Neuroehemical Research, VoL 17, No. 11, 1992, pp. 1105-1112

Influence of Exogenous Gangliosides on the ThreeDimensional Sprouting of Goldfish Retinal Explants In Vitro U. Sonnentag x, H. R6sner x, and H. Rahmann a,2 (Accepted March 10, 1992)

To investigate the 3-dimensional outgrowth of ganglion cells of normal and regenerating goldfish retina, retinal explants were cultured in a serum free 3-D fibrin matrix. Daily applications of exogenous gangliosides (GM1), injected either intraocularly (i.o.) or intraperitoneally (i.p.) had no significant effect on the sprouting activity of retinal explants prepared from lesion-activated goldfish whose corresponding optic nerve had been transected. However, in normal, unlesioned animals, a local i.o. injection of GM1 or mixed gangliosides led to a significant enhancement of the basal retinal sprouting activity as compared to controls, which were injected with a 0.9% NaC1 solution. This ganglioside related stimulation was maximal after i.o. injection of low concentrations (3 Ixg/eye),didn't occur at high concentrations (30 p~g/eye)and was similar to the response obtained after i.o. injection of NGF or insulin. I.o. injected phospholipids had no or a slightly inhibitory effect on the sprouting activity as compared to NaC1 controls. Daily in vivo i.o. injections of the monoclonal antibody Q211, specifically recognizing c-pathway polysialogangliosides, led to a dose dependent inhibition of the in vitro sprouting of goldfish retina explants. In summary, these data suggest an involvement of gangliosides in the complex process of induction of neuronal sprouting. KEY WORDS: Gangliosides; GMI; regeneration; goldfish retinal explants; sprouting; fibrin matrix.

6), suggesting an important, though still undefined, function in neuronal differentiation, growth, and synapse formation. After injury, exogenously administered gangliosides have been shown to enhance neurite outgrowth in vitro (7-10) and nerve regeneration in vivo (8,11-14). A suitable system to study the role of gangliosides during regeneration is the goldfish optic system. For example, following injury after crush (12,15,16) or transection (17,18) of the optic nerve, there are substantial metabolic changes of retinal ganglion cells including a transient increase in the synthesis of proteins and gangliosides, reaching a maximum approximately 8 days after nerve injury (14,17). Sparrow et al. (19) showed, that i.o. injection of antiserum to mixed gangliosides or GM1 inhibited the regeneration of goldfish optic axons following optic nerve crush, while Grafstein et al. (12)

INTRODUCTION Gangliosides are glycosphingolipids which are integral components of neuronal membranes and seem to play a role in regulation of neuronal development and repair. Thus, several studies have demonstrated that the ganglioside composition of nervous tissue changes, both, qualitatively and quantitatively, during development (1i Institute of Zoology (220) Garbenstr. 30 D-7000 Stuttgart 70 Tel. 0711/459-2255 Fax: 0049711 459-3450 2 To whom to address reprint requests Abbreviations used: Ganglioside nomenclature follows the IUPACIUB recommendations, 1977. Lipids, 12:455--468.

1105 0364-3190/92/1100-1105506.50/0 9 1992PlenumPublishingCorporation

1106 noticed, that daily application of exogenous gangliosides enhanced axonaI outgrowth of goldfish retinal ganglion cells in dependence from the dose and mode of application. Similarly, outgrowth of goldfish retinal explants in vitro was inhibited by anti-ganglioside antibodies, when added either to the culture medium (20) or to the retina by i.o. injection before preparation of explants (21). Thereby, antibodies to the ganglioside GM1 proved to be the most effective (20,21). The aim of the present study was to further ew'~uate the role of gangliosides in regeneration of the gol6fish optic system, using a newly developed 3-dimensional (3-D) culture system for goldfish retinal explants.

