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The Journal of Neuroscience, June 15, 1999, 19(12):4972-4983
Impairments in High-Frequency Transmission, Synaptic Vesicle
Docking, and Synaptic Protein Distribution in the Hippocampus of BDNF
Knockout Mice
Lucas D.
Pozzo-Miller1,
Wolfram
Gottschalk2,
Li
Zhang3,
Kathryn
McDermott1,
Jing
Du2,
Raj
Gopalakrishnan2,
Chikara
Oho2,
Zu-Hang
Sheng3, and
Bai
Lu2
1 Laboratory of Neurobiology, National Institute of
Neurological Diseases and Stroke (NINDS), 2 Unit on Synapse
Development and Plasticity, Laboratory of Developmental Neurobiology,
National Institute of Child Health and Human Development,
3 Unit on Synaptic Function, NINDS, National Institutes of
Health, Bethesda, Maryland 20892
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ABSTRACT |
Brain-derived neurotrophic factor (BDNF) promotes long-term
potentiation (LTP) at hippocampal CA1 synapses by a presynaptic enhancement of synaptic transmission during high-frequency stimulation (HFS). Here we have investigated the mechanisms of BDNF action using
two lines of BDNF knockout mice. Among other presynaptic impairments,
the mutant mice exhibited more pronounced synaptic fatigue at CA1
synapses during high-frequency stimulation, compared with wild-type
animals. Quantitative analysis of CA1 synapses revealed a significant
reduction in the number of vesicles docked at presynaptic active zones
in the mutant mice. Synaptosomes prepared from the mutant hippocampus
exhibited a marked decrease in the levels of synaptophysin as well as
synaptobrevin [vesicle-associated membrane protein (VAMP-2)], a
protein known to be involved in vesicle docking and fusion. Treatment
of the mutant slices with BDNF reversed the electrophysiological and
biochemical deficits in the hippocampal synapses. Taken together, these
results suggest a novel role for BDNF in the mobilization and/or
docking of synaptic vesicles to presynaptic active zones.
Key words:
BDNF; knockout mice; hippocampus; synaptic fatigue; vesicle docking; synaptobrevin; synaptophysin
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INTRODUCTION |
Neurotrophins were originally
identified as a family of protein factors that regulate neuronal
survival and differentiation. Recent studies have indicated that
neurotrophins also play an important role in the development and
function of synapses (Lo, 1995 ; Thoenen, 1995; Bonhoeffer, 1996; Lu and
Figurov, 1997). The function of brain-derived neurotrophic factor
(BDNF) in synaptic transmission and plasticity in the hippocampus has
been the focus of extensive investigation. Both expression and release
of BDNF in the hippocampus are enhanced by neuronal activity and
excitatory synaptic transmission (Zafra et al., 1990; Isackson et al.,
1991; Patterson et al., 1992; Castren et al., 1993; Goodman et al., 1996; Canossa et al., 1997). Acute application of exogenous BDNF enhances neuronal activity and synaptic transmission in embryonic hippocampal neurons in primary cultures (Knipper et al., 1994; Lessmann
et al., 1994; Levine et al., 1995), suggesting that BDNF is capable of
modulating synaptic function in the hippocampus. However, whether BDNF
enhances excitatory synaptic transmission in acute hippocampal slices
remains controversial (Kang and Schuman, 1995; Figurov et al., 1996;
Patterson et al., 1996; Tanaka et al., 1997; Frerking et al., 1998;
Gottschalk et al., 1998; Huber et al., 1998). Much more consistent
results have been obtained in elucidating the role of BDNF in the
hippocampal long-term potentiation (LTP). Application of exogenous BDNF
facilitates LTP induction in neonatal hippocampal slices (Figurov et
al., 1996), in which the endogenous BDNF levels are low (Maisonpierre
et al., 1990; Friedman et al., 1991). In contrast, treatment with
TrkB-IgG, a fusion protein that scavenges endogenous BDNF, reduces the
magnitude of LTP in adult hippocampus, in which the endogenous BDNF
levels are high (Figurov et al., 1996; Yan et al., 1997).
In addition to pharmacological approaches, the role of BDNF in
hippocampal synaptic plasticity has also been studied using BDNF
knockout mice. Although the homozygous ( /) mice exhibit growth
retardation, sensory deficits, and impairments in coordination of
movement, there is no obvious neuronal loss in the hippocampus (Ernfors
et al., 1994; Jones et al., 1994). The heterozygous (+/) mice show no
signs of behavioral abnormalities. Two independent lines of BDNF
knockout mice, however, have shown a severe impairment in hippocampal
LTP in both / and +/ mice (Korte et al., 1995; Patterson et al.,
1996). Moreover, +/ mice showed the same degree of impairment as the
/ mice, consistent with the idea that a critical level of BDNF in
the hippocampus is important for LTP. This result also suggests that
the observed defects in hippocampal LTP are unlikely to be caused by
growth retardation, neuronal death, or dysfunction in other regions of
the brain. The impairment in hippocampal LTP can be restored after
incubation with recombinant BDNF for a few hours (Patterson et al.,
1996), or by virus-mediated BDNF gene transfer (Korte et al., 1996),
suggesting that a mutation of the BDNF gene per se, rather than
cumulative developmental abnormalities, is responsible for impaired LTP
in the BDNF knockout mice.
The mechanism by which BDNF regulates hippocampal synaptic plasticity
remains unclear, although several lines of evidence support a
presynaptic locus of action. Our previous study demonstrated that BDNF
treatment significantly attenuates synaptic fatigue during a
high-frequency tetanus in immature hippocampal slices (Figurov et al.,
1996). Furthermore, manipulations known to reflect presynaptic
function, such as paired-pulse facilitation (PPF), synaptic responses
to high-frequency stimulation (HFS), and changes in extracellular
Ca2+ concentration, revealed a presynaptic
modulation of high-frequency transmission by BDNF (Gottschalk et al.,
1998). To elucidate the cellular and molecular mechanisms of BDNF
action, we have now investigated the properties of hippocampal CA1
excitatory synapses in BDNF knockout mice. We characterized the
properties of hippocampal CA1 synapses, using electrophysiological
approaches. We also performed a quantitative structural analysis of
excitatory synapses on dendritic spines in the CA1 region. Furthermore,
we determined the expression levels of several synaptic vesicle
proteins in hippocampal synaptosomes from BDNF knockout mice. Finally,
we investigated whether the synaptic deficits in BDNF knockout mice can
be rescued by treatment of the mutant slices with recombinant BDNF. Our
results suggest that impairments in high-frequency transmission in the
mutant mice are likely caused by reduced synaptic vesicle docking at the active zones of CA1 synapses, possibly attributable to a decrease in synaptophysin and synaptobrevin levels at nerve terminals.
