Control of Local Protein Synthesis and Initial Events in Myelination by Action Potentials Hiroaki Wake, Philip R. Lee, R. Douglas Fields* Formation of myelin, the electrical insulation on axons produced by oligodendrocytes, is controlled by complex cell-cell signaling that regulates oligodendrocyte development and myelin formation on appropriate axons. If electrical activity could stimulate myelin induction, then neurodevelopment and the speed of information transmission through circuits could be modified by neural activity. We find that release of glutamate from synaptic vesicles along axons of mouse dorsal root ganglion neurons in culture promotes myelin induction by stimulating formation of cholesterol-rich signaling domains between oligodendrocytes and axons, and increasing local synthesis of the major protein in the myelin sheath, myelin basic protein, through Fyn kinase-dependent signaling. This axon-oligodendrocyte signaling would promote myelination of electrically active axons to regulate neural development and function according to environmental experience. Myelin, the multilayered membrane of insulation wrapped around axons by oligodendrocytes, is essential for ner- vous system function and increases conduction velocity by at least 50 times (1, 2). Unique to vertebrates, formation of the myelin sheath must be highly regulated temporally during develop- ment and targeted specifically to appropriate axons. Many axon-derived signals regulate my- elination, but there is great interest in the pos- sibility that electrical activity could provide an instructive signal, because activity-dependent regulation of myelinogenesis could control my- elination during development according to en- vironmental experience, contribute to learning, and guide regeneration after injury according to functional efficacy (3). Electrical activity has been shown to affect proliferation and differen- tiation of myelinating glia (4–7), but if electrical activity could regulate subcellular events neces- sary for myelin induction, then myelin could form preferentially on electrically active axons. Here we test this hypothesis, beginning with the question of how electrical activity in axons might signal to oligodendrocytes to control myelination. Both neurotransmitters adenosine 5′-triphosphate (ATP) and glutamate (glu) have been implicated in signaling to oligodendrocyte progenitor cells (OPCs). Glutamatergic synapses can form tran- siently between axons and someOPCs (8, 9). It has been proposed that such synaptic communication Nervous System Development and Plasticity Section, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. *To whom correspondence should be addressed. E-mail: [email protected] Fig. 1. Release of synaptic vesicles from axons promotes myelination. (A) Synaptic vesicle release from DRG neurons was blocked by adding BnTX or TnTX to neuron cultures, and OPCs were added after washing.

1. Control of Local Protein Synthesis
and Initial Events in Myelination by
Action Potentials
Hiroaki Wake, Philip R. Lee, R. Douglas Fields*
Formation of myelin, the electrical insulation on axons
produced by oligodendrocytes, is
controlled by complex cell-cell signaling that regulates
oligodendrocyte development and myelin
formation on appropriate axons. If electrical activity could
stimulate myelin induction, then
neurodevelopment and the speed of information transmission
through circuits could be modified
by neural activity. We find that release of glutamate from
synaptic vesicles along axons of
mouse dorsal root ganglion neurons in culture promotes myelin
induction by stimulating formation
of cholesterol-rich signaling domains between oligodendrocytes
and axons, and increasing local
synthesis of the major protein in the myelin sheath, myelin
basic protein, through Fyn
kinase-dependent signaling. This axon-oligodendrocyte
signaling would promote myelination of
electrically active axons to regulate neural development and
function according to
environmental experience.
Myelin, the multilayered membrane of
insulation wrapped around axons by
oligodendrocytes, is essential for ner-
vous system function and increases conduction