EXPERIMENTAL PROCEDURE

Materials. Tricaine methanesulfonate (MS), fibrinogen from goat blood, Dulbecco's modified Eagle's medium (DMEM), penicillin/ streptomycin, tetracyclin, insulin, nerve-growth-factor(NGF), bovine serum albumin (BSA), anti-mouse Ig M conjugated to fluorescein isothiocyanate (FITC), phosphatidylserine (PS) and phosphatidylcholine (PC) were purchased from Sigma, Munich; thrombin from HoffmannLa Roche, Grenzach-Whylen;sterile plastic peN-dishes (~35mm) from Greiner, Ntirtingen; RPMI-medium, Ultroser G and fetal calf serum from Gibco BRL GmbH, Eggenstein. The ganglioside mixture Cronassial (GMix), consisting of 21% GM1, 2% GD3, 40% GDla, 16% GDlb, 19% GTlb, 2% GQlb, and the monosialoganglioside GM1 were provided by the Fidia Research Laboratories, Abano Terme. Animals. Goldfish (Carassius auratus, 8-10cm) were purchased from Tropical Center Kohlhase, Neukirchen and held at 28 - 2~ without antibiotics. Surgical Procedures. Fish were anesthetized with 0.08% MS in cold water. For transection of the optic nerve, the conjunctivalmembrane of the eye was dissected and the exposed nerve cut close to the back of the orbit. For injury of the retina, goldfish were injected intraoeularly (i.o.) into the lateral orbit with a fine needle. Drug Treatment. Regenerating fish received a daily intraperitoneal (i.p.) injection of 30 mg/kg GM1 starting one day before lesion. Unlesioned fish received on 4 days 4 injections of either GM1 (3 or 30 izg/eye and 3-30 mg/kg i.p.), GMix (3 ixg/eye), insulin (200 ng/ eye), NGF (50 ng/eye), PS (3 Ixg/eye) or PC (3 ~g/eye). Control animals were not injected or received a daily injection of a 0.9% sterile NaC1 solution. Preparation of Explants. Explantations were carried out as described previously (22), with the following modifications: Eyes were removed from fish previously adapted to darkness and sterilized by immersion in 70% ethanol for 30 see. The front of each eye was cut away, the retina was taken out of the eye cup, and the pigment epithelium was stripped from the back of the retina. Retinaewere washed in a series of sterile phosphate buffered saline (PBS, 140 mM NaC1, 26 mM KC1, 14 mM KH2PO4:,10 mM NazHP04; 10 mM glucose) to remove vitreous humor and cut into squares of about 500 ~m. Culture Conditions. For cultivation of retinal explants a novel culture technique, using a 3-dimensional fibrin matrix was developed: Dissected retinal squares were transferred to plastic petri dishes and imbedded in 20 p,1of a fibrinogen solution (25 mg/ml DMEM), which

Sonnentag, Ri~sner, and Rahmann was clotted by addition of i0 ~,I thrombin (300 units/ml). After polymerization (5', room temperature) the resulting semi-solid 3-dimensional fibrin network was supplemented with 2 ml DMEM, containing 3.7 mg/ml NaHCO3, 200 U/ml penicillin/streptomycin, 10 Ixg/mltetracyclin and 1% Ultroser G instead of serum. Explantswere incubated at 37~ 5% CO2. Evaluation of Neurite Outgrowth. Neurite outgrowth (sprouting) was quantified by counting the number of axons/explant, starting 15 h after explantation and scoring then every 24h thereafter until day 4. For evaluation, explants were divided into 5 'sprouting-classes' according to the followingcriteria: Explants without axons (1); 1-5 axons (2); 6-20 axons (3); 21-50 axons (4); more than 50 axons (5). To simplify the graphical representation, the following sprouting index was defined: Each class of explants (% proportion of total explants) was multiplied with a distinct factor. Sproutingclass 1 (withoutaxons) with the factor 0, sprouting class 2 (1-5 axons) with factor 3, sprouting class 3 with factor 10, sprouting class 4 with factor 30 and sprouting class 5 with factor 75. The sum of these resulting 5 values was defined as sprouting index (Table I). Preparation of MonoclonalAntibody (mab)Q211. Q211 producing hybridoma cells were obtained from Dr. Henke-Fahle, Tiibingen. Cells were placed in tissue culture flasks and cultured in RPMI, supplemented with 5% fetal calf serum, 200 U/ml penicillin/streptomycin, 100 g~g/mltetracyclin, 0.1% potassium-pyruvate,0.1% glutamate and 0.1% 2-mercaptoethanol. Q211 containing culture supernatant was harvested every 3 days, ammonium sulfate precipitated and used in a final dilution of 1:40. tmmunostainingof GoldfishRetinalExplants. Explantswere fixed in 3.8% paraformaldehydein PBS and incubatedwith mab Q211 (1:40 in PBS, 1% BSA). After 3 washes with PBS, they were incubated with the second antibody (anti-mouse IgM-FITC, 1:200 in PBS/1% BSA), washed again and stored in PBS. Photographswere taken with a Zeiss Universal microscope, equipped with epifluorescence.

