Experience-dependent regulation of CaMKII activity
within single visual cortex synapses in vivo
Amanda F. Mowera,1, Showming Kwoka,b,1, Hongbo Yua,2, Ania K. Majewskaa,3, Ken-Ichi Okamotoa,b,4,
Yasunori Hayashia,b,c,5, and Mriganka Sura,5
aThe Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
bRIKEN–MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, MA 02139; andcBrain Science Institute, RIKEN, Wako, Saitama
Edited* by Jon H. Kaas, Vanderbilt University, Nashville, TN, and approved November 10, 2011 (received for review May 23, 2011)
Unbalanced visual input during development induces persistent
alterations in the function and structure of visual cortical neurons.
The molecular mechanisms that drive activity-dependent changes
await direct visualization of underlying signals at individual
synapses in vivo. By using a genetically engineered Förster reso-
nance energy transfer (FRET) probe for the detection of CaMKII
activity, and two-photon imaging of single synapses within identi-
fied functional domains, we have revealed unexpected and differ-
ential mechanisms in specific subsets of synapses in vivo. Brief
monocular deprivation leads to activation of CaMKII in most syn-
apses of layer 2/3 pyramidal cells within deprived eye domains,
despite reduced visual drive, but not in nondeprived eye domains.
Synapses that are eliminated in deprived eye domains have low
basal CaMKII activity, implying a protective role for activated CaM-
KII against synapse elimination.
cortical circuits|ocular dominance plasticity|spines|excitatory synaptic
transmission|serine/threonine protein kinase
responses in the visual cortex can occur after brief periods of
monocular deprivation (MD) (1–3). These alterations are me-
diated by sequential mechanisms that transduce changes in the
amount and pattern of visual activity from the two eyes to
changes in synaptic drive (4–6). Thus, the initial loss of responses
from the closed eye is known to be rapid, on the order of hours,
as detailed elsewhere in PNAS (7), and mediated by mechanisms
that implement synaptic depression. The gain of responses from
the open eye is thought to be slower, on the order of days, and
mediated by homeostatic scaling of responses as well as homo-
synaptic long-term potentiation (LTP)-like mechanisms (8–15),
although rapid gain of responses on the order of hours also
occurs (7). However, the structural and molecular basis for these
changes at the level of single synapses are not fully understood
(16–20). Two important reasons are that the precise location and
distribution of synapses undergoing changes in the intact brain,
and the specific molecular transformations that occur at these
synapses during experience-dependent plasticity, are unknown.
Excitatory neurons in layer 2/3 receive synaptic inputs from
multiple sources, including feedforward, local, and long-range
intracortical axons (Fig. 1A), and exhibit rapid functional
changes following even brief MD (1–3). MD is known to reduce
the effectiveness of deprived eye-driven synapses as well as
rapidly eliminate synapses (16, 18). However, significant pro-
portions of synapses within deprived eye domains are also pre-
served after MD, and these synapses, depending on their origin,
serve multiple functions: potentiating open eye responses (8, 12),
acting as a scaffold for experience-dependent traces and accel-
erated shifts in response to MD, or enabling recovery of drive
when the deprived eye is reopened (7, 12). Thus, understanding
why some synapses are lost while others are preserved after MD,
and the mechanistic differences between these sets of synapses,
remains an important issue to be resolved.
uring a developmental critical period, alteration of neuronal
To address these questions, we developed a method to visu-
alize the effects of MD within identified synapses in vivo based
on activation of CaMKII, a protein kinase that is necessary and
sufficient for the induction and maintenance of synaptic plasticity
(21–23). By expressing a FRET-based optical probe for CaMKII
in neurons of the primary visual cortex (V1) in ferrets, and im-
aging individual spines within deprived or nondeprived eye zones
in vivo before and after a period of MD, we demonstrate distinct
differences between synapses that are lost and those that are
preserved. Spines that are lost after MD have low basal levels of
CaMKII, whereas spines that are preserved show increased ac-
tivation of CaMKII following MD. These data indicate that
CaMKII activation after MD could constitute a major difference
between lost vs. preserved synapses, and suggest that rapid
CaMKII activation represents a potential mechanism for synapse
preservation following reduction of input drive.
