nectin-3 y stress

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706 VOLUME 16 | NUMBER 6 | JUNE 2013 NATURE NEUROSCIENCE

A R T I C L E S

Synapses are specialized intercellular junctions that meditate the

transmission of information between neurons. Glutamatergic exci-tatory synapses are established by presynaptic axonal terminals

and postsynaptic dendritic spines, both of which anchor synapticcell adhesion molecules (CAMs)1,2. CAMs are not merely static

constituents of synapses, but are dynamic modulators of synapticactivity 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 evidenceindicates that the dysregulation of synaptic CAMs contribute to

structural modifications and cognitive deficits5,6, including thoseinduced by stress7.

Repeated exposure to severe stress exerts deleterious effects on cog-nition in different life stages8. Early adversities, such as an impover-

ished environment, impair hippocampal integrity and function, whichis 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 shownto modulate the negative effects of early-life stress on cognition andstructural plasticity 11,12. In adult animals, the influence of chronic

stress on cognition also involves hippocampal CRH-CRHR1 signal-ing13. 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 tothe presynaptic active zone and postsynaptic density (PSD)14.

Postsynaptic nectin-3 mediates heterophilic adhesion with presynap-tic nectin-1, and is indirectly connected to the actin cytoskeleton via

L-afadin. The nectin-afadin complex colocalizes and cooperates withthe cadherin-catenin complex to organize adherens junctions, and

participates in synaptic formation, maintenance and remodeling14–19.Some evidence suggests that impaired nectin-mediated adhesion

disrupts hippocampal development16 and is associated with mental

retardation20. Although expressed ubiquitously, nectin-3 is abundantin CA3 pyramidal neurons13,21 that are vulnerable to both acute22

and chronic23 stress challenge. Nectin-3 expression levels have beenshown to correlate with the observed cognitive phenotype following

chronic stress13. However, it is still unclear whether nectin-3 is caus-ally involved in mediating the effect of stress via CRHR1 signaling on

cognition and structural remodeling.We examined how stress and CRH-CRHR1 signaling might mod-

ulate nectin-3 expression in the hippocampus. Using site-specific

knockdown and overexpression of nectin-3, we then investigatedthe role of hippocampal nectin-3 in spatial learning and memoryand dendritic spine plasticity, and tested whether reinstating

nectin-3 in the adult hippocampus could reverse the detrimentalconsequences 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 Universität München, Lehrstuhl für Entwicklungsgenetik, Helmholtz Zentrum München, Neuherberg, Germany. 4Deutsches

Zentrum für 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. ([email protected]).

Received 14 January; accepted 9 April; published online 5 May 2013; doi:10.1038/nn.3395

Nectin-3 links CRHR1 signaling to stress-inducedmemory deficits and spine loss

Xiao-Dong Wang1,5, Yun-Ai Su1,5, Klaus V Wagner1, Charilaos Avrabos1, Sebastian H Scharf 1,Jakob Hartmann1, Miriam Wolf 1, Claudia Liebl1, Claudia Kühne1, Wolfgang Wurst1–4, Florian Holsboer1,Matthias Eder1, Jan M Deussing1, Marianne B Müller1 & 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 mimickedby 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.

http://www.nature.com/doifinder/10.1038/nn.3395http://www.nature.com/doifinder/10.1038/nn.3395


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NATURE NEUROSCIENCE VOLUME 16 | NUMBER 6 | JUNE 201 3 70 7

A R T I C L E S

RESULTS

Stress reduces nectin-3 levels via CRH-CRHR1 signaling

As we reported previously 13, nectin-3 is enriched in hippocampalCA3 neurons (Fig. 1a). Using male Crhr1loxP/loxP ; Camk2a-cre mice24,

in which the Crhr1 gene is inactivated postnatally in forebrain princi-pal neurons (referred to as CRHR1-CKO hereafter), we investigated

whether early-life stress (postnatal days 2–9) would lead to down-regulation of nectin-3 expression in a CRHR1-dependent manner. We

examined nectin-3 (Pvrl3) mRNA and protein levels in adult CRHR1-CKO and wild-type mice exposed to either a standard or impover-