RESULTS

Sprouting of Normal and Lesion-Activated Goldfish Retinal Explants in a Serum Free 3-D-Fibrin Matrix. To investigate the neuronal sprouting of goldfish retinal axons, explants were prepared from controls and from 1,4,8,40, and 80 days in vivo regenerating fish and cultured in a 3-D fibrin-matrix. Sprouting was scored by counting the number of axons/explant over 4 days, starting after 15h in culture. Figure 1 clearly demonstrates, that the used 3-D fibrin matrix promotes a 3-dimensional outgrowth of retinal neurites without fasciculation, thus allowing a simple and reproducible scoring of the number of outgrowing processes/explant. A comparison of the sprouting activity according to the sprouting index (Table I) indicates (i) a dependency o f the sprouting activity of goldfish retinal explants on the time interval between optic nerve lesion and explantation of the retina and (ii) an increase of neurites/explant with increasing time of culture. Maximal growth activity was observed by explants prepared from fish, whose nerve was transected 8 days before

Gangliosides and In Vitro Retinal Sprouting

1107

Fig. 1. 3-dimensional growth of retinal axons within the fibrin matrix after immunostaining of explants with the mab Q211, recognizing c-pathway polysialogangliosides.

Table I. Calculation of Neurite Outgrowth from Retina Explants According to the Sprouting Index as Described in Experimental Procedure Number of Axons

Factor (f)

% explants X, +X2+X3+X4+X5 = 100 %

0

0 (~)

x~

7

9 ..........

=~,_..~

8 days post

.~..~--~---"

lesion

40 day~ po~t lesion

6

.~

4

:,

:

, a

4 days post

/l

I day post

lesion

lesion

3

1-5

3 (~)

3[2

6-20 25-50

10 (g) 30 (h)

x~ x,

2

> 50

75 (~)

3[5

1

J """

' _

"

4

"

-

-A-~-~ ~..L~. . . . .

80 days control

post

lesion

|

"sprouting-index": ~ L.x,.10 -3 nl-5

culture, remained at a high level until 40 days after nerve transection and decreased to control level after 80 days (Figure 2).

Influence of a One-Sided Optic Nerve Transection on the Sprouting Activity of the Contralateral, Not Impaired Retina In Vitro. Explants of both eyes were prepared 8 days after transection of one optic nerve. Control explants were prepared from unlesioned fish. As demonstrated in Figure 3, transection of one optic nerve also enhanced the sprouting activity of the contralateral, not regenerating retina, as compared to untreated fish. However, this increase in sprouting activity was far below that observed for the ipsilateraI retina of

0

I

15

39

63

87

hour~ in culture Fig. 2. Sprouting activity of regenerating and control goldfish retinal explants 15-87 hours after explantation, calculated according to the sprouting index. Explants were prepared after a previous regeneration time in vivo of 1,4,8,40, or 80 days. Control explants were prepared from the retina of unoperated fish. Data represent mean values _ SD of 120-240 explants/experiment.

8 days regenerating goldfish, being directly connected to the lesioned optic nerve.