A FRET-based optical probe (24) known as Camui (Fig. 1B), in
combination with in vivo two-photon laser scanning microscopy
and intrinsic signal optical imaging in ferret visual cortex (Fig. 1C
and Fig. S1E), enabled us to detect changes in CaMKII activity
within individual spines in functionally identified regions of
cortex before and after manipulation of visual drive. The emis-
sion fluorescence spectrum of unstimulated Camui expressed in
HEK293 cells showed FRET, as evidenced by the presence of a
YFP emission peak under CFP specific excitation (Fig. S1A).
Upon Ca2+stimulation, the YFP peak decreased whereas the
CFP peak was dequenched. Chelating Ca2+only partially reversed
this change. The remaining change was attributable to autophos-
phorylation at threonine (T) 286 (24). Camui with a mutation of
T305 and T306 to aspartate (D) is deficient in Ca2+/calmodulin
binding (25, 26), and, as expected, showed no response to Ca2+
stimulation (Fig. S1 B and D). Therefore, the change in FRET, or
change in CFP/YFP ratio, is a reliable indicator of CaMKII acti-
vation (Fig. S1 A–D).
Author contributions: A.F.M., S.K., Y.H., and M.S. designed research; A.F.M., S.K., H.Y., and
A.K.M. performed research; K.-I.O. contributed new reagents/analytic tools; A.F.M., S.K.,
H.Y., A.K.M., and Y.H. analyzed data; and A.F.M., S.K., Y.H., and M.S. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1A.F.M. and S.K. contributed equally to this work.
2Present address: Center for Brain Science Research and School of Life Sciences, Fudan
University, Shanghai 200433, China.
3Present address: Department of Neurobiology and Anatomy, University of Rochester,
4Present address: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
ON, Canada M5G 1X5.
5To whom correspondence may be addressed. E-mail: email@example.com or msur@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 27, 2011
| vol. 108
| no. 52
To monitor the activity of CaMKII associated with visual
cortical plasticity, we expressed Camui in V1 neurons in ferrets
by using an HSV vector during the critical period, and repeatedly
imaged the neurons in vivo through a cranial window before and
after 4 h of MD, a manipulation known to cause functional
changes in neurons of ferret V1 (7). To precisely align imaged
spines with functional domains driven by the ipsilateral or con-
tralateral eye, the fluorescence image of spines and dendrites
obtained by two-photon imaging was matched to the ocular
dominance (OD) map obtained by intrinsic signal optical imag-
ing using the common blood vessel patterns obtained with both
techniques (Fig. 1C and Fig. S1E). To examine overall trends,
we first pooled data from spines that lay within deprived [OD
index (ODI) < −0.1], binocular (−0.1 ≤ ODI ≤ −0.1), or non-
deprived (ODI > 0.1) eye domains. CaMKII activity showed a
significant increase in both spines (Fig. 2A) and adjacent den-
dritic regions (Fig. S2A) in deprived eye domains after 4 h MD
[Kolmogorov–Smirnov (KS) test, P < 0.05 for spines and den-
drites]. CaMKII activity did not increase in the binocular domain
in dendritic spines, although there was a slight tendency (KS
test, P = 0.096; Fig. 2B). Within the nondeprived eye domain,
CaMKII activity did not show a significant change in spines or
dendritic regions (KS test, P = 0.443 for spines and P = 0.916 for
dendritic regions; Fig. 2C and Fig. S2C).
An increase in CaMKII activation in spines within a region
with reduced afferent drive was unexpected from in vitro studies,
in which increased CaMKII is correlated with the induction of
LTP and its maintenance via insertion of AMPA receptors at
potentiated synapses (21, 27, 28). In light of potentially con-
founding variables such as changes in cerebral blood flow, level of
blood oxygenation, and the differential cortical scattering of
fluorescence, we repeated the same experiments using two dif-
ferent negative control probes that would not be expected to
show a CaMKII-dependent FRET change: Camui-T305D/T306D
mutant (Fig. S1B) and a CFP-YFP (C-Y) direct fusion protein
(Fig. S1C). Neither probe produced a FRET change following
MD (T305D/T306D mutant, KS test, P = 0.658 for spines and
P = 0.953 for dendritic regions; fusion, KS test, P = 0.811 for
spines and P = 0.936 for dendritic regions; Fig. 2D and Fig. S2D).
