Neuritin produces antidepressant actions and blocks
the neuronal and behavioral deficits caused by
Hyeon Sona,b,1, Mounira Banasra, Miyeon Choib, Seung Yeon Chaeb, Pawel Licznerskia, Boyoung Leea, Bhavya Voletia,
Nanxin Lia, Ashley Lepacka, Neil M. Fourniera, Ka Rim Leeb, In Young Leeb, Juhyun Kimc, Joung-Hun Kimc, Yong Ho Kimd,
Sung Jun Junge, and Ronald S. Dumana,1
aDepartment of Psychiatry, Yale University, New Haven, CT 06508; Departments ofbBiochemistry and Molecular Biology andePhysiology, College of Medicine,
Hanyang University, Haengdang-Dong 17, Sungdong-Gu, Seoul 133-791, Korea;cDepartment of Life Science, Pohang University of Science and Technology,
Pohang, Gyungbuk 790-784, Korea; anddDepartment of Neurobiology and Physiology, School of Dentistry, Seoul National University, Seoul 110-749, Korea
Edited* by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved May 28, 2012 (received for review January 23, 2012)
Decreased neuronal dendrite branching and plasticity of the hippo-
campus, a limbic structure implicated in mood disorders, is thought
to contribute to the symptoms of depression. However, the mech-
anisms underlying this effect, as well as the actions of antidepres-
sant treatment, remain poorly characterized. Here, we show that
hippocampal expression of neuritin, an activity-dependent gene
thatregulates neuronal plasticity,isdecreasedbychronic unpredict-
able stress (CUS) and that antidepressant treatment reverses this
effect. We also show that viral-mediated expression of neuritin in
the hippocampus produces antidepressant actions and prevents the
atrophy of dendrites and spines, as well as depressive and anxiety
depressive-like behaviors, similar to CUS exposure. The ability of
neuritin to increase neuroplasticity is confirmed in models of learning
and memory. Our results reveal a unique action of neuritin in models
of stress and depression, and demonstrate a role for neuroplasticity
in antidepressant treatment response and related behaviors.
resulting in personal disability, increased rates of suicide, and
socioeconomic loss (1). Moreover, currently available anti-
depressants are only effective in approximately one-third of
patients with MDD and in up to two-thirds after multiple trials,
and they take weeks to months to produce a response (2, 3). In
addition, the mechanisms underlying the therapeutic actions of
antidepressants are poorly understood. New targets beyond
monoamine signaling are now emerging in both preclinical and
clinical reports of MDD (4, 5). These studies have focused on
key limbic brain structures, including the hippocampus, that are
significantly altered by chronic stress and depression and that are
known to regulate mood, anxiety, and cognition (6, 7). Hippo-
campal synaptic plasticity has received much attention in recent
years because human imaging and rodent studies demonstrate
that stress and depression are associated with decreased hippo-
campal volume and atrophy of neurons (6, 8).
Neuritin, also known as candidate plasticity gene 15 (CPG15),
encodes a small, extracellular GPI-anchored protein critical for
dendritic outgrowth, maturation, and axonal regeneration (9–13).
Neuritin expression in the hippocampus is induced by neuronal
activity following chemical- or electrical-induced seizures (9, 14,
15), ischemia (16), and exercise (5, 17). Neuritin has been im-
plicated in the actions of BDNF (9, 18), which is up-regulated in
the hippocampus by antidepressant treatment and is sufficient to
produce antidepressant behavioral responses (19, 20). Moreover,
chronic antidepressant treatment has been shown to increase
neuritin expression in rat brain (21). The current study was con-
ducted to test the hypothesis that neuritin is a critical downstream
mediator of antidepressant/BDNF-mediated plasticity and,
ajor depressive disorder (MDD) is a devastating and re-
current illness affecting up to 17% of the population,
conversely, that loss of neuritin could contribute to depressive
symptoms caused by stress exposure.
Neuritin Is Down-Regulated by Chronic Stress: Reversal by Antidepres-
sant Treatment. Models of chronic unpredictable stress (CUS),
which can precipitate or worsen depression, are typically used for
preclinical studies of mood disorders. We have adopted a CUS
model that results in a spectrum of cellular and behavioral abnor-
malities, notably anhedonia, a core symptom of patients with MDD
that is reversed with chronic antidepressant treatment (22, 23).
Here, we show that neuritin mRNA levels are significantly de-
creased in the major subregions of the dorsal hippocampus, in-
cluding CA1 and CA3 pyramidal and dentate gyrus (DG) granule
cell layers [Fig. 1 A and B; Fisher’s least significant difference
(LSD), P < 0.01 compared with the nonstressed control]. There
was no significant effect in the adjacent parietal cortex (Fig. 1B).
