Isolated cells from a mammalian retina

208

Brain Research, 176 (1979) 208-212 © Elsevier/North-Holland Biomedical Press

Isolated cells from a mammalian retina

P. VIJAY SARTHY and DOMINIC M. K. LAM The Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030 (U.S.A.)

(Accepted July 5th, 1979)

The highly complex network of neural connections in the vertebrate retina makes it difficult to study the physiological and biochemical properties of individual retinal cells in the absence of cellular interactions. A direct way to examine such properties is to develop techniques for dissociation of retinas into viable single cells with identifiable morphology. Using proteolytic enzymes and gentle mechanical dissociation, the major classes of cells from retinas of certain lower vertebrates such as reptiles, teleosts, and amphibians have been isolated and identified2,3,5, 7-13. In this paper, we describe a method for the isolation of viable, single cells with identifiable morphology from a mammalian retina. Male albino rats (125-150 g wt; obtained from Texas Inbred, Houston, Texas) were anesthetized with ether and decapitated. The eyes were enucleated and each eyecup was hemisected. The eyecups were incubated for 15 min at 37 °C in 1 ml of mammalian Ringer's solution containing 100 units of hyaluronidase (500 U/ml; Sigma) and 1 mg/ml collagenase (Sigma). The mammalian Ringer's solution 1 was made up of(in mM): NaC1, 125.4; KC1, 3.6; MgCI2, 1.2; NaHCO3, 22.6; NaH~PO4, 0.1; Na2HPO4, 0.4; Na2SO4, 1.2; CaC12, 1.15; and glucose, 10. The retinas were removed from the eyecups, washed in oxygenated Ringer's solution and incubated for 15-30 min at 37 °C in Ca2+-free Ringer's solution containing papain (0.10-0.3 mg/ml; Worthington, 29.8 mg/ml) and EGTA (2.5 mM). The retinas were then washed with normal Ringer's solution and transferred to a conical tube containing 1 ml Ringer's solution supplemented with bovine serum albumin (0.1 ~ ) and deoxyribonuclease I (0.1 mg/ml; Worthington, 3207 U/mg). Cells were dissociated by pipetting the tissue gently up and down through a wide bore Pasteur pipette. After the suspension had settled down for 5 min, the supernatant was removed and kept at 4 °C, and the dissociation procedure was repeated with the remaining pieces of undissociated retinas. The dissociated cell suspensions were examined under a Zeiss microscope equipped with differential interference contrast optics of Nomarski. Visual inspection under 160 × magnification showed that aggregates or undissociated tissue was seldom present as major contaminant in these suspensions. The total yield of isolated cells generally ranged from 5 × 104 to 2 x 105 cells/retina. For dissociation of rod photoreceptors, an incubation time of 15 min and a papain concentration of 0.1 mg/ml were

209

Fig. 1. Isolated cells from the rat retina: (A) rod photoreceptor; (B) horizontal cell; (C) bipolar cell; (D) amacrine cell; (E) ganglion cell; and (F) Miiller cell. All cells are at the same magnification. Bar : 10 ,urn.

