lpsilateral Retinal Projections into the Tectum during Regeneration of the Optic Nerve in the Cichlid Fish Haplochromis burtoni: A Dil Study in Fixed Tissue Claudia Wilm* and Bernd Fritzsch
+?*
Faculty of Biology, University of Bielefeld, 4800 Bielefeld, Germany
SUMMARY Retinal projections were experimentally manipulated in a bony fish to reveal conditions under which considerably enlarged ipsilateral projections developed and persisted. Three experimental groups were studied: animals after unilateral enucleation, after unilateral nerve crush, and after enucleation and crush of the remaining optic nerve. At 29 days after unilateral enucleation alone, no enhanced ipsilateral projection had developed. After nerve crush, however, large numbers of retinal fibers regenerated into the ipsilateral tectum. Retrogradely filled, ipsilaterally projecting ganglion cells were distributed throughout the entire retina. After 15 days regenerating retinal fibers covered the entire ipsilateral tectum. At
INTRODUCTION The presumably primitive condition ofbilateral retinal projections is widespread among vertebrates (Fritzsch, 199 1 ). Absence of an uncrossed projection, for example, in many bony fish (Wilm and Fritzsch, 1990) or birds (Thanos and Bonhoeffer, 1984), appears to correlate with modifications of one of the two developmental steps necessary for the formation and maintenance of an ipsilateral projection: Received August 27, 1991: accepted May 5. 1992 Journal of Neurobiology. Vol. 23, No. 6. pp. 692-707 (1992) C 1992 John Wiley & Sons, Inc. CCC 0022-3034/92/060692- 16$04.00 Present address: *E. Merck, Biologische Forschung, P.O. Box 4 I 19. 6 I00 Darmstadt I , Germany; ?Department of Biomedical Sciences. Division of Anatomy, Creighton University. Omaha, Nebraska 68 178, U.S.A. $ To whom correspondence should be addressed.
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later stages the ipsilateral projection showed progressive reduction in coverage of the tectum. Combining enucleation with nerve crush led to an ipsilateral projection that covered the tectum at 28 days and later. In this experimental situation the development of an ipsilateral projection appears to be a tno-step process: ( I ) Fibers are rerouted to the ipsilateral side at the diencephalon, and ( 2 ) ipsilatera1 fibers persist in the tectum only in the absence of a contralateral projection while they appear to be eliminated in the other cases. GI1992 John Wiley & Sons, Inc. Keywords: visual system, regeneration, optic nerve, ipsilateral projections, teleosts, ganglion cell distribution, pathfinding.
1. Some bony fish apparently never develop an ipsilateral projection in excess of a maximum of 20 fibers (Fritzsch and Wilm, 1992). Owing to the presence of monocular crossing to the contralateral diencephalon and the consequent absence of a true chiasm with the chiasmatic interdigitation of fibers of both eyes, these fish may have largely sidestepped cues shown to be important for pathfinding of many ipsilateral fibers in mammals (Godement, Salaun, and Mason, 1990) and perhaps other vertebrates. 2. In contrast, birds seem to develop an ipsilateral projection initially, but eliminate ipsilateral fibers in the tectum in a second step (Thanos and Bonhoeffer, 1984) so that adult animals have virtually no ipsilateral fibers. A comparable, likely activity-controlled process takes place in mammals (Cowan, Fawcett, O’Leary, and Stanfield, 1984), appears
Regcweruiion of the Optic Nerve
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Operation
Application
Figure 1 Surgery and application of tracers. Three experimental groups were studied. Operations were performed on 26-day-old animals. The animals survived between 4 and 60 days. NOR = nucleus olfactoretinalis; ON = optic nerve: Tec = tectum.
to cause the segregation of retinal projections in three-eyed frogs (Debski, Cline, and Constantine-Paton, 1990), and may be present in all binocularly interacting retinal projections. However, based on normal development it is unclear whether this second process is at all present in cichlid fish given that they never develop a large ipsilateral projection and do not noticeably reduce the number of ipsilaterally projecting cells with age (Fritzsch and Wilm, 1992). Despite the virtual absence of an ipsilateral retinal projection in the adult (Wilm and Fritzsch, 1990) or developing (Fritzsch and Wilm, 1992) cichlid fish Huplochromis burtoni, regenerating retinal fibers can form a larger then normal retinal
projection into the ipsilateral tectum (Wilm and Fritzsch, 1990, 1992). Comparable phenomena have been reported for the regenerating goldfish visual system (Sharma and Tsai, 1991; Springer, 1980, 198 1 ). These studies all imply that the regenerating retinal projections in fish do not recapitulate faithfully the development and may lead instead to unusually enlarged projections that are much sparser, if present at all, in undisturbed animals. Experimentally induced ipsilateral fibers may represent unusual exaggerations of ontogenetic processes. Nevertheless, given the control of timing of onset of formation and regression of induced ipsilateral projections, these projections may be particularly helpful to elucidate the factor( s ) that lead to the formation and maintenance of an ipsilateral projection in the continuously growing bony fish.
Table 1 Different Groups and Combinations of Dyes A. Experimental Groups and Anterograde Labeling of the Retinal Projection with Dil, PKH2 and FDA Groups Unilateral enucleation Days after the operation 7 and 29
Unilateral nerve crush Days after the operation 4 5 7-1 I 22-36 46 and 60
Enucleation and nerve crush Days after the operation 7 7 and 22 29
Labeling
Preparation
Left optic nerve Intact Dil
Right optic nerve Degenerating
Left optic nerve Regenerating Dil Dil Dil Dil Dil
Right optic nerve Intact
Left optic nerve Intact Dil Di I Di I
Right optic nerve Degenerating
Tccta
-
PKH2 PKH2 or FDA -
Tccta Tecta Tecta Tecta Tecta and the other retina
Tecta Tecta and retina Tecta
B. ExDerimcntal GrouDs and Retrograde Labeling of Ganglion Cells with Dil Unilateral nerve crush Days after the operation 15'
Dil into the tectum which is positioned ipsilaterally to the lesioned optic nerve
Both retinae
Dil into the tectum which is positioned ipsilaterally to the lesioned optic nerve
Remaining retina
Enucleation and nerve crush Days after the operation 152
223 I
17 animals. I I animals. I 2 animals.
In a previous study we explored this model and described some of the conditions that lead to the formation and maintenance of an unusually large ipsilateral projection (Wilm and Fritzsch, 1990). Employing tract tracing with horseradish peroxidase (HRP), ipsilateral fibers could first be detected after 7 days and no ipsilateral fibers could be detected any more after 28 days, suggesting that ipsilateral fibers may have been eliminated presumably through a process not unlike the one acting in developing birds, mammals, and three-eyed frogs. Alternatively, however, remaining ipsilateral fibers could have failed to transport HRP, and the earliest stages of regeneration may not have been investigated at all owing to rapid degeneration of the
fragile fibers. We therefore undertook the present study to analyze in detail the morphology of the first regenerating fibers and ipsilateral retinal fibers while they become eliminated employing the lipophilic tracer Dil in fixed tissue. [Preliminary results have been presented in abstract form (Wilm, 1990).]
METHODS For this study, a total of 323 specimens ofthe cichlid fish species Haplochroomis bzirtoni were subjected to surgical or postmortem procedures. The breeding animals and the larvae were kept at 25" f 1°C (lightldark cycle:
RcgcnPralion o f fkc Optic Nerve
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Table 2 Effects of Treatment on the Formation of Ipsilateral Retinal Projections A. Normal Retinal Projection ( < I 5 Fibers) into the Ipsilateral Tectum
Age (days) of the Animals
Days after the Operation
Number of Animals
Number and Percentage of Animals with Ipsilateral Fibers N
%
14 18
70
Unilateral Enucleation
I
33 55
29
20 21
86
B. Enhanced Retinal Projection (>30 Fibers) into the Ipsilateral Tectum Unilateral Enucleation
I
33 55
29
20 21
0 1
0 5
Unilateral Nerve Crush
30 31 33 and 31 41 48 55 and 57 62 12 86
4 5 land I I 15 22 29 and 31 36 46 60
14 14 35 19 26 24 10
I0 10
No labeling at all 4 29 20 51 19 I00 24 92 23 96 10 100 10 100 8 80
Enucleation and Nerve Crush
I
33 41 48 55
15
22 29
13:l 1 h). The age of the animals ( u p to 20 days) was determined according to a table of developmental stages (Wilm and Fritzsch, 1989). Free-swimming juveniles were kept at room temperature ( 2 1 5 1 "C) in small aquaria and fed with dry food for young brood (Tetramin). Animals were operated at 26 days of age (standard length: 9 mm). O
Surgery Three types of operations were performed: ( 1 ) enucleation of one eye, ( 2 ) crush of one optic nerve, and ( 3 ) enucleation of one eye and crush of the other optic nerve (Fig. I ). Prior to surgery all animals were anesthetized in 0.0 1% ethyl p-aminobenzoate (benzocaine, Sigma) following guidelines established by the German Federal Law. Operated animals were initially reared in a fish
44 10 15
II
28 9
64 90
15 II
I00 I00
Ringer solution. The salt concentration was gradually reduced by adding aquarium water over 4 days. Enucleation. The optic nerve was exposed through a caudal incision in the orbital skin and connective tissue. The eye was gently rotated rostroventrally in the orbit, and the optic nerve was cut with fine scissors. Optic-Nerve Crush. The optic nerve was exposed and then crushed with watch-maker forceps for about 5 s.
