JCB: ARTICLE

BDNF-induced recruitment of TrkB receptor into
neuronal lipid rafts: roles in synaptic modulation
Shingo Suzuki,1,2,3 Tadahiro Numakawa,4 Kazuhiro Shimazu,6 Hisatsugu Koshimizu,1 Tomoko Hara,1
Hiroshi Hatanaka,2 Lin Mei,5 Bai Lu,6 and Masami Kojima1,3
1Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka, 563-8577, Japan
Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan
3
Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan
4
National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan
5
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912
6
Section on Neural Development and Plasticity, NICHD, National Institutes of Health, Bethesda, MD 20892

B

rain-derived neurotrophic factor (BDNF) plays animportant role in synaptic plasticity but the underlying signaling mechanisms remain unknown. Here,
we show that BDNF rapidly recruits full-length TrkB (TrkB-FL)
receptor into cholesterol-rich lipid rafts from nonraft regionsof neuronal plasma membranes. Translocation of TrkB-FL
was blocked by Trk inhibitors, suggesting a role of TrkB
tyrosine kinase in the translocation. Disruption of lipid
rafts by depleting cholesterol from cell surface blocked

the ligand-induced translocation. Moreover, disruption oflipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic responseto tetanus in hippocampal slices. In contrast, lipid rafts are
not required for BDNF regulation of neuronal survival.
Thus, ligand-induced TrkB translocation into lipid rafts may
represent a signaling mechanism selective for synaptic
modulation by BDNF in the central nervous system.

Introduction
In the central nervous system (CNS), brain-derived neurotrophic factor (BDNF) not only promotes neuronal survival anddifferentiation, but also regulates synaptic transmission andplasticity. Pharmacological studies demonstrate BDNF enhances the survival of cortical neurons in culture (Ghosh et al.,1994). On the other hand, substantial experiments suggest thata major function of BDNF in the CNS is to regulate synaptictransmission and plasticity (Lu, 2003). In cultured hippocampal
or cortical neurons, application of BDNF elicits a rapid potentiation of excitatory synaptic transmission primarily by enhancing
presynaptic transmitter release (Lessmann, 1998; Takei et al.,1998). In slices, BDNF facilitates hippocampal long-term
potentiation (LTP) and enhances synaptic response to LTPinducing tetanus (Figurov et al., 1996; Patterson et al., 1996).
Both in vitro and in vivo studies demonstrate that BDNF in-

The online version of this article contains supplemental material.
Correspondence to Masami Kojima: m-kojima@aist.go.jp; or Bai Lu:
bailu@mail.NIH.gov
Abbreviations used in this paper: 4-AP, 4-amino pyridine; BDNF, brain-derived
neurotrophic factor; CNS, central nervous system; GDNF, glial cell line–derived
neurotrophic factor; GPI, glycosylphosphatidylinositol; HFS, high frequency
stimulation; KRH, Krebs’-Ringer’s-Henseleit; LTP, long-term potentiation; MCD,
methyl--cyclodextrin; PI3-K, phosphatidylinositol 3-kinase; RTK, receptor tyrosine
kinase; TrkB-FL, full-length TrkB.
The Journal of Cell Biology
http://www.jcb.org/cgi/doi/10.1083/jcb.200404106

duces complex effects on dendritic arborization of pyramidal
neurons (McAllister et al., 1995).
Despite rapid progress in this area, the molecular mechanisms remain ill defined (Lu, 2003). All the functions of
BDNF are mediated by TrkB, a receptor tyrosine kinase(RTK; Kaplan and Miller, 2000). Binding of BDNF rapidly
activates its tyrosine kinase, which in turn triggers multiple
intracellular signaling pathways. Downstream pathways include
MAPK, phosphatidylinositol 3-kinase (PI3-K) and PLC. A
critical yet poorly understood issue is how signals from thisreceptor are transduced to mediate diverse biological functionsin CNS neurons.
One idea for specific signal-function coupling is thatdifferent signaling pathways may be transduced in different
subcellular compartments. More specifically, it has been proposed that cholesterol/sphingolipid-rich microdomains called
lipid rafts make a specialized signaling platform in the plasma
membrane, and therefore can transduce signals different fromthose in the nonraft membrane (Simons and Toomre, 2000;
Anderson and Jacobson, 2002). Because both lipid components
are resistant to solubilization with nonionic detergents, lipid
rafts can be biochemically isolated as detergent-resistant membrane fractions. Raft fractions prepared from brain tissues areenriched in proteins that carry lipid modifications such as

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THE JOURNAL OF CELL BIOLOGY

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Supplemental Material can be found at:
http://www.jcb.org/cgi/content/full/jcb.200404106/DC1
Cite by DOI: 10.1083/jcb.200404106

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1 of 11glycosylphosphatidylinositol (GPI)-anchored proteins, as wellas palmitylated or myristoylated proteins such as Src-family kinases and trimeric or small G proteins, suggesting a crucial roleof lipid rafts in signal transduction in the CNS (Paratcha andIbanez, 2002). Recently, lipid rafts have been shown to serveas organizing platforms for chemotrophic guidance of nerve
growth cones (Guirland et al., 2004). Transmembrane RTKs,
including EGF receptor (Mineo et al., 1999) and FGF receptor(Citores et al., 1999) are associated with rafts. The localizationof certain signaling molecules in the rafts allows them to interact with each other more efficiently, and prevents them frominteracting with the proteins outside rafts (Simons and Toomre,
2000). Thus, entering and exiting lipid rafts of RTKs represent
a unique mechanism that transduces differential signals at thesubcellular levels. In the present study, we used brain tissues,
slices and dissociated cultures to examine whether TrkB receptor is localized in lipid rafts of the plasma membrane, and if so,
how the localization is regulated and what the functional roles
are. Our results reveal a BDNF-induced TrkB translocation
into the lipid rafts, and such translocation is important for
BDNF-induced synaptic modulation in CNS neurons.

