escapelatency during intense water maze training (6 days). (B) Dll1+/ mice (n = 23) required more time to reach the platform than their WT littermates (n = 25). There was nodifference in escape latency between, (C) Lfng+/ (n = 15) and WT (n = 14), (D) Dll1+/ /Lfng+/ (n = 8) and WT/WT (n = 11), (E) Jag1+/ (n = 11) and WT (n = 19) mice. (F–I) Spatial
memory formation as assessed in a probe trial. (F) Dll1+/ , (G) Lfng+/ and (H) Dll1+/ /Lfng+/ mice performed similarly to their WT littermates in the probe trial, spending moretime in the Target versus the Other zones of the pool (indicating robust spatial memory formation). (I) Jag1+/ mice spent less time in the Target zone than their WT controls,
indicating a spatial memory deﬁcit. Data represent mean ± SEM.p < 0.05.

D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

three other equivalent zones located in different quadrants of thepool (see Fig. 1A). We conducted an ANOVA with a between-subjects factor of Genotype (WT, HET) and a within-subjects factor ofZone (Target, Other) for each line of mice (Table S1).
In general, most lines of mice (Dll1, Lfng, Dll1/Lfng mice), spentsigniﬁcantly more time in the Target zone compared to the Otherzones of the pool, indicating formation of spatial memory. Furthermore, in these lines, there was no difference in the time spent in
the Target zone between HET and WT mice (Fig. 1F–H). That is,although there was a signiﬁcant effect of Zone, there was no significant effect of Genotype or Zone X Genotype interaction (see Table S1
for full statistical details). The exception to this general ﬁnding was
Jag1+/ mice. Jag1+/ mice spent signiﬁcantly less time in the Target
zone than their WT littermates (Fig. 1I) (Signiﬁcant Genotype X
Zone interaction [F(1, 28) = 8.1, p < 0.05], as well as signiﬁcant main
effects of Zone [F(1, 28) = 120.3, p < 0.001] and Genotype[F(1, 28) = 6.4, p < 0.05]). Post-hoc Newman–Keuls analysis of thesigniﬁcant interaction revealed that Jag1+/ mice spent signiﬁcantly less time in the Target zone than their WT littermates(p < 0.05). These results suggest that although Jag1+/ mice formed
a spatial memory, this memory was weaker than the memory
formed by their WT littermate controls.
3.1.2. Weak water maze training protocol (3 days)
To further investigate the spatial memory deﬁcit observed in
Jag1+/ mice, we trained a separate cohort of mice using a weakertraining protocol (2 blocks of 4 trials/day over 3 days). As with theintense training protocol, both Jag1+/ and WT mice showed a decrease in latency to ﬁnd the platform over the course of training(Fig. 2A). However, Jag1+/ mice took signiﬁcantly longer to reachthe platform than their WT littermate controls (Fig. 2A). An ANOVA
revealed signiﬁcant effects of Genotype [F(1, 29) = 6.8, p < 0.05] andDay [F(2, 58) = 124.1, p < 0.001], but no signiﬁcant Genotype by Day
interaction [F(2, 58) = 0.5, p > 0.05].
During the probe test, Jag1+/ mice spent less time in the Target
zone compared to their WT littermates (signiﬁcant Genotype by

43

Zone interaction [F(1, 29) = 7.7, p < 0.05], as well as signiﬁcant main
effects of Genotype [F(1, 29) = 8.0, p < 0.05] and Zone [F(1, 29) = 49.3,
p < 0.001]. Newman–Keuls post hoc analysis conducted on the signiﬁcant interaction showed that Jag1+/ mice spent signiﬁcantly
less time in the Target zone than WT mice (p < 0.001) (Fig. 2B).
Importantly Jag1+/ mice showed intact performance in a visible
platform version of the water maze (Fig. 2C, Table S1). Taken together, these ﬁndings indicate that reduced expression of Jag1
(but not Dll1 or Lfng) impaired spatial memory formation.
3.2. Fear memory formation is intact in all lines of mice
We next investigated the role of Notch signaling in different
memory tasks by examining these mouse lines in cued and contextual fear conditioning. We trained mice in a single conditioning
session in which a tone (conditioned stimulus, CS) was paired witha shock (unconditioned stimulus, US). 24 h later, mice were returned to the context in which they were previously shocked andthe amount of time spent freezing was measured (context fear).
Cued fear memory was assessed 24 h later when mice were placedin a novel context and the tone was presented. The amount of timespent freezing before and during the tone CS was measured (tonefear).
Across all lines of mice, both WT and HET showed equal and robust levels of freezing during both the context (Fig. 3A–D) and tone
(Fig. 3E–H) fear memory tests. An ANOVA conducted on each line
revealed no signiﬁcant differences between genotypes (p’s > 0.05,
Table S2). Together, these results indicate that decreasing the levels of the Notch ligands or Fringe does not impact either contextual
or cued fear conditioning.
3.3. Open ﬁeld behavior is normal in Dll1+/ , Lfng+/ and Jag1+/ mice
We next examined the effects of disrupting Notch signaling on
overall locomotor activity and anxiety-like behavior by assessing
the behavior of mice in a brightly-illuminated open ﬁeld. First,

