Synapse-Selective Control of Cortical Maturation and Plasticity by Parvalbumin-Autonomous Action of SynCAM 1

Abstract

Cortical plasticity peaks early in life and tapers in adulthood, as exemplified in the primary visual cortex (V1), wherein brief loss of vision in one eye reduces cortical responses to inputs from that eye during the critical period but not in adulthood. The synaptic locus of cortical plasticity and the cell-autonomous synaptic factors determining critical periods remain unclear. We here demonstrate that the immunoglobulin protein Synaptic Cell Adhesion Molecule 1 (SynCAM 1/Cadm1) is regulated by visual experience and limits V1 plasticity. Loss of SynCAM 1 selectively reduces the number of thalamocortical inputs onto parvalbumin (PV+) interneurons, impairing the maturation of feedforward inhibition in V1. SynCAM 1 acts in PV+ interneurons to actively restrict cortical plasticity, and brief PV+-specific knockdown of SynCAM 1 in adult visual cortex restores juvenile-like plasticity. These results identify a synapse-specific, cell-autonomous mechanism for thalamocortical visual circuit maturation and closure of the visual critical period. : Ribic et al. show that cortical plasticity is actively restricted by the synapse-organizing molecule SynCAM 1. The protein acts in parvalbumin interneurons to recruit excitatory thalamocortical terminals. This controls the maturation of inhibition and actively limits cortical plasticity, revealing a synaptic locus for closure of cortical critical periods. Keywords: synapse, SynCAM, Cadm, visual cortex, critical period, plasticity, parvalbumin, thalamocortical inputs

Full text

Article
Synapse-Selective Control of Cortical Maturation
and Plasticity by Parvalbumin-Autonomous Action of
SynCAM 1
Graphical Abstract
Highlights
dVisual plasticity selectively regulates SynCAM 1 expression
dSynCAM 1 controls thalamic inputs onto cortical parvalbumin
(PV
+
) interneurons
dPV
+
-specific SynCAM 1 knockdown restores plasticity in the
mature visual cortex
dThalamic inputs on PV
+
interneurons are synaptic sites for
critical period closure
Authors
Adema Ribic, Michael C. Crair,
Thomas Biederer
Correspondence
adema.ribic@tufts.edu (A.R.),
thomas.biederer@tufts.edu (T.B.)
In Brief
Ribic et al. show that cortical plasticity is
actively restricted by the synapse-
organizing molecule SynCAM 1. The
protein acts in parvalbumin interneurons
to recruit excitatory thalamocortical
terminals. This controls the maturation of
inhibition and actively limits cortical
plasticity, revealing a synaptic locus for
closure of cortical critical periods.
Ribic et al., 2019, Cell Reports 26, 381–393
January 8, 2019 ª2018 The Author(s).
https://doi.org/10.1016/j.celrep.2018.12.069

Cell Reports
Article
Synapse-Selective Control
of Cortical Maturation and Plasticity
by Parvalbumin-Autonomous Action of SynCAM 1
Adema Ribic,
1,
*Michael C. Crair,
2
and Thomas Biederer
1,3,
*
1
Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA
2
Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA
3
Lead Contact
*Correspondence: adema.ribic@tufts.edu (A.R.), thomas.biederer@tufts.edu (T.B.)
https://doi.org/10.1016/j.celrep.2018.12.069
SUMMARY
Cortical plasticity peaks early in life and tapers in
adulthood, as exemplified in the primary visual cor-
tex (V1), wherein brief loss of vision in one eye
reduces cortical responses to inputs from that eye
during the critical period but not in adulthood. The
synaptic locus of cortical plasticity and the cell-
autonomous synaptic factors determining critical
periods remain unclear. We here demonstrate that
the immunoglobulin protein Synaptic Cell Adhesion
Molecule 1 (SynCAM 1/Cadm1) is regulated by visual
experience and limits V1 plasticity. Loss of SynCAM
1 selectively reduces the number of thalamocortical
inputs onto parvalbumin (PV
+
) interneurons, impair-
ing the maturation of feedforward inhibition in V1.
SynCAM 1 acts in PV
+
interneurons to actively
restrict cortical plasticity, and brief PV
+
-specific
knockdown of SynCAM 1 in adult visual cortex re-
stores juvenile-like plasticity. These results identify
a synapse-specific, cell-autonomous mechanism
for thalamocortical visual circuit maturation and
closure of the visual critical period.
INTRODUCTION
Imbalanced visual input during the postnatal critical period for
development of visual function leads to a permanent reduction
in cortical responses to the affected eye and an increase in re-
sponses to the healthy eye, a phenomenon known as ocular
dominance plasticity (ODP) (Espinosa and Stryker, 2012). The
elevated potential for ODP during the critical period promotes
the extensive sensory experience-dependent refinement of syn-
apses during cortical development (Espinosa and Stryker, 2012;
Wang et al., 2010). Plasticity tapers off as the brain matures,
such that brief manipulation of visual input in adult animal models
has no effect on cortical responses (Kuhlman et al., 2013).
There is considerable evidence that elevated cortical inhibitory
neurotransmission is necessary for critical period opening and
that this involves sensory-driven maturation of excitatory drive
onto fast-spiking, parvalbumin (PV
+
) inhibitory interneurons
(Chittajallu and Isaac, 2010; Kuhlman et al., 2013). The duration
of the critical period depends on modulation of PV
+
interneuron
function by different ‘‘molecular brakes’’ (Takesian and Hensch,
2013; Trachtenberg, 2015), and it is thought that stabilization of
excitatory drive onto PV
+
cells by molecular brakes is the main
factor in critical period closure (Trachtenberg, 2015). Recent
research has demonstrated that the extracellular matrix (ECM)
protein Narp, as well as PV
+
-expressed NogoR and neuregulin
1/ErbB4 signaling, control local, intracortical excitatory inputs
onto PV
+
interneurons during ODP (Gu et al., 2013; Stephany
et al., 2016; Sun et al., 2016). Yet long-range, feedforward inputs
from the visual thalamus activate cortical PV
+
interneurons even
more strongly than pyramidal neurons (Ji et al., 2016; Kloc and
Maffei, 2014) and may be crucial in critical period closure (Trach-
tenberg, 2015). Cell-autonomous synaptic factors that organize
these thalamocortical (TC) inputs remain unknown.
Here, we identify SynCAM 1 as a cell-autonomous synaptic
organizer of feedforward TC inputs onto PV
+
interneurons in
V1. SynCAM 1 is a synaptogenic immunoglobulin that functions
in the hippocampus to assemble and maintain synapses on both
principal cells and PV
+
interneurons (Biederer et al., 2002; Park
et al., 2016; Robbins et al., 2010). Mice lacking SynCAM 1 or
with reduced SynCAM 1 expression in V1 PV
+
interneurons
exhibit immature visual function and an ODP that extends
beyond the critical period into adulthood. Remarkably, even brief
knockdown of SynCAM 1 in PV
+
interneurons restores juvenile-
like plasticity in adult V1. Together, our results reveal a SynCAM
1-dependent, PV
+
cell-autonomous, and synapse type-specific
mechanism that actively restricts cortical plasticity in the devel-
oping and adult brain and demonstrate a central role of feedfor-
ward inputs to PV
+
interneurons in critical period closure.
RESULTS
Sensory Input Selectively Regulates Expression of the
Synapse Organizer SynCAM 1 in the Visual Cortex
SynCAMs 1–4 form transsynaptic complexes throughout the
brain (Fogel et al., 2007). However, only SynCAM 1 transcripts
exhibit an increase in cortical expression after P15, when exten-
sive synaptic remodeling begins in this brain region (De Felipe
et al., 1997; Thomas et al., 2008). We performed quantitative
immunoblotting of total homogenates and quantitative immuno-
histochemistry of C57BL/6 wild-type mice V1 at four main stages
of development: postnatal day 7 (P7), start of synaptogenesis;
P14, eye opening and peak of thalamocortical remodeling; P28,
Cell Reports 26, 381–393, January 8, 2019 ª2018 The Author(s). 381
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

peak of the cortical critical period; and P45, young adult (Fig-
ure 1A) (Kuhlman et al., 2013). SynCAM 1 protein was detected
in V1 as early as P7 (Figures 1A and 1B), after which its expres-
sion in the cortex increased strongly through P14 and P28 and re-
mained high in adult mice (Figures 1A and 1B). To obtain insight
into cell-type-specific changes, we performed immunostaining
for SynCAM 1 and the neuronal nuclei marker NeuN that labels
proximal dendritic segments of pyramidal neurons (Wolf et al.,
1996), and parvalbumin (PV) that is detectable in dendrites of
fast-spiking PV
+
interneurons (Kameda et al., 2012). Imaging of
single optical sections showed dense SynCAM 1 puncta both
on NeuN and PV-labeled dendrites (Figure 1C). This was in
agreement with the reported expression of SynCAM 1 in both
pyramidal neurons and PV
+
interneurons (Fo
¨ldy et al., 2016).
V1 of mice is strongly driven by contralateral eye inputs, and a
blockade of visual input through one eye during the critical period
Figure 1. Expression of SynCAM 1 in V1 Is
Regulated by Activity in a Cell-Specific
Manner
(A) Top: time points of V1 development. Middle and
bottom: quantitative immunoblotting of SynCAM 1
in the developing mouse V1 (30 mg/lane). n = 2
animals/time point. Expression levels were first
normalized to actin and then to P7 levels.
(B) Top: representative maximum intensity pro-
jections of immunohistochemical staining of Syn-
CAM 1 in the developing mouse V1. Scale bar,
250 mm. Bottom: quantification of staining intensity.
(C) SynCAM 1 antibodies stain neuropil and den-
dritic segments of NeuN
+
pyramidal neurons (top)
and PV
+
interneurons (bottom). Single optical sec-
tions are shown. Scale bar, 15 mm.
(D) Decussation of retinal axons at the chiasm re-
sults in reduced visual responsiveness in the left
visual cortex (contralateral to the deprived eye)
after monocular deprivation (MD). The right visual
cortex (ipsilateral) continues to receive input from
the open eye and served as control in (E)–(G).
(E) Quantitative immunoblots of control and
deprived P28 V1 homogenates (30 mg/lane). Mo-
lecular weights are indicated on the left. MD
significantly increased SynCAM 1 in V1 but had no
effect on SynCAMs 2–4 or on GluA1 and
GABA
A
aR1. Measurements were first normalized
to actin and then to control (ipsilateral) levels. n =
7–10 mice/experiment; ns, not significant; *p <
0.05, paired t test.
(F and G) Representative images (F) and quantifi-
cation (G) of SynCAM 1 puncta on NeuN
+
/PV

