Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, USA
* Author for correspondence (e-mail: scohenco{at}uci.edu)
Accepted 29 July 2005
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SUMMARY |
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Key words: Xenopus laevis, Retinal ganglion cell, Axon branching, In vivo imaging
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Introduction |
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Neurotrophins, originally identified for their ability to promote neuronal
survival and differentiation, are potent modulators of synaptic connectivity
in the CNS, influencing synaptic structure and function
(Poo, 2001;
Vicario-Abejon et al., 2002
).
Specifically, BDNF influences the morphological complexity of axons and
dendrites (Cohen-Cory and Fraser,
1995
; Lom and Cohen-Cory,
1999
; McAllister et al.,
1995
), increases synapse number in the developing brain
(Aguado et al., 2003
;
Alsina et al., 2001
;
Causing et al., 1997
;
Luikart et al., 2005
;
Rico et al., 2002
), modulates
synapse maturation (Collin et al.,
2001
; Huang et al.,
1999
), controls the ultrastructural composition of synapses
(Carter et al., 2002
;
Collin et al., 2001
;
Tyler and Pozzo-Miller, 2001
;
Wang et al., 2003
) and may
regulate the incorporation of synaptic proteins into synaptic vesicle
membranes (Pozzo-Miller et al.,
1999
). Thus, BDNF is involved in multiple aspects of
synaptogenesis, from the formation to the functional maturation of synapses.
Our previous work specifically demonstrated both permissive and instructive
roles for BDNF during synaptogenesis
(Alsina et al., 2001
). In vivo
time-lapse imaging of Xenopus retinal ganglion cell (RGC) axon arbors
showed that BDNF increases arbor complexity, thereby increasing the number of
presynaptic sites in the more elaborate axons, while also influencing synapses
directly, increasing synapse density per axon branch. Although these
observations suggested that BDNF influences the formation and therefore the
stabilization of newly formed synapses, our studies did not directly
differentiate between these two dynamic events. Here, we examined the
relationship between axon branch and synapse stabilization to obtain a better
understanding of the participation of BDNF in this important aspect of
synaptogenesis. Manipulations that decrease endogenous tectal BDNF show that
the stability of axon branches and of GFP-synaptobrevin identified synapses
depends on BDNF, but that the rate of synapse turnover, a component of normal
axon remodeling, is unaffected by alterations in BDNF. Moreover, by
manipulating NMDAR transmission directly in the optic tectum, we demonstrate
that BDNF can rescue synaptic sites that would normally be affected when NMDAR
activity is altered.
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Materials and methods |
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GFP-Synaptobrevin in vivo expression and axon labeling
The method used for the simultaneous visualization of axon arbor morphology
and presynaptic sites in individual RGC axons in vivo was as described
previously (Alsina et al.,
2001), with minor modifications. In brief, a chimeric gene coding
for wild-type GFP and the complete sequence of Xenopus synaptobrevin
II was used to target GFP expression to synaptic vesicles in live tadpoles.
Retinal progenitor cells of stage 20-24 tadpoles were co-transfected with
equimolar amounts of GFP-synaptobrevin and pDsRed2 (Clontech, Palo Alto, CA)
expression plasmids by lipofection (Holt
et al., 1990
). Tadpoles were reared under filtered illumination,
in 12-hour dark/light cycles, until stage 45 when used for experimentation and
imaging. Only a few neurons per retina were transfected, with 80-90% of
transfected neurons expressing both plasmids
(Alsina et al., 2001
).
