Brain, Behavior, and Immunity 25 (2011) 10081016

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Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Astrocytes support hippocampal-dependent memory and long-term

potentiation via interleukin-1 signaling
Ofra Ben Menachem-Zidon a,b, Avi Avital c,d, Yair Ben-Menahem a, Inbal Goshen a, Tirzah Kreisel a,
Eli M. Shmueli a, Menahem Segal d, Tamir Ben Hur b, Raz Yirmiya a,
a

Department of Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel

Department of Neurology, Hadassah-Hebrew University Hospital, Jerusalem, Israel
Department of Psychology and The Center for Psychobiological Research, The Max Stern Yezreel Valley College, Israel
d
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
b
c

a r t i c l e

i n f o

Article history:
Available online 17 November 2010
Keywords:
Astrocytes
Interleukin-1 (IL-1)
Memory
Long-term potentiation (LTP)
Neural precursor cells
Hippocampus

a b s t r a c t
Recent studies indicate that astrocytes play an integral role in neural and synaptic functioning. To examine the implications of these ndings for neurobehavioral plasticity we investigated the involvement of
astrocytes in memory and long-term potentiation (LTP), using a mouse model of impaired learning and
synaptic plasticity caused by genetic deletion of the interleukin-1 receptor type I (IL-1RI). Neural precursor cells (NPCs), derived from either wild type (WT) or IL-1 receptor knockout (IL-1rKO) neonatal mice,
were labeled with bromodeoxyuridine (BrdU) and transplanted into the hippocampus of either IL1rKO or WT adult host mice. Transplanted NPCs survived and differentiated into astrocytes (expressing
GFAP and S100b), but not to neurons or oligodendrocytes. The NPCs-derived astrocytes from WT but
not IL-1rKO mice displayed co-localization of GFAP with the IL-1RI. Four to twelve weeks post-transplantation, memory functioning was examined in the fear-conditioning and the water maze paradigms and
LTP of perforant path-dentate gyrus synapses was assessed in anesthetized mice. As expected, IL-1rKO
mice transplanted with IL-1rKO cells or sham operated displayed severe memory disturbances in both
paradigms as well as a marked impairment in LTP. In contrast, IL-1rKO mice transplanted with WT NPCs
displayed a complete rescue of the impaired memory functioning as well as partial restoration of LTP.
These ndings indicate that astrocytes play a critical role in memory functioning and LTP, and specically
implicate astrocytic IL-1 signaling in these processes. The results suggest novel conceptualization and
therapeutic targets for neuropsychiatric disorders characterized by impaired astrocytic functioning concomitantly with disturbed memory and synaptic plasticity.
2010 Elsevier Inc. All rights reserved.

1. Introduction
Recent studies indicate that astrocytes are not merely the
supportive cells of the brain, but that they also play an important
integral role in neural and synaptic functioning (Haydon and
Carmignoto, 2006; Henneberger et al., 2010; Newman, 2003;
Volterra and Meldolesi, 2005). Specically, astrocytic processes ensheath most synapses in the brain, and express receptors for several
transmitters. Signaling via these receptors evokes elevations in
astrocytic Ca2+ concentration, resulting in the regulated secretion
of various gliotransmitters, which modulate neuronal excitability
and synaptic strength (Haydon and Carmignoto, 2006; Jourdain
et al., 2007; Perea and Araque, 2007). Astrocytes-to-neurons
Corresponding author. Address: Department of Psychology, The Hebrew
University of Jerusalem, Jerusalem 91905, Israel. Fax: +972 2 5882947.
E-mail address: razyirmiya@huji.ac.il (R. Yirmiya).
0889-1591/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbi.2010.11.007

communication also plays a critical role in synaptic plasticity

processes, including long-term potentiation (Nishiyama et al.,
2002; Yang et al., 2003) and synaptic scaling following prolonged
inhibition of neuronal activity (Stellwagen and Malenka, 2006).
Corroborating these ndings, in neuropsychiatric disorders that
are characterized by impaired astrocytic functioning, e.g., major
depression and neurodegenerative diseases (Bowley et al., 2002;
Cotter et al., 2001a; Mrak and Grifn, 2005; Ongur et al., 1998; Seifert
et al., 2006), behavioral and neural plasticity are also severely
disturbed (Duman et al., 2000; Selkoe, 2002).
To directly assess the possible role of astrocytes in these processes we used a mouse model of impaired learning, memory
and long-term potentiation (LTP), caused by a genetic deletion of
the receptor for the cytokine interleukin-1 (IL-1). In the brain, this
receptor is extensively (but not exclusively) expressed by astrocytes
(Ban et al., 1993; Hammond et al., 1999; Pinteaux et al., 2002; Wong
and Licinio, 1994) and plays an important role in the functioning of

