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The Journal of Neuroscience, November 15, 1999, 19(22):9996-10003
BDNF Modulates, But Does Not Mediate, Activity-Dependent
Branching and Remodeling of Optic Axon Arbors In Vivo
Susana
Cohen-Cory
Mental Retardation Research Center, Departments of Psychiatry and
Neurobiology, University of California, Los Angeles, Los Angeles,
California 90095
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ABSTRACT |
The proper development of axon terminal arbors and their
recognition of target neurons depend, in part, on neuronal activity. Neurotrophins are attractive candidate signals to participate in
activity-dependent development and refinement of neuronal connectivity. In the visual system, brain-derived neurotrophic factor (BDNF) has been
shown to modulate the elaboration and refinement of axonal arbors and
to participate in the establishment of topographically ordered visual
maps. By examining in vivo with time-lapse microscopy the effects of activity blockade and BDNF on optic axon arborization, I
show that the dynamic mechanisms by which neurotrophins and neuronal
activity regulate axon arborization differ. Acute retinal activity
blockade by intraocular injection of tetrodotoxin (TTX) rapidly and
significantly increased branch addition and elimination, thus
interfering with axon branch stabilization. The effects of activity
blockade on branch dynamics resulted in increased arbor complexity in
the long term and were prevented by altering endogenous BDNF levels at
the target. BDNF promoted axon arborization by increasing branch
addition and lengthening, without affecting branch elimination.
Activity blockade, however, did not prevent the growth-promoting
effects of BDNF, indicating that BDNF can affect axon arborization even
in the absence of activity. Together this evidence indicates that BDNF
acts as a modulator, but not as a direct mediator, of activity during
the morphological development of neurons. Consequently, neuronal
activity and BDNF use distinct but interactive mechanisms to control
the development of neuronal connectivity; BDNF modulates axon
arborization by promoting growth, neuronal activity participates in
axon branch stabilization, and together these two signals converge to
shape axon form.
Key words:
BDNF; neuronal activity; TTX; retinal ganglion cells; axon branching; Xenopus laevis
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INTRODUCTION |
In most vertebrate species the
establishment of precisely ordered neuronal connections involves a
gradual process in which afferent axonal arbors initially branch widely
over their target tissue and then gradually refine by withdrawing
branches from topographically inappropriate areas (Antonini and
Stryker, 1993a ; Roskies et al., 1995; Katz and Shatz, 1996). This
gradual remodeling of axonal arbors involves a dynamic process in which
axonal branches are constantly added and eliminated, until stable
synaptic connections are retained (Nakamura and O'Leary, 1989;
O'Rourke and Fraser, 1990). In the visual system, patterns of neuronal
activity modulate the development and refinement of visual connections.
For example, in the absence of action potential activity, axons
projecting to their target regions fail to segregate in retinotopic
specific lamina and elaborate arbors that are more complex than those
developing with normal visual input (Reh and Constantine-Paton, 1985;
Sretavan et al., 1988; Kobayashi et al., 1990; Antonini and
Stryker, 1993b). At least two mechanisms have been proposed to account
for the formation of exuberant arbors in the absence of visual
activity. One possibility is that the elimination of side branches or
the pruning of axon terminal arbors that occurs during normal map refinement is inhibited or delayed in the absence of neuronal activity
(Kobayashi et al., 1990; Shatz, 1996). Another possibility is that
neural activity blockade promotes sprouting and growth of axonal
terminal arbors by interfering with target recognition and synapse
stabilization mechanisms (Cline, 1991; Antonini and Stryker, 1993b;
Shatz, 1996). Although both mechanisms support a role for neuronal
activity as a stabilizing force during competition for synaptic inputs,
no direct evidence describes the mechanism by which action potential
activity influences axon arbor morphology.
Experimental evidence suggests that competition between axonal inputs
for common postsynaptic sites involves activity-dependent competition
for neurotrophic substances (Katz and Shatz, 1996; Snider and Lichtman,
1996). One mechanism that may explain such competition is that neuronal
activity directly regulates the production and release of a trophic
factor (Thoenen, 1995). The neurotrophins have been implicated as
candidates to mediate activity-dependent development and refinement of
synaptic connections. Increasing evidence suggests that neurotrophin
synthesis and release depend on neuronal activity (Herzog et al., 1994;
Lindholm et al., 1994; Thoenen, 1995; Blochl and Thoenen, 1996)
and that neurotrophins can modulate neuronal activity (Lohof et al.,
1993; Kang and Schuman, 1995; Thoenen, 1995; Prakash et al., 1996;
Snider and Lichtman, 1996). Moreover, the development of
topographically ordered visual maps in the cortex (Maffei et al., 1992;
Cabelli et al., 1995) and the dynamic development of optic axon
terminal arbors depend on endogenous neurotrophin function (Cohen-Cory
and Fraser, 1995).
