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Nectin-3 links CRHR1 signaling to stress-induced

memory deficits and spine loss

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2013 Nature America, Inc. All rights reserved.

Xiao-Dong Wang1,5, Yun-Ai Su1,5, Klaus V Wagner1, Charilaos Avrabos1, Sebastian H Scharf1,
Jakob Hartmann1, Miriam Wolf1, Claudia Liebl1, Claudia Khne1, Wolfgang Wurst14, Florian Holsboer1,
Matthias Eder1, Jan M Deussing1, Marianne B Mller1 & Mathias V Schmidt1
Stress impairs cognition via corticotropin-releasing hormone receptor 1 (CRHR1), but the molecular link between abnormal
CRHR1 signaling and stress-induced cognitive impairments remains unclear. We investigated whether the cell adhesion molecule
nectin-3 is required for the effects of CRHR1 on cognition and structural remodeling after early-life stress exposure. Postnatally
stressed adult mice had decreased hippocampal nectin-3 levels, which could be attenuated by CRHR1 inactivation and mimicked
by corticotropin-releasing hormone (CRH) overexpression in forebrain neurons. Acute stress dynamically reduced hippocampal
nectin-3 levels, which involved CRH-CRHR1, but not glucocorticoid receptor, signaling. Suppression of hippocampal nectin-3
caused spatial memory deficits and dendritic spine loss, whereas enhancing hippocampal nectin-3 expression rescued the
detrimental effects of early-life stress on memory and spine density in adulthood. Our findings suggest that hippocampal nectin-3
is necessary for the effects of stress on memory and structural plasticity and indicate that the CRH-CRHR1 system interacts with
the nectin-afadin complex to mediate such effects.
Synapses are specialized intercellular junctions that meditate the
transmission of information between neurons. Glutamatergic excitatory synapses are established by presynaptic axonal terminals
and postsynaptic dendritic spines, both of which anchor synaptic
cell adhesion molecules (CAMs) 1,2. CAMs are not merely static
constituents of synapses, but are dynamic modulators of synaptic
activity and plasticity. During development, synaptic CAMs are
involved in neurite growth, synaptogenesis and synapse maturation.
In the adult brain, CAMs interact with various synaptic proteins
and receptors to shape synaptic function3,4. A disruption of synaptic
adhesion may lead to functional abnormalities. Recent evidence
indicates that the dysregulation of synaptic CAMs contribute to
structural modifications and cognitive deficits 5,6, including those
induced by stress7.
Repeated exposure to severe stress exerts deleterious effects on cognition in different life stages8. Early adversities, such as an impoverished environment, impair hippocampal integrity and function, which
is manifested by progressively deteriorated cognitive performance
in the adult offspring9,10. As key mediators of neuroendocrine and
behavioral responses to stress, CRH and CRHR1 have been shown
to modulate the negative effects of early-life stress on cognition and
structural plasticity11,12. In adult animals, the influence of chronic
stress on cognition also involves hippocampal CRH-CRHR1 signaling13. Nonetheless, the molecular underpinnings of CRHR1-mediated
cognitive effects remain to be elucidated.

Nectin-3 is an immunoglobulin-like CAM, which primarily

localizes at adherens junctions in adulthood, the sites adjacent to
the presynaptic active zone and postsynaptic density (PSD)14.
Postsynaptic nectin-3 mediates heterophilic adhesion with presynaptic nectin-1, and is indirectly connected to the actin cytoskeleton via
L-afadin. The nectin-afadin complex colocalizes and cooperates with
the cadherin-catenin complex to organize adherens junctions, and
participates in synaptic formation, maintenance and remodeling1419.
Some evidence suggests that impaired nectin-mediated adhesion
disrupts hippocampal development16 and is associated with mental
retardation20. Although expressed ubiquitously, nectin-3 is abundant
in CA3 pyramidal neurons13,21 that are vulnerable to both acute22
and chronic23 stress challenge. Nectin-3 expression levels have been
shown to correlate with the observed cognitive phenotype following
chronic stress13. However, it is still unclear whether nectin-3 is causally involved in mediating the effect of stress via CRHR1 signaling on
cognition and structural remodeling.
We examined how stress and CRH-CRHR1 signaling might modulate nectin-3 expression in the hippocampus. Using site-specific
knockdown and overexpression of nectin-3, we then investigated
the role of hippocampal nectin-3 in spatial learning and memory
and dendritic spine plasticity, and tested whether reinstating
nectin-3 in the adult hippocampus could reverse the detrimental
consequences of early adverse experience on cognitive function and
structural plasticity.

1Max

Planck Institute of Psychiatry, Munich, Germany. 2Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental
Health, Neuherberg, Germany. 3Technische Universitt Mnchen, Lehrstuhl fr Entwicklungsgenetik, Helmholtz Zentrum Mnchen, Neuherberg, Germany. 4Deutsches
Zentrum fr Neurodegenerative Erkrankungen, Munich, Germany. 5Present address: Institute of Mental Health, Peking University, Beijing, China, and Key Laboratory
for Mental Health, Ministry of Health, Peking University, Beijing, China. Correspondence should be addressed to M.V.S. (mschmidt@mpipsykl.mpg.de).
Received 14 January; accepted 9 April; published online 5 May 2013; doi:10.1038/nn.3395

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Figure 1 Regulation of hippocampal nectin-3 expression by stress and the involvement of CRH-CRHR1
signaling. (a) Early-life stress downregulated CA3 nectin-3 mRNA in 78-month-old male mice (stress
effect: F1,29 = 4.373, P = 0.045, two-way ANOVA). CT, control; ES, early-life stress; WT, wild type;
CKO, CRHR1-CKO; CT-WT, nonstressed and wild-type mice. (b) At protein levels, early-life stress reduced
hippocampal nectin-3 expression in wild-type, but not CRHR1-CKO, mice (stress genotype interaction:
F1,17 = 5.507, P = 0.0313, two-way ANOVA; *P < 0.05, Bonferronis test). (c,d) Conditional forebrain
CRH overexpression mimicked the effects of early-life stress on nectin-3 expression. CA3 nectin-3 mRNA
levels (c) and hippocampal nectin-3 protein levels (d) were reduced in 78-month-old male CRH-COE
mice (t16 = 2.869, *P < 0.05; t10 = 3.336, **P < 0.01; unpaired t test). (e,f) Acute social defeat stress
disrupted hippocampal nectin-3 expression at both mRNA (stress effect: F1,70 = 4.164, P = 0.045,
two-way ANOVA) and protein levels (stress effect: F1,52 = 4.386, P = 0.041, two-way ANOVA) in
3-month-old male mice. At 4 h after the stress, nectin-3 mRNA levels (e) were markedly decreased
(t17 = 2.144, *P < 0.05, unpaired t test), which were reflected at the protein levels (f, t18 = 2.177,
*P < 0.05, unpaired t test). Data represent mean s.e.m. Scale bars in the representative in situ
hybridization images represent 500 m (a,c,e). For full-length blots (b,d,f), see Supplementary Figure 10.
In this and all subsequent figures, the number of mice is indicated in the bar graphs.

