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Investigative Ophthalmology & Visual Science, Vol. 29, No. 4, April 1988 Copyright © Association for Research in Vision and Ophthalmology Development of Neonatal Mouse Retinal Neurons and Photoreceptors in Low Density Cell Culture Luis E. Poliri, Mohamed Lehor, and Ruben Adler We describe here a culture method which allows the growth of dissociated mouse retinal neurons and photoreceptors in chemically defined medium. Neural retinas from 2-day-oId C57/BL mice were dissected from other ocular tissues, including the pigment epithelium, and dissociated into a cell suspension after brief trypsination. Most cells attached as single, unaggregated units to substrata pretreated with polyornithine and the neurite-promoting factor (PNPF). The cells were cultured in serum-free, high pyruvate Dulbecco's modified Eagle's medium containing chemically defined supplements. Under these conditions, onset of cell process development was rapid, giving rise to extensive neurite networks. Three morphologically distinct cell types were apparent during the first week in vitro. Some cells retained a circular outline and failed to produce processes, while 50-60% of the cells developed as multipolar neurons showing a large cell body and several neurites. Approximately 90% of these cells reacted with an amacrine cell-specific monoclonal antibody. Some 30% of the cultured cells expressed phenotypic properties characteristic of rod photoreceptors, including a small cell body, an apical cilium, a short neurite with a spherule-like terminal body, and immunoreactivity with antibodies against opsin as well as a rod cell-specific monoclonal antibody. No further signs of outer segment differentiation were observed in these cells. Non-neuronal "flat" cells, which represented less than 0.5% of the total cell number, reacted with an antibody against the glial fibrillary acidic protein. The number of neurons and photoreceptors remained relatively stable during the first 4-7 days in vitro. During the second week in culture, however, there was specific degeneration of greater than 90% of the photoreceptor cells, while less than 20% of the multipolar neurons were similarly affected. Consequently, in addition to providing a system for studying the differentiation of retinal neurons and photoreceptors, the specific degeneration of photoreceptors in these mouse retinal cell cultures makes this system ideal for investigating factors influencing photoreceptor survival. Invest Ophthalmol Vis Sci 29:534-543, 1988 Our knowledge of the cellular and molecular mechanisms controlling photoreceptor development and maintenance in normal animals is very limited, as is our understanding of mechanisms through which abnormal genes can lead to photoreceptor degeneration. Experience with other neural organs has shown that the investigation of these questions can be greatly enhanced by the availability of well defined in vitro systems (review in ref. 1). The chick embryo is the most frequently used source of retinal tissue for these studies, and methods have been recently developed which allow growth of low density, clump- and flat cell-free cultures of chick embryo neurons and photoreceptors.2'4 The mouse retina offers an attractive system for studies of photoreceptor cell survival and differentiation. For example, mouse strains have been described in which single gene mutations cause photoreceptor degenerations resembling human retinitis pigmentosa (review in ref. 5). Unfortunately, there is very limited experience with cell culture systems for mouse retina. Monolayer cultures have been described for retinal neurons and glial cells from other mammals, such as rat6"9 and rabbit.10 The mouse retina has been studied in explant" and reaggregation cultures,12 but low density monolayer cultures of mouse retinal neurons and photoreceptors are not available. We report here a new culture method in which neonatal mouse retinal neurons and photoreceptors are grown at low density in serum-free, chemically defined media, and express differentiated properties which can be recognized with a variety of analytical techniques. From the Retinal Degenerations Research Center, The Wilmer Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Supported by USPHS grant EY-05404, and core grant EY-01765. Dr. Politi is a fellow from the Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina. Dr. Adler is a William and Mary Greve Scholar from Research to Prevent Blindness. Submitted for publication: August 17, 1987; accepted October 16, 1987. Reprint requests: Ruben Adler, MD, Retinal Degenerations Research Center, The Wilmer Institute, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205. 