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The Journal of Neuroscience, April 15, 1998, 18(8):2891-2906
Contributions of the Optic Tectum and the Retina as Sources of
Brain-Derived Neurotrophic Factor for Retinal Ganglion Cells in the
Chick Embryo
Karl-Heinz
Herzog1 and
Christopher S.
von Bartheld2
1 Department of Neurobiochemistry, Max-Planck-Institut
for Psychiatry, D-82152 Martinsried, Germany, and
2 Department of Physiology and Cell Biology, University of
Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
Retinal ganglion cells (RGC) are supported by brain-derived
neurotrophic factor (BDNF), but it is not known if BDNF acts as a
target-derived factor or as an afferent or autocrine trophic factor.
Here we demonstrate that BDNF mRNA is expressed in the retinorecipient
layer of the chick optic tectum as well as in the inner nuclear layer
and ganglion cell layer of the retina. Amacrine cells rather than RGC
were the main source of BDNF mRNA in the ganglion cell layer, as
determined by in situ hybridization that was combined
with retrograde labeling of RGC and destruction of RGC by optic stalk
transection, followed by quantitative RT-PCR. Cells in the ganglion
cell layer as well as the retinorecipient layers of the optic tectum
were BDNF-immunolabeled. After injections into the tectum,
radio-iodinated BDNF was transported to the retina where
autoradiographic label accumulated in the inner plexiform and ganglion
cell layers. After intraocular injection, iodinated BDNF accumulated in
these same retinal layers and correlated with the distribution of p75
neurotrophin receptor protein. The majority of cross-linked
receptor-bound BDNF in the retina immunoprecipitated with p75
antibodies. No difference in the intensity of BDNF immunolabel was
observed in the experimental retina or tectum after optic stalk
transection, indicating that most of the BDNF in the RGC was not
derived from the optic tectum. These data indicate that a substantial
fraction of the BDNF in the ganglion cell layer is derived from local
sources, afferents within the retina, rather than from the optic tectum
via retrograde transport.
Key words:
retina; neurotrophin; BDNF; visual system; development; optic tectum; in situ hybridization; retinal ganglion cell; chick embryo; immunocytochemistry; neurotrophin receptor; retrograde
transport; amacrine cell
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INTRODUCTION |
During neurogenesis an excess number
of neurons is produced, which subsequently is eliminated by naturally
occurring cell death. According to the neurotrophic hypothesis, neurons
require target-derived trophic signals for their maintenance and
survival (for review, see Purves, 1988
; Oppenheim, 1991). However,
afferents also provide trophic support to developing neurons, and this
form of anterograde afferent trophism is thought to be equally
important to retrograde trophism (Oppenheim, 1991; Linden, 1994).
The retinotectal system presents a particularly advantageous system for
the discrimination of target-derived (retrograde) and local (autocrine
or paracrine) sources of neurotrophins. Most retinal ganglion cells
(RGC) degenerate after target removal or optic stalk transection in
chicken (Hughes and LaVelle, 1975; Vanselow et al., 1990). RGC can be
maintained by brain-derived neurotrophic factor (BDNF) in
vitro (Johnson et al., 1986; Rodríguez-Tébar et al.,
1989; Cohen-Cory and Fraser, 1994), and cells in the RGC layer express
the BDNF receptor trkB (Okazawa et al., 1993, 1995; Perez and
Caminos, 1995; Rickman and Brecha, 1995; Cohen-Cory et al., 1996;
Garner et al., 1996; Hallböök et al., 1996). The expression
of BDNF mRNA in target regions and its upregulation by physiological
input further support the view that BDNF derived from the target plays
a role in the maintenance of RGC (Castrén et al., 1992;
Cohen-Cory and Fraser, 1994; Herzog and Barde, 1994; Herzog et al.,
1994; Schoups et al., 1995) (for review, see von Bartheld, 1998a), but
the retrograde transport of BDNF from the tectum to the retina has not
been shown in the developing visual system.
Besides the target, local sources also provide trophic support to RGC
(de Araujo and Linden, 1993; Linden, 1994; AryPires et al., 1997), yet
the factor or factors involved have not been identified. One strong
candidate is BDNF (Cohen-Cory et al., 1996). The expression of BDNF
mRNA in the tectum (Leibrock et al., 1989) as well as in the retina
(Cohen-Cory and Fraser, 1994; Herzog et al., 1994) allowed us to
explore the respective contributions of the target and local sources of
the same trophic factor. It is important to distinguish between
different sources of trophic support (axon terminus vs dendrite/soma)
because of their potential distinct trophic effects (Clarke, 1985;
McAllister et al., 1995) and, in the case of RGC, clinical implications
for the delivery of trophic factors after injury.
Here we demonstrate which cell layers express BDNF mRNA in the retina
and optic tectum of chick embryos, where the BDNF protein is
transported and accumulates after retrograde transport, and which
receptors bind BDNF in the retina. We also demonstrate that RGC contain
significant amounts of BDNF that are derived predominantly from cells
within the retina. These data support the notion that local retinal
sources provide a considerable amount of the BDNF for RGC and that RGC
may use BDNF derived from cells within the retina in a paracrine
manner.
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MATERIALS AND METHODS |
Animals. Fertilized White Leghorn chicken eggs were
obtained from local suppliers and were incubated in humidified
incubators at 37.5-38°C. Approximately 1800 chicken eggs were used.
The ages of chick embryos were verified at the time of death according to the method of Hamburger and Hamilton (1951). Experimental procedures were conducted in compliance with the Policy on the Use of Animals in
Neuroscience Research and were approved by the local animal care
committee.
In situ hybridization. A chicken BDNF cDNA
(Maisonpierre et al., 1992) (courtesy of P. Maisonpierre and G. Yancopoulos, Regeneron, Tarrytown, NY) was subcloned into pGEM.
Single-stranded riboprobes (527 bp) were labeled with
35S-uridine 5'-triphosphate (UTP) and used for
hybridization as described (von Bartheld et al., 1991). To improve the
sensitivity of the hybridization, we labeled another riboprobe (530 bp
of the coding region; courtesy of K. Bailey, Martinsried, Germany) with
33P-UTP. Cryosections were used for the
33P-hybridizations, whereas paraffin sections were used for
the 35S-hybridizations. Cryosections (12 µm) were treated
with 0.0005% Proteinase K (Boehringer Mannheim, Indianapolis, IN) for
8 min, followed by an acetylation step with acetic anhydride in 0.1 M triethanolamine, pH 8, for 10 min. After dehydration,
sections were hybridized with 33P-labeled riboprobes
(107 cpm/µl) overnight. Subsequently, sections
were washed in 2× SSC for 15 min, followed by treatment with RNase
(100 mg/ml; Boehringer Mannheim) for 30 min at 37°C. After sections
were rinsed in RNase buffer (0.5 M NaCl, 10 mM
Tris-HCl, and 1 mM EDTA, pH 8), slides were incubated at
60°C in 2× SSC for 1 hr, followed by a stringency wash in 0.2× SSC.
Sections were dehydrated, coated in NTB-2 emulsion (Kodak, Rochester,
NY), exposed, and developed after 2 weeks.
Animals of embryonic days E13 and E15/16 were anesthetized and perfused
with 4% paraformaldehyde (PFA). Some of these embryos had undergone an
optic stalk transection at E4 as described below, whereas others
received an intraocular injection of BDNF or vehicle at E15 as
described below. Whole heads or eyes and brains were dehydrated,
cryosectioned (12 µm) or embedded in paraffin, and sectioned at 10 µm. Sections through the brain and retina were collected on
Silane-coated glass slides, together with sections through the otocysts
from 4-d-old chick embryos. These sections served as positive controls
because otocysts express high levels of BDNF mRNA (Hallböök
et al., 1993). Adjacent sections were hybridized with sense control
probes. Hybridization conditions were as described previously (von
Bartheld et al., 1991). Sections were exposed for 14-28 d, developed,
and lightly counterstained with thionine. Brain structures were
identified according to Kuenzel and Masson (1988) and tectal layers
according to the criteria of LaVail and Cowan (1971).
