Cyclic AMP Prevents Retraction of Axon Terminals in
Photoreceptors Prepared for Transplantation:
An In Vitro Study
Mohamad A. Khodair, Marco A. Zarbin, and Ellen Townes-Anderson
PURPOSE. Cell transplantation has emerged as a possible remedy
for degeneration and injury in the central nervous system
(CNS). In the retina, photoreceptor transplantation is a potential treatment for retinal degenerative disease. Graft survival
has been well documented, but evidence of functional recovery is lacking. A major obstacle to recovery of vision is lack of
synapse formation between grafted photoreceptors and host
bipolar and horizontal cells. A prior study demonstrated that
photoreceptors prepared for transplantation undergo rapid
morphologic changes, including retraction of axon terminals
toward their cell bodies, away from potential synaptic partners, a phenomenon that may interfere with graft– host synaptic interaction after transplantation. In this study, prevention of
retraction of photoreceptor axon terminals was possible by
pharmacological intervention.
METHODS. Photoreceptor sheets, prepared by vibratome sectioning, and full-thickness retinas, harvested from adult porcine
eyes, were maintained in culture and treated with either the
cyclic adenosine monophosphate analogue 8-(4-chlorophenylthio)-cyclic 3⬘,5⬘-adenosine monophosphate (CPT-cAMP), or forskolin, an adenylyl cyclase stimulant, for up to 48 hours.
RESULTS. Both CPT-cAMP and forskolin treatments successfully
blocked retraction of photoreceptor axon terminals. This effect was not due to cell toxicity and was reversed after removal
of treatment, indicating its specificity.
CONCLUSIONS. Pharmacological manipulation of photoreceptor
axonal plasticity may improve graft– host synaptic interaction
after subretinal photoreceptor cell transplantation. (Invest
Ophthalmol Vis Sci. 2005;46:967–973) DOI:10.1167/iovs.040579
W
hether adult, fetal, or stem cell populations are used in
transplantation into the CNS, restoration of function
depends in part on the formation of synapses between the graft
and host nervous tissue. Even though transplants may mediate
a trophic effect that can preserve residual host function, reestablishing normal activity demands reconstruction of synaptic networks. Photoreceptor cell transplantation is a potential
technique for restoration of vision in patients with retinal
From the Departments of Neurology and Neurosciences and Ophthalmology, University of Medicine and Dentistry of New Jersey
(UMDNJ)-New Jersey Medical School, Newark, New Jersey.
Supported by National Eye Institute Grant EY12031 (ET-A); the
F. M. Kirby Foundation (ET-A, MAZ); Research to Prevent Blindness,
Inc.; NJ Lions Eye Research Foundation; and Eye Institute of New
Jersey (MZ).
Submitted for publication May 22, 2004; revised September 30,
2004; accepted October 5, 2004.
Disclosure: M.A. Khodair, None; M.A. Zarbin, None; E.
Townes-Anderson, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Mohamad A. Khodair, UMDNJ-New Jersey
Medical School, Neurology and Neurosciences/Ophthalmology, 185
South Orange Avenue, Newark, NJ 07103; [email protected].
degenerative diseases, such as retinitis pigmentosa and agerelated macular degeneration, in which there is substantial loss
of rod and cone cells. However, the scarcity of neural process
outgrowth from transplanted photoreceptors as well as the
absence of definite synaptic contacts with the appropriate host
second-order neurons is a recurring result in all transplantation
paradigms, regardless of the type of preparation or age of
donor tissue.1–5
The apparent lack of synaptic regeneration by transplanted
photoreceptors contrasts with the growth of processes by both
rod and cone photoreceptors and by other cells of the retina
after isolation in culture,6 – 8 in experimental retinal detachment and reattachment,9,10 and during retinal degeneration in
both animal models11–15 and human disease.16 In these cases,
second-order neurons extend elaborate dendrites, and rod and
cone cells grow neurites and form numerous presynaptic varicosities. To investigate whether photoreceptors prepared as
grafts for retinal transplantation retain the ability to extend
neural processes, we developed an in vitro system for the
maintenance of adult porcine photoreceptor sheets and intact
retinas in culture. We found that photoreceptors do not extend
but instead retract their axons and terminals toward the cell
body within hours of being detached from the retinal pigment
epithelium (RPE),17 similar to what occurs shortly after retinal
detachment or photoreceptor cell isolation.8 –10 Retraction
may represent an impediment to establishing graft– host synaptic contacts.