504 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 MOUSE RETINA CELL CULTURES / Poliri er ol. No. 4 Material and Methods Materials The following sources of supplies were used: Falcon (Oxnard, CA): tissue culture plastic, 35 mm dishes; Miles Scientific (Naperville, IL): Lab Tek 8chamber culture slides; Gibco (Grand Island, NY): Dulbecco's modified, high pyruvate Eagle's Medium; Irvine Scientific (Santa Ana, CA): penicillin, trypsin, Hank's balanced salt solution and calcium-, magnesium-free balanced salt solution; Calbiochem (San Diego, CA): Hepes; Sigma (St. Louis, MO): polyornithine, bovine serum albumin, cytidine 5'-diphosphoethanolamine, cytidine 5'-diphosphocholine, DNAase, glutamine, glutaraldehyde, insulin, transferrin, putrescine, progesterone, selenium, soybean trypsin inhibitor, hydrocortisone; New England Nuclear (Boston, MA): 3H-thymidine (sp. activity 6.7 Ci/mmole); Kodak (Rochester, NY): Nuclear track emulsion NTB2, Dektol, Fixer; EM Science (Fort Washington, PA), Polaron (Hatfield, PA), American Scientific (McGraw Park, IL): electron microscopy supplies; Cooper Biochemical (Malvern, PA): fluorescein- and rhodamine-conjugated antibodies. Experimental Animals C57/BL mice were used for all these experiments. The animals were either bred in our own colony or purchased from Harlan-Sprague Dawley Industries (Indianapolis, IN). In most experiments, mice were used on the second day after birth, although other stages were used as indicated in the text. Animals were handled according to the PHS Policy on Humane Care and Use of Laboratory Animals and the ARVO Resolution on the Use of Animals in Research. Dissection Techniques Two-day-old mice were sacrificed by decapitation, and the heads were wrapped in alcohol-soaked pads and kept on ice until dissected. Under the dissecting microscope, the eyelids were removed and the eyes were enucleated using different sets of forceps to preserve sterility. While immersed in a petri dish containing Hank's balanced salt solution (HBSS), the eyes were freed of surrounding loose mesenchyme, and the cornea, choroid, sclera and pigment epithelium were dissected in one step from the optic nerve head towards the cornea using watchmaker's forceps. After removing the vitreous and lens, the isolated retinas were transferred to another petri dish with fresh HBSS. With this dissection procedure, isolated retinas are completely free of contamination with other eye tissues, including pigment epithelium (see 535 histological section in Fig. 1). Retinas from four to six eyes could be dissected in about 50 min. Dissociation Procedures Isolated retinas were transferred to calcium- and magnesium-free HBSS (CMF), cut into small fragments using tungsten needles, and incubated at 37°C for 17 min in 0.25% trypsin in CMF containing 100 ng DNAase/ml. The tissue was then rinsed twice in a 0.25% solution of soybean trypsin inhibitor in Eagle's basal medium (EBM) with Hank's salts, further rinsed twice in EBM containing 1% bovine serum albumin (BSA), and triturated 10-12 times with a narrow-tip Pasteur pipette in 1.5 ml of this solution. The resulting suspension of single cells was diluted in high-pyruvate, low-glucose Dulbecco's modified Eagle's medium (DME), and aliquots were counted with a hemocytometer. Ninety-five percent of the cells were found to exclude Trypan blue. Culture Conditions The culture medium consisted of DME supplemented with penicillin (100,000 U/l), glutamine (2 mM), cytidine 5'-diphosphocholine (2.56 mg/1), cytidine 5'-diphosphoethanolamine (1.28 mg/1), hydrocortisone (100 nM), and the N! supplement at twice the concentrations recommended by Bottenstein and Sato,13 ie, insulin (16.6 X 10"7 M), progesterone (4 X 10~8 M), putrescin (2 X 10"4 M), selenium (6 X 10"8 M) and transferrin (12.5 X 10"8 M). Culture containers were 35 mm tissue culture plastic dishes (Falcon), or chambers mounted on tissue culture plastic slides (Labtek, 8 chambers/slide), pretreated for a minimum of 12 hr with polyornithine (50 Mg/ml in borate buffer, pH 8.4), rinsed twice in DME, and incubated for a similar period at 37°C in a 25% (V/V) Schwannoma conditioned medium containing the neurite promoting factor PNPF.14 Dishes received 600,000 cells in 2 ml of medium, and slide chambers 60,000 cells in 250 /A of medium. The cultures were grown at 37°C in a humidified atmosphere of 5.5% CO2 in air. Microscopic Analysis of the Cultures Live cultures were periodically examined by phase contrast microscopy. For quantitative analysis, replicate cultures were rinsed in phosphate-buffered saline (PBS), and fixed with 2% glutaraldehyde in PBS. Total cell number was determined using an Artek image analysis system (Artek Systems Co., Farmingdale, NY). The relative frequency of different cell types was determined by visual analysis of at