REPORTSControl of Local Protein Synthesis
and Initial Events in Myelination by
Action Potentials
Hiroaki Wake, Philip R. Lee, R. Douglas Fields*
yelin, the multilayered membrane of
insulation wrapped around axons by
oligodendrocytes, is essential for nervous system function and increases conduction
velocity by at least 50 times (1, 2). Unique to
M
vertebrates, formation of the myelin sheath must
be highly regulated temporally during development and targeted specifically to appropriate
axons. Many axon-derived signals regulate myelination, but there is great interest in the pos-
day 5
day 21
+BnTX or -BnTX
or +TnTX
Plate OPC Electrical stimulation
onto DRG
5 hr
Relative expression of
myelin related protein
n.s
n.s
8
4
*
0
n.s
E
*
+TnTX
n.s
2.0
12
+BnTX
+BnTX
D
Number of myelin segments/cell
C
-BnTX
B
Collect protein
Fix the samples
-BnTX
day 1
*To whom correspondence should be addressed. E-mail:
fieldsd@mail.nih.gov
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
Nervous System Development and Plasticity Section, The Eunice
Kennedy Shriver National Institute of Child Health and Human
Development, Bethesda, MD 20892, USA.
Downloaded from http://science.sciencemag.org/ on October 23, 2020
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.
sibility that electrical activity could provide an
instructive signal, because activity-dependent
regulation of myelinogenesis could control myelination during development according to environmental experience, contribute to learning,
and guide regeneration after injury according to
functional efficacy (3). Electrical activity has
been shown to affect proliferation and differentiation of myelinating glia (4–7), but if electrical
activity could regulate subcellular events necessary 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 transiently between axons and some OPCs (8, 9). It has
been proposed that such synaptic communication
Stimulation
BnTX
-
+
PLP1
1.0
CNP
MBP
OLIG2
0.0
-
+
PLP1
-
+
CNP
-
+
MBP
-
+
GAPDH
OLIG2
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60
0
0.4s
1.8
20
*
Cell soma
H
0.8s
1648
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60
+BnTX
1.0
(10Hz)
20
1.0
40
*
0.2
I
Process
Cell soma
Process
Cell soma
60 sec
+BnTX
0.6
5 sec
Fig. 2. Electrical activity in axons is signaled to OPCs by the neurotransmitters glu and ATP. The two neurotransmitters are released through different 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)ethaneN,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 monoculture (*P < 0.005, n = five dishes in each category). (D) The peak Ca2+
40
1.4
PDGF
-BnTX
0s
NG2
*
-BnTX
Process
30
F
+BnTX
GCaMp2
(10Hz)
0
0
-BnTX
Cell soma
Ca 2+ rise in OPC
(time to peak, Ca2+ rise/sec)
0
n.s
+BnTX
Stim
0.0
n.s
1.0
-BnTX
** **
E
2.0
Process
D
0.8
G
Ca2+ rise in OPC (∆F/F0 )
1.6
+TTX
Stim OPC
monoculture
C
Ca2+ rise in OPC (∆F/F0 )
+BnTX
1.0
1.0
∆F/F
events/sec
0.02
0.01
Stim
Stim with BnTX
No stim
Glu antagonists
4s
2s
Soma
Ca2+ rise in OPC soma
(∆F/F0 normalized
to control )
0s
1.4
** ** **
0.00
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+ responses 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 specifically 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).
SCIENCE
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-BnTX
-BnTX
Process
Suramin+
glu antagonists
4s
2s 2
+BnTX
0s 0
1.8
Suramin
B
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 neurotransmitter release is blocked for at least 2 weeks
after washing out the toxin from DRG neurons
(14). OPCs were added to neuron cultures after
washing out the toxin, so that only vesicular release from axons would be impaired, and allowed
Number of Ca2+ responses
A
Ca2+ response in OPC
(fluorescence intensity,∆F/F0 )
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 provide evidence for postsynaptic specializations on
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 aggregation and the axon membrane was shown by uptake of a lipophilic FM dye (fig. S2, A to C).