and
PV
+
primary dendritic segments revealed a signifi-
cant increase of SynCAM 1 expression in the PV
+
neurons of the deprived hemisphere.
n = 4 mice/experiment; **p < 0.01, two-way RM
ANOVA. Scale bar, 20 mm. Data are presented as
mean ±SEM (A–E) and minimum-maximum (G).
for vision induces robust plasticity and re-
modeling in the contralateral V1 (Antonini
et al., 1999; Gordon and Stryker, 1996;
Heynen et al., 2003). A previous study
that sought to identify candidate regulators
of plasticity reported that monocular deprivation (MD) strongly up-
regulated SynCAM gene expression in the V1 (Lyckman et al.,
2008). To determine which of the SynCAMs is regulated by MD
and visual plasticity on the protein level, we performed quantita-
tive immunoblotting of V1 in mice that had undergone MD from
the beginning of eye opening until the peak of the critical period
(Figure 1D) (Lyckman et al., 2008). Contralateral dominance of
mouse V1 allows intra-animal comparison of changes in protein
expression, where the ipsilateral cortex serves as control (Fig-
ure 1D) (Heynen et al., 2003). Only SynCAM 1 exhibited a signifi-
cant activity-dependent change in protein expression (Figure 1E).
MD upregulated SynCAM 1 protein levels (Figure 1E; control V1 =
100 ±10.6 AU, deprived V1 = 121 ±5.1 AU; p = 0.024, paired t
test; n = 7 animals, t = 3, df = 6) but had no effect on SynCAM
2, 3, and 4 (Figure 1E; SynCAM 2: control = 100 ±7.6, deprived =
106 ±7.2; SynCAM 3: control = 100 ±5.5, deprived = 108 ±8.9;
382 Cell Reports 26, 381–393, January 8, 2019

SynCAM 4: control = 100 ±8.2, deprived = 107 ±10.8; n = 7 an-
imals; all valuesin AU). Consistent with previous reports (Lyckman
et al., 2008; Tropea et al., 2006), deprivation did not affect the
levels of glutamate or GABA receptors (Figure 1E; GluA1:control =
108 ±13.1, deprived = 107 ±12.5; GABA
A
a1: control = 106 ±5.4,
deprived = 112 ±7.7; n = 10 animals; all values in AU).
To evaluate the cell-type specificity of activity-dependent
changes in SynCAM 1 protein expression in the V1, we used
quantitative immunohistochemistry to estimate the density of
SynCAM 1 puncta in contact with NeuN
+
/PV

and PV
+
dendritic
segments after MD (Figures 1F, 1G, and S1). MD had a significant
effect on SynCAM 1 expression (interaction between cell type
and deprivation, F
[1,6]
= 7.53, p = 0.03, two-way repeated-mea-
sures [RM] ANOVA), and SynCAM 1 puncta density was elevated
on PV
+
dendrites in the deprived compared with the non-
deprived control hemisphere (Figures 1F and 1G; control =
29 ±2.3, deprived = 35 ±1.3; p < 0.01, post hoc Sidak’s multi-
ple-comparisons test; n = 4 animals). We found no change in
the density of SynCAM 1 puncta on NeuN
+
/PV

dendrites in
the deprived hemisphere (Figures 1F and 1G; control = 29 ±
1.3, deprived = 30 ±1.8; p = ns). As a control, we performed
this quantification in non-primary sensory regions on the same
coronal sections used for analysis of V1 and found that the den-
sity of SynCAM 1 puncta on PV
+
dendrites in secondary auditory
and ectorhinal cortex was not significantly different between the
groups (Figures S1A–S1C and data not shown; control PV
+
=31
±2.4, deprived PV
+
=33±1.3; control NeuN
+
/PV

=27±3.1,
deprived NeuN
+
/PV

=32±2.7; no interaction between cell-
type and deprivation on two-way RM ANOVA; F
[1.27, 3.8]
= 1.73,
p = 0.274, one-way RM ANOVA; n = 4 animals). These results
supported a cell type-specific regulation of SynCAM 1 expres-
sion in V1 during ODP and suggested a role for SynCAM 1 in
PV
+
interneurons during the visual critical period.
SynCAM 1 Limits Visual Plasticity in Both Juvenile and
Adult Brain
Brief MD during the critical period robustly depresses closed
(contralateral) eye responses in the binocular portion of mouse
V1 (bV1), resulting in a strong downward shift in the contralat-
eral/ipsilateral (C/I; closed/open) eye response ratio (Frenkel
and Bear, 2004; Gordon and Stryker, 1996). PV
+
interneurons
play a central role in this process (Kuhlman et al., 2013). As
imbalanced visual input substantially upregulated SynCAM 1
expression on PV
+
interneurons (Figures 1F and 1G), we hypoth-
esized that SynCAM 1 loss may modulate ODP in V1. To test this,
we recorded visually evoked potentials (VEPs) from the bV1 in
awake wild-type (WT) and SynCAM 1 knockout (KO) mice using
16-channel probes and a spherical treadmill setup (Figure 2A)
Figure 2. SynCAM 1 Limits Plasticity in the Visual Cortex
(A) Local field potentials (LFPs) evoked by full-field sinusoidal gratings or light
flashes were collected using 16-channel probes (inset) from animals head-
fixed over an air-suspended Styrofoam ball. Right: representative electrode
tract (DiI, pink) in the binocular V1 (bV1; DAPI, grayscale).
(B) Amplitude of visually evoked responses (VEPs; top) decreases with
increasing frequency of sinusoidal gratings in both wild-type (WT) and KO an-
imals. Visual acuity was estimated for each animal by estimating the grating
frequency at which the amplitude was equal to zero (WT = 0.52 ±0.13 cpd,
KO = 0.55 ±0.03 cpd; n = 6 WT and 7 KO animals; t = 0.25,df = 11, t test; p = ns).
(C) Experimental timelines for headpost implantation for in vivo physiology and
MD. VEPs were collected after reopening of the sutured eye. Non-deprived
(ND) animals were prepared in parallel.
(D) ND SynCAM 1-KO and WT mice had indistinguishable C/I ratios. MD during
the early CP resulted in a non-significant reduction of C/I in WT mice and a
robust C/I reduction in KO mice. MD during the CP significantly lowered the C/I
ratio in both WT and SynCAM 1-KO mice. Short MD had no effect on visual
responses in adult WT mice but significantly lowered the C/I of adult SynCAM
1-KO mice.
(E) MD during the early CP caused strong depression of the closed-eye re-
sponses only in KO mice. MD during the CP significantly depressed the
closed-eye responses in WT mice but increased the open eye responses in KO
mice. MD in adult WT mice was without effect but significantly increased open
eye responses in KO mice.
Scale bars, 250 mV and 0.5 s in (A, top), 1 mm in (A, bottom), and 100 mV and
0.2 s in (B) and (D). In (D) and (E), ns, not significant; *p < 0.05 and **p < 0.01,
one-way and two-way ANOVA (see Table S1 for details). Data are presented as
mean ±SEM; n values are indicated.
Cell Reports 26, 381–393, January 8, 2019 383

