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The Journal of Neuroscience, February 15, 2002, 22(4):1316-1327
Brain-Derived Neurotrophic Factor Modulates Cerebellar Plasticity
and Synaptic Ultrastructure
Alexandre R.
Carter1,
Chinfei
Chen2,
Phillip M.
Schwartz1, and
Rosalind A.
Segal1
1 Department of Pediatric Oncology, Dana Farber Cancer
Institute and Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115, and 2 Division of
Neuroscience, Department of Neurology, Children's Hospital, Boston,
Massachusetts 02115
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ABSTRACT |
Neurotrophins are key regulators of neuronal survival and function.
Here we show that TrkB, the receptor for brain-derived neurotrophic factor (BDNF), is located at parallel fiber to Purkinje cell (PF/PC) synapses of the cerebellum. To determine the
effects of TrkB receptor activation on synapse formation and function, we examined the parallel fiber to Purkinje cell synapses of mice with a
targeted deletion of the BDNF gene. Although Purkinje cell dendrites
are abnormal in BDNF 
/ mice, PF/PC synapses are still able to form.
Immunohistochemical analysis of mutant animals revealed the formation
of numerous PF/PC synapses with the appropriate apposition of
presynaptic and postsynaptic proteins. These synapses are functional,
and no differences were detected in the waveform of evoked EPSCs, the
amplitude of spontaneous mini-EPSCs, or the response to prolonged 10 Hz
stimulus trains. However, paired-pulse facilitation, a form of
short-term plasticity, is significantly decreased in BDNF / mice.
Detailed ultrastructural analysis of the presynaptic terminals
demonstrated that this change in synaptic function is accompanied by an
increase in the total number of synaptic vesicles in mutant mice and a
decrease in the proportion of vesicles that are docked. These data
suggest that BDNF regulates both the mechanisms that underlie
short-term synaptic plasticity and the steady-state relationship
between different vesicle pools within the terminal.
Key words:
neurotrophin; BDNF; Trk receptor; cerebellum; facilitation; ultrastructure; presynaptic plasticity
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INTRODUCTION |
Information processing depends on
the ability of neurons to modify the strength of their connections.
Considerable evidence now implicates the neurotrophins as an important
class of endogenous modulators of synaptic function. Neurotrophin
expression and release are upregulated by neuronal activity (Canossa et
al., 1997
), and neurotrophins potentiate neurotransmission and
plasticity (Lu and Chow, 1999; McAllister et al., 1999; Schuman, 1999).
Brain-derived neurotrophic factor (BDNF) acting through the receptor
tyrosine kinase TrkB has been implicated in the plasticity of
central synapses (Lu and Figurov, 1997). Adding BDNF to dissociated rat
hippocampal cultures (Lessmann et al., 1994; Levine et al., 1995)
potentiates synaptic activity. A long-lasting potentiation is seen with
the addition of BDNF to rat hippocampal slices (Kang and Schuman, 1995a,b). In addition, BDNF plays a role in short-term
plasticity, including paired-pulse facilitation (PPF) (Patterson et
al., 1996; Stoop and Poo, 1996; Gottschalk et al., 1998), and the
response to repetitive stimulation (Figurov et al., 1996; Gottschalk et al., 1998; Pozzo-Miller et al., 1999). Both long- and short-term plasticity are impaired in the hippocampus of BDNF / mice (Korte et
al., 1995), and the addition of BDNF reverses these defects (Korte et
al., 1996a,b; Patterson et al., 1996; Pozzo-Miller et al.,
1999). Because presynaptic mechanisms are thought to underlie paired-pulse facilitation (Katz and Miledi, 1968; Zucker, 1989; Schulz
et al., 1994; Atluri and Regehr, 1996) and contribute to the response
to repetitive stimulation (Kreitzer and Regehr, 2000), BDNF may
modulate presynaptic function (Li et al., 1998; Martinez et al., 1998;
Pozzo-Miller et al., 1999).
Synapses with low probability of release can undergo a pronounced
modulation of synaptic activity (Bolshakov and Siegelbaum, 1995) and
may be more likely to reveal the functional effects of neurotrophins
(Berninger et al., 1999). In the cerebellum, granule cell axons, called
parallel fibers, form numerous synapses onto Purkinje cell dendrites in
the molecular layer. These synapses are excitatory, exhibit short-term
plasticity in the form of facilitation, and characteristically exhibit
a low probability of release (Atluri and Regehr, 1996; Sabatini and
Regehr, 1997). Because granule cells synthesize BDNF (Hofer et al.,
1990) and because the receptor TrkB is expressed by both granule cells
(Klein et al., 1990; Segal et al., 1995) and Purkinje cells (Klein et
al., 1990), the parallel fiber to Purkinje cell (PF/PC) synapse is well
suited for studying the role of BDNF on short-term synaptic plasticity.
We examined the effect of BDNF deficiency at the PF/PC synapse in the
mouse cerebellum using animals with a targeted deletion of BDNF
(Ernfors et al., 1994). Numerous functional PF/PC synapses form in
these animals. The finding that TrkB is located at PF/PC synapses
supports the idea that BDNF can directly modulate short-term synaptic
plasticity. Electrophysiologic analysis of the PF/PC synapse of BDNF
/ mice revealed preserved neurotransmission but a significant
decrease in PPF. This specific impairment of PPF is associated with an
accumulation of synaptic vesicles and a lengthening of the active zone.
These observations implicate BDNF as an important endogenous synaptic
factor that regulates short-term synaptic plasticity as well as
synaptic ultrastructure. Identification of the structural changes that
accompany changes in plasticity contributes to our understanding of the
molecular mechanisms underlying activity-dependent synaptic plasticity.
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MATERIALS AND METHODS |
BDNF mutant mice. Breeding pairs of BDNF +/ mice
(Ernfors et al., 1994) were purchased from Jackson Laboratories (Bar
Harbor, ME). Genotypes of animals were determined via PCR using
three oligonucleotide primers: oIMR132: 5'-GGG AAC TTC CTG ACT AGG
GG-3'; oIMR133: 5'-ATG AAA GAA GTA AAC GTC CAC-3'; and oIMR134: 5'-CCA GCA GAA AGA GTA GAG GAG-3'.
