Neurotrophin-mediated dendrite-to-nucleus signaling
revealed by microfluidic compartmentalization
Michael S. Cohena,1, Carlos Bas Ortha,1,2, Hyung Joon Kimb, Noo Li Jeonc, and Samie R. Jaffreya,3
aDepartment of Pharmacology, Weill Medical College Cornell University, New York, NY 10065;bDepartment of Biomedical Engineering, University of
California, Irvine, CA 92697; andcSchool of Mechanical and Aerospace Engineering, Seoul National University, Seoul 151-742, Korea
Edited* by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 10, 2011 (received for review September
Signaling from dendritic synapses to the nucleus regulates impor-
tant aspects of neuronal function, including synaptic plasticity. The
neurotrophin brain-derived neurotrophic factor (BDNF) can induce
long-lasting strengthening of synapses in vivo and this effect is
dependent on transcription. However, the mechanism of signaling
to the nucleus is not well understood. Here we describe a micro-
fluidic culture device to investigate dendrite-to-nucleus signaling.
Using these microfluidic devices, we demonstrate that BDNF can
act directly on dendrites to elicit an anterograde signal that in-
duces transcription of the immediate early genes, Arc and c-Fos.
Induction of Arc is dependent on dendrite- and cell body-derived
calcium, whereas induction of c-Fos is calcium-independent. In
naling, which is MEK5-dependent, BDNF-mediated anterograde
dendrite-to-nucleus signaling is dependent on MEK1/2. Intrigu-
ingly, the activity of TrkB, the BDNF receptor, is required in the cell
body for the induction of Arc and c-Fos mediated by dendritically
applied BDNF. These results are consistent with the involvement
of a signaling endosome-like pathway that conveys BDNF signals
from the dendrite to the nucleus.
gene expression|mRNA translation
blocking antibodies impairs long-term potentiation (LTP) induced
by high-frequency stimulation (2–7). Exogenously applied BDNF
can induce LTP at medial perforant path-granule cell synapses
in vivo (8), and this effect is blocked by the transcription inhibitor
actinomycin D (9). Additionally, BDNF induces genes known to
regulate synapse function, such as activity-regulated cytoskeletal
(Arc) protein (10–12). Because the effects of BDNF on transcrip-
tion are frequently explored by bath application, it is not clear if
BDNF-mediated transcription is because of activation of TrkB in
dendrites, axons, or the cell body (13). If dendritically localized
TrkB receptors can regulate gene expression, mechanisms to con-
vey the signal from dendrites to the nucleus, a distance that can be
several hundred micrometers, would be required.
How might BDNF transduce a signal from dendrites to the nu-
envisioned, among them: (i) propagation of a calcium wave to the
soma, and subsequent activation of calcium-dependent transcrip-
tion, as has been previously suggested (14) or (ii) diffusion of sig-
naling molecules from dendrites to the nucleus, as has been
suggested for phosphorylated MAPK (6). To determine the mech-
selective stimulation or inhibition of dendritic signaling is required.
Here we describe the design of a microfluidic device to in-
vestigate BDNF-mediated dendrite-to-nucleus signaling. CNS
neurons can be cultured in this device and their dendrites can be
he neurotrophin brain-derived neurotrophic factor (BDNF)
has emerged as a key regulator of synaptic plasticity (1). De-
fluidically isolated from their cell bodies. Using this device, we
investigated intradendritic as well as dendrite-to-nucleus signal-
ing. We find that BDNF can act on dendrites to activate gene
transcription. This anterograde signaling requires selective roles
for calcium, as well as MAPK and TrkB activity, in each com-
partment. Finally, we find that anterograde neurotrophin signal-
ing from dendrites to the nucleus exhibits marked differences
compared with retrograde signaling from axons to the nucleus.
Taken together, these data identify a unique mechanism of den-
dritic signaling that may mediate the effects of dendritic BDNF.
