Experience rearranges anatomical connectivity in the brain, but such plasticity is suppressed in adulthood. We examined the turnover of dendritic spines and axonal varicosities in the somatosensory cortex of mice lacking Nogo Receptor 1 (NgR1). Through adolescence, the anatomy and plasticity of ngr1 null mice are indistinguishable from control, but suppression of turnover after age 26 days fails to occur in ngr1−/− mice. Adolescent anatomical plasticity can be restored to 1-year-old mice by conditional deletion of ngr1. Suppression of anatomical dynamics by NgR1 is cell autonomous and is phenocopied by deletion of Nogo-A ligand. Whisker removal deprives the somatosensory cortex of experience-dependent input and reduces dendritic spine turnover in adult ngr1−/− mice to control levels, while an acutely enriched environment increases dendritic spine dynamics in control mice to the level of ngr1−/− mice in a standard environment. Thus, NgR1 determines the low set point for synaptic turnover in adult cerebral cortex.

1. Neuron
Report
Anatomical Plasticity of Adult Brain
Is Titrated by Nogo Receptor 1
Feras V. Akbik,1 Sarah M. Bhagat,1 Pujan R. Patel,1 William B.J. Cafferty,1 and Stephen M. Strittmatter1,*
1Cellular Neuroscience, Neurodegeneration and Repair Program, Departments of Neurology and Neurobiology, Yale University School
of Medicine, New Haven, CT 06536, USA
*Correspondence: stephen.strittmatter@yale.edu
http://dx.doi.org/10.1016/j.neuron.2012.12.027
SUMMARY single unit electrophysiology in anesthetized mice after monoc-
ular deprivation (McGee et al., 2005). In dissociated neuron
Experience rearranges anatomical connectivity in the and brain slice studies, NgR1 has a role in acute electrophysio-
brain, but such plasticity is suppressed in adulthood. logical plasticity and synaptic morphology (Delekate et al.,
We examined the turnover of dendritic spines and 2011; Lee et al., 2008; Raiker et al., 2010; Wills et al., 2012;
axonal varicosities in the somatosensory cortex Zagrebelsky et al., 2010). Here, we examined whether the in vivo
of mice lacking Nogo Receptor 1 (NgR1). Through anatomical dynamics of adult mouse synaptic structure is
regulated by NgR1 expression. We find that NgR1 gates the
adolescence, the anatomy and plasticity of ngr1
age-dependent restriction of anatomical plasticity in the adult
null mice are indistinguishable from control, but
cortex and that NgR1 determines the set point for experience-
suppression of turnover after age 26 days fails to driven synaptic turnover.
occur in ngr1À/À mice. Adolescent anatomical plas-
ticity can be restored to 1-year-old mice by condi- RESULTS
tional deletion of ngr1. Suppression of anatomical
dynamics by NgR1 is cell autonomous and is phe- Using ngr1 mutants transgenic for Thy1-YFP-H, we assessed
nocopied by deletion of Nogo-A ligand. Whisker NgR1 regulation of dendritic spine dynamics in vivo. NgR1 dele-
removal deprives the somatosensory cortex of expe- tion does not alter the pattern of cortical dendrite branching, the
rience-dependent input and reduces dendritic spine density of dendritic spines, or the morphology of spines (see
turnover in adult ngr1À/À mice to control levels, Figures S1A–S1D available online). Repeated transcranial two-
photon confocal imaging of anesthetized mice over 2 weeks re-
while an acutely enriched environment increases
vealed dendritic spine gains and losses from layer V pyramidal
dendritic spine dynamics in control mice to the level
dendrites in the superficial 100 mm of S1 barrel field cortex
of ngr1À/À mice in a standard environment. Thus, (Figures 1A and 1B). Compared to controls, the gains and losses
NgR1 determines the low set point for synaptic turn- of ngr1À/À dendritic spines over a 14 day period are more than
over in adult cerebral cortex. doubled (p < 0.001). Similarly, increased spine turnover was
observed in M1 and V1 of ngr1À/À mice (Figure S1E). The
INTRODUCTION greater spine dynamics occur without change in total spine
density, emphasizing the necessity for time-lapse imaging.
