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The Journal of Neuroscience, September 1, 2002, 22(17):7580-7585
Brain-Derived Neurotrophic Factor Promotes the Maturation of
GABAergic Mechanisms in Cultured Hippocampal Neurons
Maki K.
Yamada*,
Kohsuke
Nakanishi*,
Shizu
Ohba,
Takeshi
Nakamura,
Yuji
Ikegaya,
Nobuyoshi
Nishiyama, and
Norio
Matsuki
Laboratory of Chemical Pharmacology, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
 |
ABSTRACT |
Brain-derived neurotrophic factor (BDNF) has been implicated in
activity-dependent plasticity of neuronal function and network arrangement. To clarify how BDNF exerts its action, we evaluated the
physiological, histological, and biochemical characteristics of
cultured hippocampal neurons after long-term treatment with BDNF. Here
we show that BDNF facilitates high K+-elicited release of
GABA but not of glutamate and induces an increase in immunoreactive
signals of glutamic acid decarboxylase, a GABA-synthesizing enzyme. The
soma size of GABAergic neurons was enlarged in BDNF-treated cultures,
whereas the average soma size of all neurons was virtually unchanged.
BDNF also upregulated protein levels of GABAA receptors but
not of glutamate receptors. These data imply that BDNF selectively
advances the maturation of GABAergic synapses. However,
immunocytochemical analyses revealed that a significant expression of
TrkB, a high-affinity receptor for BDNF, was detected in non-GABAergic
as well as GABAergic neurons. BDNF also increased to total amount of
synaptic vesicle-associated proteins without affecting the number of
presynaptic vesicles that can be labeled with FM1-43 after
K+ depolarization. Together, our findings indicate
that BDNF principally promotes GABAergic maturation but may
also potentially contribute to excitatory synapse development via
increasing resting synaptic vesicles.
Key words:
Key word: BDNF; hippocampus; plasticity; neural network; GABA; inhibitory neuron; resting pool; synaptic vesicle-associated
protein
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INTRODUCTION |
Brain-derived neurotrophic factor
(BDNF), a member of the neurotrophin family abundantly expressed in the
CNS (Lewin and Barde, 1996 ), plays a crucial role in activity-dependent
plastic changes in synaptic strength and network refinement. For
instance, BDNF is reported to participate in the induction of long-term
potentiation (Thoenen, 1995; Lu and Figurov, 1997) and long-term
depression (Ikegaya et al., 2002) of synaptic transmission and is also
implicated in the modification of neural circuits, including ocular
dominance columns (Cabelli et al., 1995; Huang et al., 1999; Lein and
Shatz, 2000). However, these two phenomena may involve distinct
mechanisms (Mataga et al., 2001; Renger et al., 2002). The contribution
of BDNF to epilepsy is well documented but contains partially
controversial arguments (Binder et al., 2001; Reibel et al., 2001).
Long-term infusion of BDNF prevents the development of kindling (Larmet et al., 1995), whereas blockade of endogenous BDNF also inhibits it
(Binder et al., 1999). Therefore, to clarify the precise mechanisms underlying physiological and pathological reorganization and functional plasticity of neural networks, the long-term effect of BDNF should be
investigated in simplified experimental systems.
We have focused the present study on the long-term effect of
brain-derived neurotrophic factor (BDNF) on neuronal functions, and we
provide a large body of evidence that BDNF primarily promotes GABAergic
maturation. BDNF induces an enlargement of the soma of GABAergic
neurons, increases the expression of GABAA
receptor subunits and glutamic acid decarboxylase (GAD; a
GABA-synthesizing enzyme), and facilitates high
K+-elicited release of GABA. In addition
to other studies that assessed the BDNF effects on synaptic vesicles
(Pozzo-Miller et al., 1999; Collin et al., 2001; Tyler and
Pozzo-Miller, 2001), our study has shown for the first time that BDNF
dramatically upregulates the expression level of presynaptic
vesicle-associated proteins without apparent changes in the active pool
of synaptic vesicles, suggesting that BDNF induces an increase in
resting synaptic vesicles. Taken together, our findings indicate that
BDNF principally promotes GABAergic maturation but may also potentially
contribute to excitatory synapse development via increasing resting
synaptic vesicles.
