mersion in Carnoy's solution. The brains were prepared for par- affin embedding and were serially sectioned in a coronal plane at 15 jum. From each brain a ...
Proc. Natl. Acad. Sci. USA Vol. 80, pp. 6131-6135, October 1983
Neurobiology
Topographic organization of certain tectal afferent and efferent connections can develop normally in the absence of retinal input (chicken embryo and chick/visual system development/isthmic nuclei/autoradiography/wheat germ agglutinin-conjugated horseradish peroxidase)
DENNIS D. M. O'LEARY AND W. MAXWELL COWAN The Salk Institute for Biological Studies and The Clayton Foundation for Research-California Division, P.O. Box 85800, San Diego, CA 92138
Contributed by W. Maxwell Cowan, July 11, 1983
ABSTRACT To test the hypothesis that the topographic organization of the connections of the optic tectum is determined during development by the "retinotopically" ordered input that it receives from the eye, we have mapped certain of the connections of the tectum in chicken embryos and chicks in which both eye rudiments were removed before the outgrowth of optic fibers. Because several of the connections of the tectum are normally organized retinotopically, we should expect, if this hypothesis were correct, that some or all of these connectional patterns would be significantly altered in such "eyeless" chickens. In fact, we have found that the connections formed between the optic tectum and two of the isthmic nuclei, with which it is reciprocally connected, show the same topographic organization in "eyeless" animals as in control chickens raised under the same conditions. This clearly indicates that the topographic organization of the chicken optic tectum is independently specified and is not contingent upon the input that it receives from the retina.
Several hypotheses have been -put forward to account for the orderly patterns of connections that develop between related populations of nerve cells in the vertebrate nervous system. At present the hypothesis that commands the widest support is the so-called chemoaffinity hypothesis first clearly formulated by Sperry (1, 2) to account for the striking order found in the connections between the retina and the major visual center of the brain of lower vertebrates, the optic tectum. This order underlies both normal, visually mediated behavior and also the capacity of certain fish and amphibians to respond to visual stimuli after regeneration of the optic nerve. Essential to this hypothesis is the notion that during development individual neurons or groups of neurons acquire distinctive chemical labels that both distinguish them from other neurons or groups of neurons within the same population and, at the same time, enable them to correctly identify their appropriate targets. The cells in the target area are thought to bear on their surfaces matching or complementary labels. Although there is now a considerable body of indirect evidence in support of this idea, a critical element in the hypothesis that remains to be tested concerns the independent acquisition of labels by the neurons in the project-
labels within the tectum but throw no light on how they were initially acquired (8, 9). It is conceivable, for example, that early in development the tectum is like a tabula rasa upon which incoming fibers from the retina impose their characteristic topographic organization and that it is from this interaction that the cells in the tectum acquire their distinctive labels (10-12). In the absence of molecular probes that could establish positively whether or not the cells in the tectum have position-determining labels on their surfaces prior to the arrival of retinal fibers, we only can address indirectly the issue of the independent specification of the tectum. One way to do this is to examine the topographic organization of certain of the efferent and nonretinal afferent connections of the tectum that are normally "retinotopically" ordered, in animals in which the optic tectum has developed in the complete absence of retinal input. The chick optic tectum provides an advantageous system for experiments of this kind. It normally receives an orderly input from the contralateral retina (13, 14) and is known to form retinotopically ordered connections with several other structures in the brainstem and, in particular, with two nuclei in the isthmic region-the nucleus isthmi pars parvocellularis (Ipc) and the nucleus semilunaris (Slu)-with which it is reciprocally connected. Because it is possible to extirpate both optic vesicles before the axons of the first-formed retinal ganglion cells leave the eye (15, 16), some 3-4 days before optic fibers normally reach the optic tectum (17, 18), one can raise chicken embryos in which the tectum has not been influenced by the retina. Neuroanatomical tracing procedures can then be used to determine whether the topographic organization of the other afferent and efferent connections formed by the tectum is similar to that seen in normal chicks or if it is significantly altered.