One requirement for a molecule to be directly involved in
activity-dependent synaptic rearrangements is its ability to influence axon terminal arborization (Snider and Lichtman, 1996). Our previous work demonstrates that the neurotrophin brain-derived neurotrophic factor (BDNF) modulates optic axon remodeling in vivo and
therefore suggests that BDNF also acts as a signal to mediate
activity-dependent synaptic rearrangement. The rapid (<2 hr) and
significant effect that BDNF exerts on retinal ganglion cell (RGC) axon
arborization and complexity in vivo (Cohen-Cory and Fraser,
1995), as well as the patterns of BDNF expression in the developing
Xenopus visual system (Cohen-Cory and Fraser, 1994;
Cohen-Cory et al., 1996), is consistent with a direct effect on optic
axons and suggested the direct involvement of BDNF in the
activity-dependent establishment of connections between RGCs and their
targets. By using the Xenopus visual system as an in
vivo model, I have now examined whether in Xenopus, as
in mammals, retinal activity plays a significant role in the
elaboration of RGC axonal arbors. By imaging the morphology of
individual RGC axon arbors over time in tadpoles in which retinal action potential activity was blocked, I examined the mechanisms by
which neuronal activity influences axon form. Moreover, I studied whether the effects of blocking action potential activity on RGC axon
terminal arbor complexity are modifiable by BDNF. These studies indicate that acute neuronal activity blockade rapidly and
significantly increases axon terminal arbor remodeling by increasing
branch addition and elimination without significantly altering arbor length. This resulted in a significant increase in RGC axon arbor complexity in the long term. These results significantly differ from
those obtained by perturbing endogenous BDNF at the target optic tectum
and are further modified by tectal BDNF. Consequently, neuronal
activity and BDNF use distinct but interactive mechanisms to modulate
axon terminal arbor complexity in vivo; neuronal activity modulates axon branch stabilization, and BDNF modulates axon growth, and together these two signals converge to shape axon form.
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MATERIALS AND METHODS |
Xenopus laevis tadpoles were obtained from in
vitro fertilization of oocytes obtained from adult females primed
by injection of human chorionic gonadotropin. Tadpoles were reared in a
modified rearing solution [60 mM NaCl, 0.67 mM KCl, 0.34 mM
Ca(NO3)2, 0.83 mM MgSO4, 10 mM HEPES, pH 7.4, and 40 mg/l gentamycin] plus
0.001% phenylthiocarbamide to prevent melanocyte pigmentation. Staging was according to the method of Nieuwkoop and Faber (1956).
Animal procedures were approved by the University of California Los
Angeles Office for Protection of Research Subjects, Animal Research Committee.
Electrophysiological recordings. Laminar field potentials in
response to visual stimulation were electrophysiologically recorded from stage 43-45 anesthetized tadpoles to test the effectiveness of
the tetrodotoxin (TTX) treatment to eliminate all retinal action potential activity. Tadpoles were anesthetized by immersion in MS-222
in modified rearing solution and placed over an agar cushion. Evoked
field potentials were recorded from the tectum using glass micropipettes filled with Ringer's solution (resistance, 20-30 M ).
A monopolar recording paradigm was used, with the electrode placed in
the tectum and a reference electrode placed in the solution-filled recording dish. Both ON- and OFF-light responses after flash
stimulation were readily recorded (50-100 µV; 100 msec delay; data
not shown) in the developing optic tectum of intact tadpoles
(n = 8 out of 8 tadpoles, each recorded at multiple
sites within the tectal neuropil). No light-evoked responses, however,
could be recorded in tadpoles after intraocular injection of 4 nl of
100 µm TTX (the concentration used in these studies), even 6-8 hr
after injection (n = 5 tadpoles; data not shown). In
only one out of the five tadpoles could a weak light-evoked response
(magnitude 10 times lower than those recorded from control tadpoles) be
recorded in 1 out of every 10 trials 8 hr after TTX injection. When
lower TTX concentrations were injected, tadpoles recovered more readily from the TTX treatment (6-10 hr after injection; swimming and twitch
behavior) and showed light-evoked responses of the same magnitude as
those of controls.