Electron microscopy
Stage 45 tadpoles with only a few RGCs expressing GFP-synaptobrevin in
their axon terminals were selected and processed for pre-embedding
immunoelectron microscopy. Tadpoles were anesthetized and fixed in 2%
paraformaldehyde, 3.75% acrolein in 0.1 M phosphate buffer (pH 7.4). Brains
were removed, post-fixed and embedded in 1% agarose. Vibratome sections (50
µm) were collected, incubated in 1% sodium borohydride in phosphate buffer,
cryoprotected, quickly permeabilized in liquid nitrogen and blocked in 0.5%
bovine serum albumin (BSA), 0.1 M Tris buffer saline (TBS) (pH 7.5). Sections
were incubated overnight in a primary mouse monoclonal antibody against GFP
(1:10 dilution in 0.1% BSA in TBS; Molecular Probes, Eugene, OR) followed by 2
hours in a secondary goat anti-mouse IgG coupled to 1 nm gold particles [1:50
dilution in 0.5% fish gelatin, 0.8% BSA in 0.01 M PBS (pH 7.4); Aurion-EMS,
Hatfield, PA]. Sections were incubated in 2% glutaraldehyde and gold particles
were enlarged using a British BioCell silver intensification kit (Ted Pella,
Redding, CA). Sections were post-fixed in 2% osmium tetroxide, dehydrated and
flat embedded in 100% Epon between Aclar sheets. Sections (70 nm) were
obtained using a Reichert ultramicrotome with a diamond knife (Diatome) and
counterstained with 2% uranyl acetate and Reynolds lead citrate.
Ultrastructural analysis was performed using a Philips CM20 transmission
electron microscope.
Drug treatment and time-lapse imaging
The behavior of individual, fluorescently labeled RGC axons was followed
with confocal microscopy in stage 45 tadpoles expressing GFP-synaptobrevin.
Only tadpoles with individual RGC axons labeled with DsRed2 showing specific,
punctate GFP labeling in their terminals were selected. Tadpoles containing
one or two clearly distinguishable double-labeled axons, with at least six
branches were imaged every 2 hours for 8 hours, then again at 24 hours.
Immediately after the first observation, 0.2-1.0 nl of anti-BDNF (330 µg/ml
of purified IgG; R&D systems, Minneapolis, MN), APV (50 µM solution;
Tocris Cookson, Ellisville, MO), MK801 (20 µM solution; Tocris Cookson),
recombinant BDNF (200 ng/µl; Amgen, Thousand Oaks, CA) or vehicle solution
(50% Niu Twitty) was pressure injected into the ventricle and subpial space
overlying the optic tectum. The specificity of the BDNF antibody versus
non-immune IgG, and its ability to influence RGC differentiation were
determined in control experiments as previously described
(Lom and Cohen-Cory, 1999).
Axon arbors in tadpoles injected with control, non-immune IgG exhibited branch
and GFP-synaptobrevin cluster dynamics comparable with those of
vehicle-treated tadpoles (data not shown). Microinjection of APV and MK801
into the optic tectum of developing tadpoles has been shown to eliminate
NMDAR-mediated synaptic currents completely
(Zhou et al., 2003
), and was
effective in blocking neuronal activity up to 8 hours after treatment (B.H.,
unpublished). To correlate GFP-synaptobrevin distribution with axon
morphology, thin optical sections (1.0 µm) through the entire extent of the
arbor were collected at 60x magnification (1.00 NA water-immersion
objective) with a Nikon PCM2000 laser-scanning confocal microscope (Melville,
NY) equipped with Argon (488 nm excitation; 10% neutral density filter) and
HeNe (543 nm excitation) lasers. A 515/30 nm (barrier) and a 605/32 nm
(band-pass) emission filters were used for GFP-synaptobrevin and DsRed2
visualization, respectively. GFP-synaptobrevin and DsRed2 confocal images were
obtained simultaneously, below saturation levels, with minimal gain and
contrast enhancements.
Data analysis
All analysis was performed from raw confocal images without any post
acquisition manipulation or thresholding. Analysis was performed blind to the
treatment group. Digital three-dimensional reconstructions of DsRed2-labeled
arbors (red only) were obtained from individual optical sections through the
entire extent of the arbor with the aid of the MetaMorph software (Universal
Imaging, West Chester, PA). To characterize the distribution of
GFP-synaptobrevin puncta to particular axonal regions, pixel-by-pixel overlaps
from individual optical sections obtained at the two wavelengths were
analyzed. Yellow regions of complete red and green overlap were identified,
counted and related to arbor morphology. GFP-synaptobrevin labeled puncta of
0.5-1.0 µm2 in size (size of smallest puncta), and hue and pixel
intensity values between 16-67 and 150-255, respectively, were considered to
be single synaptic clusters. Discrete GFP-synaptobrevin puncta classified in
this manner exhibited median pixel values 2.0 to 3.0 times greater than the
median pixel values of background non-punctate GFP within the same axon arbor.