Fig. 2. Impaired spatial memory in Jag1+/ mice was conﬁrmed using weak water maze training protocol. (A) Escape latency of Jag1+/ mice during weak water maze training(3 days). HET mice (n = 17) required more time to reach the platform than WT controls (n = 14). (B) In the probe trial, HET mice spent less time searching in Target (T) zone
than WT mice, indicating a spatial memory deﬁcit. (C) Escape latency of Jag1+/ mice trained in visible platform version of the water maze was not different in HET (n = 5)
versus WT (n = 8) mice, indicating that the spatial memory deﬁcit observed in Jag1+/ mice cannot be explained by a performance deﬁcit. Data represent mean ± SEM.
p < 0.05.

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D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

Fig. 3. Normal context and tone fear memory in all lines of Notch mutants. Freezing levels in context (A–D) and tone (CS) (E–H) fear memory tests. (A) There was nodifference in percentage of time spent freezing between Dll1+/ (n = 11) and WT (n = 19), (B) Lfng+/ (n = 9) and WT (n = 16), (C) Dll1+/ /Lfng+/ (n = 8) and WT/WT (n = 11), (D)Jag1+/ (n = 13) and WT (n = 36) mice in the context memory test. (E–H) All mouse lines showed similar freezing levels in tone fear memory test compared to WT mice, both
before (pre-CS) and during (CS) the tone. Data represent mean ± SEM.

we assessed the total distance travelled. We observed no differencein overall distance travelled in each line (HET versus WT), except in
Dll1+/ /Lfng+/ mice (Fig. 4B–E, Table S3). Dll1+/ /Lfng+/ mice traveled further than their WT/WT littermate controls [F(1, 16) = 9.2,
p < 0.05] (Fig. 4D). Importantly, the locomotor activity of Jag1+/
mice (the only line of mice to show impaired spatial memory formation) was comparable to their WT littermates (p > 0.05, Fig. 4E).
Second, as anxious rodents tend to avoid exposed spaces (Prut &Belzung, 2003), we also examined the relative time mice spent ininner versus outer zones of an open ﬁeld. Overall, most lines ofmice showed no evidence of an anxiety-like phenotype, spending
the majority of time in the outer zone of the open ﬁeld [similar
to their WT littermates] (Fig. 4F–I, Table S3). The one exception
was Dll1+/ /Lfng+/ mice, which spent less time in outer zone thantheir WT littermates (Fig. 4H). An ANOVA conducted on time spentin each zone [zone 1, outer; zone 2, intermediate; zone 3, central](see Fig. 4A), for this line yielded a signiﬁcant Genotype by Zone
interaction [F(2, 32) = 11, p < 0.001] and a signiﬁcant effect of Zone
[F(2, 32) = 308.7, p < 0.001], but no signiﬁcant main effect of Genotype [F(1, 16) = 2.6, p > 0.05]. Newman–Keuls post hoc analysis conducted on the signiﬁcant interaction revealed that Dll1+/ /Lfng+/
mice spent signiﬁcantly less time in the outer zone (zone 1,
p < 0.001) and signiﬁcantly more time in the intermediate zone
(zone 2, p < 0.05), but similar time in the central zone (zone 3,
p < 0.05) compared to their WT/WT littermates (Fig. 4H). Together,
these ﬁndings suggest that Dll1+/ /Lfng+/ mice may be less ‘‘anxious’’ than their WT/WT littermates (hyperactive, spending lesstime in the outer zone of the open ﬁeld). On the other hand,D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