Influence of a Local Perforation of the Retina on the Sprouting Activity In Vitro. Because every i.o. injection causes a local lesion of the retina, we further investigated, if a daily i.o. injection of 0.9% NaC1 or just the local perforation of the retina with a fine needle leads to a change of the sprouting activity of goldfish

1108

Sonnentag, Riisner, and Rahmann 8 days, / . . . . . . . . . . . . . . . . . . . * . . . . . . . . . . . . . . . . . . " regeneratmg retina ,/

69

/'

5 84

N a C l - t r e a t e d animals perforated retina

/' /'

'~ 4 9

/ "

.I

,/

8 days, not regenerating, contralateral retina

..=

2

..." j ...'j

control

e~ 2-

.... " control-retina

0 15

39

63

87

hours in culture Fig. 3. Enhanced sprouting activity of the contralateral, not impaired retina 8 days after transection of the ipsilateral optic nerve. Control explants were prepared from the retinae of unoperated fish. Data represent mean values - SD of 120-240 explants/experimental group.

3

b) ,~

9~ =

2 ;;"

perforated retinal-half intact retinal-half

.... oootrol

t.,

retinal explants in vitro. Goldfish received 4 i.o. injections, either with or without 0.9% NaC1 over 4 days. Both, the local perforation of the retina as well as the daily injection of 0.9% NaCI resulted in a nearly equal increase in sprouting activity as compared to unlesioned controls (Figure 4a). To investigate if this increase in neuritic outgrowth concerns only the i.o. injected, injured retinal area or the whole retina, lesioned retinae (see above) were carefully dissected into intact and injured retinal halves, cultured separately and scored over 4 days. As shown in Figure 4b, a local i.o. injection of the retina not only affects the perforated retinal half, but also the adjacent unlesioned area.

Influence of In Vivo Applied Exogenous Gangliosides on the Sprouting Activity of Regenerating Goldfish Retinal Explants In Vitro. In a next series of experiments, we investigated the influence of exogenous gangliosides (GM1) applied in vivo, on the in vitro sprouting activity of regenerating goldfish retinal explants. Explants were prepared from GM1 treated goldfish, after a previous regeneration time in vivo of I or 4 days. These times were chosen, because here the sprouting of retinal explants is still below the maximal inducible activity (compare Figure 2). GM1 was injected daily, either i.o. (3 Izg/eye) or i.p. (50 mg/kg), starting one day before nerve transection. Taken together, neither the i.p., nor the i.o. applied gangliosides remarkably influenced the regeneration related increase of sprouting activity, when compared to untreated, regenerating controls (data not shown).

Influence of In Vivo Applied Exogenous Gangliosides on the Sprouting Activity of Retinal Explants of Unoperated Goldfish: Comparison of Gangliosides with

j-

0

];; . . . . 15

39

63

8'7

hours in culture Fig. 4. Activated sprouting of goldfish retinal explants after perforation of the retina with a fine needle or after injection with 0.9% NaC1, 4 times over 4 days before explantation (a). The increase in sprouting activity concerns not only the injured, but also the intact retinal haft (b). Control explants were prepared from the retinae of untreated fish. Data represent mean values - SD of 100-120 explants/experimental group.

Different Drugs. In order to check a possible influence of in vivo applied gangliosides on the sprouting activity of normal, not regenerating goldfish retinal explants, goldfish received, on 4 days, 4 i.o. injections of either GM1 (3 ~g/eye) or GMix (3 txg/eye). Control animals remained untreated, received 4 injections of 0.9% NaC1 or of the phospholipids phosphatidylcholine (3 txg/eye) or phosphatidylserine (3 t~g/eye), respectively. Again, we observed a scarce sprouting of retinal explants from untreated controls and a slight increase in neuritic outgrowth after i.o. injection of 0.9% NaC1 (Figure 5a; compare Figure 4). A further, significant enhancement above this NaC1 induced increase in sprouting was obtained after i.o. injection of either GMix or GM1, whereas phospholipids didn't induce any increase in sprouting activity above the basal control level (Figure 5a) or were inhibitory as compared to the NaC1 injected animals. The ganglioside induced increase in sprouting activity was dose dependent and could be observed only

Gangliosides and In Vitro Retinal Sprouting

i•'i 9

,~

I

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t

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15

GMix 3 [lg/eye GMI 3 gg/eye

NaCI GM1 30 gg/eye PS 3 ttg/eye PC 3 gg/eye control

..........,~::.~.-.-..... ..