RESULTS
Stress reduces nectin-3 levels via CRH-CRHR1 signaling
As we reported previously13, nectin-3 is enriched in hippocampal
CA3 neurons (Fig. 1a). Using male Crhr1loxP/loxP; Camk2a-cre mice24,
in which the Crhr1 gene is inactivated postnatally in forebrain principal neurons (referred to as CRHR1-CKO hereafter), we investigated
whether early-life stress (postnatal days 29) would lead to downregulation of nectin-3 expression in a CRHR1-dependent manner. We
examined nectin-3 (Pvrl3) mRNA and protein levels in adult CRHR1CKO and wild-type mice exposed to either a standard or impoverished environment early in life (Fig. 1a,b). In wild-type mice with
early stressful experiences, which exhibit impaired spatial learning
and memory11, both mRNA and protein levels of hippocampal nectin3 were reduced. In comparison, control and stressed CRHR1-CKO
mice showed comparable nectin-3 mRNA and protein levels. Because
early-life stress increases CRH levels in the adult hippocampus12, we
used adult male R26flopCrh/flopCrh; Camk2a-cre mice25 (flop refers to
loxP-flanked stop) with postnatal overexpression of the Crh gene in
forebrain principal neurons (referred to as CRH-COE hereafter) to
test whether CRH overexpression would evoke similar effects on
nectin-3 expression to early-life stress (Fig. 1c,d). Compared with
the wild-type controls, stress-naive CRH-COE mice with prominent
cognitive deficits11 had lower nectin-3 mRNA and protein levels in
the hippocampus. These results indicate that early-life stressinduced
reductions of nectin-3 expression involve CRH-CRHR1 signaling.
To further elucidate the effects of stress on nectin-3 expression, we
evaluated nectin-3 levels following an acute severe stress challenge in
adulthood (Fig. 1e,f). A brief (5 min) exposure to social defeat stress
dynamically regulated nectin-3 expression in the adult hippocampus.
At 4 h, but not 1 h, 8 h or 24 h after the acute stress, hippocampal
nectin-3 levels were substantially reduced. Notably, a single treatment
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2013 Nature America, Inc. All rights reserved.

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with the glucocorticoid receptor agonist dexamethasone (10 mg per

kg of body weight) failed to alter nectin-3 levels (Supplementary
Fig. 1a), indicating that glucocorticoid receptor may not mediate the
effects of stress on nectin-3 expression.
CRH-CRHR1 signaling regulates nectin-3 expression
To dissect the involvement of the CRH-CRHR1 system in stressregulated nectin-3 expression, we manipulated CRH-CRHR1 signaling
in acute hippocampal slices. Consistent with the dynamic regulation
pattern by acute stress, 4 h (Fig. 2a), but not 1 h (Supplementary
Fig.1b), of in vitro CRH (50 nM) treatment reduced hippocampal
nectin-3 protein levels. Pre-incubating the slices with the selective
nonpeptide CRHR1 antagonist DMP696 (100 nM) reversed such
effects (Fig. 2b).
We continued to examine nectin-3 expression following CRH
administration in vivo (Fig. 2c,d). To avoid the confounding effects
of mild stress induced by handling, we anesthetized mice in their
home cages and delivered drugs intracerebroventricular (icv). At 4 h
after icv CRH (0.2 mM) infusion, total and membrane nectin-3 levels in the hippocampus decreased (Fig. 2c). Notably, icv infusion of
corticosterone (2.9 mM) did not alter nectin-3 levels (Supplementary
Fig. 1c). Co-administration of CRH with DMP696 (1 mM) prevented
CRH-induced reductions in both total and membrane nectin-3 levels
(Fig. 2d). Moreover, the selective mitogen-activated protein kinase
(MAPK) kinase (MEK) inhibitor U0126 (2.6 mM), but not the protein
kinase A (PKA) inhibitor Rp-cAMPS (4 mM), attenuated the effects of
CRH. These results suggest that the CRH-CRHR1 system modulates
hippocampal nectin-3 expression via the MAPK pathway.
Next, we examined the colocalization of CRHR1 and nectin-3 in
cortical and subcortical neurons. Because of the lack of reliable antibodies to CRHR1 (ref. 26), we used both CRHR1enhanced green
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2013 Nature America, Inc. All rights reserved.

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Figure 2 Regulation of hippocampal nectin-3 by CRH-CRHR1 signaling. (a) After 4 h of CRH application in vitro, hippocampal nectin-3 protein levels
were reduced (t8 = 2.56, *P < 0.05, unpaired t test). ACSF, artificial cerebrospinal fluid. (b) Adding the CRHR1 antagonist DMP696 to the brain slices
at 10 min before the 4-h CRH treatment prevented the effects of CRH on nectin-3 expression in vitro (interaction: F1,16 = 5.446, P = 0.033, two-way
ANOVA; *P < 0.05, Bonferronis test). (c) Intracerebroventricular administration of CRH downregulated total (t20 = 2.472, *P < 0.05, unpaired t test)
and membrane (t8 = 4.632, **P < 0.01, unpaired t test) nectin-3 levels in the hippocampus 4 h later. (d) Co-administration of CRH with DMP696
or U0126, but not Rp-cAMPS, attenuated the CRH-induced reduction of total nectin-3 in the hippocampus at 4 h after icv injection (F4,25 = 3.276,
P = 0.027, one-way ANOVA), whereas DMP696 prevented the effects of CRH on membrane nectin-3 expression (F4,25 = 2.796, P = 0.048, one-way
ANOVA). *P < 0.05, Fishers LSD test. Data represent mean s.e.m. All tested mice were 3-month-old C57BL/6N males. For full-length blots, see
Supplementary Figure 10.