The PCR was run under standard conditions. Primers 132 and 133 amplify
a ~340 bp product from the targeted BDNF allele. Primers 133 and 134 amplify a ~275 bp band from the wild-type (WT) BDNF allele. PCR of
heterozygous animals yielded both amplification products. BDNF /
pups weighed one-third to two-thirds as much as littermates, depending
on age, and were severely ataxic. BDNF / pups generally survive
until the third postnatal week.
Immunohistochemistry. Midsagittal cerebellar sections (8 µm) were obtained from postnatal day (P) 8, P15, and P24 wild-type and BDNF / mice as described (Schwartz et al., 1997). Anesthetized mice were perfused with 4% paraformaldehyde in 0.2 M phosphate buffer, and cryoprotected brains were
cut on a Leica CM 3050 cryostat. For immunohistochemistry, cerebellar
sections were incubated in a PBS blocking solution containing
5% normal goat serum and 0.1% Triton X-100. Incubations in primary
antibody were performed in the same blocking solution. The antibodies
used were as follows: anti-calbindin-D, 1:500 (CL-300, Sigma, St.
Louis, MO); mouse anti-SV2, 1:200 (kind gift of K. M. Buckley,
Harvard Medical School); anti-TrkB, 1:2000 (Chemicon, Temecula, CA);
anti-synapsin, 1:100 (Chemicon); and anti-GluR
1/2, 1:25
(Chemicon). Incubations with primary antibodies were performed
overnight at 4°C. Immunostaining was visualized using cy3-conjugated
goat anti-mouse IgG, 1:200 (Jackson Immunochemicals, West Grove, PA);
Alexa Fluor 488 goat anti-mouse IgG green, 1:500 (Molecular Probes,
Eugene, OR); or Alexa Fluor 568 goat anti-rabbit IgG red, 1:500
(Molecular Probes) for 90-120 min at room temperature. Sections were
washed and then mounted in GelTol aqueous mounting medium (Immunotech,
Marseille, France). DeltaVision restoration fluorescence microscopy was
performed on an Olympus fluorescence microscope configured with a
DeltaVision stage and software (Applied Precision Inc., Issaquah, WA).
Immunofluorescent sections were viewed with a 100× objective
(numerical aperture 1.4). Z-series (20 0.2 µm serial optical
sections) were acquired, and softWoRx imaging software (Applied
Precision) was used to perform a blind deconvolution on a Silicon
Graphics workstation. Final images were representative z-series
rendered in softWoRx Volume Viewer unless indicated otherwise.
Statistical significance for the correlation between staining patterns
was established by calculating for each wavelength the number of pixels
with an intensity 1 SD above the mean within a known area. In
double-labeled samples, synaptic structures were selected in only one
wavelength without knowledge of the second wavelength, which was then
restored after the selection of structures. Staining in both
wavelengths was considered associated only if no measurable distance
existed between the two structures within the single optical section
where they were defined or within an adjacent optical section. The
probability of two pixels for each wavelength being randomly located in
the same position or in one of the eight adjacent positions is given by: (pixels positive for wavelength 1/total pixels) × (pixels positive for wavelength 2/total pixels) × 9. Statistical
significance was determined by a two-tailed unpaired t test
assuming unequal variances.
Stensaas-modified Golgi stain. Age P15 BDNF +/+ and BDNF
/ mice were perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate. Brains were immediately
removed and immersed 48 hr in Stensaas solution containing 85 mM potassium dichromate, 151 mM chloral hydrate, 3.7% formaldehyde, and 0.5%
glutaraldehyde. Tissue was rinsed in 0.5% silver nitrate and
transferred to 1.25% silver nitrate for 4 d. After progressive
dehydrations in ethanol, tissue was transferred to low viscosity
nitrocellulose, and choloroform was added to harden the nitrocellulose.
Sections (100 µm) were cut on a sledge microtome and collected in
80% ethanol, rinsed in 95% ethanol, and transferred to terpineol.
Sections were rinsed in xylene, covered in mounting media, and
coverslipped. Images were taken with a Nikon Eclipse E800 microscope at
100× magnification. Multiple images in different focal planes were
taken of individual Purkinje cells. The focused portions of each image
were combined to bring the entire dendritic arbor into focus.
Electrophysiology. Synaptic currents were recorded from
freshly cut transverse slices of cerebellar vermis of P14-P17 BDNF / mice and their wild-type littermates as described previously (Atluri and Regehr, 1996; Chen and Regehr, 1997). Briefly, slices were
maintained in an oxygenated (95% O2/5%
CO2) external solution containing (in
mM): 125 NaCl, 2.5 KCl, 2.6 NaHCO3, 1.25 NaH2PO4, 25 glucose, 2 CaCl2, and 1 MgCl2. The
external recording solution also contained 20 µM bicuculline to block IPSCs (Sigma).
Whole-cell patch-clamp recordings of Purkinje cells were obtained using
1.0-1.5 M
electrodes containing (in mM): 35 CsF, 100 CsCl, 10 EGTA, 10 HEPES, and 0.1 D600, pH 7.4. Holding
potential was maintained at 40 mV to inactivate voltage-dependent
calcium conductances. Access resistance (<5 M after series
resistance compensation), leak current (<100 pA), and the time course
of decay of the EPSC (<6 msec) were monitored continuously, and cells
that did not meet these criteria were rejected from analysis. Parallel
fibers were stimulated with a glass electrode filled with extracellular solution and positioned several hundred micrometers from the recording electrode.
Paired-pulse facilitation protocol involved measurement of the peak
amplitude of EPSCs elicited by pairs of stimuli, separated by a
randomized interstimulus interval (ISI) ranging from 10 to 500 msec.