In contrast, chronic administration (3 wk, initiated at day 15 of
CUS) of the 5-hydroxytryptamine selective reuptake inhibitor,
fluoxetine, significantly reversed the effects of CUS exposure in
all hippocampal subregions (Fig. 1B; Fisher’s LSD, P < 0.05
compared with CUS). Chronic fluoxetine treatment alone had
no significant effect on neuritin expression in nonstressed animals
(P > 0.05). Similar effects were found with quantitative real-time
PCR of dissected hippocampus, although the combination of
CUS + fluoxetine increased neuritin mRNA compared with flu-
oxetine alone (Fig. 1C; P < 0.05). Why fluoxetine alone did not
produce a significant effect as previously reported (21) could
be due to different doses (5 mg/kg vs. 10 mg/kg) or conditions
resulting from the CUS handling paradigm.
Neuritin Increases Hippocampal Dendrite Complexity and Spine
Density. Recent studies demonstrate that atrophy of neuronal
processes contributes to the negative effects of stress/depression
and can be reversed by certain antidepressants (24, 25). We in-
vestigated the effects of viral neuritin expression in the dorsal
hippocampus on DG granule neuron dendrite branching, spine
number, and spine head diameter. An adenoassociated virus
(AAV) vector was designed to coexpress GFP and CMV pro-
moter-driven AAV-neuritin (AAV-Nrn). Control AAV (AAV-
Author contributions: H.S. and R.S.D. designed research; H.S., M.B., M.C., S.Y.C., P.L., B.L.,
B.V., N.L., A.L., N.M.F., K.R.L., I.Y.L., Y.H.K., and S.J.J. performed research; J.K. and J.-H.K.
contributed new reagents/analytic tools; and H.S. and R.S.D. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or ronald.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1201191109PNAS Early Edition
| 1 of 6
ctl) expresses only GFP. Neuritin in situ hybridization and EGFP
fluorescence immunostaining demonstrated a localized increase
of neuritin in the DG of animals infected with AAV-Nrn relative
to AAV-ctl animals (Fig. 2 A and B). Confocal microscopy
revealed a striking remodeling of granule cell dendrites following
neuritin overexpression (>4 dendritic branch points) relative to
AAV-ctl (Fig. 2 C and D; P < 0.001). In addition, AAV-Nrn
infusion significantly increased spine density and head diameter
of mushroom-like spines by ∼70% (Fig. 2 E–G; P < 0.001
compared with the AAV-ctl group).
Synaptogenesis is accompanied by up-regulation of post-
synaptic proteins, including PSD-95 and GluR1, as previously
reported (25, 26). Studies in cultured hippocampal neurons
demonstrate that AAV-Nrn increases the levels of PSD-95 (Fig.
S1A). Similar effects were observed in response to AAV-Nrn
infusions into hippocampal DG by immunohistochemical anal-
ysis of PSD-95 (Fig. S1 B and C).
Neuritin Produces Antidepressant-Like Behavioral Effects and Prevents
the Effects of CUS. To test directly whether neuritin is sufficient to
produce antidepressant-like actions, we investigated the influence
of AAV-Nrn on rodent behavioral models of depression, anxiety,
and antidepressant response. Behavioral testing was conducted
5 wk after viral infusion into dorsal DG, a time when AAV is fully
expressed and stable (27) (Fig. 3A). In the novelty suppressed
feeding test (28–30), AAV-Nrn significantly decreased the latency
to feed, similar to the actions of antidepressant treatments
(Fig. 3B; P < 0.05 compared with AAV-ctl). There was no effect
on home cage feeding, indicating that there is no general effect
on metabolic status (Fig. 3D). In the forced swim test (FST),
which responds to acute antidepressant treatment, AAV-Nrn
infusions markedly decreased immobility, a typical antidepressant
response (Fig. 3C; P < 0.001). AAV-Nrn had no effect on sucrose
preference or active avoidance (Fig. S2 A and B). Spontaneous
locomotor activity (LMA) was not different between these two
groups (Fig. 3E).
To examine the antidepressant actions of neuritin further, we
used a CUS model that decreases sucrose preference and
impairs active avoidance, a measure of despair (25, 31). AAV-
Nrn– and AAV-ctl–infused rats were randomly assigned to
nonstressed control or CUS exposure for 21 d (Fig. 4A). CUS
caused the predicted decrease in sucrose preference in control
animals (AAV-ctl) (Fig. 4B; P < 0.001), and this effect was
blocked by AAV-Nrn infusions (P < 0.001). There was no dif-
ference between AAV-Nrn–infused CUS rats and either of the
nonstressed groups (P > 0.05). AAV-Nrn infusion had no effect
in nonstressed rats. There were no differences in total fluid
consumption in any of the groups (Fig. S3C).