fluorescent protein (EGFP) reporter mice26 and CRHR1 tau-lacZ

reporter mice27. We observed that CRHR1 partially colocalized with
nectin-3 in hippocampal pyramidal neurons (Fig. 3) and neurons
in various cortical and subcortical regions (Supplementary Fig. 2),
which is suggestive of their functional interactions.
Hippocampal nectin-3 knockdown impairs spatial memory
Early-life stress and enhanced hippocampal CRH-CRHR1 signaling impair cognition 11,12 and reduced nectin-3 expression levels.
To gain insight into the behavioral and structural consequences
of nectin-3 downregulation in the adult hippocampus, we used
adeno-associated virus (AAV)-mediated nectin-3 knockdown and
investigated whether reduced levels of nectin-3 would impair cognition, thereby mimicking the early-life stressinduced phenotype.
AAV-shSCR (contains a scrambled short hairpin RNA sequence)
was chosen as the negative control and AAV-shNEC (contains the
sequence for a short hairpin RNA specific
for nectin-3) was used to suppress nectin-3
a
expression in vivo.
We found that the AAV-shNEC vector
specifically reduced hippocampal nectin-3
levels, whereas the levels of other nectins
and related molecules remained unchanged
(Supplementary Fig.3). We also examined the extent of viral transfection in the
Figure 3 Colocalization of CRHR1 and
nectin-3 in hippocampal CA1 pyramidal neurons.
(a,b) In 3-month-old female CRHR1-EGFP
reporter mice and CRHR1 tau-lacZ reporter mice,
CRHR1-EGFP (a) and CRHR1--galactosidase (b)
partially colocalized with nectin-3 in CA1
pyramidal neurons. DAPI, 4,6-diamidino-2phenylindole. Arrowheads in the representative
confocal images indicate neurons in which
the colocalization of CRHR1 and nectin-3 was
observed. Scale bars represent 20 m.

708

hippocampus (Supplementary Fig. 4). In AAV-shNECtreated mice,

nectin-3 expression levels were downregulated in CA3, dentate gyrus
and CA1 throughout the dorsal hippocampus (Fig. 4a). Short-term
spatial working memory was not altered in AAV-shNECtreated mice,
as seen by similar spontaneous alternation behavior in the Y-maze
task compared with the controls (Fig. 4b). In the spatial object recognition test, AAV-shNECtreated mice showed a markedly impaired
performance (Fig. 4c and Supplementary Fig. 5a,b). Moreover,
AAV-shSCRtreated mice discriminated the novel object from the
familiar one, whereas AAV-shNECtreated mice failed to show object
discrimination (Supplementary Fig. 5b). In the spatial training sessions of the Morris water maze task (Fig. 4d), the spatial learning of
AAV-shNECtreated mice was preserved. In the probe trial, however, control mice, but not AAV-shNECtreated mice, searched the
target quadrant longer than the other quadrants. Together, these data
indicate that suppression of hippocampal nectin-3 reproduces the

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Figure 4 Hippocampal nectin-3 knockdown impaired long-term spatial memory. (a) At 1 week after the behavioral tests, AAV-shNECinduced
hippocampal nectin-3 knockdown was verified by in situ hybridization. Nectin-3 mRNA levels in CA3, dentate gyrus (DG) and CA1 were significantly
reduced by AAV-shNEC (treatment effect: F1,30 = 96.218, P < 0.00001; F1,29 = 22.193, P = 0.00006; F1,30 = 11.486, P = 0.002; one-way
repeated-measures ANOVA; n = 16 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test. Scale bar in the representative in situ
hybridization images represents 500 m. (bd) Two cohorts of 3-month-old C57BL/6N males received an intrahippocampal viral injection and were
tested in the Y-maze and spatial object recognition tasks (the first cohort only), and then in the Morris water maze task (both cohorts) 4 weeks
later. (b) Spatial working memory was comparable between groups (t22 = 0.996, P = 0.33, unpaired t test). (c) The ratio of time spent with the
displaced (novel) object versus the non-displaced (known) object, a measure of spatial recognition memory, was significantly lower in AAV-shNEC
mice compared to the controls (t21.743 = 2.104, *P < 0.05, Welchs t test). (d) In the Morris water maze test, AAV-shNEC mice showed similar spatial
learning performance to AAV-shSCR mice (F1,43 = 1.598, P = 0.213, one-way repeated-measures ANOVA). In the probe trial, AAV-shSCR mice, but
not AAV-shNEC mice, spent more time searching the target quadrant where the platform was previously placed than the other quadrants (AAV-shSCR:
t22 = 3.38, ##P < 0.01; AAV-shNEC: t21 = 1.321, P = 0.201; paired t test). Data represent mean s.e.m.

cognitive effects of early-life stress and that nectin-3 is essential for

hippocampus-dependent long-term spatial memory.
Suppression of nectin-3 evokes dendritic spine loss
To assess the effects of long-term nectin-3 knockdown on structural
plasticity in vivo, we quantified the number and size of dendritic
spines in EGFP-expressing hippocampal neurons in AAV-shSCR
and AAV-shNECtreated mice. Similar to adult mice with a history

of early-life stress11, AAV-shNECtreated mice showed a marked

reduction in spine density in CA3 (Fig. 5a,b), dentate gyrus and
CA1 (Supplementary Fig. 6) principal neurons. Spine volume
and spine head diameter in CA3 neurons were unaffected by
AAV-shNEC (Fig. 5c).
Notably, we observed that approximately 17% of spines expressed
nectin-3 (17.41 0.89, n = 2,480 spines analyzed). The density of
nectin-3positive spines was reduced in AAV-shNECtreated mice

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Figure 5 Nectin-3 knockdown reduced dendritic

spine density in CA3 pyramidal neurons.
(a) Representative deconvolved z stacks showing
EGFP-filled dendrites and spines (green) and
nectin-3immunoreactive puncta (red) in
the stratum lacunosum-moleculare of CA3.
Arrowheads indicate nectin-3positive spines.
Scale bars represent 1 m. (b) Suppression of
nectin-3induced spine loss in CA3 pyramidal
neurons (t10 = 4.097, **P < 0.01, unpaired
t test). The number of nectin-3positive spines
was significantly less in AAV-shNEC mice
compared with AAV-shSCR mice (t10 = 10.505,
***P < 0.001, unpaired t test). Data represent
mean s.e.m. (c) Nectin-3 knockdown did not
affect spine volume (t1801.301 = 0.6693,
P = 0.5034, Welchs t test) or spine head
diameter (t2029.375 = 0.9195, P = 0.358,
Welchs t test). Male C57BL/6N mice were
3 months old when they were injected with virus
and were killed after 4 weeks of recovery. For
each mouse, 814 dendrites were analyzed.