Results
BDNF-induced translocation of TrkB into
lipid rafts

Lipid raft fraction was prepared from tissues or primary cultures of cerebral cortex according to the method of Kawabuchi
et al. (2000; Fig. S1A, available at http://www.jcb.org/cgi/
content/full/jcb.200404106/DC1). We first examined whether
full-length TrkB (TrkB-FL) and its ligand BDNF were localized in lipid rafts at different stages of cortical development. Bothproteins exhibit a gradual increase in lipid rafts after birth (Fig. S1
B). The components of the lipid rafts, such as cholesterol andraft marker proteins caveolin-2 and Fyn, also increased in rafts
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during postnatal development (Fig. S1 C), raising the possibility that the raft localization of TrkB and BDNF may depend on
the developmental expression of these components in rafts. The
parallel increase of TrkB and BDNF in lipid rafts also suggeststhat BDNF may regulate the localization of TrkB in lipid rafts.
To directly test whether BDNF recruits TrkB into lipid
rafts, we prepared the rafts in cultured cortical neurons treatedwith BDNF. In the cortical cultures used here, 93.3  2.4%
and 3.8  0.7% are NSE-positive neurons and GFAP-positive
astrocytes, respectively (n  6 independent experiments). As
shown in Fig. 1 A, application of BDNF induced an increase in
TrkB-FL in the raft fraction. There was a low amount of TrkBFL in lipid rafts in naïve neurons, suggesting that in naive cells
TrkB-FL may be associated with rafts with a low affinity. In
cultures stimulated with 200 ng/ml BDNF for 30 min, theamount of TrkB-FL was markedly increased in rafts (TrkBFLBDNF-treated/TrkB-FLcontrol: 3.4  0.8-fold, P  0.03) but decreased slightly in nonrafts (23.1  6.4%). Total proteins inboth regions were not changed by the BDNF treatment (Fig. 1
A). When expressed as TrkB-FL/total protein, BDNF increased
TrkB-FL in rafts by 3.63  0.73-fold, indicating that BDNF selectively increases the amount of TrkB-FL, but not protein concentration, in rafts.
A time course study demonstrated that the partition of
TrkB-FL into rafts was significant at 5 min, peaked at 30–120
min after BDNF application, and returned to control levels
within 6 h (Fig. 1 B). The translocation was observed 1 minafter BDNF addition (Fig. S2, available at http://www.jcb.
org/cgi/content/full/jcb.200404106/DC1). On the other hand,
TrkB-FL in nonraft fraction exhibited a gradual decrease after
BDNF exposure (Fig. 1 B). BDNF-induced recruitment of
TrkB-FL into rafts was dose dependent (Fig. 1 C). To confirm
that BDNF selectively translocated TrkB-FL into lipid rafts, we
examined whether BDNF stimulation also affects the distribution of other receptors in the rafts and nonraft membrane frac-

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Figure 1. BDNF-induced translocation of TrkBFL into lipid rafts in cultured cortical neurons.
(A) Effect of BDNF on the distribution of membrane proteins in lipid raft and nonraft fractions. Cultured neurons were treated with orwithout 200 ng/ml BDNF for 30 min. An example is shown on the left. Note that TrkB-FL,
but not EGFR or TfR, was recruited into fraction
2 after BDNF stimulation. The quantificationof TrkB-FL and total proteins in fractions 2 and6 is shown on the right. The value of densitometry unit was determined for each TrkB-FL
band, and normalized to that of “BDNF” infraction 2. (B) Time course of BDNF-induced
recruitment of TrkB-FL into rafts. Neurons weretreated with 200 ng/ml BDNF for the indicated times. (C) Dose-dependent translocationof TrkB-FL into rafts. Neurons were treatedwith the indicated concentration of BDNF for
30 min. *Indicates significantly different
from “BDNF” in fraction 2; t test; P 
0.05. n  6 preparations from five independent experiments (A), 4 preparations fromthree independent experiments (B), and 3
preparations from two independent experiments (C). Error bars in this and all other figures
represent SEM.

tions. A 30-min treatment with BDNF did not change the distribution of TfR, a nontyrosine kinase receptor, or EGF receptor,
a tyrosine kinase receptor (Fig. 1 A, left). Together, these datasuggest that BDNF rapidly and selectively induces the translocation of TrkB-FL from nonrafts into lipid rafts.
TrkB translocation: dependence on its
tyrosine kinase activity

Regulation of BDNF-induced
translocation of TrkB into rafts by
p75 NTRHow is BDNF-dependent translocation of TrkB-FL into rafts
regulated? Given that the pan-neurotrophin receptor, p75NTR
associates with lipid rafts (Higuchi et al., 2003) and forms aheterodimer with Trk family receptors (Hempstead, 2002),

Figure 2. Requirement of TrkB activation for BDNF-induced TrkB translocation into lipid rafts. Immunoblots were performed using anti-TrkB andanti–phospho-Trk antibodies. (A) TrkB-FL recruited into rafts is tyrosine
phosphorylated. Cortical neurons were treated with (200 ng/ml) or without
BDNF for 30 min. (Top) Sample blots. (Bottom) Quantification. pTrkB-FL/
total TrkB-FL was quantified in fractions 2 and 6, and shown as relative tothat of “ BDNF” in fraction 6. n  5 preparations from five independent
experiments. (B) Inhibition of TrkB-FL activation prevents TrkB-FL translocation
into lipid rafts. Neurons were treated with 100 nM K252a or 10 M
AG879 for 3 h, followed by a 30-min stimulation with 200 ng/ml BDNF.
(C) Effect of a brief exposure to BDNF on TrkB-FL translocation. Corticalneurons were treated with 200 ng/ml BDNF for only 1 min followed byincubating with medium containing no BDNF for the indicated times. Notethat once activated, TrkB-FL continued to be partitioned into rafts in theabsence of BDNF.