Environmental experience plays a major role in sculpting Separate from spine plasticity, branch extensions or retractions
brain architecture and synaptic connectivity. The malleability of are rare for pyramidal neurons and are not different in ngr1À/À
synaptic connections is extensive during adolescent ‘‘critical mice (data not shown).
periods.’’ In adults, anatomical plasticity is restricted (Grut- When spines first protrude, they are typically transient and
zendler et al., 2002; Holtmaat et al., 2005; Trachtenberg et al., quickly lost, with only a small subset becoming persistent and
2002; Yang et al., 2009; Zuo et al., 2005). Discrete rearrange- gaining the ultrastructure of synapses (Holtmaat et al., 2005,
ment of adult synaptic connections has been implicated in 2006; Knott et al., 2006; Trachtenberg et al., 2002). Learning
learning and memory for motor, sensory, and contextual tasks paradigms or sensory-enriched environments increase short-
(Hofer et al., 2009; Holtmaat et al., 2006; Lai et al., 2012; Trach- term spine turnover and also the stabilization of new spines
tenberg et al., 2002; Wilbrecht et al., 2010; Xu et al., 2009; Yama- into persistent spines (Holtmaat et al., 2006; Xu et al., 2009;
hachi et al., 2009; Yang et al., 2009). Environmentally determined Yang et al., 2009). In the adult, persistent spines are the over-
patterns of electrical activity drive patterns of synaptic change, whelming majority; a smaller pool of transient spines turns over
but the molecular basis for the set point, or gain, of anatomical frequently. Transient spines account for $80% of all spine
dynamics is not known. changes during 2 days and serve as the basis for novel connec-
Nogo Receptor 1 (NgR1) was originally identified as a mediator tivity (see Supplemental Experimental Procedures; Holtmaat
of myelin-dependent restriction of recovery from injury (Fournier et al., 2005). Here, spines were classified as persistent if they
et al., 2001). In healthy brain, NgR1 was shown to be essential in were observed on two imaging sessions at days 0 and 2. The
closing the critical period for ocular dominance plasticity by 14 day survival of persistent spines from day 2 to 16 is decreased
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2. Neuron
Nogo Receptor Limits Anatomical Plasticity In Vivo
Figure 1. NgR1 Restricts Dendritic Spine
and Axonal Varicosity Turnover in Adult
Brain
(A) Repeated transcranial two-photon imaging
of YFP-H transgenic mice reveals anatomical
plasticity of layer V dendritic spines in layer I
in vivo over 2 weeks. Orange and green arrow-
heads indicate spine gain and loss, respectively,
over 2 weeks. White arrowheads indicate
stable spines. (B) The percentage of spines
gained and lost over 2 weeks in ngr1+/À (n = 7)
and ngr1À/À (n = 8) mice at P180–P220. (C)
The survival of persistent dendritic spines
observed on both day 0 and 2 was monitored
from day 2 to 16. (D) Dendritic spine gain and
loss during a 2 day period is plotted for ngr1+/À
(n = 7) and ngr1À/À (n = 8) mice at P180–P220.
(E) Turnover of dendritic spines as a function
of age. The 2 week turnover for ngr1+/À and
ngr1À/À mice was measured starting at P26
(n = 5, n = 6, respectively), P35 (n = 5 per geno-
type), P45 (n = 5 per genotype), and P180 (n = 7,
n = 8, respectively). Turnover is unchanged
during maturation in ngr1À/À mice but decreases
significantly in ngr1+/À mice from P26 to P45.
ngr1 flx/flx mice with and without Cre (n = 4,
n = 6, respectively) were treated with tamoxifen
at P330. The 2 week turnover of dendritic spines
was measured starting at P360. (F) Repeated
transcranial two-photon imaging of en passant
axonal varicosities in S1 in vivo. Green arrowheads indicate varicosity loss over 2 weeks. (G) The percentage of axonal varicosities gained and lost over 2 weeks in
ngr1+/À (n = 5) and ngr1À/À mice (n = 6) at P180. Data are presented as mean ± SEM. **p < 0.01. Scale bars represent 1 mm.
in mice lacking NgR1, with greater persistent spine loss over age-dependent stabilization against losses is NgR1 indepen-
2 weeks, 10.6% ± 2.6% in ngr1À/À versus 3.7% ± 0.4% in dent. Overall, the development of spines is normal through P26
control, p < 0.05 (Figure 1C). In addition, the 2 day gain and without NgR1, but an adult gate on plasticity is lacking in
loss of spines is greater in mice lacking NgR1, with gains of ngr1À/À mice.