Pooling all spines from each site as comprising a single ob-
servation, we found a significant inverse correlation between the
ODI and normalized change in CFP/YFP ratio (Pearson corre-
lation coefficient R = −0.65; P < 0.05, paired t test; Fig. 2E). In
the deprived eye domains, there was an increase in synaptic
CaMKII activity, whereas in the nondeprived eye domains, there
was little or no change. Dendritic regions followed the same
tendency (Fig. S2E). Negative control constructs fell outside of
this correlation, showing almost no change in CFP/YFP ratio,
confirming that the observed changes in FRET are specific to
activation of CaMKII following 4 h MD.
Next, we examined whether the activation of CaMKII ob-
served in the deprived eye domains was related to the pre-
deprivation state of the synapse. Each spine in the deprived eye
domain was rank-ordered according to its basal CFP/YFP ratio,
from highest to lowest, and plotted together with its ratio after
4 h MD (Fig. 3A). Basal and 4 h MD CaMKII activity data were
binned in 20% increments and compared with each other at each
level. Basal CaMKII activity varied considerably; however, there
was a clear tendency for spines that had high basal CaMKII
activity to show a decrease in CaMKII activity following 4 h MD
(P < 0.05, paired two-tailed t test; Fig. 3B, first bin). In contrast,
spines with moderate to low basal CaMKII activity showed an
increase (P < 0.05, P < 0.05, and P < 0.05; Fig. 3B, third to fifth
bins). Analysis of dendritic regions adjacent to these spines
showed similar results (Fig. S3). In the binocular domain, al-
though we did not see a statistically significant change in aver-
aged CaMKII activity (Fig. 2 B and D), we observed a similar
tendency at individual spine level, namely spines with high
Deprived eye domain
domain receiving inputs from multiple sources (red). Thick red triangles represent feedforward synapses. Other synapses derive from local and longer-range
axons, including inputs from the nondeprived eye. (B) Top: Schematic drawing of the CaMKII protein. Bottom: Conformations of Camui in the inactive and
active form. (C) Alignment of two-photon microscopic images with OD map. Blood vessel and OD maps were obtained using intrinsic signal optical imaging
(Upper: A, anterior; M, medial). Gray scale indicates ODI (white, ipsilateral eye dominated; black, contralateral eye dominated). Blood vessels in low-mag-
nification optical and two-photon microscopic images were used to align two-photon images (Lower) to OD domains. A dendritic segment (red box) is
magnified (Right) and displayed as channel separated images (CFP and YFP) as well as a ratiometric image in intensity-modulated display mode, indicating the
CFP/YFP ratio. Warm hue represents high CaMKII activity.
Imaging of CaMKII activity in identified OD domains. (A) Schematic diagram of a sample pyramidal neuron (blue) in layer II/III within the deprived eye
| www.pnas.org/cgi/doi/10.1073/pnas.1108261109Mower et al.
CaMKII activity tended to decrease after MD whereas those
with lower CaMKII activity increased (Fig. 3 C and D). In con-
trast, in the open eye domain where visual input remains un-
changed, this tendency was observed in only the last bin (Fig. 3 E
and F). Thus, the basal level of CaMKII acted as an indicator of
the direction of change after MD in domains that had deprived
visual input, with most spines showing an increase in activity and
a few showing a decrease.
A key correlate of MD is spine elimination: the majority of
spines in deprived eye zones persisted after short periods of MD,
whereas a subset of spines was eliminated (16, 18). We wondered
if there was a common feature among lost spines. We found that
a small but reliable fraction of spines (seven of 116 spines) within
four deprived eye sites were eliminated following 4 h MD (Fig.