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MATERIALS AND METHODS |
Primary culture. Cultured hippocampal neurons were
prepared as described previously (Shitaka et al., 1996), with some
modifications. Briefly, whole brains were isolated from embryonic day
18 Wistar rats, and the hippocampi were dissected out and treated with
0.25% trypsin (Difco Laboratories, Detroit, MI) and 0.01%
deoxyribonuclease I (Sigma, St. Louis, MO) at 37°C for 30 min. The
cells were suspended with Neurobasal medium containing 10% fetal
bovine serum (Sanko-junyaku, Tokyo, Japan), and plated at a density of
65,000 cells/cm2 on polyethyleneimine
(Sigma)-coated 24- or 48-well plates (Costar, Cambridge, MA), 35 mm
dishes (Costar), and glass coverslips with flexiPERM (Sartorius,
Goettingen, Germany). Twenty-four hours after plating, the medium was
changed to serum-free Neurobasal medium with 2% B27 supplement (Life
Technologies, Gaithersburg, MD) and 50 ng/ml BDNF (recombinant human
BDNF, a gift from Sumitomo Pharmaceuticals, Osaka, Japan), and then the
cultures were kept for the next 6-10 d in vitro.
Determination of glutamate and GABA release. Quantification
of amino acid release was performed as described previously (Jeftinija et al., 1996), with some modifications. Briefly, cultured cells were
washed five times with Krebs'-Ringer's solution (in
mM: 130 NaCl, 3 KCl, 2 CaCl2, 0.8 MgS04, 20 HEPES,
and 10 glucose) adjusted to pH 7.4 with NaOH. Depolarizing
Krebs'-Ringer's solution containing 50 mM KCl
was applied for 1 min and collected into tubes on ice. All the assay
buffer and culture plates were kept at 37°C during the manipulations.
The samples were mixed with o-phthalaldehyde (Sigma) and
reacted for 1 min. The content of the derivatized amino acids was
determined by an HPLC equipped with the reverse-phase capillary
column C-18 (5 µm; BAS Co. Ltd., Tokyo, Japan) and the fluorescence
detector CMA280 (excitation at 340 nm, emission at 445 nm)
(CMA/Microdialysis, Stockholm, Sweden) (Lindroth and Mopper, 1979). The
mobile (60 µl/min) solution consisting of (in
mM) 100 KH2PO4, 100 Na2HPO4, and 0.1 EDTA, pH
6.0, contained 10% acetonitrile and 3% tetrahydrofuran. The peak area
at the predicted position was calibrated against the standard curves
for quantification with CMA200 software (CMA/Microdialysis).
Immunocytochemistry. Cells were fixed with 4%
paraformaldehyde at 4°C for 30 min. After treatment with 0.1% Triton
X-100 in PBS for 15 min, they were incubated in 1-2% serum-PBS for 1 hr, then with a primary antibody anti-GAD (anti-GAD for both 65 and 67 kDa; rabbit, 1:4000; AB1511 from Chemicon, Temecula, CA) or anti-GABA
(rabbit, 1:5000; Sigma) and anti-TrkB (specific for full-length
TrkB; gp145, goat, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA)
overnight at 4°C. After washes, cells were incubated with
fluorochrome-conjugated antibody (FITC-anti-rabbit IgG from Sigma or
Alexa flour 488 donkey anti-goat IgG and Alexa flour 594 donkey
anti-rabbit IgG from Molecular Probes, Eugene, OR), 1:1000 for 1 hr at
room temperature. Fluorescence images were obtained with the confocal
microscope LSM510 (Carl Zeiss, Jena, Germany) equipped with a
differential interference contrast (DIC)-microscope and processed in
the range in which negative controls showed no visible signals. For
quantification of GAD, the 8-bit images were collected with the laser
confocal microscope MRC-1000 (Bio-Rad Microscience Division, Cambridge,
MA). Ten fields (211 × 317 µm2)
from each well were randomly selected; the average of the overall fluorescence intensity values (0-255) was calculated using LaserSharp imaging software (Bio-Rad) and normalized to the control value for each
experiment. Results were obtained from 15 wells in five independent experiments.
