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The developing chick isthmo-optic nucleus forms a transient efferent projection to the optic tectum
Neuroscience Letters, 113 (1990) 241 246 Elsevier Scientific Publishers Ireland Ltd.
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The developing chick isthmo-optic nucleus forms a transient efferent projection to the optic tectum Andrea Wizenmann and Solon Thanos* Max-Planck-lnstitut fiir Entwicklungsbiologie, Tiibingen ( F.R.G. ) (Received 12 November 1989; Revised version received 2 February 1990; Accepted 9 February 1990) Key word~: Visual system; Isthmo-optic nucleus; Development; Transient projections; Fluorescent dyes; Chick The present work describes the formation of a transient efferent axonal projection from the isthmo-optic nucleus (ION) to the ipsilateral optic tectum of the chick embryo. Local application of either the carbocyanine dye Dil or rhodamine-B-isothiocyanate (RITC) to the superficial layers of the optic rectum resulted in retrograde labeling of the corresponding retinal region, and in anterograde staining of tectal axons projecting to the ION. In addition to these known projections, retrogradely labeled ION neurons appeared to be filled from the tectum. This projection, called the isthmo-tectal projection, could be characterized by means of various staining techniques: (i) It first appears at embryonic day E9 and gradually disappears after day El6. It is absent in the hatched chick. (ii) Both the cells inside the ION and those situated outside the border of the ION, the so-called ectopic cells, contribute to the formation of the isthmo-tectal projection. (iii) Double labeling from the contralateral retina (Fast blue) and from the ipsilateral tectum (DiI or RITC) revealed that some of the ION fibers projecting to the tectum are collaterals of axons normally directed to the retina. (iv) Microsurgical removal of the eye anlage early in development resulted in a numerical increase of the ION-tectal fibers. The results are discussed in terms of the role of transient projections during development.
T h e i s t h m o - o p t i c n u c l e u s ( I O N ) , the m o s t c a u d a l visual c e n t e r in birds, is the site o f o r i g i n o f c e n t r i f u g a l fibers to the retina. Its a n a t o m i c a l o r g a n i z a t i o n [7, 8, 13], f u n c t i o n a l role [10, 17] a n d d e v e l o p m e n t h a v e b e e n i n v e s t i g a t e d in the past. A c c o r d ing to these studies the I O N receives a r e t i n o t o p i c a l l y o r g a n i z e d p r o j e c t i o n f r o m the ipsilateral t e c t u m [8, 9] a n d is efferently c o n n e c t e d with the a m a c r i n e s a n d d i s p l a c e d g a n g l i o n cells o f the c o n t r a l a t e r a l r e t i n a [2, 12]. E l e c t r o p h y s i o l o g i c a l studies h a v e s h o w n t h a t the c e n t r i f u g a l i n p u t to the r e t i n a c a n e n h a n c e r e t i n a l excitability u s u a l l y by s u p p r e s s i n g i n h i b i t o r y s u r r o u n d m e c h a n i s m s o f r e t i n a l g a n g l i o n cells [17].
*Present address: Dept. of Ophthalmology, University School of Medicine, Schleichstr, 12, D-74 Tiibingen, F.R.G. Correspondence." A. Wizenmann, Max-Planck-Institut ffir Entwicklungsbiologie, Spemannstr. 35/I, D-7400 Tiibingen, F.R.G. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
242 During the period of neuronal cell death (El2 to El7) approximately 60% of the neurons degenerate within the 1ON [5]. The number of surviving cells can be experimentally manipulated by removal of either the efferent target [15] or the afferent neurons [3]. Recent studies propose a crucial role for the cell death in refining an initially imprecise projection of the ION to the retina [2]. This implies that ION-fibers are misguided to inappropriate retinal areas and are eliminated in the course of neuronal cell death: In the present work we have examined the development and disposition of a transient efferent projection of the ION to the tectum of the ipsilateral side. Fertilized white leghorn eggs were incubated at 38~C until embryonic day 3 (E3) when the embryos were placed in plastic Petri dishes for further shell-less cultivation [1]. For production of monocular embryos extirpation of one eye anlage was performed at the second day of incubation. For filling of ION cells projecting to the retina [15] we used the fluorescent marker Fast blue (FB, Illing) injected into the vitreous body (3-5/d aqueous solution, 2 3%). For labeling of ION neurons projecting to the rectum we used either DiI (D282, N,Ndioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate, Molecular