2. velocity by at least 50 times (1, 2). Unique to
vertebrates, formation of the myelin sheath must
be highly regulated temporally during develop-
ment and targeted specifically to appropriate
axons. Many axon-derived signals regulate my-
elination, but there is great interest in the pos-
sibility that electrical activity could provide an
instructive signal, because activity-dependent
regulation of myelinogenesis could control my-
elination during development according to en-
vironmental experience, contribute to learning,
and guide regeneration after injury according to
functional efficacy (3). Electrical activity has
been shown to affect proliferation and differen-
tiation of myelinating glia (4–7), but if electrical
activity could regulate subcellular events neces-
sary for myelin induction, then myelin could
form preferentially on electrically active axons.
Here we test this hypothesis, beginning with the
question of how electrical activity in axons might
signal to oligodendrocytes to control myelination.
Both neurotransmitters adenosine 5′-triphosphate
(ATP) and glutamate (glu) have been implicated in
signaling to oligodendrocyte progenitor cells
(OPCs). Glutamatergic synapses can form tran-
siently between axons and someOPCs (8, 9). It has
been proposed that such synaptic communication
Nervous System Development and Plasticity Section, The
Eunice
Kennedy Shriver National Institute of Child Health and Human
Development, Bethesda, MD 20892, USA.

3. *To whom correspondence should be addressed. E-mail:
[email protected]
Fig. 1. Release of synaptic vesicles from axons
promotes myelination. (A) Synaptic vesicle release
from DRG neurons was blocked by adding BnTX or
TnTX to neuron cultures, and OPCs were added
after washing out the toxin. Five days later, axons
were stimulated for 5 hours (10 Hz, 9 s at 5-min
intervals), and they were examined 21 days later.
(B and C) Myelin formation was greatly reduced in
cultures in which vesicular release was blocked
(MBP, green; neurofilament, purple). Scale bar,
10 mm (P < 0.005, n = 7). (D and E) OPCs had
differentiated into oligodendrocytes regardless
of whether vesicular release was blocked during
electrical stimulation (black bar, –BnTX; gray bar,
+BnTX), as indicated by protein expression (D and E)
for myelin proteins [proteolipid protein 1 (PLP1),
MBP, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase
(CNP)] and the transcription factor Olig2.
A
day 0
Plate OPC
onto DRG
day 1 day 5
Electrical stimulation
5 hr
day 21
Collect protein

4. Fix the samples
+BnTX or -BnTX
or +TnTX
B
E
N
um
be
r
of
m
ye
lin
s
eg
m
en
ts
/c
el
l
+
B

5. nT
X
-B
nT
X
+B
n
T
X
-B
n
T
X
*
4
8
12
PLP1 CNP MBP OLIG2
- + +++- - -
1.0
2.0
R
el

6. at
iv
e
ex
pr
es
si
on
o
f
m
ye
lin
r
el
at
ed
p
ro
te
in
n.s n.s n.s n.s

7. 0
0.0
*
C
+
T
nT
X
GAPDH
PLP1
CNP
MBP
OLIG2
BnTX
- +
Stimulation
D
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between axons and OPCsmight stimulate myelin
formation on individual axons that are electrically
active (10). However, glu inhibits OPC prolifer-
ation and differentiation in monoculture (11).
Electrical activity also causes nonvesicular release
of the neurotransmitter ATP from axons through
volume-regulated anion channels (12), and ATP
released from axons increases myelin formation
by regulating OPC differentiation and expression
of myelin proteins (5, 7).
Our measurements confirm that electrical ac-
tivity stimulates both vesicular release of glu and
nonvesicular release of ATP from mouse dorsal
root ganglion (DRG) neurons in a cell culture
preparation equipped with platinum electrodes
(13) (fig. S1). Immunocytochemical staining for
the postsynaptic protein, PSD-95, failed to pro-
vide evidence for postsynaptic specializations on

9. OPCs. However, punctate staining for the synaptic
vesicle glycoprotein, synaptophysin, was evident
along DRG axons (fig. S2A). Active recycling of
vesicles between these sites of vesicle aggrega-
tion and the axon membrane was shown by up-
take of a lipophilic FM dye (fig. S2, A to C).
To test the hypothesis that release of synaptic
vesicles from axons can promote myelination,
DRG neurons were treated for 18 hours with
botulinum toxin A (BnTX), which cleaves the
synaptic vesicle release protein SNAP-25 (25-kD
synaptosome–associated protein) (14). SNAP-25
is necessary for synaptic vesicle fusion, and neu-
rotransmitter release is blocked for at least 2weeks
after washing out the toxin from DRG neurons
(14). OPCs were added to neuron cultures after
washing out the toxin, so that only vesicular re-
lease from axonswould be impaired, and allowed
-BnTX
+BnTX
C
a
2+
r
is
e
in
O