ing and target populations. That such labels exist and play a role in the reestablishment of topographically ordered connections
during the regeneration of neural pathways has been demonstrated in the retinotectal system by a variety of experiments involving the rotation or translocation of portions of the optic tectum (3-7). However, it has been argued that such experiments simply confirm the persistence of position-determining
METHODS The brains of 66 chicken embryos and chicks of a White Leghorn strain were used for this study. Most of the brains were from embryos which ranged in age between 18 and 20 days of incubation, but four were from posthatched animals. The embryos were raised in an incubator at 380C and a high relative humidity. On day2 of incubation, both optic vesicles were ablated with a fine glass needle between stages 10+ and 12+ of the Hamburger and Hamilton (19) series. A second group of unoperated embryos was raised under the same conditions; these, together with the four posthatched chicks, were used to establish the normal pattern of tectoisthmal connections.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: Ipc, nucleus isthmi pars parvocellularis; Slu, nucleus semilunaris; WGA-HRP, wheat germ agglutinin-conjugated horseradish peroxidase.
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Neurobiology: O'Leary and Cowan
To map the connections between the optic tectum and the isthmic nuclei, we have used both the anterograde transport of proteins labeled with [3H]proline and the anterograde and retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Both the experimental and control embryos were injected with the appropriate tracer on day 18, 19, or 20 of incubation; the posthatched animals were injected during the first week after hatching. For the autoradiographic method, 2-5 ACi (1 uCi/10 nl; 1 Ci = 37 GBq) of L-[2, 3-3H(N)]proline (specificity activity- 20 Ci/mM, New England Nuclear) were injected into the outer layers of the tectum with a 1-Al Hamilton syringe. For the WGA-HRP experiments, 20 nl of a 1% solution of WGA-HRP (Sigma) were similarly injected. The posthatched animals were anesthetized with chloral hydrate, and a 2% solution of WGA-HRP was iontophoresed through a glass micropipette. The embryos were decapitated 6-10.hr. after the [3H]proline injections, and their brains were removed and fixed by immersion in Carnoy's solution. The brains were prepared for paraffin embedding and were serially sectioned in a coronal plane at 15 jum. From each brain a 1-in-5 series of sections covering the extent of the midbrain and isthmic region was processed for autoradiography following the protocol of Cowan et al. (20). After similar survival. periods, the embryos injected with WGA-HRP were anesthetized and perfused with 1.5% glutaraldehyde/1% paraformaldehyde/0. 1 M phosphate-buffer, pH 7.4. The posthatched animals were perfused- in the same way 24 or 48 hr after WGA-HRP injections. The perfused brains were stored overnight at 4°C in phosphate buffer .with 10% sucrose and then sectioned transversely at 50 .um on a freezing microtome. Two 1-in-2 series of sections through the midbrain and isthmic regions were mounted onto gelatin-coated slides and treated as described by Mesulam (21). To assess the completeness of the bilateral enucleations, the operated embryos were visually examined on day 6 of incubation and, when killed, their heads were carefully dissected. All cases in which the eye removals were incomplete were discarded. Before the brains were sectioned, a drawing of the optic tectum was made with the aid of a dissecting microscope and
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FIG. 2. A schematic diagram to illustrate the topographical relationships between the retina, the optic tectum, and the isthmic nuclei. The retinal quadrants -of the right eye project, in order, upon the left optic lobe; the topography ofthis projection is reflected in the reciprocal connections between the optic tectum and the ipsilateral isthmic nuclei Ipc and Slu. I, inferior; N, nasal; S, superior; T, temporal; D, dorsal; R, rostral; V, ventral; OpT, optic tectum.
drawing tube, and the point of entry of the injection needle was marked on this drawing. Tracings of selected sections were made with a microscope and an attached drawing tube; on these the extent of the injection site in the tectum and the distribution of the axonally transported label in the Ipc and Slu were plotted. To compare the results from different cases, the positions of the injection sites were normalized for variations in brain size
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FIG. 3. The appearance of two typical injection sites in the optic tectum (marked. by arrowheads). (A) [3H]Proline -injection. (B) WGAHRP injection.- Dorsal is to the top;-medial is tothe left. (Bar: 1 mm.)
dS.
