Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

RESEARCH

Open Access

Impaired spatial and contextual memory
formation in galectin-1 deficient mice
Masanori Sakaguchi1,2,3, Maithe Arruda-Carvalho1,3, Na Hyea Kang1, Yoichi Imaizumi4, Françoise Poirier5,
Hideyuki Okano4 and Paul W Frankland1,2,3*

Abstract
Galectins are a 15 member family of carbohydrate-binding proteins that have been implicated in cancer, immunity,
inflammation and development. While galectins are expressed in the central nervous system, little is known about
their function in the adult brain. Previously we have shown that galectin-1 (gal-1) is expressed in the adulthippocampus, and, in particular, in putative neural stem cells in the subgranular zone. To evaluate how gal-1 might
contribute to hippocampal memory function here we studied galectin-1 null mutant (gal-1-/-) mice. Compared totheir wildtype littermate controls, gal-1-/- mice exhibited impaired spatial learning in the water maze andcontextual fear learning. Interestingly, tone fear conditioning was normal in gal-1-/- mice suggesting that loss ofgal-1 might especially impact hippocampal learning and memory. Furthermore, gal-1-/- mice exhibited normal
motor function, emotion and sensory processing in a battery of other behavioral tests, suggesting that nonmnemonic performance deficits are unlikely to account for the spatial and contextual learning deficits. Together,
these data reveal a role for galectin-carbohydrate signalling in hippocampal memory function.
Introduction
The biological actions of carbohydrate molecules are
mediated, in part, by interactions with lectins which
recognize carbohydrate structures and bind to their specific sequences [1,2]. Galectin-1 (gal-1) is a family member of a specific class of lectins, galectins, which regulate
various biological functions through binding to lactosamine containing carbohydrate molecules [3-5]. Whileprevious cell culture studies have identified a role forgal-1 in cell death, cell adhesion and neurite outgrowth[5,6], few studies have studied the role of this lectin incentral nervous system in vivo [7-11].
Previously we have shown that gal-1 is expressed inneural stem cells (NSCs) and regulates neurogenesis in
the adult mouse brain [7-10]. In the subventricular zone
(SVZ), loss of gal-1 leads to reduced adult neurogenesis,suggesting that gal-1 usually promotes proliferation of
the adult SVZ NSCs [7,9]. Consistent with these findings, we have found that administration of gal-1 promotes adult SVZ neurogenesis and functional recovery
following ischemic brain injury [8,9]. Conversely,* Correspondence: paul.frankland@sickkids.ca
1Program in Neurosciences and Mental Health, The Hospital for Sick
Children, Toronto, Canada, M5G 1X8Full list of author information is available at the end of the article

inhibiting gal-1 blocks ischemia-induced upregulation of
SVZ neurogenesis and associated functional recovery[8]. Similarly, gal-1 potentiates the therapeutic effects oftransplanted NSCs on recovery from spinal cord injury
in non-human primates [12].
Gal-1 is also expressed in putative NSCs in the subgranular zone (SGZ) of the hippocampus [10,13]. However, in contrast to SVZ neurogenesis, we found thatloss of gal-1 led to increased levels of SGZ neurogenesis
in adult mice in a C57BL/6 background [10], suggestingthat gal-1 may usually down-regulate neurogenesis in
the adult hippocampus. As the hippocampus plays acentral role in learning and memory, this raises the possibility that gal-1 contributes to behavioral plasticity
either via neurogenic or non-neurogenic (as gal-1 is also
expressed in mature neurons in the CA1 and CA3
regions of the hippocampus) mechanisms. To evaluate
this possibility here we characterize gal-1-/- mice in arange of learning and memory tasks, and we find thathippocampus-dependent contextual and spatial learning
is deficient in these mice. These experiments reveal animportant role for gal-1 in hippocampus-dependent
learning and memory, and, more generally, represent a
first step toward understanding how lectin-carbohydratesignalling contributes to hippocampal memory function.

© 2011 Sakaguchi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

Results
gal-1 is expressed in neurogenic and non-neurogenic
regions in the hippocampus

In order to characterize gal-1 expression in the adult hippocampus, we conducted a series of immunohistochemical analyses in adult wild-type (WT) mice. We firstexamined the specificity of our gal-1 antibody by staining
WT vs. gal-1-/- mice (Figure 1A-B). In the hippocampus(and elsewhere in the brain) we found no evidence ofstaining in gal-1-/- mice [9,10], suggesting that this antibody binds only gal-1, and not, for example, other members of the galectin family which exhibit structuralsimilarity in their lectin-binding domain [5,14,15]. In WT
mice, gal-1 staining was detected in all three major subdivisions of the hippocampus (the dentate gyrus (DG), CA3
and CA1) (Figure 1A). In the DG, as shown before [10],we found NeuN-positive cells expressing gal-1 only inthe hilus, suggesting that gal-1 is not expressed in matureneurons in the granule cell layer. In the granule cell layer,
gal-1 expression was limited to GFAP-positive cells (Figure 1C-D), suggesting that gal-1 is localized to astrocytes
and/or putative neural stem cells, as we have previously
reported [10]. Within the CA3 region, the vast majorityof cells expressing gal-1 were NeuN-positive, but never
GFAP-positive (Figure 1E-F), indicating that gal-1 isexpressed in mature neurons. Within the CA1 region,
gal-1-expressing cells were either NeuN-positive (suggesting that they were mature neurons) or both NeuNnegative and GFAP-negative (Figure 1G-H). Based onmorphology and localization pattern (flattened and semilunar cell body surrounding blood vessels (Figure 1G,
arrowhead)), this latter class of cell is most likely to beendothelial cells of blood vessels as previously described
elsewhere [16].
gal-1 null mutant mice have deficient contextual
fear memory