Cyclic 3⬘,5⬘-adenosine monophosphate (cAMP) has been
shown to abolish pharmacologically induced neurite retraction
in neuronal cell lines18 and growth factor–induced growth
cone collapse as well as neurite retraction in primary neuronal
cultures.19 Furthermore, cAMP, through its downstream effector protein kinase A (PKA), blocks myelin and the myelinassociated glycoprotein inhibition of neurite outgrowth in cultured rat retinal ganglion cells, dorsal root ganglion neurons,
cerebellar neurons, and raphespinal projection neurons.20 In
the present study, we increased intracellular levels of the
second-messenger cAMP to pharmacologically manipulate photoreceptor axonal plasticity. Retraction was prevented
whether cAMP was applied exogenously, using the membranepermeable cAMP analogue 8-(4-chlorophenylthio)-cyclic 3⬘,5⬘adenosine monophosphate (CPT-cAMP) or increased by activation of endogenous adenylyl cyclase with forskolin. These
effects were reversible and did not affect cell viability. These
data have been presented in abstract form (Khodair MA, et al.
IOVS 2001;42:ARVO Abstract 4192).
MATERIALS
AND
METHODS
Experimental Animals
Twenty-eight eyes from 14 adult male and female Yorkshire pigs, 3 to
5 months old and weighing 25 to 55 kg (Animal Biotech Industries,
Danbora, PA), served as donors of retinal tissue. Animals were kept on
a 12-hour light– dark cycle and fed porcine chow, ad libitum. They
were killed at 9:00 AM by anesthetizing with telazol (7 mg/kg; 100
mg/mL), xylazine (2.2 mg/kg; 100 mg/mL), and atropine (0.02 mg/kg;
0.54 mg/mL) administered intramuscularly, followed by an overdose of
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pentobarbital sodium, 1 mL/4.5 kg, administered intravenously. Experimental procedures adhered to the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and were approved by the
University of Medicine and Dentistry of New Jersey-New Jersey Medical School Institutional Animal Care and Use Committee.
Preparation and Culture of Photoreceptor Sheets
and Full-Thickness Retinas
Photoreceptor sheets were prepared by vibratome sectioning with a
method modified from Huang et al.21 and previously described.17
Briefly, retinas still attached to the underlying RPE, choroid and sclera,
were cut from the central and midperipheral regions of the eyecup
with a trephine. Tissue was kept at 4°C at all times in Eagle’s minimum
essential medium (MEM, 10370-021; Invitrogen-Gibco-Life Technologies Inc., Rockville, MD), pH 7.4, supplemented with 0.292 mg/mL
glutamine, 10% (vol/vol) fetal calf serum, 10 ␮g/mL porcine insulin, 5.5
mM D-glucose, 1 mM pyruvate, 0.1 mM taurine, 2.0 mM ascorbic acid,
100 U/mL penicillin, 100 ␮g/mL streptomycin, and 250 ng/mL amphotericin B, and aerated with humidified 5% CO2 and 95% O2. To obtain
photoreceptor sheets, we gently detached the neural retina from the
underlying tissues and mounted it on a sterile gelatin block with the
photoreceptor outer segments (OSs) oriented downward. Inner retinal
layers were removed sequentially by vibratome sectioning. Sectioning
was terminated at the level at which one to two cells of the inner
nuclear layer (INL) remained, to ensure the intactness of the photoreceptor terminals. The sectioned retina and an underlying gelatin layer
were then placed on a glass slide, covered by another 100-␮m-thick
gelatin layer, incubated for 30 to 60 seconds at 37°C to allow the
gelatin layers to melt, and finally placed on ice to resolidify. A photoreceptor sheet embedded in a gelatin sandwich, with a total thickness
of approximately 250 to 300 ␮m, was thus produced.
For full-thickness preparations, retinas were embedded in gelatin
sandwiches according to the procedure just described, but without
sectioning the retina.