In animals infused with AAV-ctl, CUS also increased the
number of escape failures (Fig. 4C; P < 0.01), a despair phe-
notype consistent with previous findings (32). This effect was also
ization and quantitative real-time (RT) PCR. (A) Rats were exposed to CUS for
35 d and received either saline or fluoxetine (CUS + FLX) for the last 21 d, and
sections were subjected to quantitative in situ hybridization. Representative
autoradiograms from hippocampal sections from in situ hybridization are
shown for home cage control (CTR), CUS, CUS + FLX-, and FLX-treated animals
(n = 5 per group). (B) Quantified expression of neuritin mRNA from the in-
dicated subregions in A. Results are expressed as a ratio of CTR and are the
mean ± SEM, each analyzed in duplicate brain sections. Neuritin mRNA was
decreasedinCUS animalscomparedwith CTRanimals(CA1:F3, 15=9.27,**P<
0.01; CA3: F3, 15= 13.45, **P < 0.001; DG: F3, 15= 11.96, **P < 0.001). CUS
animals injected with FLX showed an increase in neuritin mRNA compared
with CUS animals (CA1: P = 0.011; CA3: P = 0.04; DG: P = 0.006). FLX in the
nonstressed CTR didnot increase neuritin mRNA compared with CTR(CA1: P=
0.075; CA3: P = 0.062; DG: P = 0.078). In the cortex, there were no differences
between groups (P > 0.2). **P < 0.01 compared with CTR and#P < 0.05 and
##P < 0.01 compared with CUS (two-way ANOVA, Fisher’s LSD post hoc anal-
ysis). (C) Rats were exposed to CUS and FLX as described above, and whole
hippocampus was subjected to quantitative RT-PCR analysis. Results are nor-
malized to cyclophilin and expressed as the mean ratio of fold change ± SEM
of six individual animals. *P < 0.05; ***P < 0.001, two-way ANOVA.
Effects of CUS on neuritin expression, determined by in situ hybrid-
Number of bifurcations
Spine density /10 µm
situ hybridization analysis was conducted to examine the expression of
neuritin mRNA, and representative autoradiograms are shown for AAV-ctl
and AAV-Nrn. (B) GFP staining is also shown and further demonstrates lo-
calization of AAV infection of the DG. (C) Representative images of GFP(+)
cells from AAV-ctl– and AAV-Nrn–injected animals. A continuous stretch of
dendritic shaft localized in the outer half of the granular layer was identified
in the outer molecular layer and imaged. (D) Immunocytochemical analysis
showing that the dendritic arborization was increased in AAV-Nrn–injected
rats compared with AAV-ctl–injected rats (***P < 0.001). The data were
expressed as the number of bifurcations per GFP(+) neurons (n = 26 cells and
n = 16 cells for AAV-ctl– and AAV-Nrn–injected groups, respectively). (E)
Representative images are shown of high-magnification Z-stack projections
of apical tuft segments of GFP(+) DG granule cells from AAV-ctl– and AAV-Nrn–
injected rats. (F) Density of dendritic spines was significantly increased in AAV-
Nrn–injected rats compared with AAV-ctl–injected rats. (***P < 0.001) (n = 10
neurons from 6 rats in each group). The data were expressed as the number
of spines per 10 μm. (G) Spine head diameterof mushroom spines was increased
in AAV-Nrn–injected rats compared with AAV-ctl–injected rats (***P < 0.001)
(n = 12–15 neurons from 6 rats in each group). Results are the mean ± SEM.
(Scale bars: A, 200 μm; B, 50 μm; C and E, 10 μm.) Student t test.
Neuritin increases dendritic arborization and synaptogenesis. (A) In
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completely prevented by AAV-Nrn infusion (P < 0.01). CUS
exposure also increased the latency to feed in the novelty sup-
pressed feeding test (NSFT) (Fig. S3A; P < 0.05) and immobility
in the FST (Fig. S3B; P < 0.01), and these effects were blocked
by infusions of AAV-Nrn (Fig. 4E). There were no changes in
spontaneous LMA after stress (Fig. S3D). In addition to these
behavioral effects, infusion of AAV-Nrn before CUS exposure
completely prevented the CUS-induced spine deficit (Fig. 4 D
and E; P < 0.01).
Neuritin Knockdown Produces Depressive-Like Behaviors. Because
CUS exposure decreases levels of neuritin in the hippocampus,
we wanted to determine if knockdown of neuritin is sufficient to
cause depressive behaviors. We used a lentivirus expressing
shRNAs targeted against rat neuritin (lenti-shNrn), and lenti-
EGFP as a control. Studies of hippocampal neurons in vitro
demonstrate that lenti-shNrn decreases both basal and BDNF-
induced neuritin levels (Fig. 5B). Infusion of lenti-shNrn into the
hippocampus also decreased levels of neuritin mRNA in
microdissections of the infused DG area (Fig. 5C; P < 0.001).
Lenti-shNrn produced behavioral effects opposite to antide-
pressant treatment, significantly increasing the latency to feed in
the NSFT (Fig. 5D; P < 0.05), increasing immobility in the FST
(Fig. 5E; P < 0.001), and decreasing sucrose preference (Fig. 5F;
P < 0.05). These effects were observed in the absence of changes
in home cage food intake or total LMA (Fig. 5 G and H). To-
gether, the results demonstrate that neuritin knockdown in the
DG is sufficient to cause depressive behaviors similar to those
observed after CUS exposure for 3 wk.