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these cells (Beskina et al., 2007; Holliday and Gruol, 1993). Previous
research indicated that mice with a genetic deletion of the IL-1
receptor type I display markedly impaired hippocampal-dependent
memory functioning and LTP, although memories that do not depend on the integrity of the hippocampus seem to be spared (Avital
et al., 2003; Goshen et al., 2007; Schmid et al., 2009). These ndings
were corroborated by other lines of evidence for the involvement of
IL-1 signaling in normal memory functioning and LTP, including the
induction of IL-1 related genes during hippocampal-dependent
memory tasks (Depino et al., 2004; Goshen et al., 2007; Labrousse
et al., 2009) and LTP (Balschun et al., 2003; Schneider et al., 1998),
the facilitation of memory consolidation by low doses of IL-1 (Brennan et al., 2003; Goshen et al., 2007; Song et al., 2003; Yirmiya et al.,
2002), as well as the interference with hippocampal-dependent
memory consolidation and LTP by IL-1 receptor antagonist (IL-1ra)
(Goshen et al., 2009, 2007, 2003; Goshen and Yirmiya, 2009; Ross
et al., 2003; Schmid et al., 2009; Spulber et al., 2009).
To test the hypothesis that astrocytes in general, and astrocytic
IL-1 signaling in particular, are critical for memory functioning and
LTP, mice with deletion of the IL-1 receptor type I (the only known
signaling receptor for IL-1) (IL-1rKO mice) and wild type (WT) mice
were transplanted intrahippocampally with neural precursor cells
(NPCs) derived from either WT or IL-1rKO mice, which differentiate
exclusively into astrocytes (Ben Menachem-Zidon et al., 2008; Raedt et al., 2009). Memory functioning in two hippocampal-dependent tasks and in vivo LTP were tested several weeks later. We
report that astrocyes derived from WT, but not IL-1rKO mice completely rescued the impaired memory functioning and partially restored LTP in IL-1rKO mice, providing direct evidence for the
essential role of astrocytic IL-1 signaling in hippocampal-dependent memory functioning and synaptic plasticity.
2. Materials and methods
2.1. Subjects
Subjects were male IL-1rKO mice and their 129/Sv X C57BL/6
WT controls (Jackson Laboratories, Bar Harbor). Mice were 9
10 weeks old, housed in an air-conditioned room (23 1 C), with
food and water ad libitum. The behavioral experiments were conducted during the rst half of the dark phase of the12-h light/dark
cycle. Newborn IL-1rKO mice and their 129/Sv X C57BL/6 WT controls were used as a source for neural precursor cells. The experiments were approved by the Hebrew University Committee on
Animal Care and Use.
2.2. Preparation of neural precursor cells and growth of neurospheres
Neural precursor cell spheres were prepared from newborn WT or
IL-1rKO mice as previously described (Ben-Hur et al., 2003; Ben
Menachem-Zidon et al., 2008). Briey, the hemispheres were dissected, followed by removal of the meninges. The tissue was minced,
digested by trypsin for 20 min, and dissociated. Following suspension in N2 medium, 10  106 cells were plated in a T-75 uncoated
ask and supplemented with 10 ng/ml FGF2 and 20 ng/ml EGF,
added daily. Un-differentiated neurospheres were collected for characterization in vitro or for transplantation after 5 days of growth.
2.3. Characterization of neural precursor cells in vitro
For the assessment of differentiation, oating spheres obtained
from WT and IL-1rKO newborn mice were adhered to polylysinecoated dishes. Spheres were allowed to differentiate for 5 days,
then xed and stained for lineage-specic markers. Experiments
were performed in triplicates and repeated three times, with at