During data analysis, we ensured that similar ratios were maintained for every
axon arbor analyzed throughout the 24 hour observation period (see
Fig. 2). Synaptic cluster
values were obtained by manual counting of yellow puncta and similar values
were obtained by digital counting. To obtain a detailed analysis of synaptic
cluster dynamics at each observation interval, several parameters were
measured: the number of clusters per branch or per unit arbor length, the
number of clusters added or eliminated, the number of clusters maintained from
one observation interval to the next, and the location of each synaptic
cluster along the axon arbor. For the quantitative analysis of axon branching,
the following morphological parameters were measured: total arbor branch
length (length of total branches), total branch number, the number of
individual branches gained or lost, and the number of branches remaining from
one observation interval to the next. Extensions from the main axon of more
than 5 µm were classified as branches. Total arbor length was measured from
binarized images of the digitally reconstructed axons. A relative measure of
cumulative length of all branches per axon terminal was obtained by counting
total pixel number from the first branch point. A total of 10-14 axon arbors
per condition were analyzed, with one axon analyzed per tadpole. Axons
analyzed had between 6-41 branches and 13-229 clusters. Data are presented as
percent increase from the initial observation interval to each subsequent
interval, or as percent increase for each 2 hour observation interval.
Two-sample unpaired t-tests, one-way ANOVA Tukey's multiple
comparison tests, and Fisher's exact and chi-square tests (Systat, SPSS) were
used for the statistical analysis of data. Results were classed as significant
as follows: *P0.05, **P
0.005,
***P
0.0005.
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Results |
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Blocking endogenous BDNF induces GFP-synaptobrevin cluster dismantling and axon branch elimination
Our previous studies show that BDNF influences the morphological maturation
of RGC axon arbors primarily by promoting axon arbor growth. Specifically,
increasing BDNF tectal levels induces axon branch addition without affecting
the degree of stabilization (Cohen-Cory
and Fraser, 1995; Cohen-Cory,
1999
). Exogenous BDNF, however, can influence axon branch
stability under conditions where stability is experimentally altered. For
example, BDNF prevents the destabilizing effects that blocking retinal
activity exerts on axon branches by maintaining the normal rates of branch
addition and elimination (Cohen-Cory,
1999
). Thus, to determine directly whether endogenous BDNF
participates in presynaptic site stabilization, we imaged RGC axon terminals
double labeled with GFP-synaptobrevin and DsRed2 in tadpoles treated with
function-blocking antibodies to BDNF. Tectal injection of anti-BDNF induced a
rapid decrease in GFP-synaptobrevin labeled synapses in RGC axon arbors
examined at 2, 4, 6, 8 and 24 hour time points following treatment
(Fig. 2). GFP-synaptobrevin
cluster number was significantly decreased 4 hours after anti-BDNF treatment
(55.8±11.0% versus 110.3±7.9% in control; P<0.0005;
Fig. 3A), an effect that was
paralleled by a significant decrease in total branch number (84.3±5.8%
versus 101.6±5.0% in control; P<0.03;
Fig. 3B). The decrease in
GFP-synaptobrevin cluster and branch number was maintained throughout the
observation period (4, 6, 8 and 24 hours;
Fig. 2 and
Fig. 3A,B). Anti-BDNF not only
decreased total GFP-synaptobrevin cluster and branch number, but also
significantly decreased GFP-synaptobrevin clusters per axon branch and per
unit arbor length (Fig. 3C,D),
indicating that endogenous BDNF significantly influences synapse density per
axon arbor. Therefore, the effects of increased tectal BDNF on synapse number,
branch number and synapse density we have previously reported
(Alsina et al., 2001
) mirror
the actions of endogenous BDNF.