Neurobiology: O'Leary and Cowan
Proc. Natl. Acad. Sci. USA 80 (1983)
and then plotted onto a standard drawing of a lateral view of the chicken brain. RESULTS The Organization of Tectoisthmic Connections in the Normal Chicken. To define the normal topographic organization of the connections between the optic tectum and the Ipc and Slu, small injections of either [3H]proline (n = 36) or WGA-HRP (n = 7) were made into the superficial layers of the optic tectum at different -sites; the [3lH]proline-treated cases served to define the efferent projections of the tectum, whereas the WGAHRP-treated cases permitted the simultaneous visualization of the efferent projections from the tectum and of the cells of origin of the reciprocal projections from the Ipc and Slu to the tectum. As the findings with the two methods were in good agreement in both the embryos and posthatched animals and because, in the WGA-HRP-treated cases, the retrogradely labeled cells in the Ipc and Slu were confined to the zone of anterograde labeling of the tectal efferents, our results can be summarized by reference to Figs. 1 and 2. In Fig. 1 we have plotted the location of the injection sites and the distribution of the resulting axonally transported label in the isthmic nuclei in a group of four of these control cases; in Fig. 2 we have extrapolated from these and the other control cases in this series
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to provide a schematic representation of the overall retinotectoisthmal projection. From this it is clear that the rostral half of the optic tectum (which receives its visual input from the temporal quadrants of the retina) sends its efferents to, and receives afferents from, the corresponding rostral parts of the Ipc and Slu; the dorsal part of the tectum (which receives its input from the inferior retinal quadrants) is reciprocally connected with the dorsal parts of the Ipc and Slu. These results are in basic agreement with the descriptions of tectoisthmal relations reported by others (22, 23). The Organization of the Tectoisthmic Connections in "Eyeless" Chickens. To determine the topographic arrangement of the connections that develop between the optic tectum and the Ipc and Slu in the absence of retinal input, both optic vesicles were removed in a series of chicken embryos on day 2 of incubation about 24 hr before the appearance of retinal ganglion cell axons (15, 16). Just before the normal time of hatching, a localized injection-of either [3H]proline (n = 15) or WGA-HRP (n.= 8) was made into the superficial layers of the optic tectum. The appearance of two typical injection sites (case BE-14, [3H]proline; and case BEH-8, WGA-HRP) are shown in Fig. 3. In both cases-the anterogradely transported tracer, marking the projection from the tectum to the Ipc and, Slu, was as well localized as in the control animals (Figs. 4 and 5). In addition, in case BEH-8 large numbers of neurons in the Ipc and Slu were
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FIG. 5. Bright- and darkfield photomicrographs, from case. BEH?-8, to show the distribution of anterogradely labeled axons and- retrogradely labeled neurons in the Ipc (A-and B) and the Slu (C and D). The bright spots outside the zones of labeling in B and D are due to debris and/or endogenous HRP containing nonneuronal cells. Dorsal is to the top; medial is to the left.t(Bar 100 um.)
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Neurobiology: O'Leary and CowanPProc. Natl. Acad. Sci. USA 80 (1983)
FIG. 6. Drawing to show the injection sites in four representative
"eyeless" cases. See Fig. 1 for abbreviations.
retrogradely labeled by the WGA-HRP; as in all of the WGAHRP-labeled cases, these neurons were confined to the zone of anterograde labeling (Fig. 5). Collectively the various injection sites in the cases analyzed covered most of the surface of the tectum, and we can briefly state that the patterns of connections between the tectum and the related isthmic nuclei that they reveal was topographically indistinguishable from that found in control animals. A few representative cases will serve to make this point. In Fig. 6 we have plotted the locations of the tectal injection sites in four embryos reared without eyes. In three of these (cases BEH-4, BEH-8, and BEH-8R), WGA-HRP had been injected into the tectum; in the fourth (case BE-14), the injection was of [3H]proline. The distribution of the axonally transported label within the Ipc and Slu in these four cases is plotted on tracings of serial sections through the two nuclei in Fig. 7. From a comparison of the two sets of figures, it is evident that each injection resulted in a fairly localized zone of labeling within the Ipc and the Slu and that the location of the labeled region