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least 30 spheres per plate. At least 1000 cells were counted for each
experimental condition to determine cell fate.
2.4. Sphere transplantation
Prior to transplantation, spheres (from either WT or IL-1rKO
newborn mice) were incubated with 25 lg/ml BrdU for 72 h. The
spheres (4000-spheres/4 ll N2 medium) were bilaterally transplanted into the hippocampus using a stereotaxic instrument. Mice
from the Sham group were injected with 4 ll of the N2 medium.
Coordinates of the injection site (in mm) relative to Bregma were
as follows: AP 2.6, L 1.4, DV 1.6, for all groups. Injections were
conducted using a 25-ll Hamilton syringe. After each injection, the
needle was left in situ for 5 min before being retracted, to allow
complete diffusion of the spheres. Four weeks after transplantation, animals were perfused with PBS followed by 4% paraformaldehyde. Tissues were deep frozen in liquid nitrogen. Serial 8 lm
brain coronal frozen sections were cut for histological analyses.
2.5. Immunouorescent staining and quantication of the
transplanted cells in vivo
The transplanted cells were detected by immunostaining for
BrdU. Sections were incubated with 2 N HCl for 30 min at 37,
washed, and incubated with 3% normal goat serum for 1 h in room
temperature (RT), followed by incubation with rat anti-BrdU (clone
BU1/75 (ICR1), serotec, 1:200 dilution) overnight at 4 C. A goat
anti rat IgG secondary antibody, conjugated to Alexa 555, was
added for 50 min at RT, and counterstaining was done with DAPI.
To determine the fate of the transplanted cells, sections were
double stained for BrdU and the astrocytic markers GFAP (rabbit
anti-GFAP:Dako, 1:100) and S100b (mouse anti S100b:Abcam,
1:100), the neuronal marker NeuN (mouse anti-neuronal nuclei:
Chemicon, 1:50), or the oligodendrocyte marker galactocerebroside
(rabbit anti-GalC:Millipore Bioscience Research Reagents, 1:100),
followed by incubation with goat anti rabbit IgG or goat anti-mouse
IgG secondary antibodies, respectively, conjugated to Alexa-488
(Molecular Probes) for 50 min at RT. To examine the phenotype of
the cells that express IL-1RI, sections were double stained using goat
anti-IL-1RI (1:100) together with rabbit anti-GFAP (1:100), mouse
anti-NeuN (1:50) or rabbit anti-GalC (1:100) antibodies, followed
by incubation with donkey anti goat IgG, goat anti rabbit IgG or goat
anti-mouse IgG secondary antibodies, respectively, (Molecular
Probes) for 50 min at RT. Images were taken using a confocal microscope (Leica) or a Nikkon E-600 uorescent microscope.
BrdU-labeled cells were counted on every sixth 8 lm coronal
section, covering the dentate gyrus in its rostro-caudal extension.
Quantication of cell numbers was based on counting immunopositive nuclei that were completely lled with the uorescent stain;
given that cells were counted in sections 48 lm apart and that the
nuclei sizes in any one dimension were smaller than 30 lm, this
procedure ensured that no cells were counted more than once.
The total number of transplanted cells was extrapolated for the entire volume of the dentate gyrus (Kempermann et al., 1997). Differentiation of transplanted cells into astrocytes, neurons and
oligodendrocytes was determined by immunouorescent staining
for GFAP, S100b, NeuN and Gal C in brains transplanted with BrdU
labeled neurospheres. As no BrdU-labeled cells were also stained
for NeuN or Gal C, quantitative analysis focused on astrocytes.
The percentage of graft-derived cells that differentiated into astrocytes was determined by dividing the number of cells manifesting
double labeling for BrdU and GFAP by the total number of BrdU positive cells. The percentage of IL-1RI positive cells that also expressed GFAP was calculated by dividing the number of cells
with IL-1RI + GFAP double labeling by the total number of IL-1RI
positive cells. All analyses were conducted by an experimenter

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who was blind with respect to the experimental and control

groups.

sured during 30 min post HFS in comparison to pre HFS responses.

The electrophysiological experiments were conducted blindly to
the genotype/treatment and the behavioral experiments.

2.6. Measurement of learning and memory in the fear-conditioning

paradigm
The fear conditioning apparatus consisted of a transparent
square conditioning cage (25  21  18 cm), with a grid oor
wired to a shock generator and scrambler (Campden instruments,
UK). Mice were placed in the cage for 120 s, and then a pure tone
(2.9 kHz) was sound for 20 s, followed by a 2 s, 0.5 mA foot-shock.
This procedure was then repeated, and 30 s after the delivery of the
second shock mice were returned to their home cage. 48 h later,
fear conditioning was assessed by measuring the time spent in
freezing (complete immobility) during exposure to the conditioned
stimuli (i.e., either the context or the tone), using an automated
system (Campden instruments, UK). The mice tested for contextual
fear conditioning were placed in the original conditioning cage,
and freezing was measured for 5 min. The mice tested for the auditory-cued fear conditioning were placed in a different context a
triangular shaped cage with no grid oor. As a control for the inuence of the novel environment, freezing was measured for 2.5 min
in this new cage, and then the tone was sounded for 2.5 min, during which conditioned freezing was measured. Freezing was also
measured during the rst 120 s of the conditioning trial, before
the tone and shock administration, in order to assess possible
strain differences in baseline freezing.
2.7. Measurement of learning and memory in the water maze
Animals were trained in a circular pool (160 cm in diameter)
lled with 23 2 C water mixed with non-toxic gouache paint to
make it opaque. In the spatial memory experiment, mice were
trained to nd the location of a hidden platform, submerged 1 cm
below the water surface, using extra maze visual cues. Training consisted of three trials per day, with a one-hour break between trials,
for 3 days. In the non-spatial memory experiment, the platform
was elevated 1 cm above the water level and therefore was visible.
Both experiments were conducted using a random protocol, in
which the entrance point to the maze was varied randomly between
trials, and the platform remained in a permanent position (see Avital
et al., 2003; Goshen et al., 2009 for additional details). To verify that
any potential differences between the groups do not stem from differences in locomotor and exploratory activity, mice were tested in
the open eld activity test. The open eld apparatus is 80  80 cm
plexiglas box with 50 cm high walls. The oor is divided into
8 cm  8 cm identical squares. Each mouse was placed in the corner
of the apparatus and left there for a period of 3 min. An observer
blind to the group assignment of the animals recorded instances of
line crossing with both hind paws in the periphery and center of
the eld, and instances of rearing with both paws up.
2.8. Measurements of hippocampal reactivity and plasticity in vivo
Perforant path (PP) evoked responses in the dentate gyrus (DG)
was conducted as described (Avital et al., 2003; Goshen et al.,
2009). Briey, mice were anaesthetized and placed in a stereotactic
apparatus. A stimulating electrode was placed in the perforant
path (PP) and a recording glass pipette was inserted into the DG
of the dorsal hippocampus. Evoked responses were amplied, ltered at 1 Hz1 kHz and stored for later analysis. Inputoutput
relations were examined using three stimulus intensities (1.1, 2.1
and 3.1 V). LTP was induced by applying high-frequency stimulation (HFS) of ve trains of eight 0.4 ms 400 Hz pulses of 2.1 V.
Ten measurements were taken and averaged every 5 min and
LTP was computed as the change in the evoked responses mea-