least 200 cells per dish. Quantitative determinations were Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 506 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / April 1988 made in triplicate dishes from at least two separate experiments, and are expressed as mean ± standard deviation. Immunocytochemistry For immunocytochemistry the cells were fixed with freshly prepared 4% paraformaldehyde in PBS for 45 min and rinsed in the same buffer. For intracellular antigens, the cells were exposed for 15 min to 0.25% Triton X-100 in Tris-saline. In all cases, the cultures were further incubated overnight in a 2% solution of unlabeled serum from the same species as the corresponding secondary antibody to reduce nonspecific binding. Cultures were incubated with polyclonal primary antibodies at 1:100 dilution for 60 min at room temperature, or to monoclonal antibodies at 1:20 at 4°C, overnight. Primary polyclonal antibodies included sheep anti-bovine rhodopsin from D. Papermaster15 and a rabbit anti-neurofilament antiserum from D. Dahl.16 Monoclonals included several retinal cell-type-specific antibodies from C. Barnstable,617 and an anti-Thy 1.2 antibody from Mark Soloski.18 Secondary antibodies were fluorescein- or rhodamine-conjugated (Cooper Biochemical). The slides were studied using an epifluorescence Nikon microscope, and photographed using Kodak Ektachrome (400 ASA) pushed to 800 ASA. Black and white prints were obtained from color slides using an intermediate black and white negative. Thymidine Incorporation and Autoradiography Mice were injected subcutaneously in the neck region on the day of birth with 10 ^Ci tritiated thymidine (New England Nuclear; sp activity 6.7 Ci/ mmole) per gram of body weight. The following day, retinas were dissected, dissociated and cultured as described above. After fixation in paraformaldehyde or glutaraldehyde and extensive rinsing in PBS, the cultures were dehydrated using increasing ethanol concentrations, air-dried, coated with a 50% solution of Kodak nuclear truck emulsion NTB2, exposed for 5-10 days at 4°C in the dark, developed in Dektol and fixed in Kodak fixer. Autoradiograms were studied with phase contrast microscopy, or with bright field microscopy after hematoxylin counterstaining. Transmission Electron Microscopy Cultures werefixedwith 2% paraformaldehyde and 2% glutaraldehyde in 0.15 M cocodylate buffer, pH 7.4. Fixation was carried out for 20 min at 22°C followed by 40 min at 4°C. Cultures were rinsed in cold buffer, postfixed for 7 min with cold 1% buffered osmium tetroxide, dehydrated and flat-embedded in Vol. 29 Epon. Cells to be sectioned were identified by phase contrast microscopy and their position in the blocks was scored with a diamond mounted in a "false" objective. Ultrathin sections were gridstained with uranyl acetate and lead citrate before examination. Results The Donor Retina Retinas from 2-day-old mice were used for most experiments. As shown in Figure 1A, mitotic figures are very abundant at this stage, and the formation of the characteristic retinal layers is still incomplete. Retinal ganglion cells and inner plexiform layer are present, and the inner nuclear layer already shows some cellular heterogeneity. The outer plexiform layer can not yet be recognized at this stage, and no signs of photoreceptor differentiation are apparent by light microscopy. The conspicuous cleavage plane between neural retina and pigment epithelium allows for the easy isolation of the neural retina completely free of pigment epithelial contamination (Fig. IB). Retinal Dissociation A variety of dissociation protocols were compared, using cell yields, presence or absence of undissociated clumps, and long-term cell survival in culture as criteria. Enzymatic treatments with papain or with collagenase followed by trypsin were less satisfactory than the mild typsinization protocol finally adopted (see Methods). With this protocol, the 2-day retina yielded viable, clump-free suspensions (2.5 to 3.5 X 106 cells/retina on postnatal (PN) day 2; 4.0 to 4.5 X 106 cells/retina on PN day 5). Mouse retinal cells appear to be extremely sensitive to proteases. Treatment with trypsin inhibitor was essential for cells to be grown in serum-free, chemically defined medium; cell survival was erratic in the absence of this treatment. Overall Development of the Cultures: Qualitative Observations Cell attachment was essentially complete within 2-4 hr after seeding, when most of the cells appeared attached as individual units; only a few cells showed processes in these early cultures (Fig. 2A). Neurite formation became more extensive by 6 hr, when many cells established contacts with other cells through their processes (Fig. 2B). The heterogeneity of the cultures increased dramatically during the first week in vitro (Figs. 2C, 3). Some of the cultured cells retained a circular outline and failed to form processes (Fig. 3D); these cells will Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 No. 4 537 MOUSE RETINA CELL CULTURES / Poliri er ol. ^•i« fcL&.