NT-3 facilitates hippocampal plasticity and learning
and memory by regulating neurogenesis
Kazuhiro Shimazu,1Mingrui Zhao,1Kazuko Sakata,1Schahram Akbarian,2,3
Brian Bates,2,4Rudolf Jaenisch,2and Bai Lu1,5
1Section on Neural Development and Plasticity, National Institute of Child Health and Human Development, National Institutes
of Health, Bethesda, Maryland 20892, USA;2Whitehead Institute for Biomedical Research, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02142, USA
In the adult brain, the expression of NT-3 is largely confined to the hippocampal dentate gyrus (DG), an area
exhibiting significant neurogenesis. Using a conditional mutant line in which the NT-3 gene is deleted in the brain,
we investigated the role of NT-3 in adult neurogenesis, hippocampal plasticity, and memory. Bromodeoxyuridine
(BrdU)-labeling experiments demonstrated that differentiation, rather than proliferation, of the neuronal precursor
cells (NPCs) was significantly impaired in DG lacking NT-3. Triple labeling for BrdU, the neuronal marker NeuN,
and the glial marker GFAP indicated that NT-3 affects the number of newly differentiated neurons, but not glia, in
DG. Field recordings revealed a selective impairment in long-term potentiation (LTP) in the lateral, but not medial
perforant path-granule neuron synapses. In parallel, the NT-3 mutant mice exhibited deficits in spatial memory tasks.
In addition to identifying a novel role for NT-3 in adult NPC differentiation in vivo, our study provides a potential
link between neurogenesis, dentate LTP, and spatial memory.
One of the two regions where adult neurogenesis occurs is at the
dentate gyrus (DG) of the hippocampus (Gage 2000). The gen-
eration of new neurons in the adult dentate is a multistep pro-
cess. Initially, stem cell-derived progenitor cells proliferate in the
subgranular zone of the DG. These cells then migrate into the
granule cell layer, where they differentiate into neurons and glial
cells (Kempermann et al. 2004a). During the process, some of the
differentiated and undifferentiated cells die. A subset of surviving
new neurons, however, gradually develop functional neuronal
properties, form new synapses, and integrate into the existing
neural network (Song et al. 2002; van Praag et al. 2002). Defining
diffusible factors that regulate this process constitutes one of the
most active areas in neural stem cell biology (Gage 2000; Gould
and Gross 2002).
Neurotrophin-3 (NT-3, also known as Ntf3) may serve as an
excellent candidate as a diffusible factor that regulates dentate
neurogenesis. The expression of the NT-3 gene in the adult brain
is highly confined to the dentate gyrus (Maisonpierre et al. 1990;
Friedman et al. 1991; Lauterborn et al. 1994). Elevated neuronal
activity, either by LTP-inducing stimuli or ischemia, enhances
the expression of NT-3 mRNA in the dentate (Lindvall et al. 1992;
Bramham et al. 1996). Cell culture experiments showed that
NT-3 antagonizes the proliferative effects of basic fibroblast
growth factor (bFGF) on progenitor cells, and enhances the dif-
ferentiation of newborn neurons (Ghosh and Greenberg 1995;
Vicario-Abejon et al. 1995; Barnabe-Heider and Miller 2003). In
addition, dietary restriction enhanced the expression of NT-3 in
dentate gyrus, paralleling the increase in the number of newborn
neurons (Lee et al. 2000). However, direct evidence for the role of
endogenous NT-3 in the proliferation, survival or differentiation
of hippocampal progenitor cells in vivo is lacking.
The functional significance of adult neurogenesis remains
one of the key unsolved issues in neuroscience (Fuchs and Gould
2000; Kempermann et al. 2004b). Although there may be a cor-
relation between neurogenesis and memory, the relationship re-
mains controversial (Gould et al. 1999b; Lu and Chang 2005).
Activities known to enhance learning and memory, such as ex-
ercise or living in an enriched environment, promotes neurogen-
esis in the adult hippocampus (Kempermann et al. 1997; Nilsson
et al. 1999; van Praag et al. 1999a,b; Cotman and Berchtold
2002). Learning itself has also been shown to enhance the sur-
vival of newly generated neurons in the dentate (Gould et al.
1999a). The most direct evidence so far was demonstrated in an
experiment in which inhibition of neurogenesis using a mitotic
inhibitor impaired hippocampal-dependent, but not hippocam-
pal-independent memory (Shors et al. 2001). Inhibition of neu-
rogenesis by radiation also impaired hippocampus-dependent,
place-recognition task, and spatial memory retention, but not
object-recognition task (Madsen et al. 2003; Rola et al. 2004). An
attractive hypothesis is that at least some forms of hippocampus-
dependent memory are mediated by the generation of new neu-
rons (Gould et al. 1999b; Kempermann 2002). How does this
model reconcile the conventional belief that hippocampal LTP is
a cellular model for hippocampus-dependent learning and
memory? It is possible that newly generated neurons could con-
tribute to the formation of new synapses, leading to alterations of
LTP in the dendritic network of the dentate granule neurons.
Indeed, running-enhanced neurogenesis is accompanied by a se-
lective increase in dentate LTP (van Praag et al. 1999a). Newly
generated granule neurons exhibit a lower threshold for LTP in-
duction, but the magnitude of LTP is smaller when stronger
stimulation is used (Schmidt-Hieber et al. 2004). The causal rela-
tionship between neurogenesis and LTP was implied in one study
showing that inhibition of NPC proliferation drastically reduced
LTP in the medial perforant path-dentate synapses (Snyder et al.