4
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varied systematically with the site of the injection. Consider first the dorsoventral dimension of the tectum. In case BEH8 the injection was located well within the caudodorsal quadrant of the tectum, whereas in BEH-8R a smaller injection was made in the caudoventral quadrant, centered at a slightly more caudal level. The resulting labeling in the isthmic nuclei in BEH8 was limited to the dorsal part of the caudal half of the Ipc and Slu, whereas in BEH-8R it was confined to the ventral halves of both nuclei over a more restricted rostrocaudal extent. The organization of the tectoisthmic connections along the rostrocaudal dimension of the tectum can similarly be shown by reference to cases BEH-8R and BE-14. As we have seen, the injection in BEH-8R was confined to the caudoventral tectal quadrant, whereas that in BE-14 was more or less in the center of the tectum. In the isthmic nuclei in these two brains, the labeling was correspondingly more rostrally located in BE-14 (as well as being at a slightly more dorsal level). The same arrangement can be shown in the dorsal part of the tectum: the injection in case BEH-8 was confined to the caudodorsal tectal quadrant, whereas that in BEH-4 occupied much of the rostrodorsal quadrant; in the isthmic nuclei this difference was reflected in the fact that in case BEH-4 the labeled fibers and cells were limited to the rostral halves of the Ipc and Slu, whereas the labeling in BEH-8 was more or less confined to the caudal portion of each nucleus. From these and our other cases of "eyeless" animals, it is clear that the general topographic organization of the tectal projections to and from the Ipc and the Slu is essentially the same as that seen in normal chicks. This can perhaps be illustrated best by comparing the findings in one of the "eyeless" chicken embryos with those in a control experiment in which the injection site in the tectum was comparable both in size and position. Cases BEH-8 and BEC-28 illustrate this point. In both brains the injection was confined to the caudodorsal quadrant of the tectum (Figs. 1 and 6) and resulted in a localized patch
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FIG. 7. Serial tracings of coronal sections through the Ipc and Slu taken from the four experimental cases whose injection sites are plotted in Fig. 6. The regions of labeling and their relative densities are indicated in each section. For the three "BEH" cases, every fourth 50-tIm section containing the Ipc and every other 50-tum section containing the Slu are shown. For case BE-14, every 20th 15-;Lm section containing the Ipc and every 10th 15-tum section containing the Slu are illustrated. C, caudal; D, dorsal; L, lateral.
Neurobiology: O'Leary and Cowan of labeling in the caudodorsal parts of both the Ipc and Slu, essentially the same rostrocaudal extent (Figs. 1 and 7).
over
DISCUSSION The principal finding in this study is clear and unequivocal: in embryos in which the tectum has developed in the complete absence of retinal input, two of its major "retinotopically" ordered sets of afferent and efferent connections form patterns of connections that are topographically indistinguishable from those seen in normal animals. It seems reasonable to conclude from this that, from the point of view of its ability to generate topographically ordered connections, the tectum is not dependent on its innervation from the retina. In this sense our findings are incompatible with the view that the topographic order recognizable in the tectum is imposed upon it by its retinal input. And, by extension, it seems not unlikely that the topographic order seen in other sensory areas of the vertebrate central nervous system will be found to be similarly independent of their peripheral input. In support of this generalization, we may cite the parallel observations of Constantine-Paton and Ferrari-Eastman (24), who have reported that the connections between the nucleus isthmi and the tectum develop with an essentially normal topography in frogs that were bilaterally enucleated at an early age, and of Kaiserman-Abramof et al. (25), who have reported that the dorsal lateral geniculate nucleus maps in a more-or-less normal manner upon the visual cortex in congenitally anophthalmic mice. Thus, our findings are consistent with the hypothesis