Exp. Eye Res. (1991)

Cold

52, 175-191

Inhibits

Neurite Outgrowth From Single Retinal Cells Isolated From Adult Goldfish A.T. ISHIDA”

Department

of Animal

(Received

Physiology,

6 March

1990

AND

University and accepted

Ganglion

M.-H. CHENG of California, in revised

Davis, form

CA 95616,

26 June

U.S.A.

1990)

We have studiedthe growth of neuritesfrom singleretinal ganglioncellsisolatedfrom adult goldfishand maintained under various primary cell culture conditions. In 10% Leibovitz’sG15 medium at 23°C. theseganglioncellsremainedviablefor up to 10 daysand generatedextensivefieldsof neurites.We found two patternsof neuritic fields.In one, a pair of neuritesexited from oppositesidesof the cell soma,forming a bipolarpattern. In the secondpattern. three to five neuritesexitedfrom severalpointsaroundthe soma, forming a multipolar pattern. Characteristically,eachneurite of this latter type taperedand branchedtwo to seventimes,whereasneuritesforming bipolar patternsshowedlessbranching and little or no taper. The fieldssubtendedby the neuritesin multipolar patternsrangedin sizefrom 33 000 to 204 000 prn2. Finally, although theseneuritesgrew asfast as35 pm hr-’ at 23°C and individually reachedlengthsof up to 735 ym. they showedessentiallyno growth at 13°C.Neurite outgrowth at 23°Cwasvigorouseven in cellswhosegrowth had previously been suppressed for as long as 8 hr at 13°C. Key words:retinal ganglion ceil: dissociatedretinal cells; neurites; dendrites; axons; regeneration; temperaturesensitivity. 1. Introduction Fish and amphibian retinae exhibit two properties thought to be unusual in the adult central nervous system-the addition of newly differentiated cells (e.g. Hollyfield, 1968; Jacobson, 1976; Johns, 1977), and the ability to recover from physical damage (e.g. Sperry, 1944 ; Grafstein. 1986). This suggeststhat fish and amphibian retinae should be useful for studying factors which influence development and repair of neuronal architecture and synaptic interactions. Within the past few years, the ability of the optic nerve to regenerate following disruption has become a focus of renewed interest in non-mammalian as well as mammalian species (e.g. Aguayo, 1985 ; Northmore, 198 7 ; Vielmetter and Stuermer, 1989 ; Carmignoto et al., 1989; Thanos et al., 1989). Virtually all of the present literature on optic nerve regeneration has, however, been obtained in situ on whole optic nerve, or in vitro with explanted portions of retina. One limitation of such preparations is that it is difficult to track the growth of individual fibers over extended periods of time. Furthermore, it is difficult to correlate stages of regeneration with possible changes in pharmacological or electrophysiological properties of individual ganglion cells. We have, therefore, initiated a study of growth and survival of single ganglion cells in vitro after their dissociation from adult goldfish retina. Recently, we described a method of identifying individual ganglion cells isolated from adult goldfish retinae, and found a GABA-activated chloride conductance, as well as voltage-activated sodium and * For

calcium conductances, in the somata of these cells (Ishida and Cohen, 1988; Ishida, 1989). The purpose of the present study was to characterize the pattern and time course of neurite outgrowth from these somata in vitro, and to test whether certain culture conditions affect this outgrowth. In this paper, we describe two properties of these neurites. Firstly, we have found that retinal ganglion cells isolated by our methods generate two basic patterns of neurites. In one pattern, three to five major neurites emerge from the soma, taper, branch, and form round-oblong fields comparable in size and shape to those of cyprinid ganglion cell dendrites in situ (cf. Cajal, 1972 ; Murakami and Shimoda, 1977; Famiglietti, Kaneko and Tachibana, 1977; Kock and Reuter, 1978b; Kock, 1982; Hltchcock and Easter, 1986). In the second pattern, one main neurite conspicuously outgrows whatever other neurites initially emerge. These singularly long neurites taper and branch less than those of cells with multiple neurites. Second, we have found that these neurites grow well at 23°C but not at 13’C, regardless of the outgrowth pattern ultimately attained. This suppression of neurite outgrowth at 13°C was reversible in that it could be relieved by returning the cell culture temperature to 23°C. To our knowledge, the results presented here are the first demonstration of this temperature effect on singlevertebrate central neurons in vitro. Some of these results have been presented in an abstract (Ishida. Cheng and Bindokas, 1990).

correspondence.

00144835/91/020175+17

$03.00/O

0 1991 Academic Press Limited

176

2. Materials

A. T. ISHIDA

and Methods

Animals The results presented below were obtained from retinal cells dissociated from adult common goldfish (Carassius auratus, 9-16 cm body length). We estimate the age of these fish to be 3-5 yr from the data of Johns and Easter (1977). These fish were maintained outdoors in a 500-gallon holding tank fed continuously by local well water, ranging in temperature from 17 to 2 l°C during the year. Selected fish were transferred to lo-gallon tanks maintained at room temperature (2 3’C) prior to the procedures described below.

Primary Cell Culture To form primary cell cultures containing isolated retinal ganglion cells, we have developed a protocol which incorporates procedures described by Schwartz and Agranoff (1981), Tachibana (1981), and Shiosaka, Kiyama and Tohyama (1984). Since the results presented below are the first to be obtained with this protocol, a detailed description of our methods is given here. Between 2 and 3 weeks prior to each retinal dissociation, two to three fish were anesthetized with tricaine methanesulfonate (Sigma: St Louis, MO) and chilled on ice, and their right optic nerves crushed intraorbitally as described by Landreth and Agranoff (1976). After being returned to fresh water, these fish recovered from the anesthesia within a few minutes, and proceeded to swim and feed actively. Optic nerve crush was performed because it induces swelling of ganglion cell nucleoli (Murray and Grafstein, 1969; Northmore, 1987), and thus allows isolated ganglion cells to be recognized in culture (Ishida and Cohen, 1988: see also Schwartz and Agranoff, 1981). At least 95 % of the somata exhibiting swollen nuclei after optic nerve crush can be backfilled via the optic nerve with horseradish peroxidase (Ishida and Cohen, 1988). For each dissociation, the operated fish (n = 2-3) were spinally transected, and the eyes with crushed optic nerves enucleated. These eyes were rinsed twice with 70% ethanol, passed through a rinse saline (Solution A; Table I), and then hemisected at the ora serata. The optic nerves were cut away from the sclerad side of the resulting eyecups, and the retinae isolated using fine forceps. The exposedphotoreceptors and any residual pigmented epithelium were ‘peeled’ (i.e. removed) with small pieces of cellulose-fiber filter (Type HA, 0.4 S-km pores; Millipore ; Bedford, MA) as described by Shiosaka. Kiyama and Tohyama (1984). [In a few experiments where photoreceptors were desired (see Results), fish were dark-adapted prior to enucleation to facilitate separation of the photoreceptors from ihe pigmented epithelium, and retinae