(Niell and Stryker, 2010). When presented with sinusoidal grat-
ings that varied in frequency, both WT and KO animals showed
the typical decrease in amplitude of VEPs evoked through the
contralateral eye as the frequency of gratings increased (Fig-
ure 2B). Visual acuity was in the expected range for mice
(WT = 0.52 ±0.13 cycles per degree [cpd], KO = 0.55 ±0.03
cpd; n = 6 WT and 7 KO animals) (Porciatti et al., 1999). We
then sutured the right eyelids of KO animals and their WT
littermates for 3–4 days during the early critical period (CP;
P21–P24), at the peak of the critical period (P25–P28), and
in adulthood (P60–P64) (Figure 2C). Non-deprived (ND) WT
and KO animals had almost identical C/I ratios (Figure 2D;
Table S1) and VEP amplitudes, as expected from normal acuity
in KO mice (Figures 2B and 2E; Table S1). For mice that under-
went MD, we reopened the sutured eyelid on the last day of
deprivation and recorded visual responses to the stimulation of
both closed (contralateral) and open (ipsilateral) eyes. Consistent
with previous studies, 3 days of MD during the early critical
period were not sufficient to significantly affect visual responses
in WT animals, but they induced a robust shift in C/I ratio and
strong depression of closed-eye responses during the peak of
the critical period (Figures 2D and 2E; Table S1)(Frenkel and
Bear, 2004; Gordon and Stryker, 1996). Short-term deprivation
had no effect in adult WT animals, in agreement with reduced
plasticity of the mature cortex (Kuhlman et al., 2013).
Distinct from WT mice, short MD decreased the C/I ratio at all
ages tested in KO mice (Figure 2D; Table S1). Three days of
deprivation in mice lacking SynCAM 1 strongly depressed
closed-eye responses already during the early critical period
and induced open-eye potentiation during its peak (Figure 2E;
Table S1). In striking contrast to WT mice, adult KO mice
exhibited robust plasticity after MD, with strong open-eye poten-
tiation. Two-way ANOVA showed a significant interaction be-
tween genotype and deprivation in the amplitude of open
(ipsilateral) eye responses (F
[3,44]
= 3.1, p = 0.035) (Table S1).
MD had no effect on adult animals heterozygous for SynCAM 1
loss, indicating that a substantial reduction of SynCAM 1 expres-
sion is necessary to permit plasticity (data not shown). Further-
more, short deprivation at P17 before the critical period opens
had no apparent effect on either WT or KO mice (WT C/I = 2.2
±0.42, KO C/I = 2.2 ±0.35; n = 4 WT and 6 KO animals). These
data demonstrated a role of SynCAM 1 in restricting the closure
of the critical period, without altering the timing of the precritical
period.
Formation of Perineuronal Nets Is Impaired in the
Absence of SynCAM 1
The closure of the critical period in V1 requires mature PV
+
-medi-
ated cortical inhibition (Fagiolini et al., 2004; Kuhlman et al.,
2013). A measure of PV
+
interneuron maturation is the formation
of proteoglycan-composed ECM structures called perineuronal
nets (PNNs) around them (Figure 3A) (Dityatev et al., 2007; Ye
and Miao, 2013). To track this maturation process, we studied
the development of PNNs in SynCAM 1-KO V1 by quantifying
the staining intensity of the PNN marker Wisteria floribunda
agglutinin (WFA) (Ye and Miao, 2013). At the start of the critical
period (early CP), WT mice already had more than 60% of their
PV
+
interneurons enwrapped with PNNs (Figures 3B and 3D)
(WT early CP/P21 = 67 ±6.3, n = 5 animals; WT CP/P28 = 70 ±
4.5, n = 4; WT adult/P60–P70 = 74 ±2, n = 4; all values in % of
PV
+
interneurons). The density of PNN puncta around PV
+
inter-
neurons that were positive for PNNs steeply increased from early
critical period to adulthood in WT mice (Figure 3E) (WT early CP/
P21 = 301 ±61.7, CP/P28 = 837 ±139.3, adult/P60–P70 = 1,271
±113.2; all values in particles/mm
2
). The overall density of PV
+
interneurons in SynCAM 1-KO mice was indistinguishable from
WT mice (Figure 3F) (early CP P21 WT = 141 ±7, KO = 140 ±
14.1; CP/P28 WT = 163 ±10.2, KO = 168 ±15.5; adult/
P60–P70 WT = 155 ±4.8 cells/mm
2
, KO = 155 ±8.9; all values
in cells/mm
2
). In contrast to the prominent enwrapping of PV
+
cells in WT mice, the fraction of PV
+
interneurons surrounded
by PNNs was significantly lower in KO mice at all ages tested
(Figures 3C and 3D) (early CP/P21 = 42 ±5.5, p = 0.008; CP/
P28 = 35 ±3.7, p = 0.0006; adult/P60–P70 = 50 ±5, p = 0.014;
F
[5,16]
= 9.9, p = 0.0002, one-way ANOVA, Holm-Sidak’s multi-
ple-comparisons test). In addition, loss of SynCAM 1 severely
reduced PNN deposition in those PV
+
cells that remained posi-
tive for WFA (Figures 3C and 3E) (KO early CP/P21 = 75 ±
47.5, n = 3, p = 0.415; KO CP/P28 = 178 ±50.4, n = 3, p =
0.003; KO adult/P60–P70 = 766 ±195.2, n = 3, p = 0.019; all
values in particles/mm
2
;F
[5,16]
= 17.4, p < 0.0001, one-way
ANOVA, Holm-Sidak’s multiple-comparisons test). We investi-
gated the distribution of SynCAM 1 in primary cultures of cortical
neurons, and its robust signal on developing and mature PV
+
in-
terneurons did not colocalize with WFA, which argued against
SynCAM 1 being a specific component of PNNs (Figure S2).
Otx2, a PNN-dependent transcription factor that directs the
maturation of PV
+
interneurons (Sugiyama et al., 2008), was un-
altered in SynCAM 1-KO PV
+
interneurons (Figure S3). These re-
sults demonstrated impaired development of PNNs in the
absence of SynCAM 1 and provided evidence that the reduced
maturation of cortical PV
+
interneurons in KO mice involves an
Otx2-independent mechanism.
SynCAM 1 Is Necessary for Recruitment of
Thalamocortical Terminals onto PV
+
Interneurons
Excitatory synaptic input can control the deposition of PNNs
around PV
+
interneurons (Dityatev et al., 2007). SynCAM 1
contributes to the development of excitatory synapses on in-
terneurons in the hippocampus (Park et al., 2016). We there-
fore studied the two main types of glutamatergic inputs on
cortical PV
+
interneurons: short range/intracortical and long-
range/thalamocortical (TC) inputs (Figure 4A), which use
presynaptic vesicular glutamate transporter 1 (vGlut1) and 2
(vGlut2), respectively (Fremeau et al., 2001; Singh et al.,
2016). WT mice exhibited intense vGlut1 signal throughout
the cortex, while vGlut2 appeared as a thick band in the thala-
morecipient layer IV, consistent with previous studies (data not
shown) (Coleman et al., 2010). We quantified the number and
size of vGlut1 and vGlut2 puncta in single optical sections of
PV
+
dendrites in V1 (Figures S4 and S5). The number of vGlut2
puncta in contact with PV
+
dendrites in layer IV of WT mice did
not vary from early critical period (early CP/P21) to adulthood
(P60–P70) in agreement with previous reports (Figures 4Band
4D) (early CP/P21 = 18 ±0.4, n = 5 animals; CP/P28 = 17 ±
0.5, n = 4; adult/P60–P70 = 17 ±0.7, n = 4; all values in
384 Cell Reports 26, 381–393, January 8, 2019

puncta/100 mm
2
)(Kameda et al., 2012). In contrast, loss of
SynCAM 1 in KO mice significantly reduced density of vGlut2
+
TC inputs to PV
+
dendrites from the onset of critical period
onward compared with WT littermate controls (Figures 4B
and 4D) (KO early CP/P21 = 16 ±0.6, n = 3, p = 0.036, 12%
reduction; CP/P28 = 14 ±0.5, n = 3, p = 0.008, 19% reduction;
adult/P60–P70 = 13 ±1.3, n = 3, p = 0.001, 25% reduction; all
values in puncta/100 mm
2
;F
[5,16]
= 8.5, p = 0.0004, one-way
ANOVA, Holm-Sidak’s multiple-comparisons test). The density
of intracortical, vGlut1
+
inputs to PV
+
dendrites was indistin-
guishable between WT and KO animals at all ages (Figures
4C and 4E) (early CP/P21 WT = 35 ±0.8, KO = 33 ±0.9;
CP/P28 WT = 31 ±1.2, KO = 30 ±1.8; adult/P60–P70 WT =
33 ±1.7, KO = 34 ±0.4; all values in puncta/100 mm
2
). Puncta
size was not significantly different between the groups for both
vGlut1 and vGlut2 (data not shown). These results strongly
supported a TC-specific input impairment in PV
+
interneurons
of SynCAM 1-KO mice.
TC axons extensively arborize in cortical layer IV during early
postnatal development, and their fine structure can be deter-
mined by injecting anterograde tracers into the dorsolateral
geniculate nucleus of the thalamus (dLGN) (Antonini et al.,
1999). To assess if arborization of TC axons was impaired,
we injected an anterogradely transporting AAV-EGFP construct
into dLGNs of adult WT and SynCAM 1-KO mice (Figure 4F)
and reconstructed single-axonal arbors (Figure 4G). Absence
of SynCAM 1 did not affect total branch length and the number
of TC branches (Figures 4H and 4I). Only their variability was
increased in KO mice, as measured by the coefficient of varia-
tion (CV
branch length
: WT = 274 ±53.98, KO = 781 ±139.8;
p = 0.015, t test; n = 4 animals, t = 3.4, df = 6; CV
branch number
:
WT = 2.2 ±0.56, KO = 6.5 ±0.42; p = 0.0008, t test; n = 4 an-
imals, t = 6.2, df = 6). Together, these results supported grossly
normal arborization of thalamocortical projections and select
aberrations in their fine synaptic connectivity in the absence
of SynCAM 1.
Figure 3. Formation of PNNs Is Reduced in the Absence of SynCAM 1
(A) PNN deposition increases parallel to the maturation of PV
+
interneurons after eye opening in V1. Pyramidal neurons (PYR; blue) lack PNNs.
(B) Double labeling for PV (red) and WFA (gray) in WT V1 showed that WFA
+
PNNs enwrapped the majority of PV
+
interneurons in the WT. Scale bar, 50 mm.
(C) PNNs surrounded fewer PV
+
interneurons in SynCAM 1-KO mice at all ages tested.
(D) Quantification of images as in (B) and (C) showed a significant difference in the density of WFA-positive PV
+
interneurons between genotypes through all ages
tested.
(E) The density of PNNs increased throughout age in WT animals but remained significantly lower from the critical period onward in the fraction of PV
+
cells of KO
mice that were positive for WFA.
(F) The density and distribution of PV
+
cells were indistinguishable between WT and SynCAM 1-KO mice.
In (D)–(F), ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA. Data are presented as mean ±SEM; n = 3–5 animals/genotype.
Cell Reports 26, 381–393, January 8, 2019 385

Figure 4. PV
+
Interneurons in V1 of SynCAM 1-KO Mice Receive Fewer Inputs from Thalamus
(A) vGlut2
+
inputs from dLGN (green) innervate both PV
+
inhibitory neurons (red) and pyramidal neurons (blue) in layers II/III and IV. Local cortico-cortical con-
nections predominantly use vGlut1 (cyan).
(B and C) Representative single optical sections of PV/vGlut2 (B) and PV/vGlut1 (C) immunofluorescence in layer IV V1 of WT and SynCAM 1-KO mice at the
indicated ages. Scale bar, 15 mm.
(D) KO mice showed a significant reduction in TC inputs in contact with PV
+
dendrites at all ages.
(E) Density of intracortical vGlut1
+
inputs on KO and WT PV
+
cells was indistinguishable.
(F) Top: anterograde AAV tracer in the dLGN. Scale bar, 500 mm. Bottom left: V1 sections from the same animal show thalamic projections in layer IV (inse t). Scale
bar, 250 mm. Bottom right: high magnification reveals thalamocortical arbors. Scale bar, 50 mm.
(G) Representative reconstructions of single thalamocortical axons from adult WT (top, black) and KO (bottom, green), arranged from simplest to most complex
(left to right).
(H and I) Overall branch length was not significantly different between WT and KO mice (H), and neither was the branching complexity (I).
In (D) and (E), ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001, one-w ay ANOVA. Data are presented as mean ±SEM (D and E) and minimum-maximum of
all data points (H and I; indicated); n = 3–5 animals/genotype, unless indicated otherwise. In (H) and (I), ns, not significant.
386 Cell Reports 26, 381–393, January 8, 2019

Figure 5. Feedforward Inhibition and Visual Circuit Function Are Immature in SynCAM 1-KO Mice
(A) Inset: example of spiking activity and peristimulus time histogram (PSTH) that marks measured parameters. Evoked firing rate was calculated as average
spontaneous firing rate subtracted from average peak firing rate. Stimulus (LED flash) is indicated in yellow. Scale bars, 100 mV and 2.5 s.
(B) Representative raster plots and PSTHs of MUA recorded from WT (top) and SynCAM 1-KO mice (bottom) at P28, the peak of CP. Scale bar, 15 spikes/s.
(C) Average spontaneous, prestimulus firing rate was comparable in SynCAM 1-KO mice with that of WT mice (left), but the evoked firing rate was significantly
increased in SynCAM 1-KO animals (right) (Table S2).
(D) Increased latency of the primary response (left) and response duration (right) in SynCAM 1-KO mice (Table S2)(n
MUA
= 39 WT and 38 KO for C and D).
(E) Mice were presented with gratings of varying orientations through both eyes, and responses of isolated binocular neurons (single unit activity [SUA]) in V1 were
isolated and compared.
(F) WT mice show overlapping binocular responses (DO), indicating mature binocular visual function. Dotted line represents Gaussian fit of normalized stimulus-
evoked spike rate (individual points). Preferred orientation was calculated as the maximum response amplitude after the Gaussian fit. Scale bar, 1 Hz.
(G) KO mice have little overlap between contralateral and ipsilateral responses (Gaussian fit in dotted line).
(H) Binocular matching or orientation preference, determined as the differe nce between preferred orientations of contra and ipsi eye responses, is significantly
different between WT and KO mice (n
SUA
= 33 WT and 28 KO).
(legend continued on next page)
Cell Reports 26, 381–393, January 8, 2019 387