In vivo microscopy imaging and drug treatment. Stage 43 tadpoles were anesthetized, and individual RGCs and their axons were labeled by microinjection of minute amounts of the lipophilic vital dye
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
[DiI; DiIC18(3); Molecular Probes, Eugene, OR] into the nasomedial
quadrant of the retina. Tadpoles were screened 18 hr later for the
presence of DiI-labeled retinal arbors in the tectum, and those
tadpoles with one to two clearly distinguishable DiI-labeled arbors,
containing at least one or two branches, were selected and used for
experimentation. Tadpoles were anesthetized and imaged with a Bio-Rad
(Hercules, CA) laser-scanning confocal microscope. Individual optical
sections were recorded at 2 µm intervals through the full extent of
the arbor within the optic tectum. After the first imaging session,
either 4 nl of a 100 µm TTX solution (Research Biochemicals, Natick,
MA ) or control vehicle solution (10% NiuTwitty) was microinjected
directly into the eye of the developing tadpoles with care to minimize
mechanical damage. Immediately after the TTX injection, 0.2-1.0 nl of
vehicle solution, 200 ng/µl recombinant human BDNF (a gift of Amgen,
Thousand Oaks, CA), specific function-blocking antibodies to BDNF (200 µg/ml; a gift of J. Carnahan) (Ghosh et al., 1994), or control IgG (200 µg/ml nonimmune rabbit IgG) in vehicle solution was pressure injected with a fine-pulled glass micropipette directly into the ventricle and subpial space overlying the caudalmost tectum (Cohen-Cory and Fraser, 1995). Tadpoles were kept in fresh rearing solution, in the
dark, after treatment and between observations. The rates and dynamics
of RGC axonal branching were followed by confocal microscopy
immediately after injection, and every 2 hr after the initial
observation, for a total of 6 hr and again at 24 hr.
Data analysis. The effects of each treatment on arbor
dynamics were examined by analyzing individual confocal optical
sections through the entire extent of the arbor at each observation
time point with the aid of the Metamorph software (Universal Imaging Corporation, West Chester, PA). Several morphological parameters were
measured. Extensions from the main axon were classified as branches
(longer than 5 µm) or spikes (shorter than 5 µm). The number of
individual branches or spikes, gained or lost, and the number of
preexisting branches that change their length by at least 20 µm from
one observation interval to the next were scored regardless of their
length change. In addition, the number of total branches per
observation time point was determined. For the analysis of arbor
complexity at 24 hr, total axon length was determined by measuring the
difference in total length of the main stem and proximal and distal
branches at 24 versus 0 hr, and the difference in total branch number
was determined by counting the total number of branches (proximal and
distal branches) at 24 versus 0 hr. Axon arbor reconstructions were
obtained by digitally tracing individual arbors from individual optical
planes with the Metamorph software. Repeated measures ANOVA (SYSTAT;
SPSS) and post hoc Tukey tests were used for the
statistical analysis of data. Significance was p  0.05.
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RESULTS |
The effect of silencing retinal activity on the dynamics of
RGC axon arborization was studied in live stage 45 Xenopus
laevis tadpoles in which action potential activity was inhibited
by intraocular injection of the sodium channel blocker TTX. Minute
amounts of the lipophilic dye DiI were injected into tadpole retinae to
visualize individual RGC axon arbors in the optic tectum. The
morphology of individual DiI-labeled RGC axon arbors was followed by
time-lapse in vivo confocal microscopy. Immediately after
the initial microscopic examination of axon arbor morphology, 4 nl of
100 µm TTX was pressure injected into the eye of each
anesthetized tadpole. This amount of TTX effectively blocked all
retinal action potential activity (see Materials and Methods). Analysis
of individual axon arbor morphology at 2 hr intervals for at least 6 hr
revealed active remodeling of axon arbors both in TTX- and
control-treated tadpoles (Figs. 1,
2). Axon arbor dynamics was
evaluated by measuring the number of branches that are added or
eliminated in each 2 hr time point and the number of preexisting
branches that lengthened or shortened. In addition, the number of
spikes (dynamic processes < 5 µm) added or eliminated in every
2 hr time period was evaluated. Blocking retinal action potential
activity significantly altered the number of branches added and
eliminated at every observation time point. On average, RGC axons in
TTX-treated tadpoles added 2.3 ± 0.23 new branches every 2 hr, whereas
RGC axons in control-treated tadpoles added 1.3 ± 0.14 new branches
(Fig. 2A). These effects were observed at the first 2 hr time point and persisted throughout the entire imaging period (data
not shown). Similarly, TTX significantly increased the number of
branches eliminated per RGC axon, compared with RGCs in control-treated
tadpoles (TTX = 1.22 ± 0.14 vs control = 0.66 ± 0.08 branches eliminated every 2 hr; Fig. 2B). TTX
treatment, however, did not affect the lengthening or shortening of
preexisting branches (Fig. 2A,B) or the addition or
elimination of spikes (Fig. 2A,B). Thus, the
increases in branch addition and elimination observed after intraocular
TTX injection indicate that RGC axonal arbors are more dynamic in the
absence of action potential activity. Moreover, these results indicate
that normal patterns of neuronal activity are required for the
stabilization of new branches but do not influence the rate at which
existing branches grow.