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Binding of BDNF to TrkB-FL induces the autophosphorylationof its tyrosine residues on the intracellular kinase domain, leading to its activation (Kaplan and Miller, 2000). To test whether
TrkB recruited into rafts is activated, we performed Western
blot analysis using anti–phospho-Trk antibody (pY490; Binder
et al., 1999). In neurons treated with BDNF for 30 min, a substantial amount of TrkB-FL recruited into rafts was tyrosine
phosphorylated (Fig. 2 A, top). In BDNF-treated cultures,
TrkB-FL tyrosine phosphorylation relative to total TrkB-FL
protein was not significantly different between in rafts and innonrafts (Fig. 2 A, bottom; P  0.64). To investigate whether
activation of TrkB tyrosine kinase was required for the translocation of TrkB-FL into lipid rafts, we performed the following
experiments. First, we treated cortical neurons with the Trk kinase inhibitors, K252a or AG879 for 3 h before BDNF stimulation (Fig. 2 B). K252a (100 nM), which reduced BDNF-dependent TrkB tyrosine phosphorylation by 68.8  12.2% in rafts
and by 49.4  7.0% in the nonrafts (n  3 independent experiments), inhibited BDNF-induced TrkB-FL translocation (Fig. 2
B). BDNF-dependent recruitment of TrkB-FL into rafts was
also blocked by another Trk kinase inhibitor AG879 (10 M).
Second, because TrkB activation could be induced by a 1-min
exposure to BDNF (Takei et al., 1998), we tested whether thisshort-term stimulation would allow recruitment and partition of
TrkB-FL in rafts. Cultured cortical neurons were stimulatedwith BDNF for 1 min, followed by incubation with medium
containing no BDNF for 5–360 min. As shown in Fig. 2 C, a
1-min stimulation with BDNF was sufficient to recruit TrkB-FL
into rafts. The amount of TrkB-FL continued to increase after
BDNF was washed out, suggesting that once activated TrkBFL can move into lipid rafts. Third, TrkB-T1, a truncated formof the TrkB receptor lacking tyrosine kinase domain, did notappear to be partitioned into rafts by BDNF (Fig. S3A, available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1).
Finally, NT-4 (200 ng/ml, 30 min), another ligand that activates TrkB tyrosine kinase, recruited TrkB-FL into rafts (Fig.
S3 B). Together, these results suggest that BDNF-induced
translocation of TrkB into rafts requires its tyrosine kinase activity. Because cultured astrocytes expressed TrkB-T1, but not
TrkB-FL (Fig. S3 C), the TrkB-FL translocation is likely to occur in neurons only.

p75NTR may facilitate BDNF-dependent association of TrkB
with rafts. To test this, we introduced p75NTR or lacZ into cortical neurons using adenovirus-mediated gene transfer. In cortical neurons expressing lacZ, p75NTR was not detectable both inraft fraction and nonraft fraction (Fig. 3, lacZ). Infection of cortical neurons with p75NTR adenovirus resulted in the expressionof the 75-kD p75NTR (Fig. 3, p75). The p75NTR did not interferewith basal association of TrkB with rafts or TrkB phosphorylation outside rafts. Interestingly, the expression of exogenous
p75NTR in cortical neurons markedly inhibited BDNF-dependent translocation of TrkB-FL into lipid rafts (Fig. 3, TrkBFLrafts). Consequently, phosphorylated TrkB-FL was not detectedin the rafts (Fig. 3, pTrkBrafts). Moreover, application of BDNF
did not increase the amount of p75NTR in the rafts (Fig. 3,
p75rafts). Together, these data suggest that a lipid raft-associated

BDNF-INDUCED TRANSLOCATION OF TRKB INTO LIPID RAFTS • SUZUKI ET AL. 3 of 11

Requirement of lipid rafts for short-term
synaptic modulation by BDNF

Figure 3. Inhibition of BDNF-induced TrkB translocation by p75NTR.
Cultured cortical neurons were infected with adenovirus expressing p75NTR
or lacZ as described in Materials and methods. Cells were treated withoutor with 200 ng/ml BDNF for 30 min. The fractions of rafts and nonrafts
were immunoblotted for the indicated proteins. (Top) BDNF-induced translocation and activation of TrkB-FL is inhibited by p75NTR expression. (Bottom)
The location and activation of TrkB-FL in nonrafts are not influenced by
p75NTR expression. BDNF did not affect the distribution of p75NTR in rafts
and nonrafts. Similar results were obtained from two separate experiments.

Distribution of signaling molecules
downstream of TrkB-FL in lipid rafts andnonrafts

Phosphorylated tyrosine residues on the TrkB intracellular domain form docking sites for the signaling proteins Shc, Grb2,
and PLC, leading to activation of MAPK (Erks), PLC, andPI3-K pathways (Kaplan and Miller, 2000). We tested
whether TrkB-FL, upon BDNF stimulation, carries these signaling molecules into lipid rafts during its translocation. Innaïve neurons, Erks extended throughout the gradient (fractions 2–5), whereas Shc, Grb2, p85 subunit of PI3-K, Akt, and
PLC were primarily localized in the bottom, nonraft fraction(Fig. 4 A, left). BDNF application did not increase the amountof any of these proteins in fraction 2 (Fig. 4 A, right), suggesting that TrkB-FL does not carry its associated signaling
molecules into lipid rafts during its translocation. When amilder detergent Triton X-165 was used to prepare rafts (Fig.
S4 A, available at http://www.jcb.org/cgi/content/full/jcb.
200404106/DC1), many of them (Shc, Grb2, Erks, and PLC)were extended throughout the gradient whereas p85 subunit of
PI3-K and Akt appeared to be still in nonraft fraction. It wasnotable that BDNF did not stimulate the translocation of thesesignaling molecules into rafts (Fig. S4 A). Parallel to these,
BDNF-induced translocation of TrkB-FL (Fig. 4 B, left) was
accompanied by a significant increase in the phosphorylationof Erks (Figs. 4 B, middle and Fig. 4 D), but not that of Akt(Fig. 4 B, right and Fig. 4 D), in lipid rafts. In nonraft regions,however, both Erks and Akt were activated by BDNF application (Fig. 4, C and D). Thus, although TrkB-FL does not movewith its associated proteins during translocation, the translocation of TrkB-FL itself into rafts may be a key event in forming TrkB signaling complex, including TrkB-FL and its associated proteins, in rafts, leading to preferential activation ofErks over Akt in neuronal lipid rafts.
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pan-neurotrophin receptor p75NTR has an inhibitory role in
BDNF-dependent translocation of TrkB-FL into rafts.

