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The Journal of Neuroscience, November 15, 1999, 19(22):9928-9938
Brain-Derived Neurotrophic Factor Differentially Regulates
Retinal Ganglion Cell Dendritic and Axonal Arborization In
Vivo
Barbara
Lom and
Susana
Cohen-Cory
Mental Retardation Research Center, Departments of Psychiatry and
Neurobiology, University of California, Los Angeles, Los Angeles,
California 90025
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ABSTRACT |
Expression of the neurotrophin brain-derived neurotrophic factor
(BDNF) and its receptor trkB in the ganglion cell layer of the
Xenopus retina during retinal ganglion cell (RGC)
dendritic arborization indicates that BDNF is spatially and temporally
available to influence RGC morphological differentiation (Cohen-Cory
and Fraser, 1994 ; Cohen-Cory et al., 1996). BDNF promotes RGC axon arborization in vivo by acting as a target-derived
trophic factor (Cohen-Cory and Fraser, 1995). To determine whether BDNF
also acts locally to regulate RGC dendritic development in
vivo, we altered retinal neurotrophin levels at the onset of
dendritic arborization and assessed the resulting arbor morphologies of RGCs retrogradely labeled with fluorescent dextrans. Injecting neurotrophins or BDNF function-blocking antibodies coupled to microspheres provided local alterations of retinal neurotrophin levels.
BDNF significantly decreased RGC dendritic arbor complexity, whereas
neutralizing endogenous BDNF levels with function-blocking antibodies
significantly increased dendritic arbor complexity. RGCs exposed to
other neurotrophins, as well as RGCs in retinae treated with BDNF but
in areas not directly exposed to the neurotrophin, developed dendritic
arbors that were indistinguishable from controls, indicating that
exogenous BDNF acts specifically and locally. In the tectum, where RGC
axons arborize, BDNF had opposite effects. BDNF significantly increased
RGC axon arbor complexity and anti-BDNF reduced RGC arborization. Thus,
BDNF reduces RGC dendritic arborization within the retina and increases
axon arborization in the tectum. These results indicate that BDNF can
differentially modulate axonal and dendritic arborization within a
single neuronal population in opposing manners and raise the
possibility that differential modulation by a neurotrophic factor
finely tunes the morphological differentiation program of a neuron.
Key words:
brain-derived neurotrophic factor; neurotrophins; retinal
ganglion cell; visual system; dendrite; Xenopus laevis; axon; retina
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INTRODUCTION |
For the nervous system to function
properly, neurons must form intricate networks of precise synaptic
connections that permit the transfer of information between individual
neurons. Numerous developmental events, including neuronal survival,
migration, arborization, and synaptic modification, must be coordinated
spatially and temporally to assure that appropriate synapses are
formed and maintained (for review, see Goodman and Shatz, 1993). The vertebrate visual system has long served as an accessible experimental model for investigating the cues and developmental events that contribute to neuronal differentiation. As the visual system develops, the morphologies of individual neurons change dramatically. The process
of dendritic and axonal arborization is a dynamic combination of branch
addition and branch elimination as well as branch lengthening and
shortening (O'Rourke et al., 1994; Cohen-Cory and Fraser, 1995; Witte
et al., 1996). It is thought that the dynamic changes in arbor
morphology reflect the discrete formation, stabilization, and
elimination of the synaptic connections of a neuron. Thus, understanding how developmental cues sculpt neuronal architecture can
provide valuable insight into the mechanisms necessary to establishing
a functional nervous system.
One family of molecular cues, the neurotrophins, has been shown to play
significant roles in diverse aspects of nervous system development from
the control of cell survival to the modulation of synaptic efficacy
(Barde, 1994; Berninger and Poo, 1996; Snider and Lichtman, 1996;
McAllister et al., 1999). The neurotrophin family includes the ligands
brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF),
neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their specific
tyrosine kinase receptors (Barbacid, 1995; Bothwell, 1995). In the
visual system the neurotrophins are potent modulators of neuronal
architecture and survival. Neurotrophins can rescue retinal neurons in
a target-dependent manner (Rodriguez-Tebar et al., 1989) (for
review, see von Bartheld, 1998) and can enhance neurite outgrowth in
vitro (for review, see von Bartheld, 1998; McAllister et al.,
1999). In vivo, neurotrophins have been shown to play a role
in shaping neuronal morphology by acting as target-derived trophic
factors (Cabelli et al., 1995; Cohen-Cory and Fraser, 1995; Riddle et
al., 1995). One direct example of this is the role of BDNF during
retinal ganglion cell (RGC) axon arborization (Cohen-Cory and Fraser,
1995). Increasing endogenous tectal BDNF can promote RGC axon
arborization in live Xenopus tadpoles, whereas neutralizing
endogenous BDNF decreases axon arborization (Cohen-Cory and Fraser,
1995). The peak in BDNF expression in the Xenopus visual
system coincides with the period of active RGC axon arborization at its
target (Cohen-Cory and Fraser, 1994), providing further support for
BDNF as a target-derived modulator of axon morphology.
In addition to its target-derived roles, BDNF may act as a local
modulator of neuronal morphology. In the visual system of most
vertebrate species BDNF is expressed at the target optic tectum as well
as locally within the retina (Cohen-Cory and Fraser, 1994; Perez and
Caminos, 1995; Cohen-Cory et al., 1996; Hallbook et al., 1996) (for
review in greater detail, see von Bartheld, 1998), and RGCs express
trkB, the specific receptor for BDNF (Jelsma et al., 1993; Cohen-Cory
and Fraser, 1994; Escandon et al., 1994; Garner et al., 1996). In the
Xenopus retina, BDNF and trkB are expressed by RGCs during
the period of active RGC dendritic elaboration (Cohen-Cory et al.,
1996), indicating that BDNF is spatially and temporally available to
influence an RGC's own morphological differentiation program. Now we
have investigated the role of retinal-derived BDNF during RGC
morphological development in vivo by experimentally altering
retinal neurotrophin levels during dendritic arborization in live
Xenopus laevis tadpoles and then assessing the resulting RGC
arbor morphologies. Applications of exogenous BDNF decreased RGC
dendritic arbor complexity, whereas neutralizing endogenous BDNF with
function-blocking antibodies increased dendritic arbor complexity.
These results contrast with the ability of BDNF to promote RGC axon
arborization at the tectum, indicating that within a single neuronal
population BDNF can modulate axonal versus dendritic arborization in
distinct and opposing manners. Thus, by acting locally or at the
target, a single neurotrophin can differentially modulate the
morphological differentiation of a neuron.