Probes, OR, U.S.A.) [11, 19] or RITC (R1755, Rhodamine-B-isothiocyanate, Sigma, St. Louis) [18]. After various times of incubation (E8/9/10/14 until E 13,/16/17 and 18) the embryos were decapitated and the brains were fixed in 4% paraformaldehyde for about 2 days. The brains were then sectioned coronally at 100/~m with a vibratome (DiI labeled neurons) or at 50/~m with a cryostat in cases of R1TC labeled specimen. The vibratome sections were mounted in 90% glycerol and the cryostat slides were air-dried for examination with the fluorescence microscope (Zeiss). The position of all DiI/ RITC and FB labeled neurons in the ION was captured with a camera lucida. For studying the projections at hatched stages, 1-4 weeks hatched chickens (n = 6) were anesthetized with an intramuscular injection of Ketanest and Rompun ( 1 ml/kg) and then labeled with Dil and FB as for the embryos. Unexpectedly, at day E9 and later, retrogradely labeled cells were observed in the ION ipsilateral to the tectum where the dye had been applied to (Fig. I ). These cells were localized both within the confines of the ION, and sometimes also outside the nucleus, namely in the region around the nuclear capsule, the so-called region with ectopic ION-neurons [4]. In 8 of 13 embryos analyzed, the labeled cells were scattered throughout the extent of the ION and there was no indication of topographic distrubtion of these cells according to the tecto-isthmic topography [3], For instance, the ION cells labeled from the anterior-ventral part of the tectum were either scattered throughout the nucleus or they were located within the ventral heminucleus. Cells labeled from more dorsal or medial parts of the rectum also showed a scattered distribution within the ION, suggesting that there is no topography of this projection. To test the hypothesis whether the newly discovered isthmotectal fibers could be collaterals of fibers projecting to the retina, a double-labeling experiment was designed: FB was injected into one eye and Dil or RITC was placed on the contrala-
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Fig. 1. Retrograde labeling in the ION with FB in the retina (a) and with DiI from the optic tectum (b) of an embryo at El6. The staining with FB from the contralateral retina outlines the entire ION (a) whereas the number of DiI labeled neurons from the ipsilateral tectum is significantly lower (b). c; drawing illustrating the pattern of double-labeling as it appears by superposition of the fluorescence photographs in (a) and (b). ©, cells labeled with FB; x , cells labeled with DiI; ®, double-labeled neurons. Scale bar: 100 pm.
teral tectum. The experimental series (n = 60) revealed that between days E9 and E ! 6/ 17 ION neurons can be double-labeled. After double-labeling, the ION was completely outlined by using FB fluorescence filter (Fig. la, c) and some of its blue cells were also fluorescent in the red RITC filter (Fig. lb,c). In addition to the doublelabeled neurons, which apparently project to both retina and tectum, also single-labeled cells of either color could be observed in 19 out of 60 embryos analyzed. On the other hand, the observation that some ION neurons were not labeled from the retina but only from the rectum indicates that either at least some part of the nucleus does not project to the retina or that Fast blue did not label all axons arriving at the retina. The labeling from the tectum was restricted to the embryonic days E9 to El6/17. In none of 6 hatched chickens could labeled ION neurons be found after application of either RITC or DiI to the tectum. Obviously, the ION projection to the tectum is eliminated after day El6. Whether only the axon collaterals degenerate or whether all those ION neurons exhibiting the double projection to the retina and tectum die, must remain an open question. In another series of experiments one eye of the embryo was enucleated. In such monocular embryos (n = 6) FB injected into the remaining retina labeled ION neurons of both sides of the brain (Fig. 2a, b). This projection is characterized by a massive increase of the ipsilateral ION-retina projection (Fig. 2b) as compared to normal animals [14]. Placement of DiI on the tectum lying ipsilaterally to the remaining eye revealed that each nucleus could be labeled (Fig. 2c,d). The ipsilateral ION, however, was massively labeled from the tectum (Fig. 2d), indicating that the absence of the
244 c o r r e s p o n d i n g c o n t r a l a t e r a l eye r e s u l t e d i n m i s g u i d a n c e o f l a r g e p o p u l a t i o n s o f I O N f i b e r s to t h e t e c t u m .