Photoreceptor sheets and full-thickness retinas were bathed in
fortified MEM (pH 7.4), with 0.1, 0.5, or 1 mM CPT-cAMP (C3912;
Sigma-Aldrich, St. Louis, MO) or forskolin (F-6886; Sigma-Aldrich) dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted with
medium to obtain final concentrations of 0.1, 1, or 10 ␮M in 0.1%
DMSO. Specimens were incubated for ⬍45 minutes, 24 hours, or 48
hours at 37°C in a humidified mixture of 5% CO2 and 95% O2. For
comparison of photoreceptor sheets and full-thickness retinas, one eye
of a donor pig was used to obtain sheets, and the contralateral eye was
used to obtain retinal preparations. Control samples were photoreceptor and retinal preparations obtained from the same retinal areas as in
the treatment groups, but incubated in fortified MEM only or with 0.1%
DMSO. Full-thickness retinas fixed immediately after detachment from
the RPE served as overall control specimens for both treated and
untreated groups.
Immunohistochemistry
At the end of incubation, specimens were fixed in 4% paraformaldehyde in 0.125 M phosphate buffer (PB; pH 7.4) overnight at room
temperature, rinsed in phosphate-buffered saline (PBS), re-embedded
in gelatin, and returned to the fixative for at least an additional 24 hours
at 4°C. Fixed, gelatin-embedded specimens were rinsed in PBS,
mounted on a tissue cutter (Sorvall, Newtown, CT), chopped into
100-␮m-thick sections, and immunolabeled as previously described.8,17
Rabbit polyclonal anti-synaptophysin (1:100; Dako Corp., Carpinteria,
CA), which localizes synaptic vesicles, was used to evaluate morphologic changes in photoreceptor terminals. The mouse monoclonal
antibody specific for rod opsin (4D2; 1:25; kindly provided by Robert
Molday, University of British Columbia, Vancouver, BC, Canada) was
used to identify rod photoreceptor membranes. Goat anti-rabbit-FITC
and goat anti-mouse-tetramethylrhodamine isothiocyanate (TRITC;
1:35) were used as secondary antibodies. Control sections were processed without primary antibodies.
IOVS, March 2005, Vol. 46, No. 3
Morphometric Analysis
One-micrometer-thick optical sections of photoreceptor sheets and
full-thickness retinas were obtained with a laser scanning confocal
microscope equipped with an argon/krypton laser and a 63⫻, 1.4
numerical aperture (N/A) oil-immersion objective lens (LSM-410; Carl
Zeiss Meditec, Thornwood, NY). Brightness and contrast were set to
obtain unsaturated images. Parameters were maintained throughout a
single experiment. Using normalized settings, we were able to detect
changes in labeling patterns between specimens. Adjustments in
brightness and contrast were performed after image analysis, exclusively for presentation purposes.
Photoreceptor axonal and terminal retraction, indicated by the area
of synaptophysin labeling in the ONL, was analyzed using NIH Image
1.62 software (http://rsb.info.nih.gov/nih-image; developed by Wayne
Rasband and provided in the public domain by the National Institutes
of Health, Bethesda, MD). Images were opened in the fluorescein
channel and a standard threshold was assigned to all images of the
same experiment to eliminate background fluorescence. The total area
of fluorescein labeling was measured within a rectangular area, outlining the ONL, as previously described.17 Measurements were in square
pixels, scaled at 6 pixels/␮m. Data were collected from nine sections
per specimen. Statistical analysis was performed with the unpaired
t-test and one-way analysis of variance (ANOVA), with Tukey’s post
hoc test for all pair-wise multiple comparisons, when appropriate.
Evaluation of Photoreceptor Viability in Culture
Photoreceptor viability was assessed by the lactate dehydrogenase
(LDH) release assay. Photoreceptor sheets and full-thickness retinal
preparations were incubated at 37°C in supplemented Eagle’s MEM (as
described earlier) without phenol red (51200-038; Invitrogen-GibcoLife Technologies, Inc.) only, or with either 1 mM CPT-cAMP or 10 ␮M
forskolin for ⬍45 minutes, 24 hours, and 48 hours. Specimens treated
with 100 ␮M ouabain were incubated under similar conditions and
served as the positive control. Tissue-free aliquots were centrifuged at
250g for 4 minutes to remove cellular debris. Supernatant was processed with an LDH in vitro toxicology assay kit (TOX-7; Sigma-Aldrich). Colorimetric absorbances were obtained with a spectrophotometer (Du series 60; Beckman Instruments Inc., Fullerton, CA) at a
490-nm wavelength. Medium-only represented the blank solution, and
medium reacted according to the LDH assay kit instructions served as
the background.