Retrograde labeling combined with in situ
hybridization. Several approaches were used to double-label
RGC, including the application of a tracer to the cut optic nerve
in vitro (Fritzsch and Hallböök, 1996) and the
in vivo injection into the optic tectum of the retrograde tracers fluorescein-horseradish peroxidase, horseradish
peroxidase-DAPA (Sigma, St. Louis, MO), or the lysine-fixable 3000 MW
dextran simultaneously labeled with biotin and tetramethylrhodamine
(Microruby, Molecular Probes, Eugene, OR). The Microruby injections
proved to be the most successful. Approximately 1 µl of a 0.1 mg/µl
Microruby solution was injected into the optic tectum of 14-d-old chick embryos with microfine "insulin" disposable syringes. The embryos were anesthetized 18 hr later and perfusion-fixed with 4% PFA; the age
of the embryos (E14-E15) was verified (Hamburger and Hamilton, 1951).
The brains were dissected, and those with a suitable injection site
were cryoprotected in 4% PFA containing 15% sucrose at 4°C. After 4 hr, the brains were frozen in OCT compound and sectioned at 20 µm to
map the injection site in the tectum and to assess the extent of
retrograde labeling in the optic tract. The eyes of animals with
suitable penetration of the tracer were processed for in
situ hybridization as described above, except that some sections
were not hybridized to evaluate the amount of quenching. Sections were
not dehydrated and cleared in xylene but were coverslipped in aqueous
mounting medium (Vectashield, Vector Laboratories, Burlingame, CA).
Sections were analyzed and photographed via epifluorescence with
rhodamine filters, dark field or bright field, and the percentages of
fluorescent and nonfluorescent cells were determined as well as the
percentages of cells labeled for BDNF mRNA (
3 grains/cell body).
Quantitative analyses were performed on six visual fields of sections
through the retina in which the fluorescent labeling and the in
situ hybridization showed normal labeling intensities and
patterns. The centralmost retina and the peripheral retina were
excluded from this analysis, because the ratios of RGC and amacrine
cells are more extreme in these regions (Ehrlich, 1981). A total of 392 cells was evaluated; statistical significance was determined by
unpaired Student's t test.
Quantification by RT-PCR. Quantification of BDNF mRNA was
performed as detailed elsewhere (Herzog et al., 1994). In brief, an
in vitro transcript of the coding sequence bearing one
nucleotide exchange was added to the tissue when RNA was extracted.
This nucleotide exchange in the recovery standard, after reverse
transcription and PCR, led to a new restriction site recognized by
BamHI. RNA was extracted by the cesium trifluoro-acetate
(CsTFA) method at ages E17 and E14, and reverse transcription followed
by PCR was performed in a one-tube reaction protocol. The PCR product
was cleaved by BamHI, separated on an 8% polyacrylamide
gel, and electroblotted. PCR products were detected by a probe, and
signals resulting from the recovery standard and the endogenous BDNF
mRNA were compared with the aid of a laser densitometer.
Optic stalk transection. Fertilized White Leghorn chicken
eggs were incubated in a humidified incubator, and, beginning with E3,
embryos were cultured in Petri dishes (Auerbach et al., 1974) or
windowed in ovo. At E4 the right optic stalk was transected just behind the eyeball with a pair of microscissors, and the embryo
was allowed to develop until tissue preparation at E8.5, E11, E14/E15,
or E17. Successful transections were verified by dissection of the
region in which the optic chiasm normally forms, and tissues were
processed for PCR, immunocytochemistry, or in situ
hybridization only when the optic nerve was absent on the operated
side.
Immunohistochemical methods. A rabbit serum was raised
against the N-terminal portion of mouse BDNF
(His-Ser-Asp-Pro-Ala-Arg-Arg-Gly-Glu-Leu-Ser-Val-Cys) coupled to KLH.
This serum was affinity-purified by the peptide coupled to Sepharose,
as described elsewhere (Jungbluth et al., 1994). For controls the
purified serum was incubated at a dilution of 1:200 in the presence of
the BDNF peptide and the neurotrophin-3 (NT-3) peptide representing an
NT-3 sequence (Tyr-Ala-Glu-His-Lys-Ser-His-Arg-Gly-Glu-Tyr-Ser-Val-Cys) coupled to Sepharose beads, respectively, in 0.1% Triton X-100 in PBS.
After 3 hr at room temperature the beads were centrifuged for 10 min at
10,000 × g, and the supernatant was processed directly for immunostaining.
After anesthesia with Nembutal, chicken embryos were perfused
transcardially with 4% PFA in PBS; tecta and retinae were
post-fixed in the same fixative for 16 hr at 4°C. Some animals were
perfused with high or low pH buffers before fixation (Zhou et al.,
1994) to dissociate BDNF from its receptors and to determine whether this may enhance antibody-antigen interaction and improve the immunolabeling. As this was not the case, animals were perfused routinely with neutral pH buffers. After sucrose impregnation of the
tissues (30% sucrose for 24 hr at 4°C), 12 µm cryosections were
thawed on gelatin-coated slides. Some sections through the tectum were
cut at 30 µm, and free-floating sections were processed. Brain
sections were cut in the transverse plane. Immunostaining was performed
according to the Vector protocol (Vector Laboratories) with some
modifications. In brief, some sections were treated with three washes
of 50% ethanol and then were incubated with diluted blocking serum for
30 min at room temperature, followed by three washes with PBS and 0.1%
Triton X-100 (PBT) and incubation overnight with primary antisera
diluted 1:200 in PBT in a humid chamber at 4°C. Sections were rinsed
three times and incubated with a biotinylated goat anti-rabbit antibody
diluted in blocking serum. For immunostaining with monoclonal p75
antibody (Tanaka et al., 1989; von Bartheld et al., 1995), a
biotinylated horse anti-mouse antibody was used. After three washes ABC
reagent was applied to the sections for 30 min, rinsed three times, and
preincubated for 10 min in 0.1% diaminobenzidine (DAB) in 0.04%
nickel in TBS (100 mM Tris, pH 7.4, and 150 mM
NaCl). Finally, the substrate was reacted in the presence of 1 µl of
glucose oxydase (type V-S, Sigma) in 1 ml of 0.1% DAB, 0.04% nickel,
and glucose in TBS until the reaction product was clearly visible.
Slides were rinsed in water, dehydrated, and coverslipped.
Injections of radio-iodinated BDNF. Human recombinant BDNF
was obtained from Regeneron (courtesy of Dr. Ronald Lindsay,
Regeneron). BDNF was iodinated by using a modification (von Bartheld,
1998b) of the method of Vale and Shooter (1985). Iodinated BDNF
migrated on SDS gels as a single band at ~14 kDa, was transported in
a receptor-mediated manner, and retained 86% of its activity in a
dorsal root ganglion cell assay (von Bartheld et al., 1996b). Injections into the tectum were made as described previously for injections into the vitreous (von Bartheld et al., 1994). Approximately 1-2 µl of iodinated BDNF was injected with microfine disposable syringes (Becton Dickinson, Franklin Lakes, NJ) in embryos at E8
(n = 2), E13/E14 (n = 5), and E17/E18
(n = 6). After 20 hr of survival, the animals were
anesthetized and perfused transcardially with 4% PFA. The amount of
radioactivity in the dissected midbrains (E14 and E18) or whole heads
(E9) was measured in a gamma counter (cpm were ~50,000-150,000 for
E9 and E14, and 200,000-300,000 for E18). The brains and the eyes were
embedded in paraffin, and a series of 1 in 15 sections (10 µm
thickness) through the brain was collected on glass slides and exposed
on x-ray film for 2-5 d; the sections were coated with NTB-2 emulsion.