We also examined the influence of neuritin knockdown on the
antidepressant response to BDNF in the FST, as previously
reported (33). Infusion of BDNF (0.25 μg per side) into DG
resulted in the expected antidepressant-like decrease in immo-
bility, and this effect was blocked by lenti-shNrn (Fig. 5I; BDNF
vs. lenti-shNrn + BDNF; P < 0.001). BDNF did not produce a
significant decrease in immobility in rats infused with lenti-shNrn
(Fig. 5I; lenti-shNrn ± BDNF; P > 0.05).
Hippocampal-Dependent Learning Is Enhanced by Neuritin. Previous
studies have implicated neural plasticity in the pathophysiology
and treatment of depression (31). To test the role of neuritin in
behavioral models of synaptic plasticity, we examined the in-
fluence of neuritin on two hippocampal-dependent learning
tasks, object recognition and contextual fear conditioning (Fig. 6).
LMA NSFT FST
Latency to feed (sec)
Home cage food intake (g)
Total distance (x103cm)
behavioral actions. (A) Experimental design. Animals were injected with
AAV-ctl or AAV-Nrn; 5 wk later, they were tested in behavioral paradigms,
and hippocampal sections were then harvested for in situ hybridization (ISH)
and immunohistochemistry (IHC). (B) NSFT. A significant decrease in the la-
tency to feed was shown in AAV-Nrn–injected animals compared with AAV-
ctl–injected animals [t(19)= 2.57, *P < 0.05]. (C) FST. AAV-Nrn–injected rats
had a shorter immobility score (time in seconds) than AAV-ctl–injected rats
[t(19)= 4.14, ***P < 0.001]. (D) Home cage feeding. There was no difference in
the home cage food intake between AAV-ctl–injected and AAV-Nrn–injected
rats [t(30)= 0.35, P = 0.73]. (E) LMA. The total distance moved in the box was
similar between groups [t(19)= 0.17, P > 0.5]. Results are the mean ± SEM
averaged from AAV-ctl–injected (n = 10) vs. AAV-Nrn–injected (n = 11) rats.
Student t test.
AAV-Nrn infusions into the hippocampus produce antidepressant
Sucrose preference (%)
Spine density /10 µm
AAV-ctl + CUS
AAV-Nrn + CUS
AAV-ctl (no CUS)
AAV-Nrn (no CUS)
icits caused by CUS exposure. (A) Experimental design. Rats were injected
with AAV-ctl (n = 16) or AAV-Nrn (n = 16). Each of the virus-infected cohorts
was split into two experimental groups and exposed to home cage or CUS
for 21 d. The efficacy of neuritin overexpression and CUS on behavioral
performances of the animals was measured for 3–4 consecutive days starting
on day 40 (D40). (B) SPT. Two-way ANOVA revealed that there was a main
effect of virus (F1, 28= 5.91, P < 0.05), a main effect of stress (F1, 28= 18.92,
P < 0.001), and interaction (F1, 28= 8.8, P < 0.01). Further analysis indicates
that CUS decreased sucrose preference compared with home cage in AAV-
ctl–injected animals (###P < 0.001). Neuritin increased sucrose preference in
CUS rats (***P < 0.001) but not in home-caged rats (P = 0.73). There was no
difference in AAV-Nrn–injected CUS animals and AAV-ctl–injected home
cage animals (P = 0.13). (C) Active avoidance test (AAT). Two-way ANOVA:
main effect of virus, F1, 28= 7.19, P < 0.05; main effect of stress, F1, 28= 5.37,
P < 0.05; interaction F1, 28= 8.19, P < 0.01. Further analysis indicates that
CUS-exposed rats had more escape failures than home cage rats in AAV-ctl
animals (##P < 0.01) and that neuritin overexpression decreased escape
failures only in CUS rats (**P < 0.01) but not in nonstressed rats (P = 0.87).
There was no difference in AAV-Nrn–injected CUS animals and AAV-ctl–
injected home cage animals (P = 0.74). (D) Representative images of high-
magnification Z-stack projections of apical tuft segments of GFP(+) DG
granule cells. (E) Density of dendritic spines. Two-way ANOVA: main effect
of virus, F3, 42= 19.05, P < 0.001; main effect of stress, F3, 42= 7.45, P < 0.01;
interaction F3, 42= 0.001, P > 0.05. The density of dendritic spines was sig-
nificantly decreased in CUS rats (*P < 0.05), and the effects were blocked in
AAV-Nrn–injected rats (**P < 0.01). n = 10–19 GFP(+) neurons from four rats
in each group. The data were expressed as the number of spines per 10 μm.
(Scale bar: 10 μm.)
AAV-Nrn infusions into the hippocampus block the behavioral def-
Son et al.PNAS Early Edition
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In object recognition, both groups readily discriminated the
novel object from the familiar object during testing (Fig. 6A;
familiar vs. novel object: AAV-ctl, P < 0.05; AAV-Nrn, P < 0.05).