Dendritic Compartmentalization Using a Microfluidic Device. To in-
to selectively stimulate dendrites. Recently, polydimethylsiloxane
(PDMS)-based microfluidic culturing devices were developed to
fluidically isolate axons from cell bodies, allowing selective stim-
ulation of distal axons (15). These culturing devices comprise two
compartments, the cell body compartmentand axon compartment,
separated by embedded microgrooves (10 μm × 450 μm). Because
axons can grow long distances in essentially straight lines, these
devices are suitable for compartmentalizing axons. However, be-
cause dendrites are branched and substantially shorter, it is not
clear whether dendrites would be compatible with a microfluidic
We designed a microfluidic device containing two separate
compartments (1.5-mm wide, 7-mm long) separated by 7 μm ×
75 μm microgrooves (Fig. 1 A and B). To determine if dendrites
would grow through the microgrooves, embryonic day 18 (E18)
rat cortical neurons were plated on poly-D-lysine (PDL)/laminin
in the cell body compartment. E18 cortical neuronal dendrites
crossed into the neurite compartment as early as day in vitro 7
(DIV7) and exhibited characteristic highly branched processes by
DIV14 (Fig. 1C). Most crossed dendrites arose from cell bodies
that were within ∼100 μm of the microgrooves in the cell body
compartment (Fig. 1C). As expected, axons also crossed and
were readily detected in the neurite compartment by DIV14
(Fig. 1D). Similar results were obtained for E18 rat hippocampal
Author contributions: M.S.C., C.B.O., and S.R.J. designed research; M.S.C. and C.B.O. per-
formed research; M.S.C., C.B.O., H.J.K., and N.L.J. contributed new reagents/analytic tools;
M.S.C., C.B.O., and S.R.J. analyzed data; and M.S.C., C.B.O., and S.R.J. wrote the paper.
Conflict of interest statement: N.L.J. is an inventor and patent holder of the microfluidic
devices described in this article and is a cofounder of Xona Microfluidics LLC, which
markets related microfluidic devices.
*This Direct Submission article had a prearranged editor.
1M.S.C. and C.B.O. contributed equally to this work.
2Present address: Excellence Cluster CellNetworks at University of Heidelberg, and De-
partment of Neurobiology, Interdisciplinary Center for Neurosciences, University of
Heidelberg, 69120 Heidelberg, Germany.
3To whom correspondence should be addressed. E-mail: SRJ2003@med.cornell.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 5, 2011
| vol. 108
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neurons, albeit less crossing into the neurite compartment was
observed (Fig. S1).
Because both axons and dendrites grow into the neurite
compartment, we examined dendrites for evidence of synaptic
connections. At DIV14, spines were observed in crossed den-
drites of GFP-expressing neurons (Fig. 1E). The average spine
density (0.461 ± 0.052 spines per micrometer) of crossed den-
drites is similar to previously reported spine densities (16). Ad-
ditionally, we detected punctate staining for α-actinin, a protein
enriched at excitatory synapses (17) (Fig. S1). Together, these
results demonstrate that dendrites can grow through the 75-μm
long microgrooves, exhibit characteristic branched morphology,
and form synaptic connections in the neurite compartment.
Fluidic Isolation of Neurites and Cell Bodies. Next, we examined
whether the neurite and cell body compartments connected by
significantly shortened microgrooves were fluidically isolated.
We incubated the neurite compartment with Alexa Fluor-647
hydrazide (20 μg/mL), a low molecular weight fluorescent dye.
After 24 h, only a trace amount (∼3%) of the dye was detected in
the cell body compartment (Fig. S2). This amount of dye leakage
was only slightly greater than that determined for the device with
450-μm long microgrooves (∼1%), indicating that despite the six-
times shorter length of the 75-μm long microgrooves, near-
complete fluidic isolation can be achieved.