The ipsilateral and contralateral retinae were sectioned at 10 µm,
and a 1 in 15 series of sections was collected on three sets of slides
and coated with NTB-2 emulsion. Sections were exposed for 10-21 d (brains) or 3-8 weeks (retinae) at 4°C. Slides were lightly
counterstained with thionine after development and were coverslipped.
After injections of iodinated proteins into the retina or into the
tectum, the amount of radioactivity in the eyes was measured in a gamma
counter. Autoradiographic silver grains were counted over retinal
layers by using 40 or 100× objectives, and grain profiles were plotted from at least 10 counts (averages). These were compared with grain counts from control retinae.
Cross-linking of BDNF and immunoprecipitation of BDNF receptors
in the retina. 125I-labeled BDNF (20-30 ng)
was injected into the eye at E15. Animals were anesthetized 20 hr later
with Nembutal and perfused with PBS; the retinae were dissected and
placed in cold lysis buffer (50 mM HEPES or Tris-Cl
and 1% NP-40 or Triton X-100) containing 1 mM
phenyl-methyl sulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and one of two cross-linkers, either
1-ethyl-3(3-dimethylaminopropyl)carbodiimide-HCl (EDC; Pierce,
Rockford, IL) or disuccinimidyl suberate (DSS; Pierce) at a final
concentration of 6 mM (EDC) or 0.1 mM (DSS).
The tissue was triturated by being drawn through 22-G needles and spun;
the supernatant was incubated at room temperature for 30 min, followed by 30 min at 4°C with 50 mM L-lysine.
Quadruplicate or duplicate samples of 100 µl were nutated for 1-24
hr at 4°C with antibodies (1 µg/100 µl) specific for the
extracellular domain of chicken p75 (ChEX; Weskamp and Reichardt,
1991), chicken trkB (von Bartheld et al., 1996b), chicken trkC (Lefcort
et al., 1996), or normal rabbit IgG as a control for nonspecific
precipitation. A washed Pansorbin cell suspension (10 µl; Calbiochem,
San Diego, CA) was added, nutated for an additional 1 hr at 4°C, and
centrifuged for 10 min. The radioactivity was counted separately for
the pellets and the supernatants, and the specific precipitation was
determined by subtraction of the counts in the nonspecific IgG pellet.
Specific precipitation was up to ~20% of total counts. The total
percentage of receptor-bound BDNF and the relative contributions of the
three different receptors were calculated. In parallel sets of control experiments the cross-linker was omitted to test the efficiencies of
cross-linking.
Control experiments to compare the precipitation
efficiencies of the antibodies. To determine whether the
efficiencies with which the antibodies immunoprecipitated the
neurotrophin receptors were approximately similar for p75 and trkB, we
cross-linked and immunoprecipitated 125I-labeled BDNF,
which was bound to Sepharose-protein A beads (CL-4B, Pharmacia
Biotech, Piscataway, NJ). Samples were loaded on 6% SDS PAGE and
blotted onto nitrocellulose (SS-NC, Schleicher & Schuell, Keene, NH).
Some blots were probed with p75 or trkB antibodies and detected with
horseradish peroxidase-conjugated secondary antibodies and an enhanced
chemiluminescent substrate (SuperSignal CL-HRP Substrate System,
Pierce). Other blots were probed directly with 125I-labeled
p75 or trkB antibodies [10 × 106 cpm/ml,
according to Hockfield et al. (1993)] and radio-iodinated with
lactoperoxidase after the preparation of Fab fragments (Harlow and
Lane, 1988). Blots were exposed to film, and the resulting bands at 75 and 140 kDa were quantified by laser densitometry. 125I-labeled blots generally rendered cleaner background,
but bands visualized with chemiluminescence gave essentially the same
results.
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RESULTS |
BDNF mRNA expression in the optic tectum
Previous studies showed that similar amounts of BDNF mRNA are
expressed in the retina and tectum during the period of developmental cell death of RGC (Herzog et al., 1994). Using in situ
hybridization, we examined the localization of BDNF transcripts at
E13-E16 when RGC are dependent on their target (Hughes and LaVelle,
1975; Nurcombe and Bennett, 1981). BDNF mRNA was expressed in the
retinorecipient layers stratum griseum et fibrosum superficiale (SGFS)
and stratum griseum centrale (SGC) of the optic tectum. The most
abundant expression was seen at E15-E16 in the sublayer SGFSi and g
(layers VI and VIII, respectively, of the embryonic tectum) (LaVail and Cowan, 1971) and the SGC (layer IV). Most of the labeled cells in SGFSi
were located in the central parts, but not in the more superficial or
the deepest parts of this lamina (Fig.
1). Many cells were labeled heavily with
6-12 grains per cell body, and it was evident in several labeled cells
that most of the silver grains were clustered at the transition zone
between the cell body and the apical dendrite (Fig.
2). The apical dendrites of most neurons
in layer SGFSi extend into the superficial layers (mainly SGFSa-f;
LaVail and Cowan, 1971; Hunt and Brecha, 1984; von Bartheld,
unpublished data), where they are contacted by RGC axons (Crossland et
al., 1975). Neurons in the SGFSg layer were labeled distinctly, but
with a lower intensity than those in SGFSi or SGC. Although many cells
in SGFSi and g appeared to express BDNF mRNA, labeling in SGC was more
heterogeneous, with some neurons expressing high levels and some
expressing very low levels of BDNF (Fig. 1). Only a subpopulation of
avian SGC neurons receive direct RGC input via their apical dendrites
(Hardy et al., 1985). BDNF mRNA could be detected in ~15-25% of
large neurons in the SGC, and there was no preferred subcellular
localization of silver grains in these multipolar neurons. BDNF
transcripts were not detected in other visual nuclei in the brain,
e.g., the lateral geniculate nucleus or the nucleus of the basal optic
root (nucleus ectomamillaris). BDNF mRNA was observed readily in
several nonvisual nuclei and ganglia, including the hippocampus, the
area hippocampalis, the nucleus inferioris hypothalami, the nucleus
nervi glossopharyngei et nucleus motorius dorsalis nervi vagi, and the
trigeminal ganglion (data not shown). In summary, these data indicate
that neurons in several layers of the tectum may release BDNF from
their dendrites in layers SGFSa-f and thus may provide BDNF for RGC
axons. When E15 embryos received an injection of 200 ng of BDNF or
vehicle only in one eye, there was no obvious difference in the
intensity of hybridization between the ipsilateral and the
contralateral tectum 20-24 hr later.
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Figure 1.
Expression of BDNF mRNA in the optic tectum of a
chick embryo at 15.5 d of incubation (E15.5). Dark-field view of a
transverse section through the central region of the tectum hybridized
with a 35S-labeled riboprobe for chicken BDNF. BDNF
mRNA-expressing layers and the corresponding Nissl staining of the same
section (camera lucida tracing) are indicated on the
right [embryonic layers III-VI,
VIII, and XII, according to LaVail and
Cowan (1971)]. Note that the expression of BDNF is most intense in the
deeper half of layer SGFSi. SGFS sublayers c,
g, i, and j are indicated.
SGC, Stratum griseum centrale; SAC,
stratum album centrale. Scale bar, 100 µm.
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Figure 2.