Number of spines per 10 m

2013 Nature America, Inc. All rights reserved.

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Figure 6 Hippocampal nectin-3 overexpression reversed early-life stressinduced cognitive deficits. (a) Following the behavioral tests, hippocampal
nectin-3 mRNA levels were determined. Nectin-3 mRNA levels in CA3, dentate gyrus and CA1 were significantly increased by AAV-OE (treatment
effect: F1,22 = 1740.24, ###P < 0.001; F1,22 = 749.003, P < 0.001; F1,22 = 68.369, P < 0.001; two-way repeated measures ANOVA). Compared with
CT-null mice, nectin-3 mRNA levels were lower in the CA3 and dentate gyrus of ES-null mice (stress effect: F1,10 = 5.581, *P < 0.05; F1,10 = 10.039,
P < 0.05; one-way repeated measures ANOVA; n = 67 mice per group). Scale bar in the representative in situ hybridization images represents 500 m.
(bd) We injected 5-month-old C57BL/6N males, with or without early-life stress, intrahippocampally with virus and subjected them to cognitive testing
after 4 weeks of recovery. (b) In the Y-maze test, early-life stress impaired spatial working memory (stress effect: F1,43 = 4.808, P = 0.0338, two-way
ANOVA). (c) In the spatial object recognition task, nectin-3 overexpression restored spatial memory in stressed mice (treatment effect: F1,42 = 4.127,
P = 0.0486; interaction: F1,42 = 4.546, P = 0.0389; two-way ANOVA; *P < 0.05, Tukeys test). (d) In the Morris water maze test, all groups of mice
performed similarly in the spatial acquisition sessions. In the probe trial, nectin-3 overexpression increased the ratio of time spent exploring the target
quadrant over non-target quadrants (treatment effect: F1,36 = 5.96, P = 0.02, two-way ANOVA). CT-null, CT-OE and ES-OE, but not ES-null, mice spent
more time searching the target quadrant than the others (CT-null: t8 = 2.72, #P < 0.05; CT-OE: t8 = 4.944, ##P < 0.01; ES-OE: t11 = 3.536, P < 0.01;
ES-null: t10 = 1.684, P = 0.123; paired t test). Data represent mean s.e.m.

(Fig.5b), whereas the numbers of spines lacking nectin-3 were comparable between groups (AAV-shSCR, 6.59 0.34 spines per 10 m of dendrite;
AAV-shNEC, 6.35 0.15; t10 = 0.637, P = 0.539, unpaired t test).

Nectin-3 overexpression rescues stress-induced memory loss

Because the suppression of hippocampal nectin-3 mimicked the
cognitive impairments by early-life stress, we examined whether

CT-OE

ES-OE
EYFP

Nectin-3

Merged

CT
ES

CT-null

ES-null

Nectin-3+

Total

10

**

**

4
2
0

3 5

4 5

Null

OE

c
100
Cumulative distribution (%)

Number of spines per 10 m

3 5

4 5

Null

OE

80
60
40
20

CT-null
ES-null
CT-OE
ES-OE

0
0.1 0.2 0.3 0.4 0.5 0.6
Spine volume (m3)
100
Cumulative distribution (%)

2013 Nature America, Inc. All rights reserved.

Null

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Opposite
Left

Figure 7 Nectin-3 overexpression rescued early-life stressevoked spine loss and spine volume changes.
80
(a) Representative deconvolved z stacks showing enhanced yellow fluorescent protein (EYFP)-filled dendrites and
spines (green) and nectin-3immunoreactive puncta (red) in the stratum lacunosum-moleculare of CA3. Scale
60
CT-null
bars represent 1 m. (b) Early-life stress reduced spine density in control mice, which was reversed by nectin-3
ES-null
overexpression (stress effect: F1,13 = 4.833, P = 0.0466; interaction: F1,13 = 9.827, P = 0.0079; two-way ANOVA;
40
CT-OE
*P < 0.05, **P < 0.01, Bonferronis test). Overexpression of nectin-3 increased the number of nectin-3positive
ES-OE
20
spines (treatment effect: F1,13 = 23.94, P = 0.0003, two-way ANOVA). Data represent mean s.e.m. (c) Nectin-3
overexpression reversed early-life stressinduced spine volume changes (interaction: F1,5365 = 15.191,
0
P = 0.0000984, two-way ANOVA). Spine volume in ES-null mice was larger than CT-null mice (P < 0.05, Tukeys
0.1 0.2 0.3 0.4 0.5 0.6 0.7
test), whereas ES-OE mice had smaller spines compared with CT-OE mice (P < 0.05, Tukeys test). In addition, nectinSpine head diameter (m)
3 overexpression increased spine volume (CT-null versus CT-OE: P < 0.001, Tukeys test) and spine head diameter in
control mice (treatment effect: F1,5880 = 12.408, P = 0.000431, two-way ANOVA; CT-null versus CT-OE: P < 0.01, Tukeys test). Male Thy1-YFPH mice
were 5 months old when they were injected with virus and were killed after 4 weeks of recovery. For each mouse, 1015 dendrites were analyzed.

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d
slm

140

120
100
80
60
40
5

ACSF

CRH

ACSF

140
120

shSCR

100
80
60
40
20
0

slm

Null

sr

sr

CRH

5 5

5 5

Vehicle

DMP696

Normalized L-afadin
puncta density
(% of shSCR)

Ratios of L-afadin to actin

(% of ACSF)

2013 Nature America, Inc. All rights reserved.

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Actin

Actin

Vehicle
DMP696
ACSF CRH ACSF CRH

L-afadin

L-afadin

20

140
120
100
80
60
40
20
0

shNEC
sr + slm

OE

shSCR

shNEC

CT
Normalized L-afadin puncta
density (% of CT-Null)

CRH

ACSF

Ratios of L-afadin to actin

(% of ACSF-vehicle)

ES
sr + slm

140
120

CT
ES

100
Figure 8 Modulation of L-afadin levels by CRH-CRHR1 signaling and nectin-3. (a) Hippocampal L-afadin
80
protein levels were reduced after 4 h of CRH application in vitro (t8 = 2.631, *P < 0.05, unpaired t test).
60
(b) Adding DMP696 to the slices at 10 min before the 4-h CRH treatment prevented the effects of CRH
40
on L-afadin levels (antagonism effect: F1,16 = 4.525, P = 0.0493; interaction: F1,16 = 5.714, P = 0.0295;
4 4
4 4
20
two-way ANOVA; *P < 0.05, Tukeys test). (c) L-afadin immunoreactivity in the stratum radiatum (sr) and
0
stratum lacunosum-moleculare (slm) of CA3 was significantly decreased in AAV-shNEC mice (t7.028 = 3.302,
Null
OE
*P < 0.05, unpaired t test). Representative coronal sections immunostained for L-afadin are shown.
(d) Overexpression of nectin-3 increased L-afadin protein levels in the hippocampus (treatment effect: F1,12 = 10.74, P = 0.0066, two-way ANOVA;
*P < 0.05, Bonferronis test). Representative transverse sections immunostained for L-afadin are shown. Note the clustered L-afadinimmunoreactive
puncta as indicated by arrows near the border between slm and sr in CT-OE and ES-OE mice. Scale bars represent 100 m. Data represent mean s.e.m.
Male mice were 36 months old. For full-length blots (a,b), see Supplementary Figure 10.