AND

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CHENG

were not peeled after isolation.] The remaining portions of retina were transferred to a flask containing 8 ml of a saline (Solution B: Table I) which included 8-10 units ml-’ of a neutral bacterial protease (Type XXIV, Sigma No. P-8038: cf. Betz and Sakmann. 1973: Vaughan and Fisher, 1987). This flask was then slowly shaken (0.5 Hz) for 20 min in a water bath maintained at 29°C. Thereafter, the retinae were rinsed by passing them through several volumes of a saline (Solution C; Table I) which contained bovine serum albumin (BSA: 0.01 “/o w/v. Sigma No. A-4378), and triturated in BSA-free rinse solution (Solution A) using a glass pipet whose tip had been broken and fire-polished to a diameter of l-l ‘5 mm. Aliquots of the resulting suspension of cells were plated in 3S-mm plastic culture disheswhose bottoms were replaced by acid-cleaned, uncoated glass coverslips (cf. Bray, 1970). Cells were initially plated in 0.5 ml of culture medium (see Table I) which was pipetted only onto the exposed area of the glass bottom. Cellswere then allowed to settle for 1 hr. after which 3 ml of culture medium were added to each culture dish. This procedure was designedto maximize usable cell yield by restricting their settling to the glass bottom through which the cells were subsequently examined. These primary cell cultures were stored until use in an incubator at 23°C. whose interior was kept humid by an uncovered reservoir of saturated CuSO,. In a few experiments, some cells were maintained in a similarly humidified incubator at 13°C. No change of culture medium was made over the period that the cultures were maintained (1-12 days). All dissociation procedures after enucleation were performed using sterile-filtered solutions, and no antibiotics were usedin any of our solutions or culture media (seealso Ishida and Neyton, 198 5). All solutions and culture media were made just prior to use from powdered ingredients, with water filtered to 18 Mohm resistivity (Milli-Q Water System, Millipore). The commercial culture media tested were Leibovitz’s I,- 1 5 and Medium M-199 (Nos 430-1300EB and 4001200, respectively ; GIBCO, Grand Island, NY ). Light Microscopy and Morphological Measurements lJsing an inverted light microscope, cells were examined through phase-contrast and Nomarski optics at various times after plating. Ganglion cells and their neurites were photographed through 40 x objectives, and drawn with the aid of a camerafucida at a final image magnification of 660 x . Smaller cells of other types (seeResults) were photographed through 6 3 x objectives. Growth of ganglion cell neurites were followed with a video camera and recorded on video tape with a time-lapse video recorder, or photographtti on 35-mm film at tie intervals ranging from 1 to 8 hr. Neurite lengths and field sizeswere measured on a digitizing tablet with the Sigma Scan system (Jandel

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TABLE I Composition of solutions used (see footnote for details) Rinse+ Solution

Enzyme

Rinse

NaCl KC1 CaCI,

140 5 0

140

W3.A

0

D-glucose Pyruvate Hepes BSA (% w/v) M-199 1% v/v, L-15 (% v/v) Glutamine Protease (unit ml-l) PH

10

140

5 5

5 5

1 10

1 10

2

2

10 0 0 0 0 9-10

10 0 0 0 0 0

7.6

BSA

2

10 0.01 0 0 0 0

7.6

7.6

10%

M-199 140 5 5

100% M-199

10% L-15

100% L-15

15

140

0

0 5

5 5

0 5

1

0

1

0

25 2

20 0

26 2

25 0

10 0.1 10 0

10 0.1 100 0

2

1.4

0

0

7.5

7.4

10 0.1 0 10 1.8 0

10 0.1 0 100 0 0

7.5

7.4

The recipe for each solution is listed (in mM. unless indicated otherwise) in the column beneath each solution name. The quantities listed are the quantities udded to make each solution. Note that these are not the Enal concentrations of each ingredient in the culture media. since the two commercial media (M-199 and L-15) contained various quantities of inorganic salts and other ingredients. The final concentrations of K+ were 5.5 mM in 10% M-199 and 10% L-15, and 5.8 mM in 100% M-199 and lOO%L-15. The fmal concentrations of Ca2+ were 5.1 mu in 10~0M-199and10’$!0L-15,and6~2 m~in100~~M-199and100~~G15.BSA,bovineserumalbumin(SigmaNoA-4378):M-199.Medium 199 (GIBCO No 400-1200); L!5, Leibovitz’s L15 (GIBCO No 430-1300EB); pyruvate. Na pyruvate: glutamine, L-glutamine: protease. Nagarse (Sigma No P-8038). The solutions labeled ‘rinse’, ‘ enzyme’, and ‘rinse +BSA’, are referred to in the Materials and Methods section as solutions A, B and C. respectively.

Scientific: Corte Madera, CA), from montages of photographs printed at a flnal image magnification of 730x. 3. Results We have shown previously that retinal ganglion cells can be identified in primary culture after having been isolated from adult goldfish (Ishida and Cohen, 1988). In the present study, we show that ganglion cell somata remain viable in culture for up to approximately 10 days, and generate extensive arborizations of neurites during this time under certain conditions. In the following sections,we describethree sets of results. Since we have modiied previous dissociation and culture methods, we begin by briefly describing the variety of morphological cell-types produced by our dissociations. We then compare the viability and growth of ganglion cells under various culture conditions. Finally, we describe the morphology of ganglion cells which have grown neuritic processesunder the best of these culture conditions. The results describedbelow were obtained from a total of 41 dissociations. Cell Types Observed In freshly plated cell cultures, we recognized six types of cells on the basis of their morphological resemblance to cells identified in situ (seeMurray and Grafstein, 1969; Cajal, 1972; Stell, 1975; Stell and Hlirosi, 19 76 ; Marc and Sperling, 19 76) : rods, cones, horizontal cells, bipolar cells, Miiller cells, and gang12