BDNF mRNA expression in neurons of tectal layer
SGFSi at E15.5. Note that silver grains (35S-labeled)
frequently accumulate over the apical half of the soma
(arrowheads) from which the apical dendrite extends into
the retinorecipient layers. The pial surface of the tectum is toward
the top of the micrograph. Scale bar, 10 µm.
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BDNF mRNA expression in the retina
In the E15-E16 retina, BDNF mRNA was distributed over several
layers. With 35S-labeled probes, discretely higher grain
densities were seen over the ganglion cell layer (GCL) and the inner
border of the inner nuclear layer (INL; data not shown). The
33P-labeled series was more sensitive than the
35S-labeled series because label over individual cell
bodies could be identified. A small fraction (5-10%) of cells in the
GCL was labeled for BDNF mRNA (Fig.
3A). The frequency of labeled
cells (as a percentage of the entire GCL population) appeared to be higher in the peripheral retina than in the central retina (data not
shown). The GCL contains RGC as well as a substantial number of
displaced amacrine cells (approximately one-third of the cells in the
GCL) (Ehrlich, 1981; Layer and Vollmer, 1982). As described below, most
of the labeled cells in the GCL are displaced amacrine cells. BDNF
expression was seen occasionally in cells of the inner INL (data not
shown) and was strong over the outer half of the INL (Fig.
3B,C), which contains the cell bodies of horizontal cells
and bipolar cells. Hybridization also was seen over the ONL, as in the
E18 retina (Hallböök et al., 1996). When embryos received
an injection of 200 ng of BDNF or vehicle in the eye at E15, the
expression of BDNF mRNA in the GCL was increased in the injected eye
after 20-24 hr, consistent with previous reports (Ballarin et al.,
1991; Mansour-Robaey et al., 1994).
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Figure 3.
Retinae of E15 chick embryos hybridized with
33P-labeled riboprobes for chicken BDNF. A,
Bright-field view of two labeled cells in the ganglion cell layer
(GCL, arrows). B,
Dark-field image of a labeled cell in the GCL (larger
arrow) and several labeled cells in the outer half of the inner
nuclear layer (INL, smaller arrows).
C, Dark-field image of E15 retina after optic stalk
transection at E4. Label in the outer half of the INL is maintained
(arrows). Scale bars: 10 µm in A; 20 µm in B, C.
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Because BDNF mRNA was expressed in layers of the retina containing RGC,
we examined the possibility that BDNF was expressed by RGC and thus may
support themselves in an autocrine manner (Acheson et al., 1995;
Cohen-Cory et al., 1996; Gao et al., 1997). RGC were retrogradely
labeled by injections of Microruby into the optic tectum in
vivo, and the sections subsequently were hybridized for BDNF mRNA.
Microruby labeled many cells in the GCL (~70%, Fig.
4A,B), consistent with
the percentage of RGC within the chicken GCL (Ehrlich, 1981). In
situ hybridization did not quench the signal (Fig.
4B), confirming a previous report (Fritzsch and
Hallböök, 1996). The percentages of fluorescent and
nonfluorescent cells in the GCL were determined as well as the
percentages of cells labeled for BDNF mRNA (Fig. 4C, Table
1). The quantitative analysis showed that
only a small percentage (<3%) of fluorescent cells expressed BDNF
mRNA, whereas a substantial fraction (
15%) of the unlabeled
(presumed amacrine cells) in the GCL expressed BDNF mRNA (Fig.
4C, Table 1). These data provide direct evidence that most
of the BDNF mRNA in the GCL is expressed by a subpopulation of amacrine
cells, and not by RGC. If BDNF mRNA expression in the GCL were altered
as a result of damage of RGC axons and terminals in the tectum when
Microruby was injected, this should have taken a longer time (>18 hr)
for manifestation and likely would increase BDNF mRNA in the RGC rather
than in the amacrine cells (Gao et al., 1997).
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Figure 4.
Identification and quantification of retinal
ganglion cells (RGC) and amacrine cells with BDNF
expression in the ganglion cell layer (GCL) of E15 chick
embryos (A-C) and quantification of BDNF mRNA in
the retina after degeneration of RGC induced by optic
stalk transection (D). A, Section
through the retina retrogradely labeled with Microruby (white
grains) and hybridized for BDNF mRNA (dark silver
grains). Note that many RGC are retrogradely labeled
(thin arrows), but most of the BDNF-expressing cells
(large arrow) are not fluorescent. Not all Microruby
grains are in the focus plane. Scale bar, 20 µm. B,
Comparison of the percentage of Microruby-labeled cells
(RGC) in the GCL before and after
hybridization. Note that there is no apparent quenching of the
fluorescent signal. The number of visual fields (with
~70 cells each) analyzed is indicated within white
squares. Error bars indicate SEM. C, Percentages
of BDNF-expressing cells for the RGC population (identified by labeling
with Microruby) and for the amacrine cells (AMA;
identified by the lack of fluorescent label). Note that <3% of RGC
express BDNF, as compared with 15% of BDNF-expressing amacrine
cells in the GCL. The number of visual
fields analyzed (with a total of 392 cells) is indicated within
white squares. Error bars indicate SEM.
D, BDNF mRNA was measured at age E17 by quantitative
RT-PCR in normal retinae (Control) and compared
with retinae lacking most RGC (Transected). BDNF mRNA
levels were not reduced significantly in retinae after the destruction
of their RGC. Error bars indicate SEM. Similar data were obtained for
retinae from age E14.
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Nevertheless, to confirm that RGC do not express a major fraction of
the BDNF mRNA in the retina, a second strategy was used: destruction of
RGC and subsequent quantitative RT-PCR for BDNF mRNA in the eye. The
optic stalk was transected at E4, before target innervation, which
eliminated nearly all RGC at later stages of development (Vanselow et
al., 1990). When levels of BDNF mRNA in the retinae were determined by
quantitative RT-PCR and compared with age-matched normal retinae, BDNF
mRNA levels were not reduced significantly (Fig. 4D).
To rule out the possibility that amacrine or bipolar cells may
upregulate BDNF expression in the absence of RGC, at E15 via in
situ hybridization we examined sections from retinae transected at
E4. There was no apparent difference in the pattern of BDNF expression
in these retinae compared with the control side (see Fig.
3C) other than a reduced number of labeled cells in the GCL.
This may represent the loss of a small fraction of BDNF-expressing RGC
or the loss of, or BDNF downregulation within, a subpopulation of
amacrine cells in the absence of RGC. There was no apparent increase in
the labeling intensity over the INL, which contains the bulk of the
amacrine and all of the bipolar cells (see Fig. 3C). Taken
together, these data demonstrate that the majority of BDNF in the
retina is produced by cells other than RGC, probably by subpopulations
of horizontal, bipolar, and amacrine cells, based on the in
situ hybridization data.
BDNF immunocytochemistry in the retina
Expression of BDNF transcripts does not necessarily reflect a
precise relationship between transcripts and translated protein (Nawa
et al., 1995; Johnson et al., 1996), and the in situ
hybridization does not allow us to evaluate which cells use and
accumulate BDNF protein. To determine the localization of BDNF at the
protein level, we used a previously characterized affinity-purified
serum raised against a peptide (B6) of the N-terminal portion of BDNF. This antibody recognizes BDNF in Western blots (Jungbluth et al., 1994)
and does not cross-react with NT-3. When this antibody was applied to
the retinae of 8.5-d-old chick embryos, the GCL, but no other layer in
the retina, was heavily labeled (Fig.