39

63

87

hours in culture

1109 tained after i.o. injection of insulin (200 ng/eye; 4 times over 4 days) or NGF (50 ng/eye; 4 times over 4 days, Figure 6) and couldn't be further enhanced by the combined application of these 3 drugs (not shown). To further evaluate the role of gangliosides during regeneration of the goldfish optic system, fish received 4 i.o. injections of the mab Q211, which has been shown to recognize specifically c-pathway polysialogangliosides (2324, compare Figure 1). Application of mab Q211 (0.33 txg/eye, 4 x over 4 days) led to an inhibition of the sprouting activity to a level significantly below that obtained after injection of 0.9% NaC1 as well as those of untreated controls (Figure 6).

.,t GMI 30 mg/kg; i.p, 3

9

/" 2

.),"

/,,~ .'"

t

...... .,.'

GMI 3 mg/k,g; i.p.

NaCI

DISCUSSION

. .:..:"

1

'"

......--.-;.--:{ ...................

o

15

39 63 hours in culture

87

Fig. 5. Sprouting activity of goldfish retinal explants after i.o. (a) or i.p. (b) injection of different doses of gangliosides (4 times over 4 days) in comparison to NaCI controls. For induction of 'submaximal' axonal sprouting, i.p. injected fish received a local lesion of the retina in parallel to the ganglioside injection. In a further series, animals received 4 injections of the phospholipids phosphafidylserine (PS) or phosphatidylcholine (PC). Control explants were prepared from untreated fish. Data represent mean values • SD from 160-240 explants/ experimental group. * p ~< 0.001 in comparison to NaC1 controls.

after application of low amounts of GM1 (optimum concentration: 3 l~g/eye). Higher concentrations (30 p~g/eye) failed to induce any response above the sprouting activity already recorded after i.o. application of NaCI (Figure 5a). To investigate if an i.p. injection of gangliosides is also effective in enhancing the in vitro sprouting activity, goldfish received a daily i.o. injection of 0.9% NaC1, to induce the basal sprouting activity observed above, while GM1 (30 mg/kg or 3 mg/kg) was injected i.p. 4 times over 4 days. Control animals received no i.p. injection or an i.p. injection of 0.9% NaCI. As demonstrated in Figure 5b, after i.p. application of GM1, a significant increase of sprouting activity above the NaC1 induced response could be observed only after application of the high doses of gangliosides (30 mg/kg). The increase of in vitro neuritic outgrowth by in vivo applied gangliosides was comparable with that ob-

Sprouting of Control and Regenerating Goldfish Retinal Explants in the 3-D Fibrin Matrix. In the present study we used a newly developed tissue culture technique consisting of a 3-dimensional serum free fibrin matrix, which ensured a close adhesion of goldfish retinal explants and promoted the 3-dimensional outgrowth of retina ganglion cell axons. A further advantage of the 3-D fibrin matrix as compared to usual 2-dimensional culture systems (25-27) was that fasciculation of neurites into fibre bundles was obviously suppressed, allowing a simple and accurate quantification of the number of neurites/explant and simultaneously the scoring of high

/

../):, 9"l','"

I immlin 200 ng/eye" GMI 3 ligleye * NOF 50 h i / e y e "

./,/,,.

3 /

i..

"~

L

"'~ "-

NaCI

.... -

2 A"

15

I...........

i d:-" ........."

..l

1[ ." ...i...I ......... /." ..........