To further assess fluidic isolation, we examined whether cy-
cloheximide (CHX) applied to the neurite compartment can af-
fect protein synthesis in the cell body compartment. Protein
synthesis was measured by metabolic labeling withthe methionine
bioisostere, azidohomoalanine (AHA), which can be labeled with
AlexaFluor-488 alkyneusingclick chemistry(18).Consistentwith
a dose-response study, application of CHX (0.5 μM) to the cell
body compartment resulted in near-complete inhibition of pro-
tein synthesis (Fig. S2). In contrast, treatment of the neurite
compartment with CHX did not affect protein synthesis in the
cell body compartment (Fig. S2). Taken together, these results
demonstrate that discrete chemical microenvironments can be
maintained with 75-μm long microgrooves.
Intradendritic mRNA Translation Is Required for Dendritic Growth. To
examine intradendritic signaling pathways, we first focused on
potential functions of dendritic mRNA translation. Dendrites
an involvement of local translation in processes such as dendritic
growth. However, because it has not been possible to maintain
selective application of protein-synthesis inhibitors to dendrites
over several days, a role for intradendritic protein synthesis in
dendritic growth has not been addressable. To test this idea, we
monitored the effect of CHX on basal dendrite growth over 7 d.
The cumulative distributions of dendritic length of crossed den-
drites after 7 d of CHX treatment demonstrated that neuritic
CHX treatment reduced the total dendritic length by 35% com-
pared with vehicle control (median, 847.2 vs. 1,308 μm, respec-
tively) (Fig.S3).To ruleoutthepossibilitythattheeffects ofCHX
on dendritic growth could reflect inhibition of intra-axonal protein
synthesis, we stained neurites for ribosomal protein S6. Immuno-
labeling of DIV9 neurons reveals discrete puncta in dendrites, but
not axons (Fig. S3), indicating the absence of ribosomes in mature
axons. This finding is consistent with previous studies demon-
strating that mature axons of the central nervous system lack the
capacity for protein synthesis (20, 21). These results indicate that
intradendritic protein synthesis is required for basal dendritic
growth in cortical neurons.
BDNF Induces a Dendrite-to-Nucleus Signal to Regulate Gene Expres-
sion. We next asked whether BDNF can elicit a signal that is
conveyed from the dendrite to the nucleus. Consistent with pre-
vious studies (10, 12), bath application of BDNF induced the
expression of the immediate early genes (IEGs) Arc and c-Fos
(Fig. S4). To determine if the transcriptional effects of BDNF can
be elicited from dendrites, we selectively applied BDNF to the
neurite compartment and measured the expression of Arc and
c-Fos in the cell body compartment. Treatment of the neurite
compartment with BDNF resulted in a significant induction of
Arc (twofold) and c-Fos (fourfold) protein (Fig. 2 A and B, white
arrowheads) and mRNA (Fig. S4) levels in a subset of neurons,
which exhibited dendrites that had crossed to the neurite com-
partment. The increase in Arc and c-Fos protein mediated by
neurite application of BDNF was abrogated by the transcription
inhibitor actinomycin D (Fig. 2 C and D). To rule out the pos-
sibility that the induction of c-Fos or Arc might reflect activation
of TrkB on axonal processes, we selectively applied BDNF to
axons of cortical neurons grown in microfluidic devices with 450-
μm long microgrooves. Application of BDNF to axons did not
induce IEG expression (Fig. S5), indicating that the effects of
BDNF do not reflect retrograde signaling from axons to the nu-
cleus. Importantly, previous studies using Campenot chambers,
where the distance between axons and cell bodies is 1 mm, have
shown that neurotrophins can convey signals from axons to the
nucleus in peripheral system neurons within the time course of
our experiments (22, 23).