What is the functional role of BDNF-induced TrkB translocation into lipid rafts? Because the translocation was observed 1
min after BDNF application (Fig. S2), we reasoned that thisprocess may be involved in the short-term actions of BDNF
(Lu, 2003). BDNF has been shown to rapidly enhance transmitter release in cultured cortical neurons (Matsumoto et al., 2001).
Therefore, we tested whether disruption of lipid rafts would interfere with BDNF modulation of depolarization-evoked transmitter release at CNS synapses. Methyl--cyclodextrin (MCD)
binds and depletes cholesterol from the plasma membrane, andthereby disrupts lipid rafts (Simons and Toomre, 2000). A 10min treatment with MCD (2 mM) removed membrane cholesterol by 33  2% (91  2 ng/well in control cultures, 61.2  2
ng/well in MCD-treated cultures, n  4 independent experiments), but did not affect the amount of caveolin-2 and Fyn inlipid rafts (Fig. 5 A, right). Treatment of the cultured corticalneurons with 2 mM MCD significantly attenuated the BDNFinduced recruitment of TrkB-FL into the rafts (Fig. 5 A, left andmiddle). Consequently, the amount of phosphorylated TrkB
was also reduced in the rafts. Importantly, TrkB phosphorylation in the nonraft fraction was not affected. Thus, cholesterol
may play a role in TrkB translocation into lipid rafts.
We reported previously that in cultured cortical neurons,
pretreatment with BDNF enhanced glutamate release evokedby 4-amino pyridine (4-AP), a K channel blocker frequently
used in biochemical assays of evoked neurotransmitter release(Matsumoto et al., 2001). As shown in Fig. 5 B, pretreatment
with BDNF for 30 min had no effect on the content ofglutamate in the medium (white bars in BDNF-pretreated cultures), but significantly enhanced 4-AP–induced glutamate release (hatched bars in BDNF-pretreated cultures). A 10-min
treatment with 2 mM MCD before BDNF exposure, however,abolished BDNF enhancement of 4-AP–evoked glutamate release. The neurons pretreated with 2mM MCD alone displayed
4-AP–evoked glutamate release, which was comparable to thatin control neurons, indicating that cholesterol depletion itself
does not impair the exocytosis machinery. Long-term treatment
with raft depleting agents such as fumonisin B1 and mevastatin
has been reported to lead to instability of AMPA receptors andloss of dendritic spines and synapses (Hering et al., 2003). In
contrast, dendritic parameters (Murphy and Segal, 1996) were
not affected by short-term treatment (30 min) with 2 mM
MCD. The dendritic diameter was about the same in control
cultures and MCD-treated cultures (1.19  0.04 m in control
cells and 1.18  0.04 m in MCD-treated cells, n  415 segments). Likewise, MCD treatment did not change spine density(spine number per 10 m dendritic segment; 2.47  0.11 incontrol cells, 2.36  0.10 in cells treated with 2 mM MCD,n  945). Furthermore, immunocytochemistry using antibodies against the presynaptic marker synaptophysin and postsynaptic marker PSD95 indicated that short-term MCD treatment
did not alter the number or the distribution of synapses (Fig. 5
C). These data support the notion that MCD selectively prevents the residing of activated TrkB in lipid rafts, leading to
impairments of BDNF modulation of glutamate release.

Figure 4. Differential activation of Erks and
Ark in lipid rafts upon BDNF-induced TrkB-FL
translocation. Cultured cortical neurons weretreated with or without BDNF (200 ng/ml) for
30 min. (A) Western blot analysis of the distribution of major signaling molecules downstreamof TrkB before and after BDNF stimulation.
Similar results were obtained from two separate
experiments. (B and C) Differential activationof Erks and Akt in lipid rafts and nonrafts after
a 30-min treatment with BDNF. Fractions 2
and 6 were immunoblotted. (D) Quantitative
analysis of BDNF effect on the activation ofErks and Akt in rafts and nonrafts. For eachprotein, phosphorylation relative to total
amount was quantified in fractions 2 and 6,
and shown as relative to that of “ BDNF” infraction 6. *Indicates significantly different
from “BDNF”; t test; P  0.05. n  4 independent experiments. In A–C, immunoblots
were performed using antibodies specific forindicated proteins and phospho-proteins.

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The biochemical assay described above measuresboth synaptic and nonsynaptic glutamate release. To determine
whether lipid rafts are important for BDNF modulation of neurotransmitter release at synapses, we measured synaptic exocytosis in cultured hippocampal neurons using a style membranedye FM1-43 (Ryan et al., 1993). A second depolarization ofthese FM dye-loaded neurons by high K solution (see Materials and methods) resulted in a rapid destaining of the FM dyelabeled spots, reflecting transmitter release at the synapses(Fig. 6, A and B). Pre-treatment with BDNF for 30 min enhanced depolarization-induced FM1-43 destaining (Fig. 6 B).
A 10-min treatment with MCD (2 mM) before BDNF application prevented the enhancement effect of BDNF (Fig. 6 C). The
neurons pretreated with MCD alone exhibited FM1-43 destain-

ing similar to that in control neurons (Fig. 6 C), indicating thatneither uptake nor destaining of FM 1-43 dye was affected by
MCD pretreatment.
Similar to the effect of BDNF in hippocampal cultures,
BDNF enhanced depolarization-induced FM dye destaining,and MCD attenuated the BDNF effect, in cultured cortical neurons (Fig. 6 D). Several experiments were performed to ensurethat the MCD effect was due specifically to removal of cholesterol and disruption of lipid rafts, rather than to a nonselective,
pharmacological effect such as removal of proteins from theplasma membrane. First, we pretreated the cultures with 50 ng/
ml filipin, a cholesterol sequestration agent with a structure distinct from that of MCD (Simons and Toomre, 2000; Ma et al.,2003). We found that, although FM 1-43 uptake appeared to be