Cell count and measurement of soma size. Cells were
immunostained for microtubule-associated protein-2 (MAP-2) (1:4000
mouse; Boehringer Mannheim Biochemica, Mannheim, Germany) or GABA
with Vectastain ABC elite kit (1:200 for secondary antibody; Vector Laboratories, Burlingame, CA). For cell count, the number of
immunopositive neurons was counted in 10 randomly chosen areas (1 mm2) of each well under a bright-field
microscope. Data were obtained from five wells from three independent
experiments. For measurement of the soma area, transmitted-light images
(211 × 317 µm2) were randomly
obtained with the confocal microscope MRC-1000 (Bio-Rad), and the soma
edge of all fully included positive cells was manually determined as a
polygonal form. The surrounded areas were measured using LaserSharp
imaging software (Bio-Rad). Data were collected from four frames in 11 wells for GABA and from one frame in three wells for MAP-2 in three
independent experiments
Western blotting. Cells were washed with PBS and collected
with a cell scraper. The cell suspension in traces of PBS was
homogenized on ice and centrifuged at 750 × g at 4°C
for 5 min to remove the nucleus. The equal aliquots of the supernatants
were separated by SDS-PAGE gel and transferred onto polyvinylidene
difluoride membranes. The membrane was incubated in PBS
containing 0.05% Tween 20 and 5% nonfat dry milk for 1 hr and then
exposed to a primary antibody against synaptophysin (1:200, mouse;
Boehringer Mannheim Biochemica), synaptobrevin (1:500, rabbit; Wako,
Osaka, Japan), syntaxin (1:300, mouse; Wako), GluR1 (1:300, rabbit;
Chemicon), NR1 (1:300, rabbit; Chemicon), or
GABAAR 2/3 (only the 3 subunit was detected
in our culture; 1:300, mouse; Upstate Biotechnology, New York, NY) in
0.05% Tween 20-PBS at 4°C overnight and then to horseradish
peroxidase-conjugated secondary antibody for appropriate IgG.
Immunoreactive protein bands were detected by enhanced
chemiluminescence using Renaissance (NEN, Boston, MA). The signals on
Hyperfilm ECL (Amersham, Buckinghamshire, UK) were digitized and
quantified using the software Scion Image (Scion Corporation,
Frederick, MD). Protein levels were normalized with blots of -actin
on the same filters. Using serial dilution of samples, we confirmed
that the signals were within the linear range of response under these conditions.
FM1-43 (Molecular Probes) imaging was performed as described previously
(Ikegaya et al., 2000b), with some modifications. The cultured cells
were loaded with the fluorescence probe FM1-43 (10 µM) by a 1 min stimulus of depolarization (25 mM KCl), and then washed gently three times.
Under the transmitted light, the frames were carefully chosen to
include nearly the same number of cell bodies. Immediately (<5 min)
after loading, FM1-43 fluorescence images were obtained with the
confocal microscope MRC-1000 (Bio-Rad). At first, the fluorescence
remaining after the second depolarizing stimulus (45 mM KCl for 1 min) was subtracted as baseline,
nonsynaptic endocytosis, but that was proven to be negligible. Thus,
four images (211 × 317 µm2) per
well were randomly collected without subtraction, and the mean value of
whole fluorescence intensity was normalized to the control value for
each experiment. The background fluorescence could be set to produce no
significant noise to the mean values.
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RESULTS |
Brain-derived neurotrophic factor alters parameters of GABAergic
synapses in cultured hippocampal neurons
After hippocampal neurons were cultured in the continuous presence
of 50 ng/ml BDNF from 1- to 7-11 d in vitro, they were briefly stimulated at a high concentration of
K+ (50 mM for 60 sec), and the amount of glutamate and GABA released into the media was
assessed by HPLC. BDNF-treated cultures displayed a higher degree of
GABA release, whereas glutamate release was virtually unchanged (Fig.
1).
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Figure 1.
BDNF enhances high K+-evoked
GABA release from cultured hippocampal neurons. After long-term
treatment with 50 ng/ml BDNF from 1 to 7-11 d in vitro,
cultures were stimulated by K+ depolarization (50 mM, 1 min), and the amount of released glutamate
(A) and GABA (B) was
quantified by HPLC. BDNF enhances high K+-evoked
release of GABA but not of glutamate. Data are means ± SEM of
seven wells. *p < 0.05 versus
control, Student's t test.
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To determine whether BDNF activates GABA synthesis, expression of
the GABA-synthesizing enzyme GAD was explored by immunocytochemical staining with anti-GAD65/67 antibody. In untreated, control neurons, GAD immunoreactivity was detectable in some neurons, but its intensity was relatively low on average (Fig.