Fig. 2. Fluorescence micrographs showing the ION projection to the retina (a, b) and on the tectum (c, d) of a monocular embryo. The remaining eye received a FB injection at day El0 and the tecta received Dil at the same time. The embryo was killed at day El3. Both ION project to the retina (a, b) although the ipsilateral (ipsi) projection is less extensive than the contralateral (contra) one. The relatively weak normal projection of the ION to the tectum (c) is dramatically increased in the brain side that was devoid of a retinal innervation by removing the contralateral eye cup (d). Scale bar: 100 pro.
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_ ) Fig. 3. Schematic drawing illustrating the connectivity relationships between the 3 main parts of the chick visual system, the retina, the tectum and the ION. The well-established projections (continuous lines) are drawn from the left retina to the right tectum and from there to the ipsilateral ION. The nucleus projects back to the retina with axons terminating on amacrines and displaced ganglion cells. The arrowheads on each line indicate the direction from the corresponding cell bodies to the axonal terminals. The neuroanatomically detected projections from the right retina on the left ION are numbered from 1 to 4. Axonal bundle No. 1 is a transient projection from the retina to the ION [17]. The projections 2 and 4 are for the first time described in the present work. For detailed description of each one of these projections see text. Abbreviations: ION, isthmo-optic nucleus; OM, nucleus oculomotorius; SGC, stratum griseum centrale; SGFS, stratum griseum et fibrosum superficiale; SO, stratum opticum; VENTR., ventricle.
In order to facilitate the understanding o f the connectivity between retina, tectum and I O N , all k n o w n neuronal projections between these main chick visual areas are summarized in the schematic drawing o f Figure 3. N o t e the retinal fibers that form a transient bundle to the contralaterai I O N (No. 1, Fig. 3). The developmental time course and arrangement along the persisting isthmo-retinal projection suggests a 'guidance template' function during embryogenesis [16] for this transient retino-isthmic bundle. It is intriguing that also the I O N seems to form a transient tectal projection (Nos. 2, 4, Fig. 3), to an area which in the adult is efferently connected with the I O N . This transient projection seems to consist o f neurons which project both to the retina and apparently via collaterals to the tectum (No. 2, Fig. 3) and o f neurons which form a projection only to the tectum (No. 4, Fig. 3). The role o f the transient I O N - t e c t u m projection remains unclear, although our results suggest that it m a y play a role as a 'template' for the persistent tectal-ischmic fibers, growing out later in development in the opposite direction. It is tempting to speculate that the transient, ipsilateral projections from the I O N to the tectum, described here, and from the retina to the I O N , described by O ' L e a r y and T h a n o s [16], m a y account for the presence o f the above mentioned, a b n o r m a l ipsilateral projections in the m o n o c u l a r chick, thereby supporting the template hypothesis.
246 T h e a u t h o r s a r e g r a t e f u l t o F. B o n h o e f f e r f o r s u p p o r t i n g t h e w o r k . W e a l s o t h a n k D r . D . D u e t t i n g f o r p r e p a r i n g t h e m o n o c u l a r e m b r y o s a n d D r s . T. A l l s o p , Y. v. B o x b e r g a n d F. B o n h o e f f e r f o r c r i t i c a l c o m m e n t s o n t h e m a n u s c r i p t . I Auerbach, R., Kubal, L., Knighton, D. and Folkman, J., A simple procedure for the long-term cultivation of chicken embryos, Dev. Biol., 41 (1974) 391 394. 2 Catsicas, S., Thanos, S. and Clarke, P.G.H., Major role for neuronal death during brain development: refinement of topographical connections, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8165 8168. 3 Clarke~ P.G.H., Neuronal death during development in the isthmo-optic nucleus of the chick: sustaining role of afferents from the tectum, J. Comp. Neurol., 234 (1985) 365 -379. 4 Clarke, P.G.H. and Cowan, W.M., The development of the isthmo-optic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons, J. Comp. Neurol., 167 (1976) 143- 164. 5 Clarke, P.G.H., Rogers, L.A. and Cowan, W.M., The time of origin and the pattern of survival of neurons in the isthmo-optic nucleus of the chick, J. Comp. Neurol., 167 (1976) 125 142. 6 Cowan~ W.M., Centrifugal fibers to the avian retina, Br. Med. Bull., 26 (1970) 112 117. 7 Cowan, W.M. and Wenger, E., The development of the nucleus of origin of centrifugal fibers to the retina in the chick, J. Comp. Neurol., 133 (1968) 207 240. 