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MATERIALS AND METHODS |
BDNF knockout mice. Our BDNF knockout mice colony was
raised from two pairs of BDNF +/ mice in C57BL/6 background (Ernfors et al., 1994), purchased from Jackson Laboratories (Bar Harbor, ME).
These mice, referred to as BL/6/BDNF, have a low rate of pregnancy and
a small number of pups per litter. Moreover, the nursing females are
poor caretakers of their pups, resulting in a high rate of mortality,
particularly for homozygotes. To raise the large number of mice
necessary for biochemical studies, and to obtain enough littermates of
the same gender from all three genotypes, we generated a new line of
mice by crossing BL/6/BDNF +/ with CD1 mice. A BL/6/BDNF +/ male
was crossed with a CD1 female. The F1 +/ mice were crossed among
themselves. The F2 +/ were then used as founders to generate all of
the mice used in the present study (referred to as CD1/BDNF). The
CD1/BDNF mice behave similarly to BL/6/BDNF knockout animals (Ernfors
et al., 1994; Jones et al., 1994). The / mice were usually smaller
than the +/+ and +/ littermates and died within 1 month. They also showed defects in coordination of movement and balance, ataxia, spinning during periods of hyperactivity, and recurrent episodes of
freezing seizures. Heterozygous mice were fertile and showed no overt
abnormalities. Within each experiment (electrophysiology, electron
microscopy, or biochemistry), only same gender, age-matched littermates
were used. In experiments using all three genotypes, littermates of
postnatal day (P) 25-44 were chosen because / animals did not live
beyond that period. In experiments in which only +/+ and +/ genotypes
were used, 2- to 3-month-old animals were chosen.
Electrophysiological recording. Transverse hippocampal
slices (400 µm) were prepared from +/+, +/, and / CD1/BDNF
littermates (young adult, P25-44), or from +/+ and +/ BL/6/BDNF
littermates (2-3 months old). The slices were maintained in an
interface chamber for both recovery (2 hr) and recording, and exposed
to an artificial atmosphere of 95% O2/5%
CO2, as described previously (Figurov et al., 1996).
Perfusion medium [artificial CSF (ACSF), 34°C] contained (in
mM): NaCl 124, KCl 3.0, CaCl2 2.5, MgCl2 1.5, NaHCO3 26, KH2PO4 1.25, glucose 10, ascorbic acid 2, pH
7.4. Field EPSPs were evoked in CA1 stratum radiatum
by stimulation of Schaffer collaterals with twisted bipolar nichrome
electrodes and recorded with ACSF-filled glass pipettes (<5 M )
using an Axoclamp-2B amplifier. Test stimuli consisted of monophasic
200 µsec pulses of constant current delivered by stimulus isolation
units. Basal synaptic transmission was monitored by alternating,
low-frequency stimulation (two per minute) of two separate pathways via
two stimulating electrodes (S1 and S2) positioned on both sides of the
recording electrode. Only slices exhibiting EPSPs of 2-3 mV in
amplitude without superimposed population spikes were used. Stimulus
intensity was adjusted to evoke EPSPs of ~1.3 mV. Input-output
curves were obtained by plotting the stimulus voltages against the
slopes of EPSPs. LTP was induced by 2 × 1 sec trains at 100 Hz
separated by 20 sec using the same test stimulus intensity. Synaptic
fatigue was induced by a 1 sec train at 100 Hz. Because the 100th EPSP was often too small to measure accurately, we used the ratio of the
slope of the 40th to that of the first EPSP to measure synaptic fatigue. In addition, single exponential equations were fitted to the
plots of EPSP slopes versus stimulus number using IGOR Pro program. The
responses to paired-pulse stimulation at different interpulse intervals
(7-50 msec) were used to measure PPF. The mean slopes of the EPSPs
during the first 3 min after the LTP-inducing tetanus were used to
measure post-tetanic potentiation (PTP). EPSPs were digitized (10 kHz),
filtered at 1 kHz (eight-pole Bessel Filter), analyzed on-line, and
stored on magnetic media using acquisition systems DaQSys 1.0 and
Process 2.0 (University of Geneva), and custom-developed software
(provided by Dr. T. Inoue, University of Tokyo). For recovery
experiments, BDNF was added directly into the interface chamber (final
concentration 2 nM), and +/ or / slices were
incubated in BDNF for 3 hr.
Electron microscopy. Eight adult CD1/BDNF littermates (two
+/+, three +/, and three /, from three different litters) and two
adult BL/6/BDNF littermates (one +/+ and one +/) were used for
electron microscopy analysis. Mice were anesthetized with ether and
perfused through the heart with 1% paraformaldehyde and 1.25%
glutaraldehyde in 100 mM cacodylate buffer, pH 7.4, for the
first 5 min, and then with 2% paraformaldehyde and 2.5% glutaraldehyde in the same buffer for an additional 5 min. After 1 hr,
the brains were removed and placed in the last perfusion fixative for
an additional 2 hr. Hippocampi were dissected, and transverse sections
were made at 400 µm thickness. These hippocampal sections were rinsed
in 100 mM cacodylate buffer, post-fixed (1 hr) with 1%
OsO4 in 100 mM cacodylate buffer, pH 7.4, rinsed in acetate buffer, pH 5, and stained en bloc with 1% uranyl
acetate (12-15 hr; 4°C). After a rinse in acetate buffer and
dehydration in a series of ethanol dilutions, slices were rinsed in
propylene oxide and flat-embedded in Araldite. Semithin sections (0.5 µm) stained with toluidine blue were used to locate and trim CA1
stratum radiatum. Ultrathin sections (60-80 nm) were cut
and stained with uranyl acetate and lead citrate.
Only complete profiles of nonperforated asymmetric synapses (identified
by the more electron-dense postsynaptic density) on dendritic spines
were photographed in a JEOL 100× electron microscope operated at 80 kV
at a final magnification of 50,000×. Perforated synapses and
symmetrical synapses on shafts were excluded from the sampling.
Dendritic spines were identified by their shape and size, their
continuity with a dendritic shaft, or their lack of microtubules and
mitochondria. Synaptic vesicles were counted only when their plasma
membranes were clearly defined for at least one-half their
circumference (Heuser and Reese, 1973). A total of 247 asymmetric
synapses on CA1 dendritic spines from 3 +/+ mice, 315 synapses from 4 +/ mice, and 257 synapses from 3 / mice were analyzed.