The time spent exploring the novel object was comparable be-
tween AAV-ctl and AAV-Nrn groups when tested 1.5 h after
training, but when the retention interval was delayed for 24 h, the
discrimination index was significantly higher for the AAV-Nrn
rats (Fig. 6B; P < 0.001). There was also a strong exploration
preference for the novel object in the AAV-Nrn rats at the 24-h
time point (Fig. 6B; familiar vs. novel object, P < 0.002). In fear
conditioning, there was no difference between the groups in
postshock (immediate) freezing (Fig. 6C; P > 0.1). When the rats
were returned to the original training context 24 h later, the
AAV-Nrn group showed significantly higher levels of freezing
compared with the AAV-ctl group, indicative of enhanced con-
textual fear conditioning (Fig. 6D; P < 0.05).
The results of the present study demonstrate that CUS decreases
hippocampallevels ofneuritinand that viralexpressionofneuritin
is sufficient to produce an antidepressant response and to prevent
the morphological and behavioral deficits caused by CUS. More-
over, shRNA knockdown of neuritin in the hippocampus causes
depressive behaviors. The mechanisms underlying the regulation
of neuritin are unclear, but neuritin expression is regulated by
pathways that mediate neuronal plasticity, including induction of
BDNF expression and signaling (9), and are decreased by stress
and increased by antidepressant treatment (19, 24).
Neuritin is enriched in synapses (10, 11), and increased ex-
pression could underlie the enhanced synaptic plasticity and
dendrite morphology reported for antidepressants (24, 34). The
induction of spine density and dendrite branching in response to
increased neuritin expression is consistent with this hypothesis.
Induction of PSD-95 is also consistent with increased synapse
formation and function. Conversely, chronic corticosterone,
which is increased by stress, reduces PSD-95 levels in the stratum
lucidum of CA3 in mouse hippocampus (35), raising the possi-
bility that neuritin induction of PSD-95 is involved in blockade of
stress-induced deficits. Although the exact mechanisms are un-
known, it is possible that a soluble form of neuritin might act as
an extracellular signal to stimulate synaptogenesis as previously
demonstrated (36). Neuritin expression occurs in progenitor
populations in the developing brain and in some differentiated
neurons during target selection and circuit formation (36). The
BDNF (10 ng/ml)
Latency to feed (x102sec)
Sucrose preference (%)
Home cage food (g)
Total distance (x103cm)
Neuritin mRNA level
Neuritin in DG
Lenti-shNrn + BDNF
Lenti-ctl + BDNF
design. Lenti-shNrn was infused into the hippocampal DG, and behavioral
testing was initiated 5 wk later. (B) Knockdown efficiency of lenti-shNrn.
Lenti-shNrn was transfected into rat primary hippocampal neuronal cells,
and neuritin and β-actin mRNA levels were measured by real-time (RT)
PCR. Treatment of cells with BDNF (10 ng/mL) for 6 h induced neuritin mRNA
expression, and the increase was reduced by lenti-shNrn in vitro in a con-
centration-dependent manner. (C) Knockdown efficiency of lenti-shNrn.
Quantitative RT-PCR of neuritin mRNA in microdissected DG from hippo-
campal sections taken from lenti-ctl–injected and lenti-shNrn–injected ani-
mals confirms that neuritin knockdown decreases neuritin mRNA in the DG
(***P < 0.001). (D) NSFT. Lenti-shNrn infusion increased the latency to feed
compared with lenti-EGFP–injected control animals (*P < 0.05, Student t test).
(E) FST. Animals injected with lenti-shNrn had higher immobility times
than those injected with lenti-EGFP when assessed by means of a 15-min test
(***P < 0.001, Student t test). (F) SPT. Lenti-shNrn decreased sucrose prefer-
ence compared with lenti-EGFP–injected animals (*P < 0.05, Student t test).
(G) Home cage feeding. There was no difference in the amount in the home
cages between groups (P = 0.8). (H) LMA. Lenti-shNrn did not influence LMA
(P = 0.16, Student t test). Results are the mean ± SEM averaged from seven
animals per group. (I) FST. (Left) Experimental design. Two-way ANOVA,
maineffectofvirus:F3, 21=25.79,P<0.001;maineffectofBDNF:F3, 21=16.50,
P < 0.001; higher immobility time by lenti-shNrn (*P < 0.05). BDNF-infused
the effect of BDNF on immobility was blocked by lenti-shNrn (***P < 0.001).
Scores are measured by means of a 15-min test. Results are the mean ± SEM
averaged from seven animals per each group.