between axons and OPCs might stimulate myelin
formation on individual axons that are electrically
active (10). However, glu inhibits OPC proliferation 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 activity stimulates both vesicular release of glu and
REPORTS
t-Fyn
3.0
*
0.00
**
**
**
No stim
Stim
+BnTX
2.0
0.0
Merge
Fig. 3. Formation of axon-oligodendrocyte signaling domains by electrical 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, neurofilament), 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
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 cholesterolrich microdomains. (D) Electrical stimulation increased phosphorylation of Fyn kinase (p-Fyn), and this was
blocked by BnTX treatment.
p-Fyn
1.0
Phospho Fyn kinase
*
Glu antagonists
D
+BnTX
0.15
BnTX DRG
No stimulation
Surface TfR
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 trafficking of membrane proteins and receptors to specialized 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 induction 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 activity can control the formation or activity of
these signaling complexes.
To test this hypothesis, we monitored trafficking 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 only after exocytosis and exposure to neutral pH
(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 stimulation of neurons pretreated with BnTX or in
0.30
No stim
+Glu antagonists
Stim
Stimulation
Relative expression of phospho-Fyn
(Phospho-Fyn/ total Fyn)
C
B
TfR surface expression
(puncta/µm)
A
ment of Ca2+ responses in OPCs independently
from responses in axons. Blocking vesicular release with BnTX or blocking glu receptors with
a combination of 6-cyano-7-nitroquinoxaline-2,3dione (CNQX), DL-2-amino-5-phosphonopentanoic
acid (AP5), and a-methyl-4-carboxyphenylglycine
(MCPG) inhibited Ca2+ responses in OPC processes 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 because the release mechanism is less restricted to
specialized sites of axo-glial contact (Fig. 2F).
Therefore, vesicular release of glu is a likely mechanism to control axo-glial signaling initiating myelination in OPC processes associated with axons.
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 specialized contacts for intercellular signaling, and
Downloaded from http://science.sciencemag.org/ on October 23, 2020
5 days to differentiate to a promyelinating oligodendrocyte. DRG axons were then stimulated for
5 hours (9 s at 10 Hz, 5-min intervals) and examined 21 days later (Fig. 1A). Similar experiments used tetanus toxin (TnTX), which blocks
vesicular fusion by cleaving a different synaptic
vesicle protein, VAMP (vesicle-associated membrane protein or synaptobrevin). Both experiments showed that myelination was suppressed
in comparison with cultures where vesicular release from axons was not blocked (Fig. 1, B and
C, and fig. S3). No differences in cell differentiation, 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 requirement for synaptic vesicle release on myelin induction. Although other mechanisms are likely
necessary for maintenance of myelin, the increased
induction of myelination after only a 5-hour stimulus has consequences that are observed as increased 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 neurotransmitters (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-
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REPORTS
the presence of glu receptor antagonists (Fig. 3,
A and B). Activated Fyn kinase (phosphorylated
at tyrosine 418) colocalized with the surface expression of TfR receptors (Fig. 3C). Electrical
stimulation increased phosphorylation of Fyn
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 oligodendrocytes 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 electrical 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 oligodendrocyte 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 converted from green to red fluorescence by ultraviolet (UV) light (23). After photoconversion,
previously synthesized MBP appears red, and
newly synthesized MBP will appear as green
fluorescent spots. OPCs transfected with a construct containing the MBP coding region showed
local MBP translation during a 40-min observation period after stimulation of DRG axons (10
Hz, 10 min), and the newly synthesized protein
was inserted into the cell membrane (Fig. 4C and
fig. S10). OPCs transfected with a construct containing 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 stimulation 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 stimulation. AMPA receptor or P2 receptor blockers
