Effect of Polyamine Depletion on Cone Photoreceptors of the Developing Rabbit Retina

Abstract

purpose. To measure the concentrations of polyamines, determine their cellular and subcellular localization, and analyze effects of their depletion in developing rabbit retina.

methods. Isolated retinas at different developmental stages were analyzed for polyamine content by high-performance liquid chromatography (HPLC). An antibody against polyamines was used to localize endogenous stores in both freshly harvested retinas and neonatal retinal explants. To determine the effects of polyamine depletion on immature retina, neonatal explants were cultured in the presence or absence of α-difluoromethylornithine (DFMO), an inhibitor of the polyamine synthetic enzyme ornithine decarboxylase (ODC). Similar studies were also performed on dissociated cell cultures. Tissue was assessed using standard histologic stains as well as cell-specific markers (peanut agglutinin for cone photoreceptors and calbindin for horizontal cells).

results. Retinal polyamine content was highest at birth, remained relatively high during the first postnatal week, and then steadily decreased to adult levels. At all ages analyzed, spermine concentration was higher than putrescine or spermidine; however, the differential was greatest in the adult. Polyamine immunoreactivity was localized to distal processes of both rods and cones during development. Strong immunoreactivity was maintained in adult cone inner and outer segments; comparatively weak staining was observed in the adult rods. Heavy staining of ganglion cells was present throughout development but was localized in the cytoplasm in immature cells and in the nucleus in the adult. Amacrine cells stained only in the adult. Polyamine depletion caused a disruption of immature cones, evident in the loss of their somata in the outer nuclear layer, in their processes in the outer plexiform layer in retinal explants, and in their decreased association with horizontal cells in dissociated cell culture.

conclusions. The relatively high concentrations of polyamines in neonatal retina and their discrete localization in developing photoreceptor outer segments and ganglion cells suggests an important role for these compounds in development. The disruption of cone-specific markers in polyamine-depleted retinas indicates a specific reliance on polyamines for expression of normal cone morphology or morphologic development. These developmental effects may involve polyamine-sensitive ion channels, which are known to exist in retina, or direct interactions with specialized cytoskeletal elements within outer segments.

The polyamines spermidine and spermine, are composed of flexible carbon chains with amino groups that are positively charged at physiological pH, thus providing opportunities for interactions with a variety of negatively charged molecules. ¹ They are synthesized sequentially from the diamine precursor, putrescine, by the enzyme S-adenosyl-l-methionine decarboxylase (SAMdc). Putrescine levels are in turn established by the activity of a highly regulated synthetic enzyme, ornithine decarboxylase (ODC). Both the availability of putrescine (reflecting the activity of ODC) and SAMdc activity can be rate limiting for polyamine synthesis. 

Spermidine and spermine are present in virtually all animal cells and have been shown to play pivotal roles in a variety of cell functions, including proliferation, differentiation, migration, and most recently, apoptosis. ^{2 3 4} Given this broad range of polyamine-dependent cell functions, it is not surprising that a number of different molecular interactions have been described. Polyamines bind to DNA, acting as regulators for transcription ⁵ ; they regulate protein phosphorylation through polyamine dependent protein kinases ⁶ ; and they are also known to regulate certain ion channels, ^{7 8} in particular the inward rectifier potassium channel. ⁹  

Recent studies in neuronal polyamines have focused on their link to various neurotransmitter systems. There is circumstantial evidence that putrescine acts as a precursor for γ-aminobutyric acid (GABA), particularly during development. ^{10 11} Other laboratories have reported effects of polyamines on GABA A receptor–mediated chloride conductance ⁷ and on the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor. ^{12 13 14 15 16 17} In the past, studies of polyamines in retina have been relatively limited compared with studies in other tissues. However, interest in retinal polyamines has increased with the discovery that glutamate-induced neurotoxicity in retina and brain can be decreased by pharmacologically blocking the polyamine binding site on the NMDA receptor. ^{18 19 20} Ifenprodil has been a particularly effective polyamine antagonist in this regard and is currently under development as a neuroprotective agent for various excitotoxicity-related disorders. ^{21 22} Also of interest is the recent report that polyamines may regulate dark adaptation through their inhibition of the cyclic nucleotide–gated calcium channel in photoreceptors. ²³  

Although a direct link between polyamines and the vision disorder gyrate atrophy of the choroid and retina has not been established, it is interesting to note that hyperornithinemia-induced toxicity is the underlying cause of this inherited retinal degenerative disease. ^{24 25} The high levels of ornithine result from functional mutations in the gene for ornithine aminotransferase. Elevated levels of ornithine would be expected to influence the rate of polyamine synthesis from ornithine and thus alter polyamine function. This alteration could contribute to the retinal toxicity and degeneration observed. 

