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Reorganization of the retinotectal pathway in rats after neonatal retinal lesions
EXPERIBIEXTAL
NEUROLOGY
40, 377-390 (1973)
Reorganization of the Retinotectal After Neonatal Retinal R. D.
LUND
Departmcrzts of Biological U>zie!ersity of Washington
AND
JENNIFER
Pathway Lesions S. LUND
Structure, Neurological School Received
of Medicine February
in Rats
’
Surgery and Ophthalmology, Seattle,
Washington
98195
24,1973
The normal retinotectal pathway of albino rats has very few uncrossed axons, limited in area of termination to a small region of the stratum opticum. If a small retinal lesion is made at birth, when not all optic axons have left the retina, there is in the adult a region in the stratum griseum superficiale of the contralateral superior colliculus which receives few or no axons from the operated eye. Peripheral to this, the colliculus receives a markedly reduced crossed optic projection. A dense aberrant ipsilateral projection is found from the intact eye to the region corresponding to the projection zone of the lesion and a lighter projection between this and the edge of the colliculus. The results are interpreted as indicating modified growth patterns of late growing optic axons which result in the aberrant uncrossed optic projection. As such, they suggest that there is a positive interaction between axons from corresponding retinal regions during their growth through the optic chiasm.
INTRODUCTION In previous anatomical studies (17-20) it was shown that unilateral eye removal from neonatal rats results in a subsequent expansion in the distribution of the optic pathway from the remaining eye to the subcortical visual centers. The additional projection is to parts of the superior colliculus and lateral geniculate body ipsilateral to the remaining eye which normally receive only crossed optic axons ; small lesion studies show this projection to originate from parts of the remaining retina which would normally 1 This investigation was supported by PHS Research Grants No. EY00491 and EY00596 from the National Eye Institute and in part by General Research Support Grant RR05432 from the National Institutes of Health, a research development Grant from Research to Prevent Blindness, Inc. and PHS Grant No. HD02274. We gratefully acknowledge the valuable technical assistance of Renee Wise, Franque Remington, and Jeri Hunter, and secretarial help of Carol Lade. 377 Copyright AU rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
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project onIy contralaterally. Despite their termination in the “wrong” side of the brain, the anomalous axons maintain an appropriate retinotopic pattern in both colliculus and geniculate. To explain these findings it was suggested [with some supporting evidence from a Golgi study (22) ] that at birth not all optic axons have grown past the optic chiasm. If one eye is removed in newborn rats, late growing axons are then misdirected, allowing the possibility that the chiasmatic crossing is the result of dynamic interaction of axons from both eyes rather than simply of growth properties determined in the retina. The possibility then arises that this interaction occurs between small groups of fibers such that a small retinal lesion at birth results in an area in each visual center devoid of a contralateral optic projection which is filled in by aberrant ipsilateral axons from the other eye. This possibility is investigated here. MATERIALS
AND
METHODS
Small retinal lesions of approximately equal size were made through the sclera in rats shortly after birth. The subsequent development of the operated eyes appeared normal in the nine successfully operated animals used in this study. The animals were allowed to survive for 2 mo, by which time all degeneration from neonatal lesions had disappeared. One eye was them removed and the animaIs allowed to survive for a further 4 days when they were perfused with 4% paraformaldehyde in phosphate buffer. Degeneration resulting from the eye removal was stained with Fink-Heimer (4) and neurofibrillar methods (21) on frozen sections cut 25 w thick. In three of the animals with temporal retinal lesions and two with nasal lesions, the eye which had originally been operated on was removed at the second operation in order to define central regions which may be free of degeneration and which would correspond to the projection zone of the damaged area. In the other four animals (two each with nasal or temporal retinal lesions), the eye contralateral to that with the lesion at birth was removed. This would allow recognition of aberrant uncrossed optic axons. In addition, control animals of four categories were studied. The first were completely unoperated normal adults ; the second were adult animals from which one eye had been removed 5 days prior to fixation; the third group were young rats 7, 14, 21, and 28 days old from which one eye had been removed at birth ; and the fourth consisted of one animal with a small retinal lesion at birth and no subsequent eye removal before perfusion at 2 mo old. All the eyes containing lesions were fixed by injection of 2% glutaraldehyde into the vitreous, and subsequently stained with cresyl violet acetate. One eye was stained as a flat mount; the others were embedded in celloidin and cut at 40 pm thickness.
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RESULTS Retinal Histology. The lesions in the eye made at birth can be recognized in each adult animal as a gap through all the retinal layers (Fig. 1A). With the exception of a region between the lesion and the retinal border, the retina appears completely normal when studied in flat preparation and in transverse section. Notable features of the normal retina are the density of packing of the ganglion cells, the wide diversity in cell body sizes (Fig. lB), and the bundles of optic axons running between groups of ganglion cells. In the region peripheral to the lesion (Fig. lC), the ganglion cell layer has fewer cells which in the flat preparation are seen to be distributed somewhat erratically, in some cases in small clusters ; the range of variation of cell body size is much less, largely because of an absence of the larger ganglion cells; and the circumscribed bundles of optic asons are not evident. Control Studies. The control animals with one eye removed at birth and short postoperative survival times show that within 21 days there is no persistent degeneration either of optic axons or terminals or of collicular cells undergoing a transneuronal reaction stainable by Fink-Heimer or neurofibrillar methods. No degeneration was found in the one control animal with a retinal lesion at birth and no subsequent eye removal. There is, therefore, no reason to believe that any of the degeneration attributed in the present studies to the second lesion is persistent from the neonatal lesion. Animals operated as adults (with no previous lesions) show that the normal crossed optic pathway is heavy throughout the colliculus. In FinkHeimer stained material, degeneration of this pathway appears as fine granules throughout the stratum griseum superficiale which are concentrated more heavily near the surface and coarser axonal debris, more prominent in the deeper part of the layer. With the neurofibrillar method used, the crossed terminal degeneration appears as rings with associated stalks. The finest of these can be confused with very occasional (about l/250,000 p3) rings found in normal undegenerate coiliculus. The uncrossed optic pathway consists of a small number of axons distributing in the anteromedial part of the stratum opticum, as well as a few asons at the posterior border of the colliculus. The last had not been seen previously (10, 16, 18) and varies from a few ambiguous granules in some animals to an obvious projection in others. In neither anterior nor posterior regions have uncrossed axons ever been seen running through the stratum griseum superficiale to the surface of the colliculus. Temporal Retinal Lesions at Birth-Operated Eye Removed as Adult. The lesions in each of these cases were in the upper temporal retinal
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FIG. 1. Nissl preparations of retina. A. Section of a lesion made at birth in the temporal retinal area of a Z-mo-old rat. X 327. B. Flat preparation showing the ganglion cell layer of a region of normal retina. X 480. C. Flat preparation showing the ganglion cell layer of the same retina as in B in a region situated peripheral to a lesion made at birth. Note the absence of large cells and generally lower packing density of cells. X 480.