2B). In cultures
exposed to BDNF for 7 d, however, the signal was more evident
(Fig. 2D). Quantitative analyses indicated that the
mean signal intensity was increased more than twofold after BDNF
treatment (Fig. 2E). Thus, the upregulation of GABA
release may be attributable, at least in part, to elevated GABA
synthesis. These results suggest that presynaptic GABAergic neurons are
a target of the BDNF action.
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Figure 2.
BDNF treatment induces an increase of the level of
GAD expression. A-D, Representative
images of cultures kept in the absence (A,
B) or presence (C, D) of
50 ng/ml BDNF for 7 d. Cultures were immunostained with
anti-GAD65/67. DIC images (A, C) were
obtained from the same microscopic fields of view as anti-GAD images
(B, D). "Hotter" colors in
B and D correspond to higher
immunoreactivity for GAD on an arbitrary pseudocolor scale. Scale bar,
20 µm. E, Quantification of GAD-immunopositive signal.
The mean fluorescence intensity of 10 frames per well was normalized to
the control. BDNF induced a significant increase in GAD
immunoreactivity. Data are means ± SEM (n = 15). **p < 0.01 versus control,
Student's t test.
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BDNF has been shown to promote neuronal survival (Lowenstein and
Arenault, 1996) and the differentiation of neuronal stem cells in
hippocampal cultures (Vicario-Abejon et al., 2000). To confirm that the
selective effect of BDNF on GABAergic neurons is not a
misinterpretation resulting from a possible alteration in the number of
surviving neurons, cultures were immunostained for MAP-2, a
pan-neuronal marker, and GABA. In our cultures, BDNF had no apparent
influence on survival of MAP-2- or GABA-positive neurons (Fig.
3A). Interestingly, however,
we noticed that BDNF-treated cultures contained GABAergic neurons with
enlarged somata. The average soma size of GABA-positive, but not
MAP-2-positive, neurons was significantly larger after BDNF treatment
(Fig. 3B). Because a positive relationship between
transcriptional activity and cell size is shown in neuronal and
non-neuronal cells (Sato et al., 1994; Schmidt and Schibler, 1995),
this observation is in accordance with BDNF-induced GAD upregulation.
Although GABAergic neurons are also positive for MAP-2 immunostaining,
this small part of neurons did not significantly contribute to the
average soma size of total neurons. Indeed, in our cultures
GABA-positive neurons were only ~20% (Fig. 3A).
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Figure 3.
BDNF-treated GABAergic neurons possessed the
enlarged soma. Neurons were cultured in the presence or absence of 50 ng/ml BDNF for 7-10 d and immunostained with anti-MAP-2 or anti-GABA
antibody. A, Lack of the effect of BDNF on the survival
of MAP-2- or GABA-positive cells. Data are means ± SEM
(n = 5). B, The soma area of each
cell was quantified. GABA-positive cells are enlarged in size after
BDNF treatment. Data are means ± SEM of 24-38 cells.
**p < 0.01 versus control,
Student's t test.
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We assumed that a lack of effect of BDNF on non-GABAergic cells
is attributable to little or no expression of TrkB, a high-affinity receptor for BDNF. To address this possibility, cultures were immunolabeled with anti-TrkB. Unexpectedly, an equivalent level of TrkB
immunoreactivity was detected in almost all neurons; that is, both
GABAergic and non-GABAergic cells (Fig.
4). More than 90% of MAP-2-positive
neurons were immunostained with anti-TrkB, but GFAP-positive astroglial
cells were negative (data not shown). These results suggest that
non-GABAergic neurons are also a possible site of action of BDNF.
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Figure 4.
TrkB is expressed in both GABAergic and
non-GABAergic neurons. The DIC photograph (A)
shows the same field as the immunocytochemical image (B) for
TrkB (green) and GABA
(red). The immunostaining was performed at 7 d in vitro for cells cultured without BDNF. TrkB
immunoreactivity was evident in both GABA-positive and GABA-negative
cells. Similar results were obtained from four independent cultures.
Scale bar, 20 µm.
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Therefore, we next tried to determine whether BDNF affects protein
levels of glutamatergic receptors. Western blot analyses revealed no
changes in the levels of the AMPA receptor subunit GluR1 or the NMDA
receptor subunit NR1 (Fig.
5A). On the other hand,
expression of the GABAA receptor subunit
GABAAR2/3 was substantially increased by BDNF
treatment (Fig. 5A). The data provide evidence that BDNF
also affects postsynaptic components of GABAergic synapses.