To gain a better appreciation of the role polyamines play in the developing retina, we measured polyamine levels and examined the cellular and subcellular distribution of polyamine immunoreactivity within retinal neurons during different maturational stages. In addition, an inhibitor of polyamine synthesis, α-difluoromethylornithine (DFMO), ²⁶ was used to identify developmental processes that may be polyamine dependent. Our results show the highest levels of putrescine and polyamines at birth, declining after the first postnatal week to reach adult levels. A localized distribution of endogenous polyamines was observed, with greatest immunoreactivity present in cone photoreceptors and ganglion cells beginning at early developmental stages and continuing through adulthood. Furthermore, the DFMO-induced reduction of polyamine levels caused a morphologic disruption of developing cones in both cultured explants and dissociated cell cultures. These data suggest an important role for endogenous polyamines in normal development of cones and perhaps ganglion cells. In addition, they set the stage for subsequent experiments designed to determine mechanisms of action and potential interactions with neurotransmitter systems in developing and mature retina. 

Materials and Methods

Rabbit Explant Preparation

All animals in this study were handled and cared for according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Dutch belted rabbits, aged 1, 3, and 7 days and adult, were obtained from a local vendor on the day of the experiment. These ages were chosen for the current studies, based on our previous work that shows that cone axogenesis and synaptogenesis occurs between postnatal days 1 and 3. ²⁷ Rabbits were anesthetized with CO₂, decapitated, and pithed by placing a blunt probe into the foramen magnum to disrupt the brain stem. Eyes were enucleated and placed in sterile, oxygenated Ames’-HEPES [N-(2 hydroxyethylpiperazine-N′-(ethanesulfonic acid)] media (pH 7.2; Sigma Chemical Co., St. Louis, MO), for transport and dissection. After enucleation, each eye was hemisected along the ora serrata to remove the anterior portion. The lens and vitreous were removed and radial incisions were made to flatten the eyecup. 

Uniform explants, 2 mm in diameter, were removed from the midperipheral retina and placed ganglion cell side down on sterile synthetic basement membrane (Matrigel; BD Biosciences, Bedford MA) matrix–coated filters. The explants and filters were immediately cultured (described later) or placed in 2.5% glutaraldehyde (Sigma Chemical Co.) in phosphate-buffered saline (PBS) and fixed overnight at 4°C. After fixation, tissue was thoroughly rinsed in buffer before being processed for immunocytochemistry. Details of this procedure have been published. ²⁷  

Dissociated Cell Culture

Dutch belted rabbits of postnatal day (P)3 were killed as described. The retina was carefully dissected from the eyecup, separated from the retinal pigment epithelium, and placed in 1 mL enzyme solution (0.2 mg/mL DNase, 21 U/mg papain, 2.7 mM cysteine; all from Sigma Chemical Co.). After 30 minutes, the enzyme solution was removed, and the enzyme action quenched by incubation in a solution of 0.1% bovine serum albumin (BSA) for 10 minutes. After three rinses in fresh AMES-HEPES, the tissue was dissociated into a single-cell suspension by trituration with a fire-polished pipette. Cells were counted using a hemocytometer and diluted in control tissue culture medium (DMEM-F12; Gibco, Grand Island, NY, supplemented with 1.5 mg/mL transferrin, 0.25 mg/mL insulin, 97 mg/mL putrescine, 0.03 mg/mL selenium, and 0.1 unit/mL progesterone; Sigma Chemical Co.) or in the same medium without putrescine and with 5 mM DFMO (Ilex Oncology, San Antonio TX). The cells (final density, 10⁶/cm²) in 250 mL were plated in each well of a 24-well tissue culture plate fitted with 12-mm glass coverslips coated with 5 mg/mL mouse laminin (Sigma Chemical Co.). They were allowed to settle for approximately 20 minutes, and 750 mL of medium was added to each well. They were grown for 3 days at 37°C in a 5% CO₂ incubator. They were then fixed with 4% paraformaldehyde with 4% sucrose (pH 7.2) for 1 hour at room temperature and rinsed several times in PBS. Cells were stained with 0.1% toluidine blue in 1% sodium borate to examine cellular morphology or processed for immunocytochemistry. 

Polyamine Depletion in Culture

For culture experiments, explants were isolated from rabbits at postnatal day 1 or 3 and maintained in serum-free tissue culture medium (DMEM-F12, supplemented with 1.5 mg/mL transferrin, 0.25 mg/mL insulin, 0.03 mg/mL selenium, and 0.1 U/mL progesterone) and incubated for up to 3 days at 37°C in a water-jacketed incubator equilibrated with 5% CO₂. To deplete endogenous polyamines, DFMO was added to the medium at concentrations of 100 μM or 5 mM. DFMO is an irreversible inhibitor of ornithine decarboxylase (ODC), a rate-limiting enzyme in the polyamine synthesis pathway, ²⁶ Explants were cultured in the serum-free medium supplemented with either 60 μM putrescine (control), DFMO in the presence or absence of putrescine, or, sequentially, in medium with DFMO for 24 hours followed by the addition of putrescine in the presence of DFMO for 48 hours (rescue). Cultured explants were fixed in 2.5% glutaraldehyde for plastic embedding and morphologic analysis and anti-polyamine antibody pre-embedding immunohistochemistry or in 4% paraformaldehyde for lectin staining. 