Pairs of EPSCs were interleaved with EPSCs elicited by a single
stimulus. The average of three to five trials was used to calculate the
percentage facilitation [100 × (A2 Al)/A1]. A1
and A2 are the average peak amplitude of the first and second EPSC,
respectively, in response to a single stimulation. At intervals shorter
than 100 msec, A2 is measured from the waveform obtained by subtracting
the average single EPSC response from the average EPSC response to a
pair of stimuli.
Mini-EPSCs (mEPSCs) were recorded as described previously (Chen and
Regehr, 1997). Extracellular solution contained 0.25 µM TTX and 20 µM bicuculline. Data were recorded in 20 sec
epochs, sampled at 2.5 kHz, and filtered digitally at 500 Hz. Inclusion criteria were an 8 pA threshold, a minimum rate of rise of 0.4 pA/msec,
and a decay time constant between 3 and 20 msec. Experiments used to
calculate the average cumulative histogram distributions of mEPSC
amplitude contained >4000 mEPSC events. Changes in mEPSC frequencies
have been used as an indicator of a presynaptic process when the same
population of synapses is studied before and after a pharmacological
manipulation. However, the frequency of mEPSCs cannot be used as a
presynaptic indicator when comparing population of synapses from
different slices. The angle of the slice and the number of intact
presynaptic fibers vary significantly from slice to slice and would
thus make mEPSC frequency comparison between BDNF/ and WT mice
difficult to interpret.
Because trains of stimuli can result in recurrent excitation and
activation of endogenous G-protein-mediated modulation of synaptic
currents, experiments using repetitive stimulation were recorded in an
external solution containing the GABAB receptor antagonist CGP55845a (2 µM), the adenosine A1 receptor
antagonist 8-cyclopentyl-1,3-dipropylxanthine (5 µM), and
the mGlu-RIII antagonist (RS)-
-cyclopropyl-4-phosphonophenylglycine (30 µM). Frequencies >10 Hz resulted in
desynchronization of EPSCs during a train of stimuli, attributable to
accumulation of presynaptic residual calcium (Atluri and Regehr, 1998;
Kreitzer and Regehr, 2000). Therefore we did not analyze the synaptic
response to higher frequency stimulation. All recordings were digitized
with a 16 bit digital-to-analog converter (Instructec, Great Neck, NY),
with pulse control software, and analyzed using Igor Pro software
(Wavemetrics, Lake Oswego, OR) and custom macros. All
experiments were performed at 25°C.
Electron microscopy. Brains were obtained from P14 wild-type
and BDNF / mice anesthetized and perfused with 4% paraformaldehyde and 2% glutaraldehyde in 0.2 M phosphate buffer.
Blocks from midsagittal cerebellar vermis in folia V were stained with
1% osmium tetroxide/1.5% potassium ferrocyanide, bathed in 1% uranyl
acetate, and ethanol dehydrated and placed in propylene oxide for 1 hr.
Infiltration with Epon/Araldite mixed 1:1 with propylene oxide was
performed overnight. Samples were embedded in Epon/Araldite. Thin
sections from sample were photographed under a JEOL 1200EX electron
microscope. Negatives were scanned into Adobe Photoshop. Images of
individual synapses were analyzed using NIH Image 3.1 (National
Institutes of Health, Bethesda, MD). Scale bars were taken from scans
of original electron micrograph negatives. All synapses were analyzed without knowledge of genotype. For each synapse the number of synaptic
vesicles and the length of the postsynaptic density (PSD) were
quantitated. Vesicle distribution was quantitated by measuring the
distance from the center of each synaptic vesicle to the closest point
at the active zone. Docked vesicles were defined as those located
within 50 nm (one vesicle diameter) of the active zone.
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RESULTS |
The TrkB receptor is located at PF/PC synapses in
wild-type mice
The BDNF receptor TrkB is expressed by both granule cells and
Purkinje cells. To determine whether TrkB is appropriately located to
mediate synapse-specific effects of BDNF, the expression pattern of
TrkB protein in the cerebellum was analyzed. In both wild-type (Fig.
1A) and mutant mice
(data not shown), significant TrkB immunostaining was seen throughout
the molecular layer and in Purkinje cell bodies and main dendrites. To
clarify the subcellular localization of TrkB, cerebellar sections of
wild-type mice were double labeled with the TrkB antibody and an
antibody against calbindin, which is expressed specifically in Purkinje
cells. Staining was visualized by high-resolution wide-field
restoration microscopy. Nanomotor-guided focusing allows for image
acquisition at precise distance intervals throughout the volume of the
sample. On the basis of the information in the stack of optical
sections, a deconvolution algorithm identifies out-of-focus light and
returns it to its source using a point-spread function. Although the
original optical sections reveal the general dendritic morphology of a
Purkinje cell (Fig. 1B), the increased contrast,
decreased background, and sharper borders of the deconvolved optical
sections (Fig. 1C) allow for the identification of details on the order of 175 nm.
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Figure 1.
The BDNF receptor, TrkB, is located at PF/PC
synapses. A, P15 cerebellum immunolabeled with
anti-TrkB. TrkB staining is seen throughout the molecular layer
(ML) and in Purkinje cell bodies (PC) and
dendrites in BDNF +/+ mice. EGL, External granule cell
layer; IGL, internal granule cell layer. B,
C, Single optical section through the molecular layer of
P15 wild-type cerebellum labeled with anti-calbindin before
deconvolution (B) and after deconvolution
(C). D-F,
Cerebellar molecular layer of P15 wild-type mouse double labeled with
anti-calbindin (D, green) and anti-TrkB
(E, red) antibodies. The merged image
(F) shows numerous TrkB puncta juxtaposed to and
sometimes overlapping with calbindin-positive Purkinje cell dendritic
spines (arrowheads). G-I,
Cerebellar molecular layer of P15 wild-type mouse double labeled with
anti-calbindin (G, green) and
anti-GluR2 (GluR delta 2) (H,
red). Merged image (I)
shows GluR2-positive overlapping Purkinje cell dendrites and capping
Purkinje cell dendritic spines (arrowheads).