/"

i/"

I

..l"

/i

c~176

~

QIII 0.3 ~g/eye"

.. ........... ,---

39

63

87

hours in culture Fig. 6. Sprouting activity of goldfish retinal explants after i.o. injection (4 times over 4 days) of either 200 rig insulin, 50 ng NGF or 3 txg GM1 in comparison to NaC1 controls. Application of the mab Q211, binding to polysialogangliosides of goldfish retina, led to a dose dependent inhibition of the retinal sprouting, below the level of untreated controls. Data represent mean values __ SD from 160-249 explauts/ experimental group. 9 p < 0.001 in comparison to NaC1 controls.

1110

numbers of explants in order to ensure statistical evaluation. Applying the 3-D fibrin culture technique, we confirmed the well-known fact that injury of the goldfish optic nerve in vivo leads to a transiently enhanced sprouting activity of retinal explants in vitro (12,22,25). Maximal activation of neurite outgrowth occurred after a lag of 8 days between injury and explantation. This time dependency coincided well with previously shown lesion induced activation of the retinal protein, and ganglioside metabolism, which leads to a transient accumulation of this newly synthesized metabolic compounds in the regenerating optic nerve (14,17). Unlike this metabolic enhancement, which decreased about 10 days post lesion, the in vitro sprouting activity of retinal explants remained at a high level for up to 40 days and reached the control level approximately 80 days post lesion. These data suggest that the lesion induced increase of retinal sprouting, which was also shown for rat retina (28), is not only due to a transient enhancement of protein- and gangIioside metabolism, but probably depends on further mechanisms as for example long lasting differential gene expression and posttranslational processing (16). Transection of one optic nerve also moderately enhanced the sprouting activity of the contralateral, not directly affected retina. Similar results were obtained after unilateral transection of the rat sciatic nerve (29) or after microinjection into the perineurium of the ipsilateral sciatic nerve (30). Menendez and Cubas (29) speculated, that the presence of electrophysiological signals, crossing the spinal cord, are responsible for the development of the centralateral effect. Similar ipsi-/contralateral connections could exist for the goldfish optic system, resulting in an increased trophic supply of both retinae after unilateral transection of one optic nerve. Because of the complete crossing of the goldfish optic nerves in the optic chiasm, most investigations published until now used the contralateral optic system as a convenient control (12,14,19,25,27). Based on the ipsi-/contralateral effect after unilateral transection of the goldfish optic nerve found in the present study, we prepared control explants only from the retinae of unlesioned or untreated fish. Enhancement of In Vitro Sprouting Following a Local Lesion of the Retina In Vitro. Injection of 0.9% NaCI into the lateral eye-bulb led to an enhanced retinal sprouting in vitro (about 10% of the maximal response obtained after total transection of the optic nerve) which was not due to the NaCI, but was induced by the lesion itself as confirmed in corresponding experiments (compare Figure 4). It is important to note that a defined

Sonnentag, Rfsner, and Rahmann

lesion of the retina within a local area induced an increase of the in vitro sprouting of the whole retina. These data suggest that a local injury promotes the production and release of trophic factors, probably by glial cells (31,32), and/or increased trophic supply of the whole injured retina via blood. Since we used serum free medium for our experiments, an injury increased sensitivity of the retinal explants to trophic factors being present in the culture medium is very unlikely (33). Influence of Exogenous Gangliosides on the Lesion Induced and Basal Sprouting Activity of Goldfish Retinal Explants. To evaluate the effects of exogenous gangliosides, most in vitro experiments have been performed using neuroblastoma cell lines (7,34-36) or embryonic primary cell cultures (34,37,38). Both, neuritogenic (7,34,38) and neuronotrophic (9,28,35,36) ganglioside effects have been reported after addition of exogenous gangliosides into the culture medium. Thereby, Byrne et al. (7) were able to show, that induction of neuronal sprouting and neurite elongation was indeed due to the applicated gangliosides and not an effect of contaminating proteins. Under our culture conditions, we did not observe significant effects of exogenous gangliosides or GM1 on the sprouting activity of goldfish retinal explants when adding the gangliosides directly to the culture medium (data not shown). This could in part be due to a binding of the added gangliosides to the 3-D fibrin matrix, which contains up to 5% fibronectin (39). A binding of gangliosides to fibronectin has previously been reported by Spiegel et al. (40) and Fishman (41). However, if gangtiosides were applicated in vivo some days before explantation of the retina, they had under certain circumstances clear cut effects on the sprouting activity in vitro. No or only a moderate influence of daily injected gangliosides (3 ~g/eye or 30 mg/kg i.p.) on the in vitro retinal sprouting activity was observed when regeneration was previously induced by total nerve transection. We suppose, that total transection of the goldfish optic nerve led to a maximal stimulation of the regeneration response. This process is likely to follow a time dependent intrinsic program as indicated by a transient severaIfold increase of retinal protein- and ganglioside metabolism (14,17). Under these circumstances, exogenous gangliosides had no obvious effects, neither on the time dependency of axonal outgrowth, nor on the degree of the above mentioned regeneration related processes. In similar experiments, in which, however, the optic nerve was only crushed instead of transected, Spirman et al. (21) reported an inhibition of the in vitro