4A). This loss after only 4 h of MD is consistent with the spine
loss described in the companion study in PNAS (ref. 7, see also
Discussion). Interestingly, the basal CaMKII activity of these
eliminated spines was lower than the average CaMKII activity of
other spines in the same imaged cortical site (P < 0.05, paired
two-tailed t test; Fig. 4B). In contrast, spines within these same
cortical sites that were not eliminated had CaMKII activity that
remained high or increased after 4 h MD. Furthermore, den-
dritic regions adjacent to eliminated spines, and spines that
persisted after 4 h MD, had basal CaMKII activity levels com-
parable to the average activity of adjacent dendritic regions (P >
0.05, paired two-tailed t test; Fig. 4C). Only one spine in the
binocular domain, and two in the open eye domain, were de-
termined to be eliminated at 4 h MD. This observation aligns
with the findings of the companion paper (7) in which spine loss
was significantly lower within the binocular domain compared
with the closed eye domain (figure 6c of ref. 7). Combined,
synapses with low CaMKII activity follow one of two fates:
a majority of spines gain CaMKII activity after MD and persist,
whereas a minority are eliminated. The eliminated spines con-
sistently have lower CaMKII activity than the average, but not all
spines with low CaMKII activity are removed.
We found no correlation between spine size and basal CaM-
KII activity (R = 0.11; P > 0.05, two-tailed t test; Fig. 5A). There
was also no correlation between the initial size of a spine and the
change in CFP/YFP ratio that resulted after 4 h MD (R = 0.08;
P > 0.05, two-tailed t test; Fig. 5B), indicating that spine size
was not a determinant of basal CaMKII activity levels or of the
change in CFP/YFP ratio after MD. Additionally, changes in
CaMKII activity were not correlated with relative cortical depth
of spines or dendritic regions (Fig. S4).
We have overcome the technical challenges of performing
chronic in vivo two-photon microscopy of a FRET probe in
single synapses in ferret V1. In contrast to in vivo imaging of
a single fluorophore, ratiometric imaging of our two-fluorophore
FRET probe has allowed us to visualize changes in the activity of
a molecular correlate of synaptic plasticity in vivo during short-
term changes in visual drive. Alignment of these ratiometric
images with identified functional domains in the visual cortex
further allowed us to define the location of these single synapses
to deprived, binocular, and nondeprived eye regions. These
technical advances open the door for detailed analysis of mole-
cular correlates and signaling pathways of plasticity processes
We observed eye-domain specific CaMKII activity changes,
specifically an overall increase in CaMKII activity in deprived
but not nondeprived eye domains. This was the result of an in-
crease in CaMKII activity in the majority of spines with mod-
erate to low levels of basal CaMKII activity and a reduction in
a few spines with high basal CaMKII activity. In addition, spines
that were eliminated constituted a subset of spines with low basal
CaMKII activity. The observation that not all spines with low
basal CaMKII activity are eliminated indicates that (i) low basal
CaMKII activity is a prerequisite for elimination but is not suf-
ficient, and (ii) there might be additional mechanisms that also
participate in the regulation of this important step of spine re-
moval. These changes in CaMKII activation in deprived zones
are consistent with the physiological consequences of MD and
indeed predict their structural basis. The initial rapid reduction
of drive from the deprived eye after MD is accompanied by
a rapid but small increase of drive from the nondeprived eye (7),
followed by a slower increase of functional drive and anatomical
% change in CFP/YFP
contra OD index ipsi
Normalized CFP/YFP ratio
Deprived eye domain
4 h MD
Normalized CFP/YFP ratio
4 h MD
Normalized CFP/YFP ratio
Normalized CFP/YFP ratio
Deprived eye domainBinocular domain
Non-deprived eye domain
domains (ODI < −0.1, n = 201 spines from seven imaging sites), (B) binocular domains (−0.1 ≤ ODI ≤ 0.1, n = 99 spines from five imaging sites), and (C) non-
deprived domains (ODI > 0.1, n = 70 spines from three imaging sites) before (basal) and after 4 h MD. CFP/YFP ratios of individual spines normalized to the
average basal CFP/YFP ratio of all spines within the respective imaged cortical site are plotted. (D) Bars show mean values of data pooled from each site imaged
in individual animals, including data from negative control constructs: Camui-T305D/T306D (n = 98 spines from three imaging sites) and C-Y fusion protein (n =
33 spines from two imaging sites). **P < 0.05 (KS test); N.S., nonsignificant. (E) Mean percent change in FRET within spines in each imaging site shown in A–C,
including negative control data sites pooled in D, plotted versus their respective ODI value (R = −0.65). Error bars represent SEM within each imaging site.