Photographic negatives were scanned at 300 dpi to make digital images.
The quantitative analysis was performed after a blind protocol: the
person who did the analysis did not know the genotypes of the mice.
Digital image analysis was performed using NIH Image. The parameters
measured in each synapse were area of the presynaptic terminal, length
of the active zone, thickness of the postsynaptic density, total number
of small (~50 nm) synaptic vesicles per terminal, and number of
docked vesicles. The docked vesicles are defined as those located up to
one-vesicle-diameter (~50 nm) distance from the active zone,
according to criteria developed by Reese and colleagues
(Dickinson-Nelson and Reese, 1983), because these vesicles have been
shown to be depleted during sustained, repetitive synaptic activity. To
avoid the bias of analyzing the larger synapses in the single sections,
only synapses with terminals and active zones of equal magnitude were
included in the comparisons between all genotypes.
The number of docked vesicles per active zone length
(DV) and the active zone length (L)
in the two-dimensional single sections are listed in Table 1. From
these numbers, it was possible to estimate the total number of docked
vesicles per whole active zone (TDV) as follows:
TDV = A/(0.866 · ADV).
The active zone was assumed to be a flat circular disk of surface area
A = 1/4 ·  · L2 (Mayhew, 1979).
The average active zone area per docked vesicle (ADV), assuming it as a square, was estimated as
(L/DV)2. Because the docked
vesicles are clustered in the active zone in a hexagonal array (Gray,
1963), ADV must be adjusted by a factor  3/2 = 0.866. The
estimated values of A, ADV, and TDV
for CD/BDNF and BL/6/BDNF mice are also presented in Table 1.
Biochemical analysis of synaptic proteins. BL/6/BDNF or
CD1/BDNF mice were anesthetized with halothane, and their hippocampi were dissected and frozen in liquid nitrogen. To determine the expression levels of synaptic proteins in the whole hippocampus in
BL/6/BDNF mice, frozen hippocampi from +/+ and +/ were homogenized in
1% SDS. Equal amounts of homogenate proteins were separated on an
SDS-acrylamide gel, transferred to a Immobilon P membrane (Millipore),
and probed with antibodies against the following synaptic proteins:
synaptotagmin, synaptophysin, synaptobrevin [vesicle-associated
membrane protein (VAMP-2)], SNAP-25, and syntaxin-1. All
antibodies were monoclonal IgG or IgM from Chemicom, except for the
those against synaptobrevin and synaptotagmin, which were polyclonal
antibodies (Dr. M. Takahashi, Mitsubishi Institute). Both monoclonal
and polyclonal antibodies were used for synaptophysin. Specific
synaptic proteins were detected by 125I-labeled IgG/M and
quantified by phosphoimaging and Storm programs (Molecular Dynamics).
Biochemical analysis of synaptic proteins in the nerve terminals was
performed only on CD1/BDNF +/+ and +/ animals, because of the
difficulties in obtaining sufficient numbers of / animals. Generally, hippocampi from four to seven animals of the same genotype, same litter, and preferably same sex were used to prepare synaptosomes. Even in the CD1 genetic background, we normally obtained only one to
three / animals per litter. The / mice are only about half the
size of the +/+ and +/ littermates, and therefore very little
hippocampal tissues can be obtained from the / mice. Furthermore,
the / mice cannot live beyond 30 d after birth, and often we
had to make a decision to kill them around P20 or we lost them. Because
of these practical difficulties, we only prepared synaptosomes from +/+
and +/ mice using the differential and discontinuous Ficoll gradient
centrifugation method (Sheng et al., 1996). Briefly, each group of
hippocampal tissue was homogenized in 10 ml cold Syn buffer (Mannitol
300 mM, EDTA 1 mM, pH 7.4). Aliquots of the
homogenates were taken to determine the levels of synaptic proteins in
the whole hippocampus. The remaining homogenates were centrifuged at
5000 × g for 10 min. The supernatants were centrifuged
at 25,000 × g for 30 min. The pellets were then
resuspended in Syn buffer, loaded on Ficoll-gradient tubes, and
centrifuged at 32,000 × g at 4°C for 90 min. The
proteins in the interfaces between 8 and 12% and 12 and 16% Ficoll
gradient were collected, diluted in Syn buffer in a ratio of 1:4, and
then centrifuged for 20 min at 25,000 × g. Pellets
were resuspended in 500 µl 1× P Buffer (KCl 5.4 mM,
MgSO4 0.8 mM, glucose 5.5 mM, HEPES
50 mM, choline chloride 130 mM, BSA 1 mM, and 0.01% CHAPS). Synaptosomes were then
solubilized with 3.5 ml buffer (NaCl 150 mM, HEPES 10 mM, and 1.5% Chaps), stirred at 4°C for 1.5 hr, and
further centrifuged at 12,000 × g for 1 hr.
Supernatants were then ready for SDS-PAGE analysis. Equal amounts of
proteins (50 µg) from either whole hippocampus or hippocampal
synaptosomes were separated by 10-20% SDS-Tricine gradient gel
electrophoresis (Novex) and immunoblotted with specific primary
antibodies. The immunoreactive bands were visualized by enhanced
chemoluminescence (ECL) (Amersham). The ECL signal intensities were
quantified using NIH Image based on standard curves of these synaptic proteins.
All data are presented as mean ± SEM (N = number
of animals; n = number of experiments). Statistical
significance was determined by either Student's t test or
ANOVA, followed by Fischer protected least significant difference as a
post hoc test. # = p < 0.05; * = p < 0.005.
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RESULTS |
To obtain a large number of mutant mice for detailed
electrophysiological, biochemical, and structural studies, and to
minimize the potential interference of genetic background to our
observations, we generated a new line of BDNF knockout mice by crossing
one BDNF line (C57BL/6 background, called BL/6/BDNF) (Ernfors et al., 1994) with an outbreed mouse line (CD1). This new CD1/BDNF line showed
several characteristics similar to other BDNF knockout lines (see
Materials and Methods) (Ernfors et al., 1994; Jones et al., 1994).
