Role of pro-brain-derived neurotrophic factor (proBDNF)to ma.docx

Role of pro-brain-derived neurotrophic factor (proBDNF)
to mature BDNF conversion in activity-dependent
competition at developing neuromuscular synapses
H. Shawn Jea,b,c,1, Feng Yanga,b,d,1, Yuanyuan Jia,e, Guhan
Nagappana,e, Barbara L. Hempsteadf, and Bai Lua,b,e,2
aSection on Neural Development and Plasticity, National
Institute of Child Health and Human Development, Bethesda,
MD 20892-3714; bGenes, Cognition,
and Psychosis Program (GCAP), National Institute of Mental
Health, Bethesda, MD 20892-3714; cProgram in Neuroscience
and Behavioral Disorders, Duke–
National University of Singapore (Duke-NUS) Graduate
Medical School, 169857, Singapore; dLieber Institute for Brain
Development, The Johns Hopkins
University Medical Campus, Baltimore, MD 21205; eR&D
China, GlaxoSmithKline, Pudong, Shanghai 201203, China; and
fDivision of Hematology, Department
of Medicine, Weill Medical College, Cornell University, New
York, NY 10021
Edited* by Richard L. Huganir, The Johns Hopkins University
School of Medicine, Baltimore, MD, and approved August 20,
2012 (received for review May
10, 2012)
Formation of specific neuronal connections often involves
compe-
tition between adjacent axons, leading to stabilization of the
active
terminal, while retraction of the less active ones. The
underlying

molecular mechanisms remain unknown. We show that activity-
dependent conversion of pro–brain-derived neurotrophic factor
(proBDNF) to mature (m)BDNF mediates synaptic competition.
Stim-
ulation of motoneurons triggers proteolytic conversion of
proBDNF
to mBDNF at nerve terminals. In Xenopus nerve–muscle
cocultures,
in which two motoneurons innervate one myocyte, proBDNF-
p75NTR signaling promotes retraction of the less active
terminal,
whereas mBDNF–tyrosine-related kinase B (TrkB) p75NTR
(p75 neu-
rotrophin receptor) facilitates stabilization of the active one.
Thus,
proBDNF and mBDNF may serve as potential “punishment” and
“re-
ward” signals for inactive and active terminals, respectively,
and
activity-dependent conversion of proBDNF to mBDNF may
regulate
synapse elimination.
neuromuscular junction | pro-neurotrophin | synapse
competition
The nervous system responds to experience by altering
thenumber and strength of synaptic connections (1). Activity-
dependent synaptic competition, a general process seen in many
parts of the developing nervous system, plays a critical role in
shaping patterns of neuronal connections (2–7). At the neuro-
muscular junction (NMJ), for example, multiple axons compete
for the same postsynaptic muscle cell during early postnatal life
until all but one is eliminated (8–10). Extensive experimental
data support the view that the more active terminal or “cartel”
gets stabilized, whereas less active ones withdraw, resulting in

canonical elimination of polyneuronal innervation (8, 11). It is
generally believed that this synaptic competition is mediated by
a “punishment” or “elimination” signal, produced by the post-
synaptic cell, that causes the retraction of the inactive
terminals,
as well as a “protective” or “reward” signal that stabilizes the
active terminal (10–12). Despite significant efforts over
decades,
the identity of the punishment or reward signals remains un-
known (4, 13). This is due at least in part, to the experimental
difficulties in manipulating gene expression selectively in one
of
the competing axons.
Brain-derived neurotrophic factor (BDNF) has been recog-
nized as a key regulator of synapse development and plasticity
(14, 15). This is because BDNF is the only neurotrophin in-
disputably secreted in an activity-dependent manner (15). In-
deed, activity-dependent secretion of BDNF has been shown to
be critical for hippocampus-dependent memory in human (16,
17). Like all neurotrophins, BDNF is initially synthesized as
a precursor (proBDNF), which is subsequently cleaved to
generate mature (m)BDNF. proBDNF interacts preferentially
with the pan-neurotrophin receptor p75 (p75NTR), whereas
mBDNF selectively binds and activates the receptor tyrosine
kinase TrkB (18, 19). Cumulative evidence supports a “yin-yang
hypothesis,” in which pro- and mBDNF elicit opposite
biological
effects by activating two distinct receptor systems (20). For ex-
ample, proBDNF, if not processed, promotes long-term de-
pression (LTD) through the activation of p75NTR in the
hippocampus (21, 22). In contrast, mBDNF-TrkB signaling is
essential for the early phase of long-term potentiation (E-LTP)
(23–25). Moreover, recent studies indicate that a significant
proportion of BDNF in the brain is secreted in the proform (26–

28), and extracellular conversion of proBDNF to mBDNF by the
tissue plasminogen activator (tPA)/plasmin protease system is
critical for late-phase LTP (29). The expression of proBDNF
and
p75NTR in rodents is developmentally regulated, with the
highest
levels in the first and second postnatal week, correlating well
with
the timings of synapse formation (28). Therefore, proteolytic
cleavage of proBDNF represents an important mechanism by
which the opposing cellular actions of proBDNF and mBDNF
may be regulated (20).
The opposing nature of proBDNF and mBDNF prompted us to
hypothesize that proBDNF and mBDNF might serve as punish-
ment and reward signals, respectively, during synaptic
competition
at the developing NMJs. In this study, we developed a triplet
sys-
tem that allows alteration of gene function in one of two
distinctly
labeled axons that innervate a single, unlabeled myocyte. Our
study suggests that proBDNF serves as a general “punishment
signal” that causes p75NTR-expressing motor terminals to
retract,
whereas at the active terminal, secretion/activation of
extracellular
protease(s) converts proBDNF to mBDNF, which serves as a re-
ward signal to stabilize the terminal.
Results
Activity-Dependent Synaptic Competition in Xenopus Nerve–
Muscle
Cocultures. Xenopus nerve–muscle coculture system was used
to
study the activity-dependent synaptic competition. We

developed
a cell-culture system, in which an unlabeled myocyte was inner-
vated by two spinal neurons (one labeled in green and the other
in red, respectively; Fig. 1A). This was accomplished by
injecting
either FITC-dextran (green) or rhodamine-dextran (red) into a
single dorsal animal blastomere at the 8- or 16-cell stage and
mixing the neural tubes from embryos that were injected with
two different fluorophores to prepare dissociated nerve–muscle
Author contributions: H.S.J., F.Y., and B.L. designed research;
H.S.J., F.Y., Y.J., and G.N.
performed research; B.L.H. contributed new reagents/analytic
tools; H.S.J., F.Y., and Y.J.
analyzed data; and H.S.J., F.Y., and B.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1H.S.J. and F.Y. contributed equally to this work.
2To whom correspondence should be addressed. E-mail:
[email protected]
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1207767109/-/DCSupplemental.
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cocultures (30). Instead of employing electrical stimulation
using
a glass electrode, which might cause mechanical damages on
neurons, we stimulated one of the spinal neurons by local pho-
tolysis of caged glutamate (MNI-glutamate; 50 μM) using
a multiphoton microscope (31). Photo-uncaging of caged gluta-
mate at a neuronal soma induced a marked potentiation of
synaptic transmission, which lasted for more than 60 min (Fig.
S1A). This synaptic potentiation was completely blocked by the
sodium channel blocker tetrodotoxin (TTX) (1 μM), demon-
strating the requirement of action potentials (Fig. S1A). Fur-
thermore, synaptic potentiation was only observed when a laser
beam was applied within 25 μm from the neuronal cell body,
suggesting that the photo-uncaging occurred locally (Fig. S1B).
A brief episode (250 ms) of photolysis of MNI-glutamate was
applied to one of the two neurons, and the resulting morpho-
logical changes in synaptic terminals from both neurons, which
were innervating a single myocyte, were monitored by dual-
color,
time-lapse confocal imaging. Upon stimulation of a red neuron,
the axon terminal of the unstimulated neuron (green terminal)
gradually withdrew from the previously innervated muscle,
whereas the terminal of the stimulated neuron (red terminal)
remained stable and occasionally extended (Fig. 1B and Movie
S1). Conversely, stimulation of a green neuron triggered the
retraction of the red terminal (Fig. S2). Because stimulation of
one neuron always resulted in the retraction of the unstimulated
neuron, regardless of whether it was red or green, we randomly
stimulated neurons in subsequent experiments based on the
convenience of photo-uncaging. We performed 11 experiments
involving preferential photolysis of one neuron. In all 11 cases,
the axon terminals of the unstimulated neurons retracted to
varying degrees. In six cases, axon terminals of stimulated neu-