RESULTS
Effect of CPT-cAMP on Photoreceptor Axon
Terminal Retraction
Two types of grafts were examined: sheets of photoreceptor
cells prepared by vibratome sectioning and full-thickness retinas. CPT-cAMP was chosen for treatment of the preparations
because it is a C-8 substituted cAMP analogue that is more
efficacious in promoting long-term survival and neurite outgrowth than N6-substituted analogues in neuronal cell culture.22 In addition, CPT-cAMP is highly membrane permeable
because of its lipophilic C-8-substituent and unlike other analogues, such as 8-Br-cAMP, it can be hydrolyzed by cAMPspecific phosphodiesterase (PDE). A potential disadvantage of
CPT-cAMP is that it is a competitive inhibitor of the cGMPspecific phosphodiesterase (PDE5a),23 which may elevate intracellular cGMP levels. However, this may have had a minimal
effect on our experiments, because the predominant PDE in
photoreceptors is PDE6.
For normal morphology, retinas fixed immediately after
removal from the eyecup (Fig. 1A) were immunolabeled with
anti-opsin and anti-synaptophysin antibodies, to delineate photoreceptor cell membranes and synaptic vesicles, respectively.
Opsin labeling was confined primarily to photoreceptor OSs,
which were relatively rectilinear. The outer nuclear layer
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FIGURE 1. Intact retinas or photoreceptor sheets, in medium alone or with CPT-cAMP, double-labeled for synaptophysin (green) and rod opsin
(red). (A) Control retina fixed immediately after removal from the eyecup. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL,
outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; and NFL, nerve fiber layer. Opsin labeling was
primarily confined to the rod OSs (arrowheads), which appeared well formed. Synaptophysin labeling highlighted presynaptic terminals in the IPL
and rod and cone terminals in the OPL. Some synaptophysin labeling was also present in the ISs (arrows). (B) Photoreceptor sheets with no
treatment after 48 hours in vitro labeled for synaptophysin (top left), opsin (top right), and double labeled (bottom). Synaptophysin labeling was
present in the OPL and ONL (arrows). Individual terminals were seen retracting toward their cell bodies, creating gaps in OPL labeling (large
arrowheads). Opsin labeling was present in the OS, cell body, and terminal of rod cells (small arrowheads). Synaptophysin and opsin colocalized
in both the retracting and stable photoreceptor terminals (yellow). OSs were distorted. (C) Photoreceptor sheet treated with 0.1 mM CPT-cAMP
for 48 hours. Synaptophysin labeling remained confined to the OPL (arrowheads), indicating the inhibition of axon terminal retraction. Opsin
labeling remained dispersed. Arrows: OSs. (D) Higher magnification of CPT-cAMP-treated photoreceptor sheet. Left: opsin labeling (rhodamine
channel) delineated the entire rod photoreceptor cell membrane, including the terminals. Middle: synaptophysin-labeled terminals (fluorescein
channel) remained in the OPL. Right: opsin and synaptophysin (both channels) colocalized in the terminals. (E) Full-thickness retina without
treatment for 48 hours. As in the untreated photoreceptor sheet, synaptophysin labeling spread into the ONL and opsin throughout the rod cells.
(F) Retina with 0.1 mM CPT-cAMP treatment for 48 hours showed little movement of synaptic labeling (arrows) but substantial opsin labeling in
the ONL (arrowheads). Scale bar: (A, E) 25 ␮m; (D) 10 ␮m. (G, H) Treatment with 0.1, 0.5, or 1 mM CPT-cAMP. (G) Photoreceptor sheets receiving
no treatment showed a significant increase in ONL labeling compared with the control by 45 minutes in vitro. The area of labeling in the ONL
increased approximately 10-fold by 24 hours and was slightly increased again after 48 hours in culture. For treated specimens, there was no
significant difference in the area of ONL synaptophysin labeling among treatment groups or in the control group for up to 24 hours. By 48 hours,
treatment groups showed small but significant increases in labeling compared with the control, but no difference compared with each other.
Labeling in the ONL of treated specimens was only 7% to 11% of that in untreated specimens. (H) Full-thickness retinas showed a similar pattern.
Untreated retinas had a significant increase in ONL labeling compared with control and treatment groups. Treated specimens showed only a small,
but significant, increase in labeling when compared with control specimens at all time points. *P ⬍ 0.001; mean ⫾ SEM; n ⫽ 10 eyes of five animals.
(ONL) was composed of five to seven layers of uniformly
spaced photoreceptor nuclei and showed no signs of pyknosis.