6.0% ± 0.6% in ngr1À/À versus 1.9% ± 0.4% in control, p < Next, we considered whether NgR1 is required during the tran-
0.005 (Figure 1D). Thus, endogenous NgR1 restricts the initial sition from juvenile plasticity to adult stability, or whether the
protrusions and retractions preceding synapse formation and protein is continuously required in the adult cortex to suppress
promotes the persistence of established spines likely to have plasticity. We used a conditionally targeted ngr1 allele, ngr1-flx
formed synapses. (Wang et al., 2011). Temporal control was provided by an actin
As WT mice transition from late adolescence to adulthood, promotor transgene that drives ubiquitous expression of a Cre
there is a dramatic increase in synaptic stability (Grutzendler fusion protein with a mutant version of the estrogen receptor
et al., 2002; Holtmaat et al., 2005; Zuo et al., 2005) that coincides (ERT2) (Hayashi and McMahon, 2002). Tamoxifen treatment
with myelination of V1 (McGee et al., 2005) and S1 (Figures S1G leads to efficient ngr1 gene rearrangement and near total loss
and S1H). Oligodendrocytic Nogo-A protein also increases of mRNA and protein within 2 weeks (Figure S1F and Wang
during this time (Wang et al., 2002b). The accelerated spine et al., 2011). Mice with floxed ngr1 alleles with or without Actin-
turnover in adult ngr1À/À mice (Figures 1A–1D) is similar to Cre-ERT2 transgene were allowed to develop with endogenous
that in juvenile WT mice (Yang et al., 2009; Zuo et al., 2005). levels of NgR1. At P330, the mice received tamoxifen to delete
We therefore tested whether NgR1-dependent restriction of NgR1 from the Cre subgroup. One month later, dendritic spine
spine turnover is age dependent (Figure 1E). Starting at postnatal stability was assessed over 2 weeks. Even at this advanced
day 26 (P26), control mice show rapid spine turnover during age, deletion of NgR1 increases dendritic spine turnover to the
2 weeks. Turnover slows by P35 and reaches a stable adult level observed in adolescent mice (Figure 1E, p < 0.001 versus
pattern by P45, which is not altered at P180 or P360, matching control, and n.s. versus P26–P40). Thus, constitutive NgR1
the progression of intracortical myelination. Dendritic spine signaling reversibly limits synaptic turnover in the adult cerebral
turnover in ngr1À/À mice is identical to controls at P26–P40 cortex.
but shows no change from P26 to P180. When gains and losses We considered whether NgR1 regulation of postsynaptic
are considered separately (Figures S1I and S1J), it is clear stability in adult cortex was coupled with similar changes in
that age-dependent restriction of gains is NgR1 dependent. presynaptic stability, or whether there was selective action in
A nonsignificant trend suggests that a minor component of dendrites. We first determined the types of presynaptic fibers
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3. Neuron
Nogo Receptor Limits Anatomical Plasticity In Vivo
Figure 2. Nogo Ligand Regulates Dendritic
and Axonal Turnover in Adult Brain
(A) Time-lapse spinning-disc confocal microscopy
of 19–22 DIV primary cortical neurons electro-
porated with myristoylated-GFP reveals dendritic
spine turnover in vitro over 6 hr. WT or ngr1À/À
cultures were imaged every hour for 6 hr. After the
first hour, cultures were treated with 100 nM
Nogo-22 or PBS. The orange arrowhead indicates
spine gain. (B) The percentage of spines gained
and lost over 6 hr (n = 6 plates, 5,806 spines) in
response to PBS or 100 nM Nogo-22 treatment at
1 hr. (C) The percentage of spines gained and lost
over 6 hr (n = 4 plates, 3,494 spines) in response to
6 day pretreatment with 100 nM Nogo-22. (D)
Repeated transcranial two-photon imaging of
dendritic spines in vivo in Nogo-A/B mutants
transgenic for YFP-H. Orange and green arrow-
heads indicate spine gain and loss, respectively,
over 2 weeks. (E) The percentage of spines gained
and lost over 2 weeks in WT (n = 3), ngr1+/À (n = 7),
nogo-A/B+/À (n = 6), nogo-A/BÀ/À (n = 4) mice,
and nogo-A/B+/À ngr1+/À adult mice. (F)
Repeated transcranial two-photon imaging of en
passant boutons (EPBs) in vivo. Orange and green
arrowheads indicate varicosity gain and loss,
respectively, over 2 weeks. (G) The percentage of
EPBs gained and lost over 2 weeks in nogo-A/
B+/À (n = 6) and nogo-A/BÀ/À (n = 4) mice
measured from P120–P134. Data are presented
as mean ± SEM. *p < 0.05, **p < 0.01. Scale bars
represent 1 mm.