rons showed slight expansion or elongation (Fig. 1 B and C).
These results suggest that a postsynaptically derived local pun-
ishment signal may trigger synaptic retraction.
Activity-Dependent Cleavage of Secreted proBDNF at NMJ.
Given
that proBDNF and mBDNF could elicit opposite effects, we
hypothesized that proBDNF and mBDNF might serve as pun-
ishment and reward signals, respectively. Furthermore, we
hypoth-
esized that the proteolytic conversion of proBDNF to mBDNF
might be essential for synaptic competition and elimination at
the developing NMJs. First, we tested whether neuronal activity
could activate proteases to process proBDNF to mBDNF using a
fluorogenic proteolytic beacon assay. The fluorogenic beacon
consisted of synthetic peptides, harboring the propeptide cleav-
age sequences (MSMRVRR↓HSD) within proBDNF, and these
synthetic peptides were flanked by a fluorophore at the N
terminus
and a quencher at its C terminus (Fig. 2A). Small size peptides
allowed both a fluorophore and a quencher within proximity,
thereby quenching the fluorescence via fluorescence resonance
energy transfer (FRET). Upon cleavage of peptides by a pro-
tease(s), which specifically recognizes the proBDNF cleavage
sequence, the fluorogenic beacon would emit the fluorescence
signal because of the separation of a fluorophore from a
quencher
(Fig. 2A). In addition, we immobilized the fluorogenic beacon
to
polystyrene beads to minimize diffusion in culture medium.
Next, we investigated whether stimulation of neurons could
activate proteases at the nerve terminals. The fluorogenic
beacons
were placed on the axonal process or termini of spinal neurons,
and neurons were subsequently stimulated either by an

electrical
stimulation or by photo-uncaging of MNI-glutamate (Fig. 2B,
bright field). After stimulation, the fluorescence intensity of the
beads on the axon terminals or axonal processes (e.g., yellow
arrows in Fig. 2B) greatly increased (Fig. 2 B and C),
suggesting
cleavage of the peptide by proteases secreted from the axon. In
contrast, no fluorescence change was observed from beads,
which
were not in contact with the axonal process or termini (Fig. 2B,
“free beads,” white arrow). Time-lapse imaging indicated that
the
increase in fluorescence occurred relatively quickly: the first
surge
came within minutes, and the florescence increase reached its
peak
within 20 min (Fig. 2C). Pretreatment with a mixture of
protease
inhibitors prevented the increase in fluorescence from beads on
axon terminals (Fig. 2D). Remarkably, inhibitors for tPA and
furin, the extracellular and intracellular proteases known to
cleave
proBDNF in the brain (29, 32), did not inhibit the fluorescence
increase (Fig. 2D). In contrast, matrix metalloprotease (MMP)
inhibitors successfully blocked the stimulus-induced increase in
fluorescence. Further analyses indicated that MMP3 and MMP9,
but not MMP2, MMP8, or MMP13, could increase fluorescence
signal on the fluorogenic beacons (Fig. 2D). Finally, when the
beacon-containing beads were placed on muscle cells,
stimulation
of muscle cells did not cause an increase in fluorescence (Fig.
2D),
N
N

M
stim.
unstim.A
N
M
N
B
C
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unstimulated neuron (11)
stimulated neuron (11)
Distance(µm)
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Elongation Retraction
M
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30 60 90 1200
Fig. 1. Activity-dependent synaptic competition in culture. (A)
Schematic
diagram (Left) and confocal image (Right) showing a triplet in
which
a spherical myocyte (M) (indicated by white dotted lines) is
innervated by
two spinal neurons (N), one labeled with FITC (green) and one
labeled in
rhodamine (red), in nerve–muscle coculture. (B) Time course of
synaptic
competition. A higher magnification of A is shown. Stimulation
of the red
neuron, by photo-uncaging of MNI-glutamate in the soma area
using a two-
photon laser, caused the unstimulated (green) axon terminal to
retract from
the synaptic target (indicated by a yellow arrow, upper row). In
contrast,
the axon terminal (red) from the stimulated neuron did not
retract, but
elongated a little (white arrows, middle row). The phase and
two-color
fluorescence images of the triplet at multiple time points (lower
row). (Scale
bar: 10 μm.) The phase (Bottom) and two-color fluorescence

images (Top
and Middle) of the triplet at “0” and “120” min are shown. (C)
Quantifi-
cation of axonal retraction and elongation measured 120 min
after stimu-
lation of one of the neurons in the triplets. *P < 0.01.
Je et al. PNAS | September 25, 2012 | vol. 109 | no. 39 | 15925
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indicating that muscle cells do not secrete proteases that could
process proBDNF.
We further examined whether postsynaptic muscle cells se-
crete proBDNF upon stimulation. Because of unavailability of
a proBDNF-specific ELISA and limited number of muscle cells
in the Xenopus cocultures (<1,000 myocytes), it was not
feasible
to measure proBDNF secretion from muscle cells using existing
biochemical techniques. Thus, we used cell surface immuno-
staining to measure proBDNF secretion, given that proBDNF is
positively charged at physiological pH and could be associated
with a negatively charged cell membrane upon secretion (27,
33).
The muscle cell cultures were depolarized by high-K+ treatment
(50 mM) for 5 min, fixed, and processed for cell surface immu-
nofluorescence staining under membrane impermeable condi-
tions. A proBDNF-specific monoclonal antibody was used for
cell-
surface staining of secreted proBDNF (27). Although proBDNF
was barely detectable on the muscle cell surface at rest
(control),
the proBDNF immunoreactivity increased dramatically (88%)
upon depolarization (Fig. S3A). This increase was even more
pronounced when extracellular cleavage of proBDNF was

blocked
by general MMPs inhibitors (indicated as MMP-In. in Fig.
S3A).
To measure the protease-mediated conversion of endogenous
proBDNF to mBDNF at the neuromuscular synapses, we per-
formed cell-surface immunostaining using a specific antibody
against mBDNF. We applied glutamate, instead of high K+, to
selectively depolarize neurons, because muscle cells do not ex-
press glutamate receptors (34). After neuronal depolarization,
cultures were subsequently fixed and processed for surface im-
munostaining using an mBDNF-specific antibody (27). In
control
conditions, there was no mBDNF staining (Fig. S3B, Left).
Upon
glutamate application (10 mM; 5 min), we observed a dramatic,
threefold increase in surface mBDNF staining at the synaptic
junction (Fig. S3B, Center). This increase in mBDNF immuno-
reactivity was observed only in myocytes that were innervated
by a spinal neuron but not in the neighboring noninnervated
myocytes (Fig. S3B, Center). This suggested that the release
and/
or activation of proteases at the nerve terminal converted
proBDNF to mBDNF at the active synaptic terminals. Further-
more, when the cultures were pretreated with the MMP inhib-
itors, the glutamate-induced increase in mBDNF staining was
completely abolished (Fig. S3B, Right). Taken together, these
results supported the notion that activity-dependent conversion
of proBDNF to mBDNF occurred at the developing Xenopus
NMJ in situ.
Synaptic Stabilization During Competition Mediated by
mBDNF/TrkB.
Previously, we have shown that exogenous proBDNF triggers
synaptic depression and subsequent retraction of axon terminal