Synaptophysin labeling was detected in both plexiform layers.
In the outer plexiform layer (OPL), however, synaptophysin
labeling was confined to the sclerad half of the layer where
photoreceptor terminals are located. Photoreceptor axons and
the ONL were mostly devoid of synaptophysin labeling.
Photoreceptor sheets and full-thickness retinas maintained
at 37°C for up to 48 hours in culture, with or without CPTcAMP, were fixed after various incubation times and immuno-
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FIGURE 2. Removal of CPT-cAMP. (A) Photoreceptor sheets that received no treatment showed the expected increase of synaptophysin labeling
in the ONL at 45 minutes, 24 hours, and 48 hours. In CPT-cAMP-treated photoreceptor sheets, synaptophysin labeling in the ONL remained at very
low levels throughout the experiment, and became significantly different from the controls only after 48 hours in culture. Removal of CPT-cAMP
was performed for two groups: one after 45 minutes and one after 24 hours. Sheets were then incubated for an additional 24 hours. A fivefold
increase in labeling occurred after 24 hours. The increase in labeling occurred regardless of the duration of treatment and was significant compared
with controls and CPT-cAMP-treated sheets. *P ⬍ 0.001; mean ⫾ SEM; n ⫽ 6 eyes of three animals. (B) Photoreceptor sheets maintained for 48
hours in medium alone (left), with 0.1 mM CPT-cAMP (middle) or with CPT-cAMP for 24 hours and then with medium alone for 24 hours (right).
Synaptophysin labeling moved into the ONL in untreated photoreceptor sheets, as expected (arrowheads). Labeling remained in the OPL in sheets
with CPT-cAMP treatment. In specimens in which treatment was reversed, labeling occurred in the ONL (arrows). Thus, retraction of terminals
toward their cell bodies appears to have resumed. Scale bar, 25 ␮m.
labeled as described earlier. In agreement with our previous
findings,17 significant morphologic changes were seen in photoreceptor sheets and full-thickness retinal preparations. Opsin
labeling, normally largely restricted to membranes of photoreceptor OSs, was observed to varying degrees in the inner
segments (ISs), cell bodies, and terminals of rod cells (Figs. 1B,
1E). The extent of opsin spread seemed to increase with longer
incubation times. Synaptic vesicle (synaptophysin) labeling
was present in the OPL, as expected, but also in the ONL (Figs.
1B, 1E). Labeling in the ONL represents retraction of axons and
terminals toward the cell bodies and has been described in
experimental retinal detachment9,24 and in porcine photoreceptor sheets.17 The retraction of synaptic terminals created
areas devoid of synaptic vesicle label in the OPL, most noticeably in photoreceptor sheets (Fig. 1B). In contrast, specimens
maintained under the same culture conditions but treated with
CPT-cAMP showed almost no labeling in the ONL and almost
continuous labeling in the OPL (Figs. 1C, 1F). Opsin labeling,
however, remained dispersed (Fig. 1D).
The change in synaptophysin localization in untreated specimens began by 45 minutes and was most pronounced by 48
hours in culture (Figs. 1G, 1H). Treatment with 0.1, 0.5, or 1
mM CPT-cAMP significantly reduced ONL synaptophysin labeling in photoreceptor sheets and full-thickness retinas to only
7% to 11% of that observed in untreated specimens after 24 to
48 hours in culture (Figs. 1G, 1H). Furthermore, in photoreceptor sheets, CPT-cAMP treatment resulted in no significant
difference in synaptophysin labeling of the ONL, compared
with control specimens for up to 24 hours, indicating a complete block of retraction. It was not until 48 hours that these
specimens showed a small (1- to 1.5-fold) but significant increase in synaptophysin ONL labeling compared with the control samples (Fig. 1G). In the full-thickness retinal preparations,
however, there was a slight (1- to 1.5-fold) but significant
increase in synaptophysin labeling as early as 45 minutes in
culture, compared with retinal control specimens, indicating a
less complete blockage of retraction (Fig. 1H). The effect of
CPT-cAMP on blockage of retraction did not show a dosedependent variation in either photoreceptor sheets or fullthickness retinas, suggesting that saturation of the response
was reached with the lowest dose.