J-L, Cerebellar molecular layer of P15
wild-type mouse double labeled with anti-SV2 (J,
green) and anti-TrkB (K,
red). Merged image (L) shows
SV2-positive puncta juxtaposed to and partially overlapping with
TrkB-positive puncta (arrowheads).
D-L were reconstructed from 5-10
adjacent optical sections. Scale bars: A, 20 µm;
B, C, 10 µm;
D-I, 5 µm.;
J-L, 5 µm.
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Double labeling for calbindin and TrkB in wild-type cerebellum (Fig.
1D-F) revealed the presence of
numerous TrkB-positive puncta (red) adjacent to and
partially overlapping with the tips of calbindin-positive dendritic
spines (green). There is a high degree of association
between calbindin-positive spines and TrkB puncta. We found that 75%
(±6% SEM) of calbindin-positive spines (n = 116) were
associated with TrkB puncta and that 79% (±1% SEM) of TrkB puncta
(n = 121) were associated with calbindin-positive spines. This degree of association is significantly higher than that
predicted by chance alone (20 ± 0.5% SEM; p < 0.01; t test).
The localization of TrkB immunostaining at spine tips is consistent
with a synaptic localization of TrkB at PF/PC synapses. Double labeling
was performed using an antibody against the glutamate receptor delta 2 subunit (GluR2) and an antibody against calbindin. GluR2 is a
useful marker for PF/PC synapses because after an initial period of
expression throughout Purkinje cells, this receptor subunit becomes
targeted specifically to the spines receiving parallel fiber afferents
(Landsend et al., 1997). Double labeling with an antibody against
calbindin (green) and an antibody against GluR2
(red) (Fig. 1G-I) yielded
numerous GluR2 puncta that overlapped significantly with
calbindin-positive dendrites and spines. We found that 74% (±5% SEM)
of calbindin-positive spines (n = 91) are associated
with GluR 2, whereas 65% (±4% SEM) of GluR2 puncta
(n = 100) are associated with calbindin-positive
spines. This degree of association is significantly higher than that
predicted by chance alone (14 ± 1% SEM; p < 0.01; t test). The finding that the degree of association
between calbindin-positive spines and TrkB puncta, and between spines
and GluR2, is comparable and that TrkB and GluR2 have similar
staining patterns relative to calbindin strongly suggests that TrkB,
like GluR2, is localized to PF/PC synapses. In fact, TrkB staining
often appeared to be even more specifically associated with the very
tips of dendritic spines than did GluR2 (stronger yellow
signal in Fig. 1I than in Fig. 1F).
The absence of antibodies of the appropriate species and sensitivity
currently make it difficult to determine conclusively the relative
positions of TrkB and GluR2.
To further confirm the synaptic localization of TrkB, double labeling
with an antibody against the presynaptic protein SV2, a component of
synaptic vesicle membranes, and the antibody against TrkB was performed
(Fig. 1J-L). We found that 75% (±6.5%
SEM) of SV2 puncta (n = 158) are associated with
TrkB-positive spines. This degree of association is significantly
higher than that predicted by chance alone (15 ± 1.1% SEM;
p < 0.01; t test). Nonetheless, the
synaptic localization of the BDNF receptor is appropriate for BDNF to
directly regulate PF/PC synapse development and function. As shown
here, there is some overlap of TrkB and SV2 puncta, and there is also
some overlap of TrkB and calbindin staining. Because of the narrow
width of the synaptic cleft (~30 nm) and the fact that the TrkB
antibody recognizes the extracellular domain of the receptor, we cannot
determine conclusively on which side of the synaptic cleft TrkB resides.
BDNF is not required for PF/PC synaptogenesis
The synaptic localization of the BDNF receptor suggests that BDNF
may play a crucial role in synapse formation and development. Therefore, the absence of BDNF could preclude PF/PC synapse formation. Previously, the addition of BDNF or the removal of endogenous BDNF with
TrkB IgG has been shown to alter Purkinje cell dendritic spine number
and morphology (Morrison and Mason, 1998; Shimada et al., 1998).
Furthermore, analysis of cerebella in BDNF / mice revealed that the
dendrites of Purkinje cells are severely atrophied and disorganized in
P8 animals (Schwartz et al., 1997). To determine whether the absence of
BDNF precludes the formation of synapses onto Purkinje cell dendrites,
we first examined Purkinje cell morphology in P8, P15, and P24
wild-type and BDNF mutant mice using an antibody against the Purkinje
cell-specific marker calbindin (Fig. 2).
Compared with Purkinje cell dendrites of BDNF +/+ animals, dendrites of
BDNF / animals remain less extensive at all ages. Accordingly, this
defect is associated with a persistently thinner molecular layer.
Nonetheless, there is continued development of Purkinje cell dendrites
even in the absence of BDNF because primary, secondary, and tertiary
branches are visible in P15 and P24 BDNF / mice.
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Figure 2.
The development of Purkinje cell dendritic arbors
in wild-type (A, C, D) and
BDNF / mice (B, D,
F). Parasagittal cerebellar sections from P8
(A, B), P15 (C,
D), and P24 (E, F)
mice were immunolabeled with anti-calbindin antibody. In the absence of
BDNF, Purkinje cell dendrites are stunted at P8 and undergo significant
development over the following 2 weeks, but remain less extensive than
in WT mice. Scale bar, 20 µm.
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Because the overlap between adjacent dendrites makes it difficult to
appreciate the morphology of individual dendritic arbors, cerebella
from P15 BDNF +/+ and BDNF / mice were stained using a rapid Golgi
method (Fig. 3) to visualize individual
Purkinje cell arbors. As illustrated, the extent of dendritic arbors in individual BDNF / Purkinje cells is reduced when compared with wild-type Purkinje cells (Fig. 3). In BDNF / cells, the primary dendrite is shorter and divides into fewer secondary and tertiary branches. Despite their abnormal morphology, BDNF / dendrites are
studded with numerous spines (Fig. 3B, inset,
C, inset) as in wild-type dendrites (Fig.