10. P
C
(t
im
e
to
p
ea
k,
C
a2+
r
is
e/
se
c)
B
*
0
0s0s 2s2s
2
4s4s

11. 0 20
C
a2+
re
sp
on
se
in
O
P
C
(f
lu
or
es
ce
nc
e
in
te
ns
ity

12. ,∆
F
/F
0
)
(10Hz)
(10Hz)
Process
Soma
40 60
P
ro
ce
ss
Cell soma
+
B
nT
X
-B
nT
X

13. NG2NG2GCaMp2GCaMp2 PDGFPDGF
0s 0.4s 0.8s
-B
n
T
X
+B
n
T
X
Cell soma
Cell soma
Process
Process
C
-B
n
T
X
-BnTX
+BnTX

14. 4s4s
1.0
1.4
1.8
1.8
1.4
1.0
0
30
60
G
S
tim
S
tim
w
ith
B
nT
X

15. N
o
st
im
G
lu
a
nt
ag
on
is
ts
N
um
be
r
of
C
a2+
r
es
po
ns
es

16. events/sec
5 sec
20 60 sec40
H I
1.0
P
ro
ce
ss
Cell soma
+
B
nT
X
C
a2+
r
is
e
in
O
P

17. C
(
∆F
/F
0
)
-B
nT
X1.0
2.0
0
n.s n.s
*
** ** **
2s2s0s0s
A
0.01
0.02
0.00
D

18. C
a2+
r
is
e
in
O
P
C
s
om
a
(
∆F
/F
0
no
rm
al
iz
ed
to
c

19. on
tr
ol
)
0 .2
0.6
1.0
S
ur
am
in
S
ur
am
in
+
gl
u
an
ta
go
ni

20. st
s
+BnTX
*
E
S
tim
+
T
T
X
S
tim
O
P
C
m
on
oc
ul
tu
re

21. ** **
C
a2+
r
is
e
in
O
P
C
(
∆F
/F
0
)
0.0
0.8
1.6 F
0
∆F/F
Fig. 2. Electrical activity in axons is signaled to OPCs by the
neurotrans-
mitters glu and ATP. The two neurotransmitters are released
through dif-

22. ferent mechanisms and produce different spatiotemporal Ca2+
responses in
OPCs. (A) Ca2+ responses were seen in the cell body of OPCs
(white arrows),
using the Ca2+ indicator Oregon Green 1,2-bis(2-
aminophenoxy)ethane-
N,N,N′,N′-tetraacetic acid 1, AM ester form (BAPTA-1 AM), in
response to
electrical stimulation of cocultures when synaptic vesicle
release was blocked
with BnTX, but responses in OPC cell processes (green box and
arrows) were
only seen when vesicular release was not blocked. Scale bar, 10
mm. (B) Plot
of Ca2+ responses in the soma (red) and cell processes (black)
of OPCs shown
in (A). Note the absence of responses in OPC cell process on
neurons when
vesicular release was blocked with BnTx. Red bar, 10 Hz field
stimulation. (C)
No Ca2+ response was produced when action potentials were
blocked with
tetrodotoxin (TTX), or when stimulation was delivered to OPCs
in mono-
culture (*P < 0.005, n = five dishes in each category). (D) The
peak Ca2+
concentration and (E) rate of Ca2+ rise after stimulation were
not statistically
different in the cell body of OPCs (red) on neurons treated with
BnTX. No Ca2+
responses were evident in the cell processes (black) after
stimulating neurons
treated with BnTX (n = 13 dishes for each condition). (F)
Somatic Ca2+ re-