Changes in synaptic CaMKII activity in spines within different eye domains after 4 h MD. (A–C) CaMKII activity of individual synapses in (A) deprived eye
Mower et al.PNAS
| December 27, 2011
| vol. 108
| no. 52
expansion of nondeprived inputs (8, 12, 29, 30). Our data suggest
that the rapid loss of deprived eye drive is mediated by Hebbian
mechanisms that include (i) a loss of some spines, with the lost
synapses having lower than average basal levels of CaMKII ac-
tivity; and (ii) a reduction of CaMKII activity at synapses that
previously had high basal levels of CaMKII activity. It is likely
that feedforward synapses (Fig. 1A) constitute a high proportion
of this latter set. More interestingly, the loss of deprived eye
drive is accompanied by a homeostatic increase of CaMKII ac-
tivity in the majority of spines that had moderate to low levels of
basal CaMKII activity. These spines are preserved following
MD; such spines likely form a substrate for response scaling via
enhanced drive from the nondeprived eye (Fig. 1A), through a
delayed process of synaptic potentiation and even reorganization
of intracortical presynaptic partners (31, 32). These spines likely
also lay a structural trace for an accelerated functional shift of
responses following later episodes of deprivation (33).
Our finding of spine loss in the deprived eye domain is con-
sistent with the findings of the companion paper (7) and indeed
provides a mechanism by which an activity-dependent rule for
spine loss and preservation following MD may be implemented.
The magnitude of spine loss in this study, however, is lower than
that in the companion study (7). This disparity might be
explained by several factors: (i) small mismatches in the OD
regions that were examined in the present and companion study
(7); (ii) overexpression of CaMKII might protect spines from
being eliminated; (iii) the possibility that the effect of spine loss
ramps up between 4 h of MD (present study) and 6 h (com-
panion study, ref. 7); and (iv) the spines analyzed in the present
study were selected conservatively and represented the most
prominent and best labeled spines, as a result of the FRET
images being dimmer than the GFP labeling in the companion
In addition, our data suggest that CaMKII activity increased
more in binocular dendritic domains (Fig. S2C) than in spines. It
is known that CaMKII has an active mechanism for being
translocated and retained in the dendritic spine in an activity-
dependent fashion (e.g., interaction with the NR2B subunit of
Rank order of spines with
decreasing basal CFP/YFP
4 h MD
4 h MD
Deprived eye domain
Non-deprived eye domain
moderate basal CaMKII activity and decreases in spines with high basal
CaMKII activity. (A) Bottom: Normalized basal CFP/YFP ratios from individual
spines in the deprived eye domain are ranked and plotted in decreasing
order along with their respective CFP/YFP ratio after 4 h MD. Top: Intensity
modulated display images of a spine with high basal and lower CaMKII ac-
tivity after 4 h MD (A) as well as a spine with low basal and higher CaMKII
activity level after 4 h MD (B). Spines a and b are indicated by their respective
arrowheads in the plot. CFP/YFP values of the spines are shown below their
images. (Scale bar: 2 μm.) (B) Data from A were binned in 20% increments
and the mean was plotted. Numbers 1–5 on the x axis denote the first to
fifth bins (**P < 0.05, paired two-tailed t test). (A and B) n = 201 spines from
seven imaging sites. (C–F) Normalized spine CFP/YFP ratios in the binocular
domain (C and D) and open eye domain (E and F) plotted in the same
manner as A and B. (C and D) n = 99 spines from five imaging sites; (E and F)
n = 70 spines from three imaging sites. Error bars represent SEM.
CaMKII activation increases in deprived domain spines with low to
4 h MD
353213 36n = 31 291331n =
Imaging siteImaging site
(A) Arrowhead shows a dendritic spine that was lost after 4 h MD. (B) Dis-
tribution of basal CaMKII activity in eliminated and spared spines. Circles
represent basal CaMKII activity in individual spines grouped according to
imaged cortical site. Only sites which contained eliminated spines are shown.