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Discussion |
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Synapse elimination is a necessary step during the remodeling of neuronal
connectivity (Goda and Davis,
2003). In most instances, the disassembly of previously functional
synapses can be correlated with presynaptic input elimination, as demonstrated
for the NMJ (Eaton et al.,
2002
; Sanes and Lichtman,
2001
; Walsh and Lichtman,
2003
). Synapse disassembly can also occur as a mechanism that
modulates the strength of connectivity between two neurons without influencing
arbor morphology (Goda and Davis,
2003
). An NMDAR mechanism that mediates synapse strengthening has
been hypothesized to influence axon branch and synapse dynamics in the
developing retinotectal system (Debski and
Cline, 2002
). Evidence that RGC axon branch stabilization relates
to the stabilization of structural synapses, however, was missing. The present
study supports the idea that synapse disassembly can occur without input
elimination (Buffelli et al.,
2003
; Hata et al.,
1999
; Hopf et al.,
2002
; Goda and Davis,
2003
). Our findings demonstrate that synaptic changes induced by
acute widespread alterations in NMDAR-mediated synaptic transmission in the
optic tectum influence presynaptic site stability, but are not sufficient to
destabilize and eliminate RGC axonal branches. The disappearance of
GFP-synaptobrevin clusters in response to NMDAR blockers may reflect an
increase in synaptic vesicle dispersion or recycling
(Bacci et al., 2001
), rather
than complete removal of the synapse. It is noteworthy that endogenous BDNF
simultaneously and independently influences the stability of both synapses and
axon branches, and that BDNF can contribute to synapse and branch stability
even when neuronal activity is altered
(Cohen-Cory, 1999
).
Neuronal activity and neurotrophins interact to modulate neuronal structure
and function (Vicario-Abejon et al.,
2002). Neuronal activity regulates gene transcription, transport
and secretion of BDNF protein (Righi et
al., 2000
; Chytrova and Johnson, 2004;
Lessmann et al., 2003
). TrkB
receptor trafficking to the membrane (Du
et al., 2000
; Meyer-Franke et
al., 1998
), and BDNF-TrkB receptor complex internalization
(Lu, 2003
) also depend on
neuronal activity. Even though BDNF signaling is tightly modulated by neuronal
activity and it is believed that neurotrophins preferentially modulate active
synapses (Lu, 2003
), BDNF can
also influence synapses and neuronal connectivity independently of whether
neurons are synaptically active
(Cohen-Cory, 1999
;
Collin et al., 2001
). The
observation that BDNF can act rapidly to maintain candidate synapses for a
significant time period following NMDAR blockade supports a role for BDNF
independent of target NMDAR activation and has important implications for
understanding the function of BDNF and potential therapeutic properties. BDNF
exerted robust and rapid effects on synapses following APV treatment, although
a single dose of BDNF was not sufficient to maintain GFP-synaptobrevin cluster
number at control levels for a prolonged period of time. The rapid effects of
BDNF observed are consistent with the localized, rapid changes in
Ca2+ signaling that BDNF elicits on axon terminals
(Zhang and Poo, 2002
) (B.H.
and S.C.C., unpublished), and with rapid, depolarizing effects of BDNF on
cultured neurons (Kafitz et al.,
1999
). The inability of BDNF to rescue synapses back to control
levels for a prolonged period of time, however, may be due to differences in
potencies or pharmacokinetics between APV and BDNF, or to rapid TrkB receptor
downregulation following NMDAR blockade
(Kingsbury et al., 2003
).
Correlated synaptic activity is thought to modulate retinotectal map
refinement by regulating presynaptic axon branch dynamics
(Debski and Cline, 2002).
Pharmacological manipulations that alter neuronal activity demonstrate that
the stability of RGC axon arbors depends on activity. For example, presynaptic
activity blockade by intraocular injection of TTX influences RGC axon branch
stabilization by increasing the rates of branch addition and elimination,
influencing arbor structure by 24 hours
(Cohen-Cory, 1999
). Chronic
NMDAR blockade in whole tadpoles decreases RGC axon branch lifetimes but only
transiently (Rajan et al.,
1999
; Ruthazer et al.,
2003
). Our observations that acute tectal administration of APV
and MK801 does not significantly influence RGC axon branching suggest that
differences in acute versus chronic effects of the inhibitors (and/or that
relative contributions of pre- and postsynaptic activity to axon branch
stabilization) may be responsible for the differential influences of activity
blockade on synapse and axon branch stabilization. In tadpoles with doubly
innervated tecta, axon branches with synchronized activity are selectively
stabilized through a NMDAR-dependent process
(Ruthazer et al., 2003
).