3A, inset). Because these dendritic spines are
the postsynaptic targets of granule cell axons, Purkinje cell dendrites
may develop sufficiently to receive synapses from parallel fibers
despite the significant effects of BDNF deficit on dendritic
morphology.
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Figure 3.
Individual Purkinje cell dendrite morphology in
the absence of BDNF. A, Modified Golgi stain resolves
the morphology of an individual Purkinje cell dendritic arbor in a P15
wild-type mouse. Inset shows a higher magnification view
of a dendritic segment studded with numerous spines
(arrowheads). B, C, Two
individual Purkinje cells from a P15 BDNF / mouse exhibit a
significant decrease in the extent of the dendritic arbor.
Insets show that dendrites nonetheless carry numerous
spines (arrowheads). Scale bars: A-C, 20 µm; insets, 5 µm.
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To determine whether synapses form on Purkinje cell dendritic spines
from BDNF / mice, immunohistochemistry was performed using an
antibody against calbindin and a second antibody against glutamate
receptor subunit GluR2. Double labeling shows puncta of GluR2
staining that cap calbindin-positive spines (Fig.
4A), suggesting that
many of these spines in fact do receive synapses as in the wild-type
mouse (Fig. 1I). Quantification of the staining patterns confirmed that 70% of spines were capped with GluR2 puncta
(n = 94) and 72% of GluR2 puncta were found at the
tips of calbindin-positive spines (n = 90). These
values are very close to those determined for BDNF +/+ cerebella. To
visualize presynaptic boutons, antibodies against the presynaptic
terminal proteins SV2 and synapsin were used (Fig.
4B). In the molecular layer of the BDNF /
cerebellum, numerous puncta immunopositive for both SV2 and synapsin
were seen, resulting in a strong yellow signal. These correspond to
individual presynaptic terminals. Quantitation revealed that 90% of
SV2 puncta were synapsin positive (n = 106) and that
92% of synapsin puncta were SV2 positive (n = 125).
This degree of association is significantly higher than that predicted by chance alone (1 ± 0.1% SEM; p < 0.01;
t test). Double-positive puncta were defined as those that
overlapped by at least 50% in the same optical section. To confirm
that these accumulations of presynaptic protein correspond to bona fide
synapses, we used the antibody against the postsynaptic marker,
GluR2. Double labeling revealed that numerous GluR2 puncta were
present and juxtaposed to SV2 puncta in the molecular layer of BDNF
/ mice (Fig. 4C). As predicted, SV2 staining and
GluR2 staining overlap noticeably less than staining for the two
presynaptic markers SV2 and synapsin, resulting in much less yellow
signal. The staining patterns for SV2, synapsin, calbindin, and
GluR2 seen in BDNF / mice were very similar to staining patterns
observed in BDNF +/+ cerebella (data not shown). This demonstrates that
even in the absence of BDNF there is development and juxtaposition of
the presynaptic and postsynaptic components of the PF/PC synapse.
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Figure 4.
Synaptogenesis in the molecular layer of BDNF
/ cerebellum. A, P15 cerebellum immunolabeled with
antibodies against calbindin (green) and the
glutamate receptor subunit GluR2 (GluR delta 2)
(red). Numerous calbindin-positive spines are capped by
puncta of GluR2 staining characteristic of functional synapses.
B, Individual presynaptic terminals in the molecular
layer immunolabeled with antibodies against the presynaptic terminal
proteins SV2 (green) and synapsin
(red) resulting in identical staining patterns.
C, Immunolabeling with antibodies against SV2
(green) and GluR2 (red) results
in staining patterns where the puncta are juxtaposed with much less
overlap, consistent with the association of presynaptic terminals with
their postsynaptic targets. Images were reconstructed from 5-10
adjacent optical sections. Scale bars: A, 5 µm;
B, C, 1 µm.
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BDNF may regulate synapse number in several systems. Therefore, a
quantitative analysis of synapse density in the molecular layer of BDNF
+/+ and / mice was performed. In cerebellar sections double labeled
with an antibody against SV2 and an antibody against GluR2, synapses
were defined by the juxtaposition or overlap of puncta of SV2 and
GluR2 staining. Synaptic density per 300 µ3 was determined from four deconvolved
optical z-series in each of three cerebellar subregions (caudal,
dorsal, and rostral) in wild-type (n = 3) and mutant
mice (n = 3). A small difference was seen in the
overall density of PF/PC synapses between wild-type mice (48.4 synapses/300 µ3 ± 4.1 SEM) and mutant
mice (40.7 synapses/300 µ3 ± 1.9 SEM;
p = 0.07). Thus, in spite of the marked effect of BDNF
on Purkinje cell dendritic morphology and molecular layer thickness,
PF/PC synapses can form, although their numbers are slightly reduced.
BDNF deficit leads to impaired short-term plasticity
BDNF enhances neurotransmission and potentiates activity-dependent
plasticity at hippocampal synapses (Kang and Schuman, 1995a,b; Korte et al., 1995; Patterson et al., 1996). We analyzed
neurotransmission across PF/PC synapses in thin cerebellar slices from
BDNF / and wild-type mice aged P14-P17. We recorded the
spontaneous mEPSC as well as the AMPA receptor-evoked EPSCs from
Purkinje cells (Chen and Regehr, 1997). Analysis of mEPSC amplitudes
showed no difference between BDNF +/+ and BDNF / synapses (Fig.
5). In addition, evoked EPSCs are similar
at BDNF +/+ and / synapses (Fig.
6A). These observations
suggest that synaptic function is preserved despite the BDNF
deficit.
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Figure 5.
A, Representative consecutive mEPSC
traces are shown from a wild-type (left) and a BDNF
/ (right) mouse. B, The normalized
cumulative amplitude distribution for BDNF +/+ (n = 4; solid trace) and BDNF / (n = 4; dashed trace) are similar
(p = 1.0 by Kolmogorov-Smirnov test).