23. sponses were inhibited by the P2 receptor blocker suramin (50
mM) and were
completely blocked by a combination of suramin and glu
receptor antagonists
AP5 (50 mM) and CNQX (20 mM). (G) The genetic Ca2+
reporter GCaMP2
transfected into OPCs (green) enabled Ca2+ responses to be
measured spe-
cifically in OPCs. Immunological staining of chondroitin sulfate
proteoglycan 4
(NG2, red) and platelet-derived growth factor receptor (PDGFR)
(purple). (H)
Ca2+ responses were not seen when GCaMP2 was used in OPC
cell processes
and vesicular release was blocked by BnTX. (I) Summary data
showing Ca2+
responses measured by GCaMP2. Glu antagonists were 20 mM
CNQX + 50 mM
AP5 + 500 mM MCPG [**P < 0.001 (black bar); n = 45 cells.]
Ca2+ responses
after 5 hours of stimulation were similar to acute responses (fig.
S8).
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5 days to differentiate to a promyelinating oligo-
dendrocyte. DRG axons were then stimulated for
5 hours (9 s at 10 Hz, 5-min intervals) and ex-
amined 21 days later (Fig. 1A). Similar exper-
iments used tetanus toxin (TnTX), which blocks
vesicular fusion by cleaving a different synaptic
vesicle protein, VAMP (vesicle-associated mem-
brane protein or synaptobrevin). Both experi-
ments showed that myelination was suppressed
in comparison with cultures where vesicular re-
lease from axons was not blocked (Fig. 1, B and
C, and fig. S3). No differences in cell differen-
tiation, amount of myelin proteins, or number of
oligodendrocytes were evident between cultures
stimulated in the presence or absence of BnTX
(Fig. 1D and fig. S4), which suggested a require-
ment for synaptic vesicle release on myelin in-
duction. Although other mechanisms are likely
necessary for maintenance of myelin, the increased
induction of myelination after only a 5-hour stim-
ulus has consequences that are observed as in-
creased myelination persisting after 16 days.
Calcium imaging showed that both glu and
ATP release can signal electrical activity in axons
to OPCs, but the spatiotemporal dynamics of
Ca2+ signaling differed for the two neurotrans-

25. mitters (Fig. 2, A and B). Stimulation (10 Hz
for 15 s) of DRG neurons induced rapid Ca2+
responses in the slender OPC cell processes
(Fig. 2, A to E), but the rise time and decay were
slower in the OPC soma. This was confirmed by
transfecting OPCs with the genetic Ca2+ indicator
GCaMP2 (Fig. 2, G to I), which allows measure-
ment of Ca2+ responses in OPCs independently
from responses in axons. Blocking vesicular re-
lease with BnTX or blocking glu receptors with
a combination of 6-cyano-7-nitroquinoxaline-2,3-
dione (CNQX), DL-2-amino-5-phosphonopentanoic
acid (AP5), and a-methyl-4-carboxyphenylglycine
(MCPG) inhibited Ca2+ responses in OPC pro-
cesses closely associated with axons, but failed to
block Ca2+ responses in the cell soma (Fig. 2, H
and I, and fig. S5). Somatic Ca2+ responses were
inhibited by the P2 receptor blocker suramin
(Fig. 2F and fig. S6). The latency of the Ca2+
response to axon stimulation provides evidence
for both ectopic release of neurotransmitter and
release of neurotransmitter localized to points of
axo-glial junctions (fig. S7). We conclude that
vesicular release of glu is a major pathway for
activity-dependent signaling between axons and
OPC processes closely associated with axons and
that ATP signaling from axons has a predominant
effect on somatic Ca2+ responses, presumably be-
cause the release mechanism is less restricted to
specialized sites of axo-glial contact (Fig. 2F).
Therefore, vesicular release of glu is a likelymecha-
nism to control axo-glial signaling initiating myeli-
nation in OPC processes associated with axons.