Because BDNF modulates RGC responses to altered activity levels by stabilizing
synapses, it is possible that BDNF may actively participate in selective
synapse and axon branch stabilization in territories where input activity is
correlated.
An important question that remains is whether the structural,
GFP-synaptobrevin identified synapses that are stabilized by BDNF are
physiologically active (Ahmari and Smith,
2002). BDNF can potentiate developing synapses in spatially
localized (Zhang and Poo,
2002
) and temporally restricted
(Kafitz et al., 1999
) manners.
Structural modifications at synapses, moreover, correlate with activation of
synaptic responses by neurotrophins
(Vicario-Abejon et al., 2002
).
For example, the number of docked synaptic vesicles and synaptic vesicle
distribution are altered in BDNF-deficient mice, an ultrastructural defect
that correlates with altered presynaptic function
(Carter et al., 2002
).
Conversely, an increase in the number of docked synaptic vesicles correlates
with the activation of synaptic responses elicited by neurotrophins in young
cultured hippocampal neurons (Collin et
al., 2001
). In this regard, loss of presynaptic function has been
correlated with the removal of synaptic vesicles and synaptic vesicle
components from individual synaptic sites
(Hopf et al., 2002
). Although
we cannot rule out the possibility that the effects that we observed relate to
the redistribution of GFP-labeled synaptic vesicles or synaptic vesicle
components (as BDNF can regulate the mobilization of vesicles from a reserve
pool to a docked synaptic pool) (Carter et
al., 2002
; Collin et al.,
2001
; Pozzo-Miller et al.,
1999
; Vicario-Abejon et al.,
2002
), the structural modifications of synapses that we observed
may represent, or eventually lead to, alterations in synaptic function
(Du and Poo, 2004
). Our
experiments demonstrating that a significant portion of GFP-synaptobrevin
clusters is eliminated following MK801 treatment suggest that active synapses
are involved in a BDNF response, as MK801 selectively blocks open NMDAR
channels. The localization of GFP-synaptobrevin to mature ultrastructurally
identified RGC synapses and the activity-dependent recycling of GFP-labeled
presynaptic sites, as determined by FM4-64 co-staining of GFP-synaptobrevin
puncta (Alsina et al., 2001
),
also suggests that GFP-synaptobrevin localizes to functional synapses.
How does BDNF influence axon arbor complexity and synapse number? While the
direct signaling mechanisms that modulate these two processes remain to be
elucidated, it is likely that BDNF signaling promotes changes in actin
polymerization and the reorganization of the actin cytoskeleton at synapses.
Actin polymerization and microtubule dynamics are necessary for growth cone
steering and axon branching (Dent et al.,
2004; Kornack and Giger,
2005
). BDNF regulates growth cone motility and filopodial dynamics
by modulating F-actin stabilization and polymerization through a Rho
GTPase-dependent pathway (Gehler et al.,
2004
; Yuan et al.,
2003
). F-actin is enriched at synapses and the integrity of the
actin cytoskeleton at pre- or postsynaptic terminals can also directly
influence the stability of developing synapses
(Dillon and Goda, 2005
;
Zhang and Benson, 2001
). It is
therefore possible that common signaling pathways that influence cytoskeletal
dynamics at both synapses and axon branches may be used by BDNF. The
identification and characterization of BDNF signaling events that coordinate
synapse formation and axon branching remain.
In conclusion, our imaging studies provide a direct link between the cellular and molecular mechanisms underlying synaptogenesis in vivo and reveal BDNF as a modulator of multiple aspects of synaptogenesis, from synapse formation to stabilization. The selective disassembly of presynaptic specializations in RGC axon arbors correlates with axon branch pruning when BDNF is withdrawn, but not when overall synaptic activity is decreased. Thus, structural rearrangements in RGC synaptic connectivity are modulated by BDNF, where BDNF influences the morphological maturation of axonal arbors and their stabilization, by a mechanism that influences both synapses and axon branches.
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ACKNOWLEDGMENTS |
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