through p75NTR in Xenopus neuromuscular synapses (35).
Based
on robust secretion of proBDNF upon synaptic depolarization,
we reasoned that active terminals might cleave proBDNF to
mBDNF, which protects these active terminals from proBDNF-
mediated synaptic depression and retraction. To test this, we
knocked down endogenous TrkB in one of neurons in our triplet
system using a morpholino (Fig. S4A; also see ref. 36). Control
experiments indicated that the expression of the TrkB morpho-
lino in presynaptic neurons blocked the mBDNF-mediated syn-
aptic potentiation (Fig. S4 B and C). When the TrkB morpholino
was selectively expressed in one of the spinal neurons in our
triplet system, stimulation of the TrkB morpholino-expressing
neuron (green) resulted in retraction of both green and red
terminals (Fig. 3A, yellow arrows). Quantitative analysis of 10
triplets indicated that both stimulated and unstimulated termi-
nals retracted ∼12 μm within 120 min (Fig. 3C).
Next, we reasoned that if the activation of TrkB by mBDNF was
critical to prevent synaptic retraction, supply of exogenous
BDNF
would prevent synaptic competition. Indeed, in a nerve–muscle
culture treated with mBDNF (25 ng/mL) for 2 h, stimulation of
the
red neuron did not cause retraction of the green terminal (Fig.
3B
and Movie S2). Moreover, application of exogenous mBDNF
even
elicited axonal elongation from the stimulated neurons (Fig. 3 B
and C, white arrowheads). These results suggest that mBDNF
plays an active role in preventing synaptic retraction of the
active
terminals through TrkB signaling during synaptic competition.
Synaptic Retraction During Competition Mediated by
proBDNF/p75NTR.

To determine whether this activity-based synaptic retraction
was
mediated through proBDNF/p75NTR signaling, we knocked
down
endogenous p75NTR by using FITC-conjugated p75NTR siRNA
that
were targeted to all Xenopus p75NTR isoforms (p75NTRa and
p75NTRb) (37). The p75NTR siRNA was introduced into a
single
neuron in our triplet system using embryo-injection techniques.
A B
C D
ROX-MSM RVRR /HSD-QXL610
Fluorophore Quencher
Proteolytic cleavage
Emit fluorescence
proBDNF cleavage sequences
0 min 6 min
Bright field Before stim. After stim.
0.8
1
1.2
1.4

1.6
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0 5 10 15 20 25 30 min
N
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fl
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in
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(F
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Control (8)
Protease In.(7)
Free beads (9)
0.8
1
1.2
1.4
1.6
1.8
9 7 7 6 5 7 8 6 98 8 6
*
*
*
*
*
*
N
or
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iz
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fl
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en
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in
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ity
(F
/F
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P
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M
M
P
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Fig. 2. Activity-dependent cleavage of proBDNF
detected by fluorogenic probes. (A) Schematic dia-
gram depicting the design of a fluorogenic in-
dicator of proBDNF cleavage. A peptide containing
the proBDNF cleavage site was placed between
a fluorophore and a quencher. Upon proteolytic
cleavage, the quencher was dissociated from the
fluorophore, leading to emission of fluorescence.
(B) Sample images of fluorogenic probes in phase
(Left) and fluorescence (Right) before and after
neuronal stimulation. Polystyrene beads soaked
with fluorogenic probes were positioned on (yellow

arrows) or near (white arrows) an axonal process
(indicated by white dotted lines), respectively, by
micromanipulation. A neuronal cell body was
stimulated by photolysis of caged-glutamate (MNI-
glutamate) with a UV laser. Note that after stimu-
lation, only beads on, but not near, the axonal
processes showed an increase in fluorescence. (C)
Time course of cleavage-dependent increase in
fluorescence intensity upon stimulation of spinal
neurons. Fluorescence intensities measured on
contacted beads or free beads were normalized to
that at “0” time point and presented as ratios (F/F0)
over time. (D) Quantification of relative fluorescence intensities
in beads 30 min after stimulation of neurons under various
conditions. Concentrations
of protease inhibitors (In.): general protease inhibitor mixture,
50 μM; pan MMP inhibitor, 60 μM; tPA inhibitor, 10 μM; furin
inhibitor, 40 μM; MMP2 inhibitor,
60 μM; MMP3 inhibitor, 50 nM; MMP8 inhibitor, 20 nM;
MMP9 inhibitor, 50 μM; MMP13 inhibitor, 80 nM. B, beads; M,
muscle cell; N, neuron.
15926 | www.pnas.org/cgi/doi/10.1073/pnas.1207767109 Je et
al.
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Western blot analysis revealed significant reduction of
endogenous
p75NTR protein in neural tubes derived from embryos injected
with
p75NTR siRNA, but not in those injected with scrambled siRNA
for p75NTR (35). We reasoned that the blockade of p75NTR
would
block synapse elimination of less active neurons. Indeed, when
photolysis was applied to the red neuron, the axon terminal of a
p75NTR siRNA-expressing neuron did not retract over a long

period
(Fig. 4A). In seven triplets, four showed no retraction, whereas
the
other three showed reduced retraction of the unstimulated termi-
nals after 2hrs (Fig. 4 A and C). Furthermore, an axon
expressing
scrambled siRNA still retracted when the competing axon was
stimulated (Fig. S5). Because p75NTR siRNA and p75NTR mor-
pholino target different regions of p75NTR, this experiment
con-
firmed that blockade of p75NTR signaling abolished synaptic
re-
traction. Taken together, these data support the hypothesis that
synaptic retraction was mediated by presynaptic p75NTR
signaling
during synaptic competition.
The results above, together with the finding that neuronal
stimulation triggered protease secretion/activation at nerve ter-
minals (Fig. 2), prompted us to speculate that active terminals
were spared from synaptic retraction because of proteolytic
cleavage of proBDNF. If this were true, then blockade of
proBDNF cleavage would trigger retraction of all axon
terminals
during synaptic competition. To test this hypothesis, we treated
nerve–muscle cocultures with a mixture of protease inhibitors
for
10 min before neuronal stimulation. These inhibitors were able
to
block proteases that could cleave proBDNF (Fig. 2D). Remark-
ably, axon terminals from red and green spinal neurons
retracted
simultaneously from the synaptic site (Movie S3). Quantitative
analysis indicated that both stimulated and unstimulated
terminals