8 Crossland, W.J. and Hughes, C.P., Observations on the afferent and efferent connections of the avian isthmo-optic nucleus, Brain Res., 145 (1978) 239-256. 9 DeLong, G.R. and Coulombre, A.J., Development of the retinotectal topographic projection in the chick embryo, Exp. Neurol., 13 (1965) 351 363. 10 Holden, A.L. and Powell, T.P.S., The functional organization of the isthmo-optic nucleus in the pigeon, J. Physiol. (Lond.), 223 (1972) 419~-447. 1! Honig, M.G. and Hume, R.I., Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures, J. Cell Biol., 103 (1986) 171 187. 12 Maturana, H.R. and Frenk, S., Synaptic connections of the centrifugal fibers in the pigeon retina, Science, 150(1965) 359 361. 13 McGill, J.I., Powell, T.P.S. and Cowan, W.M., The organization of the projection of the centrifugal fibres to the retina in the pigeon, J. Anat., 100 (1966) 35~49. 14 O'Leary, D.D.M. and Cowan, W.M., Survival of isthmo-optic neurons after early removal of one eye, Dev. Brain Res., 12 (1984) 293 310. 15 O'Leary, D.D.M., Fawcett, J.W. and Cowan, W.M., Topographic targeting errors in the retinocollicular projection and their elimination by selective ganglion cell death, J. Neurosci., 6 (1986) 3692 3705. 16 O'Leary, D.D.M. and Thanos, S., A transient retinofugal pathway in the chick embryo and its possible role in the guidance of centrifugal axons to the retina, Anat. Rec., 211 (1985) 42A. 17 Rogers~ L.J. and Miles, F.A., Centrifugal control of the avian retina. V. Effects of lesions of the isthmooptic nucleus on visual behaviour, Brain Res., 48 (1972) 147 156. 18 Thanos, S. and Bonhoeffer, F., Investigations on the development and topographic order of retinotectal axons: anterograde and retrograde staining of axons and perikarya with rhodamine in vivo, J. Comp. Neurol., 219 (1983) 420-~430. 19 Thanos, S. and Bonhoeffer, F., Axonal arborization in the developing chick retinotectal system, J. Comp. Neurol., 261 (1987) 155 164.
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The developing chick isthmo-optic nucleus forms a transient efferent projection to the optic tectum Andrea Wizenmann and Solon Thanos* Max-Planck-lnstitut fiir Entwicklungsbiologie, Tiibingen ( F.R.G. ) (Received 12 November 1989; Revised version received 2 February 1990; Accepted 9 February 1990) Key word~: Visual system; Isthmo-optic nucleus; Development; Transient projections; Fluorescent dyes; Chick The present work describes the formation of a transient efferent axonal projection from the isthmo-optic nucleus (ION) to the ipsilateral optic tectum of the chick embryo. Local application of either the carbocyanine dye Dil or rhodamine-B-isothiocyanate (RITC) to the superficial layers of the optic rectum resulted in retrograde labeling of the corresponding retinal region, and in anterograde staining of tectal axons projecting to the ION. In addition to these known projections, retrogradely labeled ION neurons appeared to be filled from the tectum. This projection, called the isthmo-tectal projection, could be characterized by means of various staining techniques: (i) It first appears at embryonic day E9 and gradually disappears after day El6. It is absent in the hatched chick. (ii) Both the cells inside the ION and those situated outside the border of the ION, the so-called ectopic cells, contribute to the formation of the isthmo-tectal projection. (iii) Double labeling from the contralateral retina (Fast blue) and from the ipsilateral tectum (DiI or RITC) revealed that some of the ION fibers projecting to the tectum are collaterals of axons normally directed to the retina. (iv) Microsurgical removal of the eye anlage early in development resulted in a numerical increase of the ION-tectal fibers. The results are discussed in terms of the role of transient projections during development.
T h e i s t h m o - o p t i c n u c l e u s ( I O N ) , the m o s t c a u d a l visual c e n t e r in birds, is the site o f o r i g i n o f c e n t r i f u g a l fibers to the retina. Its a n a t o m i c a l o r g a n i z a t i o n [7, 8, 13], f u n c t i o n a l role [10, 17] a n d d e v e l o p m e n t h a v e b e e n i n v e s t i g a t e d in the past. A c c o r d ing to these studies the I O N receives a r e t i n o t o p i c a l l y o r g a n i z e d p r o j e c t i o n f r o m the ipsilateral t e c t u m [8, 9] a n d is efferently c o n n e c t e d with the a m a c r i n e s a n d d i s p l a c e d g a n g l i o n cells o f the c o n t r a l a t e r a l r e t i n a [2, 12]. E l e c t r o p h y s i o l o g i c a l studies h a v e s h o w n t h a t the c e n t r i f u g a l i n p u t to the r e t i n a c a n e n h a n c e r e t i n a l excitability u s u a l l y by s u p p r e s s i n g i n h i b i t o r y s u r r o u n d m e c h a n i s m s o f r e t i n a l g a n g l i o n cells [17].