Our immunohistochemical analyses indicated that gal-1
is expressed in the adult hippocampus and, in particular,in putative NSCs in the DG [10]. Given the role of the
hippocampus, and, in particular, hippocampal neurogenesis, in learning and memory [17-20], we next tested gal1-/- mice in a range of learning paradigms that depend on
hippocampal function. To do this we first used a fear
conditioning paradigm, in which a tone is paired with amild foot shock in a novel context (Figure 2A). Whenreplaced in the original training context or presented
with the tone in an alternate context, mice exhibit arange of species-typical behaviors including freezing (thecessation of all but respiratory-related movement)[21].
During training, gal-1-/- and WT littermate control mice
showed similar reactivity to the shock (Figure 2B,
unpaired t-test: t(19) = 1.49, P = 0.08), indicating that the

Page 2 of 10

mutant mice were able to sense the foot shock normally.
Twenty-four hours later, freezing in the original training
context was reduced in gal-1-/- mice compared to WT littermate controls (Figure 2C, unpaired t-test: t(24) = 1.75,
P < 0.05). In contrast, WT and gal-1 -/- mice exhibited
equivalent freezing when presented with the tone in analternate context (Figure 2D; time × genotype ANOVA,
no main effect of genotype F(1, 21) = 0.16, P = 0.69, main
effect of time only F(4, 21) = 41.59, P < 0.001), exhibiting
similar levels of freezing before (planned comparison of
WT vs. gal-1-/- freezing; t(21) = 0.17, P = 0.43) and during (planned comparison of WT vs. gal-1-/- freezing; t(21) = 0.47, P = 0.32) tone presentation. As the formationof contextual, but not tone, fear memories depends on
hippocampal function [21], these results suggest that lossof gal-1 impairs the formation of hippocampus-dependent memory.
Intensive training overcomes contextual fear memory
deficits in gal-1-/- mice

We next asked whether more intensive training might
overcome these deficits in contextual fear memory in gal1 -/- mice. To do this, during training mice received 3
tone-shock pairings (Figure 3A). During training, freezing
increased incrementally, and levels were similar in both
WT and gal-1 -/- mice (Figure 3B, time × genotype
ANOVA, no main effect of genotype F(1, 20) = 0.65, P =0.43, main effect of time only F(4, 20) = 12.34, P < 0.001).
When placed back in the training context test one day
later, WT and gal-1-/- mice froze at similar levels (Figure
3C, unpaired t-test: t(20) = 0.49, P = 0.32) indicating that
more intensive training can overcome contextual fear
deficits in gal-1-/- mice. Furthermore, freezing in the tone
test was similar in both groups (Figure 3D, time × genotype ANOVA, main effect of time only F(4, 20) = 19.72,
P < 0.001), with similar levels of freezing before (planned
comparison; t(20) = 0.19, P = 0.43) and during (planned
comparison; t(21) = 1.14, P = 0.13) tone presentation in
WT and gal-1 -/- mice. These data indicate that more
intensive training may overcome these deficits in gal-1-/mice perhaps because other galectins may compensate
for the loss of gal-1 or other brain regions may berecruited to support learning when multiple shocks areused [22,23].
gal-1-/- mice have deficient spatial learning in the watermaze

The contextual fear conditioning deficits are consistent
with the idea that loss of gal-1 impacts hippocampal
learning and memory. To evaluate whether these deficits
generalize to another form of hippocampus-dependent
learning, we next trained mice in the hidden version of
the water maze [24,25]. During training, while latencies

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

Page 3 of 10

Figure 1 Characterization of gal-1 expression in adult mouse hippocampus. (A) In wild type hippocampus, gal-1 expression (red; arrows)
was found in the DG, CA3 and CA1. Blue = nuclei visualized by Hoechst 33258. (B) In gal-1-/- mice, the signal was completely absent indicating
the specificity of our gal-1 staining. (C) In the GCL of the DG, gal-1 signal was never co-localized in NeuN-positive cells. (D) In the GCL, the vast
majority of gal-1 positive cells were GFAP-positive (arrows). (E and F) In the CA3, gal-1 positive cells were always NeuN-positive (E, arrow), but
not GFAP-positive (F). (G and H) In the CA1, gal-1 positive cells were either NeuN-positive (G, arrow) or NeuN-negative/GFAP-negative (G,arrowhead, H). Scale bars: (A-B), 100 μm; (C-H), 5 μm.

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

Figure 2 Contextual fear memory deficits in gal-1-/- mice. (A)Experimental design used to fear condition WT (n = 11) and gal-1-/(n = 15) mice. (B) Shock reactivity in WT (black bar) vs. gal-1-/(white bar) mice. (C) Freezing in context test in WT (black bars) vs.
gal-1-/- (white bar) mice. (D) Freezing in tone test in context B (WT,closed circles; gal-1-/- mice, open circles). The tone was presented
after 120 s.