retracted ∼10 μm (Fig. 4C). Because MMP inhibitors,
particularly
MMP3/9 inhibitors, blocked activity-induced proBDNF
cleavage
at nerve terminals (Fig. 2D), we further tested whether
inhibition
of MMPs could perturb synapse elimination. Indeed, pretreat-
ment with a combination of both MMP3 and MMP9 inhibitors
triggered synaptic retraction upon stimulation of the competing
axon, indicating that MMP3/9 are the candidate proteases that
convert proBDNF to mBDNF at the active synaptic terminal
(Fig.
S6 and Movie S4). Taken together, these data supported the hy-
pothesis that the activation of presynaptic p75NTR by muscle-
de-
rived proBDNF triggered the retraction of less active axonal
terminals during synaptic competition.
Discussion
For over a half century, activity-dependent synaptic
competition/
elimination has been one of the central issues in developmental
neurobiology. However, the underlying mechanisms remain un-
known. Because the competition depends on activity and occurs
locally, the experimental challenge has been to selectively acti-
vate one of the competing terminals and to perform molecular
manipulation selectively in competing axons (38). In the present
study, we modified the Xenopus embryo-injection protocol, so
that we could reliably obtain triplets, in which an unlabeled
myocyte is innervated by two distinctly labeled axons. Confocal
live-cell imaging and local glutamate uncaging using a multi-
photon laser enabled us to visualize and trigger synaptic re-
traction of individual axons in triplets. To demonstrate activity-
dependent secretion of proBDNF and its conversion to mBDNF,
we performed cell surface immunofluorescence staining on
cultured cells, using specific antibodies against proBDNF and

mBDNF. FRET-based fluorogenic probes were used to monitor
the activation of protease activities locally at axonal terminals
in
real time. Moreover, embryo injection techniques were used to
selectively inhibit TrkB or p75NTR in one neuron, but not in
others,
in triplets. By combining these techniques, we provided the evi-
dence for a local and activity-dependent activation of specific
proteases that could convert proBDNF to mBDNF in neuromus-
cular synapses. Given that proBDNF and mBDNF often elicit
opposite biological effects (20, 28), this finding may help us
un-
derstand how proBDNF-to-mBDNF conversion is regulated via
neuronal activity. Moreover, we found that the blockade of
p75NTR signaling attenuated synapse elimination, whereas the
blockade of TrkB signaling, or inhibition of proBDNF cleavage
by metalloproteases, promoted synaptic retraction of both in-
nervated axon terminals in triplets. Taken together, these
findings
suggest a model for synapse elimination in which the activity-
dependent conversion of proBDNF to mBDNF selectively sta-
bilized active terminals, whereas inactive terminals were elimi-
nated in response to proBDNF and subsequent activation of
p75NTR signaling (Fig. S7).
Activity-Dependent proBDNF Cleavage Converts a
“Synaptotoxin” to
a “Synaptotrophin.” Two theories have been proposed to explain
activity-dependent synaptic competition and elimination. The
“synaptotoxin” theory suggests that the postsynaptic cell
produces
destabilizing or “toxic” signals such as proteases that
retrogradely
punish and remove presynaptic terminals. This theory (11) was
based largely on the observation that inhibition of protease ac-
tivity (39, 40), particularly thrombin, by the naturally occurring

protease inhibitor nexin I (41, 42), attenuated synapse elimina-
tion. However, specific target(s) of the proteases have not been
identified yet. More importantly, selective protection of the
active
terminal by local secretion of protease inhibitors has not been
demonstrated. It is also unclear what axons are competing for, if
the fate of an axon is dependent only on its intrinsic activity
(11).
An alternative is the “synaptotrophin” hypothesis, which
suggests
that axons compete with each other for a limited supply of
trophic
A
B
C
-10 -5 0 5 10 15 20
unstimulated neuron
stimulated neuron
BDNF (4)
TrkB morpholino (10)
Distance (µm)
unstimulated neuron
stimulated neuron
B

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5n
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m
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0 30 60 90 120
0 9030 60 120
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B
-m
or
p.
Fig. 3. Synaptic stabilization mediated by mBDNF/TrkB. (A)
Down-regula-
tion of TrkB by TrkB-morpholino (TrkB-morp.) led to retraction
of both
stimulated and unstimulated axon terminals. In a triplet system,
stimulation
of a neuron expressing TrkB morpholino (green) resulted in
retraction of the
red axon as well as the green axon. (Scale bar: 10 μm.) (B)
Treatment with
mBDNF prevented activity-dependent synaptic retraction. The
culture was
treated with mBDNF before stimulating the soma of a red
neuron. The
terminals from both red and green neurons remained in the
synaptic site
without any retraction. (Scale bar: 20 μm.) (C) Quantification of
axonal re-
traction and elongation measured 120 min after stimulation of
one of the
neurons in the triplets.

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factors derived from the postsynaptic cell. However, this model

fails to explain: (i) how the trophic factor is secreted locally
from
the postsynaptic cell only near the active terminal, but not at the
inactive terminal; and (ii) how the active terminal preferentially
binds, uptakes, or signals the trophic factor, if any. Neither
local
secretion nor preferential signaling has been reported at NMJs
to
support the synaptotrophin hypothesis. Moreover, synaptic elim-
ination is an active process that requires a punishment signal,
ultimately resulting in the loss of all, but one protected terminal
(13). Thus, the synaptotrophin theory fails to explain how
inactive
terminals initiate withdrawal and why the absence of trophic
support leads to synaptic depression and retraction.
Our data support a model that both the punishment and re-
ward signals are originated from the same molecule, namely
BDNF (Fig. S7). In this model, presynaptic activity drives the
secretion of proBDNF from postsynaptic muscle cells, which
serves as a default “punishment signal” to actively retract af-
ferent terminals through p75NTR. mBDNF, on the other hand,
serves as a reward signal for which all terminals compete. A
major conceptual breakthrough to the model is the activity-de-
pendent conversion of proBDNF to mBDNF by active axon
terminals. Locally secreted proteases at the active terminal not
only block the punishment signal (proBDNF), but also generate
a reward signal (mBDNF), which stabilizes the terminal by
activating TrkB. Further work is necessary to substantiate this
model in vivo.
proBDNF-mBDNF As Punishment–Reward Signals in Synapse
Elimination.
Using cultured neurons, computational modeling, and knockout
animals, two recent studies reported mechanisms for synaptic

competition and axonal pruning in the sympathetic nervous
system (43, 44). In this system, target-derived NGF, through
TrkA, provides protection and prosurvival of active axons,
which
are further strengthened and stabilized by increasing TrkA
transcription. On the other hand, NGF-induced synthesis and
secretion of BDNF and neurotrophin-4 (NT-4), through
p75NTR,
facilitate axonal degeneration and pruning of the competing
axons (43–45). Although these experiments were largely carried
out in cell culture, the basic premise is quite similar: TrkA
mediates reward, whereas p75NTR mediates punishment. There
are several important differences between the present study and
those published works. First, activity-dependent secretion of
the reward (NGF) or punishment signal (BDNF) in the superior
cervical ganglia (SCG) neurons was not demonstrated. In the
Xenopus neuromuscular system, we showed that BDNF is se-
creted from postsynaptic muscle cells. Thus, the target cell
(myocyte) is an important player in synaptic (as opposed to ax-
onal) competition. Second, it is widely accepted that
motoneurons
express only TrkB, but TrkC and TrkA expression is
controversial.
In addition, negligible levels of NGF are detectable in the neu-
romuscular system (46) and TrkB expression in sympathetic
neurons is extremely low (47). Therefore, application of exces-
sive BDNF, even with low affinity, can only bind to p75NTR,
trig-
gering axonal pruning. Third, and most importantly, in SCG,
two
separate ligands (NGF and BDNF) are needed for reward and
punishment signals, respectively. At the NMJ, the reward and
punishment signals are derived from the same molecule,
depending
on proteolytic cleavage. Our model suggests that the punish-
ment signal (proBDNF) acts on all competing axons to promote

