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First published online 25 May 2006
doi: 10.1242/dev.02409
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Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA.
Author for correspondence (e-mail:
scohenco{at}uci.edu)
Accepted 18 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Xenopus laevis, Synapse, Tectal neuron, PSD95-GFP, Branching, In vivo imaging, APV
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; O'Rourke and Fraser,
1990; Witte et al.,
1996). Postsynaptic dendritic arbors are also dynamic, and it is
through coordinated interactions between axon and dendritic filopodia that
target recognition and synapse formation take place
(Cline, 2001;
Cohen-Cory, 2002;
Dailey and Smith, 1996;
Deng and Dunaevsky, 2005;
Jontes and Smith, 2000;
Niell et al., 2004;
Trachtenberg et al., 2002).
Proper neuronal connections are thus specified as axons and dendrites make
initial contacts and establish synapses that are often transient. Cell-surface
adhesion molecules, inductive signals, secreted factors and signaling
molecules actively participate in cell-to-cell communication, making neurons
receptive to form synapses (Chen and Ghosh,
2005; Ethell and Pasquale,
2005; Waites et al.,
2005). Neurotrophins are potent modulators of neuronal morphology,
influencing not only axonal arborization
(Cohen-Cory and Fraser, 1995;
Singh and Miller, 2005) but
also dendritic arbor growth (Horch and
Katz, 2002; Lom et al.,
2002; McAllister,
2000). Neurotrophins can act as target-derived trophic factors to
influence presynaptic neurons, and/or as anterograde factors to influence
postsynaptic cells (Baquet et al.,
2004; Gonzalez et al.,
1999; von Bartheld et al.,
2001; Zweifel et al.,
2005). Retrograde and anterograde signaling by neurotrophins has
also been implicated in the modulation of synapse structure and function
(Du and Poo, 2004;
Elmariah et al., 2004;
Rico et al., 2002;
Vicario-Abejon et al., 2002;
Zhang and Poo, 2002).
In the visual system, BDNF modulates synaptic connectivity by influencing
presynaptic specializations in retinal ganglion cell (RGC) axon arbors.
Increasing BDNF levels in the optic tectum of live developing Xenopus
tadpoles significantly increases RGC axon arborization and synaptic density
within 4 hours of treatment (Alsina et al.,
2001). Conversely, decreasing endogenous BDNF levels destabilizes
synapses and axon branches in RGC arbors over a similar timescale
(Hu et al., 2005). The rapid
impact of BDNF on presynaptic specializations and axon arbor morphology
suggests that BDNF modulates synaptic connectivity by acting directly on
presynaptic RGCs. It is possible that BDNF influences retinotectal
connectivity by also influencing tectal neurons, the postsynaptic partners of
RGCs. The expression patterns of BDNF and its receptor TrkB also suggest that
BDNF specifically regulates synaptogenesis and the maturation of RGC axons at
the target and may also influence tectal neurons directly. BDNF is expressed
both in the retina and optic tectum during active retinotectal development.
TrkB mRNA is expressed by RGCs and is also expressed in the optic tectum,
although at significantly lower levels
(Cohen-Cory et al., 1996;
Cohen-Cory and Fraser, 1994).
Therefore, BDNF may act locally within the optic tectum to influence the
development of tectal neurons, either directly or as consequence of its
effects on presynaptic RGC axons.
Here, we have used in vivo time-lapse imaging to further explore the mechanisms by which BDNF influences retinotectal synaptic connectivity. Expression of the GFP-tagged postsynaptic density protein PSD-95 together with DsRed2 was used to simultaneously visualize postsynaptic specializations and dendritic arbor morphology in Xenopus tectal neurons in vivo. Altering endogenous BDNF levels in the optic tectum by injection of recombinant BDNF or function-blocking antibodies to BDNF demonstrates that BDNF influences tectal neuron synaptic connectivity over a longer timescale than its effects on RGC axon arbors. Surprisingly, manipulations of BDNF levels did not significantly influence dendritic arbor morphology. Time-lapse imaging also revealed a coordinated, dynamic mechanism of synaptogenesis and arbor growth in tectal neuron dendrites that closely resembles that of RGC axon terminals and that is subject to NMDA receptor activity blockade. These observations, together with the time-course of the response of tectal neurons to BDNF, suggest that this neurotrophin shapes retinotectal synaptic connectivity through a mechanism that differentially influences postsynaptic tectal neurons and presynaptic RGCs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
and animal procedures were approved by the Institutional Animal Care and Use
Committee at the University of California, Irvine.