2001). It is unclear, however, whether inhibition of neurogenesis
Present addresses:3Brudnick Neuropsychiatric Research Institute,
Department of Psychiatry, University of Massachusetts Medical
School, Worcester, MA 01613, USA;4Wyeth Research, Applied Ge-
nomics, Cambridge, MA 02140, USA.
E-mail email@example.com; fax (301) 496-1777.
Article published online before print. Article and publication date are at http://
13:307–315 ©2006 by Cold Spring Harbor Laboratory Press ISSN 1072-0502/06; www.learnmem.org
Learning & Memory
could alter synaptic plasticity in the existing synaptic network,
and if so, whether such alterations have behavioral consequences.
In the present study, the role of neurogenesis in hippocam-
pal LTP and learning and memory was examined using a line of
NT-3 conditional knockout mice, in which the NT-3 gene is se-
lectively deleted in the brain throughout development. We
tested whether NT-3 is involved in the proliferation or differen-
tiation of progenitor cells in the dentate. We further investigated
the role of neurogenesis in synaptic plasticity of specific synaptic
pathways of dentate neurons and the underlying mechanisms.
Finally, we examined the behavior deficits of the mutant mice.
Our results suggest that NT-3 specifically regulates the differen-
tiation, but not the proliferation of neuronal precursor cells in
the dentate. Such regulation appears to be important for LTP in
the lateral, but not medial perforant path-dentate synapses.
These results may help us to understand how neurogenesis could
contribute to long-term synaptic plasticity and memory.
An NT-3 conditional mutant mouse, created using the cre/loxP
system as previously described (Bates et al. 1999, 2002; Akbarian
et al. 2001), was used. To ensure that Nes-Cre is expressed in the
hippocampus, we crossed Nes-Cre transgenic mice with a Rosa-
26 reporter line that expresses ?-galactosidase upon Cre-mediated
removal of a stop codon (Soriano 1999). Strong expression was
detected in the granule cell layer of the dentate gyrus and CA1–
CA3 regions of the hippocampus (Fig. 1A). Southern blot was
performed using cortical and hippocampal tissues from various
genotypes. We generated mice with different genotypes by mat-
ing as previously described (Bates et al. 1999). We used the fol-
lowing nomenclature for the NT-3 alleles: Recombined: NT-31lox;
Floxed: NT-32lox; Wild-type: NT-3+(Bates et al. 1999). Thus, the
genotype of conditional mutant is NT-31lox/2lox, Nes-cre+/0. We
used two types of mice as controls, i.e., NT-3+/+, Nes-cre0/0, which
is the wild-type and NT-32lox/2lox, Nes-cre0/0, which is the floxed
animals. In the hippocampus, the homologous recombination
was more than 80% completed in the mutant mice (Fig. 1B).
Moreover, Northern blot revealed that NT-3 transcripts in the
hippocampus from the NT-31lox/2lox, Nes-Cre0/0mice were essen-
tially undetectable (Fig. 1C). NT-3 ELISA was used to measure the
levels of NT-3 protein in adult hippocampus. We detected
5.35 ? 0.28 pg/20 µg, and 15.50 ? 1.17 pg/100 µg NT-3 protein
from the wild-type tissues, but none from the mutant tissues.
Proliferation and survival of BrdU-positive cells
in dentate gyrus
Brains from our conditional mutant mice appeared normal (data
not shown) except for a defect in cerebellar foliation, which is
consistent with previous reports (Bates et al. 1999, 2002; Akbar-
ian et al. 2001). Examination of hippocampal sections did not
reveal any gross morphological abnormality (Fig. 2A). Since the
endogenous NT-3 gene expression in adult was relatively con-
fined to the dentate gyrus of the hippocampus (Maisonpierre et
al. 1990; Lauterborn et al. 1994), we examined cytoarchitectural
organization of this region in some detail. The volumes of the
granule cell layer (GCL), hilus, the whole dentate gyrus (DG), or
in adult hippocampus. (A) Nestin-Cre mediated recombination in adult
hippocampus (2-mo old). Coronal section from P2.5 animal [Nes-Cre/
+(male) X floxedROSA-26ref/+(female)] stained by X-gal (dark blue) to
detect cre-mediated recombination at the ROSA-26 locus and counter-
stained with Neutral red. Note the widespread and complete or near-
complete recombination in hippocampus and cerebral cortex. (B) South-
ern blot showing Cre-mediated recombination in the hippocampus and
cortex of adult mutant mice. DNA samples were prepared from brain
tissues of mice of different genotypes. (Lane 1) NT-31lox/2lox, Nes-cre+/0;
(lane 2) NT-32lox/+, Nes-cre+/0; (lane 3) NT-31lox/2lox, Nes-cre0/0; (lane 4)
NT-31lox/2lox, Nes-cre+/0; (lane 5) NT-32lox/+, Nes-cre+/0. Arrows: +: Wild-
type allele, or NT-3+; f: Floxed allele, or NT-32lox; R: knockout allele, or
NT-31lox. (C) Northern blot analysis of mRNAs isolated from adult hippo-
campi of different genotypes. (Lane 1) NT-3+/+, Nes-cre0/0; (lane 2) NT-
3+/+, Nescre+/0; (lane 3) NT-31lox/2lox, Nes-cre+/0; (lane 4) NT-31lox/2lox, Nes-
cre0/0; (lane 5) NT-3+/?, Nes-cre0/0; (lane 6) NT-32lox/+, Nes-cre+/0. (Bot-
tom) rRNA loading control is shown below. Arrows point to the positions
of 18S and 28 S rRNAs.
