Você está na página 1de 11

Intern. J.

Neuroscience, 113:1275–1285, 2003

Copyright  Taylor & Francis Inc.
ISSN: 0020-7454 / 1543-5245 online
DOI: 10.1080/00207450390212537



Neurological Sciences Institute and The Vollum Institute, OHSU
Portland, Oregen, USA

Department of Psychology, University of Arizona
Tucson, Arizona, USA

Department of Anatomy and Neurobiology
Dalhousie University
Halifax, Nova Scotia, Canada


Instituto de Biofisica Carlos Chagas Filho
Universidade Federal do Rio de Janeiro
Rio de Janeiro, Brazil

Neurological Sciences Institute, OHSU
Portland, Oregen, USA

Received 28 January 2003.

The authors would like to thank Prof. Ricardo Gattass for kindly donating the monkey retinas
for the present study. CIHR, CNPq, and the Heart and Stroke Foundation of New Brunswick
supported this work. Dr. Pinaud was an Izaak Walton Killam Scholar during the course of these
Address correspondence to Liisa A. Tremere, PhD, Neurological Sciences Institute, Oregon
Health & Science University, 505 N.W. 185th Avenue, West Campus, Building 1, Portland, OR
97006-3499, USA. E-mail: tremerel@ nims.ohsu.edu

1276 R. Pinaud et al.

In rodents, enriched environments drive the expression of the immediate

early gene NGFI-A, a regulator of the plasticity marker Synapsin I.
Both proteins have been implicated as mediators of plasticity in the rat
mammalian retina. In the present work immunocytochemistry directed
against these proteins was used to explore their basal activity in the
retina of a more visual species, the New World monkey Cebus apella. In
contrast to rat, monkey retina displayed high basal expression of both
NGFI-A and Synapsin I. The greatest number of NGFI-A-expressing
cells was observed within the inner nuclear layer, although NGFI-A
positive nuclei were also found in the ganglion cell layer. High levels of
Synapsin I were found in the inner plexiform layer and outer plexiform
layer. Our findings are consistent with the postulate that the retinas of
highly visual animals may experience ongoing reorganization as part of
normal visual processing, and that NGFI-A and Synapsin I may be well
positioned to regulate some of these changes.
Keywords NGFI-A, plasticity, rewiring, reorganization, Synapsin I, tran-

The phenomenon of neural plasticity in the adult vertebrate retina

has been used to explain the processes of light adaptation, as well
as the enhancement of local visual processing (Behrens et al., 1998;
Li et al., 2001; Baccus & Meister, 2002). The first type of plasticity
has been well characterized in photoreceptor synapses and is related
to the daily shift between rod versus cone dominant or night and
day vision, respectively (Nachman-Clewner et al., 1999; Vollrath &
Spiwoks-Becker, 1996; Haamedi & Djamgoz, 1996). A second form
of retinal plasticity, referred to as molecular plasticity, has been
hypothesized to account for genetically-driven altered retinal con-
nectivity that may accompany increased processing demands placed
on the visual system (Pinaud et al., 2002a).
While the retina demonstrates an enormous dynamic processing
range that can be achieved with pre-existing circuitry, it is also
clear that the finer connectivity of the retina is plastic and can, in
the absence of pathology, undergo reorganization (Tian & Copenhagen,
2001). There is some evidence that these mechanisms of plasticity
may be harnessed to enhance visual processing, for example, heightened
visual acuity was recently described for young adult rats that were
reared in an enriched environment (Prusky et al., 2000).
Even though a number of studies have demonstrated the adaptive
nature of the retina towards enhanced function, the molecular and
biochemical determinants of such processes remain poorly described.
High NGFI-A Basal Levels in Monkey Retina 1277

Numerous studies have indicated roles for immediate early genes

(IEGs) as both signals and regulators of neural development and
reorganizational processes in mammals (Herdegen & Leah, 1998;
Hughes et al., 1999).
Recently, we have demonstrated that exposure of animals to an
enriched sensory environment (EE), a protocol that reliably induces
plasticity in the brain, produced dramatic changes in the expression
levels of two IEGs in the rat retina (Pinaud et al., 2002a). One of
these genes, NGFI-A (also known as zif268, egr-1, krox-24, and
ZENK) belongs to a group of genes that is activated rapidly and
transiently in the initial phase of the genetic response to sensory
stimuli (Hughes & Dragunow, 1995; Herdegen & Leah, 1998). NGFI-
A is a zinc-finger transcriptional regulator whose binding site is
found in the promoter region of a great number of genes expressed
within the central nervous system. More interestingly, it has been
previously demonstrated that NGFI-A regulates the expression of
Synapsin I, a late response gene that has been identified as a corre-
late of reorganized neuronal connectivity (Thiel et al., 1994; Melloni
et al., 1994; Marti et al., 1999).
In the present work we hypothesized that in highly visual ani-
mals, such as monkeys, the biological need for rapid fine-tuning of
the retinal circuitry is reflected in the differential regulation of the
IEG NGFI-A. Indeed, our results demonstrate that unlike rodents,
where NGFI-A basal levels are relatively low, the expression of this
IEG was found at high basal levels in the monkey retina. Further-
more, expression levels of Synapsin I were also found at relatively
high levels, supporting earlier descriptions of this NGFI-A target
gene, as being involved in synaptic plasticity.