ished environment early in life (Fig. 1a,b). In wild-type mice withearly stressful experiences, which exhibit impaired spatial learning

and memory 11, both mRNA and protein levels of hippocampal nectin-

3 were reduced. In comparison, control and stressed CRHR1-CKOmice showed comparable nectin-3 mRNA and protein levels. Because

early-life stress increases CRH levels in the adult hippocampus12, weused adult male R26 flopCrh/f lopCrh ; 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) totest whether CRH overexpression would evoke similar effects onnectin-3 expression to early-life stress (Fig. 1c,d). Compared with

the wild-type controls, stress-naive CRH-COE mice with prominentcognitive deficits11 had lower nectin-3 mRNA and protein levels in

the hippocampus. These results indicate that early-life stress–inducedreductions of nectin-3 expression involve CRH-CRHR1 signaling.

To further elucidate the effects of stress on nectin-3 expression, weevaluated nectin-3 levels following an acute severe stress challenge in

adulthood (Fig. 1e,f ). A brief (5 min) exposure to social defeat stressdynamically regulated nectin-3 expression in the adult hippocampus.

At 4 h, but not 1 h, 8 h or 24 h after the acute stress, hippocampalnectin-3 levels were substantially reduced. Notably, a single treatment

with the glucocorticoid receptor agonist dexamethasone (10 mg perkg of body weight) failed to alter nectin-3 levels (Supplementary

Fig. 1a), indicating that glucocorticoid receptor may not mediate theeffects of stress on nectin-3 expression.

CRH-CRHR1 signaling regulates nectin-3 expression

To dissect the involvement of the CRH-CRHR1 system in stress- regulated 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 selectivenonpeptide 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 effectsof 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 lev-els in the hippocampus decreased (Fig. 2c). Notably, icv infusion ofcorticosterone (2.9 mM) did not alter nectin-3 levels (SupplementaryFig. 1c). Co-administration of CRH with DMP696 (1 mM) preventedCRH-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 ofCRH. 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 anti-bodies to CRHR1 (ref. 26), we used both CRHR1–enhanced green

a

W T

C K O

CT ES

0

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WT CKO

CTES

CTES

11 8 7 7

N o r m a

l i z e

d n e c

t i n -

3 m

R N A

( % o

f C T - W

T )

W T

C O E

0

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40

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80

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WT COE

*

9 9

N o r m a

l i z e

d n e c t i n

- 3 m

R N A

( % o

f W T

)

0

20

40

60

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100

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*

WT CKO

5 5 5 6

R a

t i o s o

f n e c

t i n - 3

t o a c

t i n

( % o

f C T - W

T )

b

Nectin-3

Actin

CT ES

WT CKO

CT ES

0

20

40

60

80

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R a

t i o s o

f n e c t i n - 3

/ a c

t i n

( % o

f W T

)

**

6 6

WT COE

1 4 8 24

Time after acute social defeat stress (h)

D e

f e a

t C o n

t r o

l

e

*

1 4 8 24 N o r m a

l i z e

d n e c

t i n - 3 m R

N A

( % o

f c o n

t r o

l )

0

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Defeat


9 9 1010 10 10 1010

f

c

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Actin

WT COE

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*

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t i o s o

f n e c

t i n - 3

t o a c

t i n

( % o

f c o n

t r o

l )

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100120

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Defeat


6 10 86 10 8 6 6

1 4 8 24


Nectin-3

Actin

Control Defeat Control Defeat Control Defeat Control Defeat

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 7–8-month-old male mice (stress

effect: F 1,29 = 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:

F 1,17 = 5.507, P = 0.0313, two-way ANOVA; *P < 0.05, Bonferroni’s 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 7–8-month-old male CRH-COE

mice (t 16 = 2.869, *P < 0.05; t 10 = 3.336, **P < 0.01; unpaired t test). (e,f) Acute social defeat stress

disrupted hippocampal nectin-3 expression at both mRNA (stress effect: F 1,70 = 4.164, P = 0.045,two-way ANOVA) and protein levels (stress effect: F 1,52 = 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

(t 17 = 2.144, *P < 0.05, unpaired t test), which were reflected at the protein levels (f, t 18 = 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 µm (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.