Reversibility of the Effects of CPT-cAMP
To determine whether the blockage of photoreceptor axon
terminal retraction by CPT-cAMP is caused by toxicity or irre-
versible stabilization of the cytoskeleton, photoreceptors were
examined after drug removal. Photoreceptor sheets were maintained in medium alone or with 0.1 mM CPT-cAMP, the lowest
effective concentration, for 48 hours at 37°C. In a group of
specimens, CPT-cAMP was removed and replaced by CPTcAMP-free medium after 45 minutes or 24 hours of incubation
with the drug. After an additional 24 hours of incubation in
cAMP-free culture medium, specimens were fixed. For untreated and CPT-cAMP-treated photoreceptor sheets, medium
was simply renewed. Results were compared with retinas fixed
immediately after removal from the eyecup. Untreated photoreceptor sheets showed an increase in ONL synaptophysin
labeling, whereas in CPT-cAMP-treated photoreceptor sheets,
labeling of the ONL remained at very low levels throughout the
entire course of the experiment (Fig. 2). Thus, consistent with
previous results, CPT-cAMP treatment dramatically reduced
axon terminal retraction in photoreceptor sheets. In preparations from which CPT-cAMP had been removed, a rapid and
significant increase in ONL synaptophysin labeling was seen
within 24 hours of CPT-cAMP removal, reaching 46% to 58% of
that in untreated specimens (Fig. 2). This increase in labeling
occurred regardless of whether the duration of CPT-cAMP
treatment was 45 minutes or 24 hours. Thus, retraction of
photoreceptor axon terminals toward their cell bodies resumed after removal of CPT-cAMP treatment, indicating that
the block of the retraction is reversible.
Effect of Adenylyl Cyclase Activity on
Photoreceptor Axon Terminal Retraction
To confirm that the inhibitory effect on retraction of photoreceptor axon terminals is specific to cAMP treatment, we increased the photoreceptor intracellular cAMP levels by the
stimulation of endogenous adenylyl cyclase, using forskolin.
Photoreceptor sheets were incubated at 37°C in medium
treated with 0.1, 1, or 10 ␮M forskolin dissolved in 0.1%
dimethyl sulfoxide (DMSO) or in medium with 0.1% DMSO
alone (the control), for up to 48 hours. Results were compared
with those in retinas fixed immediately after removal from the
eyecup. A significant increase of synaptophysin labeling in the
ONL was detected in untreated specimens within 45 minutes
of incubation (Fig. 3), but specimens treated with 1 ␮M forskolin showed no significant increase in labeling. In specimens
treated with either 0.1 or 10 ␮M forskolin, however, there was
a small (less than one-fold) but significant increase in labeling
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sin labeling was significantly greater (approximately two-fold)
than in specimens treated with lower doses of forskolin (Fig.
3). The less effective inhibition of axon terminal retraction by
10 ␮M forskolin applied for 48 hours may indicate a desensitization of adenylyl cyclase at high forskolin concentrations.25
Cyclic AMP and Photoreceptor Viability
FIGURE 3. Forskolin treatment. Within 45 minutes, untreated photoreceptor sheets showed a significant (approximately onefold) increase
in synaptophysin labeling in the ONL, compared with the control.
After 24 and 48 hours, specimens showed a 24-fold increase in ONL
synaptophysin labeling compared with the control. One micromolar
forskolin completely prevented the spread of synaptophysin labeling
into the ONL whereas 0.1- and 10-␮M forskolin treatments showed a
small but significant increase in ONL labeling compared with the
control at 45 minutes. After 24 hours, all forskolin-treated specimens
showed a significant increase in ONL labeling compared with the
control, but only 10% to 11% of that in untreated specimens. After 48
hours, all specimens still showed significant ONL labeling compared
with the control but less labeling than that in untreated specimens.
Lower concentrations of forskolin were more effective at reducing
labeling. *P ⬍ 0.05; mean ⫾ SEM; n ⫽ 6 eyes of three animals.