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Figure 1.
Action potential activity blockade
increases the remodeling of RGC axonal arbors in vivo.
The effects of retinal activity blockade on the dynamics of RGC axon
arborization are illustrated by reconstructions of sample arbors
followed over time after intraocular injection of TTX
(top right) or control
(top left) solution. For comparison,
tracings of a sample arbor in a tadpole with intraocular
TTX and tectal BDNF injections (bottom) are also
presented (see text and also Fig. 3). Posterior is up,
and medial is to the left.
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Figure 2.
Action potential activity blockade increases
addition and elimination of RGC axonal branches. To quantify the
effects of activity blockade on RGC axon arbor dynamics, three
morphological parameters were evaluated: addition
(A) and elimination (B) of
individual branches (>5 µm), changes in the length of preexisting
branches [lengthening (A); shortening
(B)], and addition (A) and
elimination (B) of individual spikes (<5 µm)
are expressed as the average number of changes per 2 hr time interval
in arbors followed for 6 hr (n = 20 arbors per
condition). For each parameter and condition the mean ± SEM is shown.
Statistical analysis was by repeated measures ANOVA. * indicates
significantly different from control, p 0.02.
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We demonstrated previously that BDNF modulates axon arborization by
specifically promoting growth (Cohen-Cory and Fraser, 1995). That is,
increasing tectal BDNF levels significantly increased the addition of
new branches and spikes as well as the lengthening of preexisting
branches without affecting branch and spike elimination or branch
shortening (see also Fig. 3). Thus,
although both intraocular TTX injections and perturbations in BDNF
tectal levels alter axon branch dynamics, not all parameters of axon
arbor dynamics are affected in the same manner by these two treatments.
Silencing activity with TTX significantly increased branch addition and elimination without affecting branch length and spike number, whereas
increasing BDNF tectal levels increased all parameters that reflect
axon growth (see Cohen-Cory and Fraser, 1995) (see also Fig. 3). This
suggests that BDNF modulation of RGC axon arborization might be
independent of activity. To address this issue directly, I followed RGC
axon arborization dynamics in tadpoles that were treated with both TTX
to silence activity and BDNF to increase tectal BDNF levels or with
function-blocking BDNF antibodies to neutralize endogenous tectal BDNF.
As internal controls for these experiments, individual DiI-labeled RGC
axons were followed in tadpoles in which tectal BDNF levels were
altered by injection of BDNF or anti-BDNF but the TTX treatment
was omitted (Fig. 3).
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Figure 3.
Action potential activity and BDNF differentially
modulate the dynamics of RGC axon arborization in vivo.
Comparing TTX effects on axon branch dynamics with those elicited by
alterations in tectal BDNF levels illustrates the differential response
of RGC axons to each treatment. In addition, the results of combining
intraocular TTX injections with tectal injections of BDNF or anti-BDNF
(AntiB) show that TTX influence on arbor dynamics is
modified by BDNF. The effects of each treatment on axon branch dynamics
are illustrated by quantifying the number of branches added
(A), or eliminated (C), and
the number of preexisting branches that lengthened
(B), or shortened (D), for
each treatment. These values are expressed as the average number of
changes per 2 hr time interval in arbors followed for 6 hr
(n = 20 arbors per condition for control, TTX, and
TTX + BDNF; n = 13 for BDNF; and
n = 17 for anti-BDNF and TTX + anti-BDNF). For each
parameter and condition the mean ± SEM is shown. Repeated measures
ANOVA was used for the statistical analysis of data. ** indicates
significantly different from all other groups except BDNF versus
TTX, p 0.01; * indicates significantly different
from control, p 0.05; and ~ indicates
significantly different from control by unpaired t test,
p 0.02.
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Combining intraocular TTX injections with perturbations in BDNF tectal
levels resulted in RGC axonal arbors with branch dynamics that
significantly differed from those in axons from tadpoles treated with
TTX, BDNF, or anti-BDNF alone. In agreement with previous results,
increasing the endogenous levels of BDNF by tectal injection of BDNF
alone significantly increased the number of branches added, whereas
anti-BDNF significantly decreased branch addition (control = 1.30 ± 0.15; BDNF = 2.22 ± 0.24; anti-BDNF = 0.75 ± 0.15; Fig.