5A). To confirm further the
specificity of the antibody, we incubated the serum in the presence of
the B6 BDNF peptide coupled to Sepharose beads, and, after
precipitation, sections of E8.5 retinae were incubated with the
supernatant. No labeling was observed in the retinae in these control
experiments (Fig. 5B). Immunolabeling also was abolished
when the primary antibody was omitted. In contrast, preadsorption of
the BDNF peptide serum against an NT-3 peptide resulted in labeling
similar to the serum alone (data not shown). Therefore, we consider
this antibody to be specific for BDNF.
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Figure 5.
Specificity of the BDNF antiserum in tissue
sections. A, The BDNF antiserum specifically recognizes
cells in the ganglion cell layer (GCL) of an E8.5
retina. B, No label was detected in the
GCL after preincubation and precipitation of the serum
with the B6 peptide coupled to Sepharose beads. Scale bar, 50 µm.
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BDNF-like immunoreactivity was detected at E7 in the retina (Fig.
6A); at this age all
cells in the GCL were weakly labeled. At later stages (E8.5) BDNF
labeling increased, and the intensity of labeled cells within the GCL
became more heterogenous (Figs. 5A, 6F). A
number of cell bodies in the GCL displayed strong labeling, whereas
BDNF-like immunoreactivity was barely detectable in the neuropil
[inner plexiform layer (IPL) and outer plexiform layer (OPL)]. Cells
adjacent to the IPL, presumably amacrine cells, were labeled with much
lower intensity. Furthermore, cells in proximity to the ora serrata
were BDNF-positive (Fig. 6B). This cell population
contains newly formed RGC, which presumably are not yet connected to
the tectum (Rager, 1980). Neuronal processes in the OPL were labeled
slightly above background. Naturally occurring cell death of RGC and
cell death after target removal begins after E8.5 (Rager and Rager,
1978; Hughes and McLoon, 1979). When we compared retinae after optic
stalk transection with controls at E8.5, no difference in the labeling
of the INL or the GCL could be observed (compare Figs. 6C,
5A). BDNF labeling at E11 was increased, and within the GCL
most of the large-diameter neurons displayed strong immunoreactivity.
Similar to the E8.5 retina, neurons adjacent to the IPL were labeled
weakly at E11 (Fig. 6D). In the GCL of the normal E14
retinae the distribution of labeled cells was similar to that of the
E11 eye. However, large neurons in the INL were labeled above
background levels (data not shown). These neurons may represent
displaced ganglion cells (Reiner et al., 1979). After optic stalk
transection at E4, many cells in the GCL of the operated eye had
disappeared at E14 (Fig. 6E). Only a few large-diameter neurons were BDNF-positive in the transected E14 GCL.
These cells may produce their own BDNF, or they may acquire BDNF from
neighboring amacrine cells.
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Figure 6.
BDNF immunoreactivity in the retina.
A, The ganglion cell layer (GCL) of an E7
retina is weakly labeled. B, At E8.5 newly formed cells
adjacent to the ora serrata are BDNF-positive. These cells presumably
represent retinal ganglion cells (RGC). C, In an E.8.5
retina BDNF is not visibly reduced in the GCL after optic stalk
transection (X) at E4 (compare with Fig.
5A). D, At E11 many cells in the normal
GCL are strongly labeled. E, After optic stalk
transection (X) many RGC have degenerated
at E14. Some large cells display strong BDNF immunoreactivity.
F, An E14 retina labeled with the BDNF antiserum at
higher magnification displays heterogeneity of the label within the
GCL. Scale bars: 25 µm in A-C, E; 50 µm in D; 10 µm in F.
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BDNF immunocytochemistry in the optic tectum
Many neurons in the SGC and some neurons in the SGFS of the optic
tectum were immunoreactive in the E15-E18 chick embryo (Fig. 7). Many of the labeled neurons in the
SGC were large, with clearly labeled primary dendritic processes, but
several small cells also were labeled in the SGC. Approximately
15-35% of all SGC neurons were lightly to moderately BDNF-labeled. In
the SGFS, occasional neurons were labeled in the SGFSi and g. The
labeled neurons in layer i were within the deeper third of this layer,
whereas those in layer g were slightly deeper than the more densely
packed center of this sublayer. Although BDNF-labeled neurons in the
SGFS were sparse in the normal animal, their frequency was increased
substantially, especially in layer SGFSi, in embryos in which the
contralateral eye had been manipulated by injecting either BDNF or
vehicle solution. The effect of vehicle injections in the eye also may
be attributable to BDNF, because such manipulations are known to
increase the expression of endogenous neurotrophins, including BDNF, as
described above (see also Ballarin et al., 1991; Mansour-Robaey et al., 1994). BDNF- or vehicle-injected animals showed an ~10-fold increased number of neurons that were immunolabeled clearly, and these were distributed over several sublayers of SGFS, predominantly sublayer i,
but also b, c, e, g, h, and j. The size of labeled neurons in these
layers differed, with relatively small neurons in b, c, and j, and
medium-sized neurons in layers e, g, and i. The labeled neurons in
layer i all had long apical dendrites that were labeled for ~80 µm.
In contrast to the SGFS layer, the frequency of BDNF-immunolabeled
neurons did not appear to increase in layer SGC in the tectum of
animals injected in the eye with vehicle or BDNF.
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Figure 7.
BDNF immunocytochemistry in the optic tectum of a
16-d-old chick embryo. The left panel shows a section
through the normal tectum (ipsilateral to the manipulated eye); the
right panel shows a section through the experimental
tectum (contralateral to the injection of exogenous BDNF in the eye).
The two panels are from the same tissue section. Note that many more
neurons in the i sublayer of the stratum griseum et
fibrosum superficiale (SGFSi) are BDNF-labeled after injection in the
contralateral eye. Label represents endogenous BDNF (not anterogradely
transported exogenous BDNF), because it was not apparent in this tectal
layer after the injection of radiolabeled BDNF in the eye. The
induction of endogenous BDNF in layer SGFSi was not a specific effect
of the exogenous BDNF in the eye, because intraocular injection of
vehicle elicited the same effect (data not shown). Immunolabel of
neurons in the stratum griseum centrale (SGC) or stratum
opticum (SO) was not visibly altered by manipulations of
the eye. Scale bar, 100 µm.
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Retrograde transport of BDNF from the tectum to the retina
RGC from all species that were examined respond to BDNF in
vitro (Johnson et al., 1986; Rodríguez-Tébar et al.,
1989; Cohen-Cory and Fraser, 1994), but it is not known if BDNF acts
in vivo as a target-derived factor or as a local retinal
factor. Neurons in the SGFSi and SGC layers of the tectum receive
innervation from RGC axons (Crossland et al., 1975; Repérant and
Angaut, 1977). BDNF mRNA is expressed in the optic tectum by neurons in layers that are known to receive retinal innervation, and RGC depend
for survival on their target (Nurcombe and Bennett, 1981). Therefore,
BDNF generally is assumed to be a retrograde trophic signal that is
transported from the tectum to RGC (Cohen-Cory and Fraser, 1994). To
determine whether BDNF can be transported retrogradely from the tectum
to RGC, we radio-iodinated BDNF, and ~5 ng was injected into the
tectum of chick embryos of 8, 13-14, and 17-18 d of incubation. The
transport of BDNF was examined by gamma counting of the radioactivity
in the eyes (E13-E18) and by autoradiography of sections through the
retinae (E8, E13-E18). Approximately 0.7-1.5 ng of the injected BDNF
remained in the midbrain at the time of death, as measured by gamma
counting of the dissected midbrains. The successful injections
typically were made in the dorsomedial tectum (Fig.