Horizontal bars indicate the population mean CFP/YFP ratio of each imaged
cortical site. Red circles indicate spines that were eliminated after 4 h MD.
White circles indicate spines that persisted after 4 h MD (n indicates spine
number, including spines that disappeared, in each imaged region; seven
out of a total of 116 spines were eliminated). Eliminated spines have sig-
nificantly lower basal CFP/YFP ratios (P < 0.05, two-tailed t test). (C) Basal
CaMKII activity levels in dendritic regions adjacent to spines that were
eliminated were not significantly different from the population mean of
their imaging site, shown as in B (P > 0.05, two-tailed t test).
Spines that are lost following 4 h MD have low basal CaMKII activity.
Spine width (µ m)
% Change in CFP/YFP
Spine width (µm)
0.400.65 0.90 1.15 1.40
0.40 0.650.90 1.15 1.40
MD are not correlated with spine size. (A) Basal CFP/YFP ratio in individual
spines plotted against spine width. (B) Percent change in CFP/YFP ratio in
individual spines after 4 h MD plotted against spine width. Spines shown (n =
100) are from deprived eye sites with same image brightness.
Basal CaMKII activity and changes in CaMKII activity following 4 h
| www.pnas.org/cgi/doi/10.1073/pnas.1108261109Mower et al.
the NMDA receptor or self-association) (34, 35). A typical LTP-
inducing stimulus effectively recruits this mechanism, allowing
CaMKII to be retained at the synapse (36). However, it is not
known how CaMKII behaves in response to a stimulus of in-
termediate intensity. One possible scenario is that CaMKII is
partially activated but cannot be retained at the synapse and thus
diffuses away from the synapse into the shaft. This is a possible
reason for the difference in activity of CaMKII in the dendritic
spine and shaft. The finding that high CaMKII activity within
spines, either basally or as a consequence of up-regulation after
MD, preserves and stabilizes synapses provides insight into
previous reports of the role of CaMKII in synaptic and network
plasticity. αCaMKII-T286A mutant mice show deficits in re-
sponse strengthening of the nondeprived eye in visual cortex
following MD (22) and of spared whiskers in barrel cortex after
whisker trimming (37). This is consistent with the proposal that
CaMKII activity is a critical component of a homeostatic re-
sponse to reduction of afferent drive that mediates response
scaling of spared inputs, and indeed, with CaMKII autophos-
phorylation having a key role in the induction and maintenance
of LTP (21, 27, 28); we predict that, in sensory cortex in vivo,
such a role involves nondeprived or spared inputs on neurons in
deprived zones. Furthermore, αCaMKII-T286A mutant mice
show no increase in stabilization or persistence of new spines in
barrel column borders after whisker trimming (38), consistent
with the proposal that CaMKII activation is critical for the
preservation and maintenance of spines that anchor functional
Different molecular mechanisms have been proposed to un-
derlie regulation of synaptic strength in neurons within different
functional domains (4, 6, 12, 39). However, it is still not known
how these kinds of plasticity at different synapses are linked, and
what the molecular targets of these regulatory rules might be (40,
41). Our findings demonstrate that plasticity processes governing
synaptic changes as a result of altered drive converge at least
partly onto a specific kinase, CaMKII, and that different basal
levels of CaMKII in individual synapses can lead to a predictable
change in subsequent CaMKII activation levels following an al-
teration of afferent drive. Thus, modulation of CaMKII activity
may be one mechanism by which dynamic changes in activity
from multiple sources are coordinated within a neuron.
Materials and Methods
All experiments were performed according to protocols approved by the
Massachusetts Institute of Technology Animal Care and Use Committee and
conformed to National Institutes of Health guidelines.
Construction of Camui and Controls. We extensively improved and charac-
terized a FRET CaMKII activity probe, Camui (24), resulting in a new version
used in this study. The donor and acceptor of Camui were replaced with
monomeric [alanine 206 to lysine mutation (42)] Cerulean (43) and mono-
meric Ypet (44) to prevent aggregation and improve brightness, as required
for in vivo imaging. For simplicity, this new version was not assigned a new
name because its optical properties are similar to the original version.