3A) (see also Cohen-Cory and Fraser, 1995). Surprisingly,
the number of branches added per RGC axon after TTX + BDNF treatment
did not differ from that of RGC axons in control-treated tadpoles
(control = 1.30 ± 0.15; TTX + BDNF = 1.53 ± 0.17; Fig.
3A). Similarly, the combined treatment of TTX and
function-blocking antibodies to BDNF did not alter the number of
branches added per RGC (control = 1.30 ± 0.15; TTX + anti-BDNF = 1.22 ± 0.18; Fig. 3A). Thus perturbations
in either tectal BDNF levels or activity blockade with TTX
independently caused significant changes in the number of new branches
added, but when these two treatments were combined no apparent effect
on branch addition was observed. These effects were consistent over the
6 hr time course (data not shown; for qualitative measure see Fig. 1).
Perturbations in tectal BDNF levels performed simultaneously with TTX
treatment affected other axon branch parameters in a different manner.
In those parameters in which TTX had no independent effect but BDNF significantly affected axon branch dynamics, the effects of the combined treatment with TTX and BDNF resembled those of BDNF alone. For
example, the number of branches that lengthened in each 2 hr interval
was significantly increased by the combined treatment with TTX and BDNF
to values similar to those resulting from treatment with BDNF alone
(control = 0.93 ± 0.11; TTX = 1.3 ± 0.14; BDNF = 1.74 ± 0.22; TTX + BDNF = 1.80 ± 0.17; see Fig. 3B).
Similarly, combining TTX and BDNF treatments significantly increased
the number of spikes added to values similar to those observed in RGC
axons of tadpoles treated with BDNF alone (control = 1.25 ± 0.14;
TTX = 1.5 ± 0.2; BDNF = 2.44 ± 0.33; TTX + BDNF = 2.11 x 0.24; graph not shown). Neutralizing endogenous tectal BDNF with
function-blocking BDNF antibodies elicited a small but significant increase in the number of branches shortened every 2 hr (control = 0.36 ± 0.09; anti-BDNF = 0.81 ± 0.13), an effect that was not detected as significant in our previous study (Cohen-Cory and Fraser,
1995). The differential effects that BDNF and anti-BDNF elicit on
branch lengthening and branch shortening (see Fig. 3B,D) may
be a consequence of differential threshold responses to BDNF signaling
by previously established branches (see also Song et al., 1997).
Similar to the effect of BDNF on branch lengthening, the effect of
anti-BDNF on branch shortening was not influenced by intraocular TTX
injection (control = 0.36 ± 0.09; TTX = 0.33 ± 0.1;
anti-BDNF = 0.81 ± 0.13; TTX + anti-BDNF = 0.75 ± 0.12; see
Fig. 3D). Consequently, blocking action potential activity with TTX did not alter the effects of exogenous and endogenous BDNF on
RGC axon branch elongation or spike formation. In contrast, the effects
of TTX could be blocked by perturbations in tectal BDNF levels. TTX
significantly increased branch elimination, whereas the number of
branches eliminated in BDNF- or anti-BDNF-treated tadpoles did not
differ from that of controls (control = 0.66 ± 0.08; TTX = 1.23 ± 0.14; BDNF = 0.85 ± 0.14; anti-BDNF = 0.75 ± 0.16;
Fig. 3C). In tadpoles treated with both TTX and exogenous BDNF, branch elimination did not differ from that of controls (TTX + BDNF = 0.75 ± 0.12; see above and Fig. 3C). However,
in TTX + anti-BDNF-treated tadpoles axon branch elimination
significantly increased versus control, to a value similar to that
observed after treatment with TTX alone (TTX + anti-BDNF = 1.4 ± 0.18; see above and Fig. 3C). Together, these results
indicate that exogenous BDNF can prevent most of the effects that
result from blocking action potential activity with TTX and suggest
that endogenous BDNF and neuronal activity act via distinct mechanisms
to modulate RGC axon terminal arborization in vivo.
Comparing RGC axon morphology before and 24 hr after treatment provided
a cumulative measure of the effects of neural activity blockade and
BDNF on axon terminal arbor complexity. Tracings of sample arbors
illustrate the effects of each treatment, as well as the range of
morphologies and responses to individual treatments (Fig.