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A R T I C L E S

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

reporter mice27. We observed that CRHR1 partially colocalized withnectin-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 signal-ing impair cognition11,12 and reduced nectin-3 expression levels.

To gain insight into the behavioral and structural consequencesof nectin-3 downregulation in the adult hippocampus, we used

adeno-associated virus (AAV)-mediated nectin-3 knockdown andinvestigated whether reduced levels of nectin-3 would impair cog-

nition, thereby mimicking the early-life stress–induced phenotype.AAV-shSCR (contains a scrambled short hairpin RNA sequence)

was chosen as the negative control and AAV-shNEC (contains thesequence for a short hairpin RNA specific

for nectin-3) was used to suppress nectin-3

expression in vivo.We found that the AAV-shNEC vector

specifically reduced hippocampal nectin-3levels, whereas the levels of other nectins

and related molecules remained unchanged

(Supplementary Fig. 3). We also exam-ined the extent of viral transfection in the

hippocampus (Supplementary Fig. 4). In AAV-shNEC–treated mice,

nectin-3 expression levels were downregulated in CA3, dentate gyrusand CA1 throughout the dorsal hippocampus (Fig. 4a). Short-term

spatial working memory was not altered in AAV-shNEC–treated mice,as seen by similar spontaneous alternation behavior in the Y-maze

task compared with the controls (Fig. 4b). In the spatial object recog-nition test, AAV-shNEC–treated mice showed a markedly impaired

performance (Fig. 4c and Supplementary Fig. 5a,b). Moreover,AAV-shSCR–treated mice discriminated the novel object from the

familiar one, whereas AAV-shNEC–treated mice failed to show objectdiscrimination (Supplementary Fig. 5b). In the spatial training ses-

sions of the Morris water maze task (Fig. 4d), the spatial learning ofAAV-shNEC–treated mice was preserved. In the probe trial, how-

ever, control mice, but not AAV-shNEC–treated mice, searched thetarget quadrant longer than the other quadrants. Together, these data

indicate that suppression of hippocampal nectin-3 reproduces 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-β-galactosidase (b)

partially colocalized with nectin-3 in CA1

pyramidal neurons. DAPI, 4′,6-diamidino-2-

phenylindole. Arrowheads in the representative

confocal images indicate neurons in which

the colocalization of CRHR1 and nectin-3 was

observed. Scale bars represent 20 µm.

b

CRHR1–β-gal Nectin-3 MergedDAPI

a

CRHR1-EGFP Nectin-3 MergedDAPI

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 (t 8 = 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: F 1,16 = 5.446, P = 0.033, two-way

ANOVA; *P < 0.05, Bonferroni’s test). (c) Intracerebroventricular administration of CRH downregulated total (t 20 = 2.472, *P < 0.05, unpaired t test)

and membrane (t 8 = 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 ( F 4,25 = 3.276,

P = 0.027, one-way ANOVA), whereas DMP696 prevented the effects of CRH on membrane nectin-3 expression ( F 4,25 = 2.796, P = 0.048, one-wayANOVA). *P < 0.05, Fisher’s 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.

Nectin-3

Actin

ACSF CRH

a

ACSF CRH

0

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R a t i o s o f n e c t i n - 3 t o a c t i n

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Vehicle DMP696

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CRH


( % o

f A C S F - v e h

i c l e ) *

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ACSF CRH ACSF CRH

V eh ic le D MP 69 6

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C R H

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( % o

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Total

ACSF CRH

Membrane

d

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l e

C R H C R

H +

D M P 6

9 6

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+

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M P S

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+

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l e

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H +

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9 6

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+

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M P S

C R H

+

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l e C R

H C R H

+

D M P 6

9 6 C R

H +

R p - c A M

P S C R H

+

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V e h i c

l e C R

H C R H

+

D M P 6

9 6 C R

H +

R p - c A M

P S C R H

+

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* * * *

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6 6 6 6 6

* *

*

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A R T I C L E S

cognitive effects of early-life stress and that nectin-3 is essential forhippocampus-dependent long-term spatial memory.