BDNF-INDUCED TRANSLOCATION OF TRKB INTO LIPID RAFTS • SUZUKI ET AL. 5 of 11

slightly lower, filipin, like MCD, completely reversed the effect of BDNF on depolarization-induced FM dye destaining(Fig. 6 E). Neurons pretreated with filipin alone exhibited FM
dye destaining similar to that in control neurons (Fig. 6 E). Second, if the MCD effect were due to a nonspecific removalof membrane protein, addition of MCD–cholesterol complex
should not rescue the deficits induced by MCD. We treated thecultures with MCD–cholesterol complex (1 mg/ml MCD balanced with 40 g/ml cholesterol) for 10 min (Thiele et al.,2000). Remarkably, this treatment did not affect the potentiating effect of BDNF on depolarization-induced destaining of
FM 1-43 (Fig. 6 D). We therefore conclude that lipid rafts areimportant for the modulation of neurotransmitter release andsynaptic exocytosis by BDNF.
One of the major functions of BDNF in the intact hippocampal synaptic circuits is to attenuate synaptic fatigue induced by a train of high frequency stimulation (HFS; or tetanus, 100 Hz, 1 s; Figurov et al., 1996). We next examined therole of lipid rafts in this form of synaptic modulation. In neonatal hippocampal slices (P12-13) in which the level of endogenous BDNF is low, application of tetanus resulted in pronounced synaptic fatigue at Schaffer collateral-CA1 synapses(Fig. 7 A). Consistent with our previous reports (Figurov et al.,1996), treatment with exogenous BDNF (2 nM) for 1–2 h sig6 of 11

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nificantly attenuated the synaptic fatigue (Fig. 7 B). However,pretreatment with MCD for 30 min completely abolished theattenuating effect of BDNF on HFS-induced synaptic fatigue.
Quantitative analysis indicated that treatment with BDNF
markedly increased the rate constant ( ) for synaptic fatigue
and disruption of lipid rafts with MCD completely prevented
such an increase (Fig. 7 C). It is important to note that treatment with MCD for 3 h had no effect on synaptic responses to
HFS (Fig. 7, B and C), nor did MCD affect basal synaptictransmission or tetanus induced LTP (Ma et al., 2003). These
results suggest that short-term exposure to MCD per se does
not affect the number of readily releasable vesicles in the presynaptic terminals or the number or properties of AMPA or
NMDA receptors on the postsynaptic density.
Requirement of lipid rafts for long-term
regulation of dendritic growth, but notneuronal survival, by BDNF

To test whether lipid rafts are also involved in long-term effects of BDNF, we treated cultures with cholesterol synthesis
inhibitors, mevastatin or pravastatin, which effectively deplete
rafts from cortical neurons over days (Hering et al., 2003). The
cholesterol level was significantly reduced in mevastatin- or
pravastatin-treated cortical neurons (Fig. 8 A). We first exam-

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Figure 5. Inhibition of BDNF-induced TrkB-FL translocation and evoked glutamate release by lipid raft disruption. (A) Effect of MCD on BDNF-induced TrkB
translocation. Cortical neurons were treated with or without MCD (2 mM) for 10 min. (Left) Raft (fraction 2) and nonraft fractions (fraction 6) were immunoblotted for the indicated proteins. Note that MCD blocked BDNF-induced TrkB phosphorylation in rafts and translocation to rafts. (Middle) TrkB-FLfraction 2/
TrkB-FLfraction 6 was quantified and shown as relative to “BDNF” in control cultures (n  4 preparations from three independent experiments). *Indicatessignificantly different from “BDNF” in control cultures; t test; P  0.01. (Right) Treatment with 2 mM MCD for 10 min did not influence the amount ofcaveolin-2 and Fyn in lipid raft fraction. (B) Effect of MCD on BDNF enhancement of 4-AP–evoked glutamate release from cortical neurons. Neurons werepretreated with or without 2 mM MCD for 10 min, followed by incubation with or without BDNF for 30 min. After washing four times, neurons were stimulatedwith 1 mM 4-AP. *Indicates significantly higher than all other 4-AP–treated groups; ANOVA test; P  0.05; n  4 cultures. There was no statistically significant
difference among all white bars (ANOVA test, P  0.48). Similar results were obtained from three independent experiments. (C) Effect of short-term treatment
with MCD on the number and distribution of synapses. (Left) Immunocytochemistry for the indicated proteins was done after a 30-min incubation with orwithout 2 mM MCD. (Right) The number of synapses (doubly labeled by anti–PSD-95 and anti-synaptophysin antibodies) was determined and shown as
relative to that of “Control” (n  90 dendritic segments with 50-m length from three independent coverslips and experiments). Note that there is no significant
difference in distribution and number of the immunopositive synapses between control and MCD-treated cultures. Bar, 5 m.

ined the effect of these lipid raft disrupters on BDNF regulationof neuronal survival, using the WST-1 assay, which quantitatively determines cell viability based on the cleavage activityof a soluble tetrazolium salt WST-1 by mitochondrial dehydrogenases, and by counting the number of MAP2-positive neurons in culture. 3-d treatment with BDNF significantly enhanced the survival of cortical neurons in serum-free medium(Fig. 8, B and C, left; Yamada et al., 2001). Both mevastatin
and pravastatin, however, failed to block this effect of BDNF
(Fig. 8, B and C, middle and right), indicating that lipid rafts
are not required for the regulatory effect of BDNF on corticalneuron survival.
BDNF has also been shown to be a potential regulator for
dendritic growth of CNS neurons (McAllister et al., 1995). We
next examined whether lipid rafts are important for BDNF regulation of dendritic growth. 3-d exposure of BDNF led to robust
growth of MAP2-positive primary dendrites (Fig. 8 D, BDNF).
Quantitative analysis revealed that BDNF increased the numberof primary dendrites by 50% (P  0.001). This effect of BDNF,
however, was effectively blocked by mevastatin, whereas treatment with mevastatin alone had no effect (Fig. 8 D). Thus, while
required for short-term modulation of synaptic transmission andplasticity by BDNF, lipid rafts may mediate some, but not all
long-term effects of BDNF in CNS neurons.