26. In human development, oligodendrocytes in
cerebral white matter mature 3 months before
they begin to form myelin, which suggests that
local axon-glial signaling regulates induction of
myelin on specific axons (15). Myelinogenesis
requires cell recognition, the formation of spe-
cialized contacts for intercellular signaling, and
the synthesis of large quantities of lipids and
specialized proteins necessary for formation of
the myelin sheath. Cholesterol-rich microdomains
are organizing centers for signaling molecules
and cytoskeletal elements controlling traffick-
ing of membrane proteins and receptors to spe-
cialized sites of cell-cell contact (16, 17). In
oligodendrocytes, specific axon-glial interactions—
involving activation of the Src-family kinase
Fyn (18), cell adhesion molecule L1, and integrin
(16) concentrated in lipid-raft microdomains—
regulate subcellular events necessary for induc-
tion of myelination on appropriate axons. This
includes the local translation of myelin basic
protein (MBP) from mRNA in oligodendrocyte
processes. It is not known whether electrical ac-
tivity can control the formation or activity of
these signaling complexes.
To test this hypothesis, we monitored traf-
ficking of the transferrin receptor (TfR) into the
membrane of OPCs. The TfR is enriched in
cholesterol-rich membrane domains, notably at
the postsynaptic membrane of neurons (19). OPCs
were transfected with TfR-mCherry–superecliptic
pHluorin (SEP), which becomes fluorescent on-
ly after exocytosis and exposure to neutral pH

27. (20). The results show that stimulation of DRG
axons (10 Hz, 9 s, at 5-min intervals for 5 hours)
increased TfR surface expression on OPCs (Fig.
3A). This increased trafficking, in turn, required
vesicular glu release from axons, as shown by
blocking the increased TfR trafficking by stim-
ulation of neurons pretreated with BnTX or in
A B
T
fR
s
ur
fa
ce
e
xp
re
ss
io
n
(
pu
nc
ta
/µ
m

28. )
S
tim
N
o
st
im
B
nT
X
D
R
G
G
lu
a
nt
ag
on
is
ts
*
*

29. +Glu antagonists+Glu antagonistsStimulationStimulation
No stimulationNo stimulation +BnTX+BnTX
0.00
0.15
0.30
* ** **
Surface TfR Phospho Fyn kinaseC Merge
0.0
1.0
2.0
3.0
R
el
at
iv
e
ex
pr
es
si
on

30. o
f p
ho
sp
ho
-F
yn
(P
ho
sp
ho
-F
yn
/ t
ot
al
F
yn
)
p-Fyn
t-Fyn
D

31. *
+
B
nT
X
S
tim
N
o
st
im
Fig. 3. Formation of axon-oligodendrocyte signaling domains by
elec-
trical stimulation. Trafficking of TfR into cholesterol-rich
membrane
domains of OPCs was increased by the vesicular release of glu
from
neurons induced by electrical stimulation. (A) Punctate
expression of TfR
on the membrane (yellow) was greatly increased by 5 hours of
electrical
stimulation (10 Hz, 9 s, at 5-min intervals) of neurons (purple,
neuro-
filament), but BnTX treatment or glu receptor antagonists (20
mM
CNQX + 50 mM AP5), strongly inhibited trafficking of TfR into
the membrane induced by electrical
stimulation. Scale bar, 10 mm. (B) Stimulation of neurons
treated with BnTX or in the presence of glu

32. receptor antagonists inhibited trafficking of TfR receptor into
cholesterol-rich membrane domains after
stimulation (*P < 0.005; **P < 0.001; n = 21 cells from 21
dishes in each condition). (C) Phosphorylated Fyn
kinase (red) colocalized with TfR receptor (green), consistent
with axo-glial signaling localized at cholesterol-
rich microdomains. (D) Electrical stimulation increased
phosphorylation of Fyn kinase (p-Fyn), and this was
blocked by BnTX treatment.
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the presence of glu receptor antagonists (Fig. 3,
A and B). Activated Fyn kinase (phosphorylated
at tyrosine 418) colocalized with the surface ex-
pression of TfR receptors (Fig. 3C). Electrical
stimulation increased phosphorylation of Fyn