Anterograde Tracing of the Visual Projection The short distance (only 2-3 m m in 30- to 86-day-old animals) of the retinal projection allows the application of Dil ( 1,l '-dioctadecyl-3,3,3'.3'-tetramethyl indocarbo-
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M i l r ~and Fritzsch
Figure 2 Regenerating retinal fibers into the contralateral ( A ) and ipsilateral ( B ) tectum 5 days after unilateral nerve crush. The tecta are shown as whole mounts from a dorsal aspect. The tecta were split along the rostrocaudal meridian to flatten them onto a slide. The contralatera1 tectum was opened wider at its cut than was the ipsilateral tectum. Regenerating fibers cover the rostra1 pole of the contralateral tectum and preferentially grow into the dorsomedial part of the ipsilateral tectum. C = caudal; D = dorsomedial; Tee = tectum; T r O D = tractus opticus dorsalis; V = ventrolateral. Scale bar = 200 pm.
1990). In all animals the skull was opened under anesthesia. The animals were immersed in 4% paraformaldehyde in 0.1 Mphosphate buffer( pH 7 . 3 ) at room temperature and then stored at 4°C. At I day to 4 weeks later, crystals of Dil or PKH2 were applied to the optic nerve head inside one eye. The animals were either stored in the dark at 19” to 2 1 “ C for 1 1 days (if one retinal projection was labeled with FDA) o r at 36°C for 2-3 days depending on the age (size) of the animal. For each age minimal diffusion times to label the retinotectal projection were established to avoid transcellular labeling (Fritzsch and Wilm, 1990). The material was subsequently stored at 4°C prior to examination to minimize further diffusion. The tectum together with the optic tracts were dissected. The tectum was split along the rostrocaudal meridian, sparing the rostral pole, in order to flatten the tectum onto a slide and whole mounted in 0. I A4 phosphate buffer ( p H 7 . 3 ) . Two small coverslips on each side served as spacers. The fluorescent material was viewed with an epifluorescence microscope and photographed with a rhodamine or fluorescein filter set (TMAX 100 ASA or Ektachrome 200 ASA, Kodak).
Retrograde Tracing of the lpsilaterally Projecting Ganglion Cells
Figure 3 Regenerating retinal projection I5 days after unilateral nerve crush into the contralateral half of the tectum. Contralateral fibers fasciculate (arrow) and take tortuous courses in the tectum. Shown is a “bad” regeneration with only few contralateral fibers. Otherwise, the density of the projection does not allow the demonstration of the course of individual fascicles. Abbreviations as per Figure 2. Scale bar = 200 Km.
Dil was applied into the rostral tectum of paraformaldehyde fixed animals [Table 1: Fig. I]. A shallow incision was placed transversely across the rostral tectum from dorsomedial to ventrolateral. Crystals of Dil were stuffed into the incision except at the dorsomedial margin to avoid diffusion of Dil into the other tectum. The material was stored in 4% paraformaldehyde in 0. I M phosphate buffer ( p H 7.3) at 36°C for 2 days. The retinae were dissected and the lense and iris were removed. The retina was incised at the ventral, dorsal, nasal, and temporal margin and whole mounted in phosphate buffer (pH 7 . 3 ) onto a glass slide and examined as described above.
RESULTS cyanine perchlorate, Molecular Probes, Eugene, Oregon, U.S.A.) and PKH2 (green fluorescent, lipophilic dye, gift of the Zynaxis Cell Science Inc., Malvern, PA, U.S.A.) in paraformaldehyde fixed tissue. Table I lists the different groups and combinations of the dyes. After unilateral nerve crush, the regenerating projection was traced with Dil and the undisturbed projection with PKH2 orfluorescein-isothiocyanate-coupled dextran amine [FDA ( Molecular Probes) (Fig. I )] . After unilateral enucleation alone or enucleation and crush of the remaining optic nerve, the retinal projection was traced with Dil. FDA was applied in deeply anesthetized animals onto the cut optic nerve. The animals survived 14-16 h for sufficient transport of the dye (Wilm and Fritzsch,
We considered the ipsilateral projection as an “enhanced projection” compared to unoperated animals if more than 30 fibers projected into the ipsilateral tectum, that is, if more fibers reached the tectum than did the maximal 20 fibers ever encountered in normal development (Fritzsch and Wilm, 1992). Unilateral Enucleation (Lesion 1, Fig. 1 )
After 7 and 29 days, 70% and 86% of the animals, respectively, showed few fibers ( < 1 5 ) which
Figure 4 Regenerating retinal fibers ( A ) labeled with Dil into the ipsilateral tectum 15 days after nerve crush and the native retinal projection ( B ) labeled with PKH2 into this same tectum. Half the tectum is shown. The undisturbed contralateral projection as well as the regenerated ipsilateral projection cover the entire tectum. Ipsilateral fibers course in the same layers as do the native contralateral projections. Abbreviations as per Figure 2. Scale bar = 200 urn.
coursed via the optic tract into the ipsilateral tectum (Table 2 ) . Only one animal had a prominent retinal projection (>30 fibers) into the ipsilateral tectum after 29 days (Table 2). Unilateral Nerve Crush (Lesion 2, Fig. 1 ) After 4 Days. For unknown reasons we could not label with Dil any regenerating retinal fiber in the 14 animals analyzed (Table 2).
After 5 Days. Retinal fibers had regenerated into both tecta (Fig. 2). The progress of regenerating fibers showed a high interindividual variability ranging from few fibers at the rostra1 pole of the tectum to a projection that covered nearly twothirds of the contralateral tectum. Fibers regenerated predominantly via the dorsomedial optic tract ipsi- and contralaterally; they branched more exten-
sively in the contralateral tectum. Retinal fibers coursed in 79% ( n = 1 1 out of 1 4 ) of the animals into the ipsilateral tectum with 29% ( n = 4 ) , displaying an enhanced ipsilateral projection [ >30 fibers; Fig. 2(b)]. In the remaining three animals several retinal fibers coursed in the ipsilateral optic tract presumably on their way to the tectum.
After 7-21 Days. The regenerating projection covered two-thirds to three-fourths of the contralateral tectum. In 56% of the animals, an enhanced ipsilatera1 projection had developed (Table 2 ). After 15 Days. The regenerating projection covered the entire contralateral tectum. Regenerating fibers took tortuous courses and fasciculated (Fig. 3 ) . Many fibers had growth cones. In all animals a prominent projection into the ipsilateral tectum had developed [Fig. 4(a ) ] . In 16 animals the pro-
Figure 5 Ipsilateral retinotectal projection 22 ( A ) and 29 ( B ) days after unilateral nerve crush. The density of the ipsilateral projection is decreased compared to animals with shorter postoperative time intervals. ( A ) In a few animals, ipsilateral fibers persist at the lateral and medial margin. ( B ) In most animals, the caudal tectum is free of retinal fibers. Abbreviations as per Figure 2. Scale bar = 200 pm.
Figure 6 In a few animals ipsilateral fibers course over the entire tectum even 60 days after unilateral nerve crush. Shown is a n extreme case with a dense distribution of ipsilateral fibers. Abbreviations as per Figure 2. Scale bar = 200 Fm.