enhancing nectin-3 expression in the adult hippocampus would ameliorate the deleterious effects of early-life stress. AAV-null (an empty
AAV vector) was chosen as the control vector, whereas AAV-OE (contains the sequence for nectin-3) was used to overexpress nectin-3.
We observed that AAV-OE increased hippocampal nectin-3 levels
at 4 weeks after injection, and the effects lasted for at least 8 weeks
post-injection (Fig. 6a and Supplementary Fig. 7a). The specificity of
AAV-OE (Supplementary Fig. 7bd) and the extent of viral transfection (Supplementary Fig. 8) were validated.
In the Y-maze test (Fig. 6b), early-life stress impaired short-term
spatial memory, and AAV-OE failed to reverse such effects. In the spatial object recognition task, AAV-OE restored cognitive performance
in postnatally stressed mice (Fig. 6c). In addition, early lifestressed
mice injected with AAV-null (ES-null), but not control mice injected
with either AAV-null (CT-null) or AAV-OE (CT-OE) or postnatally
stressed mice injected with AAV-OE (ES-OE), failed to discriminate
the novel object from the known one (Supplementary Fig. 9). In the
Morris water maze test, although no difference in spatial acquisition
was found among groups, ES-null mice searched the target quadrant
and the other quadrants similarly in the probe trial, indicative of
spatial memory impairments (Fig. 6d). Conversely, ES-OE mice
showed intact spatial memory.
Nectin-3 overexpression reverses stress-induced spine loss
In AAV-null and AAV-OEinjected mice, dendrites and spines could
not be visualized because the vectors did not express EGFP. Thus,
we used Thy1-YFPH transgenic mice, in which a subpopulation of
hippocampal pyramidal neurons are selectively labeled by EYFP,
and determined the effects of early-life stress and nectin-3 over
expression on dendritic spine plasticity (Fig. 7).
Postnatally stressed adult mice had reduced spine density in CA3
neurons, and nectin-3 overexpression restored the number of spines
(Fig. 7b). Notably, although CT-OE and ES-OE mice had significantly more nectin-3expressing spines than their respective controls,
the number of nectin-3negative spines was reduced (treatment effect,
nature NEUROSCIENCE VOLUME 16 | NUMBER 6 | JUNE 2013

F1,13 = 14.18, P = 0.0024, two-way ANOVA). Compared with CT-null

mice, this resulted in a shift to a higher ratio of nectin-3positive to
nectin-3negative spines, but the number of total spines remained
similar. In addition, overexpression of nectin-3 increased spine volume
and spine head diameter in control mice (Fig. 7c). ES-null mice also
had larger spine volume than CT-null mice, indicative of compensatory
enlargements of the remaining spines. In contrast, stress-induced spine
enlargement in ES-OE mice was reversed by nectin-3 overexpression.
CRHR1 signaling and nectin-3 modulate L-afadin levels
L-afadin, an F-actinbinding protein that connects nectin-3 to the
actin cytoskeleton, has been shown to modulate spine formation
and remodeling19,28,29. Thus, the effects of nectin-3 and possibly of
stress and CRH-CRHR1 signaling on spine remodeling may be mediated by L-afadin. Consistent with this reasoning, we observed that
hippocampal L-afadin levels decreased following 4 h of in vitro treatment with 50 nM CRH (Fig. 8a), which potentially induces spine loss
under similar conditions22,30. Blockade of CRHR1 by DMP696 prevented the inhibitory effects of CRH on L-afadin levels (Fig. 8b).
We observed that L-afadin immunoreactivity in the stratum radiatum and stratum lacunosum-moleculare was reduced by nectin-3
knockdown (Fig. 8c). Conversely, nectin-3 overexpression increased
hippocampal L-afadin protein levels in stressed mice (Fig. 8d). Thus,
these findings support L-afadin as a potential molecular substrate that
mediates nectin-3dependent cognitive and structural changes, which
in turn contribute to stress- and CRH-induced effects.
DISCUSSION
Unraveling the molecular machineries responsible for stress-induced
cognitive dysfunction will provide insight into the neurobiology of
learning and memory and stress-related psychiatric disorders. Here,
we identified the synaptic CAM nectin-3 as an important component
in stress- and CRH-evoked effects. Exposure to either acute severe
stress or early-life stress downregulated nectin-3 expression levels
in the adult hippocampus via CRH-CRHR1 signaling. Suppression
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a r t ic l e s
of hippocampal nectin-3 reproduced the effects of early-life stress,
destabilized synaptic contacts and hampered spatial memory,
whereas overexpression of hippocampal nectin-3 reversed such negative effects of early-life stress. Together, our findings link impaired
nectin-3driven synaptic adhesion to stress-induced structural and
functional abnormalities.
It has been shown by us and others that elevated hippocampal
CRH-CRHR1 signaling modulates the negative effects of early-life
stress11,12 and chronic stress13 on hippocampus-dependent learning
and memory in adult animals. One of the underlying mechanisms
is that excessively released CRH acts through CRHR1 and evokes
structural remodeling31,32, especially the elimination of thin dendritic spines22,30. However, the molecules mediating these structural
effects have not been fully clarified. Here, we found that hippocampal nectin-3 expression levels were regulated by early-life stress in
a CRHR1-dependent manner: stress exposure during development
or postnatal forebrain CRH overexpression reduced nectin-3 levels,
whereas postnatal forebrain CRHR1 inactivation normalized nectin-3
levels in early lifestressed mice. These findings greatly expand our
previous observations that chronic stress during adulthood reduces
hippocampal nectin-3 levels via CRHR1 (ref. 11), and pinpoint the
role of the CRH-CRHR1 system in stress-induced downregulation
of nectin-3. On the basis of our finding that acute severe stress transiently suppressed hippocampal nectin-3 expression, the influences of
stress on nectin-3 expression may shift from short-term inhibition to
enduring suppression after repeated exposure during development.
Using both in vitro and in vivo approaches, we found that the
dynamic regulation of nectin-3 expression by acute stress was mediated by CRH-CRHR1 signaling. Activation of central glucocorticoid
receptor by systemic administration of dexamethasone or intracerebroventricular infusion of corticosterone failed to reproduce the
effects of stress on nectin-3, indicating that such effects are glucocorticoid receptor independent. Taking the functional relevance and
spatial distribution patterns of CRHR1 and nectin-3 into account,
CRHR1 hyperactivity likely disrupts synaptic adhesion through indirect interactions with nectin-3. Indeed, we found that the effects of
CRH and CRHR1 could be modulated by the MAPK signaling pathway, but not by the cAMP-PKA pathway, as the inhibition of MEK,
which phosphorylates MAPK, attenuated CRH-initiated nectin-3
downregulation. Notably, CRH specifically increases the levels of
phosphorylated MAPK through CRHR1 in areas CA3 and CA1
(ref. 33). The regional characteristics of the MAPK pathway activated
by CRHR1 and the expression pattern of nectin-3 in the hippocampus
therefore provide a molecular basis for the circuit-specific effects of
stress. Nonetheless, although our data provide evidence that stress and
excessive CRH-CRHR1 signaling reduce nectin-3 levels by inhibiting
gene and protein expression, it is unclear whether other processes,
such as the cleavage and degradation of the nectin-3 protein, are influenced by stress and CRHR1. Moreover, whether acute and chronic
adult stress and early-life stress modulate nectin-3 levels through the
same molecular cascade remains an open question. Future studies are
needed to disentangle the entire molecular mechanisms responsible
for stress and CRHR1-induced downregulation of nectin-3.
Synaptic CAMs have been implicated in hippocampus-dependent
learning and memory and cognitive disorders57. The disruption of
synaptic adhesion mediated by N-cadherin, one of the key CAMs
in adherens junctions, impairs long-term, but not short-term,
hippocampus-dependent emotional memory34. However, the roles of
nectins, the other group of CAMs that organize adherens junctions, in
cognition remain unknown. We found that hippocampal nectin-3 knockdown disrupted long-term spatial memory, mimicking the cognitive
712