lion cells [Figs l(A-G)]. Rods were identified by their rod-shaped outer segments, small cell bodies, thin axons, and spherule-shaped axon terminals [Fig. l(A)]. Unlike any other cell-type, rods could be obtained in large numbers simply by shaking isolated retinae in a saline without enzyme. Cones were recognized by their relatively short, tapering outer segments, their ellipsoid bodies, and their pedicleshaped axon terminals [Fig. l(B)]. Slight cell-to-cell variations in length and caliber, as described by Stell and HQrosi (1976) and by Marc and Sperling (1976), were observed. Stellate cells [e.g. Fig. l(C)] and fusiform structures [e.g. Fig. l(D)] resembling the somata and axon terminals, respectively, of horizontal cells were observed frequently, although somaterminal pairs resembling intact cone horizontal cells (Stell, 1975) were never observed. Isolated horizontal cell axon terminals were obtained in relatively large numbers, accounting for roughly one-half of the cells in some dishes. Miiller cells [e.g. Fig. l(E)] were infrequently obtained, but were easily recognizable from their ‘endfeet’ and the stippled appearance of their membrane surfaces (Newman, 1988). Individual bipolar cells were identified by the axon and spray of dendrites which emerged from opposite sidesof their somata [Fig. l(F)]. Similar cell types have been isolated and described by other investigators (e.g. Drujan and Svaetichin, 19 72 ; Lam, 1976 ; Bader, MacLeish and Schwartz, 1978; Kaneko and Tachibana, 1986). Ganglion cells [Figs l(G), 5 and 8-101 were recovered in all dissociations. These cells were identified as ganglion cells by the presence of at least one nucleolus [e.g. n in Fig. l(G)] which had swollen to a EER 52

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AND

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CHENG

FIG. 1. Morphological appearance of six types of cells freshly dissociated from adult goldfish retina. Rod photoreceptor (A), cone photoreceptor (B), horizontal cell soma (C) and axon terminals (D; n = 2). Miiller cell (E), bipolar cell (F), and ganglion cell (G), identified as described in text. Each cell shown here was obtained from a separate dissociation, although all dissociations produced ganglion cells together with at least some of the other cell types. OS, outer segment: s. soma: at, axon terminal: e. ellipsoid : d. dendrites : n, nucleolus : Nomarski optics : calibration marks = 10 pm for all cells.

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FIG. 2. Ganglion cell viability in 10% Leibovitz’s L-l 5 Medium and 10% Medium 199 at 23°C. For each comparison, cells were obtained from one dissociation, plated in the indicated media, then stored in the same incubator. This plot showscell viability asa function of the number of days in culture, where day zero is the day on which the cells were dissociated.‘Percent cell viability’ is calculated as the number of phase-bright ganglion cells observedon a given day divided by the number of phasebright ganglioncellsobservedon day 0, multiplied by 100. Each point representsthe mean from three experiments; openandfilled circlesplot viability in 10%L- 15 and 10 %M199. respectively.Bars= S.E.M. In both media,the number of viable ganglion cells increasedduring the first day in culture, and declinedthereafter at about the samerate. In both media,cell viability droppedbelow 50% after day 8.

2-3 ,~m diameter in responseto the optic nerve crush performed prior to the cell dissociation (seeMaterials and Methods ; Murray and Grafstein, 1969 ; Schwartz and Agranoff, 1981; Northmore, 1987 ; Ishida and Cohen, 1988; Cohen, Fain and Fain, 1989). No conspicuous change in the appearance of these nucleoli was noted during the lo-12 day periods that we examined the cells described below. Almost without exception, ganglion cells observed within 1 hr of plating lacked processeswhich resemble the axons and dendrites they are known to bear in situ (e.g. Cajal, 1972; Hitchcock and Easter, 1986). Although freshly-plated ganglion cells thus appear to have lost their processesduring our dissociations, they are not inviable. First of all, these isolated somata have high resting potentials and can produce large action potentials (Ishida, 1989). Secondly, as described in detail below, these cells remain phase-bright for several days in culture, and are capable of generating extensive fields of neurites.

Viability and Growth of Ganglion Cellsin Dinerent Media and at Different Temperatures After the first day in culture, ganglion cellsproduced by our dissociations regularly exhibited the following features for approximately 1 week (range 3-l 2 days) :

179

smoothly-contoured, phase-bright somata; translucent cytoplasm ; crisp, phase-dark neurites ; and undulating, roving growth cones (see Fig. 5). Thereafter, the somata gradually became lessphase-bright, and loosenedor detached from the culture dish bottom. Eventually, these cells lost their phase-brightness and rounded shape, their cytoplasm appeared granular, and their neurites withered away. In cultures maintained for 1 month, ganglion cells were occasionally recognizable from their swollen nucleoli. However, these cellswere invariably fragile, and lacked elaborate neurites. Within the first l-6 hr after cell plating, we usually found a half-dozen ganglion cells with phase-bright somata per culture dish. In 23 dishes, from 11 different dissociations, we counted 6.4f0.4 cells per dish (meank~.~.~.; range 3-10). During the 10 days following cell plating, these ganglion cell numbers changed (see Figs 24). While quantifying these changes, we questioned whether temperature and culture media affected these changes. These experiments were motivated by the fact that almost all studies of neurite outgrowth from explants of fish retina have been performed in undiluted volumes of the commercial minimal medium Leibovitz’s L-l 5 (e.g. Landreth and Agranoff, 19 76 ; Schwartz, Mizrachi and Kimhi, 1982 ; Vielmetter and Stuermer, 1989) at room temperature. In contrast, previous electrophysiological studies of isolated ganglion and horizontal cells have been conducted using cultures maintained at reduced temperatures (1 O-l 2°C) in salines supplemented slightly with Medium M- 199 (e.g. Tachibana, 1983: Ishida and Neyton, 1985; Ishida and Cohen, 1988; Cohen et al., 1989). We therefore compared cells maintained in two different concentrations of two different culture media (Leibovitz’s L-l 5 vs. Medium 199, referred to hereafter as ‘ L15’ and ‘M-199’, respectively) at two different temperatures (13 vs. 2 3°C). To make these comparisons, we either plated cells from a given dissociation in different media, or maintained cultures in identical media at different temperatures. Then, for several days thereafter, we counted the number of cells which appeared viable (i.e. those which exhibited phase-bright somata with translucent cytoplasm), and normalized these numbers for a given day to the number observed within 2-3 hr of plating. In Figs 2 and 3, we have plotted these ratios as a function of time in different culture media at 23% These figures illustrate three basic results. Firstly, in all culture media, the number of viable cells remained at or above 100% for approximately 4 days after plating. During the first 2 days in culture, these numbers rose to as much as twofold higher than in freshly plated dishes. Over thr. subsequent 6-8 days, these numbers slowly fell: the rates at which these numbers fell were similar in all culture media examined (seeFigs 2-4). Although the reason(s) for the initial rise are not entirely certain,