Polyamine Immunocytochemistry

After fixation, retinal explants were carefully removed from the filters and saturated with 0.3% H₂O₂, to halt any endogenous peroxidase activity. The explants were incubated in 0.15 μg/mL rabbit anti-polyamine antibody (AP3; a gift from Carl Romano, Washington University, St. Louis, MO), for which the antigen is a glutaraldehyde-spermine complex, for a period of 14 days on a rotator at 4°C. The antibody recognizes spermine and, to a lesser extent, spermidine and putrescine. ²⁸ The explants were then washed in PBS for 24 hours before a 2-day incubation in biotinylated horse anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA). After incubation with the secondary antibody, the explants were washed in PBS for 24 hours before overnight exposure to an avidin-biotin-peroxidase solution (Vectastain ABC kit; Vector Laboratories). To visualize the biotinylated complex, the explants were exposed to diaminobenzidine tetrahydrochloride (DAB). The explants were then dehydrated through a series of stepped alcohol concentrations, embedded in Epon-Araldite, sectioned at 1 μm, and examined by microscope (Optiphot-2; Nikon, Tokyo, Japan). Images were captured with a microscope-mounted digital camera (Micromax 1300YHS; Roper Scientific, Princeton Scientific Instruments, Monmouth Junction, NJ; using Metaview software; Update Software, Chevy Chase, MD). 

Double-Label Cytochemistry with Peanut and Wheat Germ Agglutinins

After fixation in 4% paraformaldehyde, the retinal explants were washed in phosphate buffer. The explants were removed from the synthetic matrix–coated filters (Matrigel, BD Biosciences), placed in PBS, and pretreated with PBS containing 1 mM CaCl₂, 1 mM MgCl₂, and 1.5 mg/mL BSA. The tissue was then exposed for 15 minutes to rhodamine-conjugated peanut agglutinin (PNA) lectin and fluorescein isothiocyanate–conjugated wheat germ agglutinin (WGA; Vector Laboratories), both at a 1:10 dilution in 1 mM CaCl₂ and 1 mM MgCl₂ in PBS. PNA stains the glycoproteins on the cell membranes of cone photoreceptor cells, and WGA labels the membrane proteins on rod photoreceptor cells. ^{29 30} After the treatment, the retinal explants were washed in PBS and mounted on slides, photoreceptor side up (Fluoromount-G; Electron Microscopy Sciences, Fort Washington, PA). The retinal explants were visualized with a laser (Bio-Rad, Richmond, CA) on a confocal microscope (Olympus, Lake Success, NY). A 10-μm region of tissue was analyzed using the laser. Sequential 0.1-μm optical sections were made within the 10-μm region for a total of 101 sections. These sections were digitally compiled producing one three-dimensional image. The separate cone and rod photoreceptor images were merged into one three-dimensional image, to simultaneously visualize the double label. 

Calbindin Immunocytochemistry

After fixation, dissociated cell cultures were rinsed in PBS and incubated for 1.5 hours in PBS containing 0.5% Triton X-100, BSA and 5% serum to block nonspecific antibody binding. Cultures were incubated in mouse monoclonal anti-calbindin-D (Sigma Chemical Co.) at 1:1000 for 1 hour at room temperature, followed by secondary antibody, Cy3 conjugated anti-mouse (Vector Laboratories) 1:200 in PBS for 30 minutes. Fluorescence was visualized with the laser-outfitted confocal microscope. 

Assay of Intracellular Polyamines

The levels of endogenous putrescine, spermidine, and spermine in retina were analyzed by high-performance liquid chromatography (HPLC). ³¹ Retinas were removed from three separate rabbits of each age (P1, P3, P7, and adult) placed in 0.5 mL of 0.5 M perchloric acid, and immediately frozen at −80°C until all samples were ready for dansylation, extraction, and HPLC. Total protein content was determined with the Bradford method, ³² and polyamine content expressed as nanomoles per milligram protein. 