Neuritin knockdown causes depressive behaviors. (A) Experimental
Recognition, 1.5 hrs
Recognition, 24 hrs
FC, day 1:
Post shock Freezing
FC, day 2:
% Postshock Freezing
1 2 3
AAV-ctl AAV-NrnAAV-ctl AAV-Nrn
pendent memory. (A) Rats were injected with AAV-ctl or AAV-Nrn and then
tested in object recognition 30 d later. Both groups show comparable ex-
ploration of the novel object at 1.5 h after training (familiar vs. novel object,
P < 0.05; t < 1.00, P = 0.769). The discrimination index was not different
between the two groups (t < 1.00, P = 0.427). (B) In a separate experiment
(45 d after virus infusion), where testing was conducted 24 h after training,
rats injected with AAV-Nrn show significantly higher levels of discrimination
between the familiar and novel objects [unpaired t test, familiar vs. novel
object: t(12)= 3.47, **P < 0.005]) compared with AAV-ctl (familiar vs. novel
object: t(12)= 0.871, P = 0.4). The discrimination index was significantly higher
in animals injected with AAV-Nrn than in controls [t(12)= 4.76, ***P < 0.001].
(C) Fear conditioning (FC). The freezing behavior of animals treated with
AAV-ctl and AAV-Nrn (35 d after virus infusion) is shown. On the training
day, both groups showed similar levels of freezing in the context after each
single 2-s, 0.55-mA foot shock was delivered (P > 0.05). (D) FC, contextual
test. AAV-Nrn animals exhibit significantly higher levels of freezing when
0.05]. Results are presented as the mean ± SEM averaged from seven animals
per each group. Unpaired t-test.
AAV-Nrn infusion into the hippocampus improves hippocampal-de-
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results of the current study indicate that neuritin is sufficient to
induce synaptic remodeling of differentiated neurons in adult
brain. Further studies will be required to determine if the soluble
form of neuritin mediates this effect and serves as an activity-
dependent differentiation factor.
The results of our behavioral studies demonstrate that the
actions of viral neuritin expression are dependent on the type of
test and prior stress exposure. Neuritin expression is sufficient to
produce an antidepressant response in the FST and NSFT,
models that are responsive to acute or chronic antidepressant
administration in unstressed animals. However, there was no
effect in either the number of escapes in the active avoidance test
or preference in the sucrose preference test (SPT), models in
which the behavioral deficits are produced by prior chronic stress
and are reversed by antidepressant treatments. Only after CUS
exposure did neuritin produce an antidepressant response in
these models. These findings indicate that neuritin expression is
sufficient to produce an antidepressant response in the absence
of stress and to prevent or block the deficits caused by chronic
stress exposure, presumably by compensating for the neuritin
and synaptogenic deficits caused by stress. Conversely, the re-
sults of the lenti-shNrn knockdown experiments demonstrate
that neuritin is required for normal responding in the NSFT,
FST, and SPT consistent with the possibility that neuritin loss
could underlie the behavioral deficits caused by CUS exposure.
Moreover, analysis of postmortem tissue from depressed subjects
previously described (37) demonstrates that levels of neuritin are
decreased by 57 percent in the DG of the hippocampus com-
pared to controls (P < 0.05), raising the possibility that neuritin
deficits could contribute to neuronal atrophy and behavioral
symptoms in depressed patients. We also found that lenti-shNrn
knockdown of neuritin reverses the antidepressant effect of BDNF
in the FST, supporting the hypothesis that neuritin is induced by
and contributes to the antidepressant actions of BDNF.
The results also demonstrate that neuritin enhances memory
retention but not immediate acquisition in both the object rec-
ognition and contextual fear conditioning tasks. These effects
may be related to the role of neuritin in neurite outgrowth (9–13)
and spine formation (present study), which could facilitate syn-
aptic plasticity. The ability of neuritin to enhance memory in
these models is consistent with the hypothesis that the actions of
antidepressant treatment are mediated by increasing neural
plasticity (6, 31). This may be particularly true for hippocampal-
dependent plasticity, given the enrichment of neuritin in this
region. Our findings are consistent with recent data that neuritin
regulates synapse stabilization, resulting in efficient learning
(38). Together, the results suggest overlap of the cellular, neu-
roplasticity-related mechanisms underlying the antidepressant
and memory-enhancing actions of neuritin.
Elucidating the mechanisms for the antidepressant actions of
neuritin in the hippocampus is an important avenue of research
for future investigations. One possibility is that neuritin is involved
in regulation of newborn neurons in the adult hippocampus (39).
Recently, it has been shown that adult-born hippocampal granule
neurons buffer stress responses at both the endocrine and
behavioral levels (40). Increased expression of neuritin in the
hippocampus could play a role in adult neurogenesis, and thereby
regulate stress and antidepressant behaviors in addition to learn-
ing and memory.
Based on the findings presented here, it is possible that neu-
ritin deficits contribute to the atrophy of hippocampal neurons
during the course of lifetime stress exposures, or even during
a critical developmental period, and thereby lead to increased
vulnerability to anxiety and mood disorders. Development of
strategies that target neuritin or related signaling pathways could
represent unique approaches for improved antidepressant therapy.