PSD95-GFP in vivo expression and dendritic arbor labeling
Xenopus optic tectal neurons were visualized in vivo by methods
similar to those previously used for imaging Xenopus RGC axons
(Alsina et al., 2001). To
visualize postsynaptic specializations and tectal neuron morphology
simultaneously, brain progenitor cells were co-transfected with expression
plasmids containing a chimeric gene encoding GFP and PSD-95 (a gift from Dr D.
Bredt, UCSF) and a red fluorescent protein variant (pDsRed2; Clontech, Palo
Alto, CA). Stage 20-22 tadpoles were anesthetized and pressure injected into
the tectal primordium with 0.1-0.2 nl of 1 µg/µl of each plasmid mixed
with the lipofecting agent DOTAP (Boehringer Mannheim, Indianapolis, IN).
Tadpoles were reared until stage 45, when those tadpoles with distinct neurons
expressing DsRed2 throughout their dendritic arbor and with punctate PSD95-GFP
labeling were selected for imaging. In experiments that examined the role of
BDNF, 0.2-1.0 nl of recombinant human BDNF (200 ng/µl; Amgen, Thousand
Oaks, CA), BDNF function-blocking antibody (330 µg/ml of purified IgG;
R&D Systems, Minneapolis, MN), vehicle solution (50% Niu Twitty) or
control non-immune IgG was pressure-injected into the ventricle and subpial
space surrounding the optic tectum immediately following the first imaging
session. In experiments that established synapse and branch dynamics in
tadpoles with and without NMDA receptor activity blockade, 0.1 nl of APV (50
µM in vehicle solution; Tocris Cookson, UK) or vehicle solution alone was
injected into the optic tectum immediately following the first imaging
session. Imaging was performed on a Pascal LSM 5 (Zeiss, Germany) or a Nikon
PCM2000 (Melville, NY) laser-scanning confocal microscope equipped with Argon
and HeNe lasers as described before (Alsina
et al., 2001; Hu et al.,
2005). Thin (1.0-1.5 µm) overlapping optical sections
encompassing the entire dendritic arbor and cell body were collected
simultaneously at the two wavelengths, with minimal gain and contrast
enhancements, and at below-saturation levels. Images were acquired immediately
prior to injections, and then at various intervals up until 48 hours
post-injection.
Immunocytochemistry
For GFP and endogenous SNAP-25 co-localization, tadpoles with neurons
expressing PSD95-GFP were anesthetized and fixed by immersion in 2%
paraformaldehyde and 3.75% acrolein in 0.1 M phosphate buffer pH 7.4 (PB); the
brains were removed and postfixed with the same fixative for 1 hour.
Horizontal free-floating vibratome sections (25 µm) were obtained,
preincubated in blocking solution (1.5% goat normal serum, 0.1% Triton X-100
in 0.1 M PB), and incubated overnight with mouse anti-GFP (1:100 dilution in
0.1% Triton X-100 in 0.1 M PB; Molecular Probes) and rabbit anti-SNAP25
(1:1,000 dilution; Stressgen Biotechnologies, Victoria, Canada) antibodies
simultaneously. For endogenous PSD-95 and SNAP-25 co-localization, stage 45
tadpoles were fixed in 4% paraformaldehyde; 20 µm cryostat sections were
obtained and incubated with anti-PSD-95 (mouse IgG, 1:200 dilution; Upstate
Biotechnology, Lake Placid, New York) and rabbit anti-SNAP-25 overnight.