Page 4 of 10

to locate the hidden platform decreased in both groupsof mice, there was a strong tendency for longer latencies
in gal-1-/- mice (Figure 4A, time × genotype ANOVA,
main effect of time F(7, 27) = 22.89, P < 0.001, main
effect of genotype F(1, 27) = 4.05, P = 0.054). As the
adoption of either localized/spatially-precise (e.g., focal
searching) or some non-localized/spatially-imprecise (e.
g., chaining) search strategies may contribute to reduced
escape latencies across training [26-28], latency data canbe poor predictors of spatial memory [26]. To better
evaluate spatial memory, mice were given a probe testwith the escape platform removed from the pool. In thistest, both WT and gal-1 -/- mice searched selectively,
spending more time in the region of the pool that formerly contained the platform (target [T] zone) compared to other equivalent regions of the pool (average of
all other [O] zones) (Figure 4B, T > O for WT [planned
comparison: t(13) = 5.34, P < 0.001] and gal-1 -/[planned comparison: t(14) = 7.34, P < 0.001] mice).
However, the degree of selectivity was greater in WT
compared to mutants (Figure 4B, T WT > T gal-1-/- ;unpaired t-test: t(27) = 2.06, P < 0.05), indicating thatwhile gal-1-/- mice were able to form a spatial memory
it was nonetheless not as precise as in WT littermate
controls. Consistent with the fear conditioning analysis,
these results suggest that loss of gal-1 impairs hippocampal memory formation.
General behavior is not altered in gal-1-/- mice

Because gal-1 is also expressed outside of the hippocampus [11,29] we next evaluated whether other types of
behaviors are altered in gal-1-/- mice. First, mice weretrained in a visual version of the water maze, where theplatform location is marked by a cue. During training,
WT and gal-1-/- mice were equivalently efficient in finding the platform (Figure 5A, time × genotype ANOVA,

Figure 3 Normal contextual fear conditioning in gal-1-/- mice
after strong training. (A) Experimental design used to fear
condition (3 tone-shock pairs) WT (n = 10) and gal-1-/- (n = 12)
mice. (B) Freezing during training in WT (closed circles) and gal-1-/mice (open circles). (C) Freezing in context test in WT (black bars)
vs. gal-1-/- (white bar) mice. (D) Freezing in tone test in context B(WT, closed circles; gal-1-/- mice, open circles). The tone waspresented after 120 s.

Figure 4 Spatial learning deficits in gal-1-/- mice in the hidden
platform version of the water maze. (A) Escape latencies during
training for WT (n = 14; closed circles) and gal-1-/- (n = 15; open
circles) mice. (B) Probe test data showing percent time searchingtarget (T) zone (black bars) vs. average of three other equivalentzones (white bars) for WT and gal-1-/- mice (* < 0.05).

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

Page 5 of 10

Figure 5 General behavior in gal-1-/- mice. (A) Latencies to find platform for WT (n = 3; closed circles) vs. gal-1-/- mice (n = 3; open circles) in
the visible version of the water maze. (B) Latency to locate visible platform moved to opposite quadrant for WT (n = 3; black bar) and gal-1-/- (n= 3; white bar) mice. (C). Swim speed for WT (n = 3; closed circles) vs. gal-1-/- mice (n = 3; open circles) across training days in the visible versionof the water maze. (D) Distance travelled in open field for WT (n = 12; black circles) vs. gal-1-/- mice (n = 17; white circles). (E) Exploratory activity
in outer, middle and inner regions of an open field for WT (black bars) vs. gal-1-/- mice (white bars) in open field test. (F) Startle responses
evoked by different intensity noise bursts (0, 75-100 dB) for WT (n = 13; closed circles) vs. gal-1-/- mice (n = 8; open circles). (G) Prepulse
inhibition using three different intensity prepulses WT (n = 13; black bars) vs. gal-1-/- mice (n = 8; white bars).

no main effect of genotype F(1, 4) = 2.10, P = 0.22, main
effect of time only F(2, 8) = 39.63, P < 0.001). At theend of training, the platform and cue were moved to the
opposite quadrant, and mice were retested. As before,
latencies to reach the platform were equivalent in WT
and gal-1-/- mice (Figure 5B; unpaired t-test: t(4) = 0.01,
P = 0.50), suggesting that the ability to associate the cue
with the platform location is not compromised in gal-

1-/- mice. Furthermore, equivalent performance in thisvisual version of the water maze suggests that generalmotor function, vision and motivation are not altered ingal-1-/- mice. Consistent with this, swim speeds acrosstrials were similar in both groups (Figure 5C, time ×
genotype ANOVA, no main effect of genotype F(1, 4) =0.43, P = 0.55). Second, in an open field test, while gal1-/- mice were generally more active (Figure 5D, time ×