*'*j GC # IB Fig. 1. Toluidine blue-stained, 1 ^m plastic sections of 2-day-old mouse retinas. (A) Partially dissected retina. The existence of a natural cleavage plane between pigment epithelium (PE) and neural retina (NR) allows for the easy separation of these tissues. (B) isolated neural retina. Note absence of pigment epithelial cell contamination. Retinal differentiation isstill incomplete at this stage. Although retinal ganglion cells (GC) are already evident, the inner and outer nuclear layers have not yet become separated, and photoreceptor differentiation is not obvious. Note presence of mitotic figures (arrows). Magnification bar: 20 jtm. be referred to hereafter as "process-free cells." Other cells developed the phenotype usually associated with cultured neurons, including a relatively large cell body (10 to 15 Mm in diameter) and extensive neurite development (Fig. 3). These cells will be referred to as "multipolar neurons." Figure 3 also illustrates the broad diversity in the number, extension and branching patterns of nerve fibers derived from different multipolar neurons. For example, some neurons showed a circular meshwork of processes concentric to the cell body (Fig. 3A, C), while other neurons developed one or more long neurites (Fig. 3B, D). It was not always possible to trace the full complement of neurites and branches belonging to individual neurons because extensive neurite development resulted in the formation of complex networks. However, individual neurites as long as 150 /iin could be seen in some cases. A third cell type which developed during the first week in vitro was characterized by a small cell body (4-6 jam in diameter), and by the presence of a single, characteristically short process (5-15 jtm in length) usually terminating in a spheroid-like structure (Fig. 3A, B). These cells were polarized in that there was an apical process at the pole opposite the neurite (Fig. 3A, B). The process could be identified as a cilium by electron microscopy (see below). Also described below are immunocytochemical observations which supported the identification of these cells as developing rod photoreceptors. Non-neuronal, flat cells were frequently absent from these cultures and, when present, did not represent more than 0.5-1,0% of the total cell population. They showed an epitheloid-appearance, and were occasionally associated with small clusters of neuronal cells. Immunocytochemical analysis suggested that they are glial in origin (see Fig. 9). Quantitative Analysis of the Cultures The survival of different cell types during thefirst2 weeks in vitro is illustrated in Figure 4. Multipolar neurons represented approximately 50-60% of the total number of cells throughout the first week. The Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 538 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1988 Vol. 29 number of small monopolar, photoreceptor-like cells was low after 24 hr in vitro} but increased to approximately 30% of the total by in vitro day 7. This increase in photoreceptor-like cells was accompanied by a decrease in the relative proportion of processfree cells. During the second week in vitro the number of multipolar neurons and process-free cells remained relatively constant. On the other hand, there was an almost complete decline in the number of the photoreceptor-like cells during the same period. Immunocytochemical, Autoradiographic and Electron Microscopical Characterization of Photoreceptor-Like Cells Opsin immunocytochemistry: The presence of the visual pigment apoprotein, opsin, was investigated immunocytochemically in cultured mouse retinal cells using a sheep polyclonal antiserum against bovine rhodopsin.15 Positive immunostaining was observed in the majority of the small monopolar cells, thus supporting their identification as photoreceptors. In most of these cells, immunoreactive materials could be seen as a peripheral ring in the cell body, in the neurite and its spherule-like terminal body, and in the region occupied by the apical cilium (Fig. 5A). This pattern was observed at all the in vitro stages studied (3-7 days in culture). Opsin immunoreactivity was also observed in some small process-free "round" cells but, very importantly, multipolar neurons were consistently negative. RET-P1 monoclonal antibody: This monoclonal antibody has been shown to react selectively with rod photoreceptors in the rat.6 The pattern of immunostaining obtained with this antibody in mouse retinal