Maturation of the Visual Circuit Requires SynCAM 1
The reduced TC inputs onto PV
+
interneurons in absence of
SynCAM 1 may affect the maturation of visual responses. We
therefore analyzed spontaneous and stimulus-induced activity
of neurons (multi-unit activity [MUA]) from layer IV VEPs (Fig-
ure 5A). Feedforward inhibition in the V1 matures after eye open-
ing and strongly suppresses the primary response of pyramidal
neurons to visual stimulation (Gu et al., 2013; Shen and Colonn-
ese, 2016). During the critical period, both WT and KO animals
showed robust and transient increases in firing rate in response
to light presentation (Figure 5B). Spontaneous firing rate was
indistinguishable between KO and WT mice (Figures 5B
and 5C, left; Table S2). However, the stimulus-evoked firing
rate was significantly increased in KO animals (Figure 5C, right;
Table S2), indicating disinhibition of visual responses (Gu et al.,
2013). Detailed analysis of firing revealed a primary visual
response that was significantly delayed (Figure 5D, left) and pro-
tracted (Figure 5D, right) in KO animals (Table S2), where the
delay in firing onset likely reflected a delayed onset of retinal re-
sponses to light in KO mice (Ribic et al., 2014; Shen and Colonn-
ese, 2016). These results supported that feedforward inhibition
in V1 is impaired in absence of SynCAM 1, consistent with the
lower density of TC inputs onto PV
+
interneurons.
Visual function matures during the critical period such that
binocularly responsive neurons in adult V1 that are selective for
stimulus orientation have similar eye-specific orientation prefer-
ence (Wang et al., 2010). Binocular orientation preference is
poorly matched at the onset of the critical period in mouse V1
and improves in an activity-dependent manner until the critical
period closes (Wang et al., 2010, 2013). As visual responses
are immature in SynCAM 1-KO mice (Figures 5C and 5D), we
predicted that the binocular matching of orientation preference
might also be impaired in the absence of SynCAM 1. We isolated
responses of single cortical neurons to sinusoidal gratings that
varied in orientation (Figure 5E), and constructed orientation tun-
ing curves for responses to the stimulation of contralateral and
ipsilateral eyes (Figures 5F and 5G). Most sampled cells were se-
lective for orientation in both WT and KO, with the orientation
selectivity index (OSI) matching previous reports for critical
period mice (OSI WT contralateral = 0.52 ±0.04, WT ipsilateral =
0.38 ±0.06; KO contralateral = 0.51 ±0.05, KO ipsilateral = 0.46
±0.05; n = 33 WT and 28 KO) (Wang et al., 2013). Most cells in the
WT mice also showed matched preferred orientations between
contralateral and ipsilateral responses (DO = 18.15 ±2.77)(Fig-
ures 5F and 5H) (Wang et al., 2010, 2013). In contrast, cells in
KO mice displayed large differences between eyes in preferred
orientations (DO = 48.24 ±6.45; p = 0.0002, Mann-Whitney
t test) (Figures 5G and 5H). Binocular matching of orientation
preference was therefore significantly reduced in KO mice,
further supporting delayed maturation of V1 in these mice.
Visual stimulation suppresses PV
+
-mediated gamma power
oscillations (>40 Hz) (Cardin et al., 2009) during the critical
period in V1, which can be prevented by delaying circuit matu-
ration with dark rearing (Chen et al., 2015). As visual circuits ap-
peared immature in the absence of SynCAM 1 (Figures 5B–5G),
we hypothesized that gamma range power might be aberrant in
SynCAM 1-KO mice during the critical period. Consistent with
previous studies (Chen et al., 2015), critical-period WT mice ex-
hibited a drop in gamma power (40–70 Hz) after switching their
stimulus from blank gray screen to full-field sinusoidal gratings
(Figures 5I and 5J) (WT blank = 0.25 ±0.06 mV
2
, gratings = 0.09
±0.02 mV
2
, p = 0.026; n = 11 animals; t = 2.6, df = 10, paired
t test). Gamma suppression was most pronounced in layer IV
(Dpower
blank-gratings
at 400 mm, 0.21 ±0.08 mV
2
; layer II/III at
100–350 mm, 0.12 ±0.06 mV
2
; layers V/VI at 450–700 mm, 0.13
±0.08 mV
2
). No change in the low*frequency range (1–20 Hz)
was measured in WT mice after visual stimulation, as expected
(Figures 5I and 5K) (WT blank, 85 ±1.6 mV
2
; gratings, 85 ±
2.6 mV
2
)(Chen et al., 2015). Similar to WT mice, KO mice
showed no stimulation-induced changes in the 1–20 Hz band
(Figures 5I and 5K) (KO blank, 80 ±4.7 mV
2
; gratings, 87 ±
1.9 mV
2
; n = 8). Notably, KO mice lacked the visual stimula-
tion-induced suppression of gamma band activity (Figures 5I
and 5J) (KO blank, 0.25 ±0.07 mV
2
; gratings, 0.21 ±0.06 mV
2
).
These results provided further evidence that thalamocortical
circuitry remains immature in the absence of SynCAM 1.
SynCAM 1 Acts in PV
+
Interneurons in V1 to Control the
Maturation of the Thalamocortical Visual Circuit
Where does SynCAM 1 function to promote visual circuit matu-
ration? We addressed this question through region- and cell
type-specific manipulations. Recent studies implicated the
visual thalamus in the regulation of critical period plasticity
(Jaepel et al., 2017; Sommeijer et al., 2017). SynCAM 1 expres-
sion in the lateral geniculate nucleus (LGN) was low during
postnatal development (Figure S6A), and the organization of
LGN in SynCAM 1-KO mice was grossly normal (Figures S6B
and S6C), suggesting a cortical locus of SynCAM 1 function
during ODP. Cortical PV
+
interneurons were immature in the
absence of SynCAM 1 (Figure 3) and density and PNN coverage
of regular spiking interneurons detected with WFA and anti-
bodies against somatostatin interneurons appeared normal
(data not shown). To directly test whether the locus of SynCAM
1actionduringODParePV
+
interneurons, we cloned an
shRNA against SynCAM 1 and a control scrambled sequence
into an adenoviral vector (AAV) that allows Cre-induced short
hairpin RNA (shRNA) expression (Figure S7). We injected
AAV at P14 to deliver shSynCAM 1 (chronic knockdown
[cKD] [PV-Cre
AAV-shSynCAM 1
]) or shScramble (control [Ctrl]
[PV-Cre
AAV-shScramble
]) into the left cortex of PV-Cre mice
(I) Animals were first presented with a gray screen (marked by a gray bar) and then shown sinusoidal gratings (striped bar). LFPs were filtered to depict oscillations
and sonograms. Scale bars, 350 mV for LFP, 50 mV for 40–70 Hz, and 250 mV for 1–20 Hz and 0.5 s.
(J) Visual stimulation suppressed oscillations in the grange (40–70 Hz) in WT animals during the CP, compared with gray screen presentation. This effect was
absent in CP SynCAM 1-KO animals.
(K) No change in lower frequency bands (1–20 Hz) was detected in either WT or SynCAM 1-KO animals after stimulus presentation.
In (C)–(H), data are presented as minimum-maximum.*p < 0.05, **p < 0.01, and ***p < 0.001, Mann-Whitney rank-sum test. In (J) and (K), ns, not significant;
*p < 0.05, paired t test. Data are presented as mean ±SEM; ns, not significant; *p < 0.05; n = 11 WT and 9 KO, paired t test.
388 Cell Reports 26, 381–393, January 8, 2019

(Figure 6A), where Cre recombinase is driven by the PV pro-
moter. We deprived the right (contralateral) eyes of adult
(P60), AAV-injected PV-Cre mice and recorded VEPs and
MUA after reopening the right eye 4 days later, as well as in
ND animals (Figure 6A). No detrimental effects of the viral injec-
tion on C/I ratios were observed, and shScramble Ctrl and cKD
animals had C/I ratio and VEP amplitudes almost identical to
those of ND and non-injected WT animals during the critical
period (Figures 6B and 6C; Table S3) (Ctrl ND C/I = 2.3 ±0.2;
cKD ND C/I = 2.1 ±0.2). No gross changes in visual
responses or acuity of ND mice were observed upon KD of Syn-
CAM 1 in PV
+
interneurons (Figures 6B and 6C; Table S3) (acuity
Figure 6. PV
+
Interneuron-Specific Knockdown of SynCAM 1 in V1 Extends the Critical Period
(A) Top: experimental timeline. ND, non-implanted animals were used for immunohistochemistry (IHC). Bottom: false-colored representative section of an
AAV-injected animal with visible electrode tract (AAV transduction detected by GFP shown in turquoise, DiI in mage nta, and DAPI in grayscale). Scale bar, 1 mm.
(B) Chronic SynCAM 1 knockdown in PV
+
interneurons (cKD) had no effect on C/I ratios. Four days of MD at P60 did not affect shScramble-injected control (Ctrl)
animals, but MD robustly decreased the C/I ratio in cKD mice.
(C) Visual responses of naive, ND Ctrl, and cKD animals were almost identical. MD had no effect on Ctrl animals but significantly depr essed closed-eye responses
in cKD mice. Representative VEPs are shown on top. Scale bars, 200 mV and 0.2 s. In (C) and (D), ns, not significant; *p < 0.05 and **p < 0.01, one-way ANOVA.
Data are presented as mean ±SEM; n = 5–7 animals/group.
(D) Representative raster plots and PSTHs of MUA recorded from layer IV of P64 Ctrl (top, gray) or cKD mice (bottom, green) showed that spontaneou s firing rate
was significantly increased in cKD mice (E, right), while the stimulus-evoked rate was not significantly different between groups (E, left). Stimulus onset is
indicated in yellow in (D). Scale bar, 5 spikes/s.
(F) Latency of primary response was not different after SynCAM 1 cKD (left), but the duration was significantly increased (right). In (E) and (F), ns, not significant;
*p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney rank-sum test. Data are presented as minimum-maximum of all data points.
(G) Single optical confocal sections containing dendritic segments near the injection site were analyzed by immunostaining for PV (red) and vGlut2 (green).
(H) Quantification of data as in (G) shows that cKD of SynCAM 1 significantly reduced the density of vGlut2
+
TC terminals onto PV
+
dendrites in V1. *p < 0.05,
unpaired t test. Data are presented as mean ±SEM; n = 3 or 4 animals/group. Scale bar, 15 mm.
Cell Reports 26, 381–393, January 8, 2019 389