elimination, and the active axon is spared because it activates
ex-
tracellular protease(s) that convert the punishment (proBDNF)
signal to a reward (mBDNF) signal.
Several issues require further investigation. First, what is the
identity and source of the protease(s)? Our imaging experiments
(Figs. 2 and 3) point to metalloproteases, particularly MMP3
and
MMP9, as candidates that cleave proBDNF at the active
terminal
during synaptic competition in Xenopus NMJ. Fluorogenic bea-
con experiments suggest that the MMPs are derived from axonal
terminal, but not from muscle cells (Fig. 2D). Indeed, these pro-
teases are expressed in motor neurons and are highly enriched at
the NMJs (48–50). Further work is necessary to identify the
spe-
cific proteases required at mouse NMJs in vivo. Second,
contrary
to our findings, several previous studies have shown that
inhibition
of protease activity attenuates synaptic elimination (39, 40) and
even increases numbers of acetylcholine receptor (AChR) in the
postsynaptic muscle (51, 52). One way to reconcile these two
sets
of data is that inhibition of a protease “X,” the function of
which
is to degrade MMPs, may result in an enhanced conversion of
proBDNF to mBDNF by MMPs, leading to attenuation of syn-
aptic elimination. Moreover, our results suggest that proBDNF
is
derived from postsynaptic muscle cells but not presynaptic
motor
neurons. It remains to be established whether muscle cells are
the only source of proBDNF or whether other cell types, such as
Schwann cells, also produce and secrete proBDNF at the NMJ

in
vivo. With the currently available technologies, it is not
possible to
demonstrate the secretion of BDNF (more specifically
proBDNF)
at the NMJs in vivo. An important future experiment is to dem-
onstrate that proBDNF is both secreted and cleaved locally at
the
active terminal but not at the inactive terminal in vivo. Finally,
activation of p75NTR may be a general mechanism for activity-
dependent synapse retraction. Indeed, in sympathetic neurons,
BDNF (possibly proBDNF) acts through p75NTR to suppress
A
B
C
Distance (µm)-5 0 5 10 15 20
p75NTR siRNA(7)
Protease
inhibitors (7)
unstimulated neuron
stimulated neuron
unstimulated neuron
stimulated neuron
p7
5N

TR
-s
iR
N
A
P
ro
te
as
e
in
hi
bi
to
rs 0 120 60 30
un
st
im
.
st
im
.
90

un
st
im
.
st
im
.
0 30 60 90 120
Fig. 4. Synaptic retraction mediated by proBDNF/p75NTR. (A)
Down-regu-
lation of p75NTR by siRNA prevents activity-dependent
synaptic retraction.
Fluorescence images show a double-innervated myocyte (white
dotted lines)
by a neuron expressing p75NTR siRNAs (green) and a
rhodamine-labeled
control neuron (red). The soma of the red neuron (outside the
field) was
stimulated by photo-uncaging and the axon terminals from both
neurons
were monitored by time-lapse microscopy. (Scale bar: 20 μm.)
Even after
120 min, the axon terminals (white arrow) of both red and green
neurons
remained unchanged at the synaptic site. (B) Time-lapse images
showing
retraction of both stimulated (red) and unstimulated (green)
axons in the
presence of a mixture of protease inhibitors (50 μM). (Scale
bar: 20 μm.) (C)

Quantification of axonal retraction and elongation measured 120
min after
stimulation of one of the neurons in the triplets.
15928 | www.pnas.org/cgi/doi/10.1073/pnas.1207767109 Je et
al.
9/-
F7
inactive axons, whereas active axons (depolarized by high K+)
of
the same neurons are spared (45). It will be extremely important
to test whether protease-mediated conversion of proBDNF to
mBDNF also contributes to activity-dependent synaptic compe-
tition in the central nervous system (CNS), an example being
ocular dominance formation within the visual cortex.
Experimental Procedures
Full details are contained in SI Experimental Procedures. Use
and care of
animals in this study abided by the guidelines of the
institutional Animal
Care and Use Committee at the National Institutes of Health
NIH.
Embryo Injection and Xenopus Nerve–Muscle Coculture.
Xenopus nerve–mus-
cle cocultures were prepared as described (53). Morpholinos,
siRNAs, or cDNAs
were injected into one of the blastomeres at the 2- to 4-cell or
8- to 16-cell

stage as described (53).
Immunocytochemistry, Confocal Microscopy Image Analysis,
and Statistics.
Xenopus nerve–muscle cocultures were immunostained,
maintained, and
analyzed as described previously (35). See SI Experimental
Procedures for
details. Image analysis was performed by investigators who
were blinded to
experimental conditions.
ACKNOWLEDGMENTS. We thank Drs. Phillip Nelson, Eugene
Zaitsev, Keri
Martinowitch, Jay Chang, and Newton Woo for thoughtful
comments
and suggestions and Regeneron Pharmaceuticals for providing
recombi-
nant BDNF. We also thank Drs. Bruce Carter, Mark Bothwell,
Moses Chao,
and Phil Barker for antibodies to p75NTR and Louis Reichardt
and Moses
Chao for antibodies to TrkB. Microscopy imaging was
performed at the
Porter Neuroscience Center Light Imaging Facility with the
assistance of
Dr. Carolyn Smith (NIH). This work was supported by the
National In-
stitute of Mental Health (NIMH) and National Institute of Child
Health
and Human Development (NICHD) intramural research
programs (B.L.)
and grants from the NIH and Muscular Dystrophy Association
(MDA)
(to B.H.).

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Supporting Information
Je et al. 10.1073/pnas.1207767109
SI Experimental Procedures
DNA Constructs, Embryo Injection, and Xenopus Nerve–Muscle
Coculture. Xenopus egg laying was induced by injecting female
Xenopus with human chronic gonadotrophin (hCG) (Sigma). Re-
sulting eggs were fertilized with sperm derived from male
testis.
Morpholinos, siRNA, or cDNAs (1 μg/μL) were mixed with
fluorescence dye (FITC or rhodamine) or GFP mRNA (1 μg/mL)
in a 1:1 ratio, injected into one of the blastomeres at the 2- to 4-
cell or 8- to 16-cell stage using the Picospritzer pressure ejector
(Parker Hannifin). One day after injection, the neural tube and
associated myotomal tissues were dissected, dissociated in
Ca2+-
Mg2+-free medium [58.2 mM NaCl, 0.7 mM KCl, 0.3 mM
EDTA
(pH 7.4)] for 15–20 min, and plated on glass coverslips. Cells
were grown for 1 d in culture medium consisting (vol/vol) of
50%
(vol/vol) L-15 medium, 1% (vol/vol) FCS, and 49% (vol/vol)
Ringer’s solution [117.6 mM NaCl, 2 mM CaCl2, 2.5 mM KCl,