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quadrant about half-way between the optic disc and the peripheral border. They all consisted of a single area of damage sometimes with an irregular border. A large part of the contralateral superior colliculus appears normally innervated. However, in the anterolateral region there is a zone where both Fink-Heimer and neurofrbrillar degeneration is atypical. In the superficial half of the stratum griseum superficiale, the density of each is reduced abruptly when followed from normal to abnormal projection zones (Fig. 2-4). At part of the border between the normally and abnormally innervated zones there is a column of tissue through the stratum griseum superficiale which has no degeneration or at the most only a few degeneration granules or rings (Fig. 3A). Since the column is narrow in this material, a close section series (one in three) is necessary to demonstrate it. In the deeper half of the stratum griseum superficiale throughout the whole abnormally innervated zone, Fink-Heimer staining shows very few fine granules and only occasional coarse fibers, most of which run horizontally. A comparison of this region and of a normally innervated zone is given in Fig. 2B and C. Neurofibrillar rings are extremely rare. This deeper part of the abnormal zone occupies a larger area than the more superficial part (Kg. 2,4), and along some of the border between the normally and abnormally innervated zones there are coarse degenerating axons curving from the stratum opticum underlying the normal region into the abnormal zone as they run to the surface. They are presumed to be optic axons which supply terminals to the abnormal region since there is no clear indication in the abnormal region of axons running directly from the stratum opticum into the overlying stratum griseum superficiale. The uncrossed degeneration is distributed as in normal animals although it may possibly be somewhat heavier in the posterior region and extend into the deeper stratum griseum superficiale. However, since there is variability in the distribution in normal animals no significance can be attached to this without a larger sample size. Temporal Retinal Lesions at Birth--Normal Eye Removed at 2 Months. The crossed degeneration appears normal in density and distribution. It might be expected according to the explanation presented for the results of this study that there should be a local reduction of crossed axons from the intact eye. It is thought that the degree of reduction of density of degeneration expected could not be easily detected in the 25 pm thick sections used here. The uncrossed pathway by contrast differs considerably from normal. It is heavier in its normal region of distribution, this being particularly noticeable posteriorly where a few degenerating fibers can be traced into the stratum griseum superficiale. There are also more uncrossed axons running throughout the more medial part of the stratum opticum. These
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FIG. 2. Degeneration in the superior colliculus resulting from removal of the contralateral eye, which had been received a lesion at birth. Fink-Heimer stain. A. The
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may be supplying the posterior region but their undirected appearance suggests that some of these may be preterminal. The most striking anomaly in the uncrossed pathway, however, is a column of degeneration running to the surface in the anterolateral region (Fig. 3B). The width of the column in these animals is approximately the same as that of the column almost devoid of a projection seen in the animals with the lesioned eye removed as described above (compare Fig 3A, B). It is best seen with Fink-Heimer stains but can also be recognized in neurofibrillar preparations (Fig. 4A compared with 4B). It is usually marked by a surface depression and the area between this and the edge of the colliculus appears slightly shrunken, In this latter region there is a lighter scatter of degeneration obtainable by both Fink-Heimer and neurofibrillar methods, While exact correlation has not been attempted, it is apparent that the heavy column of degeneration lies close to or confluent with the projection region of the neonatal lesion and the region between this and the edge of the colliculus corresponds to the projection zone of the retina peripheral to the lesion. Nasal Retinal Lesion at Birth-Eye Containing Lesion Removed at 2 Months. The results from these animals are the same as after a temporal lesion at birth, except that the abnormal projection region occupies the posterolateral part of the colliculus (the lesions were made in the upper retinal quadrant). The uncrossed degeneration in these animals is similar to a control. Nasal Retinal Lesion at Birth-Normal Eye Renaoved after 2 Months. These again appear similar to the animals with temporal lesions except that the column of uncrossed degeneration extends to the surface in the posterior part of the colliculus. The uncrossed degeneration in the more anterior part of the stratum opticum appears quite normal. Degeneration in the Lateral Geniculate Nucleus. Studies on the lateral geniculate nucleus (dorsal division) show a region with no projection or with a substantially reduced projection corresponding to the projection region of the lesion and the retina peripheral to the lesion. There is clearly an enlarged uncrossed pathway from the other eye, but the logic of this must await completion of further studies in the progress on the exact region of origin of the uncrossed pathway in normal albino rats. border zone between normally and abnormally innervated regions. The region of normal innervation is to the right. Note the progressively broadening band of reduced innervation in the deeper part of the stratum griseum superficiale, moving from right to left (arrows). X 107. B. Retinotectal degeneration in the abnormally innervated zone lying peripheral to the projection area of the original lesion. X 377. C. Retinotectal degeneration in a region of normal innervation. x 377.
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FIG. 3. Superior colliculus contralateral to the eye receiving a lesion at birth, FinkHeimer stain. A. Operated eye removed as adult. Zone of minimal innervation corresponding to the region of the projection zone of the lesion. X 380. B. Intact eye removed as adult. This shows the local zone of heavy aberrant degeneration from the intact eye extending to the surface of the ipsilateral colliculus. X 387.
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FIG. 4. Adjacent section to that shown in Fig. 3B, stained by the neurofibrillar method. A. Aberrant uncrossed degenerating rings (arrows) close to the surface in the region shown in Fig. 3B. X 2536. B. Absence of ring degeneration in the more medial part of the section shown in A. In adjacent Fink-Heimer stained sections, no degeneration is stained in this region and in this preparation only normal fibers are demonstrated. X 2536.