Results

Developmental Profile of Polyamine Immunoreactivity in Rabbit Retina

Using an antibody that reacts with spermine and, to a lesser extent, with spermidine and putrescine, ²⁸ the expression pattern of these three compounds was examined in rabbit neural retina at various time points during development. Polyamine immunoreactivity was present in photoreceptors, particularly in cones, from earliest stages of development through adulthood. At P1, staining displayed a central-to-peripheral gradient, reflecting the overall developmental gradient that is present at this stage. In the periphery (Fig 1B) , which is somewhat less developed than more central regions, staining was observed in discrete cellular elements along the outer retinal surface. In the more developmentally advanced central region (Fig. 1C) , staining was also observed along the outer surface in cell bodies, with a distribution reminiscent of that in neonatal cone somata and outer segments. Thus polyamine immunoreactivity appears to be a very early marker for developing cones. At P3, staining of cell bodies was diminished, but labeling of outer segments became more prominent as outer segments increased in length (Fig. 1E) . At P7, the most active period of outer-segment genesis, we observed the most intense polyamine labeling of the outer retina of any developmental stage examined (Fig. 1G) . At this stage, immunoreactivity appeared to be present in virtually all rods and in all cone outer segments. In the adult retina, the polyamine content of rods was reduced significantly, with light staining concentrated in the area of the connecting cilia (Figs. 1I 1J) . However, robust labeling was maintained in cone inner and outer segments. 

The other cell type that showed distinct staining during development was the ganglion cell. At birth, dark staining was observed in the cytoplasm and associated processes of cells in the ganglion cell layer (Figs. 1A 1B 1C) , but was notably absent from the nuclei. At P3 (Figs. 1D 1E) and P7 (Fig. 1H) , this staining pattern was maintained. In the adult (Fig 1J) , there was a discrete shift in localization within the ganglion cell so that staining was pronounced in the nuclei, but absent in processes. Some large cells within the ganglion cell layer (presumptive ganglion cells) were unlabeled. 

Although little or no labeling of amacrine cells was observed during development, the adult retina showed staining in the nuclei of subpopulations of amacrine cells. The overall level of expression was less in this cell type than in either ganglion cells or cone photoreceptors. 

HPLC Analysis of Whole Retina

HPLC measurements of putrescine, spermine, and spermidine content in the rabbit retina at P1, P3, and P7, and in the adult retina confirmed the presence of endogenous polyamines at each age studied (Fig. 2) . Analyses were completed on samples from three separate animals at each age. At P1, the concentrations of all three compounds were roughly in the same range; however, levels of putrescine and spermidine declined dramatically thereafter. The decline in putrescine from P1 levels was statistically significant at P3 (P < 0.013) and P7 (P < 0.0007) and in the adult (P < 0.0002). The decline in spermidine from P1 levels was significant at P3 (P < 0.02) and in the adult (P < 0.005). The decline in spermine was significant only in the adult (P < 0.003). The result of this developmental pattern is that the adult contained a much higher ratio of spermine compared with spermidine or putrescine and that the levels of all three compounds were significantly lower in the adult than at any other time point measured. 

Effect of DFMO on Polyamine Immunoreactivity in Developing Rabbit Retina

We used the anti-polyamine antibody to verify that exposure to the ODC inhibitor DFMO in the absence of exogenous polyamines causes depletion of endogenous polyamines in retinal explants. Control tissue grown for 3 days in the presence of 60 μM putrescine (a constituent of our normal culture media) showed polyamine staining in the outer segments of photoreceptors (Fig. 3A) similar to that seen in intact retina, as described earlier. Explants cultured for 3 days in the presence of 5 mM DFMO with no exogenous polyamines showed a reduction in staining compared with control tissue, indicating that endogenous polyamines in the photoreceptors had been decreased under these conditions (Fig. 3B) . In tissue grown for 3 days in the presence of 5 mM DFMO and 60 μM putrescine, staining of outer segments was present, similar to that seen in control tissue (Fig. 3C) . This suggests that, as expected, DFMO (in the absence of exogenous polyamines) is effective in depleting polyamine stores in developing retina in culture. The results also confirm that explants of neonatal retina cultured under control conditions retain the cellular distribution pattern of endogenous polyamines seen in vivo and, furthermore, that exogenous putrescine can be taken up specifically by cells (photoreceptors) that normally contain endogenous polyamines. 

Effect of Polyamine Depletion on Neonatal Retinal Explants

To determine the effect that DFMO-induced polyamine depletion may have on retinal development, we used wholemount confocal microscopy to examine control and DFMO-treated explants double labeled with fluorescein-conjugated WGA lectin (which labels rod photoreceptor matrix green) and rhodamine-conjugated PNA lectin (which labels cone photoreceptor matrix red). ^{26 27} P3 retinal explants grown for 3 days in control medium containing 60 μM putrescine showed bright staining of plasma membranes of both cones (Fig. 4A) and rods (Fig. 4B) . The mosaic of both rods and cones appears to be uniformly distributed across the outer surface of the retina. Addition of 5 mM DFMO to the culture media caused a major disruption in the cone mosaic (Fig. 4C) . Far fewer labeled cone cell bodies were seen than were present in the control tissue. Rods were not as severely affected. Note that WGA labeling (green) was almost as bright and confluent as in the control explant (Fig. 4D) . To determine whether the disruption caused by DFMO is due to polyamine depletion, we examined the cone mosaic in explants that were exposed to 5 mM DFMO for 1 day in culture and subsequently treated during an additional 2 days of culture with 60 μM exogenous putrescine added to the media. With this treatment, the cone mosaic was restored to near normal. Bright PNA labeling of cones was evident, and the spacing of cone cell bodies was relatively unaltered from the standard mosaic (Figs. 4E 4F) . 