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MATERIALS AND METHODS |
Xenopus laevis tadpoles. Xenopus
laevis embryos were obtained by in vitro fertilization
of eggs obtained from females primed with human chorionic gonadotropin
(Sigma, St. Louis, MO). Embryos were reared in a 20% modified
Steinberg's solution [containing (in mM) 60 NaCl, 0.67 KCl, 0.34 Ca(NO3)2, 0.83 MgSO4, and 10 HEPES, pH 7.4 (Keller, 1991)]
supplemented with 40 mg/l gentamycin (Sigma). Embryos were staged
developmentally according to Nieuwkoop and Faber (1956). To reduce
pigmentation, we added 0.001% phenylthiocarbamide (Sigma) to
the rearing solution ~1 d after fertilization. During experimental
injections and observations the tadpoles were anesthetized by immersion
in 0.05% tricane methanesulfonate (Finquel, Argent Laboratories,
Redmond, WA) in rearing solution.
Neurotrophin-coated fluorescent microspheres. Green
fluorescent microspheres of 50-200 nm diameter (Lumafluor, New York,
NY) were prepared and coated with neurotrophins in a manner similar to
that described by Riddle et al. (1997). Microspheres were incubated overnight at 4°C in a 1:5 mix of microspheres and 100 ng/µl
neurotrophin solution, a concentration that has been used effectively
both in vitro and in vivo (Riddle et al., 1997).
After coating, the microspheres were centrifuged at 14,000 × g for 30 min and resuspended in sterile water for a final
10% microsphere concentration. BDNF was a gift from Amgen (Thousand
Oaks, CA), and NT-3, NT-4, and NGF were gifts from Genentech (South San
Francisco, CA). Neurotrophin-coated microspheres exert neurotrophic
activity comparable to that of free neurotrophins that can be retained
for at least 4 d (Riddle et al., 1997). Anti-BDNF (R & D Systems,
Minneapolis, MN) was mixed with deionized microspheres to a
concentration of 250-375 µg/ml immediately before injection. We
observed that 100 µg/ml of this antibody completely inhibited
survival in dorsal root ganglion neurons normally elicited by 2.5 ng/ml
rhBDNF in vitro and showed <2% cross-reactivity with NGF,
NT-3, and NT-4, as demonstrated by ELISA and Western assays (R & D
Systems). Control microspheres were incubated with no protein, as
described above, with 100 ng/µl cytochrome c (Sigma), or
with 100 µg/ml of nonimmune IgG. Neurotrophin-, antibody-, or
control-coated microspheres were pressure-injected into the right eyes
of anesthetized stage 38 tadpoles. Then the tadpoles were reared in the
dark until they reached stage 42-43 (~1-1.5 d later).
RGC labeling. Rhodamine-conjugated dextran or
biotin-conjugated dextran (3 kDa molecular weight; Molecular Probes,
Eugene, OR) was used to retrogradely label RGCs. Dextrans were diluted in sterile water to 100 mg/ml, purified by centrifugation in a 3 kDa cutoff microconcentrator (Amicon, Beverly, MA), and
pressure-injected into the left tecta of anesthetized stage 42-43
tadpoles. Then the tadpoles were allowed to develop to stage 45 (~1 d
later) in the dark. The tadpoles were fixed in 4% paraformaldehyde
with 4% sucrose overnight at 4°C and rinsed in PBS. The
retinae of tadpoles injected with rhodamine-dextran were prepared as
whole mounts. The pigment epithelium and lens were removed carefully from isolated right eyes; then the retinae were dehydrated by immersion
in a graded series of ethanol. Small cuts in the peripheral retina were
made so that the tissue could be mounted flat on a slide with the RGC
layer up and coverslipped with Krystalon mounting media (EM Industries,
Gibbstown, NJ). Cryostat sections (20 µm) of tadpoles injected with
biotinylated dextran were stained by the ABC method (Vector
Laboratories, Burlingame, CA). Tectal injections of rhodamine- or
biotin-dextran randomly filled a subpopulation of RGCs, with soma
distributed throughout the retinal ganglion cell layer at sufficiently
sparse densities that individual dendritic arbors were discriminated
easily. No other retinal cell type was labeled by this procedure (see
Fig. 1A).
Arbor analysis. Retinal whole mounts were coded so that RGCs
were traced and analyzed without knowledge of the treatment type. Only
dendritic arbors of rhodamine-labeled RGCs that were surrounded by
fluorescent microspheres were evaluated. The only exception to this
were rhodamine-labeled RGC arbors that did not coincide with observable
BDNF-treated microspheres. These RGCs were analyzed as a distinct
category, termed noncoincident (NC). Analysis of NC RGCs served as an
internal control to determine whether exogenous BDNF exerted effects
beyond its delineated injection site. NC RGCs were positioned at least
100 µm away from the nearest BDNF-treated microspheres. Previous work
has indicated that these microspheres do not diffuse significantly from
their injection site in vivo (Riddle et al., 1997) and
thereby provide local sources of neurotrophic factors.
Dendritic morphology was observed by epifluorescent microscopy (Nikon,
Tokyo, Japan). A 0.5 µm interval z-series was captured throughout the extent of the dendritic arbor of the RGC with a CCD
camera (Photometrics, Tucson, AZ) controlled by MetaMorph software
(Universal Imaging, West Chester, PA). The dendritic arbor of each RGC
was traced manually on transparent acetate film to represent the
dendritic arbor in two dimensions. Tracings were scanned digitally and
then analyzed with MetaMorph imaging software.
Primary dendrites were defined as direct extensions from the soma of at
least 10 µm in length. Only RGCs with at least one primary dendrite
>10 µm in total length were analyzed. Branch tips were identified as
the terminal end(s) of primary dendrites. To calculate soma area and
total dendritic arbor length, we thresholded, binarized, and
skeletonized images with MetaMorph software so that the soma perimeter
and dendrites were represented as a single pixel width. To determine
dendritic arbor complexity, we assigned each branch tip an order value
that equaled the number of branch points between the tip and the base
of its primary dendrite (also see Fig. 6A). A
dendritic complexity index (DCI) was calculated for each RGC such that
DCI = [(sum of branch tip orders + number of branch tips)(total
arbor length)/(number of primary dendrites)] (also see Fig. 6). To
correct for small variabilities in developmental staging, we normalized
values to the average control value of each parameter for each of five
independent experiments. All measurements were compared by ANOVA,
including Tukey's post hoc test (Systat, SPSS, Chicago,
IL). Significance was assigned when p < 0.05 (*), p < 0.01(**), or p < 0.001 (***).