DISCUSSION The retinal lesions made in the newborn rats have two effects. First, they destroy completely a patch of ganglion cells. Second, they damage the axons of more peripherally situated ganglion cells, and these cells may then undergo retrograde degeneration. Morest (22) has shown in newborn rats that not all the axons from more centrally situated ganglion cells, which mature earlier than those in the periphery (23), have reached the optic disc. It is likely, therefore, that the axons of someof the ganglion cells situated peripheral to the lesion area had not yet grown up to this area at birth. These cells may survive and their axons subsequently grow around the lesion site to enter the optic nerve. These direct and indirect effects of the lesion in the retina become important when assessingthe reorganization of the centripetal visual pathway, both in relation to the pattern of distribution of the aberrant uncrossed pathway from the normal eye and to the pattern of projection of the eye with the lesion to the contralateral tectum. The aberrant uncrossed axons from the normal eye terminate heavily in the projection zone of the region of the lesion and less heavily in the zone between this and the margin of the tectum. It is not known whether this aberrant projection arises from the topographically appropriate region of the normal retina. In the light of the earlier studies (17-20), a topographic distribution of the abnormal uncrossed projection might be expected, so that in the present experiment the heavy projection should arise from the retinal area homotypic with
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that containing the lesion and the lighter projection from the retinal area where there is partial ganglion cell loss in the eye containing the lesion. The conclusion derived from these earlier studies was that removal of one eye at birth has an effect on later growing axons from the remaining eye which have yet to reach the optic chiasm such that they may end atypically in ipsilateral visual centers. Three alternatives can be suggested for this redirected growth: (a) The late growing axons are all misdirected to the ipsilateral side ; (b) some axons end ipsilaterally while others may run contralaterally; and (c) the axons branch at the optic chiasm to supply both sides of the brain. Whichever occurs, the present results suggest that the interaction is a local one between individual axons or bundles of axons from corresponding regions on the two retinae. One prerequisite for such interaction is that optic axons must run through the chiasm in an orderly and predictable fashion in order to meet their partner. The mechanism of the interaction could be one of two possibilities. First, the presence of degenerating axons may inhibit crossing and cause a local ipsilateral deviation. However, since there are normally uncrossed axons related to binocular fields, the presence of which is not determined as far as is known by degenerate counterparts, it is suggested that some influence other than the degeneration caused by lesions determines the course of optic axon growth. A second explanation for the results could be that there is a positive interaction between the growing tips of axons as they pass through the optic chiasm such that contact between homologous axons results in crossing. By this theory, fibers of the normal uncrossed pathway may be prevented from meeting their partner as a result of the geometry of the optic chiasm. Anomalous crossing such as has been shown for the Siamese cat (9a) could be explained in similar terms. Axons which normally would not meet as they run through the chiasm may come to occupy an inappropriate position in the centripetal optic bundle such that in the chiasm they meet and interact with those asons arising from the corresponding region of the other eye and so cross. In addition to the specific interaction, there is suggestion after temporal retinal lesions at birth that the uncrossed pathway from the intact eye becomes increased in both density and area within its normal lamina of distribution. This implies that there is a further pattern of reorganization occurring after the small lesions. The nature of this is currently under investigation. Two previous studies (6, 7) have indicated that after cutting one optic nerve in adult frogs, the regenerating axons may distribute to the tecta of both sides. This does not happen after crushing the nerve. Gaze and Keating (7) thought this might indicate that the aberrant ipsilaterally
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directed axons escape the confines of the nerve as they regenerate, and in terms of the present experiment it may be suggested that because they bypass the chiasm they do not have the appropriate positive stimulus to cross. Although a considerable number of studies on nonmammalian vertebrates (reviewed in 15) have been concerned with the central effects of unilateral enucleation during development, none has investigated whether there is maldistribution of the fibers of the remaining eye. The recent work on bird (15) showing complete loss of the optic fiber layers after unilateral enucleation early in development would tend to preclude the existence of a major primary uncrossed pathway in this situation. Studies on cat after enucleation within the first week after birth indicate, apart from interlaminar “sprouting” in the lateral geniculate body, that there is also an aberrant ipsilateral projection to lamina A from the remaining eye (9, 12). This result may have a similar explanation to the rat studies and be the result of misrouting of the last axons to reach the optic chiasm. The restriction of the aberrant projection to part only of lamina A is reminiscent of the partial uncrossed retinotectal and retinogeniculate pathways found in rats after enucleation at 5 days postnatal (17, 18) _ This would indicate that at birth the optic outgrowth in the cat is somewhat closer to maturity than in the rat. The apparent difference between birds on the one hand and rats (and possibly cats) on the other may lie in the nature of the normal uncrossed system relative to binocular vision. Studies on owls (13), for example, where there is a large binocular field, show a completely crossed optic pathway. Perhaps in birds, unlike mammals, the choice of crossing or not crossing at the chiasm is not available, and it is possible that as in Amphibia (8) binocular coordination is attained by secondary crossing between the tecta. In Amphibia at least (8) this secondary pathway can be modified by surgical manipulation of the primary pathway in development. Another difference which may be of importance in comparing mammalian and nonmammalian experiments is the time at which the eyes are removed. In our studies the lesions were made toward the end of optic nerve outgrowth; in birds it has usually been made much earlier (3, 14, 15). It is possible that once a system has been established by very specifically programmed early growing axons, the later growing axons show more modifiable growth patterns, such that they can be disturbed by lesions as in the present experiments. The abnormal projection from the eye with a lesion to the contralateral superior colliculus has two aspects to it. IFirst, there is a column from stratum opticum to the surface where there is virtually no degeneration. This appears to correspond to the projection site of the lesion itself. The maintenance of a zone lacking innervation by the eye with the lesion is