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L

”

Time

in Culture

(days)

FK. 3. Ganglion cell viability at 23°C in two concentrations of Leibovitz’s L-l 5. This plot showscell viability as a function of the number of days in culture, where day 0 is the day of the dissociation. Percent cell viability is calculated as explained in the legend for Fig. 2. Each point represents the mean from three separate experiments: open and filled circles plot viability in 10% L- 15 and 100% L-l 5, respectively. Bars = S.E.M. Cells for each experiment were obtained from one dissociation, plated in the media indicated, then stored in the same incubator. The initial increase in the number of viable ganglion cells was greater in 10% than in 100% L-l 5. The number of viable cellsfell below 50% on day 5 in 100% L-15, but not until day 8 in 10% L-15.

daily observation of many individual ganglion cells showed that part, if not all. of this rise resulted from recovery of a healthy appearance by cells which were initially considered damaged. Maps of the position of all ganglion cells in several freshly plated dishes, and subsequent examination of these cells over several days, showed that the increase in counts of healthy cells was not due to cells which sank to the dish bottom after the initial cell counts were made. Our second finding in these comparisons was that ganglion ceils remained viable as well in L-l 5 as in M-l 99. Specifically, cell viability was as good in 10 % L-15 as in 10% M-199 (Fig. 2). and as good in 100% L-15 as in 100% M-199 (not illustrated). Our third finding was that cell viability was slightly better in 10% L-15 or 10% M-199 than in 100% L-15 or 100% M-199. For example, the ratio of viable cells observed on a given day, to the number of cells which appeared viable just after plating, remained > 50% for only 4-5 days in 100 % L-l 5, whereas this ratio remained 2 50% for 8-10 days in 10% L-15 (Fig. 3). Furthermore. the increase in viable cells observed during the first 2 days in culture was slightly greater in 10% L-15 than in 100% L-15. Although both of thesetrends were consistently observed, the cell counts in the different media did not differ drastically, and the cell counts overlapped slightly when pooled from all of the experimental trials (Fig. 3). Similar results were obtained in 10% and 100% M-199 (not illustrated). Figure 4 shows comparisons of ganglion cell viability at 13°C (0) and at 23°C (0). At both

1

z

3

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4

5

in Culture

6

7

8

9

10

-I i.

(days)

FIG. 4. Ganglion-cell viability at 13 and 23°C. This plot shows cell viability as a function of the number of days in culture, where day 0 is the day on which the cells were dissociated. Percent cell viability is calculated as explained in the legend for Fig. 2. Each point represents the mean from three experiments: open and filled circles plot viability at 2 3°C and 13°C respectively. Bars = S.E.M. For each experiment, cells were obtained from one dissociation. plated in identical media (10% Medium 199), then stored in separate incubators at the temperatures indicated. In cultures maintained at 13°C. the number of viable ganglion cells declined after a few days in culture, falling below 50% by day 5. In cultures maintained at 23°C. the number of viable ganglion cells invariably increased during the first 2 days in culture. over the numbers observed on day 0. and fell below 50% after day 7.

temperatures. the number of viable cells fell after a few days in culture. However, there are two differences in the apparent survival of cells observed at these two temperatures. First, more cells remained viable for longer periods of time at 23°C than at 13°C. At 23°C. the number of viable ganglion cells fell to half that observed in freshly prepared cultures after 7 days, whereas at 13°C this time was 4-5 days. The second difference seenin Fig. 4 is that, at 2 3°C the number of viable cells rose substantially before falling, whereas at 13°C the number of viable cells fell after a few days without showing any increase. A more striking difference between cells maintained at the two temperatures is that ganglion ceils generated elaborate arbors of neurites at 2 3°C but not at 13°C. This is illustrated qualitatively by Fig. 5. which shows cells at both temperatures within a few hours after plating, and then over a period of up to 2 days thereafter. Within the first day after plating, the cell maintained at 13°C emitted two sprays of short. thin processes[Figs 5(A) and (B)]. However, these processesfailed to exceed 10 pm in length, and began to regress by the second day in culture [Fig. 5(C)]. In contrast, four separate growth cones could be seen emerging from the cell maintained at 23°C by 9 hr after plating [Fig. S(D)]. and ultimately two major processesdeveloped from this cell [Fig. S(E)]. By 41 hr in culture, the longer of these processesreached a

I

100 ym ’

FIG. 5. Time lapse photomicrographs of two gangbon cells-one maintained at 13°C (A-C) and another maintained at 23°C (D-F). These cells were obtained from the same dissociation, and plated in 10% Leibovitz’s L-l 5 Medium. The cell in A-C was kept in an incubator at 13°C. except for the short periods required to take each photograph. Short, fine processes emerged from the cell at 13°C (A). However, these showed no growth during the 1st day in culture (B), and started to degenerate by the 2nd day in culture (C). Short, fine processes also emerged from the cell at 23°C (D) by 9 hr in culture. By the end of the first day in culture (E), two major neurites on this cell persisted. These grew to lengths of 451 and 277 pm. respectively, by day 2 in culture (F). Phase-contrast optics. Calibration bar same for all micrographs A-F.