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

genotype ANOVA, main effects of genotype F(1, 27) =10.35, P < 0.01 and time F(8, 27) = 271.5, P < 0.001),
the spatial distribution of activity was similar in WT
and gal-1-/- mice suggesting that loss of gal-1 does notalter general anxiety levels (Figure 5E, zone × genotype
ANOVA, no main effect of genotype F(1, 27) = 2.10, P= 0.16, main effect of zone only F(2, 27) = 660, P <0.001). Third, sensory-motor function, assessed using astartle reflex paradigm [30], appears to be normal ingal-1-/- mice. Startle threshold (Figure 5F, 85 dB > 0 dB
for WT [P < 0.05], 90 dB > 0 dB for gal-1-/- [P < 0.05])
and magnitude (Figure 5F, intensity × genotype
ANOVA, no main effect of genotype F(1, 19) = 0.23, P= 0.64, main effect of intensity only: F(6, 114) = 36.88, P< 0.001) were similar in WT and gal-1-/- mice. Moreover, inhibition of the startle response by auditory prepulses was unaltered in gal-1 -/- mice (Figure 5G;
intensity × genotype ANOVA, no main effect of genotype F(1, 19) = 0.02, P = 0.90, main effect of intensity
only: F(2, 38) = 94.94, P < 0.001), suggesting that theprocessing of extraneous auditory stimuli is normal following loss of gal-1. Taken together, these results suggest that impaired spatial and contextual memory
formation in gal-1-/- mice cannot be attributed to nonspecific impact of this mutation on general behavior,emotion or sensory processing.

Discussion
While galectins are expressed in the adult nervous system, little is known about how they contribute to adultbrain function. Using a gal-1 null mutant mouse model,
here we explored the role of gal-1 in hippocampal learning and memory. We found that loss of gal-1 was associated with impaired learning in two hippocampusdependent tasks, contextual fear conditioning and spatial
learning in the water maze. Additional control experiments revealed no obvious changes in motor function,
emotion and sensory processing, suggesting that nonmnemonic performance factors cannot account for thelearning deficits in gal-1-/- mice. Together, these studies
reveal a role for gal-1, and, more broadly, lectin-carbohydrate signalling in hippocampus-dependent learningand memory.
Our behavioral analyses revealed that loss of gal-1
impaired two forms of hippocampal learning. However,
the mechanisms underlying these effects are unclear andthere are several possibilities that may be evaluated infuture studies. Previously we showed that gal-1 regulates
adult neurogenesis by regulating the proliferation ofneural stem cells [9]. As adult neurogenesis in the hippocampus very likely contributes to learning and memory [17,18], one possibility is that altered regulation of
adult neurogenesis is responsible for the observed learning deficits in gal-1 -/- mice. In gal-1 -/- mice, adult

Page 6 of 10

neurogenesis is increased in the hippocampus (butdecreased in the SVZ). However, more typically a reduction (rather than an increase) in adult neurogenesis has
been associated with impaired hippocampal memory (e.
g., [17,31-34], but see:[35]). This suggests that if there isa causal relationship between gal-1-related decreases inneurogenesis and the observed contextual and spatial
learning deficits it is unlikely to be straightforward.
Nonetheless, in gal-1 -/- mice it is possible that while
additional cells are generated, they do not integrate normally into hippocampal networks and such aberrant
integration might lead to disruption of hippocampal
memory function [36,37]. To explore whether such adominant-negative effect is plausible it will be necessary
to characterize the morphological maturation of theseneurons as well as their potential to become functionallyintegrated into hippocampal memory circuits [38-40].
An alternate possibility is that loss of gal-1 affects
synaptic plasticity in the hippocampus. For example, in
adult NSCs gal-1 promotes proliferation by binding b1
integrin [7]. As (i) gal-1 is also expressed in mature neurons in both CA3 and CA1 and (ii) b1 integrin signalling plays key roles in synaptic plasticity (e.g., LTP [41])and hippocampus dependent memory [42] possibly
through AMPA receptor trafficking and glycine receptor
lateral diffusion [43], it is possible that loss of gal-1
impacts synaptic plasticity mechanisms in mature hippocampal neurons. A third possibility is that hippocampal
wiring is altered in gal-1-/- mice. For example, we havepreviously shown that gal-1 promotes neurite extension
in neurons derived from human NSCs [12]. Since gal-1
can be externalized from the cell membrane, it is possible that the gal-1 expression in the developing hippocampus affects circuit formation, which could
compromise memory stability.

Conclusions
Our knowledge of how carbohydrate molecules exert
their biological actions is currently very limited [1,2].
This is in part due to the difficulty of identifying andmanipulating carbohydrate molecules in vivo. However,such limitations may be overcome by genetically targeting upstream enzymes responsible for production of carbohydrate structures or downstream binding partners
such as lectins which recognize and bind to specificstructural portion of carbohydrates [44,45]. These
approaches have revealed critical roles of carbohydrate
molecules in many different biological contexts in animals from fertilization to the patterning of neuronal wiring [44,45]. Adopting the latter approach, our behavioral
characterization of the gal-1 -/- mice represents a firststep towards understanding how lectin-carbohydrate signalling might contribute to hippocampal learning andmemory. They reveal a specific role for gal-1 in learning

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

and memory, and, as binding of gal-1 to several lactosamine derivatives has been well-characterized [14], futurestudies may focus on mechanisms underlying theseeffects.