2.9. Statistical analysis

The results were analyzed by two-way or three-way ANOVAs,
followed by the Tukey HSD post-hoc analysis, when appropriate.

3. Results
3.1. Characterization of IL-1rKO and WT NPCs in vitro
Neural precursor cells from newborn IL-1rKO and WT mice
were expanded in spheres. Efciency of IL-1rKO neurosphere generation from dissociated brains and their growth was similar to
that of WT mice (data not shown). The potential of NPCs to differentiate in vitro was examined by immunouorescent staining, after
5 days of differentiation. Similar to our previous report (Ben Menachem-Zidon et al., 2008), WT neurospheres (n = 90) comprised
mostly astrocytes (81 3% of all differentiated cells), 17 4% of
the cells differentiated into oligodendrocytes and only 2 1% differentiated into neurons. A similar nding was obtained in IL1rKO neurospheres (n = 90), which comprised 80 4% astrocytes,
18 3% oligodendrocytes and 2 1% neurons. Thus, IL-1rKO NPCs
showed similar pattern of growth and lineage choice as WT NPCs.
These results conrmed that the population of NPCs comprised
mainly glia cells, especially GFAP-positive cells that differentiate
into astrocytes (Fig. 1A and B) .

3.2. Localization and differentiation of transplanted NPCs

The distribution and differentiation of transplanted NPCs within
the host brain were examined after completion of all behavioral
tests. Most of the transplanted NPCs were localized in the DG of
the hippocampus and were integrated into the host parenchyma,
as shown by BrdU immunourosence staining (Fig. 1C). The average numbers (SEM) of BrdU transplanted cells in the hippocampus of IL-1rKO and WT mice were 6683 334 and 6692 224,
respectively. In order to evaluate the pattern of differentiation of
the transplanted cells, slides were double stained for BrdU, together with glial or neuronal markers. Immunohistochemistry
and quantication of double-labeled cells revealed that 65% of
the BrdU transplanted cells expressed the astrocytic marker GFAP
(Fig. 1C). Confocal microscopy conrmed the co-expression of BrdU
and GFAP in these slides as shown in Fig. 1DF. To exclude the possibility that the GFAP-positive transplanted cells are un-differentiated NPCs or radial glia, additional staining was carried out with
S100b, which marks mature differentiated astrocytes. Slices from
IL-1rKO mice transplanted with WT cells were stained for BrdU
(Fig. 1G) and for BrdU with S100b (Fig. 1H), demonstrating that
most of the BrdU positive cells were also positive for S100b. In contrast, no double staining for BrdU and the neuronal marker NeuN
was observed in any cells (Fig. 1I). Similarly, no double staining
for BrdU and the mature oligodendrocyte marker Gal C was observed in any of the cells (Fig. 1J). The difference between this nding and the fractions of the cells that differentiated into
oligodendrocyes in vitro (1718% of the differentiated cells) may
be related to the localization of the transplanted cells within the
DG, which normally contains very few oligodendrocytes (Fig. 1J).
The overall pattern of differentiation was the same for either WT
or IL-1rKO transplanted cells.