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.

experimental research established by the Brazilian Society of Neuro-

science and Behavior and are in accordance with NIH guidelines.

Perfusion and Histological Procedures

Animals were deeply anesthetized by an overdose of sodium pento-
barbital and perfused transcardially with physiological saline fol-
lowed by formalin. Eyes were then removed, sectioned at the equa-
tor, and further post-fixed in buffered 4% paraformaldehyde (0.1 M,
pH 7.4) for 3 h. After fixation, eyes were rinsed in 0.1 M phosphate
buffered saline (PBS). The eye-cup preparations were then equili-
brated in a 30% sucrose solution and sectioned at 20 µm on a cry-
ostat. Sections were mounted onto Fisherbrand Superfrost Plus slides
(Fischer Scientific, USA), air-dried, and stored at –70°C.

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

of 1:100. This step was followed by another 30-min wash in PBS.

We developed these sections by incubating them in a filtered solu-
tion containing 0.03% DAB and 0.15% nickel sulfate in 0.1 M PBS
to which 0.001% hydrogen peroxide had been added. Sections were
observed under a light microscope as the reaction developed and,
after satisfactory “signal to background” was achieved, tissue was
dehydrated in a standard series of alcohols, exposed to xylenes and
coverslipped with Entellan (BDH, Darmstadt, Germany). For NGFI-
A immunolabeling, after incubation with biotinylated secondary anti-
body, tissue was washed in PBS for 30 min and incubated in a
solution of streptavidin tagged with either carbocyanine 2 or 3 (Cy2
or Cy3; Vector Laboratories), at the dilution recommended by the
manufacturer. Tissue was then mounted with Fluoromount-G (Southern
Biotechnology Associates Inc., Birmingham, AL, USA).
Controls for the immunocytochemistry procedures were carried
out by omitting the primary antibodies, which resulted in no immuno-

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.


NGFI-A Labeling

NGFI-A positive nuclei were found throughout the retinal tissue in

all levels analyzed. The majority of immunoreactive (IR) nuclei
were identified within the inner nuclear layer (INL) (Figure 1A).
The uniformity of size and tight organization of the NGFI-A IR
cells strongly suggested that these were most likely to belong to
various classes of bipolar cells (Figure 1A). However, many of the
1280 R. Pinaud et al.

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

A was not detectable in photoreceptor cell nuclei. With this excep-

tion in mind, immunolabeling could otherwise be detected in all
major cell types within the retina. It is important to note that, based
on comparisons with Nissl-stained adjacent sections, it was clear
that in each of the immunolabeled layers, NGFI-A was only present
in a fraction of the total number of cells. Furthermore, the intensity
of the nuclear labeling was highly variable, even for neighboring
No obvious differences in the relative distribution of IR nuclei
were found between central and peripheral retina.

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).


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.

in mediating neuronal plasticity result from long-term potentiation

studies or exposure of physiologically healthy rodents to enriched
sensory environments (Wallace et al., 1995; Jones et al., 2001; Bozon
et al., 2002; Pinaud et al., 2002b). Both of these protocols have
been previously demonstrated to induce plasticity within the cere-
bral cortex, resulting in both morphological and functional modifi-
cations (Rosenzweig et al., 1972; Volkmar & Greenough, 1972;
Globus et al., 1973; Bear & Malenka, 1994; Hughes et al., 1999;
Prusky et al., 2000; Toni et al., 2001). More specifically, we have
previously demonstrated that exposure of rats to an EE drives the
expression of both mRNA and protein for NGFI-A mainly over
sensory cortices, such as those of the visual and somatosensory sys-
tems, in addition to the hippocampal formation (Pinaud et al., 2002b).
While most investigations on the role of IEGs in regulating neu-
ronal plasticity have focused on the brain, our group was the first to
demonstrate that the EE produced dramatic changes in the expres-
sion levels of NGFI-A in the retinas of adult rats (Pinaud et al.,
2002a). In this earlier study, pronounced upregulation of NGFI-A
occurred only in those animals that were exposed to an EE, but not
in those controls that were handled or left undisturbed (Pinaud et
al., 2002a). It was our interpretation that the complexity of the sen-
sory environment induced the expression of this IEG and that its
expression reflected sites where the mechanism involved in mediat-
ing plastic changes in retinal circuitry had been engaged (Pinaud et
al., 2002a). These hypotheses were also supported by our findings
that EE-stimulated animals also displayed a significant upregulation
in Synapsin I levels, a target gene for NGFI-A, and a standard
marker for neuronal plasticity (Pinaud et al., 2002a; Thiel et al.,
In contrast to the low constitutive NGFI-A levels found in the
rodent retina, we show here that in primate, expression of this IEG
was found at relatively high basal levels. If one accepts that NGFI-
A plays a role in the regulation of neural plasticity, then it follows
that the high basal levels found in a visual species, but not in a
nonvisual species, are likely to reflect a greater frequency of fine-
tuning (or ongoing plasticity) of retinal circuitry for those animals
that are more dependent on vision.
The antibody directed against the NGFI-A protein identified cell
High NGFI-A Basal Levels in Monkey Retina 1283