*Present address: Dept. of Ophthalmology, University School of Medicine, Schleichstr, 12, D-74 Tiibingen, F.R.G. Correspondence." A. Wizenmann, Max-Planck-Institut ffir Entwicklungsbiologie, Spemannstr. 35/I, D-7400 Tiibingen, F.R.G. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
242 During the period of neuronal cell death (El2 to El7) approximately 60% of the neurons degenerate within the 1ON [5]. The number of surviving cells can be experimentally manipulated by removal of either the efferent target [15] or the afferent neurons [3]. Recent studies propose a crucial role for the cell death in refining an initially imprecise projection of the ION to the retina [2]. This implies that ION-fibers are misguided to inappropriate retinal areas and are eliminated in the course of neuronal cell death: In the present work we have examined the development and disposition of a transient efferent projection of the ION to the tectum of the ipsilateral side. Fertilized white leghorn eggs were incubated at 38~C until embryonic day 3 (E3) when the embryos were placed in plastic Petri dishes for further shell-less cultivation [1]. For production of monocular embryos extirpation of one eye anlage was performed at the second day of incubation. For filling of ION cells projecting to the retina [15] we used the fluorescent marker Fast blue (FB, Illing) injected into the vitreous body (3-5/d aqueous solution, 2 3%). For labeling of ION neurons projecting to the rectum we used either DiI (D282, N,Ndioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate, Molecular Probes, OR, U.S.A.) [11, 19] or RITC (R1755, Rhodamine-B-isothiocyanate, Sigma, St. Louis) [18]. After various times of incubation (E8/9/10/14 until E 13,/16/17 and 18) the embryos were decapitated and the brains were fixed in 4% paraformaldehyde for about 2 days. The brains were then sectioned coronally at 100/~m with a vibratome (DiI labeled neurons) or at 50/~m with a cryostat in cases of R1TC labeled specimen. The vibratome sections were mounted in 90% glycerol and the cryostat slides were air-dried for examination with the fluorescence microscope (Zeiss). The position of all DiI/ RITC and FB labeled neurons in the ION was captured with a camera lucida. For studying the projections at hatched stages, 1-4 weeks hatched chickens (n = 6) were anesthetized with an intramuscular injection of Ketanest and Rompun ( 1 ml/kg) and then labeled with Dil and FB as for the embryos. Unexpectedly, at day E9 and later, retrogradely labeled cells were observed in the ION ipsilateral to the tectum where the dye had been applied to (Fig. I ). These cells were localized both within the confines of the ION, and sometimes also outside the nucleus, namely in the region around the nuclear capsule, the so-called region with ectopic ION-neurons [4]. In 8 of 13 embryos analyzed, the labeled cells were scattered throughout the extent of the ION and there was no indication of topographic distrubtion of these cells according to the tecto-isthmic topography [3], For instance, the ION cells labeled from the anterior-ventral part of the tectum were either scattered throughout the nucleus or they were located within the ventral heminucleus. Cells labeled from more dorsal or medial parts of the rectum also showed a scattered distribution within the ION, suggesting that there is no topography of this projection. To test the hypothesis whether the newly discovered isthmotectal fibers could be collaterals of fibers projecting to the retina, a double-labeling experiment was designed: FB was injected into one eye and Dil or RITC was placed on the contrala-
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Fig. 1. Retrograde labeling in the ION with FB in the retina (a) and with DiI from the optic tectum (b) of an embryo at El6. The staining with FB from the contralateral retina outlines the entire ION (a) whereas the number of DiI labeled neurons from the ipsilateral tectum is significantly lower (b). c; drawing illustrating the pattern of double-labeling as it appears by superposition of the fluorescence photographs in (a) and (b). ©, cells labeled with FB; x , cells labeled with DiI; ®, double-labeled neurons. Scale bar: 100 pm.