Moreover, these mice exhibited the same defects in tetanus-induced LTP
in the hippocampus as those reported previously (Korte et al., 1996;
Patterson et al., 1996). Although 75% of the slices from +/+ young
adult mice (P25-44) exhibited LTP (>20% increase over baseline),
only 43 and 44% of the slices derived from +/ and / littermates
showed LTP, respectively (number of animals: N = 5, 5, 4; number of recordings: n = 35, 21, 27). Furthermore,
in those +/ and / slices that expressed LTP, there was a
significant reduction in the magnitude of the potentiation, when
compared with the +/+ slices (potentiation: 83% in +/+,
n = 25, 45% in +/, n = 9; 44% in
/, n = 12; ANOVA, p < 0.05). Basal
synaptic transmission was not affected by the mutation of the BDNF gene
in our CD1/BDNF line; no difference was found in the input-output
curves obtained from wild-type and mutant hippocampal slices
(input-output curve slopes: 1.2 ± 0.12 in +/+, n = 14, and 1.2 +/ 0.08 in +/, n = 14; t
test, p = 0.6949). To further study the cellular and
molecular mechanisms underlying BDNF action in the hippocampus, we
examined the properties of CA1 synapses using both CD1/BDNF and
BL/6/BDNF lines whenever possible.
BDNF knockout mice show significant synaptic fatigue
Exogenous BDNF attenuates synaptic fatigue induced by a train of
HFS in neonatal hippocampus where endogenous BDNF level is low, whereas
the BDNF scavenger TrkB-IgG fusion protein reduces synaptic responses
to the tetanus in adult hippocampus where endogenous BDNF level is high
(Figurov et al., 1996). If BDNF levels are important for hippocampal
synapses to follow HFS, synapses in the BDNF mutant mice should exhibit
more pronounced synaptic fatigue. Indeed, adult slices prepared from
both +/ and / CD1/BDNF mice showed more pronounced synaptic
fatigue in CA1 synapses during HFS compared with +/+ mice (Fig.
1a,b). The percentage of the 40th EPSP slope over the first EPSP slope in the HFS trains (from here
on termed "response to HFS") was 63 ± 3% in +/+, 43 ± 2% in +/, and 41 ± 2% in / (ANOVA, p < 0.005); N (number of animals) = 8, 8, and 6 for +/+,
+/, and /, respectively. A similar pattern in response to HFS was
also observed in BL/6/BDNF mice (Fig. 1c). HFS induced a
mild synaptic fatigue in +/+ adult BL/6/BDNF slices but much more
pronounced fatigue in slices from +/ littermates (+/+, 74 ± 3%; +/, 42 ± 3%; p < 0.05).
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Figure 1.
Pronounced synaptic fatigue in CA1 synapses during
high-frequency stimulation in BDNF knockout mice. a,
Examples of EPSPs elicited by a train of HFS (100 Hz) in hippocampal
slices from CD1/BDNF mice. Note the significant synaptic fatigue in
+/ and / mice. b, Summary of synaptic fatigue in
CD1/BDNF mice. The slope of the 40th EPSP in the train is
presented as the percentage of the first EPSP slope.
n = number of recordings. c, Summary
of synaptic fatigue in BL/6/BDNF mice.
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To better visualize the difference observed in the synaptic fatigue
between the mutant CD1/BDNF mice, we plotted EPSP slopes against the
number of stimuli in the train and compared the integrated areas under
the plots. Statistically significant differences were observed not only
between +/+ and mutants (ANOVA, p < 0.001), but also
between +/ and / synapses (ANOVA, p = 0.0243)
(Fig. 2a). Furthermore, when
the decay phases of the normalized EPSP slope/stimulus number plots
were fitted with a single exponential equation, we found that the rates
of synaptic fatigue were different among the three genotypes (Fig.
2b). The decay constants for +/+, +/, and / synapses
were 26.5, 18.2, and 13.7 stimuli, respectively. Again, the /
synapses showed a faster rate of fatigue as compared with the +/.
Interestingly, there was an initial facilitation in the first nine
EPSPs for +/+ synapses, whereas in +/ and / synapses this
facilitation ended at seventh and fourth EPSP, respectively. Thus, the
mutation of the BDNF gene leads to severe defects in synaptic
transmission at high frequencies.
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Figure 2.
Overall changes in synaptic responses during HFS
in CA1 synapses of CD1/BDNF mice. a, EPSP slopes were
plotted against the number of the stimulus in the 100 Hz train for each
of the synapses, and the integrated areas under the plots were
calculated for each synapse and compared among three genotypes. *,
Significantly different; ANOVA test with post hoc PLSD
Fischer's test. n = 8 for each genotype.
b, Rate of synaptic fatigue in +/+, +/, and /
synapses. Normalized EPSP slopes were plotted against the number of the
stimulus during HFS. The plot for each genotype was fitted with a
single exponential curve, and rate constants were obtained.
n = 8 for each genotype.
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BDNF knockout mice have impairments in presynaptic
transmitter release
Substantial evidence indicates that synaptic fatigue is a
presynaptic phenomenon (Zucker, 1989; Dobrunz and Stevens, 1997). Our
previous work demonstrated that the effect of BDNF on high-frequency transmission in the neonatal rat hippocampus is mediated, at least in
part, by presynaptic mechanisms (Gottschalk et al., 1998). Because BDNF
has been shown to attenuate GABA receptor-mediated synaptic responses
in hippocampal slices (Tanaka et al., 1997; Frerking et al., 1998), we
determined whether the pronounced synaptic fatigue seen in the BDNF
mutant mice results from elevated GABAergic inhibition. Slices treated
with the GABAA and GABAB receptor antagonists (10 µM bicuculline, 0.1 mM phaclofen,
respectively) exhibited the same degree of synaptic fatigue as compared
with untreated ones (+/: 45 ± 2%, n = 6; +/
plus antagonists: 47 ± 3%, n = 9; t
test, p = 0.4). Moreover, the pronounced synaptic
fatigue in the mutant mice was not caused by an enhanced
desensitization of postsynaptic glutamate receptors. Synaptic responses
to HFS recorded from +/ slices treated with and without the
desensitization blocker cyclothiazide (0.1 mM) showed no
significant difference (the ratio of +/ to +/ plus
cyclothiazide = 1.07, n = 6 and 9, respectively;
t test, p > 0.5). Thus, the pronounced
synaptic fatigue in the mutant slices may result from an impairment in transmitter release properties during HFS, rather than from an elevated
GABAergic inhibition or an enhanced desensitization of glutamate receptors.
To characterize the presynaptic deficits in more detail, we examined
PTP, a phenomenon thought to be caused by enhanced presynaptic transmitter release (for review, see Zucker, 1989). The magnitude of
PTP, defined as a percentage increase of the average EPSP slope 3 min
after HFS over a 20 min baseline, appeared to correlate with the levels
of BDNF in the hippocampus of CD1/BDNF mice (Fig. 3). PTP was significantly reduced in
slices from +/ mice (186 ± 14%) and / mice (142 ± 7%), as compared with the ones from +/+ animals (226 ± 12%).