Discussion
One of the conceptually difficult issues in neurotrophin research is how a single neurotrophin could elicit different effects under different conditions on the same neurons, or on certain parts of the same neurons (Bibel and Barde, 2000). How
could, for example, BDNF regulate synaptic transmission without altering the survival of neurons of adult cortex? One way toensure signaling specificity is to induce BDNF signaling inspecific subcellular compartments. In the present study, wedemonstrated that BDNF stimulation induced the translocationof activated TrkB-FL from nonrafts to lipid rafts in corticalneurons. We further showed that attenuation of such translocation by depleting cholesterol in the lipid rafts prevented the effects of BDNF on synaptic transmission and plasticity, but notneuronal survival. These results reveal a new mechanism forthe signaling specificity of BDNF, and suggest a critical role oflipid rafts in the synaptic function of BDNF in CNS neurons.
BDNF-induced translocation of activated
TrkB into lipid rafts

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Figure 6. Attenuation of BDNF enhancement of synaptic
exocytosis by lipid raft disruption. Recycling synaptic
vesicles in cultured hippocampal or cortical neurons
were labeled by FM1-43, and exocytosis was induced
by a perfusion of 50 mM KCl containing KRH buffer (highK solution). (A) Pseudo-colored images showing highK solution-dependent reduction in FM1-43 intensity. FM
1-43 images were captured at the indicated times (s) andbaseline intensity was captured at 5 s before depolarization. The monochromic image shows the neuron 5 min
before stimulation. Arrowheads indicate representative
spots with a gradual reduction of FM dye labeling after
depolarization. Bar, 10 m. (B) Representative recordingsof FM 1-43 destaining. Neurons were incubated with orwithout BDNF for 30 min, followed by a 1-min exposure
to high K solution to load FM dye. After three washes for
5 min, FM dye was destained with high K solution (blackarrow). FM 1-43 intensities at 4 s before (gray arrow) andat 15 s after (white arrow) depolarization are defined as
Fb and Fa, respectively. F (%)  (FbFa)/Fb 100. (C–E)
Summary of FM dye destaining experiments using hippocampal neurons (C) and cortical neurons (D and E). Cells
were pretreated with MCD, MCD–cholesterol complex(MCD-Chol), or filipin for 10 min, followed by BDNF
incubation for 30 min and stimulation with high K solution.
Data were collected at 4 s before and at 15 s after stimulation with high K solution. The number associated witheach column represents the number of spots recorded for each condition. A 1-min incubation with 5 mM KCl did not elicit any significant change in theintensity of FM 1-43 (not depicted). In all experiments similar results were obtained from at least two independent experiments.

One of the key findings in the present study is that TrkB translocation to rafts depends on its tyrosine kinase activity. This notion
is supported by a number of observations: (a) inhibition of the
Trk kinase activity by K252a or AG879 significantly inhibited
BDNF-induced translocation of TrkB-FL; (b) NT-4, which activates the TrkB RTK, also induced TrkB-FL translocation; (c)1-min exposure of BDNF, which activates TrkB, was sufficient
to partition TrkB-FL into rafts. Thus, TrkB activation has to occur before the translocation; and (d) TrkB-T1, the truncated formof TrkB lacking the tyrosine kinase domain, was not recruited
into rafts by BDNF. This last result, however, has to be interpreted with caution. It is also possible that the intracellular domain of the TrkB receptor may contain motifs that interact withother proteins important for the delivery of TrkB to the lipid rafts.
Another important observation is that removing of cholesterol, a major component in the lipid rafts, with 2 mM MCD
inhibited TrkB signaling, leading to impairments in synaptictransmission and plasticity. The most straightforward interpretation is that MCD disrupted BDNF-induced translocation of
TrkB-FL into rafts, and consequently impaired TrkB signaling

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in rafts. Alternatively, TrkB may need to interact with cholesterol to become fully functional, and therefore depletion ofcholesterol directly affects TrkB signaling, rather than TrkB
translocation. Consistent with this idea, BDNF failed to induce
TrkB phosphorylation on the cell surface in cultured striatal
neurons from Niemann-Pick type C mice (NPC/), whichhave abnormal cholesterol metabolism (Henderson et al.,2000). The fact that there was a small amount of TrkB in lipid
rafts in naïve cells (Fig. 1 A) also supports the idea that TrkB
interacts with rafts, perhaps with a low affinity. However, several observations argue against this idea. First, in cultured striatal neurons from NPC/, the content of free cholesterol was
normal, suggesting that the cholesterol-rich microdomains oncell surface are probably normal. Thus, the dysfunction of
TrkB on cell surface in NPC/ is probably due to reasons
other than lack of cholesterol in the plasma membrane. Second,
we demonstrated that treatment with 2 mM MCD, which removed membrane cholesterol by 33%, interfered with TrkB recruitment into rafts, but not TrkB activation outside rafts (Fig.
5 A). The basal level of TrkB localization in rafts in resting
cells was normal as well. Finally, treatment with 10 mM MCD,
which removed 95% cholesterol, did not diminish TrkB kinase activity (Fig. S5, available at http://www.jcb.org/cgi/
content/full/jcb.200404106/DC1). These data suggest that cholesterol is involved in the recruitment of TrkB into lipid rafts,
but not directly in TrkB function.
A third possible mechanism for TrkB translocation isthat BDNF signaling involves an interaction between TrkB
and protein X, which could be a protein embedded in cholesterol-rich membranes. Cholesterol depletion could in principle lead to structural changes in the protein X (Simons andToomre, 2000; Munro, 2003; Glebov and Nichols, 2004), andtherefore disturb its interaction with TrkB. Although thenature of protein X remains unknown, we tested whether
p75 NTR, which is primarily associates with rafts (Higuchi
et al., 2003) and is capable of binding to Trk receptors(Hempstead, 2002), facilitates BDNF-dependent translocation of TrkB into rafts. Interestingly, expression of exogenous
p75NTR in cortical neurons inhibited TrkB translocation into
lipid rafts, as well as the level of TrkB phosphorylation in therafts, but not the basal association of TrkB with rafts or TrkB
phosphorylation outside rafts (Fig. 3). These data are opposite
to the above prediction and suggest that p75NTR has an inhibitory function in TrkB translocation and lipid raft-mediated
BDNF/TrkB signaling. Thus, although it is unlikely that
BDNF-induced TrkB translocation into lipid rafts is mediatedby p75NTR through its interaction with TrkB, our experiments
could not rule out the possibility that a protein X is an intermediate for BDNF-induced TrkB translocation into rafts.
The dynamic behavior of TrkB into lipid rafts is mostanalogous to that of two tyrosine kinase receptors, c-Ret for
glial cell line–derived neurotrophic factor (GDNF; Tansey etal., 2000; Paratcha et al., 2001) and ErbB4 for neuregulin (Maet al., 2003). c-Ret is recruited into lipid rafts upon GDNF
stimulation by two distinct mechanisms. In cells expressing
c-Ret and GPI-anchored protein GFR 1 (GDNF family receptor1), which is primarily associated with lipid rafts, the binding