compared with control specimens (Fig. 3). By 24 hours in
culture, significantly larger amounts of synaptophysin labeling
were seen in all forskolin-treated compared with control specimens (Fig. 3); however, the amount of ONL labeling was only
between 11% and 14% of that in untreated specimens. This
pattern persisted for 48 hours in culture, where untreated
specimens showed dramatically more synaptophysin labeling
in the ONL than did treated specimens (Fig. 3). Thus, forskolin
treatment successfully reduced synaptic terminal retraction in
photoreceptor sheets for up to 48 hours. In specimens receiving the largest dose of forskolin (10 ␮M) the ONL synaptophy-
Increases in cAMP can be associated with cell death26,27; 10
␮M forskolin, specifically, has been shown to increase cell
death of isolated rod cells in culture.28 Cell death was evaluated in photoreceptor sheets as a function of LDH released into
the medium. Photoreceptor sheets were incubated at 37°C for
48 hours, with medium alone or in medium containing 1 mM
CPT-cAMP or 10 ␮M forskolin, the highest concentrations used
in our experiments. As a positive control, photoreceptor
sheets were maintained in medium containing 100 ␮M
ouabain. In all specimens, spectrophotometric absorbances
showed a statistically nonsignificant increase during the first 24
hours of incubation (Fig. 4). Levels plateaued at 48 hours, with
one exception. Medium obtained from cultures of photoreceptor sheets treated with ouabain showed a sharp and significant
increase in colorimetric absorbance after 48 hours of incubation (Fig. 4). Therefore, ouabain caused increased cell death,
whereas treatment of photoreceptor sheets with either CPTcAMP or forskolin had no significant effect on LDH levels for
up to 48 hours.
DISCUSSION
Effect of Elevation of Intracellular cAMP on Axon
Terminal Retraction
Adult porcine retina prepared for transplantation, whether as
full-thickness retina or a photoreceptor sheet, shows a redistribution of synaptophysin labeling from the OPL deep into the
ONL.17 This change in labeling most likely represents retraction of photoreceptor axon terminals toward their cell bodies,
a process that also occurs in experimental retinal detachment.9,24
We have suggested that detachment of photoreceptors or
neural retina from RPE during graft preparation is the stimulus
for photoreceptor axon terminal retraction.17 This perturbation results in spreading depression, a massive wave of depolarization known to follow metabolic, ischemic, or traumatic
FIGURE 4. LDH assay of cell viability. Cell-free aliquots of medium were collected from cultures of photoreceptor sheets maintained in medium
only or treated with 1 mM CPT-cAMP (A), 1 ␮M forskolin (B), or 100 ␮M ouabain for up to 48 hours. The only significant increase in colorimetric
absorbance occurred with ouabain treatment after 48 hours. *P ⬍ 0.05; n ⫽ 6 eyes of three animals.
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injuries to neuronal tissue,29 that presumably results in a myriad of changes in cellular homeostasis, including alterations in
ionic channel conductance, signal transduction pathways,
gene expression, and protein turnover.
In addition to axon terminal retraction, mislocalization of
opsin was seen in these specimens. Opsin labeling, which is
normally concentrated in the rod OS, was observed along the
entire photoreceptor cell membrane after 24 hours in culture.
This phenomenon has also been described previously in isolated photoreceptor cells in vitro28 and in vivo, in experimental retinal detachment,30 and in diseased retina.13,14,16,31 The
underlying mechanism for mislocalization of normal opsin is
unknown but may involve disruption of membrane targeting or
intersegmental fusion of OSs and ISs, leading to loss of the
barriers that mediate compartmentalization of opsin molecules.32,33
We hypothesize that retraction of photoreceptor axon terminals toward their cell bodies represents an obstacle to graft–
host synaptic connectivity after transplantation into the subretinal space. Indeed, one of the major problems encountered
in retinal transplantation is the absence or paucity of graft– host
synaptic interactions. In contrast, the presence of opsin in
axonal membranes is unlikely to prevent synaptogenesis, because adult rod cells in culture with mislocalized opsin can
form synapses,6,28 and developing rod cells can contain opsin
labeling throughout their membranes while the outer synaptic
layer is created.34 Opsin labeling is also found throughout
developing photoreceptors in animal models of retinal degeneration in which synapses are formed before degeneration is
severe.11,31
Applying CPT-cAMP to photoreceptors prepared as a sheet
for transplantation led to approximately 93% reduction of retraction for up to 48 hours. CPT-cAMP also blocked retraction
in full-thickness retinas. However, full-thickness preparations
were slightly less inhibited than the photoreceptor sheets after
45 minutes and 24 hours of incubation. A less effective blockage in the intact neural retinas may have occurred because of
the reduced accessibility of the cAMP analogue to the photoreceptors. Alternatively, CPT-cAMP may induce other retinal
neuronal or glial cell types to release factors that counteract
the effect of the cAMP analogue on photoreceptors. Forskolin
was less effective than CPT-cAMP, which blocked retraction
completely at both 45 minutes and 24 hours, whereas forskolin
allowed a small but significant increase in labeling in the ONL
at these times and after 48 hours. The complex interactions
that control adenylyl cyclase activity may underlie the relatively less effective blocking of retraction by forskolin. In
photoreceptors, for instance, adenylyl cyclase activity is downregulated by dopamine and light,35 whereas its expression may
be controlled by circadian rhythm.36 It is possible that detachment itself reduces adenylyl cyclase expression as it does for
other photoreceptor proteins.37 Variations in adenylyl cyclase
protein levels affect the extent of cAMP production. Inhibition
of retraction by either drug, however, was not due to toxicity
or cell death, as shown by both the ability to reverse the
blockage of retraction and by LDH release assays.