nuclei in several regions of the retina. It is noteworthy that labeling

could be detected for putative horizontal and amacrine cells. We
propose that these classes of retinal neurons may be especially well
positioned to influence states of plasticity during visual processing
and modulate these hypothetical, adaptive reorganizational changes.
Finally, the distribution of Synapsin I has been well character-
ized within the primate retina (Koontz & Hendrickson, 1993). This
protein has been implicated in governing neurotransmitter release
by regulating the size of the readily releasable pool of vesicles (Turner
et al., 1999; Hilfiker et al., 1999). In the retina, antibodies for Synapsin
I appear selectively to recognize conventional synapses, the major-
ity of which belonging to amacrine cells (Mandell et al., 1990; Koontz
& Hendrickson, 1993). Given that Synapsin I is regulated by NGFI-
A (Thiel et al., 1994) and that the heaviest labeling for Synapsin I is
localized in amacrine cells (Mandell et al., 1990; Koontz & Hendrickson,
1993), we argue that amacrine cells can be considered excellent
candidates to mediate many aspects of neural plasticity within the
adult mammalian retina.


This article demonstrates that the IEG NGFI-A is expressed at high

basal levels in the retina of the adult monkey. The greatest density
of NGFI-A immunoreactive cells was identified in the INL. Given
that sites with increased NGFI-A expression may reflect enhanced
potential for neural plasticity, the data presented in the current study
suggest that mechanisms of neuronal plasticity are primed in the
retina of the adult monkey, and that both amacrine and horizontal
cells may have a special role in mediating or facilitating changes in
retinal connectivity.


Baccus, S. A., & Meister, M. (2002). Fast and slow contrast adaptation in retinal circuitry.
Neuron, 36, 909–919.
Baekelandt, V., Arckens, L., Annaert, W., Eysel, U. T., Orban, G. A., & Vandesande, F.
(1994). Alterations in GAP-43 and synapsin immunoreactivity provide evidence for
1284 R. Pinaud et al.