Ctrl = 0.61 ±0.15 cpd, cKD = 0.51 ±0.05 cpd). Short MD from
P60 to P64 had no effect on the C/I ratio of adult shScramble
Ctrl mice, as expected (Figures 6B and 6C; Table S3) (Ctrl MD
C/I = 2 ±0.2). In striking contrast, KD of SynCAM 1 in PV
+
inter-
neurons resulted in robust plasticity in adult mice after short,
4-day MD (Figure 6B; Table S3) (cKD MD C/I = 1.3 ±0.03). This
plasticity was due to a significant depression of closed-eye re-
sponses, similar to the plasticity of WT mice during the critical
period (Figure 6C, compare with Figure 2E; Table S3).
We observed an increase in MUA firing rate in layer IV in
shScramble Ctrl mice at P64 compared with WT mice at P28
(Figure 6D, top, and Figure 6E, compare with Figures 5B and
5C; Table S4), reflecting the expected developmental increase
in neuronal firing rates in adult animals compared with critical
period mice (Chen et al., 2015). The spontaneous firing rate
was even higher after chronic KD of SynCAM 1 in PV
+
interneu-
rons (Figure 6D, bottom, and Figure 6E, left; Table S4), indicating
disinhibition (Gu et al., 2013). The latency of visual responses
was not significantly affected in cKD mice (Figure 6F, left;
Table S4). However, cKD mice had significantly protracted pri-
mary visual responses, as observed in SynCAM 1-KO mice (Fig-
ure 6F, right, compare with Figure 5D, right; Table S4). Chronic
KD of SynCAM 1 in cortical PV
+
interneurons starting at P14
was hence sufficient to maintain V1 in an immature state and
extend plasticity beyond the critical period.
To address if disinhibition of V1 after KD of SynCAM 1 in PV
+
interneurons shares a common cellular mechanism with the global
loss of SynCAM 1, we quantified the recruitment of TC terminals
onto PV
+
dendrites in V1 of adult shScramble Ctrl and cKD mice
near the AAV injection sites (Figures 6G and 6H). PV-specific KD
of SynCAM 1 in V1 reduced the density of vGlut2
+
terminals in
contact with PV
+
dendrites by 45% (Ctrl = 7 ±0.5 puncta/
100 mm
2
, n = 4 animals; cKD = 4 ±0.3 puncta/100 mm
2
, n = 3 an-
imals; p = 0.026, t = 3.1, df = 5). Intracortical inputs to PV
+
inter-
neurons did not change, as the density of vGlut1 puncta in contact
with PV
+
dendrites remained intact after SynCAM1 KD (Ctrl = 34 ±
1.7 puncta/100 mm
2
,cKD=31±7.7 puncta/100 mm
2
). Puncta size
was unaltered for both vGlut1 and vGlut2 across conditions (data
not shown). The synaptic maturation of the thalamocortical visual
circuit hence engages cell-autonomous, postsynaptic, and input-
specific functions of SynCAM 1 in cortical PV
+
interneurons.
SynCAM 1 Actively Limits Cortical Plasticity in the
Mature Brain
Chronic KD of SynCAM 1 in PV
+
interneurons retarded the devel-
opment of cortical inhibition and arrested the cortex in a plastic
state. To test if cortical plasticity is actively limited in the mature
cortex by SynCAM 1 in PV
+
interneurons, we injected KD con-
structs into left visual cortices of P45 PV-Cre animals, after the
critical period closure (Figure 7A) (Kuhlman et al., 2013). Two
weeks later, we recorded the VEPs and sutured the right eyes
of experimental animals after the recording session. Four days
later, we reopened the sutured eye and collected VEPs to the
stimulation from both eyes. Recording sites were positioned in
the center of craniotomies made for AAV injections (Figure 7A,
bottom) and craniotomies were kept small (less than 0.5 mm in
diameter) to enable precise targeting of electrodes during
the recording sessions. This approach allowed intra-animal
comparison of VEPs before and after deprivation. Spontaneous
and visually evoked activity were indistinguishable between
shScramble Ctrl and acute KD (aKD) animals (Table S5). Simi-
larly, acute SynCAM 1 KD did not affect C/I ratio or VEP ampli-
tudes (Figures 7B and 7C; Table S6). As expected, 4-day MD
at P60 had no significant effect on C/I ratios or VEP amplitudes
of shScramble Ctrl animals (Figures 7B and 7C; Table S6). In
contrast, 4-day MD after aKD of SynCAM 1 in PV
+
interneurons
significantly decreased the C/I ratio, showing robust plasticity in
adult aKD mice (Figure 7B; Table S6). This plasticity was due to
Figure 7. PV
+
-Autonomous SynCAM 1 Actively Ctrls Plasticity in Adult Cortex
(A) Top: experimental timeline. Bottom: false-colored representative section of an AAV-injected animal with visible electrode tracts through the injection site. For
section labeling, see Figure 6A. Scale bar, 1 mm.
(B) Four days of MD at P60 did not affect shScramble-injected Ctrl animals, but MD robus tly decreased the C/I ratio in aKD mice.
(C) Visual responses of animals injected with shScramble Ctrl and aKD mice before MD were almost identical. MD had no effect on Ctrl animals as expected but
significantly depressed closed-eye responses after aKD. Representative VEPs collected from Ctrl and aKD mice are shown on top. Scale bars, 200 mV and 0.2 s.
In (B) and (C), ns, not significant; **p < 0.01, one-way or two-way RM ANOVA. Data are presented as mean ±SEM. n values are indicated in (B).
390 Cell Reports 26, 381–393, January 8, 2019

depression of closed-eye responses, similar to effects of acute
reduction of inhibition in the adult cortex (Figure 7C; Table S6)
(Harauzov et al., 2010). These results demonstrated that even
brief downregulation of SynCAM 1 expression in matured PV
+
in-
terneurons robustly increased plasticity in the adult brain.
DISCUSSION
Despite extensive research, the precise synaptic mechanisms of
cortical critical period closure remain to be defined (Trachten-
berg, 2015). Our study demonstrates a key role for SynCAM
1-dependent recruitment of thalamocortical synaptic inputs
onto PV
+
interneurons in the closure of the critical period for
vision. Our study identifies a synapse type-specific function of
SynCAM 1 that contrasts with synaptic organizers of the
neuroligin family, whose loss affects both intracortical and thala-
mocortical synapses (Singh et al., 2016). Previous studies
demonstrated the requirement for a developmental increase of
PV-mediated cortical inhibition to open the visual critical period
(Fagiolini et al., 2004; Kuhlman et al., 2013), and our study
suggests a key role for TC input-driven maturation of PV
+
inter-
neurons in critical period closure.
Although less numerous than intracortical synapses onto PV
+
cells, TC synapses are much stronger so even a small reduction
in their density can result in circuit disinhibition (Cruikshank et al.,
2007; Ji et al., 2016; Kloc and Maffei, 2014). Both acute and
chronic silencing of SynCAM 1 restore juvenile-like plasticity in
the mature brain, supporting the permissive role of reduced inhi-
bition in adult plasticity (Harauzov et al., 2010; Kuhlman et al.,
2013). The differential baseline activity and plasticity of (contra-
lateral) and open (ipsilateral) eye pathways in global KO and
PV
+
cell-specific KD mouse models likely reflect additional roles
of SynCAM 1 in excitatory neurons and at other synapse types.
Excitatory synapses on pyramidal neurons are important for
open-eye potentiation in young animals (Ranson et al., 2012),
and potential contributions of SynCAM 1 to the plasticity of
closed versus open eye pathways remain to be investigated. A
recent model proposed that the maturation of inhibition during
the critical period selectively decreases spontaneous cortical
activity in favor of visually evoked activity, switching the network
learning cues to external environment (Toyoizumi et al., 2013).
The spontaneous/evoked ratio of SynCAM 1-KO mice is lower
than in WT mice already during the critical period (data not
shown), which in combination with a significantly lowered feed-
forward inhibition may shift cortical responses even further to-
ward the open eye (Bono and Clopath, 2018; Kuhlman et al.,
2013). The model proposed by Toyoizumi et al. (2013) further
predicts that MD induces a shift in ocular dominance during
the precritical period if thalamic afferents from one eye are
mostly blocked. Although this remains to be tested, the robust
shift in C/I ratio SynCAM 1-KO mice after MD during the early
critical period is consistent with this prediction as these mice
show a reduction of TC inputs already at that age.
Our results support that SynCAM 1 acts as a stabilizing factor
for feedforward inputs onto PV
+
interneurons, actively limiting
plasticity in both developing and adult brain. The elevated
expression of SynCAM 1 in adult compared with young postnatal
brain may restrict plasticity by maintaining strong TC inputs onto
PV
+
cells. SynCAM 1 may also have dynamic roles at the syn-
apse, evident in rapid increase of synaptic SynCAM 1 puncta
size after LTD induction (Perez de Arce et al., 2015). In line with
this, SynCAM 1 expression is elevated in input-deprived PV
+
in-
terneurons after MD, which may reflect a response to maintain
homeostasis and limit the extensive remodeling that occurs after
deprivation within a physiological range (Espinosa and Stryker,
2012; Takesian and Hensch, 2013).
Expression of SynCAM 1 is not restricted to thalamorecipient
layers in cortex, indicating that a presynaptic partner may confer
the synapse type-specific roles of SynCAM 1 we report here.
Although our data do not support a physical association be-
tween SynCAM 1 and ECM/PNN components, both homophilic
and heterophilic interactions between SynCAM 1 and other
SynCAM adhesion molecules across the synaptic cleft underlie
its synapse-organizing roles (Fogel et al., 2007; Perez de Arce
et al., 2015). The transsynaptic molecular partners for SynCAM 1
in the TC circuit can now be investigated in future studies.
In conclusion, these results demonstrate specific and non-
redundant roles of synapse-organizing molecules in circuit
development in vivo. Although recent research suggested
thalamic contributions to V1 plasticity (Jaepel et al., 2017; Som-
meijer et al., 2017), our study provides evidence that thalamo-
cortical inputs to PV
+
interneurons are essential for critical period
closure, in agreement with a central role of cortical inhibition in
critical period regulation (Gu et al., 2013; Kuhlman et al., 2013;
Stephany et al., 2016; Sun et al., 2016). Our work reveals that
SynCAM 1 is a PV
+
cell-autonomous brake on cortical plasticity
required for thalamocortical input-driven cortical maturation.
This sheds light on the profound impacts of excitatory-inhibitory
imbalance and regulatory feedback loops that are frequently
implicated in neurodevelopmental disorders (Nelson and Valakh,
2015).
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
dKEY RESOURCES TABLE
dCONTACT FOR REAGENT AND RESOURCE SHARING
dEXPERIMENTAL MODEL AND SUBJECT DETAILS
dMETHOD DETAILS
BAntibodies
BTissue preparation for biochemistry and microscopy
BQuantitative immunoblotting
BCulturing and immunolabeling of primary neurons
BImmunohistochemistry and confocal microscopy
BImage quantification
BBulk anterograde labeling and quantification of eye-
specific segregation
BAAV cloning, packaging, purification and shRNA vali-
dation
BEyelid suture
BIn vivo electrophysiology
BVisual stimuli, data collection and analysis
dQUANTIFICATION AND STATISTICAL ANALYSIS
dDATA AND SOFTWARE AVAILABILITY
Cell Reports 26, 381–393, January 8, 2019 391