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400 350 300 P t 5 P 9

200 -

al 2 2

150-

250 -

z

IOO-

-

23%

-

13%

150 100

50 -

0

10

20

30

Time

‘lo

in Culture

23%

-

13%/23’C

-

13%

50

x

0-k

-

f

50

60

70

60

0

(hours)

6. Comparison of neurite growth at 13 and at 23°C. The neurites observed at 23°C (n = 5) emanated from four different cells obtained from a single dissociation: two cells obtained from this same dissociation were used for measuring the length of neurites at 13°C. The third set of measurements at 13’C were obtainedfrom a cell yielded by a different dissociation.AII cells were plated in 10% Leibovitz’s L-l 5 medium. This plot shows the -length of neuritesasa function of time in culture, i.e. time after which the cellswere plated. Eachpoint (0, 2 3°C; l , 13°C) plots mean length. Bars= S.E.M. FIG.

length of 45 1 ,MI-I,while the shorter processmeasured 277,~m [Fig. S(F)]. The outgrowth of neurites from several cells maintained at these temperatures are quantitatively compared in Fig. 6, in which the mean length of five neurites from four different cells maintained at 23°C. together with the mean length of three processesfrom three different cells maintained at 13°C. are plotted against time in culture. This graph shows three results. The most obvious result is that isolated ganglion cell somata generated long neurites (measuring individually up to 735 p,u.min length) at 23°C but showed essentially no growth at 13°C. At 23°C all processesgenerally grew at their fastest rates after approximately 15 hr in culture. During the 12-hr period between 17 and 29 hr in culture, individual neurites grew at an average rate of 14 i&m hr-’ at 23°C (of, compared with 0.2 pm hr’ at 13°C (a). Within this growth period, the fastest growth rates observed (at 23’C) during any 4-hr interval measured 19 f 9 ,um hr-l (mean + s.D.. n = 9). The secondresult shown by Fig. 6 is that the growth rate at 23°C eventually fell to zero after approximately 1.5 days in culture, and that the processesstarted to degenerate (and thus shorten) after 55 hr in culture. The third result shown in Fig. 6 is that neurites emerged from ganglion cell somata after a delay of S-10 hr in

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7. Comparisonof neurite growth at 23°C and 13°C. and at 23°C after having been suppressed at 13°C. These measurements were madeas in Fig. 6, from a total of ten cellsobtainedfrom a singledissociation.Threeof thesewere maintainedcontinuously at 23°C. four were maintainedat FIG.

13°C (except for the time required for photography), and three were maintained at 2 3°C after having been maintained for 8 hr at 13°C. All cells were plated in 10% Leibovitz’s L-

15 medium. Each point plots mean length: (0 ) cells maintainedat 23°C. (0) ceilsat 13°C (a) cellsmaintained at 23°C after being chilled smaller than diameter of included. Between 16 and stored only at 23°C grew Essentially identical growth

at 13°C. Bars = S.E.M. S.E.M. is symbols where bars are not 32 hr after plating, the cells at a rate of lOi2 klrn hr-I. rates (9 f 1 pm hr-I) were

observedbetween 24 and 40 hr after plating, in the cells transferredto 23°C after beingstoredat 13°Cfor 8 hr. Note that these cells did not stop growing

even after 80 hr

in

culture, andthat the lengthsreachedby the neuritesof these cells did not significantly differ from those of the cells stored exclusively at 23°C (see also Fig. 6).

culture (see also Figs 5 and 7). Cells maintained at 2 3°C therefore did not differ dramatically in appearance from cells maintained at 13°C during this initial period. However, thereafter, there was no time at which cells exhibited more growth at 13°C than at 2 3°C. In cultures examined 1 day after plating, 43 out of 53 cells had generated arbors exceeding five cellbody diameters at 23°C whereas none of nine cells observed at 13°C showed neurites exceeding even two cell-body diameters. Reversibility of Neurite Outgrowth Suppression At 13°C light-driven changes in action potential firing can be recorded from cyprinid retinal ganglion cellsin situ (Kato and Negishi, 1978) as can responses to neurotransmitter agonists and antagonists in vitro (Ishida and Cohen, 1988: Cohen et al.. 1989). Nevertheless, having found that ganglion cells grow

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initial delay of a few hours. As also anticipated, negligible outgrowth was observed in cells (n = 7 out of seven cellsexamined) aslong asthey were incubated at 13°C. However, if cells were warmed to 23°C after being kept at 13°C for as long as 8 hr, they produced neurites like those seen in cells maintained at 23°C (n = 3 out of three cells examined). These neurites emerged roughly 8 hr after being transferred to 23”C, showed growth rates similar to those of cells stored only at 23°C (see legend, Fig. 7), and did not significantly differ in shape, length, or arborization, from the neurites of cells incubated exclusively at 23°C. We have not determined the maximum period of storage at 13% beyond 8 hr, from which neurite outgrowth can recover. However, in preliminary experiments, neurite growth was not observed in ganglion cells (n = 3) warmed to 23’C after having been kept at 13°C for 16 hr. Single Cell Morphology and Neurite Patterns Formed at 23°C in 10% L-15

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100 ym FIG. 8. Cameralucidudrawingsof ganglion cellsshowing bipolarpattern of neurite outgrowth in 10% Leibovitz’sL-l 5 Medium at 23°C. Cellsdrawn after 48 hr (A) and 69 hr (B) in culture. Nucleus in somarenderedclear: dark dots therein representnucleoli. Neuritesexit from oppositesides of the somaof each cell, producing a bipolar appearance. However, one neurite greatly outgrew the others in each cell. This longer neurite measures360 ,um in (A) and 707,~m in (B). Note that these neurites are remarkably uniform in caliber, showingessentiallyno taper along their entire length. The neurite and branchesof the cell in (B) appear to have grown with a clockwise preference, as describedpreviously by Schwartz and Agranoff (1981).

well at 23% and essentially not at all at 13% we wondered whether cellspermanently losethe ability to generate processesafter being chilled to 13°C. To test this, we incubated cells at 13°C for 8 hr, then transferred them to 23% and compared their neurite outgrowth during this entire period with that of cells from the same dissociation incubated exclusively at 23°C or at 13°C. Measurements from these cells are shown in Fig. 7. As described above (Figs 5 and 6), extensive neurite outgrowth was observed in cells (n = 3 out of three cells examined) at 23% after an

We have shown above that ganglion cells grow extensive fields of neurites at 23’C. Here, we describe the morphological properties of these cells in more detail after their processeshad reached roughly their maximum spreads, i.e. after 2-3 days in culture (see Figs 6 and 7). These observations were made on cells maintained at 2 3°C in 10% L-l 5, because we have found that their neurites arborlzed somewhat more extensively in 10% L-15 than in 10% M-199 (not illustrated). Isolated ganglion cell somata were either round or slightly oblong in shape. Round somata measured 17+3 ym (meanfs.n.; n = 5) in diameter, while oblong somata exhibited long and short axes measuring 19+4 and 15f3pm (meanfs.n.; n = 20), respectively. A histogram of the size of these somata showed peaks around 18 and 22 ,um (seeFig. 11). The nuclei in almost all ganglion cell somata were positioned eccentrically (see Figs 8-lo), as noted in earlier studies of regenerating neurons (e.g. Cajal, 1928; Murray and Grafstein, 1969). However, there was no consistent correlation between the position of these nuclei and the points at which neurites emerged from the soma. In fact, tie-lapse photographs of individual cells showed that the nuclei slowly moved around the periphery of individual cells (Ishida and Cheng, unpubl. res.). Single somata gave rise to as many as five major neurites. These neurites typically emerged from points spaced more or less equally around the cell. This resulted in two basic patterns of neurites, one which appeared bipolar and one which appeared multipolar. The bipolar pattern was formed by neurites which exited from opposite sidesof a soma, with one neurite typically dwarfing whatever other neurite(s) emerged from the cell (e.g. Figs 5, 8 and 10). Multipolar cells, in contrast, displayed three to five neurites which, on