Materials and Methods
A detailed description of the materials and methods used in this study is
provided in SI Materials and Methods.
Animals and Drug Treatment. Male Sprague–Dawley rats weighing 160–250 g
(Charles River Laboratories) were used. All procedures were in strict accor-
dance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals and were approved by the Yale University Animal Care
and Use Committee.
CUS Procedure. The CUS animals were subjected to exactly the same sequence
of 12 stressors (2 per day for 21–35 d) described by Banasr et al. (23).
Behavioral Experiments. The FST (26) and NSFT (23), as well as learned help-
lessness (33) and LMA (6) tests, were conducted as previously described.
Behavioral tests were analyzed by an experimenter blinded to the study code.
shRNA Preparation and Stereotaxic Surgery. We used shRNA constructs for
neuritin (36) and a control nontargeting shRNA (pll3.7; American Type Cul-
ture Collection). Bilateral viral injections were performed with coordinates
−4.1 mm (anterior/posterior), ±2.4 mm (lateral), and −4.1 mm (dorsal/ven-
tral) relative to the bregma.
Spine Density, Spine Head Diameter, and Dendritic Arborization Analysis.
Images were acquired through Z-stacks, which typically consisted of 10 scans
at high zoom at 1-μm steps in the z axis. Each GFP(+) granule neuron was
clearly distinguishable from other cells. Spine density, spine head diameter,
and dendritic arborization were analyzed in each section by an experi-
menter blinded to the study code.
Statistical Analysis. Statistical differences were determined by ANOVA
(StatView 5; SAS Software) followed by Fisher’s LSD post hoc analysis. The
F values and group and experimental degrees of freedom are included in
the legends of the figures. For experiments with two groups, the Student
t test was used. The level of statistical significance was set at P < 0.05 using
ACKNOWLEDGMENTS. This research was supported by National Research
Foundation of Korea Grant 2011-0028317 funded by the Ministry of Educa-
tion, Science, and Technology, Republic of Korea; by Grant 2011K000264 from
the Brain Research Center of the 21st Century Frontier Research Program
funded by the Ministry of Education, Science, and Technology, Republic of
Korea; by US Public Health Service Grants MH45481 (to R.S.D.) and 2 P01
MH25642 (to R.S.D.); and by the State of Connecticut, Department of Mental
Health and Addiction Services (R.S.D.). N.M.F. received support from a post-
doctoral fellowship award funded by the Natural Sciences and Engineering
Research Council of Canada.
1. Wong ML, Licinio J (2001) Research and treatment approaches to depression. Nat Rev
2. Berton O, Nestler EJ (2006) New approaches to antidepressant drug discovery: Beyond
monoamines. Nat Rev Neurosci 7:137–151.
3. Dulawa SC, Hen R (2005) Recent advances in animal models of chronic antidepressant
effects: The novelty-induced hypophagia test. Neurosci Biobehav Rev 29:771–783.
4. Wong ML, Licinio J (2004) From monoamines to genomic targets: A paradigm shift for
drug discovery in depression. Nat Rev Drug Discov 3:136–151.
5. Hunsberger JG, et al. (2007) Antidepressant actions of the exercise-regulated gene
VGF. Nat Med 13:1476–1482.
6. Pittenger C, Duman RS (2008) Stress, depression, and neuroplasticity: A convergence
of mechanisms. Neuropsychopharmacology 33:88–109.
7. Sapolsky RM (2003) Stress and plasticity in the limbic system. Neurochem Res
8. Fossati P, Radtchenko A, Boyer P (2004) Neuroplasticity: From MRI to depressive
symptoms. Eur Neuropsychopharmacol 14(Suppl 5):S503–S510.
9. Naeve GS, et al. (1997) Neuritin: A gene induced by neural activity and neurotrophins
that promotes neuritogenesis. Proc Natl Acad Sci USA 94:2648–2653.
10. Fujino T, Wu Z, Lin WC, Phillips MA, Nedivi E (2008) cpg15 and cpg15-2 constitute
a family of activity-regulated ligands expressed differentially in the nervous system to
promote neurite growth and neuronal survival. J Comp Neurol 507:1831–1845.
11. Karamoysoyli E, Burnand RC, Tomlinson DR, Gardiner NJ (2008) Neuritin mediates
nerve growth factor-induced axonal regeneration and is deficient in experimental
diabetic neuropathy. Diabetes 57:181–189.
12. Di Giovanni S, et al. (2005) Neuronal plasticity after spinal cord injury: Identification
of a gene cluster driving neurite outgrowth. FASEB J 19:153–154.
13. Nedivi E, Wu GY, Cline HT (1998) Promotion of dendritic growth by CPG15, an activity-
induced signaling molecule. Science 281:1863–1866.
Son et al. PNAS Early Edition
| 5 of 6
14. Nedivi E, Hevroni D, Naot D, Israeli D, Citri Y (1993) Numerous candidate plasticity- Download full-text
related genes revealed by differential cDNA cloning. Nature 363:718–722.