33. kinase (Fig. 3D and fig. S9) and increased the
surface expression of cell adhesion molecule
L1 (fig. S9A). Both responses required vesicular
release, as shown by BnTX treatment before
stimulation (Fig. 3D and fig. S9A).
These results indicate that vesicular release
of glu from axons stimulates the formation of
cholesterol-rich microdomains in oligodendro-
cytes and activates signaling pathways known to
regulate local translation of MBP. This raises the
hypothesis that local translation of MBP in
oligodendrocytes could be controlled by elec-
trical activity. We focused on MBP because it is
the major protein constituent of the myelin
sheath and because it is required for formation
of myelin. MBP mRNA is transported in RNA
granules toward the distal ends of the oligoden-
drocyte processes where the protein is translated
and delivered locally to the plasma membrane
for myelin formation (21, 22).
To visualize local translation of MBP, we
developed a genetic construct in which MBP
was labeled with a fluorescent protein, kikume
green-red (kikGR), which is irreversibly con-
verted from green to red fluorescence by ul-
traviolet (UV) light (23). After photoconversion,
previously synthesized MBP appears red, and
newly synthesized MBP will appear as green
fluorescent spots. OPCs transfected with a con-
struct containing the MBP coding region showed
local MBP translation during a 40-min observa-
tion period after stimulation of DRG axons (10
Hz, 10 min), and the newly synthesized protein

34. was inserted into the cell membrane (Fig. 4C and
fig. S10). OPCs transfected with a construct con-
taining only the 3′ untranslated region (3′UTR) of
MBP also showed local kikGR translation only
after stimulation of DRG axons, but the protein
was not targeted to the cell membrane (fig. S10).
When stimulation was delivered locally by using
bipolar electrodes separated by 350 mm, rather
than by field stimulation of the entire culture,
only oligodendrocytes adjacent to stimulated axons
showed local translation of MBP, and neighboring
oligodendrocytes outside the region of stimula-
tion showed no MBP translation (fig. S11). Either
pretreating axons with BnTX or stimulation in
the presence of glu receptor blockers prevented
local synthesis of MBP in response to stimula-
tion. AMPA receptor or P2 receptor blockers
were without effect, but AP5 or MCPG sig-
nificantly inhibited local translation of MBP,
Fig. 4. Action potentials in-
duce local translation of
MBP by vesicular release of
glu. (A) Experiments were
carried out on rat OPCs be-
fore expressing MBP pro-
tein, although MBP mRNA
was present. (B) Local tran-
slation of MBP was studied
by transfecting OPCs with
kikume protein. Actinomycin
D was used to block tran-
scription and the appearance
of newly synthesized green-
fluorescent MBP was moni-

35. tored. (C) Electrical stimulation
induced local translation in
OPCs transfected with kikume-
MBP-3′UTR (white arrows),
which was inhibited by pre-
treating axons with BnTX or
stimulation in the presence
of glu receptor antagonists
(20 mM CNQX + 50 mM AP5 +
500 mM MCPG) (see also fig.
S10). Scale bar, 10 mm. Pixel
intensity is shown on an 8-bit
pseudocolor scale. (D) Statis-
tical analysis shows that the
local translation was strongly
increased by electrical stim-
ulation and that blocking
vesicular release with BnTX sig-
nificantly decreased stimulus-
induced local MBP translation,
as did stimulation in the pres-
ence of NMDA or mGluR recep-
tor antagonists or suppressing
Fyn kinase with siRNA. AMPA
receptor and P2 receptor an-
tagonists were without sig-
nificant effect (*P < 0.005;
**P < 0.001; n = 32 cells for
each condition, one cell sampled per dish). (E) MBP mobility
was monitored using a photoactivatible GFP–MBP (fig. S12).
The mobility of newly synthesized
MBP was increased by blocking vesicular release from axons,
but not by blocking P2 receptors with suramin (P < 0.001; n =
17 cells from 17 dishes for
each condition).

