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8/17/2019 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
20
40
60
80
100
120
140
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
20
40
60
80
100
120
140
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
80
100
120
140
*
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
100
120
140
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
20
40
60
80
100
120
140 Control
Defeat
Time after acute social defeat stress (h)
9 9 1010 10 10 1010
f
c
Nectin-3
Actin
WT COE
d
*
1 4 8 24 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 o n
t r o
l )
0
20
40
60
80
100120
140 Control
Defeat
Time after acute social defeat stress (h)
6 10 86 10 8 6 6
1 4 8 24
Time after acute social defeat stress (h)
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
20
40
60
80
100120
140
R a t i o s o f n e c t i n - 3 t o a c t i n
( % o
f A C S F ) *
5 5
Vehicle DMP696
0
20
40
60
80
100
120
140 ACSF
CRH
R a t i o s o f n e c t i n - 3 t o a c t i n
( % o
f A C S F - v e h
i c l e ) *
55 5 5
Nectin-3
Actin
ACSF CRH ACSF CRH
V eh ic le D MP 69 6
b c
A C S F
C R H
0
20
40
60
80
100120
140
R a t i o s o f n e c t i n - 3 t o a c t i n
( % o
f A C S F ) *
11 11
A C S F C R H
5 5
**
Nectin-3
Actin
ACSF CRH
Total
ACSF CRH
Membrane
d
6 6 6 6 6
0
20
40
60
80
100120
140
R a t i o s o f n e c t i n - 3 t o
a c t i n
( % o
f v e h i c l e )
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
+
U 0 1 2 6
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
+
U 0 1 2 6
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
+
U 0 1 2 6
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
+
U 0 1 2 6
* * * *
*
6 6 6 6 6
* *
*
Nectin-3
Actin
Total Membrane
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NATURE NEUROSCIENCE VOLUME 16 | NUMBER 6 | JUNE 201 3 70 9
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
– 1 . 6 0
– 1 . 7 6
– 1 . 9 2
– 2 . 0 8
– 2 . 2 4
– 2 . 4 0
– 2 . 5 6
– 2 . 7 2
Bregma (mm)
10
15
20
0
25
******
***** *
N e c t i n - 3 m R N A
( a r b i t r a r y u n i t s )
DG
10
15
20
0
25
***
***** **
** **
Bregma (mm)
N e c t i n - 3 m R N A
( a r b i t r a r y u n i t s )
– 1 . 6 0
– 1 . 7 6
– 1 . 9 2
– 2 . 0 8
– 2 . 2 4
– 2 . 4 0
– 2 . 5 6
– 2 . 7 2
CA3
20
40
60
0
80
***************
*********
shSCR
shNEC
Bregma (mm)
N e c t i n - 3 m R N A
( a r b i t r a r y u n i t s )
– 1 . 6 0
– 1 . 7 6
– 1 . 9 2
– 2 . 0 8
– 2 . 2 4
– 2 . 4 0
– 2 . 5 6
– 2 . 7 2
b
shSCR shNEC S p o
n t a n e o u s a l t e r n a t i o n ( % )
0
20
40
60
80
Y-maze
12121
2
3
4
5
c
s hS CR s hN EC
N o v e l t o k n o w n r a t i o
0
Object recognition
1212
*
d
E s c a p e l a t e n c y ( s )
0
10
20
30
40
50
60 shSCRshNEC
Day 1 Day 2 Day 3
Acquisition
s hS CR s hN EC
T i m e i n q u a d r a n t s ( s )
0
5
10
15
20
25
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
40
60
80
100
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 ( % )
0.1 0.2 0.3 0.4 0.5 0.6 0.70
20
40
60
80
100
Spine head diameter (µm)
C u m u l a t i v e d i s t r i b u t i o n ( % )
shSCR
shNEC
cb
0
2
4
6
8
10
N u m b e r o f s p i n e s p e r 1 0 µ m
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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710 VOLUME 16 | NUMBER 6 | JUNE 2013 NATURE NEUROSCIENCE
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
2
3
4
5
6
c
0
N
o v e l / K n o w n r a t i o
Null OE
Object recognition
*
11 11 12 12
d
0
10
20
30
40
50
60CT-nullES-null
CT-OEES-OE
E s c a p e
l a t e n c y
( s )
Day 1 Day 2 Day 3
Acquisition
0
10
20
30
40TargetRight
OppositeLeft
T i m e i n q u a d r a n t s ( s )
Probe trial
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##
CT ES CT ES
Null OE
9 911 12
b
0
20
40
60
80
S p o n t a n e o u s a l t e r n a t i o n ( % )
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11 11 13 12
a
Null
OE
CT ES
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40
120
140
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( a r b i t r a r y u n i t s )
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*
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– 1 . 6 0
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– 1 . 9 2
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( a r b i t r a r y u n i t s )
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– 1 . 6 0
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CA1
– 1 . 6 0
– 1 . 7 6
– 1 . 9 2
– 2 . 0 8
– 2 . 2 4
– 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
0
20
40
60
80
100
CT-null
ES-null
CT-OE
ES-OE
Spine head diameter (µm)
C u m u l a t i v e d i s t r i b u t i o n ( % )
0.1 0.2 0.3 0.4 0.5 0.60
20
40
60
80
100
CT-null
ES-null
CT-OE
ES-OE
Spine volume (µm
3
)
C u m u l a t i v e d i s t r i b u t i o n ( % )
ca
EYFP Nectin-3 Merged
CT-null
ES-null
CT-OE
ES-OE
b
**
Null OE
*
N u m b e r o f s p i n e s p e r 1 0 µ m
Null OE
**
CT
ES
*
0
2
4
6
8
10
Total Nectin-3+
3
3
45 5
5 4 5
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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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 pre- vented 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
20
40
60
80
100
120
140*
R a t i o s o f L - a f a d i n t o
a c t i n
( % o
f A C S F )
5 5
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 s h S C R ) *
0
20
40
60
80
100
120140
shSCR shNEC
6 6
sr + slm
shSCR shNEC
slmsr
cb
Vehicle DMP696
0
20
40
60
80
100
120
140
R a t i o s o f L - a f a d i n t o a c t i n
( % o
f A C S F - v e h i c l e ) *
55 5 5
*
L-afadin
Actin
ACSF CRH ACSF CRH
Vehicle DMP696
ACSF CRH
d
OE
CT ES
Null slmsr
0
20
40
60
80
100
120
140
Null OE
*
4 4 4 4
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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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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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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http://research.mssm.edu/cnic/tools-ns.htmlhttp://www.r-project.org/http://www.r-project.org/http://research.mssm.edu/cnic/tools-ns.html