Morphologic examination of plastic embedded sections from retinal explants cultured in DFMO further suggests an effect of polyamine depletion on developing cones. After 1 day in culture with 5 mM DFMO, the outer plexiform layer (OPL; Fig 5B , bracket) appeared to be virtually abolished when compared with control tissue from the same animal in which a distinct OPL was present (Fig. 5A , brackets). The loss of axonal processes and terminals from cone photoreceptors could account for this observation. Alternatively, a general reorganization of the outer retina could be a factor, although other nearby cells did not seem to be disrupted. For example, horizontal cell bodies (Fig. 5 , arrowheads), which normally abut the OPL at this developmental stage, were observed in the expected position relative to the outer retinal surface in DFMO-treated tissue. In general, most other retinal cells looked relatively normal; however, additional studies are needed to confirm this initial observation. 

Effect of Polyamine Depletion on Neonatal Retinal Neurons in Dissociated Cell Culture

We have previously characterized a dissociated retinal cell culture system in which photoreceptors and their normal synaptic targets, the horizontal cells, form discrete clusters, reminiscent of the normally developing OPL (Figs. 6A 6C 6E) . ³³ In our previous study, a battery of cell-specific markers (horizontal cells: calbindin, GABA, vimentin; photoreceptors: PNA, WGA, cone opsin, rhodopsin) were used to verify the identity of these two cell types. Under control conditions, horizontal cells retain their characteristic morphology, including a large, fusiform cell body, and broad tapering neurites along which photoreceptor cell bodies seem to adhere preferentially (Fig. 6C) . In many cases, photoreceptors adhere to each other or aggregate around horizontal cells in an arrangement reminiscent of “rosettes,” as described both in intact tissue and in dissociated cells grown in suspension culture. ³⁴ The addition of 5 mM DFMO to the culture medium caused a distortion of the horizontal cell bodies and a decrease in the adherence of photoreceptors to each other or to horizontal cells (Figs. 6B 6D) . 

The staining of cultures with calbindin antibodies afforded a clearer appreciation of the morphology of horizontal cell bodies and processes. As seen in control cultures (Fig. 6E) , horizontal cells (calbindin immunoreactive, red), displayed long processes surrounded by clusters of photoreceptor cells, which appeared green due to autofluorescence. In cultures treated with DFMO, long horizontal cell processes were still present, but the cell bodies were enlarged and more spherical. Fewer clusters of photoreceptors were present; many cells remained isolated. 

One quantitative measure of the effect of DFMO in the absence of putrescine was obtained by analyzing the branching pattern of horizontal cell axons in control and treated cultures (Figs. 7 and 8) . Many thin branch points were observed along the major axonal processes of horizontal cells under control conditions (Figs. 7A 7B) . In DFMO-treated samples, major processes were still present; however, the number of small branches projecting from the major processes appeared to be significantly reduced (Figs. 7C 7D) . Extended regions of major processes were virtually devoid of branches. When 60 μM putrescine was present during DFMO treatment, the branching pattern was similar to that seen in the control. Morphometric analysis showed that the number of small branch points decreased by more than half (Fig. 8 ; P < 0.03). Addition of 60 μM putrescine along with DFMO protected against this decrease. 

Discussion

Localization of Endogenous Polyamines in Rabbit Retina

The highly specific, developmentally regulated cellular and subcellular distribution of polyamine immunoreactivity in rabbit retina represents one of the key observations in this study. Based on previous reports, ^{35 36} it was not surprising that we were able to confirm the presence of endogenous polyamines in rabbit retina; however, the discrete localization of polyamine immunoreactivity within nascent and mature inner and outer segments of cones represents an unexpected and novel finding. The polyamine antibody used in our study was originally developed by Valentino et al. ²⁸ and used by them to determine staining patterns in adult salamander retina. Their results show labeling of ganglion cells and amacrine cells, similar to that which we observed in adult rabbit retina. However, they reported only variable labeling in outer segments and were unable to draw firm conclusions. One possible confounder is that the RPE contains high levels of polyamines, ³⁷ which could have obscured staining in adjacent photoreceptor segments within the eyecup preparation used in the salamander studies. In our experiments in rabbits, all samples were taken from isolated neural retina after the RPE was removed, thus permitting a clearer assessment of staining in outer segments, particularly at early stages of development when segments are short. Additional studies are necessary to determine whether the cone-specific polyamine distribution in mammalian (rabbit) retina is also present in nonmammalian (salamander) species. 

The outer segments of rabbit cone photoreceptors are strongly immunoreactive, even at initial stages of development. From this we can infer that internal stores of polyamines accumulate in association with immature elements that are unique to the developing distal processes of cones (and ganglion cell axons; discussed later) long before functional maturity is reached. Other cone outer segment markers, such as the PNA-binding site in the cone membrane’s extracellular matrix, show a similar expression pattern. Both attributes are robustly maintained through adulthood. 