10 mM Hepes (pH 7.6)].
Western Blot Analysis. Xenopus embryos at stage 22 were
quickly
homogenized in extraction buffer I of “native membrane protein
extraction kit” according to manufacturer’s instructions (pro-
teoExtact; Calbiochem) in the presence of protease inhibitor
mixture (set III; Calbiochem). After high-speed centrifugation
(14,000 × g), the supernatants were transferred to a fresh tube
containing 300 μL of Freon (1,1,2-trichlorotrifluoroethane;
Sigma) vortexed for 1 min, incubated on ice for 5 min, and
centrifuged again to remove yolk protein. To measure proBDNF
expression, immunoprecipitation was performed. The super-
natants (cytoplasmic fraction) were measured for protein con-
centration, and an equal amount of protein was then precleared
and immunoprecipitated by chicken anti-human BDNF (de-
scribed in ref. 1; 2 μg for 500 μg of total protein) and chicken
IgY
agarose (Chemicon). The precipitated materials were subject to
Western blotting using polyclonal anti-BDNF (N-20; Santa
Cruz;
1:1,500). For membrane proteins (p75NTR and TrkB), the in-
soluble pellets after cytoplasmic extraction were solubilized in
modified RIPA buffer containing 1% SDS. Following centrifu-
gation at 10,000 × g to remove insoluble material, the superna-
tant was measured for protein concentration and 30 μg of
protein was separated on SDS/PAGE and blotted onto Im-
mobilon-P membrane (Millipore). The blots were probed with
the following primary antibodies: polyclonal antibody against
mouse intracellular domain of p75NTR (kindly provided by
Bruce
Carter, Vanderbilt University, Nashville, TN; 1:3,000); chicken
polyclonal anti-human TrkB (kindly provided by Louis Reich-
ardt, University of California, San Francisco; 1:1,000); and
polyclonal rabbit anti-tubulin (Abcam; 1:5,000). After thorough
washes, the blots were reacted with a secondary antibody con-

jugated with HRP. Signals were detected by ECL-plus kit (GE
Healthcare) exposed on HyperFilm for band intensities in the
linear range (Amersham Biosciences), scanned, and quantified
using ImageJ version 1.37.
To determine the expression of proBDNF in muscle cells, 10-
to 50-mg muscle tissues from the hindlimbs of neonatal mice or
Xenopus were homogenized in modified RIPA buffer with pro-
tease inhibitor mixture, sonicated, and extracted for 15 min by
increasing SDS and Triton X-100 to 1% and then centrifuged to
remove the insoluble material. The supernatant was estimated
for protein concentration and 30 μg of protein was separated on
4–12% Bis-Tris (NuPAGE), transferred to Immobilon-P mem-
brane, blocked using 5% BSA in TBST, probed overnight with
chicken polyclonal proBDNF antibody (Chemicon) in 3% BSA/
Tris-buffered saline (TBS) with 1% Triton X-100 (TBS-T),
washed, and developed using anti-chicken secondary antibody
conjugated to HRP (IgY-HRP; 1:5,000; Promega) and an ECL-
plus kit (GE Healthcare).
Morpholino and siRNA. Position 45–67 relative to the start
codon
of the ORF, which is shared between the two Xenopus p75NTR
genes [p75NTRa and p75NTRb (2)], was selected (5′-AGAGC-
CAUGUUGGUCAGGGCA-3′) to generate p75NTR siRNA.
The 23-nt sense and 23-nt antisense strands with two base
overhangs (AA) were chemically synthesized by Dharmacon in
deprotected and desalted form. Scrambled p75NTR siRNA (5′-
GCUGGCCGAGGCUUAAGAGAU-3′) was used as a control.
Antisense morpholino oligonucleotides, which achieve their ef-
fects by inhibiting translation initiation through its binding to
the
5′ UTR of the target mRNA, were used to down-regulate the
expression of Xenopus genes. The Xenopus p75NTR morpholino

sequence was 5′-CCATGCTGATCCTAGAAAGCTGATG-3′.
Its scrambled control was 5′-CTACTGCAAACATGGTACT-
GCTAGG-3′ (invert antisense). Anti-Xenopus TrkB morpholino
was generated as described previously (5′-CCACTGGAT-
CCCCCCTAGAATGGAG-3′) (3). Its scrambled control was
5′-CACCTAACATGGGGGGTACCTGAGG-3′ (invert anti-
sense). Anti-Xenopus BDNF morpholino (5′-CTCACCTGAT-
GGAACTTATTTTAGC-3′), which is specific to Xenopus
BDNF,
and the control morpholino oligonucleotides with a scrambled
sequence (5′-CCTCTTACCTCAGTTACAATGTATA-3′) were
synthesized by Gene Tools. To visualize their distributions, all
morpholino oligonucleotides were tagged with a fluorophore
(FITC). The effectiveness of p75NTR siRNA, TrkB morpholino,
and BDNF morpholino was confirmed by Western blotting (Fig.
S4 and see ref. 4). All of the electrophysiological and imaging
studies on synaptic depression and retraction were done in a
blind manner such that the experimenter did not know whether
or not the fluorescent cells expressed siRNAs/morpholinos for
scrambled or target genes.
Dual Color Labeling of Spinal Neurons for Synaptic
Competition. To
selectively label spinal neurons, a single dorsal, animal
blastomere
at the 8- or 16-cell stage was injected with fluorescent dyes
(FITC-
or rhodamine-conjugated dextran, 10,000 kDa). This protocol
has
been shown to selectively label spinal neurons but not other
nonneuronal cells (5). Down-regulation of gene expression in a
subset of Xenopus spinal neurons or muscle cells was achieved
by
embryo-injection techniques (6). FITC-conjugated TrkB mor-
pholino (3), FITC-conjugated BDNF morpholino, or p75NTR

siRNA was mixed with GFP mRNA (1 μg/mL) in a 1:1 ratio and
injected into the Xenopus embryos at the two-cell stage. One
day
after injection, the neural tube and associated myotomal tissues
were dissected from differently injected embryos and mixed,
and
nerve–muscle cocultures were prepared as described (7).
Time-Lapse Microscopy. Confocal imaging was performed
using an
inverted Zeiss LSM 510META laser scanning microscope with
a ×25 (1.0 NA) or ×40 (1.3 NA) oil immersion objective
(Zeiss).
For dual- or triple-color imaging, excitation lines of an argon
laser of 488 nm and two helium lasers of 543 nm and 640 nm
were used. Fluorescence was detected using a 458-/514-nm di-
chroic beam splitter and a 530- to 560-nm bandpass filter for
FITC,
a 580- to 620-nm bandpass filter for rhodamine, and a 650-nm
long-pass filter for Cy5. With the narrow band-pass filters, any
crossover or bleed-through of fluorescence was eliminated.
Time-
Je et al. www.pnas.org/cgi/content/short/1207767109 1 of 8
www.pnas.org/cgi/content/short/1207767109
lapse scanning was performed using Zeiss LSM 510 imaging
system software. Postacquisition images were processed with
the LSM 5 Image Browser (Zeiss) and Adobe Photoshop 7.0
software. One phase-contrast image and subsequent fluorescent
images were recorded every 5 or 10 min with Z-series stack at
1.0-μm intervals. Expression of fluorescent dyes or siRNA did
not affect axon morphology compared with uninjected controls.
Axons were reconstructed 3D. For time-lapse confocal imaging,

identical settings (acquisition speed, pinhole size, laser in-
tensities, dichroics, filters, size of uncaging region of interest
(ROI), uncaging iterations, etc.) were used except for the gain,
because of the heterogeneity of fluorescence signals. We also
tested the photobleaching effects. Even after 6 h, the level of
photobleaching was less than 10% of fluorescence signal. Fur-
thermore, to circumvent focal point changes during long-term
imaging, several Z-stacks (7–10 stacks) were acquired and later
projected to 2D with maximum intensity. After image acquisi-
tion, threshold was adjusted for better visualization. Finally,
we used high-powered differential interference contrast (DIC)
optics to trace the remnant of the axon terminals to ensure the
entire axon arbor was efficiently visualized.
Two-Photon Uncaging. Two-photon laser-scanning microscope
was
directly coupled to a Mira Ti:sapphire laser (100-fs pulses; 76
MHz, pumped by a 5-W Verdi laser; Coherent). For maximum
uncaging of MNI-glutamate (50 μM; Tocris), the Ti:sapphire
laser (725 nm) was beamed at the soma of a single neuron, with
a laser power of 5 mW and duration of 250 ms. The x-y
scanning
of a ROI was comprised of 25 by 25 pixels (1 pixel, 0.45 μm)
and
was performed at single z-axis planes 25 times using Zeiss
LSM510 imaging system software. Scanning speed of each pixel
was 2.5 μs.
FRET-Based Molecular Beacons. The secretion/activation of
proBDNF-
cleaving proteases was detected by a FRET-based fluorescence
beacon, which is a peptide containing the cleavage sequences
(MSMRVRRHSD) of proBDNF, flanked by a fluorophore [rho-
damine (ROX)] at the N terminus and a quencher at its C
terminus.
In the intact FRET peptide, the fluorescence of ROX is