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polar coordinates with the soma at the origin, subtended an angle 3 180” (Figs 9 and 10). Out of 35 cells which we examined in detail, 22 showed a neurite pattern which was bipolar, whereas the other 13 cells were multipolar. Furthermore, we noticed no correlation between the size or shape of a cell body and the shape of the field of neurites it generated. Aside from these patterns, a few cells displayed a lop-sided field of neurites, in which a single primary neurite (or two neurites close together) grew out from the soma and branched extensively. These cells consequently looked like multipolar cells which simply sent neurites out to only one side of their somata. and will not be described further here. Some neurites, especially those exiting cell somata in a bipolar pattern, showed little or no tapering over their entire length (see Figs 5, 8 and 10). The only

conspicuous changes in caliber observed in these processeswere at growth cones and at small varicosities (seeFigs 5, 8 and 10). Some of these neurites did not branch (e.g. Fig. 5). while others did (e.g. Figs 8 and 10). Other neurites, especially those of multipolar cells. tapered with distance from the soma, particularly between the soma and first (i.e. most proximal) branch point. This was observed in cells with either small or large somata, as can be seen in Figs 9(A) and (C) and 10(A). In the latter cell, the cell’s two major neurites (arrows) measure 4 ,um in width where they emerge from the cell body, then taper to roughly 2 /trn within the first 20 ,um of the soma. At one branch point in this cell [lower left side of Fig. 10(A)], as at the first branch points in other multipolar cells [see Figs 9(A) and CC)], the neurites thin to < 1 ,um and thereafter

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FIG. 9. Ganglioncellsdisplayinga multipolar pattern of neurite outgrowth in 10% Leibovitz’sL-15 Medium at 23°C.Camera lucida drawingsof two cells,one after 70 hr (A) and another after 51 hr (B) in culture. Nuclei and nucleoli renderedasin Fig. 8. Micrograph of a third multipolar ganglion cell after 60 hr in culture (C): arrow points to soma.Neuritesexit from several points (4 in A, 3 in B, 5 in C) around the edgeof the soma.producinga multipolar appearance.Note that someof the neurites in A and C are thickest wherethey exit the somaand taper with distancefrom the soma.whereasthosein B are relatively thin where they exit the somaand taper little. The neuritesin A and C branch many timesand intertwine with eachother, whereas thosein B branch but do not tangle. The arborsformedby theseneuritescover an area of approximatley 204000 pm2 in A, 137 100 ym2 in B, and 183000 pm2in C. Calibrationbar = 1SOpm for A, B, and C.

remain uniform in diameter except for occasional varicosities (see below). In some cells, the calibers of the neurites were difficult to measure, however, because they fasciculated and overlapped with other processes[see Figs 9(A) and (C)l. Fasciculation was not restricted to any one neurite over others in a given multipolar cell. Individual neurites of all multipolar ganglion cells examined, and of most bipolar ganglion cells, branched one to seven times (see Figs 8-10) after emerging from the cell body. In general, the neurites of bipolar ganglion cells branched less than those of multipolar cells. For example, in seven bipolar ganglion cells, the major neurite branched 1 + 05 times (mean + s.E.M.), whereas in six multipolar cells, 3 + 1

branchings (mean + s.E.M.) were observed per neurite in a total of 17 neurites. The distance between the soma and Ilrst branch-points tended to be longer in bipolar ganglion cells than in multipolar cells, measuring 89 f 18 pm and 23 f 5 ,um, respectively (mean + s.E.M.). However, this distance varied widely from cell to cell, ranging from 5 to 179 pm in bipolarshaped cells, and from 3 to 54 pm in multipolar cells. Only 10% of the neurites in all cells examined showed no clear branching. The field covered by the neurites of multipolar ganglion cells were measured as the area of the polygon formed by lines connecting the ends of adjacent neuritic branches. These fields (n = 9) ranged in area from 3 3 000 to 204 000 km2 (e.g. Fig. 9). The

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FIG. 10. Camera Zucida drawings of tapering and non-tapering neurite segments. A, C, Magnified views of the somata and primary neurite segments of the cells shown in their entirety in (B) and (D). respectively. Pairs of arrows in (A) and (C) point to pairs of tapering and non-tapering neurites, respectively. Multipolar (B) and bipolar (D) ganglion cells, drawn 50 and 44 hr, respectively, after plating in 10% Leibovitz’s L-15 medium at 23°C. Note that major neurites of the multipolar cell taper, whereas those of the bipolar ganglion cell do not. Nuclei and nucleoli rendered as in Fig. 8.

long axis of these fields measured 510 f 49 pm (mean * s.E.M., n = 9; range 283-711 ym), while the short axis measured 330 k45 pm (mean+~.~.~., n = 9 ; range 160-52 7 pm). Thus, the ratio of the long axis to short axis of the neuritic fields was roughly 1.5 in these cells. As described by Vinnikov (1946) and by Schwartz and Agranoff (198 l), the outgrowths of someganglion cells appeared to be formed by the fasciculation of two to three fine strands of neurites. As also noted in these earlier studies, varicosities, measuring a few microns in diameter, were observed in the fine, terminal branches of many cells (e.g. Fig. 9). Although we have not examined the contents of these varicosities in detail, we have noticed in time-lapse video recordings that someof these swellings moved along the neurites, sometimes toward the cell body, and at other times, away from the cell body. Growth cones decorated the tips of all growing neurites (e.g. Fig. 5). Morphologically, these structures resembled those seen capping the neurites of various cells in vitro (see Letourneau and Kater, 1985), i.e. they consisted of a fan-like array of thin processes

(filopodia and lamellipodia) emanating from a tubershaped basal structure. When viewed in time-lapse pictures, these growth cones moved in a flame-like manner, resulting from the extension and retraction of the lamellipodium and filopodia. During the lirst 12 hr of neurite extension, the filopodia of single growth cones spread over areas as wide as 3 5 km, By the 2nd day in culture, the individual filopodia tended to be shorter. Similarly, the lamellipodia were typically flared in profile during the first 24 hr in culture, whereas they were usually slim and elongated during the 2nd day in culture (seeFig. 5). 4. Discussion In this study, we have found that single retinal ganglion cells of adult goldfish generate two basic patterns of neurites in vitro, and that these cells generate these arbors of neurites relatively rapidly at 23% but virtually not at all 13%. No other cell-type in our cultures (i.e. rod, cone, horizontal, bipolar and Miiller cells) grew processesof comparable length or breadth.