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Figure 6.
Specific impairment of short-term synaptic
plasticity in BDNF / mice. A, Evoked AMPA
receptor-mediated EPSCs were elicited by stimulating parallel fibers
and recording from an individual Purkinje cell in voltage clamp. The
decay times of the evoked EPSCs are indistinguishable between WT and
BDNF / mice. Traces are the averages of 5-10 trials.
B, The mean percentage facilitation is plotted as a
function of interstimulus intervals for BDNF / ( ;
n = 6) and wild-type littermates ( ;
n = 6). BDNF / mice exhibit decreased
paired-pulse facilitation at interstimulus intervals <200 msec
(p < 0.05). C, The response
of Purkinje cells to repetitive 10 Hz stimulation is illustrated by
plotting the ratio of the amplitude of the response to the 10th and
50th pulses to the amplitude of the response to the first pulse.
Although 10th/1st ratio is significantly decreased in BDNF / mice
because of impaired PPF, there is no significant difference in the
50th/1st. This indicates that there is no difference in the response to
prolonged 10 Hz stimulation in the absence of BDNF.
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Short-term synaptic plasticity at the PF/PC synapse in BDNF/ mice
was examined using pairs of stimuli, separated by varying interstimulus
intervals. At the PF/PC synapse, pairs of closely spaced stimuli result
in the facilitation of synaptic strength (Atluri and Regehr, 1996).
This paired-pulse facilitation can be quantified as the change in the
peak amplitude of the second EPSC relative to the first (percentage
facilitation; see Materials and Methods). For brief interstimulus
intervals, the degree of facilitation in BDNF / mice is
approximately half that of control levels (p < 0.01; ANOVA) (Fig. 6B). At longer interstimulus
intervals (>400 msec), there is no facilitation in either wild-type or
mutant animals. This result suggests that there is an alteration of
short-term plasticity at the PF/PC synapse in BDNF / mice.
Response to repetitive stimulation is another index of presynaptic
function. Parallel fibers were stimulated with a 10 Hz train for 10 or
50 trials. The ratios of the peak amplitude of the 10th/1st EPSC and
the 50th/1st EPSC were determined (Fig. 6C). The similarity
in the 50th/1st impulse between wild-type (0.70 ± 0.21 SEM;
n = 6) and BDNF / mice (0.53 ± 0.03 SEM;
n = 5) indicates that the synaptic apparatus in the
mutant mouse is capable of maintaining neurotransmission over long
periods of repetitive stimulation. In contrast, after a short train of stimulation, the 10th/1st ratio is 1.40 (±0.08 SEM; n = 7) in wild-type mice and 1.11 (±0.06 SEM; n = 6) in
BDNF / mice. This difference is statistically significant
(p < 0.05; t test). Because PPF is
decreased in BDNF / mice at an ISI of 100 msec, which corresponds
to a frequency of 10 Hz, altered short-term plasticity must contribute
to this difference seen in the 10th/1st ratio. Thus the evidence for
impaired paired-pulse facilitation in the context of normal mEPSCs,
normal evoked EPSC, and equivalent response to sustained trains of
stimulation argues for a selective deficit in short-term plasticity in
BDNF / mice.
To determine whether the effects of BDNF deficit on presynaptic
function are reversible, exogenous BDNF was applied to cerebellar slices from BDNF / mice. Under the experimental conditions used (see Materials and Methods), only presynaptic effects of BDNF addition
would be detected. The functional deficit in short-term synaptic
plasticity evident in the decreased PPF was not reversed by bath
application of BDNF after either 30 min or 3 hr of BDNF exposure (data
not shown). To be certain that BDNF is capable of stimulating
responsive cells in the slice under these conditions, Trk
phosphorylation was analyzed. Treatment of wild-type or BDNF /
cerebellar slices with 200 ng/ml BDNF for 1 hr was able to induce Trk
receptor phosphorylation (data not shown).
Synaptic vesicle distribution is altered in the absence
of BDNF
Under the experimental conditions used in this study, changes in
the evoked response to pairs of stimuli are consistent with a
presynaptic mechanism that regulates synaptic vesicle exocytosis. Recent studies have revealed the existence of distinct vesicle pools
and have begun to shed light on their relationship to the dynamics of
vesicle exocytosis and endocytosis (Sudhof, 1995; Betz and Angleson,
1998; Fernandez-Chacon and Sudhof, 1999; Jahn and Sudhof, 1999; Li and
Schwarz, 1999; Doussau and Augustine, 2000; Sudhof, 2000). Because
presynaptic function is impaired in BDNF / mice, we hypothesized
that this functional impairment may also be reflected in the synaptic ultrastructure.
Ultrastructural changes have previously been associated with changes in
synaptic function. We performed a detailed analysis of the PF/PC
synapse in wild-type and BDNF / mice via electron microscopy.
Electron micrographs of the molecular layer reveal the main
ultrastructural components of the PF/PC synapse in both genotypes (Fig.
7). These include a presynaptic swelling
of the parallel fiber terminal, which contains numerous round, loosely packed synaptic vesicles. The postsynaptic dendritic spine from a
Purkinje cell dendrite is also clearly visible in cross section. The
asymmetric electron-dense band between the terminal and the dendritic
spine is characteristic of excitatory synapses and is often used to
demarcate the extent of the active zone. Although immunohistochemistry
revealed the appropriate localization of important synaptic proteins,
electron micrographs demonstrated conclusively that PF/PC synapses
display the basic structural features of synapses even in the absence
of BDNF.
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Figure 7.
Electron micrographs of the PF/PC synapse in P15
WT and BDNF / mice. Individual synapses exhibit a dendritic spine
(*), postsynaptic density demarcated by arrowheads,
and presynaptic terminal filled with numerous round, loosely packed
vesicles in both wild-type and BDNF / animals.
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A quantitative analysis revealed a significant increase in the number
of synaptic vesicles in the terminals of BDNF / mice compared with
those of BDNF +/+ mice (Table 1).