4). In addition, distribution plots of
the difference in branch number 24 hr after treatment show the means
and variability of the responses of individual arbors to all treatments
(Fig. 5). The difference in total branch
number and in total axon arbor length for each individual arbor between
0 and 24 hr provides a more quantitative measure of the effects of
individual treatments in the complexity of RGC axon terminal arbors
(Fig. 6). RGC axons from tadpoles in
which neural activity was blocked by intraocular TTX injection were
significantly more complex at 24 hr than RGC axons developing in normal
tadpoles, as shown by the significantly increased change in branch
number in the TTX-treated tadpoles compared with controls (Fig. 6; see
also Fig. 4). These effects of TTX on axon arbor complexity differ from
the very significant effects of BDNF. RGC axons in tadpoles exposed to
increased BDNF tectal levels had significantly more branches and were
significantly longer than RGC axons in control-treated tadpoles.
Although BDNF and TTX independently increased axon arbor complexity,
the overall change in complexity of RGC axon terminal arbors in TTX + BDNF-treated tadpoles was similar to that of RGC axons in control
tadpoles (Fig. 6; see also Fig. 4). In contrast, RGC axons in tadpoles treated with antibodies to BDNF alone or with antibodies to BDNF in
combination with TTX were simpler than controls 24 hr after treatment
as exemplified by the significantly lower increase in branch number
versus controls (Fig. 6; see also Fig. 4). Therefore, under all
conditions, the complexity of RGC axons at 6 hr (data not shown) and at
24 hr (Fig. 6) is in accordance with what would be expected on the
basis of the summation of all effects that each individual treatment
elicited on all parameters of axon branch dynamics in axons followed
every 2 hr.
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Figure 4.
Xenopus RGC axon
complexity is influenced by action potential activity and is modified
by BDNF. Tracings of representative axonal arbors before
and 24 hr after control, TTX, BDNF, TTX + BDNF, anti-BDNF, or TTX + anti-BDNF treatments illustrate the individual variability in arbor
morphologies for each condition tested. During active arborization, RGC
axons significantly increase their complexity as illustrated by the
difference in their morphology 24 hr after initial observation (see
controls). TTX-induced activity blockade and perturbations in tectal
BDNF levels independently influence axon arbor complexity, but when
combined, arbor complexities resembled that of controls.
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Figure 5.
Variability in individual RGC arbor responses to
activity blockade and tectal BDNF levels. The variability in responses
in arbor morphologies for each treatment is illustrated for individual
axons by the absolute difference in branch number between 0 and 24 hr
of treatment. For ease of comparison, the x-axis was
shifted to coincide with the mean change in branch number for the
control-treated axons (2.56 ± 0.41 branches/24 hr;
arrow). Within the conditions, each
symbol represents the change in branch number for an
individual axon, and the horizontal bar
indicates the mean change in branch number for that group.
AntiB, Anti-BDNF.
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Figure 6.
Action potential activity and tectal BDNF levels
significantly influence the morphology of RGC axonal arbors at 24 hr.
The effects of TTX and BDNF on arbor morphology over 24 hr were
evaluated quantitatively by measuring total arbor length and branch
number in tadpoles treated with TTX alone, BDNF alone, anti-BDNF alone
(AntiB), TTX + BDNF combined, or TTX + anti-BDNF
combined. Values are presented as the change in initial value 24 hr
after treatment. For each parameter and condition the mean ± SEM is
shown. Statistical analysis was by one-way ANOVA using multiple
comparison post hoc Tukey tests. **
indicates significantly different from control, p 0.001; * indicates significantly different from control,
p 0.05.
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DISCUSSION |
An important and long-standing question in development
neurobiology is the extent to which neuronal activity participates in
the development of neuronal connectivity (Constantine-Paton et al.,
1990; Katz and Shatz, 1996; Snider and Lichtman, 1996). Neuronal
activity may act directly to modulate neuronal connectivity or
indirectly by enabling other developmental cues, such as neurotrophic factors, to guide the formation of appropriate connections (Katz and
Shatz, 1996; Crair, 1999). By examining in vivo the events that lead to the formation of RGC axon terminal arbors, I now provide
evidence that neuronal activity and neurotrophins act via distinct but
interactive mechanisms to modulate axon arborization. A detailed
analysis of morphological parameters from time-lapse-imaged RGC axonal
arbors in live Xenopus tadpoles provides direct evidence that in the absence of neuronal activity, RGC axon terminal arbors significantly increase their complexity by actively extending and
retracting axonal branches. Thus, in young developing tadpoles, as in
mammals (Sretavan et al., 1988), neuronal activity plays an
important role in the elaboration of RGC axonal arbors. Most studies
investigating the role of neuronal activity during axon arborization
have used pharmacological agents such as TTX to block action potential
activity chronically in the developing brain but only later examined
the resulting axon arbor morphologies at a single time point (Meyer,