Figure 7 Between 22 and 60 days after unilateral nerve crush, several ipsilateral fibers end blindly ( A ) , and short segments are distributed in the ipsilateral tectum ( B ) . Fine caliber fibers often course through these segments. R = rostral; C = caudal. Scale bar = 100 pm.
jection covered the entire ipsilateral tectum without any preferential termination. In the remaining three animals ipsilateral fibers terminated at the rostral tectum or at the lateral and medial margin. The retinal projection ofthe other eye, labeled with PKH2, remained obviously undisturbed by the operation [Fig. 4( b)]. Ipsilateral fibers [Fig. 4( a ) ] coursed on top and terminated in the same layers as did the contralateral projection [Fig. 4( b)]. Between 22 and 60 Days after the Nerve Crush. In all stages examined, an enhanced ipsilateral projection occurred in 80%-100% of the animals (Table 2). But ipsilateral fibers were no longer evenly distributed, and the density of fibers appeared considerably reduced (Fig. 5 ) . The distribution of ipsilateral fibers was heterogeneous. In a few animals retinal fibers remained predominantly at the outer tectal margin [Fig. 5 ( a ) ] . But in most animals the density of fibers decreased from rostral to caudal [ Fig. 5 ( b ) ]. However, even 60 days after the nerve crush, retinal fibers coursed over the entire ipsilateral tectum in a few animals (Fig. 6 ) , but the density of the ipsilateral projection appeared lower
than 15 days after the nerve crush. Terminal branches of these ipsilateral fibers were rarely ohserved and occurred predominantly at the tectal margin. Many ipsilateral fibers ended either blindly or the fluorescence faded distally [Fig. 7 (a)]. Predominantly in the rostral tectum short segments were observed (Fig. 7b) with fine caliber fibers coursing through these segments. Examination of the contralateral retina that received no Dil application revealed no labeled ganglion cells in the two groups 46 and 60 days after nerve crush with only one exception (two ganglion cells). This suggests that ipsilaterally projecting fibers have not been transcellularly labeled by diffusion of Dil into the other optic nerve. Enucleation and Nerve Crush (Lesion 3, Fig. 1) After 7 Days. In 64% oftheanimals, enhanced ipsilateral retinal projections had developed (Table 2). After 15 Days. In 90% of the animals, enhanced ipsilateral projections had developed, which cov-
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M ilrn arid Ft2t:sc.h
Figure 8 The ipsilateral projection in the tectum 29 days after enucleation and nerve crush. After elimination of the native contralateral projection, ipsilateral fibers persist in the entire tecturn. TrOV: tractus opticus ventralis: other abbreviations as per Figure 2. Scale bar = 200 Irm.
ered the entire tectum, except for one case in which the nerve may have been inadequately crushed. We infer this because the contralateral projection did not show the tortuous courses of retinal fibers, a characteristic feature of a regenerated projection. After 22 and 29 Days. In all animals an enhanced ipsilateral projection covered the entire tectum (Fig. 8 ) . At 29 days after the operation, ipsilateral fibers exhibited complex terminal fields like the contralateral fibers (Fig. 9 ) . The density of ipsilatera1 fibers was always higher than of that in the group that received a unilateral nerve crush with comparable postoperative time interval. Distribution of lpsilaterally Projecting Ganglion Cells
15 Days after Nerve Crush. All 17 animals in which the ganglion cell population across the entire contralateral retina was labeled showed high num-
bers of ganglion cells in the ipsilateral retina (Fig. 10). There was no preferential distribution of ipsilaterally projecting ganglion cells in one retinal sector: in five animals, ganglion cells showed an even distribution across the retina, in four animals, the density was higher in the nasal retina, and in eight animals the density was higher in the ventral retina. 15 and 22 Days after Enucleation and Nerve Crush. Because the contralateral eye was absent, the completeness of labeling could not be checked. In 18 animals, ganglion cells were labeled in the entire ipsilateral retina. In seven out of these 18 animals, the density of labeled cells was highest in the nasal retina and in one animal in the ventrotemporal retina. In the remaining animals, ganglion cells were labeled either only in the dorsal (two animals) or in the nasal retina (three animals) or showed no preference (five animals).
Unilateral Enucleation
After unilateral enucleation, only one out of 2 1 animals showed an enhanced ipsilateral projection after 29 days. This confirms previous results that only 10% of enucleated animals ( 5 out of 50) exhibit an enhanced ipsilateral projection after 2 months and later (Wilm and Fritzsch, 1990). We suggested that the ipsilateral projections were induced during the surgery owing to surgical lesions of the remaining optic nerve or chiasm. This conclusion is supported by observations that an enhanced ipsilateral retinotectal projection can be induced in Xmopzis by damage to the chiasm (Taylor and Gaze, 1990). Unilateral Nerve Crush-Enucleation Nerve Crush
Figure 9 lpsilateral fibers develop complex terminal arborizations 29 days after enucleation and nerve crush. Scale bar = 100 p m .
DISCUSSION Methods
Use of diffusion of Dil in the plasma membrane of aldehyde-fixed tissue allows prior fixation of undisturbed fibers and avoids any artifacts potentially caused by the separation of the growing axons from their cell bodies (Stuermer and Raymond, 1989). As in the accompanying paper (Fritzsch and Wilm, 1992), we took great care to control the only potential artifact known for Dil, transcellular diffusion (Fritzsch and Wilm, 1990). The first developing (Fritzsch and Wilm, 1992) as well as the first regenerating fibers after the nerve crush could be labeled after 5 days with Dil, but not with other tracers. Neither with HRP nor with fluorescent dextran amines could we obtain any satisfactory tracing of regenerating fibers prior to 2 1 days postoperatively ( Wilm and Fritzsch, 1990). Similarly, we could label ipsilateral fibers with Dil even 60 days after unilateral nerve crush while fluorescent dextran amines failed to label this ipsilateral projection already after 28 days (Wilm and Fritzsch, 1990 ). The higher fluorescent yield and, in particular, the diffusion in the cell membrane independent of intracellular transport, may contribute to the apparent higher sensitivity of Dil.
and
After nerve crush, retinal fibers regenerated to both tecta indicating that crush alone creates conditions that may lead to the formation ofan ipsilateral projection. The regenerating ipsi- and contralateral projection covered the entire tecta at 15 days postcrush. Subsequently, the distribution and morphology of ipsilateral fibers depended on the persistence or absence of a native contralateral projection. If the other optic nerve remained undisturbed, then the density of ipsilateral fibers decreased, suggesting that these fibers were reduced in numbers or complexity of their arborization. However, even at 60 days postcrush ipsilateral fibers were still present but were lost after several months (Wilm and Fritzsch, 1990). In contrast, if the native contralatera1 projection had been eliminated by enucleation, the ipsilateral projection had developed complex terminals in the entire tectum 29 days later. In this experimental group the ipsilateral projection persisted up to 7 months (Wilm and Fritzsch, 1990). How are aberrant projections like the ipsilateral retinotectal fibers structurally eliminated: Do they retract? Or do they degenerate? Some in vitro observations showed retraction of fibers from unsuitable substrates (Kapfhammer and Raper, 1987) and retraction was also recently suggested for the elimination of one type of efferent fiber of the chicken retina (Fritzsch, Crapon de Caprona, and Clarke, 1990). However, the blind-ending fibers and short segments in our study on the ipsilateral projection may indicate a degeneration of retinal fibers. Comparison with the contralateral regenerating projection in the same animal showed that blind-ending fibers cannot be explained as methodological artifacts. The short segments probably
Figure 10 Flat mount of the retina with ipsilaterally projecting ganglion cells 15 days after unilateral nerve crush. The Dil-labeled ganglion cells are distributed over the entire retina. ON = optic nerve head; T = temporal; D = dorsal. Scale bar = 200 Wm.