SUPPLEMENTAL INFORMATION
Supplemental Information includes six tables and seven figures and can be
found with this article online at https://doi.org/10.1016/j.celrep.2018.12.069.
ACKNOWLEDGMENTS
We thank the members of the Biederer and Crair laboratories for valuable feed-
back, B. Carbone for preparation of neuronal cultures, T. Momoi for providing
the SynCAM 1-KO mouse line, M. Picciotto for providing AAV vectors, L.
Reijmers for sharing the PV-Cre mouse line, A. DiNardo and A. Prochiantz
for providing Otx2 antibody, and the CED support team for programming
advice. This work was supported by NIH grants R01 DA018928 (to T.B.),
R01EY015788 (to M.C.C.), R01EY023105 (to M.C.C.), U01NS094358 (to
M.C.C.), and P30EY026878 (to M.C.C.) and the Knights Templar Eye Founda-
tion (to A.R).
AUTHOR CONTRIBUTIONS
Conceptualization, A.R.; Methodology, A.R.; Software, A.R.; Investigation,
A.R.; Writing – Original Draft, A.R .; Writing – Review & Editing, A.R. and
T.B.; Funding Acquisition, A.R. and T.B.; Resources, M.C.C. and T.B.;
Supervision, T.B.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: January 16, 2018
Revised: November 5, 2018
Accepted: December 17, 2018
Published: January 8, 2019
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Cell Reports 26, 381–393, January 8, 2019 393

STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse anti-Actin, 1:4000 (IB) MP Biomedicals, Solon, OH RRID: AB_2335304
Mouse anti-GABA-A-a1, 1:1000 (IB) NeuroMab, University of California
Davis, CA
RRID: AB_2187693
Mouse anti-GluA1. 1:1000 (IB) NeuroMab, University of California
Davis, CA
RRID: AB_2315840
Mouse anti-NeuN, 1:500 (IHC) EMD Milipore Sigma, Darmstadt,
Germany
RRID: AB_2298772
Mouse anti-Otx2, 1:20 (IHC) Provided by Dr. Ariel DiNardo clone CD4; RRID: AB_2313773
Goat anti-Parvalbumin, 1:500 Swant, Belinzona, Switzerland RRID: AB_2650496
Mouse anti-Parvalbumin, 1:1000 (IHC) EMD Milipore Sigma, Darmstadt,
Germany
RRID: AB_477329
Chicken anti-SynCAM 1, 1:500 (ICC/IHC), 1:2000 (IB) MBL Laboratories, Nagoya, Japan RRID: AB_592783
Chicken anti-SynCAM 1, 1:1000 (IHC, IB) Fogel et al., 2007 RRID: AB_2721136
Rabbit anti-SynCAM 2, 1:1000 (IB) Fogel et al., 2007 RRID: AB_2721137
Rabbit anti-SynCAM 3, 1:1000 (IB) Fogel et al., 2007 RRID: AB_2721138
Rabbit anti-SynCAM 4, 1:1000 (IB) Fogel et al., 2007 RRID: AB_2721139
Mouse anti-VGlut1, 1:200 (IHC) NeuroMab, University of California
Davis, CA
RRID: AB_2187693
Guinea pig anti-VGlut2, 1:500 (IHC) EMD Milipore Sigma, Darmstadt,
Germany
RRID: AB_2665454
Anti-chicken Alexa 488, 1:1000 (IHC) ThermoFisher RRID: AB_2534096
Anti-chicken Alexa 647, 1:1000 (IHC) ThermoFisher RRID: AB_11194678
Anti-guinea pig Alexa 488, 1:1000 (IHC) ThermoFisher RRID: AB_10893081
Anti-guinea pig Alexa 647, 1:1000 (IHC) ThermoFisher RRID: AB_10894751
Anti-goat Alexa 647, 1:1000 (IHC) ThermoFisher RRID: AB_10892959
Anti-goat Rhodamine, 1:1000 (IHC) ThermoFisher RRID: AB_11148892
Anti-IgG1 Alexa 488, 1:1000 (IHC) ThermoFisher RRID: AB_2434013
Anti-IgG Alexa 647, 1:4000 (IB) ThermoFisher RRID: AB_2536165
Anti-IgG1 Alexa 568, 1:1000 (IHC, 1:4000 (IB) ThermoFisher RRID: AB_141611
Anti-IgG2a Alexa 568, 1:4000 (IB) ThermoFisher RRID: AB_2535773
Anti-chicken IRDye800, 1:4000 (IB) Rockland RRID: AB_1660856
Anti-rabbit IRDye800,:4000 (IB) Rockland RRID: AB_1660971
Chemicals, Peptides, and Recombinant Proteins
CTB Alexa 488 Invitrogen, Carlsbad, CA RRID: C-22841
CTB Alexa 555 Invitrogen, Carlsbad, CA RRID: C-22843
DAPI EMD Milipore Sigma, Darmstadt,
Germany
RRID: D9542
DiI EMD Milipore Sigma, Darmstadt,
Germany
RRID: 42364
WFA-Fluorescin Vector Laboratories, Burlingame, CA RRID: AB_2336875
Critical Commercial Assays
BCA protein assay Thermo Scientific RRID: 23225
REDExtract-N-Amp Tissue PCR Kit EMD Milipore Sigma, Darmstadt,
Germany
RRID: XNAT
KAPA Mouse Genotyping Kit KAPA Biosystems, Wilminton, MA RRID: KR0385
(Continued on next page)
e1 Cell Reports 26, 381–393.e1–e6, January 8, 2019

CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Thomas
Biederer (thomas.biederer@tufts.edu).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals. Experiments were performed on C57BL6/J WT mice (The Jackson Laboratory, Bar Harbor, ME), SynCAM 1 KO mice (Fujita
et al., 2006) and their WT littermates, and heterozygous PV-Cre mice (JAX 008069) (Hippenmeyer et al., 2005; Kuhlman et al., 2013).
SynCAM 1 KO and PV-Cre mice were maintained on a C57BL/6N background (Charles River) and KO mice had been backcrossed
more than 10 times. Animals of both sexes from P7 to P70 were used for all experiments as indicated below and stated in the figure
legends. Animals were randomly assigned to experimental groups. Littermates were compared in all experiments and the experi-
menter was blind to the genotype or group of animals used. Animals were kept on a 12/12 hour light/dark cycle with food and water
ad libitum. All experiments were performed during the light phase (7 AM-7 PM). For neuronal cultures, pregnant Sprague-Dawley rat
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Deposited Data
Synapse-selective control of cortical maturation
and plasticity by Parvalbumin-autonomous action
of SynCAM 1
Mendeley Data https://doi.org/10.17632/9wdt9rvhck.2
Experimental Models: Cell Lines
HEK293 ATCC RRID: CVCL_0045
AAV-293 Stratagene RRID: CVCL_6871
Experimental Models: Organisms/Strains
Mouse/C57BL/6 Charles River Laboratories RRID: IMSR_JAX:000664
Mouse/RA175 Dr. Takashi Momoi NA
Mouse/PV-Cre The Jackson Laboratory RRID: IMSR_JAX:008069
Oligonucleotides
17283 AAA TGC TTC TGT CCG TTT GC The Jackson Laboratory PV-Cre
oIMR9377 ATG TTT AGC TGG CCC AAA TG The Jackson Laboratory PV-Cre
oIMR8290 CAG AGC AGG CAT GGT GAC TA The Jackson Laboratory PV-Cre
oIMR8291 AGT ACC AAG CAG GCA GGA GA The Jackson Laboratory PV-Cre
AG0002 GAG TGA TTA ACA ACG TGC AGG CAA This study Ra175/SynCAM 1 KO
AG0003 ACC TGC AGG CAT GCA AGC TTG TAC This study Ra175/SynCAM 1 KO
AG0006 GAT GTG TGC TGA CTT AGG AAC GGT C This study Ra175/SynCAM 1 KO
SynCAM 1 TCC TGT TCA TCA ATA ACC TAA ACT
TCA AGA GAG TTT AGG TTA TTG ATG AAC AGG
TTT TTT C
This study; based on Faraji et al., 2012 shSynCAM 1
Scramble TAC ACC AAT CGC AAT ATT ACT TCT
TCA AGA GAG AAG TAA TAT TGC GAT TGG TGT
TTT TTT C
This study shScramble
Recombinant DNA
pCALNL-DsRed Addgene 13769
pSico Addgene 11578
AAV-GFP/Cre Addgene 49056
pAAV-CaMKII-EGFP Addgene 50469
AAV-dsRed-Sico Dr. Marina Picciotto N/A
Software and Algorithms
Mean Variance Analysis (Torborg and Feller, 2004) Dr. Michael C. Crair N/A
Spike2 Cambridge Electronics Design RRID: SCR_000903
Psychtoolbox-3 Brainard, 1997 RRID: SCR_002881
ImageJ NIH RRID: SCR_003070
Pipsqueak (ImageJ) Slaker et al., 2016 http://sites.imagej.net/PIPSQUEAK/
Cell Reports 26, 381–393.e1–e6, January 8, 2019 e2