Assuming that polyamines are stored near their site(s) of action, a number of possible molecular interactions can be proposed. Polyamines have been shown to have selective and complex effects on a variety of ion channels found in many CNS neurons, including voltage-gated calcium channels, inward rectifier potassium channels, calcium-activated chloride channels, and GABA A receptor–mediated chloride channels, ⁷ all of which could represent targets for endogenous polyamines in cones. Perhaps an even more relevant target is suggested by the recent report that putrescine, spermidine, and spermine effectively block the cyclic nucleotide–gated channel in bovine rod photoreceptors. ²³ Polyamines block this channel at an intracellular site and at concentrations equal to or below that normally found intracellularly. Thus polyamines, through their regulation of cyclic guanosine monophosphate (GMP)–regulated calcium flux, could have a major impact on the adaptation of visual transduction. Our demonstration of significant stores of endogenous polyamines in the outer segment where these polyamine-sensitive channels are concentrated makes it even more likely that active regulation of ion conductances occurs. 

Polyamines have also been shown to act directly or indirectly through second-messenger systems to maintain the cytoskeletal architecture of various cell types. ^{38 39} These effects may in part account for the potentiating effect of polyamines on cell migration. ⁴⁰ Similarly, cytoskeletal interactions could also account for the ability of polyamines to promote axonal regeneration (growth cone migration) in cultured rat hippocampal neurons ⁴¹ and for the effects we observed on branching of horizontal cell axons. Analyses of structure–activity relationships indicate that the regeneration-promoting effects of polyamines are independent of their effects on various ion channels. ⁴¹ The intense immunoreactivity present in distal photoreceptors during the period of active inner- and outer-segment genesis and in neurites of ganglion cells during their formation both suggest a role for polyamines in neurite production. That this pattern of immunoreactivity is strongly maintained in cones (and to a much lesser extent in rods) may suggest a continuing role for polyamines in maintaining the cytoskeletal structures of this specialized region in photoreceptors. 

The polyamine staining pattern in ganglion cells showed a distinct maturational shift from strong cytoplasmic labeling during development to discrete nuclear labeling in the adult. Given the well-established ability of polyamines to bind to DNA and to enhance transcription, ⁵ it is somewhat surprising that the adult ganglion and amacrine cells showed nuclear staining but cone photoreceptors did not. It has been suggested that tight interactions between polyamines and DNA-binding sites may interfere with antibody staining and that special DNase treatments may be needed to uncover the antibody-binding site. ⁴² The fact that ganglion cell nuclei stain indicates that such is not uniformly the case for all retinal cells and, furthermore, that polyamines may play a special role in the regulation of ganglion cell transcription. 

There has been a great deal of recent interest in the demonstration that NMDA receptors contain a specific polyamine regulatory site that is activated from the external membrane surface and is thus capable of responding to polyamines released in the extracellular space. ^{12 13 14 15 16 17} Neuronal release of polyamines has been reported. ⁴³ Additional interactions of polyamines at intracellular domains of the receptor, as seen for other ion channels (mentioned earlier), have not been rigorously ruled out. The molecular features of NMDA receptor–polyamine interactions are complex and may involve alterations of both glycine and glutamate binding sites on the receptor with the net result that channel activation is augmented. Drugs that antagonize the polyamine modulatory site have been shown to block NMDA receptor–mediated neurotoxicity in retina, and depletion of endogenous polyamines after treatment with the synthesis inhibitor DFMO has a similar effect. ^{18 19 20} NMDA receptors are found in abundance on bipolar, amacrine, and ganglion cells. ⁴⁴ Thus, the polyamine immunoreactivity we and others ²⁸ have observed in ganglion and amacrine cells could represent an important endogenous source of polyamines for release and subsequent regulation of NMDA receptors. 

Effect of DFMO on Retinal Development

Effects of polyamine depletion by DFMO were apparent under three different experimental conditions. First, depletion caused an apparent disruption of the normal distribution of cone cell bodies (stained with the specific cone cell marker, PNA) in the outer nuclear layer of retinal explants harvested from 3-day-old rabbits. Addition of exogenous putrescine protected against the disruption, indicating that the effects of DFMO were indeed caused by polyamine depletion. Maintenance of cellular position and distribution of cones during development is likely to result from a complex interplay of numerous factors, with any significant loss of homeostatic balance among the factors capable of triggering a disruption. In addition, our studies do not demonstrate whether the cones die, simply fail to express lectin-binding sites, or become displaced in the absence of polyamines. We can conclude, however, that polyamine depletion affects cones preferentially and not all cells globally. The absence of apparent disruption of rods (stained with the specific rod marker WGA) may indicate that separate controlling factors operate for this cell type. An alternate explanation is that because rods are generated later than cones, rods may not have reached a polyamine-sensitive state at the time chosen for these experiments. Cones become postmitotic before birth, whereas rods do so 5 to 7 days after birth in most common experimental mammals, including cat ⁴⁵ and rabbit. ⁴⁶  