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Figure 5.
BDNF upregulates GABAA receptor
(GABAAR) protein and presynaptic
proteins. Cells were cultured in the presence or absence of 50 ng/ml
BDNF for 7-10 d, and the extracts were analyzed by Western blot for
NR1, GluR1, and GABAAR 2/3 (A),
and syntaxin, synaptobrevin, and synaptophysin
(B). Immunoreactive signals were normalized to
the control. Insets are representative Western blots. A
blot for -actin for normalization is also shown. Data are means ± SEM of three independent experiments. *p < 0.05 versus control, Student's t test.
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Brain-derived neurotrophic factor upregulates the expression of
synaptic vesicle-related proteins
The synaptic vesicle-associated proteins synaptophysin and
synaptobrevin, are increased by the application of BDNF in hippocampal slice cultures (Tartaglia et al., 2001) and reduced in the hippocampal synaptosomes of BDNF knock-out mice (Pozzo-Miller et al., 1999). These
two reports suggest that these changes are associated with the ability
of transmitter release. However, in cultures of dissociated neurons we
could not find an apparent increase in glutamate release (Fig.
1A). Therefore, we suspected that BDNF does not alter
the expression of the presynaptic proteins in our cultures. This
possibility was addressed by Western blot analysis. We confirmed that
BDNF produced approximately a threefold increase in synaptophysin and synaptobrevin (Fig. 5B). In addition, we found that
synapsin, another presynaptic protein, was also increased
approximately twofold after BDNF treatment.
These results urged us to examine the effect of BDNF on the dynamics of
synaptic vesicles. The pool of synaptic vesicles can be functionally
divided into two classes, i.e., recycling and resting pools (Sudhof,
2000). After exocytosis of transmitters, a synaptic vesicle in the
recycling pool undergoes reuptake and is subsequently reused. The
styryl dye FM1-43 is co-internalized with recycling synaptic vesicles
when transmitter release occurs, and thus works as a good fluorescent
marker of these vesicles. On the other hand, the resting pool cannot be
recruited even after extensive stimulation such as 90 mM
K+ for 90 sec (Harata et al., 2001).
Therefore, K+ depolarization for 60 sec
was used here for a specific detection of recycling vesicles.
FM1-43 fluorescent punctae were widely detected in cultures (Fig.
6A). They often
overlapped each other so that we could not discriminate one from
another. Thus, the mean fluorescence intensity of the whole image was
adopted as an index of the total amount of recycling vesicles. There
was no significant difference in signal intensity between control and
BDNF-treated cultures (Fig. 6B), which suggests that
BDNF does not increase the amount of recycling vesicles in the whole
population of neurons.
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Figure 6.
BDNF induces no change in the amount of synaptic
vesicles in the recycling pool. A, Confocal FM1-43
signals are shown as black puncta superimposed on DIC
image of hippocampal culture. Scale bar, 20 µm. B,
FM1-43 fluorescence intensity in cultures exposed to 50 ng/ml BDNF for
7 d was quantitatively analyzed. The mean fluorescence intensity
of four frames per well was normalized to the control. Data are
means ± SEM of 10 wells of five independent experiments.
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DISCUSSION |
The present work has focused on the long-term effect of BDNF on
developing hippocampal neurons and has shown that BDNF promotes GABAergic maturation: long-term treatment with BDNF facilitated high
K+-elicited GABA release, upregulated the
expression of GAD and GABAA receptors, and
enlarged the soma of GABAergic neurons.
Although the role of BDNF in CNS development has been intensively
investigated, previous studies have not yet achieved a good consensus.
For example, long-term exposure to BDNF is shown to increase the
frequency and amplitude of miniature EPSCs (Vicario-Abejon et al.,
1998; McLean et al., 2000), whereas other reports indicated that
blockade of endogenous BDNF by TrkB-IgG induces an increase in
miniature EPSC amplitude (Rutherford et al., 1998). Such an inconsistency may be derived from different experimental conditions, e.g., species, animal ages, brain regions, in vivo-in
vitro, and acute or culture preparations. Even in culture
conditions, experimental data are possibly affected by many
experimental factors such as culture density (Ikegaya et al., 2000a),
culture medium, serum (Nakagami et al., 1997; Tyler and Pozzo-Miller,
2001), and the ratio of inhibitory neurons. Secondary factors may also
have a definitive influence on experimental data. For example, BDNF may indirectly affect excitatory neurons via neuropeptide Y (Greber et al.,
1994; Marty et al., 1996; Reibel et al., 2001). Therefore, it is not
surprising that BDNF exerts different effects in different experimental
systems. Rather, it is very important to determine from among them the
primary action of BDNF. The primary action must be more reliably
detectable under any experimental conditions. For this purpose, simple
experimental systems are useful to attain uniform and coherent
conclusions under well controlled conditions, which may allow us to
appreciate other data from more complex systems.