Role of cAMP in Inhibition of Retraction
Axonal retraction is dependent on changes in the cytoskeleton.
Cyclic AMP may modulate actin cytoskeletal dynamics through
interactions of its downstream effector, cAMP-dependent PKA,
with a member of the Rho subfamily of small guanosinetriphosphatases (GTPases), RhoA, which has been shown to
inhibit process outgrowth and promote neurite retraction in
vitro, in neuronal cell lines and primary neuronal cultures, and
in vivo.38 – 41 Inactivation of RhoA by ADP-ribosylation using
the Clostridium botulinum C3 exoenzyme promotes neurite
outgrowth in vitro38 – 40 and in vivo40,41 even on complex
IOVS, March 2005, Vol. 46, No. 3
inhibitory substrates.40 Furthermore, inactivation of the Rhoassociated kinase (ROK) also promotes neurite outgrowth.41
Cyclic AMP-activated PKA phosphorylates RhoA decreasing its
guanine-nucleotide exchange, possibly by increasing its affinity
to guanine-nucleotide dissociation inhibitors (GDIs). GDIbound RhoA dissociates from the membrane and translocates
to the cytosol, where it remains inactive.42 In addition, phosphorylation of RhoA reduces its affinity to its effectors (e.g.,
ROK).42
We propose that cAMP-mediated phosphorylation downregulates the RhoA signaling pathway and thus prevents retraction. Other GTPases of the Rho family, Rac and Cdc42, also
regulate the actin cytoskeleton, but in contrast to RhoA, they
serve to enhance lamellipodial and filopodial process extension, respectively, and thereby counteract the effects of
RhoA.39,43 Inhibition of RhoA, due to the increased cAMP in
our specimens, coupled with the unopposed activity of Rac
and Cdc42, may serve to shift the neuronal terminal toward a
process of outgrowth. Indeed, some neuritic processes have
been observed in full-thickness specimens, which provide
some cellular substrate for process growth, after 48 hours of
forskolin treatment (Khodair MA, Townes-Anderson E, personal observations, 2001).
Cyclic AMP-mediated phosphorylation also influences other
cytoskeletal proteins. PKA can directly phosphorylate and inactivate myosin light-chain kinase, thus preventing phosphorylation of the regulatory subunit of myosin light chain. This
inactivation reduces myosin II ATPase and motor activity and
therefore may decrease the retraction-mediating contractile
forces of actomyosin. PKA also phosphorylates microtubuleassociated proteins (MAPs) which play a key role in regulation of microtubule dynamics, neuronal morphogenesis, and
plasticity.44 MAP2c and MAP5, which are expressed in the
developing brain during periods of neurite growth and synaptogenesis, are present in adult photoreceptors.44 PKA phosphorylates MAP2c on specific serine residues located in the
tubulin-binding domain and adjacent proline-rich regulatory
region.45,46 These site-specific phosphorylations promote neuritogenesis,45 possibly by causing MAP2c to localize to the
actin cytoskeleton.46
Although cAMP through its downstream effector PKA can
play a pivotal role in regulation of both the actin and microtubule components of the cytoskeleton, it may also influence
axonal plasticity through its effects on gene expression and
protein synthesis. The relative importance of changes in gene
expression would be expected to increase with time.
In summary, experiments with CPT-cAMP have demonstrated that early morphologic changes in the axons of adult
photoreceptors that occur during preparation of grafts and
possibly after transplantation into the subretinal space can be
prevented by pharmacological manipulation. This step may be
important in the evolution of successful retinal transplantation
techniques, since these changes are thought to interfere with
the ability of the grafted photoreceptors to establish connectivity with host neurons. Similar effects of cAMP may be expected in transplants of other differentiated nerve cells.
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