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consistent with previous findings in chick embryos operated at embryonic days 3 or 4 and in fish operated as adults when the optic nerve is also cut and allowed to regenerate (2, 3, 14, 15). In none of these experiments, however, was the possibility of redirection of optic axons from the other eye into the deafferented zone considered. The second feature of the abnormal projection from the eye with the lesion is the reduced density of termination in the region of the superior colliculus peripheral to the actual lesion projection zone. This region should receive a projection from the area of retina between the lesion site and the retinal periphery (24). The optic axons appear to enter this zone of the colliculus abnormally, curving in from the normally innervated colliculus rather than entering directly from the stratum opticum underlying the abnormal zone. This might suggest that the innervation of this region is from undamaged retina adjacent to the lesion; another possibility is that the optic fibers ending in this zone arise from ganglion cells which at the time of the lesion had not yet grown their axons through the damaged area. Their axons having grown around the lesion site may then follow an unusual course to the colliculus even to their mode of final entry into the stratum griseum superficiale. Such a possibility has a precedent in that apparently normal retinotectal topography has been shown in Amphibia after optic axons have been forced to regenerate by highly abnormal routes (1,5, il). It is noticeable in the retina that there are no large ganglion cells peripheral to the lesion site. It is possible that these cells are still present but in a shrunken form, the shrinkage caused perhaps by disturbance of their normal growth process. Another possibility, which would appear more likely, is that there is a real loss of the large ganglion cell population. Sidman (23) has shown in mouse at least that the large ganglion cells complete maturation before most of the small ones. It is conceivable, therefore, that their axons may have grown farther than those of the small later developing ganglion cells and are, therefore, more likely to be damaged by the lesion, their cell bodies undergoing subsequent retrograde degeneration. Quantitative studies on ganglion cell populations in normal and damaged retinae are currently underway to try and determine the nature of these changes. It may also be suggestedthat these large ganglion cells preferentially innervate the deeper part of the stratum griseum superficiale in the normal animal and that the very limited projection to this deeper zone in the animals with lesions is due to the loss of these cells. In summary, the results suggest some elaborate patterns of interaction at the optic chiasm. The experimental lesions produce changes in the patterns of interaction and as such demonstrate influences that affect the norma developmental process. This redirected growth cannot be considered in any
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way as axonal sprouting. The abnormal connections made relate to retinotopic maps of each eye and not to visual field maps. Therefore, it would appear that unless there is compensation at further connection between sensory and motor systems, the abnormal projection patterns of the retina do not represent a functional compensation for the lesion. REFERENCES 1. ARORA, H. J., and R. W. SPERRY. 1962. Optic nerve regeneration after surgical cross-union of medial and lateral optic tracts. Amer. Zaol. 2: 389. 2. ATTARDI, D. G., and R. W. SPERRY. 1963. Preferential selection of central pathways by regenerating optic fibers. Exg. Neurol. 7 : 46-64. 3. DE LONG, G. R., and A. J. COULOMBRE. 1965. Development of the retinotectal projection in the chick embryo. Ext. Neurol. 13: 351363. 4. FINK, R. P., and L. HEIMER. 1967. Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4 : 369-374 5. GAZE, R. M. 1959. Regeneration of the optic nerve in Xenopzls laevis. Quarf. J. E.zp. Physiol. 44 : 290-308. 6. GAZE, R. M., and M. JACOBSON. 1963. A study of retinotectal projections during regeneration of the optic nerve of the frog. Proc. Roy. Sot. B. 157: 420-448. 7. GAZE, R. M., and M. J. KEATING. 1970. Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog. Brain Res. 21: 183-195. S. GAZE, R. M., M. J. KEATING, G. SZEXELY, and L. BEAZLEY. 1970. Binocular interaction in the formation of specific intertectal neuronal connections. Proc. Roy. Sot. B. 175 : 107-147. 9. GUILLERY, R. W. 1972. Experiments to determine whether retinogeniculate axons can form translaminar collateral sprouts in the dorsal lateral geniculate nucleus of the cat. J. Comp. Neurol. 146 : 407-419. ?a. GUILLERY, R. W., and J. H. KAAS. 1971. A study of normal and congenitally abnormal retinogeniculate projections in cats. J. Camp. Neural. 143: 73-100. 10. HAYHOW, W. R., A. SEFTON, and G. WEBB. 1962. Primary optic centers of the rat in relation to the terminal distribution of the crossed and uncrossed optic nerve fibers. 1. Camp. Neural. 118 : 295-322. 11. HIBBARD, E. 1967. Visual recovery following regeneration of the optic nerve through the oculomotor nerve root in Xenopus. Exf. Neurol. 19: 350356. 12. KALIL, R. E. 1972. Formation of new retino-geniculate connections in kittens after removal of one eye. Anat. Rec. 172 : 339-340. 13. KARTEN, H., and W. J. H. NAUTA. 1968. Organization of retinothalamic projections in the pigeon and owl. Amt. Rec. 160 : 373. 14. KELLY, J. P. 1970. The specification of retinotectal connections in the avian embryo. Anat. Rec. 166 : 329. 15. KELLY, J. P., and W. M. COWAN. 1972. Studies on the development of the chick optic tectum. III. Effects of early eye removal. Brain Res. 42: 263-288. 16. LUND, R. D. 1965. Uncrossed visual pathways of hooded and albino rats. Science 149 : 1506-1507. 17. LUND, R. D. 1972. Anatomic studies on the superior colliculus. &vest. OphtlzeZ. 11: 434440.
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18. LUND, R. D., T. J. CUNNINGHAM, and J. S. LUND. 1973. Modified optic projections after unilateral eye removal in young rats. Bruiti Beh.av. Evolut. (in press). 19. LUND, R. D., and J. S. LUND. 1971a. Synaptic adjustment after deafferentation of the superior colliculus of the rat. Science 171 : 804-807. 20. LUND, R. D., and J. S. LUND. 1971b. Modification of synaptic patterns in the superior colliculus of the rat during development and following deafferentation. Vision Res. 11 Szrppl. 3 : 281-298. 21. LUND, R. D., and L. E. WESTRUM. 1966. Neurofibrils and the Nauta method. Science 149 : 1506-1507. 22. MOREST, D. K. 1970. The pattern of neurogenesis in the retina of the rat. 2. Anat. EntwGesch. 131: 45-67. 23. SIDMAN, R. L. 1961. Histogenesis of mouse retina studied with thymidine-3H, pp. 487-505. In “Structure of the Eye.” Smelser, G. K. [Ed.], Academic Press, New York. 24. SIMINOFF, R., H. 0. SCHWASSMAN, and L. KRUGER. 1966. An electrophysiological study of the visual projection to the superior colliculus of the rat. I. Comb. Neural. 127 : 435-444.