8A), and the injection
site usually was restricted to <5% of the entire tectum. Measurements of the radioactivity in the eyes showed 131.6% (±8.81%; SEM) in the contralateral eye when compared with the radioactivity in the
ipsilateral eye (= 100%), indicating that the difference (equivalent to ~0.3-0.5 pg of BDNF) was transported specifically from the tectum
to the retina. Specific transport was evident by emulsion autoradiography of sections through the retinae that accumulated iodinated BDNF exclusively in the IPL and GCL layers of the
contralateral retina after tectal injections at E13/E14 and E17/E18
(Fig. 8B,C,E,F). No label was seen after
injections at E8 (Fig. 8D). Label was seen only in
the parts of the retina that are known to project to the region of the
tectum containing the injection site. 125I-label was not
seen by autoradiography in the ipsilateral retina, presumably because
neurotrophins are degraded rapidly in the vascular system
(Stöckel et al., 1976), and free 125I is removed when
the tissue is dehydrated (while the 125I incorporated into
neurotrophins remains within neurons). Profiles of the grain density
over the different retinal layers were plotted from representative
sections at ages E8, E14, and E18 (Fig.
8D-F). These profiles showed that most of the
radioactivity (>85%) accumulated over the IPL and that only ~15%
of the radioactivity was within the GCL. These data indicate that the
tectum-derived BDNF accumulates predominantly in the dendrites of RGC
rather than in their cell bodies. We cannot exclude the possibility
that some of the radioactivity in the IPL represents degradation
products of BDNF rather than intact protein. Label was not seen in the
retinae at the earliest stage examined (E8), possibly because the
density of retinal terminals in the tectum is too low at this age
(Rager and von Oeynhausen, 1979; Thanos and Bonhoeffer, 1983).
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Figure 8.
Retrograde transport of exogenous
125I-labeled BDNF from the tectum to the retina.
A, Dark-field view of an injection site in the tectum
(TeO) of an 18-d-old (E18) chick embryo. The surface of
the tectum is indicated with a dashed line. Dorsal is to
the top, and lateral is to the left.
Scale bar, 1 mm. B, Dark-field view of the contralateral
retina of the same animal showing increased grain density over the
inner nuclear layer and the ganglion cell layer, which comprises mostly
retinal ganglion cells (RGC). Scale bar, 50 µm.
C, Bright-field view of the contralateral retina of an
E13 embryo showing increased grain densities (125I-BDNF) in
the ganglion cell layer and inner plexiform layer (IPL)
after injection into the tectum. The outer nuclear layer (ONL) is not
labeled. D-F, Grain density profiles of
I125-BDNF label in the retinae after unilateral
injections into the tectum of E8 (D), E13
(E), and E18 (F) embryos.
The filled squares represent averages from 10 traces
through the contralateral retina; the open circles
represent averages from 10 traces through the ipsilateral (control)
retina (background). The retinal layers and the distances from the
pigment epithelium (PE) are indicated at the
bottom. ONL, Outer nuclear layer;
INL, inner nuclear layer; IPL, inner
plexiform layer; GCL, ganglion cell layer;
VITR, vitreous body. Note that iodinated BDNF
accumulates in the retina at E13 and E18, but not at E8, and that most
of the radioactivity accumulates in the IPL layer; a smaller amount is
present in the GCL, which contains the retinal ganglion cell
bodies.
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The RGC are not the only afferent input to tectal neurons expressing
BDNF. Several isthmic and diencephalic nuclei that innervate the SGC or
SGFS depend on an intact retinotectal circuitry during development
(Ostrach and Mathers, 1979; Wizenmann and Thanos, 1990; Clarke, 1992;
Page et al., 1993), and many of them express the BDNF receptor trkB
during development (Garner et al., 1996), including the relevant stages
during the third week of incubation for the isthmo-optic nucleus (ION;
von Bartheld et al., 1996b) and the parvocellular isthmic nucleus (Ipc;
R. Williams, Karolinska Institute, personal communication). Therefore,
BDNF produced by tectal neurons may be transported anterogradely or
retrogradely and used by isthmic or thalamic nuclei. To examine if any
of these nuclei may transport exogenous BDNF injected into the tectum, we collected sections through the brainstem, mesencephalon, and diencephalon and coated them with emulsion for autoradiography. Slightly increased grain density was observed over the ipsilateral Ipc,
but not the ION, the magnocellular isthmic nucleus (Imc), or the
lateral spiriform nucleus either ipsi- or contralateral to the
injection (data not shown). Label over the Ipc likely reflects true
accumulation of transported BDNF rather than accumulation caused by
diffusion from the injection site, because the Imc had a lower grain
density than the Ipc, although the Imc is located closer to the
injection site. These data indicate that both the RGC and the Ipc may
be recipients of tectally produced BDNF.
Accumulation of exogenous BDNF in the retina after
intraocular injection
Not only is BDNF expressed in the tectum, but similar amounts of
transcripts are produced in the developing retina (Cohen-Cory and
Fraser, 1994; Herzog et al., 1994). Because RGC depend on a presumptive
retina-derived trophic factor during development (Linden, 1994), we
examined if intraocularly injected BDNF can be taken up and can
accumulate in RGC neurons. We therefore injected radio-iodinated BDNF,
NT-3, NGF, and basic fibroblast growth factor (bFGF) into the eye, and
we examined 20 hr later the distribution of these trophic factors in
the retina by emulsion autoradiography (Fig.
9A-C). The distribution of
BDNF also was examined at 3 d after injection. Typically,
~10-15 ng of iodinated trophic factor with a specific activity of
100-200 cpm/pg was injected into the eye. All three neurotrophins
accumulated in the IPL, the GCL, and slightly in the OPL. Increased
grain densities were observed over presumptive RGC cell bodies in the
GCL (Fig. 9C). No difference was observed in the intensity
or distribution of the three neurotrophins in the retina. bFGF did not
accumulate significantly in any of the retinal layers (Fig.
9B). At 3 d after injection, the distribution of
125I-BDNF was similar to that after 20 hr, indicating the
lack of any obvious movement of exogenous BDNF from the IPL to the GCL. Although nonspecific binding in the IPL may obscure movement from the
IPL to the GCL, it is also possible that exogenous iodinated BDNF may
be internalized differently than endogenous BDNF (Rosenfeld et al.,
1993). Taken together, the autoradiographic data indicate that
neurotrophins accumulate in retinal neurons, particularly in dendrites
or axonal processes in the IPL, and that neurotrophins may be used by
retinal neurons, mainly RGC and/or amacrine cells. Thus, BDNF may be a
local retina-derived trophic factor for RGC neurons.
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Figure 9.
Accumulation of radio-iodinated BDNF
(A, C) and basic fibroblast growth factor
(bFGF, B) in the retina after intraocular
injection. A, Dark-field view of an autoradiographic
section through the E16 retina after the injection of ~10 ng of
iodinated BDNF in the eye. Scale bar, 100 µm. B,
Dark-field view of an autoradiographic section through the E16 retina
after the injection of ~10 ng of bFGF. Note that bFGF does not
accumulate significantly in any retinal layer. C,
Bright-field view at a higher magnification of the same retina shown in
A. Note that BDNF accumulates differentially in the
retinal layers, with the highest grain densities in the central part of
the inner plexiform layer (IPL), with moderate densities
in the ganglion cell layer (GCL) and the inner border of
the inner nuclear layer (INL), and with low densities in
the outer plexiform layer (OPL). Scale bar, 50 µm.