quenched
by QXL610. Upon cleavage, the fluorophore is dissociated from
the quencher, generating fluorescence, which can be monitored
at excitation/emission wavelengths of 567/591 nm. To facilitate
the detection of proteolytic cleavage locally on axonal
terminals,
polystyrene beads containing the fluorogenic peptide beacons
were prepared. Polystyrene beads (10 μm in diameter;
Polysciences)
were rinsed in 50 volumes of PBS four to six times, incubated
in
5mg/mL heparin for 1 h at room temperature, and soaked in
peptide beacons (final concentration: 250 μg/mL, from a 1
mg/mL
stock in DMSO, 0.5% BSA in PBS) for 2 h at room temperature
with gentle agitation. The beads were then rinsed in PBS three
times and applied gently near neurons in the culture dishes. The
beads were manipulated into contact with neuronal axons by a
glass
pipette controlled by a micromanipulator. Control beads were
soaked in 0.5% BSA in PBS in the same manner. Various
protease
inhibitors (Sigma) were applied 10 min before stimulation of
spinal neurons by photo-uncaging. Fluorescence changes in
beads
were monitored using Olympus IX70 inverted microscope
equip-
ped with a Hamamatsu ORCA-ER cooled CCD camera, and data
were processed off-line with the IPLab software (Scanalytics).
Quantification of Axon Elongation and Retraction. Using an
ROI
tool from the IPLab software, the optical center of mass (COM)
of a given axon terminal was identified. For single muscle-in-
nervating axons, the distances between the COM of the AChR

fluorescence signal and the COM of axon terminal were deter-
mined at different time points after proBDNF application. For
dually innervated axons, the distances between the COM of a
single
muscle cell and the COM of the two competing terminals were
measured. The extent of retraction (or elongation) was
calculated
by subtracting the distance at a given time point after neuronal
stimulation with that at before stimulation. The values for mul-
tiple axons in the same condition were pooled and averaged.
Immunofluorescence Staining of Xenopus Cultures. Xenopus
neurons
and muscle cells grown on coverslips were washed with PBS
(PBS), fixed with 4% paraformaldehyde in PBS for 30 min at
room temperature, incubated in 0.1% NaBH4 (sodium borohy-
drate) in PBS to reduce auto-fluorescence, and blocked using
5% nonfat milk in PBS for 60 min. For proBDNF surface
staining, the cultures were incubated with proBDNF antibody
(Chemicon; 1:200) in 5% nonfat milk overnight at 4 °C. For
mBDNF surface staining, cells were reacted with a newly gen-
erated antibody specific for mBDNF (1:250) in 5% BSA. The
cultures were then reacted with secondary antibody (Alexa
Fluor
antibodies from Molecular Probes) for 1 h. Following PBS
washes, the coverslips were rinsed with water and then mounted
on medium containing Mowiol 4-88 and the antifade medium
DABCO (1,4-diazabicyclo[2.2.2]octane). Images were acquired
using a 63× oil plan Apochromat lens (1.4 NA) and multitrack
option in the Zeiss confocal LSM 510 Meta for all samples on
the same day under identical conditions (laser power, pinhole,
gain, and offset for two different colors). For quantitative anal-
ysis of proBDNF or mBDNF fluorescence signals, we first sub-
tracted background and adjusted the threshold to 50% over the
background fluorescence intensity by averaging the numbers
obtained from three nonfluorescent areas. The dynamic range of

fluorescence intensity values were confined to arbitral fluores-
cence unit in 8 bits [in pixels; 0 (minimum) – 255 (maximum)]
to
normalize fluorescent signals from differently treated groups.
Average intensities from all positively stained spots along syn-
aptic area in a myocyte were obtained using the ROI tool in
IPLab software. Then, the average intensity values of fluores-
cence signal in a given synapse were averaged and presented as
mean ± SEM.
For immunofluorescence staining of TrkB, p75NTR, and in-
tracellular proBDNF, similar procedures were used except that
cells were permeabilized with 0.05% Triton X-100 in PBS for
5 min. After blocking, cells were incubated with TrkB antibody
(Santa Cruz; 1:50) or p75 NTR ICD antibody (kindly provided
by
Bruce Carter; 1:100) overnight at 4 °C.
1. Lee R, Kermani P, Teng KK, Hempstead BL (2001)
Regulation of cell survival by secreted
proneurotrophins. Science 294:1945–1948.
2. Hutson LD, Bothwell M (2001) Expression and function of
Xenopus laevis p75(NTR)
suggest evolution of developmental regulatory mechanisms. J
Neurobiol 49:79–98.
3. Du JL, Poo MM (2004) Rapid BDNF-induced retrograde
synaptic modification in
a developing retinotectal system. Nature 429:878–883.
4. Yang F, et al. (2009) Pro-BDNF-induced synaptic depression
and retraction at
developing neuromuscular synapses. J Cell Biol 185:727–741.
5. Moody SA (1989) Quantitative lineage analysis of the origin

of frog primary motor and
sensory neurons from cleavage stage blastomeres. J Neurosci
9:2919–2930.
6. Wang T, Xie KW, Lu B (1995) Neurotrophins promote
maturation of developing
neuromuscular synapses. J Neurosci 15:4796–4805.
7. Je HS, Zhou J, Yang F, Lu B (2005) Distinct mechanisms for
neurotrophin-3-induced
acute and long-term synaptic potentiation. J Neurosci 25:11719–
11729.
A
1minMNI-glutamate uncaging
60 min
Control
TTX
B
0
5 0 0
1 0 0 0

1 5 0 0
0 5 0 1 0 0 1 5 0
Distance between neuronal
soma and uncaging spot ( m)
S
S
C
fr
eq
ue
nc
y
no
rm
al
iz
ed
to
un
tr
ea
te
d

co
nt
ro
l (
%
)
25 m
MNI-glutamate uncaging
Fig. S1. Stimulation of spinal neurons by photo-uncaging of
MNI-glutamate. Caged-glutamate compound (MNI-glutamate,
50 μM) was added to the culture
medium. Synaptic currents were recorded from a myocyte
innervated by spinal neurons. A laser spot of 10 by 10 μm was
applied to the cell body region of the
spinal neurons for 250 ms (indicated by the upward line). (A)
Glutamate uncaging on spinal neuron resulted in a dramatic
increase in spontaneous synaptic
current (SSC) frequency, which lasted for more than 1 h
(Upper). The effect of photo-uncaging is completely blocked by
inclusion of TTX (1 μM) in the medium
(Lower). (B) Relationship between percentage increase in SSC
frequency and the distance of uncaging spot from neuronal cell
body. Note that uncaging further
than 25 μm from cell body did not evoke increase in SSC
frequency (n = 15).
S
tim
.