NEUROLOGY
40, 377-390 (1973)
Reorganization of the Retinotectal After Neonatal Retinal R. D.
LUND
Departmcrzts of Biological U>zie!ersity of Washington
AND
JENNIFER
Pathway Lesions S. LUND
Structure, Neurological School Received
of Medicine February
in Rats
’
Surgery and Ophthalmology, Seattle,
Washington
98195
24,1973
The normal retinotectal pathway of albino rats has very few uncrossed axons, limited in area of termination to a small region of the stratum opticum. If a small retinal lesion is made at birth, when not all optic axons have left the retina, there is in the adult a region in the stratum griseum superficiale of the contralateral superior colliculus which receives few or no axons from the operated eye. Peripheral to this, the colliculus receives a markedly reduced crossed optic projection. A dense aberrant ipsilateral projection is found from the intact eye to the region corresponding to the projection zone of the lesion and a lighter projection between this and the edge of the colliculus. The results are interpreted as indicating modified growth patterns of late growing optic axons which result in the aberrant uncrossed optic projection. As such, they suggest that there is a positive interaction between axons from corresponding retinal regions during their growth through the optic chiasm.
INTRODUCTION In previous anatomical studies (17-20) it was shown that unilateral eye removal from neonatal rats results in a subsequent expansion in the distribution of the optic pathway from the remaining eye to the subcortical visual centers. The additional projection is to parts of the superior colliculus and lateral geniculate body ipsilateral to the remaining eye which normally receive only crossed optic axons ; small lesion studies show this projection to originate from parts of the remaining retina which would normally 1 This investigation was supported by PHS Research Grants No. EY00491 and EY00596 from the National Eye Institute and in part by General Research Support Grant RR05432 from the National Institutes of Health, a research development Grant from Research to Prevent Blindness, Inc. and PHS Grant No. HD02274. We gratefully acknowledge the valuable technical assistance of Renee Wise, Franque Remington, and Jeri Hunter, and secretarial help of Carol Lade. 377 Copyright AU rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
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project onIy contralaterally. Despite their termination in the “wrong” side of the brain, the anomalous axons maintain an appropriate retinotopic pattern in both colliculus and geniculate. To explain these findings it was suggested [with some supporting evidence from a Golgi study (22) ] that at birth not all optic axons have grown past the optic chiasm. If one eye is removed in newborn rats, late growing axons are then misdirected, allowing the possibility that the chiasmatic crossing is the result of dynamic interaction of axons from both eyes rather than simply of growth properties determined in the retina. The possibility then arises that this interaction occurs between small groups of fibers such that a small retinal lesion at birth results in an area in each visual center devoid of a contralateral optic projection which is filled in by aberrant ipsilateral axons from the other eye. This possibility is investigated here. MATERIALS
AND
METHODS
Small retinal lesions of approximately equal size were made through the sclera in rats shortly after birth. The subsequent development of the operated eyes appeared normal in the nine successfully operated animals used in this study. The animals were allowed to survive for 2 mo, by which time all degeneration from neonatal lesions had disappeared. One eye was them removed and the animaIs allowed to survive for a further 4 days when they were perfused with 4% paraformaldehyde in phosphate buffer. Degeneration resulting from the eye removal was stained with Fink-Heimer (4) and neurofibrillar methods (21) on frozen sections cut 25 w thick. In three of the animals with temporal retinal lesions and two with nasal lesions, the eye which had originally been operated on was removed at the second operation in order to define central regions which may be free of degeneration and which would correspond to the projection zone of the damaged area. In the other four animals (two each with nasal or temporal retinal lesions), the eye contralateral to that with the lesion at birth was removed. This would allow recognition of aberrant uncrossed optic axons. In addition, control animals of four categories were studied. The first were completely unoperated normal adults ; the second were adult animals from which one eye had been removed 5 days prior to fixation; the third group were young rats 7, 14, 21, and 28 days old from which one eye had been removed at birth ; and the fourth consisted of one animal with a small retinal lesion at birth and no subsequent eye removal before perfusion at 2 mo old. All the eyes containing lesions were fixed by injection of 2% glutaraldehyde into the vitreous, and subsequently stained with cresyl violet acetate. One eye was stained as a flat mount; the others were embedded in celloidin and cut at 40 pm thickness.
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RESULTS Retinal Histology. The lesions in the eye made at birth can be recognized in each adult animal as a gap through all the retinal layers (Fig. 1A). With the exception of a region between the lesion and the retinal border, the retina appears completely normal when studied in flat preparation and in transverse section. Notable features of the normal retina are the density of packing of the ganglion cells, the wide diversity in cell body sizes (Fig. lB), and the bundles of optic axons running between groups of ganglion cells. In the region peripheral to the lesion (Fig. lC), the ganglion cell layer has fewer cells which in the flat preparation are seen to be distributed somewhat erratically, in some cases in small clusters ; the range of variation of cell body size is much less, largely because of an absence of the larger ganglion cells; and the circumscribed bundles of optic asons are not evident. Control Studies. The control animals with one eye removed at birth and short postoperative survival times show that within 21 days there is no persistent degeneration either of optic axons or terminals or of collicular cells undergoing a transneuronal reaction stainable by Fink-Heimer or neurofibrillar methods. No degeneration was found in the one control animal with a retinal lesion at birth and no subsequent eye removal. There is, therefore, no reason to believe that any of the degeneration attributed in the present studies to the second lesion is persistent from the neonatal lesion. Animals operated as adults (with no previous lesions) show that the normal crossed optic pathway is heavy throughout the colliculus. In FinkHeimer stained material, degeneration of this pathway appears as fine granules throughout the stratum griseum superficiale which are concentrated more heavily near the surface and coarser axonal debris, more prominent in the deeper part of the layer. With the neurofibrillar method used, the crossed terminal degeneration appears as rings with associated stalks. The finest of these can be confused with very occasional (about l/250,000 p3) rings found in normal undegenerate coiliculus. The uncrossed optic pathway consists of a small number of axons distributing in the anteromedial part of the stratum opticum, as well as a few asons at the posterior border of the colliculus. The last had not been seen previously (10, 16, 18) and varies from a few ambiguous granules in some animals to an obvious projection in others. In neither anterior nor posterior regions have uncrossed axons ever been seen running through the stratum griseum superficiale to the surface of the colliculus. Temporal Retinal Lesions at Birth-Operated Eye Removed as Adult. The lesions in each of these cases were in the upper temporal retinal
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FIG. 1. Nissl preparations of retina. A. Section of a lesion made at birth in the temporal retinal area of a Z-mo-old rat. X 327. B. Flat preparation showing the ganglion cell layer of a region of normal retina. X 480. C. Flat preparation showing the ganglion cell layer of the same retina as in B in a region situated peripheral to a lesion made at birth. Note the absence of large cells and generally lower packing density of cells. X 480.