45

Fig. 4. Open ﬁeld behavior is normal in all mice except Dll1+/ /Lfng+/ . (A) Schematic representation of the open ﬁeld. (B–E) Total distance traveled by each group of mice inthe open ﬁeld. There was no difference in total distance traveled by, (B) Dll1+/ (n = 14) and WT (n = 23), (C) Lfng+/ (n = 9) and WT (n = 16), (E) Jag1+/ (n = 16) and WT mice(n = 39). (D) Dll1+/ /Lfng+/ mice (n = 8) traveled a greater distance compared to WT/WT controls (n = 10). (F–I) Amount of time mice spent in each zone of the open ﬁeld. (F)Dll1+/ , (G) Lfng+/ , and (I) Jag1+/ mice spent comparable amounts of time relative to their WT littermates in each zone of the open ﬁeld. (H) Dll1+/ /Lfng+/ mice spent lesstime in zone 1 (outer zone) and more time in zone 2 (intermediate zone) than their WT/WT controls, suggesting that these mice might be less anxious. Data represent
mean ± SEM. p < 0.05. p < 0.001.

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D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

Jag1+/ mice showed normal locomotor activity and anxiety-likelevels in these tests (Fig. 4E and I), indicating that the impairment
in spatial memory formation observed in these mice cannot be
attributed to non-speciﬁc motor effects or an increase in anxietylike behavior.
3.4. Sensorimotor gating
Cognitive/memory impairments in many psychiatric conditions
may stem from an inability to ﬁlter distracting, trivial or non-salient stimuli (Braff et al., 2001). One way to assess this process of
sensorimotor gating is via prepulse inhibition (PPI) of the acoustic
startle reﬂex. Startle responses are evoked in every mammalian
species studied (including humans and mice) by loud, unexpected
noises. Furthermore, the startle reﬂexes exhibited by humans andmice share a number of parametric characteristics (Hoffman &Ison, 1980; Ornitz, Guthrie, Kaplan, Lane, & Norman, 1986). To
tap into the process of ﬁltering irrelevant stimuli (sensorimotor
gating), we assessed prepulse inhibition of the startle response ineach line of mice. Prepulse inhibition measures the ability to inhibit startle response when the startle-eliciting stimulus is precededby a non-startle eliciting prepulse stimulus (Braff & Geyer, 1990;
Graham, 1975; Hoffman & Ison, 1980; Ison & Hammond, 1971).
3.4.1. Habituation of the acoustic startle response
Habituation is a form of non-associative learning in which the
response to a stimulus is decreased over successive presentations(Koch, 1999). To examine habituation of the acoustic startle response in our lines of mice, we presented them with 80 trials of
the same startle-inducing stimulus (120 dB white noise burst).
Over trials, all lines of mice showed decreased startle responding (habituation). To analyze these data, we divided the trials into
4 blocks (trials 1–20, 21–40, 41–60, 61–80) and conducted an ANOVA with between-subjects factor Genotype (WT, HET) and withinsubjects factor Trial Block (Table S4). All lines of Notch mutant micetested (both HET and WT littermates) showed decreased startle
reactivity in response to the repeated presentations of startle stimulus over blocks, indicating normal habituation (Fig. 5A–D). Furthermore, habituation did not differ between WT and HET micefor any of the genotypes (Table S4). Therefore, habituation to astartling-stimulus was not disrupted in these Notch mutants.
3.4.2. Acoustic startle threshold
Next, we examined intensity of the acoustic stimuli required toelicit a startle response (threshold for acoustic startle response)(Koch, 1999). Mice were given acoustic stimuli of random intensities (0, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 dB) and the average response to each intensity of startle stimulus was measured. As
expected, all lines of mice showed increasing startle amplitudewith increasing stimulus intensity, yielding a threshold of startle
responding of roughly 95 dB (Fig. 5E–H). However, in some lines,
the startle response was altered. In the Dll1 line, HET mice showed
higher levels of startle responding to some acoustic stimuli. An ANOVA revealed a signiﬁcant Genotype by Intensity interaction[F(10, 240) = 1.9, p < 0.05], but no signiﬁcant main effect of Genotype [F(1, 24) = 2.1, p > 0.05]. Post-hoc Newman–Keuls analysis revealed that Dll1+/ mice showed a greater startle response tostimuli of 100 and 105 dB (p’s < 0.001) only over their WT littermates (Fig. 5E). In contrast, both Lfng+/ and Lfng+/ /Dll1+/ mice
showed a blunted startle response compared to their WT controls.
For Lfng mice, the results of an ANOVA revealed a signiﬁcant Genotype by Intensity interaction [F(10, 230) = 4.1, p < 0.001], and significant main effects of Genotype [F(1, 23) = 4.5, p < 0.05] and Intensity[F(10, 230) = 47.3, p < 0.001]. Newman–Keuls analysis showed that
Lfng+/ mice responded less to stimuli of 105 (p < 0.05), 110(p < 0.05), 115 (p < 0.001) and 120 dB (p < 0.05) (Fig. 5F). For