15. Newton SS, et al. (2003) Gene profile of electroconvulsive seizures: Induction of
neurotrophic and angiogenic factors. J Neurosci 23:10841–10851.
16. Rickhag M, Teilum M, Wieloch T (2007) Rapid and long-term induction of effector
immediate early genes (BDNF, Neuritin and Arc) in peri-infarct cortex and dentate
gyrus after ischemic injury in rat brain. Brain Res 1151:203–210.
17. Duman CH, Schlesinger L, Russell DS, Duman RS (2008) Voluntary exercise produces
antidepressant and anxiolytic behavioral effects in mice. Brain Res 1199:148–158.
18. Wibrand K, et al. (2006) Identification of genes co-upregulated with Arc during BDNF-
induced long-term potentiation in adult rat dentate gyrus in vivo. Eur J Neurosci
19. Nibuya M, Nestler EJ, Duman RS (1996) Chronic antidepressant administration in-
creases the expression of cAMP response element binding protein (CREB) in rat hip-
pocampus. J Neurosci 16:2365–2372.
20. Adachi M, Barrot M, Autry AE, Theobald D, Monteggia LM (2008) Selective loss of
brain-derived neurotrophic factor in the dentate gyrus attenuates antidepressant
efficacy. Biol Psychiatry 63:642–649.
21. Alme MN, Wibrand K, Dagestad G, Bramham CR (2007) Chronic fluoxetine treatment
induces brain region-specific upregulation of genes associated with BDNF-induced
long-term potentiation. Neural Plast 2007:26496.
22. Banasr M, Duman RS (2008) Glial loss in the prefrontal cortex is sufficient to induce
depressive-like behaviors. Biol Psychiatry 64:863–870.
23. Banasr M, et al. (2007) Chronic unpredictable stress decreases cell proliferation in the
cerebral cortex of the adult rat. Biol Psychiatry 62:496–504.
24. Duman RS (2009) Neuronal damage and protection in the pathophysiology and
treatment of psychiatric illness: stress and depression. Dialogues Clin Neurosci
25. Li N, et al. (2010) mTOR-dependent synapse formation underlies the rapid antide-
pressant effects of NMDA antagonists. Science 329:959–964.
26. Keith D, El-Husseini A (2008) Excitation Control: Balancing PSD-95 Function at the
Synapse. Front Mol Neurosci 1:4.
27. Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ (2003) Local gene
knockdown in the brain using viral-mediated RNA interference. Nat Med
28. Bodnoff SR, Suranyi-Cadotte B, Aitken DH, Quirion R, Meaney MJ (1988) The effects
of chronic antidepressant treatment in an animal model of anxiety. Psychopharma-
cology (Berl) 95:298–302.
29. Santarelli L, et al. (2001) Genetic and pharmacological disruption of neurokinin 1
receptor function decreases anxiety-related behaviors and increases serotonergic
function. Proc Natl Acad Sci USA 98:1912–1917.
30. Warner-Schmidt JL, Duman RS (2007) VEGF is an essential mediator of the neurogenic
and behavioral actions of antidepressants. Proc Natl Acad Sci USA 104:4647–4652.
31. Nissen C, et al. (2010) Learning as a model for neural plasticity in major depression.
Biol Psychiatry 68:544–552.
32. Banasr M, et al. (2010) Glial pathology in an animal model of depression: reversal of
stress-induced cellular, metabolic and behavioral deficits by the glutamate-modu-
lating drug riluzole. Mol Psychiatry 15:501–511.
33. Shirayama Y, Chen AC, Nakagawa S, Russell DS, Duman RS (2002) Brain-derived
neurotrophic factor produces antidepressant effects in behavioral models of de-
pression. J Neurosci 22:3251–3261.
34. Wang JW, David DJ, Monckton JE, Battaglia F, Hen R (2008) Chronic fluoxetine
stimulates maturation and synaptic plasticity of adult-born hippocampal granule
cells. J Neurosci 28:1374–1384.
35. Cohen JW, et al. (2011) Chronic corticosterone exposure alters postsynaptic protein
levels of PSD-95, NR1, and synaptopodin in the mouse brain. Synapse 65:763–770.
36. Putz U, Harwell C, Nedivi E (2005) Soluble CPG15 expressed during early development
rescues cortical progenitors from apoptosis. Nat Neurosci 8:322–331.
37. Duric V, et al. (2010) A negative regulator of MAP kinase causes depressive behavior.
Nat Med 16:1328–1332.
38. Fujino T, et al. (2011) CPG15 regulates synapse stability in the developing and adult
brain. Genes Dev 25:2674–2685.
39. Aimone JB, Wiles J, Gage FH (2006) Potential role for adult neurogenesis in the en-
coding of time in new memories. Nat Neurosci 9:723–727.
40. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA (2011) Adult hippocampal
neurogenesis buffers stress responses and depressive behaviour. Nature 476:458–461.
6 of 6
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