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RAPHAEL PINAUD
Neurological Sciences Institute and The Vollum Institute, OHSU
Portland, Oregen, USA
PETER DE WEERD
Department of Psychology, University of Arizona
Tucson, Arizona, USA
R. WILLIAM CURRIE
Department of Anatomy and Neurobiology
Dalhousie University
Halifax, Nova Scotia, Canada
LIISA A. TREMERE
Neurological Sciences Institute, OHSU
Portland, Oregen, USA
1275
1276 R. Pinaud et al.
Animals
Retinas from three adult male Cebus apella monkeys used in unre-
lated electrophysiological experiments were harvested post mortem
for use in the present study. All procedures conformed with in-
stitutional regulations and the guideline for the use of animal in
1278 R. Pinaud et al.
Immunocytochemistry
Untreated slides were removed from the –70°C freezer and tissue
sections hydrated for 30 min in PBS at room temperature (RT).
Nonspecific binding was minimized by incubating tissue in a block-
ing buffer that consisted of 0.5% albumin and 0.3% Triton X-100 in
0.1 M PBS, for 2 h (RT); this step was followed by 3 rinses in PBS
(10 min per wash). Sections were incubated in a solution containing
the polyclonal anti-egr-1 (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) or the monoclonal anti-Synapsin I (Chemicon Inc., Canada)
primary antibodies, at a dilution of 1:1000 in blocking buffer, inside
a humid chamber at 4°C, where they remained overnight. The fol-
lowing day, sections were washed for 30 min in PBS in order to
remove excess, unbound antibody. Sections selected for NGFI-A
staining were then incubated in a goat-anti-rabbit biotinylated anti-
body (IgG; Vector Laboratories, Burlingame, CA, USA), at the di-
lution of 1:200 in blocking buffer, for 2 h at RT, while tissue se-
lected for Synapsin I immunoreactivity was incubated in a solution
of biotinylated horse anti-mouse secondary antibody at a dilution of
1:400. Tissue was subsequently rinsed for 30 min in PBS. For Synapsin
I reactions, that were to be visualized using diaminobenzidine (DAB),
sections were incubated in a PBS containing avidin-biotin-complex,
(ABC; Vector Laboratories), for 2 h at room temperature at a dilution
High NGFI-A Basal Levels in Monkey Retina 1279
Imaging
Images were captured by use of a Spot camera (Diagnostics Instru-
ments Co., USA) coupled to an Axioplan microscope, and digi-
talized into a Power PC Macintosh computer. Fluorescent reactions
were converted to grayscale by Adobe Photoshop software (Pantone
Inc. USA). We also used this software to match background levels
and assemble the plate in this article.
RESULTS
NGFI-A Labeling
FIGURE 1. (A) Immunoreactive nuclei identified with the antibody directed against NGFI-
A. This reaction was visualized using streptavidin tagged with Cy3. The highest density
of nuclei was identified within the INL with scattered nuclei appearing in the GCL. The
packing density of immunoreactive nuclei appeared to be equal at all eccentricities. Scale
Bar = 30 µm. (B) Puncta and fiber labeling of the OPL and IPL with the monoclonal
antibody for Synapsin I visualized by DAB-Nickel reaction. Scale bar = 50 µm. (C) NGFI-
A immunopositive nuclei identified towards the IPL. These cells were distributed throughout
most of the retina and are likely to be amacrine cells. Scale Bar = 20 µm. (D) Equal
distribution of NGFI-A positive nuclei was also identified at basal levels near the OPL,
in positions that suggested that these were horizontal cells. Scale Bar = 20 µm. OPL =
outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL =
ganglion cell layer.
labeled cells in the outer part of the INL, adjacent to the border of
the outer plexiform layer (OPL), were distributed in an equidistant
fashion, a pattern and location that likely corresponds to horizontal
cells (Figure 1D). NGFI-A positive nuclei were also observed in
INL regions that were closer to the inner plexiform layer (IPL).
Given their relative location, these were postulated to be amacrine
cells (Figure 1C). In the ganglion cell layer, NGFI-A IR nuclei were
also detectable but were relatively modest in number (Figure lA).
By contrast to other retinal cell layers, immunoreactivity for NGFI-
High NGFI-A Basal Levels in Monkey Retina 1281
Synapsin I Labeling
The antibody directed against Synapsin I revealed puncta, within
the IPL (Figure 1B). The distribution of these immunoreactive structures
was diffuse and with an apparent homogeneous distribution for all
eccentricities of the retina (Figure 1B). Synapsin I immunolabeling
was also found in varicose fibers in the OPL and INL (Figure 1B),
a result that is in accordance with previously described data ob-
tained in the macaque monkey retina (Koontz & Hendrickson, 1993).
To the best of our knowledge, Synapsin I IR distribution has not
been previously described for this species of monkey. Although a
detailed description of the Synapsin I immunolabeling was beyond
the scope of the present work, our results are similar to other re-
ports conducted in the retina of other monkey species (Koontz &
Hendrickson, 1993).
DISCUSSION
The principle finding of the present article is that there is high basal
expression of the inducible IEG NGFI-A in the retina of the adult
primate. Both NGFI-A and Synapsin I proteins have been postu-
lated to reflect mechanisms of neural plasticity that enable reorgani-
zation of existing sensory pathways during episodes of increased
restructural pressure (Baekelandt et al., 1994; Wallace et al., 1995;
Pinaud et al., 2002a; Pinaud et al., 2002b).
Some of the clearest indications of how IEGs may play a role
1282 R. Pinaud et al.
CONCLUSION
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