consisted of several fasciculated fragments of axons. Fine caliber fibers coursed through these segments, and the dye could have diffused from these fibers into the fragments. Springer ( 1980, 198 1 ) also showed the disappearance of initially present ipsilateral retinofugal fibers after nerve crush in goldfish but could not decide with certainty whether ipsilateral fibers were eliminated or undetectable with radioautographic methods. Long-term retrograde labeling of ipsilaterally projecting ganglion cells is required to test whether or not these ganglion cells survive like the callosal cells in the developing mammalian cortex (Innocenti, 1988). Distribution of lpsilaterally Projecting Ganglion Cells In juvenile H . hzirtoni up to 20 ganglion cells projected into the ipsilateral tectum (Fritzsch and
Wilm, 1992). These ganglion cells came from the entire retina, and terminated throughout the entire tectum. Likewise, ipsilaterally projecting regenerating ganglion cells were also distributed in the entire retina without topographical preference. Therefore, all regions of the retina send at least some axons ipsilaterally. Similar results were obtained in goldfish (Sharma and Tsai, 199 1 ). It has been previously shown that only regenerating but not newly developing optic nerve fibers grow without any apparent order to the optic chiasm in goldfish (Bernhardt and Easter, 1988) and that ipsilaterally projecting ganglion cells are within the area of the retina already present at the time of optic nerve crush ( Wilm and Fritzsch, 1992). Together these data led us to hypothesize that development ofan ipsilatera1 projection may be related to the lost order in the regenerating optic nerve that causes rerouting predominant1y of regenerating fibers. Whereas in a ranid frog (Stelzner, Bohn, and
Straws, 198 1;Singman and Scalia, 199 1 ), regenerating, ipsilateral ganglion cells are coming from topographically corresponding areas of the entire retina as in normal ranid frogs (Singman and Scalia, 1990), ipsilaterally regenerating ganglion cells are located in Xenopzu mainly in the ventral retina as during normal development ( Hoskins and Grobstein, 1985a,b).These studies imply that comparable principles may operate during development and regeneration of ipsilateral projections in frogs that nevertheless lead to an apparently expanded ipsilateral projection during optic nerve regeneration (Singman and Scalia, 1990). Given these data in frogs, it is possible that the enlarged ipsilateral projection during regeneration in H. burtoni may be an exaggeration of a normal developmental process. The Formation and Maintenance of an lpsilateral Projection
This study on H. burtoni shows that a species with a small ipsilateral retinal projection develops a considerably larger ipsilateral projection during regeneration of the optic nerve. If the native contralatera1 projection to the tectum was either disturbed (by crushing the other optic nerve) or eliminated altogether, then ipsilateral fibers can persist for several months (Wilm and Fritzsch, 1990). Whereas the presence of an undisturbed contralateral projection did not interfere with the initial formation of an ipsilateral projection, then it reduced dramatically the maintenance of this projection. Therefore, the development of an ipsilateral projection is a largely independent, two-step process. These two processes will be discussed next. Pathfinding of lpsilateral Fibers in the Optic Chiasm
Evidence in mice (reviewed in Godement, Salaun, and Mason, 1990; Sretavan, 1990) suggests that the projections of the contralaterally and ipsilaterally projecting retinal ganglion cells are sorted out in a two-step process for many cells: only contralateral, but not ipsilateral fibers can cross the midline. In addition, ipsilateral fibers require crossed fibers of the contralateral eye to reach their ipsilateral target. An additional small population may navigate independently of the crossed, contralateral fibers. In frogs, the mechanisms for the navigation in the chiasm is less well studied, but is apparently influenced by thyroid hormone ( Hoskins, 1990) and also by the teratogen retinoic acid ( Manns and Fritzsch, 199 1 ) . Interestingly, if the
optic nerve was lesioned behind the eye, then an enhanced ipsilateral projection developed in Runa ( Stelzner et al., 198 I ; Singman and Scalia, I99 1 ), but not in Xmopzis (Taylor and Gaze, 1990). This indicates that the sorting mechanisms of the optic chiasm of frogs may have species-specific differences perhaps related to the size of the naturally occurring ipsilateral projection. In a previous study (Wilm and Fritzsch, 1990) we showed that retinal fibers do not grow ipsilaterally at the “chiasm” of II. hurtoni. Instead, the fibers enter the contralateral diencephalon and then, when reaching the area of “monocular fibrecrossing” (Maggs and Scholes, 1986), recross to the ipsilateral side. During their path to the ipsilatera1 side, these fibers have no contact with the contralateral nerve. Therefore, fiber interaction with the contralateral nerve is not responsible for the initial ipsilateral course in H . hzrrtoni. Moreover, we showed previously that an ipsilateral projection develops even when the nerve was crushed 1.5 years after enucleation. As verified by electron microscopy, all fibers of the contralateral nerve had degenerated by that time. Therefore, the pathfinding of ipsilateral fibers of H. bwtoni resembles the small ganglion cell population that projects in mice ipsilaterally without interaction with the contralatera1 fibers (Godement, Salaun, and Mason, 1990). Whether more ganglion cells express this capacity in the regenerating projection of H . hzirtoni or whether the regenerating nerve presents a uniquely enlarged sorting problem and thereby produces excessive numbers of ipsilateral projecting fibers as previously suggested ( Wilm and Fritzsch, 1990) remains to be clarified. Maintenance and Regression of the lpsilateral Projection
The elimination of aberrantly projecting ipsilateral fibers in mammals, for instance, is activity controlled (Cowan, Fawcett, O’Leary, and Stanfield, 1984). In bony fish, the persistence of an undisturbed contralateral projection clearly prevented ipsilateral fibers from establishing a dense projection in the tectum. Starting at 29 days, but more pronounced at 60 days after nerve crush, the density of the ipsilateral projection was less in the group that received only a crush of the optic nerve, and several months after nerve crush alone the ipsilateral projection was completely eliminated (Wilm and Fritzsch, 1990). If both nerves were crushed, then the regenerated ipsi- and contralatera1 projection developed separate patches in the tectum ( Wilm and Fritzsch, 1990). We suggest
that as with the different retinal projections of the three-eyed frog ( Constantine-Paton and Law, 1978: Debski. Cline, and Constantine-Paton, I990), ipsilateral and contralateral projection separate because of their different activity patterns. Correlated activity pattern seems to be important for the stabilization of synapses in the retinotectal projection. After nerve crush without enucleation the regenerating ipsilateral fibers are confronted in the tectum with an undisturbed and much denser contralateral projection. Potential sites for synapses should be available because the tectum is still proliferating and differentiating ( Raymond and Easter, 1983; Raymond, 1986; Wilm and Fritzsch, 1989) at its caudal margin and the whole contralateral projection is continuously shifting ( Wilm and Fritzsch, 1992). Nevertheless, the ipsilateral projection is not able to establish itself between the contralateral projection. Our study cannot explain the reason for this failure. We can only speculate that the ipsilateral projection is not dense enough to displace contralatcral terminations and stabilize their own terminations with the help of their correlated activity pattern.
tions into the tectum during development in the cichlid fish IIuploc~hromish i / r / o n / :a Dil study in fixed tissue. .I. A’cirrohiol. 23:708-7 19. GODEMENT,P.. SALAUN,J.. and MASON.C. A. ( 1990). Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers. ATcJirron5: 173186. HOSKINS.S. G. and GROBSTEIN,P. ( l985a). Devclopment of the ipsilateral retinothalamic projection in the frog Xcntip11.s lucvi.c. I. Retinal distribution of ipsilaterally projecting cells in normal and experimentally manipulated frogs. J. A’ci/ro.st,i. 5(4):9 11-9 19. HOSKINS,S. G. and GROBSTEIN. P. ( 198%). Development of the ipsilateral retinothalamic projection in the frog Xc~nopi~c /ucw.s. 11. Ingrowth of optic nerve fibers and production of ipsilaterally projecting retinal ganglion cells. J . A’rv!ro.sci. 5 ( 4):920-929. HOSKINS.S. G. ( 1990). Metamorphosis o f t h e amphibian eye. J . h‘cirrohiol. 21970-989. INNOCENTI, G. ( I988 ) . Loss ofaxonal projections in the development of the mammalian brain. In: Thr Muk/rig ( j j ’ t/w “l:c.~*or/s5 ~ : \ l c t i 7 . J. G. Parnavelas, C. D. Stern. R. V. Sterling, Eds. Oxford University Press. Oxford. pp. 283-298. KAPFHAMMER, J . and RAPER,J. (1987). Collapse of growth cone structure on contact with specific neurites in culture. ./. h‘cwrosc,i. 7 2 0 1-2 12. MANNS,M. and FRITZSCH.B. ( 199 1 ) . The eye in the This work was supported by the DFG (Fr 572). brain: retinoic acid eKects inorphogenesis of the eye and pathway selection of axons but not the differentiation of the retina. A’iwo.wi. Lc//. 