Methods
Mice

We used gal-1-/- mice that had been backcrossed > 10
generations onto the C57BL6/J background, as described
previously (Imaizumi et al., 2011). For the study, 8 to
13-week old mice were used throughout the study andkilled by anesthetic overdose at the end of each experiment. We used wild type littermates mice as controls,
and similar numbers of male and female mice were usedin each experiment. Mice were maintained on a 12-h/
12-h light/dark cycle with unlimited access to food andwater. All the experiments were performed in accordance with guidelines and regulations of The Hospitalfor Sick Children, Animal Care and Use Committee.
Immunohistochemistry

Brains were perfusion-fixed with 4% paraformaldehyde(PFA), postfixed in the same fixative overnight, and thencut into 50-μm sections on a vibratome (Leica). After
three rinses in PBS, the sections were incubated withprimary antibodies overnight, and then incubated for 60
min at room temperature with the Fab2-portion of secondary antibodies (1:500; Jackson ImmunoResearch)
conjugated with HRP or biotin (Jackson ImmunoResearch). The biotin or HRP-conjugated antibodies werevisualized using TSA (Pharmingen) with or without the
Vectastain Elite ABC kit (Vector Laboratories). For
multi-color labeling, the potential for the cross-reactivityof the secondary antibodies with off-target primary antibodies was carefully tested and excluded by using theappropriate controls (e.g., parallel staining without oneof the primary antibodies). The primary antibodies (final
dilution and source) used in this study were as follows:
goat anti-Galectin-1 (1:200, R&D Systems), mousemonoclonal anti-GFAP (1:200, Sigma); mouse monoclonal anti-NeuN (1:100, Chemicon). All representative
images were acquired using epifluorescent (Nikon
Eclipse 80i) or confocal (LSM 510 Zeiss) microscopes.
To calculate the proportion of double-labeled cells, confocal 1 μm Z-stack images were obtained using ZEN
software (Zeiss, Germany) with a minimal interval of 15
μm to prevent duplicate counts of the same cell.
Fear conditioning

The apparatus and behavioral procedures have been previously described [46]. In the fear conditioning experiments, two contexts were used. Context A consisted ofa conditioning chamber (31 cm × 24 cm × 21 cm; Med
Associates, St. Albans, VT), containing a stainless steel

Page 7 of 10

shock-grid floor. Shock grid bars (diameter 3.2 mm)
were spaced 7.9 mm apart. The grid floor was positioned over a stainless-steel drop-pan, which was lightly
cleaned with 70% ethyl alcohol to provide a backgroundodor. The front, top, and back of the chamber weremade of clear acrylic and the two sides made of modular
aluminum. For context B, a white, plastic floor covered
the shock grid bars and a plastic, triangular insert wasplaced inside the same conditioning chamber used forcontext A. One of the walls of this insert had a black/white striped pattern. The other two walls were white.
Context B was cleaned with water. As contexts A and B
were located in the same windowless room and usedcommon apparatus, they shared some overlapping features. Mouse freezing behavior was monitored via overhead cameras. Freezing was assessed using an
automated scoring system (Actimetrics, Wilmette, IL),
which digitized the video signal at 4 Hz and comparedmovement frame by frame to determine the amount offreezing.
During training, mice were placed in context A for 3
min. After 2 min of free exploration mice were
presented with a 30 s tone (2800 Hz, 85 dB) that coterminated with a 2 s footshock (0.5 mA). Miceremained in the context for a further 30 s before being
returned to their home cage. Responsivity to the shock
during training was estimated by comparing mouse
velocity immediately preceding vs. during shockpresentation using the following formula: (velocityshock velocity pre-shock )/(velocity shock + velocity pre-shock ).
Twenty-four hours after training, freezing was assessedin a 3 min test in context A. Twenty-four hours later,mice were placed in an altered context (context B).
After 2 min, the tone was presented. In the intensive
training protocol, mice received three tone-footshock
pairings. As previously, each tone (30 s, 2800 Hz, 85 dB)
co-terminated with a 2 s footshock (0.5 mA). Tones
were presented after 120 s, 180 s and 240 s. Mice werereturned to the home cage 30 sec after the final footshock.
Hidden version of the water maze

The apparatus and behavioral procedures have been previously described [25]. Behavioral testing was conductedin a circular water maze tank (120 cm in diameter, 50
cm deep), located in a dimly-lit room. The pool was
filled to a depth of 40 cm with water made opaque byadding white, non-toxic paint. Water temperature wasmaintained at 28 ± 1°C by a heating pad located beneaththe pool. A circular escape platform (10 cm diameter)
was submerged 0.5 cm below the water surface, in a
fixed position in one of the quadrants. The pool was
surrounded by curtains, at least 1 m from the perimeter

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

of the pool. The curtains were white and had distinct
cues painted on them.
Water maze training took place over 8 days. On each
day mice received 3 training trials (inter-trial interval
was ~15 s). On each trial, mice were placed into thepool, facing the wall, in one of 4 pseudorandomly-varied
start locations. The trial was complete once the mousefound the platform or 60 seconds had elapsed. If themouse failed to find the platform on a given trial, theexperimenter guided the mouse onto the platform.
Twenty-four hours following the completion of training,spatial memory was assessed in a 60 s probe test withthe platform removed from the pool. Behavioral datafrom training and the probe tests were acquired andanalyzed using an automated tracking system (Actimetrics, Wilmette, IL). Using this software, we recorded
parameters during training, including escape latency andswim speed. In probe tests, we measured the amount oftime mice searched the target zone (23.6 cm in radius,
centered on the location of the platform during training)vs. the average of three other equivalent zones in otherareas of the pool. Each zone represents 15% of the total
pool surface.
Visual version of the water maze