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Distribution of p75 receptor protein in the retina
BDNF binds to p75 receptors and to trkB receptors
(Rodríguez-Tébar et al., 1990; Barbacid, 1994; Bothwell,
1995). BDNF accumulated predominantly in the IPL and with lower
intensity in the GCL. The chicken GCL (but not the IPL) contains
full-length trkB protein (Okazawa et al., 1995; Cellerino and Kohler,
1997). Because all exogenously introduced neurotrophins accumulated in
the same layer (IPL, Fig. 9C) despite the reported
differential expression of members of the trk family of receptors in
the chicken retina (Hallböök et al., 1996), we tested
whether p75 was distributed in the same layer in which the exogenous
BDNF accumulated. Immunocytochemistry with a monoclonal antibody to p75
(Tanaka et al., 1989; von Bartheld et al., 1995) showed distinct
patterns of p75 label in the retina at E14-E16. Most neuropil label
was in the IPL with punctate label, particularly in the outer third of
the IPL (Fig. 10A).
This distribution was very similar to the distribution pattern of
exogenous BDNF. Two cell types were labeled in the INL. A very strong
label was found in regularly spaced neurons at the inner margin of the
INL (~30-40 µm apart), presumably representing a subpopulation of amacrine cells with labeled processes extending into the IPL (Fig. 10A). Another cell type was labeled with less
intensity in the central parts of the INL and may represent bipolar
cells and/or Müller glial cells (Fig. 10A). The
OPL and possibly horizontal cells were labeled with low intensity in
the central retina, but label in the OPL was more intense in the
peripheral retina (data not shown). The GCL was labeled with very low
intensity. Because cells in the GCL express p75 mRNA at E15-E16 (von
Bartheld et al., 1991) and the RGC axon terminals contain p75 receptor
protein (von Bartheld, 1997), the RGC appear to export p75 receptors
rapidly from the cell body to their dendrites in the IPL and to their axons in the optic tectum.
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Figure 10.
A, Distribution of p75 receptor
immunoreactivity in the central retina of a 16-d-old chick embryo. Note
the heavy labeling in the central parts of the inner plexiform layer
(IPL) and heavily labeled cell bodies and processes of
presumptive amacrine cells in the inner margin of the inner nuclear
layer (INL, large arrow). Faintly labeled
cell bodies are located in the center of the INL (thin
arrow). Light labeling is present in the ganglion cell layer
(GCL) and in the outer plexiform layer
(OPL). Endogenous p75 is abundant in layers in which
exogenous BDNF accumulates (see Fig. 9C). Scale bar, 50 µm. B, Relative contributions of p75, trkB, and trkC
to the fraction of BDNF that was bound to receptors and could be
immunoprecipitated with antibodies after cross-linking with EDC or DSS.
Averages of four to seven independent experiments. Note that the majority of receptor-bound BDNF binds to p75.
Error bars indicate SEM. C, Relative efficiencies of the
antibodies for immunoprecipitation of p75 and trkB. Samples of the
immunoprecipitates (pellets) and supernatants were Western-blotted (6%
SDS gel) and visualized with secondary horseradish peroxidase
chemoluminescence or 125I-labeled p75 or trkB antibodies.
The resulting bands at 75 kDa (p75) and 140 kDa
(trkB), respectively, were quantified by densitometry,
and the ratios of density values (pellet/supernatant) were compared.
Irrelevant antiserum (IgG) did not immunoprecipitate the
antigen, whereas the p75 and trkB antibodies showed approximately equal
efficiencies for immunoprecipitation (average ratios of 2.2. and 2.5).
Error bars indicate SEM. The number of independent
experiments is indicated within white squares.
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Cross-linking and immunoprecipitation of BDNF to p75 and trkB
receptors in the retina
Neurotrophins have been cross-linked to 130-140 kDa and 75 kDa
receptors in the retina, presumably trk and p75 receptors, respectively
(Escandón and Chao, 1990; Escandón et al., 1994; Cohen-Cory
et al., 1996). To determine which receptors contribute to the binding
of BDNF, we injected 125I-labeled BDNF into the retina,
cross-linked it 20 hr later with EDC or DSS to its receptors, and
immunoprecipitated it with antibodies specific for chicken p75, trkB,
or trkC (courtesy of L. F. Reichardt, F. Lefcort, and D. O. Clary, Howard Hughes Medical Institute, University of California, San
Francisco, CA). These experiments showed that a major fraction of the
BDNF was bound to receptors (~20-25%). The large majority of the
receptor-bound fraction was immunoprecipitated with the p75 antibody,
whereas a minor fraction was precipitated with the trkB antibody (Fig.
10B). These data suggest that most of the
receptor-bound BDNF was associated with p75.
A small amount of BDNF was immunoprecipitated consistently with trkC
antibodies in retinal tissues, but not in the optic nerve or the
isthmo-optic nucleus after axonal transport (von Bartheld, 1996). These
data indicate that some of the injected BDNF binds to trkC, consistent
with the report that, at 100- to 500-fold increased levels, BDNF
effectively binds to trkC and phosphorylates it (Ip et al., 1993). The
retinal concentration of BDNF after injection was ~100-500 times
higher (total BDNF = 5-10 ng) than the concentration of
endogenous BDNF at this age (total BDNF/retina 50 pg) (Johnson
et al., 1996; J. E. Johnson, Wake Forest University, personal
communication). Alternative explanations of BDNF immunoprecipitation with trkC antibodies include the possibilities that heterodimers of
exogenous BDNF and endogenous NT-3 form that bind to trkC (Jungbluth et
al., 1994; Philo et al., 1994), that trk and p75 may coprecipitate (Huber and Chao, 1995), or that the trkC antibody may recognize certain
isoforms of trkB (Garner et al., 1996) although it does not recognize
the predominant form of trkB (Lefcort et al., 1996).
When the immunoprecipitation was performed without previous
cross-linking, there was a three- to fourfold increase in nonspecific binding and a shift in the ratio with which the p75 and trkB antibodies precipitated the 125I-labeled BDNF. p75 antibodies
precipitated only 39.7 ± 12.9% (SEM; n = 6) of
the receptor-bound BDNF, whereas trkB antibodies precipitated 60.3 ± 12.9% (SEM; n = 6) of the receptor-bound BDNF. With
cross-linkers the ratio was reversed to approximately 60:30 (p75:trkB;
Fig. 10B). These data indicate that BDNF dissociates from the p75 receptor (but not from trkB) unless it is cross-linked, and the data provide circumstantial evidence for the relative efficiency of the cross-linkers. The increased pool of dissociated BDNF
may account for the higher nonspecific binding observed without the
cross-linkers.
To determine whether the efficiencies of immunoprecipitation were
approximately similar between the p75 and the trkB antibodies, we
analyzed Western blots and compared the relative amount of precipitated
p75 or trkB. Labeled bands at 75 and 140 kDa from pellets and
supernatants were taken to be p75 and trkB, respectively. When they
were precipitated with trkB or p75 antibodies, the density ratios
between the pellets and supernatants were 2.46 ± 0.38 (SEM) and
2.26 ± 0.14 (SEM) for trkB and p75, respectively. When they were
precipitated with control IgG, the densities were similar in the
pellets and supernatants (ratio of 1.12 ± 0.37; SEM). These data
indicate that the efficiencies of the p75 and trkB antibodies were
approximately similar (Fig. 10C). In conclusion, most of the receptor-bound exogenous BDNF in the retina binds to p75 receptors present in the IPL, whereas a minor fraction of BDNF binds to trkB
receptors. The distribution of p75 in the IPL is consistent with the
notion that most of the exogenous BDNF that accumulates in the retina
binds to p75 rather than to full-length trkB
receptors.
Contributions of the tectum and retina as sources of BDNF
for RGC
To distinguish between the two sources of BDNF for RGC and to
determine their contributions to the accumulation of BDNF within RGC
neurons in the retina, we compared the distribution and intensity of
BDNF immunoreactivity in E8.5 retinae after optic stalk transection (at
E4) with those in normal retinae. In addition, these experiments provided information as to whether the relative paucity of BDNF protein
in the tectum was attributable to the rapid depletion by retrograde
transport to RGC neurons; if this were the case, one would expect to
see increased BDNF immunolabel in the tectum when BDNF transport to RGC
is prevented. No difference could be observed in either the
disconnected tectum or the GCL lacking target innervation (see Figs.