whether the prototypical Nogo ligand
might regulate dendritic spines in vivo,
we exposed dissociated cortical neurons
19–22 days in vitro (DIV) to a Nogo-A
polypeptide (22 kDa, Huebner et al.,
2011) during time-lapse spinning-disc
labeled in cortical layer I of Thy1-YFP-H mice. Using described confocal imaging of myristoyl-GFP-transfected cells (Figure 2A).
morphological criteria (De Paola et al., 2006), we found that the Without added Nogo-22, spine dynamics are similar between
vast majority of labeled axons are consistent with recurrent WT and ngr1À/À cultures. This parallels what we observe in
cortical fibers from layer V and layer II/III (A3 subtype, 98.7% ± juvenile mice in vivo because 19–22 DIV dissociated cultures
0.7% of total). Presynaptic specializations along these fibers are unmyelinated (data not shown). Acute treatment with
were imaged over a 14 day interval in the S1 barrel field cortex 100 nM Nogo-22 protein reduces the appearance of new
in 6- to 7-month-old mice (Figure 1G). Consistent with previous dendritic spines by 80% (Figure 2B, p < 0.002). Acute Nogo-22
reports (De Paola et al., 2006), axonal varicosities are more treatment also leads to an increase in spine loss. In contrast to
stable than dendritic spines. Critically, axonal specializations the differential effect of acute Nogo-22 treatment on gains and
are at least twice as dynamic in ngr1À/À mice as in ngr1+/À losses, both spine gains and losses are restricted in adult mice
mice (Figure 1H; p < 0.005). The gains and losses of axonal vari- (Figure 1, Table S1). We hypothesized that chronic treatment
cosities are increased equally. While varicosity turnover is with Nogo-22 in vitro might mimic the chronic effect of myelin
increased, we observed no genotype difference in the frequency inhibition in vivo. Pretreating 19–22 DIV dissociated cortical
of rare axonal branch extensions and retractions (data not cultures with Nogo-22 for 6 days leads to a decrease in spine
shown). Overall, the requirement of NgR1 for axonal stability gains and losses (Figure 2C), mimicking adult wild-type
closely matches its role in stabilizing dendritic spines in adult spine dynamics in vivo. This suggests that NgR1 inhibition of
cortex. spine gain, paralleling inhibition of neurite extension, is the
Several ligands for NgR1 have been described, including primary event in vivo, with steady-state changes in spine loss
Nogo-A, MAG, and OMgp from oligodendrocytes (reviewed in being secondary and compensatory in vivo. Nogo-22-induced
Akbik et al., 2012), chondroitin sulfate proteoglycans (Dicken- changes are not detectable in ngr1À/À cultures (Figure 2B)
desher et al., 2012), and LGI1 (Thomas et al., 2010). To assess and are dose dependent (Figure S2).
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4. Neuron
Nogo Receptor Limits Anatomical Plasticity In Vivo
Figure 3. NgR1 Cell Autonomously Re-
stricts Anatomical Plasticity
(A) Subcellular fractionation of somatosensory
cortex in P180 mice. (B and C) WT or ngr1À/À
cortical neurons were electroporated with myr-
istoylated-GFP and then cocultured with unla-
beled neurons to assess cell autonomy in vitro at
19–22 DIV. Neurons were imaged over 6 hr.
Cultures were treated with 100 nM Nogo-22 or
PBS after the first hour. (B) Labeled WT neurons
were imaged in an excess of unlabeled ngr1À/À
neurons (n = 3 plates, 2,802 spines). (C) Labeled
ngr1À/À neurons were imaged in an excess of
unlabeled WT neurons (n = 3 plates, 2,670 spines).