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Figure 3.
Impairment of post-tetanic potentiation
(PTP) in CD1/BDNF mice. a, Examples of
EPSPs before and 1 min after tetanus in different genotypes (separated
by double slash). b, Summary of PTP in
+/+, +/, and / mice. EPSP slopes monitored within a 3 min time
window after tetanus (2 × 1 sec, 100 Hz) were averaged, and PTP
is expressed as a percentage of the baseline EPSP slope (collected
during the 20 min before tetanus). *, Significantly different from +/+;
ANOVA, p < 0.005. n = 5, 5, and 4 for +/+, +/, and /, respectively.
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Another frequently used parameter to monitor presynaptic changes is
PPF, measured as the ratio of EPSP slopes in response to two successive
stimulation pulses (Creager et al., 1980). Unlike the defects in
synaptic fatigue and PTP, a significant reduction in PPF was observed
at the shorter interpulse intervals (7 and 10 msec) only in / but
not in +/ (Fig. 4). Thus, different assays for presynaptic properties may have different sensitivities to
BDNF levels. These results indicate that presynaptic transmitter release is impaired in BDNF mutant mice.
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Figure 4.
Impairment of paired-pulse facilitation
(PPF) at short interpulse intervals in CD1/BDNF
mice. a, Sample EPSP traces during PPF at 10 and 50 msec
interpulse intervals. b, Plot of PPF at different
interpulse intervals. *, Significantly different from +/+; ANOVA,
p < 0.005. n = 5, 5, and 4 for
+/+, +/, and /, respectively.
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Synapses in BDNF knockout mice have fewer docked vesicles
Our electrophysiological studies suggest that the primary defect
in the CA1 synapses of BDNF mutant mice is in the synaptic responses to
HFS, or synaptic fatigue. To determine how BDNF regulates synaptic
fatigue during HFS, we examined the morphology of CA1 synapses in BDNF
mutant mice. The general appearance of presynaptic terminals, spines,
and postsynaptic densities at excitatory synapses on CA1 dendritic
spines was similar between wild-type and mutant animals (Fig.
5). However, quantitative analysis
revealed that there was a dramatic reduction in the number of synaptic
vesicles docked at the active zones in both +/ and / mice (Figs.
5, 6a). Docked vesicles are
defined as those located up to one vesicle diameter (~50 nm) distance
from the active zone. This definition includes vesicles in the active
zone that are discharged during sustained, repetitive synaptic
activity (Dickinson-Nelson and Reese, 1983). The mean numbers of
docked vesicles per active zone length were 3.2 ± 0.12 in +/+,
2.2 ± 0.8 in +/, and 1.9 ± 0.7 in / (ANOVA,
p < 0.005, Table 1). On
the basis of these numbers, we estimated 9.3 docked vesicles per whole
active zone in +/+ mice (see Materials and Methods). In contrast, +/
mice had 4.5 vesicles per active zone, whereas / animals exhibited
3.4 docked vesicles per active zone (Table 1). The number of reserve
pool vesicles (those located beyond ~50 nm from the active zone) was not significantly different among the three genotypes (Fig.
6b) (ANOVA, p = 0.1768). To avoid the bias
of analyzing the larger synapses in the single sections, only synapses
with terminals and active zones of equal magnitude were included in the
comparisons between all genotypes. Therefore, in our sample of synapses
on CA1 dendritic spines, presynaptic terminal area and active zone length were similar in all three genotypes (data not shown).
Presynaptic terminal area and active zone length were also similar in
all three genotypes (data not shown). Significant differences in the number of docked vesicles per active zone length were also observed between a pair of +/+ and +/ mice from the BL/6/BDNF mice (3.37 ± 0.21 in +/+, n = 46; 2.40 ± 0.18 in +/,
n = 58; t test, p < 0.005).
The total number of docked vesicles per whole active zone estimated
from those measurements was 10.3 in +/+ and 5.2 in +/ hippocampus
(Table 1). Synaptic fatigue is known to result from a gradual depletion
of the readily releasable pool of quanta or their structural correlate,
the docked vesicles, during HFS (Zucker, 1989; Dobrunz and Stevens,
1997). Thus, impairments in the docking of synaptic vesicles may
explain why more pronounced synaptic fatigue is observed during HFS in
BDNF knockout mice.
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Figure 5.
Electron micrographs of excitatory synapses on CA1
dendritic spines in hippocampal stratum radiatum from
+/+, +/, and / CD1/BDNF mice. Examples of presynaptic terminals
and adjacent dendritic spines displaying normal gross structural
features in the three genotypes. Arrows point to a
representative example of a morphologically docked vesicle as is
defined in the text; arrowheads mark the edges of the
active zone/postsynaptic density complexes. Note that +/ and /
mice have fewer docked vesicles at the active zones.
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Figure 6.
Quantitative analysis of synaptic vesicles in
excitatory synapses in BDNF mutant mice. a, Number of
docked vesicles per micrometer of active zone length in the three
genotypes. Data from CD1/BDNF and those from BL/6/BDNF mutant mice were
combined. b, Number of reserve pool vesicles per
presynaptic terminal in the three genotypes. n = number of synapses; N = number of animals.
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Synaptobrevin and synaptophysin are reduced in hippocampal synapses
from BDNF knockout mice
To examine the potential molecular mechanism(s) underlying the
regulation of synaptic vesicle mobilization and/or docking by BDNF, we
measured in the mutant hippocampus the levels of several proteins of
the SNARE complex involved in vesicle docking and fusion (Sudhof, 1995;
Calakos and Scheller, 1996). First, we determined the levels of
synaptic proteins in homogenates of whole hippocampi prepared from
BL/6/BDNF knockout mice. These included proteins on the synaptic
vesicles (v-SNAREs), such as synaptobrevin (also called VAMP-2) and
synaptotagmin, as well as proteins on the presynaptic plasma membrane
(t-SNAREs), such as syntaxin-1 and SNAP-25. We also measured the levels
of synaptophysin, a major integral membrane protein on synaptic
vesicles. Western blot analysis of the whole hippocampal homogenates
showed no differences in the levels of any of the synaptic proteins we
examined in three pairs of +/+ and +/ adult hippocampi from BL/6/BDNF
mice (Fig. 7a). We did not
analyze the / because adult littermates of the same gender from the
three genotypes of the BL/6/BDNF line were difficult to obtain.
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Figure 7.