1983; Reh and Constantine-Paton, 1985; Sretavan et al., 1988;
Kobayashi et al., 1990; Antonini and Stryker, 1993b). The mistargeting
errors and increased axonal branching observed after chronic activity
blockade suggested multiple mechanisms that may account for the effects
of interfering with activity signaling. Potential mechanisms include
interference with target recognition (Catalano and Shatz, 1998;
Dantzker and Callaway, 1998), interference with arbor pruning
(Kobayashi et al., 1990; Shatz, 1996), and/or failure to stabilize
synaptic connections (Constantine-Paton et al., 1990; Cline,
1991; Antonini and Stryker, 1993b; Shatz, 1996). By acutely blocking
action potentials via intraocular TTX injection in developing tadpoles
and examining axon branch dynamics over time, I directly demonstrate
that silencing retinal activity very rapidly affects axon branch
stabilization. Within 2 hr of TTX treatment, a significant increase in
axon branch remodeling was observed. More branches were added and
eliminated in the absence of activity, but the rate at which individual
preexisting branches grew remained the same. By 24 hr, this increased
remodeling resulted in axon terminal arbors with increased complexity,
because more branches were added than eliminated. Thus, the axon arbor morphologies resulting from activity blockade were not caused by
interference with pruning mechanisms but were rather a consequence of
the increased remodeling that may result from interference with
recognition mechanisms and/or synapse stabilization. A number of
studies indicate that postsynaptic activity, mediated by the NMDA type
of glutamate receptor (NMDA-R), is involved in the development and
topographic refinement of RGC axon arbors (Cline and Constantine-Paton, 1990; Constantine-Paton et al., 1990; O'Rourke et al., 1994; Shatz, 1996; Rajan et al., 1999). Studies that have analyzed the effects of
NMDA-R activity on axon arbor dynamics show that the dynamic remodeling
of predominantly short branches (<5 µm; termed spikes in the current
studies) is increased by NMDA-R activity blockade (Rajan et al., 1999).
However, in contrast to the effects of TTX, alterations in NMDA-R
signaling has no net effect on overall axon branch number or morphology
at 24 hr (O'Rourke et al., 1994; Rajan et al., 1999). Thus, these and
the present results support a role for neuronal activity in the
stabilization of axonal branches and further suggest that both pre- and
postsynaptic mechanisms are involved in axon branch stabilization.
The neurotrophins are attractive candidate signals for mediating
activity-dependent synaptic remodeling during development. Neurotrophins are expressed and released in an activity-dependent manner (Herzog et al., 1994; Lindholm et al., 1994; Thoenen, 1995; Blochl and Thoenen, 1996), can modulate synaptic activity both in vitro (Lohof et al., 1993; Kang and Schuman, 1995;
Schuman, 1999) and in vivo (Prakash et al., 1996), can alter
the activity-dependent segregation of inputs during development (Maffei
et al., 1992; Cabelli et al., 1995), and are potent modulators of both
axon (Cohen-Cory and Fraser, 1995) and dendritic (Cohen-Cory et al., 1991; McAllister et al., 1995) morphology. Collectively, this evidence
has raised the intriguing possibility that neurotrophins can act as
activity-dependent retrograde signals that modulate neuronal
connectivity (Katz and Shatz, 1996; Snider and Lichtman, 1996;
McAllister et al., 1999). The present study has specifically addressed
this issue by studying the combined effects of both blocking retinal
action potential activity and altering endogenous tectal BDNF levels on
the dynamics of RGC axon arborization in vivo. The results
indicate that BDNF and neuronal activity use distinct mechanisms to
modulate axon elaboration and refinement. The effects of blocking
retinal activity with TTX on axon arbor dynamics significantly differed
from the effects of altering tectal BDNF levels. TTX increased both the
addition and elimination of axonal branches, indicating reduced branch
stability, whereas alterations in BDNF tectal levels specifically
affected branch addition and elongation, parameters that reflect
growth. By following the dynamics of axon branch elaboration in detail,
it was possible to dissect out the types of interactions between
activity and BDNF in this developmental process. That different
mechanisms are in effect is supported by the evidence that the combined
treatment of tadpoles with TTX + BDNF or TTX + anti-BDNF affected axon
branch dynamics in a manner that differed from the effects of treatment with either agent alone. Tectal injection of BDNF counteracted the
effects of TTX on axon branch addition and prevented its effects on
branch elimination. Blockade of action potential activity with TTX did
not alter the effects of altered BDNF tectal levels, as is the case for
the lengthening of previously established branches and the addition of
new spikes. Therefore, the observation that activity blockade does not
alter the effects of increased tectal BDNF or of reduced BDNF signaling
indicates that the effects of endogenous BDNF are not directly
dependent on action potential activity. Moreover, the observation that
BDNF can modify the effects of activity blockade by reducing
TTX-induced branch elimination indicates that BDNF can itself modulate
the neuron's response to altered activity levels. Thus, although
activity and BDNF interact to modulate axon arborization and
remodeling, this interaction cannot simply be explained by a direct
activity-dependent control of neurotrophic function.