(D) Schematic of assessment of cell autonomy
in vivo. Labeled (green) neurons are transgenic for
both YFP and Cre-ERT2, while unlabeled (black)
neurons have neither. Tamoxifen-induced re-
combination creates a chimeric population of
labeled ngr1–/– in an ngr1 flx/flx background. (E)
Repeated transcranial two-photon imaging of
dendritic spines in vivo of SLICK-V mice over
2 weeks. Orange arrowheads represent spine gain
over 2 weeks. (F and G) The percentage of spines
(F) and EPBs (G) gained and lost over 2 weeks in
ngr1 flx/+ (n = 4) and ngr1 flx/flx (n = 6) littermates
measured at P120. Data are presented as mean ±
SEM. *p < 0.05, **p < 0.01. Scale bars represent
1 mm.
Given the acute in vitro action of Nogo-22 through NgR1 to type plated with a 24-fold excess of neurons from one or the
prevent dendritic spine gain, we utilized Nogo-A/B null mice to other genotype. The response of the imaged cell depends on
determine whether this ligand is required for NgR1 stabilization the expression of NgR1 in that cell and not on the expression
of dendritic spines in adult mice. Using the Thy1-YFP-H marker, in the majority of cells throughout the culture (Figures 3B and
dendritic spine gains over 2 weeks are increased more than 3C). Even when 96% of the culture is wild-type, rare ngr1À/À
2-fold in nogo-A/B null mice relative to control at P180 (Figures neurons do not respond to Nogo-22 (Figure 3C). In vitro, NgR1
2C and 2D; p < 0.001). A balanced increase in spine losses is acts cell-autonomously to mediate the acute action of diffusely
observed in nogo-A/BÀ/À mice (Figure 2D), and the greater applied Nogo-22.
turnover of Nogo-A/B null axonal varicosities parallels that of To determine whether NgR1 acts cell-autonomously in vivo,
dendritic spines (Figures 2E and 2F). Thus, loss of the Nogo- we utilized SLICK transgenic mouse containing a Thy1 promotor
A/B ligand phenocopies the rapid juvenile-type of synaptic turn- that drives a fluorescent marker and Cre-ERT2 in a subset of
over observed in NgR1-deficient adult mice. To examine cortical neurons (Young et al., 2008). After crossing onto the
a genetic interaction between Nogo-A/B and NgR1, we as- ngr1 flx/flx background, tamoxifen treatment provides labeled
sessed the turnover of dendritic spines in compound heterozy- NgR1 null neurons in a milieu of unlabeled WT neurons (Fig-
gotes (Figure 2E). The single heterozygotes are indistinguishable ure 3D). Compared to ngr1 flx/+ neurons, dendritic spine gains
from WT mice, but the nogo-A/B, ngr1 double heterozygous and losses for the single-cell NgR1-deleted neurons are
mice exhibit 14 day spine turnover that is similar to the increased increased (Figures 3E and 3F; p < 0.01). In this paradigm, axonal
rate in homozygous mutants, consistent with an interaction varicosity turnover for NgR1 null neurons is also increased in the
in vivo. wild-type host (Figure 3G). Thus, NgR1 acts cell-autonomously
NgR1 might alter anatomical plasticity indirectly, for example, in vitro and in vivo to limit the turnover of both presynaptic and
by changing activity patterns globally. Alternatively, the protein postsynaptic anatomical specializations.