cultures resembled that obtained with polyclonal antiopsin antibody (see above): multipolar neurons were consistently negative, while many of the small, monopolar photoreceptor-like cells were positive and showed a relatively widespread distribution of immunoreactive materials (Fig. 5B). Determination of the day ofphotoreceptor ''birth": Autoradiographic studies have shown that many mouse rod photoreceptor precursors undergo their last mitotic division on thefirstday of postnatal life,19 and that rods represent almost 75% of the cells generated postnatally.20 To determine whether some of the cells expressing a photoreceptor-like phenotype in Fig, 2. Development of mouse retinal cells in dissociated cultures, shown after 1.5 hr (A), 6 hr (B) and 7 days (C) in vitro. Note that most cells attach to the substratum as individual units. Neurite development is already underway at 1.5 hr, and becomes fairly extensive at later stages. At 7 days the cultures still remain essentially devoid of flat cells, and show multipolar neurons (long arrows), photoreceptor-like cells (short arrow), and process-free round cells. These cells are shown at higher magnification in Figure 3. Magnification bar: 40 ^m. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 MOUSE RETINA CELL CULTURES / Poliri er ol. No. 4 539 9 3C 3D Fig. 3. Mouse retinal cells after 7 days in vitro. As shown in A and B, photoreceptor-likc cells (PhR) show a short neurite ending in a spherule-like body (short arrow) and an apical structure (long arrow) that EM studies show to be a cilium (sec also Fig. 7). Multipolar neurons show different morphologies. In some cases the neurons show several long processes (B, D) while other cells appear surrounded by a circular network of processes concentric to the cell body (A, arrowhead; C). A process-free, round cell is shown in D (arrow). Magnification bars: A, B: : C , D: 5 pm. culture were still able to divide in early postnatal life, mice were injected on the day of birth with 10 ^Ci 3 H-thymidine per gram of body weight. On the next day their retinas were dissected, dissociated, cultured for 7 days as described and studied autoradiographically. As many as 5% of the cells were labeled with tritiated thymidine in a 7 day culture. Labeling was restricted to process-free, round cells and photoreceptor-like cells (Fig. 6). Although in some cases it was possible to detect RET-P1 immunoreactivity in thymidine-labeled cells, the frequency of double-labeled cells was not determined. No thymidine-labeled multipolar neurons were seen in the cultures. Transmission electron microscopy: Electron microscopical preparations showed that in the cell body of cultured photoreceptor cells the nucleus was dis- placed towards the site of origin of the neurite, while other organelles were segregated towards the opposite cell pole (Fig. 7). Moreover, cultured photoreceptors showed an apical cilium projecting from the pole of the cell opposite to the nucleus (Fig. 7). No further signs of outer segment development were observed in these cells, such as distal dilation of the cilium or formation of compacted membranous discs. The ultrastructure of the terminal spherule-like body of the photoreceptor neurite was not investigated in this study. Analysis of Multipolar Neurons Using Cell-Specific Monoclonal Antibodies The "HPC-I" monoclonal antibody, which has been shown to react specifically with amacrine Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 540 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / April 1988 A : M u l t i p o l a r neurons * ; Small monopolar cells • : Process- free cells Vol. 29 The identity of the non-HPC-1 reactive multipolar neurons has not been determined. No immunoreactivity was detected with antibodies specific for horizontal, bipolar or ganglion cells,617 but these negative results cannot be considered conclusive. Also inconclusive are our observations with the thy-1 antigen, which is selectively expressed by retinal ganglion cells in rodents.21 Mouse retinal cultures reacted with a monoclonal anti-Thy 1.2 antibody18 only rarely showed immunoreactive cells; the few positive cells that could be detected always showed some morphological signs of degeneration (not shown). Non-Neuronal "Flat" Cells 100. . Days in vitro Fig. 4. Quantitative analysis of cell survival in mouse retinal cultures. The small monopolar cells (•) can be identified as photoreceptor-like cells on the basis of immunocytochemical and electron microscopical criteria (see Figs. 5,7). neurons in rodent retinas,17 reacted with up to 90% of the multipolar neurons present in mouse retinal cultures studied immunocytochemically (Fig. 8). HPC-1 immunoreactivity could be detected without detergent pretreatment of the cultures, indicating