Morphological analysis revealed that RGCs developing in the presence of
cytochrome c-coated beads were indistinguishable from RGCs
developing in the presence of uncoated beads; thus controls include
both groups. Microspheres coated with bovine serum albumin (BSA) or
nonimmune serum served as controls to establish the specificity of the
anti-BDNF effects on RGC dendritic morphology. RGCs exposed to BSA or
to nonimmune IgG-coated beads resembled RGC exposed controls (data not
shown), indicating that the anti-BDNF effects were specific and not
attributable to other proteins or immunoglobulins present in the serum.
RGC axon arbors were visualized as described in Cohen-Cory and Fraser
(1995). Briefly, the retinae of anesthetized stage 41 tadpoles were
injected with minute amounts of the fluorescent vital dye DiI
(Molecular Probes) to label RGCs. Tadpoles with one to two
distinguishable axonal arbors branching in the optic tectum were imaged
with a Bio-Rad (Richmond, CA) laser-scanning confocal microscope before
and 24 hr after tectal application of BDNF or anti-BDNF. Vehicle
solution (10% Niu-Twitty), 200 ng/ml BDNF (Amgen), 200 µg/ml of
specific function-blocking antibody to BDNF [a gift from J. Carnahan, Amgen; antibody specificity described in Nawa et al. (1995)
and Ghosh et al. (1994)], or 200 µg/ml of nonimmune IgG was
pressure-injected into the subpial space overlying the caudal tectum
immediately after the first observation. Delivery of neurotrophins or
antibodies in solution provided a more reliable delivery mechanism of
these reagents into the optic tectum than injection of the neurotrophin
or antibody coupled to microspheres. This is attributable to the rapid
dispersion of both internalized and free microspheres that results from
active tectal cell proliferation and arbor refinement normally
occurring at this developmental stage (J. Cogen and S. Cohen-Cory,
unpublished observations). Axon arbor morphology and statistical
analysis were performed as described in Cohen-Cory and Fraser
(1995).
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RESULTS |
BDNF inhibits RGC primary dendrites
As reported previously, exogenous BDNF enhances Xenopus
RGC axonal branching, and neutralization of tectal BDNF reduces RGC axon arborization in vivo (Cohen-Cory and Fraser, 1995)
(also see Fig. 7). Thus, it is plausible that retinal BDNF also
influences RGC dendritic branching locally. To determine whether
retinal BDNF modulates RGC morphological differentiation in
vivo, we experimentally altered neurotrophin levels in developing
Xenopus retinae during the period when RGCs begin to
differentiate (stage 38) and extend short unbranched primary dendrites
within the inner plexiform layer of the retina (Sakaguchi et al., 1984;
Holt, 1989). Microspheres treated with human recombinant neurotrophins
or with function-blocking BDNF antibodies were injected directly into
the right eyes of live anesthetized tadpoles to alter retinal
neurotrophin levels. Binding neurotrophins to fluorescent microspheres
prevented the neurotrophins from diffusing from the delivery site and
enabled us to analyze only those RGCs within the treatment site. RGCs were labeled retrogradely with rhodamine-dextran at stage 42-43, and
tadpoles were reared until stage 45, the developmental stage when
endogenous retinal BDNF levels are maximal. This labeling protocol
allowed a random population of newly arborizing RGC axon terminals to
incorporate the dextran and retrogradely transport it back to the RGC
soma and dendrites. This allowed us to visualize RGC dendritic arbors
and trace their morphology (Figs. 1,
2).
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Figure 1.
Visualization of RGC dendritic arbors with labeled
dextrans. To visualize RGC dendritic arbor morphologies, we injected
biotin- or rhodamine-conjugated dextrans into the tecta of
Xenopus tadpoles during developmental stages when RGC
axons are arborizing actively in the optic tectum. Retrograde transport
of dextran specifically labels RGC soma and dendrites within the
retina. A, B, Differential interference contrast images
of transverse 20 µm retina cryostat sections of a stage 45 Xenopus tadpole that had received tectal injection of
biotin-conjugated dextran. A, Labeled neurons are
distributed throughout the retinal ganglion cell layer
(GCL) and extend dendrites up into the inner plexiform
layer (IPL). B, A higher power view of a
biotinylated dextran-labeled RGC extending its dendrites into the IPL.
C, Fluorescent image of one optical section of a
rhodamine-dextran-labeled RGC in a whole-mounted stage 45 Xenopus retina, and (D) a
corresponding overlay image showing both RGC morphology
(red) and coincident NGF-coated microspheres
(green). In C, the
asterisk denotes the axon process. E,
Low-power view of part of a retina whole mount showing
rhodamine-dextran-labeled RGCs, and (F) a
corresponding overlay image showing both the retrogradely labeled RGCs
(red) and the distribution of BDNF-coated beads in that
area of the retina whole mount. INL, Inner nuclear
layer. Scale bars: A, F, 50 µm; B, D, 10 µm.
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Figure 2.
Neurotrophins modulate RGC dendritic complexity
in vivo. Retinal neurotrophin levels were altered
in vivo by injecting fluorescent microspheres treated
with exogenous neurotrophin (BDNF, NGF, NT-3, or NT-4) to increase
retinal levels or with function-blocking BDNF antibodies to neutralize
endogenous BDNF (anti-BDNF). Control treatments
were injections of uncoated, cytochrome c-coated,
or nonimmune IgG-coated microspheres. RGCs that developed
in BDNF-treated retina, but noncoincident (NC) with the
exogenous neurotrophin, were analyzed also. The morphologies of
rhodamine-dextran-labeled RGCs were reconstructed from serial optical
sections into one plane to quantify morphological parameters that
describe dendritic arborization, such as primary dendrite number and
branch tip number (see Figs. 3-6). RGCs are depicted in whole mounts
viewed from the inner, or vitreal, surface of the retina, and axons are
not shown. For control RGCs the averages equaled four to five primary
dendrites and 16-20 branch tips, with the highest branch tip order of
4-5 and a 136-175 µm dendrite length. An overall dendritic
complexity index (DCI) was calculated by combining several of these
morphological measures (see Fig. 6 and Materials and Methods for
explanation of the calculation). The control RGCs depicted in this
figure had DCI values that ranged from 2777 to 2899; NC DCI values
ranged from 2417 to 2807; BDNF DCI values ranged between 186 and 276;
anti-BDNF DCI values ranged from 5851 to 7429; NGF DCI values ranged
from 2705 to 3204; NT-3 DCI values ranged from 2220 to 2588; and NT-4
DCI values ranged from 2213 to 4282. Scale bar, 10 µm.