might function cell-autonomously to limit synapse dynamics in While NgR1 is unique as a gene selectively titrating adult
cells expressing NgR1. If NgR1 acts cell-autonomously and dendritic spine dynamics, inflammation and experience produce
locally, an enrichment in postsynaptic densities might be ex- a similar phenotype in adult mice. We examined whether NgR1
pected. Fractionation of adult cerebral cortex reveals enrich- gene deletion is redundant or synergistic with these interven-
ment of NgR1, but not Nogo-A/B, in postsynaptic densities (Fig- tions. Surgically implanting glass windows in the skull produces
ure 3A). This is consistent with previous immunoelectron mild gliosis and greater dendritic spine turnover than does
microscopy demonstrating NgR1 in pre- and postsynaptic a thinned skull, which does not expose dura matter (Figure 4A)
structures (Wang et al., 2002b). To assess cell-autonomous (Xu et al., 2007). Our studies of M1 cortex confirm that dendritic
action in vitro, we mixed ngr1À/À and wild-type neurons in spine turnover is substantially increased by the open craniotomy
different proportions prior to Nogo-22 exposure. The GFP window relative to the thinned-skull preparation (Figure 4B; p <
marker was included in a small percentage of cells of one geno- 0.05). Importantly, NgR1 deletion increases dendritic spine
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5. Neuron
Nogo Receptor Limits Anatomical Plasticity In Vivo
Figure 4. NgR1 Regulates Inflammation-Induced and Experience-Dependent Anatomical Plasticity
(A) Photomicrographs illustrate glial fibrillary acidic protein (GFAP) staining 1 month after craniotomy for the open-skull method or 2 weeks after an initial thinned-
skull surgery. Pictures are from barrel cortex contralateral or ipsilateral to the imaging site. Scale bars represent 200 mm. (B) The 2 week turnover of dendritic
spines in EGFP-M transgenics measured using the open (n = 5 per genotype) or thinned skull (n = 4 per genotype) preparations in primary motor cortex. (C)
Turnover of dendritic spines in S1 over 2 days was assessed for mice of different genotypes and varied vibrissal sensory input levels. P180 ngr1+/À and ngr1À/À
mutants are subjected to one of four sensory paradigms: 12 days of whisker trimming (n = 5 per genotype), standard environment (n = 6 per genotype), enriched
environment (n = 7–8 per genotype), or enriched environment with whisker trimming (n = 5–6 per genotype). (D) In P180, YFP-H transgenic NgR1 mutants,
repeated transcranial imaging of dendritic spines in barrel cortex at days 0, 2, and 16 reveals a new persistent spine (indicated by the orange arrow). Scale bars
represent 1 mm. (E) The fraction of new spines at day 2 that are stabilized into persistent spines at day 16. (F) Conditionally mutant 4- to 5-month-old adult mice
with and without Cre recombinase (n = 9 and 10, respectively) were treated with tamoxifen 11 days prior to training. Mice were trained three trials per day on an
accelerating, rotating drum (Rotarod). Latency to fall off of the rod was recorded and used to generate a learning curve under one-phase decay assumptions and
compared using an extra sum of squares F-test, p = 0.0072. The half-time to saturation for Cre-positive and Cre-negative mice is 1.1 and 4.1 trials, respectively.
Except for the first trial, all points represent the average of two trials. (G) Schematic for fear conditioning and extinction protocol. Adult mice were conditioned to
an acoustic tone that coterminated with a foot shock on day 1. On days 2 and 3, a subset of mice was presented with 12 unpaired tones during a 30 min
observation period. A separate cohort was presented with four unpaired tones on day 10 to test memory of conditioning without extinction. (H) WT and ngr1À/À
mice (4 to 5 months old, n = 20 and 17, respectively) were conditioned to an auditory cue paired with a foot shock. Percentage of freezing time during the first and
last tone of acquisition is shown. (I) Extinction of conditioned fear on days 2 and 3 during exposure to 12 unpaired tones per day is presented as percentage of time
freezing (n = 15 WT, n = 13 ngr1À/À). The two genotypes differed across trials during day 3 by RM-ANOVA, bracket **p < 0.001, and differed on specific trial
blocks by one-way ANOVA. (J) Without prior extinction, freezing to the unpaired conditioned tone was assessed on day 10 (n = 4 per genotype). All data are
presented as mean ± SEM. *p < 0.05, **p < 0.01.