dams were purchased from Charles River Laboratories (Wilmington, MA). Animals were treated in accordance with Institutional
Animal Care and Use Committee guidelines.
METHOD DETAILS
Antibodies
Primary antibodies and their properties are listed in Key Resources Table. For all immunostainings, secondary antibodies were
applied in the absence of primary antibodies as a control. Secondary antibodies and reagents are listed in Key Resources Table.
Tissue preparation for biochemistry and microscopy
Animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) in saline. For protein isolation (animals aged P7-P45
for Figure 1A and P28 for Figures 1F and 1G), visual cortices were isolated according to stereotactic coordinates (0.5-1 mm anterior to
l, 2-3 mm lateral to midline) followed by sonication in 8 M urea. For LGN isolation, forebrain was flash frozen over liquid nitrogen and
later dissected on an iced platform. LGN was visually identified and isolated with a tapered scalpel blade. For GABA and glutamate
receptor immunoblots, crude synaptoneurosomes were prepared as described (Villasana et al., 2006). Protein concentrations were
determined using the BCA method (Thermo-Fisher Scientific, Holtsville, NY). For microscopy, animals (P7-P70, as indicated in figure
legends) were transcardially perfused first with ice cold PBS and then with 4% PFA (in PBS, pH 7.4). Brains were isolated and
postfixed overnight in 4% PFA and washed overnight in PBS (all at 4C). Brains were then embedded in 3% agarose in PBS
and sectioned at 40-60 mm using vibrating microtome (Leica VT1000, Leica Biosystems, Nussloch, Germany or Vibratome 1500,
Harvard Apparatus, Holliston, MA). Sections were stored in PBS with 0.01% sodium azide (Sigma-Aldrich, St. Louis, MO) at 4C.
Quantitative immunoblotting
Proteins from cortical homogenates or crude synaptosomes (10-30 mg for V1 and 60 mg for LGN, prepared as described above) were
subjected to immunoblotting using standard procedures (Fogel et al., 2007) and scanned with either Odyssey Infrared Imaging
System (LI-COR Biosciences, Lincoln, NE) or FluorChem M (Protein Simple, San Jose, CA). Antibodies used are listed in Key
Resource Table. YUC8 and 3E1 provided almost identical signal in blots, except that SynCAM 1 signal in the LGN was better visible
with YUC8, likely due to its higher affinity for different glycosylation states of SynCAM 1 (Fogel et al., 2007). For all blots imaged using
FluorChem M, milk was replaced with BSA (Sigma) for blocking and probing. For quantitative immunoblotting in Figure 1, secondary
IRDye800 antibodies or anti-IgG Alexa 647 were used. Quantification was performed using the gel analysis plugin in ImageJ, where
actin served as loading control for all samples. Quantification was always performed blind to the experimental group.
Culturing and immunolabeling of primary neurons
Cortical neurons were prepared from rats at E18 as described (Biederer and Scheiffele, 2007) with modifications. In brief, dissected
cortices were incubated in 0.05% trypsin at 37C for 20 minutes (Invitrogen, Carlsbad, CA; 25300054) and plated at a density of
30,000 cells per coverslip. Dissociated cells were plated on poly-l-lysine (Sigma P1274) and incubated in a cell culture incubator
with 5.0% CO
2
. Cytosine arabinoside (Sigma C1768) was added at a final concentration of 2 mM per well 2 days in vitro to
prevent glia cell overgrowth. Cells were washed with ice-cold PBS and fixed at DIV 7 and DIV 14 in ice-cold 4% PFA/4% sucrose
for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at RT and blocked in 5% FBS in PBS for 1 hour at
RT. Coverslips were later sequentially incubated for 1 hour at RT in anti-SynCAM 1, anti-Parvalbumin and WFA (see Key Resource
Table for more details) and their corresponding secondary antibodies. All antibodies were diluted in PBS and coverslips were
washed 3x10 minutes in PBS at RT in between all antibody incubations. Coverslips were mounted with Aqua-Mount (Polysciences
Inc., Warrington, PA) and imaged as described below.
Immunohistochemistry and confocal microscopy
Primary antibodies used in double- and triple-labeling experiments were applied sequentially and blocking steps were performed
using normal horse serum. Visual cortex sections were first washed in PBS and non-specific antibody binding sites were blocked
with 3% normal serum and 0.03% Triton X-100 (Sigma) in PBS for 1 h at RT. Primary and secondary antibodies were diluted in
3% normal serum and 0.03% Triton X-100 in PBS and incubated either for 24-48 hours at 4C (primary antibodies) or 1 hour at
room temperature (secondary antibodies). After the antibody incubation steps, sections were washed in PBS and floated on slides
in distilled water before coverslipping with mounting medium (Aqua-Polymount, Polysciences Inc., Warrington, PA, USA). Confocal
microscopy was performed on a Leica TCS SPE DM2500 microscope or a Leica TCS SP8. Images were acquired with HC PL Fluotar
10x0.30 for Figure 1B, ACS AP 40x oil lens with 1.15 NA for Figures 3,4G, S2 and S3 or ACS APO 63x oil lens with 1.3 NA for Figures 1,
4,6and Figure S1 using identical settings for each group within an experiment. For quantitative immunohistochemistry of
synaptic markers, only single optical sections were acquired. For quantification of SynCAM 1 expression during V1 development
(Figure 1B), high resolution images were taken at 5 mm intervals through the entire section. For imaging of thalamocortical axons,
bV1 area encompassing Layers II/III-V was imaged using 0.5 mm steps through the entire section. All images were acquired in binoc-
ular V1, layer IV. Low magnification images were acquired with Zeiss Axio Scope (Carl Zeiss, Jena, Germany) or BZ-X700 (Keyence,
Osaka, Japan).
e3 Cell Reports 26, 381–393.e1–e6, January 8, 2019

Image quantification
For all quantifications, only single optical sections were used, except for Figure 1B, where maximum intensity projection images were
used. For Figure 1, integrated density of SynCAM 1 signal was measured throughout the cortex using ImageJ (NIH) and later normal-
ized to NeuN integrated density to correct for differences in tissue thickness. For Figure S3, Otx
+
, WFA
+
and PV
+
cells were counted
manually using ImageJ (NIH, Bethesda, MD). Quantification of WFA
+
puncta density was performed semi-automatically using
Pipsqueak plugin for ImageJ on single optical sections (Slaker et al., 2016). Quantification of vGlut1, vGlut2 and SynCAM 1 puncta
was performed as previously described (Park et al., 2016) and as outlined in Figures S1,S4 and S5. Briefly, contours of primary and
secondary PV
+
or primary NeuN+/PV-dendrites in layer IV were manually outlined and served as ROI. vGlut1, vGlut2 and SynCAM 1
images were thresholded, binarized and the density of puncta in contact with PV
+
dendrites (within the ROI) was counted using par-
ticle analyzer tool with a vGlut1 and SynCAM 1 cutoff of 0.1 mm
2
and vGlut2 cutoff of 0.2 mm
2
, as outlined in Figures S1,S4 and S5.
On average, 10-20 dendritic segments were collected from each animal from 3-6 brain sections. For SynCAM 1 KD validation in vivo,
ROI was defined as PV
+
cell body and 20 cells on average were analyzed per animal for both SynCAM 1 KD/PV-Cre
AAV-shSynCAM 1
and
Control/PV-Cre
AAV-shScramble
.
For tracing of single thalamocortical axons, Simple Neurite Tracer plugin for ImageJ was used (Longair et al., 2011). Briefly, axons
entering layer IV were followed from their starting point to their end point by scrolling through the entire Z- stack of images. Axons with
no clear point of origin or end and axons ending abruptly were not included in analyses. On average, 10 axons per animal were
reconstructed.
Image collection and analyses were performed blind to the genotype or experimental group, where samples were usually coded by
the animal number from the animal census. All values were checked for normal distributions and averaged per animal before final
statistical analysis, unless indicated otherwise.
Bulk anterograde labeling and quantification of eye-specific segregation
Retinal ganglion cell projections from the right and the left eye were bulk labeled with CTB Alexa 488 and CTB-Alexa 555. The tracer
was diluted to 1 mg/ml in 0.9% saline. At P12/13, mice were anesthetized and injected with 1–2 mL tracer per eye using a glass pulled
pipette and Nanoject (Drummond Scientific, Broomall, PA). 48 hours later mice were transcardially perfused and the brains were fixed
overnight in 4% PFA. Coronal sections (80 mm thickness) were collected with a vibratome as described above, mounted in Aqua-
mount and imaged with a CCD camera (Zeiss). Analysis of segregation of contralateral and ipsilateral projections in dLGN was per-
formed as previously described (Torborg and Feller, 2004). Briefly, images were background subtracted with a rolling ball radius of
200 in ImageJ, and the three sections with the largest ipsilateral (Alexa 555 labeled) area were used for analysis. The logarithm of the
intensity ratio, R = log
10
(ipsilateral channel fluorescence intensity/contralateral channel fluorescence intensity), was determined for
each pixel, and a segregation index for each animal was computed as the mean of the variance of the distribution of R values. A larger
segregation index (higher variance) is indicative of better segregation (Torborg and Feller, 2004).
AAV cloning, packaging, purification and shRNA validation
For SynCAM 1 KD in vivo, sequence shSynCAM 1 (Faraji et al., 2012) was cloned into pAAV-dsRed-Sico-shRNA (Wohleb et al., 2016)
(kindly provided by Dr. Marina Picciotto, Yale University). 70% confluent AAV-HEK293 cells (Agilent, Santa Clara, CA) were trans-
fected with pHelper, AAV/DJ Rep-Cap and pAAV-dsRed-Sico-shSynCAM 1 or pAAV-dsRed-Sico-shScramble using PEI method
(Sonawane et al., 2003). Cells were collected after 72 hours and AAV was purified using iodixanol gradient (Hermens et al., 1999).
AAV was further concentrated using Amicon 15 (EMD Milipore Sigma). Titer was determined as in (McClure et al., 2011). 600 nL
of virus (3x10
12
GC/ml) was injected at 1 nl/s into layer 4 of bV1 (350 mm depth) at P14 or at P45 using stereotaxic apparatus
(Stoelting, Wood Dale, IL) and glass pipette attached to Hamilton syringe (Hamilton Robotics, Reno NV) using micro-syringe
pump (Micro4, WPI, Sarasota, FL) at following coordinates: 0-1 mm anterior to l, 2.5-3 mm lateral to midline. For targeting validation
in vitro, shSynCAM 1 sequence was cloned into pSico (Ventura et al., 2004). Confluent HEK293 cells were transfected with pCAGGS-
SynCAM 1 (Stagi et al., 2010), pSico-SynCAM 1 and pAAV-GFP-Cre (kindly provided by Dr. Dong Kong, Tufts University) using PEI
transfection. Cells were collected 72 hours later and lysed in RIPA buffer. 30 mL of protein homogenate was immunoblotted for
SynCAM 1 and quantified as described above. For targeting validation in cultured neurons, primary cortical cultures were transfected
at DIV 5 as above using Lipofectamine (Invitrogen) and fixed at DIV 14. SynCAM 1 signal was quantified as described above. For
targeting validation in vivo, animals injected with pAAV-dsRed-Sico-shSynCAM 1 or pAAV-dsRed-Sico-shScramble were perfused
as described above. SynCAM 1 immunohistochemistry was described as above and puncta were quantified using PV
+
soma signal
as ROI (as described above) with the experimenter blind to the experimental group.
For anterograde tracing of thalamocortical projections, 500 nL of pAAV-CaMKII-EGFP (purified as described above) was injected
into both left and right dLGNs (2.10 mm posterior to Bregma, 2.19 mm lateral to midline and 2.8 mm deep) of 6-8 weeks old WT and
SynCAM 1 KO mice, using Hamilton Neuros syringe (tapered, 33 G; Hamilton, Reno, NV). Animals were perfused with ice cold PBS
followed by 4% PFA as described above and brains were sliced using a vibrating microtome at 80 mm thickness.
Eyelid suture
Mice were anesthetized with isoflurane in oxygen (2% induction, 1.0%–1.8% maintenance) and placed under a surgical microscope.
Area around the right eye was sterilized with alcohol swabs and lid margins were trimmed. Three mattress stitches were placed using
Cell Reports 26, 381–393.e1–e6, January 8, 2019 e4