One intriguing observation obtained from these studies is that either
independent TTX or BDNF treatments increased axon branch addition, but
when applied simultaneously RGC axons added new branches to a value
that closely resembled that of controls. This significant result can be
explained if neuronal activity blockade and BDNF trigger different and
potentially opposing signaling mechanisms to each increase branch
addition, but the significance of the branches extended under each
circumstance differs. For example, in the absence of activity the
probability that rudimentary branches are extended may increase as a
response to failure to stabilize synaptic contacts (for review, see
Katz and Shatz, 1996). BDNF, on the other hand, may specifically
reinforce the initiation and elaboration of only those branches that
are capable of establishing successful synaptic connections (Snider and
Lichtman, 1996). Future studies that examine the relationship between
axon branch elaboration and synapse formation and stabilization will
help resolve this issue.
Observations of the morphological consequence of altering neurotrophin
and activity levels on central neurons developing in culture have led
to the suggestion that neurons must be active to respond to
neurotrophins (Cohen-Cory et al., 1991; McAllister et al., 1996). For
example, inhibiting action potential activity blocks the dramatic
increase in dendritic arborization of cortical pyramidal neurons in
culture elicited by BDNF, although activity inhibition also enhanced
dendritic growth (McAllister et al., 1996). These observations of the
combined effects of activity blockade and neurotrophins on dendritic
morphology resemble the observations presented in this study on the
final complexity of axonal arbors. However, the present study provides
further dynamic evidence that BDNF and neuronal activity interact in a
more complex way to modulate neuronal form. The combined in
vivo time-lapse analysis of axon arbor dynamics and complexity
indicates that neurotrophins can exert their effects even in the
absence of activity but that the specificity of the response to
neurotrophins and activity depends on the convergence of these two
developmental signals. That BDNF can modify the effects of activity
inhibition on dynamic morphological parameters supports a role for this
neurotrophin as a potent mediator of morphological plasticity. One
mechanism by which neurotrophins may modulate morphological plasticity
is by modulating synaptic efficacy (Lohof et al., 1993; Kang and Schuman, 1995; Wang et al., 1998; Schuman, 1999), as has been demonstrated recently by BDNF's ability to modulate synaptic strength even in the absence of action potential activity (Rutherford et al.,
1998). Thus, BDNF modulation of activity-dependent axonal branching may
involve direct activation of signaling pathways common to the control
of synaptic efficacy (Boulanger and Poo, 1999), growth cone behavior
(Song et al., 1997), and collateral sprouting (Gallo and
Letourneau, 1999).
In conclusion, the present observations demonstrate that BDNF
modulates, but does not directly mediate, activity-dependent branching
and remodeling of RGC axon arbors in vivo. Although the
results of this work favor the hypothesis that neuronal activity and
neurotrophins use distinct but interactive mechanisms to modulate morphological plasticity, it also is plausible that neurotrophin function depends on neuronal activity at other regulatory levels, such
as the expression and release of neurotrophic factors. Indeed, BDNF
expression in the Xenopus optic tectum is developmentally regulated (Cohen-Cory and Fraser, 1994), being maximal during the
period of active RGC axon arborization and synapse formation [determined both electrophysiologically (Wu et al., 1996; Zhang et
al., 1998) and by the localization of synaptic vesicle proteins to RGC
axon terminals (Lom et al., 1998)]. Thus, by interacting at multiple
levels, BDNF and neuronal activity may exert fine regulatory control of
neuronal development and function, from the control of neuronal
morphology to the formation and stabilization of individual synaptic contacts.
| |
FOOTNOTES |
Received July 12, 1999; revised Aug. 20, 1999; accepted Aug. 27, 1999.
This work was supported by National Institutes of Health Grant EY11912
and by awards from the Alfred P. Sloan, Stein/Oppenheimer, University
of California Los Angeles Frontiers of Science, and Beckman
foundations. I thank J. Cueva for assistance with initial experiments,
C. Colwell for help with electrophysiological recordings, T. Vu for
technical assistance, and R. Frostig, C. Colwell, B. Lom, and J. Cogen
for discussions and comments on this manuscript.
Correspondence should be addressed to Dr. Susana Cohen-Cory, Mental
Retardation Research Center, 760 Westwood Plaza, NPI 78-148, University of California, Los Angeles, Los Angeles, CA 90095. E-mail:
scohenco{at}ucla.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