Analysis of synaptic proteins in homogenates of
whole hippocampal preparations and in hippocampal synaptosomes from
BDNF knockout mice. a, Synaptic proteins detected in
whole hippocampal homogenates from BL/6/BDNF mice. The hippocampi were
dissected from a pair of +/+ and +/ mice, homogenized, and subject to
Western blot analysis using antibodies against specific synaptic
proteins. b, Synaptic proteins detected in whole
hippocampal preparations (right, homogenate) and
synaptosomes (left) from CD1/BDNF mice. Similar Western
blot analysis was performed. Note the significant reduction in the
levels of synaptobrevin (VAMP-2) and synaptophysin only in synaptosomal
preparations, but not of other synaptic proteins. Because
synaptotagmin, SNAP25, and syntaxin all show the same levels in the
same gel, there is no need to include loading controls in this figure.
c, Relative levels of the synaptic proteins in the
synaptosomes, quantified by scanning the blots and comparing the bands
with standard curves. In each experiment, hippocampal synaptosomes were
prepared from a large litter of CD1/BDNF mice (2-3 months old), with
three to four +/+ and three to four +/ of the same gender, and
Western blots using the same synaptosomal preparation were repeated
several times. Four such experiments were performed, and each
experiment was repeated two to three times. Data are pooled and
presented as a percentage of the wild-type levels.
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Because synaptic proteins are distributed throughout hippocampal
neurons, including cell bodies, axons, and nerve terminals, significant
differences in the distribution of synaptic proteins at presynaptic
terminals might be masked by their expression in other parts of the
cell. To investigate the potential changes in the levels of synaptic
proteins at nerve terminals, synaptosomal fractions from hippocampi of
mutant mice were prepared using Ficoll-gradient methods (Sheng et al.,
1996). Because of the difficulties in obtaining large numbers of /
hippocampi for synaptosomal preparations (see Materials and Methods),
only CD1/BDNF +/+ and +/ mice were used in the subsequent
experiments. Similar to what was observed in BL/6/BDNF mice, no
differences were found in the levels of the synaptic proteins in the
homogenates of whole hippocampi prepared from CD1/BDNF mice (Fig.
7b, right). In synaptosomes prepared from +/
hippocampi, however, a polyclonal antibody detected an 82% decrease in
the level of synaptobrevin, an integral synaptic vesicle protein
implicated in vesicle docking and/or fusion (Fig. 7b,
left, c). In addition, a monoclonal antibody
detected a 72% reduction in the amount of another vesicle protein,
synaptophysin. Similar result was obtained using a polyclonal antibody
against synaptophysin (data not shown). The level of the vesicle
protein synaptotagmin was similar in synaptosomes prepared from +/+ and +/ mice (Fig. 7b, left), consistent with the
observation that the number of synaptic vesicles in the reserve pool
was not reduced in BDNF mutant mice (Fig. 6b). The levels of
presynaptic membrane proteins, such as syntaxin-1 and SNAP-25, were
also unchanged in the mutant mice (Fig. 7b,c). Similar
results were consistently observed in four independent synaptosomal
preparations, and pooled data are shown in Figure 7c. Taken
together, these results suggest that selective reduction in
synaptobrevin and synaptophysin levels occurs at nerve terminals in the
hippocampus of BDNF mutant mice.
Treatment with BDNF reverses the synaptic defects in BDNF
mutant mice
If the synaptic deficits were attributable to a direct outcome of
the mutation of the BDNF gene rather than indirect consequences of the
lack of BDNF on hippocampal circuitry, application of BDNF to the
mutant slices should reverse the deficits. We incubated the adult
mutant slices with recombinant BDNF (2 nM) for 3 hr and
then analyzed the physiological and biochemical properties of the
hippocampal synapses. In CD1/BDNF mice, synaptic fatigue in the +/
and / slices was significantly attenuated after BDNF treatment
(Fig. 8a). In fact, the
response to the HFS seen in the +/ and / slices after BDNF
treatment returned to the levels seen in the +/+ slices. Similar
results were obtained in BL/6/BDNF mice (+/: 42%, n = 8, n = 2; +/ plus BDNF: 79%, n = 15, n = 2). In synaptosomes prepared from +/ slices,
we reproduced the selective reduction in the levels of synaptobrevin
and synaptophysin seen in those from whole hippocampus. Furthermore,
incubation of these slices with BDNF for 3 hr reversed the decrease in
the synaptosomal levels of synaptobrevin and synaptophysin (Fig.
8b,c). Taken together, these data suggest that the synaptic
deficits seen in the BDNF mutant mice are not attributable to indirect, developmental consequences of BDNF knockout. Rather, they reflect an
acute requirement for BDNF in high-frequency transmission in the
hippocampus. We could not examine the vesicle docking in mutant slices
treated with BDNF, because it was extremely difficult to obtain the
good structural preservation needed to quantify docked vesicles in the
acute slices after 3 hr in the interface recording chamber.
Interestingly, treatment of the +/+ slices with BDNF also elicited a
50.4% increase in responses to HFS (response to HFS: 63.0 ± 2.6% in control, n = 37, and 94.8 ± 5.1% in
BDNF-treated slices, n = 29), and a 151 ± 39%
and 48 ± 12% increase in the levels of synaptophysin and
synaptobrevin in hippocampal synaptosomes, respectively. These results
imply that vesicle docking in CA1 synapses is not saturated even in
wild-type animals, and BDNF has a general capacity to modulate this
process.
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Figure 8.
Effect of acute exposure to BDNF on synaptic
fatigue and the levels of synaptobrevin and synaptophysin in BDNF
mutant slices. a, Synaptic fatigue was induced by a
train of 1 sec, 100 Hz stimulation in hippocampal slices from either
+/ or / CD1/BDNF mice. The slices were then treated with
recombinant BDNF (2 nM) for 3 hr. HFS responses in +/ and
/ slices before and after BDNF treatment were normalized to that in
+/+ slices (n = 21). N = 3, 2, and 2 for +/+, +/, and /, respectively. b,
Examples of Western blots showing the levels of synaptobrevin and
synaptophysin in synaptosomes from +/+ and +/ slices and +/ slices
plus BDNF. Slices from +/ CD1/BDNF mice were treated with BDNF (2 nM) for 3 hr. Synaptosomes were prepared and synaptic
proteins were detected the same way as described in Figure 7. Note that
the same levels of actin were detected in all three lanes, suggesting
that equal amounts of proteins are loaded. c,
Quantification of the relative levels of synaptobrevin and
synaptophysin in synaptosomes prepared from +/ slices treated with
and without BDNF. Three experiments using independent synaptosomal
preparations were performed. Each experiment was repeated two to three
times, and data were pooled and presented as a percentage of the
wild-type levels.