Tissues were then rinsed and incubated with Alexa 488 anti-mouse and Alexa 568
anti-rabbit antibodies (1:200 dilution each in 0.1 M PB; Molecular Probes,
Eugene, OR). All images were collected with a LSM 5 Pascal confocal microscope
using a 63x/1.4 NA oil immersion objective. To determine colocalization
of fluorescent labels, optical sections were collected at 0.5 µm intervals
through the full extent of the PSD95-GFP positive neuron.
Electron microscopy
For electron microscopy immunostaining, 50 µm horizontal vibratome
sections were collected in 0.1 M PB. Sections were incubated in 1% sodium
borohydrade, cryoprotected in 25% w/v sucrose and quickly permeabilized in
liquid nitrogen. Sections were rinsed, incubated in blocking solution (0.5%
BSA in 0.1 M TBS), and incubated overnight with a mouse monoclonal anti-GFP
antibody (1:10 dilution in 0.1% BSA in 0.1 M TBS) followed by 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). Gold
particles were enlarged using a British BioCell silver intensification kit
(Ted Pella, Redding, CA) and sections were post-fixed and processed for
electron microscopy as described before
(Hu et al., 2005).
Ultrastructural analysis was performed using a Phillips CM20 transmission
electron microscope.
Data analysis
Data analysis was similar to that described by Hu et al.
(Hu et al., 2005) for RGC axon
arbors. All analysis was performed from raw confocal images without any
post-acquisition manipulation or thresholding. Digital three-dimensional
reconstructions of DsRed2-labeled dendritic arbors (red only) were extracted
from a stack of optical sections covering the entire extent of the arbor with
the aid of the MetaMorph software (Universal Imaging, West Chester, PA).
Pixel-by-pixel overlaps from individual optical sections obtained at the two
wavelengths were analyzed to determine the identity and position of PSD95-GFP
puncta along the dendritic arbor. Discrete PSD95-GFP labeled puncta of 0.5-1.0
µm2 in size (size of smallest puncta) with median pixel values
2.0-3.0 times greater than the median pixel values of background non-punctate
GFP within the same dendritic arbor were considered to be single synaptic
puncta. During data analysis, care was taken to ensure that similar ratios
were maintained for every neuron analyzed throughout the 48-hour observation
period. Any continuous, non-punctate GFP fluorescence in the cell body and
proximal region of a primary dendrite was excluded from the analysis.
Several parameters were measured to obtain a detailed analysis of PSD95-GFP
puncta dynamics at each observation interval: the number of puncta per branch
or per unit arbor length, the number of puncta added or eliminated, the number
of puncta maintained from one observation to the next, and the location of
each PSD95-GFP puncta along the dendritic arbor. Total dendritic arbor 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 to the next were measured for the quantitative analysis of
dendrite branching. Extensions of more than 5 µm were classified as
branches. Total arbor length was measured from binarized images of the
digitally reconstructed neurons. A relative measure of cumulative length of
all branches per dendritic arbor was obtained by counting total pixel number
from the proximalmost part of the primary dendrite. Neurons imaged were from
the mid-region of the optic tectum and had between 11 and 37 branches for
control (x=21, 20 neurons), 10 and 41 for BDNF (x=17, 16),
and 12 and 40 in anti-BDNF treated tadpoles (x=27, 12). Data are
presented as percent increase from the initial observation interval to each
subsequent interval, or as percent increase for each observation interval.
Two-sample unpaired t-tests and one-way ANOVA Tukey's multiple
comparison tests (Systat, SPSS) were used for the statistical analysis of data
and significance was *P
0.05;
**P0.005. Data are presented as mean±standard
error of the mean (s.e.m.).