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quadrant about half-way between the optic disc and the peripheral border. They all consisted of a single area of damage sometimes with an irregular border. A large part of the contralateral superior colliculus appears normally innervated. However, in the anterolateral region there is a zone where both Fink-Heimer and neurofrbrillar degeneration is atypical. In the superficial half of the stratum griseum superficiale, the density of each is reduced abruptly when followed from normal to abnormal projection zones (Fig. 2-4). At part of the border between the normally and abnormally innervated zones there is a column of tissue through the stratum griseum superficiale which has no degeneration or at the most only a few degeneration granules or rings (Fig. 3A). Since the column is narrow in this material, a close section series (one in three) is necessary to demonstrate it. In the deeper half of the stratum griseum superficiale throughout the whole abnormally innervated zone, Fink-Heimer staining shows very few fine granules and only occasional coarse fibers, most of which run horizontally. A comparison of this region and of a normally innervated zone is given in Fig. 2B and C. Neurofibrillar rings are extremely rare. This deeper part of the abnormal zone occupies a larger area than the more superficial part (Kg. 2,4), and along some of the border between the normally and abnormally innervated zones there are coarse degenerating axons curving from the stratum opticum underlying the normal region into the abnormal zone as they run to the surface. They are presumed to be optic axons which supply terminals to the abnormal region since there is no clear indication in the abnormal region of axons running directly from the stratum opticum into the overlying stratum griseum superficiale. The uncrossed degeneration is distributed as in normal animals although it may possibly be somewhat heavier in the posterior region and extend into the deeper stratum griseum superficiale. However, since there is variability in the distribution in normal animals no significance can be attached to this without a larger sample size. Temporal Retinal Lesions at Birth--Normal Eye Removed at 2 Months. The crossed degeneration appears normal in density and distribution. It might be expected according to the explanation presented for the results of this study that there should be a local reduction of crossed axons from the intact eye. It is thought that the degree of reduction of density of degeneration expected could not be easily detected in the 25 pm thick sections used here. The uncrossed pathway by contrast differs considerably from normal. It is heavier in its normal region of distribution, this being particularly noticeable posteriorly where a few degenerating fibers can be traced into the stratum griseum superficiale. There are also more uncrossed axons running throughout the more medial part of the stratum opticum. These
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FIG. 2. Degeneration in the superior colliculus resulting from removal of the contralateral eye, which had been received a lesion at birth. Fink-Heimer stain. A. The
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may be supplying the posterior region but their undirected appearance suggests that some of these may be preterminal. The most striking anomaly in the uncrossed pathway, however, is a column of degeneration running to the surface in the anterolateral region (Fig. 3B). The width of the column in these animals is approximately the same as that of the column almost devoid of a projection seen in the animals with the lesioned eye removed as described above (compare Fig 3A, B). It is best seen with Fink-Heimer stains but can also be recognized in neurofibrillar preparations (Fig. 4A compared with 4B). It is usually marked by a surface depression and the area between this and the edge of the colliculus appears slightly shrunken, In this latter region there is a lighter scatter of degeneration obtainable by both Fink-Heimer and neurofibrillar methods, While exact correlation has not been attempted, it is apparent that the heavy column of degeneration lies close to or confluent with the projection region of the neonatal lesion and the region between this and the edge of the colliculus corresponds to the projection zone of the retina peripheral to the lesion. Nasal Retinal Lesion at Birth-Eye Containing Lesion Removed at 2 Months. The results from these animals are the same as after a temporal lesion at birth, except that the abnormal projection region occupies the posterolateral part of the colliculus (the lesions were made in the upper retinal quadrant). The uncrossed degeneration in these animals is similar to a control. Nasal Retinal Lesion at Birth-Normal Eye Renaoved after 2 Months. These again appear similar to the animals with temporal lesions except that the column of uncrossed degeneration extends to the surface in the posterior part of the colliculus. The uncrossed degeneration in the more anterior part of the stratum opticum appears quite normal. Degeneration in the Lateral Geniculate Nucleus. Studies on the lateral geniculate nucleus (dorsal division) show a region with no projection or with a substantially reduced projection corresponding to the projection region of the lesion and the retina peripheral to the lesion. There is clearly an enlarged uncrossed pathway from the other eye, but the logic of this must await completion of further studies in the progress on the exact region of origin of the uncrossed pathway in normal albino rats. border zone between normally and abnormally innervated regions. The region of normal innervation is to the right. Note the progressively broadening band of reduced innervation in the deeper part of the stratum griseum superficiale, moving from right to left (arrows). X 107. B. Retinotectal degeneration in the abnormally innervated zone lying peripheral to the projection area of the original lesion. X 377. C. Retinotectal degeneration in a region of normal innervation. x 377.
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FIG. 3. Superior colliculus contralateral to the eye receiving a lesion at birth, FinkHeimer stain. A. Operated eye removed as adult. Zone of minimal innervation corresponding to the region of the projection zone of the lesion. X 380. B. Intact eye removed as adult. This shows the local zone of heavy aberrant degeneration from the intact eye extending to the surface of the ipsilateral colliculus. X 387.
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FIG. 4. Adjacent section to that shown in Fig. 3B, stained by the neurofibrillar method. A. Aberrant uncrossed degenerating rings (arrows) close to the surface in the region shown in Fig. 3B. X 2536. B. Absence of ring degeneration in the more medial part of the section shown in A. In adjacent Fink-Heimer stained sections, no degeneration is stained in this region and in this preparation only normal fibers are demonstrated. X 2536.