127: 150- 1.54. REFERENCES MAGGS. A. and SCHOLES. J . ( 1986). Glia domains and nerve fiber patterns in the fish retinotcctal pathway. J . BERNHARDT,R. and EASTER,S. S. ( I988 ). Regenerated h’cirrosc~i.6 ( 2 ) :424-438. optic fibers in goldfish reestablish a crude sectoral RAYMOND. P. A. ( 1986). Movement ofretinal terminals order in the visual pathway. .I. C ‘ o i i i p h’cwol. in goldfish optic tectum predicted by analysis of neuro277:403-4 19. nal proliferation. J . N c u w w i . 6 ( 9 ):2479-2488. CONSTANTINE-PATON. M. and LAW. M. 1..( 1978). Eye RAYMOND. P. A. and EASTER,S. S.. JR. ( 1983). Postemspecific termination bands in tecta of three eyed frogs. bryonic growth ofthe optic tcctum in goldfish. I. LocaSci(ww 202:639-64 1. tion of germinal cells and numbers of neurons proCOWAN.W. M., FAWCETT,J. W., O’LEARY.D. D. M., duced. ./. R’citro.sc,i. 3: 1077- I09 1 . and STANFIELD,B. B. ( 1984). Regressive events in SHARMA. S. C. and TSAI.C. ( I99 1 ). Retention of retinal neurogenesis. Scicww 225: 1258- 1265. axon collateral is responsible for induced ipsilateral DEBSKI.E. A.. CLINE,H. T., and CONSTANTINE-PATON, retinoteetal prqiections in adult goldfish. I l s i o r i Res. M. ( 1990). Activity-dependent tuning and the 31 (3):499-505. NMDA receptor. .I. Ncwrohiol. 21: 18-32. SINGMAN, E. L. and SCALIA.F. ( 1990). Quantitative FRITZSCH.B.. CRAPONDE CAPRONA.M. -D.. and study of the tectally projecting retinal ganglion cells in CLARKE,P. G. H. ( 1990). Development o f t w o morthe adult frog: I. Size of the contralateral and ipsilatphological types of retinopetal fibers in chick embryos. CvO/np., V ~ i / r o 302:792-809. /. as shown by the diffusion along axons of a carbocyanSINGMAN. E. L. and SCALIA,F. (1991). Quantitative ine dye in the fixed retina. .I. C’O/nli. Ncirrol. 300:405study of the tectally projecting retinal ganglion cells in 42 I . the adult frog: 11. Cell survival and functional recovery FRITZSCH,B. and WILM,C. ( 1990). Dextran amines in after optic nerve transection. ./. C‘otnp. S c i i r o l . neuronal tracing. Trcnd,s h’011ro.cci. 13( 1 ): 14. 307:35 1-369. FRITZSCH,B. ( 1991 ). Ontogenetic clues to the phylogSPRINGER, A. D. ( 1980). Aberrant regeneration in the eny of the visual system. In: 7’/w CYiungiiig L’i.sim/ $ l ~ goldfish after crushing one optic nerve. Bruin Rex i m i : , j i o t w Eurly 1 0 Lutc Stuges c!fL(fi.. P. Bagnoli, W. 199:214-218. Hodos, Eds. Plenum Press. London, pp. 33-49. SPRINGER, A. D. ( 198 1 ). Normal and abnormal retinal projections following the crush of one optic nerve in FRITZSCH.B. and WILM,C. ( 1993). lpsilateral projec-
goldfish (Curassius auratin). J . Comp. Neurol. 199s 7-97. SRETAVAN,D. (1990). Specific routing of retinal ganglion cell axons at the mammalian optic chiasm during embryonic development. J . Nezruosci. 10(6): 1995-2001. STELZNER,D. J., BOHN, R. C., and STRAUSS.J. A. ( 198 I ). Expansion of the ipsilateral retinal projection in the frog brain during optic nerve regeneration: sequence of reinnervation and retinotopic organization. .I.Comp. Neurol. 201:299-317. STUERMER,C. A. 0. and RAYMOND,P. A. ( 1989). Developing retinotectal projection in larval goldfish. J . Comp. Neurol. 28 1:6 30-640. TAYLOR, J. S. H. and GAZE, R. M. (1990). The induction of an anomalous ipsilateral retinotectal projection in Xenopio luevis. Anut. E m b r j d . 181:393-404. THANOS, S. and BONHOEFFER,F. ( 1984). Development
of the transient ipsilateral retinotectal projection in the chick embryo: a numerical flourescence-microscope analysis. .I.Comp. Nezirol. 224401-4 14. WILM, C. and FRITZSCH.B. (1989). Development of tectal neurons in the perciform teleost Zlciplochrornis hurtoni: a Golgi study. Dev. Brain Rex 41:35-52. WILM,C. ( 1990). The ipsilateral retinotectal projection in a percomorph fish: a dil-study in fixed tissue. In: Brain-Perception-Cognition. N. Elmer, G. Roth, Eds. Thieme, Stuttgart, New York, pp. 283. WILM,C. and FRITZSCH, B. (1990). Ipsilateral retinofugal projections in a percomorph bony fish: its experimental induction, specifity and maintenance. Bruin Behuv. Evol. 36:27 1-299. WILM,C. and FRITZSCH,B. ( 1992 ). Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of a fish. J . Neurohiol. 23: 149- 162.
+?*
Faculty of Biology, University of Bielefeld, 4800 Bielefeld, Germany
SUMMARY Retinal projections were experimentally manipulated in a bony fish to reveal conditions under which considerably enlarged ipsilateral projections developed and persisted. Three experimental groups were studied: animals after unilateral enucleation, after unilateral nerve crush, and after enucleation and crush of the remaining optic nerve. At 29 days after unilateral enucleation alone, no enhanced ipsilateral projection had developed. After nerve crush, however, large numbers of retinal fibers regenerated into the ipsilateral tectum. Retrogradely filled, ipsilaterally projecting ganglion cells were distributed throughout the entire retina. After 15 days regenerating retinal fibers covered the entire ipsilateral tectum. At
INTRODUCTION The presumably primitive condition ofbilateral retinal projections is widespread among vertebrates (Fritzsch, 199 1 ). Absence of an uncrossed projection, for example, in many bony fish (Wilm and Fritzsch, 1990) or birds (Thanos and Bonhoeffer, 1984), appears to correlate with modifications of one of the two developmental steps necessary for the formation and maintenance of an ipsilateral projection: Received August 27, 1991: accepted May 5. 1992 Journal of Neurobiology. Vol. 23, No. 6. pp. 692-707 (1992) C 1992 John Wiley & Sons, Inc. CCC 0022-3034/92/060692- 16$04.00 Present address: *E. Merck, Biologische Forschung, P.O. Box 4 I 19. 6 I00 Darmstadt I , Germany; ?Department of Biomedical Sciences. Division of Anatomy, Creighton University. Omaha, Nebraska 68 178, U.S.A. $ To whom correspondence should be addressed.
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later stages the ipsilateral projection showed progressive reduction in coverage of the tectum. Combining enucleation with nerve crush led to an ipsilateral projection that covered the tectum at 28 days and later. In this experimental situation the development of an ipsilateral projection appears to be a tno-step process: ( I ) Fibers are rerouted to the ipsilateral side at the diencephalon, and ( 2 ) ipsilatera1 fibers persist in the tectum only in the absence of a contralateral projection while they appear to be eliminated in the other cases. GI1992 John Wiley & Sons, Inc. Keywords: visual system, regeneration, optic nerve, ipsilateral projections, teleosts, ganglion cell distribution, pathfinding.
1. Some bony fish apparently never develop an ipsilateral projection in excess of a maximum of 20 fibers (Fritzsch and Wilm, 1992). Owing to the presence of monocular crossing to the contralateral diencephalon and the consequent absence of a true chiasm with the chiasmatic interdigitation of fibers of both eyes, these fish may have largely sidestepped cues shown to be important for pathfinding of many ipsilateral fibers in mammals (Godement, Salaun, and Mason, 1990) and perhaps other vertebrates. 2. In contrast, birds seem to develop an ipsilateral projection initially, but eliminate ipsilateral fibers in the tectum in a second step (Thanos and Bonhoeffer, 1984) so that adult animals have virtually no ipsilateral fibers. A comparable, likely activity-controlled process takes place in mammals (Cowan, Fawcett, O’Leary, and Stanfield, 1984), appears
Regcweruiion of the Optic Nerve
693
Operation
Application
Figure 1 Surgery and application of tracers. Three experimental groups were studied. Operations were performed on 26-day-old animals. The animals survived between 4 and 60 days. NOR = nucleus olfactoretinalis; ON = optic nerve: Tec = tectum.
to cause the segregation of retinal projections in three-eyed frogs (Debski, Cline, and Constantine-Paton, 1990), and may be present in all binocularly interacting retinal projections. However, based on normal development it is unclear whether this second process is at all present in cichlid fish given that they never develop a large ipsilateral projection and do not noticeably reduce the number of ipsilaterally projecting cells with age (Fritzsch and Wilm, 1992). Despite the virtual absence of an ipsilateral retinal projection in the adult (Wilm and Fritzsch, 1990) or developing (Fritzsch and Wilm, 1992) cichlid fish Huplochromis burtoni, regenerating retinal fibers can form a larger then normal retinal
projection into the ipsilateral tectum (Wilm and Fritzsch, 1990, 1992). Comparable phenomena have been reported for the regenerating goldfish visual system (Sharma and Tsai, 1991; Springer, 1980, 198 1 ). These studies all imply that the regenerating retinal projections in fish do not recapitulate faithfully the development and may lead instead to unusually enlarged projections that are much sparser, if present at all, in undisturbed animals. Experimentally induced ipsilateral fibers may represent unusual exaggerations of ontogenetic processes. Nevertheless, given the control of timing of onset of formation and regression of induced ipsilateral projections, these projections may be particularly helpful to elucidate the factor( s ) that lead to the formation and maintenance of an ipsilateral projection in the continuously growing bony fish.