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Fig. 1. Characterization of NPCs in vitro. and in vivo NPCs were characterized in vitro by immunouorescent staining, performed after 5 days of differentiation. (A) A
representative picture of one sphere shows that most of the cells (blue = DAPI) differentiated into astrocytes, expressing GFAP (red). (B) A magnication of one GFAP-positive
cell from the same sphere. (C) Transplanted cells that were labeled with BrdU before transplantation (red) are seen within the DG of the host brain. Double staining for BrdU
(red) and GFAP (green) revealed that most of the transplanted cells expressed GFAP. (D and E) Confocal microscopy conrmed the expression of BrdU (red) in the nucleus of
two representative cells, surrounded by GFAP (green) in the cytoplasm. (F) The co-localization of BrdU and GFAP within these cells is shown in the merged image. (G)
Additional staining showed that most of the BrdU transplanted cells were positive for S100b (green) (H), as shown by a larger magnication of the white rectangle in G. (I)
None of the transplanted cells (BrdU+, red) expressed the neuronal marker NeuN (green). (J) In addition, none of the transplanted cells (red BrdU+ cells, pointed to by the
white arrow) expressed the oligodendrocyte marker Gal C (green). All slides were mounted with Dapi (blue). (For interpretation of the references to color in this gure legend,
the reader is referred to the web version of this article.)

3.3. Expression of IL-1 receptor type I in the hippocampus of control

and transplanted WT and IL-1rKO mice
In the hippocampus of control WT mice, immunouorescence
staining for IL-1RI revealed the expression of this receptor in the
dentate gyrus (Fig. 2A), whereas no expression of the receptor
could be observed in IL-1rKO mice (data not shown). The IL-1RI
is expressed by neurons within the granule cell layer (GCL) and
by astrocytes, which are mainly located throughout the hilus area

as well as adjacent to the GCL. The co-expression of IL-1R and GFAP

was veried by double staining of these two markers in slices derived from WT mice (Fig. 2BD).
In IL-1rKO mice transplanted with WT cells, IL-1R1 expressing
cells could be observed within the DG (Fig. 2E). In these animals,
double staining for BrdU and IL-1RI and confocal microscopy
analysis revealed that only WT transplanted cells expressed the
IL-1RI (Fig. 2FH), while there was no such expression by IL1rKO transplanted cells (Data not shown). In order to examine

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Fig. 2. Expression of IL-1 receptor type I in the hippocampus of control and transplanted WT and IL-1rKO mice. Immunouorescence staining for IL-1RI (green) revealed the
expression of this receptor by neurons within the granule cell layer (GCL) and by astrocytes, which are mainly located throughout the hilar area as well as adjacent to the GCL
(A). Confocal microscopy showed the co-expression of IL-1R (green, B) and GFAP (red, C) in slices derived from WT mice. The co-localization of IL-1R and GFAP is shown in the
merge image (D). IL-1R1 (green) expressing cells could be observed within the DG of IL-1rKO mice transplanted with WT cells (E). In these animals, double staining for BrdU
(red) and IL-1RI (green) conrmed that the transplanted cells, which were BrdU+ (F), expressed IL-1RI (G). The co-localization of BrdU and IL-1RI within the same cell is shown
in the merged confocal image (H). NPCs transplanted cells which were positive for the IL-1RI (red, I) were also positive for GFAP (green, J). IL-1RI and GFAP co-expression (K) is
demonstrated by a merged confocal image. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

the types of transplanted cells that express the IL-1RI, slides

from IL-1rKO mice were double stained for IL-1RI together with
GFAP, demonstrating that 89% of the cells that were positive for
IL-1RI were also positive for GFAP. Confocal microscopy conrmed the co-expression of IL-1R and GFAP (Fig. 2IK) in these
cells.
3.4. Effects of NPCs transplantation on learning and memory in the
fear-conditioning paradigm
Both WT and IL-1rKO mice were divided into 3 sub-groups (i.e.,
a 2  3 design), including two control groups that were either
sham operated or transplanted with IL-1rKO NPCs, and an experimental group that was transplanted with WT NPCs (n = 510 per
group). All groups were examined in the fear conditioning (FC) test
4 weeks after the transplantation day, a period in which the cells
are known to be incorporated in the host brain (Ben Menachem-Zidon et al., 2008). Pre-conditioning baseline freezing levels were
negligible (<4%) in all groups. During the memory test, a signicant
strain by transplantation interaction (F(2,33) = 19.04, p < 0.001)
was obtained. Post-hoc tests revealed that IL-1rKO mice that were
either sham operated or transplanted with IL-rKO NPCs displayed
signicantly reduced contextual fear conditioning compared to
their respective WT controls (p < 0.05), whereas IL-1rKO mice
transplanted with WT cells performed similarly to WT mice
(Fig. 3A). In the auditory-cued FC paradigm, IL-1rKO mice did not
display any memory impairment and the transplantation in WT
mice or IL-rKO mice had no effect on freezing, either before the
tone presentation or during the tone (Fig. 3B).
3.5. Effects of NPCs transplantation on learning and memory in the
water maze paradigm
In comparison with their respective WT groups, IL-1rKO mice
that were either sham operated or transplanted with IL-1rKO NPCs
displayed severely impaired spatial learning and memory, with
markedly smaller reductions in the latencies to reach the hidden