6C, 5A), indicating that most of the
BDNF-immunoreactive material in RGC was not derived from the tectum.
Furthermore, newly generated neurons in the GCL close to the ora
serrata were immunolabeled (see Fig. 6A). At this
stage these neurons presumably do not yet have axons or growth cones that have reached the tectum (Rager, 1980) and therefore do not have
access to the target and could not have taken up and retrogradely transported BDNF from the tectum. These data indicate that the majority
of BDNF labeled with the antiserum in RGC is derived from retinal
sources rather than from the tectum.
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DISCUSSION |
The objectives of this study were to localize BDNF transcripts and
protein in the tectum and retina and to determine the contributions of
local cells and target cells as sources of BDNF for RGC. Our main claim
is that the majority of the BDNF in RGC neurons is derived from local
retinal sources, presumably amacrine cells and/or bipolar cells.
Although target cells in the optic tectum express BDNF and BDNF is
transported retrogradely from the tectum to the RGC, the target-derived
BDNF is only a minor fraction of the total BDNF in RGC. These findings
have implications for the neurotrophic hypothesis of target-derived
factors and raise intriguing questions about differences in trophic
functions of neurotrophins, depending on whether they are internalized
at the axon terminal or by dendritic processes.
BDNF as a target-derived trophic factor
Previous studies have shown that RGC depend on their target during
development. Preventing target encounter by ablation of the optic
tectum or by optic stalk transection significantly increases normal
developmental cell death of RGC (Hughes and LaVelle, 1975; Vanselow et
al., 1990), and coculturing RGC with the tectum increases survival of
RGC in vitro (Nurcombe and Bennett, 1981). BDNF promotes the
survival of RGC in vitro (Johnson et al., 1986;
Rodríguez-Tébar et al., 1989; Thanos et al., 1989;
Cohen-Cory and Fraser, 1994) and in vivo (Mey and
Thanos, 1993; Mansour-Robaey et al., 1994; Unoki and LaVail, 1995;
Weibel et al., 1995) [Voci et al. (1993), as cited in Garner et al.
(1996)]. BDNF has been suggested to be a trophic factor derived from
the tectum, and expression of BDNF mRNA in the tectum of several
different species supports this hypothesis (Leibrock et al., 1989;
Cohen-Cory and Fraser, 1994; Herzog et al., 1994). Attempts to
demonstrate retrograde transport of NGF in this system failed (Yip and
Johnson, 1983; Carmignoto et al., 1989), although BDNF is transported
from the tectum to the retina in adult rat (Fournier et al., 1997). Our data show that neurons of the retinorecipient layers of the optic tectum express BDNF, and we demonstrate, for the first time, that exogenously applied BDNF is transported from the tectum to the retina
in the developing animal. These findings are consistent with the
proposed role of BDNF as a target-derived trophic factor for RGC. RGC
axons respond to BDNF in the tectum by sprouting and arborization
(Cohen-Cory and Fraser, 1995). Thus, the role of BDNF as a
target-derived trophic molecule for RGC is well established. Surprisingly, however, our data indicate that most of the BDNF in the
RGC is not derived from the tectum, but comes from the retina
itself.
BDNF as an afferent trophic factor
Developing neurons are supported not only by trophic signals
derived from their target but also from their afferent inputs (Kelly
and Cowan, 1972; Oppenheim, 1991; Linden, 1994). Although several
target-derived trophic factors have been identified that are
transported retrogradely by their axons from the target to the cell
bodies, less is known about the trophic signals provided by afferents
or paracrine factors. Afferents are thought to have comparable trophic
effects on developing neurons as the target (Linden, 1994). Could BDNF
be this or one of these trophic factors? In the chick retina, most of
the BDNF-like immunoreactivity was found in the GCL. RGC lacking their
target showed a similar labeling intensity when compared with control
retinae, indicating that BDNF in the RGC is not attributable to
accumulation derived from the tectum. During naturally occurring cell
death, BDNF transcripts are located primarily in the outer half of the
INL, which contains the cell bodies of horizontal, bipolar, and
Müller cells. Based on the in situ hybridization of
sections in which RGC were identified by retrograde tracing, <3% of
ganglion cells express BDNF mRNA. Quantitative RT-PCR of the BDNF mRNA
levels in retinae with and without this cell population confirmed this
conclusion. Although some BDNF may be produced by RGC, the main portion
of BDNF in the retina is expressed by cells other than RGC. Besides a
subpopulation (15%) of amacrine cells in the GCL, these cells
presumably are horizontal and/or bipolar cells or Müller glia.
BDNF produced by these cells seems to accumulate in RGC, presumably
after anterograde (afferent) transport, release, and uptake by RGC
dendrites in the IPL. Anterograde transport of endogenous (Zhou and
Rush, 1996) and exogenous neurotrophins (von Bartheld et al., 1996a;
Johnson et al., 1997) has been demonstrated recently. The work of
Linden and collaborators (de Araujo and Linden, 1993; Linden, 1994;
AryPires et al., 1997) has shown that RGC respond to trophic factors
obtained from cells within the retina. RGC dendrites are thought to
compete for such a trophic factor during development (Perry and Linden, 1982; Linden, 1994). When we injected iodinated BDNF into the eye, it
accumulated in the IPL and the GCL, supporting the view that RGC can
accumulate BDNF from their surrounding tissue. The IPL showed the most
intense binding of exogenously applied BDNF, indicating that this
neuropil contains the vast majority of BDNF binding sites in the
retina. Both p75 and trkB receptor mRNAs are expressed by cells within
the GCL and at the inner border of the INL (von Bartheld et al., 1991;
Garner et al., 1996; Hallböök et al., 1996). These data are
consistent with the notion that amacrine and/or bipolar cells express
BDNF, which is transported anterogradely to the afferent terminals,
released, taken up by the dendrites of RGC, and transported to their
cell bodies.
What is the function of BDNF in the visual system?
In addition to its established survival functions in
vitro (Johnson et al., 1986; Rodríguez-Tébar et al.,
1989; Cohen-Cory and Fraser, 1994), BDNF increases in vivo
the survival of injured RGC in birds [Voci et al. (1993), as cited in
Garner et al. (1996)] and in mammals (Mey and Thanos, 1993;
Mansour-Robaey et al., 1994; Weibel et al., 1995). BDNF also regulates
the branching of developing axons in the tectum (Cohen-Cory and Fraser,
1995) and cortex (Cabelli et al., 1995) and the branching of dendritic
processes of cortical neurons in vitro (McAllister et al.,
1995, 1996). Another likely role of BDNF in the chick retina is to act
as a trophic factor for isthmo-optic neurons. Of this neuronal
population, 60% is eliminated during naturally occurring cell death
(Clarke and Cowan, 1976; Clarke, 1992). Cell death of a significant
number of isthmo-optic neurons can be delayed or prevented by
intraocular BDNF (von Bartheld et al., 1994; Primi and Clarke, 1996).
Expression of BDNF in the retina, therefore, may serve to support these
neurons.
The role of BDNF as a physiological survival factor for normal RGC
in vivo is less clear. Application of BDNF reduced the normal developmental RGC death in hamsters (Frost et al., 1997), but
not in chick embryos (Cellerino et al., 1995; Drum et al., 1996). The
number of cells in the ganglion cell layer was not visibly reduced in
BDNF 
/ mice (Jones et al., 1994), although RGC axons were
hypomyelinated in these animals (Cellerino et al., 1997
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Abstract
Introduction