were without effect, but AP5 or MCPG significantly inhibited local translation of MBP,
16 SEPTEMBER 2011
SCIENCE
+siRNA for Fyn kinase
+Negative control for siRNA
+P2R antagonist
+mGluR antagonist
+NMDAR antagonist
BnTX DRG
Glu antagonists
+BnTX
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+Suramin +AMPAR antagonist
No stim
Stim
Motility of MBP (µm/sec)
VOL 333
-BnTX
MBP local translation(puncta/µm)
No stim
Stim
(kikume-MBP-3UTR)
Stim
with BnTX
Stim
+glu antagonists
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Fig. 4. Action potentials in- A
B
DRG-OPC coculture 3 days
duce local translation of
0 day
3 day
RNA
Protein
MBP by vesicular release of
BnTX
NG2
PDGFR
glu. (A) Experiments were
Transfection
carried out on rat OPCs bePlate OPC onto DRG
Olig2
MBP
fore expressing MBP proMBP
Photoactivation
tein, although MBP mRNA
Imaging
Stimulation 10 Hz 10 min
was present. (B) Local tranGAPDH
GAPDH
40 min
0 min
slation of MBP was studied
actinomycinD
antagonists
by transfecting OPCs with
kikume protein. Actinomycin
Newly synthesized MBP Total MBP
D
D was used to block tran- C
Kikume-MBP-3UTR
scription and the appearance
0.30
of newly synthesized greenfluorescent MBP was monitored. (C) Electrical stimulation
induced local translation in
0.20
OPCs transfected with kikumeMBP-3′UTR (white arrows),
*
*
which was inhibited by pre*
treating axons with BnTX or
0.10
stimulation in the presence
of glu receptor antagonists
(20 mM CNQX + 50 mM AP5 +
**
**
**
500 mM MCPG) (see also fig.
0.00
S10). Scale bar, 10 mm. Pixel
intensity is shown on an 8-bit
pseudocolor scale. (D) Statistical analysis shows that the
local translation was strongly
increased by electrical stimulation and that blocking
vesicular release with BnTX sigE
nificantly decreased stimulus0.06
induced local MBP translation,
as did stimulation in the pres0.04
ence of NMDA or mGluR receptor antagonists or suppressing
Fyn kinase with siRNA. AMPA
0.02
receptor and P2 receptor antagonists were without sig0.00
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).
REPORTS
erentially on electrically active axons (fig. S13).
Although the mechanisms revealed here must
be confirmed in vivo, this form of activitydependent regulation could be important in modifying development of brain circuits according
to environmental experience, as myelination of
the cerebral cortex continues through at least the
first three decades of life (3). Human brain imaging has detected changes in white matter regions 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.
References and Notes
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(1996).
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6. B. Stevens, R. D. Fields, Science 287, 2267
(2000).
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8. L. M. De Biase, A. Nishiyama, D. E. Bergles, J. Neurosci.
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13. R. D. Fields, C. Yu, E. A. Neale, P. G. Nelson,
in Practical Electrophysiological Methods: A Guide
for In Vitro Studies in Vertebrate Neurobiology,
H. Kentenmann, R. Grantyn, Eds. (Wiley, New York,
1992), chap. 2.9.
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14. M. J. Welch, J. R. Purkiss, K. A. Foster, Toxicon 38, 245
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25. J. Scholz, M. C. Klein, T. E. Behrens, H. Johansen-Berg,
Nat. Neurosci. 12, 1370 (2009).
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
16 SEPTEMBER 2011
Downloaded from http://science.sciencemag.org/ on October 23, 2020
which demonstrated that vesicular release of glu
from axons acting on N-methyl-D-aspartate receptors (NMDARs) and metabotropic glu receptors (mGluRs) stimulates local translation of
MBP (Fig. 4, C and D). Suppressing Fyn kinase
activity by small interfering RNA (siRNA) transfection into oligodendrocytes completely blocked
the electrically induced local translation of MBP
(Fig. 4D).
After synthesis, myelin proteins must be restricted to the appropriate membrane regions
adjacent to the axon being myelinated. To determine whether vesicular release of neurotransmitter from axons stabilizes mobility of myelin
proteins in OPC cell processes, MBP was visualized by using a photoactivated green fluorescent 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 cultured 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-
1651
Control of Local Protein Synthesis and Initial Events in Myelination by Action Potentials
Hiroaki Wake, Philip R. Lee and R. Douglas Fields
Science 333 (6049), 1647-1651.
DOI: 10.1126/science.1206998originally published online August 4, 2011
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