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Qualitatively, RGCs that developed in regions of exogenous BDNF
appeared less complex than did RGCs exposed to other neurotrophins or
controls, whereas RGCs that were exposed to BDNF-neutralizing antibodies exhibited more complex dendritic arbors (Fig. 2). To compare
the influence of altered retinal neurotrophin levels on RGC dendritic
arborization, we quantified several morphological parameters. The
number of primary dendrites per RGC was evaluated by counting the
dendrites that extended directly from the soma of each
rhodamine-dextran-labeled RGC (Fig.
3A). RGCs that developed in
retinal areas exposed to exogenous BDNF exhibited significantly fewer
primary dendrites (BDNF = 61 ± 3.6%) than RGCs exposed to control microspheres (control = 100 ± 4.9%). To determine
whether BDNF alters RGC dendritic number only on neurons directly
exposed to the neurotrophin, we evaluated the primary dendrites of RGCs located in retinal areas devoid of BDNF-coated fluorescent
microspheres. Such RGCs were termed BDNF noncoincident (NC) and
resembled control-treated RGCs. The average number of primary dendrites
per NC RGC was indistinguishable from that for RGCs in retinae injected
with control microspheres (NC = 105 ± 6.0% vs control = 100 ± 4.9%). This result indicates that neurotrophic
regulation of RGC primary dendrite number is restricted spatially.
Consequently, exogenous BDNF applications significantly inhibited
primary dendrites only in the treatment site.
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Figure 3.
Retinal BDNF levels influence RGC dendritic
arborization in vivo. Retinal neurotrophin levels were
altered in vivo by exposure to exogenous BDNF to
increase retinal BDNF or by exposure to function-blocking BDNF
antibodies to neutralize endogenous BDNF during RGC dendritic
elaboration. RGCs noncoincident (NC) with BDNF-coated
microspheres as well as RGCs coincident with control microspheres were
analyzed also. Primary dendrite extension was evaluated by determining
the number of primary dendrites per RGC (A).
Secondary dendritic branching was evaluated by determining the total
number of branch tips per RGC (B) as well as the
number of branch tips per primary dendrite (C).
The extent of dendritic elaboration also was compared by measuring the
total dendritic arbor length (D). Primary
dendritic number (A), number of dendritic branch
tips (B, C), and total arbor length
(D) were decreased significantly by exogenous
BDNF and increased significantly by neutralizing BDNF. RGCs not
coincident (NC) with BDNF-coated microspheres did not
differ from controls in any of these morphological parameters. Plotted
values represent the averages from five independent experiments; error
bars indicate ± SEM. Numbers within the
bars indicate the total number (n)
of RGCs analyzed for each treatment. ***p < 0.001 as compared with control.
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If BDNF specifically modulates RGC dendritic elaboration in
vivo, then neutralizing endogenous retinal BDNF should alter the number of primary dendrites also. Indeed, RGCs that developed in
retinal regions treated with BDNF function-blocking antibodies had
significantly more primary dendrites than did control neurons (anti-BDNF = 135 ± 5.7% vs control = 100 ± 4.9%; Fig. 3A). Thus, retinal applications of BDNF and
anti-BDNF had opposing local effects on the number of primary dendrites
per RGC, implicating endogenous retinal BDNF as a mediator of RGC
primary dendritic development.
BDNF influences RGC dendritic branching
Our initial analysis indicated that perturbing endogenous retinal
BDNF levels significantly altered RGC primary dendrite number. To
determine whether BDNF affects dendritic arbor differentiation exclusively by reducing the number of primary dendrites or whether BDNF
also influences dendritic branching, we determined the total numbers of
dendritic branch tips per RGC in retinae injected with control-, BDNF-,
or anti-BDNF-treated microspheres. RGCs developing in the presence of
exogenous BDNF had significantly fewer dendritic branch tips than RGCs
developing in the presence of control microspheres (BDNF = 38 ± 4.8% vs control = 100 ± 7.2%; Fig. 3B). NC
RGCs branched as much as RGCs developing near control microspheres
(NC = 100 ± 8.8% vs control = 100 ± 7.2%).
These results indicated that, for BDNF to affect branch tip number,
RGCs had to be exposed directly to BDNF-coated microspheres.
Similar to its effects on primary dendrite extension, neutralizing
endogenous retinal BDNF with anti-BDNF significantly altered branch tip
number per RGC (Fig. 3B). RGC dendritic arbors had significantly more branch tips after exposure to anti-BDNF than did
control RGCs (anti-BDNF = 177 ± 12% vs control = 100 ± 7.2%). These results indicate that increasing endogenous
BDNF during active dendritic arborization decreased total dendritic
branch tip number, and neutralizing endogenous retinal BDNF
significantly increased arbor branching. These results support the idea
that endogenous retinal BDNF plays a significant role in modulating both RGC primary dendrite extension and branch number.
To determine whether the negative regulation of BDNF branch tip number
was a sole consequence of the reduction of BDNF in primary dendrite
number or whether the reduced number of branch tips per RGC was also
attributable to decreased secondary branching, we calculated the
average number of branch tips per primary dendrite (Fig.
3C). RGCs coincident with BDNF-coated microspheres had
significantly fewer branch tips per primary dendrite than did RGCs
coincident with control microspheres (BDNF = 52 ± 3.0% vs control = 100 ± 5.9%). The average number of
branch tips per primary dendrite of NC RGCs was not significantly
different from control-treated RGCs (NC = 95 ± 6.5%
vs control = 100 ± 5.9%). In addition, RGCs that
developed near microspheres treated with anti-BDNF exhibited significantly more branch tips per primary dendrite than did controls, indicating that endogenous BDNF influences RGC dendritic branching (anti-BDNF = 129 ± 8.2 vs control = 100 ± 5.9%). Thus, the opposing effects that exogenous BDNF
and anti-BDNF exert on both total branch tip number per RGC and branch
tips per primary dendrite suggest that endogenous BDNF modulates both
the extension of primary dendrites and their subsequent branching.