Cre-mediated recombination and ablation of the NT-3 gene
mice. (A) Low power (4?) images of hippocampus from an NT-3 mutant
mouse. H&E staining reveals no abnormality of morphology of the hip-
pocampus. (B) Quantitative measurement of the volumes of the subre-
gions of hippocampus. The volumes of granule cell layer (GCL), the hilar
region, the whole dentate gyrus (DG), and the whole hippocampus were
measured. n = 6 pairs of male mice; 2-mo old. (C) Summary of cell
counts. Total number of granule cells in DG is presented. n = 5 pairs of
2-mo-old male mice.
Normal morphology of adult hippocampus in NT-3 mutant
Shimazu et al.
308Learning & Memory
the whole hippocampal region (Hippo) in the mutant mice were
almost identical to that of wild-type control (Fig. 2B). For cell
counting, hippocampal sections were stained by the H&E
method. Stereological analyses showed that there was no statis-
tical difference in the number of granule neurons per dentate
gyrus between wild-type and mutant mice (Fig. 2C). Thus, inac-
tivation of the NT-3 gene does not result in obvious anatomical
changes in the adult hippocampus.
Since NT-3 has been implicated in neurogenesis (Ghosh and
Greenberg 1995; Vicario-Abejon et al. 1995), we asked whether
deletion of the NT-3 gene could affect the proliferation or sur-
vival of neuronal precursor cells (NPCs) in adult dentate. To ex-
amine NPC proliferation, we pulse labeled the proliferating NPCs
by injecting intraperitoneally bromodeoxyuridine (BrdU) 3 h be-
fore the brains were processed for BrdU immunohistochemistry.
In this method, only a fraction of dividing cells were labeled, but
the labeled cells would not have sufficient time to undergo more
than one round of mitosis. BrdU(+) cells were found in both
wild-type and mutant dentate, mostly beneath the granule cell
layer with a few also in the hilus (Fig. 3A). There was no differ-
ence in the number of proliferating cells between wild-type and
mutant mice (Fig. 3B). To examine the survival of the BrdU-
labeled NPCs, we injected BrdU twice daily for 10 d so that a large
proportion of dividing NPCs were labeled. In the remaining 2 wk
after the last BrdU injection, many NPCs underwent apoptosis.
The surviving NPCs were detected by BrdU immunohistochem-
istry. We found that there was a small decrease in the number of
BrdU(+) cells in the granule cell layer of the mutant mice, albeit
barely significant compared with the wild-type mice (Fig. 3C)
(P = 0.08, t-test). No difference was detected in the hilus area.
Taken together, these results suggest that NT-3 may regulate the
survival, but not the proliferation, of NPCs in the dentate.
To determine whether NT-3 differentially affects the differ-
entiation of the surviving NPCs into neurons and glia, we per-
formed triple staining using the same sections derived from the
NPC survival study described above. BrdU was detected by a rat
anti-BrdU antibody (green). A mouse anti-NeuN antibody (red)
was used to label mature neurons, while a rabbit anti-GFAP an-
tibody (purple) was used to label astrocytes. Fluorescence micros-
copy using three different wavelengths showed that BrdU, NeuN,
and GFAP could be clearly detected in the same section (Fig. 4A).
Cell counting using stereological techniques showed that the
number of BrdU(+) neurons was significantly reduced, while the
number of BrdU(+) astroglia was not changed in the R/f, + mu-
tant mice (Fig. 4B). Pie plots indicated that in the R/f, + mutant,
the decrease in the number of neurons was accompanied by an
increase in the number of undifferentiated cells (labeled “Nei-
ther” in Fig. 4C). These results suggest that in addition to NPC
survival, NT-3 may affect maturation, or fate determination (to
become a neuron or a glia) of NPCs.