Suppression of nectin-3 evokes dendritic spine loss

To assess the effects of long-term nect in-3 knockdown on structuralplasticity in vivo, we quantified the number and size of dendritic

spines in EGFP-expressing hippocampal neurons in AAV-shSCR–and AAV-shNEC–treated mice. Similar to adult mice with a history

of early-life stress11, AAV-shNEC–treated mice showed a markedreduction in spine density in CA3 (Fig. 5a,b), dentate gyrus and

CA1 (Supplementary Fig. 6) principal neurons. Spine volumeand 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 ofnectin-3–positive spines was reduced in AAV-shNEC–treated mice

s h S C R

s h N E C

a

–1.76 –2.08 –2.40 –2.72

Bregma (mm)

CA1

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n t a n e o u s a l t e r n a t i o n ( % )

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Object recognition

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E s c a p e l a t e n c y ( s )

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Day 1 Day 2 Day 3

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0

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30 TargetRightOppositeLeft

##

Probe trial

23 22

Figure 4 Hippocampal nectin-3 knockdown impaired long-term spatial memory. (a) At 1 week after the behavioral tests , AAV-shNEC–induced

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: F 1,30 = 96.218, P < 0.00001; F 1,29 = 22.193, P = 0.00006; F 1,30 = 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 µm. (b–d) 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 (t 22 = 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 (t 21.743 = 2.104, *P < 0.05, Welch’s t test). (d) In the Morris water maze test, AAV-shNEC mice showed similar spatial

learning performance to AAV-shSCR mice (F 1,43 = 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:

t 22 = 3.38,##P < 0.01; AAV-shNEC: t 21 = 1.321, P = 0.201; paired t test). Data represent mean ± s.e.m.

a

EGFP Nectin-3 Merged

shSCR

shNEC

shSCR

shNEC

0.1 0.2 0.3 0.4 0.5 0.60

20

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Spine volume (µm3)

C u m u l a t i v e d i s t r i b u t i o n ( % )

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Spine head diameter (µm)


shSCR

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cb

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s h S C

R

s h N E C

**

6 6

s h S C

R

s h N E C

***

6 6

Total Nectin-3+

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-3–immunoreactive puncta (red) in

the stratum lacunosum-moleculare of CA3.

Arrowheads indicate nectin-3–positive spines.

Scale bars represent 1 µm. (b) Suppression ofnectin-3–induced spine loss in CA3 pyramidal

neurons (t 10 = 4.097, **P < 0.01, unpaired

t test). The number of nectin-3–positive spines

was significantly less in AAV-shNEC mice

compared with AAV-shSCR mice (t 10 = 10.505,

***P < 0.001, unpaired t test). Data represent

mean ± s.e.m. (c) Nectin-3 knockdown did not

affect spine volume (t 1801.301 = 0.6693,

P = 0.5034, Welch’s t test) or spine head

diameter (t 2029.375 = 0.9195, P = 0.358,

Welch’s 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, 8–14 dendrites were analyzed.


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A R T I C L E S

(Fig. 5b), whereas the numbers of spines lacking nectin-3 were compara-

ble between groups (AAV-shSCR, 6.59± 0.34 spines per 10µm of dendrite;AAV-shNEC, 6.35± 0.15; t 10 = 0.637, P = 0.539, unpaired t test).

Nectin-3 overexpression rescues stress-induced memory loss

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

1

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OppositeLeft

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CT ES CT ES

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– 1 . 6 0

– 1 . 7 6

– 1 . 9 2

– 2 . 0 8

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– 2 . 4 0

– 2 . 5 6

– 2 . 7 2

Figure 6 Hippocampal nectin-3 overexpression reversed early-life stress–induced cognitive deficits. (a) Following the behavioral tests, hippocampalnectin-3 mRNA levels were determined. Nectin-3 mRNA levels in CA3, dentate gyrus and CA1 were significantly increased by AAV-OE (treatment

effect: F 1,22 = 1740.24,###P < 0.001; F 1,22 = 749.003, P < 0.001; F 1,22 = 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: F 1,10 = 5.581, *P < 0.05; F 1,10 = 10.039,

P < 0.05; one-way repeated measures ANOVA; n = 6–7 mice per group). Scale bar in the representative in situ hybridization images represents 500 µm.