We next sought to compare the magnitude of gene induction
mediated by dendritically applied versus cell body-applied
BDNF. Unlike application of BDNF to the neurite compartment,
direct application of BDNF to the cell body compartment in-
duced the expression of Arc and c-Fos in nearly all cells (Fig. 2 E
and F). However, the magnitude of c-Fos induction was nearly
top view side view
body. (A) Schematic representation of the neuronal microfluidic device. The
device is fabricated from a PDMS mold containing mirror image compart-
ments (1.5-mm wide, 7-mm long, 100-μm high) connected by microgrooves
(10-μm wide, 3-μm high). (B) Phase-contrast image of the microfluidic device
with 75-μm long microgrooves illustrating schematic representations of CNS
neurons projecting dendrites and axons into the neurite compartment. (C–E)
Immunofluorescence analysis of E18 rat cortical neurons (DIV14) cultured in
the microfluidic device. (C) Extensive crossing of dendrites (MAP2, green)
into the neurite compartment is observed for cell bodies within ∼100 μm of
the microgrooves. Cell bodies (DAPI, blue) are restricted to the cell body
compartment. (Scale bar, 50 μm.) (D) Axons (TAU-1, red) project extensively
into the neurite compartment and considerably farther from the micro-
grooves than the dendrites (MAP2, green), which remain close to the
microgrooves. (Scale bar, 20 μm.) (E) A cortical neuron expressing GFP
(green) reveals the presence of dendritic spines on dendrites (MAP2, red) in
the neurite compartment. (Scale bar, 20 μm.) (Inset) Higher magnification
image of a segment of dendrite enclosed by the white box in the merged
image. (Scale bar, 2 μm.)
A microfluidic device to fluidically isolate dendrites from the cell
Cohen et al.PNAS
| July 5, 2011
| vol. 108
| no. 27
identical in both treatment paradigms (Fig. 2H) and the magni-
tude of Arc induction mediated by neurite application of BDNF
was slightly less than that for cell body application (Fig. 2G).
Taken together, these results demonstrate that BDNF acts on
dendrites to generate a signal that is conveyed to the nucleus to
induce changes in gene expression.
Because BDNF enhances mRNA translation in dendrites (24),
we wondered if intradendritic mRNA translation is required for
BDNF-mediated dendrite-to-nucleus signaling. Selective appli-
BDNF-mediated c-Fos induction. In contrast, direct application
of CHX to the cell body compartment attenuated BDNF-induced
increases in c-Fos protein levels (Fig. 2I). Hence, intradendritic
mRNA translation is not required for gene induction mediated
by BDNF signaling at dendrites.
Dendritic BDNF-Induced Arc and c-Fos Expression Does Not Require
Glutamate Signaling. BDNF can rapidly potentiate excitatory
synaptic transmission in cultured cortical and hippocampal neu-
rons by stimulating glutamate release from presynaptic terminals
(25, 26). To determine if glutamatergic transmission is required
for dendrite-to-nucleus signaling mediated by dendritic BDNF,
cortical neurons were treated with kynurenic acid and MgCl2for
48 h before the addition of BDNF to the neurite compartment.
These conditions completely blocked c-Fos induction mediated
by the GABAAreceptor antagonist bicuculline, indicating com-
plete inhibition of glutamatergic signaling (Fig. S6). Neither Arc
nor c-Fos induction by dendritic BDNF was significantly changed
in the presence of kynurenic acid and MgCl2(Fig. S6). Thus,
glutamatergic transmission is not required for BDNF-mediated
dendrite-to-nucleus signaling. Because the basal levels of Arc and
the cell body (E and F) compartment. After 2 h, cells were fixed and immunolabeled with antibodies against Arc (green, A and E), c-Fos (green, B and F), and
MAP2 (red). (Scale bars, 20 μm.) (A and B) The cell bodies in the cell body compartment that have dendrites projecting into the neurite compartment are
indicated by white arrowheads. (C and D) Induction of Arc and c-Fos expression upon dendritic BDNF stimulation is because of new transcription. On DIV10,
actinomycin D (2 μg/mL) or vehicle control was added to the cell body compartment for 1 h followed by the addition of BDNF (100 ng/mL) to the neurite
compartment for 2 h. Cells were fixed and immunolabeled with antibodies against c-Fos and MAP2. (G and H) Quantification of results from A, B, E, and F. (I)
Local protein synthesis is not required for c-Fos induction mediated by dendritic BDNF stimulation. On DIV10, CHX (10 μM) or vehicle control was added to the
indicated compartment for 1 h followed by the addition of BDNF (100 ng/mL) to the neurite compartment for 2 h. Cells were fixed and immunolabeled with
antibodies against c-Fos and MAP2. The error bars represent SEM, ***P < 0.0001, **P = 0.006 (unpaired, two-tailed t test); n values listed above the bars
represent the number of cell bodies analyzed.