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DISCUSSION |
Our study suggests that BDNF may regulate high-frequency
transmission by facilitating synaptic vesicle mobilization and/or docking, possibly through the regulation of the distribution of specific synaptic proteins at nerve terminals. Gene targeting experiments often suffer from interference by the complexity of the
genetic background of the mice used. For example, although two
independent lines of BDNF knockout mice exhibit the same impairment in
hippocampal LTP (Korte et al., 1995; Patterson et al., 1996), only one
of the lines shows defects in basal synaptic transmission (Patterson et
al., 1996). This discrepancy in basal synaptic transmission between the
two mutant lines has been attributed to the difference in the genetic
background of the two mouse lines, rather than to the effect of BDNF
mutation. To minimize the influence of the genetic background on our
observations, we have used CD1/BDNF as well as BL/6/BDNF knockout mice
whenever possible. Knockout mice from both lines exhibit more severe
synaptic fatigue, impairments in LTP, and fewer docked vesicles in CA1
synapses. These results suggest that the described impairments in the
knockout mice were not caused by alterations in genetic background but
were attributable to the mutation of the BDNF gene.
Our previous study demonstrated that in neonatal rat, BDNF modulation
of CA1 synapses is primarily mediated through presynaptic mechanisms
(Gottschalk et al., 1998). Here we show that PPF and PTP, two forms of
short-term synaptic plasticity that reflect changes in presynaptic
properties, were also impaired in the BDNF mutant mice. These results
further support the notion that BDNF acts presynaptically. The defects
in vesicle docking may account for the severe synaptic fatigue seen in
the BDNF knockout mice. The absolute difference in the number of docked
vesicles per active zone between +/ and / mutant mice was small
(2.2 vs 1.9), although it was statistically significant. This small
difference may not be detected by our electrophysiological analysis of
synaptic fatigue, using the ratio of the 1st and 40th EPSP slopes. A
detailed analysis of the time course of responses to HFS indicates that
indeed there was a difference in the rate of fatigue at CA1 synapses
between +/ and / mice. However, the defect in PPF at short
interpulse intervals (<20 msec) was observed only in / mice.
Although this is consistent with our previous finding that exogenous
BDNF enhances PPF only at intervals of 20 msec or less (Gottschalk et
al., 1998), it is unclear why PPF is more resistant to BDNF reduction.
One possibility is that cumulative effects of multiple pulses are required to reveal the small difference in docked vesicles between +/
and / mice, and two pulses are not sufficient. Alternatively, BDNF
may modulate other presynaptic properties (such as
Ca2+ channels), in addition to vesicle docking. In
support of the latter view, slices from / mice showed more severe
impairment in PTP than those from +/ animals. Synaptic fatigue, PPF,
and PTP all have different intracellular mechanisms (Zucker, 1989) and
therefore may have different sensitivities to BDNF levels.
BDNF and the synaptic vesicle cycle
The synaptic vesicle cycle involves multiple steps, including
transport of vesicles to nerve terminals, vesicle docking, a maturation
process called "priming," Ca2+-triggered vesicle
fusion, endocytosis, and the formation of new vesicles (Sudhof, 1995;
Calakos and Scheller, 1996). In principle, modulation of any of these
steps could potentially alter the synaptic responses to repetitive
stimulation. Because the attenuation of synaptic fatigue at CA1
synapses by exogenous BDNF occurs only when the frequency of
stimulation is >50 Hz (Gottschalk et al., 1998), it is unlikely that
the modulation of vesicle transport or endocytosis would contribute to
the observed effect of BDNF. The time required for vesicle endocytosis
has been estimated to be 1 sec (Ryan et al., 1993), much too slow to
contribute to events as fast as 10-20 msec.
Our experiments suggest that BDNF modulates the vesicle docking step.
In CA1 excitatory synapses from normal mice (C57BL/6), the mean number
of morphologically docked vesicles per active zone is 10.3 based on
serial reconstruction (Schikorski and Stevens, 1997), whereas that of
releasable quanta is 8.1 based on physiological measurements (Dobrunz
and Stevens, 1997). Thus, synaptic vesicles docked at active zones in
CA1 synapses represent the readily releasable quanta, as postulated by
Katz's classical theory of synaptic transmission (Katz, 1969). In
agreement with the above assessment, we estimated that +/+ CA1 synapses
contain 10.3 docked vesicles per active zone in BL/6/BDNF line and 9.3 in CD1/BDNF line, based on measurements from single section electron
micrographs (see Materials and Methods). Substantial evidence suggests
that synaptic fatigue during sustained high-frequency transmission
results from a depletion of the readily releasable pool of quanta or
its morphological correlate, the docked vesicles (Highstein and
Bennnet, 1975; Heuser and Reese, 1979; Zucker, 1989; Dobrunz and
Stevens, 1997; Schikorski and Stevens, 1997). During 100 Hz
stimulation, the rate of fusion (every 10 msec) far exceeds that of
vesicle transport, endocytosis, or mobilization of vesicles from the
reserve pool to the releasable pool (Ryan et al., 1993). Thus the
number of docked vesicles is expected to become the rate-limiting
factor (Highstein and Bennnet, 1975). We found that CA1 synapses from
+/ CD1/BDNF mice had 4.5 docked vesicles per active zone (5.2 in +/
from BL/6/BDNF mice), whereas in / animals this number was only
3.4. A selective reduction of the docked vesicle pool in the BDNF
knockout mice resulted in more pronounced fatigue.
A reduction in the number of docked vesicles should not affect basal
synaptic transmission elicited by low-frequency stimulation (one every
30 sec). According to the measurements by Stevens and Wang (1995), an
action potential will deplete at most one quantum (or docked vesicle)
at hippocampal synapses. On the basis of our calculation, there are
three to five docked vesicles per active zone in the BDNF mutant CA1
synapses. Unless high-frequency repetitive stimulation is applied,
docked vesicles should not be depleted in the CA1 synapses of the
knockout animals. Indeed, we found no difference in the input-output
curves generated from the wild-type and mutant mice. Consistent with
this idea, BDNF rescues synaptic fatigue in neonatal rats only when the
CA1 synapses are stimulated at a frequency >50 Hz (Gottschalk et al.,
1998). These data suggest that the number of docked vesicles determines
the degree of synaptic fatigue during HFS and that BDNF modulates
high-frequency transmission, but not basal synaptic strength, by
regulating vesicle docking.