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FIG. 11. A histogramof the sizeof ganglioncell somata. Measurementsfrom 25 ganglion cellsmaintained in 10% Leibovitz’sL-l 5 medium.Somasizeis either the diameterof round cells,or the length of the long axis of elongatedcells. Peaksappearin this histogramaround 18 and 22 pm. The long axis of oblongsomatarangedfrom 12 to 27 pm, while the short axesrangedfrom 10 to 21 pm.

Comparisonwith CellsIn Situ The ganglion cells which we have been able to dissociate from adult goldfish retinae resemble ganglion cells found in cyprinid retinae in three basic respects: firstly, by the size and shape of their somata ; secondly, in that the individual shapesand collective arborizations of the neurites of some cells resemble those of intact dendrites: and thirdly, in that the shapesof other neurites more closely resemble axons. Cyprinid ganglion cell somata range in size from 5 to 20 pm in situ (Famiglietti et al., 1977; Murakami and Shimoda, 1977; Kock and Reuter, 1978a; Hitchcock and Easter, 1986) while those in our cultures ranged in size from 12 to 27 pm (Fig. 11). These ranges are remarkably consistent with each other, assuming that the ganglion cells in our cultures had hypertrophied as much as observed by Murray and Grafstein (1969) as a result of the optic nerve crush performed prior to dissociation. The majority of ganglion cell somata in our cultures were less than 2 5 km in diameter, with only 8 % of all cells exceeding 25 pm in their longest axis. On the basis of size (and relative numbers), these latter cells seemto correspond to the ‘large ’ ganglion cells discussed by Kock and Reuter (1978b), and to cells which hyperpolarize when their receptive field centers (i.e. the photoreceptors overlying their dendritic fields) are illuminated (cf. Famiglietti et al., 1977). Aside from this possibility, however, the intracellular recording and dye-injection studies published to date do not allow us to predict which functional properties other ganglion cells in our cultures might have had in situ simply on the basis of the profiles of their somata. The neurites of many cells, particularly of multipolar ganglion cells, were thickest where they exited their

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somata, and then smoothly tapered. Typically, this taper was sharpest between the cell body and first branch point, with the neurite falling abruptly in diameter and remaining uniformly thin thereafter (Fig. 9). In situ, the dendrites of at least someganglion cells exhibit very similar profiles (e.g. Boycott and Wassle, 1974; Kock and Reuter, 1978b; Hitchcock, 1989), suggesting the possibility that ganglion cell neurites which taper may be regenerated dendrites. This would be consistent with numerous studies which have classified tapering neurites in a variety of other cells in vitro as regenerated dendrites (e.g. Bartlett and Banker, 1984). Also like ganglion cell dendrites in situ, the neurites of multipolar ganglion cells branched and arborized over fields which were either round or slightly elongated in shape. Since the ratio of the long- to short-axes of these fields averaged around 1.5, the overall shape of these arbors resembled that of the dendritic fields of ganglion cells located in central and pericentral, rather than far peripheral, fish retina (Kock, 1982; Hitchcock and Easter, 1986). For example, the neuritic field in Fig. 9(A) is slightly elongated, and measures204 000 pm2 in area, like the dendritic fields of type 1.2 cells (seefig. 7 in Hitchcock, 1989). Two other common features of these cells are the virtual lack of taper after the first branch points, and the varicosities in the terminal branches. We do not know whether the size and shape of the neuritic fields which we have observed in the present study were influenced more by some intrinsic factor (Eysel, Peichl and Wassle, 1985 ; Montague and Friedlander, 1989) by the optic nerve crush performed prior to dissociation (cf. Schwartz and Agranoff, 1981) or by the culture conditions in our experiments. However, we have found no ganglion cell neurites which formed fields vastly exceeding those of ganglion cell dendrites in situ. We recognize that someof the morphological details which we have found in neurites are not unique to dendrites. For example, neurites in vitro may not be presumed to be dendrites because of their ability to branch, since branching is observed in situ in regenerating (e.g. Fujisawa et al., 1982; Murray, 1982) as well as developing (Ramoa, Campbell and Shatz, 198 7) axons. Similarly, although many neurites exhibited varicosities like those found on ganglion cell dendrites (see e.g. Hitchcock, 1989). varicosities are also found on regenerating optic nerve processes (e.g. Lanners and Grafstein, 1980; Stuermer, 1988). Furthermore, neurites which emerge from ganglion cell somata may not necessarily be identified as regenerating axons becausedendrites retain the ability to grow in postnatal (Perry and Linden, 1982 ; Eysel et al., 1985) and even adult animals (e.g. Kock, 1982: Hitchcock, 1987). Thus, the major resemblance to dendrites that we have observed in certain neurites is their tapering, and the multipolar patterns which they collectively form.