BDNF acts at dendrites to induce the expression Arc and c-Fos. On DIV10 BDNF (100 ng/mL) or vehicle control was applied to the neurite (A and B) or
| www.pnas.org/cgi/doi/10.1073/pnas.1012401108Cohen et al.
c-Fos were reduced in the presence of kynurenic acid and MgCl2
all subsequent experiments were performed with media contain-
ing these compounds.
Intracellular Calcium Is Differentially Required for Dendritic BDNF-
Induced Arc and c-Fos Expression. WenextaskedifBDNF-mediated
dendrite-to-nucleus signaling requires calcium in either the cell
body or dendrites. Induction of Arc and c-Fos by cell body appli-
cation of BDNF was blocked by cell body application of
the intracellular calcium chelator BAPTA-AM (Fig. 3A), consis-
tent with previous studies (12). When BDNF was applied to neu-
rites, Arc induction was blocked by chelating calcium in either the
neurite or cell body compartment, indicating a requirement for
calcium in both compartments in mediating dendritic BDNF
signals. However, induction of c-Fos by dendritic BDNF was
independent of calcium in dendrites and was potentiated by che-
fold increase in dendritic BDNF-mediated c-Fos induction in the
presence of cell body BAPTA-AM could reflect an inhibitory role
for intracellular calcium in regulating c-Fos expression. Taken
together, these results demonstrate a differential requirement for
intracellular calcium in dendritic BDNF-induced Arc and c-Fos
Dendritically Applied BDNF Induces Arc and c-Fos Through Trk Activity
in the Cell Body. We next examined the mechanism by which
BDNF signals are conveyed from dendrites to the nucleus. We
first asked whether the induction of c-Fos and Arc mediated by
dendritic application of BDNF reflects an increase in the release
of BDNF from the cell body. To test this idea, we used TrkB-Fc,
a membrane-impermeable scavenger of BDNF, to block the
effects of extracellular BDNF (6). Application of TrkB-Fc to the
dendritic BDNF-mediated c-Fos and Arc induction (Fig. S7). In
contrast, application of TrkB-Fc to the cell body compartment
did not affect gene expression induced by dendritically applied
BDNF (Fig. S7). These data argue against a model where den-
dritic application of BDNF leads to the release of BDNF from
the cell body.
Selective application of neurotrophins to PNS axons leads
to Trk receptor endocytosis and subsequent retrograde traffick-
ing of neurotrophin-bound Trk to the cell body, where it acti-
vates signaling pathways leading to transcription (23, 27–30). To
determine if a similar mechanism occurs in dendrites, we first
asked if endocytosis is required for BDNF-mediated dendrite-
to-nucleus signaling. Previous findings demonstrated that the
GTPase dynamin is required for TrkB receptor internalization
(31). To assess the requirement of dynamin-mediated endocy-
tosis for dendritic BDNF-induced IEG expression, we used the
selective dynamin inhibitor dynasore (32). Selective treatment of
the neurite compartment with dynasore (100 μM) before den-
dritic BDNF stimulation significantly blocked Arc and c-Fos
induction (Fig. S8).