BDNF and the molecular mechanisms of vesicle docking
The selective reduction in synaptobrevin without affecting
synaptotagmin on the synaptic vesicles in the mutant hippocampus is
consistent with the observation that the total number of vesicles was
not reduced in the mutant CA1 synapses. Moreover, the levels of
t-SNAREs syntaxin-1 and SNAP-25 were unaltered, suggesting normal
docking sites on the presynaptic membrane. Synaptic vesicles with less
synaptobrevin may fail to dock to the presynaptic active zone, leading
to the reduction of docked vesicles and pronounced synaptic fatigue.
Synaptophysin, a major integral membrane protein on synaptic vesicles,
was also downregulated in the BDNF knockout animals. Synaptophysin is
complexed with synaptobrevin in nerve terminals (Calakos and Scheller,
1994; Washbourne et al., 1995), and deletion of synaptophysin in mice
results in a decrease in synaptobrevin (McMahon et al., 1996). It is
unclear which one (or both) is the prime target of BDNF regulation. The
finding that BDNF +/ mice exhibited lower levels of synaptobrevin and synaptophysin only in synaptosomes but not in the whole hippocampus suggests that BDNF may facilitate the incorporation of these proteins into the synaptic vesicles, rather than regulating their synthesis.
Substantial biochemical evidence supports the notion that synaptobrevin
is required for vesicle docking (Sollner et al., 1993; Calakos et al.,
1994; Hayashi et al., 1994; Pevsner et al., 1994). However, cleavage of
synaptobrevin by tetanus toxin in the squid giant synapse or
Drosophila neuromuscular junction caused a slight increase,
rather than a decrease, in the numbers of docked vesicles. This is
caused by an accumulation of vesicles after fusion is blocked by the
toxin (Hunt et al., 1994; Broadie et al., 1995). One interpretation is
that docking after tetanus toxin treatment is mediated by synaptotagmin
(Schiavo et al., 1995; Reist et al., 1998). The second possibility is
that the toxin could cleave the N-terminal portion of synaptobrevin
that mediates fusion, whereas the remaining part of the molecule can
still mediate docking. Indeed, the SNARE proteins remain capable of
forming heterotrimeric complexes after cleavage of synaptobrevin by
tetanus toxin, suggesting that docking is not affected (Hayashi et al.,
1994; Pellegrini et al., 1995). Our present study provides new evidence
supporting the role of synaptobrevin in vesicle docking. We found that
basal synaptic transmission at low frequency was normal in the mutant mice. Conceivably, a reduction of synaptobrevin in the nerve terminals would lead to an impairment in docking without affecting the total number of synaptic vesicles in nerve terminals.
Acute and developmental role of BDNF in the hippocampus
Several studies have demonstrated a role for BDNF in the
development of CNS synapses (Cabelli et al., 1995; McAllister et al.,
1995). In particular, a recent study showed a marked reduction in
axonal branches, synaptic density, and total number of synaptic vesicles in the hippocampus of trkB / mice (Martinez et al., 1998).
It is therefore important to determine whether the deficits we observed
in high-frequency transmission, vesicle docking, and the levels of
synaptophysin and synaptobrevin reflect developmental consequences of
BDNF knockouts or an acute requirement for BDNF for ongoing synaptic
function in the hippocampus. In marked contrast to trkB / mice, the
morphology of the presynaptic terminals of CA1 synapses in the BDNF
knockouts is normal, suggesting that BDNF mutation does not affect the
formation of hippocampal synapses. Moreover, acute treatment of the
mutant slices with BDNF completely restored the impairment in
high-frequency transmission. In synaptosomes prepared from mutant
slices, the reduction in the levels of synaptobrevin and synaptophysin
was reversed after incubation with BDNF for 3 hr. It seems unlikely
that cumulative developmental abnormalities can be reversed within 3 hr
of BDNF treatment. For technical reasons, we could not examine whether
acute treatment of the mutant slices with BDNF can rescue the decrease
in the number of docked vesicles at CA1 synapses at the electron
microscopic level. Consistent with our findings, the defects in LTP in
slices from BDNF mutant mice can also be restored by acute BDNF
treatment (Patterson et al., 1996) or by virus-mediated BDNF gene
transfer (Korte et al., 1996). Moreover, we found that application of
BDNF to wild-type slices also elicited enhancement in high-frequency
transmission and an increase in terminal synaptobrevin and
synaptophysin. Thus, BDNF may have a general capacity to modulate
vesicle docking in CA1 synapses. Taken together, these results suggest
that BDNF plays an important role in the ongoing synaptic function
above and beyond its presumptive role in the development of CA1
synapses in the hippocampus.
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FOOTNOTES |
Received Jan. 20, 1999; revised March 16, 1999; accepted April 5, 1999.
We express our gratitude to Drs. Tom Reese, Rodolfo Llinás, Story
Landis, Chris McBain, Elaine Neale, and Serena Dudek for helpful
discussions and critical comments on this manuscript. We also thank Dr.
T. Inoue for providing acquisition and analysis software, Dr. M. Takahashi for antibodies against synaptotagmin and synaptobrevin, and
D. Abebe, J. Chludzinski, M. F. O'Connell, and Nicole
Tartaglia for technical assistance.
Correspondence should be addressed to Dr. Bai Lu, Unit on Synapse
Development and Plasticity, National Institute of Child Health and
Human Development, National Institutes of Health, Building 49, Room
5A38, 49 Convent Drive, MSC4480, Bethesda, MD 20892-4480.
Dr. Pozzo-Miller's present address: Department of Neurobiology,
University of Alabama at Birmingham, CIRC 429-A2, 1719 Sixth Avenue
South, Birmingham AL 35294-0021.
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W. G. Honer, P. Falkai, T. A. Bayer, J. Xie, L. Hu, H.-Y. Li, V. Arango, J. J. Mann, A. J. Dwork, and W. S. Trimble
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[Abstract]
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A. R. Carter, C. Chen, P. M. Schwartz, and R. A. Segal
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S. Thakker-Varia, J. Alder, R. A. Crozier, M. R. Plummer, and I. B. Black
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P. A. Heppenstall and G. R. Lewin
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W. J. Tyler and L. D. Pozzo-Miller
BDNF Enhances Quantal Neurotransmitter Release and Increases the Number of Docked Vesicles at the Active Zones of Hippocampal Excitatory Synapses
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D. S. Auld, F. Mennicken, and R. Quirion
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M. Bibel and Y.-A. Barde
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TOP
ABSTRACT
INTRODUCTION