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6. Neuron
Nogo Receptor Limits Anatomical Plasticity In Vivo
turnover by a similar proportion in both the open and thinned- If SE engages dendritic spine dynamics for NgR1 null mice
skull preparations (Figure 4B; p < 0.01). This is not explained that are similar to those of control mice in acute EE, then the
by differential degrees of gliosis, since craniotomy-induced characteristics of spine turnover should be similar. The most
inflammation is NgR1 independent (Figures S3A and S3B), robust effect of acute EE in control mice is an increase in the
consistent with previous observations using murine models of rate of stabilizing new spines into persistent spines, and NgR1
spinal cord injury or stroke (Kim et al., 2004; Lee et al., 2004; null mice in the SE fully mimic this pattern (Figures 4D and
Wang et al., 2006). These data demonstrate the robustness of 4E; p < 0.001). Thus, endogenous NgR1 restricts the initial
the ngr1À/À phenotype and additivity with inflammation to protrusions and retractions preceding synapse formation, as
increase anatomical plasticity. well as the subsequent maturation of spines leading to synapse
Next, we considered how altered sensory experience interacts formation and persistence. Mice lacking NgR1 respond to
with NgR1 deletion. Dendritic spine turnover is reported to chronic SE with a pattern of dendritic spine dynamics that
increase during the first several days after mice are exposed to matches precisely the physiological pattern of acute EE in
an enriched environment, after which turnover returns to base- control mice.
line (Yang et al., 2009). Similarly, we observed an increase in We considered whether the enhanced anatomical sensitivity
2 day spine turnover from 5.0% ± 0.6% in standard caging to experience alters behavioral responses of ngr1À/À mice.
(SE) to 13.5% ± 1.5% in a new environment strongly enriched Both motor learning (Xu et al., 2009; Yang et al., 2009) and
(EE) for whisker stimulation (Figure 4C; p < 0.001). The environ- extinction of fear conditioning (Lai et al., 2012) are reported to
ment-driven increase requires vibrissal somatosensory input correlate with cortical spine dynamics. To assess motor learning
because it is eliminated by whisker trimming at the time of rate, we examined conditional ngr1 null mice on the rotarod (Fig-
transition to the enriched environment (Figure 4C). Strikingly, ure 4F). Training improves the latency to fall more rapidly in mice
the elevated level of dendritic spine turnover in ngr1À/À mice lacking NgR1. In tone-associated fear conditioning, both geno-
is not further increased by EE (Figure 4C). types are rapidly conditioned during day 1 (Figures 4G
On the one hand, this might imply that ngr1À/À neurons are no and 4H). During the first 30 min of extinction on day 2, both
longer sensitive to experience-dependent inputs. Alternatively, groups show decreased freezing (Figure 4I). By day 3, when
the threshold for experience-dependent plasticity may be lower; new spine gains have started to form and to maintain extinction
the standard cage may provide a level of stimulation capable (Lai et al., 2012), the mice lacking NgR1 show significantly (p <
of eliciting full spine turnover in ngr1À/À but not control mice. 0.001) more pronounced extinction of freezing to the previously
To separate these possibilities, we trimmed the whiskers of conditioned tone (Figure 4I). This is due to enhanced extinction in
ngr1À/À and control mice for 12 days and then assessed the ngr1À/À mice, rather than impaired memory of the condi-
2 day S1 dendritic spine turnover in the sensory-deprived state. tioned tone, because both genotypes show freezing at 10 days
For control mice, there was a slight decrease in dendritic spine if not subjected to the extinction protocol (Figures 4G and 4J).
turnover (Figure 4C), indicating that our SE provides little effec- Thus, ngr1 mutant mice show enhanced learning in two para-
tive experiential drive to S1 dendritic spine turnover. In contrast, digms linked to cortical spine turnover.
2 day spine turnover for ngr1À/À mice is suppressed to control
levels by whisker trimming (Figure 4C; p < 0.01). We conclude DISCUSSION
that SE provides a level of vibrissal input adequate to drive expe-
rience-dependent dendritic spine changes in ngr1À/À mice, but We report that NgR1 gates the transition from rapid anatomical
not controls. turnover during adolescence to slower adult dendritic spine
Previous studies have reported activity-dependent downregu- dynamics. NgR1 is required continuously to suppress synaptic
lation of NgR1 (Josephson et al., 2003; Wills et al., 2012), raising turnover, functions cell-autonomously, and responds to Nogo
the possibility that EE might reduce NgR1 level to instruct ligand. While Nogo-A/B is required, MAG, OMgp, CSPGs, and
enhanced anatomical plasticity. To assess this hypothesis, we LGI1 are alternate NgR1 ligands (Dickendesher et al., 2012; Liu
analyzed total NgR1 levels and NgR1 enrichment in the PSD as et al., 2002; Thomas et al., 2010; Wang et al., 2002a) that may
a function of age or experience and observed no differences have coessential roles to limit synaptic rearrangement in the
(Figures S3C–S3F). While myelin and perineuronal net formation cerebral cortex. CSPGs are known to participate in electrophys-
are maturation and activity dependent (McRae et al., 2007; Wake iological plasticity of the cerebral cortex (Pizzorusso et al., 2002).