consequences of early-life stress exposure. Notably, the protein levels

of other nectins, N-cadherin and -catenin were unaffected by the
suppression of nectin-3, indicating that the observed cognitive effects
were nectin-3 specific. On the other hand, overexpressing nectin-3
in the adult hippocampus did not improve cognitive performance
per se, but reversed the negative effects of early-life stress on cognition. These results highlight the crucial role of nectin-3driven synaptic adhesion, which is susceptible to early-life stress, in long-term
spatial memory.
The cadherin-catenin complex and the nectin-afadin complex
cooperate to modulate structural and synaptic plasticity1419.
Although the roles of the cadherin-catenin complex in these pro
cesses have been investigated29,35,36, it was unclear whether nectin-3
is involved in synaptic and structural remodeling. We found that the
inhibition of nectin-3 specifically decreased the number of dendritic
spines expressing nectin-3, resulting in a reduction in the total number
of spines. Conversely, overexpression of nectin-3 reversed early-life
stressinduced spine loss. Nectin-3 knockdown did not change the
overall size of the remaining spines, whereas nectin-3 overexpression
increased spine volume and spine head diameter. Notably, early-life
stress increased the volume, but not the head diameter, of the spines,
possibly through a compensatory mechanism for the loss of spines.
However, nectin-3 overexpression normalized spine dimensions in
stressed mice. These data suggest that, although reduced nectin-3
levels do not fully mediate the effects of early-life stress on spine
morphology, enhancing its expression could rescue the abnormal
changes by stress. In addition, it should be noted that the density
of spines and synapses remains unaltered in conventional nectin-3
knockout mice, although the formation of adherens junctions is
markedly impaired16. We ascribe this to the differences in the extent
and duration of nectin-3 inhibition, as well as compensatory effects
among various CAMs1.
L-afadin links nectin-3 to the cadherin-catenin complex and actin
cytoskeleton1 and participates in spine formation and remodeling19,28,29.
We observed that hippocampal L-afadin levels could be modulated
by CRH-CRHR1 signaling. Moreover, hippocampal nectin-3 knockdown reduced L-afadin levels, whereas nectin-3 overexpression could
increase L-afadin levels in postnatally stressed mice. These findings
imply that the effects of nectin-3 on structural remodeling and cognition are likely mediated by L-afadin. However, the involvement of
L-afadin in learning and memory merits further investigations.
In summary, our findings indicate that early-life stress exposure
disrupts nectin-3mediated axodendritic adhesion in hippocampal
neurons through the enhancement of CRH-CRHR1 signaling, which
in turn destabilizes dendritic spines and compromises hippocampusdependent learning and memory. The discovery that restoring nectin-3
levels ameliorates early-life stressinduced memory deficits may
stimulate the development of therapeutic strategies for stress-related
cognitive disorders.
Methods
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary information is available in the online version of the paper.
Acknowledgments
We are grateful to D. Harbich and B. Schmid for technical assistance. This work
was supported by the European Communitys Seventh Framework Program (FP7,
Project No. 201600), the Bundesministerium fr Bildung und Forschung within
the framework of the NGFN-Plus (FKZ: 01GS08151 and 01GS08155) and by the
Initiative and Networking Fund of the Helmholtz Association in the framework of
the Helmholtz Alliance for Mental Health in an Ageing Society (HA-215).

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a r t ic l e s
AUTHOR CONTRIBUTIONS
X.-D.W. and M.V.S. designed the experiments. X.-D.W., Y.-A.S., K.V.W., C.A.,
S.H.S., J.H., M.W., C.L. and C.K. performed the experiments. X.-D.W., Y.-A.S.,
C.A. and M.W. analyzed the data. M.E., J.M.D., M.B.M. and M.V.S. supervised the
experiments. X.-D.W., W.W., F.H., M.E., J.M.D., M.B.M. and M.V.S. wrote the paper.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.

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Animals. Male Crhr1loxP/loxP;Camk2a-cre and R26flopCrh/flopCrh;Camk2a-cre mice