Synaptic transmission and plasticity
NT-3 has been shown to both induce differentiation of hippo-
campus-derived NPCs and to facilitate synaptogenesis in cultured
hippocampal neurons (Vicario-Abejon et al. 1998, 2000). We
therefore examined whether the deficits in the survival and/or
differentiation of dentate NPC in NT-3 mutant mice would result
in impairments in LTP. Two major sets of excitatory synapses on
the dendrites of dentate granule neurons were studied, i.e., one
made by lateral perforant path (LPP) and the other by medial
perforant path (MPP). By carefully positioning the stimulating
and recording electrodes, we could induce LTP at LPP and MPP
synapses separately (Fig. 5A). Application of tetanic stimulation
(4 ? 0.5 sec, 50 Hz, 30-sec interval) to MPP induced a robust LTP
in both wild-type and mutant MPP synapses, with very similar
magnitudes (Fig. 5B, bottom). In contrast, the LTP recorded at
LPP synapses in mutant mice exhibit a severe deficit, as com-
pared with that seen in wild-type mice (Fig. 5B, middle). The
magnitude of LTP measured 60 min after tetanus (4 ? 0.5 sec, 50
Hz, 30-sec interval) was 156.3 ? 9.6% in wild-type mice, but
121.2 ? 9.1% in R/f, + mice, respec-
tively (P < 0.001, t-test). As a control, we
also recorded LTP at the Schaffer collat-
eral-CA1 synapses. Tetanic stimulation
(2 ? 1 sec, 100 Hz, 20-sec interval) in-
duced identical LTP in wild-type and
mutant mice (Fig. 5B, top). Thus, dele-
tion of NT-3 gene selectively impairs
LPP-granule cell synapses in hippocam-
LTP at dentate synapses is facili-
tated by inhibiting GABAAreceptor-
mediated transmission (Hanse and Gus-
tafsson 1992). Since NT-3 has been
shown to attenuate GABAergic transmis-
sion in relatively mature neurons (Kim
et al. 1994), one potential mechanism by
which NT-3 enhances LTP is to suppress
GABAergic inputs to the granule neu-
rons. If the LPP–LTP deficit seen in NT-3
mutant mice was due to an increased
GABAergic inhibition, one would expect
no difference between wild-type and
mutant mice when GABAergic transmis-
sion is completely blocked. To test this
possibility, we recorded LPP–LTP in the
presence of GABAAantagonist bicucul-
line (10 µM). Unlike MPP synapses re-
corded in the submerged chamber
(Snyder et al. 2005), application of bicu-
culline did not seem to dramatically in-
images of BrdU-labeled cells in the wild-type (top) and mutant (bottom) dentate gyrus. The sections
were processed for BrdU immunohistochemistry followed by Nissl counter stain. (B) Effect on prolif-
eration of NPCs. Three hours after BrdU was injected intraperitoneally, the brains of the injected
animals were fixed and processed for BrdU histochemistry. The number of BrdU (+) cells in the hilus
and granule cell layer (GCL) were counted using sterological techniques. n = 3 pairs of male mice;
4-mo old. (C) Effect on survival of NPCs. BrdU was injected twice daily for 10 d, and BrdU immuno-
histochemistry was performed 2 wk after the last BrdU injection. Cells were counted the same way as
B. n = 6 pairs of male mice; 2-mo old.
Effects of NT-3 deletion on proliferation and survival of NPC. (A) Medium-power (20?)
Role of NT-3 in neurogenesis and synaptic plasticity
Learning & Memory
crease the magnitude of LTP in LLP synapses recorded in the
interface chamber (Fig. 5C). Surprisingly, the difference in LTP
between the wild-type and mutant mice was much bigger when
slices were treated with bicuculline, as compared with that with-
out bucuculline (Fig. 5B,C). As an additional control, we recorded
LTP in the floxed mice, which contains floxed NT-3, but not
Cre-recombinase (NT-32lox/2lox, Nes-cre0/0). The LTP in these mice
were almost identical to that in wild-type mice (Fig. 5C). Given
that NT-3 has also been shown to enhance GABAergic transmis-
sion, which in developing neurons is excitatory rather than in-
hibitory (Gao and van den Pol 1999), deletion of the NT-3 gene
may reduce the excitatory effects of GABA, leading to impair-
ments of LTP at the LPP synapses made by the NPC-derived cells.
We performed a series of experiments to investigate the po-
tential mechanisms underlying the LTP deficit seen at LPP syn-
apses. First, we examined the basal synaptic transmission. Field
EPSPs were evoked by stimulating LPP with increasing stimulus
intensity. The slope of EPSPs was plotted against stimulus inten-
sity to establish input–output relationships. No difference was
observed in the input–output curves in wild-type and mutant
synapses (Fig. 6A), suggesting that the deletion of the NT-3 gene
does not alter basal synaptic transmission. Next, we examined
post-tetanic potentiation (PTP), a phenomenon thought to be
due to enhanced presynaptic transmitter release (Zucker 1989).
PTP was induced by tetanic stimulation in the presence of NMDA
receptor antagonist APV. The magnitude of PTP at 1 min after
tetanus, defined as a percentage increase of the average EPSP
slope over baseline, was significantly reduced in slices from R/f, +
mice (129.1 ? 6.0%), as compared with the ones from +/+ ani-
mals (148.1 ? 6.9%, Fig. 6C). Since changes in PTP reflect pre-
synaptic problems, we further examined paired-pulse facilitation
(PPF), a form of plasticity that reflects changes in the probability
of transmitter release (Zucker 1989). PPF was measured as the
ratio of two consecutive EPSP slopes. At the LPP synapses, the
ratio is usually greater than one. PPFs at
interpulse intervals of 10, 20, and 50
msec were markedly reduced in the R/f,
+ mice as compared with wild-type mice
(Fig. 6B), suggesting an alteration in pre-
synaptic properties. This result is consis-
tent with previous reports, which used
NT-3+/?mice (Kokaia et al. 1998; Asztely
et al. 2000). Finally, to determine
whether NT-3 gene deletion also affects
the number of synaptic vesicles in the
reserved and readily releasable pools, we
measured synaptic responses to a pro-
longed, low-frequency stimulation (LFS)
and a brief, high-frequency stimulation
(HFS), respectively (Cabin et al. 2002). As
shown in Figure 6, D and E, no differ-
ences were observed between wild-type
and mutant mice in either response.