that the antigen was present at the cell surface. Although some staining could be seen in nerve fibers, it was particularly conspicuous in cell bodies (Fig. 8). Positive immunoreactivity was never seen in photoreceptors or in process-free round cells. The expression of glial fibrillary acidic protein (GFAP) was investigated immunocytochemically in these cultures using a polyclonal antiserum.16 Immunoreactive cells were rare in these cultures. In all cases, GFAP immunoreactivity was associated with non-neuronal, flat cells (Fig. 9) which, as mentioned above, represent less than 0.5% of the total cells present in these preparations. Cultures From Older Retinas The success of growing neuronal cells in culture is usually affected by the stage at which cells are isolated from the donor animal. Therefore, we investigated the behavior of retinal cells isolated from PN5 and PN7 retinas, cultured with a protocol identical to the one described in the preceding sections for PN2 retinal cells. Cell yields were approximately 4.0 to 4.5 X 106 cells/retina at both stages. By phase contrast microscopy, the cultures were qualitatively similar to those of PN2 retinas. However, the length of time that viable cultures could be maintained decreased to approximately 7-8 days in vitro for PN5 cultures, and 4-5 days in vitro for PN7 cultures (not shown). Fig. 5. Small monopolar, photoreceptor-like cells were immunoreactive with antibodies against both opsin (A) and the rod-specific antigen RET-P1 (B). Note that, in both cases, immunoreactive materials can be seen not only in the cell body, but also in the ciliary region (short arrows), and the neurite with its terminal spherule-like body (long arrow). Multipolar neurons were consistently negative with both antibodies. Magnification bar: 20 jam. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 MOU5E RETINA CELL CULTURES / Poliri er ol. No. 4 541 Thus, it appears that mouse retinal cells also show important developmental stage-dependent changes in their responses to culture. Discussion The studies reported here demonstrate the feasibility of growing mouse retinal neurons and photoreceptors in a serum-free, completely chemically denned environment. Amacrine neurons and rod photoreceptor cells are the two most abundant cell types in these cultures, but the identity of the other multipolar neurons remains undetermined. Other striking features of these cultures include the virtual absence of glial-like "flat" cells, and the presence of abundant nerve processes which give rise to complex networks. Little work has been done with stationary monolayer cultures of dissociated mouse retinal cells. Serum-containing media has been used for retinal monolayer cultures from other mammals,6"10 and it has been reported that rat retinal cells failed to survive in a serum-free medium unless brain extracts were used.8 The culture supplements used in our Fig. 6. Autoradiogram of a retinal culture from mice injected in vivo with 3 H-thymidine 24 hr before culture onset, and grown in vitro for 7 days (see Methods for details). A labeled photoreceptor can be seen (arrow). Magnification bar: 5 pun. Fig. 7. Electron micrograph of a cultured photoreceptor-like cell identified by phase contrast microscopy before sectioning as indicated in Methods. The cell nucleus (N) appears polarized towards one end of the cell, while the opposite pole shows an accumulation of organelles and a cilium (Ci). Magnification bar: 1 ^m. 7 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 542 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / April 1988 Fig. 8. Fluorescence photomicrograph of a mouse retinal culture reacted with the amacrine cell-specific monoclonal antibody HPC-1. Approximately 90% of the multipolar neurons reacted with this antibody, while photoreceptor-like cells were consistently negative. Magnification bar: 20 jim. study were chosen in pilot experiments by systematic trial-and-error, and their specific contributions to the survival and differentiation of neurons and photoreceptors still await more detailed analysis. It is interesting, on the other hand, that mouse retinal cells failed to survive in DME supplemented with different sera, including batches of fetal calf serum routinely used in our laboratory for culturing chick embryo neurons and photoreceptors.3 It has been reported that some animal sera contain inhibitory factors for mouse cells.22 Vol. 29 Mouse photoreceptor cells cultured in chemically defined medium appear similar to rat photoreceptor cells grown in serum-supplemented cultures6 in their morphology (by phase contrast microscopy) and their immunoreactivity with RET-P1 and anti-opsin antibodies. These immunoreactive materials are distributed in a diffuse manner in both