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One possible difference between the multipolar neuritic fields which we have found and diffuse dendritic fields in situ is that the neurites of single ceils often intertwined and overlapped with other neurites of the same cell over distances measuring tens of microns (see Fig. 9). Since there were no obvious physical guides or barriers which constrained the neurites to merge, some neurites thus appeared to fasciculate with each other. This is not a feature which is characteristic of adult g.anglion cell dendrites in situ (see Wfissle, Peichl and Boycott, 1981), but rather a well-known trait of ganglion cell axons (e.g. Cajal, 1972). However, dendrites of developing cat retinal ganglion cells have been found to form ringshaped structures (Dann, Buhl and Peichl, 1987), which might have arisen by the merging or fusion of pairs of dendritic processes which had branched. Also, the dendrites of some amacrine cells at least appear to overlap tightly in situ (e.g. Tauchi and Masland, 1985 ; Vaney, 1986). Thus, the ability of the neurites to fasciculate does not argue against their being dendritic. In contrast to the tapering neurites seen on multipolar cells, the major neurites forming bipolar patterns typically showed little tapering or branching. Also, these tended to be thinner than the neurites which tapered, at least where they exited their somata. All of these characteristics are shared by ganglion cell axons in situ, viz. axons differ from dendrites in that they are smaller in caliber than major dendrites where they exit the soma, taper less, and may even increase in diameter at some distance from the soma (Kock and Reuter. 1978b: see also Arkin and Miller, 1988; Dacey, 1989). It should be pointed out that there are certain morphological features of dendrites which we have not yet seen in vitro. For example, we have yet to see cells with tapering neurites arranged in a bipolar pattern, like dendrites of ganglion cells observed along the extreme periphery of the retina (e.g. Kock, 1982 ; Hitchcock and Easter, 1986). Furthermore, we generally see fewer branch points in individual neurites than has been described in dendrites in situ (e.g. Kock, 1982 ; Hitchcock and Easter, 1986), although this difference is based on counts in which we have ignored the many thin processes which were characteristically observed along the entire length of the neurites of multipolar cells. Finally, we have not yet seen a cell with a multipolar field of neurites in addition to one exceptionally long process. Since the processes which we have described emerge after a delay (see Figs 5-7) which is shorter than that after which optic nerve processes begin to regenerate in situ (McQuarrie and Grafstein. 1981), we can not exclude the possibility that ganglion cells do not regenerate axons in culture. However, we doubt this explanation because we have seen many cells with only singularly long processes with little or no taper (e.g. Figs 5, 8 and 10). An alternative explanation is that there may be

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some mechanism which inhibits dendrites and axons from regenerating simultaneously in vitro, and that the formation of either axons or dendrites delays the formation of the other beyond the time we have monitored cells in our cultures. Differences in the time of axonal and dendritic outgrowth have been observed in developing retinal ganglion cells (Cajal. 1960) as well as in regenerating hippocampal neurons (Dotti, Sullivan and Banker, 1988). Whether a cell’s outgrowth is monopolar or multipolar may depend on how much of its axon survived the dissociation procedure, as described in Aplysia neurons (Schacher and Proshansky, 1983). However, if such axonal or dendritic sturnps were retained by the ganglion cells we have studied, then these were either less than IO [drn in length (see Figs 1, 6 and 71, or they may have been resorbed by the somata and escaped our notice (see below). Neurite Outgrowth

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Vitro

It was our experience that freshly-dissociated ganglion cells almost always lacked processes which resembled their normal dendrites or axons. Similar observations have been made in other central neurons (e.g. Dotti et al., 1988). as well as in sympathetic neurons (e.g. Bray, 1973). Processes of goldfish ganglion cells may be unusually fragile, since the relatively thin axons of goldfish cones and bipolar cells can survive trituration, since cells with large dendritic arbors can be obtained from catfish retina using a dissociation protocol similar to that used in the present study (Ishida. unpubl. res), and since retinal ganglion cells from postnatal rat can be isolated with some processes intact (Leifer et al., 1984; Barres et al., 1988). The rate of individual neurite elongation which we observed (maximum over a 1-hr period: 35 pm hr-’ : average over 4-hr periods : 19 pm hr-’ : see Fig. 6) is very similar to the growth reported in various retinal preparations, e.g. 14-30 pm hr-’ for regenerating goldfish optic nerves (McQuarrie and Grafstein, 198 1). 20-30 pm hr-’ for goldfish retinal explants (Landreth and Agranoff, 19761, and 17-32 ,um hr’ for Xenopus explants and retinofugal axons (Harris et al., 1985). Furthermore, both the diffuse neurite arbors and the singularly long processes formed by cells at 23°C in our cultures resemble processes seen in other cultured retinal ganglion cells (Vinnikov, 1946; Schwartz and Agranoff, 1981 ; Leifer et al., 1984: Raju and Bennett, 1986; Wigley and Berry, 1988; Montague and Friedlander. 1989). By comparison, ganglion cells maintained at 11-13 “C (Figs 5-7; see also Ishida and Cohen, 1988) appear stunted. We have not yet investigated the mechanism(s) underlying this effect of reduced temperature. However, this effect appears not to be a peculiarity of our culture system, and thus may be mediated intracellularly. since reduced temperatures have been found to impede the regeneration

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of retinotectal connections (Springer and Agranoff, 1977), ofsciaticnerve(Carlsen. 1983) andofolfactory nerve (Cancalon, 1983). In any event, our results should be useful in planning experiments where minimal neurite outgrowth would be advantageous. For example, since ganglion cells remain rounded and do not generate large fields of neurites at all at 13%. or for several hours at 23”C, it should be possible to obtain uniform control of their membrane potential with voltage-clamp methods (cf. Taylor, Moore and Cole, 1960). This would facilitate electrophysiological studies of membrane conductances of ganglion cell somata (see Ishida and Cohen, 1988; Ishida, 1989; Cohen et al., 1989). By the same token, neurite-free cells would not be expected to yield information on the electrophysiological properties of dendrites or axons. In summary, we have demonstrated that at 23°C but not at 13% isolated adult retinal ganglion cell somata generate two patterns of neurites : large, diffuse arbors of neurites which exhibit certain similarities to dendrites seen in situ, and singularly long processes which more closely resemble axons. For several reasons, it would be useful to decide whether any of these neurites were dendritic or axonal. For example, if some of these processes could be demonstrated to be dendritic, one would wonder whether dendrites are also regenerated by retinal explants, and in turn, whether some properties attributed to regenerating axons on the basis of explant studies might be shared by regenerating dendrites. Furthermore, if dendritic and axonal processes could be firmly identified on individual ganglion cells in vitro, we may be able to identify factors which specifically affect dendritic and axonal regeneration, and begin to study how these factors exert their infuences.

Acknowledgments This work was supported by NM grant EY 08120 from the National Eye Institute (Bethesda, MD). The authors thank R. J. Leslie, B. Mulloney, R. P. Scobey and M. C. L. Wilson, for use of various pieces of equipment, Carpenter’s Goldfish Farm (Merced, CA) for a reliable supply of healthy fish, and V. P. Bindokas. R. C. Carlsen and M. C. L. Wilson, for comments on the manuscript.

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