Synaptic terminals were analyzed in longitudinal and cross-sectional
orientations. The increase in vesicle number in BDNF / terminals
was seen in both of these orientations. Investigations of the structure and dynamics of the vesicle pool have yielded evidence that it can be
subdivided into different functional pools (Rosenmund and Stevens,
1996) that may exist in a steady-state relationship. Therefore, to
determine whether the increase in the number of vesicles in BDNF /
mice was restricted to a specific region of the vesicle pool, we
plotted the number of vesicles per 25 nm bin as a function of distance
from the synaptic cleft (Fig. 8A,B).
This revealed that within the first 100 nm of the synaptic cleft there
is no difference in vesicle number. However, beyond 100 nm there is a
significant increase in the number of vesicles in BDNF / terminals
(D 
D0.01, by
Kolmogorov-Smirnov). When we calculated the fraction of all the
vesicles that was located within 50 nm of the active zone, commonly
referred to as the docked vesicle pool, we found a significant decrease
in the absence of BDNF (Table 1). These findings show that BDNF deficit
leads to a selective increase in the vesicle pool that lies distal to
the active zone.
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Figure 8.
Increased vesicle number in BDNF / synapses is
restricted to vesicles located farthest from the active zone. The
number of vesicles within consecutive 25 nm bins was plotted as a
function of distance from the synaptic cleft. Within the first 100 nm
from the synaptic cleft there is no difference in the number of
vesicles between the wild-type mice () and BDNF / mice () in
either longitudinal sections (A) or cross
sections (B). However, beyond 100 nm, BDNF /
mice exhibit a significant increase in the number of vesicles compared
with WT mice. * D D0.01
by Kolmogorov-Smirnov.
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Vesicle number may change as a function of synapse size. We found that
PSD length was significantly increased in longitudinal sections of BDNF
/ terminals, suggesting an overall elongation of presynaptic
terminals in the absence of BDNF. To correct for the effect of synapse
size, the number of docked vesicles was normalized to the length of the
active zone. Similarly, the total number of vesicles was normalized to
the total area occupied by the vesicle pool (Table 1). We found that
the density of docked vesicles was not significantly different between
BDNF +/+ and BDNF / terminals (Table 1). In contrast, the overall
packing density of vesicles was significantly decreased in the
terminals of BDNF / mice compared with terminals in wild-type mice
(Table 1). Therefore, parallel fibers terminals are not simply larger in BDNF / mice, but exhibit an altered ultrastructure and abnormal distribution of vesicles that may reflect the change in synaptic function demonstrated here.
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DISCUSSION |
In the absence of BDNF, numerous PF/PC synapses form despite
severe and persistent disturbances of Purkinje cell dendritic morphology. Although neurotransmission is remarkably well preserved, these synapses exhibit a specific defect in short-term synaptic plasticity. Although the amplitude of spontaneous mEPSCs, the waveforms
of evoked EPSCs, and the response of the synapse to repetitive
stimulation all appear normal, paired-pulse facilitation is
significantly decreased in BDNF / mice. Changes in paired-pulse facilitation are often used as a measure of changes in presynaptic function. We found that this change is associated with a significant increase in synaptic vesicle number and a significant decrease in the
overall synaptic vesicle packing density in BDNF / mice. The data
presented here support a model in which BDNF in vivo acts on
the PF/PC synapse to modify short-term synaptic plasticity and regulate
synaptic vesicle number and distribution.
Correlating vesicle pools with synaptic function
As is the case in the hippocampus in BDNF / mice (Pozzo-Miller
et al., 1999) or TrkB / mice (Martinez et al., 1998), BDNF is not
absolutely required for synaptogenesis between granule cells and
Purkinje cells to take place. In fact, in the absence of BDNF, we
detected only a slight decrease in the overall PF/PC synapse density.
Accordingly, BDNF / Purkinje cell dendrites were studded with
numerous spines as in wild-type cells, and most of theses spines were
the sites of PF:PC synapses.
Although synaptogenesis proceeds, specific structural and functional
abnormalities remain in BDNF / mice. PF:PC synapses in BDNF /
mice have more vesicles and longer PSDs than do wild-type synapses,
whereas immature synapses are characterized by fewer vesicles and
shorter PSDs. Thus the structural and physiologic differences seen in
the PF/PC synapses of BDNF / mice are not characteristics of
developmentally delayed synapses but represent an aberrant phenotype.
As demonstrated here for the cerebellum, decreased paired-pulse
facilitation has been shown to be associated with structural changes in
presynaptic terminals in the hippocampus of BDNF / (Pozzo-Miller et
al., 1999) and TrkB / mice (Martinez et al., 1998). However, the
nature of the ultrastructural changes is different in the two systems.
Pozzo-Miller et al. (1999) observed a decrease in the number of docked
vesicles in BDNF / mice. More recently, Tyler and Pozzo-Miller
(2001) have demonstrated that BDNF applied to organotypic hippocampal
slices increases docked vesicle number and neurotransmitter release. In
contrast, in the cerebellum, we observed no change in docked vesicle
density but rather an increase in vesicles distant from the active
zone. How can we reconcile these apparent differences between the
hippocampus and the cerebellum? In both systems the absence of BDNF
results in a significant decrease in the proportion of synaptic
vesicles that are docked relative to the total number of
synaptic vesicles. Different vesicle pools are likely to exist in a
dynamic steady state within the terminal (Li and Schwarz, 1999). Thus,
one interpretation that reconciles the ultrastructural findings of the
BDNF / hippocampus and cerebellum is that neurotrophins shift the
balance between the different vesicle pools in favor of the docked
pool. For example, BDNF could regulate the movement of synaptic
vesicles within the terminal by modulating the interactions between
motor proteins like myosin V, cytoskeletal actin, and
vesicle-associated proteins such as synapsin (Hilfiker et al., 1999;
Doussau and Augustine, 2000).