These results suggest that the deletion of
the NT-3 gene does not alter synaptic
To determine whether the impaired den-
tate neurogenesis and deficits in LPP–
LTP described above are associated with
behavioral consequences, we subjected
NT-3 mutant mice to the Morris water-
maze test. Briefly, mice were allowed to
swim in a circular pool filled with
opaque water and trained to find a hid-
den platform, which was kept at a constant location, using ex-
ternal visual cues. To test the learning capability, we measured
escape latency (time to find the hidden platform), which became
progressively shortened during a 7-d training period (twice daily)
in wild-type mice (Fig. 7A). In marked contrast, the NT-3 mutant
mice exhibited significantly longer latency times to find the hid-
den platform (Fig. 7A), indicating that they had a severe problem
learning. Overall, the escape latency of NT-3 mutant mice im-
proved much slower relative to wild-type mice during the 7 d of
At the end of the learning period, the platform was removed
and the animals were given a 60-sec probe trial to test their
memory, which was measured by the percentage of time they
spent in the platform quadrant (Fig. 7B). When the probe trial
was administered 3 d after the end of training, wild-type mice
spent more time in the correct quadrant (platform, 43%) than all
other quadrants. In contrast, NT-3 mutant mice spent only 9.1%
of their time in the platform quadrant (Fig. 7B), indicating that
their ability to remember the correct quadrant was severely com-
promised. Interestingly, the mutant mice also spent more time in
the left quadrant (40%). It is unclear whether this reflects a trend
in which the mutant animals tend to stay in areas closer to the
platform. The memory capacity can also be reflected by the num-
ber of times the animals crossed the platform area and the dis-
tance the animals traveled in the correct quadrant after the plat-
form was removed. Figure 7C depicts examples of the swimming
tracks by a wild-type and a mutant mouse. The wild-type mouse
swam a significantly longer distance in the correct quadrant than
the mutant mouse. Moreover, the wild-type mouse crossed the
platform areas about six times in a minute, while the mutant
crossed only once per minute.
Recently, NT-3 mutant mice generated by a different cre/
loxP system were shown to have defects in geniculocortical pro-
jections, leading to impairments in visual function (Ma et al.
injected with BrdU twice/day for 10 d. Two weeks later, the animals were processed for triple labeling
for BrdU (green), NeuN (red), and GFAP (blue), using confocal microscopy. (A) Confocal images of
BrdU-positive neurons (left) and astroglia (right). (B) Quantitative analysis of the number of
NPC-derived neurons (left) and that of glia (right) in the dentate of control and mutant mice. n = 6
pairs of male mice; 2-mo old. (*) Significantly different between control and mutant mice, P < 0.01,
Student’s t-test, two-tailed. (C) Relative distribution of cells derived from NPCs. n = 6 pairs of male
mice; 2-mo old.
Effect of NT-3 deletion on NPC-derived neurons and glia. Wild-type and mutant mice were
Shimazu et al.
310 Learning & Memory
2002). Using the same visual cliff avoidance test, we found no
such impairment in visual function in our mutant mice. Both
wild-type and mutant mice spent most of their time on the cov-
ered (bench) side of the visual cliff box without crossing the cliff
edge. The time spent on the open (suspended) side was
17.8 ? 15.3% for NT-3 mutant and 27.7 ? 12.8% for control
mice (n = 5 pairs, P > 0.05). These results suggest that our NT-3
mutant mice have relatively normal visual function. We also
measured locomotor activity of the mice using a Digiscan activity
monitor (a 16-inch square chamber equipped with two rows of
16 ? 16 infrared sensors). These sensors were used to localize the
animal’s floor position on an x-y plane, and automatically mea-
sure their horizontal and vertical movements. As shown in Figure
7D, there was no statistical difference in any of the parameters
measured between the wild-type and NT-3 mutant mice. Specifi-
cally, the mutant mice did not show any abnormality in total
distance traveled, horizontal and vertical beam breaks, or time
spent at the edge and center of the box. Thus, the cognitive
deficits in NT-3 mutant mice are not likely to be accounted for by
motor or visual deficits.
NT-3 stimulates NPCs to differentiate into neurons in culture
(Ghosh and Greenberg 1995; Vicario-Abejon et al. 1995). Dele-
tion of the NT-3 receptor TrkC reduces the number of entorhinal
afferents and the density of synaptic contacts in postnatal den-
tate (Martinez et al. 1998). However, since BrdU-labeling experi-
ments were not performed, it is unclear whether the synaptic
effects were due to facilitation of differentiation of NPC-derived
neurons by NT-3. Our study demonstrates that the number of
BrdU-labeled dentate cells in the NT-3 mutant mice are reduced.
More importantly, of the surviving BrdU cells in the NT-3 mu-
tants, the number of NeuN-labeled neurons, but not GFAP-
labeled glia, was significantly decreased, while the number of
undifferentiated cells was increased. These results suggest that
NT-3 is required to stimulate NPCs to differentiate into new neu-
rons in the adult dentate in vivo.
An important, yet unresolved issue in the neural stem-cell
field is the biological function of adult neurogenesis (Kemper-
mann 2002; Kempermann et al. 2004b). It has long been specu-
lated that adult neurogenesis is involved in learning and memory
(Gould et al. 1999b), but how newly generated neurons contrib-
ute to learning and memory is not known. Inhibition of NPC
proliferation in the adult dentate by either acute radiation (Mad-
sen et al. 2003; Rola et al. 2004) or mitotic inhibition using MAM
(Shors et al. 2001), leads to significant impairments in hippocam-
pus-dependent memory. Interestingly, spatial learning, as as-
sessed by the escape latency in the water-maze training, was not
affected. Two caveats are noted. First, drugs such as MAM may
interfere with protein synthesis or signaling mechanisms re-
quired for memory, or cause cytotoxic effects, and radiation may
damage the stem-cell niche in the subgranular zone (Monje et al.