7-0 nylon sutures and the lids were further attached using VetBond (3M). After that, ophthalmic antibiotic ointment was applied tothe
suture. Mice were monitored daily for the integrity of the sutures and signs of infection. Animals whose eyelids were not fully sutured
and animals that removed their sutures were excluded from further experiments. At the end of the deprivation period, after the head-
plate implantation (see below), the stitches were removed, and lid margins separated. Eyes were flushed with sterile saline and
checked for clarity. Mice with corneal opacities, cataracts or signs of infection were excluded from further study.
In vivo electrophysiology
Recordings were performed on awake female and male mice, ages P21 to P64, using spherical treadmill as described in (Niell and
Stryker, 2010). 4-7 days before the recording session (15 days for Figure 7), custom made titanium or aluminum (for precritical period
mice) head-plate implants were cemented to the mouse skull. Animals were anesthetized with isoflurane in oxygen (2% induction,
1.0%–1.8% maintenance), warmed with a heating pad at 38C and given subcutaneous injections of Buprenorphine SR (1 mg/kg)
and 0.25% Bupivacaine (locally). Eyes were covered with Puralube (Decra, Northwich, UK). Scalp and fascia from Bregma to behind
lambda were removed, and the skull was cleaned, dried and covered with a thin layer of cyanoacrylate (VetBond; 3M, Maplewood,
MN) before attaching the head plate with dental cement (RelyX, 3M). The well of the head plate was filled with silicone elastomer
(Reynold Advanced Materials, Brighton, MA) to protect the skull before recordings. Animals were single housed after the implantation
and monitored daily for signs of shock or infection. 1-2 days before the recording, the animals underwent 1-2 20-30 minutes handling
sessions and 1-2 10-20 minutes session in which the animals were habituated to the spherical treadmill (Dombeck et al., 2007). On
the day of recording, the animals were anesthetized as above and small craniotomies (0.5 mm in diameter) with 18G needles were
made above bV1 and cerebellum. The brain surface was covered in 2%–3% low melting point agarose (Promega, Madison, WI) in
sterile saline and then capped with silicone elastomer. Animals were allowed to recover for 2–4 h. For the recording sessions, mice
were placed in the head-plate holder above the free-floating ball and allowed to habituate for 5-10 minutes. The agarose and silicone
plug were removed, the well was covered with warm sterile saline and the reference insulated silver wire electrode (A-M Systems,
Carlsborg, WA) was placed in cerebellum. A multisite electrode spanning all cortical layers (A1x16-5mm-50-177-A16; Neuronexus
Technologies, Ann Arbor, MI) was coated with DiI (Invitrogen) to allow post hoc insertion site verification and then inserted in the brain
through the craniotomy. The electrode was lowered until the uppermost recording site had entered the brain and allowed to settle for
20-30 minutes, after which the ipsilateral eye response was checked to confirm the proper location in V1. The well with the electrode
was then filled with 3% agarose to stabilize the electrode and the whole region was kept moist with surgical gelfoam soaked in sterile
saline (Pfizer, MA). Minimum 2 penetrations were made per animal to ensure proper sampling of the craniotomy. For Figure 7, only
one penetration per recording was made, at identical sites in the center of cranitomy (location was measured by micromanipulator
using edges of the craniotomy as a reference point). Recordings sessions typically lasted 2-3 h. After the recording, mice were eutha-
nized with an overdose of ketamine and xylazine. The brains were then isolated and fixed in 4% PFA overnight at 4C. Brains were
subsequently sectioned at 100 mm using a vibrating microtome. The sections were incubated in DAPI (Sigma), floated on slides and
mounted in Aquamount. The sections were imaged on a Keyence microscope as described above to confirm the electrode location
within bV1.
Visual stimuli, data collection and analysis
Visual stimuli were generated with MATLAB (MathWorks, Natick, MA) using the Psychtoolbox extension (Brainard, 1997) and Spike2
(CED, Cambridge, UK). Varying frequencies and orientations of full-field sinusoidal gratings at 100% contrast were displayed on a
gamma corrected 17’’ LCD (Niell and Stryker, 2008) for 1.5 s with 0.2 s interstimulus interval (gray screen). Stimuli were presented
in randomized fashion and each stimulus was presented 30-50 times on average during a recording session. The screen was
centered 20 cm from the mouse’s eye, covering 80of visual space. 30-50 light-emitting diode (LED) flashes were presented before
or after the sinusoidal gratings with 10 s interstimulus interval. Non-stimulated eye was covered with custom-made blocker. Visual
response signals were preamplified 10x (MPA8I preamplifiers; Multi Channel Systems MCS GmbH, Reutlingen, Germany) and then
fed into a 16-channel amplifier (Model 3500; A-M Systems), amplified 200x and band-pass filtered 0.3-5000 Hz. The signals were
sampled at 25 kHz using Spike2 and data acquisition unit (Power 1401-3, CED). Stationary and movement stages of animal behavior
were separated using an optical mouse that tracked the movement of the styrofoam ball and was interfaced with LabView (Austin, TX)
and Spike2, or using video recordings timed to stimuli presentation (WansView, Shenzhen, PRC). Only stationary, non-running stages
were analyzed offline using Spike2 software (CED). LFPs were analyzed as waveform averages, triggered by stimulus onset. Visually
evoked potentials (VEPs) were defined as negative-going events occurring within 200 ms following stimulus onset, having an ampli-
tude of more than 3x standard deviation and having a width at half maximum of less than 50 ms (Li et al., 2013). For estimation of C/I
ratio, VEP amplitude evoked by sinusoidal gratings at 0.15 cycles per degree (cpd) was combined with amplitudes evoked by LED as
they were identical in nature. For acuity analysis, responses from 4 different frequencies from all orientations ranging from 0.05-0.6
cpd were plotted on a logarithmic scale and acuity was estimated from linear regression as the frequency where amplitude equals 0
(Porciatti et al., 1999). For multi-unit analysis, spikes were extracted from band-pass filtered data using thresholds (3x standard
deviation) and sorted in Spike2. Peri-stimulus time histogram (PSTH) analysis was performed with Spike2 using 0.01 s bins.
Spontaneous firing was calculated as average firing rate before stimulus presentation with 0.2 s offset. Spontaneous firing was
subtracted from peak poststimulus firing rate to determine evoked firing rate. For analysis of single units, spikes were isolated using
template matching and principal component analysis in Spike2. PSTHs were calculated using 0.001 s bins with 0.2 s offset, using
e5 Cell Reports 26, 381–393.e1–e6, January 8, 2019

0-180(90to 90) orientations in 30increments at 0.15-0.6 cpd as stimulus. Responses were plotted over all orientations and
preferred orientation was determined ad the orientation where maximum spiking occurred after fitting a Gaussian curve. Orientation
selectivity index (OSI) was calculated as the ratio of (Rpref – Rorth)/(Rpref + Rorth), where Rpref is the firing rate at preferred
orientation and Rorth at orientation orthogonal to preferred. Cells with OSI > 0.3 were included in the analysis. Orientation difference
(DO) of single units was calculated as the difference of preferred orientations between responses to contra and ipsi eye stimulation
(Wang et al., 2010). Spectral analysis was performed on raw LFP traces as previously described (Mohns and Blumberg, 2008), with
gamma-band oscillations defined as 40-70 Hz, as described in (Chen et al., 2015). Data collection and analyses were performed blind
to genotype or experimental group.
QUANTIFICATION AND STATISTICAL ANALYSIS
All quantitated analyses were performed with the researcher blind to the condition, as stated above. Statistical analyses were per-
formed in SigmaPlot 11 and 13 (San Jose, CA) or GraphPad Prism 7.0 (GraphPad Inc., La Jolla, USA) using t test and one or two-way
repeated-measures or regular ANOVA with post hoc comparisons (as indicated in text, figure legends and Supplementary Tables),
unless stated otherwise. When comparing two independent groups, normally distributed data were analyzed using a Student’s t test.
In the case data were not normally distributed a Mann-Whitney rank sum test was used. All data are reported as mean ±SEM, where
N represents number of animals used, unless indicated otherwise. Target power for all sample sizes was 0.8. In all cases, alpha was
set to 0.05.
DATA AND SOFTWARE AVAILABILITY
Mendeley Data repository containing all original representative images can be found under the following link: https://doi.org/10.
17632/9wdt9rvhck.2.
Cell Reports 26, 381–393.e1–e6, January 8, 2019 e6