DISCUSSION The retinal lesions made in the newborn rats have two effects. First, they destroy completely a patch of ganglion cells. Second, they damage the axons of more peripherally situated ganglion cells, and these cells may then undergo retrograde degeneration. Morest (22) has shown in newborn rats that not all the axons from more centrally situated ganglion cells, which mature earlier than those in the periphery (23), have reached the optic disc. It is likely, therefore, that the axons of someof the ganglion cells situated peripheral to the lesion area had not yet grown up to this area at birth. These cells may survive and their axons subsequently grow around the lesion site to enter the optic nerve. These direct and indirect effects of the lesion in the retina become important when assessingthe reorganization of the centripetal visual pathway, both in relation to the pattern of distribution of the aberrant uncrossed pathway from the normal eye and to the pattern of projection of the eye with the lesion to the contralateral tectum. The aberrant uncrossed axons from the normal eye terminate heavily in the projection zone of the region of the lesion and less heavily in the zone between this and the margin of the tectum. It is not known whether this aberrant projection arises from the topographically appropriate region of the normal retina. In the light of the earlier studies (17-20), a topographic distribution of the abnormal uncrossed projection might be expected, so that in the present experiment the heavy projection should arise from the retinal area homotypic with
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that containing the lesion and the lighter projection from the retinal area where there is partial ganglion cell loss in the eye containing the lesion. The conclusion derived from these earlier studies was that removal of one eye at birth has an effect on later growing axons from the remaining eye which have yet to reach the optic chiasm such that they may end atypically in ipsilateral visual centers. Three alternatives can be suggested for this redirected growth: (a) The late growing axons are all misdirected to the ipsilateral side ; (b) some axons end ipsilaterally while others may run contralaterally; and (c) the axons branch at the optic chiasm to supply both sides of the brain. Whichever occurs, the present results suggest that the interaction is a local one between individual axons or bundles of axons from corresponding regions on the two retinae. One prerequisite for such interaction is that optic axons must run through the chiasm in an orderly and predictable fashion in order to meet their partner. The mechanism of the interaction could be one of two possibilities. First, the presence of degenerating axons may inhibit crossing and cause a local ipsilateral deviation. However, since there are normally uncrossed axons related to binocular fields, the presence of which is not determined as far as is known by degenerate counterparts, it is suggested that some influence other than the degeneration caused by lesions determines the course of optic axon growth. A second explanation for the results could be that there is a positive interaction between the growing tips of axons as they pass through the optic chiasm such that contact between homologous axons results in crossing. By this theory, fibers of the normal uncrossed pathway may be prevented from meeting their partner as a result of the geometry of the optic chiasm. Anomalous crossing such as has been shown for the Siamese cat (9a) could be explained in similar terms. Axons which normally would not meet as they run through the chiasm may come to occupy an inappropriate position in the centripetal optic bundle such that in the chiasm they meet and interact with those asons arising from the corresponding region of the other eye and so cross. In addition to the specific interaction, there is suggestion after temporal retinal lesions at birth that the uncrossed pathway from the intact eye becomes increased in both density and area within its normal lamina of distribution. This implies that there is a further pattern of reorganization occurring after the small lesions. The nature of this is currently under investigation. Two previous studies (6, 7) have indicated that after cutting one optic nerve in adult frogs, the regenerating axons may distribute to the tecta of both sides. This does not happen after crushing the nerve. Gaze and Keating (7) thought this might indicate that the aberrant ipsilaterally
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directed axons escape the confines of the nerve as they regenerate, and in terms of the present experiment it may be suggested that because they bypass the chiasm they do not have the appropriate positive stimulus to cross. Although a considerable number of studies on nonmammalian vertebrates (reviewed in 15) have been concerned with the central effects of unilateral enucleation during development, none has investigated whether there is maldistribution of the fibers of the remaining eye. The recent work on bird (15) showing complete loss of the optic fiber layers after unilateral enucleation early in development would tend to preclude the existence of a major primary uncrossed pathway in this situation. Studies on cat after enucleation within the first week after birth indicate, apart from interlaminar “sprouting” in the lateral geniculate body, that there is also an aberrant ipsilateral projection to lamina A from the remaining eye (9, 12). This result may have a similar explanation to the rat studies and be the result of misrouting of the last axons to reach the optic chiasm. The restriction of the aberrant projection to part only of lamina A is reminiscent of the partial uncrossed retinotectal and retinogeniculate pathways found in rats after enucleation at 5 days postnatal (17, 18) _ This would indicate that at birth the optic outgrowth in the cat is somewhat closer to maturity than in the rat. The apparent difference between birds on the one hand and rats (and possibly cats) on the other may lie in the nature of the normal uncrossed system relative to binocular vision. Studies on owls (13), for example, where there is a large binocular field, show a completely crossed optic pathway. Perhaps in birds, unlike mammals, the choice of crossing or not crossing at the chiasm is not available, and it is possible that as in Amphibia (8) binocular coordination is attained by secondary crossing between the tecta. In Amphibia at least (8) this secondary pathway can be modified by surgical manipulation of the primary pathway in development. Another difference which may be of importance in comparing mammalian and nonmammalian experiments is the time at which the eyes are removed. In our studies the lesions were made toward the end of optic nerve outgrowth; in birds it has usually been made much earlier (3, 14, 15). It is possible that once a system has been established by very specifically programmed early growing axons, the later growing axons show more modifiable growth patterns, such that they can be disturbed by lesions as in the present experiments. The abnormal projection from the eye with a lesion to the contralateral superior colliculus has two aspects to it. IFirst, there is a column from stratum opticum to the surface where there is virtually no degeneration. This appears to correspond to the projection site of the lesion itself. The maintenance of a zone lacking innervation by the eye with the lesion is