BDNF reduces RGC total dendritic arbor length
The extent of a dendritic arbor of a neuron is thought to
influence its potential for receiving presynaptic input. We observed that increasing retinal BDNF levels decreased both the number of RGC
primary dendrites and their subsequent branching. Thus, it is possible
that a neuron could compensate for decreased dendrite number and
branching by extending longer dendrites in an attempt to achieve a
targeted total length of its dendritic arbor. To determine whether
alterations in retinal BDNF levels influenced dendritic arbor length,
we compared the total arbor length of RGCs exposed to BDNF- or
anti-BDNF-coated microspheres with RGCs exposed to control microspheres
(Fig. 3D). BDNF significantly reduced total dendritic arbor
length as compared with control RGCs (BDNF = 47 ± 6.4% vs control = 100 ± 7.4%). RGCs noncoincident with BDNF-coated microspheres extended arbors of equivalent length to
controls (NC = 102 ± 8.6% vs control = 100 ± 7.4%). RGCs in retinae exposed to BDNF antibodies had
significantly larger dendritic arbors (anti-BDNF = 151 ± 10% vs control = 100 ± 7.4%). Thus, total dendritic arbor length is decreased by exogenous BDNF and is
increased by neutralizing endogenous BDNF to values similar to those
for primary dendrite number. Moreover, the observation that the total
dendritic arbor length of RGCs exposed to control microspheres was
indistinguishable from that of noncoincident RGCs again supports our
observations that the effects of BDNF within the retina are local. The
decrease in total arbor length of BDNF-treated RGCs results from
reductions in primary dendrite formation and secondary branching
without significant compensatory increase of dendritic length.
RGC dendritic arborization is unaffected by neurotrophins other
than BDNF
To determine whether BDNF specifically regulates RGC dendritic
arborization, we similarly increased retinal levels of other neurotrophins by injecting microspheres coated with NGF, NT-3, or NT-4
into developing Xenopus retinae. Dendritic morphologies of
RGCs exposed to elevated concentrations of NGF, NT-3, or NT-4 were
compared with RGCs exposed to BDNF- or control-treated microspheres. Unlike BDNF, the neurotrophins NGF, NT-3, and NT-4 did not alter significantly the number of primary dendrites per RGC (Fig.
4A). Similarly, the
number of branch tips per primary dendrite did not differ from controls
in RGCs exposed to elevated levels of NGF, NT-3, or NT-4 (Fig.
4B), indicating that neurotrophins other than BDNF do
not affect either primary dendrite number or dendritic branching. These
results suggest that the influence of BDNF on dendritic complexity is
not a general neurotrophic response but, rather, a specific response to
BDNF.
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Figure 4.
BDNF specifically influences RGC dendritic
arborization in vivo. Retinal neurotrophin levels were
increased in vivo by injecting fluorescent microspheres
coated with control protein, BDNF, NGF, NT-3, or NT-4; the resulting
dendritic arbors of rhodamine-dextran-labeled RGCs were visualized.
Primary dendritogenesis was evaluated by counting the number of primary
dendrites per RGC (A), whereas dendritic
branching was evaluated by measuring the number of branch tips per
primary dendrite (B). Only exogenous BDNF
treatment significantly influenced primary dendrite extension
(A) or branching (B). NGF,
NT-3, and NT-4 had no significant effects on RGC dendritic arbors.
Plotted values represent the averages from five independent
experiments; error bars indicate ± SEM. Numbers
within the bars indicate the total number
(n) of RGCs analyzed for each treatment.
***p < 0.001 as compared with control.
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Neurotrophins influence RGC soma size independently of
dendritic arborization
To determine whether the reduction by BDNF of RGC dendritic
morphology was attributable to a general effect on neuronal
differentiation, we measured and compared RGC soma area for all
neurotrophin treatments (Fig.
5A). Neither exposure to
exogenous BDNF (BDNF = 90 ± 5.7%) nor to anti-BDNF
(anti-BDNF = 112 ± 6.5%) significantly altered RGC
soma area when compared with controls (control = 100 ± 4.3%). As observed for dendritic parameters, RGCs that developed in
retinae injected with BDNF-coated microspheres, but in areas
noncoincident with the microspheres, resembled RGCs exposed to control
microspheres (NC = 98 ± 7.3%). Thus, neither the
reduction of BDNF nor the increase of anti-BDNF in dendritic
elaboration parameters results from general alterations in cell size.
Although NGF, NT-3, and NT-4 did not alter RGC dendritic morphological
parameters significantly, NT-3 significantly reduced soma size (72 ± 5.6%) and NT-4 significantly increased RGC soma size
(142 ± 12%), as compared with control-treated retinae (100 ± 4.3%; Fig. 5B). Neither BDNF nor NGF
significantly affected soma area. Thus, neurotrophic influences on RGC
soma size suggest that individual neurotrophins exert distinct
biological effects on various aspects of RGC development. Neurotrophic
regulation of soma area, therefore, is likely to be independent from
the regulation of dendritic morphology.
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Figure 5.
Neurotrophic regulation of RGC soma area. To
determine whether retinal neurotrophins modulate aspects of RGC
morphology in addition to dendritic arborization, we measured the soma
area of RGCs in retinae treated with experimentally altered
neurotrophin levels. The soma area of RGCs coincident with control-,
BDNF-, or anti-BDNF-treated microspheres as well as RGCs not coincident
(NC) with BDNF-coated microspheres were compared and
found not to differ significantly from control
(A). Similarly, the effects of NGF, NT-3, and
NT-4 on soma size also were compared (B). BDNF
and NGF did not alter soma size, but NT-3-treated RGCs had
significantly smaller soma and NT-4-treated RGCs had significantly
larger soma than control-treated RGCs, indicating that NT-3 and NT-4
exert opposing influences on soma size. Plotted values represent the
averages from five independent experiments; error bars indicate ± SEM. Numbers within the bars indicate the
total number (n) of RGCs analyzed for each
treatment. ***p < 0.001 and *p < 0.05 as compared with control.
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Neurotrophins differentially regulate RGC dendritic complexity
To compare overall dendritic morphology between RGCs that
developed in altered neurotrophic environments, we calculated a dendritic complexity index (DCI) for each RGC to describe RGC complexity numerically (Fig.
6A,B). RGCs exposed to
BDNF, NGF, NT-3, NT-4, anti-BDNF, or control microspheres displayed
distributions of dendritic complexities in which RGC DCI values ranged
from very low to very high in each experimental treatment. The
distribution of DCI values, however, revealed that altering BDNF levels
regulated overall RGC dendritic complexity in vivo (Fig.
6C). Of RGCs exposed to exogenous BDNF, 65% had DCI values
in the least complex category (DCI < 250) as compared with 27%
of control RGCs and only 10% of RGCs exposed to anti-BDNF.