Table 1 Different Groups and Combinations of Dyes A. Experimental Groups and Anterograde Labeling of the Retinal Projection with Dil, PKH2 and FDA Groups Unilateral enucleation Days after the operation 7 and 29
Unilateral nerve crush Days after the operation 4 5 7-1 I 22-36 46 and 60
Enucleation and nerve crush Days after the operation 7 7 and 22 29
Labeling
Preparation
Left optic nerve Intact Dil
Right optic nerve Degenerating
Left optic nerve Regenerating Dil Dil Dil Dil Dil
Right optic nerve Intact
Left optic nerve Intact Dil Di I Di I
Right optic nerve Degenerating
Tccta
-
PKH2 PKH2 or FDA -
Tccta Tecta Tecta Tecta Tecta and the other retina
Tecta Tecta and retina Tecta
B. ExDerimcntal GrouDs and Retrograde Labeling of Ganglion Cells with Dil Unilateral nerve crush Days after the operation 15'
Dil into the tectum which is positioned ipsilaterally to the lesioned optic nerve
Both retinae
Dil into the tectum which is positioned ipsilaterally to the lesioned optic nerve
Remaining retina
Enucleation and nerve crush Days after the operation 152
223 I
17 animals. I I animals. I 2 animals.
In a previous study we explored this model and described some of the conditions that lead to the formation and maintenance of an unusually large ipsilateral projection (Wilm and Fritzsch, 1990). Employing tract tracing with horseradish peroxidase (HRP), ipsilateral fibers could first be detected after 7 days and no ipsilateral fibers could be detected any more after 28 days, suggesting that ipsilateral fibers may have been eliminated presumably through a process not unlike the one acting in developing birds, mammals, and three-eyed frogs. Alternatively, however, remaining ipsilateral fibers could have failed to transport HRP, and the earliest stages of regeneration may not have been investigated at all owing to rapid degeneration of the
fragile fibers. We therefore undertook the present study to analyze in detail the morphology of the first regenerating fibers and ipsilateral retinal fibers while they become eliminated employing the lipophilic tracer Dil in fixed tissue. [Preliminary results have been presented in abstract form (Wilm, 1990).]
METHODS For this study, a total of 323 specimens ofthe cichlid fish species Haplochroomis bzirtoni were subjected to surgical or postmortem procedures. The breeding animals and the larvae were kept at 25" f 1°C (lightldark cycle:
RcgcnPralion o f fkc Optic Nerve
695
Table 2 Effects of Treatment on the Formation of Ipsilateral Retinal Projections A. Normal Retinal Projection ( < I 5 Fibers) into the Ipsilateral Tectum
Age (days) of the Animals
Days after the Operation
Number of Animals
Number and Percentage of Animals with Ipsilateral Fibers N
%
14 18
70
Unilateral Enucleation
I
33 55
29
20 21
86
B. Enhanced Retinal Projection (>30 Fibers) into the Ipsilateral Tectum Unilateral Enucleation
I
33 55
29
20 21
0 1
0 5
Unilateral Nerve Crush
30 31 33 and 31 41 48 55 and 57 62 12 86
4 5 land I I 15 22 29 and 31 36 46 60
14 14 35 19 26 24 10
I0 10
No labeling at all 4 29 20 51 19 I00 24 92 23 96 10 100 10 100 8 80
Enucleation and Nerve Crush
I
33 41 48 55
15
22 29
13:l 1 h). The age of the animals ( u p to 20 days) was determined according to a table of developmental stages (Wilm and Fritzsch, 1989). Free-swimming juveniles were kept at room temperature ( 2 1 5 1 "C) in small aquaria and fed with dry food for young brood (Tetramin). Animals were operated at 26 days of age (standard length: 9 mm). O
Surgery Three types of operations were performed: ( 1 ) enucleation of one eye, ( 2 ) crush of one optic nerve, and ( 3 ) enucleation of one eye and crush of the other optic nerve (Fig. I ). Prior to surgery all animals were anesthetized in 0.0 1% ethyl p-aminobenzoate (benzocaine, Sigma) following guidelines established by the German Federal Law. Operated animals were initially reared in a fish
44 10 15
II
28 9
64 90
15 II
I00 I00
Ringer solution. The salt concentration was gradually reduced by adding aquarium water over 4 days. Enucleation. The optic nerve was exposed through a caudal incision in the orbital skin and connective tissue. The eye was gently rotated rostroventrally in the orbit, and the optic nerve was cut with fine scissors. Optic-Nerve Crush. The optic nerve was exposed and then crushed with watch-maker forceps for about 5 s.
Anterograde Tracing of the Visual Projection The short distance (only 2-3 m m in 30- to 86-day-old animals) of the retinal projection allows the application of Dil ( 1,l '-dioctadecyl-3,3,3'.3'-tetramethyl indocarbo-
696
M i l r ~and Fritzsch
Figure 2 Regenerating retinal fibers into the contralateral ( A ) and ipsilateral ( B ) tectum 5 days after unilateral nerve crush. The tecta are shown as whole mounts from a dorsal aspect. The tecta were split along the rostrocaudal meridian to flatten them onto a slide. The contralatera1 tectum was opened wider at its cut than was the ipsilateral tectum. Regenerating fibers cover the rostra1 pole of the contralateral tectum and preferentially grow into the dorsomedial part of the ipsilateral tectum. C = caudal; D = dorsomedial; Tee = tectum; T r O D = tractus opticus dorsalis; V = ventrolateral. Scale bar = 200 pm.
1990). In all animals the skull was opened under anesthesia. The animals were immersed in 4% paraformaldehyde in 0.1 Mphosphate buffer( pH 7 . 3 ) at room temperature and then stored at 4°C. At I day to 4 weeks later, crystals of Dil or PKH2 were applied to the optic nerve head inside one eye. The animals were either stored in the dark at 19” to 2 1 “ C for 1 1 days (if one retinal projection was labeled with FDA) o r at 36°C for 2-3 days depending on the age (size) of the animal. For each age minimal diffusion times to label the retinotectal projection were established to avoid transcellular labeling (Fritzsch and Wilm, 1990). The material was subsequently stored at 4°C prior to examination to minimize further diffusion. The tectum together with the optic tracts were dissected. The tectum was split along the rostrocaudal meridian, sparing the rostral pole, in order to flatten the tectum onto a slide and whole mounted in 0. I A4 phosphate buffer ( p H 7 . 3 ) . Two small coverslips on each side served as spacers. The fluorescent material was viewed with an epifluorescence microscope and photographed with a rhodamine or fluorescein filter set (TMAX 100 ASA or Ektachrome 200 ASA, Kodak).
Retrograde Tracing of the lpsilaterally Projecting Ganglion Cells
Figure 3 Regenerating retinal projection I5 days after unilateral nerve crush into the contralateral half of the tectum. Contralateral fibers fasciculate (arrow) and take tortuous courses in the tectum. Shown is a “bad” regeneration with only few contralateral fibers. Otherwise, the density of the projection does not allow the demonstration of the course of individual fascicles. Abbreviations as per Figure 2. Scale bar = 200 Km.
Dil was applied into the rostral tectum of paraformaldehyde fixed animals [Table 1: Fig. I]. A shallow incision was placed transversely across the rostral tectum from dorsomedial to ventrolateral. Crystals of Dil were stuffed into the incision except at the dorsomedial margin to avoid diffusion of Dil into the other tectum. The material was stored in 4% paraformaldehyde in 0. I M phosphate buffer ( p H 7.3) at 36°C for 2 days. The retinae were dissected and the lense and iris were removed. The retina was incised at the ventral, dorsal, nasal, and temporal margin and whole mounted in phosphate buffer (pH 7 . 3 ) onto a glass slide and examined as described above.
RESULTS cyanine perchlorate, Molecular Probes, Eugene, Oregon, U.S.A.) and PKH2 (green fluorescent, lipophilic dye, gift of the Zynaxis Cell Science Inc., Malvern, PA, U.S.A.) in paraformaldehyde fixed tissue. Table I lists the different groups and combinations of the dyes. After unilateral nerve crush, the regenerating projection was traced with Dil and the undisturbed projection with PKH2 orfluorescein-isothiocyanate-coupled dextran amine [FDA ( Molecular Probes) (Fig. I )] . After unilateral enucleation alone or enucleation and crush of the remaining optic nerve, the retinal projection was traced with Dil. FDA was applied in deeply anesthetized animals onto the cut optic nerve. The animals survived 14-16 h for sufficient transport of the dye (Wilm and Fritzsch,
We considered the ipsilateral projection as an “enhanced projection” compared to unoperated animals if more than 30 fibers projected into the ipsilateral tectum, that is, if more fibers reached the tectum than did the maximal 20 fibers ever encountered in normal development (Fritzsch and Wilm, 1992). Unilateral Enucleation (Lesion 1, Fig. 1 )
After 7 and 29 days, 70% and 86% of the animals, respectively, showed few fibers ( < 1 5 ) which
Figure 4 Regenerating retinal fibers ( A ) labeled with Dil into the ipsilateral tectum 15 days after nerve crush and the native retinal projection ( B ) labeled with PKH2 into this same tectum. Half the tectum is shown. The undisturbed contralateral projection as well as the regenerated ipsilateral projection cover the entire tectum. Ipsilateral fibers course in the same layers as do the native contralateral projections. Abbreviations as per Figure 2. Scale bar = 200 urn.
coursed via the optic tract into the ipsilateral tectum (Table 2 ) . Only one animal had a prominent retinal projection (>30 fibers) into the ipsilateral tectum after 29 days (Table 2). Unilateral Nerve Crush (Lesion 2, Fig. 1 ) After 4 Days. For unknown reasons we could not label with Dil any regenerating retinal fiber in the 14 animals analyzed (Table 2).