In control experiments, we examined potential effects of BDNF on
Xenopus tectal neuron morphology by altering BDNF tectal levels prior
to peak endogenous tectal BDNF expression. Stage 40/42 Xenopus
tadpoles with individual tectal neurons expressing DsRed2 were selected and
microinjected with BDNF (either in solution or coupled to green fluorescent
microspheres), or with vehicle solution or cytochrome C-treated microspheres,
as described by Lom and Cohen-Cory (Lom
and Cohen-Cory, 1999). Total dendrite number and complexity were
analyzed 48 hours after treatment, at stage 43/44. Tectal neurons in tadpoles
treated with BDNF in solution or coupled to beads had similar dendritic arbor
morphologies as neurons in control-treated tadpoles. Tectal neurons in the
BDNF-treated tadpoles had an average of 11.48±0.92 branch tips (48
neurons), while in controls, tectal neurons had 11.84±1.1 branch tips
(37 neurons) 48 hours after treatment. Thus, although under the same
experimental conditions, BDNF significantly affects RGC dendritic and axonal
arborization (Alsina et al.,
2001; Lom et al.,
2002), no significant effects were observed on tectal neuron
primary dendrite number or total branch tip number (P=0.803).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Marrs et al., 2001;
Niell et al., 2004;
Okabe et al., 2001). In
Xenopus optic tectum, PSD95-GFP had a punctate distribution along
individual DsRed2-labeled tectal neuron dendritic arbors
(Fig. 1A-C). To confirm that
PSD95-GFP was correctly targeted to postsynaptic sites in vivo, we compared
PSD95-GFP distribution with that of an endogenous presynaptic protein.
Immunostaining for the presynaptic plasma membrane protein SNAP-25 showed
that, in the tectal neuropil, endogenous SNAP-25 is distributed in a punctate
pattern that is complementary to that of PSD95-GFP punctate labeling
(Fig. 1D-G) and of endogenous
PSD-95 staining (Fig. 1H-J).
Most of the PSD95-GFP puncta co-localized with endogenous SNAP-25 punctate
staining (81.25±3.12%, 357 puncta analyzed from five neurons, one
PSD95-GFP neuron per tadpole), indicating that PSD95-GFP targets to synaptic
sites. Specific localization of PSD95-GFP at synapses was also confirmed
ultrastructurally. Immunoelectron microscopy demonstrates that PSD95-GFP
predominantly localized to the postsynaptic side of mature synaptic profiles
in the tectal neuropil of stage 45 tadpoles
(Fig. 2). Morphologically
mature synapses with presynaptic terminals containing numerous synaptic
vesicles and clearly defined postsynaptic specializations were immunopositive
for GFP. In most immunopositive profiles (84.8%, or 28 out of 33 profiles
analyzed from seven brains, one PSD95-GFP neuron per tadpole brain), the GFP
immunoreactivity was localized at or near the postsynaptic density at the
synapse (Fig. 2A-C). Therefore,
these studies directly demonstrate that PSD95-GFP is recruited to synapses,
and validate it as a marker to visualize postsynaptic sites in vivo.
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). At this stage, endogenous BDNF levels in the optic tectum
are high (Cohen-Cory and Fraser,
1994), and tectal neurons increase their complexity by an active
remodeling of their dendritic arbors
(Cline, 1998;
Cline, 2001;
Wu and Cline, 1998).
Therefore, stage 45 tadpoles were imaged by confocal microscopy at 0, 4, 24
and 48 hours to determine potential effects of BDNF on tectal neurons.
Microinjection of recombinant BDNF into the optic tectum did not alter the
growth or morphology of tectal neuron dendritic arbors imaged at any of the
observation time points when compared with controls
(Fig. 3 and
Fig. 4A,B). Similarly, BDNF did
not influence dendritic arbor complexity of neurons in young tadpoles (prior
to peak BDNF expression) imaged 48 hours post-treatment (see Materials and
methods) nor the branching of tectal neurons imaged on a time-course of once
every 2 hours (data not shown). By contrast, BDNF treatment significantly
increased the number of PSD95-GFP-labeled postsynaptic specializations per
individual arbor (Fig. 3 and
Fig. 4C,D). Quantitative
analysis of individual arbors demonstrates that the total number and density
of PSD95-GFP puncta per tectal neuron dendritic arbor were significantly
increased 24 hours after BDNF treatment when compared with controls
(Fig. 4C,D). The difference in
the number and density of PSD95-GFP puncta became more dramatic by 48 hours
(Fig. 4C,D).