Dll1+/ /Lfng+/ mice, the ANOVA showed a signiﬁcant Genotype byIntensity interaction [F(10, 170) = 2.3, p < 0.05] but no signiﬁcant effect of Genotype [F(1, 17) = 1.8, p > 0.05]. The post hoc analysis further revealed that at intensities of 115 (p < 0.05) and 120 dB(p < 0.05), HET/HET mice showed decreased startling responsecompared to WT/WT mice (Fig. 5G). In contrast to these groups,
Jag1+/ mice performed comparably to their WT littermates at all
intensities tested (p > 0.05, Fig. 5H). Therefore, some lines of Notch
mutants showed disruptions in the startle response.
3.4.3. Prepulse inhibition of the acoustic startle response
To measure PPI, we presented mice with a startle stimulus of
120 dB (pulse) preceded 100 ms by a low intensity (non-startle
eliciting) stimulus (prepulse, 70, 75 and 80 dB). We compared
the startle response of each mouse on startle alone versus prepulse
trials, thus negating any possible effect of the mutation on startle
alone. In all lines tested, startle reactivity decreased with increasing prepulse intensity, suggesting that all mice were able to gate
their response (Fig. 5I–L). That is, PPI increased with prepulse
intensity in all lines tested. An ANOVA conducted on the Percent
PPI of each line of mice [between-subjects factor Genotype (WT,HET), within-subjects factor Prepulse Intensity [70, 75, 80 dB](Table S4)], revealed that only the Dll1/Lfng line showed a difference depending on genotype. That is, PPI was higher in Dll1+/ /Lfng+/ mice compared to their WT/WT littermates, across all
pre-pulse intensities (signiﬁcant effect of Genotype [F(1, 17) = 5.2,
p < 0.05] and Intensity [F(2, 34) = 47.5, p < 0.001], but no signiﬁcant
Genotype by Intensity interaction [F(2, 34) = 1.9, p > 0.05]). Therefore, all HET mice except Dll1+/ /Lfng+/ , showed comparable levelsof PPI to their WT littermates.
4. Discussion
Components of the Notch pathway are widely expressed during
embryonic development and, in many cases, their absence has lethal
consequences. Although Notch signaling persists in the adult brain,
its precise role is unknown. A number of studies have implicated theNotch pathway in learning and memory in the adult brain. For instance, the types of neuronal activity associated with memory formation and increased expression of the activity-dependent gene
Arc (a gene important for memory formation), also engage the Notch
signaling pathway (Alberi et al., 2011). Interestingly, this activitydependent increase in neuronal Notch activation requires Arc (Alberi
et al., 2011). Furthermore, the Notch1 receptor is crucial for learningand memory in adults (Costa et al., 2003; Ge et al., 2004; Presente
et al., 2004). However, little is known about how different ligands
and/or Fringe proteins contribute to memory formation. We useddifferent lines of mice heterozygous for loss-of-function mutations
in two principal Notch1 ligands (Dll1, Jag1) and Lfng, to examine
speciﬁc roles for each of these Notch components in adult memory
formation. Since homozygous null mutants of Dll1 and Jag1 areembryonically lethal (Hrabe de Angelis et al., 1997; Xue et al.,1999) and those of Lfng have severe skeletal malformations (Evrard,
Lun, Aulehla, Gan, & Johnson, 1998; Zhang & Gridley, 1998), we took
advantage of mice heterozygous for each gene. The heterozygous
mutant mice developed normally and survived until adulthood.
Here, we show that decreased Jag1 expression is associated withspatial memory impairment. Relative to WT mice, Jag1+/ mice
showed spatial memory deﬁcits following both strong and weaktraining in the water maze. This deﬁcit cannot be attributed tonon-speciﬁc deﬁcits given that motor activity, anxiety-like behavior,and sensorimotor gating were all normal in these mice. Moreover,hippocampal-dependent non-spatial memory formation (contextual fear memory) was intact in these mice. Since an independently
developed Jag1 mutant mouse line has been reported to display eye