(b–d) 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: F 1,43 = 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: F 1,42 = 4.127,

P = 0.0486; interaction: F 1,42 = 4.546, P = 0.0389; two-way ANOVA; *P < 0.05, Tukey’s 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: F 1,36 = 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: t 8 = 2.72,#P < 0.05; CT-OE: t 8 = 4.944,

##P < 0.01; ES-OE: t 11 = 3.536, P < 0.01;

ES-null: t 10 = 1.684, P = 0.123; paired t test). Data represent mean ± s.e.m.

0.1 0.2 0.3 0.4 0.5 0.6 0.7

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ca

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3

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Figure 7 Nectin-3 overexpression rescued early-life stress–evoked spine loss and spine volume changes.

(a) Representative deconvolved z stacks showing enhanced yellow fluorescent protein (EYFP)-filled dendrites and

spines (green) and nectin-3–immunoreactive puncta (red) in the stratum lacunosum-moleculare of CA3. Scale

bars represent 1 µm. (b) Early-life stress reduced spine density in control mice, which was reversed by nectin-3

overexpression (stress effect: F 1,13 = 4.833, P = 0.0466; interaction: F 1,13 = 9.827, P = 0.0079; two-way ANOVA;

*P < 0.05, **P < 0.01, Bonferroni’s test). Overexpression of nectin-3 increased the number of nectin-3–positive

spines (treatment effect: F 1,13 = 23.94, P = 0.0003, two-way ANOVA). Data represent mean ± s.e.m. (c) Nectin-3

overexpression reversed early-life stress–induced spine volume changes (interaction: F 1,5365 = 15.191,

P = 0.0000984, two-way ANOVA). Spine volume in ES-null mice was larger than CT-null mice (P < 0.05, Tukey’s

test), whereas ES-OE mice had smaller spines compared with CT-OE mice (P < 0.05, Tukey’s test). In addition, nectin-

3 overexpression increased spine volume (CT-null versus CT-OE: P < 0.001, Tukey’s test) and spine head diameter in

control mice (treatment effect: F 1,5880 = 12.408, P = 0.000431, two-way ANOVA; CT-null versus CT-OE: P < 0.01, Tukey’s 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, 10–15 dendrites were analyzed.


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A R T I C L E S

enhancing nectin-3 expression in the adult hippocampus would amel-iorate the deleterious effects of early-life stress. AAV-null (an empty

AAV vector) was chosen as the control vector, whereas AAV-OE (con-tains the sequence for nectin-3) was used to overexpress nectin-3.

We observed that AAV-OE increased hippocampal nectin-3 levelsat 4 weeks after injection, and the effects lasted for at least 8 weeks

post-injection (Fig. 6a and Supplementary Fig. 7a). The specificity ofAAV-OE (Supplementary Fig. 7b–d) and the extent of viral transfec-

tion (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 spa-tial object recognition task, AAV-OE restored cognitive performance

in postnatally stressed mice (Fig. 6c). In addition, early life–stressedmice injected with AAV-null (ES-null), but not control mice injected

with either AAV-null (CT-null) or AAV-OE (CT-OE) or postnatallystressed mice injected with AAV-OE (ES-OE), failed to discriminate

the novel object from the known one (Supplementary Fig. 9). In theMorris water maze test, although no difference in spatial acquisition

was found among groups, ES-null mice searched the target quadrantand the other quadrants similarly in the probe trial, indicative of

spatial memory impairments (Fig. 6d). Conversely, ES-OE miceshowed intact spatial memory.

Nectin-3 overexpression reverses stress-induced spine loss

In AAV-null– and AAV-OE–injected mice, dendrites and spines couldnot be visualized because the vectors did not express EGFP. Thus,

we used Thy1-YFPH transgenic mice, in which a subpopulation ofhippocampal 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 signifi-

cantly more nectin-3–expressing spines than their respective controls,the number of nectin-3–negative spines was reduced (treatment effect,

F 1,13 = 14.18, P = 0.0024, two-way ANOVA). Compared with CT-nullmice, this resulted in a shift to a higher ratio of nectin-3–positive to

nectin-3–negative spines, but the number of total spines remainedsimilar. In addition, overexpression of nectin-3 increased spine volume

and spine head diameter in control mice (Fig. 7c). ES-null mice alsohad larger spine volume than CT-null mice, indicative of compensatory

enlargements of the remaining spines. In contrast, stress-induced spineenlargement in ES-OE mice was reversed by nectin-3 overexpression.