synaptic reorganization in adult cat dorsal lateral geniculate nucleus following retinal
lesions. European Journal of Neuroscience, 6, 754–765.
Bear, M. F., & Malenka, R. C. (1994). Synaptic plasticity: LTP and LTD. Current Opinion
in Neurobiology, 4, 389–399.
Behrens, U. D., Kasten, P., & Wagner, H. J. (1998). Adaptation-dependent plasticity of
rod bipolar cell axon terminal morphology in the rat retina. Cell & Tissue Research,
294, 243–251.
Bozon, B., Davis, S., & Laroche, S. (2002). Regulated transcription of the immediate-
early gene Zif268: Mechanisms and gene dosage-dependent function in synaptic plas-
ticity and memory formation. Hippocampus, 12, 570–577.
Globus, A., Rosenzweig, M. R., Bennett, E. L., & Diamond, M. C. (1973). Effects of
differential experience on dendritic spine counts in rat cerebral cortex. Journal of Comparative
Physiology and Psychology, 82, 175–181.
Haamedi, S. N., & Djamgoz, M. B. (1996). Effects of different patterns of light adaptation
on cellular and synaptic plasticity in teleost retina: Comparison of flickering and steady
lights. Neuroscience Letters, 206, 93–96.
Herdegen, T., & Leah, J. D. (1998). Inducible and constitutive transcription factors in the
mammalian nervous system: Control of gene expression by Jun, Fos and Krox, and
CREB/ATF proteins. Brain Research Reviews, 28, 370–490.
Hilfiker, S., Pieribone, V. A., Czernik, A. J., Kao, H. T., Augustine, G. J., & Greengard, P.
(1999). Synapsins as regulators of neurotransmitter release. Philosophical Transactions
of the Royal Society of London Series B—Biological Sciences, 354, 269–279.
Hughes, P., & Dragunow, M. (1995). Induction of immediate-early genes and the control
of neurotransmitter-regulated gene expression within the nervous system. Pharmaco-
logical Reviews, 47, 133–178.
Hughes, P. E., Alexi, T., Walton, M., Williams, C. E., Dragunow, M., Clark, R. G., &
Gluckman, P. D. (1999). Activity and injury-dependent expression of inducible tran-
scription factors, growth factors and apoptosis-related genes within the central nervous
system. Progress in Neurobiology, 57, 421–450.
Jones, M. W., Errington, M. L., French, P. J., Fine, A., Bliss, T. V., Garel, S., Chamay, P.,
Bozon, B., Laroche, S., & Davis, S. (2001). A requirement for the immediate early
gene Zif268 in the expression of late LTP and long-term memories. Nature Neuro-
science, 4, 289–296.
Koontz, M. A., & Hendrickson, A. E. (1993). Comparison of immunolocalization patterns
for the synaptic vesicle proteins p65 and synapsin I in macaque monkey retina. Syn-
apse, 14, 268–282.
Li, F., Cao, W., & Anderson, R. E. (2001). Protection of photoreceptor cells in adult rats
from light-induced degeneration by adaptation to bright cyclic light. Experimental Eye
Research, 73, 569–577.
Liu, L. O., Li, G., McCall, M. A., & Cooper, N. G. (2000). Photoreceptor regulated ex-
pression of Ca(2+)/calmodulin-dependent protein kinase II in the mouse retina. Mo-
lecular Brain Research, 82, 150–166.
Mandell, J. W., Townes-Anderson, E., Czemik, A. J., Cameron, R., Greengard, P., & De
Camjlli, P. (1990). Synapsins in the vertebrate retina: Absence from ribbon synapses
and heterogeneous distribution among conventional synapses. Neuron, 5, 19–33.
Marti, E., Ferrer, I., & Blasi, J. (1999). Transient increase of synapsin-I immunoreactivity
in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the
mongolian gerbil. Brain Research, 824, 153–160.
Melloni, R. H., Apostolides, P. J., Hamos, J. E., & De Gennaro, L. J. (1994). Dynamics of
synapsin I gene expression during the establishment and restoration of functional syn-
apses in the rat hippocampus. Neuroscience, 58, 683–703.
Nachman-Clewner, M., St Jules, R., & Townes-Anderson, E. (1999). L-type calcium chan-
nels in the photoreceptor ribbon synapse: Localization and role in plasticity. Journal
of Comparative Neurology, 415, 1–16.
High NGFI-A Basal Levels in Monkey Retina 1285

Pinaud, R., Tremere, L. A., Penner, M. R., Hess, F. F., Barnes, S. Robertson, H. A., &
Currie, R. W. (2002a). Plasticity-driven gene expression in the rat retina. Molecular
Brain Research, 98, 93–101.
Pinaud, R., Tremere, L. A., Penner, M. R., Hess, F. F., Robertson, H. A., & Currie, R. W.
(2002b). Complexity of the sensory environment drives the expression of candidate-
plasticity gene, nerve growth factor induced-A. Neuroscience, 112, 573–582.
Prusky, G. T., Reidel, C., & Douglas, R. M. (2000). Environmental enrichment from birth
enhances visual acuity but not place learning in mice. Behavioral Brain Research, 114,
Rosenzweig, M. R., Bennett, E. L., & Diamond, M. C. (1972). Brain changes in relation
to experience. Scientific American, 226, 22–29.
Thiel, G., Schoch, S., & Petersohn D. (1994). Regulation of synapsin I gene expression by
the zinc finger transcription factor zif268/egr-1. Journal of Biological Chemistry, 269,
Tian, N., & Copenhagen, D. R. (2001). Visual deprivation alters development of synaptic
function in inner retina after eye opening. Neuron, 32, 439–449.
Toni, N., Buchs, P. A., Nikonenko, I., Povilaitite, P., Parisi, L., & Muller, D. (2001). Re-
modeling of synaptic membranes after induction of long-term potentiation. Journal of
Neuroscience, 21, 6245–6251.
Turner, K. M., Burgoyne, R. D., & Morgan, A. (1999). Protein phosphorylation and the
regulation of synaptic membrane traffic. Trends in Neuroscience, 22, 459–464.
Volkmar, F. R., & Greenough, W. T. (1972). Rearing complexity affects branching of
dendrites in the visual cortex of the rat. Science, 176, 1445–1447.
Vollrath, L., & Spiwoks-Becker, I. (1996). Plasticity of retinal ribbon synapses. Micros-
copy Research and Technique, 35, 472–487.
Wallace, C. S., Withers, G. S., Weiler, I. J., George, J. M., Clayton, D. F., & Greenough,
W. T. (1995). Correspondence between sites of NGFI-A induction and sites of mor-
phological plasticity following exposure to environmental complexity. Molecular Brain
Research, 32, 211–220.