After 5 Days. Retinal fibers had regenerated into both tecta (Fig. 2). The progress of regenerating fibers showed a high interindividual variability ranging from few fibers at the rostra1 pole of the tectum to a projection that covered nearly twothirds of the contralateral tectum. Fibers regenerated predominantly via the dorsomedial optic tract ipsi- and contralaterally; they branched more exten-
sively in the contralateral tectum. Retinal fibers coursed in 79% ( n = 1 1 out of 1 4 ) of the animals into the ipsilateral tectum with 29% ( n = 4 ) , displaying an enhanced ipsilateral projection [ >30 fibers; Fig. 2(b)]. In the remaining three animals several retinal fibers coursed in the ipsilateral optic tract presumably on their way to the tectum.
After 7-21 Days. The regenerating projection covered two-thirds to three-fourths of the contralateral tectum. In 56% of the animals, an enhanced ipsilatera1 projection had developed (Table 2 ). After 15 Days. The regenerating projection covered the entire contralateral tectum. Regenerating fibers took tortuous courses and fasciculated (Fig. 3 ) . Many fibers had growth cones. In all animals a prominent projection into the ipsilateral tectum had developed [Fig. 4(a ) ] . In 16 animals the pro-
Figure 5 Ipsilateral retinotectal projection 22 ( A ) and 29 ( B ) days after unilateral nerve crush. The density of the ipsilateral projection is decreased compared to animals with shorter postoperative time intervals. ( A ) In a few animals, ipsilateral fibers persist at the lateral and medial margin. ( B ) In most animals, the caudal tectum is free of retinal fibers. Abbreviations as per Figure 2. Scale bar = 200 pm.
Figure 6 In a few animals ipsilateral fibers course over the entire tectum even 60 days after unilateral nerve crush. Shown is a n extreme case with a dense distribution of ipsilateral fibers. Abbreviations as per Figure 2. Scale bar = 200 Fm.
Figure 7 Between 22 and 60 days after unilateral nerve crush, several ipsilateral fibers end blindly ( A ) , and short segments are distributed in the ipsilateral tectum ( B ) . Fine caliber fibers often course through these segments. R = rostral; C = caudal. Scale bar = 100 pm.
jection covered the entire ipsilateral tectum without any preferential termination. In the remaining three animals ipsilateral fibers terminated at the rostral tectum or at the lateral and medial margin. The retinal projection ofthe other eye, labeled with PKH2, remained obviously undisturbed by the operation [Fig. 4( b)]. Ipsilateral fibers [Fig. 4( a ) ] coursed on top and terminated in the same layers as did the contralateral projection [Fig. 4( b)]. Between 22 and 60 Days after the Nerve Crush. In all stages examined, an enhanced ipsilateral projection occurred in 80%-100% of the animals (Table 2). But ipsilateral fibers were no longer evenly distributed, and the density of fibers appeared considerably reduced (Fig. 5 ) . The distribution of ipsilateral fibers was heterogeneous. In a few animals retinal fibers remained predominantly at the outer tectal margin [Fig. 5 ( a ) ] . But in most animals the density of fibers decreased from rostral to caudal [ Fig. 5 ( b ) ]. However, even 60 days after the nerve crush, retinal fibers coursed over the entire ipsilateral tectum in a few animals (Fig. 6 ) , but the density of the ipsilateral projection appeared lower
than 15 days after the nerve crush. Terminal branches of these ipsilateral fibers were rarely ohserved and occurred predominantly at the tectal margin. Many ipsilateral fibers ended either blindly or the fluorescence faded distally [Fig. 7 (a)]. Predominantly in the rostral tectum short segments were observed (Fig. 7b) with fine caliber fibers coursing through these segments. Examination of the contralateral retina that received no Dil application revealed no labeled ganglion cells in the two groups 46 and 60 days after nerve crush with only one exception (two ganglion cells). This suggests that ipsilaterally projecting fibers have not been transcellularly labeled by diffusion of Dil into the other optic nerve. Enucleation and Nerve Crush (Lesion 3, Fig. 1) After 7 Days. In 64% oftheanimals, enhanced ipsilateral retinal projections had developed (Table 2). After 15 Days. In 90% of the animals, enhanced ipsilateral projections had developed, which cov-
702
M ilrn arid Ft2t:sc.h
Figure 8 The ipsilateral projection in the tectum 29 days after enucleation and nerve crush. After elimination of the native contralateral projection, ipsilateral fibers persist in the entire tecturn. TrOV: tractus opticus ventralis: other abbreviations as per Figure 2. Scale bar = 200 Irm.
ered the entire tectum, except for one case in which the nerve may have been inadequately crushed. We infer this because the contralateral projection did not show the tortuous courses of retinal fibers, a characteristic feature of a regenerated projection. After 22 and 29 Days. In all animals an enhanced ipsilateral projection covered the entire tectum (Fig. 8 ) . At 29 days after the operation, ipsilateral fibers exhibited complex terminal fields like the contralateral fibers (Fig. 9 ) . The density of ipsilatera1 fibers was always higher than of that in the group that received a unilateral nerve crush with comparable postoperative time interval. Distribution of lpsilaterally Projecting Ganglion Cells
15 Days after Nerve Crush. All 17 animals in which the ganglion cell population across the entire contralateral retina was labeled showed high num-
bers of ganglion cells in the ipsilateral retina (Fig. 10). There was no preferential distribution of ipsilaterally projecting ganglion cells in one retinal sector: in five animals, ganglion cells showed an even distribution across the retina, in four animals, the density was higher in the nasal retina, and in eight animals the density was higher in the ventral retina. 15 and 22 Days after Enucleation and Nerve Crush. Because the contralateral eye was absent, the completeness of labeling could not be checked. In 18 animals, ganglion cells were labeled in the entire ipsilateral retina. In seven out of these 18 animals, the density of labeled cells was highest in the nasal retina and in one animal in the ventrotemporal retina. In the remaining animals, ganglion cells were labeled either only in the dorsal (two animals) or in the nasal retina (three animals) or showed no preference (five animals).
Unilateral Enucleation
After unilateral enucleation, only one out of 2 1 animals showed an enhanced ipsilateral projection after 29 days. This confirms previous results that only 10% of enucleated animals ( 5 out of 50) exhibit an enhanced ipsilateral projection after 2 months and later (Wilm and Fritzsch, 1990). We suggested that the ipsilateral projections were induced during the surgery owing to surgical lesions of the remaining optic nerve or chiasm. This conclusion is supported by observations that an enhanced ipsilateral retinotectal projection can be induced in Xmopzis by damage to the chiasm (Taylor and Gaze, 1990). Unilateral Nerve Crush-Enucleation Nerve Crush
Figure 9 lpsilateral fibers develop complex terminal arborizations 29 days after enucleation and nerve crush. Scale bar = 100 p m .