Correspondingly, 27% of anti-BDNF-treated RGCs were in the most
complex category (exhibited DCI values >4250), whereas only 12% of
control and none of the BDNF-treated RGCs had DCI values in this highly
complex category. Thus, this analysis of DCI distributions revealed
that exogenous retinal BDNF skewed RGC complexities toward lower DCI
values, whereas neutralizing endogenous retinal BDNF with antibodies
skewed RGC dendritic complexity toward higher DCI values. Similar to
our observations of primary dendrite number and secondary branching,
neurotrophins other than BDNF did not exert significant effects on
overall dendritic complexity. DCI distribution analysis revealed that
the complexities of RGCs in NGF-, NT-3-, and NT-4-treated retinae
varied slightly among each treatment, but DCI values generally
resembled those of controls (distribution not shown; for morphologies
and DCI values, see Fig. 2). Thus, only treatments that altered retinal
BDNF levels evoked dramatic alterations in RGC dendritic
complexity.
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Figure 6.
Retinal neurotrophin levels influence RGC
dendritic complexity in vivo. To determine the influence
of altering retinal neurotrophin levels on overall RGC dendritic
morphology, we calculated a dendritic complexity index
(DCI) that numerically describes the overall
dendritic arbor complexity for each RGC. A, The DCI
calculation is based on the number of primary dendrites, total arbor
length, and the number and order of branch tips (also see Materials and
Methods for a description of the calculation). B,
Diagram illustrating branch order analysis. Each branch tip is assigned
a number (1-4) that equals the number of times a
primary dendrite branches to produce the branch tip. C,
DCI distribution analysis reveals different distributions of RGC
complexities in control-, BDNF-, or anti-BDNF-treated retinae. RGC with
simple, medium, and complex morphologies, however, are represented in
all experimental conditions. Note that a majority of BDNF-treated RGCs
fall into the simpler complexity group (DCI values of <250), whereas
RGCs in anti-BDNF-treated retina have a skewed distribution toward
larger DCI values than controls.
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Target-derived BDNF promotes RGC axonal arborization
To compare the local effects of alterations in retinal BDNF levels
on dendritic morphology with those of tectal BDNF on RGC axon
morphologies, we experimentally altered neurotrophin levels in the
Xenopus tectum during the developmental stages when RGC axons actively arborize (Fig. 7) (also
see Cohen-Cory and Fraser, 1995). BDNF, control vehicle, or
function-blocking BDNF antibodies were injected into the subpial space
overlying the optic tectum to alter tectal neurotrophin levels (see
Materials and Methods). The morphology of DiI-labeled RGC axon arbors
was assessed immediately preceding and 24 hr after tectal injections.
The differences in total branch number between 0 and 24 hr as well as
the differences in axon arbor length at 0 and 24 hr were used as a
measure of change in axon arbor complexity (see Fig.
6A). When compared with controls, axonal arbors
became morphologically more elaborate over time in the BDNF-treated
tadpoles and became simpler in those treated with anti-BDNF. After 24 hr BDNF-treated arbors had significantly more branches (BDNF = 6.8 ± 0.87 branches vs control = 2.5 ± 0.4 branches) and increased their length significantly versus controls (BDNF = 177 ± 25 µm vs control = 107 ± 14 µm). Neutralization of tectal BDNF via
function-blocking BDNF antibodies resulted in axonal arbors that were
less complex than controls at 24 hr after treatment (increase in branch
number, anti-BDNF = 0.6 ± 0.5 branches vs control = 2.5 ± 0.4 branches; increase in branch length,
anti-BDNF = 69 ± 19 vs control = 107 ± 17 µm). Taken together, these results indicate that
endogenous tectal BDNF increases the complexity of axonal arbors,
whereas retinal BDNF decreases dendritic complexity.
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Figure 7.
BDNF enhances RGC axonal arborization in
vivo. Time-lapse analysis of DiI-labeled RGC axon arbors
reveals that the morphology of RGC axonal arbors is influenced by
tectal BDNF levels (also see Cohen-Cory and Fraser, 1995). A,
B, The effects of BDNF and anti-BDNF on RGC axon arbor
morphology after 24 hr of treatment were evaluated by measuring
quantitatively the total branch number (A) and
total arbor length (B). Values are presented as
the change from their initial value at the time of treatment. The
significant increase in total branch number and arbor length elicited
by BDNF reflects an overall increase in axon arbor complexity versus
controls. Similarly, the significantly lower increase in branch number
elicited by anti-BDNF indicates that neutralizing endogenous BDNF
prevents the increase in axon terminal arbor complexity that normally
occurs over time in normal tadpoles. Values represent the averages;
error bars indicate ± SEM. **p < 0.01 and
*p < 0.05 as compared with control.
C, Tracings of representative axonal arbors before and
24 hr after control, BDNF, or anti-BDNF injections illustrate the
effects of each treatment on RGC axon arbor complexity. Controls
include axons from tadpoles treated with vehicle solutions or with
nonimmune IgGs. Scale bar, 20 µm.
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DISCUSSION |
BDNF expression in the developing retina and tectum suggests that
BDNF may exert dual modes of action on RGC differentiation by acting
both at the target and locally within the retina. BDNF is expressed at
low levels in the retina at stage 39/40, coincident with the onset of
RGC dendritic arborization, and peaks at stage 45, when RGCs are
actively elaborating axonal and dendritic arbors (Cohen-Cory and
Fraser, 1994; Cohen-Cory et al., 1996). This report provides evidence
that retinal BDNF significantly influences the development of RGC
dendritic arbors in vivo. Surprisingly, application of
exogenous BDNF to the retina during RGC dendritic arborization resulted
in less complex RGC dendritic arbors. Exogenous BDNF significantly
reduced RGC primary dendrite extension and secondary branching without
influencing RGC soma size. Correspondingly, retinal applications of
neutralizing BDNF antibodies resulted in more complex RGC dendritic
arbors. In the developing Xenopus retina BDNF expression has
been detected exclusively on RGCs, whereas both RGCs and amacrine cells
express trkB (Cohen-Cory and Fraser, 1994; Cohen-Cory et al., 1996).
Thus, our results demonstrate that alterations in endogenous retinal
BDNF levels result in alterations in RGC dendrite elaboration in
vivo and support the idea that RGC-derived BDNF may act primarily
in an autocrine manner to modulate RGC development and differentiation. The possibility still remains that BDNF also acts on other
trkB-responsive retinal neurons, such as amacrine cells, and that
amacrine cells in turn may act on RGCs, thereby indirectly altering RGC
morphological differentiation. These two possibilities, however, are
not mutually exclusive.