D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

47

Fig. 5. Sensorimotor gating was normal in all Notch mice except Dll1+/ /Lfng+/ . (A–D) Habituation of the acoustic startle response (shown in blocks of 20 trials) are similar
between (A) Dll1+/ (n = 11) and WT (n = 15), (B) Lfng+/ (n = 9) and WT (n = 16), (C) Dll1+/ /Lfng+/ (n = 8) and WT/WT (n = 11), (D) Jag1+/ (n = 16) and WT (n = 39) mice. (E–H)
Startle threshold levels were assessed by measuring the startle response to startle stimuli with intensities of 0–120 dB. (E) Dll1+/ mice showed enhanced startle reactivity
above stimulus intensities of 100 and 105 dB, compared to WT mice. (F) Lfng+/ mice showed decreased startle reactivity at 105, 110, 115 and 120 dB stimuli compared totheir WT littermates. (G) At intensities of 115 and 120 dB, Dll1+/ /Lfng+/ mice showed lower reactivity than WT/WT mice. (H) Startle threshold levels of Jag1+/ mice werecomparable to WT littermates. (I–L) Prepulse inhibition (%PPI) in each mouse line was assessed. PPI was tested using prepulse intensities of 70, 75 and 80 dB. No signiﬁcantdifference was observed in PPI between (I) Dll1+/ and WT, (J) Lfng+/ and WT, (L) Jag1+/ and WT mice. (K) Dll1+/ /Lfng+/ mice showed enhanced PPI at all intensities tested
compared to WT/WT mice. Data represent mean ± SEM.

dysmorphology during development (Xue et al., 1999), we carriedout a visible platform version of the water maze. Jag1+/ mice
showed a comparable performance to their WT littermates in thevisible platform indicating the decreased performance in water

maze could not be attributed to visual defects. A remarkably similar
proﬁle of spatial (but not contextual) memory deﬁcits was also reported in Notch1+/ and RBP-J+/ mice (Costa et al., 2003) and micein which Notch1 was conditionally deleted in excitatory forebrain

48

D. Sargin et al. / Neurobiology of Learning and Memory 103 (2013) 39–49

neurons (Alberi et al., 2011). Together, these data suggest that achronic reduction in Jag1-Notch signaling produce speciﬁc deﬁcits
in spatial memory formation.
It is interesting to note that Jag1 mRNA is expressed at higher
levels than Dll1 in the postnatal hippocampus (Breunig et al.,2007; Stump et al., 2002). Although these studies suggest that
Jag1 may be the main Notch ligand in the adult hippocampus, consistent with its importance for spatial memory formation, a detailed analysis of Notch ligand expression throughout
development and in the adult brain will be important. In our study,
we cannot rule out compensatory effects of wild type alleles in heterozygous knockout mice. However, our ﬁnding that Jag1-Notch
signaling is important for hippocampal-dependent spatial memory
is consistent with an emerging role for Jag1 in hippocampal synaptic plasticity (Alberi et al., 2011; Wang et al., 2004). In summary,our data provide the ﬁrst in vivo evidence that Jag1-induced Notch
signaling is critical for hippocampal-dependent memory formation. Our ﬁnding agrees with several previous studies. First, application of exogenous Jag1 peptide to hippocampal slices from micewith reduced Notch levels was sufﬁcient to rescue the hippocampal LTP deﬁcit (Wang et al., 2004). Second, Jag1 has been localized
to synapses of CA1 hippocampal neurons and is upregulated in response to neuronal activity (Alberi et al., 2011).
Acknowledgements
We thank Mika Yamamoto, Russell Braybon and Antonietta(Toni) DeCristofaro for excellent technical assistance. This work
was supported by grants from the Canadian Institutes of Health Research [CIHR; MOP-74650 (SAJ), MOP-86762 (PWF)], EJLB Foundation (SAJ) and Natural Science and Engineering Research Council(NSERC, SAJ). DS received support from the Alzheimer’s Society ofCanada and LB received support from Restracomp (Hospital for
Sick Children).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.nlm.2013.03.001.
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