CRHR1 signaling and nectin-3 modulate L-afadin levels

L-afadin, an F-actin–binding protein that connects nectin-3 to theactin 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 medi-ated by L-afadin. Consistent with this reasoning, we observed that

hippocampal L-afadin levels decreased following 4 h of in vitro treat-ment 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 radia-tum 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 thatmediates nectin-3–dependent 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 oflearning and memory and stress-related psychiatric disorders. Here,

we identified the synaptic CAM nectin-3 as an important componentin stress- and CRH-evoked effects. Exposure to either acute severe

stress or early-life stress downregulated nectin-3 expression levelsin the adult hippocampus via CRH-CRHR1 signaling. Suppression

a

L-afadin

Actin

ACSF CRH

ACSF CRH0

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p u n c t a d e n s i t y

( % o

f s h S C R ) *

0

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ACSF CRH ACSF CRH

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OE

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N o r m a l i z e d L - a f a d i n p u n c t a

d e n s i t y ( % o

f C T - N u l l )

sr + slm CT

ES

Figure 8 Modulation of L-afadin levels by CRH-CRHR1 signaling and nectin-3. (a) Hippocampal L-afadin

protein levels were reduced after 4 h of CRH application in vitro (t 8 = 2.631, *P < 0.05, unpaired t test).

(b) Adding DMP696 to the slices at 10 min before the 4-h CRH treatment prevented the effects of CRH

on L-afadin levels (antagonism effect: F 1,16 = 4.525, P = 0.0493; interaction: F 1,16 = 5.714, P = 0.0295;

two-way ANOVA; *P < 0.05, Tukey’s test). (c) L-afadin immunoreactivity in the stratum radiatum (sr) and

stratum lacunosum-moleculare (slm) of CA3 was significantly decreased in AAV-shNEC mice (t 7.028 = 3.302,

*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: F 1,12 = 10.74, P = 0.0066, two-way ANOVA;*P < 0.05, Bonferroni’s test). Representative transverse sections immunostained for L-afadin are shown. Note the clustered L-afadin–immunoreactive

puncta as indicated by arrows near the border between slm and sr in CT-OE and ES-OE mice. Scale bars represent 100 µm. Data represent mean ± s.e.m.

Male mice were 3–6 months old. For full-length blots (a,b), see Supplementary Figure 10.


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A R T I C 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 nega-

tive effects of early-life stress. Together, our findings link impairednectin-3–driven 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-lifestress11,12 and chronic stress13 on hippocampus-dependent learning

and memory in adult animals. One of the underlying mechanismsis that excessively released CRH acts through CRHR1 and evokes

structural remodeling31,32, especially the elimination of thin den-

dritic spines22,30. However, the molecules mediating these structuraleffects have not been fully clarified. Here, we found that hippocam-

pal nectin-3 expression levels were regulated by early-life stress ina 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 life–stressed mice. These findings greatly expand ourprevious 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 downregulationof nectin-3. On the basis of our finding that acute severe stress tran-siently suppressed hippocampal nectin-3 expression, the influences of

stress on nectin-3 expression may shift from short-term inhibition toenduring suppression after repeated exposure during development.

Using both in vitro and in vivo approaches, we found that thedynamic regulation of nectin-3 expression by acute stress was medi-

ated by CRH-CRHR1 signaling. Activation of central glucocorticoidreceptor by systemic administration of dexamethasone or intracer-

ebroventricular infusion of corticosterone failed to reproduce theeffects of stress on nectin-3, indicating that such effects are gluco-

corticoid receptor independent. Taking the functional relevance andspatial distribution patterns of CRHR1 and nectin-3 into account,

CRHR1 hyperactivity likely disrupts synaptic adhesion through indi-rect interactions with nectin-3. Indeed, we found that the effects of