that position-dependent labels are responsible, at least in part, for the mapping of neural connections and that these labels are capable of developing independently in related neural structures. However, at present we cannot rule out the possibility that the apparent retinotopic order found in the afferent and efferent connections of the tectum in our eyeless animals is imposed upon the tectum by some other topographically ordered input. It is important to emphasize that the observation that the development of topographic order in the optic tectum is not dependent on its retinal input does not imply that the retinal afferents play no role in shaping the connectivity of the tectum. On the contrary, it is well-known that in the chicken (as in other vertebrates), eye removal results in substantial degenerative changes in the tectum (26, 24); this almost certainly has important repercussions for the connections formed by the optic lobes, and it is worth noting in this context that, although the topography of the connections between the tectum and the isthmic nuclei in "eyeless" chickens is normal, the isthmic nuclei as a group, and especially the Ipc, show a good deal of atrophy. Similarly, there is now a large body of evidence to show that the visual system is capable of substantial reorganization when confronted by abnormal retinal inputs. For example, in Siamese cats, in which an abnormally large number of retinal fibers project to the contralateral side, there can be major changes
Proc. Natl. Acad. Sci. USA 80 (1983)
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in the morphology of the lateral geniculate nucleus and in the connections of the striate cortex (27-29). Similarly, it has been shown in frogs that early rotation of one eye can lead to a substantial readjustment in the crossed isthmotectal projection, which maintains the congruence of the two visuotectal maps (30), and that visually elicited neural activity is an essential factor in this readjustment (31). We thank Mr. Kris Trulock for photographic assistance and Ms. Pat Thomas for her careful typing of the manuscript. This work was supported in part by Grant EY-03653 from the National Institutes of Health. 1. Sperry, R. W. (1950) in Genetic Neurology, ed. Weiss, P. (Univ. of Chicago Press, Chicago), pp. 232-239. 2. Sperry, R. W. (1963) Proc. Natl. Acad. Sci. USA 50, 703-710. 3. Sharma, S. C. & Gaze, R. M. (1971) Arch. Ital. Biol. 109, 357366. 4. Levine, R. L. & Jacobson, M. (1974) Exp. Neurol. 43, 527-538. 5. Jacobson, M. & Levine, R. L. (1975) Brain Res. 92, 468-471. 6. Yoon, M. G. (1975)J. Physiol. (London) 252, 137-158. 7. Romeskie, M. & Sharma, S. C. (1980) Brain Res. 201, 202-205. 8. Jacobson, M. (1978) in Developmental Neurobiology (Plenum, New York), pp. 345-433.
9. Fraser, S. E. & Hunt, R. K. (1980) Annu. Rev. Neurosci. 3, 319352. 10. von der Malsburg, Ch. & Willshaw, D. J. (1977) Proc. Natl. Acad. Sci. USA 74, 5176-5178. 11. Horder, T. J. & Martin, K. A. C. (1978) in Cell-Cell Recognition, Society of Experimental Biology Symposium, ed. Curtis, A. S. G. (Cambridge Univ. Press, Cambridge, England), pp. 275-358. 12. Schmidt, J. T. (1978) J. Comp. Neurol. 177, 279-300. 13. Crossland, W J., Cowan, W M., Rogers, L. A. & Kelly, J. P. (1974) J. Comp. Neurol. 155, 127-164. 14. Crossland, W. J. & Uchwat, C. J. (1979)J. Comp. Neurol. 185, 87106. 15. Goldberg, S. & Coulombre, A. J. (1972) J. Comp. Neurol. 146, 507518. 16. Krayanek, S. & Goldberg, S. (1981) Dev. Biol. 84, 41-50. 17. Goldberg, S. (1974) Dev. Biol. 36, 24-43. 18. Crossland, W J., Cowan, W M. & Rogers, L. A. (1975) Brain Res. 91, 1-23. 19. Hamburger, V. & Hamilton, H. L. (1951)1. Morphol. 88, 49-92. 20. Cowan, W M., Gottlieb, D. I., Hendrickson, A. E., Price, J. L. & Woolsey, T. A. (1972) Brain Res. 37, 21-51. 21. Mesulam, M.-M. (1978)J. Histochem. Cytochem. 26, 106-117. 22. Hart, J. R. (1969) Dissertation (Univ. of Wisconsin, Madison). 23. Hunt, S. P. & Kunzle, H. (1976)J. Comp. Neurol. 170, 173-190. 24. Constantine-Paton, M. & Ferrari-Eastman, P. (1981) J. Comp. Neurol. 196, 645-661. 25. Kaiserman-Abramof, I. R., Graybiel, A. M. & Nauta, W. J. H.
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26. Kelly, J. P. & Cowan, W. M. (1972) Brain Res. 42, 263-288. 27. Guillery, R. W. (1969) Brain Res. 14, 739-741. 28. Hubel, D. H. & Wiesel, T. N. (1971)J. Physiol. (London) 281, 3362. 29. Shatz, C. J. (1977)J. Comp. Neurol. 171, 205-228. 30. Udin, S. B. & Keating, M. J. (1981) J. Comp. Neurol. 203, 575594. 31. Keating, M. J. & Feldman, J. (1975) Proc. R. Soc. London Ser. B 191, 467-474.