were generated as described previously24,25 and kept on a mixed 129S2/Sv
C57BL/6J background. Female CRHR1-EGFP reporter mice26, female CRHR1
tau-lacZ reporter mice27, male CD1 and C57BL/6N mice (Charles River), and
Thy1-YFPH mice (Jackson Laboratory) were used. All mice were housed under a
12:12-h light/dark cycle (lights on at 7 a.m.) and constant temperature (22 1 C)
conditions with ad libitum access to both food and water. The protocols were
approved by the Committee for the Care and Use of Laboratory Animals of the
Government of Upper Bavaria, Germany.
Experiments. To examine the effects of early-life stress and postnatal CRH overexpression on nectin-3 expression, we killed control or stressed CRHR1-CKO
mice, stress-naive CRH-COE mice and respective wild-type mice at 78 months
of age. To study the effects of acute stress on nectin-3 expression, we killed male
C57BL/6N mice (3 months old) at 1, 4, 8 or 24 h after a single social defeat
stress. To assess the potential involvement of glucocorticoid receptor, we killed
male CD1 mice (3 months old) at 1, 4, 8 or 24 h after a subcutaneous injection
of a synthetic glucocorticoid receptor agonist dexamethasone (10 mg per kg,
Ratiopharm) or 0.9% saline (wt/vol).
To study the effects of CRH on nectin-3 expression in vitro, we incubated
acute brain slices from male C57BL/6N mice (3 months old) with either artificial cerebrospinal fluid (ACSF, containing 124 mM NaCl, 3 mM KCl, 26 mM
NaHCO3, 2 mM CaCl2, 1 mM MgSO4, 10 mM d-glucose and 1.25 mM
NaH2PO4, pH 7.3) or 50 nM CRH (Bachem) in ACSF37 in the holding chambers. At 1 or 4 h after treatment, slices were collected. To address the role of
CRHR1 in these processes, we pretreated slices with 0.01% dimethyl sulfoxide
(DMSO, vol/vol) in ACSF (vehicle) or 100 nM (ref. 38) of a CRHR1 antagonist
DMP696 (Bicoll GmbH) in vehicle for 10 min, and then incubated with or
without 50 nM CRH for 4 h. To further examine the effects of CRH-CRHR1
signaling and corticosterone on nectin-3 expression in vivo, we injected three
cohorts of male C57BL/6N mice (3 months old) icv with either (1 l per mouse)
ACSF or CRH (0.2 mM in ACSF); vehicle (1% DMSO in ACSF), CRH (0.2 mM
in vehicle), CRH (0.2 mM) and DMP696 (1 mM) in vehicle, CRH (0.2 mM) and
Rp-cAMPS (4 mM) in vehicle, or CRH (0.2 mM) and U0126 (2.6 mM) in vehicle; vehicle (5% ethanol in ACSF, vol/vol) or corticosterone (2.9 mM in vehicle).
Mice were killed and hippocampi were collected 4 h after the injection.
For the colocalization studies of CRHR1 and nectin-3, female CRHR1-EGFP
or CRHR1 tau-lacZ reporter mice (3 months old) were anesthetized and
transcardially perfused with heparinized 0.9% saline followed by buffered 4%
paraformaldehyde (wt/vol). Brains were processed for immunostaining.
To study the effects of hippocampal nectin-3 knockdown, we injected two
cohorts of male C57BL/6N mice (3 months old) with either the control or
knockdown virus and tested them in the Y-maze and spatial object recognition
tasks (the first cohort only) and the Morris water maze task (both cohorts) after
4 weeks of recovery. At 1 week after behavioral testing, mice were killed. Another
cohort (3 months old) was used to examine the roles of nectin-3 in dendritic
spine plasticity. To study the effects of early-life stress and nectin-3 overexpression, we microinjected male C57BL/6N or Thy1-YFPH mice (5 months old),
with or without early-life stress, intrahippocampally with either the control or
nectin-3overexpressing virus. After 4 weeks of recovery, C57BL/6N mice were
subjected to cognitive testing and killed 1 week later, whereas Thy1-YFPH mice
were transcardially perfused and brains processed for structural analysis.
Stress procedures. The limited nesting and bedding material procedure was
carried out as described previously9,39. The day of birth was designated postnatal day 0 (P0). On the morning of P2, control dams were provided with a sufficient amount of nesting material (two squares (4.8 g) of Nestlets, Indulab) and
500 ml of standard sawdust bedding. In the stress cages, dams were provided
with a limited quantity of nesting material (one half of a square (1.2 g) of Nestlets),
which was placed on a fine-gauge aluminum mesh platform (McNichols). All
litters remained undisturbed from P2 to P9. On P9, all dams were provided with
standard nesting and bedding material. Male offspring were weaned on P28, and
tail tips were collected and genotyped.
The acute social defeat stress procedure was performed by introducing each
male C57BL/6N mouse into the home cage of an aggressive CD1 resident mouse

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with short attack latency for 5 min (ref. 40). Control mice were allowed to explore
an empty novel cage similar to the resident cage for 5 min.
Acute brain slice preparation. Serial coronal brain slices (350 m thick) were
prepared through the hippocampus using a vibrating microtome in ice-cold ACSF
bubbled with a 95% O2/5% CO2 mixture. Slices were rapidly cut into halves and
transferred to holding chambers. At 1 or 4 h after treatment, slices were collected,
snap-frozen and stored at 20 C. Hippocampal slices were later dissected on ice,
pooled for each mouse (23 slices per treatment and time point), and homogenated for western blot analysis.
Cannulation and intracerebroventricular injection. Stereotaxic surgery was
performed as previously described41. A 23-gauge stainless steel cannula was
placed in the right lateral ventricle (0.4 mm posterior to bregma, 1.0 mm lateral
from midline, 1.6 mm dorsoventral from dura)42. Mice were allowed to recover
for 1 week. On the day of the experiment, mice were anesthetized in the home
cage by 1 ml of isoflurane, and the anesthesia was maintained using isoflurane-O2
(1~1.5:100) inhalation. Drugs (1 l) were delivered via a Hamilton syringe over a
1-min period followed by 1 min of rest. At 4 h after the injection, mice were killed,
the cannula position was examined and hippocampi were dissected.
Viral-mediated gene manipulation and intrahippocampal microinjection. To
suppress or enhance nectin-3 expression in the hippocampus, we used an adenoassociated bicistronic AAV1/2 vector (GeneDetect). AAV-shSCR (AAV1/2-U6scrambled shRNA-terminator-CAG-EGFP-WPRE-BGH-polyA), AAV-shNEC
(AAV1/2-U6-Nectin-3 shRNA-terminator-CAG-EGFP-WPRE-BGH-polyA),
AAV-null (AAV1/2-CAG-null-WPRE-BGH-polyA), and AAV-OE (AAV1/2-CAGNectin-3-WPRE-BGH-polyA) were generated and purified by GeneDetect.
Stereotaxic surgery and intrahippocampal microinjection was performed
as previously described41. Briefly, 0.5 or 1 l of the virus (1.2~11 1012 viral
genomes per ml) was injected bilaterally, aiming at the stratum radiatum of the
dorsal CA3 (1.9 mm posterior to bregma, 2.1 mm lateral from midline, 1.8 mm
dorsoventral from dura)42. The virus was delivered over a 15-min period followed
by 5 min of rest. Mice were given a 4-week period before experimentation to allow
sufficient viral infection in the hippocampus.
Behavioral testing. The tests were performed between 8 a.m. and 1 p.m. and
scored by the ANY-maze 4.50 software (Stoelting). Short-term spatial working memory was tested by recording spontaneous alternation behavior in the
Y-maze43. The apparatus was made of gray polyvinyl chloride with three symmetrical arms (30 10 15 cm3) and evenly illuminated (30 lx). Prominent intraand extra-maze spatial cues were provided. Mice were placed in the center of the
maze and allowed to explore the arms freely for 5 min. Three consecutive choices
of all three arms were counted as an alternation. The percentage of spontaneous
alternation was determined by dividing the total number of alternations by the
total number of choices minus 2.
The spatial object recognition task was performed in an open field apparatus
(50 50 50 cm3) under low illumination (30 lx)41. Prominent spatial cues were
provided. Mice were habituated to the testing environment for 10 min on 2 consecutive days before testing. During the acquisition trials, mice were presented
with two identical aluminum cubes (5 5 5 cm3) and allowed to explore the
objects twice for 10 min, separated by a 15-min intertrial interval (ITI). During
the 5-min retrieval trial, 30 min following the last acquisition trial, mice were
presented with a non-displaced object and a relocated one. The ratio of time spent
with the displaced (novel) object compared with the non-displaced (known)
object and the percentages of time exploring the novel and known objects were
calculated, with a higher preference for the novel object being rated as intact
spatial recognition memory.
The Morris water maze test was carried out in a circular tank (110 cm in diameter) filled with opaque colored water (22 1 C) and provided with prominent
extra-maze visual cues11. After day 1 with a 60-s free swim trial, mice were trained
to locate a visible platform (10 cm in diameter) above the surface of the water
for four trials. In the following spatial training sessions, mice received four trials
per day to locate the submerged platform in a fixed position over 3 consecutive
days. On the next day, reference memory was assessed in a 60-s probe trial with
platform removed, and the time spent in each quadrant was recorded. The trials in