210 used. For other cell types, a 30 min incubation in 0.2-0.3 mg/ml papain was employed. Higher enzyme concentrations resulted in cells with poor morphology. The initial incubation with hyaluronidase and collagenase was used to remove most of the vitreous. This step was essential for a good yield of single cells. The major cell types observed in our dissociated cell suspensions are presented in Fig. 1A-F. Morphological identification of the isolated cells was based on Golgi studies of mammalian retinas by Cajal 4. The most abundant class of cells (Fig. l A) could be readily identified as rod photoreceptors since they possessed an elongated cell body and a long outer segment. A second class of cells (Fig. l B) had a small cell body with an extensive dendrite structure and a long, narrow axon. These were tentatively classified as horizontal cells. Another group of cells (Fig. 1C) had a large cell body, one end of which was elaborated into a dendritic tuft with several branches. A long axon emerged from the opposite side of the cell body and terminated in two 'buttons'. These are likely to be the bipolar cells. Fig. 1D shows a putative amacrine cell body with short processes. These cells were difficult to classify and were also found only infrequently. A fifth class of cells (Fig. 1E) were probably ganglion cells since they had an elongated cell body with a long axon at one end and extensive dendritic branches at the other. One of the easiest cell types to identify are the glial (Mfiller) cells (Fig. l F). These cells are about 150/~m long and possess a small cell body with a single foot process from which short hair-like projections radiate along its length. One of these cells, which together with the photoreceptors form the external limiting membrane in the intact retina, usually contained short villous-like projections. Although the different classes of cells were present in most cell suspensions, the relative abundance of each class was, however, different. The photoreceptors were the most numerous with a yield of 1 to 2 × l04 cells per retina. There were about 2 to 5 × 10a isolated bipolar and Mfiller cells per retina. The horizontal, amacrine and ganglion cells ranged between 500 and 1000 cells per retina. In our experience, higher yields of single cells had to be sacrificed for better morphological integrity of the cells. The viability of the isolated cells was also tested by the vital dye staining technique. A drop of the cell suspension to be tested was mixed with a drop of freshly prepared trypan blue solution (0.04 ~ ) on a clean slide and incubated for 3 min at room temperature. This slide was then cover-slipped and examined under a microscope. Under our experimental conditions, ~ 95 ~ of the identified cells excluded the dye. The cells that did accumulate the dye were those with poor or rounded morphology. This result suggests that most of the identified single cells in our preparations can be considered viable by the criterion of vital dye exclusion. The viability of the isolated cells was also tested by examining the ability of the dissociated cell suspensions to synthesize and accumulate the putative transmitter candidates, acetylcholine (ACh), ~,-aminobutyric acid (GABA) and dopamine, from radioactive precursors supplied exogenously 6. These transmitters were chosen since it has been shown that of the various transmitter candidates examined, only detectable quantities of ACh, GABA and dopamine were synthesized and accumulated by vertebrate retinas 10. Retinas and dissociated cell suspensions were incubated with 100

211 TABLE I Synthesis and accumulation o f neurotransmitter candidates by retina and dissociated cells

All values are means ± S.D. of counts/min/mg protein/l h incubation (at 37 °C) for 4 experiments

Acetylcholine 7-Aminobutyric acid Dopamine

Retina

Dissociated cells

7295 ± 543 2989 ± 483 458 ± 213

8299 ± 1046 3695 i 392 297 ± 92

TABLE II Specific activities o f neurotransmitter metabolic enzymes in retina and dissociated cells

All values are means -I- S.D. of F~molproduct formed/h/g protein for 3 separate determinations. Enzymes were assayed as described earlier 11 Retina

Cholineacetyltransferase Glutamate decarboxylase Acetylcholinesterase GABA-transaminase

42.08 ± 13.74 -6876 ± 102.96 ±

Dissociated cells

4.67 1.08 531 5.46

38.91 i 14.56 -5367 ~ 88.18 ±

5.30 0.85 929 29.78

/zCi/ml each of [3H]choline, glutamic acid and tyrosine (spec. act. : 46.8, 46.1 and 91.6 Ci/mmol respectively, all from New England Nuclear) for 1 h at 37 °C. The radioactive products were analyzed by high voltage paper electrophoresis and ascending paper chromatographyg, 10. As shown in Table I, the isolated retinal cells were found to synthesize and accumulate ACh, G A B A and dopamine at levels comparable to those found for the retina. In addition, we also determined the specific activities of several enzymes in homogenates of retinas and dissociated cells to examine whether the dissociation procedure leads to leakage or partial inactivation of enzyme activities in isolated cells. Results of these experiments (Table II), show that the specific activities of 4 different enzymes measured in the dissociated cells were at least 75 ~ of those found in the retinal homogenates. Our findings therefore indicate that, at least in the case of transmitter metabolic enzymes, the cell dissociation procedure did not significantly decrease the activities or the intracellular content of these enzymes. Although the cellular integrity and physiological conditions of the isolated retinal cells must await further studies, the ability of these cells to synthesize and accumulate certain neurotransmitter candidates and the presence of significant levels of metabolic enzymes for these transmitters, indicate that isolated neurons from mammalian retinas may also be suitable for studies of synaptic chemistry such as those reported for lower vertebratesS, ~o-12. The well-preserved morphology and apparent viability of the isolated cells also suggests that these cells may be useful for examining the physiological and biophysical properties of individual neurons by intracellular

212 recordingsa,~, 7. I n a d d i t i o n , since isolated Miilter cells f r o m the rat retina can be easily o b t a i n e d , b i o c h e m i c a l studies similar to those r e p o r t e d for MiJller cells f r o m the turtle r e t i n a tl are n o w feasible f o r m a m m a l i a n retinas as well. F i n a l l y , the d e v e l o p m e n t o f a simple a n d reliable m e t h o d for the dissociation o f m a m m a l i a n retinas into identified cells s h o u l d m a k e it possible to localize a n d examine, at the single cell level, the biochemical changes t h a t occur in m a m m a l s with retinal dystrophies. W e t h a n k Mr. L a r r y Swain for assistance in p h o t o g r a p h y a n d Mrs. C o n n i e R o e d e r - G o r d o n a n d Mrs. L a u n P e a r s o n for p r e p a r i n g the m a n u s c r i p t . This w o r k was s u p p o r t e d (in part) by a F i g h t for Sight G r a n t - i n - A i d f r o m F i g h t for Sight, Inc., New Y o r k City, and by grants f r o m the R e t i n a Research F o u n d a t i o n ( H o u s t o n ) a n d the U.S. N a t i o n a l Institutes o f H e a l t h (EY 02423). D . M . K . L . is recipient o f a Research C a r e e r D e v e l o p m e n t A w a r d f r o m the N a t i o n a l Eye Institute. I Ames III, A. and Hastings, A. B., Studies on water and electrolytes in nervous tissue. 1. Rabbit retina: methods and interpretation of data, J. NeurophysioL, 19 (1956) 201-212. 2 Anctil, M., All, M. A. and Couillard, P., Isolated retinal cells of some lower vertebrates, Rev. Canad. BioL, 32 (1973) 107-119. 3 Bader, C. R., MacLeish, P. and Schwartz, E. A., Responses to light of solitary rod photoreceptors isolated from tiger salamander retina, Proc. nat. Acad. Sci, (Wash.), 75 (1978) 3507-3511. 4 Cajal, S. R. Y., The Structure o f the Retina, translated by S. A. Thorre and M. Glickstein, Charles C. Thomas, Springfield, II1., 1972. 5 Drujan, B. D. and Svaetichin, G., Characterization of different classes of isolated retinal cells, Vision Res., 12 (1972) 1777-1784. 6 Hildebrand, J. G., Barker, D. L., Herbert, E. and Kravitz, E. A., Screening for neurotransmitters: a rapid radiochemical procedure, J. NeurobioL, 2 (1971) 231-246. 7 Kaneko, A., Lain, D. M. K. and Wiesel, T. N., Isolated horizontal cells of elasmobranch retinae, Brain Research, 105 (1976) 567-572. 8 Lain, D. M. K., Biosynthesis of acetylcholine in turtle photoreceptors, Proc. not. Acad. ScL, (Wash.), 69 (1972) 1987-1991. 9 Lam, D. M. K., Biosynthesis of T-aminobutyric acid by isolated axons of cone horizontal cells in the goldfish retina, Nature (Lond.), 254 (1975) 345-347. I0 Lain, D. M. K., Synaptic chemistry of identified cells in the vertebrate retina, Cold Spr. Harb. Symp. quant. BioL, XL (1976) 571-579. I 1 Sarthy, P. V. and Lain, D. M. K., Biochemical studies of isolated glial (MOller) cells from turtle retina, J. Cell BioL, 78 (1978) 675-684. 12 Sarthy, P. V. and Larn, D. M. K., Endogenous levels of neurotransmitter candidates in photoreceptor cells of the turtle retina, J. Neurochem., 32 (1979) 455-461. 13 Sarthy, P. V., Bridges, C. D. B., Lam, D. M. K. and Kretzer, F., Lectin-receptors on cells isolated from turtle retina, Ass. Res. Vision. Ophthal., Abstr., (1979) 259.

Susceptibility of rat neostriatum to damage by kainic acid: age del:endence by J. R. Gaddy, M. D. Britt, D. B. Neilt and H. J. Haigler (Atlanta, Ga., U.S.A.) . . . . . . . . . Effect of clonidine on utilization and potassium-evoked release of adrenaline in rat brain areas by B. Scatton, F. Pelayo, M. L. Dubocovich, S. Z. Langer and G. Bartholini (Paris, France) . . . . . Development of tolerance to the effects of morphine: association between analgesia and electrical activity in the periaqueductal gray matter by G. Urca, R. L. Nahin and J. C. Liebeskind (Ramat Aviv, Israel and Los Angeles, Calif., U.S.A.) . . Isolated cells from a mammalian retina by P. V. Sarthy and D. M. K. Lain (Houston, Texas, U.S.A.) . . . . . . . . . . . . . . . . .

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