U
ns
tim
.
0 30 60 90 120
M
er
ge
d
Fig. S2. Reverse case of Fig. 1, in which the soma of a green
neuron (outside the field) is stimulated. The axon terminal of a
red neuron (yellow arrow) (Lower)
retracted, whereas the terminal of a green neuron (Upper) did
not. A myocyte (also contained a small amount of red dye) was
indicated with white dotted
lines. The phase (Bottom) and two-color fluorescence images
(Top and Middle) of the triplet at “0” and “80” min are shown.
Fl
uo
re
sc
en

ce
in
te
ns
ity
(A
U
)
Control
+ high K+/MMP-In.
+ high K+
0
0 .2
0 .4
0 .6
0 .8
1
1 .2
1 .4
*
*
30 57 53
+Glu. + Glu./MMP-In.Control
B

F
IH
C
: m
B
D
N
F
B
0
20
40
60
80
100
*
*m
B
D
N
F
st
ai

ni
ng
Fl
uo
re
sc
en
ce
in
te
ns
ity
(A
U
)
Control (9)
+ Glu./MMP-In. (12)
+ Glu. (24)
C
M
M

M
M
M
M
M
N
N
N
A
Fig. S3. Quantification of cell surface staining of secreted
proBDNF and mBDNF. (A) Cultures were treated with drugs as
indicated, lightly fixed under
nonpermeable conditions, and processed for surface
immunocytochemistry using a polyclonal antibody specific for
proBDNF. Number associated with legends
represent number of myocytes. *P < 0.01. (B) Cell surface
staining of secreted mBDNF. Cultures were treated with drugs
as indicated, lightly fixed under
nonpermeable conditions, and processed for surface
immunocytochemistry using a polyclonal antibody specific for
mBDNF. mBDNF immunoreactivities are
shown as inset images on top of DIC images. Two bigger circles
(inside of a middle, lower image) indicate the enlargement of
the areas on muscle cells. Please
note more mBDNF immunoreactivity on a muscle cell,
innervated by a spinal neuron. (Scale bar: 20 μm.) (C)
Quantification of the immunofluorescence is shown

as bar graph on the right. Number associated with legends
represents number of myocytes. “+Glu./MMP-In” means cells
were treated with glutamate (10 mM)
in the presence of MMP inhibitors (60 μM). M, muscle cell; N,
neuron. *P < 0.01.
B mBDNF
mBDNF
control
TrkB morpholino N+
5min
5min
50
0p
A
1min
0
1
2

3
4 - mBDNF
+ mBDNF
N
or
m
al
iz
ed
S
S
C
fr
eq
ue
nc
y
N-N+ M+
TrkB morpholino
*
8 9 5
*
C

A
0.0
0.2
0.4
0.6
0.8
1.0
1.2
N
or
m
al
iz
ed
in
te
ns
ity
*
S
cr
m
. M
or
ph

.
Tr
kB
M
or
ph
.
C
tl.
4
C
tl.
S
cr
m
. M
or
ph
.
Tr
kB
M
or

ph
.
TrkB
tubulin
Fig. S4. Reduction of endogenous TrkB levels by embryo
injection of morpholino. TrkB morpholinos, or its scrambled
analogs, were introduced into the
developing Xenopus by embryo injection. Neural tubes were
dissected and proteins were extracted. Western blots were
performed using TrkB specific an-
tibody. The blot was also probed with an anti-tubulin antibody
for loading controls. (A) Down-regulation of TrkB expression
by TrkB morpholino. Note
a reduction of TrkB expression in embryos injected with TrkB
morpholino but not in those with scrambled morpholino. (B and
C) Blockade of BDNF-induced
synaptic potentiation by presynaptic expression of TrkB
morpholino. BDNF (25 ng/mL) was applied directly to 1-d-old
nerve–muscle cultures expressing TrkB
morpholino either presynaptically (N+) or postsynaptically
(M+). An example (B) and the summary (C) of the effect of
TrkB morpholino on acute synaptic
potentiation induced by BDNF are shown. Each data point
represents normalized SSCs (averaged from 10 min of
recording) from a single synapse before and
after BDNF application. Note that presynaptic, but not
postsynaptic expression of TrkB morpholino prevents BDNF-
induced acute synaptic potentiation of SSC
frequency.

0
0 20 40 60 120
20 40 60 120
S
cr
am
bl
ed
p7
5N
TR
si
R
N
A
U
ns
tim
.
S
tim

.
Fig. S5. Time course showing retraction of an axon from a
neuron expressing scrambled p75NTR siRNA. Stimulation of
the red neuron resulted in the retraction
of green terminal (yellow arrow) expressing scrambled p75NTR
siRNA but not red terminal (white arrow) expressing true
p75NTR siRNA. Phase and two-color
fluorescence images at “0” min are shown on the left.
S
tim
.
U
ns
tim
.
MMP 3 & 9 In.
Fig. S6. Synaptic retraction mediated by inhibition of MMP3
and -9 metalloproteases. Inhibition of MMP3 and -9 cleavage
leads to retraction of both axon
terminals coinnervating the same myocyte. The culture was
pretreated with both MMP3 inhibitor (50 nM) and MMP9
inhibitor (50 μM) for 15 min. After
stimulating the soma of the red neuron, both red and green
terminals (yellow arrows) retracted from their prior synaptic
sites.
Win Lose
Synaptic

retraction
p75NTR
Less
active
More
active
proBDNF
protease
Trk
mBDNF
proBDNF
Fig. S7. Activity-dependent conversion of proBDNF to mBDNF:
a model for activity-dependent synaptic competition and
elimination at the NMJ. During
neuromuscular development, presynaptic innervation drives the
production and secretion of proBDNF from the postsynaptic
muscle cell. At the active axonal
terminal, proteases secreted into the synaptic cleft convert
proBDNF to mBDNF, which binds TrkB and triggers a series of
downstream signaling, leading to the
stabilization of the synaptic connection. By contrast, relatively
fewer active terminals are unable to convert proBDNF, which
binds p75NTR, to facilitate synaptic
depression and eventually retraction of these terminals.

Movie S1. Activity-dependent synaptic competition in culture.
Neurons were separately labeled with FITC-conjugated (green)
and rhodamine-conjugated
(red) dextran, respectively, by embryo injection. Stimulation of
the red neuron, by photo-uncaging of MNI-glutamate in the
soma area using a two photon
laser, caused the unstimulated (green) axon terminal to retract
from the synaptic target (indicated by a yellow arrow) (Upper).
In contrast, the axon terminal
(red) from the stimulated neuron sustained (indicated by a white
arrow) (Lower).
Movie S1
Movie S2. Treatment with mBDNF prevents activity-dependent
synaptic retraction. The culture was treated with mBDNF for 15
min, before stimulating the
soma of a red neuron. The terminals from both red and green
neurons remained in the synaptic site without any retraction.
Movie S2
Movie S3. Inhibition of proteolytic cleavage leads to retraction
of both axon terminals coinnervating the same myocyte. The
culture was pretreated with
protease inhibitors for 15 min. After stimulating the soma of the
red neuron, both red and green terminals (yellow arrows)
retracted from their prior
synaptic sites.
Movie S3

Movie S4. Inhibition of proteolytic cleavage by MMP3/MMP9
leads to retraction of both axon terminals coinnervating the
same myocyte. The culture was
pretreated with combined MMP3 inhibitor and MMP9 inhibitor
for 15 min. After stimulating the soma of the red neuron, both
red and green terminals (yellow
arrows) retracted from their prior synaptic site.
Movie S4

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