We next determined if TrkB activity in the cell body is required
for BDNF-mediated dendrite-to-nucleus signaling. To test this
idea, we sought to pharmacologically inhibit TrkB activity in the
cell body after dendritic application of BDNF. Although K252a
is commonly used as a Trk inhibitor to investigate the roles of
Trks (TrkA, TrkB, and TrkC) in retrograde axonal signaling, it is
results using a panel of structurally distinct Trk inhibitors. The
bis-indole Gö6976 is a well-described potent Trk inhibitor (35).
We also considered GW2580, a pyrimidine derivative that was
initially described as a highly selective inhibitor of the colony stim-
ulating factor-1 receptor (CSF-1R) (36), a macrophage-enriched
kinase that is not expressed at detectable levels in the cortex or
over 300 kinases using in vitro competition binding assays revealed
and TrkC (38). To determine if GW2580 can inhibit Trk activity in
cells, we treated TrkB-expressing human embryonic kidney (TrkB-
HEK) cells (39) with increasing concentrations of GW2580 before
BDNF stimulation. GW2580 inhibited BDNF-induced Tyr490
with complete inhibition achieved by 3 μM (Fig. S9).
We next tested the effects of these inhibitors on dendritic
BDNF-induced IEG expression in the microfluidic chambers.
Selective application of either K252a (1 μM), Gö6976 (50 nM)
or GW2580 (3 μM) to the cell body compartment completely
blocked Arc and c-Fos induction mediated by dendritic BDNF
(Fig. 4 A and B and Fig. S9), indicating that Trk activity in the cell
body is necessary for BDNF-mediated dendrite-to-nucleus sig-
naling. In contrast, selective treatment of the neurite compart-
ment with the Trk inhibitors before dendritic BDNF stimulation
only partially blocked Arc induction and did not significantly in-
hibit c-Fosinduction (Fig.4AandBandFig.S9).Takentogether,
these data support the idea that dendritic application of BDNF
induces TrkB endocytosis and subsequent translocation of TrkB
to the cell body, where its activity is required for c-Fos and
BDNF Uses the MEK1/2 Pathway for Dendrite-to-Nucleus Signaling.
We next examined the signaling pathways downstream of BDNF-
TrkB dendrite-to-nucleus signaling. In PNS neurons, neuro-
trophins use the MAPK kinase 5 (MEK 5) to elicit retrograde
survival signaling (40). BDNF can activate both MEK1/2 and
MEK5 in cortical neurons (41, 12); however, it is unclear which
of these kinases is required for BDNF-mediated IEG transcrip-
tion. To determine if the MEK1/2 pathway regulates dendritic
BDNF-induced IEG expression, we treated the cell body com-
partment with the selective MEK1/2 inhibitor PD 0325901 be-
fore dendritic BDNF stimulation (42). PD 0325901 significantly
inhibited Arc and c-Fos induction mediated by neuritic appli-
cation of BDNF (Fig. 4 C and D). We next sought to determine
if p90 ribosomal protein s6 kinases (RSKs), which are major
downstream effectors of MEK1/2 in CNS neurons (43), could
regulate BNDF-mediated dendrite-to-nucleus signaling. Treat-
Fluorescence intensity (AU)
Fluorescence intensity (AU)
dendrite- and cell body-derived calcium, whereas induction of c-Fos is cal-
cium-independent. (A) On DIV10, BAPTA-AM (32 μM) or vehicle control was
added to the cell body compartment followed by the addition of BDNF (100
ng/mL) to the cell body (A) or neurite (B) compartment for 2 h. Cells were
fixed and immunolabeled with antibodies against Arc, c-Fos, and MAP2. The
error bars represent SEM, ***P < 0.0001 (unpaired, two-tailed t test); n
values listed above the bars represent the number of cell bodies analyzed.