Gangliosides and In Vitro Retinal Sprouting retinal sprouting after a previous i.o. injection of antibodies to GM1 in vivo. It is possible, that crushing of the optic nerve will affect not all retinal ganglion cells to the same degree, as shown in comparable regeneration models (28,43). Thereby, a 'submaximal' induction of the regeneration activity may indeed be modulated by antibodies to gangliosides or application of exogenous gangliosides. Support of this view comes from our second series of experiments, in which only a local injury of the retina itself was performed. In this case, the resulting 'basal' sprouting activity of retinal explants in vitro was strikingly enhanced by gangliosides, applicated either i.o. or i.p. for some days before explantation. This enhancement in sprouting activity was dose dependent, could be observed only after i.o. application of low doses (3 p~g/ eye) or after i.p. application of high doses of gangliosides and was comparable with that obtained after i.o. injection of NGF (50 ng/eye) or insulin (200 ng/eye). The combined injection of GM1 + NGF, GM1 + insulin or NGF + insulin caused no further enhancement above the sprouting activity induced by each drug alone. Thus, in the goldfish optic system there seems to be no additive effects or a possible potentiation of the NGF induced axonal outgrowth after application of exogenous GM1 as has been demonstrated for other systems (37,42). I.o. application of the phospholipids phosphatidylcholine (3 Ixg/eye) or phosphatidylserine (3 ixg/eye) had no effect or were slightly inhibitory, probably due to the formation of lyso-phospholipids. Taken together, the above discussed ganglioside induced enhancement of retinal sprouting activity as well as the decrease after injection of the mab Q211 suggests a ganglioside mediated growth promoting effect on certain initial steps of regenerative neuritogenesis and probably also on the maintenance of neurite growth. This interpretation is supported by findings that polysialogangliosides of the b- and c-pathway are expressed by embryonic neurons for the first time during differentiation of mitotic neuroblasts to young neurons, starting to migrate and forming first neurites (2,24). An additional mode by which exogenous gangliosides applied in vivo enhance (suboptimal) retinal sprouting in vitro may be a modulation of the rate of neurogenesis. The generation of neurons from progenitor cells represents a general mechanism of renewal and growth of the fish retina throughout life (44,45) and was shown to be enhanced after injury (46,47). However, preliminary experiments show, that inhibition of progenitor cell proliferation in vivo by 5fiuor-2-deoxyuridine did not affect retinal sprouting activity in vitro. Thus, a contribution of 'newborn' retinal

1111 ganglion cells to retinal regeneration seems to be minor as compared to re-outgrowth of preexisting neurons.

ACKNOWLEDGMENT This work received financial support from the FIDIA company, for which we are grateful.

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Influence of exogenous gangliosides on the three-dimensional sprouting of goldfish retinal explants in vitro.

To investigate the 3-dimensional outgrowth of ganglion cells of normal and regenerating goldfish retina, retinal explants were cultured in a serum fre...
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