To control for sensory and motor impairments, wetrained mice in the visual version of the water maze. In
this version, platform location was marked by a cylindrical cue (4 cm in diameter, 4 cm in height), with a vertical black/white striped pattern. Mice were trained for 3
days (6 trials/day). On each trial, the platform location
was kept constant across trials while the start position
was varied pseudo-randomly. Forty-eight hours after thecompletion of training, the platform and cue were repositioned in the opposite quadrant of the pool, and themice were given a single trial. The latency to reach theplatform and swim speed were recorded.
Open Field

Mice were placed in the center of a square-shaped open
field (45 cm × 45 cm × 20 cm height) and allowed to
explore for 10 min. The open field apparatus was constructed of Plexiglas, and was dimly-lit from above.
Mouse location was tracked by a camera located above.
Total distance travelled and time spent in 3 different
zones (outer, middle, inner) were measured (Limelight2,
Actimetrics, Wilmette, IL). Distribution of activity in different regions of the arena was used as a measure ofanxiety-related behavior [47].
Acoustic startle experiments

Startle testing was conducted in a MEDASR-310 startle
testing system (MedAssociates, VT, USA). Mice wereplaced in a Plexiglas cylinder (3.2 cm internal diameter)

Page 8 of 10

for testing. Acoustic startle stimuli and prepulse stimuli
were delivered via a high-frequency speaker, placed at a
distance of 15 cm from the testing cylinder. Backgroundnoise levels were maintained at 65 dB throughout theexperiments. The testing cylinder was mounted on asensor platform. A piezoelectric accelerometer, attachedto the base of the sensor platform, detected and transduced all cage movements, and these were recorded by
a computer. The startle amplitude was taken to be themaximal response occurring within 100 ms of the presentation of the startle stimulus. The speakers, testing
cylinder and sensor platform were housed within asound-attenuated chamber.
Mice were initially given a habituation session to acclimate them to the testing environment. In this session,
mice were presented with 80 startle stimuli, delivered ata fixed intertrial interval of 15 s. The startle stimulus
was a 40 ms, 120 dB noise burst with a rise/fall time ofless than 1 ms. One day following this, prepulse inhibition was tested. Following an acclimation period of 5min, mice were initially presented with a total of 20
noise bursts (40 ms duration, 120 dB, 1 ms rise/falltime). In the prepulse inhibition phase, mice were presented with a total of 90 trials. Three prepulse intensities were tested: 70, 75 and 80 dB. Prepulses were 20 ms
in duration with a rise/fall time of less than 1 ms. For
each prepulse intensity, there were three types of trial:
prepulse alone, prepulse/startle stimulus and startle stimulus alone. In the prepulse/startle stimulus trial, theonset of the prepulse preceded the onset of the startle
stimulus by 100 ms. The trials were spaced 15 s apart.
Percent PPI was calculated for each mouse according to
the following formula:: %PPI = [1-(Response prepulse +startle stimulus/Responsestartle stimulus alone)] × 100)
Twenty four hours later, mice were given a startle
threshold test session. Following an acclimation periodof 5 min, mice were presented with a total of 70 trials
at a fixed intertrial interval of 15 s. There were 7 trial
types: no stimulus, and 6 types of trials where startle stimuli at a range of intensities were presented (75-100 dB;
5 dB increments). The startle stimuli were 40 ms noise
bursts with a rise/fall time of less than 1 ms. The 7 trial
types were presented in a pseudorandom order suchthat each trial type was presented once within a blockof 7 trials. Startle threshold was defined as the minimal
intensity at which responding was significantly greaterthan in the no stimulus (0 dB) trials.
Statistical analysis

Values are expressed as the mean ± standard error of
the mean (s.e.m.). An unpaired t-test (for two groups)
or ANOVA were used to detect group differences.
Because both male and female mice were used in thesestudies we initially included gender as a factor in our