36. 0.00
0.10
0.20
0.30
A B
D
0 day
Transfection
Plate OPC onto DRG
3 day
actinomycinD
Photoactivation
0 min
Stimulation 10 Hz 10 min
40 min
Imaging
BnTX
antagonists
C

37. N
o
st
im
PDGFR
MBP
GAPDH
DRG-OPC coculture 3 days
MBP
Olig2
GAPDH
NG2
Protein RNA
S
tim
(k
ik
um
e-
M
B

38. P
-3
U
T
R
)
S
tim
+
gl
u
an
ta
go
ni
st
s
S
tim
w
ith
B
nT
X

39. Kikume-MBP-3UTR
M
B
P
lo
ca
l t
ra
ns
la
tio
n(
pu
nc
ta
/µ
m
)
E
M
ot
ili
ty

40. o
f M
B
P
(
µm
/s
ec
)
0.00
0.02
0.04
0.06
Newly synthesized MBP Total MBP
+
A
M
PA
R
a
nt
ag

41. on
is
t
+
N
M
D
A
R
a
nt
ag
on
is
t
+
m
G
lu
R
a
nt
ag

42. on
is
t
S
tim
G
lu
a
nt
ag
on
is
ts
N
o
st
im
B
nT
X
D
R
G
+

43. P
2R
a
nt
ag
on
is
t
+
N
eg
at
iv
e
co
nt
ro
l f
or
s
iR
N
A

44. +
si
R
N
A
fo
r
F
yn
k
in
as
e
* *
*
******
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which demonstrated that vesicular release of glu
from axons acting on N-methyl-D-aspartate re-
ceptors (NMDARs) and metabotropic glu re-
ceptors (mGluRs) stimulates local translation of
MBP (Fig. 4, C and D). Suppressing Fyn kinase
activity by small interfering RNA (siRNA) trans-
fection into oligodendrocytes completely blocked

46. the electrically induced local translation of MBP
(Fig. 4D).
After synthesis, myelin proteins must be re-
stricted to the appropriate membrane regions
adjacent to the axon being myelinated. To de-
termine whether vesicular release of neurotrans-
mitter from axons stabilizes mobility of myelin
proteins in OPC cell processes, MBP was vi-
sualized by using a photoactivated green fluo-
rescent protein (GFP) (24). Illumination with a
spot of UV light rendered MBP fluorescent in
a discrete 1-mm region of the OPC cell process,
so that mobility of the MBP could be monitored
by time-lapse confocal microscopy (Fig. 4E and
fig. S12). Movement of newly synthesized MBP
was highly restricted to discrete regions of OPC
cell processes in comparison with OPCs cul-
tured with neurons previously treated with BnTX
(P < 0.005). Blocking P2 receptor activation
with suramin did not prevent the restricted MBP
mobility (Fig. 4E and fig. S12). This indicates
that vesicular release of glu from axons restricts
the mobility of MBP, as would be required to
wrap MBP-containing membrane selectively
around axons firing action potentials.
Our results suggest that activity-dependent
regulation of these subcellular processes in
oligodendrocytes initiates myelin formation pref-
erentially on electrically active axons (fig. S13).
Although the mechanisms revealed here must
be confirmed in vivo, this form of activity-
dependent regulation could be important in mod-
ifying development of brain circuits according

47. to environmental experience, as myelination of
the cerebral cortex continues through at least the
first three decades of life (3). Human brain im-
aging has detected changes in white matter re-
gions after learning (25), although the cellular
basis for these changes is unknown. Regulation
of myelination by impulse activity in individual
axons could regulate neurodevelopment and thus
influence information transmission in the brain.
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Acknowledgments: We thank M. D. Ehlers for the TfR
construct; J. Lippincott-Schwartz for the photoactivatable
GFP; E. A. Johnson for BnTX; D. Abebe for assistance
with experimental animals; and J. Chan, R. Chittajallu,
and J. Ou for critical comments on an earlier draft
of this manuscript. This work was supported by the
intramural research program at the National Institute
of Child Health and Human Development and by a
Japan Society for the Promotion of Science fellowship
for H. Wake.
Supporting Online Material
www.sciencemag.org/cgi/content/full/science.1206998/DC1
Materials and Methods
Figs. S1 to S13
References (26–29)
14 April 2011; accepted 13 July 2011
Published online 4 August 2011;
10.1126/science.1206998
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Control of Local Protein Synthesis and Initial Events in
Myelination by Action Potentials
Hiroaki Wake, Philip R. Lee and R. Douglas Fields
originally published online August 4, 2011DOI:
10.1126/science.1206998
(6049), 1647-1651.333Science
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