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consistent with previous findings in chick embryos operated at embryonic days 3 or 4 and in fish operated as adults when the optic nerve is also cut and allowed to regenerate (2, 3, 14, 15). In none of these experiments, however, was the possibility of redirection of optic axons from the other eye into the deafferented zone considered. The second feature of the abnormal projection from the eye with the lesion is the reduced density of termination in the region of the superior colliculus peripheral to the actual lesion projection zone. This region should receive a projection from the area of retina between the lesion site and the retinal periphery (24). The optic axons appear to enter this zone of the colliculus abnormally, curving in from the normally innervated colliculus rather than entering directly from the stratum opticum underlying the abnormal zone. This might suggest that the innervation of this region is from undamaged retina adjacent to the lesion; another possibility is that the optic fibers ending in this zone arise from ganglion cells which at the time of the lesion had not yet grown their axons through the damaged area. Their axons having grown around the lesion site may then follow an unusual course to the colliculus even to their mode of final entry into the stratum griseum superficiale. Such a possibility has a precedent in that apparently normal retinotectal topography has been shown in Amphibia after optic axons have been forced to regenerate by highly abnormal routes (1,5, il). It is noticeable in the retina that there are no large ganglion cells peripheral to the lesion site. It is possible that these cells are still present but in a shrunken form, the shrinkage caused perhaps by disturbance of their normal growth process. Another possibility, which would appear more likely, is that there is a real loss of the large ganglion cell population. Sidman (23) has shown in mouse at least that the large ganglion cells complete maturation before most of the small ones. It is conceivable, therefore, that their axons may have grown farther than those of the small later developing ganglion cells and are, therefore, more likely to be damaged by the lesion, their cell bodies undergoing subsequent retrograde degeneration. Quantitative studies on ganglion cell populations in normal and damaged retinae are currently underway to try and determine the nature of these changes. It may also be suggestedthat these large ganglion cells preferentially innervate the deeper part of the stratum griseum superficiale in the normal animal and that the very limited projection to this deeper zone in the animals with lesions is due to the loss of these cells. In summary, the results suggest some elaborate patterns of interaction at the optic chiasm. The experimental lesions produce changes in the patterns of interaction and as such demonstrate influences that affect the norma developmental process. This redirected growth cannot be considered in any
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way as axonal sprouting. The abnormal connections made relate to retinotopic maps of each eye and not to visual field maps. Therefore, it would appear that unless there is compensation at further connection between sensory and motor systems, the abnormal projection patterns of the retina do not represent a functional compensation for the lesion. REFERENCES 1. ARORA, H. J., and R. W. SPERRY. 1962. Optic nerve regeneration after surgical cross-union of medial and lateral optic tracts. Amer. Zaol. 2: 389. 2. ATTARDI, D. G., and R. W. SPERRY. 1963. Preferential selection of central pathways by regenerating optic fibers. Exg. Neurol. 7 : 46-64. 3. DE LONG, G. R., and A. J. COULOMBRE. 1965. Development of the retinotectal projection in the chick embryo. Ext. Neurol. 13: 351363. 4. FINK, R. P., and L. HEIMER. 1967. Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4 : 369-374 5. GAZE, R. M. 1959. Regeneration of the optic nerve in Xenopzls laevis. Quarf. J. E.zp. Physiol. 44 : 290-308. 6. GAZE, R. M., and M. JACOBSON. 1963. A study of retinotectal projections during regeneration of the optic nerve of the frog. Proc. Roy. Sot. B. 157: 420-448. 7. GAZE, R. M., and M. J. KEATING. 1970. Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog. Brain Res. 21: 183-195. S. GAZE, R. M., M. J. KEATING, G. SZEXELY, and L. BEAZLEY. 1970. Binocular interaction in the formation of specific intertectal neuronal connections. Proc. Roy. Sot. B. 175 : 107-147. 9. GUILLERY, R. W. 1972. Experiments to determine whether retinogeniculate axons can form translaminar collateral sprouts in the dorsal lateral geniculate nucleus of the cat. J. Comp. Neurol. 146 : 407-419. ?a. GUILLERY, R. W., and J. H. KAAS. 1971. A study of normal and congenitally abnormal retinogeniculate projections in cats. J. Camp. Neural. 143: 73-100. 10. HAYHOW, W. R., A. SEFTON, and G. WEBB. 1962. Primary optic centers of the rat in relation to the terminal distribution of the crossed and uncrossed optic nerve fibers. 1. Camp. Neural. 118 : 295-322. 11. HIBBARD, E. 1967. Visual recovery following regeneration of the optic nerve through the oculomotor nerve root in Xenopus. Exf. Neurol. 19: 350356. 12. KALIL, R. E. 1972. Formation of new retino-geniculate connections in kittens after removal of one eye. Anat. Rec. 172 : 339-340. 13. KARTEN, H., and W. J. H. NAUTA. 1968. Organization of retinothalamic projections in the pigeon and owl. Amt. Rec. 160 : 373. 14. KELLY, J. P. 1970. The specification of retinotectal connections in the avian embryo. Anat. Rec. 166 : 329. 15. KELLY, J. P., and W. M. COWAN. 1972. Studies on the development of the chick optic tectum. III. Effects of early eye removal. Brain Res. 42: 263-288. 16. LUND, R. D. 1965. Uncrossed visual pathways of hooded and albino rats. Science 149 : 1506-1507. 17. LUND, R. D. 1972. Anatomic studies on the superior colliculus. &vest. OphtlzeZ. 11: 434440.
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18. LUND, R. D., T. J. CUNNINGHAM, and J. S. LUND. 1973. Modified optic projections after unilateral eye removal in young rats. Bruiti Beh.av. Evolut. (in press). 19. LUND, R. D., and J. S. LUND. 1971a. Synaptic adjustment after deafferentation of the superior colliculus of the rat. Science 171 : 804-807. 20. LUND, R. D., and J. S. LUND. 1971b. Modification of synaptic patterns in the superior colliculus of the rat during development and following deafferentation. Vision Res. 11 Szrppl. 3 : 281-298. 21. LUND, R. D., and L. E. WESTRUM. 1966. Neurofibrils and the Nauta method. Science 149 : 1506-1507. 22. MOREST, D. K. 1970. The pattern of neurogenesis in the retina of the rat. 2. Anat. EntwGesch. 131: 45-67. 23. SIDMAN, R. L. 1961. Histogenesis of mouse retina studied with thymidine-3H, pp. 487-505. In “Structure of the Eye.” Smelser, G. K. [Ed.], Academic Press, New York. 24. SIMINOFF, R., H. 0. SCHWASSMAN, and L. KRUGER. 1966. An electrophysiological study of the visual projection to the superior colliculus of the rat. I. Comb. Neural. 127 : 435-444.