Figure 7. Effect of MCD on BDNF modulation of synaptic fatigue at hippocampal CA1 synapses. Neonatal hippocampal slices were pretreated withMCD (2 mM) for 30 min before treatment with BDNF (2 nM) for 1–2 h. A
train of HFS (100 Hz, 1 s) was applied to Shaffer collaterals and field EPSPs
were recorded from stratum radiatum. The slopes of EPSPs during the entire
recording were normalized to the first EPSP slope in each recording. (A) An
example of EPSPs elicited by HFS recorded from a BDNF-treated slice. (B)Effect of MCD on synaptic depression induced by HFS. Normalized EPSP
slopes in each condition were averaged and plotted against the number ofstimulus during HFS. (C) Summary of the MCD effect on the rate of synaptic
depression. The plot for each recording was fitted with a single exponential
curve, and rate constant ( ) for each condition was averaged and presented. *Indicates significantly higher than all other groups; ANOVA
followed by post hoc tests; P  0.001. The number associated with eachcolumn represents the number of slices used for each condition.

of GDNF to GFR 1 results in a transient recruitment of c-Ret
into the compartment. Unlike TrkB translocation, this movement is independent of the kinase activity. In cells lacking
GPI-anchored GFR 1, however, coapplication of GDNF andsoluble GFR 1 allows stabilization of c-Ret in rafts, and thiseffect is tyrosine kinase-activity dependent. These mechanisms lead to the association of c-Ret with different adaptor

proteins inside rafts and elicit neuronal survival and differentiation. Similar to TrkB-FL, ErbB4 is recruited into lipid rafts
by neuregulin. However, it is unclear whether ErbB4 translocation depends on its kinase activity. Although neuregulintranslocates the whole receptor complex, including ErbB4 andits adaptor proteins, such as Grb2 and Shc, into rafts, BDNF
recruited TrkB alone into lipid rafts, without carrying its associated proteins Shc, Grb2, and PLC (Fig. 4 A and Fig. S4 A).
However, we cannot completely rule out the possibility thatthe movement of these associated proteins into rafts was under
the detection limit of our immunoblotting analysis. Given that
Shc, Grb2, Erks, and PLC were present in lipid rafts in an experiment using a milder detergent Triton X-165 (Fig. S4 A), it
is possible that the movement of TrkB-FL into rafts allows
TrkB to interact with signaling molecules inside the rafts, a
step crucial in transmitting lipid raft-mediated BDNF/TrkB
signal transduction.
The raft localization of detergent insoluble proteins including RTKs depends on cell type and detergent stringency(Pike, 2003; Schuck et al., 2003). For example, when a lessstringent detergent (Triton X-165) was used, a larger amountof TrkB-FL was associated with rafts in naïve cells, and
BDNF-induced movement of TrkB-FL from nonrafts to rafts
became less apparent (Fig. S4 B). In PC12 cells, both TrkA
and p75NTR are constitutively localized in caveolae/lipid rafts(Bilderback et al., 1999; Huang et al., 1999) and NGF binding
does not alter the partition of TrkA and p75NTR in lipid rafts(Huang et al., 1999). This may be due to the specific cell
types they are expressed or their association with lipid raftanchored proteins.

Role of BDNF-dependent TrkB
translocation into lipid raft in synaptic
modulation

The present study has revealed the importance of TrkB translocation into rafts in short-term modulation of synaptic transmission and plasticity by BDNF. In contrast, lipid rafts are not required for BDNF regulation of cortical neuron survival. In
neurons, lipid rafts are preferentially distributed in synaptic
membranes (Ma et al., 2003). In cerebral cortex in vivo, TrkBFL is present in lipid rafts (Fig. S1 B). Moreover, the endogenous BDNF appears to be enriched in lipid raft fractions in theadult cortex (Fig. S1 B), stored in vesicles in synaptosomes(Fawcett et al., 1997), and secreted at synapses in an activitydependent manner (Hartmann et al., 2001; Kojima et al., 2001).
Thus, the translocation of TrkB into lipid rafts may imply a
BDNF-induced recruitment of TrkB into the synapses.
Because BDNF elicits the synaptic effects throughpresynaptic mechanisms (Gottschalk et al., 1998; Lessmann,
1998), activated TrkB may need to be translocated to the rafts
in the presynaptic terminals to interact with its target molecules. The specific target molecules mediating the presynaptic
effects of BDNF, however, remain to be fully identified. In purified nerve terminals (synaptosomes) from cerebral cortices,
application of BDNF facilitates evoked glutamate release andincreases the MAPK-dependent phosphorylation of synapsin I,
a synaptic vesicle-associated protein implicated in the control
of transmitter release (Jovanovic et al., 2000), suggesting a critical role in the TrkB/MAPK/synapsin signaling cascade. BDNF
also rapidly induces MAPK phosphorylation in these slices andinhibition of MAPK signaling blocks BDNF modulation of