teral tectum. The experimental series (n = 60) revealed that between days E9 and E ! 6/ 17 ION neurons can be double-labeled. After double-labeling, the ION was completely outlined by using FB fluorescence filter (Fig. la, c) and some of its blue cells were also fluorescent in the red RITC filter (Fig. lb,c). In addition to the doublelabeled neurons, which apparently project to both retina and tectum, also single-labeled cells of either color could be observed in 19 out of 60 embryos analyzed. On the other hand, the observation that some ION neurons were not labeled from the retina but only from the rectum indicates that either at least some part of the nucleus does not project to the retina or that Fast blue did not label all axons arriving at the retina. The labeling from the tectum was restricted to the embryonic days E9 to El6/17. In none of 6 hatched chickens could labeled ION neurons be found after application of either RITC or DiI to the tectum. Obviously, the ION projection to the tectum is eliminated after day El6. Whether only the axon collaterals degenerate or whether all those ION neurons exhibiting the double projection to the retina and tectum die, must remain an open question. In another series of experiments one eye of the embryo was enucleated. In such monocular embryos (n = 6) FB injected into the remaining retina labeled ION neurons of both sides of the brain (Fig. 2a, b). This projection is characterized by a massive increase of the ipsilateral ION-retina projection (Fig. 2b) as compared to normal animals [14]. Placement of DiI on the tectum lying ipsilaterally to the remaining eye revealed that each nucleus could be labeled (Fig. 2c,d). The ipsilateral ION, however, was massively labeled from the tectum (Fig. 2d), indicating that the absence of the
244 c o r r e s p o n d i n g c o n t r a l a t e r a l eye r e s u l t e d i n m i s g u i d a n c e o f l a r g e p o p u l a t i o n s o f I O N f i b e r s to t h e t e c t u m .
Fig. 2. Fluorescence micrographs showing the ION projection to the retina (a, b) and on the tectum (c, d) of a monocular embryo. The remaining eye received a FB injection at day El0 and the tecta received Dil at the same time. The embryo was killed at day El3. Both ION project to the retina (a, b) although the ipsilateral (ipsi) projection is less extensive than the contralateral (contra) one. The relatively weak normal projection of the ION to the tectum (c) is dramatically increased in the brain side that was devoid of a retinal innervation by removing the contralateral eye cup (d). Scale bar: 100 pro.
245
_ ) Fig. 3. Schematic drawing illustrating the connectivity relationships between the 3 main parts of the chick visual system, the retina, the tectum and the ION. The well-established projections (continuous lines) are drawn from the left retina to the right tectum and from there to the ipsilateral ION. The nucleus projects back to the retina with axons terminating on amacrines and displaced ganglion cells. The arrowheads on each line indicate the direction from the corresponding cell bodies to the axonal terminals. The neuroanatomically detected projections from the right retina on the left ION are numbered from 1 to 4. Axonal bundle No. 1 is a transient projection from the retina to the ION [17]. The projections 2 and 4 are for the first time described in the present work. For detailed description of each one of these projections see text. Abbreviations: ION, isthmo-optic nucleus; OM, nucleus oculomotorius; SGC, stratum griseum centrale; SGFS, stratum griseum et fibrosum superficiale; SO, stratum opticum; VENTR., ventricle.
In order to facilitate the understanding o f the connectivity between retina, tectum and I O N , all k n o w n neuronal projections between these main chick visual areas are summarized in the schematic drawing o f Figure 3. N o t e the retinal fibers that form a transient bundle to the contralaterai I O N (No. 1, Fig. 3). The developmental time course and arrangement along the persisting isthmo-retinal projection suggests a 'guidance template' function during embryogenesis [16] for this transient retino-isthmic bundle. It is intriguing that also the I O N seems to form a transient tectal projection (Nos. 2, 4, Fig. 3), to an area which in the adult is efferently connected with the I O N . This transient projection seems to consist o f neurons which project both to the retina and apparently via collaterals to the tectum (No. 2, Fig. 3) and o f neurons which form a projection only to the tectum (No. 4, Fig. 3). The role o f the transient I O N - t e c t u m projection remains unclear, although our results suggest that it m a y play a role as a 'template' for the persistent tectal-ischmic fibers, growing out later in development in the opposite direction. It is tempting to speculate that the transient, ipsilateral projections from the I O N to the tectum, described here, and from the retina to the I O N , described by O ' L e a r y and T h a n o s [16], m a y account for the presence o f the above mentioned, a b n o r m a l ipsilateral projections in the m o n o c u l a r chick, thereby supporting the template hypothesis.
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