Our previous studies demonstrate a dual function for BDNF during the
formation and stabilization of both synapses and axon branches in
Xenopus RGC arbors (Alsina et al.,
2001; Hu et al.,
2005). Increasing BDNF within the tadpole optic tectum induces new
axon branches and presynaptic specializations to be formed while decreasing
endogenous BDNF induces the destabilization of both presynaptic sites and axon
branches. These observations suggest that limiting amounts of BDNF dictate the
extent of axon arbor growth and stabilization. Thus, to determine whether the
effects of recombinant BDNF on tectal dendrites reflect the actions of
endogenous BDNF, we decreased endogenous BDNF levels by injecting a BDNF
function-blocking antibody into the optic tectum. As observed for recombinant
BDNF, the anti-BDNF treatment did not alter dendritic branch number at any
observation interval (4, 24 or 48 hours;
Fig. 3D). Total branch number
was similar for tectal neurons in control, BDNF, and anti-BDNF treated
tadpoles (Fig. 4A), indicating
that dendritic branching was unaffected by alterations in BDNF signaling.
However, neutralizing endogenous BDNF did limit the spatial extent of the
dendritic arbor. Total dendritic arbor length remained constant in tectal
neurons in anti-BDNF treated tadpoles throughout the 48-hour observation
period (105.2±9% of time zero, 6;
Fig. 4B), while in controls,
total dendrite arbor length increased to 153.5±15% of its initial value
by 48 hours (P0.05). Neutralizing endogenous BDNF with anti-BDNF
had opposite effects on GFP-labeled postsynaptic specializations to those of
recombinant BDNF, significantly decreasing the number of PSD95-GFP puncta in
the tectal dendrites (Fig. 3D).
The total number of PSD95-GFP puncta per tectal neuron was significantly lower
than controls 24 hours after anti-BDNF treatment, an effect that became more
pronounced by 48 hours (Fig.
4C). Because neutralization of endogenous BDNF influenced the
length of the dendritic arbor and the number of PSD95-GFP puncta, when
normalized, the number of postsynaptic specializations per unit arbor length
(postsynaptic specialization density; Fig.
4D) did not differ significantly from control, although the two
parameters were affected independently. Time-lapse analysis revealed, however,
that the decrease in the absolute number of PSD95-GFP puncta in the
anti-BDNF-treated tadpoles resulted in a significant decrease in the density
of postsynaptic specializations in branches that remained stable over time
(Fig. 5). Thus, like treatment
with recombinant BDNF, neutralizing endogenous BDNF within the optic tectum
affected postsynaptic specialization number in tectal neuron dendritic
arbors.
Detailed analysis of the localization and the lifetimes of individual PSD95-GFP puncta per dendritic arbor revealed that the effect of BDNF on synapse density was due to the selective addition of new postsynaptic specializations rather than stabilization of existing ones (Fig. 6). The BDNF-elicited increase in synapse addition occurred between 4 and 24 hours after treatment (Fig. 6B). Conversely, the anti-BDNF-elicited decrease in synapse number was the result of a reduced amount of newly added postsynaptic specializations. That is, significantly fewer PSD95-GFP puncta were formed by 48 hours following anti-BDNF treatment, a trend that was observed from 4 hours onwards (Fig. 6B). The proportion of PSD95-GFP puncta that remained stable in neurons in BDNF and in anti-BDNF treated tadpoles was similar to that of controls at all observation intervals (Fig. 6A). Together, our results demonstrate that alterations in tectal BDNF levels do not influence tectal neuron dendritic arbor morphology but rather influence synapse density by modulating synapse formation.