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Figure 8. Effects of raft depletion on BDNF
regulation of dendritic growth and cell survival.
Cultured cortical neurons were treated with orwithout 10 M mevastatin (Meva) or 10 M
pravastatin (Prava) in the absence or presenceof 200 ng/ml BDNF. Membrane cholesterol
content, cell survival, or the number of primarydendrites was measured 3 d later. (A) Effect ofmevastatin or pravastatin on membrane cholesterol levels. The data are expressed as thepercentage of control (nontreated cultures).
*Indicates significantly lower than control;P  0.001 (t test). n  5 cultures in each group.
(B) Effect of mevastatin or pravastatin on cell
viability. Viability of cortical neurons wasmeasured by the WST-1 colorimetric assay,
and expressed as OD450. t test; *P  0.05,**P  0.01. (C) Effect of mevastatin or pravastatin on the number of MAP-positive neurons.
Data are expressed as the percentage of control(nontreated cultures). *P  0.01, **P 
0.05. In both B and C, the number associatedwith each column represents the number ofcultures used in a single experiment, and three
independent experiments were performed. No
difference was found between the “ BDNF”
groups, nor between the “BDNF” groups(ANOVA test). (D) The effect of mevastatin on
BDNF modulation of dendritic growth. (Left)
Representative images of MAP2-stained neurons.
After 3-d incubation with BDNF and/or Meva, cells were stained with anti-MAP2 antibody. Note that the number of primary dendrites (arrowheads) is
increased by BDNF treatment, and that the effect is completely blocked by mevastatin. Bar, 10 m. (Right) The number of primary dendrites was quantified
and normalized to that of the control. *Indicates significantly higher than all other groups; ANOVA followed by post hoc test; P  0.00002. The number
associated with each column indicates the number of neurons measured in a single experiment, and three independent experiments were performed.

BDNF-INDUCED TRANSLOCATION OF TRKB INTO LIPID RAFTS • SUZUKI ET AL. 9 of 11

HFS response (Gottschalk et al., 1999). In the present study,
when TrkB was translocated into lipid rafts, Erks appeared tobe activated in the rafts (Fig. 4 B). Assuming that TrkB-FL is
rapidly translocated into the rafts in synaptic membranes, activation of Erks in rafts may lead to the phosphorylation of presynaptic molecules (e.g., synapsin, synaptobrevin, or synaptophysin) necessary to mediate BDNF effects at CNS synapses.

Materials and methods
Primary cultures
Dissociated cultures of cortical and hippocampal neurons were preparedfrom embryonic day 20 and cultured. The details were described in Online supplemental material.

Recombinant adenovirus
The rat p75NTR cDNA was supplied by E. Shooter (Stanford University, Stanford, CA). The adenovirus vector pAxCAwt was kindly provided by I. Saito(University of Tokyo, Tokyo, Japan). For adenovirus generation, see Online
supplemental material. The recombinant adenovirus was used at a multiplicity of infection of 5. After 6 d in culture, cells were infected with adenovirus
for 24 h followed by a 24-h incubation in serum-free DME to assay.
Glutamate release
For this assay, cultured neurons (6 105 cells/cm2) were prepared as described previously (Matsumoto et al., 2001). After 5 h incubation in serum-free medium, the neurons were washed four times with Krebs’Ringer’s-Henseleit (KRH) buffer containing 130 mM NaCl, 5 mM KCl, 1.2
mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, and 25 mM Hepes, pH
7.4, and pretreated with or without MCD for 10 min followed by BDNF incubation for 30 min. To induce glutamate release, 1 mM 4-AP was applied (Matsumoto et al., 2001). The glutamate released was measured using Amplex red glutamic acid oxidase kit (Molecular Probes).
Exocytosis assay using FM dye destaining
Dissociated neurons (5 104 cells/cm2) were cultured on polyethylenimine-coated glasses (Matsunami; Numakawa et al., 2002). After washing three times with KRH buffer, neurons were pretreated with or without
MCD, MCD–cholesterol complex or filipin for 10 min followed by BDNF
incubation for 30 min. FM dye (2 mM; Molecular Probes) was loaded byincubating the cells with the high K solution (KRH buffer containing 56
mM KCl and 79 mM NaCl) for 1 min at 37C. After washing the cells with
KRH buffer, fluorescence images of FM-labeled spots were taken every
second using fluorescence microscope (model IX70; Olympus) equippedwith a Cool SNAP HQ CCD camera (Roper Scientific) and a 20
0.8
NA objective (Olympus). The baseline intensity was at 4 s before thestimulation with high K solution. To induce FM dye destaining, neurons
were depolarized with high K solution. FM-labeled spots/50-m dendrite
were analyzed using a quantification menu of the MetaMorph software(Universal Imaging Co.).
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Assays of cell survival and dendritic growth
Cortical neurons (5 104 cells/cm2) were cultured in Neurobasal medium
containing B27 supplement (GIBCO BRL), 0.5 mM glutamine, for 3 d, andtreated with indicated agents in Neurobasal medium containing no B27
supplement for 3 d. Cell survival was quantitated by WST-1 assay, which
determines cell viability based on the cleavage activity of a soluble tetrazolium salt WST-1 by mitochondrial dehydrogenases. In brief, cultured
cortical neurons were incubated with a tetrazolium salt WST-1 (Roche) for
30 min and viable cells were then determined by a microplate reader at
450 nm with 650 nm as a reference wavelength. Alternatively, the number of MAP2-positive neurons was counted (Yamada et al., 2001). To
identify primary dendrites from each neuron, cells were stained with antiMAP2 antibody, followed by DAPI staining. After taking images of MAP2positive neurons with cell body of diameter (16.9  0.5 m diam, n  32
cells from four independent coverslips), the number of the primary dendrites was counted.
Online supplemental material
Fig. S1 shows localization of TrkB and BDNF in lipid rafts during corticaldevelopment. Fig. S2 shows association of TrkB-FL with lipid rafts 1 min after BDNF stimulation. Fig. S3 shows TrkB-T1 expression in rafts and nonrafts and NT-4–induced recruitment of TrkB-FL into lipid rafts. Fig. S4shows association of signaling molecules and TrkB with lipid rafts. Fig. S5shows that treatment with 10 mM MCD lead to a partial decrease in
BDNF-induced activation of TrkB-FL. Further comments on the data reported can be found in the legends. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200404106/DC1.
We thank Dr. Masato Okada for advice on lipid raft preparation, Drs. James
Q. Zheng and Eero Castren for helpful discussion, and Sumitomo Pharmaceuticals for providing recombinant BDNF.

Submitted: 26 April 2004
Accepted: 19 October 2004

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Preparation of lipid rafts and Western blot analysis
Lipid rafts were prepared according to the method of Kawabuchi et al.
(2000). Cultured neurons (3.5 105 cells/cm2) were rinsed with PBS andquickly lysed in 0.5 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1
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Slice preparation and electrophysiology
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together for 2 h before synaptic responses to HFS were tested.

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