As a second measure of the effects of polyamine depletion, we analyzed histologic sections of treated and control retinal explants with light microscopy. Results show that the OPL was significantly reduced by the treatment. At P3, this layer is composed of a monolayer of horizontal cell neurites along with axons and terminals from developing cones; rod axons and terminals appear later in development. ⁴⁶ The loss of cellular constituents from this layer may represent the absence of cone processes, consistent with observations of somatic disruptions, as described earlier. Thus, endogenous polyamines may influence the position of the entire cone cell from soma to axon terminal. 

As a third measure, we analyzed morphologic changes in dissociated retinal cell cultures harvested from 3-day-old rabbits. Polyamine depletion decreased the ability of presumptive cones (i.e., cells stained with PNA and cone opsin antibodies) to form cellular aggregates. It also caused significant changes in the shape of horizontal cell bodies (identified by calbindin immunoreactivity) and decreased the number of fine branch points along primary axons. The decrease in aggregation of photoreceptors in culture and the loss of position in explant culture could reflect similar polyamine-dependent mechanisms, which function to regulate cell recognition, cell attachment, and process growth. 

It is interesting to note the similarities between the effects of GABA receptor antagonists and polyamine depletion in these preparations in explant cultures. Both treatments cause disruption of cone cell bodies and terminals in the absence of significant effects on rods in explant cultures. ³⁰ Because putrescine has been suggested as a possible precursor for GABA synthesis in developing retina, ^{10 11} these similarities could reflect a metabolic link between these two compounds. Studies comparing the effects of polyamines and GABA in dissociated cell cultures are currently under way. 

Given these results, we are now in a position to begin investigating the specific molecular interactions responsible for the developmental effects of polyamines and to determine which effects involve direct interactions with channels or cytoskeletal elements and which effects are indirectly mediated through the GABAergic system. Results of these studies will continue to expand our appreciation of both of these important compounds in developing neurons of the retina. 

 Supported in part by Grants EY01655 and EY13080 from the National Eye Institute and Grant DK52784 from the National Institute of Diabetes and Digestive and Kidney Diseases, Research to Prevent Blindness, and International Retina Research Foundation.

 Submitted for publication August 3, 2001; revised April 15, 2002; accepted April 26, 2002.

 Commercial relationships policy: N.

 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.

 

Figure 1.

 

Polyamine immunoreactivity in sections of developing rabbit retina. (A) P1: section stained with polyamine antibody and counterstained with toluidine blue to demonstrate general morphology and position of the outer plexiform layer (OPL) and inner plexiform layer (IPL) at this developmental stage. (B, C) P1: sections stained with polyamine antibody only. Heavy staining was observed in the cytoplasm of large cell bodies in the ganglion cell layer and associated processes (arrowheads). Labeling of elements in the outermost retina (arrows) was confined to small profiles in peripheral regions of the retina (B) but included cell bodies in more central regions (C). (D, E) P3: strong immunoreactivity was maintained in the cytoplasm of large cells in the ganglion cell layer (D, arrow; E, arrowhead). Labeling was increased significantly along the outer retinal surface, primarily in association with the distal processes of developing photoreceptors (E, arrows). (F) Control tissue from rabbit at P3, processed in the absence of primary antibody and not counterstained, showed no labeling. (G, H) P7: the most intense staining in the outer retina occurred at P7. Labeling appeared to be associated with inner and outer segments of all photoreceptors, both rods and cones (G, arrowheads). Labeling of the cytoplasm of presumptive ganglion cells remained robust (H, arrows). (I, J) In the adult retina, staining was highly concentrated in the nuclei of many cells in the amacrine cell layer (I, open arrowheads) and some cells in the ganglion cell layer (I, arrows). Cone inner and outer segments were heavily stained (filled arrowheads). Lighter staining in rods was concentrated at the level of the connecting cilium (I, horizontal arrow; J, open arrows).

Figure 2.

 

Polyamine content in developing retina. HPLC analysis of isolated whole retina shows that endogenous polyamines were present in developing and adult retina. Putrescine, spermine, and spermidine were present at each age studied (P1, P3, P7, and adult). Of the three, spermine was present in the highest concentration at all ages. Concentrations of all three were generally higher at birth than at later stages, with the most striking developmental change being the dramatic decrease in putrescine from P1 to adult (P < 0.0002). Data are the mean of results from three retinas at each age; bars, SE.

Figure 3.