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;
Hu et al., 2005). It was
therefore important to determine whether presynaptic events correlate with
changes in postsynaptic tectal neurons and whether other manipulations that
affect presynaptic axons would have similar or differential effects on
postsynaptic neurons. Time-lapse imaging of tectal neurons every 2 hours for a
period of 8 hours (Fig. 7A) was
used to correlate dendrite and postsynaptic site dynamics with RGC axon branch
dynamics. Time-lapse imaging demonstrates that branch lifetimes of tectal
neuron dendritic arbors closely resemble those of presynaptic RGC axon arbors.
An average of 70.1±3.9% dendritic branches remained stable and
34.5±5.7% branches were added over each 2-hour period (five neurons;
one neuron per tadpole, Fig.
7D). Likewise, 73.3±1.6% axon branches are stable and
32.7±2.8% branches are added on RGC axon arbors similarly imaged every
2 hours (Alsina et al., 2001;
Hu et al., 2005).
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; Hu et al.,
2005). Together, these observations indicate that in the
Xenopus retinotectal system, axonal and dendritic branching and pre-
and postsynaptic site elaboration are related.
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;
Rajan et al., 1999). Our
previous work demonstrates that altering NMDA receptor activity in the optic
tectum of developing tadpoles significantly influences presynaptic site
stabilization in RGC axon arbors (Hu et
al., 2005). The number of GFP-synaptobrevin-labeled presynaptic
specializations is significantly reduced to less than 50% of its initial value
in RGC axon arbors within 2 hours of blocking NMDA receptors, a treatment that
also influences axon arbor morphology by 24 hours
(Hu et al., 2005). Analysis of
PSD95-GFP puncta and dendrite branch number in tectal neurons of stage 45
tadpoles microinjected with APV shows that the decrease in synapse number in
RGC axon arbors is paralleled by a significant decrease in the number of
postsynaptic specializations in the dendritic arbors
(Fig. 8) (see also
Hu et al., 2005). The number
of PSD95-GFP-labeled postsynaptic specializations in tectal neurons exposed to
APV decreased to less than 50% of controls 2 hours after treatment, a
reduction that became more significant by 24 hours
(Fig. 8A). Only a delayed
effect on dendritic branch number was observed at 24 hours
(Fig. 8C), similar to the
impact of NMDAR blockade on RGC axon arbor morphology that we previously
reported [Fig. 8D, adapted,
with permission, from Hu et al. (Hu et
al., 2005)]. Consequently, in vivo imaging reveals coincident
mechanisms of synaptogenesis, and pre- and postsynaptic arbor growth that are
similarly influenced by NMDA receptor activity. These coordinated,
activity-dependent changes in synaptic connectivity further underscore the
differential action of BDNF on presynaptic RGC axons and postsynaptic tectal
neurons. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Okabe et al.,
2001). Our studies provide direct ultrastructural evidence that
PSD95-GFP is transported and is specifically localized to morphologically
mature synapses in the Xenopus visual system, therefore validating
its use as a dynamic marker to study the development of synaptic connectivity
(Ebihara et al., 2003;
Marrs et al., 2001;
Niell et al., 2004;
Okabe et al., 2001). By using
PSD95-GFP to label synapses, our in vivo imaging studies revealed a direct
correlation between the dynamic remodeling of synapses and dendritic arbor
structure: in Xenopus, as in zebrafish
(Niell et al., 2004), the
active formation and stabilization of PSD95-GFP labeled synaptic sites is
related to the dynamic branching of tectal neuron dendritic arbors.
Furthermore, our studies demonstrate that the branching and synaptic
complexity of Xenopus tectal neuron dendritic arbors is closely
related to the dynamic branching and synaptic complexity of presynaptic RGC
axon arbors. Two-hour time lapse imaging of PSD95-GFP labeled postsynaptic
specializations and dendrite branch dynamics demonstrates that synapse
formation and stabilization in tectal neuron dendritic arbors parallels the
behavior of RGC axon arbors imaged at the same developmental stage and at
similar time intervals [as shown in Fig.