DISCUSSION Methods
Use of diffusion of Dil in the plasma membrane of aldehyde-fixed tissue allows prior fixation of undisturbed fibers and avoids any artifacts potentially caused by the separation of the growing axons from their cell bodies (Stuermer and Raymond, 1989). As in the accompanying paper (Fritzsch and Wilm, 1992), we took great care to control the only potential artifact known for Dil, transcellular diffusion (Fritzsch and Wilm, 1990). The first developing (Fritzsch and Wilm, 1992) as well as the first regenerating fibers after the nerve crush could be labeled after 5 days with Dil, but not with other tracers. Neither with HRP nor with fluorescent dextran amines could we obtain any satisfactory tracing of regenerating fibers prior to 2 1 days postoperatively ( Wilm and Fritzsch, 1990). Similarly, we could label ipsilateral fibers with Dil even 60 days after unilateral nerve crush while fluorescent dextran amines failed to label this ipsilateral projection already after 28 days (Wilm and Fritzsch, 1990 ). The higher fluorescent yield and, in particular, the diffusion in the cell membrane independent of intracellular transport, may contribute to the apparent higher sensitivity of Dil.
and
After nerve crush, retinal fibers regenerated to both tecta indicating that crush alone creates conditions that may lead to the formation ofan ipsilateral projection. The regenerating ipsi- and contralateral projection covered the entire tecta at 15 days postcrush. Subsequently, the distribution and morphology of ipsilateral fibers depended on the persistence or absence of a native contralateral projection. If the other optic nerve remained undisturbed, then the density of ipsilateral fibers decreased, suggesting that these fibers were reduced in numbers or complexity of their arborization. However, even at 60 days postcrush ipsilateral fibers were still present but were lost after several months (Wilm and Fritzsch, 1990). In contrast, if the native contralatera1 projection had been eliminated by enucleation, the ipsilateral projection had developed complex terminals in the entire tectum 29 days later. In this experimental group the ipsilateral projection persisted up to 7 months (Wilm and Fritzsch, 1990). How are aberrant projections like the ipsilateral retinotectal fibers structurally eliminated: Do they retract? Or do they degenerate? Some in vitro observations showed retraction of fibers from unsuitable substrates (Kapfhammer and Raper, 1987) and retraction was also recently suggested for the elimination of one type of efferent fiber of the chicken retina (Fritzsch, Crapon de Caprona, and Clarke, 1990). However, the blind-ending fibers and short segments in our study on the ipsilateral projection may indicate a degeneration of retinal fibers. Comparison with the contralateral regenerating projection in the same animal showed that blind-ending fibers cannot be explained as methodological artifacts. The short segments probably
Figure 10 Flat mount of the retina with ipsilaterally projecting ganglion cells 15 days after unilateral nerve crush. The Dil-labeled ganglion cells are distributed over the entire retina. ON = optic nerve head; T = temporal; D = dorsal. Scale bar = 200 Wm.
consisted of several fasciculated fragments of axons. Fine caliber fibers coursed through these segments, and the dye could have diffused from these fibers into the fragments. Springer ( 1980, 198 1 ) also showed the disappearance of initially present ipsilateral retinofugal fibers after nerve crush in goldfish but could not decide with certainty whether ipsilateral fibers were eliminated or undetectable with radioautographic methods. Long-term retrograde labeling of ipsilaterally projecting ganglion cells is required to test whether or not these ganglion cells survive like the callosal cells in the developing mammalian cortex (Innocenti, 1988). Distribution of lpsilaterally Projecting Ganglion Cells In juvenile H . hzirtoni up to 20 ganglion cells projected into the ipsilateral tectum (Fritzsch and
Wilm, 1992). These ganglion cells came from the entire retina, and terminated throughout the entire tectum. Likewise, ipsilaterally projecting regenerating ganglion cells were also distributed in the entire retina without topographical preference. Therefore, all regions of the retina send at least some axons ipsilaterally. Similar results were obtained in goldfish (Sharma and Tsai, 199 1 ). It has been previously shown that only regenerating but not newly developing optic nerve fibers grow without any apparent order to the optic chiasm in goldfish (Bernhardt and Easter, 1988) and that ipsilaterally projecting ganglion cells are within the area of the retina already present at the time of optic nerve crush ( Wilm and Fritzsch, 1992). Together these data led us to hypothesize that development ofan ipsilatera1 projection may be related to the lost order in the regenerating optic nerve that causes rerouting predominant1y of regenerating fibers. Whereas in a ranid frog (Stelzner, Bohn, and
Straws, 198 1;Singman and Scalia, 199 1 ), regenerating, ipsilateral ganglion cells are coming from topographically corresponding areas of the entire retina as in normal ranid frogs (Singman and Scalia, 1990), ipsilaterally regenerating ganglion cells are located in Xenopzu mainly in the ventral retina as during normal development ( Hoskins and Grobstein, 1985a,b).These studies imply that comparable principles may operate during development and regeneration of ipsilateral projections in frogs that nevertheless lead to an apparently expanded ipsilateral projection during optic nerve regeneration (Singman and Scalia, 1990). Given these data in frogs, it is possible that the enlarged ipsilateral projection during regeneration in H. burtoni may be an exaggeration of a normal developmental process. The Formation and Maintenance of an lpsilateral Projection
This study on H. burtoni shows that a species with a small ipsilateral retinal projection develops a considerably larger ipsilateral projection during regeneration of the optic nerve. If the native contralatera1 projection to the tectum was either disturbed (by crushing the other optic nerve) or eliminated altogether, then ipsilateral fibers can persist for several months (Wilm and Fritzsch, 1990). Whereas the presence of an undisturbed contralateral projection did not interfere with the initial formation of an ipsilateral projection, then it reduced dramatically the maintenance of this projection. Therefore, the development of an ipsilateral projection is a largely independent, two-step process. These two processes will be discussed next. Pathfinding of lpsilateral Fibers in the Optic Chiasm
Evidence in mice (reviewed in Godement, Salaun, and Mason, 1990; Sretavan, 1990) suggests that the projections of the contralaterally and ipsilaterally projecting retinal ganglion cells are sorted out in a two-step process for many cells: only contralateral, but not ipsilateral fibers can cross the midline. In addition, ipsilateral fibers require crossed fibers of the contralateral eye to reach their ipsilateral target. An additional small population may navigate independently of the crossed, contralateral fibers. In frogs, the mechanisms for the navigation in the chiasm is less well studied, but is apparently influenced by thyroid hormone ( Hoskins, 1990) and also by the teratogen retinoic acid ( Manns and Fritzsch, 199 1 ) . Interestingly, if the
optic nerve was lesioned behind the eye, then an enhanced ipsilateral projection developed in Runa ( Stelzner et al., 198 I ; Singman and Scalia, I99 1 ), but not in Xmopzis (Taylor and Gaze, 1990). This indicates that the sorting mechanisms of the optic chiasm of frogs may have species-specific differences perhaps related to the size of the naturally occurring ipsilateral projection. In a previous study (Wilm and Fritzsch, 1990) we showed that retinal fibers do not grow ipsilaterally at the “chiasm” of II. hurtoni. Instead, the fibers enter the contralateral diencephalon and then, when reaching the area of “monocular fibrecrossing” (Maggs and Scholes, 1986), recross to the ipsilateral side. During their path to the ipsilatera1 side, these fibers have no contact with the contralateral nerve. Therefore, fiber interaction with the contralateral nerve is not responsible for the initial ipsilateral course in H . hzrrtoni. Moreover, we showed previously that an ipsilateral projection develops even when the nerve was crushed 1.5 years after enucleation. As verified by electron microscopy, all fibers of the contralateral nerve had degenerated by that time. Therefore, the pathfinding of ipsilateral fibers of H. bwtoni resembles the small ganglion cell population that projects in mice ipsilaterally without interaction with the contralatera1 fibers (Godement, Salaun, and Mason, 1990). Whether more ganglion cells express this capacity in the regenerating projection of H . hzirtoni or whether the regenerating nerve presents a uniquely enlarged sorting problem and thereby produces excessive numbers of ipsilateral projecting fibers as previously suggested ( Wilm and Fritzsch, 1990) remains to be clarified. Maintenance and Regression of the lpsilateral Projection
The elimination of aberrantly projecting ipsilateral fibers in mammals, for instance, is activity controlled (Cowan, Fawcett, O’Leary, and Stanfield, 1984). In bony fish, the persistence of an undisturbed contralateral projection clearly prevented ipsilateral fibers from establishing a dense projection in the tectum. Starting at 29 days, but more pronounced at 60 days after nerve crush, the density of the ipsilateral projection was less in the group that received only a crush of the optic nerve, and several months after nerve crush alone the ipsilateral projection was completely eliminated (Wilm and Fritzsch, 1990). If both nerves were crushed, then the regenerated ipsi- and contralatera1 projection developed separate patches in the tectum ( Wilm and Fritzsch, 1990). We suggest
that as with the different retinal projections of the three-eyed frog ( Constantine-Paton and Law, 1978: Debski. Cline, and Constantine-Paton, I990), ipsilateral and contralateral projection separate because of their different activity patterns. Correlated activity pattern seems to be important for the stabilization of synapses in the retinotectal projection. After nerve crush without enucleation the regenerating ipsilateral fibers are confronted in the tectum with an undisturbed and much denser contralateral projection. Potential sites for synapses should be available because the tectum is still proliferating and differentiating ( Raymond and Easter, 1983; Raymond, 1986; Wilm and Fritzsch, 1989) at its caudal margin and the whole contralateral projection is continuously shifting ( Wilm and Fritzsch, 1992). Nevertheless, the ipsilateral projection is not able to establish itself between the contralateral projection. Our study cannot explain the reason for this failure. We can only speculate that the ipsilateral projection is not dense enough to displace contralatcral terminations and stabilize their own terminations with the help of their correlated activity pattern.
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