In the present study, by using a simple culture system to afford
heterogeneous neural networks, we performed comprehensive analyses on
neuronal properties. As a result, our observations revealed clearly
that BDNF promotes GABAergic maturation even in the case of no apparent
change in glutamatergic properties, suggesting that GABAergic
maturation is the primary effect of BDNF. At the same time, we found
that BDNF induces a twofold to threefold increase in levels of
presynaptic proteins; that is, synaptophysin, synaptobrevin, and
syntaxin. This intensive alteration cannot be explained merely by an
increase in GABAergic synapses because GABAergic neurons were only 20%
of the total neurons in our cultures. The upregulation of presynaptic
proteins probably occurs in both excitatory and inhibitory neurons.
Interestingly, all these proteins did not increase at an equal rate;
the levels of synaptophysin and synaptobrevin were more enhanced as
compared with syntaxin. Synaptobrevin and synaptophysin are both
localized in the membrane of synaptic vesicles, whereas syntaxin is
predominantly localized at presynaptic plasma membrane (Brunger, 2000).
Therefore, a similar degree of synaptobrevin and synaptophysin may
reflect an increase in the total number of synaptic vesicles.
Considering a lack of the effect of BDNF on FM1-43-labeled
recycling vesicles, it is possible to conclude that BDNF enlarges the
resting pool of synaptic vesicles. Therefore, BDNF may also potentially
promote the maturation of excitatory synapses via the enhancement of a latent capacity of neurotransmission. In contrast with studies by other
groups (Narisawa-Saito et al., 1999; Collin et al., 2001; Tyler and
Pozzo-Miller, 2001), we found no evidence that BDNF actually
facilitates the physiological function of glutamatergic synapses.
Therefore, we consider that BDNF-induced conversion of immature
glutamatergic synapses into active synapses depends on additional
secondary factors.
Our previous report indicated that short-term application of BDNF
rapidly induces a depression of inhibitory synaptic transmissions in
hippocampal slices (Tanaka et al., 1997), which may be mediated, in
part, by downregulation of GABAA receptors
(Brunig et al., 2001). The present study has shown the opposite, that
long-term treatment with BDNF promotes the maturation of inhibitory
synapses via upregulating GABA release and GABAA
receptor expression. A similar BDNF-mediated maturation of inhibitory
transmission was reported in the visual cortex, which is likely to play
an essential role in terminating the critical period for plasticity in
ocular dominance columns (Hensch et al., 1998; Huang et al., 1999).
Considering that BDNF is secreted from hippocampal neurons in an
activity-dependent manner (Goodmann et al., 1996), BDNF may rapidly
enhance the facility of network plasticity through GABAergic
disinhibition, but in the following late phase, it may serve to calm
the enhanced plasticity through the enhancement of GABAergic influence.
We hypothesize that this biphasic action of BDNF provides a highly
adaptive mechanism to ensure flexible temporal integration of signals,
which would help to etch an event that occurs during a limited period
into stabilized neural circuits.
In conclusion, our findings indicate that BDNF selectively promotes
GABAergic maturation at both presynaptic and postsynaptic components.
This selective effect did not depend on a long-term alteration in
glutamatergic properties. Therefore, we conclude that inhibitory
neurons are the principal target of BDNF action. Our study may provide
significant insights into functional plasticity in developing neural
networks in the CNS.
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FOOTNOTES |
Received March 19, 2002; revised May 1, 2002; accepted May 29, 2002.
*
M.K.Y. and K.N. contributed equally to this work.
This work was supported by grants from the Ministry of Education,
Culture, Sports, Science, and Technology.
Correspondence should be addressed to Dr. Maki K. Yamada, Laboratory of
Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
E-mail: maki{at}mol.f.u-tokyo.ac.jp.
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ABSTRACT
INTRODUCTION