Although exposure to exogenous BDNF dramatically and significantly
reduced the complexity of RGC dendritic arbors in vivo, some
BDNF-treated RGCs elaborated complex dendritic arbors even in the
presence of exogenous BDNF, suggesting that a subpopulation of RGCs is
unaffected by retinal BDNF. In support of a nonresponsive RGC
subpopulation, it has been shown that not all Xenopus RGCs express the trkB receptor and not all cultured RGCs respond to anti-BDNF (Cohen-Cory and Fraser, 1994; Cohen-Cory et al., 1996). Specific patterns of RGC dendritic arborization therefore might be
achieved by precise local patterns of neurotrophin and receptor expression on individual RGCs. In this way, subtle neurotrophin sculpting of RGC dendritic morphology could produce the variety of RGC
arbor morphologies that constitute the mature tadpole retinae (Sakaguchi et al., 1984). Our observations, however, do not rule out
the possibility that all RGCs may respond to BDNF or anti-BDNF treatments, but with differing degrees of responsiveness. The variety
of dendritic arbor complexities we observed in retinae exposed to
either BDNF or anti-BDNF also may be attributable to different RGC
maturities at the onset of treatment.
One indication that BDNF specifically influences RGC dendritic
development arises from our observations that applications of anti-BDNF
caused opposite effects on RGC dendritic arbor morphology than did
applications of exogenous BDNF. When endogenous retinal BDNF was
neutralized by function-blocking BDNF antibodies, RGCs elaborated more
complex arbors than control RGCs. Experiments that significantly reduce
an endogenous neurotrophin are necessary to confirm the role of that
neurotrophin during development and to assess more directly whether
exogenous neurotrophins mimic such effects (Shelton et al., 1995;
McAllister et al., 1999). Two convenient methods to neutralize
endogenous BDNF in vivo are available: trkB receptor bodies
and specific BDNF function-blocking antibodies. Trk receptor bodies are
fusion proteins of the ligand-binding domain of a specific trk receptor
with the FC domain of human IgGs that bind available neurotrophin and
thereby prevent neurotrophins from signaling via their trk receptors.
Although this approach has provided supporting evidence for
neurotrophic actions in vitro and in vivo, it
also has caused controversy. Identical treatments with two different
trkB receptor body preparations caused opposite effects on the same
neuronal population (Frost et al., 1998). Another limitation of trkB
receptor bodies is that ligand-specific neutralization cannot be
achieved; trkB receptor bodies neutralize both NT-4 and BDNF.
Function-blocking neurotrophin antibodies, however, specifically
neutralize a single neurotrophin by binding endogenous neurotrophin and
preventing it from interacting with all its receptors. The current
study used a BDNF function-blocking antibody to neutralize BDNF
in vivo without affecting retinal NT-4 levels. Therefore,
our observations of the action of anti-BDNF support a role for
endogenous retinal BDNF in modulating RGC dendritic morphology.
NT-4 and BDNF share the same specific receptor, trkB (Barbacid, 1994),
yet in our study BDNF specifically inhibited dendritic complexity and
NT-4 specifically increased soma size. This differential cellular
response to two ligands that share a common receptor suggests that BDNF
and NT-4 may be exerting their effects on RGC dendritic complexity and
soma size via distinct receptors, receptor combinations, and/or
intracellular signaling cascades. Other studies also have observed that
trkB ligands can differentially influence specific aspects of neuronal
morphology in the developing visual system. NT-4, but not BNDF, can
prevent some of the effects of monocular deprivation on lateral
geniculate neuron soma size (Riddle et al., 1995). Branching of
Xenopus RGC axon arbors in vivo is promoted by
BDNF but is unaffected by NT-4 (Cohen-Cory and Fraser, 1995). BDNF and
NT-4 also have been shown to exert differential effects on cortical
neuron dendritic morphology in vitro (McAllister et al.,
1995, 1997).
In our study BDNF was the only neurotrophin to influence RGC dendritic
morphology; NT-3 and NT-4 influenced RGC soma size without
significantly influencing dendritic complexity. RGC soma treated with
NT-3 were significantly smaller than controls, whereas NT-4-treated
RGCs had significantly larger soma. NT-4 has been shown to prevent
thalamic neuron soma shrinkage resulting from unbalanced input activity
(Riddle et al., 1995). NT-3 is known to promote early RGC
differentiation, converting retinal progenitors into differentiated
RGCs (de la Rosa et al., 1994; Bovolenta et al., 1996). Thus, our
results agree with the observations that NT-4 may affect soma size and
that NT-3 may act as a differentiation signal for RGCs. Therefore, it
is likely that individual neurotrophins differentially modulate
distinct components of RGC differentiation, such as soma size and
dendritic elaboration. In the mature retina, RGC subtype
classifications are based on both dendritic morphology and RGC soma
size (Wingate and Thompson, 1994). Neurotrophins therefore may act as
determinants of RGC subtype specification by modulating distinct
aspects of differentiation. The expression patterns of neurotrophins
and their receptors within the developing retina (Cohen-Cory and
Fraser, 1994; Hutson and Bothwell, 1998; Karlsson et al., 1998) (for
review, see von Bartheld, 1998) further support local roles for these
neurotrophins and make it plausible that alterations in retinal
neurotrophin levels may affect specific aspects of retinal development
in vivo.
The neurotrophins can exert a wide variety of influences on neuronal
morphology. Most reported neurotrophic effects on dendritic morphology
analyzed in culture indicate that individual neurotrophins promote the
arborization of specific neuronal populations (Cohen-Cory et al., 1991;
McAllister et al., 1996). For example, in slice cultures of ferret
visual cortex the pyramidal neurons from different cortical layers
respond differentially to neurotrophins (McAllister et al., 1995, 1996,
1997). Individual neurotrophins exert distinct effects on either basal
or apical dendrites, with each neurotrophin eliciting a unique pattern
of dendritic morphologies for neurons within a single cortical layer.
Although in most instances neurotrophic factors promote dendritic
arborization, in some neuronal populations they inhibit neuritogenesis.