2002). Second, the mitotic inhibitor or radiation affects only the
proliferation, but not differentiation, of the NPCs. In this study,
we show that deletion of the NT-3 gene affects the differentia-
tion, but not proliferation of NPCs. This apparently affects both
learning and memory, as assessed by spatial navigation tests.
Similarly, mice lacking methyl-CpG-binding protein 1 (MBD1)
show deficits in NPC differentiation but not proliferation, as well
as impairments in both learning and memory in water-maze tests
(Zhao et al. 2003). These results raised the interesting possibility
that the differentiation, but not proliferation of NPCs might
somehow be involved in the spatial learning process.
recording electrodes in CA1 (top) and DG (bottom). (B) LTP recordings in the CA1 region (top), the lateral perforant path (LPP) (middle), and the medial
perforant path (MPP) (bottom). Tetanic stimulation was applied at time “0”. Data from multiple slices (n) were pooled and expressed as mean ? SEM.
(C) LTP deficit in LPP synapses in the presence of GABAa antagonist bicuculline (10 µM). Mice with the NT-3 gene floxed (NT-32lox/2lox, Nes-cre0/0,
indicated as flox/flox) were studied as an additional control. Number of mice (N) and slices (n) are indicated in the figure. All mice used in electro-
physiology and behavior experiments were between 1.5 and 2.5-mo old.
NT-3 regulates synaptic plasticity at a subset of hippocampal synapses. (A) Schematic showing the arrangement of stimulating and
Role of NT-3 in neurogenesis and synaptic plasticity
Learning & Memory
The present study also provides a potential connection be-
tween adult neurogenesis and dentate LTP, and therefore a pos-
sible mechanism by which neurogenesis could contribute to hip-
pocampus-dependent learning and memory. Since dentate LTP is
believed to be critical for hippocampus-dependent memory
(Richter-Levin et al. 1995; Abraham and Williams 2003), it is
conceivable that adult neurogenesis participates in hippocam-
pus-dependent learning and memory by regulating LTP at syn-
apses in the dentate gyrus. Consistent with this idea, Wojtowicz
and colleagues have shown that inhibition of neurogenesis by
radiation selectively blocks ACSF-LTP in MPP, a form of LTP at-
tributable to newborn young neurons (Snyder et al. 2001). The
MBD1 mutant mice exhibit a parallel reduction in neurogenesis
and an impairment in LTP at the MPP synapses (Zhao et al.
2003). We have now shown that deficits in NPC differentiation
caused by a deletion of the NT-3 gene in the brain impairs LTP
selectively at the LPP synapses. While we do not know whether
the LPP–LTP is also impaired in the MBD1?/?mice, the selective
impairment of LTP at the lateral, but not medial perforant path
in the NT-3 mutant mice could be explained by a number of
mechanisms. One possibility is that NT-3 may be required for
dendritic growth and branching. Deletion of the NT-3 gene may
therefore prevent newborn granule cells from extending their
apical dendrites beyond MPP into LPP. Alternatively, NT-3 may
selectively regulate the growth of LPP afferents. The deficits in
PPF and PTP at the LPP synapses in the NT-3 mutant mice sup-
port this notion. Regardless of the mechanism, the normal LTP
seen at the CA1 synapses in the NT-3
mutant mice suggest that NT-3 does not
have a general effect on synapse forma-
tion in vivo. Consistent with this, LTP at
the CA1 synapses was normal in another
line of NT-3 conditional mutant mice
that were generated by crossing NT-3
floxed mice with Cre transgenic mice,
where the Cre gene was controlled by
the synapsin I promoter to restrict ex-
pression to postnatal neurons (Ma et al.
1999). Finally, it should be pointed out
that our results, albeit more specific than
radiation or drug treatment, are still cor-
relative in nature. DG-specific deletion
of the NT-3 gene in adult mice should
provide further insights into the rela-
tionship between neurogenesis, dentate
plasticity, and memory.
While we interpret our results as
indicating that neurogenesis plays an
important role in dentate LTP, an alter-
native is that NT-3 facilitates LPP–LTP
directly. The effect of NT-3 on hippo-
campal plasticity has been examined by
a number of groups. NT-3+/?mice ex-
hibit essentially normal short- and long-
term plasticity at the dentate synapses
(Asztely et al. 2000; Olofsdotter et al.
2000). The only deficit observed was a
reduction in PPF at the LPP synapses
(Kokaia et al. 1998; Asztely et al. 2000).
An important observation is that acute
application of recombinant NT-3 or
anti-NT-3 antibodies had no effect on
LTP at the CA1 synapses (Figurov et al.
1996; Chen et al. 1999). These results
suggest that NT-3 does not modulate
synaptic transmission or plasticity di-
rectly. Although we cannot rule out a developmental effect of
NT-3 on NPCs, since the NT-3 gene is deleted during develop-
ment, the fact that the DG volume and the total number of
granule neuron were not changed suggests that NT-3 is not es-
sential for embryonic neurogenesis or the survival of the DG
granule neurons during development.