Induction of Arc by dendritically-applied BDNF is dependent on
Cohen et al. PNAS
| July 5, 2011
| vol. 108
| no. 27
ment of the cell body compartment with the selective RSK in-
hibitor BI D1870 (44) did not block Arc or c-Fos induction (Fig.
4 C and D), but instead resulted in an increase in c-Fos induction
compared with BDNF alone. The potentiation of dendritic
BDNF-mediated c-Fos induction by BI D1870 suggests that RSK
is involved in a negative feedback loop that suppresses the
MEK1/2 pathway. Indeed, previous studies have shown that BI
D1870 can augment the kinase activity of ERK1/2, which are
direct upstream activators of RSK (44). Taken together, these
results demonstrate anterograde dendrite-to-nucleus signaling
requires MEK1/2 activity and that RSKs are not downstream
effectors in this pathway.
The transcriptional effects of BDNF have largely been in-
vestigated by bath application of BDNF to cultured neurons. As
a result, it is not clear if these effects are a result of the activation
of the BDNF receptor TrkB in the cell body or if dendritic TrkB
can generate a signal that is conveyed to the nucleus to regulate
gene transcription. By selectively applying BDNF to dendrites
using microfluidic culturing devices, we find that dendritic TrkB
can elicit a signal that is conveyed to the nucleus to induce
transcription. Dendritically applied BDNF elicited both a calcium-
dependent and a calcium-independent signaling cascade, regu-
lating Arc and c-Fos, respectively. BDNF signaling from den-
drites to the nucleus requires dynamin-dependent endocytosis in
dendrites as well as MEK1/2 and TrkB activity in the cell body.
These data suggest BDNF acting at dendrites regulates tran-
scription by inducing TrkB endocytosis and translocation to the
cell body, where it activates a MEK1/2 signaling pathway to in-
duce c-Fos and Arc. Taken together, these data demonstrate the
existence of a mechanism for conveying signals from dendrites to
the nucleus, which may mediate the long-lasting effects of BDNF
on synaptic plasticity in vivo.
The finding that dendritic BDNF-induced c-Fos expression
was calcium-independent was unanticipated, because we found
that intracellular calcium was required for the expression of
c-Fos following bath application of BDNF. Additionally, pre-
vious studies have suggested that BDNF induces c-Fos expression
in a calcium-dependent manner (14). One explanation for this
discrepancy is that the signaling pathway induced by dendritically
localized TrkB is calcium independent, and was masked by the
global activation of TrkB. Indeed, numerous signaling proteins
are expressed in specific compartments in the cell (45), and may
therefore not be accessible to TrkB depending on its localization
in cells. Our results point to the potential differences between
TrkB pathways elicited in the cell body and dendrites, and high-
light the importance of selective stimulation of dendritic TrkB
using approaches such as the microfluidic devices described here.
There are at least three differences between anterograde
to-nucleus signaling that are worth noting. First, whereas retro-
grade axonal signaling requires Trk activity in the axon (30),
dendrite-to-nucleus signaling does not necessarily require Trk
activity in dendrites. Second, whereas neurotrophin-mediated
axon-to-nucleus signaling uses MEK5 to mediate nuclear re-
sponses (40), BDNF-mediated dendrite-to-nucleus signaling uses
MEK1/2 to mediate Arc and c-Fos induction. The specific MAPK
pathway that is activated most likely determines which down-
stream signaling pathways are activated to mediate transcription
of specific genes. Finally, local mRNA translation promotes ret-
rograde signaling in axons (46), and intradendritic mRNA tran-
slation is dispensable for the induction of c-Fos by dendritically
What does the requirement for Trk activity in the cell body for
dendritically mediated BDNF induction of IEGs suggest about
the mechanism of neurotrophin-mediated dendrite-to-nucleus
signaling? A major mechanism for axon-to-nucleus signaling in
PNS neurons is the signaling endosome model (47). In this
model, the neurotrophin-bound Trk receptor is endocytosed in
distal axons and is then translocated as an endosome to the cell
body, where it activates its downstream effectors, leading to gene
transcription. Our data showing the requirement for Trk activity
in the cell body and dynamin-mediated endocytosis in dendrites
for BDNF-mediated dendrite-to-nucleus signaling is consistent
with a novel dendrite-derived signaling endosome. It will be
important to image BDNF-TrkB endosome trafficking from
dendrites to the cell body, and to determine if these signaling
endosomes contribute to BDNF-mediated changes in synaptic
plasticity in a physiological context. Previous studies have shown
that theta-burst stimulation induces the release of endogenous
BDNF from mossy fibers, as well as Schaffer collaterals onto the
dendrites of CA3 and CA1 pyramidal neurons, respectively, and
this mediates long-lasting changes in potentiation or depression
(3, 48, 6, 49). Moreover, a recent study demonstrated that re-
lease of BDNF from cortical axons onto dendrites of medium
spiny neurons in the dorsal striatum induced postsynaptic LTP
(50). It is conceivable that dendritically derived BDNF-TrkB
endosomes have a role in these pathways.