platform from the rst to the last trial. In contrast, IL-1rKO mice
transplanted with WT NPCs acquired the spatial memory task similarly to WT mice (in which NPCs transplantation had no effect)
(Fig. 3C). These ndings were reected by a signicant strain by
transplantation by time interaction (F(2,51) = 3.45, p < 0.05). To
further elucidate these differences, we conducted a specic
comparison between the groups on the last trial. IL-1rKO mice
transplanted with WT cells displayed signicantly lower latencies
to reach the platform than either sham or IL-1rKO-transplanted
mice (Fig. 3D) (F(2,51) = 3.63, p < 0.05). In contrast, no differences
were found between the IL-1rKO groups and their respective WT
control groups in the last trial of the non-spatial (visible platform)
paradigm (Fig. 3D). Similarly, there were no differences among the
groups in open eld activity (data not shown).

3.6. Effects of NPCs transplantation on hippocampal neural responses

and long-term potentiation (LTP)
To assess possible differences between the groups in the dynamic range of PP-DG synapses, we tested input/output relations.
A signicant effect was detected for stimulus intensity across the
groups (F(2,13) = 328.78, p < 0.0001), with no overall difference
between the groups (p > 0.1) (Fig. 4A). As expected based on
our previous ndings (Avital et al., 2003), IL-1rKO-Sham mice as
well as the IL-1rKO mice transplanted with IL-1rKO NPCs did
not express LTP, i.e., both groups had decreased ability to produce
potentiation and also to maintain it, as it dropped rapidly to the
basal levels of fEPSP (Fig. 4B). IL-1rKO mice transplanted with
NPCs derived from WT mice, displayed a complete restoration
in the initial potentiation phase of LTP, to a level that was not different from WT mice, and their ability to maintain the LTP was
partially restored. These ndings were reected by a signicant
group by time interaction (F(15,84) = 4.14, p < 0.001). Post-hoc
tests revealed that the IL-1rKO mice transplanted with WT NPCs
displayed signicantly elevated responses compared with the
other IL-1rKO transplanted mice at all time points after HFS
(Fig. 4B).

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Fig. 3. Effects of NPCs transplantation on learning and memory. (A) In the fear-conditioning (FC) paradigm, IL-1rKO mice that were either sham operated or transplanted with
IL-r1KO NPCs displayed signicantly reduced contextual fear conditioning (FC), as reected by reduced freezing times, compared to their respective WT controls, whereas IL1rKO mice transplanted with WT cells performed similarly to WT mice. (B) In the auditory-cued FC, IL-1rKO mice did not display any memory impairment and the
transplantation in both WT and IL-1rKO mice had no effect on memory, as shown by the similar levels of freezing in all groups. (C) In the water maze, IL-1rKO mice that were
either sham operated or transplanted with IL-rKO NPCs displayed severely impaired spatial memory, with relatively small reductions in the latencies to reach the hidden
platform from the rst to the last training trial. In contrast, IL-1rKO mice transplanted with WT NPCs acquired the spatial memory task similarly to WT mice. (D) Further
analysis of the results of the last (ninth) training trial in the spatial task (hidden platform) showed that IL-1rKO mice transplanted with WT cells displayed signicantly lower
latencies than sham or IL-1rKO-transplanted mice. In contrast, no difference was found between the IL-1rKO groups and their respective WT control groups in the last trial of
the non-spatial (visible platform) task. *P < 0.05 compared to IL-1rKO mice transplanted with WT cells and to all groups of WT mice.

Fig. 4. Effects of NPCs transplantation on long-term potentiation (LTP). (A) Baseline eld potential recordings in the DG in response to perforant path stimulation revealed
similar fEPSP responses to the three stimulus intensities in all groups, except for a slight increase in the responsiveness of the WT-Sham animals at the highest stimulus
intensity. (B) After high-frequency stimulation (HFS), WT-sham mice developed LTP of fEPSP slope. In contrast, IL-1rKO mice that were either sham operated or transplanted
with IL-1rKO NPCs displayed diminished short-term potentiation, and no LTP. IL-1rKO mice that were transplanted with WT NPCs displayed a complete restoration of the
short-term potentiation, to a level that was not different from WT mice. Their ability to maintain the enhanced response was partially restored, and was signicantly elevated
compared with the other IL-1rKO groups. Typical response patterns at baseline (left trace) and 30 min after HFS (WT-Sham: upper right trace; IL-1rKO transplanted with WT
NPCs: lower right trace) are presented in the inserts.