Camui, Camui-T305D/T306D mutant, and a C-Y direct fusion protein were
packaged into HSV as described previously (45).
Characterization of Camui and Its Mutants in Infected HEK239T Cell Lysate.
HEK239T cells were infected with HSV-Camui, HSV-Camui-T305D/T306D, and
HSV-C-Y viruses and collected after 24 h. The fluorescent profile was analyzed
as described previously (46). Further details are provided in SI Materials
HSV-Camui Injection and in Vivo Cortical Imaging Through Cranial Window. HSV
carrying Camui or negative controls were expressed in male ferrets at the
critical period for OD plasticity (postnatal days 42–50). A cranial window was
implanted post surgery to allow for multiple imaging sessions. After 2 d of
viral expression, the same spines and dendritic segments of the L2/3 pyra-
midal neurons were imaged before and after MD to capture Camui activity
changes by using a custom-built two-photon microscope (17), followed by
intrinsic signal optical imaging, performed as described previously (47), to
obtain OD. Further details are provided in SI Materials and Methods.
Analysis of Two-Photon Microscopy Images. Image analysis was performed
using Metamorph (Molecular Devices). Images were separated into CFP and
YFP channels. Regions of interest were manually placed on the CFP channel
image. After subtracting an adjacent background image area devoid of
obvious structure, the integrated fluorescence intensity at the best focal
plane (according to the CFP channel) of each manually traced spine, and
CFP/YFP ratio for each region was calculated to provide a quantitative
measurement of CaMKII activity. For calculation of spine width, we used
methods described previously (48). The majority of analyzed cells were py-
ramidal and resided in cortical layer 2/3, as determined by the shape of
dendritic arborizations and position of cell body. We also considered that
cortical depth may lead to optical scattering of one channel more than the
other; however, preliminary experiments showed that imaging depth does
not systematically affect the CFP/YFP ratio (Fig. S4).
Analysis of Intrinsic Signal Optical Imaging Data. Four single orientation maps
from stimulation of each eye were averaged to obtain a contralateral and
ipsilateral eye response map. The contralateral eye map was subtracted from
theipsilateral eye maptogeneratetheOD map. ODI was calculatedby (C−I)/
(C + I) × 1,000 per pixel, as described previously (47).
Determination of OD Value for a Specific Spine Site. Blood vessel maps
obtained during intrinsic signal optical imaging and two-photon imaging
were used to align the images (Fig. 1C and Fig. S1E). Based on the blood
vessel pattern common to images acquired from optical and two-photon
imaging techniques, the location of low-power (1×; 800 × 600 μm) FRET
images was identified in the cortex and hence within the corresponding OD
map (white rectangle, Fig. S1E, a). High-power (10×; 80 × 60 μm) FRET
images were then aligned within the corresponding 1× FRET image, and the
perimeter of this region outlined (Fig. S1E, b). The 10× image or spine site
occupied approximately 6 × 4 pixels in the OD map (Fig. S1E, c; pixel size ∼ 14
μm). The ODI of these pixels were averaged, serving as the ODI of the spines
analyzed within this site.
Statistics. Initial data analysis was performed using Microsoft Excel and
GraphPad software. Plots were made in GraphPad. Statistical differences in
values after 4 h MD were calculated in comparison with their respective basal
html) or a two-tailed paired Student t test (with Excel). Pearson correlation
coefficients were also calculated in Excel.
ACKNOWLEDGMENTS. We thank Dr. Kayi Lee for facilitating data analysis
and Dr. Rachael Neve for HSV packaging. This work was supported by a
National Institutes of Health (NIH) postdoctoral fellowship (to A.F.M.), grants
from the NIH (to M.S.) and RIKEN, NIH Grant R01DA17310, Grant-in-Aid for
Scientific Research (A), Grant-in-Aid for Scientific Research on Innovative
Area “Foundation of Synapse and Neurocircuit Pathology,” from the Minis-
try of Education, Culture, Sports, Science, and Technology of Japan (to Y.H.),
and by 973 program (2010CB327901) from China (to H.Y.).
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