cases. Photoreceptor cell differentiation has also been studied in dissociated cell culture from chick embryos in which the majority of the photoreceptors are cones, rather than rods. Under the conditions used in our laboratory,1"4 isolated chick photoreceptors develop a highly elongated, polarized phenotype. Opsin immunoreactive materials are initially distributed in a diffuse manner in these cells, but in older cultures opsin becomes clearly polarized and accumulates in the apical region of the cells which is occupied by a small, outer segment-like process.3 Considering that in vivo EM immunocytochemical studies have shown that polarized opsin distribution is only observed in rat photoreceptors when an intact outer segment is present,23 the failure of cultured mouse photoreceptors to achieve a polarized pattern of opsin distribution may be due to the lack of outer segment development beyond the presence of an apical cilium. It is also noteworthy that chick photoreceptors, when grown in vitro under conditions different from those used in our studies,3 have been found to express a less polarized pattern of morphological organization and opsin distribution.24 It is well established that the survival of many neuronal types is regulated by "trophic" factors produced by postsynaptic target cells as well as by glia and other "satellite" cells (review in ref. 1). Photoreceptor cells have not been well studied in this regard, although they are known to undergo developmental death,25 Fig. 9. Phase contrast (A) and fluorescence (B) photomicrographs of a flat cell reacted with an anti-GFAP antibody. Flat cells (which represent less than 0.5% of the cells present in mouse retina cultures) were the only cells that reacted with this antibody. Magnification bar; 20 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933139/ on 05/12/2017 MOUSE RETINA CELL CULTURES / Poliri er ol. No. 4 and to degenerate in a variety of blinding retinal diseases, including human retinitis pigmentosa. In this context, it is interesting that cultured mouse photoreceptors undergo extensive degeneration during the second week in vitro, while multipolar neurons suffer only minor losses during this same period. This selective degeneration of photoreceptor cells suggests that they may have some special trophic requirements. The recent finding that interphotoreceptor matrix preparations contain a survival-promoting macromolecular activity for cultured chick photoreceptor cells may be relevant in this context.26 Possible roles of this or similar factors can now be experimentally analyzed in cell culture, both from normal mice (as described here) and from animals with retinal degenerations of genetic origin, such as the rd mouse.27'28 Key words: mouse retina, retinal cultures, neurons, photoreceptors, neural culture, retinal development Acknowledgments The authors are grateful to Dr. A. Tyl Hewitt for his comments on the manuscript; to Drs. David Papermaster, Colin Barnstable and Mark Soloski for generous antibody gifts, and to Mrs. Doris Golembieski for secretarial help. References 1. Adler R: Trophic interactions in retinal development and in retinal degenerations: In vivo and in vitro studies. In The Retina: A Model for Cell Biology Studies, Part I, Adler R and Farber D, editors. Orlando, Academic Press, 1986, pp. 112-150. 2. Adler R, Lindsey JD, and Eisner CL: Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer culture. J Cell Biol 99:1173, 1984. 3. Adler R: Developmental predetermination of the structural and molecular polarization of photoreceptor cells. Dev Biol 117:520, 1986. 4. Adler R: The differentiation of retinal photoreceptors and neurons in vitro. In Progress in Retinal Research, Osborne N and Chader G, editors. London, Pergamon Press, 1987, pp. 1-27. 5. LaVail MM: Analysis of neurological mutants with inherited retinal degeneration. Invest Ophthalmol Vis Sci 21:630, 1981. 6. Akagawa K. and Barnstable CJ: Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies. Brain Res 383:110, 1986. 7. Berg G and Schachner M: Electron microscopic localization of A2B5 cell surface antigen in monolayer cultures of murine cerebellum and retina. Cell Tissue Res 224:637, 1982. 8. Turner JE: Promotion of neurite outgrowth and cell survival in dissociated fetal rat retinal cultures by a fraction derived from a brain extract. Dev Brain Res 18:265, 1983. 9. Sarthy PV, Curtis BM, and Catterall WA: Retrograde labeling, 543 enrichment, and characterization of retinal ganglion cells from the neonatal rat. J Neurosci 3:2532, 1983. 10. Osborne NN, Beaton DW, Vigny A, and Neuhoff V: Localization of tryosine-hydroxylase immunoreactive cells in rabbit retinal cultures. Neurosci Lett 50:117, 1984. 11. 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