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

analyses. However, we found no effects of gender, andno significant interactions between gender and genotype,
and therefore this factor was subsequently dropped fromanalysis.
Acknowledgements
We thank Mika Yamamoto for genotyping gal-1 mutant mice and membersof the Frankland/Josselyn labs for technical assistance. This work was
supported by a Canadian Institutes of Health Research grant (MOP86762) to
P.W.F. and the Funding Program for World-leading Innovative R&D on
Science and Technology to H.O. M.S. Y.I. and M.A.C. were supported byfellowships from the Japanese Society for the Promotion of Science (M.S., Y.
I.), Ontario Graduate Scholarship program (M.A.C), and the Hospital for Sick
Children (M.S., M.A.C).
Author details
1
Program in Neurosciences and Mental Health, The Hospital for Sick
Children, Toronto, Canada, M5G 1X8. 2Department of Physiology, Universityof Toronto, Toronto M5S 1A8, Canada. 3Institute of Medical Science,
University of Toronto, Toronto M5S 1A8, Canada. 4Department of Physiology,
Keio University School of Medicine, Tokyo, Japan. 5Institut Jacques Monod,
Université Paris Diderot, Paris, France.
Authors’ contributions
MS and PWF designed the research. MS, MAC, NHK and YI performed theresearch. HO and FP generated the gal-1-/- mice. MS, MAC and PWF
prepared the manuscript. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 27 May 2011 Accepted: 1 September 2011
Published: 1 September 2011
References
1. Taylor ME, Drickamer K: Introduction to Glycobiology. 2 edition. Oxford
University Press; 2006.
2. Varki A, Cummings RD, Esko JD, Freeze HH, Stanely P, Bertozzi CR, Hart GW,
Etzler ME, Eds: Essentials of Glycobiology. 2 edition. Cold Spring Harbor
Laboratory Press; 2008.
3. Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T,
Gitt MA, Hirabayashi J, Hughes C, Kasai K, et al: Galectins: a family ofanimal beta-galactoside-binding lectins. Cell 1994, 76(4):597-598.
4. Yang RY, Rabinovich GA, Liu FT: Galectins: structure, function andtherapeutic potential. Expert Rev Mol Med 2008, 10:e17.
5. Leffler H, Carlsson S, Hedlund M, Qian Y, Poirier F: Introduction to
galectins. Glycoconj J 2004, 19(7-9):433-440.
6. Perillo NL, Marcus ME, Baum LG: Galectins: versatile modulators of celladhesion, cell proliferation, and cell death. J Mol Med 1998, 76(6):402-412.
7. Sakaguchi M, Imaizumi Y, Shingo T, Tada H, Hayama K, Yamada O,
Morishita T, Kadoya T, Uchiyama N, Shimazaki T, et al: Regulation of adultneural progenitor cells by Galectin-1/beta1 Integrin interaction. J
Neurochem 2010, 113(6):1516-1524.
8. Ishibashi S, Kuroiwa T, Sakaguchi M, Sun L, Kadoya T, Okano H, Mizusawa H:
Galectin-1 regulates neurogenesis in the subventricular zone andpromotes functional recovery after stroke. Exp Neurol 2007,
207(2):302-313.
9. Sakaguchi M, Shingo T, Shimazaki T, Okano HJ, Shiwa M, Ishibashi S,
Oguro H, Ninomiya M, Kadoya T, Horie H, et al: A carbohydrate-binding
protein, Galectin-1, promotes proliferation of adult neural stem cells.
Proc Natl Acad Sci USA 2006, 103(18):7112-7117.
10. Imaizumi Y, Sakaguchi M, Morishita T, Ito M, Poirier F, Sawamoto K,
Okano H: Galectin-1 is expressed in early-type neural progenitor cells
and down-regulates neurogenesis in the adult hippocampus. Mol Brain
2011, 4:7.
11. Sakaguchi M, Imaizumi Y, Okano H: Expression and function of galectin-1
in adult neural stem cells. Cell Mol Life Sci 2007, 64(10):1254-1258.

Page 9 of 10

12. Yamane J, Nakamura M, Iwanami A, Sakaguchi M, Katoh H, Yamada M,
Momoshima S, Miyao S, Ishii K, Tamaoki N, et al: Transplantation of
galectin-1-expressing human neural stem cells into the injured
spinal cord of adult common marmosets. J Neurosci Res 2010,
88(7):1394-1405.
13. Kajitani K, Nomaru H, Ifuku M, Yutsudo N, Dan Y, Miura T, Tsuchimoto D,
Sakumi K, Kadoya T, Horie H, et al: Galectin-1 promotes basal and kainateinduced proliferation of neural progenitors in the dentate gyrus of adultmouse hippocampus. Cell Death Differ 2009, 16(3):417-427.
14. Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M,
Urashima T, Oka T, Futai M, Muller WE, et al: Oligosaccharide specificity of
galectins: a search by frontal affinity chromatography. Biochim Biophys
Acta 2002, 1572(2-3):232-254.
15. Houzelstein D, Goncalves IR, Fadden AJ, Sidhu SS, Cooper DN, Drickamer K,
Leffler H, Poirier F: Phylogenetic analysis of the vertebrate galectin family.
Mol Biol Evol 2004, 21(7):1177-1187.
16. Lotan R, Belloni PN, Tressler RJ, Lotan D, Xu XC, Nicolson GL: Expression of
galectins on microvessel endothelial cells and their involvement intumour cell adhesion. Glycoconj J 1994, 11(5):462-468.
17. Deng W, Aimone JB, Gage FH: New neurons and new memories: how
does adult hippocampal neurogenesis affect learning and memory? Nat
Rev Neurosci 2009, 11(5):339-350.
18. Shors TJ, Anderson ML, Curlik DM, Nokia MS: Use it or lose it: Howneurogenesis keeps the brain fit for learning. Behav Brain Res 2011.
19. Ming GL, Song H: Adult neurogenesis in the Mammalian brain:significant answers and significant questions. Neuron 2011, 70(4):687-702.
20. Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M,
Mori K, Ikeda T, Itohara S, Kageyama R: Roles of continuous neurogenesis
in the structural and functional integrity of the adult forebrain. Nat
Neurosci 2008, 11(10):1153-1161.
21. Kim JJ, Fanselow MS: Modality-specific retrograde amnesia of fear. Science
1992, 256(5057):675-677.
22. Wiltgen BJ, Sanders MJ, Anagnostaras SG, Sage JR, Fanselow MS: Context
fear learning in the absence of the hippocampus. J Neurosci 2006,
26(20):5484-5491.
23. Maren S, Aharonov G, Fanselow MS: Neurotoxic lesions of the dorsal
hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res
1997, 88(2):261-274.
24. Morris RG, Garrud P, Rawlins JN, O’Keefe J: Place navigation impaired inrats with hippocampal lesions. Nature 1982, 297(5868):681-683.
25. Teixeira CM, Pomedli SR, Maei HR, Kee N, Frankland PW: Involvement of
the anterior cingulate cortex in the expression of remote spatial
memory. J Neurosci 2006, 26(29):7555-7564.
26. Clapcote SJ, Roder JC: Survey of embryonic stem cell line source strainsin the water maze reveals superior reversal learning of 129S6/SvEvTac
mice. Behav Brain Res 2004, 152(1):35-48.
27. Gallagher M, Burwell R, Burchinal M: Severity of spatial learning
impairment in aging: development of a learning index for
performance in the Morris water maze. Behav Neurosci 1993,
107(4):618-626.
28. Lipp HP, Wolfer DP: Genetically modified mice and cognition. Curr Opin
Neurobiol 1998, 8(2):272-280.
29. Poirier F, Timmons PM, Chan CT, Guenet JL, Rigby PW: Expression of theL14 lectin during mouse embryogenesis suggests multiple roles during
pre- and post-implantation development. Development 1992,
115(1):143-155.
30. Frankland PW, Wang Y, Rosner B, Shimizu T, Balleine BW, Dykens EM,
Ornitz EM, Silva AJ: Sensorimotor gating abnormalities in young maleswith fragile × syndrome and Fmr1-knockout mice. Mol Psychiatry 2004,
9(4):417-425.
31. Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE,
Garcia AD, Sofroniew MV, Kandel ER, Santarelli L, et al: Ablation ofhippocampal neurogenesis impairs contextual fear conditioning andsynaptic plasticity in the dentate gyrus. Proc Natl Acad Sci USA 2006,
103(46):17501-17506.
32. Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E: Neurogenesis in
the adult is involved in the formation of trace memories. Nature 2001,
410(6826):372-376.
33. Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM: A role for adultneurogenesis in spatial long-term memory. Neuroscience 2005,
130(4):843-852.