Assuming adult neurogenesis does regulate dentate LTP,
how do newly generated neurons contribute to hippocampus-
dependent memory? An attractive hypothesis is that new neu-
rons have special properties that allow them to integrate into the
existing neural network, leading to an alteration of synaptic plas-
ticity. It has been shown that young neurons form immature
synapses at MPP (Wang et al. 2000; Snyder et al. 2001). These
synapses exhibit lower threshold for LTP induction, but the mag-
nitude of LTP is smaller when stronger stimulation is used
(Snyder et al. 2001; Schmidt-Hieber et al. 2004). Newly generated
neurons form new synapses, which are integrated into the exist-
ing neural network (Kempermann 2002). Our field-recording ex-
periments showed that LTP in the LPP, but not MPP, was selec-
tively impaired in the NT-3 mutant mice. Why alteration of NPC
differentiation affects only LPP but not MPP synapses is not
known. An important part of the neuronal differentiation pro-
gram is to grow elaborate dendritic trees. We speculate that the
newly generated neurons in the mutant mice may grow limited
dendritic trees that fail to appropriately contact the LPP. In ad-
dition, the synaptic connections made by the lateral entorhinal
afferents may also be weak. It is conceivable that impairments in
bers of slices recorded are indicated in the respective figures. (A) Input–output curves. No difference
was observed between control and mutant mice. (B) Paired pulse facilitation (PPF). NT-3 mutant mice
exhibit a substantial reduction in PPF at shorter interstimulus intervals. (C) Post-tetanic potentiation
(PTP). PTP was induced by a tetanus (100 Hz, 1 sec) in the presence of NMDA receptor blocker (Apv,
100 µM). There was a small, but significant decrease in PTP in the NT-3 mutant mice. (D) Synaptic
fatigue. Synaptic responses to a brief, high-frequency stimulation (HFS, 100 Hz, 20 pulses) were
recorded. The slopes of all EPSPs were normalized to that of the 2nd EPSP. (E) Synaptic response to a
prolonged, repetitive stimulation (LFS, 300 stimuli at 14 Hz, 280 pulses).
Impairments in presynaptic function of LPP-dentate synapses in NT-3 mutant mice. Num-
Shimazu et al.
312Learning & Memory
the formation of new synapses contribute to the deficits in field
LPP–LTP. Further experiments are necessary to test these ideas.
Materials and Methods
NT-3 conditional knockout mice
An NT-3 loxP line was crossed with a transgenic line, in which Cre
gene expression was under the control of the Nestin promoter, as
previously described (Bates et al. 2002). The following nomen-
clature was used in the present study. Wild-type, NT-3+= +;
Floxed, NT-32lox= f; Recombined, NT-31lox= R. Cre positive: +; Cre
negative, 0. For mating, we crossed NT-31lox/2lox, Nes-cre+/0males
with NT-32lox/2lox, Nes-cre0/0females to generate NT-31lox/2lox, Nes-
cre+/0offspring. We also crossed NT-32lox/2lox, Nes-cre0/0males with
NT-32lox/2lox, Nes-cre0/0females to generate more NT-32lox/2lox, Nes-
cre0/0offspring. A third type of mating used animals of purely
wild types, with the same genetic background as the first two. In
general, NT-31lox/2lox, Nes-cre+/0(MT) and wild-type (WT, in the
same genetic background) mice were
used for experiments. For some experi-
ments, NT-32lox/2lox, Nes-cre0/0(floxed, in
the same genetic background) mice were
used as further control. Only male ani-
mals were used for experiments. Geno-
typing, Southern and Northern blotting,
and X-gal staining were performed as de-
scribed (Bates et al. 2002). In all experi-
ments, male, 2–4-mo-old mice were used.
BrdU (Sigma) was dissolved in 0.9%
NaCl and sterile filtered through 22-µm
filters. The mice received a single dose of
BrdU at 100 µg/gm of body weight from
a concentration of 10 mg/mL, one intra-
peritoneal injection per day for 10 con-
secutive or for 1 d. Three hours or 2 wk
after the last injection of BrdU, the mice
were perfused transcardially with 4%
paraformaldehyde in 0.1 M phosphate
buffer. The brains were post-fixed over-
night, followed by transferring to 30%
sucrose solution. The brain was
mounted on a pedestal using Tissue-Tek
O.C.T. compound and cut at 40-µm sec-
tions using a cryostat. The sections were
stored at ?80°C until use. The 3-h time
point was selected because it is sufficient
for BrdU uptake but not for completion
of mitosis or migration.
All staining was done on free-floating
40-µm sections that were derived from
brains of BrdU-injected animals. To
count the number of BrdU-positive cells,
BrdU staining was performed using the
ZYMED BrdU staining protocol (Zymed
Laboratories Inc.) as described previ-
ously (Zhou et al. 2003). Antibodies used
for immunofluorescent triple labeling of
BrdU, NeuN, and GFAP, were rat anti-
BrdU (Accurate, Harlan Sera-Lab), 1:100;
mouse anti-NeuN (Chemicon), 1:200;
and rabbit anti-GFAP (Chemicon),
1:200, respectively. The fluorescent sec-
ondary antibodies used were anti-rat
Alexa Fluor 488, anti-mouse Alexa Fluor
568, and anti-rabbit Alexa Fluor 647
(Molecular Probe), 1:250.
Images were acquired using an Olympus IX 70 microscope
equipped with a video camera and the IPlab software (Scanalyt-
ics). A 40-µm 1-in-6 series of section from mouse was used to
quantify the area of hippocampus and the number of positive
cells using stereological methods combined with appropriate cor-
rection procedures (Gundersen et al. 1988; Coggeshall and Lekan
1996; Guillery and Herrup 1997; Kempermann et al. 1998). The
granule cell areas of each section were carefully traced in granule
cell layer (GCL), hilus, dentate gyrus, and whole hippocampus.