7 and by Alsina et al. (Alsina
et al., 2001)]. In vivo imaging studies also demonstrate that
manipulations that can directly influence synaptic interactions (for example,
by blocking retinotectal synaptic transmission with APV) have analogous
effects on branch and synapse dynamics in both RGC axons and tectal neuron
dendrites. Thus, our results are consistent with the coordinated growth and
motility of pre- and postsynaptic components, as observed for neurons
establishing synapses in culture systems
(Konur and Yuste, 2004;
Umeda et al., 2005). It will
be of great significance to observe and confirm these coordinated, dynamic
events of synaptogenesis and axon and dendritic branching in pairs of RGCs and
tectal neurons that contact one another directly in vivo.
|
).
It is only through the gradual accumulation of new branches and the
lengthening of pre-existing branches that the dendritic arbor grows to attain
a more complex morphology (Cline,
2001). In vivo time-lapse imaging in zebrafish previously
characterized two populations of postsynaptic specializations in tectal neuron
dendritic arbors (Niell et al.,
2004): a large population of PSD95-GFP puncta that is highly
dynamic, with lifetimes of less than 20 minutes; and a smaller, stable
population with lifetimes of hours. Transient puncta with lifetimes of minutes
are thought to correspond to nascent synapses that are susceptible to quick
elimination, as are the transient filopodia that often bear these transient
synapses (Cline, 2001;
Niell et al., 2004). Our
analysis focused on the long-term dynamic events that affect tectal neuron
dendritic morphology and thus predominantly considered the more stable
population of postsynaptic specializations in these neurons.
Manipulations that increased or decreased BDNF levels were used to explore
the influence of BDNF on tectal neurons and to help establish a mechanism by
which BDNF shapes retinotectal connectivity. Altering endogenous BDNF levels
within the Xenopus optic tectum influenced tectal neuron synaptic
connectivity but did not significantly reshape dendritic arbor morphology. The
effects of BDNF and anti-BDNF on synapse number emerged several hours after
treatment, becoming significant only by 24 hours. A BDNF-elicited increase in
both the number and density of postsynaptic specializations on tectal neurons
paralleled, with a relative time-delay, the previously observed increase in
presynaptic specializations on RGC axons
(Alsina et al., 2001;
Hu et al., 2005). Twenty-four
hours after exposure to BDNF, the density of GFP-labeled synaptic
specializations increased in both the pre- and postsynaptic arbors, reaching
55% and 34% greater density, respectively, than in RGC axons and tectal
neuron dendrites in control tadpoles. Illustrating the difference in the
temporal dynamics of the effects of BDNF on RGCs and tectal neurons,
postsynaptic specialization number and density in BDNF-treated tectal neurons
continued to increase, requiring 48 hours to attain levels similar to those of
presynaptic specializations in RGC axons after 24 hours [276.5±40%
increase in PSD95-GFP puncta by 48 hours,
Fig. 4C; versus
285±43.5% increase in GFP-synaptobrevin puncta by 24 hours
(Alsina et al., 2001)].
Likewise, synapse number was similarly decreased both pre- and
postsynaptically when endogenous BDNF was neutralized in vivo; more rapidly in
RGC axons than in tectal neuron dendrites
(Hu et al., 2005) (present
results). Together, these data support the idea that experimentally
manipulating BDNF levels can reflect the endogenous actions of BDNF. Moreover,
the delayed onset of the response of tectal neurons to alterations in BDNF
levels (4 to 24 hours) when compared with the rapid response by RGC axons (2-4
hours) suggests that the effects of BDNF on tectal neuron synapse number are
either direct but delayed, or induced in response to the modulation by BDNF of
RGC presynaptic differentiation and axon arbor growth.
Neurotrophins, and in particular BDNF, are potent modulators of dendritic
development, differentially influencing multiple neuronal populations.
Neurotrophins act positively to promote dendritic branching
(Horch and Katz, 2002;
McAllister et al., 1995;
Wirth et al., 2003) but they
can also limit the size of the dendritic arbor of a neuron
(Lom et al., 2002;
Lom and Cohen-Cory, 1999;
McAllister et al., 1997).