 

Effect of DFMO on polyamine immunoreactivity. P3: rabbit retinal explant cultured for 3 days in media containing 60 μM putrescine. Immunostaining shows that polyamines were localized to photoreceptor outer segments. (B) P3 explant cultured for 3 days in medium without putrescine and with 5 mM DFMO. The reduced staining in photoreceptors suggests that DFMO has decreased endogenous stores of polyamines. (C) P3 explants cultured for 1 day in the presence of 5 mM DFMO without putrescine and then replenished by the addition of 60 μM putrescine for days 2 and 3 in culture. Recovery of immunoreactivity in photoreceptors shows that addition of exogenous putrescine partially replenished intracellular stores specifically in cells that normally contain endogenous polyamines. Scale bar in (C) applies to all panels.

Figure 4.

 

Effect of polyamine depletion on the distribution of cone somata in explants of P3 rabbit retina. Explants were cultured for 3 days and stained with rhodamine-conjugated PNA lectin, which labels cone cell body plasma membranes (A, C, E, red), or double labeled with PNA and FITC-conjugated WGA lectin, which labels rod cell body plasma (B, D, F, green). (A) Explants grown for 3 days in control medium containing 60 μM putrescine. PNA brightly labeled the plasma membranes of cone cell bodies (red), showing that cones were distributed along the outer surface of the neonatal retina. (B) Control tissue double labeled PNA (red) and WGA (green) again showing a normal mosaic pattern of photoreceptors, both cones and rods, respectively. (C) P3 explant cultured for 3 days in the presence of 5 mM DFMO and no putrescine. PNA labeling (red) shows that the normal array of cones was disrupted. (D) DFMO-treated tissue double labeled with WGA (green) and PNA (red) demonstrates that the cone distribution was more severely disrupted than the rod mosaic. (E) P3 explants cultured in 5 mM DFMO plus 60 μM putrescine. PNA staining (red) shows that exogenous putrescine blocked the disruption of cones caused by DFMO. Regularity of the cone mosaic pattern was relatively normal. (F) Explants treated with DFMO and putrescine and double labeled with WGA (green) and PNA (red). The arrangement of both cones and rods was similar to that in the control. Scale bar in (F) applies to all panels.

Figure 5.

 

Effect of polyamine depletion on the developing OPL in neonatal rabbit retina. (A) P1 rabbit explants cultured for 24 hours in control medium showed a well-defined OPL (bracket) bordered by horizontal cell bodies (arrowhead). (B) P1 explants grown for 24 hours in medium containing 5 mM DFMO showed a much reduced OPL (bracket). Horizontal cells (arrowheads) were still discernible. Final magnification, ×500.

Figure 6.

 

Effect of polyamine depletion on neonatal rabbit retinal neurons in dissociated cell culture. Dissociated cell cultures of P3 rabbit retina were grown in control medium (A, C, E) or in culture medium containing 5 mM DFMO (B, D, F). (A) Control cultures stained with toluidine blue showed numerous, well-defined clusters of presumptive photoreceptors and horizontal cells. (C) Higher magnification of a horizontal cell (large cell body and large tapering neurite) in control culture with closely associated photoreceptor cells (small, darkly stained cell bodies and short processes). (E) In control cultures, the horizontal cell–specific antibody calbindin (visualized with a rhodamine-conjugated secondary antibody, red) stained only cells with characteristic horizontal cell morphology. Photoreceptors, which exhibited distinct autofluorescence (green), clustered around each horizontal cell. (B) Cultures grown in the presence of 5 mM DFMO, a blocker of endogenous polyamine synthesis, showed a dramatic decrease in the number and size of clusters formed. (D) Magnified view of an isolated horizontal cell without associated photoreceptor cells, characteristic of DFMO-treated cultures. (F) In DFMO cultures, horizontal cells stained with calbindin antibody (red) showed decreased association with photoreceptors (green). Horizontal cell processes were present, but the shape and size of somata were altered.

Figure 7.

 

Effect of polyamine depletion on neurite branching of horizontal cells from 3-day-old rabbit retina. In cultures grown for 3 days in control medium (A, B), many small branches (arrows) were observed along the full extent of the neurite. In cultures grown for 3 days in medium without putrescine and in the presence 5 mM DFMO (C, D), an overall reduction in the number of branches was seen. Branches were characteristically absent along extended regions of the neurite (D, bracket). When cultures were grown in medium with 5 mM DFMO plus 60 μM putrescine (E, F), extensive branching (arrows) was observed, similar to that seen in the control.

Figure 8.

 

Quantitative effects of polyamine depletion on axonal branching. Control and DFMO-treated cell cultures shown in Figure 7 , were stained with calbindin and subjected to morphologic analysis. The number of branch points along the primary axon of each calbindin-stained horizontal cell was determined. DFMO treatment reduced axonal branching by approximately 50% (P < 0.04) compared with the control, whereas addition of 60 μM putrescine in the presence of DFMO blocked this effect. Data are the mean of results in seven cells counted in each treatment group; bars, SEM.

The authors thank Mary Jane Viar for excellent technical assistance.