The total areas multiplied the sampled distance to determine the
reference volume. For cell counting, histological staining was
performed on sections using hematoxylin and eosin (H&E) stain-
ing method. The total granule cell number was estimated by the
mean positive cell number multiplied by the reference volume.
Triple-labeled fluorescence images were acquired using
BioRad MRC 1024 confocal microscopy and Lasersharp 2000
software (Bio-Rad Laboratories). In total, 100 ∼ 200 BrdU-posi-
tive cells per animals were analyzed for coexpression of BrdU
The animals were trained twice a day for 7 d to find the hidden platform. The latency to find the
platform is plotted against the training days. The wild-type mice rapidly learned the location of the
submerged platform, whereas the mutant mice showed a much slower learning curve (ANOVA,
P < 0.05). (B) Deficits in spatial memory. The probe test conducted at the end of training period. The
platform was removed and the animals were allowed to swim freely in the pool for 60 sec. The
percentage of time the animals spent in each of the four quadrants was measured. The mutant mice
spent less time in the platform quadrant than wild-type mice (ANOVA followed by post hoc test,
P < 0.002). (C) Representative swim paths illustrate the impairment of spatial cognition during the
probe trial in the mutant mice. (D) Open field activity. Locomotor activities were measured every 10
msec for 10 min. Unit of measurements: time, sec; distance, cm; beam break, number of times.
Deficits in spatial learning and memory in NT-3 mutant mice. (A) Deficits in acquisition.
Role of NT-3 in neurogenesis and synaptic plasticity
Learning & Memory
and NeuN for neuronal phenotype and GFAP for glial pheno-
Transverse hippocampal slices were prepared from 2-mo-old ani-
mals, maintained in an interface chamber for both recovery (>2
h) and recording, as described previously (Bramham and Sarvey
1996; Pozzo-Miller et al. 1999). Field EPSPs were evoked in CA1
stratum radiatum by stimulating Schaffer collaterals in CA1 re-
gion, or in molecular layer by stimulating perforant pathways in
DG with Teflon-insulated monopolar electrodes. A brief, high-
frequency stimulation (HFS, 100 Hz, 20 pulses) was used to study
docked vesicles, and a prolonged, low-frequency stimulation
(LFS, 14 Hz, 280 pulses) to study reserved pool vesicles. Tetanus
used to induce LTP: CA1 synapses, 100 Hz, 1 sec, two times with
a 20-sec interval; DG synapses, 100 Hz, 0.5 sec, four times, with
a 30-sec interval. Two points are worth mentioning with regard
to LTP in the dentate. First, unlike recordings obtained from the
submerged chamber (Snyder et al. 2005), the magnitude of LTP
from either lateral (LPP) or medial (MPP) perforant pathway in
the interface chamber was much bigger. We adjusted the posi-
tions of stimulating and recording electrodes to record fEPSPs for
MPP or LPP and intensity of tetanus to induce DG LTP, as de-
scribed previously (Dahl and Sarvey 1989). Second, unlike that in
the submerged chamber, the LTP recorded from LPP in the in-
terface chamber is not sensitive to inhibition of GABAergic trans-
mission (see Fig. 5C).
1. Morris water maze. A circular pool (diameter: 0.9 m) filled
with cloudy water was placed in a quiet room with easily
identifiable objects on the wall. A circular platform (12 cm in
diameter) was submerged 1.5 cm below the water surface and
placed in a specific location in the pool. Animals were sub-
jected to four trials a session, two sessions a day. A total of 6 d
of training were given. In each of the four trials, animals were
placed at four different starting positions equally spaced
around the perimeter of the pool in a random order. Animals
were allowed to find the platform within 120 sec, or else were
guided to the platform. At the end of training, each mouse was
given a probe trial (60 sec. with no platform). Memory func-
tion was evaluated by the amount of time spent on the correct
(platform) quadrant, the distance traveled, and the number of
times the animal crossed the platform area.
2. Visual cliff test. An open-topped box (60 ? 60 ? 30 cm3, with
clear Perspex bottom) was positioned on the edge of a bench
such that half of its base was suspended 1 meter above the
ground, creating a visual cliff. Mice were placed at the edge of
the cliff, and their activity monitored for 5 min by a video
camera. The percentage of time each mouse spent at the edge,
bench side, and open (suspended) area, and the direction in
which the first step outside of the edge, were measured.
3. Open-field activity. Animals were individually placed in a
Digiscan open-field box (RXYZCM, AccuScan Instruments,
Inc.) equipped with two rows of infrared beams that automati-
cally detected and quantified their activities every 10 msec for
10 min. This equipment took measurements of total distance
traveled, horizontal and vertical beam breaks, horizontal and
vertical time spent, resting time, edge and center time, edge
and center distance traveled, and time spent in the left-front,
left-rear, right-front, and right-rear areas.
We thank Eminy Lee of the Institute of Biomedical Sciences,
Academia Sinica, Taiwan, Republic of China, for initial water
maze tests of the mutant mice. We also thank Hongyuan Yan and
Vladmir Senatorov for genotyping and Daniel Abebe for behavior
experiments. We acknowledge the thoughtful comments and
suggestions of Drs. Newton Woo, Hyun-Soo Je, and Petti Pang. In
particular, we thank the late John Sarvey for help and advice on
the electrophysiological recording of the dentate gyrus. This re-
search was supported by the Intramural Research Programs of
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Received September 3, 2005; accepted in revised form February 9, 2006.
Role of NT-3 in neurogenesis and synaptic plasticity
Learning & Memory