CRH and CRHR1 could be modulated by the MAPK signaling path-way, 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 ofphosphorylated MAPK through CRHR1 in areas CA3 and CA1

(ref. 33). The regional characteristics of the MAPK pathway activatedby CRHR1 and the expression pattern of nectin-3 in the hippocampus

therefore provide a molecular basis for the circuit-specific effects ofstress. Nonetheless, although our data provide evidence that stress and

excessive CRH-CRHR1 signaling reduce nectin-3 levels by inhibitinggene and protein expression, it is unclear whether other processes,

such as the cleavage and degradation of the nectin-3 protein, are influ-

enced by stress and CRHR1. Moreover, whether acute and chronicadult stress and early-life stress modulate nectin-3 levels through thesame molecular cascade remains an open question. Future studies are

needed to disentangle the entire molecular mechanisms responsiblefor stress and CRHR1-induced downregulation of nectin-3.

Synaptic CAMs have been implicated in hippocampus-dependentlearning and memory and cognitive disorders5–7. The disruption of

synaptic adhesion mediated by N-cadherin, one of the key CAMsin adherens junctions, impairs long-term, but not short-term,

hippocampus-dependent emotional memory 34. However, the roles ofnectins, the other group of CAMs that organize adherens junctions, in

cognition remain unknown. We found that hippocampal nectin-3 knock-down disrupted long-term spatial memory, mimicking the cognitive

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

of other nectins, N-cadherin and β-catenin were unaffected by thesuppression of nectin-3, indicating that the observed cognitive effects

were nectin-3 specific. On the other hand, overexpressing nectin-3in the adult hippocampus did not improve cognitive performance

per se , but reversed the negative effects of early-life stress on cogni-tion. These results highlight the crucial role of nectin-3–driven syn-

aptic adhesion, which is susceptible to early-life stress, in long-termspatial memory.

The cadherin-catenin complex and the nectin-afadin complexcooperate to modulate structural and synaptic plasticity 14–19.

Although the roles of the cadherin-catenin complex in these pro-

cesses have been investigated29,35,36, it was unclear whether nectin-3is involved in synaptic and structural remodeling. We found that the

inhibition of nectin-3 specifically decreased the number of dendriticspines expressing nectin-3, resulting in a reduction in the total number

of spines. Conversely, overexpression of nectin-3 reversed early-lifestress–induced spine loss. Nectin-3 knockdown did not change the

overall size of the remaining spines, whereas nectin-3 overexpressionincreased 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 instressed mice. These data suggest that, although reduced nectin-3

levels do not fully mediate the effects of early-life stress on spinemorphology, enhancing its expression could rescue the abnormal

changes by stress. In addition, it should be noted that the densityof spines and synapses remains unaltered in conventional nectin-3

knockout mice, although the formation of adherens junctions ismarkedly impaired16. We ascribe this to the differences in the extent

and duration of nectin-3 inhibition, as well as compensatory effectsamong various CAMs1.

L-afadin links nectin-3 to the cadherin-catenin complex and actincytoskeleton1 and participates in spine formation and remodeling19,28,29.

We observed that hippocampal L-afadin levels could be modulatedby CRH-CRHR1 signaling. Moreover, hippocampal nectin-3 knock-

down reduced L-afadin levels, whereas nectin-3 overexpression couldincrease L-afadin levels in postnatally stressed mice. These findings

imply that the effects of nectin-3 on structural remodeling and cog-nition 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-3–mediated axodendritic adhesion in hippocampalneurons through the enhancement of CRH-CRHR1 signaling, which

in turn destabilizes dendritic spines and compromises hippocampus-dependent learning and memory. The discovery that restoring nectin-3

levels ameliorates early-life stress–induced memory deficits maystimulate 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 workwas supported by the European Community’s Seventh Framework Program (FP7,Project No. 201600), the Bundesministerium für Bildung und Forschung withinthe framework of the NGFN-Plus (FKZ: 01GS08151 and 01GS08155) and by theInitiative and Networking Fund of the Helmholtz Association in the framework ofthe Helmholtz Alliance for Mental Health in an Ageing Society (HA-215).

http://www.nature.com/doifinder/10.1038/nn.3395http://www.nature.com/doifinder/10.1038/nn.3395http://www.nature.com/doifinder/10.1038/nn.3395http://www.nature.com/doifinder/10.1038/nn.3395http://www.nature.com/doifinder/10.1038/nn.3395http://www.nature.com/doifinder/10.1038/nn.3395


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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 theexperiments. 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.

Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

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NATURE NEUROSCIENCE doi:10.1038/nn.3395

ONLINE METHODSAnimals. Male Crhr1loxP/loxP ;Camk2a-cre and R26 flopCrh/flopCrh ;Camk2a-cre mice

were generated as described previously 24,25 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 theGovernment of Upper Bavaria, Germany.

Experiments. To examine the effects of early-life stress and postnatal CRH over-

expression on nectin-3 expression, we killed control or stressed CRHR1-CKO

mice, stress-naive CRH-COE mice and respective wild-type mice at 7–8 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 arti-

ficial cerebrospinal fluid (ACSF, containing 124 mM NaCl, 3 mM KCl, 26 mM

NaHCO3, 2 mM CaCl2, 1 mM MgSO4, 10 mM -glucose and 1.25 mMNaH2PO4, pH 7.3) or 50 nM CRH (Bachem) in ACSF

37 in the holding cham-

bers. 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 µl 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 vehi-

cle; 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 andtranscardially 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 overexpres-

sion, 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-3–overexpressing 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 previously 9,39. The day of birth was designated postna-

tal day 0 (P0). On the morning of P2, control dams were provided with a suf-

ficient 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

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 µm 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 (2–3 slices per treatment and time point), and homoge-nated 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 µl) 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 adeno-

associated bicistronic AAV1/2 vector (GeneDetect). AAV-shSCR (AAV1/2-U6-scrambled 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-CAG-

Nectin-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 µl 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 work-

ing memory was tested by recording spontaneous alternation behavior in theY-maze43. The apparatus was made of gray polyvinyl chloride with three sym-

metrical arms (30 × 10 × 15 cm3) and evenly illuminated (30 lx). Prominent intra-

and 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 con-

secutive 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 spentwith 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 diam-

eter) 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


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NATURE NEUROSCIENCEdoi: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.

In situ hybridization. Coronal brain sections (20 µm 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

interest–average 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),β-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),β-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 sulfate–polyacrylamide gels, and transferred

onto nitrocellulose membranes (Invitrogen). Membranes were labeled with

primary antibodies overnight at 4 °C. Following incubation with horseradish

peroxidase–conjugated 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 immunofluo-

rescence was performed on free-floating coronal (20 µm thick) or transverse

(30 µm thick) sections11. After incubation with primary antibodies overnight

at 4 °C, sections were rinsed and labeled with Alexa Fluor 488– and Alexa

Fluor 647–conjugated secondary antibodies (1:500, Invitrogen) for 3 h at22 ± 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-afadin–immunoreactive puncta was measured (5–6 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 coro-

nal sections for dentate granule cells. In area CA3, as the stratum radiatum wasdensely packed with EGFP/EYFP-labeled commissural/associational fibers, only

the dendritic segments (20–130 µm in length, 8–15 dendrites per mouse) in

the stratum lacunosum-moleculare were analyzed. Dendrites were scanned at

0.33-µm 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 µm3. 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 µm 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-3–positive spines, deconvolved dual-channel z stacks (EGFP/EYFP andnectin-3) were merged and nectin-3–positive 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 lacunosum-

moleculare of CA1 was quantified. Dendrites (80–100 µm in length) were

scanned at 1-µm 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 µm 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 Fisher’s LSD post hoc tests as necessary. Student’s 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 Welch’s 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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of corticotropin-releasing hormone on neuronal activity propagation through the

hippocampal formation. J. Psychiatr. Res. 45, 256–261 (2011).

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antagonist DMP696: correlation with drug exposure and anxiolytic efficacy.

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partially requires forebrain corticotropin-releasing hormone receptor 1. Eur. J.Neurosci. 36, 2360–2367 (2012).

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dorsal hippocampus. J. Neurosci. 33, 3857–3864 (2013).

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nectin-3 y stress

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