We envision the investigation of other potential dendrite-to-
nucleus signaling pathways using the microfluidic device de-
scribed here. Because the microfluidic platform is compatible
with live cell imaging, intracellular trafficking of signaling mole-
cules from dendrites to the cell body can be imaged following
selective dendritic synapse activation using these devices. Re-
cently, Schuman and colleagues described a different microfluidic
device to investigate synaptic signaling (51). In this device, neu-
rons are cultured in two compartments, and project axons and
dendrites toward a narrow central perfusion chamber. Both of
these devices provide an opportunity to investigate the molecular
of Arc and c-Fos mediated by dendritically applied BDNF. (A and B) On
DIV10, K252a (1 μM) or vehicle control was added to the indicated com-
partment followed by the addition of BDNF (100 ng/mL) to the neurite
compartment for 2 h. Cells were fixed and immunolabeled with antibodies
against Arc (A), c-Fos (B), and MAP2. (C and D) On DIV10, PD 0325901 (150
nM) or BI D1870 (10 μM) or vehicle control was added to the cell body
compartment followed by the addition of BDNF (100 ng/mL) to the neurite
compartment for 2 h. Cells were fixed and immunolabeled with antibodies
against Arc (A), c-Fos (B), and MAP2. The error bars represent SEM, ***P <
0.0001, *P < 0.05 (unpaired, two-tailed t test); n values listed above the bars
represent the number of cell bodies analyzed.
Trk and Erk1/2 activity in the cell body are required for the induction
| www.pnas.org/cgi/doi/10.1073/pnas.1012401108 Cohen et al.
pathways that couple dendritic stimulation with changes in gene Download full-text
Materials and Methods
The polydimethylsiloxane microfluidic devices with 75-μm microgrooves were
fabricated as previously described (15). The preparation of the microfluidic
devices for compartmentalization of dendrites are described in SI Materials
The materials used and detailed methods are described in SI Materials
ACKNOWLEDGMENTS. We thank J. Harris (University of California, Irvine,
CA) for preparing the master for casting microfluidic devices, M. Chao (New
York University, New York) and H. Nawa (Niigata University, Japan) for TrkB-
HEK cells, and F. S. Lee (Weill Medical College, New York), J. Taunton
(University of California, San Francisco), and members of the S.R.J. laboratory
for helpful discussions. This work was supported by a Life Sciences Research
Foundation fellowship (Amgen fellow) and National Institute on Drug Abuse
Training Grant T32DA007274 (to M.S.C.), a German Research Foundation
fellowship Deutsche Forschungsgemeinschaft Forschungsstipendium (to C.B.
O), and March of Dimes Grant 12-FY07-261 and National Institutes of Health
Grants DA023581 and NS056306 (to S.R.J).
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Cohen et al.PNAS
| July 5, 2011
| vol. 108
| no. 27