et al., 2011), NgR1 is a continuously present restrictive factor for The alternate Nogo receptor PirB has also been implicated in
anatomical plasticity in the adult cortex, with instructive clues experience-dependent plasticity (Atwal et al., 2008; Syken
likely to be derived from electrical activity patterns. We also et al., 2006). Limits on recovery after injury may share a common
investigated whether NgR1 acts upstream or downstream of pathway with plasticity (Akbik et al., 2012). The transition from
experience-driven immediate early gene induction in the barrel rapid spine turnover in juvenile mice to the anatomical stability
field (Bisler et al., 2002). The rapid whisker-dependent induction of adult synapses coincides with the timing of NgR1 ligand
of c-FOS is not altered by the absence of NgR1 (Figures S3G and presentation. Intracortical myelination and perineuronal net
S3H). Thus, initial acute responses to sensory input, as marked formation both occur at the final stages of cortical maturation,
by c-FOS, are distinct from NgR1 action. In contrast, the down- near P30, when adult spine stability is achieved.
stream events required to reorganize and imprint that input As a molecular determinant for the low set point for anatomical
anatomically are achieved more easily without NgR1. This is con- plasticity in the adult, NgR1 restricts the effect of experience
sistent with NgR1 serving as a brake on anatomical dynamics. on cortical anatomy. Highly enriched artificial environments
864 Neuron 77, 859–866, March 6, 2013 ª2013 Elsevier Inc.

7. Neuron
Nogo Receptor Limits Anatomical Plasticity In Vivo
enhance spine turnover in control mice during the first 2 days, Behavior
with a subsequent return to baseline dynamics (Yang et al., Environmental enrichment, rotarod, and fear conditioning were by performed
according to standard procedures.
2009). In contrast, chronic housing under standard conditions
Detailed Methods are provided in the Supplemental Experimental
is adequate to drive rapid turnover in NgR1 null mice. From these Procedures.
observations, the cerebral cortex lacking NgR1, with its
augmented spine dynamics, is predicted to have greater sensory
SUPPLEMENTAL INFORMATION
map refinement and greater map plasticity. Indeed, the observa-
tion of adult ocular dominance plasticity of single units under Supplemental Information includes three figures, one table, and Supplemental
urethane anesthesia in adult ngr1À/À mice, but not adult wild- Experimental Procedures and can be found with this article online at http://dx.
type mice (McGee et al., 2005), is consistent with these observa- doi.org/10.1016/j.neuron.2012.12.027.
tions. Adult NgR1 null mice exhibit juvenile levels of plasticity for
acoustic preference as well as ocular dominance (Yang et al., ACKNOWLEDGMENTS
2012).
Inflammation, but not an enriched environment, is additive We thank W.B. Gan for instruction on thin skull techniques, C. Portera-Cailliau
with NgR1 deletion to increase spine dynamics, suggesting for instruction on open skull surgeries, S. Tomita for instruction on subcellular
fractionation, A. Holtmaat and A. McGee for helpful discussion, and S. Sodi
that various experience-dependent modalities will saturate at
and Y. Fu for technical assistance. F.V.A. is supported by an Institutional
different levels. For fearful responses, conditioning is associated N.I.H. Medical Scientist Training Program grant. S.M.S. is a member of the
with cortical spine loss and extinction with spine gain (Lai et al., Kavli Institute for Neuroscience at Yale University. We acknowledge research
2012). Both spine protrusion and fear extinction are age depen- support from the N.I.H. and the Falk Medical Research Trust to S.M.S. S.M.S.
dent and limited in the adult (Gogolla et al., 2009). We show that is a cofounder of Axerion Therapeutics, seeking to develop PrP- and NgR-
NgR1 antagonizes spine protrusion (Figures 1E and S1I) and based therapeutics.
stabilizes fear memories (Figure 4I) in the adult. It will be of
Accepted: December 11, 2012
interest to determine whether accelerated cortical spine loss Published: March 6, 2013
correlates with extinction of fear memories in NgR1 mutants.
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