4. Discussion
The results of the present study demonstrate that intrahippocampal transplantation of NPCs, which differentiate into astrocytes

that express the IL-1RI, can rescue the impairment in memory

functioning and LTP exhibited by IL-1rKO mice. Previous research
demonstrated that these mice do not show immunological,
physiological or neurobehavioral responsiveness to exogenous

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IL-1 administration (Bluthe et al., 2000; Labow et al., 1997), and

manifest marked disturbances in behavioral, cognitive and physiological processes that depend on endogenous IL-1 production in
the hippocampus, including learning, memory and neural plasticity (Avital et al., 2003; Goshen et al., 2009, 2008, 2007, 2003;
Koo and Duman, 2008; Yirmiya and Goshen, in press). In agreement with these ndings, we report here that IL-1rKO mice displayed severe impairments in hippocampal-dependent memory
tasks, including contextual fear conditioning and spatial memory
in the water maze, but were not impaired in tasks that do not depend on hippocampal functioning, i.e., the auditory-cued fear conditioning and the visible platform water maze paradigms.
Moreover, the immunohistochemical ndings support the notion
that the hippocampal cell type in which IL-1 signaling is critical
for memory and plasticity is likely to be astrocyte.
This notion is based on the following lines of evidence: (1) as reported earlier (Ban et al., 1993; Hammond et al., 1999; Pinteaux
et al., 2002; Wong and Licinio, 1994), we found here that the IL1RI is expressed extensively by hippocampal astrocytes in WT
mice. (2) When characterized in vitro, most of the newborn-derived
NPCs expressed the astrocytic marker GFAP, and displayed astrocytic morphology. The number of neurons was negligible (about
2%). (3) In accordance with our previous study on intrahippocampal transplantation of NPCs (Ben Menachem-Zidon et al., 2008), we
now report that when characterized in vivo (at least 5 weeks posttransplantation), most of the transplanted, BrdU-labeled NPCs (in
either IL-1rKO or WT mice) acquired an astrocytic morphology
and expressed the astrocytic markers GFAP and S100b, alongside
with the IL-1RI. In contrast, none of the BrdU-labeled transplanted
cells was co-labeled with the neuronal marker NeuN or the oligodendrocyte marker Gal C. Thus, our ndings suggest that astrocytic
functioning, in general, and astrocytic IL-1 signaling, in particular,
is critical for hippocampal-dependent learning and memory processes. Interestingly, the number of astrocytes required to support
these functions seems to be relatively small, considering that previous work, using precise stereological techniques, demonstrated
that the total number of GFAP-labeled astrocytes within the mouse
dentate gyrus ranges between 60,000100,000 (Lei et al., 2003;
Long et al., 1998), whereas in the present study only about 4300
transplanted NPCs (i.e., close to 7% of the total astrocyte number)
could be detected in this region. However, although not directly
comparable, recent ndings that addition of a small number of
new neurons during hippocampal neurogenesis (about 10% over
the lifetime of a mouse) plays a critical role in hippocampal-dependent memory (Imayoshi et al., 2008), make it clear that even a
small fraction of the cells of the hippocampus can have a substantial impact on memory functioning. It should be noted that a fraction (about 35%) of the transplanted, BrdU-labeled cells did not
express astrocytic markers. Because these cells were neither neurons nor oligodendrocytes, it is very likely that they remained as
un-differentiated NPCs. Moreover, since most (89%) of the cells
expressing the IL-1R1 also expressed astrocytic markers, it can be
concluded that only a small fraction of the transplanted un-differentiated cells expressed the IL-1R1. The possibility that this small
cell population contributed to the restoration of memory and neural plasticity can not be completely ruled out. However, it is very
unlikely that these un-differentiated cells, rather than the much
(almost 10 times) larger population of mature IL-1R1-expressing
astrocytes is responsible for the complete rescue of memory and
the partial restoration of neural plasticity.
The present ndings corroborate and extend several previous
studies on the role of astrocytes in learning and memory (Yirmiya
and Goshen, in press). For example, female mice in which the transcription nuclear factor-kappa B (NFjB) was inhibited specically
in astrocytes displayed decits in learning, memory and LTP (Bracchi-Ricard et al., 2008). Moreover, pharmacologic blockade of