doi:10.1038/nn.3395

spatial training sessions (ITI = 10 min) were terminated once the mouse found the
platform or 60 s had elapsed, and the latency to reach the platform was recorded
for each trial. Mice that did not employ a search strategy and floated in the tank
in all trials were excluded from analysis.

npg

2013 Nature America, Inc. All rights reserved.

In situ hybridization. Coronal brain sections (20 m thick) were prepared

and in situ hybridization performed as previously described13. The following
primers were used to generate an antisense RNA hybridization probe (485 base
pairs) that recognizes a shared sequence of alpha-, beta- and gamma-transcript
variants of nectin-3: AGCCGTTACATTCCCACTTG (forward primer) and
ATTGTCCATCCAACCTGCTC (reverse primer). The slides were apposed to
Kodak Biomax MR films (Eastman Kodak) and developed. Autoradiographs
were digitized, and relative expression (average optical density of the region of
interestaverage optical density of the background) was determined by Scion
Image (Scion).
Primary antibodies. For western blot, we used antibodies to nectin-3 (ab63931,
1:2,000), nectin-2 (ab135246, 1:1,000), nectin-4 (ab110387, 1:1,000), N-cadherin
(ab12221, 1:2,500), -catenin (ab22656, 1:2,000) and L-afadin (ab11337, 1:1,000)
from Abcam; to actin (sc-1616, 1:2,000), nectin-1 (sc-28639, 1:1,000) and PKR
(sc-1702, 1:1,000) from Santa Cruz Biotechnology. For immunostaining, we used
antibodies to EGFP (ab5450, 1:2,000), -galactosidase (ab9361, 1:2,000), nectin-3
(ab63931, 1:500) and L-afadin (ab11337, 1:500) from Abcam.
Western blot. Total hippocampal protein extracts were prepared and western blot
was performed as previously described13. Membrane fractions were extracted
using the Calbiochem ProteoExtract kit (EMD Millipore)44. Samples were
resolved by 10% sodium dodecyl sulfatepolyacrylamide gels, and transferred
onto nitrocellulose membranes (Invitrogen). Membranes were labeled with
primary antibodies overnight at 4 C. Following incubation with horseradish
peroxidaseconjugated secondary antibodies (1:2,000, DAKO) for 3 h, bands
were visualized using an enhanced chemiluminescence system (Amersham
Biosciences) and quantified by densitometry (Quantity One 4.2, Bio-Rad).
Immunohistochemistry and image analysis. Double-labeling immunofluorescence was performed on free-floating coronal (20 m thick) or transverse
(30 m thick) sections11. After incubation with primary antibodies overnight
at 4 C, sections were rinsed and labeled with Alexa Fluor 488 and Alexa
Fluor 647conjugated secondary antibodies (1:500, Invitrogen) for 3 h at
22 1 C. After rinsing, sections were transferred onto slides and coverslipped
with Vectashield mounting medium containing 4,6-diamidino-2-phenylindole
(Vector Laboratories).
All images (1,600 1,600 pixels) were obtained with an Olympus IX81-FV1000
laser-scanning confocal microscope (Olympus). A 10 objective (NA 0.40), a
20 objective (NA 0.75) and a 60 water-immersion objective (NA 1.20) were
used. Images were imported into the US National Institutes of Health ImageJ
software, converted to 8-bit grayscale and thresholded uniformly. The density
of L-afadinimmunoreactive puncta was measured (56 sections per mouse).
Representative images were adjusted for better brightness and contrast using the
FV10-ASW 2.0 software (Olympus).
Quantitative morphological analysis of dendritic spines. Transverse sections
were collected for analyzing the pyramidal neurons of CA3 and CA1, and coronal sections for dentate granule cells. In area CA3, as the stratum radiatum was
densely packed with EGFP/EYFP-labeled commissural/associational fibers, only

doi:10.1038/nn.3395

the dendritic segments (20130 m in length, 815 dendrites per mouse) in

the stratum lacunosum-moleculare were analyzed. Dendrites were scanned at
0.33-m intervals along the z axis using the 60 objective with a 2.5 digital
zoom, yielding a voxel size of 0.053 0.053 0.33 m3. The z stack images
were deconvolved using Huygens 4.2 software (Scientific Volume Imaging)45.
Spine density (expressed as the number of spines per 10 m of dendrite), spine
volume and spine head diameter were analyzed with NeuronStudio software
(http://research.mssm.edu/cnic/tools-ns.html)45,46. To quantify the density of
nectin-3positive spines, deconvolved dual-channel z stacks (EGFP/EYFP and
nectin-3) were merged and nectin-3positive spines were counted manually
using ImageJ.
In AAV-shSCR and AAV-shNEC mice, the background in dentate gyrus and
CA1 was higher than in CA3. Thus, only spine density in the outer molecular
layer of the suprapyramidal blade of dentate gyrus and the stratum lacunosummoleculare of CA1 was quantified. Dendrites (80100 m in length) were
scanned at 1-m intervals along the z axis using the 60 objective with a
2.5 digital zoom. For each mouse, eight dendrites from different CA1 pyramidal
neurons or six dendrites from different dentate granule cells were selected. Spines
were counted manually using ImageJ, and spine density was expressed as the
number of spines per 10 m of dendrite.
Statistical analysis. SPSS 16 (SPSS), GraphPad Prism 5 (GraphPad Software),
and R version 2.15 (http://www.r-project.org/) were used. Normally distributed
data were analyzed by ANOVA followed by Bonferroni or Fishers LSD post hoc
tests as necessary. Students t test was used to compare pairs of means. Data that
were not normally distributed were rank- and/or Box-Cox transformed to achieve
a normal data distribution. After data transformation, data were analyzed by
ANOVA followed by Tukey post hoc test when necessary, and Welchs t test was
used to compare pairs of means. The level of statistical significance was set at
P < 0.05. Data are expressed as mean s.e.m.

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