The cytokine leukemia inhibitory factor (LIF) and ciliary neurotrophic
factor (CNTF) have been shown to reduce neurite outgrowth from
dissociated sympathetic neurons (Guo et al., 1999), and BDNF has been
shown to reduce basal, but not apical, dendritic complexity of layer 6 cortical pyramidal neurons in culture (McAllister et al., 1995, 1997). Thus, combining our observations that BDNF reduces RGC dendritic complexity in vivo with previous in vitro studies
reveals a novel, additional role for neurotrophic factors as negative
regulators of neuronal arborization.
Previous studies demonstrated that Xenopus RGCs initiate
primary dendrites and elaborate dendritic arbors by target-independent mechanisms (Holt, 1989; Sakaguchi, 1989). Short, simple RGC dendrites can be observed before the first RGC axons reach the optic tectum, indicating that dendritic initiation is independent of target-derived cues (Holt, 1989). Further, the morphology and subtype specification of
RGC dendritic arbors in ectopically transplanted eyes are
indistinguishable from controls, indicating that even in the complete
absence of target interactions RGC dendritic arborization occurs
normally (Sakaguchi, 1989). Most BDNF in the retinal ganglion cell
layer is not of tectal origin, although RGCs are capable of
retrogradely transporting tectal BDNF, suggesting that most retinal
BDNF is produced locally within the retina (Herzog and von Bartheld,
1998). Thus, it is likely that retinal-derived BDNF exerts much
stronger local influence within the retina than does tectal BDNF. This report demonstrates that alterations in retinal BDNF levels before target innervation cause alterations in RGC dendritic morphology, suggesting that BDNF can act locally within the retina to influence dendritic development well before RGC axons reach the tectum where they
have the opportunity to interact with target-derived BDNF. That local
and target-derived BDNF exert distinct roles during RGC development is
supported by our unique observation that this neurotrophin is capable
of differentially modulating axonal versus dendritic arborization
within a single neuronal population. Alterations in tectal BDNF levels
significantly altered axon arbor morphology, whereas alterations in
BDNF retinal levels altered dendritic morphology, suggesting local
roles for BDNF in both locations. The possibility still exists that
target-derived BDNF also could contribute to the local effects of BDNF
on dendritic morphology, although RGC dendrites begin to develop before
their axons reach the tectum. The experiments in this study did not
eliminate this possibility. To what extent target-derived BDNF
contributes to RGC dendritic differentiation and, conversely,
retinal-derived BDNF contributes to RGC axon arborization remains to be determined.
In most vertebrate species, developing RGCs initially elaborate
exuberant dendritic arbors by extending an excess number of branches
that later are remodeled to achieve their final morphological and
maturational state (Vanselow et al., 1990; Wong et al., 1991; Yamasaki
and Ramoa, 1993). Combinations of intrinsic retinal influences and
target interactions are thought to play roles in determining RGC
dendritic form (for review, see Wingate, 1996). Interactions that
depend on neuronal activity only slightly influence the intrinsic RGC
dendritic remodeling program. For example, blocking action potential
activity does not prevent the pruning and remodeling of exuberant RGC
dendritic arbors (Wong et al., 1991; Campbell et al., 1997) but
increases RGC axonal arbor complexity (Sretavan et al., 1988). Only
manipulations in RGC density that are independent of target
interactions significantly influence RGC dendritic elaboration and
remodeling (Troilo et al., 1996). These observations suggest that
intrinsic signals within the developing retina exert significant control on RGC dendritic elaboration and remodeling. Our results demonstrating that alterations in retinal BDNF levels before full differentiation of RGC dendritic arbors (and before the peak in endogenous BDNF expression) lead to alterations in RGC dendritic morphology suggest that endogenous retinal BDNF may be one of the fine
regulatory signals that locally control RGC dendritogenesis and
remodeling in vivo. Moreover, the observation that RGCs
express both BDNF and trkB during this developmental period suggests
that a single neuronal population can finely tune its morphological differentiation program via the coordinated action of neurotrophic factors.
Our in vivo observations that BDNF differentially modulates
RGC axonal and dendritic arbor morphology in the developing visual system raise the intriguing possibility that neurotrophic signaling at
the axon terminal of a neuron can differ dramatically from signaling at
its dendrites. The mechanism underlying this differential response to
BDNF within a single neuronal population is unknown. Within the
developing visual system it is likely that BDNF acts in coordination
with other cues to direct both axonal and dendritic arborization
differentially. RGC axons and dendrites synapse with distinctly
different neuronal populations and thus may be exposed to different
cues that locally can mediate axonal and dendritic responses to BDNF in
a differential manner. Not only are the environments that the axon and
dendrites of an RGC encounter different, but the complement of cell
surface receptors may differ between its axons and dendrites. Recent
observations that neurotransmitter receptors can be differentially
targeted to axons and dendrites (Stowell and Craig, 1999) suggest that
selective delivery of cell surface receptors could explain the ability
of a neuron to respond differentially at its axons and dendrites.
Similarly, differential expression of intracellular signal transduction
machinery in axons versus dendrites also might mediate their
differential responses to a single cue. Recent work in vitro
has demonstrated that cyclic nucleotides are involved in intracellular
signaling by chemoattractive and chemorepulsive cues. Pharmacological
alterations in cyclic nucleotides caused growth cones to switch their
response to a chemoattractive molecule from attraction into repulsion
(Song et al., 1997, 1998). Thus, differentially regulated alterations in intracellular signaling mechanisms have the potential to cause opposing cellular responses within the axons and dendrites of a single
neuron. Precise patterns of neurotrophin expression, receptor
localization, and intracellular signaling mechanisms thereby may exert
spatial and temporal control over neuronal morphology and profoundly
influence nervous system development.
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FOOTNOTES |
Received June 24, 1999; revised Sept. 3, 1999; accepted Sept. 3, 1999.
This work was supported by an National Eye Institute (NEI) postdoctoral
fellowship to B.L. and awards from the NEI, Alfred P. Sloan,
Stein/Oppenheimer, UCLA Frontiers of Science, and Beckman Foundations
to S.C-C. We thank Ami Poon for developing preliminary dextran-labeling
protocols; Tilly Oren, Thuy Vu, and Ly Nguyen for technical assistance;
and Drs. Ron Frostig and Jeff Cogen for providing insightful comments
on this manuscript. Amgen (Thousand Oaks, CA) and Genentech (South San
Francisco, CA) generously provided the neurotrophins used in this study.
Correspondence should be addressed to Dr. Susana Cohen-Cory, University
of California, Los Angeles, MRRC, 760 Westwood Plaza, NPI
78-148, Los Angeles, CA 90095. E-mail: scohenco{at}ucla.edu.
| |
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ABSTRACT
INTRODUCTION