astrocytic glutamate uptake in rats was also found to impair spatial

memory (Bechtholt-Gompf et al., 2010). In addition, motor skill
learning was reported to be associated with astrocytic hypertrophy, which was reversed in the absence of continued training
(Kleim et al., 2007), and learning induced astrocytic BDNF production along the meningeal areas was suggested to underlie the benecial inuence of T cells and IL-4 on spatial memory (Derecki et
al., 2010). Finally, several studies established that astrocyte energy
metabolism is involved in memory consolidation and in the inuence of noradrenergic mechanisms on hippocampal-dependent
memory (Gibbs et al., 2008, 2006; Gibbs and Hertz, 2008).
The specic impairment in hippocampal-dependent memory
exhibited by IL-1rKO mice may be related to their defected ability
to sustain LTP (Avital et al., 2003; Goshen et al., 2009). Indeed, in
the present study IL-1rKO mice displayed reduced induction and
maintenance of LTP, which was partially rescued by intrahippocampally transplanted WT-derived NPCs. This restoration is not merely
due to a difference in the dynamic range of DG synapses, since all IL1rKO groups showed similar input/output relations. Former studies
implicated astrocytic GFAP and S-100b in regulation of LTP (Nishiyama et al., 2002; Tanaka et al., 2002). Furthermore, in both hippocampal cell cultures and slices, the induction of LTP was found to
depend on the presence of astrocytes and the secretion of D-serine
by these cells, which in turn binds to the glycine-site on neuronal
NMDA receptors and enables their critical role in LTP (Henneberger
et al., 2010; Yang et al., 2005, 2003). Astrocytes also mediate homeostatic synaptic scaling following prolonged inhibition of neuronal
activity, via secretion of the pro-inammatory cytokine tumor
necrosis factor-a (TNF-a) (Stellwagen and Malenka, 2006), which
is a known synaptic strength enhancer (Beattie et al., 2002).
Although it is not clear that all of these functions are directly related
to IL-1 signaling, these ndings, together with the results of the present study, indicate that the previously described roles of astrocytes
in neuronal functioning and synaptic transmission (Haydon and
Carmignoto, 2006; Jourdain et al., 2007; Newman, 2003; Perea and
Araque, 2007; Volterra and Meldolesi, 2005) have important functional implications for neural plasticity.
The downstream mechanisms that mediate the effects of astrocytic IL-1 signaling on memory and LTP are not known yet, but
could involve IL-1-induced modulation of the production and
secretion of several plasticity-related molecules in astrocytes,
including glutamate (Casamenti et al., 1999), neurotrophic factors,
such as BDNF (Meeuwsen et al., 2003), nitric oxide (Juttler et al.,
2007) and prostaglandins (Dayton and Major, 1996). We have recently demonstrated that although IL-1rKO mice do not display
any differences in the plasticity-related molecules GluR1 and synaptophysin, the spine size of their hippocampal neurons is smaller,
compared with WT animals (despite normal spine density in the
two strains) (Goshen et al., 2009). Moreover, we showed that exposure to environmental enrichment resulted in the normalization of
spine size in the IL-1rKO mice, concomitantly with complete recovery of memory functioning and LTP. Together with the results of
the present study, these ndings suggest that astrocytic IL-1 signaling may be involved in regulation of spine size, which is an
important mechanism of memory consolidation and neural plasticity (De Roo et al., 2008). Finally, since IL-1 is known to facilitate
glucose uptake into astrocytes (Vega et al., 2002), it is possible that
astrocytic IL-1 signaling is involved in the enhanced metabolic demands posed by the neural systems undergoing enhanced excitability and plasticity.
Impaired astrocytic functioning has been observed in many
medical conditions (Seifert et al., 2006). For example, reduced
number and altered morphology of astrocytes in several brain
areas, including the hippocampus, are among the most robust
pathophysiological nings in major depression (Bowley et al.,
2002; Cotter et al., 2001b; Ongur et al., 1998; Rajkowska and

O. Ben Menachem-Zidon et al. / Brain, Behavior, and Immunity 25 (2011) 10081016

Miguel-Hidalgo, 2007). Similarly, in animal models of depression

the number, density and somal volume of astrocytes within the
hippocampus and prefrontal cortex were markedly decreased
(Czeh et al., 2006; Leventopoulos et al., 2007), and chronic treatment with uoxetine completely revered these effects (Czeh
et al., 2006). Furthermore, astrocytic loss in the prefrontal cortex
(Banasr and Duman, 2008), as well as pharmacological blockade
of astrocytic glutamate uptake (Bechtholt-Gompf et al., 2010; Lee
et al., 2007) were found to be sufcient to induce depressive-like
behaviors. Other medical conditions, particularly neurodegenerative diseases such as Alzheimers disease, are characterized by
astrocytic over-activation and hypertrophy (Mrak and Grifn,
2005), which can also lead to impaired astrocytic functioning (Rossi et al., 2005). Obviously, both in depression and in Alzheimers
disease learning, memory and synaptic plasticity are severely impaired (Duman et al., 2000; Selkoe, 2002). Therefore, procedures
aimed at restoration of normal astrocytic functioning, in general,
and astrocytic IL-1 signaling, in particular, should be considered
as potential therapeutic targets in these diseases.
Conict of interest statement
None declared.
Acknowledgments
This study was supported by grants from the Israel Science
Foundation (Grant No. 295/07 to RY), by the legacy heritage biomedical program of the Israel Science Foundation (Grant No. 430/
09), and the Israel Foundations Trustees (OBM and RY).
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