Neurotrophins can act in anterograde, autocrine or paracrine manners to
modulate dendritic growth (Baquet et al.,
2004; Horch and Katz,
2002; Wirth et al.,
2003). Alterations in dendritic arbor morphology by neurotrophins
have therefore been taken to imply direct effects on neuronal connectivity
(Cline, 2001). Surprisingly,
our studies show that alterations that significantly impact synapse number may
not always influence dendritic branching, as arbor morphology was essentially
unchanged by manipulations in BDNF levels. The observation that dendritic
arbors continued to branch but failed to lengthen as a consequence of
decreased tectal BDNF levels suggest that BDNF-elicited expansion and enhanced
stability of presynaptic RGC arbors is responsible for the observed increases
in tectal neuron synaptic connectivity. Observations that tectal neurons
overexpressing a dominant negative form of the TrkB receptor have similar
dendritic arbor morphologies and dendritic branching and growth rates to
neurons in age-matched control tadpoles (S. Marshak and S.C.C., unpublished)
support the interpretation that changes in tectal neuron arbor growth in
response to anti-BDNF treatment are secondary to effects on presynaptic
RGCs.
Target-derived release of neurotrophins and activation of presynaptic
receptors has been considered a key mechanism of neurotrophin signaling
(Zweifel et al., 2005).
However, work over the last few years has shown that presynaptic neurotrophin
release and activation of postsynaptic Trk receptors is also a common
mechanism by which neurotrophins can regulate synaptic structure and function.
In postsynaptic neurons, TrkB signaling modulates neurotransmitter receptor
expression and function, induces depolarization and increases synaptic
transmission (Elmariah et al.,
2004; Ji et al.,
2005; Kafitz et al.,
1999; Kovalchuk et al.,
2002; Luikart et al.,
2005). Our observations that BDNF exerts coordinated, although
delayed, effects on synaptogenesis, while it differentially influences axon
and dendritic arbor structure, are consistent with BDNF acting presynaptically
at retinotectal synapses. BDNF-elicited presynaptic structural modifications
are also consistent with physiological evidence that BDNF potentiates
retinotectal synapses by enhancing neurotransmitter release rather than by
increasing postsynaptic response to RGC electrical stimulation
(Du and Poo, 2004). It remains
possible, however, that BDNF influences postsynaptic differentiation and/or
other aspects of tectal neuron development through a separate mechanism,
independent of its action on RGCs.
In summary, our work demonstrates that BDNF differentially influences presynaptic (axonal) and postsynaptic (dendritic) arbor structure in vivo. The observations that coordinated pre- and postsynaptic branching and synaptogenesis occurred under normal conditions or when retinotectal synaptic transmission was altered, but that alterations in BDNF levels elicited only delayed, corresponding changes in synapse innervation density suggest a novel mechanism by which BDNF influences the development of retinotectal connectivity in vivo (see Fig. 9). BDNF, through activation of TrkB receptors on RGCs, may induce the presynaptic RGC axons to spread their reach over a larger territory, thereby increasing the opportunity of the individual axon to come into contact with additional postsynaptic neurons. Simultaneously, BDNF may reinforce the emergent connectivity by stabilizing synapses and increasing synapse number with each and every postsynaptic partner. By modulating synapse number and strength in this manner, postsynaptic structure would remain unaffected, as the individual dendritic arbors would not need to grow to match the axon arbor. By contrast, a decline in TrkB signaling that results from the decreased availability of BDNF would negatively influence synaptic stability, reducing not only axon arbor extent but also synapse number and strength, ultimately influencing postsynaptic arbor shape. Thus, we propose that through its effects on presynaptic axons, BDNF can organize emergent retinotectal synaptic circuitry and modulate synaptic function and strength. By broadening the afferent synaptic input while simultaneously coordinating synapse formation and stabilization between pre- and postsynaptic neurons, BDNF can ultimately shape structural synaptic connectivity in the developing brain.
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ACKNOWLEDGMENTS |
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REFERENCES |
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