Sakaguchi et al. Molecular Brain 2011, 4:33
http://www.molecularbrain.com/content/4/1/33

Page 10 of 10

34. Deng W, Saxe MD, Gallina IS, Gage FH: Adult-born hippocampal dentate
granule cells undergoing maturation modulate learning and memory inthe brain. J Neurosci 2009, 29(43):13532-13542.
35. Saxe MD, Malleret G, Vronskaya S, Mendez I, Garcia AD, Sofroniew MV,
Kandel ER, Hen R: Paradoxical influence of hippocampal neurogenesis on
working memory. Proc Natl Acad Sci USA 2007, 104(11):4642-4646.
36. Jessberger S, Zhao C, Toni N, Clemenson GD Jr, Li Y, Gage FH: Seizureassociated, aberrant neurogenesis in adult rats characterized withretrovirus-mediated cell labeling. J Neurosci 2007, 27(35):9400-9407.
37. Farioli-Vecchioli S, Saraulli D, Costanzi M, Pacioni S, Cina I, Aceti M,
Micheli L, Bacci A, Cestari V, Tirone F: The timing of differentiation of
adult hippocampal neurons is crucial for spatial memory. PLoS Biol 2008,
6(10):e246.
38. Tashiro A, Zhao C, Gage FH: Retrovirus-mediated single-cell geneknockout technique in adult newborn neurons in vivo. Nat Protoc 2006,
1(6):3049-3055.
39. Kee N, Teixeira CM, Wang AH, Frankland PW: Preferential incorporation of
adult-generated granule cells into spatial memory networks in thedentate gyrus. Nat Neurosci 2007, 10(3):355-362.
40. Moore KA, Pytowski B, Witte L, Hicklin D, Lemischka IR: Hematopoietic
activity of a stromal cell transmembrane protein containing epidermal
growth factor-like repeat motifs. Proc Natl Acad Sci USA 1997,
94(8):4011-4016.
41. Kramar EA, Lin B, Rex CS, Gall CM, Lynch G: Integrin-driven actin
polymerization consolidates long-term potentiation. Proc Natl Acad Sci
USA 2006, 103(14):5579-5584.
42. Chan CS, Weeber EJ, Zong L, Fuchs E, Sweatt JD, Davis RL: Beta 1-integrins
are required for hippocampal AMPA receptor-dependent synaptictransmission, synaptic plasticity, and working memory. J Neurosci 2006,
26(1):223-232.
43. McGeachie AB, Cingolani LA, Goda Y: A stabilising influence: Integrins inregulation of synaptic plasticity. Neurosci Res 2011, 70(1):24-29.
44. Lowe JB, Marth JD: A genetic approach to Mammalian glycan function.
Annu Rev Biochem 2003, 72:643-691.
45. Kaltner H, Gabius HJ: Animal lectins: from initial description to elaborated
structural and functional classification. Adv Exp Med Biol 2001, 491:79-94.
46. Wang SH, Teixeira CM, Wheeler AL, Frankland PW: The precision of remotecontext memories does not require the hippocampus. Nat Neurosci 2009,12(3):253-255.
47. Archer J: Tests for emotionality in rats and mice: a review. Anim Behav
1973, 21(2):205-235.
doi:10.1186/1756-6606-4-33
Cite this article as: Sakaguchi et al.: Impaired spatial and contextual
memory formation in galectin-1 deficient mice. Molecular Brain 2011 4:33.

Submit your next manuscript to BioMed Central
and take full advantage of:• Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit