Rapid isolation of mammalian Mu¨ller cells

Brain Research, 261 (1983) 43-52

43

Elsevier Biomedical Press

Rapid Isolation of Mammalian Mtiller Cells MICHAEL C. TRACHTENBERG and DAVID J. PACKEY Department of Surgery, Division of Neurosurgery, and Department of Physiology and Biophysics, The University of Texas Medical Branch at Galveston, Galveston, TX 77550 (U.S.A.)

(Accepted July 27th, 1982) Key words: glia - - Mtiller cells - - bulk isolation - - retina - - mammal - - enzymatic dissociation

A procedure has been developed for the rapid isolation of relatively pure populations of glia, M~iller cells, from mammalian retina and is reported here for the rabbit. The retina, cleaned of vitreous, is dissociated by 4 interacting operations - - enzymatic digestion of extracellular matrix by means of hyaluronidase, collagenase and papain, removal of divalent cations, acidification and mild trituration. The resultant admixture consists of receptors, neurons and glia; 10% of the cells are Miiiler cells. These glial elements can be brought to 95 % purity by rapid centrifugation on convex 0-30 % Percoll gradients. Of the resultant glia, 80 % exclude trypan blue. The M011ercells are enriched in two glial specificenzymes, glutamine synthetase and carbonic anhydrase and they retain a significant fraction of the membrane bound carbonic anhydrase enzyme activity. Light and scanning electron microscopy show the cells to be well preserved and covered extensivelywith microvilli. In the outer nuclear zone, the cells are plicated and end in bulbs tufted with microvilli. The procedure we describe allows studies of a new preparation of intact, relatively undamaged, adult, isolated mammalian Miiller cells to better understand the functioning of glial cells. INTRODUCTION Neuronal-glial interactions are believed to play a vital role both in information processing and in ion, transmitter and metabolite homeostasis in the nervous system~4,5L The specific contributions of neurons and glia to such functions, however, are not fully understood. These problems are difficult to approach, in large part, because of the interdigitation o f cellular elements, which does not ease compartmental analyses6,20, 23. Retina, as a welldescribed example of laminated central nervous system tissue provides an accessible and somewhat simpler system in which to study these problems1, 2,

5,13,25,39,42,57 One approach to understanding the integrated actions o f neurons and glia is to begin by examining each in isolation, under conditions simulating those found in the intact tissue in vivo. To accomplish this goal, pure populations of the cell should be available in vitro. Tissue culture and bulk-isolation techniques have been the principal methods used to obtain such preparations. However, there are problems 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

associated with the use of these paradigms as in situ models because of cell adaptation to culture conditions on the one hand, and because of the chemical and structural changes attendant to bulk isolation on the other 1°,14,52. A method has been described recently for the dissociation of vertebrate retina into its constituent cells s2,49,5°, and for the further enrichment of the glial (M/iller) cell fraction in the turtle 49. Our interests lie in characterizing the regulation of the ionic milieu by mammalian glia 4s,55,58, and in studying the attendant biochemical alterations during ionic load. Thus, the development of a procedure for rapid bulk isolation of a highly enriched and healthy population of identifiable mammalian glial cells from a well-studied tissue, such as retina, would hold promise of clarifying many aspects of glial physiology and biochemistry. This communication is meant to describe the dissociation and enrichment procedures we have used while engaged in preliminary characterization of the isolated Miiller cells with the goal of assuring, on morphological grounds and by measurement of

44 certain enzymes, that the resultant preparations are reasonably healthy and representative of M/iller cells in vivo. MATERIALSAND METHODS Media Four variations of the artificial retinal fluid of Ames and Nesbetta have been developed for specific steps of the dissociation procedure. Since bicarbonate is known to stimulate glial swelling 11, the bicarbonate concentration of the Ames' medium was reduced from 23 to 5 mM, and the buffering capacity was restored with a combination of the amphoteric (zwitterionic) buffers developed by Good et al. 19. Calcium/magnesium-free medium (Ca-Mgfree) consists of: NaC1, 120 mM; NaHCO3, 5 mM; KC1, 3.1 mM; KH2PO4, 0.5 mM ; Na2SO4, 1.2 mM; HEPES (N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid), 10 mM; MOPS (3-(N-morpholino)propanesulfonic acid), 10 mM; bicine (N,N-bis (2hydroxyethyl) glycine), 5 mM; and D-glucose, 10 mM. The medium is bubbled with 9 5 ~ 02/5% CO2 to attain a pH of 7.3. The 3 other media used are: Ca-Mg-free medium, as above, supplemented with 2.5 mM EGTA (ethylene glycol-bis-(fl-aminoethyl ether) N,N'-tetraacetic acid); Ca-Mg containing medium, where 1.2 mM MgC12 and 0.05 mM CaClz are added to the basal medium; and acidified Ca-Mg-free medium, with 2.5 mM EGTA, adjusted to pH 6.0 with concentrated HCI. All steps are carried out at 37 °C in a cabinet; a humidified mixture of 95 % 02/5 ~ COz was passed through the cabinet. Save Percoll (Pharmacia Fine Chemicals AB), all chemicals were obtained from the Sigma Chemical Company. Adult New Zealand white giant or Dutch belted rabbits, male or female, were killed by intravenous administration of methohexital sodium (Brevital). On occasion, the eyes of cats, rats, frogs and turtles were treated similarly, with similar results. The globes were quickly removed, freed of muscle attachments, and placed in a hemisection-slicing apparatus. The vitreous was fully aspirated from the hemisected globe, after which the globe was removed to a Ca-Mg-containing medium. The globe was everted over a Teflon rod and held in place by embedded hooks, while the retina was gently dis-

sected with a glass crook2, 40. At this point, in many cases, the retinae were weighed before proceeding with the dissociation procedure. Dissociation The isolated retinae were placed in a shaking chamber and bubbled with 95 % 02/5 % CO2, where the residual vitreous was dissolved by exposure to collagenase (4 mg/ml) and hyaluronidase 200 U/ml) in Ca-Mg-containing medium for 20 rain. After a brief rinse in Ca-Mg-containing medium, they were exposed to the Ca-Mg-free, EGTA containing medium for 5 rain, followed by 5 min of exposure to the acidified medium. Then, papain (26.4 U/ml) was added, and the pH was adjusted to 6.5 by adding normal pH medium, for 10 min to dissolve extracellular matrix materials. After 3 rinses in the CaMg-containing medium, the retinal tissue was placed in a conical tube, filled with stabilizing fluid made of Ca-Mg-containing medium supplemented with a 0. i % BSA (bovine serum albumin) and 0,1 °/0 deoxyribonuclease (DNAase). Here, they could be triturated by use of a wide-bore Pasteur pipette. The whole retina was triturated 3 times, and the fragments were allowed to settle for 5 min. The supernatant (cell suspension in stabilizing medium) was removed to iced collection tubes. This process was repeated a total of 3 times. Each supernatant was examined microscopically, and the M/iller cells were counted in duplicate with a hemocytometer. Fractions enriched in MUller cells were selected for enrichment. Enrichment Percoll, a suspension of polyvinyl pyrrolidone coated silica particles, was chosen to form the gradients because of its low osmolality. In several trials, cells enriched on Percoll gradients appeared to be affected less than those recovered from gradients of BSA or Ficoll. Numerous variations in the concentrations of Percoll, and gradients of various shapes, were tested. Excellent results were obtained when the cells were spun through two successive 0-25 % linear Percoll gradients. Similar results were found, however, when a single 0-30 % convex logarithmic gradient was used, which not only simplified the enrichment procedure but increased the yield. The density profile of such convex gradients, as measured with a refractometer, is shown in Fig. 1.

45 1.020 -

•~

1.015-

¢J 1,010-

/

I M~,,o,70,,:°r~c,od

(13 1.005-

2

3

4

5

6

7

8

Fraction number

Fig. 1. Measured density profile of a convex gradient containing0--30~ Percoll in Ames'medium.Measurementswere made with a hand-heldrefractometeron 1.0 ml aliquots.The graph represents average of determinationson 4 gradients. Abscissa indicatesfraction numbers as referred to in text.

Solutions containing the appropriate amounts of Percoll were prepared by using Ca-free, Mg-containing medium such that the medium salts were at full strength and the solutions were hyperosmolar by the small amount contributed by the Percoll ( < 20 mOsm/kg). A mixture of 95% O2/5% COs was bubbled through the solutions before use. Gradients of approximately 14 ml volume were formed in 16 × 100 mm test tubes by means of a Pace gradient maker (Lab-Line). The gradient maker allows the production of either linear or logarithmic density profiles. The complete gradients were carefully stoppered and stored in the refrigerator until use. Supernatant fractions enriched in Mfiller cells, 2 ml in volume, were layered on a convex gradient of 0-30 ~ percoll in basal medium at 4 °C. The Percoll columns were centrifuged at 800 g for 20 s at 4 °C. The centrifugate was divided into eight 2 ml fractions, with care to avoid disturbing the pellet associated with the eighth (the heaviest) fraction, which has been found to contain substantial debris and clumps of undissociated cells. The Mfiller cells were seen to move to the heavier fractions, whereas the receptors, which constituted most of the remaining cells tended to stay in the lighter fractions. With the 0-30 % convex gradients, fractions 5, 6 and 7 were considered to be sufficientlypure to be pooled as the final preparation. The eighth fraction was usually included, depending on the degree of contamination by particles from the pellet. The fractions enriched in Miiller cells were counted and photographed. Samples enriched in Mfiller cells were assayed for protein by a micromodification of the procedure of

Lowry et al. an. Cell DNA was measured by the fluorimetric technique of Kissane and Robins2L The activity of two glial specific enzymes, carbonic anhydrase (EC 4.2.1.1) and glutamine synthetase (EC 6.3.1.2) were assayed for Mfiller cell enriched fractions, as well as for whole retina by the Sapirstein et al. 48 modification of the technique of Maren aS, and the Berl 7 modification of the procedure of Pamiljans et al. 41, respectively, for these two enzymes. The Mfiller cell samples were prepared for scanning electron microscopy by filter fixation with phosphate buffered 2.5 ~o glutaraldehyde. The cells were postfixed with 1 ~o osmium tetroxide. After critical point drying, they were examined by means of an AMR scanning electron microscope. RESULTS

Dissociation/enrichmentprocedure Microscopic examination of the primary retinal dissociate reveals the presence of cells, which by comparison with the Golgi stained material of Cajal (ref. 45, see also ref. 51), can be identified as receptor, horizontal, bipolar, amacrine, ganglion, and Mfiller cells. By the number counted, receptor cells are far the most prevalent, since they were present in 10 times the frequency of Mfiller cells, the next most common cell type (Fig. 2A). Typical yields of Miiller cells at this point are 2.07 4- 0.43 million cells per retina, (starting wet weight of each retina, roughly 100 mg). Some of the retina is lost in processing. Receptors are readily recognized by their maceshaped appearance, in which the handle would correspond to the inner and outer segments. Occasionally, the pedicle of the receptor is also obvious. All of the remaining retinal cell types are much less abundant; together, they account for fewer than 10 ~o of the cells. Horizontal and ganglion cells are the preponderant elements among them, with more of the former than the latter. Few amacrine or bipolar cells are evident. The relative density of cells, per ml and the ratio of receptors to Mfiller cells, vary with the dissociation fraction. The first fraction is most enriched in receptors and has high cellular density. The second fraction is almost as dense, but the ratio of receptor to Mfiller cells is more nearly equal, and the third fraction, which is of low density, has few receptors. Sheaves of Mfiller cells are rare,

46

Fig. 2. Photomicrographs of bulk-isolate preparations. A: stopped-diaphragm, bright-field micrograph of living cells which make up the primary dissociate from the second trituration. Receptors are the primary cell type seen in addition to the Mfiller cells. B: following centrifugation, the dissociate is highly enriched in Mfiller and while the number of contaminating receptors is reduced appreciably. Micrograph as in A. C: scanning electron micrograph of fixed M011ercells. The small spherical objects are receptors. A horizontal and two amacrine cells also separated with the Mfiller cells. although they can result from reducing the duration of the papain step or the concentration of enzyme. The major difficulties encountered are an occasional mucilage-like dissociate, which is probably due to residual vitreous or excess DNA. Somewhat more often, receptors adhere to the Mfitler cells, which results in a crown-like appearance. This phenomenon results from failure of the desmosomal jtmctions to be fully dissociated. The mucilaginous appearance necessitates discarding the preparation, although the problem of sheaves of Mfiller cells or receptor crowns can often be ameliorated by centrifugation, or by a second course of papain, or by reacidification. After gradient centrifugation, microscopic examination discloses that almost all of the Mfiller cells are found in fractions 5, 6 and 7 of the gradient. Few other cells are present (Fig. 2B, C). Contamination from rods accounts for less than 50~i of the cell

bodies, and rare horizontal cells can be seen. By volume, the differences are even more striking; the contaminant rods make up less than 2 ~,, of the mixture as determined by planimetric determinations. The yield of Mtiller cells at this point is 1.18 ± 0.12 million cells per retina, which corresponds to 57 ~k 5.6 o / o f the initial dissociate. This yield is equivalent to 1. l0 mg protein and contains 0.23 #g of DNA. Exposure of these cells to 0.05 ~ trypan blue for 10 rain shows that at least 80 ~ of Miiller cells will exclude the marker.

Glial enzymes Glutamine synthetase catalyzes the ATP-dependent conversion of glutamate to glutamine, with either free NHa or another amino acid used as the amine donor TM. Histochemical evidence has indicated that this enzyme can serve as a marker for glia in retina 46 as well as in brain 36. Localization of

47 TABLE I Glial marker enzymes Total ( U/m4~protein)

Carbonic anhydrase(EC 4.2.1.1.) 549 Miillercells 270 Retina Glutaminesynthetase(EC 6.3.1.2.) Mfillercells Retina

Soluble ( U/mg protein)

Soluble (%)

297 118

54.1 43.7

0.470 0.227

glutamine synthetase in Mfiller cells has been shown in turtle retina by use of dissociated cells49. We observe a two-fold increase of the specific activity of this enzyme in our M/flier cell preparations as compared to homogenates of otherwise intact retinas (Table I). Carbonic anhydrase, like glutamine synthetase, is believed to be a glial specific enzyme, although in brain there is some controversy as to which glial cell type exhibits this enzyme (see Discussion). In retina, carbonic anhydrase has been localized histochemically to Miiller cellsS,9,30,z4,35,4z. In the absence of pigment epithelium or red blood cells, it can be considered as a glial marker. The specific activity of this enzyme is enriched two-fold, as one proceeds from retinal homogenates to bulk isolated M/flier cell fractions (Table I). In nervous tissue, carbonic anhydrase isolates on centrifugation with both the particulate or membrane-bound, and supernatant or soluble fractions5s. Fractionation of retinal homogenates by high speed centrifugation discloses that about 44 % of the enzyme activity is in the soluble fraction. Centrifugation of the M/flier-cell preparation shows a slight increase in this fraction to 54 (Table I). Morphology

Mfiller cells are easily recognized in the light microscope. They clearly correspond to the Golgistained examples from Caja145 as the largest, most distinctive elements of the retina (Fig. 3A). The cells are 6-8/zm in diameter and 80-100/zm long. The vitreal end of the cell has 5-6 finger-like projections with knob-like endings. These projections extend the thickness of the ganglion cell and nerve fiber layers, and the knob-like endings make up the

internal limiting membrane. The projections emanate from a bulbous expansion of the cell body. The length of the finger-like projections is highly variable and they are not infrequently retracted into the bulbous tip under the stress of isolation. In contrast to the variation in the finger-like projections, the remainder of the cell morphological features are constant. The Miiller cells are covered with microvilli-like processes of various lengths throughout their length, although distinct differences are found that are characteristic of each retinal layer. The most prominent microvilli are along the shaft in the inner plexiform layer. These were indicated by Caja145 (Fig. 3A) as small extensions, but are seen to be much more extensive in the light micrographs (Figs. 3B, C). Also, a large concentration of microvilli is found in the outer portion of the inner nuclear layer and in the outer plexiform layer. They emanate from the region adjacent to the soma. That portion of the Mfiller cell which invests the receptor somata in the outer nuclear layer exhibits long indentations and foldings. The indentations probably correspond to the cascade of receptor nuclear regions. The folds, perhaps between groups of receptors, are studded with microvilli, particularly at their crest. These plicae are capped by bulbous feet, each of which ends in a maze of microvillous tufting (Fig. 3E). These bulbs will help to form the outer limiting membrane. The microvilli extend towards the receptor outer segments. DISCUSSION Tbe development of a technique for isolation of Miiller cells from lower vertebrates 49 has provided

48

Fig. 3. A: Golgi impregnated appearance of two MOiler cells from the ox eye. These are typical of Mfiller cells in all mammals; from Caja145. The cornea would be above and the receptors downward; several ganglion cell somata are shown in stiple. B: brightfield stopped-diaphragm photomicrograph of a Mfiller cell after centrifugation. The finger-like processes of the ganglion cell layer are slightly shortened. Microvilli are most apparent in the outer retina. C: differential interference contrast (Nomarski) photomicrograph of a Mfiller cell in the primary dissociate. The pticae and bulbous endings of the receptor cell layer are clear. The finger-like processes of the outer retina have been retracted. D: scanning electron micrograph of 3 Mfiller cells. The central cell shows full extension of the finger-like processes of the ganglion cell layer and their bulbous endings. At the receptor end of the cell the plicae and their tufted bulbs can be seen. E: higher power scanning electron micrograph of the outer nuclear layer portion of a Mfiller cell. The plicae would surround receptor cell bodies as thin lamellae ending in microvilli. The plicae terminate distally as spheres covered with microvillus tufts, surrounding the inner segment of lhe receptors and forming the outer limiting membrane. the framework for the development of the procedures described herein for preparation of isolated healthy Mfiller cells from mammalian retina. These procedures differ from the adaptation by Sarthy and Lam 50 for rat retinae in several ways. The use of an amphoretic buffer system allows reduction of medium bicarbonate to 5 mM (bicarbonate, is thought to be stimulatory to glial cell swelling11). The amphoteric buffer system also allows pH stability even in the absence of CO2. The dissociation is carried out at 37 °C, which was found to yield morphologically more representative cells than was the case at 4 °C, 15 °C or 23 °C. Exposure to papain, presumably the most damaging of the enzymes used, has been reduced, and another step has been included, a short incubation of the retinae in acidified Ca-Mg-free medium, to facilitate the disruption of desmosomal junctions between the cells 15. Preparation of purified M/iller-cell fractions from dissociates of mammalian retina has not been reported previously; rather, Sarthy and Lam 5° had to select ceils manually for enzyme studies. We have achieved cell purification by replacing the time-con-

suming unit gravity sedimentation procedure, previously used on lower vertebrates 49, with a very short gradient centrifugation procedure at relatively low centrifugal fields, Keeping the gravity x minute product roughly equal to the sedimentation time used by Sarthy and Lam 49, many variations of the spin speed, as well as the content and profile of the gradients were tested. The gradient parameters reported are those resulting in the greatest yield and purity of morphologically undamaged cells. The introduction of a continuous, convex, Percoll gradient, prepared in the isolation medium, allows rapid centrifugation, which is the key to the speed of the procedure, the extreme purity ( > 9 5 ~o), and the high recovery we have achieved. Thus, in 2-3 h, a population of at least 1.2 × 106 and up to 2.5 × 106 M/iller cells per retina, containing about 75,000 very small receptor cell bodies, can be isolated for physiological or biochemical study. Maintaining morphological integrity is a second key to this procedure. It eliminates the need for corroborative studies of cell identity and population homogeneity. Preliminary experiments indicate that this procedure is

49 applicable to other species, to young as well as adult, to normal as well as diseased retinae. Two considerations may help to explain the success of gradient centrifugation over the sedimentation procedure. First, since it is impractical to oxygenate the gradients used in either procedure, the enrichment is performed at 4 °C. If one assumes that cells from the retina of a homeotherm are more sensitive to the resultant metabolic inhibition than are those isolated from poikilothermic lower vertebrates, the advantage of the more rapid centrifugation procedure follows directly. Second, since excessive leakage of calcium into many cell types appears to damage the cell 43, we have taken steps to prevent such increases in intracellular calcium activity. Calcium has been omitted from the gradients to prevent leakage across the plasmalemmal membrane during the period of metabolic inhibition. Release of calcium from intracellular stores, particularly mitochondria, which is linked to the temporary loss of metabolism, is more difficult to prevent. It seems likely, however, that the repeated exposure of the tissue to Ca-Mg-free, EGTA containing medium during dissociation would deplete the intracellular calcium stores to some degree. When the cells are reexposed to calcium in the stabilizing medium at the completion of the dissociation, the concentration of calcium is kept low (5 × 10-5 M) to stabilize the cell membranes while minimizing reaccumulation of the ion. Decreasing the period of metabolic inactivity serves to minimize the accumulation of calcium and its detrimental effects. M/Jller cells have been shown by histochemical procedures to exhibit strong reactions for glutamine synthetase and carbonic anhydraseS,9,~s,3o,34,37,4o, 4s,46. These findings are confirmed by biochemical measure of isolated Mfiller cells in the turtle and as we report in the rabbit 40. For carbonic anhydrase, but not glutamine synthetase, the situation for retina is in contrast to that for brain, where some controversy exists as to whether the enzyme is present in both astrocytes and oligodendrocytes or is localized only to the latter17,81,47. In retina, carbonic anhydrase may be found in several other cells besides those of Mfiller. Pigment epithelial cellsS,34,37,43 cones 37,4a the mitochondria of rabbit photoreceptors a4 and the oligodendrocytes and their myelin around the emerging ganglion cell axons all show

carbonic anhydrase activity. We have applied the dissociation procedure described to rabbits with pigmented eyes, as well as to the retinae of other pigmented species and can report that there are virtually no pigment epithelial cells contaminating the preparations. Myelin is also excluded from the preparations as it migrates to fraction 8. Were myelin fragments to remain their contribution would be small. Yandrasitz et al. 60 report 30 ~ of the membrane bound carbonic anhydrase to be in myelin, whichcorresponds to less than 17~ of the total 4s. Furthermore, their data are taken from the rat, an animal with perhaps the highest myelin specific carbonic anhydrase activity (ref. 48). Thus the only non-Miiller contaminant as regards carbonic anhydrase activity comes from the small fraction of photoreceptors which remain in the preparation. At present their contribution is unknown though it is likely to be small became they are only a few percent of the total cell number. In the M/iller cell preparations from rabbits, glutamine synthetase is enriched two-fold in comparison to intact retina. This compares well with the 2.5fold enrichment seen for turtle Mfiller cells49 while the specific activity of the enzyme in rabbit Mfiller cells is 60 ~ greater than in the turtle. In contrast, the enzyme activity is 1000-fold less in primary cultures of cerebral astrocytes differentiated with dibutyryl cyclic AMP 51. Also, human glial tumors show less than 2.0 ~ of the enzyme activity reported here 54. Insofar as the glial glutamine synthetase activity appears to be dependent on trophic interactions with glutamatergic neurons 26, the lack of activity in cultured glia is not surprising, and the level of enzyme activity in dissociated M/iller cells would support, indirectly, other evidence which indicates that glutamate is a retinal neurotransmitterlz. Carbonic anhydrase activity also showed a twofold increase in isolated rabbit Mfiller cells in comparison to intact retina, and a five-fold enrichment is found for the turtle ag. In the intact rabbit retina 56 ~ of this activity is in the membrane fraction. This decreases to 46 ~ in the isolated Miiller cells which may represent membrane damage due to the use of proteolytic enzymes. Measurements of carbonic anhydrase activity in primary cultures of brain astrocytes range from 1.4 to 6.0 U/mg proteine6, 38, 51 in comparison to a value of 8.0 U/mg protein 50 in

50 bulk isolated Mfiller cells from the turtle and the higher activity seen in the rabbit. Furthermore, the subcellular fractionation data reveals a dramatic shift in the ratio of soluble to membrane bound enzyme in the cultured cells. Narumi et al. 3s, for example, find 2770 of the enzyme is membranebound, a value about half that found for intact nervous tissue or isolated M/iller cells. Does a comparison of enzyme activity from intact and isolated tissues provide any insight into relative cell density? Ghandour 17 et al. suggested on the basis of their studies with markers for carbonic anhydrase and glial fibrillary acidic protein that such inferences may be drawn, reporting that the glial cell population is 15-2070 of the neuronal population (the number of cells, not cell volume). The doubling of enzyme activities we see for both glutamine synthetase and carbonic anhydrase implies that M/iller cells may occupy up to 50 ~ of the weight of the retina. This is likely to be an overestimate since we have undoubtedly lost some of the activity originally present, but if one considers the extensive branching of M/iller cell processes and the paucity of any other glia in the retina, an estimate between 35 and 4070 seems justified. Rough estimates by Pope 4~ of the volume profile of neoeortical cells places the neuronal portion at 35--40 ~ and the astrocytic portion at 20-25 ~ . If one considers a tissue comprised of only neurons and one principal glial type, Pope's estimates would result in the same range of 35-4070 glial volume on a cellular basis. The original description of M/iller cell morphology by Caja145 has been reinforced by a variety of modern histological techniques applied to both intact retina and retinal dissociates4,15,~6,~2,a3,~°,51,55. The detailed correspondence between such. descriptions and the morphology of the cells seen in our preparations leaves little doubt as to the identity of the cells. Short processes, which emanate radially

REFERENCES 1 Ames, A., llI and Gurian, B. S., Measurement of function in an in vitro preparation of mammalian central nervous tissue, J. NeurophysioL, 23 (1960) 676--691. 2 Ames, A., III and Hastings, A. B., Studies on water and electrolytes in nervous tissue. I. Rabbit retina: methods and interpretation of data, J. Neurophysiol., 19 (1956) 201-212.

from the entire length of the cell, are consistently observed and appear to be resistant to damage from the isolation procedures. This observation also applies to the microvilli which would extend from the outer limiting membrane into the receptor layer of the intact retina. In contrast, the finger-like processes that extend into the ganglion cell layer and which originate from an enlarged bulb at the boundary of the inner plexiform and ganglion cell layers, are greatly variable in appearance (compare Fig. 3B and C). Beyond these previously described features we now report that the distal end of the cell has a bulb-like protuberance upon which microvilli are studded. In this paper, we have described modifications of the retinal dissociation techniques of Sarthy and Lam49, 50, which have resulted in the first available homogeneous preparation of normally differentiated, non-transformed, healthy glial cells from a mammalian retina. This preparation should be valuable in describing the neuronal-glial interactions that occur not only in retina, but in other parts of the mammalian central nervous system as well. The unique aspects of the functioning of mammalian glia, particularly in the interactions of ion homeostasis and metabolism, may now be studied through cells which appear accurately to represent their counterparts in vivo. ACKNOWLEDGEMENTS This work was supported by grants EYO 3479-01, NS 07377-10 and NS 07377-11 from the N.I.H. We are very grateful to Ms. B. L. Johnson who provided continuing laboratory support. Scanning electron micrographs were kindly provided by Ms. M. Ackerson. Drs. D. M.-K. Lam, L. Partlow, D. Redburn, A. Ames, III and K. M. Hokanson provided helpful discussions.

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