This study of BDNF / cerebellar synapses provides a new example in
which changes in presynaptic function correlate with alterations in
vesicle number or vesicle distribution. In various systems, the degree
of paired-pulse facilitation is inversely related to the number of
docked vesicles (Dobrunz and Stevens, 1997; Murthy et al., 1997; Jiang
and Abrams, 1998; Schikorski and Stevens, 1999). There are several
examples in which mutations that alter the distribution of synaptic
vesicles also lead to changes in presynaptic function. For example,
mutations in synaptotagmin, the presynaptic calcium sensor, impair
evoked neurotransmitter release (DiAntonio and Schwarz, 1994) and
decrease the number of morphologically docked vesicles in
Drosophila (Reist et al., 1998). Hippocampal synapses
deficient in synapsin I, an abundant presynaptic phosphoprotein,
exhibit increased paired-pulse facilitation (Rosahl et al., 1993) and a
significant decrease in the number of vesicles distal to the active
zone (Takei et al., 1995). The unconventional myosin, myosin Va, is a
motor protein that may contribute to actin-based vesicle movement in
synaptic terminals (Prekeris and Terrian, 1997). In mice, mutations in
myosin Va lead to progressive ataxia, loss of balance, seizures, and
death (Evans et al., 1997), and in humans they lead to a rare
immunodeficiency and pigmentation disorder, Griscelli syndrome (Kumar
et al., 2001). Cerebellar granule cells in myosin Va mutant animals
have enlarged synaptic terminals with increased numbers of vesicles
suggesting the abnormal accumulation of presynaptic components
(Bridgman, 1999). Acute changes in synaptic proteins can likewise alter
both vesicle distribution and presynaptic function. The injection of anti-synapsin antibodies into lamprey axons depletes the distal pool of
vesicles and results in a pronounced decrease in EPSP amplitude in
response to high-frequency but not low-frequency stimulation (Pieribone
et al., 1995). These studies are essential in helping to elucidate the
relationship between synaptic vesicle distribution and synaptic
function at various types of synapses. Here we have shown that
facilitation is impaired in enlarged terminals where vesicles
accumulate in the absence of BDNF. The increase in parallel fiber
terminal size and decrease in overall vesicle density in the absence of
BDNF are similar to the changes observed in the terminals of
hippocampal afferents in TrkB / mice (Martinez et al., 1998) as
well as in parallel fiber terminals of myosin Va mutant mice (Bridgman,
1999) and suggest a common underlying abnormality.
Chronic and acute effects of BDNF
The changes in BDNF / mice reported here could reflect either
the long-term effects of BDNF deficit on synapse development or the
acute effects of BDNF deficit on synaptic function. Neurotrophins are
powerful differentiation factors and may guide the recruitment and
assembly of the molecular components required for synaptic function
and/or plasticity. Thus alterations in short-term plasticity and
synaptic ultrastructure may represent abnormal development. This idea
is consistent with the finding that exogenous BDNF fails to reverse the
defect in PPF. If the changes in BDNF / mice are caused by
long-term BDNF deprivation, then BDNF rescue may require days.
Alternatively, the effects of BDNF on synaptic plasticity could be very
acute and depend on the timing of endogenous BDNF release at active
synapses. Bath application of BDNF may fail to reproduce the in
vivo time course and hence fail to reverse the defect in PPF.
Recent studies have demonstrated that BDNF is located within vesicles
at the presynaptic terminal (Haubensak et al., 1998) and can be rapidly
released in response to activity (Canossa et al., 1997; Marini et al.,
1998). BDNF can also potentiate neurotransmitter release from
cerebellar granule neurons (Numakawa et al., 1999; Yamagishi et al.,
2000) and can cause a rapid depolarization of Purkinje cells (Kafitz et
al., 1999). These rapid and local effects of BDNF may not be mimicked
by prolonged bath application of neurotrophin.
Finally, the inability of exogenous BDNF to alter facilitation could
reflect a postsynaptic role of BDNF. The experimental conditions used
here detect changes in presynaptic function exclusively (see Materials
and Methods). Thus, if endogenous BDNF acts on postsynaptic TrkB
receptors and induces the release of a trans-synaptic retrograde signal such as a cannabinoid (Kreitzer and Regehr, 2001;
Maejima et al., 2001) or adenosine to alter paired-pulse facilitation,
we would not detect any change in PPF after application of exogenous BDNF.
Taken together, the data presented here suggest that BDNF can regulate
the degree of short-term synaptic plasticity that can be elicited at a
synapse. Given that neurotrophin expression and release are activity
dependent, neurotrophins may be important mediators of hebbian
plasticity by providing neuronal circuits with a mechanism by which
plasticity is favored at more active synapses. Such second-order
plasticity, or metaplasticity, constitutes a powerful additional
dimension in which neurons can adjust the strength of their connections
to achieve their information-processing goals.
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FOOTNOTES |
Received March 19, 2001; revised Oct. 26, 2001; accepted Nov. 27, 2001.
This work was supported by grants from National Institutes of Health
(NIH) (NS37757 to R.A.S.) and the Howard Hughes Medical Institute
(Postdoctoral Research Fellowship for Physicians to C.C.). A.R.C. was
supported by a fellowship from NIH (5F31LM00040-05), and R.A.S. was
supported by a Klingenstein Fellowship. In accordance with the Harvard
conflict of interest guidelines, we note that R.A.S. has a financial
interest in Curis Inc. We thank Dr. Tom Schwarz and Dr. Wade Regehr for
their helpful comments. We thank Maria Ericsson and Louise Trakimas for
tissue preparation and training for electron microscopy, Pieter Dikkes
for Golgi staining, Anita Bhattacharyya and John Alberta for training
for deconvolution microscopy, and Erin Berry for technical assistance.
Correspondence should be addressed to Rosalind A. Segal, Department of
Pediatric Oncology, Dana 620, Dana Farber Cancer Institute, 44 Binney
Street, Boston, MA 02115. E-mail:
rosalind_segal{at}dfci.harvard.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
